SPECIAL PIJRLICATION NO.

30

TRAINING MANUAL ON
SHRIMP FARMING

COURSE MATERIAL FOR THE TRAINING CONDUCTED

DURING 11.1 2.2006 TO 15.1 2.2006

CENTRAL INSTITUTE OF BRACKTSHWATER AQUACULTURI

(INDIAN COUNCIL OF AGRlCULTURAL RESEARCTT'

-
CHETYNAJ -
 TRAINING MANUAL ON
SHRIMP FARMING

COURSE MATERIAL FOR THE TRAINING CONDUCTED

DURING 11.12.2006 TO 15.12.2006

CENTRAL INSTITUTE OF BRACMSHWATER AQUACULTURE

(INDIAN COUNC
 Training Programme on
'Shrimp farming'
LIST OF RESOURCE PERSONS

1 S.No I Name I
Address 1
1 I
I II
!
j
I
1. Dr.P.Ravichandran / Principal Scientist and Scientist - in charge (SIC),
! j Crustacean Culture Division, CIBA, Chennai-28.

/ 2. 1 Shri.M.Kathilve1 1 Principal Scientist: Crustacean Culture Division, 1

1 1' Dr.S.Kulasekara Pandian ( CIBA, Chennai-28 1
1/
! 3. 1 Principal Scientist, Crustacean Culture Division, j
i CIBA, Chennai-28 1
4.

5. I
1
Dr.S.M.Pi1lai

Dr.T.C.Santiago
Principal Scientist, Crustacean Culture Division,
CIBA, Chennai-28
Principal Scientist and SIC, Aquatic Animal Health
I
I
and Environment Division, CIBA, Chennai-28
1 Dr.S.Ahmed Ali
1
1
I
6.
I
, Principal Scientist and SIC, Nutrition, Genetics and
' Biotechnology Division, CIBA, Chennai-28
1
7. Dr.B.P.Gupta Principal Scientist, Aquatic h i m a l Health and
I Environment Division, CIBA, Chennai-28 I
1
1 8 Dr.N.Kalaimani Principal Scientist, Aquatic Animal Health and
I j Environment Division, CIBA, Chennai-28
9. Dr.C.Gopa1 Senior Scientist, , Crustacean Culture Division,

II
1
10. 1
Dr.K.K.Krishnani
CIBA, Chennai-28
Senior Scientist, Aquatic Animal Health and
'
Environment Division, CIBA, Chennai-28

11 ll.
12.
Dr.M.Muralidhar

Dr.M.Jayanthi
1 Senior Scientist, Aquatic Animal Health and
Environment Division, CIBA, Chennai-28
Senior Scientist, Crustacean Culture Division,
CIBA, Chennai-28
'i 14.
I
Dr.S.A.Alavandi Senior Scientist, Aquatic Animal Health and
Environment Division, CIBA, Chennai-28
13. Dr.C.P.Ba1asubramanium Senior Scientist, Crustacean Culture Division,
CIBA, Chennai-28
15. Dr.P.Nila Rekha Scientist (SS), Crustacean Culture Division, CIBA,
Chennai-28
Scientist (SS), Aquatic Animal Health and
Environment Division, CIBA, Chennai-28
 TRAINING MANUAL ON SHRIMP FARMING

CONTENTS

1 SI.No I Topic 1 Page

NO.
Site selection, designing and construction of shrimp farms -
1 1
P.Ravichandran and M. Jayanthi
2 Suitability of soils for brackishwater shrimp farming - 8
M.Muralidhar and R.Saraswathy
3 Pond preparation and bottom soil management - 14
M.Muralidhar and B.P.Gupta
4 Wate quality requirements - 21
K.K.!&ishnki, B.P. Gupta and S.M.Pillai
5 Water quality management - 28
K.K.Krishnani, B.P. Gupta and S.M.Pillai
6 Selection characteristics of shrimp postlarvae - 31
C. P. Balasubramanian
7 Seed transportation, acclimatization and stocking - 37
C.
- Gopal
S. Ahamed Ali
9 Feed management in shrimp aquaculture - 46
S. Ahamed Ali
10 Shrimp diseases - General aspects - 50
S. V. Alavandi and T.C. Santiago
11 Viral diseases with special reference to Indian shrimp farming- 53
T.C. Santiago, S.V. Alavandi and N. Kalaimani
12 Bacterial and fungal diseases of shrimp - 58
S.V. Alavandi
13 Methodology of shrimp disease investigation - 62
S.V. Alavandi, T.C. Santiago and N. Kalaimani
14 Molecular diagnosis in shrimp disease with special reference 65
to PCR of Indian White Spot Virus -
T.C. Santiago, S.V. ~ l a v i dand i N. Kalaimani
15 Shrimp farm management with special reference to social, 71
1 / environmental, health and food safety issues - I
S.M. Pillai and P. Nila Rekha
16 Harvest and post-harvest handling of cultured shrimps - 75
I M. ~ a t h i r v eand
i S. ~ u l a s e k a r a ~ k d i a n
 1. SITE SELECTION, DESIGNING AND CONSTRUCTION OF SHRIMP FARMS

P. Ravichandran and M. Jayanthi

1. Introduction
The success or failure of shrimp farming depends on the environmental conditions of
the location of the farms. The social and environmental impacts like soil and drinking water
salinisation and nutrient loading which are attributed to shrimp farming, mainly arise due to
improper location of the shrimp farms. A vast majority of problems affecting the shrimp
culturists as well as the environment could be avoided by proper site selection. The following
criteria are recommended for consideration during site selection.

2. Shrimp farm siting

2.1 Social and Environmental considerations
Location of shrimp farms in relation to other land uses and human habitation
assumes greater importance in view of the various social and environmental conflicts reported
due to shrimp farming. The following aspects should be kept in mind while deciding on a site
for shrimp farming.
m Mangrove forests play a very important role in coastal ecosystem. They are a source
of livelihood for the coastal population and it protects the coastal settlements. They
also act as habitat and nursery for a variety of marine organisms. Hence, destruction
of mangroves for any purpose will have far reaching social and environmental
impacts. Further mangrove areas are generally acidic in nature and are not suitable
for shrimp farming. In view of these facts, shrimp farms should not be located in
mangrove forest area.
a Similarly, shrimp farms should not be located near ecologically sensitive areas like
marine parks and sanctuaries to avoid any disturbance to the otherwise, fragile
ecosystem.
a Establishment of shrimp farms by converting productive agricultural lands and
saltpan will have social consequences since these are essential commodities for
human beings and involve the livelihood of many farmers. Use of unproductive
agricultural lands located in the tail end of the river systems could be used for
setting up of the shrimp farms, but only after getting it reclassified by the concerned
Government authorities1agencies.
LQ The nearness of shrimp farms to various other land uses may have some negative
impacts due to the seepage of water, which will increase the salinisation of land and
water resources. To avoid such salinisation impacts, buffer zones should be
provided in such areas depending on the soil conditions. Sandy andor porous soils

e;l Locating shrimp farms close to one another prevents access to the traditional users
of the water front. Hence it is advisable to leave enough space between the f m s
for free access to the water front. Smaller farms of 2-5 ha should leave a minimum
of 20 m between the farms. Larger farms of above 5 ha should design their f m in
1
 clusters of atleast 5 ha. each with free access provided between clusters.
m Shrimp farms should not be located on natural flood drains. Construction of shrimp
farms adjoining each other without any space between them will lead to flooding in
human habitations.
a Water spread area of a farm should not exceed 60 per cent of the total area of the
land. The rest of the 40 per cent could be used approximately for other purposes.
Q Wherever the intake and outfall are in the same creek, over crowding of the farms
should be avoided. The total area of shrimp farms that could be supported by a
creek depends on the water flow, tidal amplitude, water retention time, and Ievel of
intensification of culture systems. This is defined as the 'carrying capacity' of the
particular creek and can be estimated taking all these parameters into account. New
farms can be permitted only after an assessment of the carrying capacity of the
creek.
m Integrated Coastal Zone Management Plans have been prepared by all the maritime
states in the country and the states are expected to develop master plans for the
development of aquaculture farms taking all the environmental and social issues
into account.
m Remote Sensing and Geographical Information Systems are being used to delineate
the suitable sites based on the above said criteria.

2.2. Soil quality

Soil is the most important component in a culture system. The quality of soil should be
ascertained for pH, permeability, bearing capacity and heavy metal content. Soil with low pH
of below 5 and acid-sulfate soils should be avoided. Similarly soils with high concentrations
of heavy metals also should be avoided. The soil characteristics suitable for a shrimp culture
farm are
PH 7-8
Organic carbon - 1.5 - 2.5%
Calcium carbonate - >5%
Available nitrogen - 50-75 mg/100 g soil
Available phosphorus - 4 - 6 mg/100 g soil and
Electrical conductivity - > 4 mmhos/cm
Generally clayey loam soils are preferred. Sandy soils are seepage prone and will lead
to problems of salinisation of adjoining land and water resources. Further, maintenance of a
farm in sandy area needs high capital and operational costs. Hence, sandy areas should be
avoided. A best site is the one, which involves lesser capital investment for constructing filly
drainable ponds.
2.3. Water Quality
Availability of good quality water in required quantities is one of the most
important prerequisite for sustainable aquaculture. While locating the farm site, careful study
should be made on the source of water, quantity of water available during the different seasons
and the quality of water. The optimal levels of various water quality parameters required for
the best growth and survival of cultured shrimps are presented below.
 ........................................................................
Water quality parameters Optimal level
-----------------
1. Temperature (OC)
2. Transparency (cm)
3. pH
4. Dissolved oxygen (pprn)
5. Salinity (ppt)
6. Total alkalinity (ppm)
7. Dissolved P. (pprn)
8. Nitrate - N (pprn)
9. Nitrite - N (pprn)
10. Ammonia - N (pprn)
11. Cadmium (pprn)
12. Chromium (pprn)
13. Copper (pprn)
14. Lead (ppm)
15. Mercury (pprn)
16. Zinc (pprn)

2.4. Site elevation

Since drying of the pond bottom and proper water exchange form integral part
of the technology of shrimp farming, ponds that are drainable by gravity are essential for a
successful venture. Hence, the elevation of the site from the lowest low water level of the
supplying creek should be given due consideration while selecting the site. A minimum
elevation of 0.45 to 0.6 m is essential to ensure proper drainage.

2.5 Hydro-meteorological parameters

The hydro-meteorological data of the proposed area is very important to
develop the design of the farm. The most important data required are rainfall, tidal fluctuation,
wind direction and velocity, flood levels, frequency and time of occurrence of natural
calamities such as storm, cyclone, hail storm etc., Construction of farms in cyclone prone areas
should be avoided.

2.6 Infrastructure facilities

The infrastructure facilities like roads, electricity, proximity to hatcheries, ice plants,
processing plants should be considered while choosing the site for a shrimp farm since these
play very important roles in the economics of culture operations.

3. Pond design, Layout and Construction

The earthwork in the construction of the ponds, canals, levees alone comprises 35-50%
of the construction costs. If capital costs can be minimized while still maximizing productivity
and reducing the risks, the farming operation will be more profitable. A proper layout of the
farm+ significantly reduces the cost of construction and ensures the smooth and trouble free
opera&on of the various facilities and structures allowing proper management of production
.activities
 Proper designing and construction of farms are essential for their efficient
management and for promoting environmental protection. Good site selection and
incorporation of mitigative features in the design of the farm are the best ways to avoid
problems related to flood levels, storms, erosion, seepage, water intake and discharge points.
Proper planning during the construction can prevent or greatly limit the probable
environmental impacts. Since site characteristics vary greatly from place to place, a site-
specific approach to design and construction is necessary. A well experienced construction
team under the supervision of a qualified aquaculture engineer should be employed to ensure
proper construction. Earthmoving equipments like bulldozers, scrapers, hydraulic shovel etc.
can be used to execute the work expeditiously.
In areas where soil productivity is less, the top fertile soil layer should be removed and
re-laid after the construction of the ponds. Construction of sluices and supply channel should
be done carefully to avoid fbture problems in water management.
The following aspects of the design and lay-out of the farm should be given importance
to avoid major problems during culture.

3.1 Peripheral dyke

The peripheral dyke of a f m is the most important structure since it protects the farm
against flood, tidal thrust and cyclone. The structure of pond dykes depends on the load
bearing capacity of the soil and its compactability. In areas with sandy soil, impervious
materials like concrete, clay or sand bags should be used as the core of the dyke
Puddle core -
claylconcretelsandbags
pond bottom

Wherever the outer side of the dyke faces the water front, it should contain a berm and
stone pitching or a retaining wall should be constructed.

The height of the pond dyke should be atleast 1.5 m so as to retain a maximum of 1.0
m water in the pond. The height may vary depending on the highest flood level and highest
hightide level (Spring tide). A free board of 0.6 to 0.7 m is required above these levels.

The slope of the dyke may range from 1:l for clayey soil and 3:l for sandy soil. The
top width of the dyke (crest) should be large enough to hold the supply channel and also to be
used as a road around the farm.
3.2. Water intake system
The design of the supply canal mainly depends on the daily water requirement
of the farm. Depending on the soil quality, earthern or lined or concrete supply canals are
designed. In small farms of 3 to 5 ha, PVC pipelines with valves are used for the supply. The
supply inlets can be simple PVC pipes or concrete structures with suitable screens to prevent
the entry of pests and predato~s.

Water inlet (Masonry

structure) with Net
screen

The outlet is generally made up of wood or concrete with provisions for harvest
bags, strain nets and wooden shutters
For efficient and best possible water exchange, the outlet should be located
diagonally opposite to the inlet. The wooden shutters should be made of small planks so that
the draining of water either fiom surface or bottom could be effected easily. The width of the
outlet sluice may vary fiom 0.3 m to 1.0 m depending on the size of the pond and the daily rate
of water exchange. The bed of the drainage canal should be atleast 30 cm below the pond bed
level with adequate slope (1:2000) towards the main outlet. The size of the drainage canal
.depends on the maximum amount of water to be let out in a day. A bottom width of about 1 m
will be normally sufficient
3.3 Culture pond
Rectangular or square ponds are appropriate for shrimp culture. Natural aeration
through wind action could be maximised by designing the longest axis of the pond parallel to
the wind direction. But in places where the wind action is very high and there is a need to
reduce the wave action in the ponds, the ponds should be positioned with the longest axis
perpendicular to the wind direction.
The rearing pond must have a minimum depth of 1 m and a maximum of
atleast 1.5 m. The pond bottom should have a slope of 1:2000 towards the outlet with an
overall drop of 20 to 30 cm for a 1 ha pond. This will facilitate easy draining and drying of the
pond bottom.

3.4. Reservoir/Effluent Treatment pond

In areas where the source water is very turbid with suspended solids, a
reservoir pond is required to act as settlement pond. Two such reservoirs are needed for
alternate use. In places where outbreak of diseases has been noticed in the past, these reservoirs
could be used for chemical treatments. Following are some of the site specific design
characteristics essential in pond construction

i) Source water clean and clear, good water current; Intake and outfall into different
creeks:

ii) Source water turbid; good water current, intake and outfall into different creeks

Source creek Reservoir

iii) Source water turbid, low water current or intake and outfall into the same creek

Source creek and

outlet into the
same creek

Effluent treatment pond (ETP) becomes an essential part of a semi-intensive

farm, if the drainage water has to be released back into the source creek. Where there are
number of small f m s located in a cluster, there is a need to have a Common ETP to avoid
nutrient loading. Culture of molluscs and sea weeds can be taken up in ETP to act as biological
purifiers of suspended solids and dissolved nutrients, respectively.

3.5 Pond lining

Ponds constructed with good design and lining materials prevent saline water from
seeping out and contaminating freshwater reserve and adjacent agricultural lands different
methods such as clay lining, compaction, cementing are commonly used. The drainage canal
lining is most important in big farms, to prevent the water entering other ponds and infecting
the other animal health when the particular pond water is discharged out due to some disease
problems,

3.6 Aeration in brackishwater ponds

Dissolved oxygen (DO) is considered as one of the most critical water parameter in the
shrimp ponds. Aeration devices are used in shrimp farms to increase the DO level and paddle
wheel aerators are widely used due to better circulation efficiency. The average oxygen
transfer efficiency is 2.13Kg Oz/Kw-hr. Usually paddle wheel require approximately 1 KW
power for each 50 cm of paddle wheel length for a speed of 80-90 rpm and paddle depth of 10-
15 cm.
In a rectangular pond, the best position is to place the aerator in the middle of one of
the long sides of the pond.

4. Construction
A well experienced construction team under the supervision of a qualified aquaculture
engineer should be employed to ensure proper construction. Earthmoving equipments like
bulldozers, scrapers, hydraulic shovel etc. can be used to execute the work expeditiously. In
areas where soil productivity is less, the top fertile soil layer should be removed and relaid
after the construction of the ponds. Construction of sluices and supply channel should be done
carefully to avoid future problems in water management.
 M.Muralidhar and R.Saraswathy

The properties of soils should be considered in selecting a site for shrimp f m i n g .

Interactions between soil and water that influence water quality in ponds must not be ignored,
because poor soil condition in ponds can impair survival and growth of aquaculture species.
For example, acidic soils can cause low pH and total alkalinity in ponds, and unless lime is
applied, ponds may be unsuitable for aquaculture. A satisfactory pond soil is the one in which
mineralization of organic matter takes place rapidly and nutrients are absorbed, held and
released slowly over a long period.

1. TYPES OF SOILS
In India shrimp ponds are located in salt affected soils or coastal soils and hence it is
necessary to have a broad knowledge of these soils in order to understand the problems of
shrimp production in relation to the soil conditions. The total area of salt affected soils in India
is about 8 million ha, out of which 3.1 million ha are coastal saline soils including 0.5 million
ha of mangrove areas. Most of the coastal areas in India are saline much before the
development of scientific brackishwater f m i n g practices.

The soils are classified as saline if the solution extracted from a saturated soil paste has an
electrical conductivity value of 4 or more dS/m at 25oC, the amount of exchangeable sodium
less than 15% and the pH below 8.5. Saline soils usually have a surface crust of white salt,
especially in the dry season. The soluble salts that are measured and represented as electrical
conductivity consist of cations and anions. The cations are: C a t t , Mg*, Nat, K+ and anions:
C03--, HC03--, C1-, S04--.

2. SOIL PROPERTIES CONSIDERED FOR SHRIMP FARMZNG

Before initiating aquaculture operation, one should be well acquainted with the nature of
soil as it affects the shrimp production. The properties of soil selected to aquaculture are
described below.

Texture
Soil texture refers to the relative percentage of sand, silt and clay in the soil. It has direct
bearing on the productivity of the ponds. In brackishwater ponds, benthic productivity is more
important than the production of plankton. A clayey soil rich in organic matter encourages the
growth of benthic blue algae, which along with the associated microorganism form the main
food of most of the brackishwater animals.

Aquaculture ponds usually are located in mineral soils that contain less than 5% organic
carbon. In mineral soils, texture is important because light texture (sandy or loamy) facilitates
the exchange of dissolved oxygen and other substances between water and sediment. Also,
when ponds are drained and their bottoms dried between crops, light-textured soils dry quicker
and aerate better than heavier-textured soils. Clayey soils are best suited for constructing bunds
and have good water retention properties. The soils with even 5% clay are ideal for
aquaculture. Sandy clay, sandy clay loam and clay loam are some of the textured names
suitable for aquaculture.
pH
The pH gives an idea whether the soil is acidic (17) or alkaline (>7). This is one of the
most important soil quality parameters since it affects the pond condition. In general, slightly
acid to slightly alkaline soil pH is favourable for higher production. The availability of
nutrients, rate of mineralization of organic matter, bacterial activities and fixation of
phosphorus are greatly influenced by soil pH. The soil pH ranging between 6.5 and 7.5 are best
suited for brackishwater environment. Under this pH range, the availability of nitrogen,
phosphorus and potassium, sulfur, calcium and magnesium is maximum. The availability of
microelements, iron, manganese, boron, copper, chlorine and zinc is more in the acid range
than in neutral or alkaline range. Since the requirement of these microelements is small, the
quantities available at pH 6.5 to 7.5 are usually enough for brackishwater environment.

Organic matter
Soil organic matter is an important index of soil fertility. Its presence in various
proportions influences the productivity of the pond. It also helps in prevention of seepage loss,
increases arability of pond soil bottom and supplies nutrients. Organic matter helps in reducing
the turbidity of pond water and acts as antitoxicants. The microbial activity is entirely
dependent on the organic matter.

Though excess amount of readily decomposable organic matter may cause problems, but
in brackishwater ponds, a high level of organic matter is always desirable. It is possible to
name the soil for productivity based on the organic carbon content. The soil which has organic
carbon content less than 0.5% is low productive, 0.5 to 2% medium production and above 2%
high production.

Calcium carbonate
The soil with no CaC03 content will show acidic reaction. Such soil can be improved
to neutral soil pH or alkaline by the application of lime. By doing so, the harmhl actions of
certain substances like sulphides and acids can be reduced. The soil rich in CaC03 content
promotes biological productivity as it enhances the breakdown of organic substances by
bacteria creating more favourable oxygen and carbon reserves. It precipitates suspended or
soluble organic materials, decreases biochemical oxygen demand (BOD) and enhances
nitrification due to the requirement of calcium by nitrifying organisms. The productive soil
should have calcium carbonate more than 5%.

Soil salinity
Saline soils are usually barren but potentially productive soils. These soils do not support
plant growth primarily because of excessive salt in the soil solution. These saline soils
commonly occur in arid and semi-arid regions and areas near to sea. Salinity plays an
important role in the transformation of nitrogen, both native as well as added in the form of
fertilized. The available nitrogen content in water increases with the increase in salinity.
However, at higher salinity greater amount of nitrogen is held in the soil complex and
nitrification rate is slow. Rate of decomposition of organic manure is also affected under
different water salinity levels and is comparatively lower under higher water salinity levels.

The optimum range of soil characteristics suitable for shrimp farming with minimum
and maximum values are mentioned in Table 1. ,
3. SOIL LIMITATION RATINGS CONCEPT IN SHRIMP AQUACULTURE
A system of limitation ratings and restrictive features for soil properties was offered for
use in shrimp aquaculture. Ranges for classes and degree of limitation for each property were
based on literature, experience and best iudment.

Soils were placed into three classes according to their limitations for excavated ponds,
pond levels, dikes or embankments. The rate class is given in forms of limitations and
restrictive features. Only the most restrictive feature should be listed when a limitation class is
given. If the rating is slight there is no need for restrictive feature.

Excavated ponds
Interpretation of soil limitations for excavated ponds (Table 2) considered soil properties
to a depth of 150 cm.

Pond embankments, dikes and levees

Embankments, dikes and levees are raised structures of soil material constructed to
impound water. The soil material is considered as being mixed and compacted to medium
density. Soil used for these applications must resist seepage and erosion. The final material
should not cause toxic leachate to enter ponds. The ratings given in Table 3 for an in-place soil
from the surface to the depth of 100 cm, with the assumption that all soil layers will be mixed
in dozing, loading, dumping and spreading. The major properties considered are erosion,
stability and permeability.

Definition of limitation ratings

Soils should be rated in-place. Soils are rated to have a slight, moderate or severe
limitation for a particular property. A moderate or severe limitation does not mean that a soil
cannot be used for aquaculture. Developers can modify soil features, adjust plans and redesign
to compensate for many moderate and severe soil limitations. Managers can implement
management practices to overcome many severe water limitations. However, the initial cost
of pond and dyke construction and cost of maintenance must be considered when on-site soils
have a restrictive feature. Limitation ratings are for single properties; consequently, efforts to
overcome limitations are different depending on the property and local conditions. The
following are limitation-rating definitions essentially used;

(1) Slight - This rating indicates that on-site soils have properties favourable for use. No
unusual construction, design, management or maintenance will be required for the
designated use.
 -
(2) Moderate This rating indicates that on-site soils have one or more properties that will
require special attention for the designated use. This degree of limitation can be overcome
or modified by special planning, design management or maintenance.

(3) Severe - The severe rating is given when one or more properties of on-site soils are
unfavorable for the rated use. Major reclamation and modifications in design,
management or maintenance will be required for the designated use and sometimes, it may
not be economically feasible.

In our study conducted at Gopalapuram area of Nellore District, Andhra Pradesh some of
the properties of farm area such as low pH, high sand content and low organic carbon comes
under moderate rating according to the classification mentioned above i.e., these soils have one
or more properties that will require special attention for the designated use. This degree of
limitation can be overcome or modified by special planning and management such as liming,
organic manuring and additional compaction of soils. The soils may be considered suitable for
shrimp farming upon managing these moderate limitation properties.

4. ACID SULPHATE SOILS

Acid coastal sediments known as acid sulphate soils (ASS) have been identified
recently as a major cause of shrimp production problems in many countries. Acid sulphate
soils are extensive on coastal plains in the tropics but are less common in temperate regions. It
has been estimated that India has over 2 million ha of ASS that have the potential to cause
long-term production problems, if excavated.
Table 2. Soil limitation ratings for excavated ponds

Table 3. Limitation Ratings for pond embankments, dikes and levees

Property Limitation rating Restrictive feature
Slight / Moderate 1 Severe
Clay content (%) >3 5 / 18-35 1<18 Too sandy
Clayey Loamy Sandy
Depth to sulfidic or sulfbric >I00 50-100 <50 Toxicity; potential
1 material (cm) ! I I acidity
1 slope
(%) <8 i 8-15 1 >15 Siope
?hichess of organic material <15 i 15-50 Subsides; excess
1 humus; difficult to
I compact
Depth to water table (cm) >I00 50-100 <50 Wetness
Fraction >8 cm diameter (%) 4 5 25-50 >50 Large stones
Depth to bedrock (cm) >I00 50-100 <50 Depth to rock
Shrink - swell potential Low Medium Very high Shrink - swell
1 to high 1
Erodibility (K) ( <0.1 1 0.1-0.3 ( >0.3 ( Erosion
Source : Boyd, C.E. (1 994)

Acid sulphate soil is the common name given to soil and sediment containing oxidisable, or
already oxidised sulphides. The principal form of sulphides is iron pyrite (cubic FeS2) If the soil
remains in a reduced condition so that no oxidation of the sulphide occurs, the soil is referred as a
potential acid sulphate soil (PASS) or sulphudic soil. Where the sulphides are exposed to air, so
that oxidation can take place, the soil is called an actual acid sulphate soil (AASS) or sulphuric soil
(field pH of 3.8 and chloride to sulphate ratio in soil solution < 2 mg/L). Oxidising conditions
frequently overlie reducing conditions in the same profile so that AASS and PASS apply to
different parts of the same profile. As long as sediments containing pyrites are submerged and
anaerobic, they remain reduced and change little. However, if they are exposed to the air,
oxidation results and sulphuric acid is formed. Soils are termed as acid sulphate soils once
oxidation of pyrite occurs. When aerobic, acid sulphate soils will have a pH below 4.0. The pH of
acid sulphate soils often will decrease as much as 3 units upon drying. The positive test is to
measure pH before and after drying.

Indicators of ASS

a) Water chemistry as an indicator

C1-/S04-2 ratio by mass of less than four and certainly less than two is a strong indicator
of an extra source of sulphate.

b) In situ field soil pH testing

The pH probe should be inserted at 0.1 m intervals down the soil profile and the pH and
depth are to be recorded. Below the surface soil, actual AASS show pH less than 4 and
increases to mere neutral pH in the unoxidised PASS horizon.

c) Hydrogen peroxide field test

1 ml of 30% hydrogen peroxide is added to the soil and the degree of effervescence may
be used as a visual indicator of pyrite concentration and should be noted as none, weak or
vigorous.

Reclamation of acid sulphate pond soils

In general, there are five options viz., avoidance, prevention of oxidation, neutralisation
, oxidation and leaching and removal of pyritic materials for managing ASS. The
particular strategy or mix of strategies adopted will depend on the situation. ASS
formation in existing ponds can be avoided by following correct pond preparation. Excessive
12
turning over of pond bottom should be avoided since this will expose sites of potential acid
soil to become actual and problem soil. The most beneficial but expensive method of using
ASS for shrimp aquaculture is to employ plastic liners presently used in SE Asia, particularly
in Malaysia and Indonesia. However, the procedure for rapid reclamation of ponds with acid
sulphate soils for shrimp culture is as follows:

? In the early part of the dry season, dry the pond and harrow thoroughly. Fill with
brackishwater. Measure the pH of the water frequently. The pH will drop from that of
seawater to below 4. Once the pH has stabilized, drain the pond. Repeat the process until
the pH stabilizes above 5. Oflen three or more drying and filling cycles may be required.
(OR) Do not till the pond bottom and bunds have to be constructed with the soil ffom
outside.

? When the pond is being reclaimed, acid must be removed from the surrounding levees. To
achieve this, build smafll bunds along each side of levee tops to produce shallow basins.
Fill the basins with backwater. When the pond is drained for drying, also drain the small
basins on the levee tops for drying. Repeat if necessary. Finally remove the bunds and
broadcast agricultural lime (CaC03) over the tops and sides of levees at 0.5 to 1.0 kglm2.
Once the last drying refilling cycle is completed, broadcast CaC03 over the pond bottom at
500 kgiha.
 3. POM) PREPARATION AND BOTTOM SOIL MANAGEMENT DURING
SHRIMP CULTURE
M.Muralidhar and B.P.Gupta

Successful shrimp culture depends on good bottom soil condition. Some soils may have
undesirable properties like potential acid sulphate acidity, high organic matter content or
excessive porosity. On the other hand, even if the site is good, problems may still crop up by
the large quantity of inputs like feed and fertilizers, which lead to excessive phytoplankton
production, low dissolved oxygen, high ammonia, poor bottom soil condition and other
problems. Most of these problems can be avoided by proper management practices during
pond preparation and culture period.

1. POND PREPARATION
There are various aspects in the pond preparation, which should be carried out before the
pond is used for shrimp culture for the first time and before each subsequent crop. The main
objectives of pond preparation are to provide the shrimp with a clean pond base and
appropriate stable water quality. Pond preparation is generally dealt in two categories viz.,
newly constructed ponds and existing culture ponds.

1.1 Newly constructed ponds

In newly dug out ponds, the characteristics of the soil has to be understood first before
adopting the various measures to prepare the pond. Soil samples taken from different locations
of the pond are thoroughly mixed together and a representative portion has to be taken for
analysis. Soil deficiencies should be identified and treated in new ponds instead of waiting
until poor bottom soil quality develops later. For example, if soil in a new pond is acidic, it
should be limed before initiation of aquaculture. Understanding of the soil parameters helps to
decide the management strategies to be followed in terms of liming, manuring, fertilization,
water management etc.

1.2 Pond preparation after harvest

Before initiating a second crop, the pond has to be prepared for stocking the shrimp post
larvae. In this case, pond preparation is entirely different fi-om that of a newly dug-out pond.

Cleaning
During production cycle, considerable quantity of waste accumulates in the ponds
depending upon the culture practices. This waste must be removed to ensure sustained
production in the pond. Removal of waste by draining and drying of the pond bottom after the
production cycle are some of the steps to be followed for keeping pond environment clean.
Two systems are commonly used to clean the pond after a production cycle. One is to allow
the pond to dry out and then remove the waste; the other is to wash away the waste before it
dries off.

Dry method
In this method afier the final drain harvest, the pond bottom is allowed to dry and crack,
primarily to oxidize the organic components left after the previous culture. The pond bottom
14
should be dried for at least 7-10 days or until it can support a man's weight without subsiding
and the soil should crack to a depth of 25 - 50 mm. After drying, the waste can either be
removed manually or with machines. Drying and cracking of pond bottom enhances aeration
and favours microbial decomposition of soil organic matter. Soil respiration measured in a
pond bottom increased drastically during first 3 days after drying. The optimum moisture
content for drying is 20%, but it might vary among soils from different ponds. Pond drying
certainly enhances the mineralisation of organic phosphorous but mineralised phosphorus is
subjected to available for water column as well as to pond mud. It is an effective method for
elimination and control of undesirable species in the pond. This method has some advantages,
for example, the solid waste can be easily handled and transported away from the ponds.
However it needs site for dumping of the removed waste. In cases, where complete drying is
not possible, organic, biodegradable, piscicides such as Mahua oil cake (100-150 ppm) and tea
seed cake (15-20 ppm) can be used.

Wet method
In this method, after the final drain harvest, the accumulated black material on the pond
bottom is flushed in the form of thin slurry using a pump and a pressure. It is quick and more
efficient process than the dry method, reducing the period between production cycles. The
advantage of this method is that waste is removed in suspension. This method needs a settling
pond where waste is removed from the water and treated repeatedly to avoid polluting the local
environment.

Pond maintenance
The pond dike is strengthened with soil wherever it has become weak and the inner slope
of the dike is consolidated with soil. Tunnels and holes caused by burrowing organisms are to
be closed/plugged. Reconditioning of the bottom trench levelling of pond bottom, repairs of
sluice structures and sluice screens are also to be attended.

Liming
The reason for liming aquaculture ponds is to neutralize soil acidity and increase total
alkalinity and total hardness concentrations in water. This can enhance conditions for
productivity of food organisms and increase aquatic animal production. Either total alkalinity
or soil pH may be used to estimate the agricultural limestone dose. If both are available but
values are not in agreement, use the variable that gives the greatest agricultural limestone dose.
Brackishwater ponds with total alkalinity below 60 mg I-', and any pond with soil pH below 7
usually will benefit from liming.

The amount of different lime materials required to raise the pH to 7 is given in Table I.
Agricultural limestone should be spread uniformly over bottoms of empty ponds up to the top
-
of the dike and left for 10 15 days, or alternatively, it may be spread uniformly over water
surfaces. A large proportion of the lime should be spread on the feeding areas and any part of
the pond that has remained wet. Agricultural limestone will not react with dry soil, so when
applying over the bottoms of empty ponds, it should be applied while soils are still visibly
moist but dry enough to walk on. Tilling after liming can improve the reaction of agricultural
limestone with soil.
 Table-1. Amount of lime (tonsha) to raise the soil pH to 7.0.
I Quantity of lime material (tonsha)
Soil pH I Dolomite ' Agricultural lime Quick lime
6 to 6.5 5.7 to 2.8 5.5 to 2.8 4.6 to 2.3
5.5 to 6.0 8.5 to 5.7 8.3 to 5.5 6.9 to 4.6
5.0 to 5.5 11.3 to 8.5 11.1 to 8.3 9.2 to 6.9
4.5 to 5.0 14.2 to 11.3 13.9 to 11.1 11.5 to 9.2
4.0 to 4.5 17.0 to 14.2 16.6 to 13.9 13.8 to 11.5

Tilling
Tilling bottom soils can enhance drying to increase aeration and accelerate organic
matter decomposition and oxidation of reduced compounds. Soil amendments such as
agricultural limestone or burnt lime can be mixed into soil by tilling. Accumulations of organic
matter of other substances in the surface layer of soil also can be mixed with deeper soils to
reduce concentrations of the substances in the surface layer. Pond bottoms should not be tilled
when they are too wet to support tillage machinery. Ruts caused by machinery will fill with
soft sediment and be likely sites for anaerobic conditions. Ruts also interfere with draining and
increase the difficulty of drying pond bottoms. Depth of tillage usually should be 5 to 10 cm,
so mould board plows, often called turning plows, can be used to turn soil over. Tilling can be
counterproductive in ponds where heavy mechanical aeration is used. Tilling will loosen the
soil particles and aerator-induced water currents will cause severe erosion of the pond bottom.
Thus, if bottoms of heavily aerated ponds are tilled, they should be compacted with a heavy
roller before refilling.

Fertilisation
Decomposition in organic soils is slow because pH usually is low and the amount of
carbon relative to nitrogen (C:N ratio) is high. Nevertheless, because of high organic matter
content, such soil often becomes anaerobic during shrimp culture. Application of agricultural
limestone to increase pH and inorganic nitrogen fertilizers to supply nitrogen will increase soil
organic matter degradation during fallow periods between crops. Urea can be spread over pond
bottoms at 200 to 400 kg ha-' at the beginning of the fallow period to accelerate decomposition
of organic soil. Agricultural limestone should not be applied until a few days after urea is
applied to prevent a high pH. Sodium nitrate can be applied @ 20 to 40 g rn-' to wet soil to
encourage organic matter decomposition in wet areas. However, nitrate fertilizers are more
expensive and are not recommended where soils can be adequately dried.

-
The rate of application of inorganic fertilizers ranges from 25 100 kg/ha as a basal
-
dose during pond preparation with minimum water depth of 10 15 cm. When the shrimp
culture progresses, depending upon the phytoplankton density as exemplified by turbidity of
the pond water, required quantity of the fertilizers may be applied in split doses at short
intervals for sustained plankton production. The main nutrient limiting phytoplankton
production in brackishwater ponds is phosphorus. Hence both phosphorus and nitrogen should
be applied in the ratio of 1:1. Excessive application of urea and ammonium fertilizers may
cause ammonia toxicity to shrimps and also may lead to algal blooms reducing of dissolved
oxygen.
 Shrimps being bottom dwellers, benthic organisms constitute their main food items.
Hence fertilization of soil instead of water is more effective. Productivity of benthic organisms
may be low in ponds with concentrations of organic carbon below 0.5 to 1.0%. Organic
fertilizer can be applied to such soils to enhance organic matter concentration. Chicken and
other animal manures have been applied at 1,000 to 2,000 kg ha-' to pond bottoms during the
fallow period. In brackishwater conditions decomposition of cattle dung is slow and hence
application of chicken manure, if available, is advisable. The rate of chicken manure is one-
third of cattle dung. However, application of a higher quality organic matter such as plant
meals--e.g., rice bran, soybean meal, and crushed corn-or low-protein-content animal feed at
500 to 1,000 kg ha-' is more efficient. When organic fertilisation of pond bottoms is practiced,
ponds should be filled with 10 to 20 cm of water and allowed to develop a dense plankton
bloom. In shrimp farming, both organic manures and inorganic fertilizers are supplementary to
each other and one cannot be exchanged for the other. It is always better to apply both organic
and inorganic fertilizers together as a basal dose during pond preparation for optimum result.

Raising of water level

The pond is filled with brackish or seawater by pumping or by opening the sluice with
proper screens to prevent entry of unwanted organisms into the pond. The water level is
maintained to 30 - 40 cm and allowed to remain for 10 - 15 days. By this time, the colour of
water may turn dark green with algal bloom and a layer of benthic algae along with associated
food organisms will form at the bottom. Subsequently small doses of organic and inorganic
fertilizers are applied based on the observations (transparency with secchi disc 30 - 40 cm) of
algal production. The water level is then raised to 100-125 cm. Now the pond is ready for
stocking post larvae of shrimps.

2. MANAGEMENT OF POND BOTTOM DURING CULTURE

All aquaculture pond bottoms become covered with sediment, and this sediment can be
considered as aquaculture pond soil. In describing various physical, chemical and biological
processes occurring in the pond bottom, it is convenient to refer to bottom deposit as sediment.
A core taken through the sediment and extending into the original bottom soil is called a
profile. Layers in the profile are known as horizons (Fig.1). For practical purposes, the F and S
horizons are most important in aquaculture because they exchange substances with overlaying
water to influence water quality.

Oxidized Layer
The oxidized layer at the sediment surface is highly beneficial and should be
maintained throughout the shrimp culture. Metabolic products of aerobic decomposition are
carbon dioxide, water, ammonia, and other nutrients. In anaerobic sediment, some
microorganisms decompose organic matter by fermentation reactions that produce alcohols,
ketones, aldehydes, and other organic compounds as metabolites. Other anaerobic
microorganisms are able to use oxygen from nitrate, nitrite, iron and manganese oxides,
sulfate, and carbon dioxide to decompose organic matter, but they release nitrogen gas,
ammonia, ferrous iron, manganous manganese, hydrogen sulphide, and methane as
metabolites. Some of these metabolites, and especially hydrogen sulfide, nitrite, and certain
organic compounds, can enter the water and be potentially toxic to shrimp. Methane and
nitrogen gas pass through the layer and diffuse from the pond water to the atmosphere. These
two gases do not cause toxicity to aquatic organisms under normal circumstances.

Aquaculture pond soil profile

P F
Flocculent layer

d Mixed Sediment
layer
S C Reduced (anaerobic) SR
e

Mature stable
sediment
e
FJ n

11
t
0

Soil Horizons
0
I
Transitional layer

Original undisturbed pond bottom

Fig.1 Pond soil profile showing different horizons
MT

I p

F - Water with high concentrations of mineral and organic solids, aerobic

-
S Sediment with high water content and low dry bulk density, abundant organic matter, well stirred by physical
and biological agents,
-
So Thin aerobic surface (Oxidised)
-
Sg Anaerobic below (Reduced)
M - Sediment with medium water content and intermediate dry bulk density, abundant organic matter, not stirred,
anaerobic
T -
Transition between M (MT) and P (PT) horizons with characteristics intermediate between M and P
horizons, not stirred, anaerobic
P - Low water content and high bulk density, usually compacted, low organic matter, not stirred, anaerobic
The oxidized layer at the sediment surface prevents diffusion of most toxic metabolites
into pond water because they are oxidized to non-toxic forms by chemical and bioIogica1
activity while passing through the aerobic surface layer. Nitrite will be oxidized to nitrate,
ferrous iron converted to ferric iron, and hydrogen sulfide will be transformed to sulfate. Thus,
it is extremely important to maintain the oxidized layer at the sediment surface in shrimp
culture ponds.Loss of the oxidized layer can result when soils accumulate large amounts of
organic matter and dissolved oxygen is used up within the flocculent layer (F horizon) before it
can penetrate the soil surface. F and S horizons are most important that influences overlying
water quality. Even in ponds without high concentrations of organic matter in sediment, high
rates of organic matter deposition resulting from large nutrient inputs and heavy plankton
blooms can lead to oxygen depletion in the F horizon. Ponds should be managed to prevent
18
large accumulations of fresh organic matter in the F horizon at the soil surface, or in the
upper few millimeters of soil. Toxic metabolites entering well-oxygenated pond water will be
quickly oxidized. However, if the rate of release of toxic metabolites into water exceeds the
rate that metabolites that are oxidized, equilibrium levels of metabolites in the water may be
high enough to have detrimental effects on culture animals.

Nutrient exchange between soil and water

The two most important nutrients in pond aquaculture are nitrogen and phosphorus
because these two nutrients often are present in short supply and limit phytoplankton growth.
These two nutrients are added to ponds in fertilizers, manures, and feeds. Fertilizer nitrogen
usually is in the form of urea or ammonium, and urea quickly hydrolyzes to ammonium in
pond water. Ammonium may be absorbed by phytoplankton,
converted to organic nitrogen, and eventually transformed into nitrogen of shrimp protein via
the food web. Ammonium may be oxidized to nitrate by nitrifying bacteria, and nitrate may be
used by phytoplankton or denitrified by anaerobic microorganisms in the sediment. Nitrogen
gas formed by denitrification diffises fi-om sediment to pond water to the atmosphere.
Ammonium is in equilibrium with ammonia, and ammonia also can diffuse from pond waters
to the atmosphere. A small amount of ammonium may be adsorbed on cation exchange sites in
pond bottom soils. Organic nitrogen in plankton and in aquatic animal feces may settle to the
bottom to become soil organic nitrogen. Nitrogen in soil organic matter may be mineralized to
ammonia and recycled to the pond water, but the rate is slow.

Phosphorus usually is present in fertilizer as calcium or ammonium phosphate.

Phytoplankton can rapidly remove phosphate from water, and phosphorus in phytoplankton
may enter the food web culminating in shrimp. Pond soil strongly adsorbs phosphorus, and the
capacity of pond soil to adsorb phosphorus increases as a function of increasing clay content.
Most of soil phosphorus was tightly bound, and only a small amount was water soluble. Pond
soils are not a major source of phosphorus to water because soil-adsorbed phosphorus is highly
insoluble. Phosphorus released by decomposition of organic matter in pond bottoms is rapidly
adsorbed by soil and little of it enters the water. Soils that are near neutral in pH have less
capacity to adsorb phosphorus and a greater tendency to release phosphorus than do acidic or
alkaline soils Nevertheless, even neutral soils remove phosphorus from the water and are a sink
rather than a source of phosphorus. Once dissolved in the water, nitrogen and phosphorus
originating from manures and feed also will enter the same pathways as nitrogen and
phosphorus applied in chemical fertilizers.

Application of microbial products for improvement of soil quality

A number of products are promoted to enhance beneficial chemical and biological
processes and to improve soil quality. These products include cultures of living bacteria,
enzyme preparations, cornposted or fermented residues, plant extracts, and other concoctions.
There is no evidence from research that any of these products will improve soil quality.
Nevertheless, they are not harmful to the culture species, surrounding environment, workers, or
quality of aquaculture products.

Monitoring of soil parameters during culture period

 Monitoring of soil quality condition can be valuable in shrimp culture pond
management. During culture the carbonaceous matter, suspended solids, faecal matter and dead
plankton etc. also settle at the pond bottom. Major concerns in pond bottom soil management
are low soil pH, high soil organic matter, loss of the oxidized layer, and accumulation of soft
sediment. Pond managers should still strive to prevent severe soil quality problems from
developing. In older ponds with impaired soil quality, problems should be corrected and
prevented from recurring. These materials have combined effect on the environment of the
pond bottom. To understand the condition of the pond bottom, the following parameters are to
be monitored regularly;

Soil pH
This is one of the most important soil quality parameters since it affects the pond
condition. Generally, soil pH ranging between 6.5 and 7.5 is the best suited where availability
of nitrogen, phosphorus, potassium, calcium and magnesium is maximum. The micronutrient
whose requirements are very small is also available in this pH range. The low pH of bottom
sediment indicates unhygienic condition needs regular check up.

Organic matter
The change in the bottom in terms of increasing organic load should be recorded
regularly for the management of the pond bottom.

Redox-potential
Anaerobic condition can be developed in pond, when input of organic matter exceeds
the supply of oxygen needed for decomposition of organic matter. This reducing condition can
be measured as the redox potential (Eh). Redox potential indicates whether the water or soil is
in reduced condition (Eh with '-I value) or oxidized (Eh with '+' ve value) condition. Reduced
or anaerobic sediments may occur at the pond bottom of heavily stocked pond with heavy
organic load and poor water circulation. Under anaerobic condition of the pond bottom,
reduced substances such as H2S, NH3, CH4 etc. are formed which are toxic to benthic
organisms. In shrimp ponds, development of highly reducing conditions at the surface of the
pond mud is highly undesirable. Water circulation by water exchange, wind or aeration helps
to move water across mud surface and prevent the development of reduced condition. Draining
at the centre of pond, as is being practiced by some farmers, is an ideal remedy for the
prevention of formation of highly reducing condition during the last phase of culture period.
Bottoms should be smoothened and sloped to facilitate draining or organic waste and toxic
substances. The redox potential (Eh) of mud should not exceed -200 mV.
 4. WATER QUALITY REQUIREMENTS FOR SHRIMP FARMING

K.K.Krishnani, B.P. Gupta and S.M.Pillai

Shrimp farming in brackishwater ponds is an economically attractive and a rapidly

growing industry in many tropical nations. Shrimp farming promised quick and high returns
with little investment. However, production of shrimp in ponds is often limited by the water
quality degradation. Disease outbreak in shrimp culture system is related to the environment
factors such as deterioration of water quality and sedimentation. The water management and
control of water quality are therefore key factors for regulating success in brackishwater
shrimp culture. Basic aspects that can lead to serious problems are

S.NO. FACTORS
1. Lack of proper monitoring of water characteristics
2. Excess of phytoplankton growth.
3. Use of chlorine in grow out ponds.
4. Excessive Liming
5. High alkalinity/excessive phosphorous
6. High suspended solids/High organic load.
7. High toxic nitrogenous and sulphurous compounds
8. Lack of proper aeration and water exchange
9. Over Feeding
WATER CHARACTERISTICS
The physical variables such as pH, Salinity, Turbidity and Total Suspended Solids (TSS)
and chemical variables such as Oxygen level (DO), Alkalinity, Hardness, Ammonia, Nitrite,
Chemical oxygen demand (COD), Biochemical oxygen demand (BOD), Residual chlorine and
hydrogen sulfide are the most important parameters for the management of the pond water.
The best growth performance of animals can be achieved only in optimum condition of these
environmental factors.

PHYSICAL CHARACTERISTICS
Normal, optimum and critical ranges of physical characteristics for brackishwater
shrimp culture are presented in the Table 1.
Physical Shrimp farm pond water
Parameters Normal 1 O ~ t i m u m/ Critical
Temperature (OC) 17-33 28-32 4 4
PH 7.0-9.0 7.5 - 8.5 c6.0 (Daily fluctuation 0.5) & > 11
'Salinity (ppt) 7.5-34 15-25 < 5 and > 40 (Daily fluctuation 5 ppt)
Transparency (cm) 25-40 30-40 <20 and>60
TSS (ppm) < 100 ---- ----
Temperature
Water temperature plays a very important role in regulating the activities of cultured
animals. The optimum level of temperature for most of the brackishwater penaeid shrimp is
28-32OC. In brackishwater shallow ponds, where regular exchange between the tidal water and
the pond water is not maintained during the hot dry months, the temperature of pond water
may shoot up beyond the tolerance limit causing mortality of reared shrimp. The high rate of
evaporation may also occur with the result of increase in salinity beyond the tolerance level.
Similarly, during the winter season, the low temperature will have a chilling effect reducing
metabolic and growth rates of cultured shrimp. On account of unequal distribution of
temperature with higher temperature near the surface layer and decreasing temperature with
depth, thermal stratification can occur in deeper ponds. This can result in reduced heat budget
for the pond and formation of methane, hydrogen sulphide and ammonia can occur causing
degradation of water quality. The planting of trees on pond dikes to give shade will reduce
stratification but at the same time reduce the beneficial effects of wind mixing and restricts
solar energy for photosynthesis. Operation of aerators during warm and calm afternoons help
to break thermal stratification by mixing warm surface water with cool sub surface water.

pH
The pH indicates acidic or basic nature of water. It is an index of the presence of
metabolites, photosynthetic activity and the fertility of the polid water. Low pH is reported to
be harmful to crustaceans and higher pH can lead the alkaline death. Effects of pH on the
growth of shrimp is shown below:

PH EFFECT
<4 Acid death point
4-6 Slow growth .
7-9 Best for normal growth
9-1 1 Slow growth
> 11 Alkaline death point

The normal range for the growth of penaeid shrimp is 7.0 to 9.0. Above or below this
range, the water should immediately be changed. It is at maximum when photosynthetic
activity is vigorous. High pH value means water is too fertile, therefore, there is the possibility
of plankton bloom, which remove carbon dioxide for use in photosynthesis and more oxygen is
formed. This result in an increase in carbonates concentration (Ca and Mg hardness), which
react with water to form hydroxyl ions, which in turns increase, the pH. High pH can
dramatically reduce the ammonia ionisation constant increasing % of the toxic unionized
portion. As a medium for shrimp culture, brackishwater has many advantages. It contains a
high concentration of nutrient salts and is perfectly buffered medium against abrupt changes in
pH. Nonetheless, It can fluctuate between 7.5-9.5 with the accumulation of residual feed, dead
algae and excreta over a 24-hour period with lowest pH occurring near dawn and the highest
pH occurring in the afternoon. Low variation in pH values will indicate stable phytoplankton
blooms. The pH should be in optimum level of 7.5 to 8.5. It should not vary more than 0.5 in a
day.
Salinity

The term salinity refers to the total concentration of all dissolved ions in grams contained
in lkg of seawater. Following major ions contribute to the saline nature of water.
 Salinity as a single factor plays an important role in shrimp farming as it is responsible
for many functions such as metabolism, growth, osmotic behaviour, reproduction etc. Shrimp
have an optimal range of salinity for better growth and survival, depending on the species. If
the salinity is allowed to go beyond the optimal limit, the shrimp refrain from taking normal
food and hence are emaciated and become susceptible to disease. In pond condition, Pemeus
monodon can tolerate wide range of salinity from as low as 5 ppt to a high of 40 ppt, but white
shrimp P. indicus and banana shrimp P. nzerguiensis generally prefer brackishwater (Salinity: 5
to 25 ppt). Salinity above 45 to 60 ppt can be lethal. Most species will grow best at salinities
of 15 to 30 ppt.
Due to high evaporation rate in summer, salt concentration in ponds gradually
increases. Salinity may increase to beyond 40 ppt, which can affect the growth of shrimps. In
such cases, water should be exchanged fiequently either by pumps or through tidal exchange.
The groundwater with low salinity (2-5 ppt) can be utilized for reducing the salinity. Seawater
(35 ppt) mixed with groundwater can be used for preparing water with required salinity for use
or exchange. Sudden fluctuations in the salinity associated with the heavy rains result in heavy
mortality. Shrimp larvae are produced in waters with salinities of 28-35 ppt but advanced post
larval stages often are stocked in ponds where salinity is much lower. At the time of stocking
they should be acclimated gradually to the salinity of pond water so as to reduce stress and
mortality. The acclimatization rate should not exceed 1 or 2 ppt per hour.

Total Solids
Organic and inorganic, settleable, suspended (TSS) and dissolved matter is termed as
total solids. Portion of organic and inorganic solids that settles in l h in an h h o f f cone is
known as settleable solids and dissolved solids are portion of organic and inorganic solids
which is not filterable. Settleable solids more than 20 mlll result in rapid silting of the pond and
decreasing of water depth. Portion of inorganic and organic solids that are not dissolved are
suspended solids (TSS). Deforestation, poor soil management practices in agriculture and
erosion in drainage basins of rivers are major causes for heavy load of suspended solids (silt
and clay) in intake water. Optimum level of TSS for most of the shrimp is < 100 ppm.
Excessive TSS led to increased sedimentation of eco-system.

Turbidity & Transparency

Turbidity can be caused either by planktonic organisms or by suspended soil particles.
The turbidity due to silt and clay particles is also known as inorganic turbidity and can interfere
with the penetration of light and by absorbing nutrients present in the water and in turns affects
the growth of benthos. This can cause uneasiness and stress to the shrimp leading to disease.
Suspended clay particles (>4% by volume) damage the gills of shrimp by clogging it. In
certain cases, oxygen deficiency has also been reported as a result of sudden increase in
turbidity.
 Turbidity due to both plankon density and suspended silt and clay particles can be
measured in terms of transparency using Secchi disc. High value of transparency (>60 cm) is
indicative of poor plankton density and therefore water should be fertilized with right kind of
fertilisers. Low value indicates high density of plankton and hence fertilization rate and
frequency should be reduced. The optimum range of transparency is 25-35 cm. Transparency
less than 20 indicates that the water is unsuitable for shrimp culture and should be changed
immediately to flush out excess bloom.
Inorganic turbidity should be removed at intake point, if not, sedimentation
tankslcanals or basins have to be used before water can be taken into production ponds.
Following sedimentation, water should be passed through sand filters, as sedimentation is not
adequate to rid the water to remove fine suspended particles. Unless the source of turbidity is
eliminated, no lasting benefit can be expected. Generally saline water facilitates the
flocculation and sedimentation of suspended soil particles, and water retention times 1 to 2 hrs
is adequate. The major factor favouring rapid sedimentation is reduction of velocity and
turbulence of water. Baffle levies can be used to reduce velocity and turbulence where only a
small area is available for sedimentation.

CHEMICAL CHARACTERISTICS
Chemical water quality parameters required for shrimp farming are given in the Table below:
Cliemical Parameters Shrimp farm pond water
Normal / Optimum / Critical
Total alkali& 50-200 1 100-200 1 <20

Dissolved Oxygen
DO is the most important and critical water quality parameter because of its direct effect
on the feed consumption and metabolism of shrimp as well as indirect influence on the water
quality. DO should be maintained in the range of 3-10 mgll. For penaeid shrimp, optimum
concentration of DO for maximum growth rate is 6 ppm. Prolonged exposure to low oxygen
content causes low feed consumption which leads to slow growth and the culture organisms
become inactive and they are susceptible to disease. Furthermore, many or even all organisms
may die fiom lack of oxygen. DO in the waters come fiom two sources: 1. As a by-product of
photosynthesis and 2. Diffusion of atmospheric air. Photosynthesis is the primary source of DO
in brackishwaters. As photosynthesis occurs most rapidly in the surface layer of water and DO
concentration decline with depth, in deeper ponds, DO may fall to o ppm at depth of 1.5 m or 2
m. Hence it is advantageous to have fairly shallow ponds (75 cm to 150 cm deep) for shrimp,
because they dwell mainly on the bottom and low DO at the pond bottom would be harmful.
DO can be affected by many factors particularly water temperature, respiration of plants
and animals and the level of organic matter. In tropical waters the DO level is normally low
because of higher temperature. The rate of chemical and biological reactions is said to double
at every 10°C increase in temperature. This means that aquatic organisms will use twice as
much dissolved oxygen and chemical reactions will progress twice as fast at 30°C than 20°C.
Thus the dissolved oxygen requirement of aquatic species is higher in warmer than in cooler
water. The concentration of toxic substance such as unionized / reduced form (NH3), sulphur
(H2S) and carbon metabolites (methane) increases when low DO level exists. However, in the
presence of optimum level of oxygen the toxic substances are converted into their oxidized and
less harmful farms. The use of aerators result in mixing of water at surface and bottom and
breakdowns DO stratification and also can eliminate black mud formed at interface of pond
water and bottom mud. Water exchange is the best solution to prevent low DO problem in the
pond where aeration is not practiced.
Over feeding should be avoided in order to maintain the DO level. One of the effects of
overfeeding is to decease the feed conversion efficiency. Uneaten feed gets decomposed,
releasing nutrients into the water. Consequently, phytoplankton abundance increases as a
function of increasing feeding rate. DO concentration decline more rapidly with depth as
phytoplankton abundance increases in response to higher feeding rates. Besides, the
phytoplankton die-off is greater in ponds with high feeding rate and abundant phytoplankton.

Alkalinity and hardness

Total alkalinity is a measure of the concentration of carbonates, bicarbonates and
hydroxide ions present in the water and is usually expressed as mgfl equivalent calcium
carbonate. It indicates the buffering capacity of the water. Alkalinity primarily determines the
magnitude of dial fluctuation of pH of water. Waters with low alkalinity (20 mgil) has low
buffering capacity against pH changes. This results in wide fluctuations in pH value from 6 or
7.5 at dawn to 10 or even higher in the afternoon. Very high alkalinity (200-250 mgll) coupled
with low hardness (<20 mgil) results in rise in the afternoon pH beyond 11 and cause alkaline
death. Very high alkalinity water may also suffer from poor productivity due to a limitation of
carbon dioxide for photosynthesis. pH of water with moderate to high alkalinity values (20-150
mgll) normally fluctuates between 7.5 or 8 at dawn and 9 or 10 in the afternoon.
Total hardness is defined as the total concentration of divalent cations such as caU and
~ g ions
* in waters, also expressed as mg/l of calcium carbonate. The importance of hardness
is closely related to alkalinity. However, low hardness water contains insufficient calcium ions.
Hardness and alkalinity are more important for the exo-skeleton of shrimp. Alkalinity and
hardness can be increased by addition of agricultural lime. In brackishwater, alkalinity and
hardness are usually high, so these variables are seldom important in management of shrimp
farms.

Carbon dioxide
Free carbon dioxide refers to the concentration of C02-kH2C03. Concentration of COz
below 20 mg/l probably is not harmhl to shrimp, provided DO is high. DO concentrations
declines when photosynthesis is not proceeding as rapidly as respiration, thus COz accumulates
because it is not removed for use in photosynthesis. COz concentrations are normally high
when DO concentrations are low. Because of the necessity of light for photosynthesis, C02
concentrations increase at night and decrease during the day. High concentrations of C02 also
occur in ponds during cloudy weather. However, it is seldom practical to remove C02 from
pond waters.
Residual Chlorine
Chlorine in various forms is commonly used in shrimp hatcheries to disinfect seawater.
This may cause toxicity problems. The residual chlorine if allowed to remain in water can form
highly toxic chloramines with nitrogenous compound present in hatchery water. Permissible
level of chlorine residuals in treated water for use in shrimp grow-out ponds is less than 0.001
ppm and for hatcheries, it is nil or traces.
Chlorination should be done before tilling the pond and applying fertiliser. It is safer to
manipulate water quality in reservoir before release to grow-out ponds. Sufficient number of
paddle wheel aerators must be placed to ensure sufficient aeration for complete
dechlorination. Physical aeration is the cheapest and the best available method of
dechlorination available to shrimp farmers. Without effective dechlorination and monitoring,
chlorination of source water for hatchery purpose should be avoided.

Nutrients- Nitrogen, Phosphorus, Nitrates and Phosphates

Total N includes organic nitrogen, ammonia, nitrite and nitrate. Organic nitrogen is
bound nitrogen into protein, amino acid and urea. Nitrate is the final product of nitrification of
ammonia and is a major phytoplankton nutrient in marine environments. It is the least toxic of
inorganic nitrogen compounds. Total P exists in organic and inorganic form. Organic P is
bound in organic matter and inorganic form of P exists as orthophosphate and polyphosphate.
Nitrogen and phosphorous along with carbon and other trace elements serve as nutrients thus
accelerate the growth of phytoplankton, which is the base of the food web in culture system.
Optimum nutrient concentrations are undefined. Since coastal waters are polluted slowly, there
is a possibility that shrimp farms are supplied waters that are contaminated with domestic
sewage. This water contains moderate to high concentrations of nitrates, ammonia and
phosphate. When held in ponds, this water will usually produce phytoplankton blooms.

Toxic metabolites ;Ammonia, Nitrite, Hydrogen Sulphide

Ammonia is the most common toxicant in high stocking density culture system.
Ammonia is the principal excretory product of crustaceans. Ammonia exists in aqueous
solution as highly toxic unionized ammonia and non-toxic ionized form m+). The toxicity
of ammonia is known to be affected by water pH; its toxicity increases with increasing pH.
Obviously ammonia toxicity will be a greater problem of high pH. Ammonia increases oxygen
consumption by tissues, damages gills and reduces the ability of blood to transport oxygen.
Hence, it is very harmful to aquatic organisms and should be below 0.1 mgil. Concentration of
unionized ammonia, greater than 0.1 mgll may adversely affect growth of shrimp and above 1
mg/l are potentially lethal. In high stocking density culture systems, ammonia and nitrite
increases exponentially over time in both hatchery and grow-out ponds even with frequent
water exchange. High concentration of ammonia and nitrite reduce shrimp growth and in
extreme cases cause mortality.
Various techniques for reducing total ammonia nitrogen (TAN) concentration may be
implemented. Some common procedures are water exchange, aeration and application of
zeolites and bacterial products. Water exchange can dilute ammonia concentration and it can
be effective if enough water is available to rapidly exchange a large volume of the pond with
water of much low TAN concentration. Zeolites are ion exchange media which removes
ammonia from water but there is no evidence that zeolite can effectively reduce ammonia
concentration in ponds. While, this is technically true, a very large amount of zeolite would be
26
required to significantly lower ammonia concentration. Besides, bioaugmentation materials
are claimed to improve water quality by reducing ammonia concentration. Since, bacteria in
these products already occur naturally in ponds, application of these commercial preparations
of bacteria are unnecessary. In addition, research has failed to demonstrate benefits; they don't
affect much on reduction of TAN concentration. The toxic effect of ammonia may be
minimized in following ways: Maintaining sufficient level of DO facilitates oxidation of
ammonia to harmless nitrates by nitrifying bacteria. Periodic partial removal of algal blooms
by flushing or scooping out the scum, which facilitates optimum density and prevents sudden
die-off of the bloom.
Nitrite is also one of the common toxicants in high stocking density culture system. It is
an intermediate product in the bacterial nitrification of ammonia to nitrate, a process called
nitrification. It is toxic to fish and crustaceans and therefore is important for aqua culturists.
The toxicity of nitrite is known to be affected by water pH and the presence of chloride and
calcium ions. Nitrite toxicity increases with increasing pH. It decreases with increasing
calcium and chloride concentrations. Hence nitrite is more toxic in freshwater than in seawater.
Nitrite in shrimp ponds is seldom at concentrations great enough to kill shrimp, but the growth
may be adversely affected by concentrations above 4 mgtl. The desired level is less than 0.2
mgil for maximum production in brackishwater culture system. In ponds effective removal of
organic waste, adequate aeration and correct application of fertilizers are the methods to
prevent the accumulation of nitrite to toxic levels. In hatcheries, control of nitrite may be
accomplished by installing biological filters.
H2S is a by-product of decomposition of dead plants, animals and organic residues in
the ponds. This accumulates on the pond bottom and turns the soil black. The pH regulates the
distribution of total sulphides among its forms (HzS, HS, S-). Unionized H2S is toxic to
aquatic organisms : the ionic form, however, have no appreciable toxicity. The percentage of
hydrogen sulphide decreases as the pH increases. Therefore, presence of sulphides is
considered as indicator of organic pollution under reducing conditions. Heavy accumulation of
H2S results in oxygen depletion leading to mortality of shrimp. The concentrations of 0.01 to
0.05 mgil of hydrogen sulphide may be lethal to aquatic organisms. The safe levels of H2S are
less than 0.003 mgil for shrimp. Toxic H2S levels can be controlled either by adopting central
drainage system, higher rate of water exchange and other management practices like effective
removal of organic waste from the pond bottom.
 5. WATER QUALITY MANAGEMENT RV SHRIMP CULTURE

KICKrishnani, B.P. Gupta and S.M.Pillai

Maintenance of water quality within the optimal levels during the culture is very
essential for ensuring good growth and survival of shrimps. The following management
methods are used which help in maintaining water quality requirements of the shrimps.

Liming
The problem of low alkalinity (<20 mg/l as CaC03) can be abated through application
of liming materials. The use of agricultural lime such as dolomite and limestone for pond
preparation is strongly recommended over the use of hydrated or quick lime. Use of latter two
limes can destroy the initial inoculum of decomposing bacteria, which will slow down the
mineralisation processes during culture operations and will increase nutrientlorganic load on
pond bottom, which in turn will favour the growth of fouling bacteria. Excessive liming should
be avoided, because it can be harmhl by removing COz, precipitating dissolved phosphate and
raising pH, which, in turn, favours ammonia toxicity to shrimp. As a medium for shrimp
culture, brackishwater has many advantages. It contains a high concentration of nutrient salts
and have alkalinities of 50-75 ppm or higher. Hence, it is a perfectly buffered medium against
abrupt changes in pH and liming normally is unnecessary. Liming is done whenever the pH
drops below 7.5 or the daily fluctuation in pH is more than 0.5. Further liming has to be done
after every water exchange and heavy rains.

Fertilization
In addition to the initial fertilization done during pond preparation, additional
fertilization is required during the course of culture to maintain good algal bloom. 10% of the
original dose of fertilizer should be applied at weekly or fortnightly intervals depending on the
percentage of water exchange and the algal bloom. Sometimes, ponds, which develop clear
water condition, are repeatedly fertilised with high doses of inorganic fertilisers with the hope
to produce bloom. Once the benthic algae develop, it is useless to fertilise the ponds. Badly
prepared ponds can pose problems of maintaining plankton blooms, stable water conditions
and eventually lead to disease syndromes. The disadvantage of various types of algae in shrimp
ponds is known. Some species of benthic algae are highly toxic and hinder movement of
shrimp and compete for oxygen and algae aid sedimentation and pond bottom degradation.
These are all potential factors contributing to stress on the shrimp and invites disease outbreak.
Dense phytoplankton blooms as net consumers of oxygen should be of greater concern than
their role as oxygen producers. Hence, it is advisable to check the inherent organic load,
plankton and possible contamination in the water source. Plankton density, suspended silt and
clay particles can be measured in terms of transparency using Secchi disc. High value of
transparency is indicative of poor plankton density and therefore water should be fertilised.
Low value indicates that the plankton density is high or the water is turbid due to suspended
particles and hence fertilisation rate should be reduced.
 Water exchange
Regulation of environmental factors is mainly achieved by controlling water exchange
either by the use of tidal flow or pumping depending on the system. In the case of low stocking
density, water exchange is not required in well prepared ponds for first 30 days. Only water
level has to be maintained due to evaporation loss. Thereafter, water exchange should be
should only be used when necessary, in order to maintain the optimum water quality and
reducing the levels of toxic metabolites. Routine water exchange does not improve water
quality, because this can discharge carbon, nitrogen and phosphorus substances fiom ponds
before they can be assimilated. This should only be used under the following pond conditions :
- (i). Transparency greater than 50 cm or less than 20 cm, (ii), pH less than 7.5 or greater than
8.5, (iii). pH fluctuation in a day greater than 0.5, (iv). Shrimp are noticed on the water surface
or on the sides of the pond. (v). Free ammonia level is greater than 0.1 mg/l

Aeration
In culture systems with a stocking density of 6-10 no/m2, aeration is generally required
during the last 30-45 days depending on the water quality conditions. Normally aeration will
be required during the following situations ; cloudy and rainy days; dissolved oxygen level
dropping below 4 mgil especially during early morning hours when the DO level will be at its
minimum; excessive algal blooms & application of water treatment additives. Paddle wheel
aerators @ 4-6 noha are generally used in shrimp ponds. The positioning of the aerators in the
ponds should be done properly to ensure maximum water flow within the pond. For high
stocking density culture, long arm aerators are recommended.

Feeding
Proper feed management is indispensable for successful and profitable shrimp culture.
Shrimp feed is an organic input into the pond. Overfeeding is more dangerous than under
feeding as over feeding rate can degrade the pond bottom and can lead to excessive
phytoplankton, DO depletion and toxic concentration of metabolites. Well prepared ponds with
low stocking juveniles will not require initial feeding for a few days, as naturally generated
food organism form nutritious foods for post larvae. As aerators are not used in extensive
system to keep feeding areas clean, it is advisable to broadcast feed evenly in all areas of the
pond to avoid bottom degradation in specific areas.

Crisis management in water quality

Effects of various abiotic factors on shrimp farming and their remedial measures are
summarized in the following- Table
PHYSICAL FACTORS
FACTORS EFFECT REMEDY
i. TEMPERATURE Cramped body, DO depletion, Use of Aerators & Planting of trees on Pond
Excessive :- respiratory trouble, Thermal Dikes
I stratification
/ ii. Excessive
TURBIDITY
:
Asphyxiation & Osmo- Use of Sedimentation tanks following use of sand
regulatory Stress filters
iii. TSS Sedimentation & damage the
Excessive :- gills by clogging & DO
Depletion
iv. TRANS- Excessive Poor PlanMon density Fertilisation
Low value High PIanWon Density Reduction of Fertilisation, water exchange to
remove excessive blooms
 v. SALNITY : MetabolisdGrowth WaterPTidal Exchange
L Excessive :- EmaciatiodStress

CHEMICAL FACTORS
EFFECT REMEDY
Low pH (c4) Acidic Death Point Application of Lime
""" :P
High pH (>11) Alkaline Death Point Reduce fertilization 1 Water Exchange
DO Depletion Slow Growth, Increase in NH3,S- Aerationi'ater Exchange
& C metabolites; Stress Shallow Ponds (75-100 cm deep)/
Diseasehfortality Remove scumiAvoid Overfeeding
1 ALKALINITY Poor Productivity & Alkaline Water exchange
I High (200- 250ppm)+ death
/ ACCUMULATION OF NH3 ; Increase O2Consumption Effective removal of organic waste;
ORGANIC MATTER & Damage the gills Adequate aeration ;correct application
of fertilizers ;Use only water treatment
effective products; Periodic Removal of
Bloom;

i NO2
Water Exchange
Adequate Aeration, Removal of Organic

i1 Excessive CODBOD;
Destroy gills; Inhibit normal
waste
Water Exchange; Removal of Organic
waste
i respiration
EXCESSIVE N & P High Algal bloom+ Reduce Fertilisation.
DO Depletion
I INDISCRIMINATEUSE Mutation, Resistant pathogens & Avoid the use of such chemicals, prevent
I OF CHEMICALS Bioaccumulation the entry of such chemicals
GASEOUS FACTORS

Hzs Poor Water Quality Water Exchange; Central Drainage System, Effective
DO Depletion bioaugmentors;Remove Organic waste.
clz Excessive Chloramines Use of Aerators, Dechlorination

The shrimp farmers should try to maintain the water quality variables within the
optimum range as far as possible by suitable management techniques. It is high time to adopt
the old Chinese proverb " To culture fish, one has to culture water. Thus, by aiming well all
round water quality management practices towards reduction of stressors, the occurrence of
disease and mortality can be efficiently prevented and the magnificent industry can be made
sustainable.
 6. SELECTION CHARACTERISTICS OF SHRIMP POST LARVAE

C. P. Balasubramanian

Shrimp aquaculture has undergone revolutionary development over the past few
decades and it is one of the world's fastest growing agri-industry. Although this growth in
production is impressive, there have been several problems and impediments that have yet to
be resolved. In recent years, problems have continued to mount as outbreaks of disease and
crop losses. Furthermore, variability in post larval (PL) quality has made culture technology
unreliable and limited the commercial feasibility of shrimp culture (Castle et a1 1993). Even
with good husbandry practices, stocking the pond with poor quality fry results low yields at
harvest. These low yields are often related to poor survival rate or slow growth resulting in
unprofitably high fed conversion ratio. As a consequence to unpredictable production in grow
out; farmers and researchers are more concerned today about the quality of PL entering the
rearing system.

The importance of PL quality evaluation has been acknowledged since the early phase
of the development of shrimp culture industry (Bauman and Jamandre, 1990). Factors affecting
the PL quality are determined by the intrinsic properties of the PL itself and environment in
which PL is subsequently reared. Post laraval quality is extremely variable; some of the factors
affecting PL quality are known, but many (probably most) are unknown. The components of
PL quality may be endocrine status of oocytes in the ovary, or sperm, diet of the broodstock or
genetics of the broodstocks. Indeed, several criteria for larval quality assessment have been
proposed, and some of them are widely used by aqua culturists. However, most of these criteria
are based on anecdotal evidences rather than the empirical data. The experimental studies to
establish the link between the predictive criteria used to assess the larval quality in hatcheries
and the actual performance in grow - out system is extremely limited (Racotta et a1 2004). The
objectives of this article were to summarize the various procedures currently used to evaluate
the larval quality in shrimp hatcheries and grow out system. Based on the available information
a practical set of criteria have also been proposed.

Criteria for offspring quality

Generally the term larval quality refers to the physiological conditions, performance
during cuIture and resistance to stress test. Although there is no accepted method of
determining PL quality, many different criteria have been suggested for the evaluation of the
PL quality (Samocha et a1 1998). Larval qualities are generally assessed following several
criteria and methodological tools that can be broadly categorized into: 1) morphology 2)
behavior, 3) survival to stress test, 4) time needed to complete larval metamorphoses, 5)
Survival during larval development 6 ) PL size 7) Screening of PL for major Pathogens (Bray
and Lawrence 1992; Racotta et a1 2004)

Morphology
In this category large sets of variables have been used such as size, weight, occurrence
of deformities, colour, muscle/gut ratio, gill and digestive system morphology etc. (Racotta et
a1 2003). The most simple and widely used criterion is the visual observation of fry. Active fry
with dark colour is considered to be best for stocking. The PL with clean carapace should be
selected and it indicates the animal is growing fast and moulting frequently. Slow growth is
indicated by the presence of pathogens and necrosis.
Muscle gut ratio:
It was reported that the wild fry has a tail muscle generally exceeds their hind gut
diameter by a ratio of at least 4:l. Based on this, muscle gut ratio is used widely to assess the
PL quality in many hatcheries. The measurement is taken half way between the telson and last
abdominal segment. The muscle should completely fill the shell form the gut down to the
ventral side. Poor quality fry will often have muscle gut ratio less than 4:l. This method is
proved to be very successfhl. However, it should be noted that this procedure is limited to
stages before PLZ0, as in older fry (>22 days) it is hard to measure due to the prominent
pigmentation.

Post larval size:

Increased growth and reduced variability in size during post larval stage is proved to be
related to further growth to juvenile stage (Castle et a1 1993). Uniformity of length of hatchery
reared PL is widely used as an early indicator of PL quality. Studying the production
characteristics of Litopenaeus vannamei Clifford (1999) proposed a scale (Fig 1). Length
uniformity was evaluated using the coefficient of variation (C. V.), which is calculated as the
standard deviation divided by the mean. If the C. V is lesser than 10% the population is
considered to be excellent for stocking, and if C. V. is greater than 15% the population may
have been infected.

Gill area:
Gill area is proposed to be a predictive criteria for the good quality and survival
(Racotta et a1 2004), although their potential application in commercial rearing system has yet
to be established. They confirmed that large gill area of PL is positively correlated with high
survival rate.

Behaviour
Larval behaviour has often been regarded as a predictive criterion for good larval
quality. The positive phototrophic behaviour of nauplii is widely used to harvest nauplii, and it
is also thought to be an indirect indicator of larval quality (Bray and Castle 1991). However,
later it was proved that this trait would last only up to the protozoeal stage and after this stage
there would not be any significant difference bemeen larvae had higher phototropism and
remnant larvae (Ibarra et a1 1998). Swimming activity is also used widely as an indicator for
larval quality (Clifford 1992). Poor or erratic swimming activity obviously suggests a lower
physiological activity.

Survival to stress test

Stress tests have been proposed as a meaningful tool for assessing fish and shrimp
larval quality in aquaculture industry and environmental research management (Wedemyer and
McLeay 1981, Clifford 1992, Fegan 1992). Defining stress is a difficult task. Selye (1973)
defined stress is a response of organism to any demand placed on it such that it causes an
extension of physical state beyond the normal resting state. Philosophy behind the stress test is
that high resistance of PL to stress may be a security for their growth/survival performance in a
grow out ponds. It is based on the principle of exposing an organism to a short but extreme
external condition in which physiological state of the animal determines its chances of survival
(Fegan 1992). In other words, batches of shrimps in poor health will be less able to tolerate
such stresses than the batches of healthy shrimps, and the tolerance can be determined by a
stress test before any impact on the growth can be noted. Salinity stress test and formalin stress
tests are the most widely used stress tests used in the shrimp seed production industry.
Another test proposed with potential application is ammonium stress test.

Salinity stress test:

Osmotic stress has been used as a tool to evaluate the quality or hardness of post larvae
produced in shrimp hatcheries (Samocha et a1 1998). It is one of the most widely used criteria
for assessing the larval quality. High survival from this test is supposed to be associated with
high performance in grow-out systems. This stress test can be applied to PLI and PLm. The test
for PL, would be used to select a best of batches of PLI for further larval rearing in the
hatchery where as PL20would be used to evaluate grow-out performance. The methodology
used for salinity stress test varies from hatcheries to hatcheries. The most common procedures
are summarized below.

1. The post larvae are abruptly transferred to dilute media (in the case of PLI it can be
15-18 ppt and for PL 26 it should be below 3ppt) for 30 minutes then back to the
original sea water for another 30 minutes. At the end of the test survival is measured
by counting dead organism.
2. The larvae are held at reduced salinity for a period of 4 hours and then mortalities are
recorded.

Clifford (1994) suggested a scale for assessing larval quality based on salinity stress test (Fig
2). According to this scale survival rate above 70% is acceptable. Even though this test is
widely used in Penaeid hatcheries world wide, there are still controversies regarding the
eficacy of this challenge test. The shrimp species like L. vannamei and P. monodon are
euryhaline, and, there fore less susceptible to low salinity stress. More over, the survival to
salinity stress test is an individual measure of diverse complex process such as nutritional
status, mobilization of nutrients under a sudden stress, the degree of development of PL,
osmoregulatory capacity of gills etc (Racotta et a1 2003). Therefore, the likelihood for
obtaining positive correlation between the low salinity tolerance and better grow-out
performance is almost unlikely. Recent experimental studies show that the salinity stress test is
usehl for predicting of survival during stocking and, however, it is not a reliable predictive
indicator of performance (survival and growth) during grow-out.

Formalin stress test:

Acute formalin stress test has also been widely used in Penaeid hatcheries. Samocha et
a1 (1998) proposed formalin stress test methodology for L. vannamei. The methodology
proposed by them is summarized below.
Place a pre determined number of PL in seawater containing formalin, and count the
number of live PL after 2h. If the number of live PL is above the expected number then the PL
used are hardier and can be accepted for firther stocking. For example, 100 animals are kept in
200 ppm formalin for lh, and if survival is >90% 20 points can be given. If survival is between
80 and 89% 10 points should be given.
Ammonia stress test:
A challenge test using ammonia has also been proposed as a predictive criterion to
evaluate the larval quality (Racotta et a1 2004), although this test is not being used in
commercial Penaeid hatcheries. The feasibility of using this test in commercial hatcheries has
yet to be evaluated. The methodology proposed by Racotta et a1 (2004) is summarized below.
 The test is used at pro~ozoealstage. A total of 30 protozoea are exposed to ammonia
for 24 h and survival of protozoea is assessed after 24 h. They observed a positive correlation
between survival to ammonia stress test and survival of nauplii to PLI

Screening of post larvae for major pathogens

Owing to the frequent viral epizootics (white spot syndrome virus) and associated crop
losses many shrimp aquaculturists are keen to evaluate the presence of pathogen before
stocking. Therefore, larval screening for pathogens has become a pivotal part of the larval
quality evaluation. Testing for virus using molecular diagnostics such as Polymerase Chain
Reaction (PCR) has become increasingly common.

Principle of the PCR:

Basically PCR is a method of amplifying DNA of the target organisms (in this case
white spot syndrome virus). For this purpose a small, complementary fragment of the virus
DNA (= primer) is used as a template to amplify the target DNA. The primer along with
sample tissue is placed in the PCR vial with reagents and enzyme taq polymerase. Repeated
cycles of heating and cooling causes the DNA to separate and anneal, resulting the doubling
the quantity of targeted DNA in the process. Thus, the concentration of DNA increases enough
to be detected by electrophoresis. If band correspond to the primer are found in the gel, the
virus is present.
Apart from the anecdotal evidences, very few empirical studies have been conducted to
evaluate the association of WSSV negative seed and the successful grow out performance.
Peng et a1 (2001) surveyed the production characteristics of 27 P. monodon culture ponds. The
ponds were grouped into 3 groups based on the WSSV infection level at the initial PL level;
group 1 consists of ponds with >50% animals are infected, group 2 with 1-49% animals are
infected and group 3 with WSSV negative PL They reported highest proportion of successful
harvest in those ponds where the post larvae were WSSV free, whereas none of the ponds
stocked with population having > 50% WSSV positive PL was cultured through the harvest. In
another study conducted in Thailand Withychumnarnkul, 1999, reported almost similar results.
In his study, he used a farm comprised of 188 earthen ponds following semi-closed intensive
culture system. The ponds were classified into 5 groups. One group is stocked with WSSV
PCR positive PL and all other group were stocked with WSSV PCR negative PL While only
5% ponds only survived in the ponds stocked with PCR positive seeds, -70% ponds survived
in the ponds stocked with PCR negative seeds. In conclusion, initial detection of WSSV rates
can be used to predict the fate of cultured shrimp population.

Recommended criteria for evaluation of Penaeus monodon post larvae in commercial

hatcheries
Decision to stock or not to stock a batch of post larvae is ultimately an assessment of
risk. It is extremely difficult to suggest a fixed guideline or standards. We suggest the
following guideline that should be finalized after a series of discussion with hatchery
managers, farmers and scientists. The following tentative guideline is proposed
I FRY Analytical Procedures
MBV By malacite green straining.
Absence of occulusion bodies is negative
WSSV By nested PCR method. Minimum 100 post larvae
to be taken for the PCR analysis
Stress test with Formalin 100 animals are subjeted to 200 ppm formalein for one hour.
Survival above 90% = 20 points
Survival 80-89% = I0 points
Stress test with It can be done by increasing or decreasing the salinity
salinity by 10 ppt for one hour
Survival above 90% = 20 points
Survival 80-89% = I0 points
Muscle Gut Ratio(M G R ) Compare the thickness (Ventrodorsal distance) of the mid
portion of the sixth abdominal segment with the width of the
gut at the same position. 4:l Ratio. Above 75% carries I0
points 1
Body length (BL) Distance between the tip of the Rostrum to the tip of the
Telson.
Gut Empty l Half I Full
Swollen hind Gut ( S H G ) The hind gut portion swollen due to bacterial infection.
Chromotophore I I I1 I 111
PL Age It is determined based on the Rostra1 spine
4to5
5 to 6
6to7
7to8
Size variation Uniform size 90 - 100 animals 10 points
Uniform size 80 - 89 animals 8 points
-
Uniform size 70 79 animals 6 points 1

References
Alvarwz, A. L., Racotta, I. S., Arjona, O., E. Palacios. 2004. Salinity stress test as a predictor
of survival during growout in Pacific white shrimp (Litopenaeus vannamei). Aquaculture 237:
237-249.

Arcos, F. G., Ibarra, A. M., Palacios, E., Vazquez-Boucard, C., Racotta, I. S. 2003. Feasible
criteria for reproductive performance of white shrimp Litopenaeus vannamei: Egg quality and
female physiological condition. Aquaculture. 228: 335-349.

Bauman, R. H. and Jamandre D. R. 1990. A practical method for determining quality of

Penaeus monodon (Fabricius) fiy for stocking in grow-out ponds. In: New M. B. Saram H.
Sigh, T. (Ed) Technical and economic aspects of shrimp farming. Proceedings of the Aquatech
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Bray W. A. and A. L. Lawrence. 1992. Reproduction in Penaeus species in captivity. In: Fast,
A. W. and Lester L. J. (Eds.), Marine shrimp culture: Principles and Practices. Elsevier,
Amsterdam, Netherlands, pp, 93-1 70.
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Variability in growth and survival of early postlarval shrimp (Penaeus vannamei Boon 1931).
Aquaculture 113: 65-8 1

Clifford, H. C. 1992. Marine shrimp farming: A review. In: Wyban, J. (Ed.) Proceedings of the
special session on shrimp farming. World Aquaculture Society, Baton Roug, LA. USA, pp,
110 Clifford, H.C. 1994. Semi-Intensive sensation: A case study in Marine shrimp pond
management World Aquaculture 25(3): 6-12; 98-104.

Clifford, H. C. 1999. Prevention of shrimp white spot virus disease: Water management
strategies. The Advocate .4ugust-October: 58-63

Fegan, D. F. 1992. Recent developments and issues in the Penaeid shrimp hatchery industry.
In: Wyban, J. (Ed.) Proceedings of the special session on shrimp farming. World Aquaculture
Society, Baton Roug, LA. USA, Pp. 55-70.

Ibarra, A. M., Ramirez, J. L., and C. L. Perez-Rostro. 1998. Effect of positive phototropism
selection in nauplii of white shrimp, Penaezis vannamei, on growth, survival and family
variance of later developmental stages. Aquaculture '98, World Aquaculture Society, Las
Vegas, USA, p. 26 1.

PengS.E.,Lo,C.F.Lin,S.C.,Chen,L.L.,Chang,Y.S.,Liu,K.F.,Su,M.S.andG.H.Kou.
2001. Performance of WSSV- infected and WSSV- negative Penaeus monodon post larvae in
culture ponds. Disease of Aquatic Organisms 46: 165-172.

Racotta, I. S., Palacios, E. and A. M. Ibarra. 2003. Shrimp larval quality in relation to
broodstock condition. Aquaculture.227: 107-130.

Racotta I. S., Palacios, E., Hernandez-Herrera, R., Bonilla, A. Perez-Rostro, C. I. and J. L.

Ramirez. 2004. Criteria for assessing larval and post larval quality of Pacific white shrimp
(Litopenaeus vannamei, Boone 1931)

Samocha, T. M., Guajardo, H., Lawrence, A. L., Castille, F. L., Speed, M. McKee, D. A. and
K.I. Page. 1998. A simple stress test for Penaeus vannamei post larvae. Aquaculture. 165:
233-242.

Selye, H. 1973. The Physiology and pathology of exposure of stress. Acta Montreal.

Wedemyer, G. A., and D. J. McLeay 1981. Methods for determining the tolerance of fishes to
environmental stress. In: Pickering, A. D. (Ed.) Stress and fish, Academic Press, New York,
pp, 247-275

Withyachumnarnkul, B. 1999. Results fiom black tiger shrimp Penaeus monodon culture
ponds stocked with post larvae PCR-positive or -negative for white-spot syndrome virus
(WSSV). Disease of Aquatic organisms 39: 21-27
 7. SEED TFWUSPORTATION, ACCLZiMATIZATION AM)STOCKING

1. Introduction
Selection of disease free quality shrimp seed is the foremost requirement for the
success of shrimp farming operation. It has a direct relationship with the survival and growth
of the cultured shrimps. The seed should be transported to the farm site without much stress
and conditioned to the pond conditions before stocking to avoid physiological shock of
changed water quality conditions.

2. Packing and Transportation

B On the day of transportation, the PL should be carefully harvested, held in tanks

containing clean: disinfected and filtered seawater with aeration at no more than 1
millionimt.

B Then the temperature should be decreased gradually (by adding bagged ice) until the
desired transport temperature is reached. Decreasing the temperature fkom 28-30°C to
23°C should take at least 30-40 minutes to help reduce stress. Water salinity should be
the same as the water in the larval rearing tanks

3 Transportation temperature reduction is required to lower the metabolic rate of the PL

so that they will use less oxygen, excrete less waste and remain calm during
transportation.

B The temperature used will depend upon the duration of the transportation. Typically, no
temperature reduction is required if the hatchery is within one hour of the farm.
Temperature should be reduced to 26-28OC for transportation times of 1-3 hours, 25-
26'C for 3-12 hours or 23-25oC for over 12 hours.

B Transportation should be done in the evening or night in insulated vehicles to avoid

excessive temperature during transport.

P The postlarvae should be transported with minimal stress.

P Packing of seed under oxygen in polythene bags is an accepted practice. It can also be
transported in large closed tanks with continuous oxygen supply.

> The polythene bags should be used in double to prevent bag breakage and should be
filled to 113 level

B Optimal packing density PL20 - 500-1000 no./lit.; PL 15 - 1000-2000 nollit.

3 Live Artemia nauplii should be disinfected, washed (with fresh water) and added @ 15-
20 no./PL as feed for each 4 hours of transport to prevent cannibalism during
transportation
 9 The bags should be placed in thermocol or polystyrine boxes with packets of ice
placed outside the bag to maintain the required temperature.

3. Acclimatization

> The postlarvae should be acclimated to the pond water pH, salinity and temperature
before stocking to avoid stress and shock.
> Salinity acclimation should be done very gradually over 2-3 days period. Hence, it is
advisable to inform the hatchery operators the prevailing pond salinity and the
acclimation can start at the hatchery itself. SaIinity acclimation should be done only
after the PLlO stage with well developed gills.
O Acclimation to temperature is easily achieved by floating the seed bags for about 30
minutes in the pond before opening it.
9 Acclimation to pH is done by mixing equal volume of pond water into the seed bags
and keeping for 30 minutes before release.
9 Weak and dead postlarvae should be removed before stocking. This is achieved by
keeping the postlarvae in FRP tanks and treating them with 100 ppm formalin for 30
minutes under strong aeration. After the treatment the strong and active postlarvae
should be segregated and stocked in the ponds.
3 Stocking should be done during early morning when the temperature is the lowest.

4.On-farm Nursery Rearing

In view of the existing disease conditions, it is advised to stock nursery reared juveniles
to reduce the chance of disease occurrence. Since harvesting and transportation involved stress
for the shrimps, it is better to have on-farm nurseries, where they can be easily transferred from
nursery ponds to grow-out ponds with minimal stress.

9 Nursery ponds of area 500 to 1000 m2 are suitable.

9 The ponds should be prepared as is being done for the grow-out ponds.
9 PL15 should be stocked @ 50-75 no/m2
9 Supplementary feeding is done with 200-500 micron crumbled diet.
9 Nursery rearing is normally carried out for 15 - 20 days, by which time the juveniles
reach 1 to 1.5 g size.
> On-farm nursery rearing may be carried out in 'Hapa' or net enclosures in the grow-out
pond itself for 10-15 days to evaluate the capacity of the PL to adjust to the pond
conditions.

5. Stocking density
The rate at which the shrimp seed is stocked in the pond depends on many factors.
Overstocking may lead to high mortality and poor growth and at the same time under stocking
might lead to uneconomical culture with less profit. The optimal stocking density is generally
dependent on the type of management system followed, number of culture days possible and
the expected size at harvest. High density culture requires intensive water quality management
and any failure of the machinery during the course of culture will lead to heavy mortality of
shrimps and associated problems. Further, high density cultures will lead to serious
deterioration in pond soil conditions. Presently, due to the prevalence of viral diseases and
environmental issues, low- density cultures are only permitted in the country. A maximum
density of 6 ndm2 within the CRZ and a maximum of 10 no/ m2 outside the CRZ, is permitted
by the Aquaculture Authority.

6. Close linkage with hatchery operators

Shrimp farmers need to have close linkage with the hatchery operators to ensure that
the shrimp larvae are produced as per their requirements.
P The seed should be booked well in advance and the process of larval rearing could
be monitored.
> The seed testing can be carried out at any stage of the larval rearing. Acclimation
of seed to the pond water salinity could be done at the hatchery itself.
 8. NUTRITION AND FEEDING OF PENAEID SHRIMP

S. AHAMAD ALI

INTRODUCTION

Shrimp farming has shown phenomenal growth in the last decade in India producing
protein rich health food and earning valuable foreign exchange. Feed is a major input in
shrimp farming. The development of nutritionally balanced feed involves understanding the
dietary requirements of candidate species, selection of feed ingredients, formulation of feeds
and appropriate processing technology for producing water stable pellet feeds. Depending
upon the type of farming, a wide range of feeds is used for feeding stocked shrimp. While no
feed is used in traditional farming systems, supplementary and balanced feeds are used in
extensive and semi-intensive aquaculture.

NUTRITIONAL REQUIREMENTS OF SHRIMPS

Shrimp diet should have adequate energy, not only to meet the needs of body
maintenance called basal metabolism, but also for growth. In nature shrimp feeds on a variety
of food items and derive their balanced nutrition for healthy growth. When shrimps are
cultured in confined systems (ponds), they should be provided with a balanced diet as close to
natural food as possible. It is for this reason understanding the nutritional requirements of
candidate species is essential.

Protein requirement
Protein is the most important and essential nutrient in the diet of shrimp. It also
contributes a major share to the cost of feed. The requirement of protein varies with size of
shrimp and also with the source of protein used in diet. The dietary requirement of protein for
tiger shrimp Penaeus monodon ranges from 35 to 45% and for P. indicus it ranges f?om 30 to
43%, which are the most sought after species for culture. It has been demonstrated that
postlarvae and juveniles require higher protein in diet and the requirement decreases, as the
shrimp grows larger in size.

Amino acids
The growth of shrimp is directly related to the quality of protein in terms of amino acids.
Out of the twenty-five odd amino acids that are generally found in proteins, ten are essential
amino acids (EAA). These are arginine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine threonine, tyrosine, tryptophan and valine. Shrimps are not capable of
synthesizing these amino acids and should be provided through diet and hence they are termed
as essential. It is found that if the amino acid composition of the protein in the feed matches
with the amino acid composition of shrimp body tissue, such feed promotes good growth. The
quantitative requirement of EAA in the diet is related to protein level in diet and their
recommended levels in shrimp feeds are given Table 1.
Table 1: Essential Amino Acid requirement in shrimp feed
...............................................................................
% of feed at protein level in feed
Amino acid As % of protein --------------------------------------
36.0 38.0 40.0 45.0

Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Valine

Source: Modified from Akiyama and Dominy, 1989

Lipid requirement
Lipid is a complex mixture of simple fat, phospholipids, steroids, fatty acids and other fat
soluble substances such as pigments, vitamins, A, D, E and K. The quantitative requirement of
fat in the diet of shrimp is in the range of 5 to 10%. However, the quality of fat in terms of
fatty acids is more important.

Fatty acids
Fats are triesters of glycerol. Among the long chain fatty acids polyunsaturated fatty acids
(PUFA) such as linoleic acid (18:2n6), linolenic acid (18:3n3), eicosapentaenoic acid (205113)
(EPA) and docosahexaenoic acid (22:6n3) (DHA) are essential for growth, survival and good
feed conversion ratio (FCR) for P. monodon and other penaeid shrimps. The n3 fatty acids are
more essential than the n6 acids (also known as w fatty acids). The fatty acids, EPA and D M ,
which are known as highly unsaturated fatty acids (HUFA) of n3 series, are particularly
important. Quantitatively EPA and DHA are needed at 0.5% to 1.O% in the diet of larvae and
juvenile shrimp. Studies in P. indicus have shown that oils rich in PUFA such as fish (sardine)
oil, squid oil and prawn head oil produce superior growth when incorporated in its diet. These
oils are rich in HUFA.

Phospholipids
The phospholipid, phosphatidylcholine (lecithin), is essentially required in the diet of
shrimp for fast growth and good survival. Soya lecithin is a good source of phospholipid for
shrimps. It is required at 2% level in the diet. The development and survival of larvae is
significantly improved when the diet contained lecithin. It was established that phospholipids
having choline and ethanolamine are only effective. Those phopholipids having other groups
such as serine are not as effective as these derivatives. Phospholipids are found to be involved
in the transport of lipid, especially steroids in the haemolymph.
Steroids
Shrimps grow through the process called moulting in which they periodically shed body
skin (shell). Steroid hormones called, ecdysones, are responsible for moulting. To synthesize
these hormones, the steroid cholesterol is required in the diet. Shrimps are not capable of
synthesizing cholesterol in their body and hence must be supplied through diet. The
requirement of cholesterol in shrimp diet was shown to vary from 0.5% to 1.0%. For P.
monodon and P. indicus the dietary requirement of cholesterol is 0.5%. Plant sterols, such as
phytosterol, ergosterol and p-sitosterol were also tested for shrimp P. japonicus. Though these
sterols support growth and survival, the performance of cholesterol is superior to these sterols.
24-methylcholesta-5, 22-dienol was also found to be as effective as cholesterol. Many natural
feed ingredients, such as prawn head waste and squid are good sources of cholesterol which
can be included in the feed formulations.

Energy requirements
The major components of shrimp diet are protein, fat and carbohydrate, which are the
main sources of energy to animals. One gram of protein is approximately equal to 5.5 kcal of
energy for shrimp, while fat is the highest energy source equal to 9.5 kcallg. The energy
equivalent of carbohydrate is 4.5 kcalig. The total digestible energy content of a diet varies
with the proportion of protein, fat and carbohydrate. While keeping minimum essential levels
of these nutrients, the energy requirement in the diet of penaeid shrimp was found to be 2800
kcal to 4300 kcal/kg for tiger shrimp, Penaeus monodon and 3500 to 4000 kcaykg for P.
indicus.

Carbohydrate
Carbohydrate is an inexpensive source of energy in shrimp diet. Among the different
types of carbohydrates available, shrimp are found to utilize disaccharides and polysaccharides
better than monosaccharides. The Indian white shrimp P. indicus showed superior growth on
diets containing maltose and starch than those containing glucose, fructose, galactose and
glycogen. Tiger shrimp P. monodon showed preference for trehalose, sucrose and glucose for
growth. However, diets containing maltose and molasses gave inferior results.
The quantitative requirement of carbohydrate in the diet of shrimp is related to dietary
protein and lipid levels. Depending upon the total energy content required in the diet,
carbohydrate can be used from 10 to 40% level. Carbohydrate has protein sparing effect in.
Using starch as source of carbohydrate in diet has dual advantage. Besides being energy
source, it can act as binder if gelatinized by cooking with moisture and improves water stability
of diet. Corn flour, wheat flour, tapioca flour and other grain flours are good sources of starch
in shrimp feeds.
Another polysaccharide, cellulose (also known as crude fiber) is also found to be
required in shrimp diet. The enzyme cellulase is detected in digestive tract of penaeid shrimp.
But the digestibility of cellulose in shrimp is negligible. However, it is needed in the diet as
roughage for improving the feed efficiency. Cellulose levels in shrimp diet should be in the
range of 1 to 3% for best results and should not exceed 6%. However, in good quality feeds the
crude fiber levels are maintained below 3% level.
The dietary requirements of major nutrients for tiger shrimp and Indian white shrimp are
summarized in Table 2.
Table 2: Requirement of major nutrients of shrimps cultured in India
__-_---_-^_-__*____----~*-~--------~-----------m--------------------

Dietary requirement
Nutrient P. monodon P. indicus
________________^__-------------------------------------------------

1. Energy (Kcalkg) 2800 - 4300 3500 - 4000

2. Protein % 35.0 - 45.0 30 - 43
3. Lipid ,, 5-15 6 - 10
4. Carbohydrate ,, 20 - 25 25- 30
5. Phospholipids ,, 0.1 -2.0 0.1 -2.0
6. Cholesterol ,, 0.5 0.5
....................................................................
SHRIMP FEEDS FOR GROW-OUT CULTURE
Shrimps are slow feeders and have a very short digestive system. While the larval stages
are filter feeders, the postlarvae, juveniles and adult shrimp can catch hold of the feed and
consume by nibbling slowly. Because of this feeding behavior of shrimps, particle (pellets)
type feeds of different sizes are convenient and suitable at different growth phases.

Feed formulations

Feed formulation is essentially recipe making by a skillful nutritionist. Properly

selected ingredients are mixed together to obtain nutritional balance to meet the requirements
of a candidate species. Of course, the cost of the final feed and the economics of its use are
also taken into account. Feeds formulated with multi-ingredient mixtures give better
performance than the single or limited ingredients formula.

Feed ingredients

Since protein is the most important and expensive component in shrimp feeds, selection of
protein sources for formulating feeds should be carehlly done. To achieve higher protein
levels and supply essential amino acids, animal proteins, especially marine protein sources, are
necessarily used in shrimp feeds. For balancing the aminoacids, plant protein sources are also
blended with animal proteins. Among the marine protein sources, fish meal, shrimp
head meal, mantis shrimp (Squilla), silkworm pupa and clam meal, and among the plant
protein sources, soybean cake, groundnut cake, gingelly cake, coconut cake and single cell
protein Spirulina are the useful sources. By mixing plant and animal protein sources in proper
proportions a balanced amino- acid composition can be achieved. Among the plant protein
sources, soybean meal is an excellent protein source. However, it is generally recommended at
15 - 20% in shrimp diets, because it contains anti-digestive factor called trypsin inhibitor. This
can be destroyed by heating soybean meal at llO°C for 10 - 15 minutes. Cotton seed cake,
though a high protein material, contains anti-nutritional factor gossypol which cannot be
removed easily. Caution should be exercised in using this material in shrimp feeds. Single cell
proteins Spirulina and yeast are good quality materials containing vitamins and pigments. The
non-conventional ingredients, meat meal, blood meal and poultry feather meal are not tested in
shrimp feeds systematically and data is not available regarding their suitability and efficiency
in shrimp feeds. Fish oil fkom oil sardines, shark liver and also body oils of other mixed fish
are good sources of PUFA, especially EPA and DHA. Soya lecithin is used as the source of
phospholipid. However, vegetable oils are deficient in EPA and DHA.
Wheat flour, maida flour, rice flour, tapioca powder, corn flour and other cereal flours are
good sources of carbohydrate (starch) in shrimp feed.

Feed processing technology

Apart from the nutritional quality of the feed, shrimp feed pellets should have good
water stability. This can be achieved by adopting appropriate processing and production
technology.

Processing of ingredients
The dry ingredients selected for the feed formulation are finely powdered to uniform
particle size passing through 0.3 mm mesh sieve (or screen). These are then mixed according
to the formula and thoroughly homogenised in a horizontal mixer. The feed mixture is made
into pellets by using either extruder or pelletiser.

Pelletisation process

Extruder technology
An extruder consists mainly of a barrel and screw assembly fitted to a high speed
motor. It has direct steamlwater injection facility into the feed mix and works at high
temperature (1 10°C) and Pressure (14 - 98 kg/cm2). As a result of this, feed gets well cooked
improving the feed digestibility and gelatinization of starch which imparts good water stability
to the pellets. Because of high working pressure, the extruded pellets expand and hence float
or sink very slowly in water and the outer finish of the pellets is rough.

Pelletiser technology
Ring-Diet pelletiser technology is extensively used for commercial shrimp feed
manufacture. This technology uses a combination of high pressure between two rotating
rollers and a ring die for compacting pellets. The pressure ranges from 42 - 1800 kgicm2 and
the feed is steam conditioned at a pressure of 0.5 to 3.5 kg/cm2. The pelletiser works at a low
feed moisture of 15-16% and the working temperature ranges from 75 - 95°C. Selection of
feed binder, proper mixing and reaction between binder and feed mixture are critical factors for
obtaining water stable pellets. The pellets obtained through pelletiser are very compact with
smooth surface and attractive finish.

Water stability of feed pellets and binders

Shrimp feed pellets when dispersed in water should absorb water, become soft and retain
the shape for some time without disintegration. This is known as the water stability of feed
pellets. If the feed dissolves faster, it will pollute water and depletes dissolved oxygen in the
culture system. This may lead to mortality of shrimps. The pellets should be stable under
water for at least two hours. The water stability of commercial shrimp feed pellets show wide
variation. Feed pellets with 2 to 4 hour water stability with frequent feeding are more
desirable.
Water stability of feed pellets is achieved by using binders. Right combination of
pelleting conditions such as moisture, steam conditioning temperature and pressure, play an
important role in binding the feed. For good water stability, wheat gluten and synthetic binders
are used. Making the pellets too hard is also not desirable as it affects the digestibility of feed.
Feed attractants and growth promoters
Shrimps are attracted to feed with help of some sensor organs called chemoreceptors.
These are situated all over the body of shimrp. These chemoreceptors recognise certain
chemicals released by feed and guide the shrimp to the feed. Shrimp feeds, therefore, should
have these attractants and leach out when they are dispersed in water. Free aminoacids such as
glycine, glutarnic acid, methionine and lysine are found to be some of the feed attractants in
shrimp feeds. Fish meal has free amino acids which act as feed attractants. Betain, a derivative
of aminoacid, glycine is also used as feed attractant in shrimp feeds.
Natural materials which can promote faster growth in shrimps may be included in feeds
as growth promoters. Squid proteins, clam meat, yeast, Spirulina, alfalfa etc are some of the
materials which have growth promoting properties. Squid meal, clam meal, prawnlprawn head
meal also have flavours attractive for shrimps. However, steroid hormones and antibiotics
should not be used as growth promoters in shrimp feeds. These substances accumulate in
shrimp body and cause bad effects in huinan beings. Such shrimps with antibiotic residues are
not accepted in international market.

Feed grades
Shrimp feeds are produced at least in three different grades. These are 'Starter', 'Grower'
and 'Finisher' feeds. Nutritionally, there are minor differences among these grades, for
example, starter may have slightly higher protein than grower and finisher. However, there are
differences in physical shape and size to suit the different sizes of growing shrimps. Starter
feeds are granular in shape ranging from 0.2 mm to 1.0 mm size. Grower feeds are pellets of
1.8mm to 2.3 mm in diameter and finisher feeds are pellets of 2.0 to 2.5 mm in diameter.
Using appropriate size feeds suitable to the size of shrimp helps in better utilization and
minimizes wastage of feed. There are also sub-grades in each grade.

Feed conversion ratio and economics

Feed conversion ratio (FCR) is the ratio which indicates the quantity of feed (dry
weight) is needed to produce one kilogram of shrimp (wet weight).

Weight of feed given in dry weight

FCR = ..................................................
Weight of shrimp produced in wet weight
The factors effecting the economics of shrimp production is the cost of feed and FCR. Poor
quality feed with high FCR will enhance the cost of production. The FCR of a feed depends
upon many factors such as the nutritional quality of the feed, the water stability, rate of feeding
and water quality in culture pond. Proper feed and water management are important to obtain
good FCR. Feed with FCR ranging from 1.5 to 2:l may be considered as good. Feeds with
high FCR are not only poor in quality but also lead to higher organic waste excretion into the
culture system leading to pollution.
 9. FEED MANAGEMENT IN SHRINIP AQUACULTURE

S. AHAMAD ALI

What is feed management?

Feed management means control and use of feed for aquaculture operation in such a
manner that the utilization of feed is optimum with minimum wastage, negligible impact on
environment, achieving best feed conversion ratio (FCR) and maximum growth of fish and
shrimp and production. Such feed management practice if adopted, aquaculture production will
be not only economical and profitable but also sustainable and eco-friendly. A best feed can
produce poor results if the feed management is poor. On the other hand a moderate feed can
produce best results under good feed management.
Most of the feed suppliers provide feeding charts for feeding shrimp during the period
of culture operation. These tables may be prepared based on either some experiences or
theoretical models. Since most of the feeding charts are based on size of shrimp and biomass in
the culture pond, still errors occur because accurate estimation of biomass in a pond is very
often not possible correctly. In many farms excess feeding may occur due to this error. In
some cases farmers may be over enthusiastic in achieving faster growth may over feed the
stock leading to poor feed management.
Quality of feed
The nutritional and physical quality of the feeds is important. As per the nutritional
requirements of shrimp, the feeds for shrimp are expected to have the following nutrient levels
in the feeds. Feed manufacturers generally declare the nutrient composition of the feeds on the
bags. These may be checked for their confirmation to requirements of shrimp.

Table 1: Expected Nutrient levels in different grades of shrimp feeds

Tiger shrimp White Shrimp
Nutrient Starter Grower Finisher Starter Grower Finisher
Crudeprotein(%) 40-45 38-40 35-38 40-42 35-38 32-35
Lipid (%) 6-8 8-10 8-10 6-8 8-10 8-10
Carbohydrate (%) 10-16 15-20 20-25 10-15 15-25 20-30
Crude fiber (%) 1-2 1-3 2-4 1-2 2-4 3 -5
Ash (%) 10-12 10-15 12-18 10-15 10-16 10-18
Energy(Kca11100g) 350-400 380-420 380-420 350-400 350-400 350-400

The physical quality is the freshness of the feeds and also the appropriate grade of
the feed to suit the growing stages of shrimp. Freshness of the feeds can be checked by smell.
Typical fishmeal smell is an indication of freshness of feed. Stale and musty smell indicates old
stock of the feed. Proper selection may be made based on the observations and past experience.
Following are the specifications of different grade feeds produced:
Pre-starter - 0.2 mm; Starter-I - 0.5 mm; Starter-I1- 1.0 mm; Grower - 1.8- 2.0 mm;
Finisher - 2.3 - 2.5 mm

Rate of feeding
Even though there are some investigations on the quantities of requirements of feed in
relation to size and stage of the growing shrimp still research on these aspects is needed for
making the feeding tables more accurate. Generally the method of calculating the daily ration
is based on the body weight of shrimp. Suppose if W grams is the average weight of the
stocked animal and if there are A number of animals in the pond then the total biomass in the
pond is W x A grams which is equal to W x A11000 kg. If feed is to be given at 10% of body
weight then the quantity feed required per day is
WxA 10
----------x ------ kg
1000 100
To estimate the biomass accurately in a pond is not possible. Generally periodically
(once a week or 10 days) using a suitable net, sampling of the shrimp and the average weight
of the animal is calculated. Total biomass is calculated by multiplying the average weight by
the number of animals surviving at that time. This is mainly done by counting the numbers of
animals caught per each netting and estimating the total number of animals taking into account
the area covered by each netting and the total area of the pond. Some times the number of
animals surviving in the pond is approximately estimated by giving a margin of 10% mortality
in the first month and 3-5% per month subsequently on the total number of animals initially
stocked.
Schedule and frequency of feeding shrimp in grow-out ponds
The quantity of feed required in a day for feeding shrimp is estimated based on biomass
in the culture pond. To start with feed is offered at 15 - 20% of body weight. As the shrimps
grow, it is gradually reduced and brought down to 2-3% towards the end of the culture period.
A model chart for feeding is given in Table 2. The entire quantity of feed required for a day in
-
a pond should not be put at one time. The shrimps should be offered feed at every 3 4 hours
in small doses. Shrimps are active feeders during night, hence large doses may be offered in
the evening and during night. Observations and experiences show that frequent feeding of
small portions of the ration seems to help in better utilization of the feed and there by lead to
efficient FCR. The daily ration can be offered at every 2- 4-hour interval in divided doses.
There must also a mechanism in each case to monitor the feed consumption and offering of the
next scheduled dose should be regulated according to the consumption fiom the previous feed
offered. Regular observations and experience help in mastering the management of feeding in a
culture farm. Keeping the feed in bamboo or velon screen trays kept inside the pond at
different locations is a good practice (Fig. I). These are known as feed-check trays.
Periodically these check trays can be lifted up to check the feed consumption. A part of the
feed may also be broadcasted for proper distribution. Instructions of the feed supplier with
regard to feeding may be followed. Excess feeding leads to uneaten feed at the pond bottom.
This will cause pollution of pond water and stimulates algal blooms, which may cause stress to
shrimp. Under these conditions mass mortality of shrimp may occur. Feeding a little less does
not do any harm, but feeding a little excess may be harmful and can cause heavy loss. Feed
management needs experience and skill to obtain best results. Water quality in culture pond is
also linked to feed management. If the water quality (such as dissolved oxygen, ammonia,
nitrite, nitrate, hydrogen sulphide) in the pond is poor, even the best feed may give poor
performance.
Shrimp feeds should be stored properly. Absorption of moisture during storage leads to
mould growth and lowers the quality. Certain kinds of Eungi (Aspergillus sp) produce
aflatoxin, which is toxic to shrimps. Feed stocks required for use of one month may be
purchased at a time and stored in a cool and well-ventilated place. For longer shelf-life, the
feed may be stored at lower temperature of 10' C.
Farmers should look for feeds that are as fresh as possible. Fresh feeds generally give
good fishy smell. Stale smell indicates that the feed is not fresh. Water stability of feed also
affects the performance of the feed. It will not only disintegrate fast but also causes water
pollution leading to economic loss. The feed should be stable under water at least for 2 hours.
Feed should not be too hard also as it not properly assimilated the animal. Feed with poor
water stability leads to poor FCR and higher cost of production.

Table 2: Rate of feeding of shrimp and quantity of feed to be given in culture pond

Week Weight of Survival Rate of feeding Quantity of feed to

after shrimp expected % of body weight* be given per day (kg)
stocking (g) % 5/mZ* l01m2 5/m2 * 10/m2 *

nil - nil 2 .O
nil -- nil 4.0
4.0 6.0 3.5 10.5
3.8 5.5 4.8 13.9
3.6 5.0 5.9 16.6
3.4 4.8 7.1 20.2
3.2 4.6 8.3 23.9
3.0 4.4 9.3 27.4
3.0 4.0 11.0 295
3O. 3.8 13.2 33.4
2.8 3.4 15.2 37.1
2.5 3.2 15.2 38.9
2.4 2.8 16.2 38.9
2.3 2.7 17.0 40.0
2.2 2.5 18.0 41.0
2.0 2.3 18.0 41.4
2.0 2.1 19.8 41.7
2.0 2.0 21.7 43.4
1.9 2.0 22.0 46.2
1.8 1.9 22.0 46.2

The above figures are only a guideline. The actual figures should be calculated by periodic sampling and recording
the average weight and estimated survival.
Fig.1: Arrangement of feed check trays in a shrimp farm for monitoring the feed
consumption

REFERENCES FOR FURTHER READING

Akiyama, D.M. and N.L.M Chawng. 1995. Shrimp feed requirements and feed management. Aqua International,
August-September, 1995: 14-27.

Guillaume, J. and S.J. Kaushik. 1995. Fish nutrition and the protection of the environment. Proceedings AADCP
Workshop, 25-27 October 1994, Bangkok, Tahiland: 294-31 1.

Kaushik, S.J. and C.B. Cowey. 1991. Ammoniogenesis and dietary factors affecting nitrogen excretion. In:
Nutritional Strategies & Aquaculture Waste, (C.B. Cowey and C.Y. Cho,eds.)
University of Guelph, Guelph, Canada. pp 3-19.

Watanabe, T., 1991. Past and Present approaches to aquaculture waste management in Japan. In: Nutritional
Strategies & Aquaculture Waste, (C.B. Cowey and C.Y. Cho,eds.)
University of Guelph, Guelph, Canada. pp 137-154..
 10. SHRIMP DISEASES: GENERAL ASPECTS

S. V. Alavandi and T.C. Santiago

Introduction

Coastal aquaculture, especially shrimp aquaculture has undergone a fast growth in

recent times in India. While traditional type of shrimp farms were being improved, new
extensive and semi-intensive farms were being established at rapid pace. Majority of the
investors ventured into aquaculture by initially familiarising themselves with technical aspects
of site selection, pond design, feeding techniques, intensive stocking etc. More often, the
significant impact of disease was overlooked. However, concomitant with the rapid expansion
and intensification of shrimp farming activities serious disease outbreaks were of frequent
occurrence.

Attention to disease problems was paid only when widespread outbreak of disease
alarmingly reduced the profit from shrimp farming projects. It has become essential for shrimp
farmers to understand the biological and environmental factors that lead to disease
development, the maladies that can cause considerable loss to cultured shrimp, the early
detection of incidence of diseases and drawing up farming strategy that would minimise or
prevent the onset of diseases.

What is disease and how diseases develop?

As any other living organisms, shrimp also have specific physiological functions for
growth and development, which is greatly influenced by various factors of the environment in
which they are living. Any impairment in the physiological functioning may lead to abnormal
condition of an organism, and this phenomenon is known as disease. However, many experts
consider that there are 3 factors, which interact with each other and result in the occurrence of
disease. These factors are the host (shrimp), the environment and disease-causing organism
(pathogen). Therefore, disease can be described as an expression of complex interaction of
host, pathogen and environment (Fig.1).

A decline in host's immunity is the main cause of disease. A lot of factors will impair
shrimp health and the most important pre-disposing factors leading to diseases in shrimp
culture are:
I. Adverse environment
11. High stocking density with limited water exchange facilities
111. Nutritional deficiencyipoor nourishment
IV. Accumulation of unused feed
V. Inadequate aeration
VI. Sub-optimal or heavy algal blooms in the pond
VII. Physical injury and
VIII. Presence of virulent pathogens in high count.

In these, changes in the physical or chemical factors will be obvious, but the biological factors
will be subtle and complicated. This can be explained by micro ecology. This refers to the
interaction of biological factors and it explains the interaction between normal
microorganisms and its environment.

Host

Like any other crustaceans, shrimp host's body is covered by exoskeleton, which is
regularly replaced by a new one during moulting. The moulting process exerts energy
requirement on the shrimp and renders the shrimp susceptible to disease agents or cannibalism.
In addition, the shrimp's nutritional well being, size and immune response determine its degree
of resistance to disease agents. Behavioural characteristic such as burrowing at the pond
bottom also exposes the shrimp condition prevailing in the pond.

Environment

The term environment in aquaculture comprises the pond soil, rearing water and the
various living organisms in it. The living organisms include not only shrimp but also other
aquatic fauna and flora including pathogenic organisms. The survival and growth of the
organisms is largely influenced by various physico-chemical parameters such pH, dissolved
oxygen, temperature, light etc. Any abnormal change in these factors will adversely affect
shrimp in the culture system. For example, high ammonia level, low dissolved oxygen etc. are
stressful and may affect the survival of shrimp.

Pathogen

Various pathogenic organisms may be present in the aquaculture system. They may be
the part of the natural flora and fauna of the rearing water or pond soil. Various disease causing
organism of shrimp have been reported. Mere presence of these organisms may not cause any
disease condition. However, when present in large numbers these may readily invade the
injured tissues get established and multiply resulting in disease and death. Nevertheless, the
quantitative level of pathogen is influenced largely by prevailing culture condition such as
availability of food source, temperature, dissolved oxygen, pH etc.
 11. VIRAL DISEASES WITH SPECIAL REFERENCE TO 1NDIA.N SKRIMP
FARMlNG

T.C.Santiago, S.V. Alavandi and N. Kalaimani

Viruses are ultramicroscopic, infective agents capable of multiplying in the host living
cells causing improper cell knction or cell destruction leading to the death of the host. Viral
diseases constitute the most serious problems of shrimp culture due to the high infectivity,
pathogenicity and total lack of curative measures. Worldwide, shrimp aquaculture has
suffered substantial economic losses due to pathogenic viruses, and the Indian shrimp
farming is no exception. So far, 15 viruses infecting cultured shrimps have been recorded
across the shrimp farming countries of the world (Table). Till today, only five viruses have
been recorded from Indian farms.

Monodon baculovirus (MBV)

Kature of infection:
Monodon baculovirus (MBV) is the first viral pathogen to be recorded from the
cultured penaeids of India. Presently the virus is enzootic in Indian hatcheries and farms,
infecting both P. monodon and P. indicus. MBV infections have been observed in the
hepatopancreatic cells of all life stages of the prawn except egg, nauplius and protzoea 1 and
2 stages. Postlarvae and farmed shrimps of all sizes with severe MBV infections appear
normal and healthy. The virus, widely distributed in the cultured populations is well tolerated
by the shrimps, as long as rearing conditions are optimal. Hence, under good culture practices
the impact of the MBV infection can be minimal. However, under adverse environmental
conditions, MBV may predispose infected shrimp to infection by other pathogens, causing
poor growth, secondary infections and mortality.

Pathogenisis and diagnosis:

MBV is a single-enveloped, rod shaped, occluded double stranded DNA virus
belonging to the group baculovirus. The virus occurs freely or within proteinaceous
polyhedral occlusion bodies in the nucleus, with virions measuring 75-300nm. The presence
of MBV in the prawn can be detected by direct microscopic examination of impression
smears of infected hepatopancreas (HP) or midgut tissue, stained with 0.05 to 0.1% of
malachite green by demonstrating the usually multiple spherical intranuclear inclusion
bodies. Histological preparations of the infected HP can be used for further confirmation due
to the presence of prominent eosinophilic single to multiple spherical bodies within the
hypertrophied nuclei of the hepatopancreatic tubule or midgut epithelial cells. Transmission
electron microscopy (TEM) can also used to show the presence of MBV virions. DNA based
rapid diagnostic tools, polymerase chain reaction (PCR) and DIG-labelled DNA probes are
also available for the early diagnosis of MBV.

Prevention and controk

MBV infection may be prevented only through avoidance by quarantine methods,
destruction of contaminated stocks, and disinfection of contaminated facilities. There is no
treatment for MBV, however good farm management can minimize this disease.
Infectious Hepatopancreatic and Lymphoid organ Necrosis Disease (IHLN)

Nature of infection:
The first ever shrimp epizootic reported from India is the IHLN disease, from shrimp
farms located along the Kandaleru Creek, Nellore, Andhra Pradesh during July 1994. This
was a localized epizootic confined to the watershed areas of the Kandaleru Creek. The IHLN
affected the crops of culture duration ranging from 60 to 100 days weighing 3-28 g. The
onset of the disease was sudden and within 3-5 days post infection, more than 90 % of the
stock in the farms was lost. The disease prevailed in a virulent form for about three months.
Gross signs of the diseased shrimp were: light yellow or pinkish cephalothorax, reddish
discoloration of the body and appendages, empty gut, lethargy, poor escape reflex, secondary
bacterial infection and mortality. Dead shrimps were found scattered all over the pond
bottom. Only P. monodon was affected, P.indicus was found refractory to the disease.

Pathogenisis and diagnosis:

The most prominent feature of the disease was the highly melanized and shrunken
HP. Acute damages were observed in the HP, manifested by multi-focal necrosis of the
tubule epithelium marked by hemocytic infiltration and encapsulation resulting in
melanization. Densely stained, globular, basophilic bodies were observed in the HP cells and
lymphoid organ (LO). The mortality pattern and external signs of infection (yellow
cephalothorax) of the disease suggested resemblance to yellow head virus (YHD) in the P.
monodon. However, the prominent necrotic changes in the HP and LO and absence of
pathological changes in the gills, indicated that the disease clearly differed from YKD,
however, the etiology of the disease was of viral nature. The peculiar host specificity to P.
monodon, presence of basophilic globular structures resembling viral inclusion bodies in the
HP and LO, sudden and mass mortality, are the diagnostic features of IHLN disease.

Prevention and control:

None

Hepatopancreatic Parvo Virus (HPV)

Nature of infection:
The HPV has been observed in the heptopancreas of cultured P. monodon and P.
indicus. However, the infected shrimps did not show any external signs of the disease.
Further, the virus was not associated with any mortality. Gross signs of HPV may not be
specific, but in severe infections may include an atrophied HP, poor growth rate, anorexia
and secondary infections by pathogenic Vibrios. Only few samples (3-5 samples) of shrimps
collected during a 12 months period showed the presence of HPV, indicating the low
incidence of this virus in Indian shrimp farms.

Pathogenisis and diagnosis:

Histopathologically, basophilic inclusion bodies of HPV can be seen in necrotic and
atrophied hepatopancreatocytes. The HPV is a single stranded-DNA virus of 22-24 nm size.
DIG-labeled HPV gene probes are also available for the sensitive diagnosis of HPV.

Prevention and control:

Avoid the occurrence of the disease by quarantine methods and destruction of the
infected stocks. There is no treatment for HPV.
White Spot Disease (WSD)

The nature of infection:

The first incidence of White Spot Disease (WSD) in India was noticed in December
1992 in P. monodon and P,indicus from a few seawater based farms near Tuticorin,
Tamilnadu. Infected shrimps with prominent white spots on the cephalothorax region of
exoskeleton succumbed to death. The incidence was a localised one and did not cause alarm
due to the limited impact and localised nature. Since then far about one-and-half-years, there
was a temporary reprieve from the disease. However, during November 1994, the disease
staged a comeback in the shrimp farming belts of Andhra Pradesh and Tamilnadu. The
virulence of the disease was such that the cumulative mortality reached 100% after the
appearance of clinical signs in most of the infected farms, within a period of 3-10 days.
Disease affected the shrimps of all ages and sizes, extensive to intensive farming conditions
and all range of salinities. The most important fact about the WSD is its wide range of hosts,
i.e. it infects all cultured penaeids, crabs, lobsters and other crustaceans like copepods and
amphipods. Acutely affected shrimps showed lethargy and anorexia. The moribund shrimp
showed up on the water surface and gathered on the edges of the pond. By September 1995,
the disease spread to the shrimp farms in Kerala, Karnataka, Goa, Maharashtra, Gujarat,
Orissa and West Bengal. The impact was so severe that it forced the closure of many farms
creating a total chaos in the Indian shrimp aquaculture industry. During the period 1994-1995
alone, the shrimp loss due to the disease was about 15000 tonnes valued at Rs 500 crores.
Even now, the shrimp farms in the country are under the grip of this epizootic with changing
virulence.

Pathogenesis and diagnosis:

The causative agent of the WSD was found to be a rod-shaped virus, the white spot
virus (WSV). This non-occluded, enveloped, nuclear virus infects shrimp tissues of
ectoderamal and mesodermal origin. Typical clinical sign in infected shrimp is the
appearance of white spots or patches of 0.5 to 3mm in diameter on the inner surface of the
exoskeleton. In many cases, moribund shrimps displayed reddish to pinkish coloration
without any white spots. Histologically, the infection is characterized by eosinophilic to
progressively more basophilic inclusion bodies in the hypertrophied nuclei of infected cells,
due to the development and accumulation of intranuclear virions. Histopathological study
demonstrates that WSV targets various tissues originating from mesoderm and ectoderm,
particularly cuticular epidermis, gills, stoamch, lymphoid organs, hematopoietic and antenna1
gland. The disease could be diagnosed by histology and confirmed by TEM. New generation
DNA based diagnostic tools like gene probes and PCR are also available for the
asymptomatic detection of WSD.
Though morphological features, histopathological response, and mode of infection are
similar, the white spot disease has been named differently by various authors from different
countries: rod-shaped nuclear virus of Penaeus japonicus (RV-PJ) and penaeid rod shaped
DNA virus (PRDV) in Japan; systemic ectodermal and mesodermal baculovirus (SEh4.W)
and white spot syndrome virus (WSSV) in Thailand; hypodermal hematopoietic necrosis
baculovirus (WSBV) in China and white spot b a c u l o v i r u s ' ( ~ S and
~ ~ )white spot disease
(WSD) in Taiwan. The name white spot disease (WSD) for the disease, and white spot virus
(WSV) for the pathogen has been used for the Indian strain.

Prevention and control:

There is no treatment for WSD. Preventive measures include avoidance of the
disease by quarantine methods, destruction of known contaminated stocks, and disinfection
of the culture facility can help to remove the possibility of infection. Use of W radiation
and Ozone (physical disinfectants) and sodium hypochlorite, Benzalkonium chloride and
povidone iodine (chemical disinfectants) at proper doses has been found useful in inactivating
the WSV from the rearing systems.

Another interesting aspect of WSV infection in Indian shrimp farms is the changing
virulence status. During the last two years, many farmers were able to make a reasonable
harvest of 1-2 tonnes of 15-30g prawns, inspite of observing a few specimens with gross
signs of WSV infection present in their ponds during the initial phase of culture and then later
throughout the cultivation cycle. Similar observations were reported from other shrimp
farming countries of Asia like Thailand. This stands out against the situation of massive and
total mortality during the initial phase of the epizootic. It appears that either the shrimp is
learning to live with the virus (viral accommodation) or the virus itself is changing its
virulence to a less lethal level. However this phenomenon is not uniform across the country
and the incidence of WSD mortality is still common. It is essential to resolve the scientific
details of this phenomenon through research, which may be usefil in the control of the
white spot epizootic.
 12. BACTERIAL AND FUNGAL DISEASES OF SHRIMP

S.V. Alavandi

The bacteria causing diseases of penaeid shrimp constitute part of the natural microbial
flora of seawater. Accumulation of un-utilized feed and metabolites of shrimp in the culture
tanks1 ponds enrich the water with organic matter that supports the growth and multiplication
of bacteria and other microorganisms. Bacterial infections of shrimp are primarily stress
related. Adverse environmental conditions or mechanical injuries are important predisposing
factors of bacterial infections and disease. The most .common shrimp pathogenic bacteria
belong to the genus Vibrio. Other Gram-negative bacteria such as Aeromonas spp.,
Pseudomonas spp., and Flavobacterium spp., are also occasionally implicated in shrimp
diseases.

Bacterial Septicaemia (Vibrio disease)

Signs and Symptoms:

This is one of the severe systemic diseases caused by bacteria. The affected shrimps are
lethargic and show abnormal swimming behaviour. The periopods and pleopods may appear
reddish due to expansion of chromatophores and the shrimps may show slight flexure of the
abdominal musculature. In severely affected shrimps the gill covers appear flared up and
eroded. In more severe cases extensively melanised black blisters can be seen on the carapace
and abdomen.

Cause:
Bacteria such as Vibrio alginolyticus, V. anguillarum, ?i parahaemolyticus, Vibrio spp.

Diagnosis:
The bacterial septicaemia or systemic vibriosis is diagnosed based on the gross signs and
symptoms, and confirmed by isolation of pathogen from haemolymph by standard
microbiological methods and histopathology.

Prevention :
Maintain good water quality and reduce the organic load by increased water exchange.

Control:
Increase water exchange with good quality seawater. Feed shrimps with antibiotic fortified
feeds (only after ascertaining 'in-vitro sensitivity of the pathogen). e.g., feeds containing
oxytetracycline @ 1.5g K g , fed at 2-10% of body weight for 10-14 days along with
proper water and pond management. Sufficient withdrawal period (about 25 - 30 days) should
be allowed for the antibiotic to become inactive or harmless.

Luminescent Bacterial Disease

The luminescent bacterial disease is a serious problem in the hatcheries. Occasionally,
the juveniles and adult shrimp may also be affected in the grow-out farms.

Signs and Symptoms:

The infected larvae appear luminescent in darkness, and suffer heavy mortality.
Cause:
Luminescent bacteria, viz., Vibrio haweyi.

Diagnosis:
Goss signs and symptoms and microscopic demonstration of swarming bacteria within the
haemocoel of moribund shrimp larvae would confirm luminescent bacterial disease. The
luminescent bacteria can be readily isolated on Zobell's Marine Agar or a selective medium.
Identity of the isolates could be confirmed based on their morphological and biochemical
characteristics.

Prevention:
Use ultraviolet irradiated and chlorinated (calcium hypochlorite 200ppm for 1 h.) water.
Clean the debris collected at the bottom of the culture tanks daily.

Control:
Exchange 80% of water daily with UV sterilised / sand filtered seawater.

Brown spot disease (Shell disease or Rust disease)

Signs and Symptoms:

The affected animals show presence of brownish to black eroded areas on the body surface
and appendages.

Cause:
Bacteria such as Vibrio spp., Aerornonus spp., and Flavobacteriuin spp., with chitinolytic
activity.

Diagnosis:
Diagnosis of brown spot disease is achieved by simple observations on the gross signs and
symptoms and confirmed by isolation of the bacteria fiom the site of infection on Zobell's
Marine Agar and identification of the pathogen.

Prevention:
Reduce organic load in water by increased water exchange. Avoid unnecessary handling and
overcrowding to minimise chances of injury and infection.

Control:
Induction of moulting by applying tea seed cake may be useful. Improve water quality by
increasing water exchange. Although antibiotics may be useful their use in the culture sytem
is not recommended.

Necrosis of appendages

Signs and symptoms:

The tips of walking legs, swimmerets and uropods of affected shrimp undergo necrosis and
become brownish and black. The setae, antennae and appendages may be broken and
melanised.
Cause:
The epibiotic bacteria such as Vibrio spp., Pseudolnonas spp., Aeromonas spp. and
Flavobacterium spp.

Diagnosis:
Based on gross signs and symptoms.

Prevention:
Maintain good water quality. Stock shrimp at optimum density. Avoid unnecessary handling
of the shrimp, which may lead to injuries, leading to infection and necrosis.

Control:
Induction of moulting by applying 0.5 - 1 ppm tea seed cake may be of help.

Vibriosis in larvae

Signs and Symptoms:

The affected larvae show necrosis of appendages, expanded chromatophores, empty gut,
absence of faecal strands and poor feeding. Cumulative mortalities may be very high reaching
up to 80% within few days.

Cause:
Bacteria, viz., Vibrio alginolyticus, Zparahaemolyticus, and Zanguillarum.

Diagnosis:
Microscopic demonstration of motile bacteria in the body cavity of moribund shrimp larvae,
and isolation and identification of pathogenic bacteria would help in the diagnosis of the
disease.

Prevention:
Maintain good water quality and reduce organic load in the water by increased water
exchange.

Control:
10-15 ppm EDTA to the rearing water.

Filamentous Bacterial Disease

Signs and Symptoms:

The affected shrimp larvae show fouling of gills, setae, appendages and body surface.
Moulting of affected shrimps is impaired and may die due to hypoxia.

Cause:
Filamentous bacteria, such as Leucothrix mucor.

Diagnosis:
Diagnosis of filamentous bacterial disease could be achieved based on gross signs and
symptoms and by microscopically demonstrating filamentous bacterial fouling of body
surface and appendages of shrimp larvae.
Prevention :
Maintain good water quality with optimal physico-chemical parameters.

Control:
0.25 - 1 ppm Copper sulphate bath treatment for 4-6 hrs.

FUNGAL DISEASES

Larval Mycoses
It is one of the most devastating diseases in shrimp hatcheries. However, larval mycoses
have been successfully controlled during the recent years with better management practices.
Signs and symptoms: Affected larvae appear opaque followed by sudden mortality. The
protozoeal and mysis stages are highly susceptible. Within 1-2 day's, whole stock of shrimp
larvae may suffer mortality.

Cause:
Oomycetous fungi, Lagenidiunz spp, Sirolpidium spp, and Haliphthoros spp. These fingi are
filamentous, non-septate and coenocytic. Upon infection, the hngal mycelium replaces the
larval tissues and ramifies into various parts of the body. Vegetative propagation of these
hngi is through production of bi-flagellate zoospores, which are released into the rearing
medium. These zoospores further infect fresh shrimp larvae. These hngi can be isolated on
peptone yeast extract glucose (PYG) agar or Saboraud's dextrose agar.

Diagnosis:
Microscopic demonstration of presence of extensively branched non-septate, fungal hyphae
within the body cavity of the shrimp larvae.

Prevention:
Remove bottom sediments and dead larvae periodically. Disinfect the tanks and other
equipment in the hatchery from time to time. Treat spawners with 5 ppm treflan bath for 1 h.

Control:
When the disease is detected in early stages, Treflan (Trifluralin) 0.1-0.2 ppm bath for 1 day
may help in reducing mass mortality.
Other fungi such as Fusarium spp. cause infections in nauplii, protozoea, juveniles
and adults. Black gill disease is often caused by this fkngus. The hngus can be identified by
microscopic examination of its characteristic canoe shaped micro-conidia. Other oomycetous
fungi such as Saprolegnia spp. and Leptolegnia spp. are also known to affect shell of shrimp
and produce dark necrotic lesions causing gradual mortality.
 13. METHODOLOGY OF SHRlMP DISEASE INVESTIGATION

S.V. Alavandi, T.C. Santiago and N. Kalaimani

Proper and accurate diagnosis of diseases forms the cardinal step in any disease
control and prevention programme. To diagnose a shrimp disease problem, history of the
farms, the soil and water conditions of the ponds, incidence of any disease problem in the
adjoining areas, possibility of disseminating disease through birds or other carriers, are of
great importance. These are the significant information related to the epidemiology of the
disease. This section stresses the need to examine general information on the farming activity
and the information regarding disease on site and some points on collection of samples for
laboratory investigation.

1. Background information about the farming practices

A. Examination of ponds
This involves the various parameters of ponds such as methods followed in the
preparation of ponds, depth, nature of the bottom, water treatment methods, nature of water
inlet and outlet procedure etc. Apart fiom these, colour of water, algal blooms, turbidity of
water and presence of bioluminescence during night are also important criteria, which
determine the'health of the shrimp.

B. Stocking parameters
These parameters include origin and source of seeds, health status of spawners and the
larvae, survival rate of larvae within the hatchery and nursery, whether any antibiotic1
disinfectant used for larval rearing, stocking density, time of stocking etc. These parameters
are very much important in assuring the healthy or resistant nature of the larvae and in the
diagnosis of any possible disease problem.

C. Management practices
These include data generated out of the close monitoring of the system for the growth,
survival and occurrence of diseases. These also include the quality of the feed, feeding
regime, consumption of feed by the shrimp, time and rate of water exchange and the use of
chemicals, immunostimulants or bioremedial measures also to be recorded.

D. Environmental parameters
Diseases may be of infectious and non-infectious eteologies. Majority of the non-
infectious diseases are due to nutritional deficiency or due to abnormal environmental
conditions. Hence, a close examination and recording of the various parameters of water and
soil quality should be done periodically.

2. Field observation for signs and symptoms

A. Observation of behaviour of shrimp

Critical observation on the behaviour of shrimp will give an indication of the health of
the animals. Significant behaviour includes escape reflex, swimming at the surface, moulting
behaviour, feeding behaviour etc.
 Healthy shrimp will have quick reflexes i.e.. shrimp will respond instantaneously to
any outside disturbances or artificial stimulation; Shrimp showing poor escape reflex may not
be in a healthy condition.
Swimming at the surface is an indication of either inadequate oxygen level, or
respiratory impairment. Disease conditions, such as fouling, white spot disease etc always
show such behavioural changes.
Moulting behaviour is another important factor, which has to be observed. Regular
and continued moulting indicates continuous growth. Abnormal moulting indicates a diseased
condition. Chronic condition due to hepatopancreatic infection may cause abnormal
moulting.
Feeding behaviour is also an important indicator of health of the shrimp. Diseased
shrimp normally will show reduced appetite (e.g. white spot disease, protozoan fouling).
However, it has been reported that juveniles and sub-adult affected with yellow head disease
show an abrupt abnormal increase in feeding rate for several days.

B. Observation of external signs and symptoms

To assess the health status of a shrimp, following gross signs should be examined.

i. Colour and nature of exoskeleton: Healthy shrimp will have pale blue coloured,
bright, smooth and clear cuticle with proper hardness. Exoskeleton will show
brownish discoloration and occasional mat-like appearance (muddy cuticle) in
protozoan fouling. Moulted shrimp and shrimp with soft shell syndrome will show
soft exoskeleton. However, the shrimp with soft shell syndrome will have a hard
rostra1 spine.
ii. Apart from these, visible blisters or brown or black eroded areas on the exoskeleton
may indicate possible bacterial infection. Presence of white spots or patches on the
carapace indicates the visual disease, white spot disease.
iii. Appendages: Tips of walking legs, swimmerets and uropods may show necrosis and
become brownish black indicating a possible bacterial infection. Often, physical
injury may be a predisposing factor for bacterial infection and resultant necrosis and
melanisation.
iv.Musculature: White muscular opacity may indicate muscle necrosis due to
environmental stress or a microsporean infection.

C. Examination of internal organs for pathological signs

i. m: Gills are normally clean, semi-transparent and colourless. External fouling due
to epicommensals will cause dark yellow discoloration. Bacterial infection can cause
blackening of gills. Vibriosis may cause yellow discolouration of branchiostegites.
ii. Heuato~ancreas:Hepatopancreas of normal shrimp will be obvious, with proper size
and shape. Colour of top half is brown and the bottom has a white membrane cover.
Abnormal colour, enlargement or atrophy etc. are indication of bacterial or viral
infection, or the presence of toxic substances or nutritional deficiency.
iii. Haemolvmuh: Haemolymph of normal shrimp have slight blue colour. It will easily
coagulate in 1 min. after taking it out from shrimp. Some of the bacterial and viral
infections cause the haemolymph non-coagulable, colourless or light reddish or muddy
in nature.

3. Collection of samples
For accurate diagnosis of the disease, typical and representative sample of infected
animals should be collected. Very often, in one pond itself there will be multiple infections.
All the dead animals may not be a representative sample. Instead of dead animals, moribund
animals will be suitable for analysing the symptoms, for pathological studies and for isolation
of pathogens. Moribund shrimp may have secondary infection also, and more often, shrimp
with disease in the initial stage may not exhibit the real symptoms. All these factors should be
taken into account while collecting the required sample.
According to many experts, there are four methods to collect the samples of shrimp:
i. Picking from the sides around the ponds
ii. Catching from the middle of the pond
iii. Using cast net
iv. From the feed trays.
These samples will really reflect the actual disease status in pond. Samples of
moribund shrimp, which are collected fiom the sides around the ponds, will be mostly at the
terminal stage of infection. The samples collected &om the middle may be in an intermediate
stage. Cast net will give a random sample and is preferable, while the samples from the feed
tray will be usually healthy.
 14. MOLECULAR DIAGNOSIS IN SIlRZMP DISEASE WITH SPECIAL
REFERENCE T O PCR O F INDIAN M I T E SPOT VIRUS
T.C. Santiago, S.V. Alavandi and N. Kalaimani

Introduction
Increasing disease problems mars the growth of promising aquaculture industry both
nationally and internationally. Vulnerability of the aquaculture production system to disease
is due to the co-existence and close interaction of host, pathogen and environment. A small
shift of equilibrium between these three can trigger a disease outbreak leading to mortality
resulting in crop failure. The latest viral disease problem in shrimp farming arena due to the
white spot virus exposed the vulnerability of aqua-business.
Disease problems are inevitable, as aquaculture has to look forward to produce more
animal protein, more jobs and more revenues for the people. To tackle the menace of disease
problems, a scientific health management approach has to be developed emphasizing the
conventional wisdom - 'prevention is better than cure'. An integral part of such a program is
the use of diagnostic tests at the strategic point of production cycle to eliminate or control the
disease causing pathogens.

Conventional diagnostic methods

The conventional diagnostic methods practiced in aquaculture are mostly adapted
fiom the field of human health and veterinary sciences. Among the diagnostics mentioned
(Table I), visual examination, microscopic, histological examination and bacterial
examination are the most widely used and still form the essential part of disease diagnosis.
But these methods often fail to deliver data in time to support a decision making in the
health management to salvage the crop. This is mainly due to the time consuming and
laborious methodologies and the inability of these tests to detect sub-clinical I latent / carrier
state of infection.

DNA - based diagnostics

Developments in molecular biology enabled researchers to collect information on the
genetic material that serves as the blueprint for all living organisms. The most recent
development in diagnostics have utilized molecular biology to design new generation of
diagnostics tools, the Polymerase Chain Reaction (PCR) and Gene Probes. These DNA-based
diagnostic tools stand out among other conventional diagnostic methods with its speed,
sensitivity and simplicity. Polymerase Chain Reaction and Gene probes capable of
identifying a number of viral, bacterial and parasitic pathogens are finding their way into the
area of infectious disease diagnosis in aquatic species.
Table 1: Methods available for disease diagnosis and pathogen detection

Method Tests and data obtained

History History of disease at facility or region, facility design, source of seed, type of
feed used, environmental conditions etc.
Gross, clinical signs, lesions visible, behaviour, abnormal growth, feeding or
food conversion efficiency, etc.
Direct microscopy Bright-field, phase contrast, or dark field examination of stained or unstained
tissue smears, whole-mounts, etc. of diseased or abnormal specimens
Histopathology Routine histological or histochemical analysis of tissue sections
Electron microscopy Ultrastructural examination of tissue sections, negatively stained virus
preparations, or sample surfaces
Culture and biochemical Routine culture and isolation of bacteria and identification using biochemical
characterization reactions
Enhancement Rearing samples of the appropriate life stages under controlled conditions to
enhance expression of latent or low grade infections
Bioassay Exposure to potential pathogens
Serological methods Use of specific antibodies as diagnostic reagents in immunoblot,
agglutination, ELISA, IFAT, or other tests.
Tissue culture In vitro culture of pathogens in cell lines
DNA probes Detection of unique portion of a pathogen's nucleic acid using a labelled
DNA probe
PCR Amplification of unique portion of a pathogen's genome to readily detectable
concentrations using specific primer pairs

The key to the DNA based diagnostics is the generation of unique genetic information of
the target pathogen through recombinant DNA technology. This is done by purifying the
infectious agent of interest (Fig. 1) and isolating the nucleic acid. Isolated DNA is then
subjected to restriction digestion and cloning. From the selected clones, desired DNA
fragment has to be sequenced. Once the adequate genetic information (sequence information)
is generated, the information can be used in PCR or gene probes.

Polymerase Chain Reaction (PCR)

PCR is relatively a simple technique by which a DNA or cDNA template is amplified
many thousand or a million fold quickly and reliably in a short period of 3-4 hours. So far no
other technique has equalled PCR in sensitivity, which is about one DNA target molecule.
A typical amplification reaction includes
I. The sample of the target DNA
11. Two oligonucleotide primers
111. Four Deoxynucleotide triphosphates (dNTPs)
IV. Reaction buffer
V. Magnesium and optional additives
VI. Taq-DNA polymerase
vIr. ~ ~ H Z O

The components of the reaction are mixed and placed in an automated instrument
called thermocycler that takes the reaction to a series of different temperatures for varying
amounts of time. This series of temperature and time is referred to as one cycle of
amplification. In each cycle of amplification the quantity of target DNA doubles, and as few
as 20 cycles would generate approximately a million times the amount of target DNA which
was present initially.
The first step of PCR involves thermal denaturation of the double-stranded target
DNA molecules. The next step is the annealing of oligonucleotide primers to the
complementary target sequences by temperature reduction. Thereafter, primer directed DNA
synthesis reaction will follow with the help of thermostable DNA taq polymerase, resulting in
the doubling of the amount of target sequence in the sample. By repeating the cycle of
denaturation, primer annealing and DNA synthesis (primer extension), the copy number of
the target DNA is increased exponentially.
Once a product is obtained, it can be analyzed in a number of ways like agarose gel
electrophoresis. The products will be readily visible by UV transillumination of an ethidium
bromide stained gel.

Nested PCR
Nested PCR or two-step PCR is useful in reducing or eliminating unwanted products
simultaneously increasing the sensitivity significantly. An aliquot of the first PCR product is
then subjected to an additional round of amplification using primers complementary to the
sequences internal to the first set of primers. Only the legitimate product is amplified in the
second round. This approach of two-step amplification is often successful even if the
designed product is initially below the level of detection by ethidium bromide staining.

Optimization of PCR
Designing of an ideal primer pair, optimisation of the concentration of Mg, primer
and template DNA, buffer pH and cycling conditions are important for the success of a PCR.
Ideally PCR primers should have 40 - 60 % G+C content, which generally range in length
from 15-30 bases. Negative controls are mandatory in each PCR run to rule out any false-
positive results caused by contamination. However, every step should be taken to avoid the
possible contamination during the setting up of a PCR.

Nucleic Acid Probes

Nucleic acid probes are segments of DNA or RNA that have been labelled with
enzymes, antigenic substances, chemiluminescent substances or radioisotopes. Probes can be
directed to either DNA or RNA targets. Probes can bind with complimentary sequences of
pathogenic DNA during the detection process providing a signal (like colour change) that can
be identified or measured. Nowadays, non-radioactive probes (eg: digoxigenin (DIG) labelled
probes) are gaining importance due to their high level of sensitivity and safety as compared to
the radioactive probes. The in-situ hybridization and dot blot hybridization are examples of
gene probes, which are finding its use in aquatic disease diagnostics. However, PCR has
advantages over the gene probes in its sensitivity to be used for direct detection in clinical
specimens.

-
PCR based diagnosis of Indian white spot virus
In the aquaculture of penaeid shrimps, White Spot Disease (WSD) caused by White
Spot Virus (WSV) is the major cause of morbidity and mortality today in Asia, resulting in
huge economic losses for shrimp farmers. Among all the recorded viral diseases in cultured
penaeids, WSD is the most widespread. The rapid onset and lethality of WSV has put Asian
shrimp farming as a whole at the breaking point. The WSV was first reported in 1992 from
Taiwan, subsequently from most shrimp farming countries in Asia and recently firom the
western hemisphere.
White spot disease has diminished the prospects of shrimp farming in India. This is
the most virulent virus known to affect cultured shrimps. Till date, no treatment is known to
control the White Spot Disease. Hence, early diagnosis followed by suitable management
practices is the only alternative in tackling this disease. Diagnosis of WSD can be done by
methods such as histopathological techniques. A presumptive diagnosis can also be done by
observing clinical symptoms such as the presence of white spots. These methods can detect
the WSD only in the late stage of infection. The PCR is a powerfit1 and sensitive diagnostic
tools for identification of viral pathogens even at a very early stage (asymptomatic / carrier
stage) of infection.

Purification of Virus, DNA extraction and sequencing

In the case of white spot virus, ectodermal or mesodermal tissues of the shrimp
infected with WSV can be used for viral purification. The purity of the virus is checked using
2 % PTA stained TEM. Viral DNA isolation is done using proteinase K and CTAB treatment
followed by phenol-chloroform extraction and ethanol precipitation. After checking the
purity of the DNA using electrophoresis, sequence information of WSV is generated
following cloning and sequencing of WSV genome.

Simplified scheme of diamosis of the Indian white-spot disease by volvmerase chain

reaction in 4-6 h.
I Broodstock/ post-larvael field sample I

C
I Eve-stalk1 Pleooodl haemolvmoh 1
f
1 Homoaenise with buffer

I Boil for 10 rnin I

Cool, Centrifuge and Collect Supernatant

(Template DNA)
I
+
PCR 13 hours)

+I
I Electrophoresis (45 min.)
I

PCR Primers for Indian WSV

e Diaanosis

Two sets of PCR primers have been designed from the sequencing information of a
fragment of Indian White Spot Virus, for the lStstep and Second step (nested) PCR
amplification by scientists of CIBA, with products of 600 bp and 300 bp respectively.
PCR - Protocol
PCR and agarose gel electrophoresis are used in conjunction to determine the presence
or absence of WSV virus in shrimp. The standard operation procedure consists o f

I. DNA - template preparation 1 nucleic acid extraction

Genomic DNA extracted from the infected animal tissue or DNA-template
preparations using simple methods like boiling of the sample with a suitable buffer can be
used as a template for WSV PCR. Samples used for template preparations can be stored
frozen or preserved in 70-95 % ethanol. The template can be stored frozen at - 20 to -70 OC.
11. PCR preparation and reaction
1. Prepare a master mix of the following components aliquot into individuaI 25 p1 PCR
reaction vials.
dd-H20 12 p1 X number of samples
10 X buffer 2.5 1.11 X number of samples
dNTP solution* 4 11.1 X number of samples

Prepare solution by adding 10 pM stock solutions of dNTPs into the ratio of

One part dATP: 1 part dGTP: 1 part dCTP: lpart dTTP: 4 parts ddH20
Primer A (pM) 1p1 X number of samples
Primer B (pM) 11.11 X number of samples
25 m M MgClz 1.51-11
22 pl master mix per vial
To each PCR reaction vial containing 22 pi master mix, add template DNA (5ngIpl): 2p1

2. Program thermal cycler as follows:

1 Cycle of: 95 OC for 3 minutes
30 Cycles of: 95 OC for 0.5 minute
58 OC for lminute
72 OC for lminute
1 cycle of: 72 OC 5 minutes

3. Place the reaction tubes in thermal cycler, heat to 95 OC and hold at that temperature.
4. Add lpl(1 pl of a 0.5 U solution) Taq polymerase to each sample. Resume
program.When program is complete, remove samples, add 5p1 of loading buffer to each
sample mix well and spin briefly.

Agarose gel electrophoresis

1. Assemble large gel electrophoresis box to run a maximum of 20 samples.
2. Prepare a 2.0 % agarose gel by adding 1.46 grams of agarose to 73 ml of 1 X
TBE buffer(* 1 X TBE buffer is prepared by diluting 5 X TBE. 5 X TBE is
prepared by adding 54.0 grams Tris base and 27.5 grams boric acid to
980 ml of dHnO. Add 20 ml of 0.5 M EDTA (pH 8.0). Filter solution
through a 0.45-micron filter).
3. Cover gel with 1 X TBE and load samples. Load 5 p1 of PCR marker into
one lane of the ge1.Run the gel at 80 volts for about 1.5 hours, or until the
loading dye lane runs off the anode side of the gel.
 4. Place gel in a 0.5 pglml ethidium bromide dye solution for 15 to 20
minutes.(A 0.5 pglml ethidium bromide solution is prepared by adding 10 pl
of a 1% stock solution to 200 ml of 1 X TBE buffer).
5. Remove the gel from ethidium bromide solution and rinse TBE buffer. Place
gel on a W transilluminator with filter 312 nm to visualize PCR product and
photograph.

Conclusion
Molecular diagnostic technique like PCR provides a fast, sensitive and specific tool
for disease diagnostics in aquaculture. The selective amplification of a small segment of
pathogenic DNA from the mass of unrelated host DNA sequences to the detectable level, and
that too fiom an extremely small quantity of the starting material, makes PCR a revolutionary
technology. The PCR has a wide and significant use in checking the entry of lethal pathogens
like virus to the hatchery and growout system by helping to select healthy broodstock and
seeds. This can also be used in the epizootiological study of the pathogen in an effort to draw
disease controi measures. In the coming days, DNA based diagnostic tools are going to play a
major role in the newly evolving aquatic animal healthcare.
 15. SHRlMP FARM MANAGEMENT WITH SPECIAL REFERENCE TO SOCIAL,
EEVIRONMENTAL, HEALTH AND FOOD SAFETY ISSUES

S.M. Pillai and P. Nila Rekha

1. Introduction
Shrimp farm management has two important aspects - a) Technical management to
assure good production and productivity, and b) General management to insure that the farming
practice is socially acceptable, environmentally safe and conform to the food safety standards of
the importing countries. Increasing focus is being placed on the safety of foods being sold in the
world markets. These concerns include not only ensuring that foods for human consumption are
free from excesses of harmful or undesirable chemicals, but also that the workers producing
these foods and the environment surrounding the production facility have been protected from
negative effects of the use of these chemicals. Increasing calls for total traceability of food
products are also affecting the food production industry such that consumers can be assured that
the product has been produced without the use of transgenic technologies, without addition of
undesirable or harmful chemicals or additives and that all the environments and ecosystems
affected by the production facilities has not been compromised in any way.

2. Social issues
Shrimp farming has led to many debates and discussions on issues revolving around
socio economic fabric of the coastal communities. Most of the issues like salinisation of
agricultural lands and drinking water could be solved by selecting suitable site as discussed
under site selection. The other major issue is employment. Shrimp farming, no doubt, enhances
employment opportunities but the following aspects should be taken into account to avoid social
unrest.

Minimize conflicts with local communities that may result from shrimp farm
development and operation and ensure that aquaculture developments are mutually
beneficial.
Take measures to ensure shrimp farming benefits the communities in shrimp f m areas.
Ensure shrimp farm workers welfare and fair working conditions. Minimum wages
should be provided to the labourers.
* Services of women labourers should be utilized.
Child labourers should not be engaged.

3. Environmental issues
Aquaculture can be considered as an integral part of the natural environment, and the
interactions between aquaculture and the environment are inevitable. The major natural resources
used for aquaculture are land, water and biological inputs such as seed and feed. The level of
environmental impacts of shrimp aquaculture are dependent on site condition, geographical
location ,tidal characteristics, species cultured, type of farming practice, degree of intensification
and quality of inputs

The various environmental issues in shrimp farming are destruction of mangrove

ecosystem, conversion of agricultural land, soil salinisation, nutrient loading from shrimp farm
to the open water bodies, self pollution and salinisation of freshwater aquifers. Perhaps the most
important one to be managed at the farm level is the wastewater management.
The waste from shrimp pond contains mainly suspended solids, comprising of
unconsumed feed, faecal matter and plankton, dissolved nutrients such as ammonia, nitrite,
phosphorous, carbon-di-oxide and hydrogen sulphide. Proper designing of the farm with
independent intake and outfall, adequate compaction of bunds with vegetative cover, appropriate
pond preparation measures and apt water and soil quality management in the culture ponds will
reduce the nutrient loading of wastewater. Above all responsible feed management will reduce
feed wastage which in turn reduce the nutrient loading .The following guidelines are to be
adopted for better waste management.

The wastewater should be treated before discharged into open waters or in the drainage
canal. The drainage canals may be designed in such a way that are wide enough to
slow down the flow of water from ponds so as to allow the settlement of these suspended
sediments. Larger farms above 5.0 ha should have an Effluent Treatment Pond (ETP), to
reduce the nutrient load in the wastewater. The ETP may include settlement tanks and
bioremediation tanks.
During harvest, water should be drained carefully avoiding re-suspension of sediment
into the waste stabilization ponds and kept for a few days for settlement before releasing
into the open water.
Planting of mangroves in drain canals and effluent treatment ponds could improve the
water quality by absorbing nutrients and other organic pollutants. The mangrove
plantations along the bunds help to stabilize and prevent soil erosion and reduce the
sedimentation of ponds during flood.
Shrimp farmers should form cooperatives, associations or self help groups in order to
exchange technology and to achieve cooperation in wastewater use and waste
management.

4. Sanitation and Phytosanitation (SPS) issues

The SPS issues have assumed greater importance with concerns for human, animal and plant
health safety. The presence of human pathogens in imported shrimp leads to immediate rejection
of the imports. The introduction of such pathogens into the culture area could come only
through improper sanitation. The following guidelines should be followed to ensure human
health safety.
All solid waste materials should be scientifically disposed, away from the farm site.
No septic runoff from human or other animals should enter the farming area. Facilities
for proper disposal of such wastes should be incorporated while designing the farm itself
and it should be located away from the culture area.
Farm ponds should not be allowed to be used for any human or other animal activities
like bathing or washing etc.
Post-harvest handling of the shrimps like washing and immediate icing should be done
as per the requirements of the processors.
 5. Shrimp health issues
Biosecurity has become an essential requirement in shrimp farming systems due to the
continued viral disease outbreaks. Viral pathogens could enter the culture system through a)
seed, b) water and c) carriers. It spreads into the system through water, personnel and
equipments. The following guidelines should be strictly followed.

No outside vehicle should be allowed near the pond area or precautions like tyre bath
should be introduced.
No pets or live stocks should be allowed on site.
Water should be filtered through double filter bags to avoid the entry of aquatic carriers.
Disinfection of water in reservoirs can be adopted in large farms.
Bird scares are provided so that dead shrimps are not brought in by the birds.
Feed and other inputs should be procured as per the requirement and stored in dry,cool
place. Long-term storage should be avoided.
Nets and other materials should be disinfected before use in any pond to avoid the
horizontal transmission of virus. Even the personnel and labour should be careful to
disinfect themselves when they work in different ponds.

6. Good Management Practices

Adoption of paper management strategies depending on the environmental characteristics
of sites, system of culture, type of management and the needs of the local population will
ultimately lead to sustainable development of shrimp farming. The objectives of Goodl Better
Management Practices (GMP/ BMP) are a) environment protection, b) improved shrimp health,
c) improved food safety, d) socio-economic sustainability and e) profitable farming.
GMPJBMP s includes every aspect of shrimp farming starting from site selection to post-
harvest handling of the produce. Implementation of GMPsI BMPs are presently meant for
voluntary adoption of the farmers. But with the strict standards that are being imposed by
importing countries, these might become mandatory requirements very soon.
The requirement of total traceability from production to consumption is becoming a
necessity in view of the rejection of imports due to non-compliance of food safety standards. In
shrimp exports the major food safety issues are antibiotic, heavy metal and pesticide residues. It
is necessary to locate the points of introduction of these materials into shrimp farming system.
To establish product traceability, the following data shall be recorded for each pond and each
production cycle
pond identification number
pond area
pond preparation
stocking date
source of postlarvae (hatchery)
quantity of postlarvae stocked
seed acclimatization
antibiotic and drug use
herbicide, algicide and other pesticide use
manufacturer and lot number for each feed used
 soil, water and feed management
6 growth assessment
4 harvest date
* harvest quantity
processing plant or purchaser

7. Organic farming
Organic shrimp farming is one of the future options for fetching higher premium price.
The designation "organic" refers to a production process, not food safety or residue testing.
Organic farming delivers the highest quality, best-tasting food, produced without artificial
chemicals or genetic modification with respect to animal welfare and environment protection,
while helping to maintain the landscape and the welfare of the rural communities. The main
principles of organic shrimp farming are:
* integration of natural plant communities in the farm management
utilisation of natural breeding methods, without the use of hormones and antibiotics
* absence of GMOs in stocked animals and feed
limits on stocking density
* feed and fertilizer must be from certified organic agriculture
* use of feed ingredients which are not appropriate for human food
there are certain criteria for fish meal sources and limits on its use
the use of synthetic pesticides, herbicides, growth promoters, colouring agents, etc is
avoided
restrictions on energy consumption (eg., for aeration)
cultured organisms should not be subjected to any kind of mutilation
discharge water utilization to raise productive crops and enhancing the biodiversity

8. Cluster1 Group farming

In India more than 90% of the farmers are small holders owning less than 2 ha of water
spread area and in most cases they share the same water source with other small farmers. Further
in most cases the intake and outfall from the shrimp farms are in the same creek. With such a
system, all the farms located within a source creek should be considered as a single unit for the
purposes of management to avoid disease spreading and environment safety. In view of this,
globally small farmer groups are encouraged for collective management of the resources.
NACA-MPEDA study on Group farming in Andhra Pradesh has successfully demonstrated that
group farming and implementation of BMPs reduced the disease incidence and increased the
productivity.
Group farming has the following advantages
r Uniform stocking time with disease free seed from a single contract-hatchery
Water intake and discharge regulated by the group
Disease incidence reported and water disinfected within the pond
Collective bargaining power for inputs like seed and feed
r Planned harvesting and demand for correct price from the processor, eliminating the
middle-men
 16. HARVEST Alrc?) POST-HARVEST HANDLING OF CULTURED SHRIMPS

M. KATHIRVEL AND S. KULASEKARAF'ANDIAS

INTRODUCTION
Harvest and post-harvest handling are the final phases of a culture operation. The
economy of the culture operation depends on the quantity harvested and that of marketing on
the quality achieved. In the traditional shrimp culture practices in India, harvest is carried out
by nets, bamboo-screen traps and complete draining of pond water and subsequent hand
picking, For burrowing species like Kuruma shrimp (Marsupenaeus japonicus), a
mechanically driven drag net is operated for harvest in Japan. The present paper deals with
the nets and other methods used for harvest of shrimps from traditional and the present-day
culture operation in India and the criteria to be adopted for harvest and post-harvest handling.

TRADITIOAN.4L SHRIMP FARMING

NETS
Conical bag net (Fig.1):

Fig. 1. Harvest by a conical shaped net from a seasonal shrimp-cum-paddy field

It measures 5 to 9 m in length with a mesh size of 5 to 13 mm and is fixed to the mouth of the
sluice gate by means of a rectangular bamboo frame. The net is operated during the low tide
when the water is let out through the sluice gate at night. A light is installed near the mouth
of the sluice to attract more shrimps. The shrimps trapped in the cod ned of the net are
removed periodically into a stationed canaoe in the outer canal. Fishing by the net is carried
out for 7 to 8 nights in a fortnight, i.e. 3 or 4 nights on either side of the full moon and new
moon. This type of gear is extensively used in the traditional paddy-cum-shrimp cultivation
of Kerala as well as in the bheries of West Bengal.
Cast net (Fig. 2):

T>

Fig. 2. Cast net in operation

If the water depth is one metre, it should be reduced to 0.5 metre and then cast can be
operated to catch the cultured shrimp. Two cast netters can be employed for the harvest from
a 0.4 ha pond.

Drag net (Fig. 3):

Fig. 3. Drag net in operation

When the water level in the culture pond is knee-deep, drag net can be operated. The drag net
is more suitable to tiger shrimp (Penaeus monodon) than the Indian white shrimp
(Fenneropenaeus indicus) because the tiger shrimps tend to cling to the net, while the white
shrimps will tart back from the net and escape.
TRAP
Bamboo trap (Fig. 4):

Fig. 4. BIteries in West Bengal where bamboo traps are used for catching shrimps inside
the field

In the bheries of West Bengal, traps made of bamboo are laid in the bottom of the pond near
the sluice gate to catch the cultured shrimp, mostly during the low tide .

HAND-PCIKING
In the final harvest of shrimp farming in paddy fields, the water is drained through the
sluice during the low tide and hrther draining is effected by pumping. When the water level
is almost nil, the cultured shrimps are hand-picked easily.

PRESENT-DAY SHRIMP FARMING

In tide-fed ponds, the maximum water is drained through sluice and the harvest is
done by conical net fixed to the sluice. While the water is drained through sluice, the
maximum number of shrimps which move along the water current are caught by conical net.
Later, bag nethast net are used and finally, after pumping out the remaining water, shrimps
are collected by hand-picking. In pump-fed ponds, the water is partially pumped out and the
shrimps are harvested by bag nethast net and after complete draining, the remaining shrimps
are collected by hand-picking. In those ponds where the complete draining is not possible,
the cast net is carried out for 3 to 4 days for harvest of shrimps.

CRITERIA FOR HARVEST AND POST-HARVEST HANDLING

To retain the freshness and quality and without muddy smell in the cultured shrimps,
the following criteria are suggested.
Harvest
1. Before the harvest, removal of algae and keeping the bottom of pond free fiom dirty
materials are to be done.
2. Frequent exchange of water is to be done to remove algal water, in case of heavy
bloom is present.
3. If moulted shrimps exceed 10 %, the harvest should be postponed. Also do not
exchange or reduce water 3 to 4 days from harvest.
4. Do not feed the shrimps 6 hours prior to the harvest, in order to keep the stomach
empty, thus improving the shell life.
5. Draining of water and harvest should be completed within 6-8 hours, mostly between
6 P.M. and 6 A.M.
6. Harvest and packing of shrimps should not be done during hot days.
7. Usage of bag net in knee-deep water will catch more shrimps than cast net.
8. Wherever, the complete draining is not possible, lead screens may be fixed in deeper
areas of the pond, which will guide the shrimps towards the bag net from where
shrimps can be collected.

Post-harvest handling
1. Shrimps harvested by bag net and hand-picking should be separately kept. Those
hand-picked shrimps should be thoroughly washed and packed separately.
2. Clean water should be used for washing shrimps and ice made up of potable water
should be used for packing.
3. The washed shrimps should be immersed in slurry of ice for not less than 15 minutes,
which enhances freshness of shrimps and increasing their weight by 5 %.
4. Do not use any chemicals while washing the shrimps or chill killing without
knowledge of processors.
5. Adequate use of ice will ensure the chilling of shrimps.
6. Harvested shrimps can be packed in plastic tubs with crushed ice at 1:l ratio (i.e. 1 kg
of shrimps versus 1 kg of ice) for better preservation.
7. Before stacking the packed plastic tubs one above the other, the cleanliness of the
bottom of each tub should be attended to.
8. The packed tubs should reach the processing plants quickly without any delay, which
will ensure better quality.