COLDWATER FISHERIES AND

AQUACULTURE MANAGEMENT
Technology for Sustainable Food Production
 COLDWATER FISHERIES AND
AQUACULTURE MANAGEMENT
Technology for Sustainable Food Production

Edited by
Mohd. Ashraf Rather, PhD
Faisal Rashid Sofi, PhD
Adnan Amin, PhD
Kawkabul Saba, PhD
First edition published 2024
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Library and Archives Canada Cataloguing in Publication

Title: Coldwater fisheries and aquaculture management : technology for sustainable food production / edited by Mohd.
Ashraf Rather, PhD, Faisal Rashid Sofi, PhD, Adnan Amin, PhD, Kawkabul Saba, PhD.
Names: Rather, Mohd. Ashraf, editor. | Sofi, Faisal Rashid, editor. | Amin, Adnan (Lecturer in aquatic environmental
management), editor. | Saba, Kawkabul, editor.
Description: First edition. | Includes bibliographical references and index.
Identifiers: Canadiana (print) 20230474950 | Canadiana (ebook) 20230474985 | ISBN 9781774913420 (hardcover) |
ISBN 9781774913437 (softcover) | ISBN 9781003369905 (ebook)
Subjects: LCSH: Fish culture. | LCSH: Freshwater fishes. | LCSH: Sustainable aquaculture—Management. | LCSH:
Sustainable fisheries.
Classification: LCC SH159 .C65 2024 | DDC 639.3/1—dc23
Library of Congress Cataloging-in-Publication Data

CIP data on file with US Library of Congress

ISBN: 978-1-77491-342-0 (hbk)

ISBN: 978-1-77491-343-7 (pbk)
ISBN: 978-1-00336-990-5 (ebk)
 Dedication
Dedicated to Fish Farmers

“If you give a hungry man a fish, you feed him for a day,
but if you teach him how to fish, you feed him for a lifetime.”
—Lao Tsu
About the Editors

Mohd. Ashraf Rather, PhD

Assistant Professor, Division of Fish Genetics and Biotechnology Faculty
of Fisheries, Rangil, Ganderbal, Sher-e-Kashmir University of Agricultural
Sciences and Technology–Kashmir, India
Mohd. Ashraf Rather, PhD, is working as an Assistant Professor in the
Division of Fish Genetics and Biotechnology at the Faculty of Fisheries,
Rangil, Ganderbal, Sher-e-Kashmir University of Agricultural Sciences and
Technology, Kashmir. He graduated from the College of Fisheries, Ratnagiri,
Maharashtra, and obtained master’s and doctoral degrees in Fisheries
Biotechnology from the Division of Genetics and Biotechnology, Central
Institute of Fisheries Education (CIFE), Mumbai. His area of research
interest is reproductive physiology and molecular endocrinology of fish
proteins. His major research contribution is the identification, characteriza­
tion, and expression profiling of more than 25 reproductive genes in fish,
including kisspeptin genes in Indian major carp. He has more than eight
years of experience working on the molecular endocrinology of fish.
Dr. Rather is a recipient of various accolades such as Prof. K. H. Alikunhi
Gold medal from the Central Institute of Fisheries Education and Dr. Karu­
nasagar Best Post-Graduate Thesis (PhD – Indian category) award from
the Professional Fisheries Graduates Forum (PFGF). He received a Young
Scientist Award in 2017 from the Society of Fisheries and Life Science,
College of Fisheries, Mangalore. He has also received an Asia-Pacific Alltech
Young Scientist Award–2012 (first rank) in Agriculture and Allied Field from
Alltech, USA. He is a recipient of various national and international fellow­
ships, including the Prince Songkla Fellowship from the Prince Songkla
University of Thailand, the National Post-Doc fellowship (NPDF) from the
Science and Engineering Research Board (SERB), Department of Science
and Technology–New Delhi, Indian Science Academy Fellowship, Senior
Research Fellowship (SRF, first rank) and Junior Research Fellowship (JRF,
first rank) from the Indian Council of Agricultural Research (ICAR), New
Delhi.
He has published more than 40 research publications in both national and
international peer-reviewed research journals. In addition, he has published
two books, two conference proceedings, and various chapters. He functions
viii About the Editors

as an editorial board member in various international and national research

journals. To date, Dr. Rather has received three best research paper awards
from various international and national institutes and organizations. He has
mentored several master’s and doctoral students pursuing higher studies in
fisheries biotechnology/fisheries science.

Faisal Rashid Sofi, PhD

Assistant Professor, Division of Post-Harvest Technology (Fisheries),
Faculty of Fisheries, Sher-e-Kashmir University of Agricultural Sciences
and Technology–Rangil, Kashmir, India
Faisal Rashid Sofi, PhD, is an Assistant Professor in the Division of Post-
Harvest Technology (Fisheries), Faculty of Fisheries, Sher-e-Kashmir
University of Agricultural Sciences and Technology—Rangil, Kashmir. He
graduated from the College of Fisheries, Veraval, Junagadh Agricultural
University, Gujarat, and obtained a master’s degree from the same univer­
sity in the Department of Post-Harvest Technology. Later on, he obtained a
doctoral degree in Fish Processing Technology from the College of Fisheries,
Mangalore. His area of research interest is phenolic compounds from natural
sources and their efficacy for the preservation of fish and fishery products.
His major research contribution is increasing the shelf life of Indian mack­
erel with the effect of grape seed extract. He has received a best paper award
at the International Conference on Profits on Aquaculture. He has published
more than 25 research publications in both national and international jour­
nals. In addition, he has published 12 chapters.

Adnan Amin, PhD

Assistant Professor, Division of Aquatic Environmental Management,
Faculty of Fisheries, Rangil, Ganderbal, Sher-e-Kashmir University of
Agricultural Sciences and Technology–Kashmir, India
Adnan Amin, PhD, is presently working as an Assistant Professor in the
Division of Aquatic Environmental Management at the Faculty of Fisheries,
Rangil, Ganderbal, Sher-e-Kashmir University of Agricultural Sciences and
Technology—Kashmir. He is an undergraduate from the College of Fishery
Science Muthukur, Nellore SVVU, Tirupati, Andhra Pradesh. He did his
master’s from the College of Fisheries, Mangalore, in the Department of
Aquatic Environment Management. He completed a doctoral degree from
the College of Fisheries Ratnagiri in the Department of Aquatic Environment
Management. His area of research interest is aquatic toxicology and limnology.
About the Editors ix

His major research contribution is heavy metal and pesticide toxicity in

aquatic organisms, impact of climate change on aquatic biodiversity.
He is a recipient of a university fellowship, i.e., merit fellowship for
pursuing his master’s. He is associated with the Society of Fisheries and
Life Science, College of Fisheries, Mangalore. He has published more than
16 research publications in both national and international peer-reviewed
research journals. In addition, he has published five chapters, six review
papers, and 18 magazine articles. Dr. Amin has received one best research
paper award from a national organization.

Kawkabul Saba, PhD

Assistant Professor and Scientist, Division of Fish Nutrition and
Biochemistry, Faculty of Fisheries, Sher-e-Kashmir University of
Agricultural Sciences and Technology, Rangil, Kashmir, India
Kawkabul Saba, PhD, is an Assistant Professor Scientist in the Division
of Fish Nutrition and Biochemistry, Faculty of Fisheries, Sher-e-Kashmir
University of Agricultural Sciences and Technology, Rangil, Kashmir. She
graduated from the College of Fisheries, Veraval, Junagadh Agricultural
University Gujarat, and obtained a master’s degree from the same university
in the Department of Fisheries Resource Management. She obtained a
doctoral degree in Fisheries Resource Management from the Faculty of
Fisheries, SKUAST-K. Her area of research interest is the development of
low-cost feed from slaughterhouse waste. She is a recipient of INSPIRE
fellowship during her doctoral program from the Department of Science and
Technology (DST), New Delhi. She has published more than 20 research
publications in both national and international journals. In addition, she has
published five chapters.
Contents

Contributors ...........................................................................................................xiii
Foreword 1 ............................................................................................................. xix
Foreword 2 ............................................................................................................. xxi
Preface .................................................................................................................xxiii
Acknowledgments.................................................................................................. xxv
Abbreviations ...................................................................................................... xxvii
Introduction ........................................................................................................ xxxiii

1. The Rainbow Trout Genome: A Significant Milestone for

Aquaculture Development ..............................................................................1
Syed Umair Ahmad, Imran Zafar, Shabana Bibi, Zainab Jan, Anwar Ullah,
Irfan Ahmad, Qurat Ul Ain, and Mohd. Ashraf Rather

2. Economics of Coldwater Fisheries ...............................................................35

Khemraj Bunkar, Udai Ram Gurjar, and Suman Takar

3. Nutritional Requirements of Coldwater Fishes ..........................................51

Seemab Zehra, Asaad H. Mohamed, Edoardo Pantanella, and Ramzy A. Yousif

4. Bacterial Diseases of Finfish Prevalent in Coldwater Aquaculture...........91

Priyanka Ashwath, Ramya Premanath, Rajeshwari Vittal, Deekshit,
Prarthana Aithal, Feroz A. Shah, and Akhila Dharnappa Sannejal

5. Biotechnological Interventions in Coldwater Aquaculture

Health Management ....................................................................................147
Md. Idrish Raja Khan

6. Coldwater Fish Diversity: Issues and Sustainable

Management in India ..................................................................................179
Udai Ram Gurjar, Suman Takar, Khemraj Bunkar, Dinesh Mohan, and N. N. Pandey

7. The Zebrafish: A Trending Model for T-Cell and Thymic

Development Along with Prevailing Challenges .......................................195
Sneha Sabu, Sofia Khanam, and A. Jothilin Subitsha

8. Plant-Based Proteins in Fish Diets for Sustainable

Coldwater Fisheries .....................................................................................217
K. T. Hafeef Roshan, Mohammed Meharoof, and K. A. Sajina

9. Small-Scale Fisheries by Indigenous Fishing Methods ............................249

Shabir Ahmad Dar, Gohar B. Wani, Faisal Rashid, and Kawkabul Saba
xii Contents

10. Reproductive Physiology and Breeding Biology of Rainbow Trout

(Oncorhynchus mykiss) ................................................................................259
Younis Ahmad Hajam, Disksha, Rajesh Kumar, and Mohd. Salim Reshi

11. Feeding Carbohydrates to Fish: Utilization and Looking

Beyond Energy Nutrition ............................................................................279
Garima Anand, Puspa Kumari, Tincy Varghese, and Showkat Ahmad Dar

12. Application of Plant-Derived Natural Preservatives for

Shelf-Life Extension of Freshwater Fish ...................................................291
Hafsa Maqbool, Mudassir Azhar, and A. A. Zynudheen

13. Perspective of eDNA Application to Control Pollution in

Freshwater Ecosystems ...............................................................................305
Adnan Amin, Monisa M. Malik, D. Pamanna, Imtiyaz Qayoom, and
Adnan Abubakr

14. Nutritional Composition of Coldwater Fishes...........................................319

K. Sravani, Faisal Sofi, Tariq Hussain, Kawkabul Saba, S. Vijay Kumar Reddy,
S. Devadharshini, P. Ganesan, K. Dhanapal, and Shabir A. Dar

15. Genotoxicity in Fishes: With Special Reference to

Micronucleus Formation in Hematocytes..................................................339
Imtiyaz Qayoom, Sameena Khan, Monisa M. Malik, Adnan Amin, and
Adnan Abubakr

16. Infectious Diseases of Coldwater Fishes: Focus on Viral and

Fungal Infections .........................................................................................351
Syed Shariq N. Qadiri, Inain Jaies, Feroz A. Shah, Shabir A. Dar, and Asifa Wali

17. Nutrigenomics: Boost for Aquaculture Research and Development ......369

Kawkabul Saba, Faisal Sofi, K. Sravani, S. Vijay Kumar Reddy, Oyais Aismi,
Ashwini Kumar, Tariq Hussain, S. Devadharshini, P. Ganesan,
Shabir A. Dar, and Neeraj Pathak

18. Immune Components and Defense Mechanism in Fish: An Overview ..383

Salik Nazki, Munazah Shahzad, Inain Jaies, Syed Shariq N. Qadiri, and Feroz A. Shah

19. Comprehensive Transcriptomics Analysis of Coldwater Fish .................397

Deepak Agarwal and Mohd. Ashraf Rather

20. Predictable Threats to Coldwater Fisheries from the Unpredictable

Nature of Climate Change: An Indian Perspective ..................................407
Ankur Jamwal, Vikas Phulia, and Syed Talia Mushtaq

Index .....................................................................................................................419
Contributors

Adnan Abubakr
Division of Aquatic Environmental Management, Faculty of Fisheries, Sher-e-Kashmir,
University of Agricultural Sciences and Technology (SKUAST) of Kashmir, Rangil, Ganderbal,
Jammu and Kashmir, India

Deepak Agarwal
Tamil Nadu Dr. J. Jayalalithaa Fisheries University, IFPGS, OMR Campus, Chennai, Tamil Nadu, India

Irfan Ahmad
Division of Fish Genetics and Biotechnology, Faculty of Fisheries, Sher-e-Kashmir University of
Agricultural Sciences and Technology, Jammu and Kashmir, India
Syed Umair Ahmad
Department of Bioinformatics, Hazara University, Mansehra, Pakistan

Qurat Ul Ain
Department of Chemistry, Government College Women’s University Faisalabad, Pakistan

Oyais Aismi
Division of Fish Nutrition and Biochemistry, Faculty of Fisheries, SKUAST-K, Jammu and Kashmir, India

Prarthana Aithal
Coldwater Fisheries Research, Nainital, Uttarakhand, India

Adnan Amin
Division of Aquatic Environmental Management, Faculty of Fisheries, Sher-e-Kashmir,
University of Agricultural Sciences and Technology (SKUAST) of Kashmir, Rangil, Ganderbal,
Jammu and Kashmir, India

Garima Anand
ICAR–Central Institute of Fisheries Education, Mumbai, Maharashtra, India

Priyanka Ashwath
Nitte University Center for Science Education and Research, Paneer Campus, Deralakatte, Mangalore,
Karnataka, India

Mudassir Azhar
Department of Fisheries Science, Doon PG College of Agriculture and Allied Science, Uttarakhand, India

Shabana Bibi
Yunnan Herbal Laboratory, College of Ecology and Environmental Sciences; International Joint Research
Center for Sustainable Utilization of Cordyceps Bioresources in China and South-East Asia,
Yunnan University, Kunming, Yunnan, China

Khemraj Bunkar
ICAR–Central Institute of Fisheries Education, Mumbai, Maharashtra, India

Shabir A. Dar
Division of Fishery Engineering, Faculty of Fisheries, SKUAST-K, Jammu and Kashmir, India
xiv Contributors

Shabir Ahmad Dar

Division of Aquatic Animal Health Management, Faculty of Fisheries, Sher-e-Kashmir University of
Agricultural Sciences and Technology (SKUAST) of Kashmir, Rangil, Ganderbal,
Jammu and Kashmir, India

Showkat Ahmad Dar

Department of Aqualife Medicine, College of Fisheries and Ocean Studies, Chonnam National University,
South Korea
Deekshit
Nitte University Center for Science Education and Research, Paneer Campus, Deralakatte, Mangalore,
Karnataka, India
S. Devadharshini
Department of Fish Processing Technology, Fisheries College and Research Institute, TNJFU,
Thoothukudi, Tamil Nadu, India

K. Dhanapal
Department of Fish Processing Technology, College of Fisheries, Muthukur, Andhra Pradesh, India

Disksha
Department of Life Sciences and Allied Health Sciences, Sant Baba Bhag Singh University,
Khalia Padhiana, Jalandhar, Punjab, India
P. Ganesan
Department of Fish Processing Technology, Fisheries College and Research Institute, TNJFU,
Thoothukudi, Tamil Nadu, India

Udai Ram Gurjar

ICAR–Central Institute of Fisheries Education, Mumbai, Maharashtra, India

Younis Ahmad Hajam

Department of Life Sciences and Allied Health Sciences, Sant Baba Bhag Singh University,
Khalia Padhiana, Jalandhar, Punjab, India; Department of Biosciences, Division Zoology,
Career Point University, Hamirpur, Himachal Pradesh, India

Tariq Hussain
Division of Post-Harvest Technology, Faculty of Fisheries, SKUAST-K, Jammu and Kashmir, India

Inain Jaies
Division of Aquatic Animal Health Management, Faculty of Fisheries, Sher-e-Kashmir University of
Agricultural Sciences and Technology (SKUAST) of Kashmir, Rangil, Ganderbal,
Jammu and Kashmir, India

Ankur Jamwal
College of Fisheries and Center of Excellence on Water Management,
Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, Bihar, India

Zainab Jan
National Center for Bioinformatics, Qauid-i-Azam University, Islamabad, Pakistan

Md. Idrish Raja Khan

Department of Aquatic Health and Environment, College of Fisheries, Central Agricultural University (I),
Lembucherra, Tripura, India

Sameena Khan
Division of Aquatic Environmental Management, Faculty of Fisheries, Sher-e-Kashmir,
University of Agricultural Sciences and Technology (SKUAST) of Kashmir, Rangil, Ganderbal,
Jammu and Kashmir, India
Contributors xv

Sofia Khanam
MPharm, Department of Pharmacology, Calcutta Institute of Pharmaceutical Technology and AHS,
Uluberia, Howrah, Kolkata, West Bengal, India

Ashwini Kumar
Division of Fish Nutrition and Biochemistry, Faculty of Fisheries, SKUAST-K, Jammu and Kashmir,
India
Rajesh Kumar
Department of Biosciences, Himachal Pradesh University, Shimla, Himachal Pradesh, India

Puspa Kumari
ICAR–Central Institute of Fisheries Education, Mumbai, Maharashtra, India

Monisa M. Malik
Division of Aquatic Environmental Management, Faculty of Fisheries, Sher-e-Kashmir,
University of Agricultural Sciences and Technology (SKUAST) of Kashmir, Rangil, Ganderbal,
Jammu and Kashmir, India

Hafsa Maqbool
Division of Aquatic Animal Health Management, Faculty of Fisheries, Sher-e-Kashmir University of
Agricultural Sciences and Technology of Kashmir, Jammu and Kashmir, India

Mohammed Meharoof
PhD Scholars, ICAR–Central Institute of Fisheries Education, Mumbai, Maharashtra, India

Asaad H. Mohamed
Beacon Development, King Abdullah University of Science and Technology, Thuwal, Jeddah,
Saudi Arabia

Dinesh Mohan
ICAR–Directorate of Coldwater Fisheries Research, Bhimtal, Uttarakhand, India

Syed Talia Mushtaq

Division of Fisheries Resource Management, Faculty of Fisheries, Sher-e-Kashmir University of
Agricultural Sciences and Technology, Jammu and Kashmir, India

Salik Nazki
The Pirbright Institute, United Kingdom

D. Pamanna
College of Fishery Science, Muthukur, Nellore, Andhra Pradesh, India

N. N. Pandey
ICAR–Directorate of Coldwater Fisheries Research, Bhimtal, Uttarakhand, India

Edoardo Pantanella
Beacon Development, King Abdullah University of Science and Technology, Thuwal, Jeddah, Saudi Arabia

Neeraj Pathak
Department of Quality Assurance and Management, College of Fisheries, Thoothukudi, Tamil Nadu, India
Vikas Phulia
Krishi Vigyan Kendra, (HQ: Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana),
Punjab, India

Ramya Premanath
Nitte University Center for Science Education and Research, Paneer Campus, Deralakatte, Mangalore,
Karnataka, India
xvi Contributors

Syed Shariq N. Qadiri

Division of Aquatic Animal Health Management, Faculty of Fisheries,
Sher-e-Kashmir University of Agricultural Sciences and Technology (SKUAST) of Kashmir,
Rangil, Ganderbal, Jammu and Kashmir, India

Imtiyaz Qayoom
Division of Aquatic Environmental Management, Faculty of Fisheries, Sher-e-Kashmir,
University of Agricultural Sciences and Technology (SKUAST) of Kashmir, Rangil, Ganderbal,
Jammu and Kashmir, India

Faisal Rashid
Division of Post-Harvest Technology, Faculty of Fisheries, SKUAST-K, Jammu and Kashmir, India

Mohd. Ashraf Rather

Division of Fish Genetics and Biotechnology, Faculty of Fisheries, Sher-e-Kashmir University of
Agricultural Sciences and Technology, Jammu and Kashmir, India

S. Vijay Kumar Reddy

Department of Fish Processing Technology, College of Fisheries, Gadvasu, Ludhiana, Punjab, India

Mohd. Salim Reshi

Toxicology and Pharmacology Laboratory, Department of Zoology, School of Biosciences and
Biotechnology, Baba Ghulam Shah Badshah University, Rajouri, Jammu and Kashmir, India
K. T. Hafeef Roshan
PhD Scholars, ICAR–Central Institute of Fisheries Education, Mumbai, Maharashtra, India

Kawkabul Saba
Division of Fish Nutrition and Biochemistry, Faculty of Fisheries, SKUAST-K, Jammu and Kashmir,
India
Sneha Sabu
MSc, Department of Microbiology, CMST, Manonmaniam Sundharanar University, Rajakkamangalam,
Kanyakumari, Tamil Nadu, India
K. A. Sajina
PhD Scholars, ICAR–Central Institute of Fisheries Education, Mumbai, Maharashtra, India

Akhila Dharnappa Sannejal

Nitte University Center for Science Education and Research, Paneer Campus, Deralakatte,
Mangalore, Karnataka, India

Feroz A. Shah
Division of Aquatic Animal Health Management, Faculty of Fisheries,
Sher-e-Kashmir University of Agricultural Sciences and Technology (SKUAST) of Kashmir,
Rangil, Ganderbal, Jammu and Kashmir, India

Munazah Shahzad
Department of Veterinary Epidemiology and Public Health, University of Surrey, United Kingdom

Faisal Sofi
Division of Post-Harvest Technology, Faculty of Fisheries, SKUAST-K, Jammu and Kashmir, India

K. Sravani
Department of Fish Processing Technology, Fisheries College and Research Institute, TNJFU,
Thoothukudi, Tamil Nadu, India
Contributors xvii

A. Jothilin Subitsha
MSc, Department of Microbiology, CMST, Manonmaniam Sundharanar University, Rajakkamangalam,
Kanyakumari, Tamil Nadu, India

Suman Takar
TNJFU–Fisheries College and Research Institute, Thoothukudi, Tamil Nadu, India
Anwar Ullah
The Second Affiliated Hospital, Institute of Cancer Stem Cell, Dalian Medical University, Dalian,
People’s Republic of China
Tincy Varghese
ICAR–Central Institute of Fisheries Education, Mumbai, Maharashtra, India

S. Vijay Kumar Reddy

Department of Fish Processing Technology, College of Fisheries, Gadvasu, Ludhiana, Punjab, India

Rajeshwari Vittal
Nitte University Center for Science Education and Research, Paneer Campus, Deralakatte, Mangalore,
Karnataka, India

Asifa Wali
Division of Aquatic Animal Health Management, Faculty of Fisheries,
Sher-e-Kashmir University of Agricultural Sciences and Technology (SKUAST) of Kashmir,
Rangil, Ganderbal, Jammu and Kashmir, India

Gohar B. Wani
Division of Fishery Engineering, Faculty of Fisheries, SKUAST-K, Jammu and Kashmir, India

Ramzy A. Yousif
Department of Fisheries and Wildlife Science, Sudan University of Science and Technology, Khartoum,
Sudan
Imran Zafar
Department of Bioinformatics and Computational Biology, Virtual University of Pakistan, Pakistan

Seemab Zehra
Beacon Development, King Abdullah University of Science and Technology, Thuwal, Jeddah, Saudi Arabia

A. A. Zynudheen
Fish Processing Division, Central Institute of Fishing Technology, Kochi, Ernakulam, Kerala, India
Foreword 1

India, being one of the mega biodiversity hotspots, harbors the greatest number
of endemic freshwater fishes in continental Asia. Among the freshwater fishes,
the coldwater fisheries harbor 258 species belonging to 21 families and 76
genera. Out of about 258 cold water fish species (both indigenous and exotic)
reported from Indian uplands, snow trout, brown trout, Chinese carps, and
rainbow trout are commercially important species. The trout and salmon are the
world’s second most traded fish species in terms of value, accounting for 14%
of global trade, following shrimp which accounts for 15% of the total. Trout
and salmon fish accounts for more than 6% (5 million tons) of the total global
aquaculture production (SOFIA FAO, 2020). In the Himalayan states, rainbow
trout is becoming a source of livelihood and food security for the hill population.
The aquaculture sector is considered the fastest-growing food-producing
sector and can meet the protein needs of an increasing world population. India
is the second largest producer of fish in the world. The prospects for eradi­
cating malnutrition and fulfilling the Sustainable Development Goal of the
UN partly lie with our efforts to increase aquaculture production. To achieve
this, we must adopt species diversification, and developing seed production
and feed technology is a prime requirement. Understanding physiology and
adaptation in coldwater fishes by means of different tools, such as transcrip­
tomics, proteomics, bioinformatics, etc., would help in understanding key
mechanisms in the physiology of coldwater fishes. This new information can
be used for both guiding new innovations and formulating policies for the
coldwater fisheries sector.
This book brings together an informative compilation of different
aspects of coldwater fisheries and aquaculture management technology for
sustainable food production. I congratulate the editorial team and all the
authors for bringing out a comprehensive compilation in the form of this
book, Coldwater Fisheries and Aquaculture Management: Technologies for
Sustainable Food Production.
—S. Ayyappan
Former Secretary, DARE and Director General, ICAR,
Chairman, Karnataka Science and Technology Academy,
Bangalore, Karnataka, India
Foreword 2

Globally, aquaculture has expanded significantly during the past millennia.

India continues to be the world’s second-largest aquaculture fish grower
with a production of 14.16 million metric tonnes (MT) in 2019–2020. The
industry has significantly increased fish production, with an average annual
growth rate of 7.53% during the previous five years. However, given its
abundant natural resources, India has tremendous potential and the ability to
become a global leader.
Increased production in coldwater aquaculture can be attained by making
the most use of available resources and using high-quality inputs. More than
6% (5 million tonnes) of the world’s aquaculture production is made up of
salmon and trout fish. Through the efficient application of new technologies
and scientific management techniques, the factor cost of production may
be rationalised. Few species are currently used in coldwater aquaculture
operations, thus immediate species heterogeneity is needed to fully use
the available ability. Quality fish seed and feed are essential for improving
aquaculture productivity, which is one of the main concerns with regard to
species variation. Establishing reproduction and seed production techniques
requires an understanding of the physiology of coldwater fishes. Modern
technologies including genomes, transcriptomics, proteomics, and metabo­
lomics are all grouped under the name “omics” technology. These methods
hold enormous promise for uncovering novel mechanisms underlying a
coldwater fish’s intricate thermal adaption, breeding, and reproductive
behavior. The chapters in this book cover a wide range of important topics
related to coldwater fish reproductive biology and endocrinology, including
gonadal development and maturation, vitellogenesis, steroidogenesis, fish
whole genome information, transcriptomics, proteomics, artificial intel­
ligence, etc. I’m confident that the book will be of great use to readers of
all types, including faculty members and students, in learning the processes
involved in fish physiology in cold water and how they might be used in
aquaculture.
I commend the authors, editors, and collaborators for compiling Coldwater
Fisheries and Aquaculture Management: Technologies for Sustainable Food
Production into such a thorough collection of top-notch chapters. I want to
xxii Foreword 2

invite all of this book’s readers to join the editors in making the most of this
intriguing, difficult, and fruitful project.

—Pramod Kumar Pandey

Director, Anusandhan Bhawan, Industrial Area,
Bhimtal, Distt. Nainital, Uttrakhand, India
Preface

India has a coastline of about 8,129 km, 0.506 million sq. km of continental
shelf and 2.02 million sq. km of exclusive economic zone. Total fish
production of India for the year 2019–2020 is estimated to be 14.16 million
metric tons (MT). Currently, India is the second largest fish producer across
the globe. The sector has presented extraordinary development with fish
production recording an average annual growth rate of 7.53% during the
course of the last five years.
India, being one of the hotspots of aquatic animal biodiversity, harbors the
greatest number of endemic freshwater fishes in continental Asia. Amongst
the freshwater fishes, the coldwater fisheries harbor 258 species belonging
to 21 families and 76 genera. Among 258 coldwater fish species reported
from Indian uplands, rainbow trout, brown trout, snow-trout, and Chinese
carps are commercially vital species. The trout and salmon are the world’s
second most traded fish species in terms of value contribute 14% of world
aquaculture trade. Trout and salmon contribute greater than 6% of the total
world aquaculture production (FAO, 2020). In the Indian Himalayan region,
rainbow trout is nowadays becoming a basis of livelihood and food security
for the hill population.
The whole globe is looking for sustainable source of nutritional security and
aquaculture is standing firmly with the expectations. Today’s food production
industry scenario clearly indicates the dominancy of fish and fishery products
in terms of growth rate and future potential of providing nutritional security.
According to SOFIA 2020, total fish production in 2020 crossed the mark of
178.5 million tons, of which around 46% was contributed by aquaculture. A
major portion of total fish production, i.e., around 88%, was used for direct
human consumption (SOFIA, 2020).
India has witnessed tremendous growth in aquaculture during the last 10
years and is also geared to move up through adopting advanced aquaculture
technologies along with species diversification to utilize the diverse potential
water resources. Current coldwater aquaculture practices are limited to very
few species and require urgent species diversification to harness their existing
potential. Quality fish seed and feed technology is the prime requirement
for coldwater aquaculture, and it is the major area of concern for species
xxiv Preface

diversification. Understanding the physiology of coldwater fishes is essential

for developing breeding and seed production technology.
Modern technologies like metabolomics, transcriptomics, genomics, and
proteomics will be essential players in understanding the molecular basis of
coldwater fishes. These techniques offer immense potential to unravel novel
mechanisms underlying the complex thermal adaptation, breeding, and
reproductive behavior of a coldwater fish. This book presents an extensive
collection of articles on different key aspects associated with the reproductive
biology and endocrinology of coldwater fishes, such as gonadal development
and maturation, vitellogenesis, steroidogenesis, whole genome information
of fishes, transcriptomics, proteomics, artificial intelligence, etc. The book
has a recent compilation, and it will offer immense benefits to readers of
all categories, viz., students and faculties, for understanding the underlying
mechanisms of coldwater physiology of fishes and their potential application
in aquaculture.
This book, “Coldwater Fisheries and Aquaculture Management:
Technologies for Sustainable Food Production,” is in its current shape due to
the contribution made by several researchers working at different levels with
reputed institutes across India and having research experience on coldwater
fisheries and aquaculture. This book will provide invaluable support to
scientists, teachers, researchers, students, etc., who are interested in the field
of coldwater fisheries and aquaculture.
—Editors
Acknowledgments

First and foremost, we are grateful to the Almighty for enabling us to

complete this book. We would like to express our heartfelt gratitude and
respect to all authors who have contributed to this book in the form of their
chapters. Indeed, it is our utmost pleasure to acknowledge all the people
behind the completion of this book. It was not possible to complete this
work without their continuous encouragement and support. We are eternally
thankful to all authors.
We are very much thankful to Padma Shree. S. Ayyappan Chancellor
CAU, Former Secretary, DARE, and Director General, ICAR, Chairman,
Karnataka Science and Technology Academy, Bengaluru, and Dr. Pramod
Kumar Pandey, Director, ICAR–Directorate of Coldwater Fisheries
Research (DCFR) Bhimtal, Uttarakhand, India, for their compliment, praise,
and blessings to the entire team of young researchers for their contributions
in this book.
We are thankful to the Vice-Chancellor of Sher-e-Kashmir University
of Agricultural Sciences and Technology-Kashmir and the Dean Faculty
of Fisheries, Rangil Ganderbal–Kashmir, for their constant support and
encouragement.
We will also take this opportunity to express our sincere gratitude to
Apple Academic Press (AAP) exclusive co-publishing with CRC Press, a
Taylor and Francis Group, Canada, for publishing this work.
Support of our students, colleagues, and family members is highly
appreciated.
Finally, we would like to put our head down in front of the Almighty, who
gave us courage, passion, and strength for completing this work.

—Editors
Abbreviations

AA amino acid
ADC apparent digestibility coefficient
ADHD attention-deficit/ hyperactivity disorders
AFLP amplified fragment-length polymorphism PCR
AFPs antifreeze proteins
AHLs acylated homoserine lactones
AI autoinducer
AMPs antimicrobial peptides
ANF anti-nutritional factor
APCs antigen-presenting cells
BFC Behning fertilizing coefficient
BFW Bankfull width
BGD bacterial gill disease
BHT butylated hydroxytoluene
BKD bacterial kidney disease
BPE beetroot peel extract
CAI-1 Coholarae quorum-sensing autoinducer 1
CEAA conditionally EAA
CFU colony forming units
CHD coronary heart disease
CNS central nervous system
CPC canola protein concentrate
CRISPR-Cas9 clustered regularly interspaced short palindromic
repeat-associated nuclease
CRP C-reactive protein
CSM cottonseed meal
CWD chronic wasting disease
DCFR Directorate of Coldwater Fisheries Research
DCs dendritic cells
DHA docosahexaenoic acid
DNA deoxyribonucleic acid
DNV densovirus
dpf days post-fertilization
DSBs double-strand breaks
xxviii Abbreviations

EAA essential amino acids

EC NFDB Enzyme Commission National Fisheries Development Board
eDNA environmental DNA
EFA essential fatty acids
ELISA enzyme-linked immunosorbent assay
EOs essential oils
EPA eicosapentenoic acid
ERM enteric red mouth
ESTs extended performance tasks
EU European Union
EUS epizootic ulcerative syndrome
FAA functional amino acids
FAO Food and Agricultural Organization
FAT fluorescent antibody test
FCR feed conversion ratio
FDA Food and Drug Administration
FE fennel extract
FM fish meal
FOS fructooligosaccharide
G-DAA gluconeogenic discardable AA
GDP gross domestic product
GE gene-editing
GIBs growth-inhibiting bacteria
GMS Gomori methenamine silver
GnRH gonadotropin-releasing hormone
GR gonadosomatic report
GRAS generally regarded as safe
GSDF gonadal soma-derived growth factor
HNE hydrononenal
HP histopathological
HPF hours post-fertilization
HPG hypothalamic-pituitary-gonadal
HPP high-pressure processing
HTST high-temperature short time
HUFA highly unsaturated fatty acids
i.e. that is
IBW initial body weight
IFNs interferons
Ig immunoglobulin
Abbreviations xxix

IHN infectious hematopoietic necrosis

IHNV infectious hematopoietic necrosis virus
Inos inducible nitric oxide synthase
IP intraperitoneal
IPN infectious pancreatic necrosis
IPNV infectious pancreatic necrosis virus
ISAV infectious salmon anemia virus
KDM kidney disease medium
KEGG Kyoto Encyclopedia of Genes and Genomes
LA linkage approaches
LAMP loop-mediated isothermal amplification
LBT luminescent bacterial disease
LD linkage disequilibrium
LLM Leucaena leaf meal
LPS lipopolysaccharides
LSNV Laem-Singh virus
MALDI-TOF mass spectral fingerprinting
MDA malonaldehyde
MG mycotic granulomatosis
MGC multinucleated giant cell
MH maximum height
MHC major histocompatibility complex
miRNA microRNA
MISA micropylar sperm attractant
MNT micronucleus test
MOS mannan-oligosaccharides
MT metric tons
MUFA monounsaturated fatty acids
NADPH nicotinamide adenine dinucleotide phosphate
NCA National Commission on Agriculture
NCDs non-communicable diseases
NCE new chemical entities
NF chromosomal arm number
NFDB National Fisheries Development Board
NGS next-generation sequencing
NHEJ non-homologous enjoining
NNV nervous necrosis virus
NO nitric oxide
NPN non-protein nitrogen
xxx Abbreviations

NRC National Research Council

NRCCWF National Research Center on Coldwater Fisheries
NSP non-starch polysaccharide
OAL overall length
OP organophosphorus
OTUs operational taxonomic units
PA protected area
PAHs polyaromatic hydrocarbons
PAMPs pathogen-associated molecular patterns
PCA principal component analysis
PCR polymerase chain reaction
PD pancreatic disease
PFGE pulsed feel gel electrophoresis
PIBO pacfish in fish biological opinion
PKU phenylketonuria
PTSD post-traumatic stress disorder
PUFA polyunsaturated fatty acid
QTLs quantitative trait loci
QTNs quantitative trait nucleotides
REP-PCR repetitive extragenic palindromic-PCR
RNA ribonucleic acid
RNAi RNA interference
RNA-Seq RNA-sequencing
ROS reactive oxygen species
RSD red spot disease
RTGE rainbow trout gastroenteritis
RT-MPCR reverse transcription-multiplex PCR
SBM soybean meal
SCFA short-chain fatty acids
SCI stream condition inventory
SFA saturated fatty acids
SFB segmented filamentous bacteria
SHBG sex hormone-binding globulins
SMIF sperm motility- initiation factor
SPC soybean protein concentrate
SPI soy protein isolate
SSO specific spoilage organisms
SVCV spring viremia of carp virus
TALENs transcription activator-like effector nucleases
Abbreviations xxxi

TBHQ tertiary butyl hydroxy quinone

TCR T-cell receptor
TEC thymic epithelial cells
TL total length
TLR toll-like receptors
TMAO trimethylamine oxide
TN total nitrogen
TP total phosphorus
TSGs tumor suppressor genes
TSV Taura syndrome virus
UM ulcerative mycosis
VHS viral hemorrhagic septicemia
VHSV viral hemorrhagic septicemia virus
VN virus neutralization
WAFI Williams absolute fecundity indices
WGD whole-genome duplication
WHO World Health Organization
WPF week post-fertilization
WSSV white spot syndrome virus
YHW yellow head virus
YRD Yangtze river delta
ZFNs zinc finger nucleases
Introduction

Coldwater fisheries play a significant role in the world’s freshwater fisheries.

Since aquaculture productivity in coldwater regions is much lower than in other
inland areas, there has been an exciting development of coldwater fisheries in
recent years. Aquaculture, ornamental fish rearing, and eco-tourism all have
possibilities in the coldwater sector. The coldwater environment is under
serious stress from anthropogenic activities due to rising population growth,
urbanization, and industrialization, which has resulted in changes in stream
flow regimes, habitat loss, and species extinction. However, a combination
of anthropogenic and natural (climate change) stressors is harming coldwater
fishery resources, causing a drop in overall productivity.
This book focuses on various elements of coldwater fisheries, as well as
technology created for the breeding, culture, and management of economi­
cally important species in order to increase production in a sustainable
manner. This book covers the effects of anthropogenic activities on coldwater
fishes and management solutions for aquatic ecosystems. All of these topics
are updated, and new study findings are included.
The chapters in this volume will evaluate and highlight various key
issues regarding the potential of coldwater aquaculture, fish species suitable
for coldwater culture, the economics of coldwater, genomic perspectives
and nutritional requirements of coldwater fishes, reproductive physiology
and breeding biology of different coldwater species, coldwater fish disease
management, climate change and anthropogenic pollution on the ecological
health, genotoxicity in fishes, nutrigenomics, responsible approach towards
coldwater fish stock, post-harvest management of coldwater fishes.
This compilation will be valuable for scientists, teachers, researchers,
students, and policymakers when they look for a blue economy and blue
revolution. It will be the first of its kind in the field of coldwater fisheries.
CHAPTER 1

The Rainbow Trout Genome: A Significant

Milestone for Aquaculture Development
SYED UMAIR AHMAD1, IMRAN ZAFAR2, SHABANA BIBI3,4,
ZAINAB JAN5, ANWAR ULLAH6, IRFAN AHMAD7, QURAT UL AIN8, and
MOHD. ASHRAF RATHER7
1
Department of Bioinformatics, Hazara University, Mansehra, Pakistan
2
Department of Bioinformatics and Computational Biology,
Virtual University of Pakistan, Pakistan
3
Yunnan Herbal Laboratory, College of Ecology and Environmental Sciences,
Yunnan University, Kunming, Yunnan, China
4
International Joint Research Center for Sustainable Utilization of
Cordyceps Bioresources in China and South-East Asia, Yunnan University,
Kunming, Yunnan, China
5
National Center for Bioinformatics, Qauid-i-Azam University,
Islamabad, Pakistan
6
The Second Affiliated Hospital, Institute of Cancer Stem Cell, Dalian
Medical University, Dalian, People’s Republic of China
7
Division of Fish Genetics and Biotechnology, Faculty of Fisheries,
Sher-e-Kashmir University of Agricultural Sciences and Technology,
Jammu and Kashmir, India
8
Department of Chemistry, Government College Women University
Faisalabad, Pakistan

ABSTRACT

Rainbow is a salmonid species that benefit aquaculture, wildlife fisheries,

and commercial fish farming. Because of their economic worth, rainbow
Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
2 Coldwater Fisheries and Aquaculture Management

trout are extensively used as model fish in various scientific fields.

From an evolutionary genomic standpoint, the rainbow genome is truly
intriguing because of a whole duplication event along salmon fishing
lines 88–96 million years ago. Because of the development of next-
generation sequencers, a wide variety of genomic technologies, including
whole-genome sequencing, are now accessible in this species. Several
instances of whole-genome duplication (WGD) have affected the history
of vertebrate animals, and they are often related to adaptive radiation or
developmental progress. Due to an additional cycle of WGD, the rainbow
genome offers a chance to study the early evolutionary destiny of the same
vertebrate genome, and researchers found that the subgenomes of the
two forebears remained remarkably identical after 100 million years of
evolution. MiRNAs, on the other hand, were essentially wholly duplicated
copies of their genome. The completion of genome sequencing by
researchers opens up new opportunities for aquaculture, which is a critical
step toward addressing specific issues in trout farming, such as disease
resistance and identifying specific strains with higher growth potential or
better adapted to marine growing conditions. The presence of various viral
diseases in the salmon and trout industry has highlighted the possibility of
naturally resistant rainbow trout reared in seawater for the generation of
smoked filets from larger fish weighing over 3 kg. This chapter presents a
synoptic assessment of current knowledge on rainbow trout genomics and
what molecular evidence has already been discovered due to functional
genomics.

1.1 INTRODUCTION

Salmonids families are found worldwide, and several of these are essential
for fish farming, wild population fishing, and the leisure sportfishing
industry (Clavelle, Lester, Gentry, & Froehlich, 2019). In 2018, overall,
aquatic output purse to reach over 3 million metric tons (MT), with such a
worldwide economic interest of above US $1,800,000,000,000 including
Wild salmon, seemed to be the most significant managed salmonid species,
contributing for 3,000,000 MT (Cabral et al., 2020). Rainbow is native to
the western, northern United States, but it is also one of the most imported
fish. According to Fish-Base (Akhtar, Ghazanfar, & Shafi, 2021), rainbows
are currently found in at least 70 different countries, with a population
spanning almost all of the world’s largest hemispheres. Although it has
The Rainbow Trout Genome 3

a lower prominent place than wild salmon aquaculture, the productivity

levels for trout are pretty good. World total production peaked at 5 million
MT for perhaps the first time in 2001 and had been over 9,00,000 MT in
2015 with either total export revenues of about $3.5 billion (Adusei, 2015)
(Figure 1.1).

FIGURE 1.1 Rainbow trout production across different countries (Adusei, 2015).

The chinook salmon is a member of the salmonid (Salmonidae) family,

which includes three subfamilies: the Salmoninae (primarily trouts, chars,
and sockeye), the Thymallinae (graylings), and the Coregoninae (salmons)
(whitefish and ciscos). Salmonids separated from its closest sister group,
Esocidae (pikes and mud minnows), circa 110–150 Mya, while the last
common ancestor of contemporary salmonids is estimated to get along with
58–63 Mya (Bobe et al., 2016). Salmonid complete genome duplication
(Ss4R) is thought to have occurred between 88 and 96 million years ago
(Bobe et al., 2016; Nugent, 2019).
More than 20 other European countries share the remainder of manu­
facturing (Chavanne et al., 2016). The bulk of America’s production takes
place in Chile. In contrast, while minor, development in the USA was worth
$80 million in yearly sales in 2014, without including the refurbishment,
management, and leisure industries.
4 Coldwater Fisheries and Aquaculture Management

1.2 RAINBOW TROUT: A MODEL ORGANISM FOR LABORATORY

EXPERIMENTS

Apart from its commercial significance, the rainbow has often been used as a
conceptual framework in a variety of domains such as disease studies, toxi­
cological, comparative immunotherapeutic, epidemiological, physiological,
nutritional, including fertility and adaptation research. Rainbow is one of
the most studied aquatic organisms, with various practical advantages such
as the availability of different natural populations and monoclonal lines,
the simplicity through which breeding can be achieved and the ability to
perform exceptionally accurate gene ablation (Rajabi, Ramazani, Hamidi, &
Naji, 2015). Rainbow trout’s relatively sizeable physical growth compared
to mimic fish such as zebrafish or service fish makes it an excellent surrogate
model for conducting biochemical and genetic studies of specific cellular
components, which are much more challenging to assess in native fish models
(Lutfi, 2017). Various cytogenetic materials have been adopted during the
last two decades to facilitate its utility in scientific research and aquacul­
ture, including morphological and molecular mappings, BAC libraries and
BAC end sequences, as well as several simple sequence markers. When the
very first Rainbow trout entire genetic sequencing effort began in 2010, the
majority of the required genetic materials for the which was before stage
were accessible (Schill, Heindel, Campbell, Meyer, & Mamer, 2016).

1.2.1 RAINBOW TROUT LIFE CYCLE

Rainbow trout is a subspecies of North USA’s Northwest coast, with a

geographic distribution that extends from Alaska to the area to the north
(Taylor & May-McNally, 2015). Although steelhead trout species may be
found in the ocean and eventually return to rivers and lakes to spawn, this
is mostly a freshwater species found in streams and ponds. Freshwater
populations are often found with wells, lakes and rivers with referendum
temps ranging from 10 to 16°C (Bobe et al., 2016; Boulajfene et al., 2019)
(Figure 1.2).
They reproduce fast throughout the spring, with spawning occurring in
cold, well-oxygenated, shallow rivers with good gravel bottoms (Stoessel,
Raadik, & Ayres, 2015). A single adult rainbow trout can lay 1–8,000 eggs
(3.5–6 mm) in one or more batches, which are promptly fertilized by sperm
from either a single male or a pair of males. Eggs usually fall amid rocks and
fissures and hatch 4–5 weeks after implantation. During vitellus breakdown,
The Rainbow Trout Genome 5

embryonic fish emerge from either the sandy bottom or the muddy substrate
and feed on zoobenthos and phytoplankton prey. Rainbow trout seems to be
an adventurous eater in its mature form, dependent on various food sources,
from microscopic insects to crayfish (Lopez-Santamarina et al., 2020).

FIGURE 1.2 Trout life cycle.

1.2.2 GENETIC ARCHITECTURE AND GENOMIC ASPECTS OF RAINBOW

TROUT

From the standpoint of adaptive genetics, the freshwater fish DNA (deoxy­
ribonucleic acid) is particularly intriguing (Anastasiadi, Díaz, & Piferrer,
2017). The Rainbow trout genome is of significant size, 2.4 109 bp, six
times larger than just the buffer genome or even more effective than the
goldfish’s DNA, 1.7 109 bp. Second, the freshwater fish genome is complex,
with a chromosomal arm number (NF) of 104 and a variable double set
of chromosomes ranging from 57 to 63 due to Robertsonian reconfigura­
tions. An important cause of this complication is a tetraploidization event
(whole genuine duplication, WGD) that occurred in the rainbow line (Ss4R:
Salmonfish-specific fourth round of WGD). Fish genomics is therefore still
believed to undergo rediploidization, which involves evolutionary algorithms
or reduction of gene duplication, a process known as “widely differing settle­
ment,” which claimed to reduce hybrid conditions and encourage species
6 Coldwater Fisheries and Aquaculture Management

formation. Such a phenomenon may well have played a significant role in

the astonishing diversity of species found in fish (Salzburger, 2018).
Moreover, differential settlement in marine invertebrates has been described
following an ancient genomic multiplication (triple WGD or Teleost-specific
third round after WGD = Ts3R) between 225 and 330 million years ago (Mya).
Still, only extremely variable individuals have been evaluated. Salmonid
genomics gives a chance to study the evolutionary algorithms of recently
divergent tetraploidized genomic and the further development of replicated
genomes during the initial stages of diploidization (Brunet et al., 2017).

1.2.3 RAINBOW TROUT GENOMIC DIFFERENTIATION AND

STRUCTURAL DIVERSITY

Salmonids possess the fundamental features of a highly vital socioeconomic

organism and a species of significant scientific value, with a critical place in
the evolutionary tree of ray-finned fish. Such integrated research and practice
objectives have greatly influenced a well-structured worldwide scientific
world in developing a significant number of genomics techniques that are
now accessible in so many rainbow trout species (Krajcik & Czerniak, 2018).

1.2.4 RAINBOW TROUT GENOMIC SEQUENCING REVEALS NEW

INSIGHT ON VERTEBRATE EVOLUTION AFTER WHOLE-GENOME
DUPLICATION (WGD)

Whole-genome duplications (WGDs) are uncommon but spectacular occurrences

that result in abrupt duplication integrity of the entire genomic sequences
(Pophaly, 2017). Although WGDs are uncommon among mammal species,
these have had a significant impact on vertebrates evolution and serve as critical
evolutionary milestones from which certain main bloodlines have diverged. For
example, the ancient genomic of all teleost’s experienced a WGD, known as the
effectiveness 3rd WGD (Ts3R), which occurred between 225- and 332-million­
year-old (Mya) (Lien et al., 2016). This Ts3R signature is still relevant to
contemporary vertebrates’ genomics, and it was accompanied by two prior
WGD events shared by all skeletal vertebrates. Throughout WGD, the resultant
replicated genomics maintain just a minuscule fraction of replicated DNA,
whereas superfluous duplicates are immobilized through a mechanism known
as gene fractionated (Stephens, 2019). Too far, the form of genetic separation in
vertebrates has received little attention since all well-characterized WGDs are
The Rainbow Trout Genome 7

exceedingly old, and gene fractionation is assumed to be finished in such species.

Salmonids are of particular relevance in this context since gene fractionation
may still be occurring in their lineage as a result of an extra as well as fairly
new WGD occurrence (the salmonid-specific 4th WGD or Ss4R) dated 25 to
100 Mya (Minkley, 2018). Rainbow trout (Oncorhynchus mykiss) is a salmonid
that is of tremendous environmental relevance across the globe. It is among the
most researched fish species, with substantial research in various categories
including malignancy, toxicity, immunological, ecological, physiological, and
nourishment (Mamdouh, Mohamed, Mohamed, & Fahmy, 2022). This is also a
significant aquatic organism with massive financial relevance that is cultivated
for both climates and on all geographies.
Because of its new WGD, the rainbow trout offers a different chance to
comprehend the early stages of genome fractionated (Symonová & Howell,
2018). Our findings, followed by an analysis of the rainbow fish pretty much
the entire sequencing, demonstrate that the 2 ancient subgenomes had main­
tained very coincident throughout 1 billion years of human history. Approxi­
mately 50% of the nutrient genomes have been preserved as duplicated copies,
with the remainder lost primarily via pseudogenization (Rendón-Anaya et
al., 2019). In stark contrast to nutrient sequences, miRNA genetic mutations
have virtually all been preserved as doubled versions. Our findings point to a
long and incremental rediploidization mechanism, which calls into question
the current idea that WGD is accompanied by huge and fast chromosomal
restructurings and genetic losses (Pérez-Sánchez et al., 2019).

1.2.5 PREEXISTING GENOMIC RESOURCES AND FIRST STEP TO

RAINBOW TROUT GENOMIC SEQUENCING

The freshwater fish genomic is approximately 2.4 109 kb in length, with a

G + C composition of 42% (Nguinkal et al., 2019). One of the issues that
hampered the early scanning and formatting of the goldfish genome was
the degree of heterogeneity among the members of the overall population
used to contribute DNA to such research. Essential selection criteria and
androgens were employed to successfully create double homozygous adults
in the rainbow, which was used to establish homogeneous cloned lines
that have been carefully documented at genomic and phenotypic levels.
Completely homogeneous samples were required to develop rainbow
genomic approaches to cope with both the considerable genomic variation
predicted due to the initial WGD of such an organism (Du et al., 2020). The
Nucleotide Sequence from such a unique twice haplotype trout (Swanson
8 Coldwater Fisheries and Aquaculture Management

YY doubly haplotype clonal line) utilize to create numerous rainbow trout

BAC collections. Several BAC libraries use to make the first generations, and
2nd gen BAC was physically mapping (Książkiewicz et al., 2015). Multiple
lake trout frameworks that rely on doubly haploid adults and outbreeding
communities have been created utilizing microsatellite and SNP markers.
A synthesized connection map was created by combining earlier ones, and
it had 2,226 characteristics at a moderate concentration of 1.0 marker/cM.
In the rainbow trout, cDNA pools from several tissue and morphogenesis
have been generated and processed simultaneously. As of June 2015, 2,90,406
extended performance tasks (ESTs) sequencing were publicly accessible in
the GenBank database. These ESTs served as the foundation for various
rainbow trout cDNA, and research has focused on hybridization systems
(Cassé, Richetin, & Toni, 2018). Segments through both endpoints of 96,000
fish BACs also were accessible, and all these BAC-end sequencings use to
generate the first trout repeat database, demonstrating that repeated DNA
accounts for almost 58% of the genomes. These BAC-end transcripts were
used to create either the first, second, or third-generation consolidated maps
after testing microsatellites (Clouse et al., 2016).

1.2.6 NGS SEQUENCING TECHNOLOGIES AND RAINBOW TROUT

GENOME SEQUENCING

The rainbow trout genetic sequencing technique was designed using NGS
sequence alignment capabilities accessible in 2010 at the start of this genome
research (Juanchich et al., 2016). This genomic sequence was assembled
using a whole-genome scattergun approach utilizing genetic material from
a single duplicated haplotype (YY) freshwater fish male. The accessibility
of DNA from such an utterly homogeneous organism was a critical resource
that greatly aided in constructing the freshwater fish genomic (Abdelrahman
et al., 2017). For the entire acoustic technique, solitary reading collections
and 8 kb, 12 kb, and 20 kb buddy pools were processed using 454 Titanium
technique up to something like a 20-fold sequence coverage and a 70-fold
coverage utilizing Solexa-Illumina sequences (Yang & Sun, 2017). Solexa-
Illumina segments operate to fix the features present and scaffolding
segments that resulted from assembling the 454 Titanium reads, which are
recognized as a blunder, particularly in copolymer regions. This method was
supplemented by extensive coverage of BAC-end genomes, which aided in
scaffolding contigs and anchoring the genome onto combined maps (Argyris
et al., 2015). The entire size of this initial iteration of the freshwater fish
The Rainbow Trout Genome 9

genomic sequences was 1.9 Gb, with a framework N50 of 384 kb (half of
the assembly is contained in 1,014 scaffolds longer than 384 kb). Scaffolds
were fixed onto chromosomal at 898 different loci utilizing correlation and
topological mapping knowledge. The genetic sequence was annotated, and
46,585 nutrient genetic categories were discovered (Salem et al., 2015),
corroborating protein data from some other mammals and transcriptome
information from 15 organs of a doubling haplotype lake trout adult collected
by RNA-seq. Compared to other vertebrates, this large number of projected
nutrient genes is consistent with the current Ss4R. 495 microRNA (miRNA)
loci belonging to 84 distinct families and 164 matured sequencing were also
found. Transposable elements make up roughly 38% of the rainbow trout’s
entire genome (Rodriguez & Arkhipova, 2018).

1.2.7 RAINBOW TROUT STRUCTURAL GENOMICS INFORMATION

Approximately 300 million years ago, the teleosts’ primordial genomic

sequence experienced a WGD entity known as the teleosts-specific third
WGD (Ts3R) (Mya) (Bobe et al., 2016). This Ts3R characteristic appears
to play a role in the contemporary teleost genomes sequence. Two other
ancient WGD processes that all musculoskeletal animals inherit (vertebrate
genome duplications 1 and 2, VGD1 and VGD2) (Lorin, Brunet, Laudet,
& Volff, 2018). While WGD occurrences are uncommon amongst animal
races, they are major technological milestones from which several major
lineages have diverged. Multiple identical genomics ultimately maintains
just a miniscule fraction of replicated genomes, while apparently superfluous
versions are inhibited in a mechanism known as gene separation, which is
unexplained. Rainbow trout genetic structure is especially interesting in this
context since salmonids have undergone an additional relatively fresh WGD
occurrence (the salmonid-specific fourth WGD or Ss4R), which has been
firmly predicted to occur between 25 and 100 Mya (Colgan et al., 2021). As
a result, genetic separation in that species may still be happening, providing
a novel paradigm for understanding the underlying factors controlling this
rediploidization process.
The lake trout genomes permitted the restoration of the primordial
karyotype of salmonids before the Ss4R multiplication by rebuilding the
Ss4R alternatively spliced areas (Bobe et al., 2016; Shavalier, 2017). The
current lake trout genomic is arranged in 38 prominent pairings of replicated
sections, as well as 14 of the 30 chromosomes come out from the merger of
different separate post-Ss4R chromosomes, which is consistent paralogies
10 Coldwater Fisheries and Aquaculture Management

deduced from trout association studies (Du et al., 2020; Lin, 2019).
Other genomes include more complicated mosaics of distinct post-Ss4R
chromosomes, indicating additional cross-functional and cross translocations
since Ss4R event. In these Ss4R alternatively spliced areas, several ohnologs
(paralogous genes produced by a WGD process) were found (Chen et al.,
2019). The estimated dispersion of modest nucleic acid replacements (dS)
throughout several sets of Ss4R ohnologs found in freshwater fish, as well as
between pairs of orthologs found in Atlantic salmon and lake trout (Carruthers
et al., 2018) (representing the species deviation time, i.e., roughly 30 Mya,
between most of the two species) allowed us to calculate the timeframe of
the Ss4R at 96 Mya, in the top perimeter of such 25–100 Mya from prior
response but in the above-mentioned 25–100 Mya This contradicts the
estimated history of the Salmonidae family of 50–60 Mya (Osinov, Volkov,
& Mugue, 2021), indicating that the Ss4R evolved far earlier (> 30 Mya)
than the earliest single ancestor of modern salmonids. This is consistent
with the WGD Radiological Lag-Time Model, which proposes considerable
lagtimes between WGDs and the consequent adaptive radionuclides, which
are also generally linked with WGD incidence (Tank et al., 2015).
The genomic expansion includes the loss of one genetic duplicate among
most homologous genes via gene fractionation after just a few million years
following a WGD (Inoue, Sato, Sinclair, Tsukamoto, & Nishida, 2015).
Even though all WGD investigations seem too ancient to acquire such data,
this process has never been detected in any vertebrate throughout the whole
genomic scale. Because the Ss4R is younger, alternatively spliced sites in the
rainbow trout genome can be identified (Sadd et al., 2015). Approximately
half of the Ss4R double identical domains have undergone gene partitioning
and returned to a diploid pattern over the whole genome, while the remainder
have preserved both ohnologs. Gene dispersion is a fairly slow approach
in fish genomics since genomes were inactivated at a rate of roughly 170
genomes every million years.
Furthermore, even though most singletons are often dysfunctional,
the majority of singletons may still be connected with evident paralogous
sequences emanating from the Ss4R (pseudogenes). In contrast to the 50%
retention of homologous histone genes in employees, we observed that post­
Ss4R retention of genes encoding miRNAs is total (Taylor, Daniels, Morata,
Gundappa, & Macqueen, 2022), with nearly all miRNA ohnologs maintained
as repeat Ss4R copies. This higher conservation appears to be attributable
to more subtle selection processes rather than shorter miRNA-coding
sequences. Overall, the greater consumer retention frequency of homologous
duplicates and pseudogenes demonstrates that the fractionation process in
The Rainbow Trout Genome 11

trout is still mostly finished and ongoing (Lu & Luo, 2020). The lake trout
genomes’ phylogenetic tree, even during the Ss4R WGD event, is depicted
schematically. Since the Ss4R WGD resulted in a complete duplication of the
genetic material, each genetic Code (and genes associated with its regulatory
domains) were available in multiple genetically identical instantly after the
Ss4R. Following the Ss4R WGD, chromosomal fusions and specialized
framework rearrangements decreased the total number of chromosomes
to 30, which was similar to the preduplication karyotype (Murat, Armero,
Pont, Klopp, & Salse, 2017). The reduction of replicated genes following
WGD by alterations and omissions to revert to a predominantly diploid
form, known as rediploidization or gene fractionated, is an evolving issue
in the lake trout genomic sequence (Perreault-Payette et al., 2017). At the
time, the duplicated sections of the gene were still primarily collinear, with
around half of the repeated homologous genes retaining dual ohnologs.
Ohnologs that are still present in both copies are constantly diverging and
may take on new or auxiliary tasks (neo- or sub-functionalization) or be
deleted due to gene fractionation. Prior WGD occurrences predicted that
only around 20–25% of duplicated genes would be preserved in homologous
pairs (Li et al., 2015). There are 36 genomes in the aquatic biological
cultured. The genome organization level of research of the Ss4R duplicated
regions revealed a good collinearity among haplotypes exome sequencing,
consistent with a conserved order of ohnologs, and therefore no compelling
evidence of a concentration of single parents versus ohnologs throughout
the genomes. These findings imply that gene fractionation will exclude
severe genomic structural changes such as inversions or chromosomal
rearrangements, which might disrupt the order of genes in the genome, as
well as large deletions, which will also contribute to the lengthy cluster
of singletons. Ss4R ohnologous protein coding domains and mRNAs are
also evolutionarily related, with 92.9% amino-acid and 96.4% nucleotide
similarity, respectively. Furthermore, the similarity between the Ss4R
protein-coding singletons and their associated nucleotide sequences is quite
great (average amino acid (AA) identity 79.0%), indicating that the majority
of these genetic illnesses and disorders occurred (Lien et al., 2016).

1.3 THE RAINBOW TROUT GENOME–AN IMPORTANT LANDMARK

FOR AQUACULTURE

The rainbow trout genome’s first iteration unified maps are made up of 238
BAC contigs joined to the chromosomal map areas (Wiernasz et al., 2021).
12 Coldwater Fisheries and Aquaculture Management

It covers more than 10% of the genome and contains sequences from all 29
chromosomes. This map is a starting point towards a more detailed composite
genomic map. Extensive comparative genome analysis, precise mapping of
QTL, positional cloning, selection of positioned genetic markers for economi­
cally significant characteristics, and incorporation of MAS into rainbow trout
breeding strategies will all be possible with the availability of an integrative
individual genetic map. A fully integrated map might provide a minimal
tiling strategy for genetic research and a basis for whole-genome assembly
(Chapman et al., 2015). The phylogenetic tree of the rainbow trout genome
is practical implication during Ss4R WGD phase for Ss4R WGD results in a
complete doubling of the genetic Code, providing access to all chromatids in
a large number of genetically related individual people especially following
the Ss4R. After Ss4R WGD, chromosomal conglomerations and specialized
framework reworkings decreased the total number of chromosomal instabili­
ties to 30, which was similar to the preduplication karyotype. Rediploidization
or gene stratification is an ongoing process in the rainbow trout genome that
involves the loss of duplicated genes following WGD through alterations and
replacements to revert to a predominantly diploid state (Waples, 2015). The
repeated parts of the genome are still substantially collinear at present, with
around half of the duplicate homologous genes maintaining both ohnologs.
Ohnologs that are still extant, including both copies, slowly diverge at the
sequence level. They may ultimately assume new or auxiliary roles (neo- or
sub-functionalization), or they may be lost due to gene fractionation (Lamb,
2021). Based on previous WGD cases, only around 20–25% of duplicated
genes are expected to be kept in multiple copies. As the recent update of
NCBI current gene set of rainbow trout is given in Figure 1.3.

1.3.1 IMPORTANCE OF GENOME FOR GENETIC AND FUNCTIONAL

INVESTIGATIONS

Since the rainbow trout exome sequencing is finalized (Lyons, Turnbull,

Dawson, & Crumlish, 2017), the key difficulty is determining how to interpret
the information encoded in the DNA sequence. Throughout several genome-
wide investigations, the task of determining the expression of certain genes,
regulatory genes, and their interactions remain unsolved. Because alterations
in the entire genes have a high probability of causing clinical diseases,
functionality evaluation is crucial for breeding and aquaculture development
and production (Palti et al., 2015). For many years, a range of methods and
tools have been employed in functional genomic analysis. Meanwhile, quick
The Rainbow Trout Genome 13

transformative innovation and improvement in high-throughput methods,

extending from classic real-time PCR to far more complex processes like as
next-generation sequencing or NMR spectroscopy, has occurred just in the
last decade (Walter et al., 2020). Furthermore, proper bioinformatic research
is crucial for credible scientific outcomes, in addition to experimental inquiry.
These approaches provide accurate and complete holistic assessment across
several areas of study, including genomes, metagenomics, proteome, and
interact omics (Pinu et al., 2019). This is critical for addressing information
gap in dynamic cellular mechanisms at both the cellular and organismal
levels. Furthermore, every approach has benefits and limits that should be
considered before identifying the appropriate technique for certain particular
research in assuring an effective analysis.

FIGURE 1.3 Current gene set distribution of rainbow trout genome (NCBI, 2022).

1.3.2 DECIPHERING PROCESS AND THE GENETIC ARCHITECTURE

OF RAINBOW TROUT TRAITS

Quantitative trait loci (QTLs) are genomic areas (loci) that are linked to
morphological characteristics within a trait (Khedikar et al., 2018). A QTL
is typically associated with or contains the gene(s) influencing the target
characteristic. As a result, using polymorphism connected with QTL regions
to discover the main variables that influence the unpredictability of the target
14 Coldwater Fisheries and Aquaculture Management

trait is a strategy to improve breeding effectiveness (dos Santos et al., 2020).

Determining QTLs is also a step toward the molecular deconstruction of
many characteristics, as well as the finding of genes that govern processes of
interest (causative genes) and their regulation (Price et al., 2018). Many QTL
for a range of phenotypes in rainbow trout were discovered using generally
known intermediate resolution genomic maps and linkage approaches (LA)
in family QTL designs (Salem et al., 2018), emphasizing adaptation and pest
resistance traits.
Furthermore, the likelihood of QTL placements is typically high, making
their utilization problematic (Fraslin et al., 2020). With the fast growth of
NGS technology, high-density genotyping approaches are now accessible.
For example, limitation linked DNA tagging (RAD-tags) sequencing allows
for the low-cost detection of hundreds of SNP (Feng et al., 2018).
Linkage maps based on RAD tags were created in rainbow trout,
leading to enhanced marker density (Zhang et al., 2018). RAD-sequencing
was also employed in a prolonged SNP discovery project, which revealed
almost 1,45,000 SNPs, permitting the construction of high-density genotype
sequencing chips such as the 57 K SNP chip recently created for rainbow
trout and now commercially accessible. In such investigations, possessing
a benchmark genomic information as a model helps organize newly found
SNPs into maps or create local haplotypes (Juanchich et al., 2016). These
improved genotyping technologies enable a drastic change in QTL mapping
methodologies, allowing the use of linkage disequilibrium (LD)-based methods
and population-level association analysis and genome wide association study
(Hérault, Damon, Cherel, & Le Roy, 2018). This offers new possibilities for
understanding complicated features, identifying genomic regions beneath
selection, and implementing genomic selection in aquaculture production in
the hereafter (Casu et al., 2022).

1.4 FUNCTIONAL CHARACTERIZATION AND INTEGRATIVE

FUNCTIONAL ANALYZES IN RAINBOW TROUT

Nutrition Rainbow trout aquafeeds have been created during the last three
decades based on the species’ well-known nutritional requirements (Hardy,
Kaushik, Mai, & Bai, 2022). New aquafeeds must be created to replace
nutritious protein sources and fish oil (sourced from fragile ocean biodiver­
sity) with phytoconstituents or alternative materials to achieve sustainable
aquaculture (Turchini, Trushenski, & Glencross, 2019). This shift in dietary
composition necessitates a full understanding of intermediary metabolic
The Rainbow Trout Genome 15

pathways, mainly based on molecular markers. The molecular LC-PUFA

synthesis route is functional (Pewan et al., 2020). Considering the restriction
of fish oils in aquaculture diets, one of the most pressing challenges is main­
taining the highest levels of long-chain polyunsaturated fatty acids (PUFAs)
in meat (Barta, Coman, & Vodnar, 2021; Roy et al., 2020). Several studies on
fish have revealed that this organism contains the structural characteristics to
produce these LC-PUFAs from plant oil precursors (Bermúdez et al., 2021).
When fish oil is eliminated from the diet, mRNA encoding system enzymes
and elongase increase; this incentive is insufficient to achieve the same
levels of LC-PUFA in muscle as fish given fish oil. LC-PUFA biosynthesis in
fish has been demonstrated to be substrate-restricted, despite some fish lines
having more significant quantities of fatty biosynthetic molecular capacity
(Ampong, 2020).
Rainbow trout are recognized to be weak dietary carbohydrate users,
limiting the integration of starch in aquaculture feeds (Liu et al., 2020; Song
et al., 2018). Recent research (Steinberg, 2022) indicates that trout fish has
the transcriptional ability to catabolize dietary glucose (Cadonic, 2019).
In contrast, the molecular control of hepatic glucose synthesis appears
to be unusual, which may be one of the causes for trout’s poor metabolic
utilization of carbohydrates. A reduction in feed intake linked with a lower
plasmatic insulin/glucagon ratio (primarily due to the pursuit of hepatic
gluconeogenesis) is frequently reported in fish, followed by the consump­
tion of novel aquaculture feeds, potentially having a significant influence on
feed conversion ratio (FCR). Recent research (Karras, Koufakis, Mustafa, &
Kotsa, 2019) indicates that glucose and triacylglycerol sensing mechanisms
in the hypothalamus and endocrine pancreas are present in nutritional regula­
tory frameworks of nutrient-sensing routes in these organs. In rainbow trout
served alternate diets, fine nutritional modulation of these pathways occurs
at the molecular level (Rimoldi, Antonini, Gasco, Moroni, & Terova, 2021).
There has been a decrease in nutritional digestibility and a decrease in the
plasmatic leptin ratio in fish, followed by the consumption of novel aqua­
culture species, which may significantly influence digestibility (Xu et al.,
2021; Ferosekhan et al., 2014). Recent research (Sanjana Sharma, Aroura,
Gupta, & Priyadarshini, 2022) shows that in rainbow trout served alternate
diets, fine nutritional modulation of these pathways occurs at the molecular
level. Some transcriptomics studies were followed with proteolytic analysis,
which appeared to support the transcript assessment but was limited by the
inability to detect polypeptides in rainbow trout due to technical limitations
(Beemelmanns et al., 2021; Sobhkhez, Krasnov, & Robertsen, 2018).
16 Coldwater Fisheries and Aquaculture Management

1.5 NUTRITIONAL MODULATION OF THE ACTIVATION OF

HEPATIC GLUCOSE-6-PHOSPHATASE GENES IN RAINBOW TROUT:
HOW TROUT GENOTYPING BRINGS CREATIVE APPROACHES TO
EXISTING CONCERNS?

The rainbow trout is considered glucose-intolerant due to sustained hyper­

glycemia after a carbohydrate-rich meal or hypoglycemia tolerance tests
(Panserat, Marandel, Seiliez, & Skiba-Cassy, 2019). One explanation for
such a phenotypic is a lack of restriction of endogenous insulin synthesis
via the glycogen synthesis pathway (Chen et al., 2021). Hyperglycemia
has already been postulated to be a major player in glycemic richness at
high blood sugar levels or after caloric intake by attempting to establish
a meaningless glucose/G6P cycle with hexokinase, the first glycogen
synthesis protease that initiates the transcriptional activity of glucose in
G6P (Cho, Kim, Mansfield, & Chou, 2018; Wang & Dong, 2019). In 2000,
a gene encoding for G6pc in fish was sequenced mainly for the first time
(Juanchich et al., 2016). Its expression’s nutritional/hormonal control was
subsequently thoroughly examined using northern blot analysis, gradually
supplanted by modern real-time quantitative PCR research. Therefore, the
amount of g6pc mRNA was demonstrated to be slightly higher compared
to high dietary fats but not, interestingly (Rabot et al., 2010), by high
carbohydrate food consumption comprising digestible starch. In vivo and in
vitro studies further revealed that g6pc mRNA levels were favorably linked
with protein and AA pool levels (Mullarky & Cantley, 2015). Furthermore,
particular AA like glutamine, one gluconeogenic discardable AA (G-DAA),
or lysine and methionine were implicated in the g6pc gene (Rolland et al.,
2016). Finally, insulin regulation of this genotype was investigated and
found to have a significant effect on g6pc mRNA levels in vitro and in vivo
studies and the ability to counteract the increase in mRNA levels caused by
glycemic supplementation in tissue culture fluids or high-diet fish carbohy­
drate (Balasubramanian et al., 2007).
Nonetheless, when compared to enzyme activity data, several of these
overexpression results were questioned. Insulin, for example, appeared to
work at the biological level to reduce g6pc mRNA levels but had no influence
on G6pc hepatic or intestinal function (Hong et al., 2014). Similarly, only
one G-DAA (alanine) reduced g6pc mRNA levels, while enzyme activity
was decreased in trout fed three G-DAA swapped meals (alanine or aspartic
acid or glutamic acid).
The Rainbow Trout Genome 17

Furthermore, until finding a second specific gene for G6pc in EST data­
bases in 2014 (Hong et al., 2014), no convincing justification could be an
offer to justify why G6pc activity did not alter when a single serving with
glucose suppressed g6pc. Indeed, the study of this novel gene provided addi­
tional information to interpret the results reported above, suggesting that the
rainbow trout genome’s complexity accounts for a better understanding of
dietary control. Data revealed that not only were both g6pc genes differently
regulated by nutritional status, but they also demonstrated divergent regula­
tion in terms of the relative percentage of carbohydrates in the diet (Panserat
et al., 2019). The current editing of the lake trout genome advanced molecular
understanding of g6pc/G6pc dietary control to the next level (Juanchich et al.,
2016). In fact, in silico studies indicated that five orthologous genes encoding
for G6pc were preserved in the lake trout genome following Ss4R. Two of
them, previously unknown, demonstrated an unanticipated up-regulation of
respective mRNA in fish given a high carbohydrate diet. Overall enzyme
activity in the livers of these fish revealed that proteins transcribed in the
latter two genes were engaged in this activity (Morro et al., 2019). It was
postulated that these two genes, together with glucokinase, would participate
in the reactive hypoglycemia phenotypic by generating glucose in the blood
via creating a futile cycle. Previous findings (Juanchich et al., 2016), relating
the regulation of g6pc genes by insulin or G-DAA can now be reinterpreted
in light of the current discovery (Carr, Turner, & Pirmohamed, 2021), taking
into account that g6pc combining information is possibly variably governed
but that they all contribute to total enzyme activity.
The instance of g6pc loci exemplifies how genomic breakthroughs might
benefit our understanding of dietary control of metabolism and bring new
solutions to old concerns (Dowaidar, 2021). However, as is frequently the
case, closing a window frequently opens a door (Kiepas, 2021). There are
numerous concerns concerning the significance of divergent expression of
g6pc orthologous genes and their associated contributing to one or another
more phenotypes arise.

1.6 RAINBOW TROUT GENOMICS FOR HEALTH

The Rainbow trout genomics mapped, “Immunomet,” a nearly comprehen­

sive repertoire of genes involved in the immune system, became available.
In both normal and pathological settings, large amounts of data on genes
implicated in human and mouse immunity were already available at the
18 Coldwater Fisheries and Aquaculture Management

biochemical, structural, cellular, and molecular levels; this data merged

into specialized immunogen and pathway databases and tools. When the
immunome of other vertebrate animals are transliterated, then three main
methods apply to identify it: a functional knowledge of immunogens from
other species, such as humans or mice, can be attached to their orthologs
since phylogenetic haplotypes in distinct species often have similar roles,
and datasets from high-throughput approaches for gene expression under
infectious and pathological conditions can recognize the genes affected in
diseases and during responses. Finally, the newly accessible genome use
to direct genomic and operational analysis of genes or groups of genes.
Immunological annotation of freshwater fish is a difficult task that cannot
achieve simply by transferring knowledge from animal models and human
databases. The WGD occurred during salmonid growth, resulting in many
ornithological genes that are susceptible to subfunctionalization. Some gene
families, such as chemokines and adornments, have undergone significant
specialized expansion in salmonids. Accordingly, a well-composed genome
with tightly linked doublets should be an essential resource for defining the
immunome, especially from high-throughput transcripts. A sophisticated
trout immune system will serve as a solid foundation for future research,
such as studying genetic variance and epigenetic techniques, which will be
helpful for marker-based selection systems.

1.6.1 THE CASE OF ANTIGEN-SPECIFIC RECEPTORS, IGS, AND TCRS

As modern sequencing methods enabled researchers to access a complete

variety of immunoglobulin (Ig), or T-cell receptor (TCR) patterns expressed
in a tissue or even a whole individual—research on immune repertoires has
grown dramatically in the last five years. These studies give a thorough over­
view of innate and adaptive immunity against infections and examine the
immune system’s condition in various illnesses (Liu et al., 2021). Based on
Ig and TCR sequences accessible in these species, such techniques develop
in freshwater fish and zebrafish. The availability of a whole trout genome
sequence, as well as the opportunity for extensive tagging of TCR and Ig
loci in this organism, will offer researchers a much-enhanced mechanism
for accessing the full range of immunological repertoires and responses.
The rainbow trout genome provides the initial step—and a vital resource
—in developing such techniques, which will help evaluate future vaccines
(Adams, 2019).
The Rainbow Trout Genome 19

1.6.2 A GENOME ASSEMBLY ENGAGES IN LINKAGE STUDIES,

INCLUDING GENE CLUSTERS AND LINKAGE RESEARCH

The prevalence and functional significance of immune-related gene clusters

are still unknown. Several selection forces involving regional gene ampli­
fication and co-regulation of expressions can represent such clusters (Lal,
2017). The trout genome offers an exciting opportunity to investigate such
problems, one of which is the development of the large histocompatibility
complex (MHC). TCR identifies antigens as short peptides (9–22 AAs)
linked to membrane proteins known as MHC class 1 or class 2 molecules,
which are produced on the surface by “antigen-presenting cells (Juanchich
et al., 2016). MHC restriction occurs when TCR identifies both the MHC
polypeptide and the polypeptide antigen provided by the MHC molecule
(Collins & Riddle, 2008). The genetic “MHC” is a section of the genome
that is determined by the presence of MHC class 1 and 2 molecules and
genes involved in peptide antigen production and charge for MHC presenta­
tion. In practice, the MHC encodes many genes, about half of which are
engaged in immunity; hence, the genes in this area contribute significantly
to immunome and defense mechanisms (Torres et al., 2016). The origin and
evolutionary process of MHC are still unknown. MHC is found in all jaw
vertebrates, including sharks and mammals. According to Ohno’s theory, two
rounds of WGD happened after the development of urochordates and even
before the radiation of jawed vertebrates, resulting in four sets of paralogic
zones, as previously described. Several critical genomic regions, including
the HOX and MHC complexes, have been discovered to have such tetrads
(Cañestro, 2012). Two additional tetrads have been discovered in vertebrate
genomes, which appear to be derived from outlying areas on another ances­
tor’s genetic material containing “proto-MHC” from great grandparents of
vertebrate species. These MHC-related tetrads contain a gene engaged in
vertebrate immunogenicity and several humongous epigenetic frameworks
like the innate immunological component and the intricate granulocyte
target proteins and many of the body’s preventative gene families like B7
receptors, TRIMs, and so on (Suurväli et al., 2014).
In addition to fish, where the regions expressing MHC class 1 and MHC
class 2 locate on distinct chromosomes, MHC has been preserved as a defined
genetic unit by vertebrates from sharks to mammals (Kulski, Shiina, Anzai,
Kohara, & Inoko, 2002). The functional relevance of this conservation and
the distinctive pattern identified in fish are still unknown. Co-regulation of
many immune-related genes is an appealing concept, but it has gathered
20 Coldwater Fisheries and Aquaculture Management

little direct evidence and contradicts the pattern reported in fish. Fish are
helpful models for dealing with such problems and learning about the
immunological consequences of a “broken” MHC. StarCraft and pipe fish
are excellent examples of fish groups that have lost MHC class II genes and
classic T-helper responses (Segner, Verburg-Van Kemenade, & Chadzinska,
2017). With additional cycles of genetic code multiple copies and continued
rediploidization, the salmonids represent another context of great interest
in confronting and fully understanding the fate of the MHC region and
its paralogs or common carriers. Rainbow DNA is a valuable resource for
fish biologists and comparable immunologists (Xu et al., 2017). As a basis
for defining a comprehensive immune system, it is a critical resource for
future innovations that require monitoring of immune responses, including
identifying more resistant animals. Given the DNA from the strongly linked
Atlantic salmon, it also provides an opportunity to answer general and evolu­
tionary concerns about which tele cheeses have distinct properties. Finally,
a high-quality screening will be crucial for future trout gut (or any other)
microbiota investigations.

1.6.3 THE REPRODUCTION OF RAINBOW TROUT USING A GENOMIC

SEQUENCE

The Web of Science database searched for over 8,000 papers about rainbow
trout reproduction (Liu et al., 2020). A comparable search for zebrafish
reproduction (a commonly known biological model) generated just 3,500
references (Hill, Teraoka, Heideman, & Peterson, 2005). This demonstrates
the significance of the scientific community working on rainbow trout
reproduction and the potential influence of the rainbow trout genome and
related genomic resources on the study of repetition in this species. As with
other scientific domains, the recent publication of the rainbow trout genome
opens up new avenues for studying gene synteny and elucidating previously
unresolved evolutionary histories of genes involved in reproduction, as
seen below. Sex hormone-binding globulins (SHBG) are blood proteins that
transport sex steroids in plasma and regulate their availability to target tissues
(Selby, 1990). Fish have two genes, SHBGA and SHBGB, with SHBGA
becoming the ortholog of the SHBG gene sequence (Juanchich et al., 2016).
SHBGB has been detected in rainbow trout and salmon but has yet identi­
fied in any non-salmonid teleost species. While this may imply that SHBGA
and SHBGB are the results of Ss4R WGD, the topology of the SHBG
tree, as well as new analysis utilizing the rainbow trout genome sequence,
The Rainbow Trout Genome 21

indicate that this duplication is considerably older than previously assumed.

Gonadal soma-derived growth factor (GSDF) is a newly discovered member
of the TGF-beta superfamily that plays a critical role in primordial germ
cell and spermatogonia proliferation (Kaneko et al., 2015). It is likewise
geographically and temporally connected to early testicular differentiation
in medaka. GSDF gene expression is confined to Sertoli and Granulosa cells
in medaka gonads. The fate of GSDF following Ss4R WGD is unknown,
however, preliminary studies show that Ss4R produced two GSDF genes in
rainbow trout with potentially divergent expression patterns. The rainbow
trout genome will enable detailed research of GSDF ohnologs, including any
regulating the expression or sub-/neo-functionalization following duplica­
tion (Juanchich et al., 2016).
With the rainbow trout genome sequence, it will be feasible to perform
genome editing without depending on current resources (e.g., BAC libraries)
to select critical genes and characterize the phenotypes associated with
matching knockouts. While the rainbow appears to have a relatively long
lifespan compared to some other fish model organisms (e.g., zebrafish,
medaka), genetic knockout is sometimes required to sort out the function
of unique salmonid genes such as the sex-determining gene SDY. Rainbow
trout might be an intriguing model to do genetic modifications in a well-
characterized teleost model demonstrating group-synchronous oogenetic
development due to its long-term usage as a model for reproduction. A
recent study showed that the sound and widely used Crispr/Cas9 technology
is employed successfully on salmonids (Sumana & Petsalaki, 2018). Finally,
the latest rainbow trout publication offers many possibilities in reproductive
biology, with significant consequences for the control of sex, adolescence,
fertility, and sterility (Pewan et al., 2020).

1.7 CONCLUSIONS AND FUTURE TRENDS: RESOURCES ARE NOW

BEING EXPENDED

Next-generation sequencing (NGS) is a fast-growing discipline. NGS tech­

nologies are now available to more extended reading techniques and larger
volumes of sequence data produced. NGS has expanded the resources of
the rainbow transcriptome in many studies that sequenced cDNA in various
organs, enabling therapeutic or diagnostic situations. miRNA sequencing
throughout the rainbow has resulted in vast databases of miRNA compo­
nents, in addition to cDNA transcripts. Complete genome sequencing of
new rainbow organisms from multiple genetic origins, including duplicate
22 Coldwater Fisheries and Aquaculture Management

haploids, will be possible with current NGS technology at a minimal cost.

As a result, more markers and polymorphisms identification, allowing for
further QTL identification and functional analysis refinement. The Rainbow
Genetics framework is structured on NGS’s most recent significant advance­
ments in Illumina’s genomic technology, allowing for the development
of massive amounts of 2,250 bp data. The new feature of Code has been
disclosed, and it will be available in 2016. This composite-enhanced rainbow
rendering is 2.17 Gb in size, with N50 compatibility of 1,700 kb. Each of these
emerging capacities is now taken into account to generate comprehensive
cited maps of all functional sections of the rainbow genome, for example,
through multinational efforts like the Computational Investigation of Animal
Genomes, which would take the place of ENCODE Human. Together, these
efforts will lead to a better knowledge of the lake trout genome, allowing it
to use in genomic breeding, comprehensive epigenetic regulatory study, and
other comprehensive studies.
Future opportunities and challenges Environmental and genetic interac­
tions Aquaculture production occurs in various environments. The GxE
exchange evaluates the animals’ sensitivity to changes in their environ­
ment and genetic performance in different situations. Genotypes can be
reclassified in other contexts as a result of this change. This means that the
largest genotypes in one environment (such as freshwater) may not be the
best genotypes in another. As a result, GxE can wreak havoc on selective
breeding by making EBV predictions more difficult and lowering selection
efficiency. As a result, GxE must be included in genetic assessment models
regularly. Variations in nesting conditions resulted in significant misclassifi­
cation for Atlantic salmon survival and moderate to severe classification for
rainbow growth, according to a comprehensive study of the GxE interaction
between aquatic species. Additional evidence for specific environmental
factors affecting trait variability and complete characterization of the GxE
interaction for growth and other traits of the rainbow has still required water
temperature, population density, salinity, photoperiod, feeding, etc.
Trout raised for breeding has long been assumed to thrive in watery envi­
ronments. Consequently, the selected applicants’ development was assessed
using the same criteria. New legislation permitted most breeding species to
relocate to freshwater facilities on land following the sanitary disaster created
by the ISA virus in Chile between 2007 and 2009. As a result, some breeding
animals are grown in environments other than those found in maritime areas,
and GxE should be present. If there is a GxE link between growth measured
at inland facilities and development measured at sea, Chilean salmon farming
The Rainbow Trout Genome 23

strategies need to be adapted to consider this interaction. Establishing marine

test stations that simulate one or more production conditions and testing
siblings and cousins as selection candidates can help achieve this. As a result,
the sample index may include lineage or genetics for production growth.

1.8 RESEARCH AND DEVELOPMENT

Enormous technological advances in genomics have enabled progress in

selective breeding strategies over the past decade (Boudry et al., 2021). For
example, GS increased sampling accuracy in salmon and trout compared
to traditional pedigree alternatives. Although metrological research on GS’s
efficacy in aquaculture programs is published (Engle, Kumar, & van Senten,
2021), the implementation of practical genotype indexing techniques will
facilitate the cost-effective application of GS in samplers for difficult-to­
measure traits (Boudry et al., 2021). Understanding inherent conformational
changes, also known as quantitative trait nucleotides (QTNs), would improve
population genomic reliability and eliminate the need for phenotypic data
for each generation and group (Stein et al., 2021; Zafar, Iftikhar, Ahmad,
& Rather, 2021). Identifying QTN for genomic characteristics by incor­
porating data sets such as GWA, transcriptomic data, or sequencing of the
entire genome is a critical step in global functional annotation (Naserkheil
et al., 2022), which aims to understand the meaning of current genetics and
recognize QTN in a variety of salmonids.
The gene-editing method (GE) has the power to revolutionize precise
methods of genetic modification for a myriad of applications in all living
organisms (Rasheed et al., 2021; Zafar et al., 2021). In salmonids, GE is
more likely to improve the frequency and stability of beneficial alleles that
segregate at critical QTLs that affect economically significant properties
(Valente et al., 2013). On both sides, the growth of high-GE technologies
can enable the regulation of multiple QTNs with moderate power sizes. In
vitro, GE platforms (cell lines) were able to identify and confirm impor­
tant fundamental properties of QTN efficiently. In salmonids, targeted GE
research; However, the CRISPR-Cas9 (clustered regularly interspaced short
palindromic repeat associated nuclease) method (Zafar et al., 2021) is used
to effectively inactivate the GFP gene in CHSE cell lines remove the dnd
gene, which induces sterility in Atlantic salmon. Regulators have not yet
approved the actual use of GE’s precision processing techniques. A key
question is whether the new organism should not be classified as anything
24 Coldwater Fisheries and Aquaculture Management

other than a genetically modified plant, as the European Union (EU) recently
did. Although GMOs are not formally controlled in Chile, the main target
markets for Chilean rainbows are more susceptible to the processing and
commercialization of genetically modified products (Regan et al., 2021).
As a result, the enormous potential of genomics is driving rapid progress
and discoveries of the underlying variables and processes that underpin the
essential characteristics of salmon and trout.
Significant advances in science and technology tend to exacerbate
existing problems. With cross-functional teams, infrastructure, and “internal”
resources, it is unlikely that breeding companies will be at the forefront.
As a result, a partnership between companies and technology is required
to create fast solutions and remain competitive. Financial authorities must
adopt appropriate technologies to increase the links between innovation and
marketing.

1.9 MARKET SHARE DIRECTION

Significant financial investment in breeding animals is required to apply

genomic technology and develop inland facilities to ensure exceptional egg
quality through improved pedigrees. These investments led the market to
package specialized egg suppliers with substantial production volumes,
which diluted the cost of eggs. Customers will notice an increase in the value
of eggs and find out what product quality and efficiency they can expect from
the egg supplier. In the context of market research, the trial occurrences and
sample frequencies as per earlier researchers (Soh, Lim, & Chua, 2021) were
used to see whether they could be adjusted to meet the specific demands of
each potential egg buyer. Consequently, every egg supplier and other large
livestock enterprises formed massive managed service units. As a result, the
practices to achieve the greater power to manipulate genetic information. As
a result, logistical support will become a great marketing tool for standing
out among the top egg suppliers (Hosain, Ullah, Al Sayam, Mohiuddin, &
Rahman, 2021) in many countries to increase the economy. Benchmarking
programs implemented to demonstrate added value to increase production
costs and mitigate the effects, breeding companies must investigate nutrient
digestibility and the ability to withstand high temperatures. In the coming
years, we expect technological advances in genomic screening to increase
market share, with fewer companies competing for positions in the Chilean
and global sectors for better salmon and trout eggs.
The Rainbow Trout Genome 25

CONFLICT OF INTEREST STATEMENT

The authors reported no potential conflict of interest related with present

work.

FUNDING

Not applicable.

KEYWORDS

• antigen-specific receptors
• aquaculture
• gene clusters
• genome duplication
• genome sequencing
• rainbow trout
• whole genome

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CHAPTER 2

Economics of Coldwater Fisheries

KHEMRAJ BUNKAR1, UDAI RAM GURJAR1, and SUMAN TAKAR2
1
ICAR–Central Institute of Fisheries Education, Mumbai, Maharashtra,
India
2
TNJFU–Fisheries College and Research Institute, Thoothukudi,
Tamil Nadu, India

ABSTRACT

The total fish production during 2019–2020 is recorded at 14.15 million

tons. The inland fishery sector contributes more than 73% of total produc­
tion. Coldwater fisheries occupy a key place among India’s freshwater
fishes, contributing significantly to nutritional security and job creation in
rural hilly areas. More than 1.40 lakh fishermen in India directly depend on
coldwater fisheries in hilly areas. Manipur has the highest fish population
with 47,711 fishers’ population. Total fry production is 4,388.9 fish fry in
hilly states in which Manipur produces the highest fish seed with 2494.8
lakh fish fry production. In 2019–2020, total coldwater fish production was
estimated at 1.07 lakh tons; Manipur has the largest share of 32,000 tons. It
also observed that ecotourism based on fisheries is a sustainable resource
use option and a means of environmental conservation. In Jammu and
Kashmir, the annual total cost and gross revenue per farm were estimated
to be Rs. 2.36 lakh per farm and Rs. 4.30 lakh per raceway, with a benefit-
cost ratio of 1.83, indicating the business economic viability. In Sikkim, a
trout raceway of 51 m3, which costs around 1.20 lakhs, has been observed
to produce 500 kg of trout fish at an average price of 800 per kg, yielding
an income of 4 lakhs. The net profit from a single raceway would come to
₹ 2.7 lakhs, which is a very venturing and reasonable profit to the farmers.

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
36 Coldwater Fisheries and Aquaculture Management

Major challenges in coldwater fish farming have been stated available water
resources for culture, unawareness of fish farming, lack of facilities like cold
storage, quick transport, and other marketing facilities, and lack of modern
technological knowledge.

2.1 INTRODUCTION

During 2019–2020, the total fish production is recorded at 14.15 million tons.
The fisheries sector plays a significant role in the country’s socio-economic
development and provides a source of income for a large proportion of
the economically disadvantaged rural population (Ayyapan & Krishnan,
2004). It ensures food security while also addressing unemployment in
these regions, which rural residents primarily populate. It has grown in
importance and is recognized as a rich source of inexpensive, nutritious
food (Kumar et al., 2007). The inland fishery sector contributes significantly
to nutritional security and employment opportunities in comparison to the
0.24 million tons of fish production in 1950–1951, the country’s inland fish
production during 2019–2020 reached 10.43 million tons, this increases
in the fish production the country became the second-largest producer
of inland fish. The fisheries sector accounts for approximately 1.24% of
the country’s GDP (gross domestic product) and 7.28% of its agricultural
GDP. The annual export earnings from the fishery sector are ₹ 46,662.85
crores (2019–2020). This sector provides a living for more than 14 million
people who work in it full-time, part-time, or in ancillary capacities.
Furthermore, the sector provides an important source of ancillary jobs for
the rural population, particularly in marketing, retailing, and transportation.
However, the industry is still largely unorganized, owing to the dispersed
and diffuse nature of activities (DoF, 2020).
Inland fishery resources of India are diverse and plentiful which is
illustrated in Table 2.1. The vast and varied resources of Indian fisheries
accounted for 1,73,285 km of rivers and canals, 1.098 million hectares of
swamps and other wetlands, 0.202 million hectares of the floodplain, 0.072
million hectares of uplands lakes, 2.414 million hectares of ponds and tanks,
0.357 million hectares of mangroves and 0.285 million hectares of estuaries
resources, 0.191 million hectares of lagoons, 3.150 million hectares of
reservoirs, and 1.240 million hectares of brackish water provide excellent
opportunities for livelihood. The country’s freshwater culture resources
include 2.36 million ha of ponds and tanks (DoF, 2020).
Economics of Coldwater Fisheries 37

TABLE 2.1 Current Scenario of Indian Fisheries

SL. No. Particulars Details
1. World rank 2nd in aquaculture, 3rd in fisheries
2. Total fish production (2019–2020) 14.15 MMT
3. Export value (2019–2020) ₹ 46,662.85 crore
4. Fisheries contribution to agricultural GDP 7.28%
5. Contribution to national GDP 1.24%
Source: DoF (2020).

2.2 COLDWATER FISHERIES IN INDIA

The term “coldwater” refers to the aquatic ecosystem that maintains thermal
and oxygen levels for the well-being of trout, mahseers, snow trout, and other
minor species. The temperature is generally within the trout and salmons
tolerance limits in the Salmonidae family, which is less than 20°C. Various
physical, chemical, geochemical, and biological parameters of different
water bodies, such as water temperature, dissolved oxygen, velocity,
turbidity, substratum, trophic status, food availability, and so on, influence
the distribution and abundance of various coldwater fisheries species. Cold-
water fishes inhabit the Himalayan and sub-Himalayan zones in the north
and watersheds draining the southern slope of the Deccan plateau in the
Indian subcontinent (Sunder et al., 1999). As per Anon (2011), area-wise
different types of aquatic resources available in hilly regions of India, such
as reservoir water spread area (2,65,000 ha), Deccan Plateau and Himalayan
Riverine Resources (10,000 km), Natural Freshwater lakes (1,500–2,000
m asl) 18,150 ha, Brackish water lakes (above 3,000 m asl) 2,340 ha, High
altitude lakes of Kashmir (above 3,000 m asl) 400 ha, Central Himalayas
freshwater lakes (Kumaon region) 355 ha, Shivalik Himalayan lakes (74
ha), and Wetlands (3,000 ha).
India’s coldwater fisheries sector is small but vibrant, with significant
growth potential. Thrust areas have been identified for the holistic devel­
opment of all segments in order to improve coldwater fisheries production
through cluster-based farming and natural resource conservation. The vision
is to significantly expand coldwater fisheries through the use of new and
innovative production technologies, management, and utilization of unde­
rutilized water resources, and proper market setups. Fish culture has been
carried out successfully in Jammu and Kashmir, Himachal Pradesh, Sikkim,
38 Coldwater Fisheries and Aquaculture Management

Uttaranchal, Nagaland, Meghalaya, Arunachal Pradesh, Tamil Nadu, and

Kerala in lakes, ponds, and reservoirs. The Indian government established
the National Research Center on Cold Water Fisheries (NRCCWF). It had
played a crucial role in the improvement and conservation of indigenous
species as well as the establishment of exotic species in this region. As one
of the promising sectors, the DADF intends to improve and strengthen cold-
water fisheries. As a result, the Mission Coldwater Fisheries Development
Action Plan–2022 was created in order to conserve and improve coldwater
fisheries in an economically, socially, and environmentally responsible
manner, as well as to promote fish farming in the hill region. The Mission
Coldwater Fisheries Development Action Plan is expected to improve food
and employment security in coldwater areas in rural and urban areas (Chettri,
2020; DAHDF, 2018).

2.3 ECONOMIC IMPORTANCE OF COLD-WATER FISHERIES IN INDIA

The coldwater fisheries in India occupy a vast region that is uneven and
versatile, with a rich biological floral and faunal diversity. These regions
are broadly classified as the eastern Himalayas, the central Himalayas,
and the western Himalayas. Coldwater fisheries have the employment
generation capacity in the hilly area where the employment opportunities
are significantly less and play a crucial role in employment in such areas
(Chetrri, 2020). According to the DoF (2020), the total fishermen population
in major hilly areas is estimated at 1.40 lakh fishermen who directly
depend on coldwater fisheries. Among the states, Manipur has the highest
fish population with 47,711 fishermen population followed by Arunachal
Pradesh (24,015 fishermen), Jammu and Kashmir (17,396 fishermen),
Meghalaya (16,567 fishermen), Himachal Pradesh (11,806 fishermen),
Uttarakhand (8,352 fishermen), Nagaland (7,958 fishermen) and least hilly
fishermen population lived in Mizoram (6,289 fishermen) among the major
hilly coldwater fisheries states of India which shown in Figure 2.1.

2.4 COLDWATER FISH PRODUCTION

Total seed (fry) production is 4,388.9 fish fry in hilly states namely Himachal
Pradesh, Jammu and Kashmir, Manipur, Meghalaya Mizoram, Nagaland,
Uttarakhand, and Arunachal Pradesh.
Economics of Coldwater Fisheries 39

FIGURE 2.1 Fishermen population in hilly states of India.

Source: DoF (2020).

The state Manipur produces the highest fish seed with 2494.8 lakh fish
fry production among all hilly states, followed by Nagaland (795 lakh fish
fry), Mizoram (400 lakh fish fry), Uttarakhand (289.6 lakh fish fry), Jammu
and Kashmir (209.7 lakh fish fry), Arunachal Pradesh (190 lakh fish fry) and
Meghalaya is the least fish seed producing state with only 3 lakh fish fry
(Figure 2.2).
The fish production of all the hilly states of India shows an increasing
trend (Figure 2.3). Jammu & Kashmir is the largest producer of coldwater
fishes in India. During 1995–1996, coldwater fish production was 16.52
mt in Jammu & Kashmir, which increased to 19.15 mt in 2005–2006 and
20.08 mt in 2015–2016. Himachal Pradesh is the second-largest cultivator
and producer. Its production increased from 5.94 mt in 1995–1996 to 7.3
mt in 2005–2006 and 11.8 in 2015–2016. In 2005–2006, Uttarakhand’s fish
production was 2.79 mt, which increased to 4.14 mt in 2015–2016 with a
growth of 38%. The production of fish in Sikkim was the lowest amongst
these states because it is the smallest state in terms of area as well fewer areas
are under cultivation than other states. However, production reached from
0.11 mt in 1995–1996 to 0.15 mt in 2005–2006 and 0.4 mt in 2015–2016 with
a growth of 166% during this decade. During 1995–1996, fish production in
Arunachal Pradesh was 1.85 mt, which increased to 2.75 mt in 2005–2006
and 4.05 mt in 2015–2016.
40 Coldwater Fisheries and Aquaculture Management

FIGURE 2.2 Coldwater fish seed production in hilly states of India (2020).
Source: DoF (2020).

FIGURE 2.3 Coldwater fish production in the Himalayas of India (in ‘000’ tons).
Source: DAHDF (2018); DoF (2020).

In 2019–2020, total coldwater fish production is estimated 1.07 lakh tons. The
states mentioned above showed an increasing trend except for Sikkim, which
offers a minor reduction in production. Among the above-mentioned states,
Jammu and Kashmir have the leading position among the states with 21,000
Economics of Coldwater Fisheries 41

tons of fish production, followed by Himachal Pradesh (14,000 tons), Arunachal

Pradesh (5,000 tons), Uttarakhand (5,000 tons), and Sikkim (0.39,000 tons).
The majority of fishery resources in upland Himalayan regions are
currently exploited through capture fisheries, though fish production through
culture practices is gaining traction. At the moment, total fish production
from upland areas accounts for about 2% of total inland fish production in
India, with a negligible contribution to total fish production. The coldwater
aquaculture production potential has yet to be fully realized. Except for a
few hill states like Kashmir Valley and Himachal Pradesh, other mountain
regions of India are still underdeveloped or underexploited in terms of
coldwater fisheries development. These hill states, which are endowed with
natural lakes and reservoirs, could benefit from a culture-based capture fish­
eries program to increase fish production. DCFR has taken up open water
cage culture initiatives in Himalayan lakes for stock enhancement.
Total coldwater fish production is shown in Figure 2.4 which is 1.07 lakh
tons from hilly states, namely Manipur, Jammu and Kashmir, Meghalaya,
Mizoram, Himachal Pradesh, Arunachal Pradesh, Nagaland, and Uttarakhand.
Manipur is the leading state among the coldwater fish producing states in India
with 30% coldwater fish production, followed by Jammu and Kashmir (20%),
Himachal Pradesh (13%), Meghalaya (13%), Nagaland (8%), Mizoram (6%),
Arunachal Pradesh (5%) and Uttarakhand (5%) (Figure 2.2).

FIGURE 2.4 Coldwater fish production in hilly states of India (2020).

Source: DoF (2020).
42 Coldwater Fisheries and Aquaculture Management

2.5 SWOT ANALYSIS OF COLDWATER FISH FARMING IN INDIA

In order to formulate a future action plan for the development of coldwater

fishery, SWOT analysis has been performed by the Department of Animal
husbandry, Dairying and Fisheries–2018, to illustrate ground realities as
shown in Table 2.2.

TABLE 2.2 SWOT Analysis of Cold-Water Fish Farming in India

Strength:
• Basic culture technologies, supported with field-oriented projects.
• With assured financial returns.
• An adequate gap in demand supply for local consumption, besides scope for export.
• Existing schemes for the production of coldwater fisheries.
• Presence of unexploited coldwater resources in the form of rivers, streams, and lakes.

Weaknesses:
• Insufficient allocation of funds.
• Poor extension machinery in potential states, except Jammu and Kashmir.
• Lack of confidence among the rural population.
• Absence of cold chain, processing, value addition and marketing channels.
• Lack of hygiene protocol in culture ponds.
• Lack of high-tech breeding and capital-intensive culture methods.

Opportunities:
• Employment avenues in the rural area.
• Eco-tourism-based fisheries.
• Presence of breeding/seed production infrastructure.
• Adequate demand for consumption.

Threats:
• Environment degradation, damming of rivers, tributaries, and streams.
• Non-availability of low-cost indigenous substitutes.
• Absence of linkages for large growth expansion among research institutes, government
departments and other development agencies.
• Spread of infectious diseases.

Source: DAHDF (2018).

Economics of Coldwater Fisheries 43

2.6 COLDWATER FISHERIES FOR SPORTS, RECREATION, AND

LIVELIHOOD

Coldwater fishery encompasses three major objectives: resource assessment

and sustainable utilization, upland livelihood and nutritional security, sports,
and ecotourism for job creation (Chettri, 2020). Brown trout (Salmo trutta
fario), rainbow trout (Oncorhynchus mykiss), and certain species of large-
scaled barbels are the main sports species in Kashmir, Himachal Pradesh,
Uttarakhand, Sikkim, North Bengal, Nilgiris, Kodai hills, and Munnar
ranges, which attract a large number of Indian and foreign tourists each year.
The sport fishery is an important source of revenue in some areas. Trout
alone accounts for 40–50% of the state’s fisheries revenue in Jammu and
Kashmir (Chettri, 2020).
Ecotourism based on fishing is both a sustainable resource use and a
means of environmental conservation. It is a non-consumptive use of biolog­
ical resources that provides people with socioeconomic benefits. Through
tourism generation, the hill region provides an emerging area of employ­
ment generation. Many picturesque valleys, riverbanks, and mountains are
available for the development of trout or mahseer-based sport fisheries, as
well as opportunities for the development of restaurants, transportation,
parking, boating, and angling facilities. The government has been working
on developing suitable models for the development of fish-based ecotourism.
Important locations for the development of sports and angling facilities have
been identified (Chettri, 2020).

2.7 COSTS AND RETURN ANALYSIS OF TROUT CULTURE IN

JAMMU AND KASHMIR

Costs and return analysis of trout culture on a sample farm of 30 trout

farmers in Jammu and Kashmir have been analyzed by Gawa et al. (2017).
They analyzed and presented the costs and returns in trout farming per farm
and raceway for the sample trout farms in Table 2.3. The annual total cost
and gross revenue per farm were estimated to be Rs. 2.36 lakh and Rs. 4.30
lakhs, respectively. Total variable and fixed costs were Rs. 1.77 lakhs and
Rs. 0.58 lakhs, respectively, with a share of the total cost of 75.32% and
24.68%. Feed was the most expensive component, accounting for 45.35% of
the total cost and playing an important role in determining the profitability
of trout farming. Similar results have been obtained by Bozoglu & Ceyhan
44 Coldwater Fisheries and Aquaculture Management

(2009); and Bozoglu et al. (2006). They conducted a comprehensive study

on trout culture in Turkey. They discovered that the variable cost in trout and
sea bass farming was 74.02% and 67.49%, respectively, of the total cost.

TABLE 2.3 Costs and Return of Trout Culture in Sample Farms

Particulars Cost (Rs/farm) Cost (Rs/raceway) Percent Share
Variable Costs
Seed 49550.00 21543.48 21.04
Feed 106787.50 46429.35 45.35
Medicine and chemicals 1093.42 475.40 0.46
Transportation 9496.67 4128.99 4.03
Hired human labor 365.83 159.06 0.16
Miscellaneous 2648.00 1151.30 1.12
Total working capital 169941.42 73887.57 72.16
Interest on working capital 7434.94 3232.58 3.16
A. Total Variable Cost 177376.35 77120.15 75.32
Fixed Costs
Depreciation 14725.09 6402.21 6.25
Interest on fixed capital 34971.71 15205.09 14.85
Annual repair and maintenance 7422.33 3227.10 3.15
Land rent 1000.00 434.78 0.42
B. Total Fixed Cost 58119.13 25269.18 24.67
Total cost (A+B) 235495.48 102389.34 100.00
Total production (Kg) 1105.67 – –
Cost of production (Rs/Kg) 212.99 – –
Selling price (Rs) 389.17 – –
Farmer’s margin (Rs/Kg) 176.18 – –
Gross revenue 430291.67 – –
Net revenue 194796.19 – –
B:C ratio 1.83 – –

The second most crucial factor that influences the cost of trout farming
is seed cost, which accounted for 21.04% of the total cost of trout farming at
Rs. 21,543 per raceway. The government-recommended stocking density is
3,000 fingerlings per raceway, followed by most trout farms. Feed and seed
transportation costs accounted for 4.03% of total expenses. It was discovered
that trout farms were dispersed throughout the valley. Due to a lack of retail
Economics of Coldwater Fisheries 45

outlets for feed and seed, farmers were forced to purchase them directly from
the producing centers, incurring significant transportation costs. Bozoglu &
Ceyhan (2009) stated that feed cost accounted for 45.53% and 47.73% of
the total cost of trout and sea bass culture, respectively. This was a crucial
factor in determining profit margin. The feed was the most expensive aspect
of trout production (Gawa et al., 2017). Under fixed cost, interest on fixed
capital accounted for 14.85% of the total cost, followed by depreciation,
which accounted for 6.25% of the total cost. The other cost components were
interesting on working capital, annual repair and maintenance, medicine, and
chemicals, land rent, hired human labor costs, and miscellaneous charges,
which accounted for 3.16, 3.15, 0.46, 0.42, 0.16, and 1.12% of the total cost,
respectively. The benefit-cost ratio was estimated to be 1.83, indicating the
business’s economic viability, which is consistent with the findings of Olaoye
(2013), who estimated that variable costs account for 86.68% and fixed costs
account for 13.32% with a BCR of 1.69, indicating the economic viability
of trout farming in Nigeria. They discovered a significant difference in fixed
costs between earthen and concrete ponds, with earthen ponds being less
expensive. Gawa et al. (2017) stated that because trout farming is practiced
in concrete raceways in Kashmir, the cost of building a raceway is very high.
Trout farming was profitable even without subsidies, so subsidies were not
accounted for in profit when calculating costs and returns. There is a strong
need to encourage Kashmiri youths to pursue trout culture in raceways as
a means of earning a living. This can be accomplished by improving their
technical and managerial skills in trout farming and facilitating easy access
to institutional credit.

2.8 A CASE STUDY ON THE ECONOMICS OF COLD-WATER FISH

(TROUT) FARMING IN SIKKIM

Trout farming is one of the oldest commercial fish production methods. It has
over 400 years in Europe and about 150 years in the United States (Hinshaw,
1990). Chettri (2021) recently examined trout farming in Sikkim and found
it to be a significant and beneficial employment opportunity for the people of
the hilly region. He conducted a cost-benefit analysis, as shown in Table 2.4.
As per the latest specifications and design of the DOF of Sikkim, the
ideal size of the trout raceway is 51 m3 which costs around ₹ 1.20 lakhs.
Chettri (2021) observed that stocking 1,500 rainbow trout fingerlings at Rs
20 per each cost 0.30 lakhs, and the cost of feed, including transportation,
is around 1.00 lakh. As a result, the total operating cost is 1.30 lakhs. On
46 Coldwater Fisheries and Aquaculture Management

average, 500 kg of trout fish at 800 per kg would generate Rs 4.00 lakhs in
revenue. The net profit from a single racetrack is 2.7 lakhs, which is a very
volatile and reasonable profit for the farmers.

TABLE 2.4 Cost and Benefit Analysis of Trout Fish Farming

SL. Particulars Details Indian Rupee
No.
1. Capital cost Construction of trout raceways as per design/ 1.20 lakhs
specification. Size 17 m × 2 m × 1.5 m = 51 m3
2. Species Rainbow trout 0.30 lakhs
Number of stocks – 1,500 fingerlings 1.00 lakhs
Operating cost: (Recurring)
i) Cost of seeds @ Rs 20/-per each for 1,500
numbers including transportation cost.
ii) Cost of feed including transportation cost.
3. Benefits (B) Trout fish average growth 400–600 grams per year. 4.00 lakhs
production Mortality 20%. Total production 500 kg @ Rs 800
per kg.
4. Net profit B-C Rs. 4.80 lakhs–1.30 lakhs 2.7 lakhs

2.9 CHALLENGES AND PROSPECTS OF TROUT FARMING

The perennial springs and streams in the Himalaya are flowing from the
uplands to lowlands are not adequately and fully utilized. The coldwater
fisheries sector faces problems such as poor accessibility, difficult hilly
terrain, a lack of transportation and an inefficient market, a lack of aquacul­
ture infrastructure, etc. As a result, this sector could not foster to the expected
and extensive level. The major problems faced by the trout farmers in hill
region are the non-availability of fish feeds, high cost of feeds, and lack of
required fish seeds on time, etc. Even though such problems exist, there is
a massive prospect of fisheries and fish farming in the hilly state and can
enhance production level if some of the following strategies are considered:
• The available water resources could be brought into fish farming and
aquaculture development by utilizing these resources efficiently and
adequately rather than going into waste.
• Awareness of the importance and role of fisheries and aquaculture
for rural development and nutritional value can motivate the younger
generations, resulting in greater participation of people in fish farming.
Economics of Coldwater Fisheries 47

• Fish is a highly perishable product; proper accessibility, cold storage,

adequate transportation, and marketing facilities in the rural areas
could foster this sector so that local fish can be readily available in the
market.
• The use of modern technology and training for farmers can increase
production and productivity.
• The retail local fish shop should be started at the district level so that
the local fish will be readily available in the market, and this will also
encourage fish farmers to produce local fish.
• Although water resources are plentiful, many problems need imme­
diate attention from the government and the management teams to
get better results in steady growth in production, productivity, profit­
ability, sustainability, etc.
• To minimize the major problems faced by the farmers, such as the
nonavailability of feed on time and high feed costs, the government
should frequently monitor or establish at least some feed mill plants
within the states to redress such problems.

2.10 FUTURE PROSPECTIVE

Our country’s hill regions are endowed with valuable indigenous fish germ-
plasm and pristine water resources with a wide range of thermal regimes.
The fish can play an important role in supplementing protein requirements
for poor people living in remote Himalayan regions and providing a source
of income for a subset of people who overexploit natural resources due to
resource constraints in terms of cultivable lands in hills. At high altitudes,
there is enormous potential for the development of low volume, high value
species such as trout, particularly the rainbow variety. Production of 150 tons
is possible with a 4% growth rate in rainbow trout farming, which can be
increased to 200 tons with an 8% growth rate. According to the study, our
domestic demand is estimated to be 800 tons; therefore, steps must be taken to
meet this target. This can even help the feed industry and preservation units at
high altitudes. Similarly, sport fishing ancillary units will be operational, such
as short and long-distance trout transportation. It will all pave the way for the
formation of self-help groups in hill regions. However, it will be possible if the
seed production centers and table fish production units for trout are separated,
and the private sector is involved in fish production. In contrast, quality seed
and feed supply are guaranteed through state channels. To maintain sanitary
standards, all private units should be registered (Sarma et al., 2018).
48 Coldwater Fisheries and Aquaculture Management

Training in coldwater fishery and hill resource management is another

issue that needs to be addressed. Once upon a time, the confluence of rivers
in the northeast and the central Himalayas was reported to be one of the best
mahseer angling spots in the world. As a result, creating adequate facilities
for anglers for sport fishing is critical. Similarly, streams in Kashmir and
Himachal Pradesh are world-renowned for brown trout angling and fishing.
Commercial farming through Jhora fishery in sub Himalayan West Bengal
has a good potential for generating rural income. It will help to improve the
socioeconomic conditions of the local population in this hill region when
combined with sport fishery development. Another requirement for hill states
is the establishment of carp hatcheries and farms at lower altitudes to meet
the demand of local farmers engaged in small-scale fish farming. Running
water fish culture based on exotic carp has enormous potential. This should
be encouraged by providing farmers with knowhow and seed to supplement
their income. Despite the fact that some effective efforts have been launched
to develop and popularize fisheries in our country’s hill region, there is still
much work to be done. A holistic approach required for overall fisheries
development includes expanding fish culture activities in all potential areas,
integrated aquaculture, stock diversification, implementation of sustainable
production, enhancement measures in lakes and reservoirs, development of
ornamental fish, and promotion of fishery-based ecotourism at suitable sites,
in addition to conservation of valuable fish species (Mahanta et al., 2011).
Depending on the natural aquatic resources in the hills, the fishery, if
developed scientifically, will contribute significantly to the rural economy in
remote hilly zones. It is suggested that fishery development begins focusing
on available resources, including both exotic and indigenous fishes. There
is a pressing need to replenish depleted stocks. Stocks of some of the most
important commercial and recreational fishers. The indiscriminate use of
chemicals in orchards/tea gardens may impact the water quality of catch­
ments in regional river/stream systems (Sarma et al., 2018).

2.11 CONCLUSION

The Himalayan regions of India have an abundance of water resources in

the form of rivers, lakes, tanks, and reservoirs, etc., which have diverse
varieties of fish species that are exotic and indigenous, cultivable, and
non-cultivable. These water resources are suitable for the capture as well
as culture fisheries. Jammu & Kashmir have the most prominent river or
canals, i.e., 27,781 km. In major hilly areas, the total fishermen population
Economics of Coldwater Fisheries 49

is estimated at 1.40 lakh fishers who directly depend on coldwater fisheries.

Total coldwater fish seed production is 4388.9 lakh fry from all hilly states
of India, Manipur producing the highest fish seed with 2494.8 lakh fish fry
production. In 2019–2020, the states mentioned above showed an increasing
trend except for Sikkim, which offers a minor reduction in production. The
annual total cost and gross revenue per farm in Jammu and Kashmir were
estimated to be Rs. 2.36 lakh per farm and Rs. 4.30 lakh per raceway, with a
benefit-cost ratio of 1.83, indicating the business’s economic viability (Gawa
et al., 2017). A trout raceway of 51 m3 in Sikkim, which costs around 1.20
lakhs, has been observed to produce 500 kg of trout fish at an average price
of 800 per kg, yielding a 4 lakhs income. The net profit from a single trout
raceway (51 m3) would come to ₹ 2.7 lakhs, which is a very venturing and
reasonable profit to the farmers (Chettri, 2021). Significant challenges in
coldwater fish farming are the availability of water resources for culture,
unawareness of fish farming, lack of cold storage, quick transport, and other
marketing facilities, and lack of new technical knowledge. Future plans
also suggested to improve coldwater fisheries in the country are technology
demonstration, establishing a brood banking facility for the availability of
brood through the year, and implementation of diversifying culture systems
and open water cage culture to use water resources efficiently. Training in
coldwater fishery and hill resource management needs to be addressed for
further development.

KEYWORDS

• benefit-cost ratio
• coldwater
• ecotourism
• fish farming
• marketing
• socio-economic development
50 Coldwater Fisheries and Aquaculture Management

REFERENCES

Anon. (2011). Handbook of Fisheries and Aquaculture (2nd edn.). Directorate of knowledge
management in agriculture–ICAR, New Delhi.
Ayyappan, S., & Krishna, M., (2007). Changing consumption pattern. The Survey of Indian
Agriculture, 6(6).
Bozoglu, M., & Ceyhan, V., (2009). Energy conversion efficiency of trout and sea bass
production in the Black Sea, Turkey. Energy, 34, 199–204.
Bozoglu, M., Ceyhan, V., Avni, C. H., Demiryürek, K., & Kilic, O., (2006). Evaluation of
different trout farming systems and some policy issues in the Black Sea Region, Turkey. J.
Appl. Sci., 6, 2882–2888.
Chettri, K. B., (2020). Ecology and Economics of Coldwater Inland Fisheries in Sikkim (p. 261).
Ph.D. thesis.
Chettri, K. B., (2021). Current status of rainbow trout culture in Sikkim: A sustainable farming
system in the hills. Indian J. Hill Farm., 34, 126–133.
Chettrih, K. B., & Kundu, R., (2020). Evaluation of socio-economic conditions of coldwater
fish farmers: A case study of Sikkim. Indian J. Econ., 16, 62–71.
DAHDF, (2018). Mission Cold Water Fisheries Development National Action Plan – 2017–
2022. Ministry of Agriculture and Farmers Welfare, Government of India.
DoF., (2020). Handbook on Fisheries Statistics. Ministry of Fisheries, Animal Husbandry and
Dairying. Government of India. New Delhi. https://dof.gov.in/sites/default/files/2021-02/
Final_Book.pdf (accessed on 3 February 2023).
Ganesh, K. B., Shyam, S. S., & Katiha, P. K., (2014). Coldwater Fisheries (pp. 129–154).
Livelihood Status of Fishers in India.
Gawa, S., Kumar, N. R., Tiwari, V. K., Prakash, S., Yadav, V. K., & Wani, G. B., (2017).
Trout culture in Kashmir: An opportunity for profitable enterprise. Social Entrepreneurship
in Aquaculture, 381–389.
Hinshaw, J. M., (1990). Trout Production: Handling Eggs and Fry (p. 220). Southern Regional
Aquaculture Center SARC.
Kumar, R., Umesh, K. B., & Rajesh, E., (2007). Economics of inland fish production Karnataka.
J. Rural Dev., 26.
Mahanta, P. C., Moza, U., & Joshi, K. D., (2011). Coldwater fisheries and aquaculture.
Handbook of Fisheries and Aquaculture. Published by DKMA, ICAR, New Delhi.
Olaoye, O., (2013). Assessment of socioeconomic analysis of fish farming in Oyo State,
Nigeria. Glob. J. Sci. Front. Res. D: Agric. Vet., 13, 45–53.
Sarma, D., Akhtar, M. S., & Singh, A. K., (2018). Coldwater Fisheries Research and
Development in India, 93–133.
Shyam, S., Raina, H. S., & Joshi, C. B., (1999). Fishes of Indian Uplands (p. 64). Bull. No.2,
NRC on Coldwater Fisheries, Bhimtal.
Tyagi, B. C., Sundar, S., & Mohan, M., (2005). Coldwater Fisheries Research and Development
in Northeast Region of India–a Report of National Research Center on Cold Water Fisheries.
Bhimtal.
CHAPTER 3

Nutritional Requirements of Coldwater

Fishes
SEEMAB ZEHRA1, ASAAD H. MOHAMED1, EDOARDO PANTANELLA1,
and RAMZY A. YOUSIF2
1
Beacon Development, King Abdullah University of Science and
Technology, Thuwal, Jeddah, Saudi Arabia
2
Department of Fisheries and Wildlife Science, Sudan University of Science
and Technology, Khartoum, Sudan

ABSTRACT

Aquaculture is one of the fastest-growing food-producing sectors globally,

and coldwater fish species are the important aquaculture species farmed
in different parts of the world. Coldwater fish are an important source of
protein for the hill people, and there is an urgent need for coldwater fisheries
development with the assistance of international organizations. Nutrition is a
crucial factor for fish production as it plays a vital role in boosting aquaculture
production. Successful and sustainable aquaculture depends on nutritionally
balanced, environmentally friendly, and economically viable practical feeds.
The literature on the nutritional requirements regarding coldwater fisheries
is sparse. To improve fish productivity, it is important to provide specific
and efficient diets based on satisfying the requirements of coldwater fish
species for various nutrients. This chapter explores the literature related to
the protein, amino acid (AA), lipid, fatty acid, vitamin, and mineral require­
ments of coldwater fishes which will be useful for boosting the aquaculture
of these fishes.

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
52 Coldwater Fisheries and Aquaculture Management

3.1 INTRODUCTION

Nutrients are compounds in foods, dispensable to life, providing energy, the

building blocks for repair and growth, and substances essential to regulate
several physiological activities. Due to the ever-increasing population
globally, many people are suffering from malnutrition, and thus, there is a
dire need to fill the global food basket. Aquatic food is a crucial constituent
of the worldwide food basket to over the people’s malnutritional problems.
Aquaculture can be an alternative to meet the growing global demand for
nutritious food fish for a rapidly expanding population and contribute to
the growth of national economies. Fish can make a unique contribution to
improve and diversify dietary intake and promoting nutritional well-being
among most population groups. Fish and other seafood forms provide
a balanced diet and reduce the protein gap. Fish have a highly desirable
nutrient profile providing an excellent source of high-quality animal protein
containing all the 10 essential amino acids (AAs) in desirable concentrations
that are easily digestible and of high biological value. Feeds and feeding are
essential elements in the aquaculture of organisms. Hence, knowledge of
nutrition and practical feeding of fish is essential for sustainable aquaculture.
In aquaculture systems, feed is responsible for more than one-half of the
variable cost, often ranging from 50–60% depending upon its intensity.
Any reduction in the feed cost through their development improved
husbandry and, therefore, is necessary for the industry’s development
and well-being. Production of eco-friendly feeds depends on fish species’
nutritional requirements and meeting those requirements with balanced feed
formulations and appropriate feeding practices.
Coldwater fisheries occupy an important place amongst the freshwater
fishes of India. The coldwater fisheries deal with fisheries activity in the water
where water temperature ranges from 5°C to 25°C. India’s coldwater fisheries
resources are mainly in upland streams, rivers, high, and low altitudinal lakes,
and reservoirs located in different hill states of India. Diverse populations of
indigenous and a few exotic coldwater fish species (Tables 3.1 and 3.2) in
these mountain water bodies form an immense potential to generate income
in rural areas and provide food and nutritional security. The value of fish
as a health food is well known but poorly documented. Fish is the cheapest
animal protein accessible to the poor and is aptly called the ‘rich food of
the poor.’ Apart from essential fatty acids (EFAs) and protein, fish is rich in
vitamins and minerals. Fish as a protective food assumes greater significance
in India’s different regions, including Himalayan coldwater regions due to
 Nutritional Requirements of Coldwater Fishes
TABLE 3.1 Important Cold-Water Fishes
Snow Trout Minor Carps Mahseer Exotic Minor Carps Barils/Minnows/
Catfishes/Loaches
Schizo thorax richardsonni Labeo dyocheilus Tor putitora Onchorhynchus mykiss Labeo dyocheilus Barilius bendelisis
Schizothoraichthys curvifrons Labeo dero T. tor Salmo trutta fario Labeo dero B. bakeri
S. longipinnis Crossocheilus latius T. khudree Salvelinus fontinalis Crossocheilus latius latius B. vagra
S. esocinus Gara gotyla T. malabaricus Cyprinus carpio var. Gara gotyla B. bariia
specularis
S. niger G. hughi Neolissochilus C. carpio var. G. hughi Raimas bola
hexagonolepis communis
S. plannifrons Puntius – C. carpio Var. nudus Puntius ophicephalus Danio divario
ophicephalus
S. micropogon – – Tinea tinea – Botia birdi
S. progastus – – Carrasius carrasius – Glyptothorax
pectinopterus
S. nasus – – – – G. conirostre
conirostre
S. hugeiii – – – – –
Lepidopygopsis typus – – – – –

53
 54
TABLE 3.2 Important Edible Fishes of India Coldwater Fish Species
Ornamental Brachydanio Danio devario Labeo nandina Lepidocephalus Gageta Conta Carassius C. auratus
Fish Species rerio guntea cenia conta carassius
Edible fish Snow trout Mahseer Exotic trout Minor carps Barils/minnows/catfishes Fish species transported
species loaches from plain area
Schizothorax Tor putitora Onchorhynchus Labeo dyocheilus Barilius spp. Hypopthalmichthys
richarsonii; mykiss molitrix

Coldwater Fisheries and Aquaculture Management

S. longipinnis T. tor Salmo trutta Labeo dero Raimas bola Ctenophraryngodon Idella
fario
S. esocinus Neolissochilus Saivelinus Gara gottjla Danio divario –
hexagonolepis fontinalis
S. niger – Cyprinus carpio Puntius spp. Botia birdi –
var. specularis
S. progastus – C. carpia var. – Glyptothorax spp. –
commums
S. nasus – H. molitrix – – –
C. idella
Sports fish Tor putitora T. tor T. khudree T. malabaricus Salmo trutta fario –
species
Nutritional Requirements of Coldwater Fishes 55

the rich fish and fisheries resources of the country, which can play a pivotal
role in mitigating protein deficiency and malnutrition. But despite all these
advantages of consuming fish, the quantitative nutritional requirements of
coldwater fishes from India are not all known, which is the major impediment
in their culture. Also, the lack of consistent data on the essential nutrients
requirements of different growth stages of coldwater fish species makes it
difficult to correctly formulate diets that maximize growth to their full genetic
potential. Hence, it is essential to develop nutritionally balanced quality feeds
for different growth stages to develop aquaculture of coldwater fish species.
To formulate nutritionally balanced feeds, there is a dire need to determine the
nutritional requirements of concerned species and scan the nutrient profile of
locally available feed ingredients. This information will enable us to increase
the production of nutritionally balanced feeds. Any balanced formula for fish
diet should include essential AAs, energy sources, EFAs, specific vitamins,
and minerals to support life and promote growth.

3.2 PROTEIN AND AMINO ACID (AA) REQUIREMENTS

The proteins are essential constituents representing the most extensive

group of chemicals in the organism. Generally, 16% protein is contained
by the whole fish carcass. Proteins are a crucial component of the cell and
accordingly account for the bulk of the muscle tissues, internal organs,
brain, nerves, and skin. As scarcity and excess of dietary protein affect
growth performance and general body maintenance, the generation of data
on the actual protein requirement of fish is vital to formulate well-balanced,
low-cost, and environmentally friendly feeds (Zehra & Khan, 2012). The
study of dietary nutrient requirements in fish has been almost based on reviews
comparable to those performed on terrestrial farm animals. Therefore, it
follows that nearly all the existing data on the dietary nutrient requirements
of aquaculture species is derived from laboratory-based experiments. The
animals are kept in a controlled environment at high density and have
no availability of natural food organisms (FAO, 2014). Dietary protein
requirements are usually expressed in terms of a dietary percent or as a ratio
of protein to dietary energy. There is not much information documented
on the protein nutrition of cold water fishes. A study has been conducted
on the performance of locally available low-cost feed ingredients on Tor
putitora to introduce this species into commercial aquaculture (Islam, 2002).
Also, aqua-culturists have been trying to find out nutritionally balanced
diet by incorporating different ingredients in varied proportions to realize
56 Coldwater Fisheries and Aquaculture Management

better production levels. The incorporation of silkworm pupae as a protein

source in the diet of Deccan Mahseer was tried by Shyama (1990), who
reported no harmful effect on flesh quality, the optimum level of inclusion
being 60%. Spirulina was used as a significant protein source for the species
by Keshavanath et al. (1986). Several experiments and trials have been
conducted at National Research Center on Coldwater Fisheries (NRCCF)
to formulate diets for various life stages of golden Mahseer by using local
ingredients like soya meal, silkworm pupae, rice/wheat starch, etc. Based on
these investigations, it was observed that the early rearing stages of Mahseer
up to advance fry/fingerlings (45–55 mm) require about 45% protein (Mohan,
2002). The information on protein nutrition of Tor khudree (Shankar, 1988);
Tor putitora (Hossain et al., 2002; Mohan, 2002; Sawhney & Gandotra,
2010; Akram & Swapna, 2014) and Tor tambroides (Misieng et al., 2011)
has been documented. Table 3.3 summarizes the known quantitative protein
requirements of some of the coldwater fish species.

TABLE 3.3 Protein Requirements of Cold-Water Fish Species

Fish Species Protein Requirement (%) References
Rainbow trout 35–50 NRC (2011)
Tinca tinca 50 Garcia (2015)
Cyprinus carpio 28–47 Nose (1979); NRC (2011)
41 Ahmed & Maqbool (2017)
Ctenopharyngodon idella 38 Wang (2005)
33 Koprucu (2012)
35 Shang (2009)
Tor putitora 40 Hossain (2002)
45–50 Islam (2004)
Schizothorax richardsonii 45 Wagle et al. (2016)

Fish do not have a dietary need for protein per se; they need AAs that
comprise protein. The AA is made up of the basic amino group (NH2), the
acidic carboxyl group (COOH), and the R. The AAs structure units form
proteins. They combine to form short polymer chains called peptides or
long chains called polypeptides or proteins. In nutrition, AAs are classified
as essential or non-essential. Essential AAs, also known as essential AAs,
are a group of AAs that humans and other vertebrates can synthesize from
metabolic intermediate. These AAs must be provided in food because the
human body and other vertebrates do not have the necessary health science
Nutritional Requirements of Coldwater Fishes 57

methods to make these AAs. Non-essential, also known as available AAs,

can be eliminated from food.
The 20 to 22 AAs that comprise proteins are as follows:

Essential Amino Acids for Fish Non-Essential Amino Acid

Arginine Alanine
Histidine Asparagine
Isoleucine Aspartic acid
Leucine Cysteine
Lysine Glutamic acid
Methionine Glutamine
Phenylalanine Glycine
Threonine Proline
Tryptophan Serine
Valine Tyrosine
– Selenocysteine
– Pyrrolysine

From a quantitative perspective, protein utilization and muscle protein

growth efficiency are the most critical issues and improving the inclusion
of AAs in line with demand and improving protein utilization in the growth
of body proteins with minimal environmental impact is common in all
fish production systems (Kaushik & Seiliez, 2010). AAs are the important
component to synthesize proteins and other nitrogenous compounds and serve
as important regulators of metabolic pathways. Deficiency of any essential
AA will impair protein synthesis and suppress fish growth (Masagounder
et al., 2009). Moreover, feeding excess AA concentrations can increase
ammonia release and spoil water quality (Hart et al., 2010). It is necessary
to know the fish species’ specific AA requirements and prepare mixtures of
protein supplemented with the deficient AAs and achieve maximum growth
and protein efficiency. Based on feeding techniques developed earlier for
other animals, the dietary AA requirements of fish were first investigated
by Halver (1957). Fish were fed a balanced diet containing graded levels
of a test AA (casein: gelatin mixture supplemented with crystalline AAs to
simulate the whole hen’s egg protein). Halver (1957) determined the arginine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine,
tryptophan, and valine requirements for chinook salmon and concluded the
58 Coldwater Fisheries and Aquaculture Management

essentiality of 10 AAs. The dietary needs of 10 essential AAs for various

cultivable fish species have also been confirmed by several researchers (Khan
& Zehra, 2020). Most of the AA requirements have been estimated using
dose-response and carcass deposition methods in which replicate groups of
fish were fed diets containing graded levels of the test AA until significant
differences appear in fish growth. The AAs requirement of coldwater fish
species is given in Table 3.4. Quantitative dietary requirements for all
10 essential AAs have been estimated for only Atlantic salmon, Chinook
salmon, Chum salmon, Coho salmon, Common carp, and Rainbow trout
amongst cold water fish species.

3.3 LIPIDS REQUIREMENTS

Lipid is chemically defined as soluble in water and soluble in alcohol, ether,

and chloroform. Lipids are an essential part of living cells. Lipids (fats) are
very powerful nutrients that can be used to save part of the protein in the diet
of marine animals. Lipids provide twice as much energy as proteins and carbo­
hydrates. Lipids usually comprise about 15 g/kg of fish diet, provide essential
oils (EFA) and act as a medium of soluble vitamins. The latest practice of
feeding fish is to use high amounts of lipids in the diet. Although the increase
in lipids you eat can help diminish the escalating cost of food by keeping a
portion of the protein in the diet, problems such as excess fat in the liver can
reduce the quality of life and the fish market. Light lipids include fatty acids
and triacylglycerol. Fish usually need fatty acids in the omega 3 and 6 families
(n–3 and n–6). Fatty acids can be: (i) saturated fatty acids (SFA, no double
bonds); (ii) polyunsaturated fatty acids (PUFA, > 2 double bonds); or (iii) satu­
rated fatty acids (HUFA);> 4 double bonds). Marine fish oil is naturally high
(> 30%) in omega 3 HUFA (highly unsaturated fatty acids) and is an excellent
source of lipid in fish processing. The lipids from these oily oils can have
favorable effects on human health of the heart and blood vessels (Craig, 2009).
Dietary lipids are a crucial source of energy that can be easily digested
and are the only source of essential oils (EOs) needed by fish for maximizing
growth and further development. They are also a carrier and help to absorb
fat-soluble nutrients such as sterols and vitamins A, D, E, and K. Due to the
incapability of organisms to combine de novo fatty acids in the n–6 and n–3
series, these fatty acids should be supplemented in the diet. In terrestrial
animals, the linoleic series (n–6) has been found to have the highest activity
of EOs (EFA), with the linolenic series (n–3) having only partial EFA
activity. Thus, it follows that most of the fatty acids (PUFA) in terrestrial
Nutritional Requirements of Coldwater Fishes 59

TABLE 3.4 Amino Acid Requirements of Cold-Water Fish Species

Arginine Requirement
Species CP% REQ % REQ % References
Diet Protein
Atlantic salmon 40 1.6 4.1 Lall et al. (1994)
42 2.0–2.2 4.8 Berge et al. (1997)
45 – 4.6 Rollin (1999)
Chinook salmon 40 2.4 6.0 Klein & Halver (1970)
Coho salmon 40 2.3 5.8 Klein & Halver (1970)
45 2.2–2.5 4.9–5.5 Luzzana et al. (1998)
Common carp 48 1.7 4.3 Nose (1979)
Rainbow trout 45 1.6–1.8 3.6–4.0 Walton et al. (1986)
40 1.4–1.7 3.5–4.2 Chiu et al. (1988)
43 1.6 4.0 Fournier et al. (2003)
Isoleucine Requirement
Atlantic salmon 45 – 3.2 Rollin (1999)
Chinook salmon 41 1.0 2.6 Chance et al. (1964)
Chum salmon – – 2.4 Akiyama & Arai (1993)
Coho salmon – – 1.2 Aria & Ogata (1993)
Common carp 48 1.0 2.5 Nose (1979)
40 – 2.3 Ogino (1980)
33 1.29 3.84 Zhao et al. (2012)
Grass carp 35 1.4 4.0 Shang et al. (2009)
Lake trout 35 0.7–0.9 1.5–2.1 Hughese et al. (1983)
Rainbow trout 40 – 2.4 Ogino (1980)
33 0.7–1.4 1.5–2.8 Rodehutscord et al. (1997)
Leucine Requirement
Atlantic salmon 40 0.81 2.0 Scott et al. (1996)
45 1.8 – Rollin (1999)
Chinook salmon 40 0.7 1.8 Klein & Halver (1970)
Chum salmon 40 0.7 1.6 Akiyama et al. (1985)
– – 1.6 Akiyama & Arai (1993)
Coho salmon 40 0.7 1.8 Klein & Halver (1970)
– – 0.9 Aria & Ogata (1993)
Common carp 48 0.8 2.1 Nose (1979)
40 – 1.4 Ogino (1980)
Rainbow trout 40 – 1.6 Ogino (1980)
34 0.5–0.6 1–1.2 Rodehutscord et al. (1997)
60 Coldwater Fisheries and Aquaculture Management

TABLE 3.4 (Continued)

Arginine Requirement
Species CP% REQ % REQ % References
Diet Protein
Histidine Requirement
Atlantic salmon 45 – 5.2 Rollin (1999)
Chinook salmon 41 1.6 3.9 Chance et al. (1964)
Chum salmon – – 3.8 Akiyama & Arai (1993)
Coho salmon – – 3.4 Aria & Ogata (1993)
Common carp 48 1.3 3.3 Nose (1979)
40 – 4.1 Ogino (1980)
Lake trout 35 1.3–1.7 2.7–3.7 Hughese et al. (1983)
Rainbow trout 40 – 4.4 Ogino (1980)
43 3.4 9.2 Choo et al. (1991)
34 1.1–1.4 2.3–2.9 Rodehutscord et al. (1997)
Valine Requirement
Atlantic salmon 45 – 3.9 Rollin (1999)
Chum salmon – – 3.0 Akiyama & Arai (1993)
Coho salmon – – 2.2 Aria & Ogata (1993)
Common carp 48 1.40 3.60 Nose (1979)
40 – 2.9 Ogino (1980)
Jian carp 34 1.3 4.0 Dong et al. (2012)
Lake trout 35 0.8–1.0 1.8–2.2 Hughese et al. (1983)
Rainbow trout 40 – 3.1 Ogino (1980)
34 0.8–1.6 1.7–3.4 Rohutscord et al. (1997)
36 1.4 3.85 Bae et al. (2012)
Lysine Requirement
Atlantic salmon 50 2.0 4.0 Anderson et al. (1993)
– – 3.2–3.6 Berge et al. (1997)
45 – 6.1 Rollin (1999)
43 2.2 5.0 Espe et al. (2007)
50 2.6 3.0 Helland et al. (2011)
Chinook salmon – – 5.0 Halver et al. (1958)
Chum salmon 40 1.9 4.8 Akiyama et al. (1985)
– – 5.0 Akiyama & Arai (1993)
Coho salmon – – 3.8 Arai & Ogata (1993)
Common carp 48 2.2 5.7 Nose (1979)
40 – 5.3 Ogino (1980)
Nutritional Requirements of Coldwater Fishes 61

TABLE 3.4 (Continued)

Arginine Requirement
Species CP% REQ % REQ % References
Diet Protein
Grass carp 38 2.1 5.4 Wang et al. (2005)
Rainbow trout 40 – 5.3 Ogino (1980)
47 – 6.1 Ketola (1983)
45 1.9 5.3 Walton et al. (1984b)
45 1.9 4.3 Walton et al. (1986)
35 1.3 3.7 Kim et al. (1992b)
34 2.5 5.3 Rodehutscord et al. (1997)
45 1.8 5.1 Cheng et al. (2003)
40 2.3 5.8 Encarnação et al. (2004)
50 2.7 8.4 Wang et al. (2010)
36 2.2 6.0 Yun et al. (2016)
Methionine Requirement
Atlantic salmon 45 – 3.1 Rollin (1999)
42 0.7 1.7 Espe et al. (2008)
Chinook salmon – – 4.6 Halver et al. (1959)
Coho salmon – – 2.7 Aria & Ogata (1993)
Common carp 48 0.8 2.0 Nose (1979)
40 – 1.6 Ogino (1980)
40 0.8 2.1 Schwarz et al. (1998)
Rainbow trout 40 – 1.8 Ogino (1980)
– – 2.2 Walton et al. (1982)
– – 3.0 Rumsey et al. (1983)
40 0.8 1.9 Cowey et al. (1992)
45 0.5 1.5 Kim et al. (1992a)
34 0.9 1.9 Rodehutscord et al. (1995)
Grass carp 28 0.6 2.1 Wu et al. (2017)
Phenylalanine Requirement
Chinook salmon 41 1.7 4.4 Chance et al. (1964)
Chum salmon – – 6.3 Akiyama & Arai (1993)
Coho salmon – – 4.5 Aria & Ogata (1993)
Common carp 48 1.3 3.3 Nose (1979)
40 – 4.9 Ogino (1980)
Juvenile grass carp 40 2.4 6.5 Gao et al. (2015)
Nile tilapia 28 1.1 3.8 Santiago & Lovell (1988)
62 Coldwater Fisheries and Aquaculture Management

TABLE 3.4 (Continued)

Arginine Requirement
Species CP% REQ % REQ % References
Diet Protein
Rainbow trout 40 – 5.2 Ogino (1980)
35 0.7 2.0 Kim (1993)
Threonine Requirement
Atlantic salmon 45 – 3.2 Rollin (1999)
40 1.1 2.6 Bodin et al. (2008)
Chinook salmon 40 – 2.2 Delong et al. (1962)
Chum salmon 40 1.2 3.0 Akiyama et al. (1985)
Coho salmon – – 2.0 Aria & Ogata (1993)
Common carp 48 1.5 3.9 Nose (1979)
40 – 3.3 Ogino (1980)
Rainbow trout 40 – 3.4 Ogino (1980)
– – 3.2 Rodehutscord et al. (1995)
40 1.1 2.6 Bodin et al. (2008)
36 0.95 1.07 Yun et al. (2015)
Tryptophan Requirement
Atlantic salmon 45 0.7 1.5 Rollin (1999)
Chinook salmon – – 0.5 Halver (1965)
Chum salmon fry 40 0.2 0.7 Akiyama et al. (1986)
Coho salmon – – 0.5 Halver (1965)
Common carp 48 0.3 0.8 Nose (1979)
40 – 0.6 Ogino (1980)
– – 0.3 Dabrowski (1981)
Rainbow trout 40 – 0.5 Ogino (1980)
42 0.2 0.6 Poston & Rumsey (1983)
55 0.3 0.9 Walton et al. (1984a)
45 0.3 0.7 Walton et al. (1986)
– – 0.6 Kim et al. (1987)
33 0.2 0.6 Rodehutscord et al. (1997)
Sockeye salmon – – 0.5 Halver (1965)

animal tissues are part of the linoleic series, such as 18: 2n–6 (linoleic acid)
and 20: 4 n–6 (Arachidonic acid).
Generally, cold water freshwater fish have a specific dietary requirement
for n–3 PUFA (18:3 n–3, 20:5 n–3, 22:6 n–3), i.e., salmonids, Ayu, while
Nutritional Requirements of Coldwater Fishes 63

warm freshwater fish have either a requirement for both the n–3 series and
n–6 series PUFA, i.e., carps, eel, and possibly channel catfish, or for the n–6
series alone, i.e., tilapias, and possibly the snakehead C. micropeltes; for
review see Kanazawa (1985). In the case of marine carnivorous fish species
(i.e., black sea bream M. macrocephalus, opaleye G. nigricans, puffer fish F.
rubripens, yellow tail S. quinqueradiata, plaice P. platessa, gilthead bream
S. auratus, turbot S. maximus. Since the food organisms consumed are rich
in 22:6 n–3 and 20:5 n–3, they have lost the ability to chain elongate and
further desaturate 18:3 n–3 to the corresponding HUFA.
Numerous reviews of fish diets have been published containing informa­
tion on lipid and fatty acid requirements (NRC, 2011). The lipid and fatty
acid requirements for coldwater fish species are summarized in Table 3.5.

3.4 VITAMIN REQUIREMENTS

Vitamins are organic components required in small amounts from an

external source for optimal growth, health, proper body metabolism and
normal physiological functions. These micronutrients are classified as
fat-soluble and water-soluble. Vitamins A, D, E, and K are the fat-soluble
vitamins that function independently of enzymes, or in some cases such as
vitamin K, may have co-enzyme roles. Eight of the water-soluble vitamins
viz. riboflavin, pantothenic acid, thiamine, pyridoxine, nicotinic acid,
biotin, cyanocobalamin, and folic acid are required in trace amounts, have
mainly co-enzyme functions, and are known as vitamin B complex. Three
of the water-soluble vitamins, Choline, Inositol, and vitamin C are required
relatively in larger quantities and have the functions other than co-enzymes.
Water-soluble vitamins are not stored in optimal amounts in the animal’s
body; body stores quickly collapse due to a lack of soluble vitamins in the
water. Therefore, toxicity of soluble vitamins in water is not possible. Several
deficiency signs include corneal vascularization, cloudy lens, hemorrhagic
eyes, dark coloration, poor appetite, anemia, poor growth, clubbed gills,
necrosis, and scarring, cellular atrophy, gill exudate, sluggishness, lesion
in colon, difficult motion, weakness, edema of stomach and colon, muscle
spasm while resting fragmentation of erythrocytes, scoliosis, lordosis,
impaired collagen formation and altered cartilage have been reported in
various cultivable fish species. In order to avoid the prevalence of such
diseases and ensuring faster growth provision of vitamin-balanced practical
feed is a prerequisite to successful aquaculture. The vitamin requirements
have been reported for coldwater fish species and given in Table 3.6.
TABLE 3.5 Lipid and Fatty Acid Requirements of Cold-Water Fish Species

64
Lipid Requirement of Cold-Water Fish Species
Common Name Scientific Name Requirement (% dry diet) References
Gibel carp Carassius auratus gibelio 14.05 Pei et al. (2004)
Grass carp Ctenopharyngodon Idella 2 Du et al. (2008)
Juvenile tench Tinca tinca 8.5–12 Royuela et al. (2015)
Malaysian Tor lambroides 5 Ng & Andin (2011)
mahseer
Manchurian trout Brachymystax lenok 17.38–19.50 Chang et al. (2017)

Coldwater Fisheries and Aquaculture Management

Dietary Fatty Acid Requirement of Cold-Water Fish Species (Expressed as Percentage of Dry Diet)
Common Name Scientific Name Fatty Acid Percentage of Dry References
Diet (Requirement)
Atlantic salmon Salmo salar n–3 PUFA 1% Ruyter et al. (2000)
n–3 HUFA 1% (showed better
growth than n–3 PUFA)
Common carp Cyprinus carpio (i) n–6 PUFA (i) 1 (0.25% LA) Radunzneto et al. (1996);
(ii) n–3 PUFA (ii) ~0.05 Tian et al. (2016)

Coho salmon Oncorhynchus kisutch ALA 1% Yu & Sinnhuber (1979)

Chum salmon Oncorhynchus keta ALA+LA/n–3 HUFA 1% Takeuchi, Watanabe, & Nose (1979)
Grass carp Ctenopharyngodon idella (i) ARA 0.3% Tian et al. (2014); Li et al. (2015);
(ii) n–3 HUFA (Fish oil fish oil performed Zeng et al. (2016)
and lard oil) better.
(iii) ALA:LA 1.08, 1.19, 1.05%
TABLE 3.5 (Continued)

Nutritional Requirements of Coldwater Fishes

Dietary Fatty Acid Requirement of Cold-Water Fish Species (Expressed as Percentage of Dry Diet)
Common Name Scientific Name Fatty Acid Percentage of Dry References
Diet (Requirement)
Juvenile fat cod Hexagrammos otakii n–3 HUFA 1.2–1.6 (squid liver oil Lee & Cho (2009)
performed better than
corn, linseed oil and
lauric acid)
Rainbow trout Oncorhynchus mykiss n–3 HUFA 1% Castell et al. (1972)
Common carp Cyprinus carpio Citric acid 30 g kg–1
Khajepour et al. (2012)

65
TABLE 3.6 Vitamin Requirements of Cold-Water Fish Species

66
Common Name Scientific Name Source Requirement Reference
Thiamin
Common carp Cyprinus carpio – 0.5 mg Aoe et al. (1969)
Grass carp Ctenopharyngodon idella – 1.3 mg, 5 mg Huang et al. (2014)
Jian carp Cyprinus carpio var. Jian – 1.02 mg Huang et al. (2011)
Pacific salmon Oncorhynchus spp. – 10–15 mg Halver (1972)
Rainbow trout Oncorhynchus mykiss – 1–10 mg McLaren et al. (1947)
Rainbow trout Salmo gairdneri – 1 mg Morito et al. (1986)

Coldwater Fisheries and Aquaculture Management

Riboflavin
Common carp Cyprinus carpio – 4 mg Aoe et al. (1967a)
– – 6.2 mg Aoe et al. (1967a)
– – 7 mg Takeuchi et al. (1980)
Grass carp Ctenopharyngodon Idella – 6 mg Serrini et al. (1996)
– – 6.65 mg Chen et al. (2015)
Jian carp Cyprinus carpio var. Jian – 5.0 mg Li et al. (2010)
Pacific salmon Oncorhynchus spp. – 20–25 mg Halver (1972)
Rainbow trout Oncorhynchus mykiss – 5–15 mg 6 mg McLaren et al. (1947); Takeuchi et al. (1980)
– – 3 mg Hughes et al. (1981)
– – 2.7 mg Amezaga & Knox (1990)
Spotted snakehead Channa punctatus – 5.7–7.7 mg Zehra & Khan (2017)
Niacin
Brook trout Salvelinus fontinalis – 95.0 mg Phillips & Brockway (1947)
TABLE 3.6 (Continued)

Nutritional Requirements of Coldwater Fishes

Common Name Scientific Name Source Requirement Reference
Thiamin
Common carp Cyprinus carpio – 28 mg Aoe et al. (1967b)
Grass carp Ctenopharyngodon Idella – 34.01, 42.08 mg Li et al. (2015)
– – 25.5 mg Wu et al. (2007a)
Pacific salmon Oncorhynchus spp. – 150–200 mg Halver (1972)
Rainbow trout Oncorhynchus mykiss – 1–5 mg McLaren et al. (1947)
– – 10 mg Poston & Wolfe (1985)
Common carp Cyprinus carpio – 30–50 mg Ogino (1967)
Grass carp Ctenopharyngodon Idella – 25 mg Liu et al. (2007)
Jian carp Cyprinus carpio var. Jian – 23 mg Wen et al. (2009)
Pacific salmon Oncorhynchus spp. – 40–50 mg Halver (1972)
– – 17 mg Leith et al. (1990)
Rainbow trout Oncorhynchus mykiss – 10–20 mg McLaren et al. (1947)
– – 20 mg Cho & Woodward (1990)
Pyridoxine
Atlantic salmon Salmo salar – 5 mg Lall & Weerakoon (1990)
Common carp Cyprinus carpio – 5–6 mg Ogino (1967)
Gibel carp Carassius auratus gibelio – 7.26–11.36 mg Wang et al. (2011)
Grass carp Ctenopharyngodon Idella – 1.13–3.73 mg Yang et al. (2017)
Jian carp Cyprinus carpio var. Jian – 6.07 mg He et al. (2009)
Pacific salmon Oncorhynchus spp. – 10–20 mg Halver (1972)

67
TABLE 3.6 (Continued)

68
Common Name Scientific Name Source Requirement Reference
Thiamin
Rainbow trout Oncorhynchus mykiss – 1–10 mg McLaren et al. (1947)
– – 2 mg Woodward (1990)
– – 3–6 mg Woodward (1990)
Biotin
Common carp Cyprinus carpio – 1 mg Ogino et al. (1970a)
Channel catfish Ictalurus punctatus – R Robinson & Lovell (1978)

Coldwater Fisheries and Aquaculture Management

Jian carp Cyprinus carpio var. Jian – 0.15 mg Zhao et al. (2012)
Lake trout Salvelinus namaycush – 0.1 mg Poston (1976b)
– – – 0.5–1 mg Poston (1976b)
Pacific salmon Oncorhynchus spp. – 1–1.5 mg Halver (1972)
Rainbow trout Oncorhynchus mykiss – 0.05–0.25 mg McLaren et al. (1947)
– – 0.08 mg Woodward & Frigg (1989)
Vitamin B12
Common carp Cyprinus carpio – NR Kashiwada et al. (1970)
Grass carp Ctenopharyngodon idella – 0.094 Wu et al. (2007b)
Pacific salmon Oncorhynchus spp. – 0.015–0.02 mg Halver (1972)
Rainbow trout Oncorhynchus mykiss – R Phillips et al. (1964)
Folic Acid
Common carp Cyprinus carpio – NR Aoe et al. (1967c)
Grass carp Ctenopharyngodon idella – 3.6–4.3 Zhao et al. (2008)
TABLE 3.6 (Continued)

Nutritional Requirements of Coldwater Fishes

Common Name Scientific Name Source Requirement Reference
Thiamin
Juvenile abalone Haliotis discus hannai – 2.62–5.29 mg Miao et al. (2013)
Pacific salmon Oncorhynchus spp. – 6–10 mg Halver (1972)
– – – 2 mg Leith et al. (1990)
Rainbow trout Oncorhynchus mykiss – 1.0 mg Cowey & Woodward (1993)
Choline
Common carp Cyprinus carpio – 1,500 mg Ogino et al. (1970b)
Grass carp Ctenopharyngodon idella – 3,000 mg Wang et al. (1995)
Lake trout Salvelinus namaycush – 1,000 mg Ketola (1967)
Pacific salmon Oncorhynchus spp. – 600–800 mg Halver (1972)
Rainbow trout Oncorhynchus mykiss – 50–100 mg McLaren et al. (1947)
– – 714–813 mg Rumsey (1991)
Myoinositol
Common carp Cyprinus carpio – 440 mg Aoe & Masuda (1967)
Grass carp Ctenopharyngodon idella – 166–214 mg Wen et al. (2007)
Gibel carp Carassius auratus gibelio – 165.3 mg Gong et al. (2014)
Pacific salmon Oncorhynchus spp. – 300–400 mg Halver (1972)
Rainbow trout Oncorhynchus mykiss – 250–500 mg McLaren et al. (1947)
Vitamin C
Atlantic salmon Salmo salar ECAA 50 mg Lall et al. (1990)
– – AMP 10–20 mg Sandnes et al. (1992)

69
TABLE 3.6 (Continued)

70
Common Name Scientific Name Source Requirement Reference
Thiamin
Coho salmon Oncorhynchus kisutch AA 60 mg Lim & Lovell (1978)
– LAPP 11 mg El Naggar & Lovell (1991)
– LAPP 15 mg Robinson (1992)
– AA 50 mg Halver et al. (1969)
Common carp Cyprinus carpio AA NR Sato et al. (1978)
– LAPP R Dabrowski et al. (1988)

Coldwater Fisheries and Aquaculture Management

– – 45 mg Gouillou-Coustans et al. (1998)
Grass carp Ctenopharyngodon idella LAPP 25 mg Henrique et al. (1998)
Jian carp Cyprinus carpio var, jian AMP 60–100 mg Teshima et al. (1993)
– – 40.9 mg Liu et al. (2010)
Rainbow trout Oncorhynchus mykiss AA 50–100 mg Halver (1969)
– AA 40 mg Hilton et al. (1978)
– ECAA 10 mg Cho & Cowey (1993)
Vitamin A
Common carp Cyprinus carpio – 1.2–6 mg Aoe et al. (1968)
Gibel carp Carassius auratus – 0.53 mg Yang et al. (2017)
– – – 0.80 mg Shao et al. (2016)
Rainbow trout Oncorhynchus mykiss – 0.75 mg Kitamura et al. (1967)
Vitamin E
Atlantic salmon Salmo salar – 35 mg Lall et al. (1988)
TABLE 3.6 (Continued)

Nutritional Requirements of Coldwater Fishes

Common Name Scientific Name Source Requirement Reference
Thiamin
Common carp Cyprinus carpio 50 mg Wilson et al. (1984); Watanbe et al. (1970)
100 mg
Grass carp Ctenopharyngodon idella – 200 mg Takeuchi et al. (1992)
– – 62.9 mg Wu et al. (1980)
Juvenile grass carp Ctenopharyngodon idellus – 100.36 mg Li et al. (2014)
Pacific salmon Oncorhynchus spp. – 30 mg Woodall et al. (1964)
– – 40–50 mg Halver (1972)
Rainbow trout Oncorhynchus mykiss – 30 mg Woodall et al. (1964)
– – 25 mg Hung et al. (1980)
– – 50 mg Cowey et al. (1983)
– – 100 mg Watanbe et al. (1981)
Vitamin D
Pacific salmon Oncorhynchus spp. – NR Halver (1972)
Rainbow trout Oncorhynchus mykiss – 40–60 µg Barnett et al. (1982)
Vitamin K
Atlantic salmon Salmo salar – < 10 mg Krossoy et al. (2009)
Atlantic cod Gadus morhua – 0.2 mg Grahl-Madsen & Lee (1997)
Grass carp Ctenopharyngodon idella – 1.9 mg Jiang et al. (2007)
Lake trout Salvelinus namaycush – 0.5–1 mg Poston (1976a)
Pacific salmon Oncorhynchus spp. – R Halver (1972)

71
72 Coldwater Fisheries and Aquaculture Management

3.5 MINERALS REQUIREMENTS

Minerals, or inorganic elements, are needed by organisms to maintain the

metabolic processes and essential components of major structural elements.
A dietary source of 23 minerals has been demonstrated as essential in one
or more animal species. Around 10 minerals (Ca, P, Mg, K, Cu, Fe, Zn, Mn,
Si, and I) have been identified as essential nutrient in fish feed. Out of these
calcium, phosphorus, chlorine, magnesium, potassium, and sodium, are the
most commonly recognized macro-minerals. Therefore, fish must obtain these
essential minerals via food or water supplied to them for optimal growth and
development. Fish must obtain these essential minerals via food or water
supplied to them for optimal growth and development. The main function of
these essential minerals in the body of fish includes the formation of skeletal
structure, maintenance of colloidal system and regulation of acid base equilib­
rium. They are also important components of hormones, enzymes, and activa­
tions of enzymes. Minerals are required for the transmission of nerve impulses
and muscle contraction. They also serve as structural constituents of soft
tissues (Kaushik, 2002; Lall, 2002). A number of reviews have been compiled
by different authors and National Research Council (NRC, 1993, 2011) and
summarized the information on mineral and trace element requirements of
fish. Other periodic reviews on minerals and trace element nutrition in fish
by Schwarz (1995); Davis & Gatlin (1996); Watanabe et al. (1997); Kaushik
(2002); Lall (2002); Prabhu et al. (2013, 2016); Hossain & Yoshimatsu (2014)
are also worth mentioning. These workers have reviewed different aspects
of the mineral and trace element nutrition in fish. Mineral requirements of
coldwater fish species are compiled and presented in Table 3.7.

3.6 CONCLUSION

Nutrition plays a vital role in aquaculture development. Undoubtedly significant

progress on the nutrient requirements of aquatic species has been made in the
past decades. At present, there is plenty of information available on the nutrient
requirements of fish. Inadequate availability of quality seed primarily because
of unavailability of nutritionally balanced starter feeds due to fragmented
information on nutrients requirements is one of the important factors limiting
their production. In this chapter, available information on nutrient requirements
of coldwater fish has been compiled. This chapter of the book is a valuable
database that will be beneficial in formulating nutritionally balanced, quality
protein feeds for boosting the aquaculture production of cold water fish species.
Nutritional Requirements of Coldwater Fishes 73

TABLE 3.7 Minerals Requirements of Cold-Water Fish Species

Common Name Scientific Name Requirement References
(g/100 g diet)
Dietary Calcium Requirements
Common carp Cyprinus carpio Dispensable FW Ogino & Takeda (1976)
(20 mg Ca/L)
Rainbow trout Oncorhynchus Dispensable FW Ogino & Takeda (1978)
mykiss (20–23 mg Ca/L)
Japanese flounder Paralichthys Required SW Hossain & Furuichi
olivaceus (2000)
Chum salmon Oncorhynchus keta Dispensable FW Watanabe et al. (1980)
(20 mg Ca/L)
Grass carp Ctenopharyngodon 1.0 FW (35 mg Ca/L) Liang et al. (2012)
idella
Dietary Phosphorus Requirements
Common carp Cyprinus carpio 0.6–0.7 FW Ogino & Takeda (1976)
(0.002 mg P/L)
Rainbow trout Oncorhynchus 0.7–0.8 FW Ogino & Takeda (1978)
mykiss (0.002 mg P/L)
0.54–0.61 FW Ketola & Richmond
(1994)
Chum salmon Oncorhynchus keta 0.5–0.6 FW Watanabe et al. (1980)
(0.002 mg P/L)
Atlantic salmon Salmo salar 0.6 (available P) Ketola (1975)
FW (< 0.5 mg P/L)
0.83–0.93 FW Vielma & Lall (1998)
1.0 (0.9 available P) Asgard & Shearer (1997);
FW Lall & Bishop (1977)
Jian carp Cyprinus carpio 0.52 (available P) Xie et al. (2011)
var. Jian FW
Grass carp Ctenopharyngodon 0.85 (available P) FW Liang et al. (2011)
idella (0.02–2.27 mg/L)
Dietary Magnesium Requirements
Rainbow trout Oncorhynchus 0.06–0.07 FW Ogino et al. (1978)
mykiss (3.1 ppm Mg)
0.05 FW Knox et al. (1981)
(1.2 mg Mg/L)
0.06 FW Shearer (1989)
(1.3 mg Mg/L)
0.03 FW Shearer & Asgard (1992)
(1.4 mg Mg/L)
74 Coldwater Fisheries and Aquaculture Management

TABLE 3.7 (Continued)

Common Name Scientific Name Requirement References
(g/100 g diet)
Common carp Cyprinus carpio 0.06 FW Dabrowska et al. (1991)
(9.4 mg Mg/L)
0.04–0.05 FW Ogino & Chiou (1976)
(3.5 mg Mg/L)
Dietary Phosphorus Requirements
Atlantic salmon Salmo salar 0.03 FW El-Mowafi & Maage
(54 mg Mg/L) (1998)

Grass carp Ctenopharyngodon 0.05–0.07 FW Wang et al. (2011b)

idella (5.6 mg Mg/L)
Dietary Potassium Requirements
Chinook salmon Oncorhynchus 0.80 FW Shearer (1988)
tshawytscha (<1 mg K/L)
Rainbow trout O. mykiss 1.10 FW Kalantarian et al. (2013)
Grass carp Ctenopharyngodon 0.47–0.73 Zhu et al. (2014)
idellus FW
(1.86–3.10 mg K/L)
0.95–1.00 Liang et al. (2014)
FW
(1.71–8.18 mg K/L)
0.74 Chen et al. (2016)
FW (6.86–9.10 mg
K/L)
Dietary Sodium/Chloride and Sodium Requirements
Sodium Chloride
Rainbow trout Salmo gairdneri Dispensable FW; Salman & Eddy (1988)
11.6%
Common carp Cyprinus carpio Required FW; Level Nasir & Hamed (2016)
up to 1.5% for better
growth and FE
Copper Requirement
Common Name Scientific Name Requirement References
(mg/kg)
Juvenile Atlantic Salmo salar 5–10 Lorentzen et al. (1998)
salmon
Juvenile Atlantic 37 Berntssen et al. (1999)
salmon
Nutritional Requirements of Coldwater Fishes 75

TABLE 3.7 (Continued)

Common Name Scientific Name Requirement References
(g/100 g diet)
Juvenile common Cyprinus carpio 3 Ogino & Yang (1980)
carp
Juvenile rainbow Oncorhynchus 3 Ogino & Yang (1980)
trout mykiss
Young grass carp Ctenopharyngodon 4.70–4.95 Tang et al. (2013)
idella
Zinc Requirement
Juvenile Atlantic Salmo salar 37–67 Maage & Julshamn
salmon (2001)
Common carp Cyprinus carpio 15 Ogino & Yang (1979)
15–30 Satoh et al. (1992)
Jian carp Cyprinus carpio 43.2–48.7 Tan et al. (2011)
(var. Jian)
Rainbow trout Oncorhynchus 15 Ogino & Yang (1978)
mykiss 20 Satoh et al. (1987)
40 Satoh et al. (1987)
80 Satoh et al. (1987)
Selenium Requirement
Atlantic salmon Salmo salar <1.2 Lorentzen et al. (1994)
Atlantic salmon Salmo salar 0.14 Poston et al. (1976)
Coho Salmon Oncorhynchus 8.6 Felton et al. (1996)
kisutch
Juvenile golden Tor putitora 0.68 Khan et al. (2016)
Mahaseer
Iron Requirement
Atlantic salmon Salmo salar 60–100 Andersen et al. (1998)
60 Naser (2000)
Juvenile common Cyprinus carpio 147.4 Ling et al. (2010)
carp
Grass carp Ctenopharyngodon 300 Su et al. (2007)
idella
Manganese Requirement
Atlantic salmon Salmo salar 7.5–10.5 Maage et al. (2001)
Juvenile common Cyprinus carpio 12.0–13.0 Satoh et al. (1987)
carp
76 Coldwater Fisheries and Aquaculture Management

TABLE 3.7 (Continued)

Common Name Scientific Name Requirement References
(g/100 g diet)
Juvenile grass carp Ctenopharyngodon 20.6 Liang et al. (2015)
idella
Rainbow trout Oncorhynchus 19 Satoh et al. (1991)
mykiss
Young grass carp Ctenopharyngodon 16.91 and 18.21 Tang et al. (2016)
idella

KEYWORDS

• amino acid
• coldwater fishes
• essential oils
• minerals
• nutrient
• protein
• vitamin

REFERENCES

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CHAPTER 4

Bacterial Diseases of Finfish Prevalent in

Coldwater Aquaculture
PRIYANKA ASHWATH1, RAMYA PREMANATH1, RAJESHWARI VITTAL1,
DEEKSHIT1, PRARTHANA AITHAL2, FEROZ A. SHAH3, and
AKHILA DHARNAPPA SANNEJAL1
1
Nitte University Center for Science Education and Research,
Paneer Campus, Deralakatte, Mangalore, Karnataka, India
2
Coldwater Fisheries Research, Nainital, Uttarakhand, India
3
Division of Aquatic Animal Health Management, Faculty of Fisheries,
Sher-e-Kashmir University of Agricultural Sciences and Technology
(SKUAST) of Kashmir, Rangil, Ganderbal, Jammu and Kashmir, India

ABSTRACT

Coldwater aquaculture is a rapidly growing industry functioning

worldwide. The challenges faced by this sector are sophisticated and
multilateral. Among the constraints encountered, infectious diseases
take a lion’s share, creating a huge loss annually. Bacterial diseases of
salmonids are considered to be important in causing severe loss in the cold
water fisheries. Therefore, preventive strategies based on scientifically
recommended principles are advocated. These strategies should ideally
focus on preventing the occurrence of the disease rather than treating the
diseased ones. As a single approach is not always successful, a combination
of different schedules is recommended. The current chapter highlights
some of the important bacterial diseases of coldwater aquaculture and their
preventive approaches.

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
92 Coldwater Fisheries and Aquaculture Management

4.1 INTRODUCTION

Coldwater aquaculture holds an important place in the aquaculture sector

throughout the globe. The term ‘cold water’ refers to the aquatic ecosystem
where the temperature of the water body remains below 20°C which offers
the predominance of fish belonging to Salmonidae, particularly the salmons
and trout. Coldwater aquaculture encompasses the fisheries activity in water
where the temperature does not go beyond 20°C throughout the year. Cold-
water aquaculture is prevalent in upland rivers, lakes, and streams found in
high altitudes. In India, cold water fisheries are found in the Himalayan and
the sub-Himalayan regions (Mahanta & Sarma, 2010).
The major constraint in aquaculture that results in severe loss annually
is mainly due to the infectious diseases caused by various bacteria, viruses,
fungi, and parasites. Cold water fisheries are also hampered by a serious
loss that causes concern to aqua-culturists. Various abiotic and biotic factors
create an ideal environment for the disease outbreak. Furthermore, the higher
density of a single species of fish also adds to the rapid disease spread. Some
of the most devastating and insidious diseases in coldwater aquaculture are
due to the bacterial pathogens. To overcome the losses often, antibiotics are
excessively used which creates selective pressure on the bacteria to develop
resistance. The emergence of antibiotic resistant bacteria possessing the
resistance genes is of concern to food safety as these genes could be transferred
to human pathogens. Thus, management practices become very important in
reducing the use of antibiotics. As vaccines are not available in the market
for a few bacterial cold water diseases, it becomes imperative to prevent
the disease occurrence. The implementation of scientifically proven and
recommended management practices such as the use of genetically resistant
stocks, dietary supplements, probiotics, nonspecific immunostimulants,
water disinfection and movement restrictions are contemplated to be the best
approaches in controlling infectious bacterial cold water diseases (Kumar
et al., 2016; Pokhrel et al., 2018). This chapter emphasizes the important
bacterial diseases of the cold water fish, the clinical manifestation of these
diseases, diagnostic approaches, and management practices.

4.2 ENTERIC RED MOUTH (ERM) DISEASE

Yersinia ruckeri is one of the important fish pathogens that causes enteric red
mouth (ERM) disease of yersiniosis in rainbow trout. A Gram-negative rod-
shaped bacterium, first isolated from Onchorynchus mykiss in USA (Ross
Bacterial Diseases of Finfish 93

et al., 1966). Although infections are frequently reported in other fishes

throughout the world, rainbow trout are found to be highly susceptible to
ERM (Tobback et al., 2007; Shaowu et al., 2013). It has also been isolated
from the tissues of major carps in West Bengal (Manna et a., 2003; Surendr­
araj et al., 2009; Pajdak-Czaus et al., 2019) and in Andhra Pradesh (Ummey
et al., 2021). The bacterium belonging to the Enterobacteriaceae family is
a facultative anaerobe with a genome size of 3.7 Mb (Navas et al., 2014).
Based on the production of serological markers, there are four serotypes of
Yersinia of which serotype 1 has two subgroups O1a and O1b while serotype
2 has three subgroups O2a, O2b, and O2c. However, majority of the Yersinia
infection in salmonids is associated with serotype O1a (Romalde & Toranzo,
1993). Based on the biochemical characteristics, they are also categorized as
biotype 1 that shows positive for motility and lipase secretion while biotype
is negative for both the tests (Tobback et al., 2007; Ormsby et al., 2016).

4.2.1 PATHOLOGY

Yersinia ruckeri causes chronic infections in adult fishes, while acute infec­
tions are highly prominent in young fry and fingerlings. The infected fishes
usually become lethargic with loss of appetite and swim near the surface. In
addition, exophthalmia, darkening of the skin, subcutaneous hemorrhages in
and around mouth and throat, petechial hemorrhages on the liver, pancreas,
swim bladder and in the lateral muscles were also seen (Horne & Barnes,
1999; Tobback et al., 2007). Histopathological (HP) examinations show the
inflammation in most of the organs including kidney, liver, spleen, heart,
and gills. In addition, edema, and desquamation of epithelial cells of the
secondary lamellae of the gills were also seen in severely infected fish
(Tobback et al., 2009). Multiple virulence factors are known to contribute
to the clinical signs associated with the hemorrhagic form of the disease
(Romalde & Toranzo, 1993). This include several extracellular products
such as iron regulated Serratia like hemolysin, an azocasein protease and the
47 kDa metalloprotease that usually degrades fibronectin, actin, and myosin
(Secades & Guijarro, 1999).

4.2.2 EPIDEMIOLOGY AND TRANSMISSION

Infection caused by Y. ruckeri can be transmitted by direct contact of an

infected fish with non-infected fishes. As an enteric pathogen, the bacterium
94 Coldwater Fisheries and Aquaculture Management

also gets excreted through feces and survives at least four months outside
the host (Busch & Lingg, 1975). This is an important for the successful
transmission of the pathogen to another host. In certain cases, fishes exhibit
carrier state of infection (Rodgers, 1992; Tobback et al., 2007). Such asymp­
tomatic carriers when unstressed were unable to transmit the bacterium to
a healthy recipient fish. However, when asymptomatic carriers are stressed
with elevated temperatures, the bacterium was successfully transmitted
from carrier to recipient fish causing recipient fish to undergo carrier state
with mild lower intestinal infections without any mortality (Hunter et al.,
1980). In addition, birds and mammals can also act as important vectors that
contribute to the spread of the disease (Kumar et al., 2015). The bacterium
is known to form biofilm by over expressing flagellar proteins, which is
important for the initial development of the biofilm. This further enhances
the survival of the pathogens on surfaces of aquatic systems (Coquet et al.,
2002). Such bacterial biofilms form a frequent source of recurrent infections
in rainbow trout farms (Coquet et al., 2002). The progression of the infection
usually starts from the gills which is an important portal of entry. Thereafter,
it spreads to other organs through blood stream (Tobback et al., 2009). The
optical projection tomography and immunohistochemistry confirms that the
gut is the major affected organ upon immersion treatment. It has been found
to reach the gut within 30 mins of post-infection and kidney at 3 days post-
infection while other organs after 7 days post-infection (Mendez & Guijarro,
2013; Ohtani et al., 2014).

4.2.3 DIAGNOSIS

Several diagnostic techniques have been developed for Y. ruckeri. However,

classical bacteriological assays are mainly based on culturing of Y. ruckeri
on Columbia blood agar and MacConkey agar. They grow as circular,
elevated, shining, and whitish colonies at a temperature range between
20°C and 28°C (Pajdak-Czaus et al., 2019). In addition, commercially
available API-20E strips can be used to detect the bacterium based on the
biochemical characteristics (Kumar et al., 2015). Serological tests such as
ELISA (enzyme linked immunosorbent assay), agglutination, and immuno­
fluorescence tests can be used to detect Y. ruckeri antibodies (Smith et al.,
1987). Molecular methods include PCR to detect 16 rRNA gene (Tobback
et al., 2007), restriction fragment length polymorphism (Gracia et al., 1998)
and loop mediated isothermal amplification (LAMP) to amplify yruR gene
Bacterial Diseases of Finfish 95

(Saleh et al., 2008). Most often specimens include internal organs such as
gills, spleen or intestinal swabs. However, for PCR tests blood samples are
usually preferred (Altinok, Grizzle, & Liu, 2001). A PCR test developed by
Gibello et al. (1999) could detect low levels of Y. ruckeri, thus making it
possible to detect the pathogen even in asymptomatic carriers. To improve
sensitivity, a qPCR assay was also developed by Bastardo et al. (2012), that
target recombinase gene (recA). Most recently, nanoparticle-based detection
methods that detect DNA or RNA hybridized with pathogen specific probes
that are attached with gold nanoparticles have been developed (Saleh et al.,
2015). This rapidly emerging technology detects hybridization at a specific
temperature and time with the aggregation of nanoparticles and concomitant
appearance of red or blue color.

4.2.4 PREVENTION AND CONTROL

The treatment of ERM mainly based on antimicrobial therapy. Antibiotics

such as oxolinic acid, amoxicillin, oxytetracycline, sulfadiazine in combina­
tion with trimethoprim and florfenicol are most commonly used (Michel et
al., 2003). However, emergence resistance towards oxytetracyclines, oxolinic
acid and sulfadiazine has been identified recently (Austin & Austin, 2016).
To overcome the problem of antimicrobial resistance, alternative methods
such as probiotics can be developed. For instance, it was found that oral
administration of Bacillus subtilis and Bacillus licheniformis could protect
the rainbow trout from subsequent infections with Y. ruckeri (Raida et al.,
2003). This might be due to the secretion of antimicrobial agents by the
probiotic bacteria or due to imuunostimulating effect. These results further
encouraged scientists to look for several other bacteria such as Carnobac­
terium maltaromaticum B26, Carnobacterium divergens (Kim & Austin,
2006) and Lactobacillus lacti (Balcázar et al., 2008) showed antagonistic
effect to Y. ruckeri.
The economic loss caused by ERM in the rainbow trout farm can be
controlled to some extent by effective vaccination. The vaccine contains
monovalent inactivated Y. ruckeri serotype O1 and biotype 1 whole cell
suspension (Soltani et al., 2014; Villumsen et al., 2014). Newer vaccines
based on Y. ruckeri Yrp1 protease, aroA gene and lipopolysaccharide are
known to provide good protection against ERM (Temprano et al., 2005;
Ispir & Dorucu, 2014). However, these monovalent vaccines are not effec­
tive in controlling infections caused by biotype 2. Thus, a bivalent vaccine
96 Coldwater Fisheries and Aquaculture Management

containing formalin inactivated biotype 1 and 2 was found to show good

protection against infection caused by biotype 2 (Deshmukh et al., 2012).

4.3 FURUNCULOSIS

The causative agent furunculosis is Aeromonas salmonicida which is an

obligate fish pathogen belonging to the family Aeromonadaceae. The
organism was first isolated from a German freshwater brown trout hatchery
by Emmerich & Weibel (1894). It is a gram-negative, nonmotile, facultative
bacterium that grows well at temperature range between 22°C and 25°C.
It has five subspecies, salmonicida, achromogenes, smithia, masoucida,
and pectinolytica (Graf, 2015). Among these, A. salmonicida subspecies
salmonicida is known to cause furunculosis in marine fish species (Valder­
rama et al., 2017). The bacterium is positive for catalase and cytochrome
oxidase and ferments and oxidizes glucose.

4.3.1 PATHOLOGY

A. salmonicida produces several extracellular factors that includes 25

proteins, enzymes, and toxins. These extracellular virulence determinants
contribute to the pathogenesis of infection caused by the bacterium that
can be further correlated with the stress of the susceptible fish (Austin &
Austin, 2016). In addition, surface polysaccharides such as capsule, glucan,
and lipopolysaccharide, iron binding systems, secretory systems, fimbriae,
and flagella also contributes to the pathogenesis of the disease. However, it
is the combination of all the factors that is responsible for clinical signs of
the disease during infection (Ali et al., 1996). Further, the type III secretion
system that translocates the effector proteins into the host cell is responsible
for subsequent immune response and inflammation (Ebanks et al., 2006; Frey
& Origgi, 2016). Based on the severity, the infection can be acute, subacute,
chronic or latent. The subacute form of the disease usually presents with few
clinical signs such as darkening of the skin, exophthalmia, and distended
abdomen (Cipriano & Austin, 2011). The acute form of the disease shows the
same clinical signs as that of subacute form, in addition to anorexia, petechia,
and hemorrhages (Cipriano & Austin, 2011). The pathological signs chronic
form include boil like lesions referred to as furuncles that initially seen
at the internal organs and then to the external surfaces. This further leads
Bacterial Diseases of Finfish 97

to hemorrhages in the fin bases, mouth, viscera, and reproductive organs

(Roberts, 2012) (Figure 4.1).

FIGURE 4.1 Fishes showing the caudal lesion.

4.3.2 EPIDEMIOLOGY AND TRANSMISSION

Transmission of furunculosis from fish to fish mainly occur through direct

contact or via ingestion. Horizontal transmission of the pathogen from
seawater to freshwater or vice versa has been reported in sea reared rainbow
trout farms in Denmark (Bartkova et al., 2017). Water plays an important
role in transmission of A. salmonicida from infected fish to healthy fish
(Skrodenytė-Arbačiauskienė et al., 2012). Further, changes in the environ­
mental conditions such as pH, temperature, oxygen content, etc., stimulate
the replication and spread of A. salmonicida infection through water bodies.
However, vertical transmission has not been reported in A. salmonicida
infection.

4.3.3 DIAGNOSIS

Routine culture-based identification includes growth of the bacterium on

blood agar wherein they produce brown diffusible pigmented large colonies
within two to four days at 25°C. While on TSA it gives rise to colonies
with dark brown water-soluble pigments when incubated at 22°C for three
days (Austin & Austin, 2007). Biochemically the strains are positive for
oxidase, catalase, lysine decarboxylase, gelatin hydrolysis and methyl red.
Molecular confirmation can be done by REP-PCR and 16 rRNA sequencing
(Skrodenytė-Arbačiauskienė et al., 2010) and by qPCR (Bartkova et al.,
2017). More rapid serological tests such as serum agglutination, ELISA,
98 Coldwater Fisheries and Aquaculture Management

fluorescent antibody test (FAT) can be performed on tissue or isolated bacte­

rial samples (Austin et al., 1986). A PCR based DNA probe test developed by
Mooney et al. (1995) was found to be successful at a rate of 88% in detecting
furunculosis in Atlantic Salmon. Further, a reverse transcription-multiplex
PCR (RT-MPCR) developed by Rattanachaikunsopon & Phumkhachorn
(2012) was able to discriminate between viable and nonviable cells with
a sensitivity of detection of 30 CFU (colony forming units) bacterium.
MALDI-TOF is another technique which could be successfully used to iden­
tify and discriminate between various subspecies of A. salmonicida (Benagli
et al., 2012; Jansson et al., 2015).

4.3.4 PREVENTION AND CONTROL

The control of furunculosis outbreaks mainly relay on the distribution of

antibiotics. However, the extensive use of antibiotics such as oxytetracy­
cline, sulfadiazine/trimethoprim has led to the development of drug resis­
tance (Schmidt et al., 2001; Krikan et al., 2003). To combat the problem of
antimicrobial resistance, the aquaculture industries have now widely used
alternatives such as bacterial vaccines, which are generally administered
as intraperitoneal injections (Midtlyng, 2014). These vaccines result in the
nonspecific activation of macrophages in the kidney that can eliminate A.
salmonicida (Midtlyng, 2014). However, intraperitoneal injection vaccina­
tion has also been linked with side effects such as impaired growth and
inflammation. To avoid such side effects, vaccination through immersion
method has been demonstrated though with short lived protection (Midt­
lyng, 2014). The ability of recombinant vaccines using iron regulated outer
membrane proteins and L-forms of the bacterium have been investigated for
its use against A. salmonicida infection (Lutwyche et al., 1995; Robertson
et al., 2005). The use of disease resistance fish strains through selective
breeding has also received considerable attention recently. For instance,
higher bactericidal activity was found in sera of rainbow trout (Hollebecq
et al., 1995) and salmon families (Zhang et al., 2011) that are bred for their
resistance to A. salmonicida. Use of probiotic bacteria viz Carnobacterium
sp. (Robertson et al., 2000), Lactobacillus plantarum and L. fermentum
(Balcazar et al., 2008) were found to be either antagonistic or prevent the
adhesion of A. salmonicida. The immunostimulant substances such as
glucan and chitosan were also found to reduce mortality caused due to A.
salmonicida infection by improving the nonspecific immunity and thereby
survival of the fishes (Anderson & Siwicki, 1994). Finally, specific lytic
Bacterial Diseases of Finfish 99

bacteriophages can also be used as an effective alternative to antibiotics.

For instance, Kim et al. (2012) identified 2 lytic bacteriophages (phiAS5
and PAS-1) against A. salmonicida was found to reduce mortalities during
challenge with A. salmonicida (Kim et al., 2015).

4.4 BACTERIAL COLD-WATER DISEASE

Bacterial cold water disease is a disease of freshwater fish, particularly the

salmonid fish that results in significant mortality in the salmon and trout
populations. It causes considerable economic losses and difficulties are
encountered during commercial aquaculture and in conservation hatcheries
(Antaya, 2008). The disease is referred by other names including cold water
disease (Lumsden et al., 2006), fin rot disease (Post, 1987), peduncle disease
(Davis, 1947), saddleback disease (Borg, 1960), fry mortality syndrome
(Toranzo & Barja, 1993), and rainbow trout fry mortality syndrome (Barnes
& Brown, 2011).

4.4.1 CAUSATIVE AGENT

Flavobacterium psychrophilum, belonging to the family Flavobacteriaceae

and previously known as Cytophaga psychrophila is the etiological agent of
cold water disease is a serious fish pathogen. F. psychrophilum is prevalent
in the aquatic environment, particularly freshwater (Groff & LaPatra, 2001).
The bacterium was discovered in 1948 from Oncorhynchus kisutch from
the Pacific Northwest United States. It is a Gram-negative, aerobic, catalase-
positive, oxidase-positive, psychrophilic bacilli with growth optimum below
between 18°C and 20°C. The size of the bacterium is approximately 1.5
to 7.5 μm in length and 0.75 μm in breadth. It can resist the action of the
lysozyme for up to 2 mg/mL and survive at 100 ppm of povidone-iodine for
30 min. By changing its morphology, it can withstand starvation for months
(Vatsos et al., 2001). The recovery of F. psychrophilum from the brain of
a newt, a non-fish host, from algae (Amita et al., 2000), and from benthic
diatoms (Izumi et al., 2005) indicates its adaptability (Brown et al., 1997)
and perhaps indicates the reservoir of this bacterium. The bacterium exhibits
gliding motility which is influenced by the nutrient concentrations. More­
over, the extent of gliding motility varies between strains (Perez-Pascual et
al., 2009). It can tolerate a salt concentration of up to 0.5% (Bernardet &
Kerouault, 1989).
100 Coldwater Fisheries and Aquaculture Management

The chromosome of a virulent F. psychrophilum has been mapped.

It shows 2,861,988 base pairs which is comparatively smaller than other
bacteria within the family (Duchaud et al., 2007). The percentage G+C
content has been found to be 33% (Bernardet & Grimont, 1986). The
pathogen produces a wide array of virulence factors including fibronectin
type adhesins, proteases, cytolysins, and hemolysin like proteins which help
the bacterium in attachment and invasion (Duchaud et al., 2007). There are at
least three main serotypes of F. psychrophilum with distinct genetic variation
which has an influence on the virulence of the pathogen (Izumi et al., 2003).

4.4.2 EPIDEMIOLOGY

The disease is prevalent throughout the world. Although the fatal disease
outbreaks are linked to water temperatures below 10°C, the commercial trout
industry has seen a severe disease at constant temperatures of 15°C. The
younger fish (fry and fingerling) are found to be at risk of contracting an
infection (Brown et al., 1997).
Horizontal spread of the infection occurs in the presence of predisposing
conditions such as damage to the skin or lesion (Madetoja et al., 2000).
Invasion into fish is most likely to occur if there is a break in the tegument
(Miwa & Nakayasu, 2005). It has been indicated that the bacterium can
be transmitted within eggs making the control using povidone-iodine
disinfectant difficult (Cumagai et al., 1998). Studies by Brown et al. (1997);
and Ekman et al. (1999) has shown the vertical transmission of the pathogen
from adult to progeny. It has been recovered from intraovum, ovarian fluids,
milt, mucus, egg surfaces, and kidneys from sexually mature trout and
salmon (Taylor, 2004; Cipriano, 2005).
Studies by Dalsgaard & Madsen (2000); and Chen et al. (2008) has
reported the susceptibility of coho salmon and juvenile rainbow trout to
F. psychrophilum. The pathogen can survive for months to years in fresh­
water outside a fish host (Michel & Garcia, 2003). Overcrowding of fish
has been found to be a predisposing factor for disease outbreaks (Davis,
1947). Water hardening in aquaculture ponds free from the pathogen seems
to prevent surface contamination (Kumagai et al., 2000). Spleen size has
been correlated with the innate immunity of the fish. Hadidi et al. (2008)
proved that specimens with a larger spleen index were notably more resistant
to F. psychrophilum. A greater number of bacteria are released from dead
fish (Madetoja et al., 2000). Bacteria have an increased affinity for the fin,
lower jaw, and caudal peduncle (Martinez et al., 2004). The presence of
Bacterial Diseases of Finfish 101

ectoparasites has shown an increase in the invasiveness of F. psychrophilum

(Busch et al., 2003). High nitrite or organic content in water may play a
role in the disease incidence (Nematollahi et al., 2003). Human activities are
likely the reason for the spread of the clonal complexes of F. psychrophilum
(Nicolas et al., 2008). The mortality rate is shown to be varied in different
countries with an average mortality of 70% in trout from Western Europe
(Santos et al., 1992), 35% from Denmark (Jensen et al., 2003), and 10–30%
from the UK (Bruno, 1992). The variation in the mortality rates could be
attributed to water temperature.

4.4.3 CLINICAL SIGNS

The disease exhibits varied manifestations with the most frequent or the
classic form of the disease (CWD: chronic wasting disease) giving rise to
typical open lesions on the exterior surface of the fish. Initially, these lesions
may be observed as areas of rough appearing skin or with a fraying fin tip
along with loss of appetite and lethargy. The regions of bacterial colonization
on the fins appear as pale white areas with separation of the fin rays as found
in some fish. Necrosis erupts at the location of the bacterial colonization
as the disease progress, manifesting in the dorsal region with adipose fin
pathology. Systemic infection sets in along with the external symptoms and
causes substantial damage. Other signs of the disease may involve abdominal
enlargement with a higher volume of ascites, and pale gills. In acute infec­
tions, the presence of exterior lesions will be less prevalent and presents
with an extensive systemic infection (Starliper, 2011). In chronic infections,
the infected fish reveals erratic or spiral swimming, darkened black caudal
regions and/or spinal abnormality with spinal compressions in the posterior,
mid, or anterior of the fish (Kent et al., 1989; Madsen et al., 2001).
In advanced infection, the damage to the caudal region will be severe that
progresses to the caudle vertebra which gets exposed. The bacterial load will
be more in the liver, spleen, intestine, peritoneum, heart, and pancreas indi­
cating septicemia (Decostere et al., 2001; Ekman & Norrgren, 2003). Lesions
can also be found on the snout jaw region, lateral sides, and muscular tissue
between the back of the head and the dorsal fin. Necrosis of liver, spleen,
and kidneys, elevated hemosiderin in the kidney and eosinophils, higher
vacuolar degeneration, pyknosis, and lymphocyte infiltration in the lateral
musculature of the skin lesions and dermis (Nematollahi et al., 2003). The
bacteria is also observed in the retina and choroid gland of the eye eventually
leading to blindness (Ostland et al., 1997).
102 Coldwater Fisheries and Aquaculture Management

The other disease manifestation set forth is known as the rainbow trout
fry syndrome. As the name suggests, it attacks the sac fry to the early feeding
stages of the fish. It is an acute form of the disease resulting in a higher
mortality rate (50% or more). There is a development of bacteremia in addi­
tion to ample internal pathology causing pale liver and kidneys along with
anemia. Additional characteristic features of the disease are exophthalmia,
dark skin pigmentation, pale gills and dullness (Toranzo & Barja, 1993).
The clinical signs of the disease vary with the age of the fish as well as
the outbreaks. In general, the disease severity of the disease is greater in
younger fish.

4.4.4 HOST RANGE

Cold water disease is believed to affect nearly all species of salmonids.

Steelhead and rainbow trout and coho salmon are found to be infected the
most. Although the disease is considered primarily to be of the young fish,
the old fish can also be infected. The bacterial pathogen has been isolated
from a number of cultured and free ranging salmonid fish and also from non­
salmonid fish hosts. The broad host range has been represented in Table 4.1.

TABLE 4.1 Host Range of F. psychrophilum

Hosts References
Salmonids
Atlantic salmon, sockeye salmon, chinook salmon, Rucker et al. (1953); Bullock et al.
cutthroat salmon, chum salmon, brook trout, brown (1971); Holt et al. (1993)
trout
Rainbow trout Rangdale et al. (1997)
Arctic char, coho salmon Hesami et al. (2008)
Grayling, sea trout, whitefish Madetoja et al. (2001, 2002)
Amago salmon, Yamame salmon, Iwana salmon Amita et al. (2000)
Non-Salmonids
Carp, crucian carp, eel, tench, ayu Lehmann et al. (1991); Wakabayashi
et al. (1994); Lee & Heo (1998)
Japanese eel Izumi et al. (2003)
Pale chub Iida & Mizokami (1996)
Perch, roach Madetoja et al. (2002)
Sea lamprey Elsayed et al. (2006)
Zacco platypus Fujiwara-Nagata et al. (2019)
Goby Amita et al. (2000)
Bacterial Diseases of Finfish 103

4.4.5 DIAGNOSIS

A presumptive diagnosis of the disease can be made on the basis of case

history, rearing condition for the fish, the pattern of mortality, characteristic
clinical signs, age, and water temperature. Culture and characterization of
the pathogen from the affected tissues, including spleen, brain, reproduc­
tive parts, liver, kidney, and skin lesion is considered to be the definitive
diagnosis of the disease (Cipriano & Holt, 2005).
Infected tissue examination reveals the presence of thin, rod-shaped F.
psychrophilum with an approximate size of 0.75–1.0 µm wide by 3–5 µm
long. The cells may be found attached end to end, giving the appearance
of a lengthy structure. Histological specimens exhibit osteitis, meningitis,
periostatis, and gangloneuritis (Bruno, 1992).
The culture involves the use of various infected tissues. However, not all
infected fish will have adequate viable cells in the internal tissues for primary
culture. The isolation of the pathogen from skin lesions is often challenging
as compared to the internal specimen due to the presence of oomycetes fungi
and environmental bacteria that will easily grow on the culture media. The
recovery of bacteria is enhanced by increasing the number of samples from
a greater number of fish and homogenization of tissue samples. Inoculation
can be carried out either by using direct streak plating or serial dilution. Poor
growth or no growth is found on nutrient rich media such as blood agar,
brain heart infusion agar, and tryptic soy agar. As the bacterium is some­
what fastidious, it might require specific nutrients in the culture medium
(Alvarez & Guijarro, 2007). Various culture media may be used to grow
F. psychrophilum. Variations of cytophaga medium are routinely used in
diagnostic laboratories (Lorenzen et al., 1997; Daskalov et al., 1999). Pale
yellow colonies with a diameter of 2–3 mm develop after a period of 2–3
days of incubation which later transforms into a characteristic fried egg
colony with a raised center and irregular margin. The suspected colonies are
subjected to various biochemical tests for the confirmation of the bacterium.
They are catalase and oxidase-positive, hydrolyze casein and gelatin, break
down tyrosine, produce flexirubin like pigments, and lyse killed Escherichia
coli cells. Variability in the results for nitrate reduction and elastin hydro­
lysis is found which could be attributed to the difference in the strains.
Supplementation of cytophaga medium with 10% calf serum, the addition
of glucose, galactose, and skimmed milk, increasing the concentration of
tryptone and beef extract resulted in improved growth of F. psychrophilum
(Obach & Laurencin, 1991; Daskalov et al., 1999; Rangdale et al., 1997)
Tryptone-yeast extract-salts agar, tryptone-yeast extract agar, and antibiotic
104 Coldwater Fisheries and Aquaculture Management

containing media have also been used (Wiklund et al., 2000; Madetoja et
al., 2002; Kumagai et al., 2004). A study by Cepeda et al. (2004) found that
addition of glucose to tryptone-yeast extract-salts agar was effective at 18°C
in isolating F. psychrophilum. Tryptone-yeast extract-salts agar in combina­
tion with activated charcoal also improved the growth of F. psychrophilum
(Alvarez & Guijarro, 2007).
Immunological techniques have increased the speed, sensitivity, and
specificity of identifying F. psychrophilum. Immunohistochemistry and
immunofluorescence antibody techniques have been employed to confirm the
culture results (Lorenzen & Karas, 1992; Lindstrom et al., 2009). Detection
of the pathogen on the cell surface components by using enzyme-linked
immunosorbent assay has been developed (Crump et al., 2003). Colony
blotting and immunostaining for the detection and quantification have been
used (Misaka et al., 2008).
Nucleic acid based techniques are increasingly being used for the detec­
tion of F. psychrophilum or to confirm the culture based results. Polymerase
chain reaction (PCR) using DNA gyrase subunit gene, gyrB, and the peptidyl­
prolyl cis-trans isomerase C gene, ppiC are the preferred primers for the
identification of the pathogen (Starliper, 2011). Nested and multiplex PCR
techniques have been described for the characterization of F. psychrophilum
(Amita et al., 2000; Del Cerro et al., 2002).

4.4.6 TREATMENT

An accurate diagnosis is of utmost importance before treating any fish for

cold water disease. Oxytetracycline was widely used for the control of the
disease (Lumsden et al., 2006). In Europe, amoxicillin and oxolinic acid
were extensively used (Bruun et al., 2000). The development of resistance
to these antibiotics has made florfenicol the antibiotic of the current use
(Gultepe & Tanrikul, 2006). The drug is given orally to affected fish through
medicated feed at a concentration of 10 mg/kg of fish for a period of 10 days.
To prevent the development of antibiotic resistance in the strains, indiscrimi­
nate antimicrobial therapy should be avoided. Before the administration of
antibiotics, it is desirable to isolate, confirm, and check the susceptibility of
the pathogen to the antimicrobial agent intended to be used.
The use of non-antibiotics and chemicals has also been reported. Gultepe
& Tanrikul (2006) detailed the use of hydrogen peroxide bath followed by
feeding florfenicol medicated diets. A similar approach was followed with
quaternary ammonium baths and potassium permanganate treatment in
Bacterial Diseases of Finfish 105

combination with antibiotics (Schachte, 1983). The treatment should always

be used in combination with better environmental conditions that reduce the
stressors to fish. Maintaining clean holding tanks and removal of dead fish
minimizes the transmission of the pathogen in the water column.

4.4.7 PREVENTION

The ubiquitous nature of the pathogen makes prevention difficult. Good

management practices can reduce the transmission or introduction of patho­
gens, and minimize the severity of the disease outbreaks. The best preventive
measure often involves reducing stress and decreasing the damage to the
fish skin. Removing the sick, dying, and dead fish from aquaculture ponds is
extremely important as it reduces the bacterial load and risk of transmission
of disease. Good management practices in conjunction with the available
treatments are the only methods for the control of cold water disease.
Iodophor disinfection of eggs just prior to hatching is the most frequently
used preventive method (Taylor, 2004). As the pathogen is found in the fluid
surrounding the eggs in sexually mature salmonids, surface disinfection
of eggs reduces the contamination. Treatment with 100 ppm of the active
ingredient for 10 to 30 min is preferred. However, studies have indicated
the tolerance of iodine by the pathogen by the virtue of which it can survive
even after treatment (Brown et al., 1997; Kumagai, 1998). The relative
ineffectiveness of iodine treatment as evident in some studies might be one
of the reasons for the spread of virulent strains around the world. Although
treatment with povidone-iodine is not 100% effective under all situations,
it can reduce the egg associated pathogen transmission. Sodium chloride
approved by the FDA (Food and Drug Administration) as a “low regulatory
priority” can be effectively used for treating external infection.
To prevent the disease outbreaks, it is very important to reduce physical
handling and stress (Cipriano & Holt, 2005). As physical handling increases
the chance of skin abrasions that serve as the entry point for the pathogen,
it is highly recommended to minimize physical handling. Decreasing the
rearing densities is also preferred (Taylor, 2004). Early detection reduces
the chance of disease spread. It is very important to take care while the fish
is being moved between culture facilities in case of disease suspicion or
if the facility has a disease history. As dead fish serve as the reservoirs for
F. psychrophilum it becomes very important to remove them from rearing
areas. The introduction of novel or wild fish into the existing fish stocks
should be avoided.
106 Coldwater Fisheries and Aquaculture Management

High quality fish diet to a certain extent can help in preventing the disease.
Malnutrition has been listed as a probable cause for the disease. Daskalov et
al. (2000) demonstrated a link between diet and cold water disease. In their
study, high oxidized lipid diet and control diet were fed to rainbow trout. They
observed higher mortality in fish fed with a high oxidized lipid diet when
challenged with F. psychrophilum. Better protection was observed in rainbow
trout fed with a diet naturally contaminated with deoxynivalenol compared
to controls (Wald et al., 2016). A health enhancing diet could increase the
effectiveness of a vaccine. A probiotic Enterobacter sp. administered orally
protected fry from disease outbreaks with F. psychrophilum (Ghosh et
al., 2016). Probiotic treatment of water also reduced the populations of F.
psychrophilum (Boutin et al., 2013).
Increasing the quality of water used in aquaculture can reduce disease
outbreaks. Decreasing the nitrite and organic content of water helps in
reducing the infectivity of F. psychrophilum. Use of filtration, UV treatment,
or ozonation for pathogen free water was suggested by Cipriano & Holt
(2005). Increasing the water temperature may also reduce F. psychrophilum
infections.
Broodstock screening may serve as a tool for the prevention of cold water
disease. The use of FATs and ELISA has been suggested for broodstock
selection (Lindstrom et al., 2009). In the absence of potent vaccines, selective
breeding has shown a significant promise in reducing the incidence of the
disease (Henryon et al., 2005).
There are currently no available vaccines for cold water disease. Various
groups are working on developing an effective vaccine for the disease.
Researchers have tried using live attenuated strains and formalin killed
strains of F. psychrophilum as vaccine candidates. A study by Alvarez et
al. (2008) demonstrated a significant disease resistance in fish injected with
attenuated strains. Similarly, Kondo et al. (2003) found a notable survival
efficiency in fish challenged with virulent strains. Specific antigens of F.
psychrophilum particularly of the outer membrane identified by LaFrentz
et al. (2007) could provide better protection against the disease. Another
problem hampering the vaccination strategy is the delivery method by which
it can be administered effectively to a large number of fish.
The use of F. psychrophilum bacteriophages has shown promising
results in laboratory experiments. They have been shown to survive the fish
stomach, penetrate the intestine, and enter the circulatory system after oral
delivery. This indicates the possibility of delivering phages through coated
feed pellets (Christiansen et al., 2016).
Bacterial Diseases of Finfish 107

Other preventive measures include maintaining a clean fish holding

tank with water free from pathogens, regular health inspection on statisti­
cally significant numbers of fish, sanitization of equipment, and appropriate
storage of quality fish food.

4.5 RAINBOW TROUT GASTROENTERITIS (RTGE)

Rainbow trout gastroenteritis (RTGE) is characterized by the presence of large

numbers of “Candidatus arthromitus,” a segmented filamentous bacterium
within the intestine. Several reports have recorded the mortality rate of fish
infected with RTGE in the affected sites. Around 10–40% proportion of the
productive units remain affected by the RTGE (Branson, 2003; Michel et al.,
2002; McCarthy et al., 2016). RTGE is frequent in fishes larger than 800 g
(Michel et al., 2002). The presence of outbreaks lasted for 2–4 weeks, with
the peak mortality range on the second week after the onset of the infection
(Branson, 2003; Michel et al., 2002).
This syndrome was seen preferentially in summer wherein the tempera­
ture of water might be a major factor in its development. Several studies
suggested that despite the water temperature being an important factor, there
is no such constant temperature threshold for RTGE (Branson, 2003; Michel
et al., 2002; McCarthy et al., 2016; Del-Pozo et al., 2009; Toranzo et al.,
2004). In the disappearance of clinical RTGE, changes in the environment and
diet played a major role (Michel et al., 2002). Factors like low-energy diets
could be added as an advantage with RTGE related infections (McCarthy et
al., 2016).
Filamentous bacteria seen in the distal intestine of the RTGE infected
fishes have been recognized as a part of the Candidatus arthromitus group
of segmented filamentous bacteria (SFB even though culturing of the SFB
could not be possible outside the gastrointestinal tract of the host, populations
were maintained as a mono-associations with mice (Schubert et al., 2021).
The term Candidatus is a provisional status given to describe prokaryotes
which are incompletely termed (Murray et al., 1995). Arthromitus in Greek
meant joint thread. Basically, C. arthromitus represent bacterial group with
identical morphological and ecological niches. The endospore-forming
SFB are Gram-positive and have found to be attached to the intestine of
a wide range of vertebrates (Urdaci et al., 2001; Snel et al., 1995; Murray
et al., 1995; Smith, 1997; Lowden & Heath, 1995; Angel et al., 1990) and
invertebrates (Klaasen et al., 1991; Margulis et al., 1999).
108 Coldwater Fisheries and Aquaculture Management

The presence of the pathogen by light microscopy in the intestine of

healthy individuals including the carps was observed (Klaasen et al., 1991).
This demonstrates the presence of SFB in the animal kingdom is ubiquitous.
Despite repeated trials to transform the rat or chicken SBF to mice, gave a
clue that the host specificity of the SFB was slightly different depending
on the host species (Allen, 1992; Tannock & Archibald, 1984). Subsequent
phylogenetic 16S rRNA analysis of C. arthromitus from crab eating monkey,
mice, rats, and chickens further validated that they were in fact different
species (Tannock & Archibald, 1984; Imaoka et al., 1997). These findings
stated that different species of SFB colonize different host species specifi­
cally, and the fact has been accepted even till date. The same theory holds
good as far as the trout SFB is concerned, indicating they represent different
species within the C. arthromitus group.
Several authors studied the life cycle and multiplication of C. arthromitus
(Snel et al., 1995; Smith, 1997; Ferguson & Birch-Andersen, 1979; Chase &
Erlandsen, 1976).
The endospore formation in the C. arthromitus is accompanied with
the production of active intracellular offspring for dispersal/reproduction
through unfavorable environments like aerobic conditions. The spores
have been shown to be actively responsible for the spread of SFB amongst
susceptible mice and chickens (Ivanov et al., 2009; Herbert et al., 2016).
The same mechanism of spread has been reported in trout C. arthromitus
(Michel et al., 2002).
Whole growth cycle of the SFB in mice was noted to occur within 20–30 h
villous transit time of epithelial cells, except if the attachment is not hindered
or there is inhibition in the cell migration (Klaasen et al., 1993; Pamp et al.,
2012). This may also be accepted to be the same in the case in C. arthromitus
infecting the trout.
Arguments arise in the role of C. arthromitus as an etiological agent in
RTGE in trout as these have never been reported in the digestive system
of healthy trout. There are reports suggesting its involvement and recovery
from the RTGE-affected trout (Michel et al., 2002; Toranzo, 2004; Urdaci et
al., 2001). Yet, there are no reports to prove the association of C. arthromitus
in causing diseases in other animal species, though it was shown to cause
malabsorption, diarrhea, gas, and fluid-filled intestines in poultry (Goodwin
et al., 1991).
The same scientists reported that the bacteria might be just a part of
normal microflora of the intestine and may not be necessarily a pathogen
(Umesaki et al., 1999).
Bacterial Diseases of Finfish 109

Fragility of trout SFB could be a result of impaired C. arthromitus

membranes in the intestine of RTGE-infected trout (Michel et al., 2002).

4.5.1 PATHOLOGY

Fishes affected with RTGE shows loss of appetite and lethargy. Affected
fishes tend to gather on the surface of the ponds with occasional uncoordinated
swimming and reminiscent of neuropathological toxic mechanisms (Branson,
2003; Michel et al., 2002; McCarthy et al., 2016; Del-Pozo et al., 2009).
Mucous content of the intestine extruded from the anus and abdominal
distension are some of the gross lesions. In few cases the infected fishes
presented yellowish mucoid excretions from the vent, dark streaks scattered
along the flanks and dyschromia (Branson, 2003; McCarthy et al., 2016;
Michel et al., 2002; Toranzo, 2004). Few other cases reported an internal lesion
in diarrheic trout including acute hemorrhagic enteritis more significantly in
the hind gut accompanied with edematous appearance of the intestinal mucosa
and hemorrhage. Straw-colored mucoid material was seen to fill the entire
digestive tract, including the pyloric caeca and enlarged stomach causing a
dense occluding plug in the terminal portion (Branson, 2003; Del-Pozo et al.,
2009; Toranzo, 2004).
The most significant and defining clinical feature of the RTGE syndrome
is the large quantity of SFB which could be observed in the smears of the
intestinal contents of the infected fishes (Del-Pozo et al., 2010). SFB seen
within the digestive tract of the RTGE-affected trout were characterized
to be nonbranched and found to be approximately 1 µm (0.6–0.12 µm) of
width and up to 60 µm in length. However, the length of the bacteria varied
between 1.2 and 2.6 µm in segments (Urdaci et al., 2001).
In the wet mount preparations stained with metachromatic toluidine blue,
several forms corresponding to different stages of maturation of the SFB
was observed. The bacteria are gram-variable, but usually Gram-positive
producing spores that readily stain with malachite green (Del-Pozo et al.,
2009, 2010; Ericssonl et al., 2014).
Reports suggest varied locations of the filamentous bacteria harboring
the digestive system of the diseased trout. Few reports (Michel et al., 2002)
observed their presence throughout the digestive system, while others
demonstrated its presence more frequently in the distal intestine (Branson,
2003; Michel et al., 2002; Del-Pozo et al., 2009, 2010).
Affected trout exhibited HP lesions in the digestive system, severe lesions
were seen in the caeca and pyloric stomach (Del-Pozo et al., 2010; Urdaci
110 Coldwater Fisheries and Aquaculture Management

et al., 2001; Ericssonl et al., 2014). Congestion was observed in the mucosa,
thickening of the intestinal wall by edema. A most significant change was
epithelial necrosis with extensive mucosal detachment (Ronza et al., 2009).
There are no reports on the HP changes in the other organs.

4.5.2 DIAGNOSIS

Progression from the culture-dependent techniques like the phenotypic

characterization including the morphology, biochemical, and physiology,
molecular techniques, in particular the 16S rRNA gene sequencing provides
insights into the phylogenetics. The culture-independent approaches
have resulted in the identification of new uncultured bacterial groups like
Candidatus. Sequencing of the 16S rRNA gene is more reliable in allocating
names to the bacterial pathogens. It is marked advantageous over the
phenotyping identifications (Jolley et al., 2012).
Antibody-based procedures are effective in detecting the exposure
of fish to viruses, while the identification of bacterial pathogens becomes
complicated due to cross reactivities unless the specific known molecules/
antigens are used to coat the ELISA plates rather than whole pathogens
(Adams & Thompson, 2006). However, these techniques are specific,
sensitive, reliable, and rapid.
Chemotaxonomy comprehends the use of chemical constituents of
bacteria for the investigation. In particular, in the study of Gram-positive
bacteria. Polar lipids, lipopolysaccharide, fatty acids, ubiquinones, mena­
quinones, naphthoquinones, mycolic acids, teichuronic, and teichoic acids,
peptidoglycan, can be used in the detection of the RTGE related pathogens
(Austin, 2011). Molecular methods employing techniques like PCR-based
gene sequencing, reverse transcriptase-sequencing and 16S rRNA gene
sequencing have been additional advantages to the armamentarium of
methods applicable to the bacterial taxonomy (Yin et al., 2013). However,
the DNA hybridization introduced into bacterial taxonomy during the 1960s
is marked the “gold standard” to determine the absence or presence of new
species.
Nucleic acid fingerprinting methods that include pulsed field gel
electrophoresis (PFGE), amplified fragment-length polymorphism PCR
(AFLP), REP-PCR (repetitive extragenic palindromic-PCR), ERIC-PCR
(enterobacterial repetitive intergenic consensus sequences-PCR), rep-PCR
(repetitive element primed PCR) and ribotyping may provide some data at/
below the subspecies level. Amongst all of the above, ribotyping, and AFLP
Bacterial Diseases of Finfish 111

are considered as standards and are extremely useful (Laing et al., 2011).
The methodologies might be culture-independent, those enable the study of
uncultured organisms, but there are issues with genomic fluidity. Candidatus”
reports uncultured prokaryotes for which phylogenetic relationships have
been discussed, and the authenticity has been confirmed by methods such as
in situ probing (Austin, 2011). Above all, the molecular techniques allowed to
apprehend unculturable pathogens that belong to new groups of Candidatus.

4.5.3 PREVENTION AND CONTROL

Anecdotal observation is the only possible route of possible management

option for the RTGE. Changes in the environment, diet, and feeding restric­
tions have been found effective in reducing RTGE losses (Branson, 2003;
Michel et al., 2002). Treatment of the affected sites with RTGE infection is
a trial and error-based experiment, due to lack of knowledge and literature
on the etiology and pathogenesis. Addition of antibiotic treatments to the
feed liquid paraffin or sodium chloride at varying concentrations. Keeping
the affected fishes on fast for a week or more has also been used as one of
the treatment options in Italy (Rashidian et al., 2020). Tested drugs for the
control of RTGE includes bacitracin, clindamycin, amoxycillin, doxycycline,
cotrimoxazole, streptomycin, gentamicin, trimethoprim, neomycin, cipro­
floxacin, vancomycin, cefotaxime, polymyxin, and metronidazole (Singh et
al., 2013). All of these drugs were observed to reduce the number of SFB in
the ileum to different degrees, nevertheless suggesting the sensitivity of the
SFB to the antimicrobial drugs (Caselli et al., 2010). The cause of RTGE
will not be completely eliminated from the population by the treatment, the
affected populations will be further susceptible to the disease on continuous
exposure.

4.6 BACTERIAL GILL DISEASES (BGD)

Bacterial gill disease (BGD) at first was demonstrated by Davis (1926) as a

colonization of the gill by thread-like filamentous bacteria, causing a rapid
increase in the epithelium ultimately leading to lamellar fusion. Coloniza­
tion of the lamellae of the gills by the bacteria results in a decreased rate of
exchange of gas. BGD is a bacterial infection of the gills that impairs the
oxygen uptake of the fish (Wakabayashi, 1980). It is predominately believed
to be a disease of hatchery-bred juvenile salmonids and is not a considerable
112 Coldwater Fisheries and Aquaculture Management

problem in the wild fish populations (Starliper & Schill, 2011). The disease
is prevalent among salmonids, fish of all ages and species including Rohu
(Labeo rohita), Sheatfish (Silurus glanis), Silver carp (Hypophthalmichthys
molitrix), Walleye (Sander vitreus) and Catla (Catla catla).
The main causative agent of the BGD is the Flavobacterium bran­
chiophilum (Touchon et al., 2011). At first, BGD was thought to be caused
by various forms of gliding bacteria, referred to as “myxobacteria” (Bullock,
1972). Subsequently, the agents of BGD, including Flavobacterium and
Cytophaga were grouped into a general category, a yellow-pigmented
bacteria, isolated on Anacker and Ordal’s medium referred to as Cytophaga
Agar (Anacker, 1959). Flavobacterium branchiophilum was first isolated
from gill lesions of a rainbow trout (Oncorhynchus mykiss) and Yamame
(Oncorhynchus masou) suffering from BGD in hatcheries in Gunma, Japan
in 1975 and 1977, respectively (Wakabayashi et al., 1989).
Bacteria belonging to the genus Flavobacterium are Gram-negative,
exhibit aerobic yellow-pigmented rod. They are capable of gliding motility
and can degrade many polysaccharides except for cellulose (Ko & Heo,
1997). Molecular techniques namely DNA-DNA hybridization, whole-cell
protein, fatty acid profiles and DNA-rRNA hybridization were the basis of
classification of the classified the Flavobacterium species. Distribution of the
species are commonly soil environments and the freshwater.
F. branchiophilum, F. psychrophilum, and F. columnare are three impor­
tant fish pathogens belongs to the members of the Flavobacterium genus.
F. columnare is said to be the etiological agent of the columnaris disease
which generally occurs in water temperatures of 20°C, affecting many fishes
of wild and cultured species (Tort, 2000). Rainbow trout fry syndrome is
caused by F. psychrophilum, a thin rod-shaped bacterium, growing at colder
temperatures (10–16°C) (Mohammed, 2015). It also causes diseases in many
fishes including tail rot, salmon species, wild, and cultured trout (Tort, 2000).
Both F. psychrophilum and F. columnare cause systemic and co-infections
in both hatchery-raised and wild fish (Skulska, 2014).

4.6.1 PATHOLOGY

Increased brachial movements, decreased interest in feeding lethargy and

fatigue leading to fish floating near the water surface are some of the signs
associated with BGD (Good et al., 2015). However, the conditions for the
onset of BGD are undiscovered, it is speculated that BGD outbreaks are
associated with stress factors, such as poor water quality, overcrowding,
Bacterial Diseases of Finfish 113

and increased turbidity in the water supply low oxygen availability (Tort,
2000).
Good et al. (2009) observed that the hatcheries with low water exchange
and poor quality of water could be considered a risk factor for the BGD
outbreaks. Mortality of the BGD affected fishes could be reduced by the
addition of ozone to the water supply, thus by improving its quality (Tort,
2000). Increased amount of feed at the hatcheries could be a possible reason
for the infection by F. branchiophilum, as observed by Good et al. (2009).
This may be because the water quality being affected by the increased feed,
by an increase in the amounts of ammonia concentration and suspended
solids. While, increased ammonia and decreased dissolved oxygen in the
water are thought to be involved with BGD outbreaks (Tort, 2000). It was
observed that the effect of fish feeding was a factor for consideration than
the food itself, because the fishes unable to feed required feeding through
stomach tubes for BGD to manifest, suggesting that a physiological change
triggers the disease. Presence of the feed in water directly may not be respon­
sible for the BGD outbreaks but definitely could amplify the effects of the
disease (Faisal et al., 2011).
The fishes affected with BGD exhibit lethargy, in appetent, and seem to
swim on the surface of the water. Bilaterally flared opercula and gasping are
some of the common symptoms. Mortality rate depends on the age, size,
stocking density, and environmental conditions of the fishes (Loch & Faisal,
2015). Around 10–15% of the mortality has been recorded within 24–48 h
post-infection. Since, F. branchiophilum is non-invasive pathology remains
limited to the gills. Flared arches or ragged gills and lamellar congestion,
excessive debris/mucus are quite evident. It elicits fusion of the primary
lamellae, lamellar fusion and epithelial hyperplasia in severe cases. This in
turn causes a reduction in the respiratory surface area leading to the inhi­
bition of gaseous, ionic, and osmotic homeostasis (Loch & Faisal, 2015)
(Figure 4.2).
Infections of the flavobacteria are diagnosed depending on several factors
like the behavioral changes, case history, histopathology, gross pathology
with subsequent isolation, phenotypic characterization (Austin & Austin,
2016).
Cytophaga Agar was commonly used for culturing of Flavobacterium
(Loch & Faisal, 2015). Light microscopy can be carried out for the identi­
fication of F. branchiophilum colonies on culture. Casitone-yeast agar and
Modified Sheih’s medium have been used to culture F. branchiophilum, of
which Casitone-yeast agar has worked best (Kolton et al., 2013).
114 Coldwater Fisheries and Aquaculture Management

FIGURE 4.2 Fishes affected with bacterial gill disease caused by Flavobacterium.

Though the isolation of F. branchiophilum has been proved successful,

culturing it is a challenge. For the same reason, as a presumptive diagnosis,
examination of gill clippings under light microscopy revealing the presence
of long filamentous bacteria on the gill surface has been a better alternative.
In addition to the above-mentioned methods, confirmation is achieved best
when preformed the molecular and serological assays. FATs, in situ hybridiza­
tion, Whole-cell agglutination, enzyme-linked immunosorbent assays, PCR,
quantitative PCR, loop-mediated isothermal amplification (LAMP), DNA
array-based multiplex assay, immunomagnetic separation in conjunction with
flow cytometry are some of the molecular bases techniques employed for
the identification and detection of the Flavobacteria species (Loch & Faisal,
2015; Racheal, 2017; Loch, 2012; Soltani et al., 2019; Noble & Summerfelt,
1996). However, it should be noted that some of the diagnostic reagents
specific for the fish associated flavobacteria are lesser known, making their
identification hard and time-consuming.

4.6.2 PREVENTIVE AND CONTROL

Optimizing the husbandry conditions by maintaining good water quality,

minimizing stress, providing adequate water flow, reduction of rearing
densities can help to reduce infections caused by the Flavobacteria (Long
et al., 2014). Low amounts of dissolved oxygen and elevated amounts of
ammonia are the predisposing factors for BGD outbreaks. Suboptimal
exchange of water is considered a risk (Wahli & Madsen, 2018).
For the prevention infection by F. psychrophilum the eggs of the salmonid
must be disinfected with povidone iodine (50 ppm) in the pre-water hardening
phase (fertilized eggs before the chorion becomes rigid due to exposure to the
water) has proven promising (Oplinger et al., 2015). Disinfecting the sperm
Bacterial Diseases of Finfish 115

of the rainbow trout with streptomycin and penicillin was found successful
in the subsequent reduction of the F. psychrophilum infection (Pérez-Pascual
et al., 2021). However, this needs to be further investigated because there
are some drawbacks like lower egg fertilization and survival. Usage of
the above-mentioned antibiotics in addition to 0.5% NaCl to the hatchery
water prior to the hardening phase, significantly reduced F. psychrophilum
(Webster & Thompson, 2015). The combination of streptomycin and peni­
cillin with elevations in the water temperature killed the pathogen under
laboratory conditions (Gonçalves et al., 2019). However, there might be a
contradiction among the in vitro results and in vivo effectivity as far as flor­
fenicol, enrofloxacin, and doxycycline in treating RTFS-affected trout was
concerned. Copper sulfate when used for the control of F. psychrophilum
infections was not found successful as it was found to be toxic to the eggs.
Broodstock screening was considered a potential method for the evaluation
of F. Psychrophilum infection levels (Perez-Pascual et al., 2021).
Various substances related to the fish feed were tested which were efficient
in preventing the infections by Flavobacteria. In this regard, trout by-product
hydrolysates, produced by trout pepsin, recorded the highest antibacterial
activity against F. Psychrophilum. Restrictions on the feed intake served as
a protection for the rainbow trout infected with the pathogen (Ghosh et al.,
2016). Additionally, rainbow trout fed a diet naturally contaminated with
deoxynivalenol also yielded better results. When the fishes were fed a health
enhancing” diet, the effect of the vaccine was seen to be enhanced (Jarau et
al., 2019). Oral or intraperitoneal administration of the Enterobacter species
was an effective strategy to combat disease outbreaks with F. psychrophilum
(Castillo et al., 2014). Prebiotic supplements with 30% soy bean exhibited
minimal effect on the F. psychrophilum infected cutthroat trout by a cohabi­
tation method. It is noteworthy that the probiotic treatment decreased the F.
psychrophilum population in the water (Madsen et al., 2013).
Phage based therapy of the F. psychrophilum was suggested and also
laid promising hopes when experimented in vitro. Ability of the phages
to survive passage through the stomach of the fish, penetrate the intestinal
barrier and gain entry to the circulatory system after oral delivery consti­
tuted an efficient method to treat as well as prevent the diseases caused by F.
psychrophilum (Lahnsteiner, 2021; Farmer et al., 2017). For the prevention of
the infections by F. columnare various chemicals like the chloramine-T and
hydrogen peroxide treatment were effective. They also were able to control
the mortality rates associated with the bluegills and external columnaris in
bass (Beck et al., 2015). By the addition of extra copper sulfate to the feed,
116 Coldwater Fisheries and Aquaculture Management

a positive response was observed in regard to the survival of several fish

species affected by columnaris disease (Yang et al., 2015).
Chitosan can be a promising candidate and an alternative to antibiotics.
Inhibition of the growth of F. Columnare could be achieved by Nigella sativa
(black cumin) seeds when added to the fish feed, its oil has been effective
in enhancing the survival rates of zebrafish and catfish (Perez-Pascual et al.,
2021). Resistance against F. Columnare infections could progress when the
Grass carp fed diets supplied with Ficus carica polysaccharide (Laanto et
al., 2015).
In addition, the ungeremine and its analogs were efficient as bactericides
against F. Columnare (Perez-Pascual et al., 2021). The activity of Flavone
(Wogonin) in vitro was effective against F. columnare. Optimal dietary levels
of vitamin C in the diet improve the barrier functioning while its deficiency
was found to depress the gill physical and immune barriers in grass carp
infected with F. columnare (Tie et al., 2021). Susceptibility towards
columnaris infection could be achieved by the use of different prebiotic dietary
additives. Increased survival of fishes infected with F. columnare disease in
walleye was achieved when two strains of Pseudomonas fluorescens isolated
the from skin and gut of healthy walleye (Sander vitreus) was used as a
probiotic (Perez-Pascual et al., 2021). As an alternative to chemotherapy,
administration of phages against columnaris disease in aquaculture as
demonstrated in controlled experiments (Laanto et al., 2015; Pedersen &
Lazado, 2020).

4.7 COLD WATER VIBRIOSIS

In the late 1970’s there were low levels of mortalities observed in Atlantic
salmon smolts, in the Northern part of Norway (Egidius et al., 1984; Kent,
1982). Initially, mortalities were related with environment and nutritional
status of fishes. Later observation of abnormal fishes had the necrosis in
mucosa of the caeca and intestine, sloughing into the lumen. Hemorrhages in
lamina propria, with extensive vasodilatation, congestion of the blood vessels
resulting in increase in numbers of eosinophilic granular cells. Regions in the
kidney responsible for giving rise to red blood cells were sparse, with large
areas of necrosis filled with macrophages with grayish cytoplasm predomi­
nated. Spleen had considerable enlargements with signs indicating inflamma­
tion densely populated with macrophages (Soleim, 1985). But identification
of isolates from internal regions of moribund fishes indicated bacterial
infection. Cold water vibriosis or Hitra disease or hemorrhagic syndrome is
Bacterial Diseases of Finfish 117

a bacterial infection caused by motile gram-negative rod Vibrio salmonicida

mainly affecting Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus
mykiss) and Atlantic cod (Gadus morhua) (Egidius et al., 1981, 1986).
Vibrio salmonicida is gram-negative motile rod. Tryptic soy with added
sodium chloride could be used for its growth. For the primary isolation
nutrient agar with supplements like, 1 to 1.5% sodium chloride and 5% sheep
blood are often used. Optimum growth of the bacteria could be achieved at a
medium salinity (1.5%) and temperature of 15°C (Egidius et al., 1986). The
biochemical properties of these isolates, however, did not resemble those
of Vibrio anguillarum or V. ordalii. The isolates were sensitive to all of the
antimicrobial tested, including oxytetracycline, furazolidone, tribrissen, and
oxolinic acid (Bruno et al., 1985).

4.7.1 EPIDEMIOLOGY

Vibrio salmonicida has more frequently reported in the marine waters of

Eastern Canada, Scotland, and Norway. Specifically, infecting host Species
Atlantic salmon and rainbow trout. The bacteria turn pathogenic at a water
temperature of below 10°C. Smolts, juveniles, and adults are said to be
affected by the pathogen. Water is the primary root of fish-to-fish transmis­
sion of Vibriosis (Bruno et al., 1985).
A temperature range 5–20°C is usually optimum for the growth of
Salmonids. At a temperature range of 13–17°C (Wallace, 1993), efficient
growth of salmon is achieved. Unfortunately, this range of water temperature
also favors the growth of Vibrio salmonicida.
The bacteria establish latency period until it proliferates in large numbers
to show the signs of the symptoms. The clinical manifestation include
hemorrhage to intestines, body cavity, spleen, and muscle, distended mucoid
and necrotic intestine and petechiation, erosion, and darkened coloration to
the skin and fins. Changes to the eyes include distension and cloudiness and
periorbital swelling. White/gray lesions can be found on the intestines and
spleen with swollen spleen. Isolation of this bacterial pathogen from ascites
and a variety of internal organs could be achieved (Totland et al., 1987).

4.7.2 PREVENTION AND CONTROL

Reduction and control of vibriosis could be achieved by implicating the

following methods. Oxolinic acid and florfenicol are commonly used in
118 Coldwater Fisheries and Aquaculture Management

the treatment of vibriosis Norway (Frans et al., 2011). Flumequine and

quinolones are some of the widely used antibiotics in the control cold water
vibriosis (Shao, 2001; Defoirdt et al., 2011). Erythromycin, cotrimoxazole,
streptomycin, and chloramphenicol are some of the reported antibacterial
to combat V. harveyi infection in giant tiger prawns. Use of a wide range
of chemicals like benzalkonium chloride, copper sulfate, formalin, and
malachite green could help eliminate the opportunistic pathogens affecting
the fishes. Overdose of these chemicals have adverse effect on fishes
including gill damages (Punitha et al., 2008). Hypochlorite’s were used by
the farmers of Malaysiato control vibrios (Mohamed et al., 2015).

4.8 MYCOBACTERIOSIS

Mycobacteriaceae is a Gram-positive bacterium family that includes a huge

number of harmful bacteria. Fish mycobacteriosis is a chronic, progressive
condition caused by nontubercolous mycobacteria, which are acid-fast in
nature (Chinabut, 1999). Mycobacterium was initially discovered in a diseased
marine aquarium fish in 1897. Formerly, the organism was named as M. piscium.
Even though unproven, the organism originally referred to as M. piscium is
alleged to be the same thing as the one named as M. marinum currently (Noga,
2010). The genus Mycobacterium contains more than 150 species, including
the obligate pathogens that cause tuberculosis in mammals and environmental
saprophytes that can cause opportunistic infections. Members of the
Mycobacterium tuberculosis such as M. tuberculosis, M. bovis, M. caprae, M.
pinnipedii cause tuberculosis in mammals, and non-tuberculous mycobacteria
are typically separated into two families (Antuofermo et al., 2014). The
organisms in the latter group include environmental saprophytes, which can
cause opportunistic infections, and other species such as M. marinum, which
are common pathogens. Nontuberculous mycobacteria can be further divided
between fast-growing mycobacteria such as M. fortuitum, M. chelonae, M.
abscessus and slow-growing mycobacteria such as M. marinum, M. ulcerans,
M. haemophilum, M. gordonae which might take weeks or even months to
develop visible colonies on solid agar. At least 20 species of nontuberculous
mycobacteria, including both fast and slow growers, can cause disease in
fish. They include Mycobacterium marinum, M. shottsii, M. pseudoshottsii,
M. ulcerans, M. haemophilum, M. gordonae, M. fortuitum M. fortuitum, M.
peregrinum, M. saopaulense, and the M. chelonae M. abscessus, M. chelonae,
M. salmoniphilum, M. chesapeaki, M. montefiorense, M. neoaurum, M. simiae,
M. scrofulaceum and M. stephanolepidis. Some of these species, including M.
Bacterial Diseases of Finfish 119

marinum and M. haemophilum, appear to be very harmful to fish, while others

are often carried sub clinically (Asakura et al., 2016).
M. marinum, M. ulcerans, M. haemophilum, M. abscessus, M. chelonae,
M. fortuitum, M. peregrinum, M. gordonae, M. iranicum, M. neoaurum, M.
florentinum, and M. arupense are some of the mycobacteria that have been
isolated in fish and are known to affect humans (Bercovier et al., 2001).
Mycobacteriosis was produced by M. senegalese, a bacterium generally
associated with cattle, in cuts from a shattered fish tank in a toddler who had
no apparent link to cattle. Other organisms that can cause piscine mycobac­
teriosis should almost certainly be termed zoonotic. M. pseudoshottsii and
M. shottsii, in particular, are closely related to M. marinum and are difficult
to distinguish from this organism (Bhatty et al., 2000).
M. marinum has been defined in at least 150 species of freshwater and
marine fish, including eels and syngnathid fish namely; seahorses, pipefish,
sea dragons. Outbreaks appear to be more common in captive Anabantidae
(bettas and gouramis), Characidae (tetras and piranhas), and Cyprinidae
(danios and barbs), though this could be related to their lengthy lifetimes
rather than unique susceptibility. There is no much information available
about the members of the M marinum complex. M. shottsii has been found in
striped bass Morone saxatilis, which is the host for M. pseudoshottsii and M.
chesapeaki. Yellow tail (Seriola quinqueradiata), sea bream (Sparus aurata),
white perch (Morone americana), Atlantic menhaden (Brevoortia tyrannus),
and bay anchovy (Anchoa mitchilli) are among the marine species known to
be infected with M. pseudoshottsii. The majority of cases have been linked to
fish farms. M. ulcerans is generally associated with stagnant water, and while
it has been identified in fish, it does not appear to serve as a reservoir. M.
marinum is one of the most prevalent Mycobacterium species found in sick
amphibians. Captive bullfrogs (Rana catesbeiana), Japanese forest green tree
frogs (Rhacophorus arboreus), leopard frogs (Rana pipiens), a red-eyed tree
frog (Agalychnis callidryas) and Hong Kong warty newts (Paramesotriton
hongkongensis) it has caused clinical cases. It has also been found in reptiles,
including Egyptian spiny-tailed lizards, a Fly River tortoise and a bearded
dragon. M. marinum has been reported in marine animals, pigs, cattle, camels,
a European hedgehog (Erinaceus europaeus), a bilby (Macrotis lagotis), and
an armadillo, as well as a blue-fronted Amazon parrot (Amazona aestiva). In
mice and bats, experimental infections have been established.
Many organisms found in fish, such as the M. fortuitum group and the
M. chelonae/M. abscessus complex, are prevalent environmental organisms
in soil, water, and plants. They are not specifically associated with fish,
but have been documented in a variety of species, including amphibians,
120 Coldwater Fisheries and Aquaculture Management

reptiles, mammals, and birds, with or without clinical indications. Few of

them may favorably affect certain piscine hosts. For instance, M. chelonae
and M. salmoniphilum have been reported mainly in coldwater fish, specifi­
cally salmonids. M. salmoniphilum also cause disease in burbot which is
a freshwater species, and experimentally infected Atlantic cod (Gadus
morhua). M. haemophilum has been encountered in zebrafish (Danio rerio)
and other aquarium fishes such as Cuchu’s blue tetra, Boehlkea fredcochui;
peacock gudgeon, Tateurdina ocellicauda.

4.8.1 PATHOLOGY

Piscine mycobacteriosis is frequently a long-term illness, with infected fish

living for months or even years. It is characterized by granulomatous inflam­
mation in internal organs and muscles as well as the integument. Clinical
indications vary and are sometimes nonspecific, reflecting those of other fish
diseases. Affected fish may appear in appetent, malnourished, or less active
than usual; they may swim erratically and/or isolate themselves from other
fish, seeking out corners of the holding facility. In the group, there may be
a decreased growth rate or increased mortality. Some fish develop shallow,
irregular, nonhealing skin ulcers; nodular skin lesions, such as farmed
Atlantic salmon infected with M. chelonae; cutaneous hemorrhages from
muscle lesion rupture; or nonspecific changes in cutaneous pigmentation,
such as fading in tropical fish or bright colors in salmonids (Bouricha et al.,
2014). Exophthalmos (bulging eyes), pale gills, profuse cutaneous mucus,
elevated scales, abdominal distention, skeletal abnormalities such as spine
curvature or stunting deficiencies, and fin and tail rot are among of the other
apparent symptoms. External indications of disease in fish with internal
granulomas are not always visible. Acute cases have only been described
following experimental injection, and they appear to be uncommon. Experi­
mentally infected fish perished within 2–3 weeks of infection, with few
clinical indications other than edema, fin erosions, and/or fluid accumula­
tions in the coelomic cavity (Antuofermo et al., 2014).

4.8.2 DIAGNOSIS

Mycobacteria can be isolated from internal organs to make a definitive diag­

nosis. External lesions may also include these organisms; however, these find­
ings should be interpreted with caution because they could be contaminants.
Bacterial Diseases of Finfish 121

Fast-growing species (e.g., M. chelonae, M. fortuitum, or M. abscessus)

can grow on non-selective conditions and appear possible in as short as 5
days. Slow-growing mycobacteriums like M. marinum, M. haemophilum,
and M. ulcerans are more difficult to recover and are frequently isolated on
mycobacterial selective medium (Middlebrook 7H10 agar or Lowenstein-
Jensen slants). M. haemophilum is one of the few bacteria that needs iron
(heme supplementation) in its media. The optimal growth temperature varies
by species, although most organisms that influence fish prefer temperatures
between 20 and 30°C. M. marinum thrives best at temperatures about 30°C.
M. marinum can take several weeks to isolate and cultures containing this
organism should be preserved for at least 6–8 weeks. M. haemophilum and
M. ulcerans can take even longer to produce visible colonies, thus PCR may
be a better way to detect them. Biochemical tests can distinguish mycobacte­
rial species to a limited extent; however, genetic approaches are now favored
for identification. Smears or tissue sections of lesions stained with Ziehl-
Neelsen (or its variants), Fite’s acid fast stain, or fluorochrome may include
acid-fast bacilli (auramine). Histopathology can also be beneficial.
PCR-based approaches, such as multiplex PCR assays, are effective for
detecting mycobacteria, but they cannot discriminate between some species,
such as the M. marinum complex’s individual members. M. marinum
complex has also been detected using a loop-mediated isothermal amplifi­
cation (LAMP) assay. If necessary, DNA sequencing, DNA fingerprinting,
MALDI-TOF (mass spectral fingerprinting), and other techniques can be
performed to confirm the species (Sanguinetti et al., 1998).

4.8.3 PREVENTION AND CONTROL

Quarantines for new stock (though many mycobacterial illnesses do not

manifest clinically within this time), egg disinfection, frequent cleaning and
disinfection of tanks and equipment, and water treatment with a germicidal
UV light are also preventive measures. Since mycobacteria can be found in
water, food, and other fomites, screening eggs and fish is the possible option
to prevent the infection (Enzensberger et al., 2002). Sentinel procedures are
in place at some facilities to detect agents as soon as they are introduced. If
trash fish or fish tissues (salmon viscera) are utilized as a protein source in the
feed, they should be pasteurized or heated for 30 minutes at 76°C (169°F).
Any ill or dead fish should be removed and destroyed as soon as possible.
Reptiles and other animals that are prone to mycobacteriosis should not have
fed them. When mycobacteriosis is introduced into a commercial facility, the
122 Coldwater Fisheries and Aquaculture Management

standard reaction is to cull the fish, thoroughly clean and disinfect the tanks
and equipment, and then replenish. In recreational aquaria or valuable fish in
displays, depopulation may or may not be maintained. If mycobacteria aren’t
completely eradicated, husbandry practices, including reducing stress and
crowding and ensuring adequate water quality can help lower the likelihood
of infection (Delghandi et al., 2020).
Treatment is rarely undertaken because it looks unlikely to completely
eradicate Mycobacterium from infected fish colonies, and it is also imprac­
tical in fish meant for human consumption. Clarithromycin, doxycycline,
minocycline, trimethoprim sulfamethoxazole, fluoroquinolones, rifampin,
rifabutin, and ethambutol have all been used to treat M. marinum in humans.
Some of these agents have been used in fish and other vertebrates in various
combinations, with variable degrees of success. Isoniazid, pyrazinamide,
and streptomycin resistance are common in M. marinum.

4.9 BACTERIAL KIDNEY DISEASE (BKD)

Bacterial kidney disease (BKD) of salmonid fishes is a slowly progressive,

systemic illness with a long course and an insidious nature. The bacterium that
causes BKD is a small Gram-positive diplobacillus known as Renibacterium
salmoninarum. Initial reports in the 1930s recorded a new complex disease
mainly affecting the kidneys from the rivers of Aberdeenshire Dee and Spey-
Spey in Scotland (Furunculosis Committee, 1933; Smith, 1964), brown trout
(Salmo trutta), rainbow trout (Oncorhynchus mykiss) in the Western United
States (Belding & Merrill, 1935), salmon (Salmo salar) from the rivers Aber­
deenshire Dee and from hatchery reared brook trout (Salvelinus fontinalis).
Lesions describing metabolic disturbances and reactions against the
etiological agents were traced. From the smears of the kidney, Gram-posi­
tive rods were recovered. Unfortunately, with the available conventional
techniques for fish pathogens, propagation could not be studied (Weins,
2011). Later on, the Gram-positive bacteria was cultivated by the injection
of an emulsion from internal organs, Koch’s postulate could be fulfilled.
The disease was termed as white boil disease or Dee disease or Kidney
disease (Sanders & Fryer, 1980).
The identical etiology of the disease was confirmed by the comparison
of the clinical picture, pathological lesions and its characterization (Ordal &
Earp, 1956; Smith, 1964). The disease was finally known to be as Renibac­
terium salmoninarum (Sanders & Fryer, 1980). Bacteria R. salmoninarum is
an obligate pathogen, which has been proved by its limited survival in water
Bacterial Diseases of Finfish 123

up to a few weeks, either in a diseased or in a latent carrier fish (Evelyn,

1993). Propagation of R. salmoninarum has been observed in a few other
species of fishes like sablefish Anoplopoma fimbria (Bell et al., 1990) and
cyprinids (Sakai et al., 1989).
BKD is a major threat to the wild and cultivated salmonid fishes. The
disease accounted in several European countries like Germany, Scotland,
England, France, Denmark, Norway, Iceland, and Finland. And from South
and North America, Japan (Kinkelin, 1974; Fryer & Sanders, 1981; Hoff­
mann et al., 1984; Kimura & Awakura, 1977; Fryer & Lannan, 1993). Serious
implications of the disease were observed in areas where Pacific salmon occur,
i.e., the Great Lakes region of the United States and the Pacific Northwest.
R. salmoninarum is a Gram-positive rod, non-motile bacteria of length
0.3–1.0 by 1.0 to 1.5 μm. They often occur in pairs. It is a non-alcohol fast
bacterium and does not produce endospores (Goodfellow et al., 1986).

4.9.1 PATHOLOGY

Diseased fish may look normal. The presence of several small, open sores
in the epidermis that reveal the underlying musculature can give rainbow
and brown trout a “buckshot” appearance. In brook trout and coho salmon
fingerlings and yearlings, massive boils filled with a pinkish milky fluid
containing vast quantities of Renibacterium salmoninarum bacteria can be
discovered on the flanks of the fish.
Exophthalmia (popeye) is a common symptom of BKD caused by osmo­
regulatory disturbance. However, because other reasons, such as gas super-
saturation (gas bubble disease), enteric redmouth (ERM) infections, some
viral disorders, or parasites, might induce this symptom, it is not diagnostic.
As indicated by its name, BKD affects the kidneys and, to a lesser extent,
the spleen and liver. In contrast to the smooth, concave surface of healthy
kidneys, the kidneys are frequently enlarged, convex, and have a corrugated
or bumpy surface. Massive populations of the pathogenic organism are
represented by creamy, soft, off-white cysts. Cysts of this type are prevalent
in the posterior kidney, and their size and number might vary. They are not
to be confused with normal stannous masses in the mid-kidney or nephro­
calcinosis (kidney stones) that can fill the kidney’s excretory tubules. In the
abdominal cavity and around the heart, a bloody, turbid, or yellow-brown
fluid frequently accumulates. The appearance of other internal organs and
visceral fat may be normal or exceptionally white. A white or yellow viscous
fluid may be present in the intestine.
124 Coldwater Fisheries and Aquaculture Management

4.9.1.1 EXTERNAL LESIONS

Loss of balance occurs in the fishes infected with R. salmoninarum. Super­

ficial blisters on skin and cavities in the musculature are described in the
infected salmonids. Cavitation’s and blisters are narrated to contain a white
or yellowish hemorrhagic fluid. Hemorrhagic areas around the fins and
the lateral line, petechiae, distended abdomen are a few common external
indications of BKD (Belding & Merrill, 1935; Ordal & Earp, 1953; Bruno,
1986; Evelyn, 1993).

4.9.1.2 INTERNAL LESIONS

Macroscopic lesions are one of the most common signs observed in connec­
tion to the fishes infected with R. salmoninarum. A swollen kidney, white-
grayish nodules of various sizes on the surface of the kidney are some of the
additional characters of the disease. Nodules of similar appearance could
be seen in the heart, liver, and spleen. An increase in size of the spleen and
bright coloration of the liver could be noted. Accumulation of ascitic fluid
in the abdominal cavity and petechial hemorrhages of the muscle lining the
peritoneum are described. In complicated cases, necrosis, grayish discolor­
ation, extensive kidney damage is experienced (Belding & Merrill, 1935;
Wood & Yasutake, 1956; Bell, 1961; Fryer & Sanders, 1981; Bruno, 1986).
Histologically, BKD is described as a granulomatous, chronic, and
systemic inflammation (Snieszko & Griffin, 1955; Wood & Yasutake, 1956).
Chronic infections in the infected fishes are distinguished by dispersed
melanin (Bruno, 1986) and increased amount of melanomacrophages.
Some of the typical observations are focal necrosis edged by granulomatous
tissue of epithelioid and lymphoid cells with varied stages of encapsulation.
Epithelioid appearance due to the development of granulomas as an activa­
tion of macrophages adhering to each other could be observed. Activated
macrophages could fuse to a multinucleated giant cell (MGC) (Holland et
al., 2003) demonstrated in chronic infections in fishes of parasitic or fungal
origin (Ferguson, 1989), and also in BKD infected fish (Snieszko & Griffin,
1955; Wood & Yasutake, 1956). Tissue necrosis occurs as a result of the
release of huge amounts of MGCs and activated macrophages.
Salinity (Fryer & Sanders, 1981), stress (Mesa et al., 1998) and tempera­
ture (Sanders et al., 1978) are few of the environmental factors responsible
for mortality and pathogenesis in connection with BKD. Morphological and
Bacterial Diseases of Finfish 125

macroscopical lesions are due to the dual effect of the bacteria itself and a
result of the immunological response of the fish to the infection (Young &
Chapman, 1978; Fryer & Lannan, 1993). Complications in the pathogenesis
of BKD arise due to the differences in susceptibility among the salmonid
species. Species considered most susceptible and associated with higher
mortality rates are chinook salmon (O. tshawytscha), salmon (O. nerka)
and Pink salmon (Oncorhynchus gorbushca). Rainbow trout (O. mykiss),
Atlantic salmon (Salmo salar) and brown trout are considered fairly resistant
to the BKD infection (Sanders et al., 1978; Sakai et al., 1991; Dale et al.,
1997; Starliper et al., 1997).

4.9.2 DIAGNOSIS

Advanced cases of BKD are the occurrence of white nodules in internal

organs such as heart, liver, kidney, spleen, and enlarged grayish kidney.
Gram-staining of smears, immunological techniques could be used to identify
the characteristic lesions, since the bacterial growth is time consuming. The
pathogen is aerobic and fastidious in its growth requirements. Ever first
achieved propagation of the bacteria was on Chicken embryo among the
various media tested for growth and sub-cultivation.
Dorset’s medium was proven successful. Increased possibility of isolation
of the kidney disease bacterium was by the addition of L-cysteine to the
Dorset’s medium. Though modifications of several different agar media for R.
salmoninarum like kidney disease medium (KDM) Evelyn, 1981) Charcoal
agar (Daly & Stevenson, 1985) and L-cysteine supplemented Muller Hinton
medium (Wolf & Dunbar, 1959) were performed, secondary bacterial
infections were a serious issue in the diagnostics. These contaminations by
other bacteria and fungi could be eliminated, by the use of (SKDM) containing
cycloserine, polymyxin B sulfate, oxolinic acid and cycloheximide (Austin et
al., 1983). Optimal temperature for the cultivation of R. salmoninarum was
15–18°C, no growth was observed at 30°C. Primary isolation and incubation
time for the initial growth of the organism was reported to be as long as 19
weeks post-inoculation of the kidney sample (Benediktsdóttir et al., 1991).
Serum-free broth, consisting of 0.1% L-cysteine, 1% peptone and 1% yeast
extract are suitable for the growth of R. salmoninarum in large batches (Daly
& Stevenson, 1993). Cultivation of R. salmoninarum is possible by cell culture
techniques. The bacteria were propagated intracellularly in the fish cells lines
(EPC, RTG-2, CHSE) (McIntosh et al., 1996; González et al., 2004).
126 Coldwater Fisheries and Aquaculture Management

The API-ZYM test has been found appropriate, as it is an enzymatic test

for which propagation of the bacterium is not necessary (Goodfellow et al.,
1986; Austin & Rayment, 1985). Proteolytic activity and a strong catalase­
positive reaction are described (Ordal & Earp, 1956; Goodfellow et al.,
1986). The β-Hemolytic activity of the bacterium declines after prolonged
cultivation (Bruno, 1986b).
BKD is defined as chronic, a systemic granulomatous inflammation.
Recommended staining methods to distinguish the pathogen are the standard
gram-staining, Lillie’s allocrome stain and modified Ollet’s gram-stain
(Midtlyng et al., 2000).
In addition to the observations made on the morphological lesions,
immunohistochemical techniques could be used for the identification of
the BKD pathogen. Dispersed melanin and melanomacrophages are the
characteristic feature of chronic inflammations in fishes (Bruno, 1986).
Differentiation among the melanin pigments from the intact bacteria is
difficult by the immunohistochemistry techniques due to a similarity in size
and in color of the oxidation product of commonly used chromogens. This
problem could be avoided by the use of strong hydrogen peroxide solution
for bleaching, without any negative effects in the immunoreactivity of the
identified epitopes (Jansson et al., 2002).
The utilization of antigen-antibody affinity in the naked eye detection
of antigens is one the common immunological technique (de la Pena,
2001). Stability of the antibody-antigen complex (avidity) and strength of
the antibody-epitope interaction (affinity) are the two crucial factors in the
immunological technique. However, the sensitivity and specificity of the
technique depends on the quality of the antibodies employed. Specificity
of the monoclonal antibodies, produced by a single B-cell, was considered
more reliable, until the polyclonal antibodies, from several B-cell lines, was
used for the identification and was completely reliable (Lu et al., 2020).
Indirect fluorescent antibody technique (Laidler, 2006), Real time PCR
(Jansson et al., 2008). ELISA (Laurin et al., 2019) was reported to be more
sensitive, in comparison to the culture based or the has been FAT for detec­
tion of R. salmoninarum in the affected fishes.

4.9.3 PREVENTION AND CONTROL

Temporary effect observed upon antibiotic treatment was not recommended

as a therapy, due to the possibility of intracellular survival of the bacteria.
Frequent screening in order to control the vertical and horizontal transmission
Bacterial Diseases of Finfish 127

of BKD has been the most effective prevention strategy (Elliott et al., 1989;
Fryer & Lannan, 1993). Separation of the rearing eggs and juvenile fishes
into separate groups have helped in areas of prevalence of BKD (Elliott et
al., 1995).
Injection of erythromycin to the brood stock, before spawning was
observed in reducing the transmission of R. salmoninarum to the eggs.
Destruction of gametes by brood stock culling has been reported to reduce
the prevalence of the disease (Gudmundsdottir et al., 2017). This stage
requires a sensitive procedure in identifying the carriers and a careful
separation of the eggs until further confirmation of the diagnosis has been
established. Administration of R. salmoninarum expressing low levels of cell
associated p5 orally have resulted in increased the duration of time from
challenge of infection to death (Piganelli et al., 1999). However, this protec­
tion was not associated with the humoral antibody level. This is supportive
of the previous HP indications which suggested the involvement of the cell-
mediated immune response in recovery of the diseased fish.
Advancements in the control and prevention of the BKD depends on the
knowledge with regard to the basics of the immune functions in fish and the
pathogenesis of the infection.

KEYWORDS

• coldwater aquaculture
• diseases
• polymerase chain reaction
• preventive
• rainbow trout gastroenteritis
• segmented filamentous bacteria

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CHAPTER 5

Biotechnological Interventions
in Coldwater Aquaculture Health
Management
MD. IDRISH RAJA KHAN
Department of Aquatic Health and Environment, College of Fisheries,
Central Agricultural University (I), Lembucherra, Tripura, India

ABSTRACT

Over the years, owing to the adoption of scientific practices aquaculture

industry has flourished and achieved new horizons of production; however,
with progression, fish also became vulnerable to an array of pathogens,
including aquatic organisms from cold water environment. Concurrently,
the fish health management approaches have also advanced then also the
episode of disease outbreaks are still frequent and widespread, and leading
to fettered aquaculture production. The biotechnological approach has
shown promising responses either to improve the health status of cultured
organisms or to strengthen the diagnosis of diseases while safeguarding the
aquatic environment. Such biotechnological interventions led to avoidance
of the pathogen by strengthening the immune response of fish as well as
avoiding any possible use of chemical remedial agents. Recently with the
development of anti-microbial resistance and the emergence of superbugs,
the employment of biocontrol strategy through biotechnology-based tools
have gained momentum. The present chapter is a brief account of different
biotechnological approach and their application in aquaculture to augment
the immune response with appropriate disease diagnosis to achieve sustain­
able aquaculture production.

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
148 Coldwater Fisheries and Aquaculture Management

5.1 INTRODUCTION

Aquaculture has arrived as one of the most flourishing agri-based commodi­

ties with a significant share in the global food basket with a noteworthy
contribution to total animal protein supply, i.e., 17%, globally (FAO, 2020).
When it comes to global fish production India stands at second place just after
China with a significant annual growth rate of over 7%. These attainments
are the collective bequest of adaptation of intensive aquaculture practices
with scientific advancement. India is bestowed with vast water resources
ranging from freshwater, brackish water and marine environment. Moreover,
coldwater resources stretched over the Himalayan and peninsular regions
also behold as a major resource to capitalize. Indian coldwater resources
harboring about 272 fish species belonging to 21 families and 76 genera
(Singh & Akhtar, 2015). These species may contribute to India’s 2030 dream
of self-sufficient for all kinds of fish products and by-products with the adop­
tion of modern and scientific culture practices. However, the low productivity
level of the aquatic systems and higher degree of resource unpredictability
roots to unique challenges including pathogenic infection caused by an array
of pathogens. However, fish production through advanced approaches has
led more often than not irresponsible culture practice with indiscriminate
application of chemical therapeutics which very often made fish vulnerable
to various resistant pathogens. Such detrimental circumstances with negli­
gence to sustainable practices often led to deprived growth, severe mortality
and eventually fettered aquaculture production.
Over time, there has been significant development in animal biotech­
nology and its application in various fields, including aquaculture health
management. Biotechnology is the practice in which biological organisms
or systems are utilized for enriched production. Increased demand for fish
food and declining natural available habitats have encouraged researchers
to explore ways that biotechnology can offer to enhance the production and
make aquaculture one of the rising avenues in animal research. Biotech­
nology offers a “give and take management system” where biotechnological
tools can be employed for healthy aquaculture practices ranging from seed
rearing to table size fish production (Vijayan et al., 2013). Biotechnology
is already making important contributions to the world, and it perceives
that biotechnological tools should be employed as an adjunct and not as
a replacement to the conventional approaches in resolving issues, as well
as their application, should be need-driven rather than technology-driven.
Aquaculture sector requires novel biotechnological approaches to tackle
the challenges emerging in sustainable fish production, particularly disease
Biotechnological Interventions in Coldwater Aquaculture 149

prevention, diagnosis, and their management in the aquatic animal culture

systems. In the recent past, there are several examples of biotechnology-
based tools that are employed in aquaculture health management to avoid
any probable disease outbreak (Figure 5.1).

FIGURE 5.1 Biotechnology-based tools in aquaculture health management.

5.2 DISEASES–A CONSTANT THREAT FOR AQUACULTURE

Disease outbreak is often documented as a major risk factor for the growth of
both fish as well as shellfish industry. Globally, numerous pathogenic micro­
organisms affected the aquaculture industry and often led to catastrophic
results. It has been observed that about 10% of all cultured fish die because
of disease outbreaks (Plumb, 1999). Moreover, in catfish culture, about 75%
of newly hatched swim-up fry die before they reach marketable size. And, in
the case of rearing freshwater fish, the final survival is about 30% in which
majority of loss is contributed by varied etiologies including diseases that
have often gone undocumented. The frequent episode of disease outbreak
150 Coldwater Fisheries and Aquaculture Management

is a multifarious interaction between the host, disease-causing agent and

the condition of the aquatic environment. The poor management practice
in intensive and semi-intensive farming favored the occurrence of disease
outbreak due to varied reasons which includes high stocking density and
intensive feeding regime with inadequate water exchange leading to
increased stress, and ultimately making them vulnerable to an array of patho­
genic agent (Kaoud, 2015). Therefore, there is a need for the employment
of standard culture practices with scientific developments to minimize any
potential disease outbreak.

5.3 BIOTECHNOLOGICAL TOOLS FOR HEALTH MANAGEMENT IN

COLDWATER AQUACULTURE

5.3.1 VACCINES

Vaccination approach in aquaculture is a well-established and cost-effective

approach to mitigate infectious diseases. A vaccine is any biological
preparation that can establish or improve the immunity of the host upon
administration and provide protection against a specific pathogen. The
recipient’s immune system develops a primary immune response following
exposure to the antigen either killed, attenuated, sub-unit, recombinant, etc.
(Adams & Thompson, 2006). A vaccine not only ease the severity of disease
losses but also reduce the need for chemotherapeutics, leave no residues
in the organisms and does not lead to the emergence of resistance in the
microorganisms (Subasinghe, Bondad-Reantaso, & McGladdery, 2001). An
optimal vaccine must be efficient enough to rouse innate immune response
and sufficient antibody titer with specific immune memory in the host for
next exposure (Mishra et al., 2020). Vaccine application has arrived as the
most effective and significant approach to prevent diseases outbreak in
aquaculture. Presently, the policy is more effective against bacterial and viral
diseases than against parasitic infections. Eventually, numerous momentous
developments have been documented to develop efficient vaccines with
superior efficiency and easy to administer over diverse culture environment.
However, until now very few vaccines are commercially available in the
market against viral and bacterial pathogenic agents.
Bacterial pathogens are the most common and significant threat to
aquaculture sector with an approximate contribution of 34% to total disease
outbreaks in fish culture systems (Mishra et al., 2020). Owing to the
frequency of bacterial disease outbreaks and losses incurred by them, most
Biotechnological Interventions in Coldwater Aquaculture 151

of the commercialized vaccines are developed against bacterial pathogens.

Which includes Aeromonas salmonicida, Vibrio anguillarum V. salmonicida,
Renibacterium salmoninarum, Yersinia ruckeri, Flavobacterium columnare,
F. psychrophilum, Edwardsiella ictaluri Pseudimonas salmonis, Lactococcus
garvieae, Streptococcus iniae, Photobacterium damselae subsp. Piscicida,
etc. (Haenen, 2017). There are several commercial vaccines available against
viral agents as well such as infectious pancreatic necrosis virus (IPNV),
infectious salmon anemia virus (ISAV), spring viremia of carp virus (SVCV),
infectious hematopoietic necrosis virus (IHNV), nervous necrosis virus
(NNV), viral hemorrhagic septicemia (VHS), etc.
Most of the commercial vaccines available in the market are inactivated
(killed) vaccines. However, few reports suggest the failure of such vaccines,
particularly against viruses, which lead the foundation for the development
and adoption of live attenuated vaccines (Adams & Thompson, 2006).
However, the application of live vaccines also brings several concerns that
the attenuated pathogen might back-mutate and revert to the virulent nature
and may lead to catastrophic results (Thompson & Adams, 2004). There are
several commercially available vaccines which include attenuated (against
A. salmonicida and E. ictaluri infection), killed or inactivated (against
ISAV and IHNV infection), recombinant protein (against IPNV and SVCV
infection), DNA vaccine (against IHNV, IPNV, VHSV, and NNV infection),
synthetic peptide vaccine (VHS), etc. (Table 5.1).

5.3.2 IMMUNOSTIMULANTS

Several alternative immune-enhancing practices to avoid any possible disease

outbreak has been adopted globally, among them administration of immu­
nostimulants has gained momentum enormously as it can non-specifically
strengthen the immune response of cultured organism. Immunostimulants
can be defined as any natural or chemical compound, action or stressor that
augments the non-specific immune response of fish and crustaceans by acti­
vating various immune cells of the organism (Mishra et al., 2020; Barman,
Nen, Mandal, & Kumar, 2013). Immunostimulants protect organisms without
any side effects and with no or minimal detrimental impact on the environ­
ment; however, the approach is still limited to shrimp culture practices only.
Yet various immunostimulants are commercially available in the market
which depicts the worth of the approach and practical utility as a substitute
to improve survival response against myriads of diseases (Apines-Amar &
Amar, 2015). Through studies, it has been revealed that the most established
TABLE 5.1 Biotechnology-based Tools Employed in Aquaculture

152
Type of Antigen Commercial Host Organism Mode of Remarks References
Vaccine Name Application
A. Commercially Available Vaccines
Attenuated Aeromonas Brivax II Rainbow trout Intraperitoneal The cellular, humoral, Liu et al. (2015)
salmonicida (Oncorhynchus mykiss) (IP) injection and mucosal immune
Edwardsiella ictaluri NA (in vitro) Channel catfish IP injection response of host got Liu et al. (2015)
(Ictalurus punctatus) elevated

Killed or Infectious pancreatic Alpha Ject® Salmon IP injection Autogenous Biering et al. (2004)
inactivated necrosis virus (IPNV) 1000

Coldwater Fisheries and Aquaculture Management

Spring viremia of Bioveta Carps IP injection Easy administration. Salgado-Miranda et
carp virus (SVCV) Safe for use V. al. (2013)
A. salmonicida and MULTIVaC, Salmonids IP injection Salmonicida Salgado-Miranda et
Vibrio anguillarum Microtek, al. (2013)
Vector based Infectious salmon NA (In vitro) Atlantic salmon (Salmo IP injection Apoptosis was detected Wolf et al. (2012)
vaccine anemia virus (ISAV) salar) in the infected cells;
Infectious NA (In vitro) Atlantic salmon (S. IP injection additionally further Adams &
hematopoietic salar) field level efficacy must Thompson (2006)
necrosis virus need to be performed
(IHNV)
Recombinant IPNV/VP2 Microtekz Salmon IP injection Economically viable De Kinkelin (1994)
protein and safe to host.
Sufficient production of
immune proteins. Safe
and low-cost method
TABLE 5.1 (Continued)

Biotechnological Interventions in Coldwater Aquaculture

Type of Antigen Commercial Host Organism Mode of Remarks References
Vaccine Name Application
Spring viremia of International Carp IP injection Salgado-Miranda et
carp virus (SVCV) Inc. al. (2013)
Salmon rickettsiae Bayovac 3.1, Salmonids IP injection Adams &
Pharos, S.A. Thompson (2006)
DNA Viral hemorrhagic NA (in vitro) Salmonids IM injection Rouses cellular as Meeusen et al.
vaccine septicemia virus well as humoral (2007)
(VHSV) immunological indices
IPNV Novartis Rainbow trout (O. IM injection in host. Ballesteros et al.
mykiss) (2014)
IHNV Aqua Health Salmon IM La Patra et al.
Ltd. (intramuscular) (2001)
injection
Pancreatic disease NA (in vitro) Rainbow trout (O. IM injection Kurath (2008)
(PD) mykiss)
Nervous necrosis NA (in vitro) European sea bass Oral feeding Valero et al. (2016)
virus (NNV) (Dicentrarchus labrax)
Genetically A. salmonicida Brivax II Rainbow trout (O. IP injection Elevation in Liu et al. (2015)
attenuated mykiss) immunological
pathogen response of host
Synthetic Viral hemorrhagic NA (in vitro) Rainbow trout (O. IP injection Possibility of a vector Coeurdacier,
peptide septicemia (VHS) mykiss) encoding several Laporte, & Pepin
vaccine antigens to provide (2003)
multifarious protection

153
TABLE 5.1 (Continued)

154
B. Commercially Available Immunostimulants
Commercial Name and Company Contains Method of Application References
Nutri-care by Nutricorp Animal Bio Vitamins, extracts of herbs, enzymes, For salinity stress of below 30 ppt–10 g/ Mishra et al. (2020)
Solutions, India amino acids and organic minerals, kg of feed for 5 days, whereas above 30
probiotics, etc. ppt 15 g kg–1 of feed for 5 days
Lysozyme by Hydrochloride by Hen egg lysozyme As a dietary supplement
Belovo, Belgium
Selenium Yeast by Alko, Finland Selenium yeast As feed ingredient

Coldwater Fisheries and Aquaculture Management

Lactoferrin DMV by International, Bovine milk lactoferrin As feed ingredient
Netherland
Macroguard Biotec by Mackzymal, Yeast β (1,6) and β (1,3) glucan With vaccines through injection;
Norway through-feed for 6–8 weeks continuously
DS 1999 by International Bacterin Direct incorporation in the culture
Aquaculture Biotechnologies, Ltd. medium
Levamisole by Janssen Tetrahydro-6-phenylimidazolthiazole As a dietary supplement
Pharmaceutica, Belgium hydrochloride
Immustim by Immundyne, USA β(1,6) branched β(1,3) glucan from yeast Immersion for larvae/PLs and feed
ingredient for grow-out shrimp
C. Commercially Available Probiotics
Commercial Name and Composition Indications or Result Dose References
AQUAGEN PRO (probiotic strains) Induces ammonia-oxidation 2-to-3-liter acre–1 in 3 to 5 feet depth Mishra et al. (2020)
ALGUTPRO (active/inactive forms of Enhanced proliferation of 2 to 3 kg per ton feed
Saccharomyces spp., Candida spp. and several beneficial microorganisms
growth enhancers)
TABLE 5.1 (Continued)

Biotechnological Interventions in Coldwater Aquaculture

Commercial Name and Composition Indications or Result Dose References
SUPERGUT (combination of multi-strain of Limit the proliferation of 10 to 20 ml kg feed
–1

probiotics along with seaweed extract and pathogenic bacteria and

molasses) maintain a balance of gut
inhabiting microflora
PROBAC-G (Cerevisiae, Lactobacillus spp., B. Improve feed conversion 500 g per ton of feed
subtilis, fungal diastase, vitamin B12, pepsin, ratio (FCR) and provide
papain, etc.) resistance against diseases
Pro marine (probiotics mixed with vitamin C Eliminates harmful 5 g per kg of feed twice daily
and calcium) pathogens and enhances
growth
Thiomax (probiotics and micronutrients) Improve the pond bottom 2 to 3 kg per acre
environment
Spark-PS (probiotic mixture) Produces various hydrolytic 2 to 3 liter per hectare
enzymes to digest complex
organic substances
VIBRION (probiotic strains, lipase, alkaline Eliminates harmful 250 to 500 g per acre for 7 to 10 days
protease, etc.) pathogens like Vibrio spp.
Environ (mixture of probiotics) Improves aquatic 250 to 500 g per acre
environment for healthy
growth
GENTECH P.S. (probiotics) Facilitates degradation of 5 liter per hectare
organic substances

155
TABLE 5.1 (Continued)

156
D. Common Prebiotic Used in Aquaculture
Prebiotic Dose and Length of Administration Host Results References
Oral feeding with 20 g per kg for one Siberian sturgeon Increased growth rate Mahious, Van, &
month (Acipenser baerii) Ollevier (2006)
Oral feeding with 20 g per kg for one Turbot (Psetta maxima) Increased growth rate and proliferation Mahious et al. (2006)
month larvae of gut microbiota such as Bacillus spp.
5 and 10 g per kg for one week Gilthead seabream Reduces the phagocytosis and Cerezuela et al.
(Sparus aurata L.) respiratory burst activity of leucocytes (2008)
Oral feeding with 0.8% for 114 days Sharpsnout sea bream No significant change in growth and Piccolo et al. (2011)

Coldwater Fisheries and Aquaculture Management

(Diplodus puntazzo) digestibility
FOS Oral feeding with 20 g per kg for one P. maxima larvae Enhanced growth and proliferation of Mahious et al. (2006)
month gut microbiota (Bacillus spp.)
Oral feeding with 10 g kg for 4 Atlantic salmon No significant effect on feed intake, Grisdale-Helland et
months (Salmo salar) growth, or digestibility al. (2008)
SCFOS 0.8 or 1.2 g kg–1 for 8 weeks Hybrid tilapia Enhanced growth rate, FCR along with Hui-Yuan et al.
(Oreochromis aureus x significant proliferation of beneficial (2007)
O. niloticus) bacteria such as Lactobacillus spp.
1 g kg–1 for 56 days Hybrid tilapia Increased proliferation of gut Zhou et al. (2009)
endosymbionts
Fish meal (FM) based diets enriched European sea bass No significant change in growth and Guerreiro, Oliva-
with 1% SCFOS for 7 weeks (Dicentrarchus labrax) survival Teles, & Enes (2015)
MOS Oral feeding of 0.2% White sea bream No significant effect on growth, survival Dimitroglou et al.
Artemia enriched by A1 DHA (Diplodus sargus) larvae (2011)
(docosahexaenoic acid) SelcoTM with
MOS addition for 23–43 days
TABLE 5.1 (Continued)

Biotechnological Interventions in Coldwater Aquaculture

D. Common Prebiotic Used in Aquaculture
Prebiotic Dose and Length of Administration Host Results References
FM-based diets enriched with 20 g European sea bass (D. Increased growth Torrecillas et al.
MOS @ 0.16% for 8 weeks labrax) (2015)
FM-based diets enriched with MOS European sea bass Increased growth and disease resistance Salem et al. (2016)
@ 0.21 g 0.05%, 0.1%, 0.2%, 0.3%
and 0.4% till 75 days
E. Synbiotics Used in Aquaculture
Synbiotic Combination Aquatic Organism Effects References
Enterococcus faecalis + MOS Rainbow trout (Oncorhynchus mykiss) Increased growth and immune response Rodriguez-Estrada
and polyhydroxybutyrate acid in et al. (2009)
different combination for 12 weeks
B. clausii + MOS, FOS in different Japanese flounder (Paralichthys Increased growth performance and Ye, Wang, & Sun
combination for 56 days olivaceus) digestibility performance (2011)
Biomin IMBO (E. faecium + FOS) Rainbow trout (O. Mykiss) Increased growth and disease resistance Mehrabi,
in different combinations for 60 capacity Firouzbakhsh, &
days Jafarpour (2012)
B. subtilis + FOS in different Juvenile large yellow croaker Increased growth and disease resistance Ai et al. (2011)
combination for 10 weeks (Larimichthys crocea) capacity
B. subtilis + chitosan in different Cobia (Rachycentron canadum) Increased growth response and immune Geng et al. (2011)
combination for 8 weeks response
L. plantarum 7–40 + Pacific white shrimp (Litopenaeus Increased growth response and Huynh et al. (2018)
galactooligosaccharide vannamei) digestibility
B. lincheniformis + MOS Pacific white shrimp (L. vannamei) Elevate the dietary assimilation, intestinal Chen et al. (2020)
SCFAs content, and immune response of

157
host to confer healthy growth
TABLE 5.1 (Continued)

158
F. Paraprobiotic Application in Aquaculture
Paraprobiotic Organism Under Study Mode of Post-Administration Responses References
Administration
L. plantarum Giant freshwater prawn In vivo Significant rise in immune response and Dash et al. (2015)
(Macrobrachium rosenbergii) diseases resistance
B. pumilus SE5 Orange-spotted grouper In vivo Significant rise in immune response Yan et al. (2016)
(Epinephelus coioides)
Pseudomonas Labeo rohita In vitro Significant expression of immune genes Giri et al. (2016)
aeruginosa VSG2 and immune cell activity
L. plantarum Pacific white shrimp (L. In vivo Elevation in growth, immune response, Zheng et al. (2017)

Coldwater Fisheries and Aquaculture Management

vannamei) and tolerance towards salinity stress
B. amyloliquefaciens Catla catla In vivo Significant rise in immune responses Singh et al. (2017)
FPTB16
Lactobacillus Whiteleg shrimp (Penaeus In vivo Positively modulated the gastrointestinal Zheng et al. (2020)
plantarum vannamei) microbiota

G. Effect of Quorum Quenching on the Virulence Capacity of Pathogens

Pathogens Quorum Quenching Virulence Host Challenge Bacterial Density References
Method Capacity Method
V. harveyi luxS, luxO, luxP gene Abolished Brine shrimp artemia Bath challenge 104 CFU ml–1 Defoirdt et al.
mutation (2004)
A. hydrophila ahyR gene mutation Reduced Epithelioma papillosum Coculture 30 min MOI 1 Bi et al. (2007)
of carp (Cyprinus
carpio) cells
Swordtail Intraperitoneal 104–10 CFU ml–1
(Xiphophophorus injection (0.1 ml)
helleri)
TABLE 5.1 (Continued)

Biotechnological Interventions in Coldwater Aquaculture

Pathogens Quorum Quenching Virulence Host Challenge Bacterial Density References
Method Capacity Method
ahyI gene mutation Reduced Epithelioma papillosum Coculture 30 min MOI 1 Chu et al.
cyprini (EPC) cells (2011)
Goldfish (Carassius Intraperitoneal 104–10 CFU ml–1
auratus gibelio) injection (0.1 ml)
V. parahaemolyticus opaR gene mutation Increased Chinese hamster ovary Coculture 5 h 1.5 × 106 CFU ml–1, Gode-Potratz
cells MOI 15 & McCarter
(2011)
V. alginolyticus luxT gene mutation Slightly Zebrafish Intramuscular 10 –10 CFU per
4 7
Liu et al.
reduced injection fish (2012)
V. harveyi HAH Probiotic application, Reduced P. vannamei In vitro and in vivo Immersion @ Yu et al. (2019)
Cobetia sp. tests 104–106 CFU ml–1
A. hydrophila Probiotic application, Reduced Zebrafish Injected 2.6 × 108 CFU ml–1 Chen et al.
B. licheniformis T-1 intraperitoneally (2020)
H. Commercially Available Phage-based Products in Aquaculture
Name of the Company/Institute Bacterial Host Product Description References
Intralytix Escherichia coli, V. tubiashii and V. Phage cocktail to control infections in Intralytix
coralliitycis oyster (2018)
Fixed Phage Ltd. V. parahaemolyticus aquaPHIX™ is a phage-based solution Mattey (2016)
that binds with feed pallets applied
directly in the aquaculture system
ICAR-CIBA Vibrio spp. LUMIPHAGE for biocontrol of ICAR-CIBA
luminescent bacterial disease (LBD) (2017)
in shrimp larvae
Phage Biotech Ltd. V. harveyi Phage therapy to treat vibrio infections Phage Biotech

159
in shrimp (2017)
TABLE 5.1 (Continued)

160
Name of the Company/Institute Bacterial Host Product Description References
ACD Pharma Yersinia ruckeri CUSTUS®YRS is phage-based solutions ACD Pharma
against yersiniosis, or enteric red (2017)
mouth disease (ERM) of Atlantic
salmon
Proteon pharmaceutical Pseudomonas spp. and Aeromonas spp. BAFADOR® is a phage-cocktail Grzelak (2017)
applied via immersion
Mangalore Biotech Laboratory Vibrio spp. LUMI-NIL MBL is a phage Mangalore
formulation to control luminous Biotech
vibriosis in shrimp Laboratory

Coldwater Fisheries and Aquaculture Management

(2019)
I. Applications of RNAi against Viral Infection in Shrimps
Target Species Target Gene Delivery Method Pathogen RNAi Inducer References
Giant tiger prawn (P. YRP65 Transfection Yellow head virus In vitro transcribed dsRNA Ongvarrasopone
monodon) (YHV) et al. (2011)
vp15 and vp28 Injection White spot syndrome siRNA Ho et al. (2011)
virus (WSSV)
ns1 and vp Injection Densovirus (DNV) Bacterially expressed dsRNA Sellars et al. (2011)
PmRab7 Injection Laem-Singh virus Bacterially expressed dsRNA Sudhakaran et al.
(LSNV) (2011)
GAV, b-actin Oral Gill-associated virus Bacterially expressed dsRNA Posiri,
(GAV) Ongvarrasopone,
& Panyim (2011)
Pacific white shrimp Rab7 Injection Taura syndrome virus Bacterially expressed dsRNA Mejía-Ruíz et al.
(L. vannamei) (TSV) (2011)
rr2 and DNApol, Injection WSSV-TSV In vitro transcribed dsRNA Hirono et al. (2011)
ORF
TABLE 5.1 (Continued)

Biotechnological Interventions in Coldwater Aquaculture

J. Antimicrobial Peptide Application in Aquaculture
AMPs Administration Pathogen Infected Organism Results References
Method
CEME, Intraperitoneal V. anguillarum Coho salmon Increased survival percentage Jia et al. (2000)
cecropin-melittin injection (O. kisutch)
Synthetic hepcidin Intraperitoneal V. anguillarum European bass Significant reduction in the Alvarez et al.
injection (D. labrax) mortality (2016)
FSB-AMP Oral V. Pacific white shrimp Significant reduction in the Cheng et al.
parahaemolyticus (L. vannamei) mortality (2017)
SCY-hepc Oral A. hydrophila Sea bream (Sparus Increased survival percentage He et al. (2018)
macrocephalus) and
hybrid grouper
Synthetic hepcidin Intraperitoneal Edwarsiella tarda Mudskipper Significant increase in survival Chen, Nie, &
injection (Boleophthalmus Chen (2018)
pectinirostris)
Hepcidin Oral and Flavobacterium Grass carp Significantly increased survival Wei et al.
intraperitoneal columnare (Ctenopharyngodon (2018)
injection idellus)

161
162 Coldwater Fisheries and Aquaculture Management

mode of action of immunostimulants is to through the proper functioning of

phagocytic cells and to enhance their anti-microbial response (Barman, Nen,
Mandal, & Kumar, 2013). A wide variety of compounds has been found to
have immunostimulatory nature, including synthetic chemicals (FK-565,
Levamisole, etc.), bacterial derivatives (β-glucan, lipopolysaccharides or LPS,
EF203, etc.), polysaccharides (chitin, chitosan, schizophyllan, etc.), nutritional
factors (vitamin C, vitamin E, etc.), cytokines, hormones along with several
plant and animal-derived products/by-products have been extensively studied
in aquaculture (Barman, Nen, Mandal, & Kumar, 2013) (Table 5.1).

5.3.3 PROBIOTICS

Probiotics are live microbial feed supplements that confer health benefits
in the host organism through improved microbial balance. Colonization of
healthy gut flora helps the host to resist invasion of a broad range of virulent
microorganisms (Kesarcodi-Watson et al., 2008). Numerous probiotics or
probiotic-derived products from different potential bacterial species are
available in the market for use in aquaculture, globally. Throughout the year
probiotics have established themselves as an alternative to chemical thera­
peutics in preventing infectious diseases outbreak in aquaculture throughout
the world. There are several well established beneficial effects that have been
observed with the application of probiotics in aquaculture such as overall
health status of the host, increased survival percentage, bioremediation of
nitrogen and phosphorus related metabolites in pond which confer improved
water quality and ultimately increased yields with minimal application of
chemical remedial agents (Wang, Xu, & Xia, 2005; Shefat, 2018; Khan,
Choudhury, & Kamilya, 2020).
Application of probiotics in intensive and semi-intensive aquaculture
practices has surfaced as one of the most appropriate and promising alternatives
to meet the nutritional needs by producing a premium quality animal protein
with minimal application of chemicals. In the last two decades, the exploratory
research and probiotic application in aquaculture have expanded a great deal.
According to recent studies majority of potential probiotic candidates belongs
to 20 bacterial genera where most of the isolates were identified as members
of either Bacillus or Lactobacillus genera (Khan, Choudhury, & Kamilya,
2020). Other commonly administered probiotic candidates in the aquaculture
system include Bifidobacterium spp., Enterococcus spp., Streptomyces spp.,
Carnobacterium spp. and yeast (Van et al., 2019) (Table 5.1).
Biotechnological Interventions in Coldwater Aquaculture 163

5.3.4 PREBIOTICS AND SYNBIOTIC

The concept of prebiotics is quite recent, which serves as a source of nutrient/

food for probiotic microorganisms (Panigrahi & Azad, 2007). Prebiotics can
be defined as non-digestible food ingredients that are metabolized by specific
bacteria which selectively stimulate the proliferation of gut inhabiting
symbiotic bacteria (Ringø et al., 2010; Ganguly, Dora, Sarkar, & Chowdhury,
2012). They are believed to be resistant to endogenous digestive enzymes and
hence can reach the site of proliferation of microflora in the gastro-intestinal
site and promote their proliferation. Primarily, prebiotics is fragments of
carbohydrate or complex oligosaccharides (3–10 sugar moieties) such as
galactose, fructose or mannose. Additionally, short-chain fatty acids (SCFA)
were also reported as prebiotic later as they also have a positive influence
on colonic health (Ringø et al., 2010; Ganguly, Dora, Sarkar, & Chowdhury,
2012). Some of the common prebiotic compounds that are presently used
in feed are fructo-oligosaccharide (FOS), mannan-oligosaccharides (MOS),
mixed oligo-dextran, etc. (Panigrahi & Azad, 2007). Bacterial cell surface
have certain lectins (glycoproteins) that recognize specific sugars to allow
attachment of cells with sugar moieties and utilize the same for multiplication
and further proliferation (Panigrahi & Azad, 2007). Over the years, despite the
potential benefits of prebiotic compounds in health and growth performance
of aquatic animals, the application of prebiotics in aquaculture is still under-
explored and requires further investigation (Table 5.1).
Gibson & Roberfroid predicted that a combination approach of probiotic
with prebiotic can be adopted to achieve elevated proliferation of potential
probiotic, which later termed “synbiotic” (De Vrese & Schrezenmeir, 2008).
The supplementation synbiotic lead to a positive influence on feed utiliza­
tion, immunomodulation, survival against varied infection with superior
growth performance (Cerezuela, Meseguer, & Esteban, 2011). Intestinal
probiotic microorganisms selectively ferment dietary prebiotic compounds
to produce short-chain compounds which eventually enhance the bioavail­
ability of nutrients (Breves, Sztkuti, & Schrder, 2001; Bongers, & Van, 2003).
Additionally, the synbiotics application has also shown increased fermen­
tation and production of numerous SCFA, vitamins, and peptides leading
to reduced intestinal pH thereby eliminating the chances of any probable
infection of certain pathogenic bacteria while triggering the augmentation
of various endosymbionts (Breves, Sztkuti, & Schrder, 2001; Bongers, &
Van, 2003). Furthermore, there is evidence that prebiotic augmentation can
directly enhance the health status of fish by inducing the immune response
(Ringø et al., 2010) (Table 5.1).
164 Coldwater Fisheries and Aquaculture Management

5.3.5 PARAPROBIOTICS AND POSTBIOTICS

The term paraprobiotics was proposed by Taverniti & Guglielmetti (2011)

and defined as non-viable beneficial microbial cells or cell fragments or cell
extracts, which upon administration confer health benefit to the host organism.
The prefix ‘para’ is adopted from the Greek word, which means ‘atypical’ or
‘alongside of’ (Choudhury & Kamilya, 2019). Because of their crude nature,
they are also called “ghost probiotic” (Choudhury & Kamilya, 2019) whereas;
Villamil et al. (2002) were the first who observed the immune augmentation
potential of paraprobiotics, Lactococcus lactis in turbot. This study leads the
foundation for future research, which revealed that paraprobiotic preparations
may play a vital role in mediating growth and immune responses, resistance
from various diseases, etc. Several other studies have revealed the capability
of paraprobiotic in uplifting the immune response. The studies advocates for
the potency of paraprobiotic to augment immunity levels significantly through
enhanced myeloperoxidase content, nitric oxide (NO) production, respiratory
burst, phagocytic response, proliferation of leukocytes in fish head-kidney,
etc. (Villamil et al., 2002; Salinas et al., 2006; Román et al., 2012; Kamilya
et al., 2015) (Table 5.1).
The term ‘postbiotics’ evolved recently and refers to “soluble factors such
as metabolic products or by-products secreted/released by bacterial lysis”
(Aguilar-Toalá et al., 2018). These compounds include different enzymes,
peptides, vitamins, SCFA, and various cell surface proteins (Tsilingiri &
Rescigno, 2012; Konstantinov, Kuipers, & Peppelenbosch, 2013). There are
reports suggesting immune-enhancement through postbiotic, however exact
mode of action is yet to be known (Kareem et al., 2016; Nawaz et al., 2018)
(Table 5.1). A recent study has shed some light on the mode of action of
postbiotics led immune response on host, where study has shown that dietary
prebiotics and their gut microflora led fermentation radically increase the
levels of SCFA including propionate, acetate, and butyrate (Hoseinifar et al.,
2015). Reports also suggest that the SCFA are capable of rousing non-specific
immune as well as inflammatory response in the host organism (Maslowski
& Mackay, 2011).

5.3.6 QUORUM QUENCHING OR QUORUM SENSING INHIBITION

Quorum sensing is a mechanism of bacterial cell-to-cell communication, in

which they produce, release, and detect small signaling molecules to coor­
dinate the production or expression of certain proteins or genes (Defoirdt,
Biotechnological Interventions in Coldwater Aquaculture 165

Boon, Bossier, & Verstraete, 2004). The mechanism was first reported in a
bacterium, V. fischeri (Nealson, Platt, & Hastings, 1970). Later on, similar
biological communication systems were also reported in many other bacteria,
where they produce Cholerae quorum-sensing autoinducer-1 (CAI-1), auto­
inducer-2 (AI-2), acylated homoserine lactones (AHLs), etc., as communica­
tion molecules (Defoirdt, Boon, Bossier, & Verstraete, 2004; Nealson, Platt,
& Hastings, 1970). Bacterial phenotypic characteristics that are coordinated
by the quorum sensing mechanism include nodulation, antibiotic production,
biocorrosion, swarming, conjugation, bioluminescence, sporulation, and
most importantly expression of virulence proteins (toxins, lytic enzymes,
adhesion molecules, siderophores, etc.), and biofilm formation (Defoirdt,
Boon, Bossier, & Verstraete, 2004; De Kievit, & Iglewski, 2000; De Windt
et al., 2003). Quorum sensing probably increases the probability of pathogen
infection to their host successfully by expression and co-ordination of
these virulent factors to overcome the host’s immune system (Donabedian,
2003). Over the years, it has been reported that the disruption of bacterial
communication through probiotic, chemical administration, gene mutation,
etc., can significantly reduce the expression of virulence factors and may
provide novel avenues for disease control in aquaculture (Swift et al., 1999)
(Table 5.1).

5.3.7 BACTERIOPHAGE

Bacteriophages or phages are the viruses that lyse the specific bacterial host
cell and release new progenies (Al-Sum & Al-Dhabi, 2014). Harnessing this
precise killing capability of phages to control virulent bacterial pathogens
is called ‘phage therapy or phagotherapy.’ Phages employ the bacterial host
bio-machinery for all kinds of metabolic support such as different enzymes
and proteins to survive (Al-Sum & Al-Dhabi, 2014). As the natural environ­
ment is replete with loads of bacterial hosts, the occurrence of bacteriolytic
phages is natural. It can grow up to 107–108 virions g–1 in soil and approxi­
mately 107 virions ml–1 in water either fresh or saline environment (Park
et al., 2020; Ninawe et al., 2020). The life cycle of phages can be catego­
rized into two stages, the first is lytic (virulent) and the second, temperate
(dormant). For phagotherapy, lytic phages are preferred because of their
bacteriolytic activity. Although bacteriophages were discovered way back
at the beginning of the 19th century, the focus on their therapeutic potential
against bacterial diseases was limited because of the poor understanding
of phage life cycle and phage-bacteria interactions (Almeida et al., 2009).
166 Coldwater Fisheries and Aquaculture Management

However, the emergence of multi-drug resistant bacteria has substantially

encouraged researchers to explore the potential of phage therapy. The strain-
specific lytic phages can be employed as bioagents against a wide range of
bacterial pathogens. Owing to the specificity of phages, the probability of
disturbing the natural microbiota of aquatic environment or host inhabiting
beneficial bacteria will be null, which is very unlikely with the administra­
tion of chemical therapeutics such as common broad-spectrum antibiotics
(Fortuna et al., 2008). Nowadays, work associated with phage therapy
against bacterial pathogens in aquaculture has been accepted worldwide and
encourages researchers to explore the isolation, application, and efficacy of
phage therapy in different circumstances under various cultural conditions
(Table 5.1).

5.3.8 RNA INTERFERENCE (RNAI)

RNAi is a post-transcriptional gene silencing method that is evolutionally

conserved, where dsRNAs are introduced into cell to cause sequence-specific
degradation of desired homologous mRNAs (Almeida & Allshire, 2005).
The method is a natural defense strategy adopted by fungus, plants, and
invertebrates to counter the introduction of unwanted nucleic acids (viruses,
etc.). However, the technique has been also reported in several other eukary­
otic organisms revealing that the mechanism corresponds to control the
endogenous gene expression (Voinnet, 2002). In the RNAi machinery, the
post-transcriptional activity to degrade the cytoplasmic RNAs can be adopted
as key to advance towards antiviral strategy in aquaculture (Molnár et al.,
2005). Recent advancements in the application of RNAi-based technology
promise as a potential molecular tool for silencing particular genes involved
in the expression of virulence factor or validation of potential drug targets
in various organisms (Golding et al., 2006; Hong-Geller & Micheva-Viteva,
2010) (Table 5.1). In this perspective, RNAi-based silencing or molecular
scissoring could help in protection from viral infection.

5.3.9 GENE THERAPY

Gene therapy is a genome editing technology for precise genetic modifica­

tions, enabling knocking-out or introduction of a specific gene by engineered
nucleases (Li et al., 2019). There are four major ways of gene editing by
Biotechnological Interventions in Coldwater Aquaculture 167

programmed nucleases: transcription activator-like effector nucleases

(TALENs), zinc finger nucleases (ZFNs), mega-nucleases, and clustered
regularly interspaced short palindromic repeat-associated nuclease Cas9
(CRISPR-Cas9) (Li et al., 2019). These nucleases precisely recognize
specific sites in the genome and generate DNA double-strand breaks (DSBs).
Production of DSBs will lead to activation of natural DNA repair mecha­
nism, non-homologous end joining (NHEJ) which will cause direct rejoining
and ultimately lead to insertions or deletions of gene (Cox, Platt, & Zanhg,
2015). As a result of this mutation, the gene may lose undesired function
or gain desired property, this particular approach offers a novel perspective
in disease management in aquaculture as we can design the specific nucle­
ases and precisely achieve predictable substitution, insertion, or deletion of
desired genes (Osakabe & Osakabe, 2014) (Table 5.1).

5.3.10 ANTIMICROBIAL PEPTIDES (AMPS)

AMPs are a group of conserved oligopeptides (>5–100 amino acids (AAs))

that play a key role in innate immunity and are known for exhibiting broad-
spectrum antimicrobial responses. They are cationic, which can precisely
target bacterial cell membranes because of their negative charge and cause
the destruction of the organism by disruption of the cellular membrane and
impaired cellular functioning (Reddy, Yedery, & Aranha, 2004). Owing to
their broad-spectrum anti-microbial capacity, these molecules are serving
and may provide advanced alternative biodegradable anti-microbial agents in
aquaculture without any concerns towards the development of anti-microbial
resistance (Lai & Gallo, 2009) (Table 5.1).

5.3.11 BIOREMEDIATION

Bioremediation techniques are well established as environment-friendly,

efficient, and cost-effective tools in improving the quality of the aquatic
environment by reducing the waste and associated environmental damage
caused by the accumulation of various organic and inorganic wastes. Unlike,
terrestrial organisms, aquatic animals are comparatively more susceptible to
change in their surrounding environment. The aquatic environment serves as
a carrier of excretory products of aquatic animals and pollutants from nearby
sources, which makes aquatic organisms more prone to diseases caused by
168 Coldwater Fisheries and Aquaculture Management

different pathological agents under the influence of deteriorated environ­

mental conditions. So, biotechnology-based bio-remediation approaches
can resolve this circumstance to sustain the aquaculture activities. In biore­
mediation, the environmental pollutants are breakdown by friendly micro­
organisms particularly bacteria to produce a cleaner environment (Ghosh &
Pal, n.d.).
There are several biotechnological approaches that can be employed for
bioremediation purposes such as biosorption, biofilters, bioaugmentation,
etc. Biosorption is a rapid process of elimination of toxic metals ions from
wastewater by living or non-living (dried) biomass (Gavrilescu, 2009).
The most common example of biosorption is through the algal mass as it is
economically feasible and well effective in waste clean-up from polluted
sources (Brinza, Dring, & Gavrilescu, 2007). Biological or biological filters
allow swift diffusion of oxygen and provide proliferating ground for the
aerobic bacteria responsible for nitrification and denitrification of various
excretory products (Rijn, 1996). This type of system is commonly used in
recirculatory or zero water exchange system to remediate generated waste.
The captured waste is drained as thick effluents which later decomposed
(up to 90%) by the bacterial microflora present in the biological filter (Rijn,
2013). Bioaugmentation deals with the incorporation of naturally isolated
bacterial strains or certain enzymes or both in combination to enhance
the degradation process carried out by the different indigenous bacterial
flora. The most common example of augmenting agents belongs to genus
Bacillus as they are known for the production of different enzymes to
facilitate mineralization and breakdown of biomolecules (Singh, Khati, &
Chauhan, 2020).

5.3.12 DIAGNOSTICS

A modern and scientific biotechnology-based diagnostic test enables early

and precise identification of pathogens. The advanced epidemiology based
on recent nucleotide sequencing or whole genome sequencing enabled
tracing or pinpointing the origin of any infection. Over the years, numerous
modern technologies are being employed for aquaculture health manage­
ment in cold water practices including prevention as well as diagnostics in
aquaculture. These modern approaches are based on interactions of particular
biomolecules of the pathogenic agent such as nucleic acids, virulent protein,
Biotechnological Interventions in Coldwater Aquaculture 169

etc. Such biotechnological methods such as enzyme-linked immunosorbent

assay (ELISA) and polymerase chain reaction (PCR) have become a gold
standard for diagnosis and monitoring of diseases, globally. Molecular means
of diagnosis have significantly transformed disease diagnostic programs. The
technologies are much more reliable than conventional methods as they are
fast, specific, precise, and preceding cultivation of pathogenic microorgan­
isms especially slow-growing pathogens is not required (Subasinghe et al.,
2001). A number of antibody-based approaches are now being employed in
fish and shellfish diagnosis. Serology based methods, used for screening of
particular antibodies, have been a valuable indicator of previous exposure of
a specific pathogen and are being regularly used in clinical science (Camp­
bell & Landry, 2006). The major advantage of serology is the rapid and
precise detection which is not possible in culture-based or any other method
(Kaoud, 2015). Over the years, the advancement in molecular methods has
also led to the creation of global databases or repositories that allow the
verification and exchange of molecular data throughout the world which has
further strengthened the diagnosis as well as providing a global platform for
the identification of pathogens.

5.4 CONCLUSION

Aquaculture sector including cold water fisheries is suffering from severe

loss caused by diseases of diverse origins. Several strategies either prophy­
lactic or therapeutic were adopted to resolve the issue; however, the problem
remains the same. On the other side, biotechnology is growing rapidly and
has endowed us with several new tools and technology that availed capa­
bility to create new horizons in aquaculture, including health management.
Biotechnological research are rising at a very fast rate and have gained vast
importance in disease management in recent time. Some of the advanced
biotechnological approaches such as vaccination, anti-microbial peptides,
gene editing, etc., have shown promising results in managing the health of
cultured organisms over different agro-climatic conditions, globally. Biotech­
nology has enabled us with such tools and power which we can precisely
manipulate the genes and can create novel genotypes as per requirement.
However, there is a lot of scope in the optimization and commercialization of
these methodologies at different culture conditions so that these technologies
can be developed into farmer-friendly tool.
170 Coldwater Fisheries and Aquaculture Management

KEYWORDS

• aquaculture
• biotechnology
• diagnosis
• immune response
• infectious pancreatic necrosis virus
• nervous necrosis virus
• short-chain fatty acids

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CHAPTER 6

Coldwater Fish Diversity: Issues and

Sustainable Management in India
UDAI RAM GURJAR1, SUMAN TAKAR2, KHEMRAJ BUNKAR1,
DINESH MOHAN3, and N. N. PANDEY3
1
ICAR–Central Institute of Fisheries Education, Mumbai, Maharashtra,
India
2
TNJFU–Fisheries College and Research Institute, Thoothukudi,
Tamil Nadu, India
ICAR–Directorate of Coldwater Fisheries Research, Bhimtal,
3

Uttarakhand, India

ABSTRACT

Coldwater fish diversity is necessary for stabilizing aquatic ecosystems,

protecting general environmental water quality, and understanding the
inherent value of every species. Coldwater fish diversity holds a lot of
promise for sport fishing and ecotourism in hilly areas. The distribution of
fish species in Himalayan streams is influenced by water temperature, food
availability, flow rate, and substratum composition. From the northwestern to
the north-eastern Himalayan region and some portions of the Western Ghats,
India offers abundant and diverse coldwater fish resources for food, sport,
and ornamental value, spanning around 12 states. Upland rivers, streams,
high and low-altitude natural lakes, and reservoirs provide coldwater
fisheries resources throughout the Himalayan and peninsular regions. As a
result of natural processes and manmade actions, there has been a significant
change in climate, as measured by temperature and rainfall patterns, over a
long period of time. However, increasing anthropogenic activities, climate

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
180 Coldwater Fisheries and Aquaculture Management

change, and changing streamflow regimes are wreaking havoc on coldwater

resources and fisheries, resulting in lower overall productivity. The exten­
sive biomonitoring of all coldwater resources in time and space is required
to assess threats perspectives in relation to fish diversity for utilizing the
resources sustainable manner.

6.1 INTRODUCTION

Mountains are the highest repositories of biological diversity throughout

the world. The availability of cold waters considers mountain regions,
many of which are home to fish and are primarily supported by subsistence
fishing. India is also one of the world’s mega biodiversity hotspots, ranking
ninth in terms of freshwater mega biodiversity (Mittermeier et al., 1997).
Biodiversity is critical for ecosystem stability and general environmental
quality and for recognizing the intrinsic wealth of all species (Jena & Gopal­
akrishnan, 2012). The coldwater commonly refers to the aquatic ecosystem,
which keeps thermal and oxygen levels for well-being of mahseers, trout,
snow trout, and other species. The temperature mostly falls in the toler­
ance limits of trout and salmons, which belongs to the family Salmonidae
(<20°C) (Sarma et al., 2018). Remarkably, the mean temperature of water
in all Kumaon lakes during the summer season remains above the 20°C
thresholds; the maximum temperature rises well above 25°C (Johri et al.,
1989). The country is blessed with an abundance of diverse coldwater
fisheries resources dispersed across the Himalayan and peninsular areas in
the form of streams, upland rivers, reservoirs, and natural lakes (Table 6.1).
The potential coldwater areas in India include the Himalayas’ stretched of
about 2,500 km from east to west and 200–400 km from north to south,
covering an area of 5,94,400 km2. In the high altitude, there are about 8,243
km rivers and streams, 50,000 ha of reservoirs, both natural and manmade,
20,500 ha natural lakes and 2,500 ha brackish water lakes (Mahanta &
Sarma, 2010). Even though there are some of the limitations of coldwater
fisheries such as accessibility, tough terrain, and lack of a viable market,
the current achievement of growing aquaculture in mountainous regions
of India displays that fishermen in the rural villages can become the direct
beneficiaries of the implementation of low-cost aquaculture techniques. As
a result, they accomplish substantial development in their living standard.
As fisheries sector plays a significant role in supplying food and a source
of income to the mountain people. The Coldwater streams and rivers are
recognized for their high-velocity waterfall, deep pools, rapid flows, and
Coldwater Fish Diversity 181

substratum comprising bedrock, boulder, and sand. These enormous and

diverse water resources in the uplands support a diverse ichthyofauna, with
large populations of native and exotic, culturable, and non-cultivable fishes
(Sehgal, 1999; Mahanta & Sarma, 2010; Raveendar et al., 2018). The low
productivity and increased degree of resource seasonality and volatility
result in a unique aquatic diversity, which is generally susceptible to a wide
range of perturbations (Bhatt et al., 2012; Jena & Gopalakrishnan, 2012;
Singh et al., 2014; Raveendar et al., 2021).

TABLE 6.1 The State-Wise Aquatic Resources in the Himalayan Region of India
Length of Rivers/Canals (1,000 km) Water Bodies (hectare) State
27.78 30,000 Jammu and Kashmir
3.0 43,000 Himachal Pradesh
2.69 20,000 Uttaranchal
2.0 4,000 Arunachala Pradesh
5.60 10,000 Meghalaya
0.90 3,000 Sikkim
3.36 46,000 Manipur
1.20 17,000 Tripura
1.40 2,000 Mizoram
1.60 67,000 Nagaland

6.2 COLDWATER FISH DIVERSITY OF INDIA

The water resources of the Himalayan region support diverse ichthyofauna.

In the Indian context, 17% of fishes were reported from the mountain region,
establishing the main origin and development of biotic resources (Ghosh,
1997). The coldwater fisheries support 258 species which belong to 21 fami­
lies and 76 genera. Out of these, a total of 255 species have been reported
from North-East Himalaya, followed by the west and central Himalaya with
203 and the Deccan plateau with 91 species. The commercially important
coldwater fishes of India are shown in Table 6.2. The eastern Himalayan
region has a higher coldwater fish diversity as compared to the western
Himalayan region. Few of these species contribute to the capture fishery,
while others are cultured on the farm at various altitudes based on their
temperature tolerance limits (Table 6.3). The most common and important
fish species inhabiting the mountain waters of India are mahseer, snow trout,
and Indian hill trout. Mahseer is a group of sport fish that predominantly
182 Coldwater Fisheries and Aquaculture Management

TABLE 6.2 Common Cold-Water Fishes of India

Neolissochilus hexagonolepis Tor putitora (Hamilton, 1822)
(McClelland, 1839)
Onchorhynchus mykiss (Walbaum, 1792) Schizothorax richardsonii (Gray, 1832)
Barilius bendelisis (Hamilton, 1807) Raiamas bola (Hamilton, 1822)
Labeo dyocheilus (McClelland, 1839) Labeo gonius (Hamilton, 1822)
Labeo pangusia (Hamilton, 1822) Glyptothorax spp. (Blyth, 1860)
Chagunius chagunio (Hamilton, 1822) Bangana dero (Hamilton, 1822)

TABLE 6.3 Common Indigenous and Exotic Cold-Water Fishes of India

Groups Species
Indigenous
Mahaseer Tor tor, T. putitora, T. khudree, T. mosal, T. mosal mahanadicus,
T. mussulah, T. nelli, T. progeneius, Neolissochelius hexagonolepis,
N. wynaadensis, and Naziritor cheylinoides
Snow trout Schizothorax richardsonii, S. kumaonensis, S. progastus,
S. plagistomus, S. molesworthi, Schizothoraichthys esocinus,
S. curvifrons, S. niger, S. micropogon, S. labiatus, Lepidopygopsis
typus, Ptychobarbus conirostris, and Diptychus maculatus.
Indian hill trout/ Raiamas bola, Barilius bendelisis, B. bakeri, B. vagra, B. barila and
Barils B. gatensis.
Minor carps Labeo dero, L. daycheilus, L. gonius, L. pangusia, Osteobrama
belangeri, Garra lamta, G. gotyla, G. hughi, Chagunius chagunio,
Crossochelius latius, Semiplotus semiplotus and Puntius
ophicephalus
Catfishes Glyptothorax pectiniopterus, Pterocryptis wynaadensis,
Pseudechenesis sulcatus, Sperata sp., Heteropneutes sp.,
Mastacembelus sp.
Loaches Botia birdil, B. almorhae, Nemacheilus rupecola, N. arafi,
N. montanus, N. fascimaculatus, N. multifasciatus, Triplophysa
choprai, T. gracilis, T. griffthi, T. ladacensis, T. microps and T.
tenuicauda
Exotic
Trout Salmo gairdneri gairdneri, Salmo tutta fario, S. lavensis, S. salar,
Onchorhynchus mykiss, and O. nerka
Tench Tinca tinca
Carps Ctenopharyngodon idella, Hypophthalmichthys molitrix, Carassius
carassius, Cyprinus carpio, Cyprinus carpio communis (scale carp),
Cyprinus carpio nudus (leather carp) and Cyprinus carpio specularis
(mirror carp).
Coldwater Fish Diversity 183

belongs to the genus Tor, also conjointly consisting of some species from the
genus Neolissochilus (Neolissochilus hexagonolepis, chocolate mahseer).
Schizothoracines (family Cyprinidae and sub-family schizothoracinae) are an
economically important group of coldwater fishes throughout the Himalayan
zone, particularly in Kashmir. Out of concerning 258 cold water fish species
(both native and exotic) described from Indian uplands, various authors have
been identified 17 members of the snow-trout (locally called Sela, Rasella or
Asela in Uttarakhand, Koushargad in Kashmir, and Gulgali in Himanchal)
(Sehgal, 1999, 2012; Sunder et al., 1999). Among these, 10 species belong
to genus Schizothoraichthys (species being S. esocinus, S. niger, S. longipin­
nias, S. curvifrons, S. micropogon, S. hugelli, S. planifrons, S. labiatus, S.
progastus, S. nasus), two species in the genus Schizothorax (S. kumaonensis
and S. richardsonii) and one each in the genera Gymnocypris (biswasi),
Diptychus (maculates), Schizopygopsis (stoliczkae), Lepidopygopsis (typus),
Ptychobarbus (conirostris). Lapidopygopisis typus is exclusively found
in Periyar lake (Western ghats), Kerala. All other species are found in the
Jammu and Kashmir region. The most widespread and commonly dispersed
species in the Himalayan region is Schizothorax richardsonii (Sehgal, 1999).

6.3 MAJOR ISSUES AND THREATS TO COLD-WATER FISH

DIVERSITY

The aquatic habitats are facing major threats to biodiversity and environment
constancy, necessitating a variety of tactics and priorities to tackle this crisis
(Jena & Gopalakrishnan, 2012; Singh et al., 2014). Regardless of the critical
role of fish biodiversity in an ecosystem, all fish species that collectively
contain fish biodiversity suffer unique risks and threats. The risks could be
natural or anthropogenic, or both with interconnected impacts. Such threats
are diverse, resulting in habitat changes, reduction of resources, shrinkage
of habitat area, loss of germplasm, and so on as a result of reduced water
discharge in rivers, overfishing, siltation, pollution of water bodies, the
introduction of exotic species and climatic variability, among other things.
The following serious threats to the diversity of coldwater fishes are:
1. Overfishing/Overexploitation: Overexploitation of any species
leads to loss of genetic diversity as well as a decrease in the relative
abundance of species as individuals and/or groups of interrelated
species. The size of the population can be decreased due to the distur­
bances in sex composition and age structure (Sarkar et al., 2008).
184 Coldwater Fisheries and Aquaculture Management

Overfishing also has an effect on heritable life-history traits such as

sexual maturity, age, and growth. Proficient fishing gear removes
fast-growing larger aquatic individuals. Subsequently, the proportion
of slow-growth individuals increases, while the mean size of indi­
viduals in a population drops. In its report on fisheries, the National
Commission on Agriculture (NCA, 1976; Nautiyal, 2013) specified
that there was an overall decrease in the mahseer fishery because of
indiscriminate fishing of juvenile and brooder fishes. The overexploi­
tation of resources is not only a single pressure to decline the mahseer
population but also caused by an increase in development activities,
particularly the rising number of hydroelectric-cum-irrigation proj­
ects, which leads to fragments disturb its natural habitats (Nautiyal &
Singh, 1989). Therefore, the mahseer population has dropped rapidly
in several locations (Kashmir, Nainital); over the last few decades,
it has been designated as ‘threatened’ in India (Khan & Sinha, 2000;
Mahanta & Sarma, 2010; Akhtar et al., 2014). In spite of this, the use
of destructive fishing methods such as bleaching powder, dynamite,
damming, and pesticides which result in mass mortality of brooders
and juveniles has continued unabated, particularly in small streams,
which are the spawning grounds of mahseer (Nautiyal, 1994), hence
adversely affecting the rates of recruitment.
2. Introduction of Exotic Species: The introduction of non-native
aquatic species, mainly fishes, led to a global alarm because it caused
a slew of issues, including the extinction of native species (Singh &
Lakra, 2011; Singh et al., 2013). The exotics compete with native
species for environs and food (Singh et al., 2013). They may also
predate on indigenous fishes, transmit new parasites and diseases,
produce hybrids, cause genetic ‘erosion’ of native species, and
degrade the physicochemical characteristics of aquatic environments
(Singh et al., 2013). All of this could lead to a loss of biodiversity
(Singh & Lakra, 2011). Numerous studies have been conducted on
the disastrous effects of alien imports on ecosystems and species
(Singh & Lakra, 2011; Singh et al., 2013). The possible hazards
influence the standard or amount of biodiversity and the economic
aspects of the local people, which rely upon aquatic environments
for nourishment (Singh & Lakra, 2011). Over 300 exotic fish species
have been introduced into our country at various times for trial
aquaculture, mosquito control, aquarium keeping, and sport fishing
(Kumar, 2000; Singh & Lakra, 2011). Numerous exotic fishes are
Coldwater Fish Diversity 185

now well recognized in India’s natural water resources. The intro­

duction of Cyprinus carpio var. specularis into Loktak lake and Dal
lakes have influenced the native population of Osteobrama belangeri
and Schizothorax sp., respectively (Singh & Lakra, 2011). After the
introduction of silver carp significantly reduced the population of
indigenous mahseer and catla in Govind Sagar reservoir (Singh &
Lakra, 2011).
3. Habitat Destruction/Modification: Anthropogenic activities are
considered to be the primary cause of habitat alteration, which is
directed to the depletion of aquatic resources (Takar & Gurjar, 2020).
Natural disasters such as floods and landslides, biological invasion,
changes in environmental flow, and other factors have significantly
impacted the Himalayan coldwater ecosystem, posing a serious
threat to coldwater fish diversity. Reduced ichthyofaunal diversity
in Asian countries is attributed to pollution, increased flow altera­
tion, sedimentation, water diversion, and new species (Nguyen &
De Silva, 2006; Singh & Lakra, 2011; Singh et al., 2013, 2014).
Because of recurrent floods and the resulting ecological degradation,
the original wild stock of valuable fish species such as snow trout,
mahseer, minor carps, and others in coldwater rivers and lakes has
been greatly affected in recent years.
4. Climate Change: Climate variability is an important aspect in
regulating the distribution and quantity of aquatic animals as well
as the structure of the ecosystem (Jena & Gopalakrishnan, 2012).
The impact of climate change on fish is determined by the amount of
the shift as well as the sensitivity of specific species or ecosystems.
The influence of temperature changes caused by climate change will
impact the biological capabilities of aquatic organisms, as most of
them are cold-blooded in nature. Coldwater fishes are key ecological
indicators for climate change in temperate zones because they are
very sensitive to variations in water temperature and other envi­
ronmental parameters. Many Himalayan coldwater fish species are
endangered; therefore, they may be the most vulnerable to climate
change or warming. They currently exist at the upper limit of their
thermal tolerance range in many regions, and ecological models
predict that there will be significant losses of temperate fish species
as climate change reduces fish habitat and variety (Mohseni et al.,
2003).
186 Coldwater Fisheries and Aquaculture Management

6.4 METHODS FOR CONSERVATION AND SUSTAINABLE

MANAGEMENT OF COLD-WATER RESOURCES

Coldwater fish diversity is under threat due to various factors or anthropogenic

activities, such as resources overexploitation, habitat degradation, exotic
species introductions, and others. Therefore, quick action and strategies
for protecting aquatic biodiversity are necessary to conserve preserve
these threatened ecosystems and species for coming generations (Jena &
Gopalakrishnan, 2012). All aquatic ecosystems face grave risks to ecosystem
stability and biodiversity. Generally, aquatic conservation techniques should
encourage sustainable development and management of biological resources
in ways to conserve ecosystems and habitats. Several strategies and priorities
have been presented for coldwater fish diversity conservation in order to
make it effective are discussed in subsections.

6.4.1 IN-SITU CONSERVATION

The definition of in-situ conservation is “the conservation of ecosystems and

natural habitats and the maintenance and recovery of the feasible popula­
tion of species in their natural habitat and, in the case of domesticated or
cultivated species, in the nature or habitat where they have developed their
distinguishing properties.” This conservation approach integrates research on
fish and habitat diversity, life history features, habitat utilization, and human
intervention. The fish breeding grounds need to special safeguard by asserting
them as ‘no-fishing zone’ or ‘protected area (PA). In-situ conservation is
accomplished by designating certain places as PAs and designating them as
Wildlife Sanctuaries, Biosphere Reserves and National Parks. PAs conserve
threatened, rare, depleted, or endangered species and populations and protect
their environments.

6.4.2 EX-SITU CONSERVATION

In this approach, species are protected outside of their natural environments

by keeping populations in genetic resource and breeding centers or preserving
in gene banks, germplasm banks, and gamete storage. As in the case of fishes
and other animals, fast freezing of gametes to ultralow temperatures was
discovered to be fruitful. The storage of fish sperm (milt), eggs, and embryos
Coldwater Fish Diversity 187

without loss of viability is significant for conservation as well as nourishing

of aquaculture.

6.4.3 AQUA RANCHING

This method is designed to enrich resources by seed stocking in open waters

with desired aquatic organisms and providing suitable manmade shelters for
the animals to protect themselves from natural threats, allowing them to grow
to a size where predation and juvenile mortality are significantly affected
decreased. This approach is used to replenish the depleted fish stocks of lakes,
reservoirs, rivers, streams, and estuaries as a result of captive breeding and
hatchery programs (Rout et al., 2007). Juvenile fish are frequently removed
from their original habitat and allowed to mature sexually and reproduce
within the safe limits of an aquaculture or lab setting, with the young ones
raised in captivity being returned or ranched to natural resources (Lakra et al.,
2007). The DCFR has stocked the fingerlings of golden mahseer in various
rivers and lakes of different hill states of Arunachal Pradesh, Uttarakhand,
Sikkim, Meghalaya, and Assam. The Directorate has also given thousands of
golden mahseer seeds to the Madhya Pradesh and West Bengal Departments
of Fisheries for their reservoir/wetlands stocking augmentation program.
The DCFR stocked golden mahseer at Shyamlatal Lake in Kumaon, India, in
2001, and they have thrived, growing to mature sizes, and becoming a tourist
attraction. On 4th November 2020, DCFR organized a seed ranching program
of endangered golden mahseer in three different lakes Bhimtal, Naukuchiatal,
and Sattal lakes of the Kumaon region (Figure 6.1). On this occasion, 10,000
mahseer fingerlings were released into each lake for the sustainability of
these resources. However, as a step forward for other diminishing coldwater
fish species, stock enhancement programs for other declining species should
be prioritized.

6.4.4 CONSERVATION AQUACULTURE AND CAPTIVE BREEDING

Conservation aquaculture is becoming more important in the restoration

of threatened/endangered fishes (Moyer et al., 2009). The use of captive
multiplication to sustain endangered species or populations and conserve
local traits in the face of severe loss is known as conservation aquaculture
(Schreier et al., 2012). It entails aquaculture in endangered fish population
188 Coldwater Fisheries and Aquaculture Management

restoration programs by boosting the threatened species’ effective popula­

tion size (Ne).
Captive breeding programs have become a popular method for compen­
sating for falling fish populations while also supplementing and increasing
the yields of wild fisheries. In this regard, the Directorate of Coldwater
Fisheries Research (DCFR) has developed breeding and seed growing tech­
niques for golden mahseer and a regular producing golden mahseer seeds for
aquaculture and conserving the resources.

FIGURE 6.1 Ranching of mahseer fingerlings into the Bhimtal lake (ICAR-DCFR, Bhimtal).

DCFR has further developed flow through mahseer hatcheries at Iduli

fish farm in Roing, Arunachal Pradesh; Eco-camp, ABACA, Nameri
National Park, Tezpur, Assam, and Bagua fish farm in Sikkim to meet
the entire need for mahseer seeds. The NBFGR developed techniques for
captive breeding and larval rearing of various fishes, which have been
extremely successful (Jena et al., 2011). White sturgeon populations have
been restored using conservation aquaculture tools (Schreier et al., 2012),
and the same technology has also been used to conserve Pacific salmon.
Coldwater Fish Diversity 189

Similarly, in India, conservation aquaculture could be an efficient tech­

nique for the conservation of coldwater fish species whose populations are
declining.

6.4.5 SUSTAINABLE FISH HARVEST

Harvesting fisheries resources from conventional fishing grounds is requisite

to regulate and manage appropriately. To the sustainable management of
stock, brooders, and juveniles should be protected. The strict mesh size
regulations must be imposed to conserve or protect juvenile resources. The
use of explosives or poison to kill fish needs to be strictly prohibited (Rout
et al., 2007).

6.4.6 MASS AWARENESS

Increasing awareness among the public is one of the most imperative ways
to sustain and manage aquatic resources and biodiversity. It is commonly
understood that the success of biodiversity protection is ultimately dependent
on public understanding of the various ecological, cultural, socio-economical,
nutritional, recreational, esthetic, pharmaceutical, and other services avail­
able to people. Hence, protection of diminishing diversity is the account­
ability of each citizen. This can be achieved by implementing incentive
schemes, educational initiatives, and volunteer monitoring programs. The
DCFR, Bhimtal, undertook various awareness programs to protect the golden
mahseer, which raised public knowledge and demonstrated the concept of
“feeling of belongingness” and made local people understand the need for
conservation. The respective state governments should also step forward to
organize awareness programs and activities with local residents’ participation
to reach out to a larger public in the coldwater states.

6.4.7 HABITAT RESTORATION/MITIGATION MEASURES

Habitat restoration is a powerful strategy for reversing ecological damage.

Situation-specific habitat restoration programs want to be carried out based
on the type of degradation. Depending on the type of degradation, situation-
specific habitat restoration programs should be implemented that can be
restored by habitat restoration management (Ali, 2010). For example, in the
190 Coldwater Fisheries and Aquaculture Management

event of siltation/sedimentation, deforestation efforts must be immediately

halted, along with a massive afforestation campaign in erosion-prone catch­
ment areas. Polluted water bodies require immediate attention, which can
be achieved by maintaining strict adherence to pollution control legislation.
Due to recurring floods and resulting ecological degradation, the original
wild stock of valuable fish species such as mahseer, trout, snow trout, minor
carps, and others in cold water lakes and rivers has considerably diminished
in recent years. For coldwater fish diversity management, several manage­
ment measures such as the development of riparian buffer zones and the
restoration of natural flow patterns and discharge commands will be used.

6.4.8 REGULATORY/LEGAL MEASURES

To protect biodiversity and avoid the exploitation of ecologically sensitive

areas under the Wildlife Protection Act of 1972 and the Environmental
Protection Act of 1986, the Indian government established a network of PAs.
Since 1st February 1982, India has been a signatory member of the Ramsar
Convention for the conservation of wetland resources. In order to conserve
and maximize the use of its bioresources, India adopted the Biological
Diversity Act in 2002. The Indian Fisheries Act, 1897 (as amended in 1956)
is a revolutionary moment for fish protection. Aside from requiring that
mesh sizes, gears, and fishing or closed seasons be observed, the legisla­
tion also forbids the use of poisons or explosives to kill fish in any water.
However, the Act’s enforcement is minimal, particularly in coldwater states.
As a result, state governments in the coldwater region should ensure that the
act is effectively enforced to ensure the long-term management of coldwater
fishes. Furthermore, the act is quite old and needs to be updated to include
all necessary legal issues in order to safeguard the states’ fish germplasm
resources, with special provisions for coldwater fisheries.

6.5 CONCLUSION

Coldwater fishery resource conservation and management require concerted

efforts that integrate cultural, capture fisheries, and environmental
monitoring. The expanding population and economic expansion will
continue to put pressure on natural resources, resulting in biodiversity loss.
Advanced environmental legislation with stringent enforcement measures
must be adopted to conserve fish biodiversity and habitat. However, greater
Coldwater Fish Diversity 191

biodiversity monitoring, risk assessment, and a more structured approach to

biodiversity protection are all critical parts of effective risk management (Jena
& Gopalakrishnan, 2012). Extensive biomonitoring of all coldwater resources
in time and space is required to assess danger perspectives concerning fish
diversity. Because the country’s upland regions are vulnerable in nature, they
must be conserved and used sustainably.

KEYWORDS

• biomonitoring
• climate change
• coldwater
• fish diversity
• Himalayan region
• in-situ conservation
• upland rivers

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CHAPTER 7

The Zebrafish: A Trending Model for

T-Cell and Thymic Development Along
with Prevailing Challenges
SNEHA SABU1, SOFIA KHANAM2, and A. JOTHILIN SUBITSHA1
MSc, Department of Microbiology, CMST, Manonmaniam Sundharanar
1

University, Rajakkamangalam, Kanyakumari, Tamil Nadu, India

2
MPharm, Department of Pharmacology, Calcutta Institute of
Pharmaceutical Technology and AHS, Uluberia, Howrah,
West Bengal, India

ABSTRACT

Recently, the Danio rerio (zebrafish) has become a trending genetic model
for the evolution of vertebrates, offering new research opportunities that
are not readily available in other model organisms. The research with D.
rerio has elucidated the molecular and cellular mechanisms underlying these
biological processes. T-cell and thymic growth are strongly maintained
mechanisms that have been throughout the evolution of vertebrates. D.
rerio, like mammals, have an adaptive immune system composed of B
lymphocytes, as well as the αβ and γδ lineages of T cells that form in the
thymus. Zebrafish are a highly recommended research model since it has
plenty of advantages over others such as easy and convenient manipulation
of genome, rapid embryonic development, less generation time, external
fertilization, etc. While using zebrafish as a prominent research tool, it is
significant to consider the prevailing challenges. This review chapter points
out identifying the similarities and differences between the immunobiology
of D. rerio and mammalian T-cells, accentuating the benefits of using D. rerio

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
196 Coldwater Fisheries and Aquaculture Management

as a genetic model to discover T-cell and thymic development, application of

zebrafish as a promising research tool in the biomedical sector and prevailing
challenges as well.

7.1 INTRODUCTION

The zebrafish is a rapidly growing and very important model organism for
understanding vertebrate developmental biology. The brain, digestive tract,
muscle, vasculature, and innate immune system of zebrafish are all quite
similar to those of humans (Gore et al., 2012; Kanungo et al., 2014; Kalueff
et al., 2014; Guyon et al., 2007; Weinstein, 2002; Lieschke et al., 2001; Zhao
et al., 2015). Furthermore, 70% of human disease genes share functional
similarities with zebrafish genes (Santoriello et al., 2021). Mammals and
zebrafish have similar blood cells and use similar molecular pathways to
regulate the production of blood cells such as thymocytes (Burns et al., 2005;
Rhodes et al., 2005; Bajoghli et al., 2009). T-cell development always takes
place in the thymus’ epithelial microenvironment (Boehm, 2011).
The zebrafish (Danio rerio) has lately emerged as a significant genetic
model of vertebrate development, opening up new areas of investigation not
available in other model species. Although invertebrate genetic models have
provided enormous insight into the mechanisms that control developmental
pathways, Caenorhabditis elegans, and Drosophila melanogaster do not have
the same blood-cell lineages as vertebrates and lack an adaptive immune
system, making dissection of lymphocyte development impossible in these
organisms (Chitwood, 1974; Schulenburg et al., 2004; Evans et al., 2003).
Furthermore, forward genetic screenings in zebrafish are substantially less
time-consuming than those in mice (Appleby & Ramsdell, 2003), and
zebrafish have conserved genetic programs that underlie vertebrate blood
formation (Thisse & Zon, 2002). Zebrafish have both T and B cells, just like
mammals, birds, and reptiles (Hansen & Zapata, 1998; Trede et al., 2001;
Trede & Zon, 1998), which enables the investigation of these lymphoid-
cell populations in this model. Like mammals, zebrafish have an adaptive
immune system that includes B lymphocytes as well as T-cells from both the
lineages that mature in the thymus. Furthermore, the molecular pathways
underpinning T-cell development in zebrafish appear to be strikingly similar
to those in humans. As a result, discoveries from the zebrafish model will be
very relevant to similar mechanisms in humans.
Several aspects of T-cell and thymic development have been found to
be retained from teleosts to mammals in investigations conducted over the
The Zebrafish: A Trending Model for T-Cell and Thymic Development 197

last two decades. Work with zebrafish (Danio rerio) in particular has given
information on the cellular and molecular mechanisms that underlie these
biological processes. The simplicity with which these species may be imaged
non-invasively in vivo allows for direct viewing of all events connected with
these processes, which are technically challenging in mice. In this study, we
will define the role of zebrafish in T-cell development and thymus organo­
genesis, as well as their applications and challenges.

7.2 ROLE OF ZEBRAFISH AS A RESEARCH MODEL

The norms to choose animal models for biomedical research are directly
connected to the final goal line of the research. The usage of zebrafish
as a research model was recommended by George Streisinger and fellow
workers at the University of Oregon, who launched the contemporary era
for zebrafish in the field of biomedical research (Clark & Ekker, 2015).
Zebrafish are prevalent animal models since they have abundant benefits
than other species. The utmost valuable characteristics of zebrafish are
an entirely sequenced genome, trouble-free manipulation of its genome,
elevated fertility, quick generation period (about 3 months), quick embryonic
growth (24 hr.), and exterior fertilization. The luminous zebrafish embryo
allows investigations of the various developmental phases initiated from the
initial phase of embryogenesis. Furthermore, zebrafish embryos form whole
organ systems, together with the heart, intestine, and blood vessels in 48 hr.
after reproduction. More than 10,000 mutants in protein-coding genes were
created (Howe et al., 2013) also several transgenic lines of zebrafish have
been accomplished to study human diseases. The obtainability of numerous
species of zebrafish is an additional significant benefit of this species.
Besides, it is also very inexpensive to maintain a huge number of zebrafish in
a comparatively small capacity of laboratory space. Though zebrafish need
comparatively easy management, distinct care must be paid to guaranteeing
a healthy diet and suitable water quality to enhance fish health and growth.
Several laboratories use zebrafish to investigate human diseases, together
with neural disorders, tumors, infectious diseases, cardiac diseases, kidney
diseases, diabetes, loss of sight, deafness, intestinal diseases, hematopoiesis,
and muscle disorders. Zebrafish are too significant for emerging new
treatments or screening novel drugs to treat or avoid human diseases.
Although zebrafish are a vital research model, they have some boundaries,
together with the variation of some organs like the respiratory system and the
reproductive system. Therefore, it is hard to practice zebrafish for disorders
198 Coldwater Fisheries and Aquaculture Management

in respiration or reproduction in humans. Furthermore, since zebrafish live in

an aquatic habitat, screening of some water-solvable medicines in zebrafish
is another restriction (Figure 7.1).

FIGURE 7.1 Zebrafish as a research model.

7.3 T-CELL DEVELOPMENT IN ZEBRAFISH

For in vivo genetic studies of human development Zebrafish is becoming an

influential craniate model (Amatruda & Zon, 1999; Lieschke & Currie, 2007;
Trede et al., 2004). Zebrafish and warm-blooded creatures share compara­
tive blood cells and utilize common molecular pathways to control the blood
cell and thymocytes (Burns et al., 2005; Rhodes et al., 2005; Bajoghli et
al., 2009). In zebrafish, the primary authoritative HSCs originate from the
ventral wall of the dorsal aorta at around 28–30 hours post-fertilization (hpf),
at that point move to the caudal hematopoietic tissue, and finally domestic to
the thymus and the kidney, where T-cell growth and mature hematopoiesis
occur, correspondingly (Murayama et al., 2006). T-cell progenitors marked
by ikaros expression show up in the thymus by 3 days post-fertilization (dpf)
(Murayama et al., 2006). As in warm-blooded creatures, zebrafish T-cell
development gives rise to two T lineages, γδ, and αβ and is escorted by the
reordering of four T-cell receptors (TCR) loci (TCRα, TCRβ, TCRγ, and
TCRδ) (Schorpp et al., 2009). As in warm-blooded creatures, TCR genes in
The Zebrafish: A Trending Model for T-Cell and Thymic Development 199

zebrafish are accumulated by a V(D)J recombination technique that is reliant

on the recombination activating genes, rag1 and rag2 (Willett et al., 1997).
The T lymphocyte-specific nonreceptor tyrosine kinase lck is similarly found
within the lymphoid precursors and developing T lymphocytes within the
bilateral thymic lobes of zebrafish (Trede et al., 2004; Langenau & Zon,
2005). A current investigation summarizes extra genes that play a vital role
in zebrafish and serve as markers of T lymphocyte development (c-myb,
ikaros, rag1, rag2, lck, TCRα, TCRβ, TCRγ, TCRδ, IL7R, jak3, ccr9a, ccr9b,
zap70, gata3, runx1, foxn1, etc.) (Ma et al., 2013).

7.3.1 STAGES IN T-CELL DEVELOPMENT

In this segment, we parted the development of T-lymphocytes into four wide

stages (Figure 7.2).
• The entry of lymphoid progenitors into the thymus;
• Commitment of thymocytes;
• Choice of functional also non-self-reactive thymocyte; and
• Egress of thymocytes.
In the initial step, lymphoid progenitors inhabit the thymus; the second
spans T-cell commitment and the divergence of αβ and γδ T-cells lineages.
The third step is the choice of functional also non-self-reactive T-cells. Finally,
developed T-cells exit the thymus into the periphery (Bajoghli et al., 2019).

FIGURE 7.2 The development of T-lymphocytes.

200 Coldwater Fisheries and Aquaculture Management

7.4 THYMIC DEVELOPMENT IN ZEBRAFISH

The thymus is one of the lymphoid organs where thymocytes differentiate

and mature, which is present in all vertebrates (except for jawless fish like the
lamprey). It plays an essential role in the development of T-cells repertoire,
which is required for immunocompetency (Boehm & Bleul, 2007; Boehm
et al., 2012). The formation of the thymic primordium, its colonization by
lymphocyte precursors, the expansion of thymocytes, and the histologic
regionalization of this organ have all been proposed as four successive
processes in the thymus histogenesis (Lam et al., 2002). The structure and
function of the thymus in mammals change over the lifespan (Peel & Belov,
2017). Thymus development in humans begins during embryogenesis and
achieves its maximal size and output of naive T-cells throughout early life
(Palmer, 2013). Following that, the thymus starts a period of slow decline
marked by remodeling of the histoarchitecture, shrinkage, and the production
of naive T-cells (Manley et al., 2011; Rezzani et al., 2014). The origination
of thymus involution was formerly thought to occur during puberty, in
conjunction with increased levels of sex steroid hormones and decreased
growth hormone synthesis. Human thymus regression is generally widely
considered to begin during childhood; however, it may become more visible
throughout puberty (Haynes et al., 2000; Manley et al., 2011).
Thymus function comes at a high price in terms of physiological
expenses. This is due to the thymus’s stringent selection processes because
the output of T-cells to the periphery accounts for <10% of all produced
thymocytes (Quaglino et al., 2014). The loss of thymus function may be
bearable for aged organisms after the development of peripheral T-cell
populations, which happens throughout early life because peripheral T-cells
are long-lived and can undergo clonal growth (Kernen et al., 2020). Shanley
et al. (2009) also addressed the potential life cycle tradeoffs associated with
a strong immunological response. To assist successful reproduction, natural
selection may favor a robust immune response early in life and a finely
regulated inhibition of immune activities later in life.
In teleosts, the thymus matures as a bilaterally paired organ near the
operculum on the dorsal side of the branchial cavity. The expression of foxn1
(Schorpp et al., 2002; Hess & Boehm, 2012; Bajoghli et al., 2009), a tran­
scription factor necessary for the development of TECs (thymic epithelial
cells) (Boehm, 2008), determines when thymopoiesis begins in zebrafish
(Figure 7.3). Foxn1’s role in defining the thymic anlage has been preserved
throughout evolution in jawed vertebrates (Swann, 2014). In zebrafish, foxn1
regulates the expression of two significant factors for T-cell development,
The Zebrafish: A Trending Model for T-Cell and Thymic Development 201

viz. the delta like 4b (dll4b) and the c-c chemokine ligand 25a (ccl25a). The
former gene is required for lymphoid progenitor attraction into the thymus,
while the latter gene is required for lymphoid progenitor specification into
T-cells (Koch et al., 2008; Hozumi et al., 2008; Ferrero et al., 2013). Teleosts,
unlike mammals, have TECs that express the foxn4 paralog gene from the
larval stage forward (Swann, 2014). On the other hand, foxn1-deficient in
mice and do not develop thymus (Schlake et al., 1997). Because the ortholog
gene does not express in the murine thymus, it has been suggested that foxn4
thymic expression may have been lost during vertebrate evolution. This
is further confirmed by the finding that ectopic foxn4 expression in TECs
cannot compensate for foxn1 deficiency in nude mice (Swann, 2014).
From one-week post-fertilization (wpf) to four weeks post-fertilization
(wpf), the thymus grows from a small spherical shape and develops into a
more complicated conical shape (Lam et al., 2002). A comparable change in
thymus form has been seen in sharp snout seabream (Romano et al., 1999),
showing that this process is conserved among teleosts. The adult thymus of
teleosts has a cortical and medullary organization similar to that of mammals,
however, it can vary from one lobule to numerous lobules (Flajnik, 2018;
O’Neill, 1989). Each side of the body has only one thymic lobule in zebrafish.
Thymic compartmentalization occurs later in zebrafish juveniles, between 2
and 3 wpf (Lam et al., 2002). During adolescence, the thymus reaches its
peak output, following which it begins to decline with age. In mammals,
this age-related thymus involution is well-known (Boehm & Swann, 2013;
Lynch et al., 2009). This mechanism starts at 15 wpf in zebrafish (Lam et
al., 2002). The point at which the thymus begins to shrink in teleosts, on the
other hand, varies greatly (Deanesly, 1927). The thymic structure in some
teleosts can even last their entire lives (O’Neill, 1989).

FIGURE 7.3 The timeline of zebrafish T-cell and thymic development.

7.5 APPLICATION OF ZEBRAFISH AS A RESEARCH MODEL

The criteria for selecting animal models for biomedical research are linked
to the research’s ultimate purpose. George Streisinger and colleagues at the
University of Oregon proposed using zebrafish as a biomedical model, which
202 Coldwater Fisheries and Aquaculture Management

ushered in a new age for zebrafish in biomedical research (Clark & Ekker,
2015). Zebrafish are widely used as animal models due to their multiple
benefits over other species. A fully sequenced genome, facile genome
manipulation, high fecundity, short generation period (approximately 3
months), rapid embryonic development (24 hours), and external fertilization
are the most favorable traits of zebrafish. The transparent zebrafish embryo
allows researchers to examine several developmental phases, beginning with
the initial stages of embryogenesis. Furthermore, after 48 hours of fertiliza­
tion, zebrafish embryos create complete organ systems, including the heart,
gut, and blood arteries. More than 10,000 protein-coding gene mutants have
been generated as well as multiple zebrafish transgenic lines for research
into human diseases (Howarth et al., 2013).
In recent years, the Zebrafish model has indeed been frequently used in
animal and human health investigations, as well as aquaculture. Regardless
of the fact that rodents are the most often used study model in the world, the
zebrafish (Danio rerio) model has grown in popularity among academics
in recent decades. It adheres to the 3Rs (replacement, reduction, and
refining) principle, as mandated by a number of national and international
regulatory agencies. Furthermore, as compared to the models of more estab­
lished animals, the usage of the zebrafish model saves time and resources.
When compared to in vitro outcomes, it also delivers more information
and predictability (MacRae & Peterson, 2015). As a result, utilizing the
zebrafish model, it is possible to replace and limit the usage of mammals in
research while also addressing issues connected to their welfare. Further­
more, zebrafish are utilized as confirmatory models of previously obtained
favorable results, allowing researchers to fine-tune their findings (Bailone
et al., 2019).
Another significant advantage of zebrafish is the availability of various
strains. Furthermore, keeping a high number of zebrafish in a limited quan­
tity of laboratory area is quite cost-effective. Many of these labs research
human diseases such as metabolic disorders, brain disorders, cancer, infec­
tious diseases, cardiovascular diseases, kidney diseases, diabetes, blindness,
deafness, digestive diseases, hematopoiesis, and muscular problems using
zebrafish (Taeme et al., 2019), For aquaculture species, toxicity, and drug
discovery (Khan & Alhewairini, 2018). Gene Expression Analysis, Forward
Genetic Screens, Transgenesis, Protein Overexpression, Gene Knockout
in Zebrafish, Cell Transplantation and Cell-Cell Interactions, and Testing
Chemical Compounds in Zebrafish Embryos are all employed as model
organisms in developmental pediatric research (Veldman & Lin, 2008).
The Zebrafish: A Trending Model for T-Cell and Thymic Development 203

7.5.1 ZEBRAFISH AS A MODEL FOR HUMAN DISEASES

Except for the lungs and prostate and mammary glands, most tissues and
organs in humans and zebrafish are identical. The cloning of mutated genes
screened for specific symptoms in zebrafish is analogous to the cloning of
mutated genes screened for specific phenotypes in humans, therefore it can
be used as a model for human disease and to explore underlying mechanisms.
The first human disease discovered using zebrafish was a blood condition
caused by a mutation in the ALAS2 gene (Chitramuthu, 2013), which caused
a specific failure in hemoglobin formation. A large number of additional
mutants with phenotypic parallels to human disease have been found. Neuro­
logical problems (Sosa et al., 2012), hematopathological disorders (Berman
et al., 2012; Brownlie et al., 1998), cardiovascular diseases (Sehnert et al.,
2002), muscular disease (Pappalardo et al., 2013), malignancies (Liu &
Leach, 2011; Patton et al., 2005), Parkinson’s disease (Sarath et al., 2016),
anxiety-posttraumatic stress disorders (Chakravarty et al., 2013) are only a
few examples.

7.5.2 ZEBRAFISH AS A RESEARCH MODEL FOR DIFFERENT

METABOLIC DISEASES

Duchenne muscular dystrophy, human melanoma, acute lymphoblastic

leukemia, polycystic kidney disease, nephronophthisis, acute kidney injury,
Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, myocardial
infarction, and some metabolic diseases are examples of human diseases
that have been successfully modeled in zebrafish. Zebrafish are a good
model for studying metabolic disorders because they have all of the organs
involved in energy balance and metabolism, such as hunger and insulin
control, as well as a lipid storage system that is similar to that of humans
(Nishio et al., 2012). Cardiovascular disease is the most common cause of
death among metabolism-related human illnesses, as per the World Health
Organization (WHO) study (Lozano et al., 2012). Obesity (Ng et al., 2014),
type 2 diabetes mellitus (LaBrecque et al., 2014), and non-alcoholic fatty
liver disease (Ng et al., 2014) all raise the risk of cardiovascular disease.
Zebrafish is a prominent model for studying metabolic problems because
they have similar metabolic organs to humans (such as the digestive organs,
adipose tissue, and muscle). In particular, various new tools and methods,
including as TALENS, CRISPR/Cas9 (Wu et al., 2018), compound treatment
204 Coldwater Fisheries and Aquaculture Management

(Poureetezadi et al., 2016), mass spectrometry-based polar metabolomics

and lipidomics (Zhang et al., 2018), and in vivo imaging of fluorescent dyes
(Minchin et al., 2018), make it feasible to explore the molecular pathways.
Researchers have also utilized zebrafish as a model organism to investigate
a variety of metabolic problems, including congenital metabolic abnormali­
ties, hyper-, and hypothyroidism, disorders of the hypothalamus-pituitary­
adrenal axis, circadian clock dysregulation, and cancer metabolism (Gut et
al., 2017).

7.5.3 ZEBRAFISH AS A SYSTEMATIC CANCER RESEARCH MODEL

Vertebrate models are essential for better understanding the development,

growth, and dissemination of malignant tumors thus zebrafish are a good
model for cancer studies. Although humans and fish have a common ancestor,
the biology of cancer is the same in both groups of creatures (Amatruda et
al., 2002). The zebrafish is adroitly exploited for carcinogenic treatment,
transplantation of mammalian tumor cells, and transgenic regulations due to
the tremendous benefits of using zebrafish as a research model (Mizgirev &
Revskoy, 2010) and malignancies (Liu & Leach, 2011; Patton et al., 2005),
Parkinson’s disease (Sarath et al., 2016), anxiety, and PTSD (Chakravarty et
al., 2013).
Cancer is a disorder in fishes, just like it is in people, as evidenced by
melanomas that occur in Xiphophorus hybrids (Walter & Kazianis, 2001;
Patton et al., 2005). Using zebrafish as a model organism in cancer studies
serves numerous advantages. With zebrafish, highly conserved cancer
pathways can be genetically tested. Cancer is primarily an adult illness,
although cell cycle phenotype in fast-developing transparent zebrafish
embryos could be investigated using mutagenesis screens.
To examine tumors caused by environmental carcinogens, a variety of
fish have been employed as models. The zebrafish has proven to be the
finest model for studying embryogenesis, organogenesis, and the impact
of environmental carcinogens on cancer formation (Bailey et al., 1996).
Histopathologically (HP), chemically generated tumors are comparable
in both humans and zebrafish (Amatruda et al., 2002), and orthologous
oncogenes and tumor suppressor genes (TSGs) have been discovered in
both fish and people (Lam et al., 2006). Hepatic transcriptomic profiles in
humans and zebrafish at various stages of tumor aggressiveness were shown
to be similar in these two phylogenetically distant species (Mirbahai et al.,
The Zebrafish: A Trending Model for T-Cell and Thymic Development 205

2011). Pancreatic cancer, lung cancer, ovarian carcinoma, breast cancer,

prostate cancer, retinoblastoma, leukemia, and other tumors have already
been implanted into zebrafish (Zhao et al., 2015).

7.5.4 ZEBRAFISH IN TOXICOLOGY AS WELL AS DRUG DISCOVERY

RESEARCH

The zebrafish model has been utilized to determine organ function assays
and drug effect assessments (Kari et al., 2007). The effects of medicines
on specific organs have also been investigated, with organ toxicity being
found to be similar in both zebrafish and mammals. Gentamicin, cisplatin,
vinblastine, quinine, neomycin, doxorubicin, dexamethasone, cyclosporin
A, caffeine, camptothecin, MPA, fluorouracil, and other medications were
employed to assess organ damage (Zhang et al., 2003; Daroczi et al., 2006;
Langheinrich et al., 2002; McAleer et al., 2005; Ton et al., 2005; Wu et al.,
2006). The zebrafish is quickly establishing itself as a promising model
animal for pharmacological and chemical toxicology research (Spitsbergen
& Kent, 2003; Rubinstein, 2006). The toxicity of some important medica­
tions has been investigated using the zebrafish model. For instance, using
susceptible zebrafish, Amanuma et al. (2000) developed a test to detect
small molecule-induced mutagenesis. The developmental damage caused by
ethanol or acetaldehyde exposure was compared using zebrafish embryos
(Reimers et al., 2004). The toxicity of antirheumatic medications like diclof­
enac was studied using zebrafish. The zebrafish now is widely acknowledged
as a promising animal model for drug toxicity and environmental toxicology
research (Chakraborty et al., 2009).
The zebrafish model has been used to investigate the effects of neuro­
toxic, ototoxic, and neuroprotective drugs, as well as drug discovery.
Lead compound screening, target selection, target validation, and assay
development are the four main components of the drug discovery process
(Handen, 2002). Target identification is the process of discovering a gene
or protein that, when altered by treatment, can have a beneficial effect on
disease progression. Following the discovery of a possible target, the target’s
validation approach begins with the determination of protein function and a
draggability assessment (Eckstein, http://www.alzforum.org/drg/tut/tutorial.
asp; Lindsay, 2003; Frank, 2005). For zebrafish, each of these drug discovery
domains is extremely important.
206 Coldwater Fisheries and Aquaculture Management

7.6 OUTLINING SOME CHALLENGES

In biomedical research, the zebrafish (Danio rerio) is currently an exten­

sively utilized model organism. The zebrafish has long been used to research
heart formation. Exploring the operational inputs and their accompanying
physical stimuli, such as mechanical or calcium cues, in greater depth in
vivo will soon follow, thanks to the expanding number of genetic tools and
technological improvements.
Since some genes with a single copy in humans may have two copies in
zebrafish, genome duplication is a concern for several zebrafish models (Lu
et al., 2012; Semon & Wolfe, 2007; Alsop & Vijayan, 2009). On just one
hand, this significantly raises the overall complexity of the genetic analysis
required for proper data analysis. Other investigators, on the other hand,
see possibilities in this genetic uniqueness, because zebrafish models, for
example, might be used to investigate the impact of genes where null mutations
induce developmental death when expressed in a single copy (Stewart et al.,
2015). The lack of a large amount of possible and characterized zebrafish
genetic strains is related to genetic assessments and stems not only from the
relative novelty of zebrafish in biomedical and neuroscience research but
also from quick fertility loss due to inbreeding in teleosts (Stewart et al.,
2014; Nasiadka & Clark, 2012). There are presently no definite inbred lines
whereby all animals are similar and homozygous (Nasiadka & Clark, 2012),
and the level of genetic, physiological, and behavioral characterization of
such strains does not match that of laboratory rodent strains (Stewart et al.,
2014). Near-homozygous zebrafish lines were created utilizing heat shock
and early pressure, but they were too hard to maintain (Johnson & Zon,
1999), while ongoing research may soon help alleviate this problem (Hou
et al., 2015).
In vertebrate animal and human studies, gender is widely recognized as
an important biological characteristic that should be considered in research
designs, analysis, and reporting. However, because zebrafish lack sex
chromosomes and instead rely on a polygenic sex determination, its status
is still “complex” for zebrafish models (Nguyen et al., 2013; Lawrence et
al., 2008; Liew et al., 2014). As a reason, sex ratios in colonies vary widely,
and fish models often display negligible or abnormal sexual dimorphism
when contrasted to mammals (Nguyen et al., 2013; Lawrence et al., 2008;
Liew et al., 2014). As a result, the ability to obtain sex-dependent variations,
which are typically crucial for numerous human diseases, may be hampered.
Furthermore, in zebrafish investigations, under-reporting of sex ratio
determination lowers research repeatability, which should be addressed by
The Zebrafish: A Trending Model for T-Cell and Thymic Development 207

increasing data recording standards in the field. Other physiological variances

could also be a problem that needs to be addressed.
The blood-brain barrier, metabolism, thermoregulation, the formation,
localization, and function of neurotransmitter signaling pathways, and
brain anatomy (e.g., cortex and hippocampus) are all examples of species
differences (Stewart et al., 2015; Kalueff et al., 2014). Additionally, certain
methodological inconsistencies in zebrafish behavioral testing may have
an impact on data repeatability (Facciol et al., 2017). Furthermore, despite
significant improvements in mechanization and recording equipment,
population-level analysis and movement pattern identification remain
technological challenges, particularly in larvae studies (Rennekamp et al.,
2015; Ahmad et al., 2012) 29. Finally, individual and contextual factors
can influence CNS (central nervous system) models and drug screens in
zebrafish (Nichols et al., 2004). As a result, more research is needed to
better understand the role of sex, individual, strain, and age disparity in
zebrafish models, as well as housing circumstances, nutrition, testing time,
and social standing (Hart et al., 2016).
Arrhythmias and ventricular hypertrophy, both of which are underlying
illnesses that contribute to sudden cardiac arrest, can be caused by ion channel
dysregulation, such as LTCC. Future research that focuses on functionality
input modulation will aid in the development of new therapies for common
cardiac illnesses such as cardiac hypertrophy, heart failure, and arrhythmias.
Many questions about zebrafish vascular and lymphatic research remain
unresolved. In parallel to VEGF and Wnt, other signaling pathways such as
the Hippo system are being investigated in vascular development (Nagasawa-
Masuda & Terai, 2017; Nakajima et al., 2017; Astone et al., 2018). Similarly,
hemodynamic factors appear to have a role in the cardiovascular system’s
growth and differentiation (Chen et al., 2012, 2017; Goetz et al., 2014).
The species is also being used more regularly to examine specifications
and repair, as well as to gain a better understanding of human skeletal
diseases. When it comes to observing and assessing the skeleton, the model
organism’s small size is both a plus and a burden. Using the unique methods
of zebrafish skeletal tissue imaging and evaluation that we have described
in this work can overcome a significant restriction of the zebrafish model
for skeletal research: the extreme microscopic size of skeletal structures,
particularly in early developmental stages. Technological innovations for
imaging live-image-labeled skeletal cells and visualizing small skeletal
elements in three dimensions will aid our understanding of the cellular and
molecular mechanisms that underlie skeletal development and remodeling
(Bruneel & Witten, 2015).
208 Coldwater Fisheries and Aquaculture Management

The embryonic zebrafish is a vertebrate model that has proved useful

in uncovering fundamental neurodevelopmental pathways and may be used
for outstanding chemical screening. In zebrafish, gene editing can also be
employed to add genetic vulnerabilities. Despite the fact that the zebrafish
model has become more widely used in toxicology studies in recent years, the
various tools for imaging structural differences in the developing zebrafish
brain have yet to be widely used in studies of the impact of gene-environment
interplay on neuronal interconnection in the developing zebrafish brain
(Figure 7.4) (Miller et al., 2018).

FIGURE 7.4 Some limitations of zebrafish as a model organism in biomedical research.

7.7 CONCLUSION

Zebrafish have significant advantages compared to other vertebrate models

used in modeling human diseases, particularly for large-scale genetic mutant
The Zebrafish: A Trending Model for T-Cell and Thymic Development 209

and therapeutic compound screenings, as well as other biomedical research

applications. The zebrafish provides a unique developmental and genetic
model for immune system development, and the thymus has been the focus
of study because it is the first primary lymphoid organ to form throughout
zebrafish ontogeny. Future studies should look at the role of sex steroids in
teleost species’ in thymus involution. We know that sex steroids affect both
thymus differentiation and thymus involution in mammals. In recent years,
various methodologies and technologies have been used to shed light on
the mechanisms controlling thymus homing, intrathymic cell trafficking,
and the identification of hitherto identified T-cell gene regulators. Although
the zebrafish model has some advantages over current T-cell and thymic
development models, it also has certain drawbacks. Antibodies specific for
blood-cell lineages have yet to be shaped, making it hard to distinguish
between diverse T-cell populations at present. The gene-knockout technique
for zebrafish has yet to be developed. The zebrafish is fast developing as a
new model system for studying T-cell development and vertebrate immuno­
biology, adding to the elegant research in mice, chickens, and fetal thymic
organ culture that have characterized key elements of thymic and lymphoid-
cell development.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

KEYWORDS

• biomedical applications
• chronic unpredictable stress
• Danio rerio
• hours post-fertilization
• T-cell receptors
• T-cells development
• thymic development
210 Coldwater Fisheries and Aquaculture Management

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CHAPTER 8

Plant-Based Proteins in Fish Diets for

Sustainable Coldwater Fisheries
K. T. HAFEEF ROSHAN, MOHAMMED MEHAROOF, and K. A. SAJINA
PhD Scholars, ICAR–Central Institute of Fisheries Education, Mumbai,
Maharashtra, India

ABSTRACT

Coldwater fisheries are a crucial component of aquaculture that has been

progressing over the years meeting the supply needs and contributing
towards the economy. The increase in human population will generate a huge
demand for fish and coldwater aquaculture has the potential to gracefully
sustain this demand. However, for a sustainable culture, the selection and
use of a feed with the required nutrient profile and cost-effective nature is
one of the key factors that decide the success and growth. Fish meal (FM)
and fish oil are the excellent sources of protein, lipid, and other essential
nutrients which are mainly used in aqua feed formulation, but the limited
availability of these resources necessitates to look for alternative protein
and nutrient sources. While using feed ingredients to supplement fishmeal,
it is imperative to consider the key elements such as nutritional value,
acceptance, palatability, efficacy, availability, cost, and growth performance
of animal. The conventional plant sources like terrestrial plant-based proteins,
non-conventional plant sources are being investigated for potential feed
contributions to substitute fishmeal but the anti-nutritional factors (ANFs),
amino acid (AA) profiles, fatty acid profiles, mineral profiles and palatability
are some of the challenges being faced. Several studies demonstrated that the
use of feed additives reduces the negative effects of plant-based proteins and

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
218 Coldwater Fisheries and Aquaculture Management

increases the feed performance. Fish oil requirements also can be replaced
using oils from genetically engineered plants that are rich in omega-3 fatty
acids. Future research must be streamlined towards developing alternative
plant-based proteins that maximize sustainable yield and triggers mariculture
sector growth. The chapter touches on some important principles, practical
implications, and potential challenges for the use of plant-based proteins in
fish feed.

8.1 INTRODUCTION

Fisheries and aquaculture remain a significant sector and plays a vital role
as a source of food, nutrition, income, and livelihood for people around the
world. Aquaculture sector now accounts for around half of all food fish eaten
and provides substantial food security and economic development and FAO
(2014) revealed that the prospect of feeding human population to grow to
9.6 billion by 2050 which further ascertains the importance of the sector. To
deal with the rising population and food security, coldwater fisheries could
aptly fit as the next feasible alternative for expanding fish production and
thus meeting the global demand and providing livelihood security. The word
‘coldwater’refers to an aquatic ecosystem that maintains a temperature of less
than 20°C and enough oxygen levels for fishes like salmon, trout, mahseers,
snow trout, and other minor species (Sarma et al., 2018). Coldwater aquacul­
ture is a specialized branch of aquaculture involving the culture of organisms
that inhabits in fairly low water temperature in an enclosed section of the
river (cages/pens), or in tanks, ponds or raceways for food and alternative
products. Globally, the culture produces many high-value fishes of which the
main finfish classes encompass the salmons, trout, Mahseer, and common
carps. Within the coldwater body, particularly in exposed areas around the
world, there is a huge potential for culture. The experts estimate that, if
coldwater aquaculture is developed and promoted responsibly which can
expand the production and greatly contribute to global nutritional and liveli­
hood security (Sarma et al., 2018; Singh, 2018). This allows for a roadmap
that may enable aquaculture to turn out to be a serious provider of food for
humans in the 21st century and in the near future, in line with agriculture.
Despite the growth in aquaculture production, the economic gains of
this sector have been systematically affected by increased competition for
production factors and higher feed costs (Meharoof et al., 2020). In aqua­
culture, the cost of feed typically accounts for 60–80% of the operational
Plant-Based Proteins in Fish Diets 219

expenses (Hasan, 2010) which varies with the type of feed (e.g., live, wet,
moist, and dry feeds), quality of individual feed ingredients, feed production
and the methodology of feed delivery. This will not only influence produc­
tion, but also will have a huge effect on the composition of the species, where
fish that need less and cheaper feed will produce more than a good candidate
species. According to FAO (2020), the increase in feed cost coupled with
increase in demand of fish will escalate the price to 22% by 2030. In order
to ensure the development of the industry and to meet consumer require­
ments, it is therefore crucial to address these challenges in this sector with
environment friendly and economical aquaculture feed ingredients. The use
of plant-based resources for aquaculture production can strengthen the sector
by reducing the cost of feed, thus meeting the increasing demand of fish
without a rise in cost.
Significant progress has been made over the last decade to make a better
use of plant-based feed resources for fish and crustaceans (Gatlin et al., 2007;
Naylor et al., 2009; Aklakur et al., 2016). The limited accessibility of FM
supplies, as well as lower production prices obtained from plant resources,
motivated this transition. For aquaculture, the strategy to accelerate the
fraction of plant product in existing pellet feeds was successful; a feed
supply crisis in cold water fisheries has likely been met and continued to
be supported with increased use of plant products as feed ingredients must
be correlated with the key means of bringing the cultured species to a lower
biological process level, as well as the biological process level of the many
cultured species in nature (Kaushik & Troell, 2010; Tacon et al., 2010). FM
is one of the most important ingredients required for aqua feed due to its rich
content of extremely digested and well-balanced proteins, but it is a limited
resource. In parallel, coldwater fisheries production is likely to increase soon
and requires more protein derived from FM to be incorporated in feed. As
a result, with a high demand for FM and fish oil with limited availability
poses a challenge to the development of the rapidly growing and sustainable
coldwater fisheries sector. This situation has prompted the exploration of
sustainable aquaculture feed alternatives. Therefore, one of the driving forces
behind the hunt for cheaper alternatives is the dramatic increase in the price
of fishmeal since the beginning of the millennium. This review highlights the
short-and long-term alternative feed resources that can be offered and takes a
stand on how the feed requirements for coldwater fisheries are often met by
and on the far side of 2050.
220 Coldwater Fisheries and Aquaculture Management

8.2 REPLACEMENT OF FISH MEAL (FM) IN COLD-WATER

FISHERIES WITH PLANT DERIVATIVES: HURDLES AND PROSPECTS

Aquaculture has become the primary means of producing many of the candi­
date fish species and the use of compounded (formulated) feed to intensively
produce farmed fish has increased. For the culture in both marine and inland,
fishmeal is mainly used to be the source of protein in compounded feed.
Until 2006, several developments had been made in substituting alternative
protein sources for fishmeal proportions in aquafeeds and especially in
salmon, trout, sea bream and sea bass feeds decreased by 25–50%, depending
on species and phase of life history. Similarly, the percentage of fishmeal
in feed for omnivorous fish species, especially in grow-out feeds, also
declined. However, as aquaculture production and thus aquafeed produc­
tion continued to increase, the use of fishmeal in the aquafeed sector grew
gradually, accounting for 10% of annual fishmeal production in the early
1980s, compared with approximately 29% in 1995 as well as 50% of annual
fishmeal production in 2005. Despite significant breakthroughs in farmed
fish feed formulations, the substantial increase in FM prices, as well as the
ongoing expansion of the spectrum of business coupled with increased feed
prices and production costs, has been noticed. While prices have declined,
feed costs remain the most urgent concern of the aquaculture industries are
under enormous compulsion to develop cost effective formulations that
sustain efficient growth at lower price per unit yield. It was relatively easy to
substitute plant protein ingredients with fishmeal to provide approximately
half the dietary protein, but it is difficult to replace higher percentages of
fishmeal. Dependence of aquaculture on agricultural and fishery resources
as fertilizer and feed inputs and increasing competition for such resources
between aquaculture, human, and livestock sectors. The development of
fish feed is mainly dependent on the use of FM in aquafeeds developed as
the primary source of dietary protein. A significant number of studies have
been undertaken recently to minimize reliance on fishmeal given the limited
supply and increasing costs by research institutions and the aquaculture
feed industry (Rana et al., 2009; Tacon et al., 2006). Promising candidates
to substitute fishmeal as a protein source in aquafeeds are a large variety
of plant protein ingredients, and they are easily accessible and moderately
priced, facilitating the use of aquafeed plant protein sources. In order to
sustain rapid growth rates and feed quality values at higher levels of fishmeal
replacement, a range of obstacles need to be addressed. Recognized nutrient
deficiencies in plant protein contribute to the central issue as it moves to
replace higher levels of FM with plant proteins. A significant alternative
Plant-Based Proteins in Fish Diets 221

source of protein already widespread in aquafeeds is corn gluten meal, but

its amino acid (AA) profile and insoluble carbohydrate content counterpart
to FM have disadvantages. The crude protein content of corn gluten meal,
however, is slightly above its minimum guaranteed level of 60% implies
that 40% of the gluten meal of maize is composed of non-protein material,
primarily carbohydrates that are not soluble. In fish, non-soluble carbohy­
drates have no nutritional value (Stone, 2003) and are not added back to
the protein fraction during processing, corn gluten meal can be processed to
contain higher protein levels, but this method leaves producers with no outlet
for the non-soluble carbohydrate fraction.
Nutritional limitations of plant proteins compared to fishmeal is another
obstacle faced by the aquafeed industry as it transfers greater replace­
ment of FM with plant proteins. Fishmeal is a complicated product that
contains essential nutrients as well as a large number of biologically active
compounds. Feed formulators combine plant protein concentrates and
AA supplements to ensure that the AA content of feeds with reduced FM
levels meets or exceeds the criteria for AAs in farmed fish. They can also
supplement feed with mineral supplements such as di calcium phosphate
or double the trace mineral premix to increase the levels of calcium, phos­
phorus, and trace mineral feed when FM is extracted from formulations of
fish feed. This may not, however, be sufficient to resolve other deficiencies
or imbalances that occur when the amount of FM in feed is lowered. Over
time, researchers have detected a variety of dietary constituents that have
been added to poultry feed, allowing formulators to reduce FM as a feed
ingredient and eventually the removal of it. It’s not the same as in aquafeeds
because the undiscovered growth factors required for fish are less likely to be
trace elements and amines like taurine and possibly steroids. However, like
with nutritional deficiencies in all-plant diets, macro, and trace mineral defi­
ciencies cannot be eliminated. Anti-nutritional compounds in plant proteins
are the major challenge replacing fishmeal with plant protein concentrates.
In general, proteins produced from oilseeds contain more anti-nutrients
that are important to fish than proteins produced from grains. Some of the
anti-nutrients were inactivated by processes involved in the processing of
products or during pelleting by extrusion.

8.2.1 ANTINUTRITIONAL FACTORS

Anti-nutrient factors may be identified as substances which, by themselves

or through their residual metabolic products, interfere with the use of feed
222 Coldwater Fisheries and Aquaculture Management

and have an impact on the health and production of animals (Mohanta,

2012). Antinutritional factors can be classified narrowly into a few catego­
ries such as: (i) those that influence the digestion and use of proteins such
as protease inhibitors, lectins (hemagglutinins), tannins, etc.; (ii) those that
affect the use of minerals such as phytates, gossypol (pigment), oxalates,
glucosinolates, etc.; and (iii) miscellaneous substances, such as mycotoxins,
mimosin, cyanogens, alkaloids, phytoestrogens, saponins, etc. (Francis et
al., 2001a). Inclusion of plant protein at improved degrees or entire substi­
tute of animal proteins by plant proteins have resulted in negative boom and
feed performance, which are chiefly approved to the existence of ANFs and
beside the point AA balance (Francis et al., 2001). Awareness of the essence
of antinutrients, their effects, potential modes of action and ways of reducing
the effects or removing potent feed ingredients of antinutrients of plant
origin have been studied in detail. The following gives a brief understanding
of several important anti-nutritional variables:
1. Protease Inhibitors: These are the entities found in the entire plant
kingdom, especially among legumes such as soybean with the ability
to inhibit the proteolytic activity of certain enzymes (Norton, 1991).
There may be several protease inhibitors within a single plant, showing
variations in their structure, molecular weight, and mode of action. For
example, soybeans show as many as five protease inhibitors that are
primarily binds to digestive enzymes such as chymotrypsin or trypsin,
rendering them partially or entirely inactive. All protease inhibitors
are heat-labile and are easily destroyed by roasting or cooking.
2. Lectins or Hemagglutinins: Phytohemagglutinin or lectins are
commonly distributed glycoproteins in legumes and certain oil
seeds (including soybeans) that have an affinity for particular sugar
molecules and are distinguished by their ability to treat antinutri­
tional factors of plant origin. Hemagglutinins, the name came since
they activate RBC agglutination (red blood corpuscles) and are
shown to be present in several plant seeds. The growth-retardant
properties of raw legumes due to protein digestion inhibition and
pancreatic hyper-activity resulting in increased development of
trypsin and chymotrypsin and consequent cystine loss have been
reported to be partly responsible for these inhibitors. Lectins are
non-immune proteins or glycoproteins, especially abundant in plants
such as maize, beans, quinoa, peas, etc., that are capable of binding
carbohydrates or glyco-conjugates without altering them. However,
they are heat-labile, and are destroyed by heat processing or roasting.
Plant-Based Proteins in Fish Diets 223

3. Tannins: These are water-soluble phenolic compounds with a

molecular weight greater than 500 Daltons, capable of precipitating
proteins in the aqueous solution. Tannins that are hydrolysable and
condensed (leguminous forage and seeds) are two different groups.
They are heat stable and decrease the digestibility of proteins in
animals, either by partially making protein inaccessible or by inhib­
iting digestive enzymes and increasing fecal nitrogen. It is evident
that in plant feed, they inhibit the activities of trypsin, chymo­
trypsin, amylase, and lipase, decrease the content of protein in
foods and interfere with the absorption of dietary iron. The activity
of microbial enzymes, including cellulose and intestinal digestion,
may be inhibited if the concentration of tannin in the food becomes
too high. Tannins also form protein-insoluble complexes and these
complexes may be responsible for the anti-nutritional effects.
Tannins can establish a less digestive complex with dietary proteins
and can bind and inhibit endogenous proteins, such as digestive
enzymes and have documented intestinal disturbance, interference
with iron absorption and the possibility of carcinogenic effects
(Butler, 1989).
4. Phytates: Phytic acid, often known as phytate, is a type of vitamin
that is found in plant seeds and is chemically known as vitamin
myo-inositol hexa-phosphates. Soybean oilcake, sesame oil cake,
rapeseed oilcake, and other regularly used feed components contain
levels ranging from 10–75 g/kg. Matyka et al. (1993) estimated
that organically bound phytin phosphorus accounts for 62–73% of
total phosphorus (TP) in cereal grains and 46–73% in legume seeds,
respectively. Phytic acid functions as a strong chelator, forming
complexes of protein and mineral-phytic acid; decreased protein and
mineral bioavailability are the net result (Khare & Nakajima, 2000).
The phosphorus retained as phytic acid can be released by the diges­
tive enzyme phytase. They form a less digestible complex of phytate
protein, thereby limiting dietary protein availability and inhibits the
function of gastrointestinal tyrosinase, trypsin, pepsin, lipase, and
amylase (Liener, 1980; Hendricks & Bailey, 1989).
5. Gossypol: This is a secondary metabolite dependent on terpene,
mainly present in cottonseed protein flour (Anderson et al., 2016)
and plays an important role in the defense mechanisms of the
cotton plant against pests and diseases (Romano & Scheffler, 2008).
Gossypol is stored in the roots, seeds, leaves, and green bolls of
224 Coldwater Fisheries and Aquaculture Management

the plant in structures called “glands,” and is considered to have

anti-nutritional effects on warm-blooded animals and fish fed with
cottonseed products (Romano & Scheffler, 2008). The average
gossypol content of cotton seeds with gland ranges from 0.4–2.4%
to less than 0.01% free gossypol in certain low-gossypol cottonseed
meals (Liener, 1980; Robinson & Brent, 1989). It is hypothesized
that its anti-nutritive effect is due to its binding to lysine and thus
rendering this essential AA less accessible (Wilson et al., 1981) and
thus adversely affecting the use of protein and the production of
nitrogen waste. This cottonseed meal antinutrient may be minimized
by genetic selection or alteration for “glandless” seeds with low
levels of the lysigenous glands (Alam et al., 2018; Anderson et al.,
2016). An alternative approach is the extraction of acidified polar
solvents, which eliminates about 90–95% of gossypol (Pelitire et al.,
2014).
6. Oxalates: Oxalic acid is a metabolic product produced in plants
and animals through many pathways. Monovalent oxalates formed
with divalent ions, such as calcium, magnesium, and iron, are almost
insoluble in water, whereas ions such as sodium, potassium or ammo­
nium are well soluble in water (Libert & Franceschi, 2000). These
essential ranges are included in aquaculture diets: saltbush (Atriplex
halimus), 1.0–3.0% 1.0–2.5% and alfalfa (Medicago sativa); 2.4%.
In peptic digestion, oxalates impair calcium and magnesium metabo­
lism and combine with proteins to form complexes that have an
inhibitory effect.
7. Glucosinolates: In the Brassica nigra plant species of the family
Crusiferae, these compounds are usually present. Glucosinolates
are the main antinutritional factors present in rapeseed oilcake and
mustard oilcake which are potential sources of protein in fish feed.
These compounds have been shown to impair the proper func­
tioning of thyroid glands in fish with problems such as decreased
feed intake, feed utilization, metamorphosis, and maturation
(NRC, 1993). Thyroid dysfunction is shown by continuous intake
of feed-containing glucosinolate ingredients such as mustard
oilcake ultimately affecting metabolism and fish development.
It is difficult to set a glucosinolate threshold level in different
formulated fish feeds because of the lack of data and the quantities
of toxic products such as isothiocyanates and/or nitriles which are
generated.
Plant-Based Proteins in Fish Diets 225

8. Saponins: There are naturally occurring steroid or triterpenoid

glycosides in some feed ingredients derived from plants, such as
legumes (18–41 mg/kg in various legume seeds) (Francis et al.,
2001). The administration of soybean saponins has reported negative
growth performance outcomes, but this appears to depend on either
the raw material administered, potential interactions between dietary
ingredients or species tolerance (Chen et al., 2011; Couto et al., 2015;
Kokou et al., 2012). Saponins were treated as toxic because they
appeared to be particularly toxic to fish and cold-blooded animals
and in many of them, they had high hemolytic activity. Saponins,
in high concentrations, add a bitter taste and astringency to dietary
plants which is the key factor that limits its use. In Atlantic salmon,
major effects of soy saponin with pea protein concentrate in feed have
been observed by reducing feed intake, evident lipid digestibility,
AAs and ash (Chikwati et al., 2012). Saponin levels greater than 2 g/
kg will decrease feed palatability and result in the accumulation of
unconsumed feed pellets.
9. Non-Starch Polysaccharides (NSPs): These are the key plant
ingredients which contribute towards digestive disability and waste
production. Since these ingredients primarily affect fecal physico­
chemical properties, their effects should be eliminated by in vitro
digestion or the addition of degrading NSP enzymes as a result of
waste processing as well. Experiments involving cell wall NSPs
from potential sources of vegetable protein and NSP-degrading
feed enzymes at the same time may help to explain the fundamental
mechanism by which their effects are exercised by NSPs and to
provide a means to mitigate possible anti-nutritional effects. The use
of NSP-degrading enzymes has been studied to reduce their effects on
red sea bream (Pagrus major), golden sea bream, Japanese sea bass
(Lateolabrax japonicus), rainbow trout and Atlantic salmon (Castillo
& Gatlin, 2015; Ai et al., 2007; Carter et al., 1994; Dalsgaard et al.,
2009; Farhangi & Carter, 2007; Ogunkoya et al., 2006). Enhancing
the utilization of NSP-degrading enzymes and the carbohydrates
by fish is a major nutritional bottleneck for the sustainability of the
industry, while a mixture of pure protein concentrates will increase
the retention of nutrients and minimize the release of waste to the
environment.
10. Alkaloids: These are present in many legumes, but only seeds
of the genus Lupinus have concentrations high enough to cause
226 Coldwater Fisheries and Aquaculture Management

problems (Petterson et al., 1997; Wasilewko and Buraczewska,

1999). They are chemical compounds formed by plants and have a
bitter taste which serves as a barrier to food. Experiment on Lupinus
luteus, which is one of the feed ingredients that have the highest
protein and sulfur AA content (van Barneveld, 1999) which is fed
to rainbow trout and observed a decrease in feed consumption. It is
realized that the reason for the decrease in feed consumption is due
to the presence of alkaloids like gramine, lupinine, and sparteine
(Glencross et al., 2006). In order to prevent palatability concerns,
potential inclusion should concentrate on the alkaloid levels in raw
materials (Figure 8.1).

FIGURE 8.1 Anti-nutritional factors.

8.3 MEETING THE CHALLENGES IN A CONSTRUCTIVE MANNER

In the case of plant-based products, such as soy or various types of grain,

there is an especially broad and promising range of alternative raw materials.
Plant-Based Proteins in Fish Diets 227

Generally, plant proteins are available in much greater amounts than fishmeal.
Increasing nutritional quality through plant genetic manipulation can help
in the enrichment of protein which also reduces the antinutritional factors.
For aquafeeds, the nutrient and antinutrient profiles of plant products are
not currently optimal. Fortunately, it is possible to change specific traits of
grains, such as protein and oil content, by breeding and genetic modification.
The molecular pathways associated with the expression of various nutrient
traits have been determined through genomic and genetic studies. Genetic
manipulations have improved over the last decade, with the ability to further
enhance some specific nutrients in plants. The following are examples of
improvements in many primary antinutrients and nutrients:
• Genetic materials with high lysine;
• Manipulation of starch-structure;
• Enhancing the use of fish through genetic selection;
• Increased content of micronutrients.
There is a great concern about the abundance of anti-nutritional factors
(ANFs) and harmful effects in plants used as animal feed and ways and
means should be found to remove or reduce their rate to the minimum
(Soetan & Oyewole, 2009). By developing innovative processing tech­
niques and technologies to increase the digestibility/nutritional value of
nutrients and consistency may help in this regard. Feed-processing methods
have been employed to improve the physical qualities and nutritional
content of feed for several years, and the outcomes of different production
methods are very well documented (Booth, Allan, & Warner-Smith, 2000;
Cheng & Hardy, 2003; Venou et al., 2003). Cooking extrusion, for example,
enhances carbohydrate digestibility (Allan & Booth, 2004) and creates a
more durable pellet that can be managed to make the pellet sink (Barrows &
Hardy, 2001). Until combining the ingredients together into a full feed, pre­
processing refers to the treatment of particular ingredients. Pre-processing
of ingredients using both biological enhancement and mechanical alteration
is being studied to improve the nutritional value of plant derived ingredients
for fish. Fermentation by yeasts, bacteria, and fungus has been investigated
and found to have a lot of potential for removing antinutrients while also
providing key nutrients like protein and AAs (Mukhopadhyay & Ray, 1999;
Bairagi et al., 2002). The ultimate purpose of these processes is to increase
the concentration of protein and decrease antinutrient levels. The reduc­
tion of foreign or fermentable carbohydrates and phytate is well within this
approach’s capabilities (Ng et al., 2002; Skrede et al., 2002). Microorganisms
228 Coldwater Fisheries and Aquaculture Management

absorb and transform carbohydrates into cell mass that serves as a digestible
source of both protein and energy from unavailable energy. The nutrient and
antinutrient profile of the initial product will be determined by the type of
microorganisms and plant substrates used in the fermentation. The primary
factor influencing their use in future aquafeed formulations would be the
costs of processing biologically enhanced plant products. An extension
of existing pre-processing methods is the mechanical alteration of plant-
derived ingredients. Until grinding and mixing, these techniques may be as
easy as de-hulling barley to reduce the fiber content and thereby improve
the protein content (Hardy & Barrows, 2000). Processing by extrusion can
reduce the antinutritional factors in feed products. Extrusion is a short-term
processing of high temperature (HTST) that incorporates many methods,
including heat and mass transfer, mixing, shearing, reduction of particle
size, melting, texture, caramelization, and forming. As growth agents (e.g.,
cinnamic acid derivatives) and contaminating microorganisms are more
effectively eliminated or reduced, HTST processing is considered an effi­
cient approach in terms of nutrient retention. This method has been used
to inactivate or completely eliminate aflatoxins, which normally involve
with conditions such as high shear and elevated temperatures (Saalia &
Phillips, 2011). The benefits of extrusion include ANF degradation, starch
gelatinization, increased soluble dietary fiber, and decreased lipid oxida­
tion (Nikmaram, Kamani, & Ghalavand, 2015). Extrusion is also a very
effective method of inactivating the function of a-amylase, trypsin, chymo­
trypsin, and hemagglutinin inhibitors without altering the amount of protein
in food products (Soetan & Oyewole, 2009). Several other methods have
been implemented in recent years, such as high-pressure processing (HPP),
microwave and extrusion, as alternatives to minimize ANF levels (Zarei
& Kafilzadeh, 2013). These manufacturing processes, which included non-
thermal, limited heating or high temperature cooking over a short period of
time, provided a greater capacity to manufacture finished products with a
substantial increase in nutritional quality compared to traditional methods
(Linsberger-Martin et al., 2013). In addition, some beneficial improvements
in the functional properties of legumes and cereals are anticipated through
these processing technologies and, in particular, the elimination or reduc­
tion of ANFs could be achieved (Ohlsson & Bengtsson, 2002).
Enhancing the palatability of feedstuffs from plants is imperative for
the development of plant-based feeds as fish exhibit distinct taste prefer­
ences. Fish tastes and preferences are largely species-specific, and the
distinctions between fish species may be seen when comparing the width
Plant-Based Proteins in Fish Diets 229

and content of spectra for both stimulants and deterrents. There is a strong
link between the evolution of the ontogenic gustatory system in fish and
its ability to differentiate between the taste properties of food products.
Bioassay and electrophysiological data comparisons suggest that palat­
ability in the gustatory system is not associated with excitability. However,
these studies examining the impact on filet consistency of different plant
proteins, including SBM, SPC, soy flour, corn gluten, wheat gluten, peanut
meal, canola meal, extruded peas, lupin seed meal and CSM, suggests that
in about 40% of the studies, product quality was significantly affected by
the source of protein. Color was the characteristic most affected by the
source of protein, as calculated by instrumental and sensory methods. In
some instances, texture, and flavor were significantly affected by the source
of protein, particularly when large substitutions were made. Other research,
however, in which 30–60% of the diet consisted of plant protein meals,
identified no differences between filets in consumer acceptability (Gatlin et
al., 2007). Clearly, further research is needed into the effects of plant protein
meals on product quality, particularly those subject to different degrees of
concentration and/or isolation.
Apart from this, monitoring the effect on the quality of fish products
prepared out of fish fed with plant-based feeds and consumer health is very
important. Market research on aquatic foods consistently shows that the
single most significant factor influencing fish-buying behavior is product
consistency. Consumers are becoming more aware of the health implications
linked with eating fish and if we move forward towards increasing the use
of plant products in aquafeeds, it is important to assess the impact of these
newly developed diets on the quality of fish products and consumer health.
Salmonids, namely rainbow trout and Atlantic salmon, were the subject of
the vast majority of research, particularly those that used sensory approaches
to assess quality. Gulf sturgeon, Acipenser sp. comprise non-salmonid
species sporadically assessed for dietary effect on sensory quality. In most
of the studies taste, color, odor, and/or texture have been significantly
influenced by diet (Clydesdale, 1993) Given the physiological, nutritional,
environmental, and compositional differences among cultured finfish, it is
not possible to automatically apply conclusions drawn on the product quality
of one species to another. In order to explain the effect of dietary ingredients
on the quality of aquaculture species of interest, further research is needed.
Although numerous studies have evaluated the effects on fish growth and
feed efficiency of different alternative plant proteins, relatively few have
tracked the dietary impact on the quality of fish.
230 Coldwater Fisheries and Aquaculture Management

8.4 PLANT-BASED FISH FEED SOURCES

For multiple species for commercial fish culture, the use of plant protein
in fish feed has been investigated as these feed components are unique to
species specifically based on their requirements. Grasses, veggies, aquatic
weeds, plant leaves, roots, seeds, and seed extracts, leaves, fruits of certain
plants, grains, oil cakes can be used for feed formulation (Mondal & Payra,
2015). Commonly use fish feed additives includes linseed, safflower,
sunflower, soybean, cereals, and cereal products, broken rice, rice polish,
sweet potato tubers, wheat bran, maize, cassava, and sorghum.

8.4.1 CANDIDATE PLANT PRODUCTS FOR USE IN AQUAFEEDS

8.4.1.1 SOYBEANS (GLYCINE MAX LINNAEUS)

Soybean is a leading oilseed crop that has an estimated production for

2018–2019 is 358.2 mmt and most of this production is used in the extrac­
tion of oil, making a high-quality protein cake. This is processed to yield a
large variety of soybean products, such as soy flour, soybean meal (SBM),
soybean protein concentrate (SPC) and soy protein isolate (SPI) that have
been tested in fish. The predominant type of soybean used was SBM and
is available either as dehulled (48% crude protein) or with added hulls
(44% crude protein) (NRC, 1993). Soy products are known to be inex­
pensive and nutritious feed products with a high content of crude protein
and a relatively balanced profile of AAs. In general, the concentrations
of 10 essential amino acids (EAA) and tyrosine in SBM are lower than
in FMs, with the exception of cystine, which is present in SBM at higher
concentrations. Lysine, methionine, and threonine are the EAA of concern,
which can be restrictive in soy-based diets fed to aquatic animals. With the
processing of soy flakes, concentrations of these EAAs increase in SPC
and SPI which exceed those found in FM. However, these items are not
yet economical for large-scale use in aquafeeds due to the manufacturing
costs. Crude concentrations of solvent extracted SBM fat, ash, and other
soy products appear to be lower than those in the meal, but concentrations
of carbohydrates tend to be higher. Appropriate supplementation with
mineral premixes and lipids may resolve lower ash and fat concentrations
in soy products, but high carbohydrate concentrations remain an area of
concern.
Plant-Based Proteins in Fish Diets 231

8.4.1.2 CANOLA (BRASSICA RAPA LINNAEUS)

Canola is made from cultivars of rapeseed that have been developed to

have low levels of erucic acid and glucosinolates. The crop of canola is
an oilseed, and the primary product of its production is canola oil. For
salmonids and other carnivorous species of farmed fish, Canola protein
concentrate (CPC) has been widely evaluated as a protein source and
supports growth rates comparable to those fed with FM-based diets. As
long as AA supplements are employed to address restricted AA levels and
feeding stimulants, such as betaine, are introduced to the diet to overcome
reduced intake, this is the principal source of protein in aquafeeds. The
protein concentrate of Canola has a comparable protein content to a high-
quality FM, but it is not widely available and no market prices have been
identified.

8.4.1.3 CORN (ZEA MAY LINNAEUS)

Corn oil is the primary food product of maize production, and while most
of the maize grown is fed as an energy source to livestock. Corn starch,
a commonly used sweetener in foods and drinks, is used to make over
400 products, including ethanol, paper coatings and corn syrup. The corn
kernel is divided into its main components, bran fiber, germ, gluten, and
starch, in the wet-milling process. The oil is extracted from the germ and
applied to the corn gluten feed; the remaining corn germ meal is used.
To form corn gluten meal, the gluten protein is condensed, filtered, and
dried, and a minimum of 60% protein on a ‘as-is’ basis is guaranteed to
include the widely traded corn gluten meal. The crude protein content of
processed and purified maize gluten protein can be 70–73% crude protein,
but there are limitations in the commercial production process, so that
these amounts cannot always be met. Although the price will be higher
than commercially traded corn gluten meal, a higher crude protein corn
gluten meal would be a more acceptable commodity for use in aquafeeds.
To decide whether the development of a high-protein corn gluten meal
is warranted, economic, and marketing analysis is required. Corn gluten
meal is being utilized in fish feed for salmon and other marine animals
like European sea bass, Dicentrarchus labrax Linnaeus, and Sparus
aurata Linnaeus.
232 Coldwater Fisheries and Aquaculture Management

8.4.1.4 COTTON SEED (GOSSYPIUM HIRSUTE LINNAEUS)

The third leading legume seed by weight (after soya and rapeseed) used
worldwide is cottonseed and cottonseed meal (CSM) have an enormous
potential for use in high protein aquafeeds due to its high protein content for
human consumption and animals, as well as its low market price relative to
other legumes and FM. Multiple studies evaluating CSM in catfish, salmonid,
and tilapia diets have found that 40% of solvent retrieved may be utilized in
aquaculture diets without causing growth decline. However, the technique
of replacing one plant protein source with another was most commonly used
in these experiments, while replacing FM or other animal protein sources
would be more difficult and profitable. Lee et al. (2002) used a method with
exceptionally positive results to fully substitute FM with a combination of
animal by-products and both SBM and CSM in the juvenile rainbow trout
diet. They also pointed out that the use of CSM in fish diets is affected by
the source of CSM and associated processing. Lee et al. (2006) summarized
a series of studies in rainbow trout in which CSM completely substituted
FM over the three year period without significantly affecting female and
male rainbow trout growth rates, while CSM diets had significantly lower
assimilation of protein and phosphorus. In one study, Ictalurus punctatus
Rafinesque, a channel catfish, was raised in earthen ponds at high densities
and fed a diet containing 51% CSM with lysine supplementation to satisfy
the diet (0.65%). Results showed that the rate of growth, dressing percentage
and chemical composition of the filets did not vary significantly from the
SBM-containing fish fed diets (42%). Fowler (1980) demonstrated that for
two Pacific salmon species, up to 34% of CSM can be used in the diet to
replace SBM without growth depression.

8.4.1.5 PEAS/PEAS LUPINS

Pisum sativum Linnaeus and Lupinus sp. Linnaeus, already used or

considered for use in aquafeed for marine and freshwater fish worldwide,
are traditional plant ingredients throughout the planet, both goods are
manufactured in large amounts (Allan et al., 2000; Glencross, Boujardand, &
Kaushik, 2003; Thiessen, Campbell, & Tyler, 2003). The nutrient profile of
peas and lupin shows that they are capable of replacing a large proportion of
FM protein in aquafeeds. The protein content of peas and lupin is moderate
compared to animal meals such as FMs, meat, and bone meals, and poultry
Plant-Based Proteins in Fish Diets 233

meals, while lysine and methionine are small and both contain high levels of
carbohydrates (Allan et al., 2000). Starch is the primary carbohydrate energy
reserve in peas, and can make up around 55% of the total crop. Lupins,
on the other hand, contain negligible starch levels (0.3%), with non-starch
polysaccharides (NSPs) such as b-(1,4)-galactan serving as the primary
carbohydrate storage energy (Van Barneveld, 1999). For a variety of pea
products for rainbow trout, Thiessen, Campbell, & Tyler (2003) recorded
protein apparent digestibility coefficient (ADC) values in excess of 90%
and also showed that dietary levels of 25% dehulled peas could be used to
replace soyabean meal. Glencross et al. (2003a), however, published protein
ADC values of 495% for a variety of red sea bream lupin varieties, Pagrus
auratus Forster. When fed with lupin at up to 60% of the diet, Glencross
et al. (2003a) have reported acceptable growth of juvenile red sea bream.
Energy ADCs for pea and lupine products were poor in all of the studies
described above (51–78%).

8.4.1.6 WHEAT

Wheat (Triticum aestivum Linnaeus and T. dicoccoides, var. durum) unlike

other cereals has at least six broad subdivisions of cultivars that are based
on the grain’s end-use properties. The presence of large seed storage protein
that forms the cross-linking networks of gluten when hydrated four is mixed
is the functional characteristic of wheat which distinguishes it from other
cereal grains. Gluten gives the dough elastic characteristics and the ability to
hold leavening agent-generated gasses. Owing to its strength and restricted
water solubility, gluten is also an important binder in aquafeeds. Flaking (roll
milling) of whole grains or pelleting of mill feed and grains has been used
in conventional wheat preparations for diets. In composition publications,
the wheat kernel is frequently believed to be 12% protein by weight at 12%
humidity (Bushuk & Wrigley, 1974). Like other cereals, whole grain wheat is
mainly an energy source rather than a source of protein based on its high starch
composition. In addition to protein and starch, wheat kernels are about 2%
crude fiber and about 2% ash (Martin, Leonard, & Stamp, 1976). Potassium
(0.40%), phosphorus (0.38%) and sulfur (0.20%) are the main components
of the ash fraction, with calcium (0.04–0.05%) as a comparatively minor
mineral portion (Guttieri et al., 2004). In the aleurone layer in phytin, phytic
acid with chelated metal ions, phosphorus in wheat is mainly sequestered.
Phytic acid mutants in wheat will dramatically reduce the concentration of
234 Coldwater Fisheries and Aquaculture Management

phytic acid and increase the nutritionally more readily available amounts
of inorganic phosphorus in the diet (Guttieri et al., 2004). Wheat embryo
proteins contain up to 8% lysine and 2% methionine (Pomeranz et al., 1970).
Mill feed, however, has many water feed disadvantages. Second, since most
of the digestible carbohydrates in the processing of flour are eliminated, the
energy quotient is decreased. Secondly, in the milling process, the glutenin
and gliadin storage proteins that form gluten are also extracted, with the
remaining bran proteins usually of lower value for use as a feed binder. The
remaining mill feed for monogastric animals is high in crude fiber (10%)
with minimal digestibility and has a more concentrated phytic acid composi­
tion that reaches almost 0.5% of total weight (Guttieri et al., 2004).

8.5 LEAF PROTEIN SOURCES

8.5.1 MORINGA LEAF MEAL (MORINGA OLEIFERA)

A highly nutritious supplement to the diet of plant-eating fish such as tilapia,

barbs, fancy cars, etc., is the fresh leaves of Moringa oleifera or ‘drumstick.’
They contain rich proteins, lipids, vitamins, and minerals, and hence the
leaves, kernels, and pods are also used in the feed industry for aquaculture.
Except for saponins and phenols, the leaf is free of other antinutritional
variables. Ogbe & Affiku (2021) reported that moringa is nutritionally rich
and can be used at low levels in the fish diet. Fresh Moringa leaves provide
additional protein, vitamins, and AAs such as methionine, cystine, tryptophan
that can promote fish growth and health. Amisah et al. (2009) stated that the
optimum amount of inclusion of 20% of moringa leaves can be used for
African catfish in the formulation diet. Afuang et al. (2003) also reported that
30% of FM in Oreochromis niloticus can be substituted by solvent extracts
of moringa leaves. However, in contrast, Tilapia fed with raw moringa leaf
meal revealed that no adverse effect on growth output was caused by 10%
substitution of FM-based dietary protein (Ritcher et al., 2003). Magouz et
al. (2016) concluded that the addition of 12% of Moringa oleifera leaves
can be recommended as a partial feed replacement in the Nile tilapia diet
without any adverse effects on perfect growth. Moringa leaves have been
successfully used to partially replace traditional diets without compromising
the growth potential of Nile tilapia.
Puycha et al. (2017) recorded that moringa leaf could be included at 10%
without a negative impact on growth, feed utilization, digestibility, and serum
biochemistry in the Bocourti catfish (Pangasius bocourti) diet. Adewumi
Plant-Based Proteins in Fish Diets 235

(2014) reported that Moringa oleifera leaf meal can be used without adverse
effects on survival and growth efficiency to substitute up to 10% of the FM
in the C. gariepinus fry diet. Yuangsoi & Masumoto (2012) recorded that
up to 20% soybean protein replacement without adverse effect on growth
and digestibility could be used for fancy carp (Cyprinu carpio) diets with
moringa leaf diet containing ingredients.

8.5.2 SWEET POTATO LEAVES

Sweet potato, Ipomoea batatas is widely cultivated around the world and
is one of the most important food crops in tropical regions as well. Due
to the high protein and fiber content, the leaves were used as forage for
the cattle. Adewolu (2008) stated that the content of crude protein in sweet
potato leaves ranged from 26.5 to 32.5%. According to the study, the plant’s
leaf meal contains 26–33% crude protein, as well as significant levels of
AAs, minerals, and vitamins. He also mentioned that one of the advantages
of utilizing this plant meal in fish feed is that it is accessible multiple times
a year, making it easier and less expensive to obtain. Oven or sun-drying,
boiling or steaming and grinding will eliminate the presence of ANFs such as
invertase and protease inhibitors prior to use in fish feed, thereby improving
palatability. Experiments with Tilapia zilli showed that, sweet potato leaf
meal could be used in diets at up to 15% without sacrificing growth and feed
quality (Adewolu, 2008).

8.5.3 MULBERRY LEAF MEAL

Due to the high protein content and mineral elements, Morus alba or mulberry
leaves grown for the silkworm industry are often used in fish feed formula­
tions. Deficiencies in essential AAs, the presence of ANFs and complex
carbohydrates are responsible for limiting its use (NRC, 1993). Bairagi et al.
(2002) proposed that fermentation may be a simple and inexpensive way of
growing the ANFs in it. Mondal et al. (2012) stated that a possible protein
source in the diet of Labeo bata may be mulberry leaf meal. For Nile tilapia,
Cruz & Laudencia (1978) used mulberry leaf meal, resulting in increased
growth and lower FCR at 60% inclusion in combination with 40% rice bran.
Bag et al. (2012) found that including leaf meal into the stinging catfish
diet improved growth and survival, increased fish acceptance, and improved
immunity to common illnesses.
236 Coldwater Fisheries and Aquaculture Management

8.5.4 AZOLLA

An underwater free-floating fern that has the capacity to fix and assimilate
atmospheric nitrogen is a mosquito plant or Azolla. It grows rapidly and in
2–3 days it has the ability to double its weight. It is also known as a “super
plant” because of its high nutrient content, which includes vital AAs, vitamins
(vitamin A, vitamin B12, and Beta-carotene), growth boosters, and minerals
such as calcium, phosphorus, potassium, ferrous, copper, magnesium, and
others (Sheeno & Sahu, 2006). It has a high protein content (19–31%), as
well as a low lignin content, making it a popular element in animal feed.
Azolla pinnata has been identified as a possible source of protein in the fish
feed industry (Micha et al., 1988). When azolla was added to the fish diet,
Antoine et al. (1986) observed enhanced feed usage in Tilapia mossambica
and higher growth in Rohu, Nile tilapia, Common carp, Silver carp, and
Mrigal. Azolla powder can be used to substitute up to 25% in tilapia fishmeal
(El-Sayeed, 1992), and Etroplus suratensis (Joseph et al., 1994), whereas
45% in Cirrhinus mrigala fry (Gangadhar et al., 2014), and 42% in Oreo­
chromis niloticus fry (Santiago & Lovell, 1988).

8.5.5 WATER HYACINTH

A rapidly growing, herbaceous, free-floating aquatic plant native to the

Amazon Basin is the water hyacinth, Eichhornia crassipes. Owing to
their invasive existence and the culture of it is limited in some parts of the
world, this plant is considered to be the world’s worst aquatic weed.” Lareo
& Bressani (1982) stated that for various animals, fresh, and composted
water hyacinth is used as feed. They recorded 38% protein content and
17–26% minerals in the dry matter of the leaves. Many researchers did
not recommend the use of water hyacinth as feed for large-scale fish diets
unless the hyacinths were composted or fermented (Wersal & Madsen,
2012). El-Sayed (2003) stated that up to 25% inclusion level of fermented
water hyacinth can be integrated into the diet of Nile tilapia. He recom­
mended fermentation in order to increase the fish’s palatability and better
consumption. They indicated that composted hyacinth water can be used to
substitute up to 75% of the Nile tilapia diet for fishmeal. The inclusion of
up to 18.9% inclusion amount of water hyacinth meal (dry and ground) did
not compromise the growth and survival of Matrincha fish (Brycon sp.),
according to Saint-Paul et al. (1981). They did say, however, that high-level
Plant-Based Proteins in Fish Diets 237

inclusion was not recommended since the tannin in the plant interferes with
the protein’s digestion.

8.5.6 DUCKWEEDS

Duckweeds are a floating aquatic plant that grows luxuriously in tropical

and subtropical areas in freshwater bodies. It is made up of four genera,
viz. Spirodela, Lemna, Wolfilla, and Wolffiella. Duckweeds contain 15–43%
crude protein, 5–30% fibers, 5% lipids, and are frequently used in the animal
feed industry (Cheng & Stomp, 2009; Mohapatra & Patra, 2013). Duckweed
was suggested for use in fish diets because of its rich protein, trace minerals,
K and P, and colors, particularly carotene and xanthophyll. Common carp,
Thai sharputi, raj puti, silver carp, mrigal, and tilapia diets have all benefited
from the usage of new duckweed (Kabir et al., 2009). When duckweed was
included in the diet of common carp, Mohapatra & Patra (2013) discovered a
substantial difference in growth when 45% duckweed was supplied together
with feed. Fishmeal may not be replaceable with duckweed meal, but can be
used to supplement the diet with up to 15% inclusion level to decrease costs
without sacrificing growth (Ogello et al., 2014). Das & Ray (1989) reported
that without affecting the growth and survival of the fish, dried duckweed
(Lemna polyrhiza) could be potentially used as a feed ingredient for Labeo
rohita fingerlings. Yilmaz et al. (2004) suggested lemna leaf meal fermenta­
tion for the formulation of fish feed. They also recorded that duckweed in the
feed of common carp can replace up to 20% of commercial FM. And Fasakin
et al. (1999) outlines that the addition of up to 30% solar-dried duckweed
(Spirodela polyrrhiza) FM substitute in the Nile tilapia diet improves healthy
growth and is cost-effective. Mbagwu et al. (1990) reported improved growth
and survival of diet-fed mango tilapia fingerlings containing 10% duckweed
(Lemna paucicostata).

8.5.7 ALFALFA MEAL

Alfalfa (Medicago sativa) is a pea-family flowering plant (Fabaceae),

cultivated as a major forage crop. It is known in the United Kingdom,
Australia, and New Zealand as Lucerne and in southern Asia as Lucerne grass.
Because of its high protein content, Alfalfa is widely cultivated throughout
the world as a forage for cattle. Physicians used young alfalfa leaves in
238 Coldwater Fisheries and Aquaculture Management

early Chinese medicine to treat digestive and kidney-related disorders.

Dried alfalfa leaves in powder form have been used in the preparation of
fish feed and tested under laboratory conditions on the diet of a freshwater
fish, Cirrhinus mrigala (Mrigal Carp) and found better growth nutrient
utilization (Maity & Patra, 2008). The sole skill of Medcago sativa is not to
replace conventional FMs. It is therefore used along with the conventional
ingredients as a supplementary fish feed ingredient; however, the level of
incorporation of traditional ingredients such as groundnut oilcake, rice bran,
etc., was held at a lower level of inclusion.

8.5.8 SUBABUL (LEUCAENA LEUCOCEPHALA)

Wee & Wang (1987) have shown that the inclusion of up to 25% of the total
protein with soaked Leucaena leaf meal (LLM) has no adverse effects on
O. niloticus development. In Indian Major Carp, with Leucaena as a dietary
protein source, improved growth performance (Ghatnekar et al., 1983).
Osman et al. (1996) reported that the higher percentage of dried or cooked
LLM in tilapia diets significantly improved weight gain, basic growth rate,
feed conversion ratio (FCR) and protein consumption parameters. Man
et al. (2017) have suggested that in diets for Macrobrachium rosenbergii,
leucocephala meal could substitute 25% for FM in juveniles. Amisah et
al. (2009) concluded that Leucaeana leucocephala leaf meal can be up to
30% included in Clarias gariepinus at inclusion diets. Tiamiyu et al. (2015)
recorded that at 20% levels without adverse impact, Leucaeana leucocephala
leaf meal can be included in the diets of Clarias gariepinus.

8.5.9 CASSAVA WASTE (MANIHOT ESCULENTACRANTZ)

Kana et al. (2012) found that body weight was highest in birds fed diets in
which 50% of the maize was replaced by cassava flour meal. It was found
that 15% cassava meal can replace coconut meal in broiler diets with no
adverse effect on growth performance (Ravindran et al., 1986). In 10% of
Nile tilapia fingerling diets, cassava leaf meal produced the best growth,
FCR and survival ratio (Nnaji et al., 2010). Lacerd et al. (2005) reported
that the 30% inclusion level of cassava root meal can replace up to 100%
maize grain with no detrimental effect on growth efficiency in the diet of
Ctenopharyngodon idella fingerlings.
Plant-Based Proteins in Fish Diets 239

8.5.10 CHROMOLAENA ODORATA (SIAM WEED/DEVIL WEED/

FRENCH WEED/COMMUNIST WEED/COMMUNIST PACHA)

Chromolaena odorata leaves are of high nutritive value and might have the
potential to be used as a protein supplement to ruminants (Apori et al., 2000).
Igboh et al. (2009) also suggested that C. odorata is a source of high-quality
protein which could serve as a potential source of protein supplement. Apori
et al. (2000) reported the crude protein content of C. odorata leaf is above
194 g kg–1 dry matter. The nutrient composition of C. odorata leaf meal
and AA composition, mineral composition and its availability is comparable
with cassava leaf meal (Aro et al., 2009). C. odorata leaf meal can effec­
tively include up to 30% in in the diet of Labeo rohita fingerlings as an
alternate ingredient to improve the growth (Sajina et al., 2021). There are
some reports that C. odorata leaf meal can be incorporated into the feed
of rabbits up to the level of 30% (Bamikole et al., 2004). There are studies
which found that dietary supplementation of C. odorata leaf extract powder
improved feed utilization and growth rate and survival of Clarias gariepinus
(Dada & Sonibare, 2015).

8.6 CONCLUSION

Slow growth rate of fish and high-cost feeds have been seen as major
obstacles in the rapid development and expansion of coldwater fish produc­
tion. Plant based fish feeds are gaining attraction in aquaculture because it
is one of the sustainable and cost-effective methods for future cold water
aquaculture practice. On the one hand, it can be cost effective, and moreover,
it can lead to the sector’s sustainability. Many aspects of the study can be
evaluated, and innovative methods and tactics for effective incorporation of
plant-based nutrients in fish diets can be developed, paving the way for a
prosperous future for the aquaculture sector. A better understanding of the
nutritional and physiological effects of possible plant-based feeds on fish
would aid in the development of corrective techniques to counteract the
negative effects. Further studies on bioactive compounds in plants and its
effect in animal body could help in improved acceptance and utilization of
plant stuffs by the animal. There should be some strategies for improving
the effective use of plant-based diets in coldwater aquaculture. Research
has to be streamlined in these aspects, and the improved understanding of
this field leads to several applications and development of plant-based feed
240 Coldwater Fisheries and Aquaculture Management

technologies in the coldwater fisheries sector. Slowly but surely the sector
will achieve sustainability in the near future.

KEYWORDS

• alternative feed
• anti-nutritional factors
• coldwater fisheries
• fish meal
• high-pressure processing
• non-starch polysaccharide
• nutrition
• plant protein
• sustainability

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CHAPTER 9

Small-Scale Fisheries by Indigenous

Fishing Methods
SHABIR AHMAD DAR1, GOHAR B. WANI1, FAISAL RASHID2, and
KAWKABUL SABA3
1
Division of Fishery Engineering, Faculty of Fisheries, SKUAST-K,
Jammu and Kashmir, India
2
Division of Post-Harvest Technology, Faculty of Fisheries, SKUAST-K,
Jammu and Kashmir, India
3
Division of Fish Nutrition and Biochemistry, Faculty of Fisheries,
SKUAST-K, Jammu and Kashmir, India

ABSTRACT

The fishing craft and gears operated in the lake were simple tools used by local
fishermen for commercial catches of fish production. Against this background,
the study was attempted to explore the different fishing methods operated by
fisher folk of Dal Lake, Srinagar, Kashmir. During the present investigation
one type craft and six types of gears were observed in the lake. The craft was
plank built wooden boat, while the gears were long line, Cast net, Narchoo,
Scoop net, Gillnet, and Rod and Line. The fishers use indigenous materials
and methods for construction, fabrication, and maintenance of fishing gears
and crafts. A total of nine species of fishes were recorded during the present
study viz., Cyprinus carpio, Carassius carassius, Schizothorax niger, S.
esocinus, S. curvifrons, Crossochielus diplochilus, Trplophysa spp., Puntius
conchonius, and Ctenppharygodon idella. The dominant species during the
study period was C. carpio which contributed 63.7% of the total catch by
biomass and 41.64% by number on the whole.

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
250 Coldwater Fisheries and Aquaculture Management

9.1 INTRODUCTION

Fishing methods of Kashmir valley include a wide range of fishing gears.

The gears mainly used for fishing in Kashmir valley are Cast net, Gill net,
long line, Hook, and Line, Scoop net, Trap, and Harpoon displayed in Figure
9.1(a) (Dar et al., 2014). Among various fishing nets, cast net is the main
type of gear used in the valley (Sunder et al., 1978; Yousuf, 1996; Shabir,
Najar, & Ashraf, 2010) and these existing fishing methods in the Dal Lake
may be termed as primitive (Anonymous, Annual Report, 1977). The main
fishing gear used in the Dal Lake is the cast net which is made of six pieces
each with different mesh sizes (Anonymous, Annual Report, 1977). Cast
nets are used by hand force to enclose and catch fish as shown in Figure
9.1(b) and (c). These nets are used from the shore, dam, or boat (Nedelec &
Prado, 1989).
Gillnet is one of the oldest types of fishing gear and is widely used to
harvest diverse fish species (Sainsbury, 1996). Modern nets are made of
synthetic fibers such as polyamide and can be monofilament or multifila­
ment or a combination of both in case of more than one panel as shown in
Figure 9.1(d) and (e). But the gillnets are in general, considered as having a
high degree of selectivity, in terms of fish species, as well as the size of the
fish, which directly depends on the size of the mesh (Anonymous, 2000).
Technologically, gillnets are simple, easy to mend, require little in the way
of on-board equipment and are relatively cheap to purchase (Hovgard &
Lassen, 2000).
The other methods of fishing operated in Kashmir are the Scoop net,
Narsoo (multi-headed spear), rod & line and long lines (Anonymous, 1977;
Shabir, Najar, & Ashraf, 2010). Sunder et al. (1978); and Yousuf (1996) also
reported about the fishing techniques employed in the Kashmir Valley. They
reported about the use of nets mostly the traditional ones like Long Line
locally called wael raz, Hook & line and Harpoon (Narsoo). There has been
no improvement in such gears in Kashmir and as such efforts are needed
towards improvement of the gears and crafts used for making them more
economic and sustainable. The main fishing crafts used in the Dal Lake are
the wooden boats which are not more than 10 meters in length and one meter
in depth. The total fishermen population in the State is presently estimated
at 91,984 (Anonymous, 2013). About 15,000 professional fishermen derive
their livelihood from natural water resources (Anonymous, 2013).
The work done on gear and craft in Kashmir has been limited. Also, the
work on the efficiency of fishing gears including the catch composition is
Small-Scale Fisheries by Indigenous Fishing Methods 251

also negligible. The changing ecological condition of the Lake over the years
has also affected the fish catch and composition. It was with this background
that the present research work on the operation of different types of gears and
their fish catch composition were carried out.

(a) (b) (c)

(d) (e)
FIGURE 9.1 Representation of (a) harpooning; (b) & (c) operation of cast net; (d) and (e)
operation of gill net.

9.2 CATCH COMPOSITION

Catch composition of any fishing operation gives an idea about the varieties
of fish availability in that region, which in turn helps in better understanding
of the biodiversity. In addition to this type, fish quality and quantity landed
by any gear play an important role in determining its economic viability.
A total of nine species of fishes were recorded in the catches of cast net
and gillnet during the study period in Dal Lake. These included Cyprinus
carpio var. communis, Cyprinus carpio var. specularis, Carassius carassius,
Schizothorax niger, Schizothorax esocinus and Schizothorax curvifrons,
Crossochielus diplochilus, Tryplophysa spp., Ctenopharyngodon idella, and
Puntius conchonius presented in Figure 9.2. The commercially important
species were Cyprinus carpio var. communis, Cyprinus carpio var. specu­
laris, Carassius carassius, Schizothorax niger, Schizothorax esocinus, and
252 Coldwater Fisheries and Aquaculture Management

Schizothorax curvifrons. As many as 17 species of fish were identified in the

Lake by (Das, & Subla, 1963). A presence of Cyprinus carpio, Schizothorax
niger, Schizothorax esocinus, and Schizothorax curvifrons in Dal Lake. Along
with these some other fish species reported by Anonymous (1977) were
Barbus conchonius, Crossochielus latius, and Botia birdi (Shafi et al., 2005)
also reported the presence of seven species in Dal Lake namely Cyprinus
carpio var. communis, Cyprinus carpio var. specularis, Carassius carassius,
Schizothorax niger, Schizothorax esocinus and Schizothorax curvifrons,
Crossochielus diplochilus and Puntius conchonius. Bhat et al. (2010) reported
10 species from the Lake: Cyprinus carpio var. communis, Cyprinus carpio
var. specularis, Carassius carassius, Crossocheilus diplochilus, Schizothorax
esocinus, Schizothorax curvifrons, Schizothorax niger, Schizothorax labiatus,
Botia birdi, Puntius conchonius, and Gambusia affinis.

FIGURE 9.2 Image depicts species wise contribution to the total catch.

The analysis of fish catches during the study period has shown that
common carp is gaining predominance over the more acceptable indigenous
fishes of the sub family Schizothoracinae. The catch was dominated by
common carp, throughout the study period. By weight it contributed 63.45%
at Hazratbal and 56.88% at Bod dal basin. Due to prolong spawning period,
plenty of facilities (aquatic vegetation) to spawn and due to excessive rate
of reproduction of Common carp, the indigenous fish are losing their ground
Small-Scale Fisheries by Indigenous Fishing Methods 253

in the lake (Anonymous, 1977). Common carps have adaptive advantages

to utilize Lake resources for its growth and dominate over other species due
to its high fecundity (Das & Subla, 1963). The highest catch on the basis of
weight was seen in the month of May in both the basins using both the gears.
In May its percentage composition by weight was 72.89% (Cast net) and
86.83% (Gill net) at Hazratbal basin while as at Bod dal it was calculated
to be 67.65% (Cast net) and 76.14% (Gill net), respectively. This could
be attributed to the higher temperature and better food availability in the
summer months (Sunder et al., 1978; Yousuf, 1996; Shafi et al., 2005; Bhat
et al., 2010; Das, & Subla, 1964) reported similar observation in their study.
Carassius carassius was the second highest catch on the basis of weight
with average biomass of 43.04% at Hazratbal and 38.76% at Bod dal during
the study period. Highest catch in Hazratbal basin using cast net was recorded
in the month of March (28.75%) and in the month of Feb. (34.22) using
gill net. While in the Bod dal basin highest catch of 27.1% was recorded in
the month of May using cast net and 21.99% in the month of March using
gill net, respectively. Shafi et al. (2005) reported the presence of Carassius
carassius with 12.41% biomass and 20.55% by number. Their percentage in
the Lake has increased from the last decade.
Schizothorax niger was seen to contribute 3.86% by biomass during
the study period in Hazratbal basin while a higher proportion of 9.56%
was observed in Bod dal basin. The highest catch was seen in the month
of February in both the basins. The catch of S. niger declined towards the
summer months. In Hazratbal basin highest catch observed was 8.74% while
in Bod dal a slightly higher catch of 13.84% was observed. S. niger spawns
during March-April. S. niger does not show much breeding migration unlike
the other Schizothoracids but it becomes quiescent and inactive and thus,
is not much seen in the catches from March onwards. Anonymous (1977)
observed similar trends.
Schizothorax curvifrons showed a rare presence in Hazratbal basin while
it was caught in better proportion in Bod dal basin. Six samples of the fish
were seen in the catches of February in Hazratbal basin while no other
specimens were recorded for the rest of the study period in the said basin
contributing only 0.3% by biomass. However, a slightly higher contribution
of 5.64% was observed in Bod dal basin. Schizothorax esocinus was seen
to contribute 2.88% by biomass in Hazratbal basin and 4.40% at Bod dal
basin, respectively. This trend indicates the over exploitation of the fish in
Hazratbal basin. Also, the catch of the fish seemed to decline from February
onwards. The probable reason could be the breeding migration showed by
254 Coldwater Fisheries and Aquaculture Management

certain snow trout. Das & Subla (1963) also discussed that breeding migra­
tion of the fish starts from March to April towards the incoming streams
hence decreasing their catch in the Lake.
In the present study, common carps were seen to dominate the catch by
63.69% by biomass and 41.6% by number. Among these carps Cyprinus
carpio var. communis contributed 40.51% and Cyprinus carpio var.
specularis contributed 23.45%. The report by Anonymous (1977) also
indicates that Common carp dominated the catches in Dal Lake. Similar
trends can be seen in the study conducted by Shafi et al. (2005). According to
Shafi et al. (2005) Common carp dominated the catches by 69.13%. The two
varieties; Cyprinus carpio var. communis contributed 59.2% while Cyprinus
carpio var. specularis formed 9.11% of the total catch by weight. Sunder et
al. (1978) also found Cyprinus carpio to be the dominant contributor to the
total catch from this water body. The cyprinid species reported by Shafi et al.
(2005) included Carassius carassius which contributed about 12.41% of total
catch, C. diplochilus with 4.35% of the total catch. The three Schizothoracids
contributed 14.6% with S. niger being the main contributor with 10.37%.
In the present study, Carassius carassius contributed 20.50% by weight
showing increased abundance in the water body. Crossochielus diplochilus
was found to contribute 3.25% showing a slight decline comparatively
while Schizothoracids were seen to contribute 10% of the total catch with
S. niger being the dominant contributor with 5.25% by weight. The native
Schizothoracids show a declining trend. The deterioration of the Lake water
has shown a profound effect on the ichthyofauna of the Lake, registering
a sharp decline in the fish catches to the extent of 25–35% (Sunder et al.,
1978). The introduction of common carp (Cyprinus carpio) also has severely
affected the indigenous schizothoracid population of the Lake (Saxena &
Koul, 1966; Das & Subla, 1967; Qureshi et al., 1971). More than 65% of the
present day fish catches from the Lake comprises of common carp, whereas,
the endemic schizothoracids contribute only about 20% (Sunder, 1995;
Shafi et al., 2005). Their abundance is seen to be diminishing in the water
body. Month wise analysis showed that the maximum productive month in
the Lake in both the basins was May using both the gears. Thus, it can be
concluded that the same varieties of fish were caught all along the two basins
by gillnet as well as cast net and the dominance of Common carps persists
and our natives species are diminishing in catches.
During the study, wooden plank-built boats were recorded with an overall
length (OAL) range between 5 m and 10 m and width range between 0.5 m
and 0.75 m. These crafts were made up of locally available wood-Deodar,
Small-Scale Fisheries by Indigenous Fishing Methods 255

which is costly but durable. It does not decompose in water and remains
sturdy throughout. Locally it is known as Naav. They are often navigated by
two boatmen. No modifications have been seen as such in the crafts used.
The gears and methods mainly used for fishing in Kashmir valley are
long line, Hook and Line, Scoop net, Rod & line, Narsoo, Cast net, Gill net,
and Trap. Among various fishing nets, cast net is the main type of gear used
in the valley (Sunder et al., 1978; Yousuf, 1996; Shabir, Najar, & Ashraf,
2010). The main fishing gear used in the Dal Lake is the cast net which
is made of six pieces each with different mesh sizes (Anonymous, 1977)
Similar observations were seen in the current study.
Several types of fishing gears were seen in Dal Lake for fish harvesting,
such as, Cast net is a throw net made up of three parts: the upper section
(net band), the middle section (a conical-shaped net mesh), and the lower
section. The lower section of the net was seen to have pockets with fixed
iron weights. One net is operated by one person. No fish attractive device
used. The size range used is between 1.0 and 2.0 m in diameter. Length of
cast net, when hung is 4.0 m and diameter of the net when spread is 4.8 m.
The mesh size varies between 1.2 and 3.0 cm bar to bar. The net is provided
with iron or lead sinkers of about 8–10 kg weight around the peripheral cord.
They are cylindrical in shape with a length of 17 mm and diameter of 10
mm. This net can only be operated from a boat. The life expectancy of a cast
net is 3–4 years. It costs around Rs. 2,500 for its construction. Depending
upon the number of meshes the cast net is known by different names such
as: guran zaal (1,200 meshes), thapthat zaal (1,100 meshes), dal zaal (1,000
meshes), Naushath zaal (900 meshes), nuchkul zaal (800 meshes), pouchkul
zaal (500 meshes) and Ara zaal (400 meshes). Among these noushath zaal,
guran zaal and dal zaal are used in Dal Lake. They locally call it duph or
zaal. No detailed work had been done in this regard. Sunder et al. (1978);
Yousuf (1996); and Shabir et al. (2010) mentioned cast net as the main gear
being used in water bodies of Kashmir (Anonymous, 1977) gave some
further details regarding the structure which have not changed much till date.
Anonymous (1977) mentioned a six-piece cast net each with different mesh
sizes as the main fishing gear being used in Dal Lake.
Gill nets locally called shaitan zaal in Dal Lake are 15 to 40 m long and
1.5 to 3 m wide with mesh size ranging between 45 mm and 75 mm. Gill nets
used in Dal Lake are made up of thin nylon monofilaments usually sea green
in color. They have a short life expectancy of just two to four months, after
which they have to be replaced. Locally made sinkers are used. The use of
these nets has seriously affected the regenerative capacity of the fish fauna. It
256 Coldwater Fisheries and Aquaculture Management

has been observed that the communities themselves had imposed restrictions
on the use of lower mesh size nets due to decline in fish catch. Promotion
of gill nets was also undertaken by the State Government Department at
select locations but due to declining fish catch in the Lake, the Department
of Fisheries has imposed a ban on gill nets. Despite the fact, gill netting is
still being practiced in some parts of the water body, especially in the winter
months due to low catch.
Long lining locally known as ‘wael razz’ is a primitive method of fishing
in which a nylon line measuring about 1,000 meters in length is generally
used in the Lakes. In Dal Lake long lines of length 30–100 m is used. In Dal
Lake the line fishing is mainly done from June to September months. Rod
and line (Bislai) is also an important gear used in Dal Lake. It is usually
practiced in the warmer months of May, June, July, and August.
In fishing accessories fishers use a long wooden pole called Shum, which
is made up of wood mainly Daevdor timber (budhul), it is used to handle
the net to catch the fish; its length is 10–12 ft. Fishers use it while operating
cast nets as well as gill nets. It is an essential tool used while fishing. It helps
in searching suitable fishing grounds for using cast net and also in reaching
back the net once it is thrown in the water body.

9.3 CONCLUSION

For increasing fishing efficiency in reservoirs and lakes, replacement of age

old fishing crafts and gears by motorized ones. However, no mechanized
fishing methods could be recommended because of scientific reasons. Most
of the fishermen of Dal lake are still using traditional fishing craft and
gears because of which are obsolete because of their inefficiencies. Fishers
complained of a decline in revenue generation during the last few decades.
The most important problem faced by the fishers was decline in fish catch
and that was the greatest problem to the fishers, because they do not have
enough fish to catch. The second vital problem was lack of capital for the
purchase of fishing crafts and gears. It can be concluded that cast net is an
appropriate fishing gear operating in the Lake throughout the year. Gill net
can also be an economic gear provided the appropriate mesh size is regulated.
It can prove to be more time and energy saving. It may be summarized that
fish catch in the Lake has declined gradually due to pollution, indiscriminate
gear use, and lack of management policy.
Small-Scale Fisheries by Indigenous Fishing Methods 257

KEYWORDS

• biodiversity
• catch composition
• Dal Lake
• fishing gear
• indigenous fishing gear
• overall length
• Schizothorax niger

REFERENCES

Anonymous, (1977). Annual report, Published by CIFRI. Report on Dal Lake for Development,
Srinagar, Kashmir with Suggestions for Development. Bulletin no. 24.
Anonymous, (2000). Fisheries Research, International Council for the Exploration Sea (ICES).
In: Walsh. S. J., Engas, A., Ferro, R., Fonteyne, R., & Marlen, B. V., (eds.), Improvement of
Fishing Technology to Catch (or conserve) More Fish: The Evolution of the ICES Fishing
Technology and Fish Behavior Working Group During the Past Century.
Anonymous, (2013). Published by Economic Survey J&K, p. 124.
Bhat, F. A., Balkhi, M. H., & Yousuf, A. R., (2010). Fish diversity in Jammu and Kashmir and
conservation measures. Kashmir Speaks, 104–115.
Dar, S. A., Desai, A. Y., Rather, A. M., Sayani, A. N., Parmar, R., Arjamand, S., & Chesti, A.,
(2014). Fishing gears operated along the Wular Lake, Jammu and Kashmir, India. Ecology
Environment and Conservation, 2, 45–47.
Das, S. M., & Subla, B. A., (1963). The ichthyofauna of Kashmir—History, topography,
origin, ecology and general distribution. Ichthyologica, 1, 68–106.
Das, S. M., & Subla, B. A., (1964). The ichthyofauna of Kashmir, Part II. The speciation of
Kashmir fishes. Ichthyologica, 3, 57–62.
Hovgard, H., & Lassen, H., (2000). Manual on Estimation of Selectivity for Gillnet and Long
Line Gears in Abundance Surveys (p. 397). FAO, Fisheries Technical Paper No. 2000.
Nedelec, C., & Prado, J., (1989). FAO Catalogue of Small-Scale Fishing Gear (p. 224).
Blackwell Science Ltd., Oxford.
Qureshi, M. Y., Singh, J. P., & Das, S. M., (1997). On the Problem of Depletion of Endemic
Fishes of Kashmir by the Introduction of Exotic Carp (Cyrprinus carpio) (pp. 118, 119). All
India symposium on icthyology and hydrobiology and fisheries.
Sainsbury, J. C., (1996). Comemercila Fishing Methods (3rd edn., p. 310). Oxford: Osney Mead.
Saxena, D. B., & Koul, D. N., (1966). Fish and fisheries of Jammu and Kashmir State, part 1.
fisheries resource and problems. Ichthyologica, 5, 45–52.
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Shabir, A. D., Ferooz, A. B., & Balkhi, M. H., (2015). Study on different fishing methods (gear
& craft) used in Manasbal Lake of Kashmir, Himalaya. J. Himalayan Ecol. Sustain. Dev., 10.
Shabir, A., Najar, A. M., & Ashraf, M., (2010). Indigenous Technical Knowledge (ITK) in
Fisheries Sector in Central and Northern Region of India (p. 165). Narendra Publishing
House, New Delhi.
Shafi, S., Bhat, F. A., Parveen, M., & Yousuf, A. R., (2005). Catch composition of fish from
Dal Lake, Kashmir. Journal of Research and Development, 5, 111–114.
Sunder, S., (1995). Some conservation and management strategies for Dal Lake Fisheries.
Punjab Fisheries Bulletin, 19, 53–63.
Sunder, S., Bhagat, M. J., Joshi, C. B., & Ramakrishna, K. V., (1978). Fishing methods and
fish catch composition of Dal Lake, Srinagar (J&K) during 1969–1972. J. Inland Fish Soc.
India, 10, 9–18.
Yousuf, A. R., (1996). Fishery resources of Kashmir. In: Khan, A. H., & Pandit, A. K., (eds.),
Ecology, Environment and Energy (pp. 75–120).
CHAPTER 10

Reproductive Physiology and Breeding

Biology of Rainbow Trout (Oncorhynchus
mykiss)
YOUNIS AHMAD HAJAM1, DISKSHA1, RAJESH KUMAR2, and
MOHD. SALIM RESHI3
1
Department of Life Sciences and Allied Health Sciences, Sant Baba Bhag
Singh University, Khalia Padhiana, Jalandhar, Punjab, India
Department of Biosciences, Himachal Pradesh University, Shimla,
2

Himachal Pradesh, India

3
Toxicology and Pharmacology Laboratory, Department of Zoology,
School of Biosciences and Biotechnology, Baba Ghulam Shah Badshah
University, Rajouri, Jammu and Kashmir, India

ABSTRACT

The rainbow trout (Oncorhynchus mykiss) is a trout and species of salmonid

native to coldwater tributaries of the Pacific Ocean in Asia and North
America. Freshwater forms that have been introduced into the Great Lakes
and migrate into tributaries to spawn are also called steelhead. Rainbow
Oncorhynchus mykiss trout likes to live in cold water and are mostly found in
lake, reservoirs, rivers, etc., and rainbow trout are also considered as fluvial
populations in the whole forest. The population of fluvial of rainbow trout is
not figured out clearly because they do not refer to the migration into rivers
to spawn lives into larger drainage. Female partner prefers to make their nest
where male partner protects it from other interested males and predators. The

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
260 Coldwater Fisheries and Aquaculture Management

female digs the nest (a redd) by using her anal fin and then descends sits in a
way so that her vent and anal fin goes into the deepest part of the redd. The
male partner joins her parallelly so that their vents become opposite to one
another. Both the partners open their mouth, arch their backs, and deposit the
eggs and milt (fish sperm) at the same time. The eggs are enclosed by a cloud
and the fertilized. However, the reproductive processes of the Rainbow trout
are affected by various environmental factors and changes the internal endo­
crine environment of the fish. Therefore, Rainbow trout, has received wide
research consideration. This chapter summarized the reproductive patterns
of Rainbow trout and impact of environmental and hormonal stress on the
reproduction of Rainbow trout.

10.1 INTRODUCTION

Aquaculture is also known as aquafarming. Fish, crustacean algae, etc.,

are the organism that lives in this, and the high quality of both progeny
and gamete shows its success rate (Bromage, 1995). At the time of brood
selection, some characters should be analyzed such as eggs spawned, size
of an egg and its mortality, size, and mortality percentage of fingerlings
(Leitritz & Lewis, 1980). High-quality gametes are required but some­
times it creates a stressful environment for the broodstock (Billard et al.,
1981). Transportation, handling, cleaning, crowding, water quality, etc.,
are some general practices. But sometimes, unfavorable water also affects
the reproduction rate by acting as stressors (Billard et al., 1981; Bromage,
1995). Pottinger & Pickering (1990) reported that the fish reproductive
system is also influenced due to both acute and chronic environmental
stresses. The gamete quality is also affected due to all these stressors
(Campbell et al., 1992, 1994). The various researcher reported in previous
years that there is significant decrease was observed in the survival of
Oncorhynchus mykiss and egg size as well constantly (Campbell et al.,
1992, 1994). Bromage (1995) studied that the proper management of
broodstock also helps in increase in the progeny performance and eggs
quality by providing husbandry activities. The period, in which females
use huge energy for gamete growth till its maturation and the process is
known as vitellogenesis (Tyler et al., 1990; Rather et al., 2016). Therefore,
stressor affects the reproductive processes during vitellogenesis because
lots of energy is required for this process and stressor divide this energy
in two ways.
Reproductive Physiology and Breeding Biology of Rainbow Trout 261

10.2 BIOLOGY AND ECOLOGY

10.2.1 SYSTEMATICS/TAXONOMY

The detailed study of rainbow trout and other salmonids including taxonomic
description was described by Behnke (2002), and for this, these rainbow
trout were named Oncorhynchus mykiss. On the other hand, they have about
three ecological forms (anadromous, stream resident, and lake-dwelling) of
rainbow trout, among these anadromous also classified as Oncorhynchus
mykiss irideus (Behnke, 2002). Behnke (2002) reported that there are some
subspecies of rainbow trout, i.e., red-band trout, coastal rainbow trout, and
Gulf of California (Mexico) based on their appearance and life history as
well (Behnke, 2002).

10.2.2 IDENTIFICATION

Behnke (2002) studied the identification of rainbow trout. It is silver-gray

tom greenish brown in color on the back. It has a white color belly on the side
in stream-resident form. It has lateral strips of pink or reddish color. But in
some cases, lavender and connotations from gill covering (the whole length of
fishtail). Behnke (2002) also reported that in caudal fins small dark spots are
having on both dorsal and adipose fins as well and the lower fin color is pale
pink. In some cases, black spots saw on the head and their sides. The color
becomes deep in males with intensive red side strip at the stage of spawning.

10.2.3 MOVEMENTS AND ACTIVITY PATTERNS

Generally, rainbow trout likes cold water, so they are found in lakes,
reservoirs, rivers, etc., and rainbow trout are also considered as fluvial
populations in the whole forest. The population of fluvial of rainbow trout
is not figured out clearly because they do not refer to the migration into
rivers to spawn lives into larger drainage such as upper Taylor River, lower
Taylor River, Cimarron River, San Miguel River, etc., researchers studied
the salmonids life-history adaptation mostly Anadromous fish species. The
various researcher reported that the movement of juvenile and adult straying
play an important role that allows anadromous populations for catastrophic
turbulences continuity (Reeves et al., 1995). In anadromous adaptation,
fluvial behavior plays an important role. It is an important mechanism by
262 Coldwater Fisheries and Aquaculture Management

which a healthy population of rainbow trout forms. Generally, fishes like

dynamic conditions and are patchy as well so they are preferably found in
that particular forest. Sustainability during the drought periods is successful
by proper movement of rainbow trout and its straying when spawning allows
“meta-population.” It also forms due to change in habitats during mainte­
nance. There are no restriction forms when fishes move in the water bodies.
But in some cases, like man-made barriers or sometimes natural barriers
trouble them. In 2004, in the forest, about 200 streams were found in crossing
sites and about 11% found in elegiacal due to the aquatic organisms’ move­
ments, also increase and decrease in velocity and depth, respectively make
trouble in fish movement. However, annually about 1–2 stream “crossing
improvement projects” were established in the forest, and in 2001–2007
habitat intensive inventories were covered completely up to 62.3 miles
from 224 reaches. There are three protocols for stream habitat assessment
were designed for the measurement of its parameters. The parameters are
stream condition inventory (SCI), R1/R4 method of fish habitat inventory,
and Pacfish In fish Biological Opinion (PIBO) (Frazier et al., 2005; Overton
et al., 1997; Heitke et al., 2006), and in material and methods they are all
slightly different from each other. But a core set of habitat variables equally
measured that already identified by hydrologists and fisheries scientists and
habitat conditions were also recorded from the entire forest that helps to
represent the area of the stream where rainbow trout could not occupy. There
are different ways to studied about the rainbow trout habitat conditions and
the requirements forms during reproduction, and about 1–7% gradient found
in a stream in the forest found in previous data. Also, another data indicates
less Bankfull width (BFW), i.e., about 5.5 m (n = 216), in which about 90%
samples show the minimum range of BFW, i.e., 1–10 m. Two trout forms,
i.e., brook trout and cutthroat trout are well documented found in headwa­
ters. However, the distribution of trout occurs in western mountainous areas.
Meanwhile, brown trout and rainbow trout are the forms of trout that are
found in mid and lower elevation sections or large rivers (Rahel & Nibbelink,
1999; McHugh & Budy, 2005), and in these rainbow trout are distributed in
lower gradient and elevation sections with large width in the whole forest.
About 3–100 mm range forms in spawning gravel in rainbow trout (Orcutt
et al., 1968; Raleigh et al., 1984), however, substrate size depends upon the
spawned size. So, according to that, we can say that rainbow trout have about
50 cm length and the diameter of spawning gravel about 15–100 mm ranges
(Orcutt et al., 1968). A data (pebble count) was collected from 210 stream
reaches that showed the spawning substrate size ranges, i.e., 3–100 mm that
play an important role to constitutes about 56% of substrate because the
Reproductive Physiology and Breeding Biology of Rainbow Trout 263

substrate size is ranging from 15–60 mm particularly that refers the composi­
tion of substrate, i.e., 44%. In this study, the biologists reported that there
is an availability of spawning gravel in the entire forest that are adapted by
trout mainly in large streams. A data that the measurements of fine sediment
from pool tails revealed that “percent fines” lower than 2 mm that comprise
a huge percentage of typical spawning sites and also 189 reaches showed
results in its favor that areas suitable for Spawning comprise (20% fines
less than 2 mm). Raleigh et al. (1984) reported that for rainbow trout, the
optimum spawning condition may be less than 5% fines because generally,
fines have about 30% spawning gravel, decreasing the embryo survival rate
found (Raleigh et al., 1984). So, for the survival of rainbow trout, the fine
sediments are the limited factor revealed in the data.
About 12–18°C temperature is required for Oncorhynchus mykiss
(Rainbow trout) reported in different studies. Also, the increase in mortality
occurs with an increase in temperature (25°C) (Raleigh et al., 1984).
Oncorhynchus mykiss (Rainbow trout) requires a particular temperature and
water temperature in June–September period on which different data are
available. After September, the water temperature gets decreases, i.e., 0°C
from November to March. In the winter season, the feeding process in fishes
(in adult rainbow trout) decreases with a decrease in metabolic activities.
Due to this they could not grow properly or were negligible in extremely
cold water studied by various workers (Raleigh et al., 1984). In the winter
season for the survival of trout, the proper density of the pool and its depth
play a significant role. Mainly, fish survive in the pool in small streams that
seem a key factor that affects its diversity and trout abundance as well, the
pool depth is about 1–2 m to maintain its good quality (Raleigh et al., 1984).
Generally, in the system, 0.02–1.61 depth of pool ranges, an average it is
0.32 m. In large streams, about 1 m depth of the pool is required minimum
and if it became less than it (≥1 m) then trout survival rate also become
decreased. It particularly occurs when low flow conditions are there in the
winter season. For the maintenance of trout, the population pool should have
a suitable depth. Different streams on the forest could not have efficacy suit­
able depth of the pool. It is formed due to water production, basin area, etc.

10.3 BREEDING BIOLOGY

Raleigh et al. (1984) reported that spawning occurs at particular ‘natal

streams’ that form comparatively low straying incidence. The maturity of
rainbow trout occurs when they are 2–3 years old and the length of the trout is
264 Coldwater Fisheries and Aquaculture Management

100–150 mm revealed from a data. At the stage of maturity, females dig nests
commonly known as redds. Females used it for the deposition, fertilization,
and covering with gravel of egg. Mostly female forms these redds having
proper depth and velocity and bottom configuration that induced flow of
water through stream substrate (Young, 1989). Spawning of rainbow trout
season, i.e., mid-April to late June having temperatures 7–12°C, respectively
(Raleigh et al., 1984). About 28–49 days is the incubation period of rainbow
trout that further varies according to the temperature of water (Raleigh et
al., 1984). Bjornn & Reiser (1991) reported that after the hatching of larvae
they stay up to a weak in redds, and in that period they develop continuously.
There are two stages in which rearing is processed in a different way, i.e.,
summer rearing stage and the winter rearing stage in which various methods
of behavior are displayed. The various researcher reported that adult rainbow
trout come out from the gravel riffles in late winter and early springtime
(temperature get increased). They seek out appropriate spawning sites
(Raleigh et al., 1984; Bjornn & Reiser, 1991). There is no feeding activity
found in the winter season due to which trout gets aggregated rapidly, mostly
at limited pool habitat (Bjornn & Reiser, 1991). The temperature of the water
plays an important role in behavior change. It inhibits the energy and its
metabolic rate. In summer, due to rearing habitat destruction, the survival
rate of fish becomes decreased as compared to another season also the repro­
duction rate is very low and the fecundity rate varies and at the early stage,
predation is severe (Root, 1994). About 3% rates of survivorship were found
in trout at post-emergence period reported in a previous study (Pender &
Kwak, 2002).
In the Salmonidae family, Oncorhynchus mykiss is the species that
increase their population rapidly because they adapt themselves according
to the system (Bud et al., 2007). Cocan et al. (2013) reported that rainbow
trout breed artificially in Romania. So, to evaluate all the systems of artificial
breeding, all the technology and activities should be streamlined according
to the selection of the broodstock program. The various researcher studied
that in the salmonid unit, breeding plays a significant role because it is a
method of technological growing flow that effect by environmental condi­
tions and nutrition also (IHUŢ et al., 2015). Age, weight, and structure also
affect the formation of broodstock because brood quality is related to the
survival rate of offspring, its development, and disease resistance (Kayam,
2004). So, it was also reported by different researchers that for random
selection, there is a requirement of breeding stock (Mireşan et al., 2013).
For the better improvement in the selection aspect, utilization of correlation
Reproductive Physiology and Breeding Biology of Rainbow Trout 265

occurs in phenotypic characters and breeding indices as well, and in between

breeding indices and somatic features (Cocan et al., 2013).

10.4 REPRODUCTIVE ENVIRONMENT IN RAINBOW TROUT

10.4.1 OVARIAN FLUID OF RAINBOW TROUT A STANDARD MEDIUM

FOR FERTILIZATION

The various researcher reported salmonids in-vitro fertilization that shows a

positive effect on ovarian fluid for Caspian brown trout (S. trutta caspius) and
brown trout (S. trutta f. fario) as well (Hatef et al., 2009; Lahnsteiner, 2002).
But some researchers recommend that the ovarian fluid can be washed out
when the fertilization process occurs artificially especially in rainbow trout,
because some negative effects are also seen by the researchers in the case
of sub-fertile females (Hugunin et al., 2008). They explained as fertilization
can be fertilization by impeding the movement of sperm and contact with the
egg. Rime et al. (2004) reported that the substance released from it is associ­
ated with this released substance from the overripe egg. Different authors
also accredited the outcomes of fertilization discrepancies towards the post-
copulated effects that designed to select the best sire, i.e., cryptic female
choice and especially Butts et al. (2012) reported the spermatozoa perfor­
mance when ovarian fluids get activated in females. Various researchers also
studied that some factors affect ovarian fluids such as pH of maternal fluids
and almost 40% variability caused by the factors like iron, carbohydrates, and
proteins (Wojtczak et al., 2007). It was also reported by other researchers that
in Chinook salmon the performance of protozoa affected in different female
ovarian fluids (Rosengrave et al., 2008), and similarly a case was reported
by the researchers that the ovarian fluid in the fertilization medium does not
form towards “cryptic female choice” in Arctic charr (Kleppe et al., 2018).
The experiment that was carried out showed that the fertilization medium
prepared by ovarian fluid also helps to increase sperm performance. At the
initial stage, this medium was kept in the water as experimental work and
that showed about 20% low motility in female albino rats and the number of
fertilized eggs was 1,50,000:1 (spermatozoon-egg ratio) which was very low.
Rosengrave et al. (2008); and Myers & Cassinelli (2020) also studied this and
present observation supports their results that if experiment occurs in water,
in which, in salmonids, it emphasized the inadequacy of pre-fertilization
motility estimation. In both activation medium and water, the motility rate
of sperm was observed and resulted that the rate being low in regular rate as
266 Coldwater Fisheries and Aquaculture Management

compared to the albino rats and higher in water, respectively. The number of
colored eggs embryos increased significantly when ovarian fluids are washed
in albino female eggs, but on the other hand, if this fluid was utilized as an
activation solution with the use of isosmotic saline, then showed positive
results in both males and this showed that the performance of spermatozoa
was triggered through non-uniformity of fertilization environment through
ovarian fluid confining the batch of eggs. Also, when the fertilization medium
is ovarian fluid then it also increases the embryos from these males. Because
the ovarian fluid helps to enhance the spermatozoa performance that further
wins the competition among the sperm. In the fertilization medium, the ratio
of spermatozoa and egg was 1,50,000:1, and the ratio increase 10-fold to
1,500,000 spermatozoa per egg shown in the cases of different experiments. It
was also observed in in-vitro fertilization conditions, the association of small
and dependencies which is due to huge male gametes and the formation of any
relationship without proper knowledge about the condition required for it may
cause an effect on gametes (the gamete ratio), so the in-vitro process shows
positive effects and shows maximum fertilization. Some aspects considered
below by us on the sperm behavior having potential that understands the
mechanisms of the process.

10.4.2 RAINBOW TROUT OVARIAN FLUID ENHANCES THE KINETIC

TRAITS OF SPERMATOZOA

Male gamete plays an important role in successful fertilization. It carries

genetic material (DNA). It fuses with the female gamete that further changes
the features of spermatozoa. Different researchers reported that this change
becomes a true velocity in both (single male and the male having no completion
among them) and also in case of competition in sperm (Lahnsteiner et al.,
1998; Levitan, 2000). The survival rate of spermatozoa is more among
them that have huge genetic contributions and vertebrates that are fertilized
externally showing sperm competition worldwide, particularly in the fish
(Taborsky, 1998). Salmonids are species that have very tough completions
in sperm due to variations in the tactics from spawning in pairs. Various
researchers reported significant velocity of spermatozoa in a salmonid (Gage
et al., 2004; Liljedal et al., 2008). Moreover, Gage et al. (2004) studied that
for sperm completion, sperm velocity plays an important role by increasing
the winner spermatozoa velocity that penetrated more frequently with the egg
micropyle. Different researchers reported that ovarian fluid plays an important
role to enhance the performance of spermatozoa in the activation medium
Reproductive Physiology and Breeding Biology of Rainbow Trout 267

with an increase in velocity (Lahnsteiner, 2002; Poli et al., 2019; Butts et al.,
2012; Litvak & Trippel, 1998; Turner & Montgomerie, 2002; Rosengrave et
al., 2009). Various workers studied that there was an increase in the number
of spermatozoa in 20% water solution of ovarian fluid as compared with
unrelated male gametes or lake trout Salvelinus namaycush (Butts et al.,
2012). But in a similar study carried out by Rosengrave et al. (2009) cannot
find that any kind of clues represents the absence of correlation in water and
ovarian fluid. In the present work, it was confirmed that the ovarian fluid
plays a significant role that affects the kinetic features of spermatozoa than
the water activation in rainbow trout. Also, Turner & Montgomerie (2002)
reported that there was no clue of an increase in curvilinear velocity (10 s
post-activation) for salmonid and Arctic charr species, but Poli et al. (2019)
reported the same case in Zebrafish (Danio rerio). When spermatozoa
velocity decreases then the post-activation process also gets reduced in the
presence of ovarian fluid. When dilution forms in plain water then only some
cases are there that show differences in about 5% aqueous solution of ovarian
fluid. Similarly in isotonic saline, the velocity of spermatozoa activated that
different from ovarian fluid, in which the initial velocity was higher in saline
than that of water or in ovarian fluid as well. However, the spermatozoa can
activate in ovarian fluid much faster than that of water solution. Moreover,
there was no significant difference in the velocity when ovarian fluid was
diluted with the isotonic saline. In the propagation of spermatozoa, motility
tracks show its essential role in addition to the velocity, in which ovarian
fluid shows an effect on male gametes’ path. Also, this was observed by
different workers that the activation of spermatozoa in the ovarian fluid found
in rainbow trout (Dietrich et al., 2008). There was symmetrical propagation
shown with the flagellum and in water, it shows the non-symmetrical flagellar
wave of spermatozoa, with a decrease in the ovarian fluid concentration
the specific trajectories also get increased as turn and run pattern. These
were present in almost even 2%, 5%, and 10% but they do not recognize in
“non-diluted ovarian fluid” (up to 50% dilution), so it was observed that it
was not the features of sperm when activated in isotonic saline. The ovarian
fluid molecular weight cut-off fraction was only 100+kDa that induced a
pattern of motility and showed no significant difference. There were circular
trajectories in other molecular weight cut-off fractions. In which they show
significant results in both (ovarian fluid and 100+ kDa fraction). Alavi et al.
(2019) reported that osmolarity plays a significant role as a driver of fish
spermatozoa motility that presents in freshwater because both (osmolarity
and calcium ion concentration) have a cross effect on motility traits. They
also reported that there was a difference between the outer media initializer
268 Coldwater Fisheries and Aquaculture Management

and inner media initializer (fraction membrane orchestra of channels) thus

motility becomes triggered. Alavi & Cosson (2006) also studied that in
spermatozoa, calcium ions (an integral part of some physiological processes)
are responsible for the initiation of motility and their progress.
In our experiments on the cross-effects of osmolarity and Ca2+ concentra­
tion, we varied these indices to identify which among these isolated factors
or combination of them may be responsible for the chemokinetic effects
of spermatozoa when contacting the ovarian fluid. Some examples show
that osmolarity and calcium ion concentration both play their role to affect
the path linearity (individually as well as in combination form), i.e., when
calcium ion is absent then osmolarity increases (0–300 mOsm/L). When the
calcium ion concentration was intermediated the path linearity depending
upon the medium of osmolarity and both Calcium ion concentration and
osmolarity corresponding towards the ovarian fluid, controlled motility traits
that further showed the same ovarian fluid effect but osmolarity signally
shows more effective results.

10.4.3 THE OVARIAN FLUID CAUSES THE ATTRACTION AND

TRAPPING OF SPERMATOZOA

Inactivated spermatozoa media, with the help of fluid-filled micro-injection,

play an important role to copy exactly interface found in between water and
ovarian fluid as well which mainly surrounds the egg, and when male gamete
and maternal fluids interact with each other than male gamete behavior get
changed, it showing different behavior as is in a uniform environment. The
experiment also shows that spermatozoa can show a positive response for
non-uniformity in the medium. It gets trapped and found some conditions such
as pH, osmolarity, and ionic content also and all these conditions are caused
by ovarian fluid. It is also helpful to detect the factors that affect mostly the
response of spermatozoa. The positive results were found in a plain area of
trapping and taxis in the experimental trial. And the spermatozoa of rainbow
trout are activated in both water and as well as in isosmotic saline and the
effects form more effective when the molecular weight becomes decreased
and towards thermos-treated ovarian fluid. Somewhere it is caused by active
agents’ spatial distribution from injected fluids having low viscosity from
the ovarian fluid. In some cases, it was noted that in both mediums (isotonic
medium or low molecular fraction) in the ovarian fluid was injected in
water. After that spermatozoon get separated into different populations, in
which some of them showed a “hyper activation-like-motility pattern” (close
Reproductive Physiology and Breeding Biology of Rainbow Trout 269

towards the border of the injected cloud or near to microcapillary tip). About
1 mmol/L Ca2+ was observed in isosmotic saline and distilled water at the
time when the motility pattern changes. Finally, the positive results were
observed of taxis for ovarian fluids from a complex set of reactions of the
spermatozoa of rainbow trout for the different factors present in the fluid
and also the change in the movement of spermatozoa comes in an optimal
environment and non-optimal temperature as well. When ovarian fluid and
isotonic saline-injected then due to its trapping behavior, this is also exhibited
(where optimal motility conditions have been developed) and it increases
straight motion period. On the other hand, negative behavior (negative taxis)
found when distilled water injected in the medium (in this, males avoided
entry in the medium), so it can be concluded from this behavior that the
spermatozoa of rainbow trout are very sensitive to the provided environment
that allows to change its direction also and only live in the conditions that
provided by ovarian fluid. Different researchers reported that there were
similar effects on both the effect of ovarian fluid on spermatozoa and in
extreme fertilized species. Various researchers reported that rainbow trout
spermatozoa show good performance when in contact with female fluids as
compared to the invertebrates found in marine habitats such as sea urchins,
ascidians, and squids (Hirohashi et al., 2013). The behavior is considered
more spectacular as it is similar which is in rainbow trout form turn and run
loops through asymmetric flagellar bending. It also occurs in a segment of
flagella that is bent and forms like a burst rise in Ca2+ concentration. A study
was carried out by different researchers that this process also helps to notice
the chemotactic activity of ascidian spermatozoa attached with chemotactic
receptors of the membrane (Yoshida et al., 2018). The symmetry of the
flagellum of spermatozoon is changed when in rainbow trout spermatozoa
the intraflagellar Ca2+ increased in all marine invertebrates (sea urchins,
ascidians, and squids) and in path curvature. The concentration of Ca2+ found
constant when path curvature is constant (either when in the ovarian fluid
it run straight-forward in water. Kaupp (2012); and Yoshida et al. (2018)
reported that all the theories and experiments of “spermatozoon chemotaxis”
are mainly based upon certain investigations that were carried out in marine
invertebrates. The researchers also studied that in different fishes that are
discussed above, the Pacific herring was well studied in sperm-egg encounters
only that further works on the model for better activity and sperm entry into
the egg (Yanagimachi et al., 2017). The spermatozoa have a unique feature
that it survives for an hour to some days till the activation. Mainly, males
release their sperm into the water. Further, it is attached to the female and
then fuses with the egg. A specific protein “sperm motility-initiation factor”
270 Coldwater Fisheries and Aquaculture Management

(SMIF) was used to activate the male gamete. The protein is attached to the
ovarian fluid and in the egg membrane (Inagawa et al., 2001). The different
researchers reported that interaction occurs in the membrane receptor and
activator molecules that are followed by activation of different molecules
(adenyl cyclase, potassium, and calcium), and also the attachment of Camp
and influx of potassium and calcium ions (Yanagimachi et al., 2017), when
it was activated, through micropylar sperm attractant (MISA) protein the
spermatozoa bind with micropyle area (Yanagimachi et al., 2013). Also,
Yanagimachi et al. (2017) reported that this binding occurs due to the action
of Ca ion channels and proton pumps. These help to control the calcium ion
concentration. As similar to the pacific herring, the activation of spermatozoa
in water is less in rainbow trout. But in an actual way, it occurs in the majority of
freshwater species. Female produces eggs with ovarian fluid and the motility
of rainbow trout spermatozoa is changed due to ovarian fluid. The “turn and
run” process is carried out by the spermatozoa in diluted ovarian fluid and
forms positive taxis. Therefore, the cell that includes it gets trapped. When
a cell with plenty of water reached the border then they back to its position
also. The asymmetric band that is present in the flagella helps to process the
activity. In the bent segment, the concentration of calcium ions is increased
and presumably formed attachment in spermatozoa and eggs. In Pacific
herring and marine vertebrates (sea urchins, etc.), some same similarities
were found that are described for them. This also helps to suppose that some
basis for some chemotactic response was recognized in the broader spectrum
that already formed. The interaction between the egg and sperm in rainbow
trout has been attached through particular characteristics of the spawning
process.
Some tests were carried out on freshwater spawning fish and rainbow
trout to study the gamete features and the results showed that female fluids
play an important role in it by increasing male gamete performance showing
spawning behavior in trout species. It does not show any agent particularly
that affects the motility of spermatozoa. The main aim of this is to identify
the natural agents that are present in the male gametes and the different
mechanisms formed in the process of reproduction that show a significant
role in evolutionally development biology. The processes are well adapted
for the particular environmental conditions with modification (tactics and
reproductive strategies) due to their different ecological niches. Fishes are
good for experimental work in both in-vivo and as well as in in-vitro condi­
tions especially for the freshwater species because they have limited time
for fertilization. Therefore, the future study of gamete encounters helps to
understand the reproduction evolutionary aspects, behavior, and vice versa.
Reproductive Physiology and Breeding Biology of Rainbow Trout 271

10.5 REGULATION OF REPRODUCTION BY ENDOCRINE SYSTEM

IN RAINBOW TROUT

Environmental cues are processed through endocrine signal by activating

the hypothalamic-pituitary-gonadal (HPG) axis. Hypothalamus releases
gonadotropin-releasing hormone (GnRH). GnRH shows their expression
in various tissues; however, it is produced in the hypothalamus (typically
termed GnRH1) and primary contributes in the activation of the HPG axis by
stimulating the synaptic gonadotropin-producing cells (gonadotrophs) of the
pituitary (Rather et al., 2020). Further modulation of GnRH synthesis and
release occurs through the more recently discovered Figures 10.1(a) and (b).

FIGURE 10.1 The role of environmental cues in the activation of endocrine signaling and
its contribution in the maturation of gametes and sexual behavior.

10.5.1 EFFECTS OF STRESS ON THE REPRODUCTIVE ENDOCRINE

SYSTEM

In female brown, and male rainbow trout, two weeks of confinement cortisol
level increase in circulatory plasma, inhibits testosterone level in circula­
tory plasma, but does not change the level of estradiol and level of plasma
VTG decreases in female rainbow trout. On the other hand, plasma E2
level decreases after 2–24 h of imposed confined stress given to rainbow
trout. During the post-capture period the level of testosterone and estradiol
decreases in migratory wild rainbow trout kept in stream cages for 24 hrs.
272 Coldwater Fisheries and Aquaculture Management

after capture, more it has been observed that cortisol level increases. All the
changes induced by stress on reproductive performance has been summa­
rized in Figures 10.2(a) and (b).

FIGURE 10.2 The effect of stress on the reproductive performance of rainbow trout.

10.5.2 REPRODUCTIVE INDICES OF RAINBOW TROUT

(ONCORHYNCHUS MYKISS) FEMALES FROM A TROUT FARM

About three-year-old Oncorhynchus mykiss were carried out to study the

reproduction rate and the performance of the production of rainbow trout,
so based on their weight and samples after spawning and before spawning
the relative fecundity (335.88±17.723 g) was determined to have 52.77%
variability coefficient. It was the first generation that have high variability
coefficient. This activity was performed without any improvement activity
or any selection process in the nursery. A criterion in production that is
used most of the time is the total number of eggs and egg diameter (TNE
& Ed), as the weight of broodstock increases that helps to increase the
eggs diameter and their number (Ahmadnia et al., 2014). The value of the
total number of eggs was 8127.44±478.532. Also, there was an increase in
variability coefficient (58.88) (Kayam, 2004), in the future, the spawn has
a good quality of the individual. Different researchers reported that some
factors affect the production of rainbow trout eggs (among breeding stock,
their weight, quality of water, etc.) (Ozgur & Bayir, 2013). In this, the
weight of the egg and its diameter were 0.06±0.001 g and 4.16±0.029 mm,
Reproductive Physiology and Breeding Biology of Rainbow Trout 273

respectively. Similarly, Ozgur & Bayir (2013); and Kocaman et al. (2009)
reported the diameter of eggs in four-year-old broodstock, i.e., 5.58±0.18
mm and 5.33±0.10 mm, respectively. The data concluded that both the egg
diameter and their number increased with an increase in the broodstock age
and some reproduction indices were also calculated (gonadosomatic report
(GR); Behning fertilizing coefficient (BFC) and Williams absolute fecundity
indices (WAFI). Different studies represent different results. The volume of
the egg was 63.63±1.223 mm3 and the values of the GR were 14.82±0.587.
But according to the Coroian et al. (2013); and Bud et al. (2009) the GR
values (15.218±0.438 and 22.54±1.13) were decreased in the female who
was four years old. So only those females were selected in the future that
has higher GR but some researchers reported that the female having 6–8
years old is good because in both the rainbow trout and other females of the
species showing good performance with an increase in their age (Cocan et
al., 2013). In this, the study was carried out on the females that were three
years old at the starting period of reproduction. Various workers reported
that 16.82±0.563 was BFC having 33.48% variability. Coroian et al. (2013)
reported that the total number of the egg was increased with an increase in
Behning coefficient value with age. Coroian et al. (2013) studied that the
value of Williams’s absolute fecundity is directly proportional to the age of
females. The higher value was recorded, i.e., 5.77 ± 0.333 in the study than
the previous one (4.278±0.279’) in the same age group females. The positive
correlation was represented by the GR which was 0.621. It has a higher initial
body weight and maximum height (IBW and MH), in which the correlation
value decreased, i.e., 0.415 and it was lower from previous results obtained
in the same aged female by Cocan et al. (2013). The correlation coefficient
value shows that to improve the features, the selection can be done, and
it results from an increase in correlation value. In contrast, the GR shows
the negative correlation for the total length (TL) that was lower from the
value reported by Cocan et al. (2013), i.e., –0.116 and –0.027, respectively. It
shows that with an increase in the TL both the coefficient and indices values
and breeding get decreased. The positive value has been represented by the
BFC for TL, i.e., r = 0.264 which was higher than the value represented in
the Cocan et al. (2013), i.e., r = 0.130. On the other hand, it also shows a
negative correlation for the initial bodyweight, i.e., r = –0.258. A strong and
positive value well was represented by Williams absolute fecundity index for
both MH and IBW (r = 0.635 and r = 0.562 which was lesser than previous
results (r = 0.562) obtained by Cocan et al. (2013). The negative value of
correlation occurs –0.054 for TL but Cocan et al. show a positive correlation
274 Coldwater Fisheries and Aquaculture Management

value (r = 0.162). It needs further evaluation for better results. There are
some strong correlations and positive correlations found in the FBW (final
body weight) and IBW, i.e., r = 0.887. Also, in relative fecundity and MH (r
= 0.752) and 0.696 in the total number of eggs and MH. It was concluded
that for the better selection of female breeders the above correlation showed
much better results. It gives the best egg quality and quantity as well at the
particular range of age.

10.6 CONCLUSION

Reproduction in rainbow trout are affected by various environmental factors

and changes the internal endocrine environment of the fish. Therefore, it can
be concluded environmental cues are processed through endocrine signal
by activating the hypothalamic-pituitary gonadal (HPG) axis. Hypothalamus
releases GnRH and gets expressed in various tissues and contributes to
the activation of the HPG axis by stimulating the synaptic gonadotropin-
producing cells (gonadotrophs) of the pituitary. It was concluded that for the
better selection of female breeders the above correlation showed much better
results. It gives the best egg quality and quantity as well at the particular
range of age.

KEYWORDS

• gamete maturation
• gonadotropin-releasing hormone
• micropylar sperm attractant
• rainbow trout
• reproduction
• stress

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CHAPTER 11

Feeding Carbohydrates to Fish:

Utilization and Looking Beyond Energy
Nutrition
GARIMA ANAND1, PUSPA KUMARI1, TINCY VARGHESE1, and
SHOWKAT AHMAD DAR2
1
ICAR–Central Institute of Fisheries Education, Mumbai, Maharashtra, India
2
Department of Aqualife Medicine, College of Fisheries and Ocean Studies,
Chonnam National University, South Korea

ABSTRACT

Carbohydrates are one of the most prevalent organic compounds in the

biosphere and are known for the cheapest source of energy. Even though
there is an unlimited carbohydrate source present in nature, only a few have
nutritional value in animal nutrition. However, in fish nutrition carbohydrates
are not indispensable in fish diets, although it constitutes an inexpensive
source of energy. In fishes, it is found that there is no dietary requirement
for carbohydrates; however, if carbohydrates are not provided in the diet,
other nutrients such as protein and lipids are catabolized for energy and
metabolic intermediates for the synthesis of other biologically important
compounds. The use of carbohydrate-rich ingredients in fish feed is gaining
day in day importance because of their ability to spare the more expensive
protein. So various methodologies and techniques have been used to utilize
and uses of carbohydrates in fish feed range from highly digestible and
indigestible hemicellulose and cellulose recovered from various sources,
including seaweed, algae, and plankton, to refined grain and other numerous
products. The present chapter gives the idea about the importance, methods,

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
280 Coldwater Fisheries and Aquaculture Management

mechanism, and ways to utilize more carbohydrate sources in fish nutrition

and reduce the cost of fish feed.

11.1 INTRODUCTION

Nowadays, the majority of the developing countries and many developed ones
have extended the use of artificial compounded aquafeeds for farmed fishes
and other invertebrates. The increased use of aqua-feeds has stimulated the
rapid development of the production sector, which became one of the fastest
expanding agricultural industries globally. The success of culture-based fishery
is partially and or wholly dependent on the quality and cost of feed delivered as
the cost of feed depends on the protein source used in the feed. So, it is necessary
to use low-cost protein source ingredients for feed formulation, which reduce
the overall cost of the feed. Research studies showed that carbohydrate has
a protein-sparing effect (Shiau & Peng, 1993; Zhou et al., 2014; Lin et al.,
2018) and is the least expensive dietary energy source for humans and animals
(Stone, 2003; Knudsen et al., 2012; Ludwig et al., 2018). Carbohydrate-rich
ingredients are available in desirable quantities at low prices. Carbohydrates
in fish feed range from highly digestible and indigestible hemicellulose and
cellulose recovered from various sources, including seaweed, algae, and
plankton, to refined grain and soybean products. However, carbohydrates
are not indispensable in fish feed, although it constitutes an inexpensive
source of energy. In fishes, it is found that there is no dietary requirement for
carbohydrates, however, if carbohydrates are not provided in the diet, other
nutrients such as protein and lipids are catabolized for energy and metabolic
intermediates for the synthesis of other biologically important compounds.
Thus, it is necessary to provide the appropriate carbohydrate level in the diet
of the fish species being cultured (Wilson, 1994; Kamalam & Panserat, 2016).
However, certain fish species, when fed with carbohydrate-free diet, showed
in reduced growth rate whereas high levels of dietary carbohydrate resulted
in increased glycogen content in body and vital organs and liver size in fishes
(Hilton & Atkinson, 1982; Boonanuntanasarn et al., 2018; Singh et al., 2012)
optimum levels of dietary incorporation are still unclear.

11.2 SOURCES OF CARBOHYDRATES

Fish are not able to utilize all types of carbohydrates. The relative utilization
of dietary carbohydrates by fish varies and appears to be associated with
Feeding Carbohydrates to Fish 281

the complexity of carbohydrates. For example, starch is more efficiently

used compared to glucose in both marine and freshwater species: Carp, red
seabream (Furuichi & Yone, 1982), yellowtail (Furuichi et al., 1986), Tilapia
(Shiau & Peng, 1993), white sturgeon (Deng et al., 2005). On the other side,
juvenile grass carp and rainbow trout (Hung & Storebakken, 1994) utilize
glucose better than starch. However, in grouper (Shiau & Lin, 2001), starch
seems to be used efficiently as glucose (Wilson & Poe, 1987). It has been
found that channel catfish can efficiently use starch or dextrin but cannot
utilize dietary mono and disaccharides as an energy source. The exact mech­
anism in fish that is responsible for the difference in carbohydrate utilization
is not known. Still, Pieper, & Pfeffer (1980) suggested poor performance
with simple sugar such as glucose saturation. That might be because simple
sugar like glucose requires no digestion and is rapidly absorbed from the gut,
whereas complex carbohydrates like starch must undergo enzyme hydrolysis
before absorption. Thus, free glucose appears at gut absorption sites more
rapidly than glucose from a complex carbohydrate source. Various factors
affect the digestible energy provided by complex carbohydrates in fish diets
(Bergot, 1993). Some of the factors include their source and treatment of
starch.

11.3 CARBOHYDRATE REQUIREMENT OF FISH

The nutritional value of carbohydrates varies among species. In the case

of warm water fish, they can utilize much higher levels of dietary carbo­
hydrates than coldwater and marine fish. Grass carp (Ctenopharyngodon
idella) is herbivorous fish in which the optimum dietary level of digestible
carbohydrate is significantly higher than carnivorous categories, or even in
other carp (Lin, 1991; Kamalam & Panserat, 2016). It has been reported that
larger fish utilize dietary carbohydrates better than fish with small body sizes
(Pen-Hsing & Shi-Yen, 1993; Hatlen et al., 2005). No specific requirement
of carbohydrates in the fish diet is known. Still, some studies showed that
excess carbohydrate levels in the fish diet reduce the growth rate are often
accompanied by poor feed utilization (Hemre et al., 2002; Han et al., 2021).

11.4 EFFECT OF CARBOHYDRATE ON GROWTH OF FISH

Carbohydrates are one of the most widespread organic compounds in the

biosphere. Although there is an infinite carbohydrate source present in nature,
282 Coldwater Fisheries and Aquaculture Management

only a few have nutritional value in animal nutrition. Some of them are
hexoses (glucose), disaccharides, and some homopolysaccharides, including
starch. Fish do not require dietary carbohydrates for their sustenance, they
have a higher capability to make glucose through gluconeogenesis for their
energy needs than other animals. However, fish nutritionists want to include
carbohydrates in aquafeed, as it is the cheapest energy source (Kamalam
& Panserat, 2016). Anderson & Coworkers (1984) found that Oreochromis
niloticus fed diets containing 40% carbohydrate (approximately 30% of
gross dietary energy) did not retard growth. An increase in glucose level
up to 15% in the rations positively affects weight gain, food efficiency, and
PER in juvenile starry flounders (Lee & Lee, 2004). But at the same time,
some studies showed the negative effect of carbohydrates on the growth of
C. batrachus fry when the levels were maintained at more than 15% (Mollah
& Alam, 1990). Furuichi & Yone (1980) noted depression in growth and
feed efficiency in red sea bream, yellowtail, and common carp fed diets high
in carbohydrate content. Tung & Shiau (1991) studied how meal frequency
affects carbohydrate utilization in tilapia. Fish were fed with diets containing
44% of different carbohydrates sources (starch, dextrin, and glucose). They
found that fish fed starch, dextrin, or glucose diets six times a day gained
significantly more body weight than fish fed these diets two times a day.

11.5 PROTEIN-SPARING EFFECT BY CARBOHYDRATE

The use of carbohydrate-rich ingredients in fish feed is gaining day in day

importance because of their ability to spare the more expensive protein
component of the diet to be used for growth (Shiau, 1997; Zhu et al., 2014;
Lin et al., 2018). Although lipids serve as an important source of non-protein
energy for fish (Kaushik et al., 1989), they are more expensive than carbo­
hydrates. Protein cost contributes to a large part of the cost of most prepared
feeds, so protein fraction must only be utilized for growth, not energy.
This knowledge of the optimal protein level and protein-sparing effects of
non-protein nutrients such as carbohydrates can be used effectively to help
reduce feed costs. Shiau & Peng (1993) conducted a study to evaluate the
possible protein-sparing effects of carbohydrates in juvenile tilapia. Three
dietary protein levels (32, 28, and 24%) were achieved by substitution with
three levels (33, 37, and 41%) and three sources (glucose, dextrin, starch) of
dietary carbohydrates. Results indicated that fish fed starch or dextrin had
significantly higher weight gain and FER than that fed glucose for all the
protein levels, except for weight gain for the 24% protein level, in which the
Feeding Carbohydrates to Fish 283

difference was not significant. Fish fed starch and dextrin had similar weight
gain. Decreasing the dietary protein level from 28% to 24% by increasing the
starch or dextrin content in the diet from 37% to 41% did not reduce weight
gain and FER, suggesting that starch or dextrin could spare some protein
when the dietary protein level was low. Fynn-Aikins et al. (1992) also did
not observe a protein-sparing effect when sturgeon were fed diets with an
optimum protein (42%) and different levels of D-glucose (0–35%), which
were effectively utilized by sturgeon (Hung et al., 1989). These studies show
that carbohydrates can be used in fish feed at an optimum level to fulfill the
energy requirement of fish.

11.6 CARBOHYDRATE UTILIZATION IN FISH

Fish, in general, utilize dietary carbohydrates poorly. Apart from being the
most economical source of energy in the diet, Carbohydrate-rich ingredients
also have good availability throughout the world. But despite all these
benefits, fishes have poor ability to make use of dietary carbohydrates
because of the scarcity of carbohydrates in the aquatic environment. Two
problems are related to carbohydrate nutrition in fish. Firstly, as a general
rule, digestion of complex carbohydrates is low in the aquatic animal.
Secondly, fishes are poorly adapted to the metabolic utilization of simple
sugars (monosaccharides or disaccharides). It is also found that carbohydrate
utilizations in fish vary with their feeding habits and habitat.
Warmwater fish can utilize much higher levels of dietary carbohydrates
than cold water and marine fish. There are certain reports of limited ability
to utilize carbohydrates in carnivorous fish like cod Atlantic salmon (Hemre
et al., 1995) and white sturgeon (Deng et al., 2005). However, improved
growth performance was observed in different species of fish upon feeding
high carbohydrate diets viz., eels (Degani & Viola, 1987), rainbow trout
(Grisdale & Helland, 1997), catfish (Hung et al., 2002) and carp (Mohapatra
et al., 2003).

11.6.1 IMPROVING CARBOHYDRATE UTILIZATION IN FISH

Several promising strategies have been explored to overcome the challenges

in carbohydrate utilization in farmed fishes, especially the carnivorous fish.
Therefore, tailoring metabolic pathways to improve carbohydrate stimulus
at critical early developmental stages may imprint an adaptive ability to
284 Coldwater Fisheries and Aquaculture Management

withstand with high carbohydrate diets in progressive life stage (Kamalam &
Panserat, 2016). Further dietary modifications of carbohydrates may improve
its utilization in fish. These modifications include: (1) pre-gelatinization,
which are made by applying starch water slurry to heated drum dryer; (2)
chemical modification of starch by treatment with acids or with enzymes;
and (3) Starch dextrinization with heat treatment. Starch is a valuable source
of energy, and its utilization is enhanced by gelatinization (Bergot & Breque,
1983) due to its increased digestibility (Bergot, 1991; Podoskina et al., 1997;
Mohapatra et al., 2003). Gelatinization of starch results in hybridization and
swelling of starch granules (Hofer & Sturmbauer, 1985) and thus improves
digestibility by increasing the susceptibility of starch granules to the enzyme.
Digestibility of starch varies with the source of starch. For example, it has
been reported that the digestibility of tuber starch (potato and tapioca) is
lower than that of cereal starch in rainbow trout (Bergot, 1991) and Carp
(Schwarz & Kirchgessner, 1993). Pigs fed with Corn were found to have a
higher feed conversion ratio (FCR) than wheat fed groups (Han et al., 2005).
Corn-based rations are also commonly used in poultry for high available
energy (Wang et al., 2005). The inclusion level of starch also plays an impor­
tant role in their digestion; it is found that low digestibility of starch at higher
inclusion level has (Hemre et al., 1989). However, a significant increase in
digestibility was reported with an increase in gelatinized carbohydrate level
in the diet of a carp L. rohita fry (Mohapatra et al., 2003).

11.7 IMPACT OF CARBOHYDRATE ON IMMUNE SYSTEM OF FISH

Compared to the immense number of studies in nutritional immunology in

humans and livestock, the evaluation of the nutritional impact on fish health
is in its infancy. The mechanisms by which the single nutrient modulates
immunity are frequently not understood, even if experiments indicate the
mode of action. Among all the macronutrients, carbohydrate is considered
one of the cheapest source nutrients, but at the same time, it has their limita­
tions. Fish have limited ability for digestion and metabolism of carbohydrates,
and hence excessive intake of this nutrient may adversely affect fish health
through metabolic disorder and physio-clinical signs such as hyperglycemia,
increment of glycogen deposition, liver hypertrophy, and histopathological
(HP) development (Lall, 1991; Li et al., 2019; Azaza et al., 2020). Some of
the studies show that fish with excess glucose is found to be under constant
metabolic stress which may lead to suppressed immune functions (Wiik et al.,
Feeding Carbohydrates to Fish 285

1989; Talukdar et al., 2019; Li et al., 2020; Han et al., 2021). Stress is one of
the important factors that increase fish’s susceptibility to infectious diseases
(Mishra et al., 2017; Sarkodie et al., 2019). Thus high dietary carbohydrates
may increase the susceptibility and incidence of infectious. Fewer studies
are conducted on the health implications of high dietary carbohydrates on
fish. High carbohydrate in the diet of rainbow trout (Oncorhynchus mykiss)
shows intracellular damage due to deposition of glycogen particles and thus
reduce the liver detoxification capacity by influencing the metabolism and
cause glucose metabolism disorders (Hilton & Hodson, 1983; Azaza et al.,
2020; Wang et al., 2020; Zhou et al., 2021).

11.8 CONCLUSION

Being the cheapest source of energy, carbohydrates are an integral part

of aquafeeds. However, carbohydrate utilization in fish depends on many
factors such as inclusion level, molecular complexity and physical state of
fish. Another factor impacting carbohydrate utilization is species’ habitat, as
warmwater fishes can more efficiently utilize carbohydrates than cold water
and marine fish. They have a role in modulating fish immunity, and they
also affect the metabolic fate of other nutrients by sparing action. Thus, the
importance of carbohydrates in fish nutrition is beyond that of an energy
substitute.

KEYWORDS

• agricultural industries
• carbohydrates
• carbohydrates
• energy
• feed
• fish nutrition
• macronutrients
• metabolic utilisation
• protein-sparing effect
286 Coldwater Fisheries and Aquaculture Management

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CHAPTER 12

Application of Plant-Derived Natural

Preservatives for Shelf-Life Extension of
Freshwater Fish
HAFSA MAQBOOL1, MUDASSIR AZHAR2, and A. A. ZYNUDHEEN3
1
Division of Aquatic Animal Health Management, Faculty of Fisheries,
Sher-e-Kashmir University of Agricultural Sciences and Technology of
Kashmir, Jammu and Kashmir, India
2
Department of Fisheries Science, Doon PG College of Agriculture and
Allied Science, Uttarakhand, India
3
Fish Processing Division, Central Institute of Fishing Technology, Kochi,
Ernakulam, Kerala, India

ABSTRACT

Quality loss in fish happens shortly after death, throughout the processing and
storage and is linked to enzymatic, microbiological, and chemical reactions.
As a result, food additives are required to preserve the shelf life, nutritional
content, texture, and flavor of both raw and processed materials. Following
the advent of “green consumerism” which resulted in a decrease in consumer
demand for synthetic food additives, extensive research is being done on
natural additive applications. The natural extracts isolated from various plant
sources such as spices and herbs, fruits, and vegetables, seaweed, and so on
comprises of bioactive compounds that have demonstrated remarkable free
radical inhibitor and antibacterial activities. Plant phenolic compounds are
of particular interest due to their antioxidant and antimicrobial properties.

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
292 Coldwater Fisheries and Aquaculture Management

Agricultural handling factories produce profuse volume of phenolic-rich

by-products, which could be valuable natural sources of antioxidants and
anti-bacterial. Some of these by-products have been studied and found to be
effective sources of phenolic antioxidants and anti-bacterial. Based on this,
plant extract treatments have been effectively practicable to chilled, frozen,
and dried fish, fish filets and mince as a natural additive. This chapter will
discuss the mode of action and applications of these natural preservatives
for maintaining quality and extending the shelf life of freshwater fish and its
products.

12.1 INTRODUCTION

Due to the high content of polyunsaturated fatty acids (PUFAs) and protein,
fish, and fish products are considered a valuable part of human nutrition
(Kykkidou et al., 2009) long-chain polyunsaturated fatty acids (PUFAs) in
these products have gained attention because of their prevention of human
cardiovascular diseases (Ozogul et al., 2006). These products have a short
shelf-life, despite their nutritional significance. This is due to the large
amounts of free amino acids (AAs), volatile nitrogen bases and unsaturated
fatty acids that are highly susceptible to oxidation (Mexis et al., 2009), which
can affect the flavor, texture, aroma, and shelf-life of fish (Banerjee, 2006).
In recent years, consumers have demanded fresh, natural, and minimally
processed foods, as well as an increase in food safety and quality. This
perspective has prompted research into alternative natural antimicrobials
and antioxidants, as well as pressure on the food sector to gradually phase
out chemical preservatives (Cox et al., 2010). Some plants and vegetables
have been found to contain beneficial antioxidants and antibacterial with
nutritional and medicinal benefits. Antimicrobial activities of various plant
extracts and essential oils (EOs) have been evaluated against a variety of
foodborne bacteria (Boziaris et al., 2011).

12.2 SPOILAGE MECHANISM IN FISH

Fresh fish can quickly deteriorate after being harvested and acquire rigor
mortis, a condition in which fish lose their elasticity due to muscle stiff­
ening a few hours after death. Enzymatic autolysis, oxidation, and microbial
growth are the three mechanisms that cause spoilage.
Application of Plant-Derived Natural Preservatives 293

12.2.1 ENZYMATIC ACTIVITY

Autolysis, the process of destroying proteins by native enzymes, begins

immediately after rigor mortis is complete. This procedure provides an
environment that is conducive to bacterial growth (Ghaly et al., 2010). The
degradation process in fish starts quickly after collection owing to enzy­
matic deterioration that causes chemical and biological changes and thus
the breakdown of basic molecules. Although the autolytic enzymes had a
significant impact on textural quality, resulting in spoilage, they did not cause
the typical spoilage with off-flavor and off-odor. The presence of gastroin­
testinal digesting and endogenous muscular enzymes in the fish is found to
be responsible for post-mortem changes in fish muscles during processing
and storage, resulting in sensory changes. On the other hand, autolysis of fish
muscle proteins may lead to the deterioration of fish meat and the generation
of biogenic amines. During autolysis, the degree of freshness of the fish must
be determined before it begins to decay (Ocanohiguera et al., 2011). It has
been suggested that maintaining the low activity of endogenous autolytic
enzymes in muscle during storage can be achieved by keeping the tempera­
ture and water activity (aw) low (Pachecoaguilar et al., 2010).

12.2.2 MICROBIAL ACTIVITY

Trimethylamine oxide (TMAO) is found in many fish species and promotes

bacterial growth over a broad temperature range and has a high content of free
AA, a high pH after death, and a high-water content (Chaillou et al., 2015).
The growth and metabolism of microorganisms involved in the production
of biogenic amines, alcohols, histamine, putrescine, sulfides, organic acids,
aldehydes, and ketones are thought to be the primary cause of deterioration
of fish quality (Kuley et al., 2017). The majority of the fish microflora from
humid sea waters consists of psychotropic Gram-negative bacteria. On the
other hand, continuous processing or extended storage of products allows
Gram-positive bacteria to take over and ruin the product (Al-Bulushi et al.,
2010). Specific spoilage organisms (SSOs) are smaller portions of the fish
microbiota that are considered the major causes of seafood spoilage, such
as Shewanella, Photobacterium phosphoreum, and Pseudomonas (Gram
& Dalgaard, 2002). The SSOs consume the metabolites produced from the
breakdown of non-protein nitrogen (NPN) of fish muscle causing the unac­
ceptable off-flavors and thus the rejection by the consumer.
294 Coldwater Fisheries and Aquaculture Management

12.2.3 CHEMICAL ACTIVITY

In general, seafood is high in lipids, particularly fats containing long-

chain PUFAs (Venugopal & Gopakumar, 2017). Lipids have a key role in
the production of off-flavor and off-odor, as well as the nutritional value
of seafood (Mariutti & Bragagnolo, 2017). Lipid oxidation also causes a
depletion of fat-soluble vitamins and other substances (Souza & Bragagnolo,
2014). Lipid oxidation has a three-stage free radical mechanism: initiation,
propagation, and termination, which might lead to spoilage and degradation
due to the production of reactive aldehydes such as malonaldehyde (MDA)
and hydrononenal (HNE) as depicted in Figure 12.1 (Shahidi & Botta, 1994).
Discoloration, protein lipoxidation, cytosolic enzymes, photolytic oxidation,
and hydrogen peroxide are some of the processes that initiate and propagate
lipid oxidation in fish (Schneider, 2009). There are two types of lipid oxida­
tion: enzymatic and non-enzymatic, together it can result in deterioration of
fish quality. The reaction of oxygen with the double bonds of fatty acids is
known as oxidation. Fish contains high amount of unsaturated fatty acids
and pro-oxidant compounds, both of which are easily oxidized, resulting in
rancidity and quality loss (Losada et al., 2007). The smell, color, texture, and
nutritional value of these compounds all are affected due to these substances.
Protein denaturation, alteration of protein electric field characteristics,
nutrient deterioration, and intracellular antioxidant system deficits, and the
production of fluorescent substances have all been linked to lipid oxidation
products (Lugasi, 2007). Several pro-oxidants (hemoglobin, myoglobin, and
cytochrome c) can cause lipid oxidation (Ghaly et al., 2010).

12.3 FOOD ADDITIVES

Codex alimentarius defines additive as any substance that is added in foods

for the purposes of restoring esthetic appearance degraded during production
and improving aroma or taste, rather than nutritional improvement (WHO/
FAO, 2004). Food additives are used in the food sector because of the variety
of new technologies and consumer preferences. These can be used to reduce
losses, improve quality, extend shelf life, and develop novel formulas. Food
preservers, dietary ingredients, food dyes, artificial sweeteners, texturizing
agents, and other additives are all types of food additives. The majority of
additives have multiple functions (Carocho et al., 2015). Antimicrobials
such as nitrites, sulfites, and organic acids, as well as antioxidants such as
tertiary butyl hydroxy quinone (TBHQ), butylated hydroxy toluene (BHT)
Application of Plant-Derived Natural Preservatives 295

and butylated hydroxy anisole (BHA) were employed in fish for a long time
to limit the microbial growth and lipid oxidation (Manju et al., 2007). The
moieties of these synthetic additives have been considered as potentially toxi­
cological, in spite of their great activity (Naveena et al., 2008). The practice
of functional food ingredients has grabbed a lot of attention, due to the rapid
development of the social economy. Because of the negative perception of
synthetic preservatives, researchers are focusing on finding safe, effective,
and acceptable natural preservatives for suppressing the microbiological and
chemical pathways that cause fish to decay.

FIGURE 12.1 Mechanism of lipid oxidation in fish.

Source: Shahidi & Botta, 1994.

12.4 PLANT EXTRACT

Plant extracts and EOs derived from plant petals, leaves, fruits, peels, stems,
and roots are now considered as natural preservatives or food additives
with potent antibacterial, antifungal, and antioxidant properties, and are
used in the fish industry to preserve raw and processed fish (Chouliara et
al., 2007). Phytochemicals are chemical compounds found in plants that
have an impact on the bacteriological and esthetic perception of foods. The
296 Coldwater Fisheries and Aquaculture Management

efficiency of plant extract varies due to qualitative and quantitative variances

in the presence of bioactive phytochemicals. Polyphenolic compounds like
flavonoids, terpenoids, isothiocyanates, and polypeptide are illustrations
of phytoconstituents. Extracts are dissolved portion of organic materials
such as spices, fruits, and vegetables that are produced by solubilizing the
components of interest in an aqueous, alcohol, lipid, solvent, or supercritical
CO2 phase and then removing it (Brewer, 2011). Plant extracts are generally
regarded as safe (GRAS) due to their long history of usage with no reported
adverse from several specialized toxicological investigations (Smid &
Gorris, 1999). Plant based natural antioxidant and antimicrobials have been
long recognized and are been used as preservatives in a variety of food
systems (Kanatt et al., 2007).

12.4.1 EFFECTIVE COMPOUNDS IN PLANT EXTRACTS AND ITS

SYSTEM OF ACTIVITY

Plant extracts have bioactivities due to the presence of phytochemicals,

particularly polyphenolics. Polyphenols are organic molecules found in
plants that have one or more aromatic ring structures with multiple hydroxyl
groups (Boudet, 2007). They are largely divided into four groups—
flavonoids, phenolic acids, hydroxycinnamic acids and lignans as shown in
Figure 12.2 (Maqsood et al., 2013). Flavonoids are categorized as flavanol,
flavanones, isoflavones, and anthocyanins based on the degree of oxidation
of the heterocyclic ring. Quercetin is a flavonol found in onions and apples;
catechin is flavanol mostly exist in tea and some fruits; naringenin is the main
flavanone found in grapefruit; and cyanidin-glycoside is an anthocyanin found
in berry fruits (black currant and blackberry); and cyanidin-glycoside is an
anthocyanin found in berry fruits (black currant and blackberry); and genistein
and glycitein are the main isoflavones found in soybean (D’Archivio et al.,
2007). There are two categories of phenolic acids: benzoic acid derivatives like
gallic acid and cinnamic acid derivatives like coumaric, caffeic, and ferulic
acid. The most abundant phenolic acid is caffeic acid, found in many fruits
and vegetables, which is often esterified with quinic acid like in chlorogenic
acid, the most important phenolic ingredient in coffee. Ferulic acid, found in
grains, is another prevalent phenolic acid, while coumaric acid is plentiful in
citrus fruits. Polyphenols contain active hydroxyl groups that can interact with
free radicals to reduce lipid oxidation (Mitsumoto et al., 2005). Polyphenols
can also act as free radicals and metal scavengers, reducing the generation
of metal catalyzed free radicals (Rice-Evans et al., 1996). The antioxidant
Application of Plant-Derived Natural Preservatives 297

capacity is determined by the presence of “–OH” as the functional group in

the structure and its position either on the phenolic or flavonoid molecular
ring.

FIGURE 12.2 Classification of phenolic compounds.

12.4.2 PLANT EXTRACT AS ANTIMICROBIAL AND ANTIOXIDANT

AGENT

Plant extracts exhibit antibacterial activity due to phytochemical compo­

nents that may be deadly to microbial cells or impede secondary metabo­
lite formation. The outer cell membrane, also known as the cytoplasmic
membrane, is a major site of bacterial contact; damage to this important
membrane can cause the bacterium to die in one of several ways: (i) epithe­
lial interruption (Shimamura et al., 2007); this leads in the loss of macro­
molecules from the inside; (ii) contact with protein, which causes structural
and functional deformation; and (iii) disrupting membrane-associated
activities such as electron transport and generation of new protein (Bajpai
et al., 2009). Antimicrobial activity has been found in a variety of plant
extracts against bacteria, yeast, and molds. Phenols, alcohols, aldehydes,
ketones, ethers, and hydrocarbons are among the active chemicals that can
298 Coldwater Fisheries and Aquaculture Management

improve storage stability. Various research have documented antimicrobial

properties of plant extracts produced from herbs, spices, vegetables, and
fruits (Al-Habib et al., 2010; Hayek et al., 2012). Antioxidants are food
additives that delay or prevent food from becoming rancid due to oxidation,
hence extending the shelf-life. Natural antioxidant molecules are abundant
in plants. Plant-based antioxidants, such as BHT and BHA, have a protec­
tive impact. Different vegetables and fruits might be considered intriguing
sources of functional additives for food fortification and stability due to
their antioxidant characteristics. Antioxidants can slow the oxidation of
lipids by preventing the generation of free radicals or delaying their spread
by one or more mechanisms: (a) scavenging species that cause peroxida­
tion; (b) chelating metal ions so they do not generate reactive species that
cause oxidation; (c) quenching O2 and so preventing peroxide generation;
(d) stopping the auto-oxidative chain reaction; or (e) lowering the O2
concentration. The ability of antioxidants to disrupt free-radical chain
reactions is the most important factor for determining their efficacy. The
hydroxyl groups of phenolic compounds are efficient hydrogen donors.
The polyphenol becomes a radical after reacting with the original reactive
radical species. These radical intermediates are stabilized by the creation
of quinone structures and thus cause delocalization of electrons within the
aromatic ring. Plant polyphenols can thus inhibit lipid oxidation in seafood
(Maqsood et al., 2014).

12.4.3 APPLICATION OF PLANT EXTRACT FOR FISH AND FISH

PRODUCTS PRESERVATION

The use of agricultural produce and their peels, seed extract, or powders
having antioxidant and antibacterial qualities as organic component utilized
to enhance the storage period of fish and their products at chilled temperatures
is a potential approach. Hafsa et al. (2019) evaluated the combined effect of
beetroot peel extract (BPE) and chilled condition on the biochemical proper­
ties of fresh mahseer (Tor khudree) steaks and found that BPE dip treatment
enhanced the storage stability and extended the shelf life of mahseer by six
days compared to control steak during the chilled storage.
Tea polyphenols are gaining popularity as a natural food preservative
among researchers. Li et al. (2012) used 0.2% tea polyphenol to treat
entire un-gutted crucian carp and found that it effectively inhibited micro­
biological growth, hindered chemical deterioration, and thus extended the
Application of Plant-Derived Natural Preservatives 299

shelf life by 6–8 days as compared to control during refrigerated storage.

The white radish (Raphanus satavis) extract, a rich source of antioxidant,
therapeutic, and antimicrobial properties was found to limit the progress of
fish spoilage bacteria and thus extended the shelf life of fish steaks over 15
days of storage (Hafsa et al., 2020). BPE was found to have a significant
role in the free radical process and in restricting the bacterial growth in
frozen steaks. Control steaks had a shelf-life of about 5 months, according
to sensory analysis scores, whereas BPE treated steaks were found to be
acceptable for more than 6 months (Hafsa et al., 2021). In vacuum packed
sardine filets held at 31°C, Ozogul et al. (2011) found that rosemary and sage
tea extracts reduced ammonia and biogenic amine production. Turmeric, a
good source of antibacterial substance was found to preserve quality param­
eters of rainbow trout during vacuum storage of 20 days under refrigerated
conditions when used singly or combined with shallot (Allium cepa) extract
(1.5% each, v/v) (Pezeshk et al., 2011). In comparison to the pure extract
treatment and control, Mazandrani et al. (2016) found the least lipid oxida­
tion, TVB-N content, and microbial load during storage of silver carp filets
at 4°C treated with liposomal encapsulated fennel extract (FE). Silver carp
filets had their shelf life prolonged from 6 to 15 days when treated with 0.5%
encapsulated FE and 12 days when treated with 0.5% pure extract. When
compared to standard icing, including plant extracts into ice was found to
be effective in preventing microbiological and biochemical deterioration
of fresh fish. This unique icing approach has been found to be particularly
effective in increasing the quality of fish housed on board and transported to
interior locations. Chemical, sensory, and microbiological approaches were
used by Bensid et al. (2014) investigated the effect of ice extracts containing
thyme (0.04% w/v), oregano (0.3% w/v), and clove (0.2% w/v) extracts on
anchovy quality parameters (Engraulis encrasicholus). When compared to
the standard ice batch, these icing techniques resulted in a substantial anti­
oxidant impact as well as a considerably slighter decrease of aerobic and
low temperature loving bacteria in anchovy muscle. Oxidation is a major
cause of seafood quality decline during frozen storage. To delay oxidation
reactions in frozen storage fish and fish products, antioxidant chemicals were
applied after freezing, and the fish and fish products were dipped in organic
suspension before sub-zero temperature. In recent years, biodegradable
protein-based films have become increasingly important in food packaging,
acting as a specific hurdle to water movement, aerobic metabolism and the
loss of odor components (Maqsood et al., 2013). Most of the edible packages
possess barrier properties to gas, organic vapors, and oils when compared
300 Coldwater Fisheries and Aquaculture Management

to plastic wraps (Jiang et al., 2007). The texture and physical qualities of
surimi seafood are mostly determined by the gel strength of fish protein.
Exogenous substances of chemical and biological origin can improve the
texture of fish protein (Chiou et al., 2006). Plant phenolic compounds have
also been demonstrated to have the ability to cross-link proteins, making
them potential gel enhancers (Rawel et al., 2002). When phenolic chemicals
interact with proteins, changes in protein secondary structure occur, resulting
in increased heat stability (Ali, 2002).

12.5 CONCLUSION

Polyphenolic constituents of plant extracts are most active as natural

antioxidants and antibacterial in seafood. These ingredients have the
potential to replace synthetic additives in food systems to avoid oxida­
tion and quality degradation, particularly in fish and fish products. As a
result, plant extracts broaden the range of multifunctional natural food
components that can be used in the fish processing industry for a variety
of purposes.

ACKNOWLEDGMENT

I am thankful to A. A. Zynudheen for his valuable help, Ashraf Rather and

Adnan Amin for providing me with the opportunity to write this chapter.

KEYWORDS

• antimicrobial properties
• antioxidants
• malonaldehyde
• natural preservative
• non-protein nitrogen
• polyunsaturated fatty acids
• shelf-life
• specific spoilage organisms
Application of Plant-Derived Natural Preservatives 301

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CHAPTER 13

Perspective of eDNA Application

to Control Pollution in Freshwater
Ecosystems
ADNAN AMIN1, MONISA M. MALIK1, D. PAMANNA2, IMTIYAZ QAYOOM1,
and ADNAN ABUBAKR1
1
Division of Aquatic Environmental Management, Faculty of Fisheries,
Rangil, SKUAST-K, Jammu and Kashmir, India
2
College of Fishery Science, Muthukur, Nellore, Andhra Pradesh, India

ABSTRACT

The continual loss of biodiversity on earth is a serious concern and task

for the 21st century. This mission is affected in large part by a dearth of
understanding about the condition and distribution of biodiversity both
aquatic and terrestrial. The advancements made in the stream of molecular
biology over the last decade have transformed the way we think about
biodiversity on the planet. Aquatic ecosystems must be monitored and
assessed quickly and accurately. Environmental DNA (eDNA) sequencing
has been proved to be a reliable and sensitive technology for detecting and
monitoring single or multiple species in a variety of aquatic samples. In this
chapter, we look at how eDNA can be used to control and mitigate pollution
in aquatic ecosystems, particularly freshwater, as well as how it can be used
to monitor aquatic biodiversity, survey species communities, and provide
instructions for environmental conservation and when used in conjunction
with other sophisticated techniques and conventional ways.

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
306 Coldwater Fisheries and Aquaculture Management

13.1 INTRODUCTION

Environmental DNA (eDNA) refers to the genetic material found in

environmental materials such as substratum, H2O, and air, as well as complete
cells, extracellular DNA, and maybe whole species (Ficetola et al., 2008).
eDNA may be taken from a variety of ecosystems, recognized, improved,
sequenced, and categorized depending on the sequence (Deiner et al., 2015).
By gaining this information about particular environment, it will be easy
to detect and classify various species. eDNA can be of different source
such as water, mucous, saliva, sperm, eggs, fishes, benthos, macrophytes,
microorganisms’ urine, blood, roots, leaves, vegetation, and decomposing
remain of larger species, etc. (Bohmann et al., 2014).
Although being a moderately new technique of surveying, eDNA has
holds promising potential in biological monitoring of different ecosystems.
Traditionally used techniques for evaluating richness and abundance are
restricted in terms of taxonomic identification, leading to possible habitat
alteration or loss. eDNA can overcome these limitations by targeting various
species, sampling larger diversity, and enhancing taxonomic resolution
(Deiner et al., 2017). Furthermore, eDNA has ability to identify rare species,
however not determining qualitative population traits such as male female
ratios and health status, making it suitable replacement for conventional
research (Goldberg et al., 2016; Deiner et al., 2017).
In past decades, eDNA investigation has drawn much limelight as a
potential and responsive approach for studying biodiversity (Kelly et al.,
2016; Pikitch, 2018), which in turn can help in evaluating the changes of
different species in various eutrophication levels. Because of the breakthrough
and significant growth of high-throughput Sequencing technology, it is now
possible to utilize this approach to quickly detect the community structure
of organisms (Hamady et al., 2008). Additionally, eDNA metabarcoding was
seen to be useful for both qualitative and quantitative monitoring of diverse
communities in various habitats (Hanfling et al., 2016). In determining the
efficacy of eutrophication, pollution, and HAB control, assessing diversity
of species, community architecture, and species change patterns is critical
(Figure 13.1). For example, the complete biomass and species compositions
of planktonic organisms in aquatic environment are important factors that
indicate the water quality of lentic or lotic body (Chen et al., 2017). The
analysis of species compositions can aid in the discovery of ecological
indicators that can be used to mitigate the problems associated with nutrient
enrichment and excessive algal blooms in aquatic environments. The most
Perspective of eDNA Application to Control Pollution 307

recent technology of employing eDNA as a molecular marker for species

recognition and biodiversity assessment should provide comprehensive
and useful data for ecosystem monitoring and conservation (Schwartz et
al., 2007).

FIGURE 13.1 Application of eDNA for controlling pollution in aquatic ecosystem.

Eutrophication of rivers, lakes, and oceans due to significant nutrient

intake has become a pervasive issue in the aquatic environment (Smith,
1998). Increased anthropogenic activities lead to nutrient inputs in aquatic
ecosystem which includes population explosion, urban development,
agriculture, forestry, and fishery industries (Carpenter et al., 1999). The
most important factors which leads in eutrophication of any water body is
increasing nitrogen (N) and phosphorus (P) inputs (Conley et al., 2009; Ma
et al., 2015) and thus, cutting short supply of N and P concentrations can help
in improvement of water quality and ecological status (Sondergaard et al.,
2013; Wu et al., 2017). The harmful effects of eutrophic or polluted waters
might end in murky water, HABs, anoxia or toxicity of water bodies which
can lead to a decrease in species diversity (Smith & Schindler, 2009).
There are various methods which can help in halting cyanobacterial
blooms and enhance water quality, such as coagulation and use of copper
sulfate (Kenefick et al., 1993; He et al., 2016). These methods, on the other
hand, can be time-consuming, costly, and harmful to the environment.
Furthermore, due to the strength and residues of chemical reagents, the
use of chemical agents may induce heavy metal deposition and secondary
pollution of the aquatic environment (Lin et al., 2019). For the protection
of aquatic ecosystem, natural approaches are helpful tool in cyanobacterial
bloom control (He et al., 2016). It is now widely established that algicidal
microorganisms can restrain cyanobacterial blooms efficiently and with
specificity (Ahn et al., 2003; Zeng et al., 2015).
308 Coldwater Fisheries and Aquaculture Management

eDNA is said to be the most helpful tool for tracking species distributions
and classifying different organisms (Deutschmann et al., 2019). eDNA is
extracellular DNA released into the environment by organisms by a variety
of discharges such as secretions, excrement, sperms, eggs, sloughed cells, or
biological remnants (Furlan et al., 2016; Epp et al., 2012). eDNA was introduced
in the area of microbiology by Ogram et al. in 1987. eDNA is an important
tool, and study of eDNA is more and more being used to identify delicate
and nonnative or invasive species, identify low abundance or endangered
species and approximate biodiversity (Coble et al., 2019; Robson et al., 2016).
In addition to it, earlier studies revealed that eDNA investigation could be
more suitable, capable, and complete than conventional techniques used for
evaluating diverse taxa across spatial and temporal scales (Jo et al., 2019).
Conventional methods, such as physical identification, are ineffective in
aquatic environment for reflecting true population configuration and detecting
nonnative species early (Ardura et al., 2015), because living organisms in aquatic
ecosystems may be hidden underwater and hard to detect using conventional
methods. eDNA is more applicable to a broader spectrum of aquatic bodies,
besides other animals and plants (Hanfling et al., 2016). Consequently, eDNA
analysis could be a sensitive and effective assay for discovering species that
would otherwise go undetected by typical survey methods (Rees et al., 2014;
Stoeckle et al., 2017). Furthermore, in aquatic ecosystem, eDNA analysis aids
in the discovery of specific groups from multipart samples. Rudko et al. for
example, established four species-specific identification methods in aquatic
samples from Northern Michigan lakes (Rudko et al., 2019). eDNA is already
employed in the numerous studies of both freshwater and marine ecosystems
(Table 13.1). DNA metabarcoding, as proposed by Banerji et al. could be used
to enhance traditional biological approaches to facilitate to get more thorough
profiles of freshwater planktonic organisms commune structure and dispersion
(Banerji et al., 2018). The use of eDNA for quick surveillance of biodiversity
changes offers considerable potential for monitoring the projected changes
as a due to climate change, particularly in vast lake ecosystems (Wagner &
Adrian, 2011). eDNA analysis has been employed to investigate and examine
microorganisms, fish, and other organisms in marine water samples (Sogin et
al., 2006; Thomsen et al., 2012).

13.2 COLDWATER MONITORING

Studies pertaining to assessment of Biodiversity using eDNA are relatively

same to those in marine and estuarine ecosystems, among the prime areas
Perspective of eDNA Application to Control Pollution 309

of attention is biofilms, bulk specimens, sediment, and water samples

(Figure 13.2). In aquatic environment particularly freshwater ecosystem the
animal communities are sensitive towards environmental change, making
them potential indicators of environmental health, but traditional monitoring
is require huge man power. eDNA has the capability to enhance aquatic
biodiversity monitoring; however, more research into the characteristics of
species and life cycle traits that may affect identification and quantification
is required (Evans et al., 2016).

TABLE 13.1 Usage of eDNA for Different Species in Different Habitat

SL. eDNA Usage Habitat Evaluating References
No. Method
1. Mekong giant catfish Riverine qPCR Bellemain et al.
(Pangasianodon gigas) (2016)
2. Crown of thorns Coral reef qPCR+Sanger Doyle et al. (2017)
(Acanthaster cf. solaris) sequencing
3. Tilapia Freshwater Pond qPCR+Sanger Robson et al. (2016)
sequencing
4. Mollusks Ballast water Metabarcoding Ardura et al. (2015a)
5. Eukaryotic plankton Freshwater metabarcoding Banerji et al. (2018)
community
6. Plant DNA Lake sediments Metabarcoding Alsos et al. (2018)
7. Brown trout (Salmo Freshwater stream Metabarcoding Deutschmann et al.
trutta) (2019)

FIGURE 13.2 Schematic diagram of biosurveillance of aquatic ecosystem with eDNA.

310 Coldwater Fisheries and Aquaculture Management

13.3 SEDIMENTS

eDNA is being used on sediments from both freshwater and marine water
ecosystem to analyze plant communities. Alsos et al. (2018) estimated the
biodiversity of vegetation in lake and vascular plant eDNA at 11 different
lakes by using both methods eDNA and traditional sampling technique to
assess the level to which new taxa are reported in sediment eDNA samples.
They revealed 489 taxa by conventional method, of which 17–49% were
identified with the help of eDNA, while 47 plant taxa were determined
using eDNA, 73% of the samples were found to be in agreement with those
documented using traditional methods. They summarized that the taxonomic
groups which were available in eDNA were skipped during the sampling
and those skipped in eDNA were revealed with increased sampling effort
during traditional technique. Additionally, they reported that identification
rates differentiated among plant groups, and land based plant DNA because
of dependence upon sampling distance (Alsos et al., 2018).

13.4 EDNA AND EUTROPHICATION

The monitoring and management of freshwater habitats, such as lakes,

ponds, and rivers, has been the subject of several research (Thomsen et al.,
2012; Li et al., 2018). By properly monitoring changes in the microbial,
phytoplanktonic, and zooplanktonic population structure in lakes, eDNA
could be utilized to identify environmental stressors and analyze the trends
of lake ecosystems (Schulz, 2004). It has been suggested that eDNA data
could be useful in predicting pollution levels and identifying the major
drivers influencing ecological networks. Li et al. used eDNA metabarcoding
and principal component analysis (PCA) to determine species distribu­
tions and major community stressors such as total nitrogen (TN) and total
phosphorus (TP) in the Yangtze River Delta (YRD) (Li et al., 2018). Based
on operational taxonomic units (OTUs) and multivariate linear regression
models, these findings could be utilized to forecast pollution status (MLRs).
Many chemical pollutants have been linked to a reduction in eukaryotic
microbial diversity and a disruption of geochemical element cycling in water
(Yergeau et al., 2012). In situ collection of microbial communities in sedi­
ments from the Nanfei River in Anhui Province, China, was reported by Xie
et al. can be utilized to show chemical contamination from various land-use
types, such as agricultural and industrial districts (Xie et al., 2017).
Perspective of eDNA Application to Control Pollution 311

13.5 EDNA AND HABS

Cyanobacteria are the dominating species in eutrophicated water and can

create cyanobacterial blooms with fast cell multiplication (Vardaka et al.,
2005; Otten et al., 2012). Harmful blooms, such as cyanobacterial blooms,
may be induced by global warming and eutrophication (Davis et al., 2009).
By depleting oxygen and limiting nutrient cycling and light, cyanobacterial
blooms can have a severe impact on the environment and aquatic biodiver­
sity (Paerl, 2013). Additionally, cyanobacteria-produced toxins have been
reported to be harmful to animals, plants, and humans (Imai et al., 2010).
Bell et al. discovered changes in the eukaryotic and bacterial populations
in Lake Velence, as well as their correlations with environmental variables
(Bell et al., 2018).
In freshwater ecosystems, a large number of microorganisms, such as
bacteria, viruses, and fungi, play a key role in managing and mitigating
HABs (Yamamoto, 2013). Heterotrophic bacteria have also been identi­
fied as a tool for evaluating and reducing cyanobacteria’s harmful effects.
Around 460 bacterial strains were recovered from lentic, lotic, and drinking
water samples by Berg et al. (Berg et al., 2009). They used 16S rRNA DNA
sequencing to determine the taxonomic features of these heterotrophic
bacteria. Many growth-promoting and growth-inhibiting cyanobacterial
strains were discovered in the study’s findings, including Flavobacterium
and Pedobacter. Other strains capable of degrading poisons or chemical
substances, such as Sphingomonas, were also discovered. Cyanobacteria’s
secondary metabolites may have an impact on water quality and be toxic to
plants, animals, and humans (Briand et al., 2003; Funari et al., 2008).
Although eDNA is largely valuable tools for detecting diverse species,
as well as looking into the link between biodiversity and environmental
stress, few studies have looked into using it to find algicidal microbes in
freshwater environments. The majority of the research has been on isolated
and cultivated bacteria or fungus from freshwater habitats that can directly or
indirectly suppress HABs. Growth-inhibiting bacteria (GIBs), which hinder
Microcystis aeruginosa growth, have been isolated from the biofilm surface
of various aquatic plants, including Trapa japonica in Japan (Miyashita
et al., 2019). Imai et al. found bacteria from the biofilm surface of aquatic
plants (Egeria densa and Ceratophyllum demersum) in Lake Biwa that
induced mortality in Microcystis and GIBs (Imai et al., 2010). Most algicidal
microbes are undetectable by conventional procedures, which could create
a bottleneck in the termination of HABs. As a result, putting more focus on
312 Coldwater Fisheries and Aquaculture Management

hiring should be prioritizing the eDNA approach to discover and analyze

algicidal microbes.

13.6 SPECIMENS AND BIOFILMS

Sample acquisition for metabarcoding is a good choice for documenting

freshwater biodiversity, similar to what is done in marine habitats, with
many of these research concentrating on plankton. In a study of the influence
of prefiltering on the finding of microeukaryotic planktonic groups, Liu et al.
(2017) discovered that about 25% of readings in both categories were from
metazoans.
They also discovered no significant variations in richness, diversity, or
spatiotemporal trends, implying that prefiltering has little effect on eukaryotic
plankton size categories in freshwater ecosystems. Wurzbacher et al. (2017)
released a study that year which investigated eDNA metabarcoding for
dynamics in total plankton community through time, including eukaryotes
and prokaryotes. They found changes in community composition over
time that followed the same trends as morphological methods, although
morphological methods missed a bloom of a rare dominating bacterium
that was affecting the planktonic population. These findings demonstrate
the value of microbial and macrobial population metabarcoding research
for gaining a better knowledge of whole ecosystems, species interactions,
and community dynamics. Banerji et al. (2018) authored a similar study
in which they used metabarcoding to identify over 1,300 taxonomic units
from samples collected over 4 months from multiple sites in a reservoir,
including metazoans, protists, chlorophytes, and fungi. They discovered that
estimates of diversity varied between sites and taxonomic groups, implying
that distinct plankton groups respond to various environmental factors.
Freshwater biofilms have also been employed for broad microbial
metabarcoding. Besemer et al. (2013) tested fluvial biofilms using eDNA
metabarcoding to evaluate the variety of ecologically important benthic
bacteria’s distribution. They discovered that microbial richness dropped
downstream, particularly near confluences, based only on metabarcoding
data, implying that environmental and biotic factors may influence biofilm
connectivity and diversity. Furthermore, they discovered a large degree of
diversity in species composition between streams that was not explained by
geographic distance, implying that microbial dispersion is constrained by
fluvial networks (Besemer et al., 2013).
Perspective of eDNA Application to Control Pollution 313

13.7 CONCLUSION

eDNA has shown to be a highly effective tool for tracking biodiversity and
monitoring species populations, different communities, thanks to the rapid
advancement of DNA sequencing technologies. For the control and mitiga­
tion of Pollution and eutrophication in lentic and lotic water bodies, it is
vital to monitor and detect changes in coldwater habitats on a consistent
basis. The eDNA procedures when combined with other techniques, such as
physical and chemical methods, extensive data can be obtained. When these
new technologies are combined with traditional field methods for estimating
population structure, abundance, biomass, or individual condition, as well as
the extent of pollution, the utility of eDNA as a tool to better understand and
predict their impacts on the aquatic environment will be greatly enhanced.

KEYWORDS

• aquatic biodiversity
• environmental DNA
• freshwater
• growth-inhibiting bacteria
• nitrogen
• pollution
• principal component analysis

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CHAPTER 14

Nutritional Composition of Coldwater

Fishes
K. SRAVANI1, FAISAL SOFI2, TARIQ HUSSAIN2, KAWKABUL SABA3,
S. VIJAY KUMAR REDDY5, S. DEVADHARSHINI1, P. GANESAN1,
K. DHANAPAL4, and SHABIR A. DAR6
1
Department of Fish Processing Technology, Fisheries College and Research
Institute, TNJFU, Thoothukudi, Tamil Nadu, India
2
Division of Post-Harvest Technology, Faculty of Fisheries, SKUAST-K,
Jammu and Kashmir, India
3
Division of Fish Nutrition and Biochemistry, Faculty of Fisheries,
SKUAST-K, Jammu and Kashmir, India
4
Department of Fish Processing Technology, College of Fishery Science,
SVVU, Muthukur, Andhra Pradesh, India
5
Department of Harvest and Post-Harvest Technology, College of Fisheries,
Gadvasu, Punjab, India
6
Division of Fisheries Engineering, Faculty of Fisheries, SKUAST-K,
Jammu and Kashmir, India

ABSTRACT

Nowadays, the consumption of fish, as food has been increasing as fish

contains almost all major and minor nutrients and show positive health
benefits. Sensory attributes like texture, taste, flavor, appearance, and quality
are the main concern for the consumers which are due to the net result of

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
320 Coldwater Fisheries and Aquaculture Management

complex reactions between major and minor nutrients. The geographical

location, morphology, season, ecosystem, culture practices are the different
components that play a significant role play in representing the quantity of
these nutrients. When compared with amino acid (AA) composition, fatty
acid composition is mostly affected by food. In one way or other, all the
coldwater fishes are good in their nutrient composition. Most of the important
cold water food fishes are superior in expressing the ratio of n–6/n–3 fatty
acids. All the amino and fatty acid composition in coldwater fishes show
good effect on the cardiovascular system of adults and prenatal development
in the case of children. The present chapter is aimed to compile and
complement to the available nutritional composition of coldwater fishes in
view of proximate, fatty acid and AA phrases along with medicinal benefits.

14.1 INTRODUCTION

Coldwater fishes play a significant role as a part of the diet of the upland
people of India and ensure good nutritional profile comprising of Essential
amino and fatty acids, minerals, and vitamins. Coldwater fishes have
immense prospects, a great potential in creating livelihood opportunities
and occupy an important place in higher upland areas. Cold water fishery
resources dispersed over the Himalayan and peninsular areas of India,
where these are known as a candidate species for sports, ornamental, and
food. In India, a number of Coldwater streams, rivers, lakes, and reservoirs
are present. The overall lengths (OALs) of the upland rivers and coldwater
streams are distributed to 10,000 km. Climate variations are seen in different
parts of the country due to attribution, slopes of mountains, variation in
geography, vegetation, and river valley expansion, on account of which the
water resources harbor rich species diversity in upland areas. In total the
coldwater fishes constitute 258 species, which are dispersed in 21 families
as 76 genera. Out of which, 91 from the Deccan plateau, 203 from West
and Central Himalaya and 255 species are from Northeast Himalayas.
The variation in dispersion of hill stream fishes will vary depending on is
primarily flow of water, characteristics of substratum, temperature profile,
quality of water and the availability of food naturally. Salmons, trout,
mahseers, and the mirror carp comprise the major group of the economically
important coldwater fishes. As per economic importance, few of coldwater
species are used for sports, some as food and a few others are as ornamental
fishes.
Nutritional Composition of Coldwater Fishes 321

14.1.1 SPORT FISHES

Tor tor (Golden mahseer), Tor mossal (Copper mahseer), Mazirifor chely­
noides (Black mahseer), Neolissochilus hexagonolepis (Chocolate mahseer),
Raiamas bola (Indian trout) among indigenous species and Oncorhynchus
mykiss (rainbow trout) and Salmo trutta fario (brown trout) among exotics
are considered as important sport fishes.

14.1.2 FOOD FISHES

The important genera among food fishes are–snow trout (Schizothorax),

Garras (Garra sp.), minor carps (Labeo sp.), Bariels (Barilius sp.), exotic
carps (Ctenopharyngodon idella, Hypothtalmichthys molitrix, Cyprinus
carpio), eels (Mastacembalus armatus) are known as food fishes.

14.1.3 ORNAMENTAL FISHES

Colorful and captivating species have been recognized as ornamental

fishes. The crucial ornamental fish species are Carrassius auratus, Puntius
gelius, Puntius conchonius, P. ticto, P. sophore, Badis badis, Barillus vagra,
Brachydanio rerio.

14.2 POTENTIAL BENEFITS OF FISH CONSUMPTION

Fish is being increasingly preferred due to health consciousness among the

people all over the world and is playing a crucial role in human nutrition
as low caloric foods. Fishes are best source of Omega-3 poly unsaturated
fatty acids which proved to have beneficial results in reducing the chance
of cardio-vascular diseases and are shown to have positive benefits in
many other pathological situations especially, few types of cancers and
arthritis.
The superiority of sea food is due to:
• More amount of poly unsaturated fatty acids;
• High amount of essential amino acids (EAAs);
• Easily digestible proteins;
• Abundance of several micro-nutrients.
322 Coldwater Fisheries and Aquaculture Management

14.2.1 NUTRIENT CONTENT

Seafoods reflect as an excellent source of major nutrients. Fish provides a

significant range of nutrients on a caloric unit basis, though more intake of fish
is compatible with a reduction of both saturated fatty acid and caloric intakes.
Fish nutrients are having a lot of advantages and some curative benefits for
contemporary health problems like osteoporosis, obesity, coronary heart
disease (CHD), cancer, hypertension, iron, protein deficiency, and arthritis.

14.2.1.1 SIGNIFICANCE OF FISH PROTEINS AND AMINO ACIDS (AAS)

The major nutrient of fish muscle is protein, a 100 gr of cooked fish in most
species serve approximately 11–24 gr of proteins, which is about one third
of the average daily recommended protein intake, thus playing a beneficial
role in eliminating the protein-calorie malnutrition. Fish protein digestibility
is more due to the presence of very less stroma protein fraction and has an
outstanding range of essential AAs as per the requirement of the body for
growth and repair of the tissue. Some proteins are incomplete and must be
supplemented with other protein foods, containing all most all AAs as per
the requirement of the body. The protein from vegetable resources and fish
will show a lesser burden in renal hemodynamics when compared with the
animal protein sources.
There is proven evidence that the fish protein has the capacity to improve
lipid profiles of blood in humans. Fish proteins also play a role as heart
protective major nutrient as they are participating in reducing the cholesterol
in plasma due to less fraction of lysine to arginine and methionine to glycine.
The proteins in fish protein exerts beneficial effects on some of the nutritional
abnormalities related to body weight.
The knowledge on composition of AAs of fish is important because of
their role in setting up probable nourishment as they provide AAs for protein
biosynthesis, which is important for growth of infants and children and
also for the replacement of body proteins in adults, Work as precursors of
hormones, other biomolecules, secondary metabolites and provide a signifi­
cant fraction of the total daily energy requirement of the body.
Conventionally, the AAs are categorized as non-essential (NEAA), essen­
tial (EAA) and conditionally EAA (CEAA). Newly, the group called the
functional amino acids (FAA) has been initiated. The group of AAs which
regulate the crucial metabolic pathways for survival, improving health,
Nutritional Composition of Coldwater Fishes 323

development, growth, reproduction of an organisms and lactation are called

functional AAs. Examples for these functional AAs are Arginine, methionine,
leucine, cystine, proline, tyrosine, aspartic acid, tryptophan, glycine, gluta­
mine, glutamic acid, and taurine. Arginine, methionine, cystine, histidine,
isoleucine, leucine, lysine, proline, phenylalanine, threonine, tryptophan,
tyrosine, valine are considered as EAA whereas alanine, asparagine, aspartic
acid, serine as non-essential AAs and glycine, glutamic acid, glutamine, and
taurine as CEAA (Wu, 2013).
Arginine plays a key role in healing of wounds, cell division, immune
properties, hormone release and removal of ammonia. It works as a key
signaling molecule in a various of biological processes because it is a
precursor of nitric oxide (NO), it has a role in the treatment of conditions
where vasodilatation is required. It can be given as a supplement for treat­
ment of diseases like hypertension, pre-eclampsia, sepsis, erectile dysfunc­
tion, anxiety, etc. In case of coldwater fishes O. mykiss (6.5±0.3 gm 100 g–1
protein) T. putitora, N. hexagonolepis were reported to contain great amount
of arginine, when compared with the other species studied and hence, these
fishes can be suggested for arginine deficiency (Sarma et al., 2013).
Leucine, is the AA that plays a role in stimulation of protein synthesis
by muscle (Etzel, 2004) and is also having curative role in stress conditions
like sepsis, burn, and trauma (DeBandt & Cynober, 2006). Leucine content
is found to be high marine fishes like S. waitei and R. kanagurta (10.4±0.4
and 10.3±0.410.4 g 100–1 g protein, respectively), carps C. mrigala, L. rohita
and catfishes C. batrachus and H. fossili.
The AA Methionine finds it way for treatment of psychosis, depression,
liver disorders, improving wound healing, dipsomaniac, hypersensitivity,
branchial asthma, copper poisoning, radiation side effects, drug withdrawal,
and Parkinson’s disease (Mischoulon & Fava, 2002). Methionine content
is higher in mutton when compared with fish. The marine fish S. waitei
(4.0±0.4 100–1 g protein) and coldwater fish T. putitora (3.6±0.3 g 100–1 g
protein) was reported to have good quantity of methionine than other fishes
(Loest et al., 1997).
Glutamic acid is having a crucial role in the metabolism of AAs as it is
involved in transamination reactions which are required for the synthesis of
important molecules, called glutathione. Glutamic acid also plays an impor­
tant role regulation of metabolism, tissue injury prevention, anti-oxidant
activity enhancement, promotion of protein synthesis, wound healing,
increasing immunity and in curing of obesity, cardio vascular disorders,
certain types of cancers, and some inflammatory diseases (Wang et al., 2013).
324 Coldwater Fisheries and Aquaculture Management

Tryptophan is a precursor for serotonin, a brain neurotransmitter which

plays a role in reducing pain (Segura & Ventura, 1988). It is also a precursor of
kynurenine, tryptamine, and melatonin and has a major role in the functioning
of neurotransmitters like nor-epinephrine and dopamine. Tryptophan can also
be used as a supplement to get relief from pain, sleeplessness, dejection,
seasonal affective disorder, bulimia, premenstrual dysphoric disorder, ADH
disorder and chronic fatigue (Richard et al., 2009). T. putitora was found to
contain the highest amount of tryptophan among the fishes studied coldwater
fishes.
Histidine, plays a role as a precursor during the biosynthesis of histamine
and carnosine. The stability of proteins has been found to be increasing
during thermal stress due to the presence of histidine which is responsible for
tissue repair and growth, myelin sheaths maintenance and in heavy metals
removal from the body (Heimann, 1982).
Glycine is considered as an essential and functional AA for fish and plays
key roles in a wide range of metabolic processes and chronic insufficiency
of glycine leads to insignificant growth, immune response impairment and
various bad effects on nutrient metabolism and health.
The essential AA that is broadly important for optimal growth is Lysine
and its deficiency results in immunodeficiency (Chen et al., 2003). Lysine
can be used as an oral supplement or directly to the skin for reducing or
preventing cold sores. Lysine content is recorded to be very high in S.
commersoni (16.1±0.9 g 100–1 g protein) and T. putitora (9.4±0.6 g 100–1 g
protein).
Threonine finds its role in curing different disorders related to brain
which includes spinal spasticity is used for to treating various nervous system
disorders including spinal spasticity, weakness, amyotrophic lateral sclerosis
and familial spastic paraparesis (Hyland, 2007). S. waitei can be suggested
as source as natural supplement for threonine.
Isoleucine is the AA that is important for correct growth and formation
of muscle (Charlton, 2006). Leucine, isoleucine, and valine are reported to
be in low in plasma of patients with chronic renal failure. Alanine is a major
fraction of the antifreeze proteins (AFPs) where it is present as alanine­
alanine-threonine in the blood plasma of polar region fishes and animals to
prevent crystallization in blood. Alanine content is observed high in patients
with hypertension, more cholesterol and BMI.
Non-essential AAs are having role in gene expression regulation and
animal cell metabolism. Aspartic acid which is considered as functional AA,
works as a precursor to methionine, threonine, isoleucine, and lysine and
also regulates the hormonal secretion. Similarly, for glycine, cysteine, and
Nutritional Composition of Coldwater Fishes 325

tryptophan, serine acts as a precursor and also important for cell signaling.
Most commonly serine is used as a supplement in curing of schizophrenia.
Taurine, conditionally essential AA present in the most of the fishes is
helpful for healing people having edema, high blood pressure, cardiac disor­
ders, proper eye functioning, eagerness, and arthrosclerosis and is important
for better usage of sodium and along with protective effect on brain.
Particularly some fishes are considered as good source of various AAs
viz., Oncorhynchus mykiss, Tor putitora, Neolissochilus hexagonolepis are
rich in Arginine, Rastrellger kanagurta, Catla catla, Stolephorus waitei,
Amblypharyngodon mola, Puntius sophore are high source of Histidine,
Oncorhynchus mykiss, Labeo rohita, Stolephorus commersonii are good
sources of Isoleucine and Leucine, Stolephorus commersonii, Thunnus
albacares, Tor putitora are considered as rich source of Lysine, Stolephorus
waitei, Tor putitora, Rastrelliger kanagurta are source of Methionine,
Cirrihus mrigala, Catla catla, Labeo rohita are nice source of Phenyl alanine,
threonine content is more in Thunnus albacares, Nemipterus japonicus,
Stolephorus waitei, Stolephorus commersoni, Tyrosine concentration is
more in Oncorhynchus mykiss, Tor putitora whereas Nemipterus japonicas,
Cirrihus mrigala, Rastrelliger kanagurta are good source of Valine, Tor
putitora, Cirrihus mrigala, Catla catla, Labeo rohita are showing high
volume of Tryptophan, Glutamic acid, Glycine, and Proline is present in
Oncorhynchus mykiss, Tor putitora. The nonessential AAs like Alanine is
present in Nemipterus japonicus, Labeo rohita, Catla catla, Stoleophorus
commersonii, Heteropneustes fossilis, Clarias batrachus, Nemipterus
japonicas, Thunnus albacares are a rich source of Aspartate and Serine
(AICRPs/Network Projects/Outreach Activities, 2014; Mohanty et al., 2014).

14.2.1.2 LIPIDS

Most fish are considered excellent because of their low-fat content. The
majority of fish consumed are low in fat, containing 1–2% fat present
mostly as phospholipids components of muscle membranes. A few popular
finfish contain more than 2–3% fat. A few species contain more than 10%
fat, located mostly in the sub-epithelial layer. The fat in finfish is composed
mostly of triglycerides which are fatty acid esters of the trihydric alcohol,
glycerol. The composition of fatty acid in fish varies with species, season of
catch and diet. The eicosapentenoic acid (EPA) and docosahexaenoic acid
(DHA) are the two important fatty acids observed in fish oils out of seven
omega-3 fatty acids.
326 Coldwater Fisheries and Aquaculture Management

14.2.1.2.1 Functions of Ω-3 Fatty Acids

The Ω-3 fatty acids play a major role in various body functions viz.:
• Notably decrease the chance of heart attack and stroke by:
o Reducing the hypertension by inhibiting the body’s production of
eicosanoids that cause inflammation in blood vessels.
o Reduces the levels of triglycerides in the blood, decreases the
growth rate of artery narrowing plague.
• Reduction of rheumatoid arthritis when supplemented through diet,
i.e., reduction of swelling, pain, and redness of joints.
• Help in curing people with psoriasis or eczema by blocking the action
of arachidonic acid.
• Omega 3 fatty acid (DHA) helps to improve the mental health, especially
depression and attention deficit/hyperactivity disorders (ADHD).
• Reduce the tumor growth in people with cancer.
• Thrombosis and ameliorate ischemic heart diseases can be lowered by
consuming a good quantity of triglycerides.
• For the many years only Ω-6 fatty acids, arachidonic, and linoleic are
considered necessary (at 1–2% of dietary calories) for normal growth
and health, especially from the skin.
• Proofs shown that Docosahexaenoic acid (C22: 6n–3), is a component
of phospholipid membrane in retinal receptors, cerebral gray matter
and sperm and it is required for regular retinal functions. Compara­
tively more amount of DHA is required for normal brain development
and learning ability in newborn. Hence, the mechanism of Selective
incorporation is practiced supplying high levels of DHA to the fetal
brain.
• The role of DHA is suggested to be one of regulation of membrane
function. The fetus has specific requirements for Ω-3 fatty acids. DHA
is preferentially taken by brain cell membrane phospholipids. Liver,
forebrain, and retinal cells for the developing fetus show proportional
increase of Ω-3 and decrease of Ω-6. A high Ω-3: Ω-6 ratio can be
damaging.

14.2.1.2.2 Cholesterol

Seafood is not a major dietary source of cholesterol. Finfish generally

contain modest amounts of cholesterol, i.e., 50–90 mg/100 g muscle. The
Nutritional Composition of Coldwater Fishes 327

cholesterol content of fish tends to increase with fat content. Shellfishes are
reported to contain moderately high quantity of cholesterol; so, crustaceans
(crabs, lobsters, and shrimps) are having 60–100 mg/100 g and mollusks
(clams, oysters, and mussels), 40–110 mg/100 g. Squid and fish roe contain
relatively high amount of cholesterol.

14.2.1.2.3 Squalene

Shark liver oils contain more amount of Squalene which is an isoprenoid

molecular protein, which works as a lipid lowering agent (antilipidemic),
reduces or prevents the rate of oxidation (antioxidant) and membrane stabi­
lizing agent. Squalene is included in cholesterol metabolism as a precursor
in the biosynthesis of cholesterol and inhibitor of HMG CoA reductase. It is
also having the property of suppressing the action of carcinogenic tumor, on
exposure to squalene over a period of time. Squalene improves the effect of
cholesterol reducing drugs.

14.2.1.3 MINERALS

14.2.1.3.1 Sodium

There has been probably more publicity about this element in these past
few years than virtually any other nutrient. This is largely because sodium
is strongly linked to blood pressure or hypertension. Controlling the amount
of sodium consumption can be helpful in lowering high blood pressure. In
other words, one should consume mainly foods naturally low in sodium.
Fresh seafood is one best choice for curbing sodium intake since all fish
varieties are low in sodium. Most fresh fish range from about 60 to 110 mg
of sodium per 100 g of raw muscle. Most shellfishes, on the other hand,
have more sodium, usually in the range of 200–400 mg/100 g. Squid falls in
between at about 160 mg. In case of processed fish like freezing, canning,
smoking, and curing, sodium content is found to be high, which varies from
300 to 900 mg/100 g. The reason behind the more content of sodium is due
to conventional processing and on-board handling procedures like brine
treatment and stowage in refrigerated sea water system, treatment of filets
with sodium tripolyphosphate to reduce drip loss, etc., which results in the
infusion of sodium. Frozen battered seafood products contain an average
of 400 mg/100 g. In the majority of these products, the battered/breaded
328 Coldwater Fisheries and Aquaculture Management

coatings have more sodium than the meat portion. The increased popularity
of surimi may require the use of salts other than sodium.

14.2.1.3.2 Potassium

Potassium is well-known as an electrolyte, one of the minerals that body’s

cells to function properly and to help regulate fluid balance. Potassium
has other functions that help in critical activities like regulating heartbeat,
muscle contractions and nerve signaling. It’s required to help some enzymes
to function, aids in bone health, lowers the occurrence of some heart related
disorders, and can lower the risk of kidney stones, too. Normally, fresh fishes
will have more amount of potassium, ranging from 250–320 mg/100 g. The
safe daily intake of potassium is between 1.8 and 5.6 grams.

14.2.1.3.3 Calcium

Calcium is necessary for the formation of strong bones and bone strength.
Proper absorption of calcium is associated with vitamin D. Calcium plays an
important role in the proper functioning of muscles, nervous system, bold
clotting process. Small fishes can be consumed along with bones, which
forms a good source calcium rather than discarding. Calcium deficiency is
associated with a condition called rickets in case of young children and in
case of adults leads to osteomalacia where bones will become soft. Fluorine
is also important for strong bones and teeth. Calcium is vital for the growth
of children, women, especially during reproductive years to immense subse­
quent osteoporosis. Intakes of 1.25 g/day are recommended, where milk is
the major source. Seafoods can also be useful as source of calcium, which
are having a calcium range of 6 to 120 mg/100 g, depending on the species
and processing.

14.2.1.3.4 Iron

This can be a limiting element in the diets of young women. The level found
in seafood range from 0.3 to 7 mg/100 g. Seafoods, especially shellfish and
dark fleshed fish (mackerel, tuna, and sardine) are reasonably good source of
iron, supplying 1–2 mg/100 g muscle.
Nutritional Composition of Coldwater Fishes 329

14.2.1.3.5 Zinc

This is an essential component of enzymes involved in the biosynthetic

reactions. There is some indication that the dietary intake can fall below
the daily recommended allowances of 15 mg per day. Seafoods, particularly
shell fishes are major source of zinc, and also a probable source of copper
and iodine. Seafoods are also rich source of selenium which, as an integral
component of peroxidase, provides protection against the toxicity of heavy
metals.

14.2.1.4 VITAMINS

Vitamin A is very important in the diet of man. It is required for vision,

epithelial membrane integrity and perhaps, as an anticancer retinoid. It may
also function on an anti-oxidant capacity as a free radical quencher, but it is
toxic when consumed in large amounts. Fish liver oils contain more amounts
of vitamin A, and small amounts occur in fish muscle. Hence, fishes are not
considered as extensive source of this vitamin. Fatty fish contain a modest
amount of vitamin A, in contrast to lean fish which contain very little of
it. Fish liver oil contain vitamin which is associated with calcium transport
and calcification of bones. In its active form, vitamin D promotes synthesis
of the protein involved in the transport of calcium. The high fat species of
fish such as mackerel and sardines are having high amount of D vitamin in
the tissues. Vitamin E is a natural antioxidant that helps in protecting cell
membranes and strengthening the immune system. Vitamin K plays a role in
blood coagulation by preventing internal bleeding.
Fish contain varying concentration of B-vitamins, depending upon the
specific vitamin and the fish species. Although a poor source of thiamine,
fish is having moderate amount of vit B9 (folic acid), B7 (biotin), B3 (niacin)
and B5 (pantothenic acid). Dark fleshed fishes are considered as moderate
source of Riboflavin. Fish and shellfishes contain a reasonable quantity of
Pyridoxine and are rich source of vitamin B12.

14.3 NUTRITIONAL COMPOSITION OF COLDWATER FISHES

Most of the population from hill stations consumes fish as daily food.
Geographical location, seasons, ecosystem, culture practices show their
330 Coldwater Fisheries and Aquaculture Management

effect on proximate composition. For example, when compared with the

other freshwater fishes coldwater fishes are having high amount of PUFA.
Nutritional composition of golden Mahseer changes based on the seasonal
changes and geographical changes (Sarma et al., 2015). The average protein
value varied from 15.59 to 17.29 g/100 g fish, whereas ash, crude fat and
moisture values observed to be varied as 1.23–1.55, 0.62–1.52, and 76.24–
79.24 g/100 g fish, respectively. Seasonal changes influence on mean protein
content of Mahseer where highest protein content and moderate fat content
was reported during the months of June to September, as it is breeding
season for the particular species. Mahseer is having high quantity of calcium,
which is reported as 219.33 ± 9.018–1951.67 ±3.510 mg/100 g followed by
potassium (755.33±14.740–1523.0±3.000 mg/100 g) but the average sodium
concentration is low comparatively (99.67±2.082–253.33±4.163 mg/100 g).
The average micro-mineral contents like iron, manganese, zinc, and sele­
nium are 0.83 ±0.208, 0.17 ±0.036, 1.43 ±0.153, 0.87 ±0.306 mg/100 g,
respectively.
In Oncorhynchus mykiss and Schizothorax richardsonii proximate
composition also varied along with geographical locations and seasonal
variations. In O. mykiss, the protein, ash, fat, and moisture content on
dry matter basis are reported as 62–75.81%, 4.34–6.21%, 10.46–13.76%,
72.67–77.14% respectively and the macro-and micro-minerals as sodium
(116.66–216.66 mg/100 g), Potassium (868–1538.67 mg/100 g), Calcium
(299.66–488 mg/100 g), Iron (0.429–5.171 mg/100 g), Manganese (0.077–
0.193 mg/100 g), Zinc (0.479–1.789 mg/100 g), Selenium (0.268–1.660
mg/100 g). Whereas S. richardsonii showed an average protein content of
56.96–72.45%, ash content of 5.49–10.68%, fat content of 11.18–20.59%
and a moisture content of 70.12–77.34% and is abundant source of Potassium
(608–1246.67 mg/100 g), followed by Calcium (208–417 mg/100 g), sodium
(60–241 mg/100 g), Zinc (1.197–2.705 mg/100 g), Selenium (0.826–1.032
mg/100 g), Iron (0.461–0.892 mg/100 g) and Manganese (0.204–0.345
mg/100 g) (Das et al., 2012).
The habituated coldwater fishes of kosi river viz., Labeo dero, Labeo
dyocheilus, Labeo pangusia, Tor putitora and Schizothorax rechardsonii
forms a major contribution to total catch. The Proximate composition of
the fish has been reported by Sarma et al. (2011). The Average moisture,
crude protein, crude fat and ash of Labeo dero are noted as 76.92%, 70.24%,
32.53%, 8.86% respectively. The macro- and micro-mineral content is
reported as sodium (138 mg/100 g), Potassium (1,233 mg/100 g), Calcium
(342 mg/100 g), Iron (1.016 mg/100 g), Zinc (1.239 mg/100 g), magnesium
Nutritional Composition of Coldwater Fishes 331

(0.213 mg/100 g) and selenium (0.265 mg/100 g) and in Labeo dyocheilus

the average moisture was found to be 76.32%, crude protein as 68.53%,
crude fat as 12.66% and ash content as 6.51% whereas the macro- and micro-
mineral content was reported as sodium (100 mg/100 g), Potassium (925
mg/100 g), calcium (263 mg/100 g), Iron (0.984 mg/100 g), Zinc (0.963
mg/100 g), magnesium (0.076 mg/100 g) and selenium (0.442 mg/100 g).
In Labeo pangusia the values were observed as 81.54%, 73.01%, 24.19%,
7.39% for moisture, crude protein, crude fat and ah content, respectively
and is a rich source potassium (784 mg/100 g) and calcium (270 mg/100 g)
followed by low level of sodium (103 mg/100 g), iron (0.263 mg/100 g),
zinc (0.769 mg/100 g), magnesium (0.164 mg/100 g) and selenium (0.565
mg/100 g). Tor putitora is having a moisture content of 72.68%, crude protein
52.43%, crude fat 19.25% and ash as 6.23%. The macro- and micro-mineral
content is shown as sodium (241 mg/100 g), potassium (1,228 mg/100 g),
calcium (397 mg/100 g), iron (0.628 mg/100 g), zinc (1.407 mg/100 g),
magnesium (0.128 mg/100 g), and selenium (1.311 mg/100 g). Snow trout
(Schizothorax spp.) are distributed from Jammu and Kashmir to Arunachal
Pradesh. Of these S. niger, S. progastus, S. plagiostomus, S. curvifrons,
and S. esocinus are important food fishes. In all the five species the protein
content ranged from 15.43–17.17%, crude fat from 2.21 to 7.98%, Ash
content from 0.89–1.18% and moisture content from 75.20 to 82.74% on
wet weight basis and the macro- and micro-mineral content was reported as
sodium (241 mg/100 g), potassium (1,246 mg/100 g), calcium (406 mg/100
g), Iron (0.464 mg/100 g), zinc (1.497 mg/100 g), magnesium (0.221 mg/100
g), and selenium (0.121 mg/100 g).
Some important coldwater fishes in Himalayas are Semiplotus semiplotus,
Labeo dero, Labeo dyocheilus, Sanguina sanguine, Barilius bendelisis, Garra
mullya, L. pangusia, Tortor, Salmo trutta, and Neolissochilus hexagonolepis
(Sarma et al., 2014). These fishes are mainly consumed by the people in
entire North Eastern India and among all the 10 species the moisture content
ranged from 71.21 to 78.22%, crude protein content varied from 16.3 to
20.5%, fat content from 1.51 to 9.65% and ash content differed from 0.91
to 3.54%. Labeo dero is rich source of Potassium (1435.56 mg/100 g) and
Calcium (1423.31 mg/100 g) whereas Sanguina sanguine is considered as
a good source of Sodium (309.12 mg/100 g) and Selenium (177.52 mg/100
g). The other species Barilius bendelisis shown high amount of Iron (8.74
mg/100 g) and Manganese (1.24 mg/100 g) and Semiplotus semiplotus
showed high amount of Zinc (1.72 mg/100 g).
332 Coldwater Fisheries and Aquaculture Management

14.4 AMINO ACID (AA) COMPOSITION OF COLDWATER FISHES

AAs are having good nutritive value and are having good health benefits
(Chyun & Griminger, 1984). The reference AA score (mg/g protein) for
histidine, Isoleucine, leucine, lysine, methionine-cysteine, phenylalanine-
tyrosine, threonine, tryptophan, valine was 14, 28, 66, 58, 25, 63, 34, 11, 35,
respectively.
Sarma et al. (2013) reported the AA composition of five coldwater fishes
which are most important and the results were discussed below:
1. Tor putitora: The golden mahseer is a freshwater fish inhabiting
along streams, rivers, and lakes and is benthopelagic in habitat. It had
amino composition (mg/g protein) with more quantity of Tryptophan
(618 mg/g protein) followed by phenylalanine-tyrosine (187 mg/g
protein), lysine (170 mg/g protein), Methionine-cysteine (149.78
mg/g protein), Isoleucine (137.73 mg/g protein), leucine (120 mg/g
protein), valine (114.15 mg/g protein) and Histidine (36.15 mg/g
protein).
2. Neolissochilus hexagonolepis (McClelland, 1839): The chocolate
or copper mahseer is having highest amount of histidine (183.43 mg/g
protein) followed by tryptophan (149.98 mg/g protein), methionine-
cysteine (143.30 mg/g protein), valine (126.69 mg/g protein), lysine
(92.83 mg/g protein), leucine (77.34 mg/g protein), threonine (74.67
mg/g protein), isoleucine (72.84 mg/g protein), and phenylalanine-
tyrosine (67.16 mg/g protein).
3. Oncorhynchus mykiss (Walbaum, 1792): i.e., rainbow trout had 17
AAs of which the most abundant AA (g/100 g of muscle) was Proline
(1.67), Aspartic acid (1.48), Tyrosine (1.44), Glycine (1.21), Serine
(1.16), Arginine (1.13), Isoleucine (1.12) and tryptophan (1.07). In
total 20, AAs were identified with Cyprinus carpio (Linnaeus, 1758)
with highest amount of Aspartic acid (2.721) followed by Serine
(2.441), Glutamic acid (1.903), Alanine (1.553), Glycine (1.497),
and Arginine (1.011).
The composition of AAs (mg/g protein) in trout were analyzed of which
all the species showed that they are good sources of alanine, Leucine, and
Phenyl alanine. S. curvifrons is the first highest source of Alanine (37.48
g/100 g protein) and second highest source of Leucine (10.38 g/100 g
protein), whereas S. esocinus is first highest source of Leucine (12.51 g/100
g protein) and second highest source of Alanine (22.62 g/100 g protein). S.
labiatus reflected with a high amount of Leucine (12.51 g/100 g protein) and
Nutritional Composition of Coldwater Fishes 333

Phenyl Alanine (16.93 g/100 g protein) and S. niger showed large amount of
Alanine (15.63 g/100 g protein) and Leucine (14.63 g/100 g protein).

14.5 COMPOSITION OF FATTY ACIDS IN COLDWATER FISHES

The composition of fatty acids in coldwater fishes is categorized under the

groups as saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs)
and polyunsaturated fatty acids (PUFAs) and the fatty acid composition of
fishes varies depending on type of species, period of maturity, age of fish,
size of fish, seasonal variations, and location.
The fatty acid profile for cold water fishes like T. putitora, N. hexagono­
lepis, O. mykiss, S. richardsonii, C. carpio were analyzed by Sarma et al.
(2013). The total saturated fatty acid content, total monounsaturated fatty
acid content, total n–3 PUFA and the total n–6 PUFA in Tor putitora were
analyzed and the results were 52.97%, 28.06%, 8.69%, 9.67% whereas in N.
hexagonolepis the fatty acid components were shown as 44.29%, 23.95%,
21.48%, 9.72% respectively. The ratio of n–3 to n–6 PUFA in golden
Mahseer was 0.9% and in N. hexagonolepis was 2.21%. The saturated fatty
acid content was the most dominant fatty acids in both species. From the
SFAs, palmitic acid (C16:0) is major where stearic acid (C18:0) consists
of 5.87–9.58% of the total fatty acids. Next to these MUFAs were high in
quantity which are reported as 23.95% and 28.06% in N. hexagonolepis and
T. putitora, respectively. The major contributions from poly unsaturated fatty
acids includes Linolenic acid (C18:3n–3), Linoleic acid (C18:2n–6), EPA
(C20:5n–3), DHA (C22:6n–3) and Arachidonic acid (C20:4n–6).
The total saturated (TSF) and monounsaturated fatty acid, total n–3
PUFA and n–6 PUFA content of O. mykiss was reported as 34.53%, 34.7%,
as 13.61% and 17.77% respectively. The fraction of n–3 to n–6 PUFA was
found to be 0.77%. In S. richardsonii, the TSF, total MUFA, total n–3 PUFA
and the total n–6 PUFA were found to be 42.55%, 37.32%, 16.18%, 3.25%
respectively and the ratio of n–3 to n–6 PUFA was 4.98%, whereas, in case
of C. carpio the values are 46.19%, 31%, 9.81%, 13.91% respectively and
the fraction of n–3 to n–6 PUFA was 0.7%.
In both rainbow trout and common carp species, MUFA was found to
be dominant followed by SFAs and PUFAs. Among MUFA, Palmitic acid
(C16:0) was abundant followed by stearic acid (C18:0). Oleic (C18:1) and
Palmitoleic (C6:1) acids and from PUFA the highest to lowest quantity of
acids are Linoleic acids (C18:2n–6), DHA (C22:6n–3), Linolenic (C18:3n–
3), Arachidonic acid (C20:4n–6) and EPA (C20: %n–3) in both species.
334 Coldwater Fisheries and Aquaculture Management

14.6 FATTY ACID COMPOSITION OF SOME CULTURED AND

NATURAL COLDWATER FISHES

14.6.1 SALMON

Atlantic salmon is the major consumed coldwater species by the people.

The total lipids in farmed and wild Atlantic salmon were 7.4 and 7 g/100 g
whereas the total fat content was about 4.1 g/100 g in farmed and 4 g/100 g in
wild salmon, respectively. In farmed Atlantic salmon the total PUFAs were
abundant (41%) later to that monosaturated fatty acids (33.4%) and SFAs
(25.6%). The percentage of n–6 PUFA was 9.8%, n–6 HUFA was 1.7%,
C18:2 (linoleic acid) was 7.4% and C20:4 (arachidonic acid) was 0.9%. The
percentage of n–3 PUFA was 31.1% and n–3 HUFA was 28.3%, C 18:3
(α-linolenic acid) was 1.6%, C 20:5 (EPA) was 7.9% and C 22:6 (DHA) was
15.2% respectively. In case of wild Atlantic Salmon, the total MUFAs were
more (53.7%) trended by PUFAs (27.3%) and SFAs (19%). The percentage
of n–6 PUFA was 2.3%, n–6 HUFA was 0.8%, C18:2 (linoleic acid) was
1.2% and C20:4 (arachidonic acid) was 0.4%. The percentage of n–3 PUFA
was 25% and n–3 HUFA was 23.8%, C 18:3 (α-linolenic acid) was 0.5%, C
20:5 (EPA) was 6.6% and C 22:6 (DHA) was 13.1% respectively. The ratio
for n–3 to n–6 value was high in wild Atlantic salmon (11%) in contrast to
Farmed Atlantic salmon (3.6%). Comparatively the fatty acid profile was
high in farmed species to that of wild species (Blanchet et al., 2005).

14.6.2 RAINBOW TROUT

The total fatty acid and lipid in cultured and natural rainbow trout were 3.2
g/100 g, 0.6 g/100 g and 5.6 g/100 g, 1 g/100 g, respectively. Farmed rainbow
trout has the highest amount of PUFA (40.6%) followed by MUFA (32.5%),
Saturated fatty acid (26.9%). Wild rainbow trout has also contained the
highest amount of PUFA (58.6%) followed by SFAs (24.4%), MUFAs (17%).
In Farmed rainbow trout the percentage of n–6 PUFA, n–6 HUFA, C18:2
(linoleic acid), C20:4 (arachidonic acid) was 8.5%, 1.7%, 6.2% and 0.9%
respectively. The percentage of n–3 PUFA, n–3 HUFA, C 18:3 (α-linolenic
acid), C 20:5 (EPA), C 22:6 (DHA) was 32.2%, 30.1%, 1%, 7.3%, 18.7%
respectively. Natural rainbow trout the percentage of n–6 PUFA, n–6 HUFA,
C18:2 (linoleic acid), C20:4 (arachidonic acid) was 12.5%, 7.9%, 4.2%
and 5.4% respectively. The percentage of n–3 PUFA, n–3 HUFA, C 18:3
Nutritional Composition of Coldwater Fishes 335

(α-linolenic acid), C 20:5 (EPA), C 22:6 (DHA) was 46.2%, 44%, 1.7%,
8.1%, 32.2% respectively. Cultured rainbow trout contain high levels of n–3
HUFA and reported to show more health benefits to the consumers (Blanchet
et al., 2005).

14.6.3 DICENTRARCHUS LABRAX

In case of wild Dicentrarchus labrax, the total SFAs, total monoenoic, n–6,
n–3, total polyenoic and ratio of n3:n6 is 33.4, 19.4, 11.8, 35.6, 47.4, and
3.02% where as in case of Farmed species the values were noted as 29.3,
34.6, 9.3, 26.8, 36.1, and 2.88, respectively. In comparison between the wild
and farmed one, earlier one is showing high fatty acid content than the later
(Nettleton & Exler, 1992).

14.6.4 ONCORHYNCHUS KISUTCH

In this species the total SFAs, total monoenoic, n06, n–3, total polyenoic, n3:
n6 and total PUFA are observed as 0.77, 1.02, 0.06, 0.9, 0.96, 14, and 2.9,
where as in case of farmed one the values are 1.84, 2.88, 0.46, 1.42, 1.87,
3.1, and 6.88, respectively (Nettleton & Exler, 1992).

14.6.5 ICTALURUS PUNCTATUS

In case of wild Ictalurus punctatus, the total SFAs are 0.56%, total monoenoic
acids are 0.82, n–6 series is 0.22%, n–3 series is 0.28%, total polyenoic acids
are 0.5%, the ratio of n3:n6 is 1.3 and total PUFA are 1.98%, whereas in case
of Farmed species the values are recorded as 2.48, 5.72, 1.52, 0.37, 2.02, 0.2,
and 10.4% respectively and farmed species is showing good fatty acid profile
(Nettleton & Exler, 1992).

14.6.6 SALMO GAIRDNERRI

Salmo gairdnerri, wild species is observed with 1.22% of total SFAs and
farmed one is 1.5%, the total monoenoic acid content was found to be
1.27% in wild and 1.49% in farmed species. The total PUFA of wild and
farmed species are recorded as 4.17 and 4.92% respectively and the ration
336 Coldwater Fisheries and Aquaculture Management

of n3:n6 as 4.2 and 1.4% respectively in wild and farmed one (Nettleton &
Exler, 1992).

14.7 MEDICINAL AND THERAPEUTIC VALUE OF FISHES

Fish is having great medicinal and therapeutic value and will be utilized
as medication for preventing various disorders. Fish proteins are generally
superior to other plant or animal proteins. Oils produced from fishes are
having good therapeutic values, especially liver oils can extensively use for
medical purposes. Oils from fish are great flavor enhancers and are a root
of essential fatty acids (EFAs) such as omega-3 and omega-6. Most of the
diseases like Alzheimer’s, asthma, cancer, bronchitis, heart diseases, arterio­
sclerosis, blood pressure can be cured with oils derived from seafood. These
omega 3 oils play a vital role in brain and eye development of infants. Hence,
pregnant women are prescribed to have omega 3 fatty acids available in fish.
The cartilage of shark has the capacity to fight with cancer. Halibut is a
good source of vitamins (folic acid) and minerals (Magnesium) source and
provides protection against ovarian and digestive tract cancers. Folic acid in
halibut fishes will reduce the homocysteine compound, which is responsible
for damaging artery walls. Magnesium increases blood flow in the body
which indirectly helps to supply oxygen and nutrients. Consumption of fish
roe is also shown to have medicinal properties as roe is a good source of
vitamins, minerals, and long chain fatty acids. Salmon roe is prescribed for
consumption to the people suffering with chronic liver diseases as it contains
phosphatidylcholine. Haddock and cod, are low calorie foods that help in
weight loss. Shellfishes are having more fraction of zinc, and hence can be
used for healthy skin, good muscle and proper fertility. To supplement iodine
deficiency, in treating the people with goiter marine sea foods like cod, sea
bass, perch, and seabass are consumed in olden days. Chondroitin sulfate
is known for its capacity to treat arthritis pain, which is abundantly present
in sea cucumber. Along with chondroitin sulfate oils from sea cucumber
consists of anti-inflammatory fractions, which can be used as an alternate to
fish oil and as a potential inhibitor of 5-LOX (lipoxygenase) enzyme system.
These lipoxygenase enzyme system inhibitors play an important role in
modern drug development for treating asthma, ulcers, and arthritis. Bacteria
present in sea cucumbers, sponges, and tunicates are able to produce some
fatty acids that help in inhibiting cancer effects which have been reported in
initial research with prostate cancer cells line and other human cancer cells.
Nutritional Composition of Coldwater Fishes 337

14.8 CONCLUSION

In the nutrition of humans, fish occupies a unique position along with fish
takes a vital role in the reduction of poverty and hunger. Fish as food, will
nourish the households, especially for young children by reducing malnutri­
tion and improving the overall growth, health, and maintenance of the body.
All the coldwater fishes are best in their nutritive value in one or other way.
Though the cold water fishes were widely distributed, their consumption
is limited because of a lack of information about the nutritional profile.
The coldwater fishes Oncorhynchus mykiss, Tor putitora, Schizothorax
richardsonii, and Neoliccochilush exagonolepsis are very good sources of
omega-3/6 PUFAs. Besides, these fishes are rich sources of quality proteins
(16–18% of body weight) and functional AAs arginine, methionine, tyrosine,
and proline.

KEYWORDS

• antifreeze proteins
• coldwater fishes
• docosahexaenoic acid
• eicosapentaenoic acid
• functional amino acids
• tyrosine

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Das, P., Sarma, D., Bisht, H. C. S., & Das, P., (2012). Nutritional quality of exotic rainbow
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Nutritional quality in terms of amino acid and fatty acid of five Coldwater fish species:
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CHAPTER 15

Genotoxicity in Fishes: With Special

Reference to Micronucleus Formation in
Hematocytes
IMTIYAZ QAYOOM, SAMEENA KHAN, MONISA M. MALIK, ADNAN AMIN,
and ADNAN ABUBAKR
Division of Aquatic Environmental Management, Faculty of Fisheries,
Sher-e-Kashmir, University of Agricultural Sciences and Technology
(SKUAST) of Kashmir, Rangil, Ganderbal, Jammu and Kashmir, India

ABSTRACT

Toxic insults of xenobiotic compounds pose a threat to non-target organisms in

aquatic biota. Contaminants like pesticides, heavy metals, sewage, polycyclic
hydrocarbons, plastics, and persistent organic pollutants are known to induce
physiological dysfunctions in fishes under sub-lethal exposure. Among various
injuries induced by them, environmental toxins are also potent genotoxins that
damage, modify or transform the genetic makeup of fishes by altering the struc­
ture of DNA. Moreover, the formation of micronuclei in fish hematocytes has
been a long and basic tool to quantify the level of genetic injuries caused in fish
due to continuous xenobiotic exposure in nature. Hence this chapter identifies
the mechanism of genotoxicity in fish and the role significance of micronuclei
evaluation in fish blood cells for the quantification of genotoxic abuses.

15.1 INTRODUCTION

Fishes live in an environment that is susceptible to toxic insults by the

contaminants. Waterbodies usually lie in the depression and act as ultimate
Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
340 Coldwater Fisheries and Aquaculture Management

sinks of all the substances coming to from their watershed. Hence the
environmental toxins, which include pesticides, heavy metals, plastics,
persistent organic pollutants, and sewage coming as a part of municipal
left over pose threat to fishes under natural conditions. These toxicants get
washed away through leaching, drift, and percolation to aquatic ecosystems
(Miyamoto et al., 1990) and induce toxicity in the non-target organisms like
fishes which are badly hit by their exposure, even though the xenobiotics
are bioavailable in minute quantities. Apart from various physiological
dysfunctions, environmental toxins cause alterations in the structure and
function of DNA resulting in genotoxicity and DNA damage.
Genotoxicity or genetic toxicity refers to some total cumulative damages
or destructive changes in the genetic material (DNA and RNA) of fish cells
distressing its integrity. Genotoxin, a substance inducing genotoxicity has an
ability to prompt genetic damage in the cells by interacting with DNA. They
have common physical and chemical properties that enable them to interact
with nucleic acids and cause damage in them (Shugart, 1995; Sharma,
2016). Genotoxicity and mutagenicity are different terms. Every mutagen
is necessarily a genotoxin but the reverse of it is not true. In definition,
genotoxicity only refers to the potential of genotoxins to cause damage to DNA
or RNA. They may be physical or chemical substances that have the ability to
alter gene sequences and cause changes in the genetic information of fishes.
If a genotoxin affects the genetic material of a somatic cell, it will not be
transferred to the progeny, hence heredity will not be witnessed. Conversely,
the effect of genotoxic substance on germ cells can get transferred to the
progeny as hereditary. Moreover, the genotoxic effects can be diminished
by DNA repairments, majorly by the enzyme activity of cells. Various
contaminants such as polyaromatic hydrocarbons (PAHs), more particularly
benzo-pyrenes and aldehydes are well known genotoxins and cause DNA
adducts while binding with a nucleotide in DNA, more particularly guanosine
base (Nikinmaa, 2014). An increase in the frequency of mutation is directly
dependent on the type of DNA adduct and keeps on increasing if damaged
DNA is not repaired by the replacement of adducted base by unmodified one.
Unlike a genotoxin, mutagen causes permanent and transmissible change
in DNA, resulting in mutations. Mutagens can be physical which include
different types of ionizing and non-ionizing radiations known to disrupt
DNA structure. The transitions and transversions of the DNA sequence can
be produced by chemical and biological mutagens. Base similarities, NO
species, and intercalating agents are mediators of chemical mutagens that
leads to the formation of apurinic and apyrimidinic sites, creating alterations
in DNA. However, various viruses that cause host mutations by incorporating
Genotoxicity in Fishes 341

their DNA into the host cell, causing the change in the sequence of DNA
of host cell are included in biological mutagens. There is a broad range of
adverse effects on genetic components of the cell which may include disrup­
tion of genetic materials of cellular components that regulates the fidelity
of the genome such as the spindle apparatus, topoisomerases, DNA repair
systems and DNA polymerases WHICH can be used in Experimental struc­
tures that measures the capability of genotoxins in genotoxicity testing.

15.2 MECHANISM OF GENOTOXICITY IN FISHES

Various mechanisms for the induction of genotoxicity in fishes have been

studied. Important among all are point mutations, clastogenicity, and aneu­
ploidy. All these changes occur in DNA structure after genotoxin-DNA
interactions. Genotoxins cause lesions, breakage, fusion, deletion, mis-segre­
gation, or non-disjunction leading to damage and mutation (Anonymous).
Genotoxins likely affect actively transcribed and readily dividing cells rather
than uncoiled genomes. It manifests that toxic insult in embryos lead to tera­
togenic effects more quickly and profoundly than adults on account of their
actively diving cells to form an organism. Exposure to genotoxins can cause
change in nucleotide sequence either by addition of a base, deletion, insertion
of a new gene sequence or replacement of one base by another. All these types
are called point mutations. In addition to the point mutations, toxicants can
be clastogenic, i.e., they lead to breakage of DNA strands which are inherited
into daughter cells and cause genotoxicity. It is known that exposure of fishes
to arsenic and benzene containing compounds lead to clastogenicity in them.
Formation of micronuclei in fish blood cells is the best studied clastogenic
effects till date.

15.3 MICRONUCLEUS AS AN INDICATOR OF GENOTOXICITY

During the process of cell division, the cytoplasmic chromatin-containing

bodies which are formed when acentric chromosome fragments fail to get
incorporated into daughter cell nuclei during anaphase are called micronu­
clei (Kirsch-Volders et al., 2011). The delay during anaphase arises due to
genotoxic exposure leading to genetic damage either by the lack of centro­
meres or by abnormalities in the formation of mitotic spindle, which triggers
and accomplishes the micronuclei formation (Schmid, 1975). During cell
division, micronuclei are formed regardless of the type of DNA damage
342 Coldwater Fisheries and Aquaculture Management

(Salvadori et al., 2003; Nandanpawar et al., 2018). And the formation of

micronucleus is related to the number of cells under n division. The major
mechanism of MN formation resulting from chromosome mal-segregation
is hypo-methylation of centromeric and para-centromeric regions satellite
repeats However, the satellites are hyper-methylated, and loss of methyla­
tion lengthens repeat areas resulting of lowering tension in kinetochores and
resulting in incorrect connections between mitotic spindle microtubules and
chromosomes in most of the cases (Fenech et al., 2011). Due to tubulin depo­
lymerization, chromatids/chromosomes are sometimes unable to segregate
as the mitotic spindle cannot force them apart. Moreover, chromosomes
lag behind at telophase due to the absence of kinetochore or centromeric
abnormalities. A dicentric chromosome having two centromeres could
get attached to two opposite spindle bodies thereby pulling chromatids in
opposite directions. When breakage does not occur, the nuclear membrane
surrounds both nuclei creating nucleoplasmic bridges between them. The
nucleoplasmic bridge is eventually disrupted during cytokinesis, ending in
the creation of a micronucleus. Boller & Schmidt (1970) proposed the term
micronucleus test (MNT) in the early 1970s. MNT is used to identify the
genomic damage induced by genotoxins in fishes and is the simplest of all
techniques. This procedure targets interphase cells of any proliferating cell
population regardless of its karyotype and is technically easier. Hence, this
biomarker is widely utilized in environmental bio-monitoring programs. The
potential toxicity of a compound in fishes is indicted by frequency of micro-
nuclei formation which must meet the following criteria like the diameter of
MN should be less than one-third of the main nucleus; there should be clear
identification between the nuclear boundary of MN and the main nucleus
and MN and the main nucleus should be identical in staining Fenech et al.
(2003). As mentioned above, the micronuclei can be different as per their
shape, like notched, blebbed, lobbed, budded, fragmented or bi-nucleated.
All are considered as high quality indicators of cytotoxicity irrespective of
their shapes observed in fish hematocytes (Ayllon & Vazquez, 2000, 2001).
The health status index of different species cab be evaluated by Hematology
as it provides a steadfast estimation via non-lethal means (Satheeshkumar
et al., 2012). Though due to several internal and external factors, it can be
quite challenging, yet hematological parameters serve as a valuable indicator
to evaluate the health conditions of aquatic organisms. Blood parameters
of fish are closely related to environmental and biological factors, also, the
conditions like physiological changes could be a consequence of stress,
and can be identified by using a number of hematological tools as studied
by Fernandes & Mazon (2003). Establishment of hematological status
Genotoxicity in Fishes 343

based on data achieved from healthy animals is considered fundamental to

illustrate a normal range variation. However, to establish a reference index
before using hematological parameters is considered necessary to evaluate
the physiological condition of different species of fish (Thrall et al., 2007;
Leatherland et al., 1998).

15.4 GENOTOXICITY INDUCED DUE TO PESTICIDES

Pesticides induce the formation of micronuclei in fish hematocytes which

indicates DNA damage on account of their exposure. A dose and time depen­
dent increase of micronuclei is reported in almost all fishes such as Cyprinus
carpio specularis and Cyprinus carpio communis (Al-Sabti, 1986; Nepomu­
ceno et al., 1997; Gustavino et al., 2001; Buschini et al., 2004). Thiometon,
an organophosphorus (OP) pesticide is also known to induce micronuclei in
peripheral erythrocytes of fish along with other OPs. Similarly, chlorpyrifos
is rated as a dangerous pesticide in India to induce micronuclei in peripheral
erythrocytes are often dot-shaped and near to the major nucleus, with size
and form varying from cell to cell (Bhatnagar et al., 2016). Various other
classes of pesticides have been found to induce micronuclei in peripheral
blood erythrocytes of different fishes such as Oreochromis mossambicus
(Naqvi et al., 2016), Chanos chanos (Palanikumar et al., 2014), Cirrhinus
mrigala (Anita et al., 2016), Cyprinus carpio var. communis and Cyprinus
carpio var. specularis (Malik & Ganaie, 2011; Dar et al., 2014). Induction of
micronuclei due to pesticides in various fishes is given in Table 15.1.

15.5 GENOTOXICITY INDUCED DUE TO HEAVY METALS

Heavy metals are potential water contaminants which includes lead (Pb),
chromium (Cr), zinc (Zn), copper (Cu), cadmium (Cd), nickel (Ni), etc.
(Patel et al., 2017; Kumar & Singh, 2018) and pose threat to fish life.
They find their way into the water supply through industrial and consumer
materials. Also, from acidic rain that breaks down soils thereby, releasing
heavy metals into streams, lakes, rivers, and groundwater. They are point of
concern worldwide because of their persistent, bio-accumulative, and toxic
nature (Ali et al., 2019) the induction of morphological, behavioral, repro­
ductive, genotoxic, and mutagenic damages to the aquatic biota is caused
by Heavy metals (Pulley et al., 2016). When the heavy metals are taken
up, they are stored at a faster rate than they are metabolized or excreted,
344 Coldwater Fisheries and Aquaculture Management

hence, they get accumulated in living organisms. The three most dangerous
heavy metals identified in fishes are lead (Pb), mercury (Hg) and cadmium
(Cd). Several studies have shown that fishes exposed to water and sediments
contaminated by heavy metals can exhibit genotoxic effects (Turan et al.,
2020; Kontas & Bostanc, 2020) and DNA damage as reported in Geophagus
brasiliensis exposed to Fe, Mn, Cd, Cu, and Pb (Gomes et al., 2019). Da
Silva et al. (2020) reported the effect of heavy metals on Astyanax lacustris
as a genotoxic effect due to various heavy metals in the order of Mn > Ba
> Zn > Ni > Ti > Cr > Cu. There are possibilities of synergistic effects of
heavy metals that lead to profound effects in fishes when compared with
the individual toxic potential of any metal (Enserink et al., 1991). Some
studies have shown that nanoparticles of Titanium dioxide exposure caused
significant genotoxic changes in the blood when it reaches the blood stream,
gets accumulated in the muscles and brain of fishes and also lead to oxidative
stress by the generation of ROS (Carmo et al., 2019). Studies on Oreochromis
niloticus, Poronotus triacanthus and Puntius altus lead (Pb), copper (Cu)
and cadmium (Cd) induced MN formation and genetic damage in the order
of Pb > Cu > Cd (Jiraungkoorskul et al., 2007). Induction of micronuclei due
to pesticides in various fishes is given in Table 15.2.

TABLE 15.1 MN Nucleus Frequency Induced by Pesticides in Different Fish Species

SL. Species MN per Causative Pesticide References
No. 1,000 Cells
1. Channa 0.02 Dimethoate, dichlorvos, chlorpyriphos, Kumar et al.
punctatus and malathion, methyl parathion, (2010)
fenvalerate, cypermethrin, and carbaryl
2. Channa 0.028–0.048 Pesticide toxicity Ali et al. (2008)
punctatus 0.72–4.11 Pesticide toxicity Farah et al. (2002)
3. Carassius 3.17 Herbicide Canvas & Konen
auratus (2007)
4. Cheirodon 0.0–1.0 Pesticide toxicity Campana et al.
interruptus (1999)
5. Clarias 0.47–3.95 Pesticide toxicity Ateeq et al.
batrachus (2002)

15.6 GENOTOXICITY INDUCED DUE TO OTHER SOURCES

Various other substances are also known to induce genotoxicity in fishes. Some
chemicals like colchicine and radiations like X-rays are reported to induce
Genotoxicity in Fishes 345

clastogenic and mitoclastic (spindle poisoning) in Cyprinus carpio species

(Gustavino et al., 2001). Moreover, discharge of armament factory wastes is
also known to cause progressive genotoxic insults by micronuclei induction
(Ayllon & Vazquez, 2001). Even intermittent heat shocks at variant temperature
have also been reported as external genotoxins to induce nuclear anomalies
like micronuclei formations, chromosomal aberrations, DNA damage and cell
proliferation in Carassius auratus (Anitha et al., 2000). Induction of micronu­
clei due to pesticides in various fishes is given in Table 15.3.

TABLE 15.2 MN Nucleus Frequency Induced by Heavy Metals in Different Fish Species
SL. Species MN per Causative Pesticide References
No. 1,000 Cells
1. Carassius auratus 2.26 Metal toxicity Cavas (2008)
2. Carassius auratus gibelio 13; 10; 5.2 Metal toxicity (Cr) Al-Sabti et al. (1994)
3. Carassius sp. 1.8 Metal toxicity (Cu ions) Hayashi et al. (1998)
4. Cyprinus carpio 1.2 Metallic mercury Nepomuceno et al.
(1997)
5. Oncorhynchus mykiss 0.1 Cadmium injections Castano et al. (1998)
6. Oreochromis niloticus 0.21 Heavy metals Barbosa et al. (2010)
7. Phoxinus phoxinus 0.3–0.7 Metal toxicity Ayllon & Garcia-
Vazquez (2000)
8. Poecilia latipinna 0–0.4 Metal toxicity Ayllon & Garcia-
Vazquez (2000)

15.7 SIGNIFICANCE OF GENOTOXICITY TESTING

Genotoxicity testing in fishes provides information about damage caused to

their DNA. The endpoints of genotoxic insults resulting due to unscheduled
DNA synthesis, sister-chromatid exchange, adduct formation in DNA,
mitotic recombination, chromosomal aberrations studies and micronuclei
formation are evaluated and quantified by genotoxicity testing tools. Thus,
toxic endpoints of all the substances, chemicals, pesticides, and drugs, etc.,
can be measured by toxicity testing. Safety evaluation of new chemical
entities (NCE) and their efficiency are one of the mandates of toxicity testing.
similarly, evaluating natural waterbodies for the presence of genotoxins in
order to generate baseline data for evaluation of potable waters is mandatory.
Moreover, its safety for commercially significant flora and fauna is need of
an hour for human safety.
 346
TABLE 15.3 MN Nucleus Frequency Induced Due to Other Sources in Different Fish Species
SL. No. Species MN per 1,000 Cells Causative Pesticide References
1. Carassius auratus 0.18 Mutagenic chemicals (orange (AO) Ueda et al. (1992)
fluorescent staining)
2. Carassius auratus 0.26 Heat shock Anitha et al. (2000)
3. Clarias lazera 0.31 Industrial waste ethyl methane Odeigah & Osanyinpeju (1995)

Coldwater Fisheries and Aquaculture Management

sulfonate
4. Cyprinus carpio 0.02 X-rays and colchicine Gustavino et al. (2001)
5. Cyprinus carpio 0.52 Chemical industries and/or a nuclear Llorente et al. (2002)
power plant waste
6. Cyprinus carpio 0.9 Sewage treatment plant discharges. Grisolia & Starling (2001)
7. Odontobutis oscura Obscura 0.181 Wastewater Ayllon & Garcia-Vazquez (2001)
0.33 River pollution De Flora et al. (1993)
1.0 Radiation Schultz et al. (1993)
8. Oreochromis niloticus 0.8 Sewage treatment plant discharges Grisolia & Starling (2001)
9. Salmo trutta 0.4–2.3 Waste discharge Ayllon et al. (2000)
10. Pseudopleuronectes 0–1.30 Wastewater Hughes & Hebert (1991)
Americanus
Genotoxicity in Fishes 347

15.8 CONCLUSION

At different biodiversity levels, multiple consequences are produced by

aquatic pollutants, the most dangerous being carcinogenic and mutagenic
compounds as their effects may not be limited to that of the individual but
can reach to the several generations. Pesticides, heavy metals, sewage, and
PAHs are known genotoxins in fishes and are considered as environmental
toxins. Moreover, some other sources like radiations, heat shocks, factory
wastes and chemicals like colchicine are documented as genotoxins in vide
rage of fish species leading to deleterious effects in them. The application of
genotoxicity biomarkers allows assessing the mutagenic hazards in sentinel
organisms and for the contaminants fate and sources identification.

KEYWORDS

• genotoxicity
• micronucleus
• micronucleus test
• new chemical entities
• organophosphorus
• toxicants

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CHAPTER 16

Infectious Diseases of Coldwater Fishes:

Focus on Viral and Fungal Infections
SYED SHARIQ N. QADIRI, INAIN JAIES, FEROZ A. SHAH, SHABIR A. DAR,
and ASIFA WALI
Division of Aquatic Animal Health Management, Faculty of Fisheries,
Sher-e-Kashmir University of Agricultural Sciences and Technology
(SKUAST) of Kashmir, Rangil, Ganderbal, Jammu and Kashmir, India

ABSTRACT

Intensification of aquacultural practices results in disease outbreaks of

prevalent pathogens alongside new and emerging diseases. Water as a
medium in an aquatic environment facilitates the spread of disease from
infected specimens to naïve fish. The transmission of disease can be either
horizontal (fish to fish) or vertical (parent to offspring). Stress is always a
predisposing factor to a disease outbreak. The pathogenicity and virulence of
the invading pathogen will determine the severity of infection. In addition,
the immune status and genetic make-up of fish will define whether the host
succumbs to the invading pathogen or not. It is to mention that the immune
system of a coldwater fish is generally weak in comparison to a warm water
fish given the low temperature which makes fish more susceptible to infec­
tious diseases. Infectious diseases of coldwater fishes are either of bacterial,
viral, parasitic, or fungal etiology. In the context of the present chapter, the
focus here will only be on viral and fungal pathogens prevalent in cold water
aquaculture.

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
352 Coldwater Fisheries and Aquaculture Management

16.1 INTRODUCTION

Any detrimental divergence from the standard structural and functional

state of an organism is termed as disease. It is generally coupled with
certain signs and symptoms which are indicative of the abnormal state
of the host. Being always immersed in the aquatic environment, fish is
very sensitive to its surroundings which contains myriad of pathogenic
organisms (Rombout et al., 2014; Qadiri et al., 2019a). Under normal
conditions, fish is in equilibrium with pathogen and once this equilibrium
deviates, disease outbreak occurs. Although, the defense mechanisms in
fish are well developed but whenever these pathogens breach the host
defenses, fish gets infected. Study of disease (pathology) involves the
identification of causative agent (etiology), understanding the mechanism
of disease development (pathogenesis) and the changes associated with the
disease process. The epidemiological triad (Figure 16.1) which involves
the interaction of host, environment, and pathogen determine disease
process and pathogen ecology (Kelly & Renukdas, 2020). The common
viral and fungal diseases of finfish prevalent in cold water fish are enlisted
in Table 16.1.

Indigent sanitation

Water quality fluctuation

Influx of fauna
Organic load

Age dependent Water flow

Resistant
immune strains
response
Increament of
Species virulence
Lack of
appropriate
Stressful vaccines
conditions
Diagnostics

Susceptibility Monitoring

FIGURE 16.1 Epidemiological triad (host-pathogen-environment).

TABLE 16.1 Commonly Prevalent Viral and Fungal Diseases in Cold Water Fish

Infectious Diseases of Coldwater Fishes

SL. No. Disease Type Causative Agent Clinical Signs
1. Viral hemorrhagic septicemia (VHS) Viral VHSV • Anorexia
• Lethargy
• Petechial hemorrhages
• Dark color
2. Spring viremia of carp (SVC) Viral SVCV • Exophthalmia
• Edema
• Abdominal distension
• Hemorrhages
3. Infectious hematopoietic necrosis (IHN) Viral IHNV • Ascites
• Scoliosis
• Dark coloration
• Pale gills
4. Infectious pancreatic necrosis (IPNV) Viral IPNV • Anemic gills
• Swelled abdomen
• Pop eye
5. Saprolegniasis Fungal Saprolegnia sp. • Depigmented lesions
• Whitish brown cotton-like patches
• Lethargy
• Bloating

353
TABLE 16.1 (Continued)

354
SL. No. Disease Type Causative Agent Clinical Signs
6. Branchiomycosis Fungal Branchiomyces sp. • Pale and rotten gills
• Fishes become weak
• Gasping for air
• Mouth wide open
7. Epizootic ulcerative syndrome (EUS) Fungal Aphanomyces • Lesions on the lateral sides
invadans • Protruding scales
• Scale loss

Coldwater Fisheries and Aquaculture Management

• Skin erosion
• Reddened skin
Infectious Diseases of Coldwater Fishes 355

16.2 VIRAL DISEASES OF COLD-WATER FISH

16.2.1 VIRAL HEMORRHAGIC SEPTICEMIA (VHS)

16.2.1.1 ETIOLOGICAL AGENT

The viral hemorrhagic septicemia virus (VHSV) causes viral hemorrhagic

septicemia (VHS) infection in cultured rainbow trout (O. mykiss) and is one
of the most lethal finfish viral diseases. VHSV is a member of the Novirhab­
dovirus genus in the Rhabdoviridae family (Ammayappan & Vakharia, 2009),
with a single linear, negative sense ssRNA of roughly 11.1 kb as genetic
material. The disease was first described by Schäperclaus (1938), and the
etiology was later identified as Egtved virus (Jensen, 1963) as it initially
occurred in rainbow trout near the Danish village of Egtved. Therefore, VHS
disease is also known as “Egtved illness.”

16.2.1.2 CLINICAL SIGNS AND PATHOLOGY

Affected fish show anorexia and are lethargic. Dark color and pale gills
are common. Petechial hemorrhages are seen on the skin, fins, and internal
organs. Infected fish shows irregular swimming and heavy mortality. In the
liver, kidney, spleen, and skeletal muscle, the majority of tissue alterations
are observed which usually indicate moderate to significant necrosis or cell
death (Nishizawa et al., 2006). The primary site of infection is the hema­
topoietic (blood-forming) regions of the kidney and spleen which display
severe necrosis. The virus shows a strong tropism for blood and is frequently
localized in organs such as the heart due to high blood supply and likely
gains entry into fish via gill (Qadiri et al., 2019b, 2020). Thickened lamellae
in the gills and pyknotic nuclei in the liver are also apparently visible. Blood
collects in skeletal muscle, although it is not severely damaged. Due to the
tropism of virus for brain tissue, living fish infected with VHS may appear
listless or limp, hang just beneath the surface, or swim very erratically, such
as continual flashing circling.

16.2.1.3 DIAGNOSIS

Histopathological (HP) investigation and tissue observation under a micro­

scope are used to get a preliminary diagnosis. To diagnose VHS according
356 Coldwater Fisheries and Aquaculture Management

to OIE guidelines, the virus must first be isolated in cell culture and then
neutralized using serum. Commonly used fish cell lines are BF-2, EPC,
and FHM (OIE, 2019). The bullet-shaped rhabdovirus can be seen using
electron microscopy, but it is insufficient for a definite diagnosis. RT-PCR
(Snow et al., 2004) and standardization of various qRT-PCR protocols are
in routine use for molecular diagnosis of VHSV (Garver et al., 201; Kim
et al., 2014).

16.2.1.4 CONTROL AND TREATMENT

Utilization of virus-free water supplies (water from wells or spring sources

that are separate from surface water sources), sourcing eggs from certified
virus-free stocks, and disinfection of eggs prior to introduction to the produc­
tion facility are all measures to prevent VHS outbreaks in cultured fish.
Modern aquaculture operations have a facility biosecurity plan that defines
site-specific equipment and disinfection procedures for workers and equip­
ment that transit between sites. Disease outbreaks have been demonstrated to
be reduced when processed (pelleted) feed is used instead of raw fish-derived
feed. The use of all in-all out production systems is one of the management
techniques for marine culture systems. Disinfection of hatchery, specific
pathogen free stock and other management practices should be employed.
Common disinfectants such as formalin, sodium hydroxide and sodium
hypochlorite are to be used.

16.2.2 SPRING VIREMIA OF CARP (SVC)

16.2.2.1 ETIOLOGICAL AGENT

It is caused by SVC virus belonging to the genus Vesiculovirus and family

Rhabdoviridae (Carstens, 2010). The name refers to a viremia that mostly
affects common carp (Cyprinus carpio), particularly in the spring (Kibenge,
2016). The term was coined to distinguish virus-induced disease from infec­
tious dropsy of carp which has a complex etiology (Fijan et al., 1971, 1984).
Viral genome is a single strand of non-segmented, negative-sense RNA
of 11,019 nucleotides that codes for five proteins: a nucleoprotein (N), a
phosphoprotein (P), a matrix protein (M), a glycoprotein (G), and an RNA-
dependent, RNA polymerase (L).
Infectious Diseases of Coldwater Fishes 357

16.2.2.2 CLINICAL SIGNS AND PATHOLOGY

Exophthalmia, edema, petechial hemorrhages, and abdominal distension are

commonly observed in SVCV infection. The virus quickly spreads to the
liver, kidney, spleen, and alimentary tract, resulting in viremia and peritonitis
(Dikkeboom et al., 2004). Edematous and enlarged kidneys with tubular
degeneration and enlarged spleen are frequently observed with hemorrhages
in the swim bladder. Vasculitis is frequently caused by SVCV multiplication
in blood vessel endothelium and is the most likely source of both exterior and
interior hemorrhages (Ahne et al., 2002). Diseased fish move slowly, gather
at sides, and are sluggish to any stimulus. Moribund fish have been observed
lying on their sides, frequently on the tank’s bottom, and swimming up but
then returning to the bottom when frightened.

16.2.2.3 DIAGNOSIS

The virus can be isolated using OIE recommended cell lines like FHM or
EPC (Fijan et al., 1983; Clark et al., 1974). If the virus is present, the cells
will degenerate and round out. One or more indirect test procedures should
be done by a recognized laboratory to confirm that SVC virus is present in
the cell line. Definite diagnosis of SVCV requires isolation of virus followed
by neutralization assay, IFAT, ELISA, and RT-PCR (OIE, 2015).

16.2.2.4 CONTROL AND TREATMENT

SVC-free providers and farms should be used to obtain new fish. Surface
water should be disinfected before being supplied to the farm. Maintain a
separate set of SVC-susceptible species-specific equipment and disinfect it
between ponds or tanks. Birds, livestock, pets, and other animals should not
be allowed access to ponds or tanks.

16.2.3 INFECTIOUS HEMATOPOIETIC NECROSIS (IHN)

16.2.3.1 ETIOLOGICAL AGENT

This fish virus belongs to the genus Novirhabdovirus and family Rhabdo­
viridae and is negative sense single stranded RNA molecule. It is bullet
shaped containing a unique non-virion protein gene. IHNV isolates are
358 Coldwater Fisheries and Aquaculture Management

classified into three genetic categories based on their geographic distribu­

tion (Lakshmi et al., 2019). Isolates from Alaska, British Columbia, coastal
Washington watersheds, and the Columbia River basin, as well as a few from
Oregon, California, and Japan, belong to the U genogroup. Most viruses
from California and Oregon’s coast belong to the L genogroup. Isolates from
Idaho, the Columbia River basin, and Europe, as well as a virus from the
Washington coast, make up the M genogroup. The genetic diversity of the M
genogroup is much higher than that of the L or U genogroups.

16.2.3.2 CLINICAL SIGNS AND PATHOLOGY

Ascites, dark color, exophthalmia, pale gills are seen in infested specimens.
Petechial hemorrhages are seen near fins and mouth. Scoliosis is also seen in
surviving specimens and infected specimens swim in a corkscrew manner.
IHN may be suspected based on clinical indicators and a history of past
outbreaks. Depending upon the viral strain, environmental conditions, and
the condition of the host, it may cause 10% to 90% mortality in salmons. The
main symptom in some cases is a large increase in mortality in juvenile fish
with little clinical indications. The majority of outbreaks take place in the
spring and early summer. With a broad viremia and concomitant necrosis in
all tissues, this type of the illness leads to a deadly necrosis of the hematopoi­
etic tissues of the kidney and spleen. Renal failure induced by an electrolyte
imbalance can result in fish death (Rodriguez et al., 2003).
IHNV is spread by asymptomatic carriers and clinically unwell fish. This
virus can be found in feces, urine, sexual fluids, and mucus. The disease is
spread mostly from fish to fish, mostly through direct contact, but also through
the water. IHNV can persist for at least a month in water, especially if it contains
organic material. Although the gills and the digestive tract have been proposed
as significant virus entry points, research reveals that IHNV may enter near
the base of the fins (Harmache et al., 2006). There is also “egg-associated”
(vertical) transmission; whether IHNV can be found inside the egg as well as
on the surface is debatable. It is possible that invertebrate vectors exist.

16.2.3.3 DIAGNOSIS

To confirm a diagnosis, procedures such as staphylococcal agglutination,

virus neutralization (VN), indirect fluorescent antibody detection, ELISA,
RT-PCR, qRT-PCR, and DNA probe technology can be utilized (Dhar et
Infectious Diseases of Coldwater Fishes 359

al., 2008; Purcell et al., 2006; Barlic-Maganja et al., 2002). Alternatively,

histology can be utilized to diagnose infection by identifying degeneration
and necrosis of granular cells in the lamina propria, stratum compactum, and
stratum granulosum of the gastrointestinal tract.

16.2.3.4 CONTROL AND TREATMENT

IHNV infection prevention relies on avoiding viral exposure through the

establishment of stringent control rules and good hygiene measures. To
prevent infection with IHNV at a fish production site, proper cleaning of
fertilized eggs, the use of virus-free water supplies for incubation and rearing,
and the operation of facilities under established biosecurity procedures are
all essential. Disease incidence is reduced by good husbandry practices such
as excellent water quality, moderate stocking density, and no batch mixing.
To eradicate the disease, highly rigorous protocols regarding mobility, water
supplies, and stock replacement must be in place which is still difficult to
execute and comes at a hefty expense.

16.2.4 INFECTIOUS PANCREATIC NECROSIS (IPN)

16.2.4.1 ETIOLOGICAL AGENT

IPNV was the first isolated birnavirus in rainbow trout (Wolf et al., 1960).
IPN virus, a non-enveloped, bisegmented, double-stranded RNA genome
member of the genus Aquabirnavirus and family Birnaviridae, is the caus­
ative agent of IPN disease. Serologically, IPNV strains are separated into
groups A and B, and further classified based on deduced amino acid (AA)
similarities of VP2. Infectious pancreatic necrosis (IPN) is a highly conta­
gious viral infection that primarily affects trout and salmon, although the
virus has also been found in a range of other fish species. It is a contagious
disease that mostly affects the Salmonidae family (Dopazo, 2020). IPN is a
highly infectious viral disease with a significant mortality rate in adult fish
and a 100% mortality rate in fry and fingerlings.

16.2.4.2 CLINICAL SIGNS AND PATHOLOGY

The virus enters the fish body through gills, intestine, and skin, then spreads
through the bloodstream to the internal organs (Munang’andu et al., 2012).
360 Coldwater Fisheries and Aquaculture Management

The head kidney, spleen, and pancreas are predilection locations for primary
IPNV replication in internal organs. Dark coloration, distended abdomen,
exophthalmia, pale gills, petechial hemorrhages on the ventral surface and
weak respiration are common signs. Very young farmed salmonids develop
clinically acute or chronic illness conditions. Only a small percentage of
older fish are harmed. Variations can be seen in the outbreak’s clinical
manifestations. Infected specimens show corkscrew manner of rotation and
large mortality generally occurs. The loss of exocrine pancreatic acinar tissue
is the only persistent HP hallmark of this illness. The presence of “McKnight
cells” in the lamina propria of the gut in surviving fish is commonly observed
in IPNV infection (McKnight & Roberts, 1976). The necrotic alterations are
caused by infected cells rupturing and releasing zymogen granules. Internal
organs including the spleen, heart, liver, and kidney can also seem unusually
pale with empty stomach and white fecal casts from their anal orifice
(Roberts, 2012).

16.2.4.3 DIAGNOSIS

Virus isolation in any of a variety of standard fish cell cultures confirms the
diagnosis of infectious pancreatic necrosis in fish with characteristic clinical
or microscopic lesions. Because substantial amounts of virus are present in
the kidneys of fish with either clinical or subclinical infections, kidney is
the tissue of choice for sampling. Plaque assay can be used to titrate virus in
fish cells. The standard cell lines for isolation of aquatic birnavirus are FHM,
RTG-2, CHSE-214, BF-2 and EPC. For direct detection of viral antigens in
internal organs and confirmation of virus identity from cell cultures, immu­
nofluorescence (frozen sections or tissue smears) using virus-specific mono­
clonal or polyclonal antibodies can be utilized. Neutralization, enzyme-linked
immunosorbent assays, and RT-PCR assays can all be used to determine the
identity of infectious pancreatic necrosis virus (IPNV). Antiviral antibodies
are seen in fish with subclinical infections, although serology is not employed
as a diagnostic method very often (Pérez-Prieto, 2003).

16.2.4.4 CONTROL AND TREATMENT

The most effective control measure is avoidance. In order to do so, virus-

free eggs must be incubated, and IPN-free stock must be propagated in an
uncontaminated water supply and this approach is the preferred method. To
Infectious Diseases of Coldwater Fishes 361

avoid the introduction or unintentional spread of IPN, a thorough fish health

inspection program is essential. Good sanitation and proper management
techniques reduce the disease incidence.

16.3 FUNGAL DISEASES OF COLD-WATER FISH

16.3.1 SAPROLEGNIASIS

16.3.1.1 ETIOLOGICAL AGENT

The disease saprolegniasis is caused by water mold (oomycetes) mostly

belonging to the genus Saprolegnia (Van West, 2006). This fungus is charac­
terized by having non-septate multinucleate hyphae and branched mycelium
can be easily identified on substratum. Morphological, physiological, and
molecular analysis of Saprolegnia isolates has shown several species misas­
signments apparent for S. diclina, S. australis, S. litoralis or S. ferax due to
similar oogonial morphology (Diéguez-Uribeondo et al., 2007).

16.3.1.2 CLINICAL SIGNS AND PATHOLOGY

Small, round, depigmented lesions appear first, sometimes with hemorrhagic

edges. Lesions can become ulcerative in advanced cases, extending through
the skin and into muscular tissue (Willoughby et al., 1983). Whitish brown
cotton-like patches appear on the surface of the skin and/or on the gills.
Hemorrhage, necrosis, exophthalmia, lethargy are commonly seen in affected
specimens. Bloating caused by gut obstruction may progress to perforation
of the abdominal wall in small juvenile fishes (Jaies et al., 2020).
Infectious biflagellated zoospores secreted from hyphal sporangia
transmit external mold infections through ambient water. S. parasitica
prefers skin and gill as anatomical predilection sites in rainbow trout and
any damage to the structural base of these organs results in loss of body
fluids and imbalance in osmoregulation (Jaies et al., 2021). In cultured fish,
systemic infections are caused by the intake of uneaten food that has been
colonized by mold hyphae. Environmental stress is a significant contributor
to the etiology of external disease. Outbreaks are most common after minor
injuries from handling or in crowded environments with poor environmental
quality. Adult salmon traveling to spawning grounds have compromised
immune systems and are frequently infected with Saprolegnia on the outside.
362 Coldwater Fisheries and Aquaculture Management

Cold water temperatures also make fish more susceptible to mold sickness
because zoospores and sexual stages thrive in cold water, while host tissue
healing and the inflammatory response are hampered by the lower host
metabolism.

16.3.1.3 DIAGNOSIS

Gross clinical signs of white, cottony tufts of hyphae on the skin, gills, and
other surfaces of infected fish or eggs are common diagnostic measures.
Branching and non-septate hyphae are seen in the wet mounts of fungal
mycelium. Polymerase chain reaction (PCR) is essential for species identi­
fication and can be accomplished by using published protocols (Eissa et al.,
2013; Touhali et al., 2018).

16.3.1.4 CONTROL AND TREATMENT

The easiest way to avoid Saprolegnia is to keep your water clean and feed
your fish a healthy diet. This is the finest way to boost their immune system,
which is the best Saprolegnia deterrent. Maintaining a proper maintenance
schedule, removing of trash from the gravel, and maintenance of a hospital
tank on hand to confine any potentially sick fish are some measures to
reduce fish saprolegniasis. The effect of this disease can be further reduced
by improving water quality and minimizing stress, especially in the late
summer and fall. Saprolegniasis can be treated with saltwater baths (10–25
g/l for 5–30 min SID), benzalkonium chloride (2 mg/l bath for 10–60 min),
malachite green (1–3.3 mg/l for 1 hour or 0.15–0.2 mg/l aap prolonged bath),
methylene blue (10–20 mg/l for 15 min), formalin, potassium permanganate,
and copper sulfate.

16.3.2 BRANCHIOMYCOSIS

16.3.2.1 ETIOLOGICAL AGENT

It is also known as “gill rot” and was first reported in striped bass (Meyer
& Robinson, 1973). It is caused by two Branchiomyces species, namely, B.
sanguinis and B. demigrans. Carps are the fish that are most afflicted. Bran­
chiomycosis is most commonly found in fish in waters with a high organic
Infectious Diseases of Coldwater Fishes 363

content and at temperatures of 20°C or above. This deadly disease can cause
up to 50% morbidity in infected fish, and they can die quickly (Harrell, 1997).

16.3.2.2 CLINICAL SIGNS AND PATHOLOGY

Gills appear pale and anemic, while as fungus development can be seen in
gill tissue with naked eyes. Fishes become weak and gather in groups at the
inlet/outlet of the water body. Infected specimens are seen gulping oxygen
at the water surface with wide open mouth. Infected fish have respiratory
symptoms as well as a loss of balance. Histologically, hyperplasia, fusion
of gill lamellae, and areas of massive necrosis due to thrombosis of vessels
by fungal hyphae are apparent. Affected fish may succumb to death within
two days after infection, and morbidity can reach to 50% (Devashish, 2016).

16.3.2.3 DIAGNOSIS

Gross examination of clinical signs, wet mount preparation and examina­

tion and the molecular identification of pathogen are important for disease
diagnosis. The hemorrhages and necrosis result in a patchy marbled look
of the gills. A diagnosis can be made using wet mounts or histology of the
lesions. Typical hyphae will be evident within the gill vessels or piercing
the gill tissue. Light brown, slightly refractile, branching, and non-septate
hyphae are routinely observed. To identify the fungal elements, special stains
such as the Gomori methenamine silver (GMS) can be used. B. denigrans
appears to impact the entire gill, whereas B. sanguinis appears to harm only
the blood vessels of the gill. Strict sanitation, disinfection is essential for
controlling the disease.

16.3.2.4 CONTROL AND TREATMENT

For damaged fish, greater water flow and adequate hygiene are recommended
which includes removing dead fish, boosting water supply, and avoiding
overfeeding, especially at high water temperatures. Branchiomycosis can
be prevented using good water quality management practices. However,
keeping fish in a malachite green solution for 24 hours has also been reported
to be effective. Infected specimens are treated with malachite green @ 0.1
mg/l for extended time or 0.3 mg/l for 12 hours.
364 Coldwater Fisheries and Aquaculture Management

16.3.3 EPIZOOTIC ULCERATIVE SYNDROME (EUS)

16.3.3.1 ETIOLOGICAL AGENT

Aphanomyces invadans is the causative agent of EUS. Aphanomyces

species have typhoid mycelium that is cylindrical and coenocytic. EUS is
also known as red spot disease (RSD), mycotic granulomatosis (MG) and
ulcerative mycosis (UM). It was first reported in Japan in goldfish and ayu
in the year 1971 (Egusa & Masuda, 1971). Hyphae in infected fish have
minimal branching and a diameter ranging from 6 to 27 mm, but they are
much narrower in culture (Roberts et al., 1993).

16.3.3.2 CLINICAL SIGNS AND PATHOLOGY

Infested specimens have lesions on their body surface. Lesions are most
commonly found on the lateral sides, however they can develop elsewhere on
the body. Pinpoint red spots or hemorrhagic spots or localized inflammation
is common. Protruding scales, scale loss, skin erosion, reddened skin and
ulceration are the common clinical signs (Roberts et al., 1993). Because of
the blood loss induced by the hemorrhagic lesions, fish with EUS have a
considerable increase in white blood cells (particularly neutrophils) due to a
local inflammatory response, while the quantity of red blood cells, hemoglobin
concentration, and hematocrit level are all reduced.

16.3.3.3 DIAGNOSIS

Speculative diagnosis is based on sickening appearance and aseptate hyphae

in squashed preparations. For confirmatory diagnosis, histological analysis
is a must approach. There are no viable serological approaches for detecting
and identifying A. invadans in EUS specimens.

16.3.3.4 CONTROL AND TREATMENT

Stocking numbers should be kept as low as possible during high-risk periods,

such as when EUS prevalence is high in nearby wild fish populations, and
farmed populations should be subjected to minimal stress. Similarly, the
pond environment might be monitored to ensure that abiotic elements like
Infectious Diseases of Coldwater Fishes 365

as low DO concentrations, which can cause skin injury, are kept within safe
ranges (Devashish, 2016). Disinfection of fish eggs and larvae against water
molds is effective. Disease eradication, good husbandry practices, proper
surveillance and biosecurity measures are necessary. Because mycelium and
zoospores of A. invadans are killed at very low concentrations, treatment
with Malachite green and formalin is currently the most effective strategy to
prevent EUS.

16.4 CONCLUSION

Disease can be a major threat to cold water aquaculture in terms of economic

loss to fish farmers. The focus should always be on prophylaxis rather
than therapeutics to prevent the disease outbreak at a fish farm. Ensuring
a water source that is free of pathogens, maintenance of proper hygiene at
farm, disinfection of nets and equipment at the farm site, vaccination of the
stock, procuring of the disease-free seed, proper stocking density, and use
of immunostimulants are some of the reliable measure to reduce disease
outbreaks in cold water aquaculture.

KEYWORDS

• cold water aquaculture

• cold water fishes
• fungal and viral diseases
• infectious hematopoietic necrosis
• infectious pancreatic necrosis
• stocking density

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pancreatic necrosis in trout. Proceedings of the Society for Experimental Biology and
Medicine, 104(1), 105–108.
CHAPTER 17

Nutrigenomics: Boost for Aquaculture

Research and Development
KAWKABUL SABA1, FAISAL SOFI2, K. SRAVANI3, S. VIJAY KUMAR REDDY4,
OYAIS AISMI1, ASHWINI KUMAR1, TARIQ HUSSAIN2, S. DEVADHARSHINI3,
P. GANESAN3, SHABIR A. DAR5, and NEERAJ PATHAK6
1
Division of Fish Nutrition and Biochemistry, Faculty of Fisheries,
SKUAST-K, Jammu and Kashmir, India
2
Division of Post-Harvest Technology, Faculty of Fisheries, SKUAST-K,
Jammu and Kashmir, India
3
Department of Fish Processing Technology, Fisheries College and
Research Institute, TNJFU, Thoothukudi, Tamil Nadu, India
4
Department of Fish Processing Technology, College of Fisheries, Gadvasu,
Ludhiana, Punjab, India
5
Division of Fishery Engineering, Faculty of Fisheries, SKUAST-K,
Jammu and Kashmir, India
6
Department of Quality Assurance and Management, College of Fisheries,
Thoothukudi, Tamil Nadu, India

ABSTRACT

Nutrigenomics is a broad field of science that focuses on the interaction

between genes and the activity of common dietary nutrients, as well as their
function in achieving a balance between disease and health by altering the
structure or expression of a person’s genetic composition. Nutrigenomics is
a field that uses postgenomic and related technologies to study an individual

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
370 Coldwater Fisheries and Aquaculture Management

organism’s reaction to food contents. Nutrigenomics is an area that requires

more rigorous investigation and evaluation, and it can be thought of as a
new tool for nutrition research in understanding concerns related to animal
production and health. Even while more research is being done, public
awareness is not being raised to the point where science can be used to its
full potential. As a result, efforts must be made to investigate the existing
state of knowledge, practices, and abilities linked to the use of nutrigenomics
to improve animal health and production. So, the purpose of this chapter
is to explore the importance of nutrigenomics, how it works, and what its
applications are in the animal sciences field.

17.1 INTRODUCTION

Scientific interventions are playing a major role in fishery because of which

aquaculture industry in India is showing enormous growth for the last 40
years with 10–11 folds increase in fish production. It is targeted to produce
10 million metric tons (MT) by the end of 2030 year, for which more research
has to be planned for understanding the basic process of growth necessary
for improving the aquaculture productivity (Anonymous, 2013). Growth is
considered as the conversion of feed into flesh, which is commonly calculated
as an increase in biomass production, i.e., the variation between the initial
and final biomass of fish, which is nothing but the accumulation of muscle
mass, especially the accumulation of protein. Many complex reactions are
involved in individuals’ bodies for the transformation of protein in the diet
to tissue protein and revealing the molecular mechanisms of these complex
reactions will help to provide a picture of the growth phenomenon taking
place inside the body.
In the nutrition of fish, especially those that are maintained in aquacul­
ture, approximately 31.5 MT of cultured fish are depending on the external
feeds that may be farm-made, fresh materials, or commercially manufac­
tured feeds. The aquaculture feed production has increased to 39.9 million
tons in the year 2016, when compared to 7.6 million tons in 1995, which
is showing a growth of more than three folds, with an average production
rate of 12% per annum (All Tech, 2016). The formulated feeds are notably
different from naturally available diets, as the nutrients in formulated feeds,
particularly oil and protein sources are dominated by land-based ingredients.
Nowadays, new feeds, known as functional feeds, are prepared by incorpo­
rating nutrients, believing that the nutrients will help in enhancing the health
of fish, reducing outbreaks of diseases, or increasing post-infection recovery.
Nutrigenomics: Boost for Aquaculture Research 371

By knowing the new technologies like nutrigenomics, nutritranscriptomics,

epigenetic interactions, neuroproteomics, and nutrimetabolomics the level
of fish nutrition can be increased which boosts the production and health of
farmed fishes (Figure 17.1).

FIGURE 17.1 Nutritional genomics in association with fish nutrition

17.2 NUTRIGENOMICS

Nutrigenomics is a science that deals with the relation between diet, nutrients,
and the expression of genes. It also deals with the association between human
nutrition, genome, and health. The researcher in the field of nutrigenomics
works on the responses of the entire body towards a single nutrient or particular
food component as well as a gene. Nutrigenomics or Nutritional genomics
deals with the occurrence of the role of natural ingredients or chemicals present
in food that will alter the expression of individual genetic information. The
part of nutrients on the expression of genes and regulation was highlighted
by nutritional studies. There are so many advanced techniques like genomics,
metabolomics, and proteomic techniques that provide a facility to know about
the constituents of the diet, and the crucial factors involved in gene-nutrient
interactions at the cellular level. During gene expression or activation, that
particular gene will have some biochemical and physiological function at the
cell level as a result of which proteins will be produced. With the advent of
nutrigenomics, it is possible to identify that particular gene responsible for
372 Coldwater Fisheries and Aquaculture Management

producing nutritionally required proteins and cofactors at the site of use and
enzymes required for digestion. Thus, genetic interactions include both the
effect of genes and the effect of genetics on genetic metabolism.
The field of Nutrigenomics is popularly identified as a tool, for the func­
tion of diet which is having an important role in the prevention of cancer,
balancing homeostasis, avoiding the risk of chronic disease, or reduction
of disease progression. The outlook of nutrition research may currently be
unrewarding.
In today’s world, nutrition research has focused on health problems and
malnutrition. The beginnings of genomics can be described as a collection of
advanced technologies for the use, processing, and production of scientific
information about the functions and structure of genomes, leading to
unique opportunities to successfully understand how genes modify genetic
expression and protein, ultimately affecting the environment and cellular
metabolism. Nutrigenomics, often known as nutritional genomics, is the
study of the relationship between diet, health, and genomics. It’s a hybrid
of molecular nutrition and genetics, to put it another way. Post-translational
modifications (proteome), metabolite profiles (metabolome), chromatin
structure (epigenome), and gene-expression patterns are all part of the
protein-expression patterns (transcriptome).

17.3 NUTRITRANSCRIPTOMICS

Sometimes, nutrients behave as transcription factors during the interaction

with a receptor, which can induce gene expression after binding to DNA. In
the recent past, various studies were conducted to demonstrate the interac­
tion of nutrients with genes in cultured fish species. Others have reported
the influence of nutrients in genetic expression through written features
(Afman & Muller, 2006; Fuks, 2005) – called “nutritional nerves.” The
nuclear receptor superfamily of transcription factors binds to many recep­
tors, fatty-binding receptors (PPAR) or cholesterol, and metabolites that bind
to receptors (X receptors), which bind directly to the nucleotide sequence
(reactions elements) to the facilitator of various genes (Afman & Muller,
2006). Nuclear receptors begin to function in the binding of ligands due to
coherent flexibility driven by the systematic division of co-activators and the
recruitment of co-activator proteins. As a result, these factors act as nutrient
sensors by altering the genetic code of certain genes in response to changes
in nutrition.
Nutrigenomics: Boost for Aquaculture Research 373

17.4 EPIGENETIC INTERACTIONS

Gene expression is altered due to the alteration in the DNA or histone protein
structure by the nutrients. The process of DNA methylation or methylation
and acetylation of histones or by both ways mediates the epigenetic effects
(Holiday & Grigg, 1993). These mutations result in genetic mutations that
last throughout the life of an animal or even for generations. DNA methyla­
tion on cytosine bases influences gene registration and genomic stability,
and these mutations in genetic modifier regions modify genetic expression
(Kersten, 2002).
Methylated 5′ CpG 3′ islands attract capping proteins, which prevent tran­
scription factors from gaining access because of the increased methylation.
Once these islands in genes have been methylated, the methylation is repli­
cated each time the gene is copied. The methylation effects are maintained in
this manner. When DNA is wrapped around proteins, it inhibits access to gene
promoter regions when they are firmly packed. The channels will be produced
by the methylation and acetylation of histones, which will allow transcription
factors to pass through and activate gene promoters. Some nutrients, including
vitamin B6, methionine, vitamin B12, folate, and choline, operate as methyl
donors in one-carbon metabolism. Numerous research and publications show
that nutrients interact epigenetically with genes to alter biological processes
including growth, breeding, and immune response.

17.5 NUTRIPROTEOMICS

Nutriproteomics is the study of molecular and cellular changes in protein

structure and expression in response to food intervention using the dynamics
of proteomic methods. Proteins play a crucial part in a cell’s structure and
function. The proteome is the cell’s large and constantly changing network
of proteins. The proteome changes depending on the cell type, while the
genome stays the same. The large amount of proteins derived from the
genome is attributable to the following factors:
• Splicing of mRNA;
• Protein processing;
• Post-translation editing.
Dietary supplements and non-nutritious food components act as signals
that the written material and nutrients are beginning to emerge. The sensors
of nutrition change the gene. However, there is another way the dietary
374 Coldwater Fisheries and Aquaculture Management

component can affect the proteome: disruption at the level of posttranslational

transformation. Nutritional interventions cause two types of changes in the
proteome: changes in quantity and function. Various tools for detecting these
changes are also being developed. However, there are a few common types
of protein separation and purification processes. Mass spectroscopy, peptide
digestion, and isotope labeling are some of the methods used in quantitative
proteomics. 2D PAGE, mass spectroscopy, and antibody-based methods are
all used in active proteomics. The structure and function of proteins should
be determined by active proteomics. Proteins do not move to the immune
system and are acquired after the release of 2D PAGE in this area. Allows
precision specification of protein micro-heterogeneity. Active proteomics
can also be illuminated by real-time protein kinetics and protein sequence.

17.6 NUTRIMETABOLOMICS

The study of the distinct chemical fingerprints created by specific biological

processes as a function of the animal’s nutritional condition or in response
to nutritional intervention. Oliver coined the word “metabolome” in 1998
to describe a collection of low-molecular-mass chemicals found in cells.
Genetics, environmental variation, and food all have an impact on a cell’s or
organism’s metabolome. Nutrimetabolomics testing is usually done on bodily
fluids like blood, urine, or feces, but it can also be done on other tissues. The
following approaches are being used in nutrimetabolomic studies:
i. Metabolite Fingerprinting: It is used to identify not only the
metabolites but the identification of overall nature. It involves the
physiochemical characteristic too.
ii. Fingerprinting: It is known as the output of the sensors in response
to the sample.
Research on different chemical fingerprints created by specific biological
processes such as the function of an animal’s nutritional status or in response
to a nutritious food intervention. Oliver coined the term “metabolome” in 1998
to describe a set of low-cell chemicals found in cells. Genetics, biological
diversity, and diet all contribute to the cell or biological metabolome.
Nutrimetabolomics tests are usually done on body fluids such as blood, urine,
or feces, but can also be done on other tissues. The following methods are
used in nutrimetabolomic studies:
1. Metabolite Fingerprints: It is used to identify not only metabolites
but to identify the whole environment. It includes the physiochemical
Nutrigenomics: Boost for Aquaculture Research 375

aspect as well. Fingerprints are known as nerve output in response to

a sample.
2. Metabolite Profiling: The analysis identifies the metabolites using
chromatographic and spectrophotometric methods.
3. Metabolome Analysis: It is defined as a time-dependent resolution-
based analysis of metabolite molecules. It entails using a combina­
tion of analytical tools to investigate the full range of metabolites
present in a sample.
4. Systems Biology: It performs partial or complete integration of
transcriptomics, proteomics, and metabolomics info.
There are two sorts of tools utilized in nutrimetabolomic investigations:
• Metabolites were separated using the following tools: Thin layer
chromatography, gas chromatography, and capillary electrophoresis
are some of the techniques used; and
• Metabolites are detected using the following tools: It involves nuclear
magnetic resonance and mass spectroscopy.

17.7 GENERAL OVERVIEW OF NUTRIGENOMICS

Conventionally more attention is towards the effect of macro- and micro-

nutrients like proteins, lipids, carbohydrates, vitamins, and minerals. The
main objective of nutritional research is mainly based on their requirements,
presence in excess or deficiency. Modern-day research is incomprehensible
that most people have identical dietary requirements. Mostly every individual
will not get benefit from the same dietary regime as they possess different
nutritional phenotypes. The human genome is 99.9% identical with some
0.1% variations and this variation is due to their responsibilities towards their
diet or food. In the case of having a meal, a few dietary compounds may get
metabolized whereas some don’t as they get attached to the cellular recep­
tors by acting as a ligand. For an instance, the meal contains some ligand
it can able to turn on disease-fighting genes or turn off the genes capable
of causing disease in future nutritional research. According to reports, the
human genome project began in the 1990s and finished in 2003, with the
primary goal of identifying the human genetic code by mapping out every
single gene. Nowadays, a new field of research has emerged in relation to the
human genome project, which comprises nutrigenetics and nutrigenomics,
both of which are concerned with the interaction between human genes and
nutrition. Similarly, Nutrigenetics scientifically describes about the result
376 Coldwater Fisheries and Aquaculture Management

of genetic differences in dietary response, ensuring differences in nutrients

metabolism. Although the term Nutrigenomics,’ is defined as the technologi­
cally how the nutrients adjust the gene expression, it is often measured as a
critical excessive-throughput genetic tool inside the field of nutrition studies.
Excessive throughput is applied to display nutrients research that enables us
to find in what manner nutrients have an effect on the lots of genes expressed
in the human genome. This chapter focuses more on Nutrigenomics and the
many aspects of nutrigenomics and nutrition research, as well as their overall
importance.
Nutrigenomics adheres to the following principles:
i. Nutrition may directly affect gene expression or indirectly induce
genetic mutations that alter gene expression or gene silencing in
metabolic pathways.
ii. Depending on inherited genetic variants, nutrient outcomes in health
can also be affected by the biochemical reaction involving enzymes
and their cofactors as well as the metabolism of nutrients.
iii. Health outcomes can be improved by taking into account inherited
and acquired genetic characteristics based on age, gender, and health
status.
Nutrigenomics explains how certain foods affect the genes in our body. Our
diet determines what genetic messages our bodies receive. These messages,
in turn, regulate the molecules that make up our metabolism: genes are
molecules that control the body to burn or store calories. We can take control
of our bodies and digest our food if we learn to understand our genes and
control their messages and instructions. By changing the way food interacts
with our bodies, we can lose weight and improve our health (Mark Hyman,
2006). Nutrigenomics began with the idea that food affects health. It includes
the knowledge that food affects genes, which have long been used in dietary
modification. Phenylketonuria (PKU) is one of these, and it is caused by a single
genetic mutation. People with this condition should avoid foods that contain the
amino acid (AA) phenylalanine. Because of a gene that encapsulates lactase,
an enzyme that breaks down lactose and is often eliminated after weaning,
most adults in the world suffer from lactose intolerance, which means they are
unable to digest milk proteins. About 10,000 to 12,000 years ago, the people
of Northern Europe developed a polymorphism from a single nucleotide of
DNA. This polymorphism caused the lactase gene to continue maturing. The
genetic makeup of cells helped scientists to identify other genes that interact
with genes in the late 20th century, making this SNP beneficial to people living
in areas with shorter growth periods.
Nutrigenomics: Boost for Aquaculture Research 377

Nutrigenomics was marketed in the 1980s. The science of nutrigenomics

was started in the 1990s by a human genetic project, which traced all the
DNA bases to the human genome. In 2007, a number of genetic, nutritional,
and disease-related interactions were identified. Nutrigenomics has intro­
duced many new ideas, discourses, and experimental processes in healthy
food research, such as high-throughput technology that allows global
research into genetic expression in a cell or organism. Geneticists, public
health professionals, nutritionists, and cooks will need to work together on
nutrigenomics. It is not difficult to prepare delicious food. If you add butter
or butter to it, it will taste better. We need to find a way to cut fat while
making nutritious and delicious dishes. Considering nutrigenomics, as well
as the increase in obesity and chronic diseases such as type 2 diabetes, this
could be a panacea for the future.
Chronic diseases are largely ignored in the global health agenda,
despite rising rates of chronic diseases worldwide. As a result of dietary
and lifestyle changes, developing countries are now facing an epidemic
of non-communicable diseases (NCDs) linked to global trade, as well as
competing for health problems. Globalization has led to something that can
be linked to global health issues and must compete with competing health
priorities. In 2005, NCDs killed an estimated 35 million people worldwide,
mainly cancer, respiratory disease, heart disease, and diabetes. Over the next
decade, deaths from NCDs are expected to increase by another 17%. NCDs
are expected to deal with 80% of the global disease burden by 2020 and will
be responsible for seven out of 10 deaths in developing countries and 60% of
global deaths by 2005 (approximately 35 million deaths). As a result, limited
healthcare funds, especially in less developed countries, face a (double)
burden. Therefore, the promises of nutrigenomics should be considered
in view of the increasing epidemic in developed and developing countries
(Gobard & Hurlimann, 2009). In India, the death toll from deadly diseases
is lower than in Western lands. These rates, however, are rising as a result
of urbanization and lifestyle changes (Rao, 2001; Shetty, 2002; Sharma &
Majumdar, 2009). The consumption of rice and wheat has changed from
rough grains to refined grains in recent decades. In India, there are very high
rates of coronary heart disease (CHD), with urban rates three times higher
than in rural areas. Obesity and diabetes are also increasing in both remote
and high-income sub-regions (Sinha et al., 2003). Many chronic illnesses,
such as type 2 diabetes, high blood pressure, cardiovascular disease, cancer,
and mental health problems are associated with obesity. Lifestyle changes
and eating habits are the major contributors to its development. Diet appears
to be associated with higher levels of diabetes, heart disease, and obesity
378 Coldwater Fisheries and Aquaculture Management

(Kaput et al., 2007; Hossain et al., 2007), although genetic factors may play
a role. Therefore, general awareness of food-related problems that lead to
genetic mutations is needed, where nutrigenomics should be studied in more
detail.

17.8 BENEFITS OF NUTRIGENOMICS TO AQUACULTURE INDUSTRY

Research on nutrigenomics will benefit aquaculture industry in following

modes:
1. To assess the reaction of an animal to nutrients. Nutrigenomics research
will help to assess the different reactions between the animals and
particular nutrients. For example, some animals will show particular
reactions to carbohydrates, whereas other animals will not be affected
by that. Nutrigenomics will explore the knowledge about the response
between animals and nutrients. This can be explained in a better way
as more dietary carbohydrates in mammals generally decrease gluco­
neogenic activity whereas the same reaction is not seen in fishes.
2. For enhancing the diet. Nutrigenomics will inform the researcher
about the potential diet requirements of a particular animal and their
interactions with that particular nutrient, which helps in formulating
or enhancing a diet as per the dietary requirement of the animal.
Dietary carbohydrates, in the case of herbivorous fish, will maintain
the activity and appearance of digestive enzymes of carbohydrates
whereas in the case of carnivorous fish, its activity is less (Polakof et
al., 2012).
3. Nutrigenomics will provide a better idea regarding the response of
nutrients in cell.
4. Nutrigenomics research will help to synchronize the research results
for improving husbandry practices (Volkoff et al., 2005).
5. Nutrigenomics studies will help to recognize the aspects that
influence energy metabolism. In fish, the increase in lipase gene
expression of lipoprotein of adipose tissue will initiate catabolism of
fat, and released energy will be utilized for growth. And hence, the
amount of protein and lipids in the feed has to be increased during
this phase (Mead et al., 2002; Nurnberg et al., 1998; Hocquette et al.,
2010; Caballero et al., 2002).
6. It enables to know the organ and tissue specificity for utilizing
various nutrients. In the fish, Glucokinase is different from the other
Nutrigenomics: Boost for Aquaculture Research 379

three hexokinase enzymes (Moon, 2001; Hemre et al., 2002). The

nutrigenomic studies confirmed that the expression of glucokinase
is not inhibited by glucose-6-phosphate but it is able to restrict the
expression of hexokinase in other tissues (Rocha et al., 2015). Insulin
causes an increase in adipose lipoprotein lipase content but the same
reduces the muscle LPL and fat deposition in the tissue (Bouraoui et
al., 2011).
7. Specific changes in metabolism. In the adult stage, the amount of
beta-oxidation in the red muscle of salmon is 10% but in the smolt
stage, it is 60% (Jordl et al., 2006). Nutrigenomics will elucidate the
reasons behind this change.
8. Regulation of basic metabolism by dietary factors. Zambonino
& Cahu (2010) cite the retro-inhibition of delta-6 desaturase by
DHA as an example of metabolic regulation by dietary variables.
Nutrigenomics contributes greatly to a greater comprehension of
metabolic regulation. Studies of nutrigenomics in the transportation
of nutrients.
The development of different transporters in response to diverse forms
of the same nutrients improves nutrition utilization. Hexokinases, for
example, respond to dietary glucose levels, whereas glucokinase does not
(Wilson, 1994). Furthermore, when dietary phosphate is limited, Na-PO4
co-transporter mRNA expression is up-regulated, and when dietary phosphate
is supplemented, it is down-regulated (Reed, 2014). The nutrigenomic
technique can be used to confirm the type of phosphate that best up-regulates
activity.

KEYWORDS

• coronary heart disease

• co-transporter mRNA
• fatty-binding receptors
• non-communicable diseases
• nutrigenomic technique
• nutrigenomics
• phenylketonuria
380 Coldwater Fisheries and Aquaculture Management

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CHAPTER 18

Immune Components and Defense

Mechanism in Fish: An Overview
SALIK NAZKI1, MUNAZAH SHAHZAD2, INAIN JAIES3,
SYED SHARIQ N. QADIRI3, and FEROZ A. SHAH3
1
The Pirbright Institute, United Kingdom
Department of Veterinary Epidemiology and Public Health,
2

University of Surrey, United Kingdom

3
Division of Aquatic Animal Health Management, Faculty of Fisheries,
Sher-e-Kashmir University of Agricultural Sciences and Technology
(SKUAST) of Kashmir, Rangil, Ganderbal, Jammu and Kashmir, India

ABSTRACT

During microbial invasion, an effective clearance and degradation system

may be required to prevent severe manifestations and, eventually, mortality.
Surface barriers, lysozyme, complement, and lectins are non-specific
defense components that operate as the first line of defense and are critical
at all stages of infection. During microbial invasion, non-specific defense
systems may be potentiated, resulting in more efficient clearance and
elimination of pathogens or other hazardous chemicals. Humoral immunity,
cell-mediated immunity, and memory are three crucial characteristics of a
specific defense system. Each component has its own inherent protective
value, and the final combination is likely to be linked to the immunological
history and evolutionary development of the fish. The present chapter will
present a brief overview of innate and adaptive components of fish defense
system.

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
384 Coldwater Fisheries and Aquaculture Management

18.1 INTRODUCTION

Network of cells, tissues, and organs make up the immune system. The
innate and adaptive immune system are two branches of the fish defense
system. The innate immune system is the first line of defense against
infections, and it has no memory of prior reactions. If a pathogen over­
comes the innate immune response, the adaptive immune system will
target it with precision and memory. Immunoglobulins (Igs) and T-cell
receptors (TCR) in conjunction with major histocompatibility complex
(MHC) are components of the adaptive immune system. The adaptive
immune system is highly specific to a specific antigen and can provide
long-term immunity (Alberts et al., 2002). The lymphoid organs in the
vertebrate immune system are classified as main or secondary based on
their ontogeny and functional properties. As the majority of the infectious
agents affect or initiates the process of infection in the mucous surfaces,
the mucosal immune response plays a crucial role in the course of the
infection (McNeilly et al., 2008). Despite certain differences, the immune
systems of fish and higher vertebrates are biologically comparable. Fish is
in close contact with water which can contain high quantities of bacteria,
viruses, and other pathogens. During this phase, they are primarily reliant
on a non-specific immune system to survive (Sahoo et al., 2021). While
the non-specific immunity is the more primitive defense mechanism,
acquired immunity is also important for maintaining homeostasis. The
various innate and adaptive immune components in fish are presented in
Figure 18.1.

18.2 NON-SPECIFIC DEFENSE MECHANISM IN FISH

18.2.1 SURFACE BARRIERS

The first line of defense against infection in fish includes skin mucus, gills,
epidermis, and gastrointestinal tract. The constant sloughing of skin mucous
prevents microbial invasion. In addition, fish mucus is constantly secreted
and renewed, preventing the steady colonization of potentially pathogenic
microbes as well as metazoan parasite invasion (Ángeles, 2012). Hyperplasia
of Malpighian cells is the first response shown by non-keratinized epidermis
against pathogens. Mucus production and a highly proliferative epithelium
protect the gills.
Immune Components and Defense Mechanism in Fish 385

FIGURE 18.1 Different components of fish defense system.

18.2.2 LYSOZYME

Lysozyme is an enzyme that can lyse bacterial cell membranes, making it an

effective antibacterial agent. The level or activity of lysozyme is a significant
indicator of innate immunity in fish, and it is found in all living species.
The lytic activity of fish lysozyme against Gram-positive and Gram-negative
bacteria has been thoroughly characterized. Most fish species have lysozyme
in their mucous, lymphoid tissue, and serum (Nigam et al., 2012). Lysozyme
bacteriolytic action in fish skin mucus and other tissues aids in the host
defense against bacterial infection (Subramanian et al., 2007).

18.2.3 COMPLEMENT

Complement is a group of roughly 35 proteins that make up an important part

of the innate immune system. Fish complement proteins have many similari­
ties to their mammalian counterparts, and their activation pathways appear
to be similar to those found in mammals. Fish complement can lyse foreign
cells and opsonize alien organisms for phagocytic destruction. Complement
fragments may possibly play a role in inflammatory reactions, according to
some evidence. Several complement proteins, such as C3 and factor B, have
numerous isoforms in fish (Holland & Lambris, 2002).
386 Coldwater Fisheries and Aquaculture Management

18.2.4 LECTINS

Non-immune carbohydrate binding proteins are known as lectins. Lectins are

components of the innate immune system that have an affinity for carbohydrate
moieties and can cause cell agglutination and/or glycol-conjugate precipitation.
Lectins bind to pathogenic surface features, causing opsonization, increased
phagocytic activity or complement pathway activation (Matsushita et al.,
2004; Ottinger et al., 1999).

18.2.5 ANTIMICROBIAL PEPTIDES (AMPS)

Antimicrobial peptides (AMPs) are becoming more well recognized as

an important component of the host defense. They are effective against a
wide range of pathogenic organisms, including Gram-positive and Gram-
negative bacteria, yeast, fungus, enveloped viruses, and parasites, with little
or no damage to host cells (Ángeles, 2012). Fish have a lot of alpha-helical
amphipathic peptides (Smith & Fernandes, 2009). The piscidin family, which
comprises the pleurocidins and piscidins, contains the majority of fish-
helical peptides. Pardaxin, parasin 1, hipposin, oncorhyncin III, oncorhyncin
II, SAMP-H1 are a few examples of AMPs found in fish epidermal mucus
(Smith & Fernandes, 2009; Fernandes et al., 2003; Thompson et al., 1986).

18.2.6 TRANSFERRIN

Transferrin is made up of a single polypeptide chain with roughly 700 amino

acid (AA) residues that is divided into two lobes, each with two domains where
iron is coordinated (Reyes-López et al., 2015). Iron is required for the formation
of infection by many pathogens, but due to its strong affinity for the blood
protein transferrin, iron availability in vertebrate tissue fluids is exceedingly
low. Importance of transferrin in the immune system stems from its ability to
control serum free-iron levels, resulting in low-iron conditions wherein the
potential of pathogenic bacteria to cause infection is limited (Chen et al., 1999).

18.2.7 INTERFERON

Interferons (IFNs) are proinflammatory, antiviral, and immunomodulatory

cytokines that play a key role in the host response to infection (Martin et
Immune Components and Defense Mechanism in Fish 387

al., 2007). Two of the three IFN families found in higher vertebrates are
now known to play a role in teleost fish antiviral defense (Zou & Secombes,
2011). Once IFN binds to the receptor, signal transduction activates IFN-
responsive genes through conserved signal transduction pathways, causing
the cell to change function (Schroder et al., 2004).

18.2.8 C-REACTIVE PROTEIN (CRP)

C-reactive protein (CRP) was discovered in the serum of a marine teleost fish,
Pleuronectes platessa (White & Fletcher, 1985), and has since been isolated
and described primarily from teleost fish. It binds to the phosphoryl choline
found on the cell walls of microorganisms. Because its concentration rises in
reaction to harmful substances and bacterial infections, CRP in trout is clas­
sified as an acute phase protein (Kodama et al., 2004). Salmon CRP is a non-
acute phase protein of 208 AAs, and only one of five CRP types is activated
by cytokines (Pathak & Agrawal, 2019). When tissue is injured, traumatized,
or infected, the levels of these proteins rise. These proteins are involved in the
immune system (Cook et al., 2003), the classical complement pathway (De
Haas et al., 2000), and in the clearance of apoptotic cells (Nauta et al., 2003).

18.2.9 PHAGOCYTOSIS

In fish, neutrophils and macrophages are the major cells involved in phagocytosis
(Secombes & Fletcher, 1992). Because it is the process that is least affected by
temperature, phagocytosis is one of the most critical processes in poikilothermic
species (Magnadottir et al., 2005). During a respiratory burst, these cells primarily
eliminate microorganisms by producing reactive oxygen species (ROS).

18.3 CELLS INVOLVED IN NON-SPECIFIC DEFENSE MECHANISM

18.3.1 MACROPHAGES

The best-studied macrophage phenotype in teleosts is one that is similar

to the M1 activation state, which plays an important role in host defense.
M1 macrophages produce a wide range of cytokines, chemokines, and lipid
mediators that help to enhance and fine-tune inflammatory and adaptive
immune responses. These cells can kill pathogens quickly by engulfing them
388 Coldwater Fisheries and Aquaculture Management

and producing toxic reactive intermediates, phagolysosomal acidification,

and nutrition limitation (Neumann et al., 2000; Rieger et al., 2010; Grayfer
et al., 2014). The synthesis of multicomponent enzyme nicotinamide adenine
dinucleotide phosphate (NADPH) subunits at the plasma membrane during
the macrophage respiratory burst results in the transfer of electrons from
NADPH to molecular oxygen, culminating in the superoxide anion (Briggs
et al., 1975). Superoxide is quickly converted to ROS such as hydrogen
peroxide (H2O2), hydroxyl radical (OH), and hyperchlorous acid (HOCl)
after it is formed (El-Benna et al., 2008). In comparison to those described in
mammals, the inducible nitric oxide (NO) system of teleost macrophages is
largely preserved. The activation of inducible nitric oxide synthase (iNOS/
NOS2), which catalyzes the conversion of l-arginine to l-citruline, culmi­
nating in the generation of NO, a strong antibacterial molecule, distinguishes
classically activated macrophages (Nathan & Xie, 1994).

18.3.2 NEUTROPHILS

Teleost neutrophils are terminally differentiated leukocytes that defend

the host and mount rapid and effective antimicrobial responses against
infections. They are usually the first leukocytes to arrive at a location of
inflammation (Havixbeck et al., 2015). Using toxic intracellular granules
(Flerova & Balabanova, 2013), the generation of ROS (Katzenback &
Belosevic, 2009), and the deployment of extracellular traps (Pijanowski
et al., 2013), neutrophils become powerful killers. NO is required for
neural transmission, cell growth inhibition, vasodilation, and intracellular
signaling (Napoli et al., 2013). It also has strong toxic properties and is
an important component of antimicrobial defenses. NO has been proven to
have powerful antibacterial properties against a variety of diseases found
in fish (Wiegertjes & Forlenza, 2010). Trypanoplasma borreli, a parasite
hemoflagellate, has similarly been demonstrated to promote NO generation
in carp head kidney neutrophils (Scharsack et al., 2003).

18.4 SPECIFIC DEFENSE MECHANISM IN FISH

18.4.1 B-CELL AND ANTIBODY

B1-B cells, which are implicated in innate activities, respond to stimula­

tion in-vitro via toll-like receptors (TLR) (Rawlings et al., 2012), causing
Immune Components and Defense Mechanism in Fish 389

proliferation and differentiation of B1-B cells into Ig-secreting cells. After

innate activation, B1-B cells also exhibit a quick capacity to release large
levels of the immunomodulatory cytokine interleukin-10. Some fish species
exhibit interesting B cell characteristics, such as the lack of pathogen-specific
IgM in gadoids following successful pathogen immunization (Magnadottir
et al., 2009), and the absence of the entire IgM gene in a coelacanth species
(Amemiya et al., 2013).
Plasma blasts and plasma cells produce secretory Igs, which are important
for maintaining mucosal homeostasis. Until recently, it was thought that
teleost fish B cells only expressed two types of Igs, IgM, and IgD (Hordvik
et al., 1999), with IgM being the only one that responded to pathogens in
both the systemic and mucosal compartments. However, a third teleost Ig
class, IgT/IgZ, was recently found to be the most common Ig in gut mucosal
immune responses (Salinas et al., 2011). IgM, IgD, and IgT are the three Ig
isotypes produced by teleost B cells. Antibodies found in teleost cutaneous
mucus and skin play an important part in the host preventive defense
against surface infections (Dickerson et al., 1998). The primary isotype in
teleost fishes is IgM, which has one variable and four constant domains
and is detected in plasma, bile, and skin mucus (Flajnik et al., 2003). The
most common Ig found in teleosts is an IgM tetramer with eight antigenic
combining sites. Salmon and Atlantic cod have different amounts of IgM
depending on their size, temperature, and water quality season (Uribe et
al., 2011). Ig has two identical heavy (H) chains and two identical light (L)
chains as its basic structure (Mashoof & Criscitiello, 2016). The constant
section of both heavy and light chains consists of one N-terminal variable
domain (VH or VL) and one or more C-terminal constant domains (CH or
CL). Complementarity-determining regions are three hyper variable regions
found within each VL and VH domain. The antigen binding site of the Ig
molecule is made up mostly of the three CDR segments of the VL domain
and the three CDR segments of the VH domain (Abbas et al., 2010). Typical
structure of a monomeric antibody is shown in Figure 18.2.

18.4.2 MEMORY COMPONENT

Before a second antigen encounter, fish acquire a memory response (Whit­

tington et al., 1994). The memory mechanism is based on the generation
of long-lived T and B memory cells, as well as alterations in the nature of
reacting cells, resulting in the selection of clones with high antigen affinity.
Both humoral and cell-mediated immunity are present in fish. Memory
390 Coldwater Fisheries and Aquaculture Management

formation in carp appears to take longer than in mammals, roughly 3–6

months and lasts for 8–12 months after initial stimulation. Increased antigen
sensitivity is another sign of memory (Lamers et al., 1985). For example,
trout that had been primed with an ideal dose of antigen and then boosted
with a sub immunogenic dosage had a larger antibody titer than those that
had been primed with the optimal dose. T-dependent antigens require two
exposures before the fish responds to the second dose, but T-independent
antigens just require one exposure (Uribe et al., 2011).

FIGURE 18.2 Structure of a monomeric antibody.

18.4.3 ANTIGEN PRESENTATION

Professional antigen presenting cells (APCs) play an important role in

pathogen identification by PRRs and adaptive immunity activation by antigen
presentation to naïve T cells. Dendritic cells (DCs), monocyte/macrophages,
and B cells ingest pathogens and convert antigen to peptides as professional
APCs. MHC class I molecules present intracellular antigens to cytotoxic T
cells (CD8+ T-cells), while MHC class I molecules present extracellular anti­
gens to helper T cells (CD4+ T-cells) (Keech et al., 2010). DCs are the most
common and strong APCs in vertebrates, and they play an important role in
bridging and modifying both innate and adaptive immune responses (Mildner
& Jung, 2014). Macrophages are massive mononuclear cells found in nearly
Immune Components and Defense Mechanism in Fish 391

every animal tissue. Macrophages are phagocytes with germline-encoded

PRRs that recognize pathogen-associated molecular patterns (PAMPs) like
lipopolysaccharide, peptidoglycan, and LTA from Gram-positive bacteria’s
cell wall (Silva & Correia-Neves, 2012). Teleost B cells, like B-1 cells in
mammals, can swallow particles and eliminate ingested microorganisms
intracellularly (Sunyer, 2013). B lymphocytes in zebrafish, for example, can
phagocytize both soluble and particulate antigens (Zhu et al., 2014).

18.5 LYMPHOID ORGANS IN FISH

18.5.1 THYMUS

The thymus is found near the gill cavity in most teleosts and is present even
in adult fish, however its volume decreases with age or sexual development.
The thymus is the first lymphoid organ to grow and become lymphoid in many
teleost species throughout development. The thymus can be thought of as a
collection of macrophages that stimulate T cell encapsulation and proliferation
(Davis et al., 2002). The thymus is thought to play a crucial role in the
immunological response of higher vertebrates in fish. Fish have been shown to
exhibit immunological reactions that are dependent on thymus-derived cells.
T-like cells are the cells that have a role in these tasks (Chilmonczyk, 1992).
In teleosts, the differentiation of the thymic structure is exceedingly diverse,
and in many species, there is no apparent distinction between the cortex and
medulla as seen in higher vertebrates (Bowden et al., 2005).

18.5.2 KIDNEY

The teleost kidney is divided into two sections: the head kidney, which makes
up about 20% of the anterior region of the kidney, and the trunk kidney,
which makes up the remaining 80% and runs down the dorsal wall. The head
kidney is mostly made up of steroidogenic interrenal cells, chromaffin cells,
and hematopoietic tissue, whereas the trunk kidney is primarily made up of
filtering nephrons, as well as hematopoietic and pigmented cells. The head
kidney is a secondary lymphoid organ rich in monocytes and macrophages
that are involved in phagocytosis of invading pathogens and secretion of
cytokines such as the pro-inflammatory cytokines interleukin-1 and tumor
necrosis factor, thus playing a key role in the initiation of the immune
response in fish (Soulliere & Dixon, 2017).
392 Coldwater Fisheries and Aquaculture Management

18.5.3 SPLEEN

The spleen is the fundamental secondary lymphoid organ, and it is found

in almost all gnathostomes, where adaptive immune responses are formed
(Flajnik, 2018). Fish have melano-macrophages in their spleens, which can be
grouped in clusters or more loosely diffused inside the white pulp, depending
on the species. Several studies on antigen retention have suggested that such
areas function as “dumping grounds” for various types of waste material
(Agius & Roberts, 2003).

18.6 CONCLUSION

Fish make up a large part of the aquatic biodiversity. The variation in

immune system adaptability in different fish species may have resulted from
evolutionary pressure from pathogens. Immunological parameters have
equivocal functions at the individual, species, and population levels, and
the interactions between immune parameters are poorly understood. Beyond
the field of immune evolution, a better understanding of fish immune
components could be beneficial in fish farming and possibly human health.
Innate protective systems are the first line of defense against disease, and
they provide adequate disease protection in healthy populations of fish.
Moribund and dead fish emit significant amounts of germs into the water,
putting the remainder of the population in danger. As a result, the unique
defense mechanism counteracts the increased infection pressure.

KEYWORDS

• adaptive immunity
• antibody
• antimicrobial peptides
• defense system
• immune organs
• immunoglobulins
• innate immunity
• interferons
Immune Components and Defense Mechanism in Fish 393

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Defenses (Vol. 1, pp. 241–275). Science Publishers, Enfield, NH, USA.
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Soulliere, C., & Dixon, B. (2017). Immune system organs of bony fishes. Reference Module
in Life Sciences Amsterdam: Elsevier Ltd. https://doi.org/10.1016/B978-0-12 809633-8.
12179-X.
Subramanian, S., MacKinnon, S. L., & Ross, N. W., (2007). A comparative study on
innate immune parameters in the epidermal mucus of various fish species. Comparative
Biochemistry and Physiology B., 148(3), 256–263.
Sunyer, J. O., (2013). Fishing for mammalian paradigms in the teleost immune system. Nature
Immunology, 14(4), 320–326.
Thompson, S. A., Tachibana, K., Nakanishi, K., & Kubota, I., (1986). Melittin-like peptides
from the shark-repelling defense secretion of the sole Pardachirus pavoninus. Science,
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Uribe, C., Folch, H., Enríquez, R., & Moran, G. J. V. M., (2011). Innate and adaptive immunity
in teleost fish: A review. Veterinarni Medicina, 56(10), 486.
White, A., & Fletcher, T. C., (1985). The influence of hormones and inflammatory agents
on C-reactive protein, cortisol and alanine aminotransferase in the plaice (Pleuronectes
platessa L). Comp. Biochem. Physiol., 80(1), 99–104.
Wiegertjes, G. F., & Forlenza, M., (2010). Nitrosative stress during infection-induced
inflammation in fish: Lessons from a host-parasite infection model. Curr. Pharm. Des., 16,
4194–4202.
Zhu, L., Lin, A., Shao, T., Nie, L., Dong, W., Xiang, L., & Shao, J., (2014). B cells in teleost fish
act as pivotal initiating APCs in priming adaptive immunity: An evolutionary perspective
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2699–2714.
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Developmental & Comparative Immunology, 35(12), 1376–1387.
CHAPTER 19

Comprehensive Transcriptomics Analysis

of Coldwater Fish
DEEPAK AGARWAL1 and MOHD. ASHRAF RATHER2
1
Tamil Nadu Dr. J. Jayalalithaa Fisheries University, IFPGS, OMR Campus,
Chennai, Tamil Nadu, India
2
Faculty of Fisheries, Rangil-Ganderbal SKUAST, Jammu and Kashmir, India

ABSTRACT

Transcriptomics is a highly performed experimental technique recently used

in fishes to enrich the molecular data associated with several vital physiology
such as reproduction, immune system, growth, and adaption to different
environmental conditions. Transcriptomics has been transformed into a
powerful, cost-effective, and user-friendly tool with the advent of technology.
The study starts with RNA isolation which represents the complete set of
genes expressed at the point in time. Transcriptome analysis help to aid the
differential expression of important genes at various developmental stages
and time points before or after some pathogenic or immunogenic stimulation.
Transcriptomics generates a huge number of biological data which demand
computational systems and various bioinformatics tools to process, analyze,
and store the data for further deep analysis. Several applications of transcrip­
tomics have been done in freshwater aquaculture but very few in cold water
fisheries. Transcriptomics is a recent state-of-the-art technique to develop
molecular markers in huge numbers for important commercial fish species.
Transcriptomics helps in enriching the genetic resources of candidate and
novel fish species to make better fruitful breeding strategies of fishes.

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
398 Coldwater Fisheries and Aquaculture Management

19.1 INTRODUCTION

In molecular biology, transcripts expression is one of the most frequently

performed experimental techniques in identifying a cell or an organism at the
molecular level and studying physiology before and after some stimulation.
Transcriptomics is defined as the complete set of complementary mRNAs
produced in a cell or an organism. Transcript sequencing is done by
RNA-sequencing (RNA-Seq) in which the RNA from different tissues at
different time points are first isolated and then analyzed on their expression
level. The development of the RNA-Seq method allows an unprecedented
opportunity to analyze the expression of protein-coding, noncoding RNA and
also de novo transcript assembly of a new species or organism (Chatterjee
et al., 2018). Comprehensive transcriptome analysis using high throughput
sequencing techniques is believed to provide a resource for genome
annotation, candidate gene identification and molecular marker development
(Agarwal et al., 2016). Recent advancements in next-generation sequencing
technologies like high-throughput mRNA sequencing (RNA-seq) facilitate
deciphering the functional complexity of the whole transcriptome of an
organism. RNA-Seq generates a huge volume of data and accurate analysis of
this huge data is always challenging and involves different steps and several
bioinformatics tools. In the recent era, we are blessed with some advanced
technologies and supercomputers. By using these supercomputers and
advanced bioinformatics tools, the data generated from NGS platforms can
reveal several hidden and significant information regarding the physiology
and biology of the fish at the micro-level.
Temperature is a crucial environmental factor affecting the survival
and growth of aquatic organisms. Although cold water fish habitat to
low-temperature water, however, the global warming caused by the growth
of greenhouse gas emission has severely changed the climate which
ultimately exerts varying degrees of physiological stress to the fishes through
knocking the metabolic pathways closely associated with homeostasis and
physiological adaptation to the changing environment. Several endocrine
receptors have been reported which are regulated at the transcription level
in the cell and control the reproduction in fish (Sundaray et al., 2021). Such
a mechanism of adaptation brings a substantial change in the transcriptome
profile of the different organs/tissues which can be identified through RNA
sequencing. The genes associated with changing temperature and salinity
conditions have been identified in many fish including cold water species.
The transcriptome profiling provides a better understanding of the adaptation.
Comprehensive Transcriptomics Analysis of Coldwater Fish 399

Exploration of such differentially expressed genes and associated pathways

helps to understand the adaptation at the micro-level.

19.2 THE WORKFLOW OF TRANSCRIPTOMICS THROUGH

ILLUMINA SEQUENCING

The RNA sequencing starts from sample collection which is a vital step in
transcriptome analysis. Sample collection is the most significant step of any
kind of research so needs high level precautions. Samples should be stored
in RNA later and properly preserved in liquid nitrogen. To decrease the
number of samples, the tissues from different fishes of the same experiment
are pooled in equimolar concentration before the sequencing. Transcripts
represent the RNA expressed inside the cell which needs to be extracted
from the sample through the most appropriate and suitable method such as
Trizol method. The integrity of purified RNA is a significant factor to assess
the quality of RNA which is measured using Bioanalyzer. The samples that
have RIN (RNA Integrity Number) ≥8 is considered best for library prepara­
tion. RNA samples are stored at – 80°C until further use. After purifica­
tion and fragmentation, the mRNA is converted into cDNA using random
hexamers. Following the remaining steps of Transcriptomics as described in
Figure 19.1, the library is prepared and validated using the same Bioanalyzer
instrument. The library can be sequenced on any high throughput sequencer,
but for the Transcriptomics study, Illumina is the most preferred and recom­
mended platform.

19.3 BIOINFORMATICS ANALYSIS

After sequencing, a huge data is received in raw form which needs accurate
and reliable scanning. Numerous bioinformatics tools are available in the
public domain to analyze the data. Some of the tools are freely available
and some of the tools need subscription charges. In this section, the
primary tools required for transcriptome data analysis are discussed. The
raw reads generated in the FASTQ file format are checked using FastQC
software which determines the quality of reads like quality score, per base
sequence content, per base GC content, sequence length distribution and
duplicate sequences. Then raw reads are subjected to a quality filter to trim
and remove unwanted or low-quality sequences using different software
such as PRINSEQ and Kraken. The short quality reads are then assembled
400 Coldwater Fisheries and Aquaculture Management

FIGURE 19.1 Flow chart of Illumina RNA-Seq.

into a reference transcriptome (Contigs). Two approaches can be followed

depending on the availability of reference genome availability. Reference-
guided assembly if a reference genome sequence is available otherwise, de
novo RNA-Seq assembly in the absence of a reference genome sequence.
Different software and packages are available like Trinity, Velvet, and
CLC genomics workbench, TopHat-Cufflinks, etc. Once the assembly is
completed, the next task is to find out the homology to determine putative
gene descriptions of all the de novo assembled contigs. Often, local BLAST
is used to perform the homology search which provides all needed details
of the contigs such as gene id, protein id, coordinates of genes, hit numbers,
expectation (E)-value, etc. For local blast, it is best to download the database
first from the NCBI and then using the Linux operating system, BLASTX of
the query sequences is done against the local nr (non-redundant) database.
All contigs are also essential to be annotated functionally. GO (Gene
Ontology) assignments were used to classify the predicted coding sequences
based on their functions: Biological process, molecular function, and
Cellular component. Usually, Blast2GO is the most common and popular
tool for functional annotation. Several other tools have been developed for
GO analysis like GOstat, EasyGO, Gorilla, etc. For the prediction of gene
pathways, the assembled annotated contigs are also compared to the Kyoto
Comprehensive Transcriptomics Analysis of Coldwater Fish 401

Encyclopedia of Genes and Genomes (KEGG) database using the Blast2GO

program. As in transcriptome data, all the contigs are expressed genes, their
EC (Enzyme Commission) number is often given with the help of several EC
prediction tools such as E-zyme, ECOH, ECPred, etc.

19.4 TRANSCRIPTOMICS APPROACH FOR COLD-WATER FISH

Transcriptome profiling has been used to decipher the novel mechanisms

behind several physiological processes of the fishes. Although the appli­
cation of RNA-seq in fish Transcriptomics is at the nascent stage, the
results of a PubMed literature search (with keywords, “fish” and “RNA­
seq”) indicated that the number of publications in this field has increased
considerably in the last five years. The transcriptome analysis of several
cold water fish species has not been done so far. The transcriptome can
be used in a wide range of applications such as identifying a collection
of protein-encoding genes with alternative splicing and post-translation
modifications, molecular markers, generation of genetic resources and
importantly measuring differential expression of thousands of genes within
an individual at different environmental conditions or before and after the
stimulation (Chandhini & Rejish, 2019).
The most basic application of transcriptomics is to enrich genetic
resources and molecular data. Several researchers have been utilized this
technique to enhance the data availability of many cold water fishes to
understand the molecular mechanisms associated with important traits such
as growth, immune response, reproduction, and sex determination. Lau et al.
(2021) performed transcriptome analysis of Javan mahseer (Tor tambra) for
the first time and suggested the data will be useful to gain insight into tran­
scription regulation and biomarker discovery for the subsequent improve­
ment of this species for aquaculture purposes. Barat et al. (2016) generated
organ-specific transcriptome profiles of the golden mahseer (Tor putitora)
using next-generation sequencing and suggested that the database will be
helpful to understand the local adaptation, genome evolution, and also future
functional studies on the immune system of the golden mahseer. Efforts
have been made to obtain a comprehensive genome sequence for rainbow
trout. Transcriptome sequencing was done to generate comprehensive de
novo transcriptome information of rainbow trout suitable for functional and
comparative genomics studies (Saleem et al., 2010, 2015).
One of the important objectives of transcriptome analysis is the identi­
fication of genes regulated during reproductive development and spawning
402 Coldwater Fisheries and Aquaculture Management

in fish (Yang et al., 2019). Several genes and hormones are up and down-
regulated during fish maturation and spawning. These genes are essential
for inducing oocyte maturation, ovulation, and spermiation in domesticated
fishes (Agarwal et al., 2020; Chutia et al., 2021). Numerous studies have
been conducted to determine the transcriptional profile of gene programming
in GnRH or GnRHa treated fish to provide better strategies for inducing
breeding (Agarwal et al., 2020; Ahmad et al., 2108). RNA sequencing of gill
and liver tissues of wild migrating sockeye salmon (Oncorhynchus nerka)
was done to identify differentially expressed genes associated with changing
salinity, temperature, pathogen exposure and dissolved oxygen indicate
that these environmental variables most strongly impact physiology during
spawning migrations (Evans et al., 2011). A similar kind of study responding
to climate change with shifts in migration timing and habitat was done in
sympatric sister species of salmon species (McKenzie et al., 2021).
Cold water fishes can survive at low temperatures due to the adaptation
of fish to low temperatures which is the result of long-term evolution. The
tolerance to temperature determines the habitat, distribution, and migration
of fish and affects its reproduction, growth, and survival. Comparison of tran­
scriptome profiles of fishes in different tissues at different temperatures may
help to understand the physiology more precisely and will aid in deciphering
the genetic basis of ecological and environmental adaptations of fishes. In
Amur carp, cold-adaptive responses were identified through transcriptome
profiling using high-throughput sequencing technology and suggested the
strategies of gene induction during their six-month overwintering period
(Liang et al., 2015). The molecular mechanisms of temperature adaptability
of old-freshwater codfish (Lota lota lota) were studied and gene expression
at the transcriptome level was revealed to decipher the response to acute
temperature acclimation (Yang et al., 2021).
Change in seasons modulates the fish growth through the circannual
rhythms in which the expression of genes associated with breeding show
fluctuation in the brain and gonads. Danzmann et al. (2016) conducted the
muscle transcriptome analysis of rainbow trout during different seasons
unveiled that seasonal changes are correlated with a change in gene expres­
sion. They investigated the effects of a declining photoperiod regime on
white muscle compared to an increasing photoperiod regime in fast and
slow-growing rainbow trout through transcriptomics.
Recently transcriptome approaches are being widely utilized in the nutrig­
enomics field of the fisheries sector. The gene expression profile of genes
involved in metabolism aid to determine the effect of feeding on growth,
survival, and productivity. The ingredients given in diet play a major role
Comprehensive Transcriptomics Analysis of Coldwater Fish 403

in controlling physiology at the molecular level (Gora et al., 2018; Mir et

al., 2020). Nowadays, studies are being done to replace the non-active or
less available feed ingredients with another more active and easily available
feed ingredient. Callet et al. (2018) performed the whole transcriptome of
rainbow trout fry and detected the new pathways involved in the acceptance
and the utilization of a plant-based diet in isogenic lines (Quillet et al., 2007).
The whole-body transcriptome of trout alevins was done to determine the
effects of long-term feeding of fish meal (FM) and fish oil free diet to the
brooder females of rainbow trout and revealed the metabolic capacities of
progeny (Lazzarotto et al., 2016). The level of micro- and macro-nutrients
present in diet also influences fish performance and reproduction. Further
transcriptomics studies have been provided valuable information for under­
standing the molecular mechanism and potential physiological pathway of
dietary nutrients. Nazari et al. (2021), conducted the transcriptome profile
of rainbow trout fed different zinc sources for the first time. Their findings
provide molecular support to the notion and suggested that dietary zinc is
useful material in commercial diets for brood stock rainbow trout.
Transcriptome profiling has been used to understand the novel mecha­
nisms behind the immune responses of the fishes. Several studies have been
done to decipher the molecular mechanism underlining immune response
in fishes through a transcriptome approach. In one of the commercially
important cold water fish golden mahseer (Tor putitora), infected with A.
hydrophila, the transcriptome responses were recorded to understand the
immune response of the fish (Kumar et al., 2017). Several studies have been
conducted to enrich the transcriptome data and to determine the involvement
of the immune system in response to bacterial infection in rainbow trout (Ali
et al., 2014; Wang et al., 2021; Syahputra et al., 2019). The Immunogenomics
data produced through several studies can be used in clarifying the origin
and evolution of the immune systems (Kaiser et al., 2008). Transcriptome
analysis studies have also been performed to better understand the immune
response in fishes immunized with some widely used vaccines (Lim & Hong,
2021; Chinchilla et al., 2020).

19.5 CONCLUSION

Sequencing technologies have a tremendous impact on life science research

and development work. The technology has evolved a lot over the period
starting from classical first-generation Sanger’s sequencing to high throughput
and technologically advanced third and fourth generation sequencing, making
404 Coldwater Fisheries and Aquaculture Management

it better than earlier in terms of the ability of throughput, error rate, long
read lengths and of course the cost of sequencing. The availability of these
technologies has enabled a more complete understanding of whole-genome
and transcriptome in shorter time intervals with high accuracy. Transcrip­
tomics has better advantages over other gene expression approaches such as
microarray. Although, the transcriptome data have been generated for many
commercially important fishes in the case of cold water fisheries the research
progress is still in its initial stage. Very few cold water fishes have been
sequenced and the data available in the public domain need more attention
to be focused on. Several physiological activities have not been studied using
high throughput sequencing so far. As transcriptomics expands endlessly and
its cost keeps decreasing, within the next few years transcriptomics will be
exploited to a larger extent without a doubt and lead to many more exciting
discoveries in the cold water fisheries sector.

KEYWORDS

• Kyoto Encyclopedia of Genes and Genomes

• mRNA sequencing
• RNA integrity number
• RNA isolation
• RNA-sequencing
• transcriptomics

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CHAPTER 20

Predictable Threats to Coldwater

Fisheries from the Unpredictable Nature
of Climate Change: An Indian Perspective
ANKUR JAMWAL1, VIKAS PHULIA2, and SYED TALIA MUSHTAQ3
1
College of Fisheries and Center of Excellence on Water Management,
Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur,
Bihar, India
2
Krishi Vigyan Kendra, SAS Nagar, (HQ: Guru Angad Dev Veterinary and
Animal Sciences University, Ludhiana), Punjab, India
3
Fisheries Resource Management, Faculty of Fisheries, Sher-e-Kashmir
University of Agricultural Sciences and Technology, Jammu and Kashmir,
India

ABSTRACT

The coldwater fishery of India contributes to only 2% of the total fisheries of

the nation; however, it plays a vital role in food security and nutrition of people
living in the remote Himalayan and peninsular regions. Since the Himalayas
and the Peninsular Ghats of India are sensitive to the vagaries of climate
change, the fishery and aquaculture practices along these regions are also
vulnerable. The coldwater fisheries of India is susceptible to the hydrological
and rheological changes caused by receding glacial headwaters, altered
habitat range and increased severity of natural disasters. Changing climate
is also expected to favor invasive species that can replace the indigenous
fauna upon which the local populations are dependent culturally and for their
nutritional requirements. Warmer temperatures and environmental stress also

Coldwater Fisheries and Aquaculture Management: Technology for Sustainable Food Production.
Mohd Ashraf Rather, Faisal Rashid Sofi, Adnan Amin, & Kawkabul Saba (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
408 Coldwater Fisheries and Aquaculture Management

pose a risk to the fishery and aquaculture industry from increased disease
virulence. A thorough assessment of risks to the coldwater fisheries of India
from climate change is required. Policy intervention at both state and national
levels can help in the mitigation of some risks from climate change. Improved
disaster management infrastructure and better engineering interventions
in aquaculture can also help in the prevention of loss of human lives and
aquaculture infrastructure from flooding and other natural disasters.

20.1 INTRODUCTION

The world population is estimated to cross 9.7 billion by 2050 and providing
them with nutritious food will be one of the biggest challenges before
humankind (FAO, 2018). At the present rate of agricultural growth and
plateauing arable land area, it would be difficult to meet the growing demand
for nutritious food (Bommarco et al., 2013; Hunter et al., 2017). Therefore,
necessitating the development of other resources, such as meat and dairy.
Further, it is estimated that improvement in economic status of Asian and
Latin American countries will induce a shift in dietary pattern towards
inclusion of more meat and other animal products in the diet increasing the
demand for meat and dairy products in the near future (Gerbens-Leenes et
al., 2010; Lange et al., 2018). In this context, there lies an opportunity for
the fisheries sector to grow and play an important role in meeting the global
demand for nutritious food.
Currently, fisheries have outpaced all other meat-producing sectors and
is expected to remain the leading supplier of animal meat even in the future
(FAO, 2018). People all over the world have started to recognize fish as an
essential component of a healthy diet. This perception is also substantiated by
a growing body of scientific literature that suggests multiple health benefits
of fish in the human diet. For example, lower collagen content in fish meat
makes it easy to digest and thus increases its nutritional value (Listrat et al.,
2016). Fish meat is also rich in various other essential nutrients such as ω-3
fatty acids, vitamins, and minerals (Domingo, 2016). It is because of the
superior nutritional quality of fish meat, its inclusion in the human diet has
shown to help against various modern lifestyle ailments such as coronary
heart disease (CHD), diabetes, and obesity (Domingo, 2016). The demand
for fish is therefore expected to rise further as the awareness about the
health benefits of eating fish increases. Although the future holds immense
opportunities for this sunrise sector, it is not insulated from various threats
such as climate change.
Predictable Threats to Coldwater Fisheries 409

20.1.1 UNDERSTANDING CLIMATE CHANGE

Climate is the average of weather conditions prevailing over a long period

of time and all the biological processes depend upon a delicate balance of
temperature, rainfall, light, wind pattern, humidity, and many other abiotic
factors that exist during a season in a specific climatic condition. It is the
rhythmicity of the combination of abiotic factors that determine the growth
and reproductive cycle of animals and plants. For example, Indian major
carps breed primarily between June and August; when the subcontinent
experiences monsoon-like conditions. Similarly, trees blossom and bear
fruits in specific months only. Further, on a much larger and complex scale,
all biological processes also depend on each other for the flow of energy and
form a trophic hierarchy – breeding of fish mostly coincides with abundance
of phyto and zooplankton (base of the aquatic trophic chain), which in turn
depends on the availability of nutrients through upwelling, floods, and rain
in a season (rhythmicity of abiotic factors). Interestingly, and contrary to the
common belief, the climatic conditions on this planet have never remained
stationary. For example, the Earth has transitioned from colder Pleistocene
(Ice Age) to warmer Holocene epoch. Such transitions result from natural
factors and processes such as the changes in Sun’s intensity, volcanic
eruptions, change in the Earth’s orbit around the Sun, and change in ocean
current circulation. However, the change in climate due to natural factors
is slow and subtle, which allows enough time for the organisms to adapt.
In contrast, climate change induced by anthropogenic activities leading to
CO2 accumulation, aerosol emission, and deforestation resulted in a sudden
change in global climatic conditions. The adaptive processes, which is slow,
cannot occur in response to drastic and sudden changes in climate. As per an
estimate, the period after the Industrial age has resulted in increased global
temperatures and more instances of extreme weather conditions leading
to “exceptionally rapid loss of biodiversity over the last few centuries,”
indicating the sixth mass extinction event (Ceballos et al., 2015). A substantial
amount of scientific evidence suggests that the global average temperatures
will increase by 1.5°C before 2040 if serious interventions are not made to
stop it (IPCC, 2018).
The effects of climate change are manifold, and their comprehensive
discussion is beyond the scope of this review; however, some of the most
apparent effects being currently experienced are altered precipitation
patterns, increasing number of hot days, severe cyclones, extreme heatwaves,
flooding, and desertification. As far as fisheries is concerned, it is mostly
threatened by:
410 Coldwater Fisheries and Aquaculture Management

• Acidification of oceans;
• Coral bleaching;
• Reduced glacial cover;
• Loss of habitat to invasive species;
• Oceanic cyclones;
• Floods;
• Drought;
• More instance of disease and stress;
• Poor agricultural output resulting in scarcity of ingredients for fish
feed.
The above-mentioned effects of climate change, in the context of cold-
water fisheries of India, will be discussed in this review.

20.2 EFFECTS OF CLIMATE CHANGE ON COLD-WATER FISHERIES

The coldwater fishes of India live in some of the most vulnerable habitats
on this planet – the Himalayas and the Nilgiris. Both these ecosystems are
threatened by: (i) changes on account of natural causes; (ii) climate change
due to anthropogenic factors; and (iii) exploitation of resources to sustain
modern civilization. The recession of glaciers and altered flow of Himalayan
rivers for hydroelectricity projects or other developmental activities pose the
biggest threat to both capture and culture fisheries of coldwater species.

20.2.1 RECEDING GLACIERS AND ALTERED HABITAT RANGE

Geo-spatial studies indicate that almost all the glaciers outside the Karakoram
range in the Himalayas are shrinking at a much faster rate over the last
two decades (Bolch et al., 2012; Scherler et al., 2011). Glaciers form the
headwaters for most coldwater streams and rivers, and their recession by
excessive melting can result in floods in the short run and drought over
the extended period. Unfortunately, the effects of receding glaciers have
already become visible in India. As per the report published by NITI Aayog,
nearly 50% of springs in the Indian Himalayan Region have either dried up
or have become seasonal (NITI Aayog, 2018). In contrast, the population
dependent on these springs is increasing exponentially. Therefore, there is
extreme exploitation pressure on the cold freshwater resources for domestic
consumption, electricity generation, fishery, and industrial purpose. Since
the exploitation of water resources for domestic consumption and industrial
Predictable Threats to Coldwater Fisheries 411

applications takes precedence, the fisheries sector is left starving. Moreover,

the detrimental effects of climate change can be accentuated by altered
rheological features, habitat fragmentation from constructions, illegal mining
activities, and unsustainable tourism activities leading to reduced carrying
capacity of rivers and severe food security concerns in regions where fish
forms a part of the diet (Sarkar et al., 2012, 2015; Ziv et al., 2012). Lack of
in-depth and systematic studies on the dynamics of coldwater fish populations
of India remains a significant bottleneck in assessing the impact of climate
change and needs to be addressed with urgency.
Climate change is not only reducing glacial volume but with rising global
temperatures, the habitat of native coldwater species is also receding and
moving upwards (Shepard et al., 2016). Glacial melt in summers provides a
thermal buffer to streams and rivers from a drastic change in water tempera­
ture. However, with reduced glacial volumes and higher atmospheric varia­
tion, the rivers are experiencing high variability in temperature – resulting
in native species being outcompeted by other fish that are better suited for
changed conditions (Shepard et al., 2016). For example, mahseer and snow
trout (Schizothorax spp.), which prefer colder waters with high dissolved
oxygen content, are being outcompeted by voracious and fastidious catfish
that can survive better in warmer waters with slightly lower dissolved oxygen
content (Sarkar et al., 2015).
In addition to the fast-flowing rivers, the Himalayan states of Himachal
Pradesh, Sikkim, Uttarakhand, Arunachal Pradesh, and the union territories
of Ladakh and Jammu and Kashmir also have 4,699 lakes (see Table 20.1)
that are often ignored from the fisheries point of view (Panigrahy et al., 2012).
Scientific literature also suggests that high-altitude lakes are shrinking due
to increased evaporation and sedimentation from melting glaciers (Philip &
Mazari, 2000). Since lakes are enclosed waterbodies, with no escape routes
for fish, change in thermal regime can be disastrous for its inhabitants. The
ichthyofaunal status of most high-altitude lakes in the Indian Himalayan
Region is still unknown and is an impediment in assessing the effects of
climate change on their fish biodiversity.

20.2.2 CLIMATE CHANGE AND NATURAL DISASTERS

Recent devastation due to floods, cloud bursts and landslides in Indian

Himalayan Regions and the Western Ghats are not some isolated events that
could be explained merely by chance. In contrast, various climatological
models had already predicted increased penetration of monsoon into the
412 Coldwater Fisheries and Aquaculture Management

Himalayas with consequences such as higher incidences of floods, cloud

bursts and increased riverine discharge (Wasson et al., 2013). These events
negatively affect the fisheries industry. Flash floods and natural disasters
not only keep fishers from going out to fish, but the organized coldwater
aquaculture is also affected by such disasters. Most coldwater fishery infra­
structure, such as hatcheries and farms, are situated along the banks of rivers/
rivulets because these water bodies provide a continuous flow of water for
raceways. Therefore, flash floods or abnormal discharge in these waterbodies
can damage or wash-out the infrastructure – directly affecting the coldwater
aquaculture industry. Although the Governments (e.g., Government of
Himachal Pradesh) provide insurance coverage for the infrastructure but
the rebuilding of structures and establishment of fish stocks can take years.
Even worse, the cultured gene stock can contaminate the wild population if
escaped during such disasters.

TABLE 20.1 List of High-Altitude Lakes in the Indian Himalayan Region Classified based
on Their Area
SL. No. Class Range No of Lakes Area (ha)
1. Very large > 500 ha 12 95,499
2. Large 100–500 ha 31 4,993
3. Medium 25–100 ha 177 7,366
4. Small 10–25 ha 498 7,679
5. Very small 2.25–10 ha 1,985 8,592
6. < 2.25 ha < 2.25 ha 1,996 1,996
Total 4,699 1,26,125
Source: Reprinted from Panigrahy et al., 2012. © Space Applications Centre, ISRO, 2012.

20.2.3 LOSS OF HABITAT TO INVASIVE SPECIES

It is predicted that coldwater fish could lose about 31% of their habitat to
warm water fish species in the US because of climate change (Mohseni et
al., 2003). A similar situation is not out of the picture for India. The scientific
community agrees that warmer water temperatures, shorter winters, altered
rheological patterns, and increased demand for water can result in the
establishment of non-native species by eliminating native coldwater fish
(Rahel & Olden, 2008). Since the reservoirs will be used for the introduction
of novel fish species for aquaculture practices, they could serve as hotspots
for entry of invasive species. The introduction of silver and mirror carp in
Indian reservoirs is an example of how the introduction of exotic species can
Predictable Threats to Coldwater Fisheries 413

affect the survival of native fish species. Silver carp (Hypophthalmichthys

molitrix), after its introduction into the Gobind Sagar reservoir (Himachal
Pradesh), has formed a breeding population that has outcompeted native
mahseer by completely dominating the lower trophic food chain. Silver carp
is a coldwater fish but grows at a much faster rate and matures much earlier
(within one year) than the native fish species – thereby replacing native fish
stocks. Similarly, mirror carp, which is also a coldwater fish, was introduced
in various Himalayan lakes and reservoirs to augment fisheries. However, the
introduction of mirror carp has again proven to be destructive for native fish
species in Gobind Sagar reservoir, upland lakes of Kashmir and Kumaon,
and Loktak Lake in the northeast (Sugunan, 2000).
Increasing demand for fish can motivate various resource managers to
introduce fish species for outdoor culture in pens and cages. Warm water
fish species have better growth rates and are often preferred over native
species for such aquaculture operations. For example, the National Fisheries
Development Board (NFDB) of India recommends culture of exotic cichlids
(Tilapia) and catfish (Pangasianodon spp.) for reservoir-based aquaculture,
especially in the scenario where warmers temperature may favor such
species. Although the regulatory guidelines prohibit the culture of individual
fish species that may establish a reproducing population, accidental intro­
duction (as in the case of silver carp in Himachal) of a breeding pair can be
detrimental and difficult to contain.

20.2.4 CLIMATE CHANGE AND INCREASED DISEASE VIRULENCE

Alien species or their invasion can bring with them disease and parasites not
previously known to coldwater species–resulting in an epizootic outbreak in
native species (Marcogliese, 2001a). Increased instances of whirling disease
in North American and European salmonid species due to higher virulence of
myxosporean parasite, Myxobolus cerebralis, has been observed and linked
with climate change (Marcogliese, 2001b). Infection from Streptococcus is
observed in temperatures above 30°C; therefore, a rise in global temperatures
or increased number of warmer days in a year can result in Streptococcal
outbreaks. Similar, threats to Indian coldwater fish stocks cannot be ruled
out. Moreover, stress due to sudden changes in environmental variables is
expected to reduced disease resistance capabilities in fish, hence increasing
their vulnerability. It has been shown experimentally that finfish undergo meta­
bolic depression when reared at temperatures close to their thermal maxima.
Metabolic depression often results in suppression of disease response in fish.
414 Coldwater Fisheries and Aquaculture Management

20.3 MITIGATION AND ADAPTATION STRATEGIES

20.3.1 RISK ASSESSMENT

Climate change presents a situation of uncertainty and making decisions in

the face of this ambiguity is the biggest challenge. However, accumulation
and analysis of time-series data on the changing environmental variables,
its effects on water bodies and fish caught in the past can help in risk
assessment. Lack of baseline studies and continuous use of rivulets and
rivers for developmental projects in ecologically fragile Himalayas and
peninsular ghats puts the coldwater fisheries of India in a highly precarious
situation. Therefore, the first steps towards mitigation of risks from climate
change should begin with the initiation of cross-disciplinary risk assessment.
Development of computer-based models to predict the effects of climate
change on fish production, understanding of interactions between human
activities and fish production, and assessment of losses due to inaction
should also form part of mitigation policies.
Robust fish-disease monitoring and reporting system, based on the
principles of one-health, can go a long way in preventing loss of fish in
culture systems and natural waters.

20.3.2 CREATE A POLITICAL WILL AND ENGAGE SCIENTISTS TO

PREPARE POLICIES

Since it is apparent that climate change and its consequences are inevitable,
the governments will need to fund scientific data gathering and constitute
policies to adapt coldwater fisheries infrastructure. The policies can be reac­
tive or anticipatory (Smith & Lenhart, 1996). The reactive policies propose
remedial measures in response to the changes and effects already visible.
For example, relocation of fish hatcheries and farms constructed in sites
vulnerable to flooding and landslides. Ranching operations to replenish the
wild stocks can also be undertaken. Responsible tourism and strict policy-
based development activities to restrict the emission of greenhouse gases
should also be incorporated into the reactive policy measures. The coldwater
fisheries of India on its own has a very little carbon footprint. Hence policies
should be drafted to keep the fisheries clean in terms of fossil fuel use. The
anticipatory policies, on the other hand, include preemptive measures based
on sound scientific principles and predictions using computer-based models.
Regulation of fishing with only selective, non-destructive gears is one such
Predictable Threats to Coldwater Fisheries 415

example of anticipatory measure. Currently, various states have their laws

to regulate capture fishing activities; however, the regulations on the aqua­
culture industry are still missing, especially in the states along the Indian
Himalayan region. Inclusion of the aquaculture industry, in consultation
with stakeholders, for the development of policies towards climate change
resilient coldwater fishery should be promoted.

20.3.3 DIVERSIFICATION OF AQUACULTURE INDUSTRY

While the coldwater capture fisheries of India is primarily dependent upon

three groups of fishes, the mahseers, snow trout (Schizothorax spp.), and the
rainbow trout (Oncorhynchus mykiss), the culture of rainbow trout forms
the mainstay of coldwater aquaculture. Therefore, any detrimental effects of
climate change on the survival or wild recruitment of rainbow trout, mahseer,
and snow trout can crash the entire coldwater fishery of India. Efforts are
required to diversify the species of the profile of coldwater aquaculture in
India. The captive breeding and culture techniques of mahseer and snow
trout have already been standardized but have yet to be popularized.
Efforts for selection fish strains that are resilient to disease and sudden
change in climate should be selected and used for aquaculture and ranching
purposes. Research into the diversification of coldwater aquaculture by
introducing more fish species should also be explored. However, stringent
measures to prevent escapement of exotic fish species and their reproductive
proliferation in the wild must be formulated. Monosex culture or culture of
sterile fish can prevent the indigenous gene pool from contamination. Research
into improved techniques for fish cultured is also required. Currently, trout is
primarily cultured in the raceways system that requires a continuous flow of
water. Consequently, trout culture has a high-water requirement. Adoption of
alternate fish species or fish culture techniques, such as the recirculatory aqua­
culture system that is less water-intensive is required. Research efforts are also
required to reduce the dependency of aquaculture on FM as a protein source.
Even better, fish species that feed on lower trophic levels should be preferred.

20.3.4 DISASTER MANAGEMENT

Loss of aquaculture infrastructure built along the banks of rivers or rivulets

can be prevented by the construction of flood-control measures such as
embankments. The installation of early warning systems can also help in
416 Coldwater Fisheries and Aquaculture Management

the prevention of loss of human lives. Aquaculture techniques that are less
water-intensive or do not require a continuous water supply offer the flex­
ibility to move aquaculture infrastructure away from flood-prone regions.

20.4 CONCLUSION

Climate change is expected to affect the aquatic system the most. Altered
environmental variables are predicted to shift the ecological balance against
the native species and will favor natural disasters, disease outbreaks and
invasion of exotic fish species. The threat from climate change is particularly
imminent for coldwater fishes that are stenothermal and stenohaline.
Disasters associated with climate change are expected to destroy natural fish
breeding grounds and manmade aquaculture infrastructure. Additionally,
altered thermal regime, concomitant with an invasion of exotic species, may
result in loss of habitat range for cold water fishes. Unfortunately, there is a
severe dearth of scientific literature on the vulnerability of Indian coldwater
fisheries to climate change and there is an urgent need to address this lacuna.

KEYWORDS

• aquaculture industry
• climate change
• coldwater fisheries
• disaster management
• mitigation strategies
• natural disasters

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Index

α Ammonia concentration, 113

Amoxicillin, 95, 104
α-linolenic acid, 334, 335
Amplified fragment-length polymorphism
PCR (AFLP), 110
β
Amylase, 223, 228
β-hemolytic activity, 126 Amyotrophic lateral sclerosis, 324
Anabantidae, 119
A Anadromous
Abdominal distension, 109, 357 adaptation, 261
Achromogenes, 96 fish species, 261
Acidic carboxyl group, 56 populations, 261
Active proteomics, 374 Anatomical predilection sites, 361
Acute hemorrhagic enteritis, 109 Anchoa mitchilli, 119
Acylated homoserine lactones (AHLs), 165 Anecdotal observation, 111
Adaptive immune, 18, 390, 392 Anoplopoma fimbria, 123
responses, 387, 390, 392 Anthocyanin, 296
system, 195, 196, 384 Anthropogenic
Adhesion molecules, 165 activities, 179, 185, 186, 307, 409
Adipose fins, 261 factors, 410
Advanced environmental legislation, 190 Antibacterial substance, 299
Aerobic Antibiotic, 92, 98, 99, 104, 105, 115, 116,
bacteria, 168 118, 166
conditions, 108 production, 165
Aeromonadaceae, 96 resistance, 104
Aeromonas salmonicida, 96–99, 151 bacteria, 92
infection, 97, 98 Antibody, 126, 150, 169, 358, 374, 389,
Agalychnis callidryas, 119 390, 392
Agglutination, 94, 97, 114, 222, 358, 386 antigen complex, 126
Agri commodities, 148 Antifreeze proteins (AFPs), 324, 337
Agricultural industries, 280, 285 Antifungal, 295
Aldehydes, 293, 294, 297, 340 Antigen
Alfalfa, 237 presenting cells (APCs), 19, 390
Algicidal microbes, 311, 312 specific receptors, 25
Alkaloids, 222, 225, 226 Antimicrobial
Allium cepa, 299 activities, 292
Alpha-helical amphipathic peptides, 386 agent, 95, 104
Alternative feed, 219, 240 peptides (AMPs), 167, 386, 392
Amazona aestiva, 119 properties, 291, 298–300
Amblypharyngodon mola, 325 resistance, 95, 98, 147, 167
Amino acid (AA), 11, 16, 19, 51, 52, 55–59, therapy, 95, 104
76, 167, 217, 167, 221, 222, 224–227, Antinutrient profiles, 227
230, 231, 234–236, 239, 292, 293, 320, Anti-nutritional
322–325, 332, 337, 359, 376, 386, 387 compounds, 221
420 Index

effects, 223–225 Artificial sweeteners, 294

factor (ANFs), 217, 222, 226–228, 235, 240 Aseptate hyphae, 364
variables, 222 Aspartic acid, 16, 323
Anti-oxidant, 292, 294, 298, 300 Astyanax lacustris, 344
capacity, 329 Asymmetric flagellar bending, 269
characteristics, 298 Asymptomatic carriers, 94, 95, 358
chemicals, 299 Atriplex halimus, 224
properties, 295 Attention deficit-hyperactivity disorders
Antirheumatic medications, 205 (ADHD), 326
Anxiety-posttraumatic stress disorders, 203 Autoinducer-2 (AI-2), 165
Aphanomyces invadans, 364, 365 Auto-oxidative chain reaction, 298
Apoptotic cells, 387 Azocasein protease, 93
Apparent digestibility coefficient (ADC), 233 Azolla pinnata, 236
Aquaculture, 1–4, 12, 14, 15, 22, 23, 25,
35, 37, 46, 48, 51, 52, 55, 63, 72, 91, B
92, 98–100, 105, 106, 116, 147–150,
152, 162, 163, 165–170, 179, 180, 184, BAC-end transcripts, 8
186–188, 195, 202, 217–220, 224, 229, Bacillus, 95, 162, 168
licheniformis, 95
232, 234, 239, 249, 259, 260, 279, 291,
subtilis, 95
305, 319, 339, 351, 356, 365, 369, 370,
Bacterial
378, 383, 397, 401, 407, 408, 412, 413,
cell-to-cell communication, 164
415, 416
cold water disease, 92, 99
health management, 148, 149, 168
colonization, 101
industry, 147, 149, 370, 378, 408, 412,
derivatives, 162
415, 416
diseases (salmonids), 91
infrastructure, 46, 408, 415, 416
gill disease (BGD), 111–114
production, 14, 22, 51, 72, 147, 148,
kidney disease (BKD), 122–127
218–220
pathogen, 92, 102, 110, 117, 150, 151,
sector, 92, 150, 239 165, 166
systems, 52 agents, 150
Aquafarming, 260 phenotypic characteristics, 165
Aquafeeds, 14, 220, 221, 227, 229–233, Bacteriolytic
280, 285 activity, 165
Aquatic phages, 165
animal culture systems, 149 Bacteriophages, 165
biodiversity, 186, 305, 309, 311, 313, 392 Badis badis, 321
biological cultured, 11 Barbus conchonius, 252
birnavirus isolation, 360 Barilius, 321
conservation techniques, 186 bendelisis, 182, 331
ecosystem, 37, 92, 179, 180, 186, 218, vagra, 321
305, 307–309, 340 Beetroot peel extract (BPE), 298, 299
environment, 99, 147, 150, 166, 167, 184, Behning
283, 306–309, 313, 351, 352 coefficient value, 273
organisms, 4, 147, 167, 185, 187, 262, fertilizing coefficient (BFC), 273
342, 398 Benzalkonium, 118, 362
Ara zaal, 255 Benzoic acid derivatives, 296
Arachidonic acid, 62, 326, 333, 334 Beta-oxidation, 379
Arginine, 57, 59–62, 323, 325, 332 Bifidobacterium spp., 162
Arthrosclerosis, 325 Bioaugmentation, 168
Index 421

Biochemical Breeding
characteristics, 93, 94 biology, 259
properties, 117, 298 indices, 265
reaction, 376 Brevoortia tyrannus, 119
tests, 103 Brine treatment, 327
Biocorrosion, 165 Broad-spectrum anti-microbial capacity, 167
Biodiversity, 14, 180, 183, 184, 186, 189, Broodstock
190, 251, 257, 305–308, 310–313, 347, program, 264
409 screening, 106, 115
protection, 189, 190 Brycon sp., 236
Biogenic amine production, 299 Budhul, 256
Bioinformatics tools, 397–399 Butylated hydroxy
Biological anisole (BHA), 295, 298
active compounds, 221 toluene (BHT), 294, 298
diversity, 180, 374
mutagens, 340, 341 C
Bioluminescence, 165
Biomedical Cadmium, 343, 344
applications, 209 Caenorhabditis elegans, 196
sector, 196 Calcium, 73, 268, 328, 330, 331
Biomonitoring, 180, 190, 191 deficiency, 328
Bioremediation, 167 ion concentration, 267, 268, 270
nitrogen, 162 Camptothecin, 205
Biosecurity Cancer metabolism, 204
measures, 365 Candidatus, 107, 110, 111
procedures, 359 arthromitus, 107–109
Biosphere reserves, 186 Canola, 231
Biosynthetic reactions, 329 protein concentrate (CPC), 231
Biotechnological, 1, 147–149, 152, Capillary electrophoresis, 375
168–170, 259 Caramelization, 228
approach, 147, 148, 168, 169 Carassius
bio-remediation approaches, 168 auratus, 344, 345
diagnostic test, 168 carassius, 182, 249, 251–254
interventions, 147 Carbohydrate, 17, 58, 221, 222, 225, 227,
tools, 147, 148 228, 230, 233, 234, 265, 279–285, 375, 378
Biotin, 63, 329 digestibility, 227
Biswasi, 183 rich
Bocourti catfish, 234 ingredients, 279, 280, 282, 283
Boehlkea fredcochui, 120 meal, 16
Bold clotting process, 328 utilization, 281–283, 285
Botia birdi, 182, 252 Carcinogenic
Brachial movements, 112 effects, 223
Brachydanio rerio, 321 tumor, 327
Brain Cardiac disorders, 325
disorders, 202 Cardiovascular
heart infusion agar, 103 disease, 202, 203, 292, 377
neurotransmitter, 324 system growth, 207
Branchiomyces species, 362 Carnobacterium, 98, 162
Branchiomycosis, 362, 363 divergens, 95
Brassica nigra plant species, 224 maltaromaticum B26, 95
422 Index

Carp head kidney neutrophils, 388 fusions, 11

Carrassius auratus, 321 mal-segregation, 342
Casitone-yeast agar, 113 rearrangements, 11
Catechin, 296 Chronic
Catla catla, 112, 325 environmental stresses, 260
Caudal infections, 93, 101, 124
hematopoietic tissue, 198 unpredictable stress, 209
peduncle, 100 wasting disease (CWD), 101
Cefotaxime, 111 Chymotrypsin, 222, 223, 228
Cell Cinnamic acid derivatives, 228, 296
atrophy, 63 Ciprofloxacin, 111
growth inhibition, 388 Circadian clock dysregulation, 204
mediated immunity, 383, 389 Circulatory plasma, 271
transplantation, 202 Cirrhinus mrigala, 236, 238, 323, 325, 343
Central nervous system (CNS), 207 fry, 236
Centromeric abnormalities, 342 Cisplatin, 205
Ceratophyllum demersum, 311 Clarias
Cereals, 228, 230, 233 batrachus, 282, 323, 325, 344
Chanos chanos, 343
gariepinus, 238, 239
Characidae, 119
fry diet, 235
Chelated metal ions, 233, 298
Classical
Chemical
bacteriological assays, 94
administration, 165
first-generation Sanger sequencing, 403
deterioration, 298
Clastogenic, 341
modification of starch, 284
effects, 341
remedial agents, 147, 162
screening, 208 Climate
therapeutics, 148, 162, 166 change, 185, 191, 308, 402, 407–416
Chemokinetic effects, 268 variability, 185
Chemotactic receptors, 269 Climatological models, 411
Chemotaxonomy, 110 Clinical manifestation, 92, 117, 360
Chemotherapeutics, 150 Cloudy lens, 63
Chitosan, 116 Clubbed gills, 63
Chloramphenicol, 118 Clustered regularly interspaced short palin­
Chlorine, 72 dromic repeat-associated nuclease Cas9
Chlorogenic acid, 296 (CRISPR-Cas9), 23, 167
Chocolate mahseer, 183 Cod Atlantic salmon, 283
Cholerae quorum-sensing autoinducer-1 Codex alimentarius, 294
(CAI-1), 165 Coenocytic, 364
Cholesterol, 326 Cold water
reducing drugs, 327 aquaculture, 41, 91, 92, 127, 217, 218,
Chromatids, 12, 342 239, 351, 365, 412, 415
Chromium, 343 fish, 37, 39, 51, 55, 76, 181, 183, 185,
Chromolaena odorata, 239 190, 320, 323, 324, 330–333, 337, 351,
leaf meal, 239 365, 401, 404, 410, 416
Chromosomal diversity, 179, 186
aberrations, 345 fisheries, 35, 38, 52, 91, 92, 169, 217,
arm number, 5 219, 397, 404
conglomerations, 12 sector, 404
Index 423

resource conservation, 190 C-reactive protein (CRP), 387

resources, 42, 148, 180, 190 Crossochielus
Colony diplochilus, 249, 251, 252, 254
blotting, 104 latius, 252
forming units (CFU), 98 Crude concentrations, 230
Columnaris disease, 112, 116 Crusiferae, 224
Commercial, 24, 169 Cryptic female choice, 265
farming, 48 Ctenopharyngodon idella, 56, 73, 74, 182,
fish 238, 249, 251, 281, 321
farming, 1 fingerlings, 238
production methods, 45 Cultured
trout industry, 100 independent approaches, 110
Community composition, 312 rainbow trout, 335
Comparative immunotherapeutic, 4 Cutaneous pigmentation, 120
Complex Cyanidin-glycoside, 296
carbohydrates, 235, 281, 283 Cyanobacterial
oligosaccharides, 163 blooms, 307, 311
Comprehensive transcriptome analysis, 398 produced toxins, 311
Conditionally EAA (CEAA), 322, 323 Cycloheximide, 125
Congenital metabolic abnormalities, 204 Cycloserine, 125
Conirostris, 182, 183 Cyclosporin A, 205
Conservation Cyprinidae, 119, 183
aquaculture tools, 188 Cyprinus carpio, 56, 73–75, 182, 185, 235,
ecosystems, 186 249, 251, 252, 254, 321, 332, 333, 343,
hatcheries, 99 345, 356
Contemporary vertebrates genomics, 6 communis, 182, 343
Continual flashing circling, 355 specularis, 182, 343
Conventional var
fishing grounds, 189 communis, 251, 252, 254, 343
methods, 308 specularis, 185, 251, 252, 254, 343
wheat preparations, 233 Cyprinids, 123
Copper, 115, 118, 236, 307, 323, 329, 332, Cytochrome c, 294
343, 344, 362 Cytochrome oxidase, 96
sulfate, 115, 118, 307, 362 Cytogenetic materials, 4
Coral bleaching, 410 Cytolysins, 100
Coregoninae, 3 Cytophaga, 99, 112, 113
Corn psychrophila, 99
oil, 231 Cytoplasmic chromatin-containing bodies,
rations, 284 341
Corneal vascularization, 63 Cytosolic enzymes, 294
Coronary heart disease (CHD), 322, 377, Cytotoxic T cells, 390
379, 408
Co-transporter mRNA, 379 D
Cotrimoxazole, 111, 118 Dal zaal, 255
Cottonseed Damming, 42, 184
meal (CSM), 224, 229, 232 Danio rerio, 120, 195–197, 202, 206, 209,
antinutrient, 224 267
products, 224 Dark skin pigmentation, 102
424 Index

Days post fertilization (dpf), 198 Disease, 2, 12, 18, 63, 91, 92, 108, 112, 115,
Defense system, 383, 392 120, 127, 147, 149–151, 164, 165, 167,
Deforestation efforts, 189 169, 184, 197, 202, 203, 206–208, 223,
Degree of 321, 323, 326, 336, 351, 352, 355, 370,
heterogeneity, 7 377, 388
oxidation, 296 eradication, 365
Dendritic cells (DCs), 390 free seed, 365
Denitrification, 168 Docosahexaenoic acid (DHA), 325, 326,
Deoxynivalenol, 106, 115 333–335, 337, 379
Deoxyribonucleic acid (DNA), 5–8, 12, 14, Dopamine, 324
20, 95, 98, 104, 110, 112, 114, 121, 151, Double
167, 266, 306, 308–311, 313, 339–341, homozygous adults, 7
343–345, 358, 372, 373, 376, 377 strand
methylation, 373 breaks (DSBs), 167
repair systems, 341 RNA genome member, 359
Desertification, 409 Doxycycline, 111, 115, 122
Development Drosophila melanogaster, 196
bacteremia, 102 Duchenne muscular dystrophy, 203
biotic resources, 181 Duckweeds, 237
riparian buffer zones, 190 Dynamite, 184
Dexamethasone, 205
Dextrin, 281–283 E
Diagnosis, 103, 104, 114, 120, 127, 147, Ecological
149, 169, 170, 355–358, 360, 363, 364 degradation, 185, 189
laboratories, 103 indicators, 185, 306
Dicentrarchus labrax, 231, 335 Economic
Dietary aquaculture feed ingredients, 219
carbohydrate, 15, 280–283, 285, 378 viability, 35, 45, 49, 251
constituents, 221 Ecotourism, 35, 43, 48, 49, 179
incorporation, 280 Edwardsiella ictaluri, 151
ingredients, 225, 229, 294 Egeria densa, 311
nutrient requirements, 55 Egg
protein associated pathogen transmission, 105
availability, 223 iodophor disinfection, 105
requirements, 55 micropyle, 266
Egtved illness, 355
supplements, 92
Egyptian spiny-tailed lizards, 119
Diet-fed mango tilapia fingerlings, 237
Eichhornia crassipes, 236
Digestibility
Eicosapentaenoic acid (EPA), 325, 333–335,
nutritional value of nutrients, 227 337
starch, 284 Electricity generation, 410
tuber starch, 284 Electrophysiological data, 229
Diploidization, 6 Embryogenesis, 197, 200, 202, 204
Dipsomaniac, 323 Employment
Diptychus, 182, 183 generation, 38, 43
Directorate of Coldwater Fisheries Research opportunities, 36, 38
(DCFR), 41, 179, 187–189 Endangered species, 186, 187, 308
Disaster management, 408, 416 Endogenous
infrastructure, 408 autolytic enzymes, 293
Index 425

digestive enzymes, 163 Erythrocytes, 63, 343

insulin synthesis, 16 Escherichia coli cells, 103
muscular enzymes, 293 Essential
proteins, 223 amino, 320
Endospore formation, 108 acids (EAA), 52, 57, 230, 321–323
SFB, 107 fatty acids (EFAs), 52, 55, 58, 336
Endosymbionts, 163 nutrients, 55, 217, 221, 408
Energy, 52, 55, 58, 107, 203, 228, 231, 233, oils (Eos), 58, 76, 292, 295
234, 256, 260, 264, 279–285, 322, 378, Estuaries resources, 36
409 Ethambutol, 122
requirement (fish), 283 Etroplus suratensis, 236
Engineering interventions, 408 Eukaryotic microbial diversity, 310
Engraulis encrasicholus, 299 European Union (EU), 24
Enteric red mouth (ERM), 92, 93, 95, 123 Eutrophication, 306, 307, 311, 313
Enterobacter sp., 106, 115 Excrement, 308
Enterobacteriaceae, 93 Exocrine pancreatic acinar tissue, 360
Enterobacterial repetitive intergenic Exogenous substances, 300
consensus sequences-PCR (ERIC-PCR), Exophthalmia, 93, 96, 102, 123, 357, 358,
110 360, 361
Enterococcus spp., 162 Exotic
Environmental fish species invasion, 416
adaptations of fishes, 402 species, 38, 183, 186, 412, 416
bio-monitoring programs, 342 Extended performance tasks (EPTs), 8
conservation, 35, 43, 305 Extensive
DNA (eDNA), 305–313 comparative genome analysis, 12
parameters, 185 systemic infection, 101
saprophytes, 118 External fertilization, 195, 202
toxicology research, 205 Extracellular antigens, 390
toxins, 339, 340, 347 Extreme heatwaves, 409
Enzymatic Extrusion, 228
autolysis, 292
Commission (EC), 401 F
deterioration, 293 Facile genome manipulation, 202
linked immunosorbent assay (ELISA), Farmed fish feed formulations, 220
94, 97, 104, 106, 110, 114, 126, 169, Fatty
357, 358, 360 acid composition, 320, 333
Eosinophilic granular cells, 116 binding receptors, 372, 379
Epidemiology, 383 biosynthetic molecular capacity, 15
Epigenetic interactions, 371 Fecal physicochemical properties, 225
Epithelial Feed, 5, 15, 45–47, 52, 55, 63, 72, 104,
cells desquamation, 93 106, 111, 113, 115, 116, 121, 162, 163,
hyperplasia, 113 217–231, 233–239, 279–285, 356, 362,
interruption, 297 370, 378, 403, 410, 415
membrane integrity, 329 containing glucosinolate ingredients, 224
Epizootic alcerative syndrome (EUS), 364, conversion ratio (FCR), 15, 235, 238, 284
365 formulation, 52, 230, 280
Erectile dysfunction, 323 processing methods, 227
Erinaceus europaeus, 119 utilization, 163, 224, 234, 239, 281
426 Index

Fennel extract (FE), 74, 299 Flood

Fertilization control measures, 415
discrepancies, 265 prone regions, 416
environment, 266 Fluid
medium, 265, 266 accumulations, 120
Fibronectin, 93, 100 filled micro-injection, 268
Ficus carica, 116 Flumequine, 118
Filamentous bacteria, 107 Fluorescent antibody test (FAT), 98, 126
Fluoroquinolones, 122
Final body weight (FBW), 274
Fluorouracil, 205
Fingerprinting, 374
Fluvial populations, 259, 261
Fish Folic acid, 63, 329, 336
associated flavobacteria, 114 Food
biodiversity, 183, 190, 411 borne bacteria, 292
breeding grounds, 186, 416 Drug Administration (FDA), 105
catch composition, 251 Formalin, 96, 106, 118, 356, 362, 365
defense system, 383–385 Forward genetic screenings, 196
diversity, 179–181, 185, 186, 190, 191 Foxn4 thymic expression, 201
farming, 2, 36, 38, 46, 49, 392 Free radical inhibitor, 291
feed formulations, 235 Freshwater, 4, 5, 7, 8, 10, 18, 22, 35–37, 52,
hematocytes, 339, 342, 343 62, 63, 96, 97, 99, 100, 112, 119, 120,
maturation, 402 148, 149, 180, 232, 237, 238, 267, 270,
meal (FM), 217, 219–221, 230–232, 234, 281, 292, 305, 308–313, 330, 332, 397,
235, 237, 238, 240, 403, 415 402, 410
nutrition, 279, 280, 282, 285, 371 ecosystem, 309, 311, 312
processing industry, 300 fish genomic, 7, 8
populations, 4
production, 35–37, 39–41, 47, 51, 57,
species, 4, 120, 270, 281
148, 218, 239, 249, 359, 370, 414
Frozen battered seafood products, 327
species nutritional requirements, 52 Fructo-oligosaccharide (FOS), 163
spermatozoa motility, 267 Fry mortality syndrome, 99
Fisheries, 1, 35, 37, 38, 42, 51, 52, 91, 147, Functional
179, 187, 188, 190, 195, 217, 218, 249, amino acids, 322, 337
256, 259, 279, 291, 305, 319, 339, 351, food ingredients, 295
369, 383, 397, 407 genomics, 2
development, 48 Fungal
resources, 41, 220, 320 development, 363
Flagella, 96, 269, 270 pathogens, 351
proteins, 94 viral diseases, 365
Flavobacterium, 99, 112–115, 151, 311 Furazolidone, 117
branchiophilum, 112–114 Furunculosis, 96, 98, 122
columnare, 112, 115, 116 transmission, 97
psychrophilum, 99–106, 112, 114, 115,
151 G
infection, 106, 115 G6pc orthologous genes, 17
species, 112 Gadus morhua, 117, 120
Flavobacteriaceae, 99 Gambusia affinis, 252
Flavonoid, 296 Gamete
molecular ring, 297 maturation, 274
Flexirubin, 103 storage, 186
Index 427

Gangloneuritis, 103 Gentamicin, 111, 205

Garra, 321 Geographic
mullya, 331 distribution, 4, 358
Gas chromatography, 375 location, 320, 330
Gastrointestinal Geophagus brasiliensis, 344
tract, 107, 359, 384 Germplasm banks, 186
tyrosinase, 223 Gillnet, 249, 250
Gelatin hydrolysis, 97 Global
Gelatinization, 284 climatic conditions, 409
starch, 284 food basket, 52, 148
Gene functional annotation, 23
banks, 186 Globalization, 377
clusters, 25 Gluconeogenic discardable AA (G-DAA),
dispersion, 10 16, 17
editing (GE), 23 Glucose, 15–17, 96, 103, 104, 281–285, 379
environment interplay, 208 6-phosphate, 379
expression, 18, 21, 166, 324, 371–373, metabolism disorders, 285
376, 378, 402, 404 Glucosinolates, 222, 224, 231
analysis, 202 Glutamic acid, 57, 323, 325, 332
fractionation, 7, 10–12 Glutathione, 323
knockout technique, 209 Glycol-conjugate precipitation, 386
mutation, 165 Glycoproteins, 163, 222, 356
nutrient interactions, 371 Gobind Sagar reservoir, 413
ontology (GO), 400 Goldfish genome, 7
Generally regarded as safe (GRAS), 296 Gomori methenamine silver (GMS), 363
Genetic Gonadal soma-derived growth factor
contributions, 266 (GSDF), 21
illnesses, 11 Gonadosomatic report (GR), 273
information (fishes), 340 Gonadotropin-releasing hormone (GnRH),
makeup (fishes), 339 271, 274, 402
manipulations, 227 Good husbandry practices, 359, 365
metabolism, 372 Gossypol, 222–224
modification, 21, 23, 166, 227 Government-recommended stocking
products, 24 density, 44
mutations, 373, 376, 378 Gram
resource, 186, 397, 401 negative bacteria, 385
vulnerabilities, 208 positive rods, 122
Genome staining (smears), 125
duplication, 9, 25, 206 Granulocyte target proteins, 19
sequencing, 2, 21, 25 Granulomatous inflammation, 120, 126
wide association study, 14 Granulosa cells, 21
Genotoxic, 339–341, 344, 345, 347 Gross
effects, 340, 344 domestic product (GDP), 36, 37
exposure, 341 examination (clinical signs), 363
mechanism, 339 Group-synchronous oogenetic development,
testing, 341, 345 21
Genotoxin, 340, 341 Growth
DNA interactions, 341 inhibiting
428 Index

bacteria (GIBs), 311, 313 Hill resource management, 48, 49

cyanobacterial strains, 311 Hipposin, 386
retardant properties, 222 Histamine, 293, 324
Guran zaal, 255 Histidine, 57, 60, 323–325, 332
Gut mucosal immune responses, 389 Histocompatibility complex, 19
Gymnocypris, 183 Histologic regionalization, 200
Histones acetylation, 373
H Histopathological (HP), 93, 109, 110, 127,
Habitat 204, 284, 355, 360
destruction-Mmodification, 185 Hitherto identified T-cell gene regulators,
fragmentation, 411 209
utilization, 186 Homologous histone genes, 10
Haplotypes exome sequencing, 11 Homopolysaccharides, 282
Hatchery Hormonal stress, 260
bred juvenile salmonids, 111 Hours post-fertilization, 198, 209
programs, 187 Human
Healthy gut flora colonization, 162 melanoma, 203
Hemagglutinins, 222 thymus regression, 200
Hematological parameters, 342, 343 Humoral antibody level, 127
Hematopathological disorders, 203 Hyacinths, 236
Hematopoiesis, 197, 198, 202, 355, 358, 391 Hydrogen peroxide (H2O2), 104, 115, 126,
Hemodynamic factors, 207 294, 388
Hemoglobin, 203, 294, 364 treatment, 115
Hemolysin, 93, 100 Hydrononenal (HNE), 294
Hemolytic activity, 225 Hydroxycinnamic acids, 296
Hemorrhages, 96, 97, 120, 355, 357, 358, Hyper activation-like-motility pattern, 268
361, 363 Hyperchlorous acid, 388
eyes, 63 Hyperglycemia, 16, 284
spots, 364 Hypersensitivity, 323
Hepatic glucose synthesis, 15 Hypertension, 322–324, 326, 327
Herbivorous fish, 281, 378 Hypophthalmichthys molitrix, 112, 182, 413
Heritable life-history traits, 184 Hypothalamic, 15, 204, 271
Heteropneustes fossilis, 323, 325 pituitary-gonadal (HPG), 271, 274
Heterotrophic bacteria, 311 Hypothtalmichthys molitrix, 321
Hexokinase enzymes, 379
High density genotype sequencing chips, 14 I
High diet fish carbohydrate, 16 Ichthyofauna, 181, 254
High pressure processing (HPP), 228, 240 Ictalurus punctatus, 232, 335
High protein corn gluten meal, 231 Illegal mining activities, 411
High quality Immune
gametes, 260 organs, 392
screening, 20 response, 20, 96, 127, 147, 150, 151, 163,
High throughput 164, 170, 200, 324, 352, 373, 384, 391,
sequencing technology, 306, 402 401, 403
technology, 377 related gene clusters, 19
transcripts, 18 Immunofluorescence
Highly unsaturated fatty acids (HUFA), 58, antibody techniques, 104
63, 334, 335 tests, 94
Index 429

Immunogenic, 18 Intrathymic cell trafficking, 209

stimulation, 397 In-vitro fertilization, 265, 266
Immunoglobulin (Ig), 18, 389, 392 Ion channel dysregulation, 207
Immunohistochemical techniques, 126 Ipomoea batatas, 235
Immunohistochemistry, 94, 104, 126 Iron, 75, 328, 330, 331, 386
Immunological reactions, 391 Isoleucine, 57, 323, 324, 332
Immunomodulation, 163 Isoniazid, 122
cytokine, 386 Isosmotic saline, 266, 268, 269
interleukin-10, 389 Isothiocyanates, 224, 296
Immunostimulant, 151, 162, 365
substances, 98 J
In vivo genetic studies, 198 Juvenile resources, 189
Incremental rediploidization mechanism, 7
Indigenous K
fauna, 407
Ketones, 293, 297
fishing gear, 257
Kidney
schizothoracid population, 254
disease, 197, 202
Indigestible hemicellulose, 279, 280
medium (KDM), 125
Inducible nitric oxide synthase (INOS), 388
related disorders, 238
Infectious
Kyoto Encyclopedia of Genes-Genomes
biflagellated zoospores, 361
(KEGG), 401
diseases, 42, 91, 92, 150, 162, 197, 202,
Kynurenine, 324
285, 351
hematopoietic necrosis (IHN), 151, 357,
L
358, 365
virus (IHNV), 151, 357–359 Labeo, 321
pancreatic necrosis (IPN), 151, 170, bata, 235
359–361, 365 dero, 182, 330, 331
virus (IPNV), 151, 359, 360 dyocheilus, 182, 330, 331
salmon anemia virus (ISAV), 151 pangusia, 182, 330, 331
Ingredients pre-processing, 227 rohita, 112, 237, 239, 284, 323, 325
Laboratory experiments, 55
Initial body weight (IBW), 273, 274
Lactobacillus, 95, 98, 162
Inland fishery resources, 36
lacti, 95
Innate immune, 100, 167, 385, 392
plantarum, 98
system, 196, 384–386
Lactococcus
Innovative production technologies, 37 garvieae, 151
In-situ conservation, 186, 191 lactis, 164
Interferons (IFNs), 386, 392 Lake trout genomic sequence, 11
Intestinal Lamellar
diseases, 197 congestion, 113
disturbance, 223 fusion, 111, 113
probiotic microorganisms, 163 Lapidopygopisis typus, 183
Intracellular Lateolabrax japonicus, 225
antioxidant system, 294 Lead compound screening, 205
signaling, 388 Lectins, 222, 386
Intraovum, 100 Lemna, 237
Intraperitoneal, 98, 115 paucicostata, 237
injection vaccination, 98 polyrhiza, 237
430 Index

Lepidopygopsis, 182, 183 Macro-nutrients, 284, 285

Lethargy, 101, 109, 112, 113, 361 Macrophage, 98, 116, 124, 387, 388, 390–392
Leucaeana respiratory burst, 388
leaf meal (LLM), 238 Macrophytes, 306
leucocephala, 238 Macroscopic lesions, 124
Leucine, 57, 323, 332 Macrotis lagotis, 119
Light microscopy, 108, 114 Maculates, 183
Linkage Magnesium, 72, 224, 236, 330, 331
approaches (LA), 14 metabolism, 224
disequilibrium (LD), 14 Mahseer population, 184
Linolenic acid, 333 Maize production, 231
Linux operating system, 400 Major histocompatibility complex, 384
Lipase, 93, 223, 378, 379 Malabsorption, 108
secretion, 93 Malachite green, 109, 118, 362, 363
Lipid, 58, 294 Male gamete performance, 270
oxidation, 228, 294–296, 298, 299 Malnutrition, 106
Lipopolysaccharide (LPS), 95, 96, 110, 162, Malonaldehyde (MDA), 294, 300
391 Mammalian T-cells, 195
Liver Management
detoxification capacity, 285 freshwater habitats, 310
hypertrophy, 284 policy, 256
Local inflammatory response, 364 practices, 92, 105, 356, 363
Long-distance trout transportation, 47 Mannan-oligosaccharides (MOS), 163
Loop mediated isothermal amplification Marine
(LAMP), 94, 114, 121 ecosystems, 308
Lordosis, 63 environment, 148
Lotic water bodies, 313 invertebrates, 6, 269
Low-cost aquaculture techniques, 180 Marker selection systems, 18
Low-molecular-mass chemicals, 374 Masoucida, 96
Luminous zebrafish embryo, 197 Mass
Lupinine, 226 mortality (brooders), 184
Lupinus spectral fingerprinting (MALDI-TOF),
luteus, 226 98, 121
sp. linnaeus, 232 spectrometry polar metabolomics, 204
Lymphocyte infiltration, 101 spectroscopy, 374, 375
Lymphoid Mastacembalus armatus, 321
progenitor, 199 Maximum height (MH), 273, 274
specification, 201 Mazirifor chelynoides, 321
tissue, 385 Meat-producing sectors, 408
Lysine decarboxylase, 97 Medicago sativa, 224, 237, 238
Lysozyme, 99, 383, 385 Melanomacrophages, 124, 126
bacteriolytic action, 385 Melatonin, 324
Menaquinones, 110
M Meningitis, 103
Macrobial population metabarcoding Mental health problems, 377
research, 312 Metabarcoding data, 312
Macrobrachium rosenbergii, 238 Metabolic
Macro-minerals, 72 capacities (progeny), 403
Index 431

disturbances, 122 Mitotic

pathways, 57, 283, 322, 376, 398 recombination, 345
rate, 264 spindle microtubules, 342
regulation, 379 Modern real-time quantitative PCR
utilisation, 285 research, 16
carbohydrates, 15 Modified Sheih medium, 113
Metabolite Molecular
fingerprinting, 374 biology, 305, 398
profiling, 375 confirmation, 97
Metabolomics, 371, 375 deconstruction, 14
Metachromatic toluidine blue, 109 induced mutagenesis, 205
Metagenomics, 13 mappings, 4
Metal scavengers, 296 markers, 15, 397, 401
Metamorphosis, 224 Mono-associations, 107
Meta-population, 262 Monoclonal antibodies, 126
Methionine, 16, 57, 230, 233, 234, 322–324, Monofilament, 250
332, 337, 373 Monogastric animals, 234
Metric tons (MT), 2, 3, 370 Monosex culture, 415
Metronidazole, 111 Monounsaturated fatty acid (MUFAs), 333,
Microbial 334
dispersion, 312 Monovalent
growth, 292, 295 oxalates, 224
invasion, 383, 384 vaccines, 95
Microbiological, 291, 295, 298, 299 Moribund fish, 357
Microcystis aeruginosa, 311 Moringa
Microeukaryotic planktonic groups, 312 leaf diet, 235
Micronuclei, 342, 347 oleifera, 234, 235
evaluation, 339 Morone
formation, 341, 342, 345 americana, 119
test (MNT), 342, 347 saxatilis, 119
Micronutrients, 63, 227, 375 Mucosal
Micropylar sperm attractant (MISA), 270, homeostasis, 389
274 immune response, 384
MicroRNA (miRNA), 7, 9, 10, 21 Multi-drug resistant bacteria, 166
genetic mutations, 7 Multifarious interaction, 150
splicing, 373 Multinucleated giant cell (MGC), 124
Microsatellites, 8 Multiple identical genomics, 9
Microscopic Multivariate linear regression models, 310
insects, 5 Murine thymus, 201
lesions, 360 Muscle contraction, 72, 328
Mineral, 52, 55, 72, 73, 76, 222, 234–237, Mutagenic
320, 328, 330, 336, 375, 408 compounds, 347
bioavailability, 223 damages, 343
phytic acid, 223 Mycobacteriaceae, 118
requirements, 51, 73 Mycobacterium, 118–120, 122
Mineralization, 168 abscessus, 118, 119, 121
Mis-segregation, 341 arupense, 119
Mitigation strategies, 416 bovis, 118
432 Index

caprae, 118 Natural

chelonae, 118–121 additive applications, 291
chesapeaki, 118, 119 antimicrobials, 292
florentinum, 119 antioxidants, 300
fortuitum, 118, 119, 121 aquatic resources, 48
gordonae, 118, 119 disasters, 407, 408, 412, 416
haemophilum, 118–121 preservative, 292, 295, 300
iranicum, 119 resource conservation, 37
marinum, 118, 119, 121, 122 water resources, 185, 250
montefiorense, 118 Naushath zaal, 255
neoaurum, 118, 119 Near-homozygous zebrafish lines, 206
peregrinum, 118, 119 Necrosis, 63, 101, 110, 116, 124, 355, 358,
pinnipedii, 118 359, 361, 363, 391
piscium, 118 Nemipterus japonicas, 325
pseudoshottsii, 118, 119 Neoliccochilush exagonolepsis, 337
salmoniphilum, 118, 120 Neolissochilus, 182, 183, 321, 325, 331, 332
saopaulense, 118 hexagonolepis, 182, 183, 321, 323, 325,
scrofulaceum, 118 331–333
senegalese, 119 Neomycin, 111, 205
shottsii, 118, 119 Nephrocalcinosis, 123
simiae, 118 Nephronophthisis, 203
stephanolepidis, 118 Nervous necrosis virus (NNV), 151, 170
tuberculosis, 118 Neuronal interconnection, 208
ulcerans, 118, 119, 121 Neuropathological toxic mechanisms, 109
Mycobacteriosis, 118, 119, 121 Neuroproteomics, 371
Mycotic granulomatosis (MG), 364 Neuroscience research, 206
Myelin sheaths maintenance, 324 Neurotransmitters, 324
Myeloperoxidase content, 164 Neutralization, 360
Myocardial infarction, 203 assay, 357
Myoglobin, 294 New chemical entities (NCE), 345, 347
Myo-inositol hexa-phosphates, 223 Next-generation sequencing, 13, 21, 398,
Myosin, 93 401
Myxobacteria, 112 Niacin, 329
Myxobolus cerebralis, 413 Nicotinamide adenine dinucleotide phos­
Myxosporean parasite, 413 phate (NADPH), 388
Nigella sativa, 116
N Nile tilapia diet, 234, 236, 237
Naphthoquinones, 110 Nitric oxide (NO), 164, 323, 340, 388
Na-PO4 co-transporter mRNA expression, Nitrification, 168
379 Nitrogen, 223, 224, 236, 292, 307, 313, 399
National Non-alcohol fast bacterium, 123
Commission on Agriculture (NCA), 184 Non-communicable diseases (NCDs), 377,
Fisheries Development Board (NFDB), 379
413 Non-conventional plant sources, 217
Research Non-digestible food ingredients, 163
Center on Cold Water Fisheries Non-homologous end joining (NHEJ), 167
(NRCCWF), 38, 56 Non-immune carbohydrate binding proteins,
Council (NRC), 56, 63, 72, 224, 230, 235 386
Index 433

Non-ionizing radiations, 340 deficiencies, 221

Non-keratinized epidermis, 384 digestibility, 15
Non-native aquatic species, 184 genomics, 371
Non-nutritious food components, 373 immunology, 284
Non-protein modulation, 15
energy, 282 nerves, 372
nitrogen (NPN), 293, 300 quality, 227, 228, 408
nutrients, 282 requirements, 14, 51, 55, 407
Non-self-reactive thymocyte, 199 security, 35, 36, 43, 52
Non-septate multinucleate hyphae, 361 status (fishes), 116
Non-soluble carbohydrate fraction, 221 value (seafood), 294
Non-specific Nutritranscriptomics, 371
immune system, 384 Nylon monofilaments, 255
immunostimulants, 92
Non-starch polysaccharides (NSPs), 225, O
233, 240 Oceanic cyclones, 410
degrading enzymes, 225 Ohnologs, 11, 12
Non-tuberculous mycobacteria, 118 Omega-3
Normal brain development, 326 fatty acids, 218, 325
Noushath zaal, 255 poly unsaturated fatty acids, 321
Novirhabdovirus genus, 355 Oncorhynchus
Nuchkul zaal, 255 gorbushca, 125
Nuclear magnetic resonance, 375 kisutch, 75, 99
Nucleoplasmic bridges, 342 masou, 112
Nucleotide mykiss, 7, 43, 74, 75, 92, 112, 117, 122,
sequence, 11, 341, 372 125, 259–261, 263, 264, 272, 285, 321,
similarity, 11 323, 325, 330, 332, 333, 337, 345, 355,
Nutrient, 7, 9, 15, 24, 52, 55, 72, 76, 99, 415
103, 117, 163, 217, 220, 221, 227, 228, irideus, 261
232, 236, 238, 239, 284, 294, 306, 307, nerka, 125, 182, 402
311, 320, 322, 324, 327, 371, 372, 376, Oncorhyncin II, 386
378 Oncorhyncin III, 386
digestibility, 24 Ontogenic gustatory system, 229
metabolism, 376 Oocyte maturation, 402
requirements, 72 Oogonial morphology, 361
retention, 228 Oomycetes, 103, 361
Nutrigenomic, 369–372, 375–379, 402 Operational taxonomic units (OTUs), 310
studies, 379 Opportunistic
technique, 379 infections, 118
Nutrimetabolomic, 371, 374 pathogens, 118
investigations, 375 Opsonize alien organisms, 385
studies, 374 Optical projection tomography, 94
testing, 374 Oreochromis
Nutriproteomics, 373 mossambicus, 343
Nutritional niloticus, 234, 236, 238, 282, 344, 345
abnormalities, 322 Organic acids, 293, 294
composition, 330 Organophosphorus (OP), 343, 347
coldwater Fishes, 319 Ornamental fishes, 320, 321
434 Index

Osmolarity, 267, 268 Phagotherapy, 165

Osteobrama belangeri, 182, 185 Phenolic-rich by-products, 292
Osteoporosis, 322, 328 Phenotypic
Ovarian fluid, 100, 265–270 characterization, 110, 113
Overall length (OAL), 254, 257, 320 characters, 265
Overfishing-overexploitation, 183 Phenylalanine, 57, 323, 332, 333, 376
Oxalates, 224 Phenylketonuria (PKU), 376, 379
Oxalic acid, 224 Phosphatidylcholine, 336
Oxidase-positive, 99, 103 Phosphoprotein, 356
Oxolinic acid, 95, 104, 117, 125 Phosphorus, 72, 162, 221, 223, 232–234,
Oxytetracycline, 95, 98, 104, 117 236, 307
Photobacterium
P damselae subsp. piscicida, 151
Pagrus phosphoreum, 293
auratus, 233 Photolytic oxidation, 294
major, 225 Photoperiod, 22, 402
Pancreatic cancer, 205 Phylogenetic
Pangasianodon spp., 413 haplotypes, 18
Pangasius bocourti, 234 relationships, 111
Pantothenic acid, 63, 329 Physicochemical characteristics, 184
Paramesotriton hongkongensis, 119 Physio-clinical signs, 284
Paraprobiotic, 164 Physiological, 4, 7, 52, 63, 113, 200, 206,
Parasites, 92, 123, 184, 386, 413 207, 229, 239, 268, 339, 340, 342, 343,
Pardaxin, 386 361, 371, 398, 401, 403, 404
Particle size reduction, 228 Phytates, 223
Pathogen Phytic acid mutants, 233
agent, 150, 168 Phytochemical components, 297
associated molecular patterns (PAMPs), Phytoconstituents, 14, 296
391 Phytoestrogens, 222
elimination, 383 Phytohemagglutinin, 222
infection, 148 Phytoplanktonic, 310
microorganisms, 149, 169 Piscidins, 386
organism, 123, 352, 386 Piscine mycobacteriosis, 119, 120
Pathogenesis, 96, 111, 124, 125, 127, 352 Pisum sativum Linnaeus, 232
infection, 96 Plankton, 279, 280, 309, 312
Pectinolytica, 96 organisms, 306, 308
Peptide antigen production, 19 population, 312
Peptidoglycan, 110, 391 Plant
Periorbital swelling, 117 antioxidants, 298
Periostatis, 103 nutrients, 239
Peripheral erythrocytes, 343 phenolic compounds, 291, 300
Peritoneum, 101, 124 polyphenols, 298
Persistent organic pollutants, 339, 340 protein, 217, 218, 220–222, 227, 229,
Petechia, 96 230, 232, 240
hemorrhages, 93, 124, 357, 360 ingredients, 220
Petechiation, 117 meals, 229
Phagocytosis, 387, 391 Pleurocidins, 386
Phagolysosomal acidification, 388 Pleuronectes platessa, 63, 387
Index 435

Poikilothermic species, 387 Proline, 323, 337

Pollution, 183, 185, 189, 256, 305–307, Prophylactic, 169
310, 313 Protease, 100
Poly aromatic hydrocarbons (PAHs), 340, inhibitors, 222, 235
347 Protected area (PA), 186, 374
Poly cyclic hydrocarbons, 339 Protein, 9, 11, 14, 16, 47, 51, 52, 55–58, 72,
Poly unsaturated fatty acids, 321, 333 76, 112, 121, 148, 151, 162, 168, 197,
Polymerase chain reaction (PCR), 13, 94, 202, 205, 217, 219–239, 269, 270, 279,
95, 98, 104, 110, 114, 121, 126, 127, 169, 280, 282, 283, 285, 292, 294, 297, 299,
356–358, 360, 362 300, 322–324, 327, 329–333, 356, 357,
Polymorphism, 13, 94, 376 370, 372–374, 378, 386, 387, 398, 400,
identification, 22 401, 415
Polyphenolic compounds, 296 calorie malnutrition, 322
Polysaccharides, 96, 112, 162 denaturation, 294
Polyunsaturated fatty acids (PUFAs), 15, 58, expression patterns, 372
62, 63, 292, 294, 300, 330, 333–335, 337 insoluble complexes, 223
Population density, 22 lipoxidation, 294
Poronotus triacanthus, 344 micro-heterogeneity, 374
Post-activation process, 267 processing, 373
Post-infection recovery, 370 sparing effect, 280, 282, 283, 285
Post-transcriptional activity, 166 Proteolytic activity, 222
Post-translation Proteome, 13, 372–374
editing, 373 Pseudimonas salmonis, 151
modifications, 401 Pseudogenes, 10
Potassium, 72, 74, 104, 224, 233, 236, 270, Pseudomonas, 116, 293
328, 330, 331, 362 fluorescens, 116
permanganate treatment, 104 Psychosis, 323
Potent feed ingredients, 222 Psychotropic gram-negative bacteria, 293
Potential Ptychobarbus, 182, 183
molecular tool, 166 Pulsed field gel electrophoresis (PFGE), 110
pathogenic microbes, 384 Puntius
Pouchkul zaal, 255 altus, 344
Povidone-iodine, 99, 100, 105 conchonius, 249, 251, 252, 321
Prebiotic compounds, 163 gelius, 321
Pre-eclampsia, 323 sophore, 321, 325
Pre-fertilization motility estimation, 265 Purified maize gluten protein, 231
Pre-gelatinization, 284 Putrescine, 293
Premenstrual dysphoric disorder, 324 Pyknosis, 101
Pre-water hardening phase, 114 Pyrazinamide, 122
Principal component analysis (PCA), 310, Pyridoxine, 329
313
Probiotic, 92, 95, 162 Q
application, 162 Quantitative
treatment, 106 nutritional requirements, 55
Profuse cutaneous mucus, 120 proteomics, 374
Proliferation trait
epithelium, 384 loci (QTLs), 13, 14, 23
microflora, 163 nucleotides (QTNs), 23
436 Index

Quaternary ammonium baths, 104 wetlands stocking augmentation program,

Quercetin, 296 187
Quinine, 205 Resource seasonality, 181
Quinolones, 118 Respiratory disease, 377
Quorum sensing, 164, 165 Retinoblastoma, 205
Reverse
R transcriptase-sequencing, 110
Radiation side effects, 323 transcription-multiplex PCR (RT-MPCR),
Raiamas bola, 182, 321 98
Rainbow, 1–5, 7, 9, 14, 15, 17, 20–22, 46, Rhabdoviridae, 355–357
56, 58–62, 73–76, 102, 107, 112, 125, Rhacophorus arboreus, 119
259, 260, 263 Riboflavin, 329
genomic approaches, 7 Rifabutin, 122
trout, 2, 4, 6–18, 20, 21, 25, 43, 45, 47, RNA integrity number, 404
92–95, 97–100, 102, 106, 112, 115, RNA interference (RNAi), 166
117, 122, 127, 225, 226, 229, 232, 233, silencing, 166
259, 261–265, 267, 269–274, 281, technology, 166
283–285, 299, 321, 332–334, 355, 359, RNA isolation, 397, 404
361, 401–403, 415 RNA polymerase, 356
entire genome, 9 RNA sequencing (RNA-Seq), 398–400,
fry mortality syndrome, 99 402, 404
fry syndrome, 112 Robertsonian reconfigurations, 5
gastroenteritis (RTGE), 107–111, 127 Robust fish-disease monitoring, 414
genome, 1, 10–13, 17, 18, 20, 21 Routine culture identification, 97
Rana pipiens, 119
Rapeseed oilcake, 223, 224 S
Raphanus satavis, 299 Salmo
Rapid embryonic development, 195, 202 gairdnerri, 335
Rastrelliger kanagurta, 323, 325 salar, 73–75, 117, 122, 125
Reactive trutta, 43, 122, 309, 321, 331
hypoglycemia phenotypic, 17 caspius, 265
oxygen species (ROS), 344, 387, 388 f. fario, 265
Red spot disease (RSD), 364 fario, 43, 321
Rediploidization, 5, 9, 11, 12, 20 Salmon, 75, 98, 334, 336, 387, 389
Reduced glacial cover, 410 Salmonicida, 96–99, 151
Refurbishment, 3 Salmonid, 2, 3, 6, 7, 102, 117, 229, 266
Renal hemodynamics, 322 complete genome duplication, 3
Renibacterium salmoninarum, 122–127, 151 families, 2
Repetitive fish, 99, 102, 122, 123
element primed PCR (rep-PCR), 110 life-history adaptation, 261
extragenic palindromic-PCR (REP-PCR), species, 1, 2, 125, 229, 413
97, 110 Salmonidae, 3, 10, 37, 92, 180, 264, 359
Reproduction, 20, 21, 108, 197, 198, 200, Salvelinus
252, 260, 262, 264, 270, 272–274, 323, fontinalis, 122
397, 398, 401–403 namaycush, 267
organs, 97 Sander vitreus, 112, 116
Reservoir Sanguina sanguine, 331
aquaculture, 413 Saponins, 225
Index 437

Saprolegnia, 361, 362 fluids, 358

deterrent, 362 hormone-binding globulins (SHBG), 20
Saprolegniasis, 361, 362 mature, 184
Saturated fatty acids (SFAs), 58, 333–335 salmonids, 105
Scavenging species, 298 trout, 100
Schizophrenia, 325 steroids, 20, 209
Schizophyllan, 162 Shaitan zaal, 255
Schizopygopsis, 183 Shelf-life, 292, 298–300
Schizothoracids, 253, 254 Shewanella, 293
Schizothoracinae, 252 Short-chain fatty acids (SCFA), 163, 164, 170
Schizothoraichthys, 182, 183 Shrimp culture practices, 151
hugelli, 183
Shum, 256
longipinnias, 183
Siderophores, 165
micropogon, 182, 183
Silurus glanis, 112
nasus, 183
niger, 182, 183, 253, 254, 331, 333 Single duplicated haplotype, 8
planifrons, 183 Sister-chromatid exchange, 345
progastus, 182, 183, 331 Site-specific equipment, 356
Schizothorax, 56, 182, 183, 185, 249, Situation-specific habitat restoration
251–253, 257, 321, 330, 331, 337, 411, programs, 189
415 Slow-growing mycobacteriums, 121
curvifrons, 182, 183, 249, 251–253, 331, Smithia, 96
332 Socio-economic
esocinus, 183, 249, 251–253, 331, 332 development, 36, 49
labiatus, 182, 183, 252, 332 organism, 6
niger, 249, 251–253, 257 Sodium, 72, 74, 105, 111, 117, 224, 325,
richardsonii, 56, 182, 183, 330, 333, 337 327, 328, 330, 331, 356
kumaonensis, 182, 183 consumption, 327
Scoliosis, 63 hydroxide, 356
Seafoods, 322, 328, 329 hypochlorite, 356
Seaweed, 279, 280, 291 tripolyphosphate, 327
Secondary Solexa-Illumina sequences, 8
lamellae, 93 Somatic features, 265
lymphoid organ, 391, 392 Soy
metabolite formation, 297 protein isolate (SPI), 230
Secretory systems, 96
bean, 222, 223, 225, 230, 235, 280, 296
Sedimentation, 185, 189, 411
bean meal (SBM), 229, 230, 232
Segmented filamentous bacteria (SFB),
containing fish fed diets, 232
107–109, 111, 127
bean protein concentrate (SPC), 229, 230
Selenium, 329–331
Semiplotus semiplotus, 182, 331 Sparteine, 226
Sensory methods, 229 Sparus aurata, 119, 231
Sequencing technologies, 403 Spawning process, 270
Serine, 57, 325, 332 Specific
Seriola quinqueradiata, 63, 119 spoilage organisms (SSOs), 293, 300
Serratia, 93 vitamins, 55
Serum free-iron levels, 386 Spectrophotometric methods, 375
Sex Spermatozoa, 265–270
determination, 206, 401 chemotaxis, 269
development, 391 non-symmetrical flagellar wave, 267
438 Index

performance, 265, 266 Sweet potato tubers, 230

rainbow trout, 268, 269 Synaptic gonadotropin-producing cells, 271
Sphingomonas, 311 Synbiotics application, 163
Spinal compressions, 101 Synthetic additives, 295, 300
Spirodela, 237 Systemic infection, 101
polyrrhiza, 237
Sport T
fishery development, 48
fishing, 47, 48, 179, 184 Tor
Sporulation, 165 khudree, 56, 298
Spring viremia of carp virus (SVC), 151, mossal, 321
356, 357 putitora, 55, 56, 75, 182, 323–325,
susceptible species-specific equipment, 357 330–333, 337, 401, 403
Squalene, 327 tor, 182, 321, 331
Ss4R double identical domains, 10 Tannins, 223
Starch Target
dextrinization, 284 bacterial cell membranes, 167
gelatinization, 228 selection, 205
Stenohaline, 416 Tateurdina ocellicauda, 120
Stenothermal, 416 Taurine, 325
Stock Taxonomic groups, 310, 312
density, 113, 150, 359, 365 T-cell
diversification, 48 development, 196–198, 200, 209
Stolephorus receptor (TCR), 18, 19, 198, 209, 384
commersoni, 324, 325 T-dependent antigens, 390
waitei, 323–325 Tea polyphenols, 298
Stoliczkae, 183 Teleosts, 9, 196, 200, 201, 206, 387, 389, 391
Stratum Terpenoids, 296
compactum, 359 Terrestrial plant proteins, 217
granulosum, 359 Tertiary butyl hydroxy quinone (TBHQ), 294
Straw-colored mucoid material, 109 Testing chemical compounds, 202
Stream condition inventory (SCI), 262 Thapthat zaal, 255
Streptococcal, 151, 413
Therapeutic compound screenings, 209
iniae, 151
Thermal maxima, 413
outbreaks, 413
Thermos-treated ovarian fluid, 268
Streptomyces spp., 162
Streptomycin, 111, 115, 118, 122 Thin layer chromatography, 375
Subcutaneous hemorrhages, 93 Threonine, 57, 230, 323–325, 332
Subfunctionalization, 18 Thunnus albacares, 325
Subgenomes, 2, 7 Thymallinae, 3
Substratum, 37, 179, 181, 306, 320, 361 Thymic
Sulfadiazine, 95, 98 compartmentalization, 201
Sun-drying, 235 development, 196, 201, 209
Survey species communities, 305 epithelial cells, 200
Sustainability, 47, 187, 225, 239, 240 primordium, 200
aquaculture Thymocytes, 196, 198–200
feed alternatives, 219 expansion, 200
production, 147 Thymus, 200
production implementation, 48 differentiation, 209
utilization, 43 organogenesis, 197
Index 439

Tilapia Trout
mossambica, 236 by-product hydrolysates, 115
zilli, 235 farming, 45
Tissue production, 45
injury prevention, 323 Trypanoplasma borreli, 388
samples homogenization, 103 Tryplophysa spp. , 249, 251
Titanium dioxide, 344 Trypsin, 222, 223, 228
Toll-like receptors (TLR), 388 Tryptamine, 324
TopHat-cufflinks, 400 Tryptic soy agar, 103
Topoisomerases, 341 Tryptone-yeast extract-salts agar, 103, 104
Tryptophan, 57, 62, 234, 323–325, 332
Topological mapping knowledge, 9
Tumor
Total
aggressiveness, 204
length (TL), 273
suppressor genes (TSGs), 204
nitrogen (TN), 310
Turbidity, 37, 113
phosphorus (TP), 223, 310 Typus, 182, 183
Toxic intracellular granules, 388 Tyrosine, 103, 199, 230, 323, 332, 337
Toxicants, 340, 341, 347
Toxicity, 7, 63, 202, 205, 307, 329, 340, U
342, 344, 345
Traditional Ubiquinones, 110
biological approaches, 308 Ulcerative mycosis (UM), 364
ingredients, 238 Underwater free-floating fern, 236
sampling technique, 310 Unsustainable tourism, 411
Transcription activator-like effector nucle­
ases (TALENs), 167 V
Transcriptome Vacuolar degeneration, 101
analysis studies, 403 Vasodilation, 388
data analysis, 399 Ventricular hypertrophy, 207
information, 9, 401 Vertebrate
profiles (fishes), 402 evolution, 6
profiling, 401, 403 immunogenicity, 19
Transcriptomics, 15, 375, 397–399, Vesiculovirus, 356
401–404 Vessels thrombosis, 363
Transferrin, 386 Vibrio
anguillarum, 117, 151
Trapa japonica, 311
salmonicida, 117
Treatment, 105, 111, 122
Vinblastine, 205
starch, 281
Viral hemorrhagic septicemia (VHS), 151,
Triacylglycerol sensing mechanisms, 15 355, 356
Tribrissen, 117 virus (VHSV), 151, 355, 356
Trimethoprim, 95, 98, 111, 122 Virus
sulfamethoxazole, 122 free water supplies utilization, 356
Trimethylamine oxide (TMAO), 293 induced disease, 356
Triterpenoid glycosides, 225 neutralization (VN), 358
Triticum aestivum Linnaeus, 233 Vitamin, 51, 63, 76, 116, 162, 223, 236, 328,
Trizol method, 399 329, 373
Trophic Vitamin B complex, 63
hierarchy, 409 Vitamin B12, 236, 329, 373
status, 37 Vitamin E, 329
440 Index

Vitamins A, 63 Y
Vitellogenesis, 260
Yersinia, 92, 93, 151
W infection, 93
ruckeri, 92–95, 151
Warm-blooded creatures, 198 antibodies, 94
Water-soluble phenolic compounds, 223 Yrp1 protease, 95
Week post-fertilization (wpf), 201
Yangtze River Delta, 310
Wet-milling process, 231
Yersiniosis, 92
Wheat, 233, 234
Whole
Z
body transcriptome of trout alevins, 403
genome, 10, 25, 168 Zebrafish, 4, 18, 20, 21, 116, 120, 195–209,
duplications (WGDs), 2, 5–7, 9–12, 18–21 391
scattergun approach, 8 application, 196
sequencing, 2 embryos, 197, 202, 204, 205
Williams absolute fecundity indices model, 202
(WAFI), 273 Zinc, 75, 329–331
Wolffiella, 237 finger nucleases (ZFNs), 167
World Health Organization (WHO), 203, 294 Zooplankton, 409
population structure, 310
X Zoospores, 362, 365
Xanthophyll, 237 Zymogen granules, 360
Xenobiotic compounds, 339