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CtSA SPL PUBLlCAnON

ARTEMIA CULTURE

CENTRAL INSllTUTE OF BRACKISHWATER AQUACULTURE

(Indian Courlcll of Agricultural Research)
75, Santhome High Road, R.A. Puram, Chennai-600 028

wim
IC'AR

October 2003

No. 19

CiBA SPL PUBLlCA TiON

Training course material

A R T M CULTURE

CENTRAL lNSTlTUTE OF BRACKISHWATER AQUACULTURE

(Indian Council of Agricultural Research)
75, Santhome High Road, R.A. Puram, Chennai-600 028
October 2003

No. 79

CONTENTS

S. No.

Title of the paper

Page No.

1.

Biology and distribution of Avtemia.

2.

Pond culture of Artemia for biomass and cyst production.

12

3.

Artemia biomass production in controlled systems.

25

4. Artemia cyst hatching, separation of hatched nauplii,

cyst decapsulation and cyst quality analysis

BIOLOGY AND DISTRIBUTION OF ARTEMIA

BY

S. KULASEURAPANDIAN and M. KATHIRVEL

Central institute of Brackishwater Aquaculb.ure, Chennai

Introduction
Artemia is commonly known as the brine shrimp. It is a crustacean.
It lives in high saline waters. It is widely distributed through out the world. It is
the most important live feed organism. The genus Artemia is comprised of both
bisexual and parthenogenetic strains. All parthenogenetic strains of Arfemia are
commonly designated as Artemia parthenogenetica even though they have
important differences in ploidy and isoenzyme pattern. Among the bisexual
strains, mainly six sibling species have been described so far.

They are Artemia salina (reported from England but now extinct), A.
tunisiana (from Europe), A. franciscana (from North, Central and South
America),A. monica, (from Mono Lake of California, U.S.A.), A. persimilis (from
Argentina) and A. urmiana (from Iran).

Biology of Artemia
Structure
Adult

Hatching

Fig. t . Life cycle of the brine shtimpdrtemia sp. A. Antennule; B. Antenna;

C. Compound eye; D. Thoracopods; E. Uterus; F. Eggs; G . Abdomen;
H. Caudal furca; I . Mandible; J . Median eye; K. Developingthoracopods.

In general, the adult Artemia measures about 10 mm in total

length and about 1 mg in weight. However, in some polyploid parthenogenetic
strains, the total length goes upto 20 mm. It has an elongated body which is
divided into three parts, namely head, thorax and abdomen (Fig.1). The head
region consists one pair each of antennules, antennae and stocked eyes (Fig.1.
A, B & C). Thorax has eleven pairs of appendages which are known as
thoracopods (Fig.1.D). The abdomen ends in a furca, covered with spines
(Fig.1.H). The antennules and antennae are sensory in function in females.
However, in males of the bisexual strains, the antennae are developed into a pair
of clasper (Plate 1 .A). Further, male has penis while female possesses a uterus
or ovisac (Fig.1 .E & Plate 1. B). These features form as secondary sexual
characters.

Reproduction

The eggs develop in paired ovaries, situated on both sides of the

digestive tract behind the thoracopods. Fully developed oocytes are transferred
via the oviducts to the ovisac

In the bisexual strain, precopulation in adult

Artemia is initiated by the male in grasping the female between the uterus and
the last pair of thoracopods with its muscular claspers. The couples can swim in
this so called "riding position" for long periods of time, beating their thoracopods
in a synchronized fashion. During copulation, the male transfers the sperm to

the uterus of the female. Fertilization takes place in the uterus. Further
embryonic development occurs in the uterus. In the case of parthenogenetic
strain, there is no such fertilization. Embryonic development starts as soon as
the eggs from ovaries reach the uterus (Fig. 1.F).

In the uterus, the embryo develops into nauplius. Otherwise, it is

coated with shell to form as cyst. This changeover is caused by the prevailing
environmental conditions. If conditions are favourable, nauplii will be released.
This mode of reproduction is called as ovoviviparity.

During unfavourable

conditions, the embryo will be released as cyst after being coated with shell.
This mode of reproduction is known as oviparity. The liberated cysts are about
200-300 microns in size (Fig. 1 & Plate - 1 .C).

Feeding and development

The newly emerged nauplius is about 400-500 microns in length

and 0.002 mg in weight (Fig. 1. & Plate - 1.D). It can not feed for want of mouth
parts. Hence, utilizing the reserve yolk material present in the body, it grows and
after 12 hours, it moults into second larval stage, lnstar I1 nauplius, which can

Plate - 1

A. Adult male.

Dry cysts

D. Nauplii

feed through the newly formed mouth. Artemia is a continuous filter feeder. It
feeds on particles, which are less than 50 microns in size. The second instar
nauplius uses its second antennae for filtering the water. During development,
thoracic appendages are differentiated in relation to their function as locomotary,
respiratory and filter feeding appendages. Artemia grows from nauplius to adult
stage by passing through metanauplius (Juvenile) (Plate-2.A). post-metanauplius
and post-larval stages during which period, it has to undergo about 15 moults.

Plate-2.A. Metanauplius
(Juvenile)

Plate-2.B. Altemia
Adult Biomass

From 1 0 ' ~larval stage onwards, important morphological as

well as functional changes take place. Thoracopods are now differentiated for
locomotion, respiration and filter feedig. Adult stage reaches in about two weeks.
During this short period of development, 20 fold increase in dimension and 500

fold increase in biomass is ensured. Brine shrimp can live for about 6 months.
After attaining adult stage, the brine shrimp produces about 300 naupliilcysts per
batch (according to the prevailing environmental conditions) at an interval of 5-7
days during its life period. Under favourable food availability and environmental
conditions, Arfemia multiplies by ovoviviparous mode of reproduction (releasing
nauplii directly) and attains a stage with thick biomass (Plate-2.B). It goes for cyst
production under unfavourable environmental conditions to ensure the survival
for future generation (Fig. 2).

.-."""..'.-----

Dry cysts

CJ

hour S e a

water

i-iydrated c y s t s

2 4 hours S e a w a t e r

opZimcli b a t c h i p 9 conditions
E-7

Cysfn

b r e a k i n g rtaga

'

up f n 9PC3
every 5 days

up to SCaO
every Sdays

Fig. 2. Schematic diagram of Artemia life cycle

DISTRIBUTION OF ARTEMlA
Geographic Distribution

= Area of occurrence

Fig. 3. World distribution of Artemia

The

distribution of Artemia is limited to biotopes where

salinity is always sufficiently high. Artemia can live very well in natural sea
water or in Brackishwater (up to 5 ppt salinity). It does not have any defense
mechanism and hence it can be eliminated by predation from sea water 1
Brackishwater. However, it has a high osmoregulating capacity. Because of
this feature, it can live in high saline waters where only very few organisms can
survive. In addition, it is capable to synthesize very efficient respiratory pigments
(haemoglobins) to cope with the low oxygen levels that prevail at high saline
condition.

Arfemia is found in natural salt lakes as well as in man-made

salterns.

Different geographical strains have adapted to widely fluctuating

conditions with regard to temperature (6 - 35OC) and the ionic composition of the
medium (chloride, sulphate as well as carbonate rich waters).

So far over 350 natural Artemia biotopes, spread over the

five continents have been identified (Fig.3). Several populations, which were
reported in the oldest literature on Artemia distribution, do not exist at present.

In recent times, man does Artemia transplantation in Soutb

America, Australia and South-East Asian countries either for salt improvement or
for aquaculture production purposes.

Distribution of Arfemia in India

Didwan

Samblz

i* 11

r
i..

t?

Vedaranyam

*0
U

Karsewar Island

Fig 4. Distribution of Natural Population of

Artemia in India

All Indian strains of Artemia which are present in nature, are

parthenogenetic. Its occurrence was reported in the saltpan areas in the Gulf of
Kutch in

Gujarat, Sambhar and Didwana Lakes in Rajasthan, saltpans near

Mumbai in Maharashtra and saltpan areas in'Karsewar island and Veppalodai

near Thoothkudi, Vedaranyam and Kelambakkam in Tamil Nadu. Sambhar and
Didwana Lakes are inland in origin. All other areas are in the coastal belt. These
areas are present in the migratory route of flamingos. Hence, it is believed that
flamingos caused the distribution of Ariemia in these areas.

POND CULTURE OF ARTEIMIA

FOR BIOMASS AND CYST PRODUCTION

BY
S. KULASEKARAPANDIAN and P. RAVICHANQRAN

Central Institute of Brackishwater Aquaculture, Chennai

Introduction

Arfemia becomes an important input for the success of

aquaculture, which has tremendously developed in recent years. As aquaculture
is likely to develop manifold in future, the demand for Arfemia would likely to
increase in conjunction with its phased development. At present, the availability
of Arfemia from its natural resources is very much limited. To meet the everincreasing demand for Arfemia biomass and cysts, culture of Arfemia in the
ponds is as an alternative source of their availability.

Suitable site and infrastructure required for pond culture

Because of its high osmoregulatory capacity, Arternia lives in

high saline environment where its

predators cannot survive. Hence, Arfemia

can be effectively cultured in saline waters. Such water bodies are found in
solar saltpans, which occur all along our coast.

In a classic type of solar saltpan, water is pumped into the

evaporation pond from the reservoir as a first step. It is retained for a fixed
period of time. Subsequently, it is allowed to flow from one evaporation pond to
many of them in the series (Fig.1.A). During the time of retention in each
evaporation pond, salinity of the water goes on increasing by solar evaporation.
This transfer of water continues till the water becomes brine, i.e. saturated with
sodium chloride. The brine is then introduced to the crystalline ponds where
sodium chloride precipitates. Arfemia can be intensely cultured in evaporation
ponds of the solar saltpans (Fig.1 .C). Only minimum inputs are required for the
above culture.

Culture site should have a high evaporation rate with little rainfall. It
should be closer to the sea for easy intake of water, either by pumping or through
tidal influence. Water should be free from pollution. The pond should maintain
the water level without leakage or seepage. The infrastructure

such as

water-pumps etc., available in the saltpan for salt production, can also be used
for Arfemia culture.

water

Ievet-

G
Pond Modification
Fig.1 .A. Conventional evaporation ponds of the saltpan

Fig.?.B.Conversion of conventional evaporation pond to Arfemia culture pond.

Fig.1.C. Modified evaporation ponds of the saltpan for Artemia culture.

Pond preparation
The culture pond has to be deepened to hold a desirable
water depth of 70-100 cm (Fig.1.Bj. This high water level minimizes the chances
of water temperature reaching the iethal level of 40C. Deepening can be
achieved either by pond excavation or by increasing the dyke height. Initially, the
pond has to be dried and exposed to sunshine for a period of 7-10 days.
Optimum soil pH is 7.0-7.4. If soil pH is less than 7 (acidic), soil has to be treated
with lime at the rate of 1,000 kg per ha.

Water intake

Salinity of the pond water has to be maintained at about 100

ppt during the growth and multiplication of the stock (Fig.2.). Hence, 70-75 ppt
water is taken initially. Further, the intake of water has to be screened to prevent
the entry of predators. pH of the water should be above 8. If it goes below 8,
lime (CaO) has to be added at the rate of 500 kg per ha.

Fertilization

Pond water should be fertilizedlmanured in order to produce

enough primary (phytoplankton) food. For this, fertilizers or manures or both are
used. Among manures, chicken droppings are preferred. It should be manured
at the rate of 1,000 kglha as basal dose. Half of the above rate should be given
as subsequent doses, Interval between successive doses is 10-15 days. In the
case of inorganic fertilizers, urea, superphosphate and diammonium phosphate

form as satisfactory combination. They should be used at the rate of each 50

kglha as basal dose and half of it as subsequent dose. Manurelfertilizer should
be broadcasted. The basal dose is provided 3-5 days prior to stocking. If both,
organic and inorganic, are going to be used, manure should be given as single
basal dose (at 1,000 kglha), while inorganic urea, superphosphate and
diammonium phosphate will be given as basal (each 50 kgiha) and subsequent
(each 25 kgiha) doses. In this case, manure has to be immersed in water after
keeping in gunny bags in different areas of the pond. This operation allows the
manure to leach out slowly. In addition to these fertilizers, mono ammonium
phosphate (NPK ratio of 16:20:0) at the rate of 100-200 kglha, together with
ammonium nitrate (NPK ratio of 30:O:O) at 50-100 kglha, can also be used. In
this case, 50 kg of the former and 25 kg of the latter have to be applied
periodically once in a week.

Manuringlfertilization is to be regulated with

reference to water fertility and intensity of phytoplankton bloom in the pond.

Stocking
Stocking is done with the strains of Artemia which yield
small sized cyst and nauplius and possess high fecundity and desirable growth
rate. San Francisco Bay strain of Arfemia has all the above features. Hence, it
is the most suitable candidate strain for stocking. it can be transplanted in a
newly constructed culture pond which is devoid of any natural population.
However, many saltpans have natural populations of the local strain. These local

Arfemia have better survival by adapting themselves to the prevailing

environmental feature. If Artemia culture pond is a converted evaporation pond

of such saltpans, stocking is preferred with the existing local strain.
Stocking in the culture pond is done at nauplius stage. 35-40 nauplii per litre of
water is the optimum stocking density. Data on the water level and the hatching
efficiency of the cyst to be used, are necessary to calculate the quantum of
nauplii, required for stocking. Cysts can be kept for hatching either at the culture
site or in the laboratory. Hatched nauplii are transferred to the culture pond and
stocked. Late evening or early hours of the day is the suitable time for stocking.
Nauplii can be stocked at any salinity without any acclimatization.

Water management

Mode of reproduction in Artemis is mainly influenced by the

dissolved oxygen content of the water. Salinity has an inverse relationship with
the oxygen content. Hence, the simplest way of manipulating oxygen content of
the water is by correspondingly changing the salinity.

100-120 ppt salinity is

retained to maintain ovoviviparity through which population multiply. This is done

during the first two months of the culture period. Third culture month is for
inducing the shift from ovoviviparity to oviparity. To achieve this, the salinity is to
be slowly increased to above 150 ppt (Fig.2). For continuous maintenance of cyst
production, the salinity is retained above 150 ppt but below 200 ppt. Last two
culture months are assigned for cyst production and harvest. Artemia become
weak and die at above 250 ppt (Fig.2).

Growth monitoring

Growth of the population is monitored by collecting data on

the population composition.

For this, population samples are analysed by

grouping as nauplii, juveniles and adults.

Overall production stages of the

population can be correlated with the change in its composition. For example,
presence of only adults reflects the status of nil recruitment. Reproductive status
of the adults can be found out by observing the presence of naupliilcysts and
shell gland in the broodsac. It also indicates whether the population is in growth
phase or in stationary phase. Generally, Arfemia shows heterogenic distribution.
During early morning or at night, Arlemia is present spreading out uniformly
throughout the pond.

Hence, population density can be measured through

sampling method, to certain extent, during these hours. Information on rainfall,

salinity, water turbidity and water temperature are to be regularly collected.
These information are required to regulate water management, harvest and
fertilization and thereby monitor the population structure.

Biomass harvest

7
elastic

Fig.2.

Double screen dip net

Biomass is harvested when the majority of the population is

composed of adults.

At the time of harvest, population density should be

approximately above 200 numbers per litre. Dip net or drag net, with 120 micron
mesh size at its cod end, is suitable for harvest (Fig.2). A hand net with 800
microns can also be used in harvesting the biomass (Plate I.A.). Biomass
harvesting has to be stopped when the population reaches to a density of about
100 numbers per litre.

Plate 1.A. Biomass harvest.

Cyst harvest

Plate 1. B. Cyst harvest.

When the salinity is above 150 ppt but below 200 ppt, the
population switches to oviparous mode of reproduction, resulting in the liberation
of cysts. The cysts float and are driven towards the shore due to wind action.
Liberated cysts should be harvested as soon as possible. Othewise, they will
/

accumulate in the shore and get conta'minated with impurities (Fig.2). If they are
left out in the shore for a long period without harvesting, they will face high
humidity and

rainfall.

This causes repeated hydration and

dehydration.

Because of this, they will loose their energy reserve and thereby become
non-viable. High wind action creates waves in the pond which in turn causes
foam formation and cysts normally get trapped and lost. To prevent foam
formation by wave action, wave-breakers should be installed parallel with cystbarrier in 2 or more rows at about Imetre distance from each other (Fig.3).

Fig.3. Reduction of foam formation by installing wave breakers.

-wooden
+pond

Fig.4. Cyst barrier

support
bottom

To avoid all these, cysts should be harvested when they are

floating in the water surface (Plate 1. B). Installation of cyst-barriers (Fig.4), close
to shoreline, retains the cysts on the water surface from where cysts can be
effectively harvested.

Rate of production

On an average, 20 kg dry Arfemia cysts and 200 kg live

biomass can be produced from one hectre pond per year.

PROCESSING AND STORING THE HARVESTED PRODUCT

Biomass storage

The harvested biomass can be cleaned by washing with

freshwater. Washing removes adhering salt. After cleaning, biomass can be
spread out in thin layers in plastic bagslice trays. Afterwards, it has to be transferred to a quick freezer maintained at -25OC.

Cyst storage

Harvested cysts can be stored temporarily in saturated brine.

For long period of storage, they have to be cleaned, processed and dehydrated.
Collected cysts are contaminated with dead Arfemia, algae, debris, etc.
These contaminants should be removed to ensure the purity of the cysts.

Harvested cysts

have to be initially cleaned. Different mesh-sized sieves are

used to remove the corresponding

sized impurities while cleaning.

Contaminants, which are equal in size with the cysts. cannot be removed by
sieving. To remove them, cysts are to be initially suspended in brine. Cysts and
lighter debris float in brine while heavy dirt particles sink. Floating cysts along
with lighter debris, can be collected with 100 micron net and transferred to
freshwater1 seawater. In this treatment, lighter debris and non-viable cysts float
in freshwaterlseawater. On the other hand, viable cysts sink thereby ensuring
complete separation from all contaminants. Settled cysts are collected and
stored. In addition to purity, water content of the cyst determines cyst's quality.
If it is retained less than 10% of moisture, cyst-viability is most satisfactorily
maintained. To bring down the water content to minimum (i.e. less than

lo%),

purified cysts are dried until there is no change in their weight. This dehydrated
condition has to be maintained by packing them in vacuum containers or in
containers with nitrogen atmosphere.

ARTEMI'A BIOMASS PRODUCTION IN CONTROLLED SYSTEMS

BY
S.KULASEKARAPANDIAN and P. W V I C H A N D M N
Central Institute of Brackishwater Aquaculture, Chennai
Introduction

Arfemia juveniles and adults form as suitable diet for shellfishlfinfish

juveniles in their nursery phase of development. Further, reproductively active
adult Artemia, when offered as food, induces maturity in crustacen/fish
broodstock. Artemia grows from nauplius to adult in 2 weeks, increasing in length
by a factor of 20 and in biomass by a factor of 500, thereby offering very high
rate of production within a short period. Artemia can be intensively cultured
under controlled conditions in the same finfishJshellfish hatcherieslnurseries in
order to have a ready availability of feed for juvenilesladults and hence Artemia
biomass production under controlled environment gains importance.
Under controlled conditions, Artemia nauplii are stocked at high
density and reared. Because of intensive stocking and rearing, care has to be
taken for ensuring adequate oxygen and food availability in the rearing medium
and elimination of waste materials from the medium during the culture. Air-waterlift (AWL) operated raceway, which ensures continuous aeration, homogeneous
circulation of the medium, uniform distribution of the added feed within a short
time and maintenance of all particulate matter in suspension, is used for Arfemia
production under cpntrolled conditions.
Arfemia biomass production under controlled conditions can be
carried out in batch or in flow-through culture systems. In batch culture, the same
water will be used and no water change will be given whereas in the flow-through
system, the water will be renewed continuously.

Construction of air-water-lift raceway

Air supply l~nc

I

]+Air distributing cylinder

Fig.1. Raceway

Fig.2. Top view of the raceway

An ovallcircular/rectangular tank with central partition is

required to make a raceway. If rectangular tanks are to be used, it will be

better to curve their corners. In order to have optimum circulation, the
central partition should not be very close to the sides and should be kept 2
to 5 cm off the bottom by suspending it from the top ( F i g l )

The most

important parameter for the configuration of the tank is the heighvwidth

ratio which should be kept close to 1 and the water depth should not exceed
1 metre. To construct air-water-lifts, PVC pipes and elbows are used.

Various systems such as plastic tubes with screw, PVC-ring with screw and
piece of plastic with rubber band can be used to attach the air-water-lifts
with central partition. To have optimum circulation in the raceway, the
opening of the elbow should be an angle of 30 to 45' with the central
partition (Fig. 2).
Optimal aeration and circulation will be obtained if the
pipes of the AWLs are installed at 25 to 40 cm intervals (Fig.1 & 2). In order
to have maximum water-lift, diameter of the AWL and the depth of the water
are to be taken into account. (If the water level is 40 cm, the inner diameter
of the AWL should be 40 mrn which will provide 6.6 IitrelminuteIAWL of
water.). Small polythene tube of 3 to 6 mm diameter will form as aeration
line and it can be mounted in the AWL through a hole at the top of the PVC
elbow. The aeration line can be raised or lowered at will and it should be
fixed tightly through the elbow in order to prevent undesired displacement.
To ensure satisfactory water-lift, the aeration lines should extend as deep
as possible in the AWLs. Foam formation and formation of tiny air-bubbles
are to be avoided by properly fixing the AWLs as foam traps the brine
shrimp thereby causing their mortality and ingestion of tiny air bubbles or
trapping of tiny air bubbles between thoracopods makes Artemia to float
thereby leading to mortality. Hence, air-stones are to be avoided as they
cause foam formation and the formation of tiny air bubbles. All the aeration
lines will take lead' from a central air distributing container so as each
aeration line need not have a separate regulating valve system. It is always
better to keep the outflows of the air-water-lifts half submerged to prevent
water falling from height and formaton of tiny air bubbles. An identical and
constant aeration is obtained by adjusting all air tubes to the same
hydrostatic depth in the AWLs.

Arternia culture in raceway

Culture medium

Artemia culture in raceway system can be carried out with

the culture medium of any salinity. However, for easy accessibility, seawater will
be used. It is safer to do production under controlled conditions in 50-100 ppt in
order to avoid contamination with ciliates and other competitors and predators.
Stocking
lnstar I Artemia nauplii will be stocked as they remarkably
tolerate the salinity stress, if any, developed at the time of stocking. Rate of
stocking depends upon the feed availability and water management. In batch
culture system, stocking can be safely increased to 10,000 instar I nauplii per
litre if feeding is maintained at 15-20 cm transparency. In the case of flowthrough system, 25,000 nauplii per litre can be stocked and reared.
Feed and feed preparation

As Arternia is a non-selective filter feeder, it can be cultured

by feeding them with a wide range of feed, both live and inert materials.
However, the particle size of the feed is very important and it should be less than
50 micron. Hence, the feed should be squeezed (if inert feed) or passed (if algal
feed) through 50 micron sieve. Rice bran is a very good inert feed for Arfemia.
Soluble products in the feed material are not taken up by Arternia and they will
be decomposed by bacteria in the culture medium thereby deteriorating the water
quality. Hence, the feed should be properly prepared to get rid 'off dissolved
matter. This can be achieved by aerating the feed solution for 1-2 hours and
allowing the feed particles to settle by cutting off the aeration for one hour.
Dissolved matter will be in the solution and it will be discarded. Particulate feed
will be settled and it is used for feeding by making into solution with required
quantum of seawater.

Feeding
Arfemia is filter feeder and it will go on continuously filtering

the culture medium. Hence, medium must contain adequate food at all times and
food distribution is very important. It is noted that the transparency of the
medium is found to be a very useful parameter for determining the food level
present in the medium and it is measured with the help of a transparency stick
(Fig.3) which is a graduated stick of about half a metre in length with a disc
attached at one end. Transparency stick is a type of Secchi disc in which letters
(for example - hungry) will be painted on the disc portion and the transparency of
the culture medium is measured by submerging and lowering the stick in the
culture medium until the letters will be just visible.

stick

the word ' ~ u n g r 'y

painted on white plate

Fig.3. Transparency stick

If the stocking rate is about 10,000 nauplii per litre in the
batch culture system, feeding should be continued until the medium reaches a
transparency of 15-20 cm.

Water quality maintenance

As the animal grows, Arfemia moults and the exuviae

and the faecal pellets hamper the food uptake by Arfemia. Further, they affect the
water quality. These particulate wastes should be continuously removed from
the culture medium from day 4 onwards. In batch culture system, culture medium
is partly purified and reused with the help of filter-screen system (Fig.5) and plate
separator (Fig.4).

Woler bock to
culiurc tank

Polytthyltnr tubr

Fig.4. Plate separator

ia------ 4 5 C m -------I4

Air supply

-~ir

+20 cm --c(

collar

Fig.5. Filter frame for filter-screen system

The plate separator is a rectangular sedimentation tank

which is divided into three interconnected compartments by two perforated
vertical sheets. In the middfe compartment, plates with coarse surface will be
placed horizontally or vertically with specific intervals. In the batch culture
system, water from the culture tank will be pumped to the first compartment by

AWL which will pass through the plates and leave the separator from the last
compartment after a retention time of 20-30 minutes. The larger particles, faecal
pellets and exuviae settle down at the bottom of the separator and on the plates
while water with small food particles in suspension drain back into raceway.
In order to assure that Atfemia is not pumped to the plate
separator, a filter-screen system with aeration collar (Fig.5) is used. The filterscreen system is made of a filter-frame over which the filter screen bag is tied
and an air-collar is attached to its bottom. The width in the upper part of the filter
frame (15 cm) is more than that of the lower part (10 cm) and the length at
anterior end (45 cm) is cbnsiderably more when compared to the bottom length
(20 cm). During operation, this special shape of the filter frame helps to reduce

clogging by raising a continuous curtain of air bubbles against the sides of the
filter bag. However, it is better to clean the filter bag daily to have an effective
prevention from clogging. As the brine shrimps grow, the filter bags should be
changed by new ones with progressively larger mesh width, Initial size of the
filter bag is 100 micron and it should be changed progressively as 200, 350,
450/500' and 800 microns in correspondence to the growth of the stock.
pH of the culture medium

When inert products will be given as feed, the pH of

the culture medium will go down and if it goes below 7.5, the pH should be raised
by adding NaHC03 at the rate of about 300 gm per ton of water.

Monitoring growth

Frequent observation of Ariemia under microscope

will enable an operator to judge the progress of the culture. Well fed Artemia will
have full digestive track. Futher, Artemia will not feed when its mouth is blocked
by the stick nature of the feed provided (for example uncleaned rice bran).
Similarly, Arfemia has to starve when its filtering surface of the thoracopods are
covered by larger waste particles. These adverse conditions will be clearly
detected when observed under microscope.
The growth of the animal should be periodically
checked by estimating the biomass. By taking wet weight of Artemia, present in
the sample of 1 litre medium and calibrating to the total volume of medium,
biomass can be calculated.
Culture duration

Culture period varies with the temperature and the

strain selected. Generally, Artemia nauplii will reach the harvestable adult size
(about 8 mm in total length) within 2 weeks time.

Harvesting the biomass

Harvesting the brine shrimp is facilitated by taking

advantage of the special surface respiration behaviour in Arfemia.

When

aeration is cut off; the oxygen concentration of the medium drops to a critical
minimum after about 30 minutes and

because of the anaerobic condition,

developed especially in the bottom of the container, Arfemia concentrate in

dense numbers at the surface from where they can be easily scooped out with a
net.

Rate of production
A production of 5 kg Artemia biomass (wet weight)

per cubic metre of water during a period of 2 weeks will be obtained when
stocking is done at the rate of 10,000 nauplii per litre of the medium under batch
culture system and feeding with rice bran at 15-20 cm transparency.

By

flow-through culture system, a production of 25 kg biomass I m31 2 weeks will be

achieved by stocking 20, 000 nauplii per litre and feeding with rice bran or algae
at 15-20 cm transparency.

ARTEMIA CYST HATCHING, SEPARATION OF HATCHED NAUPtll,

DECAPSULATON AND CYST QUALITY ANALYSIS.

BY
S. KULASEKARAPANDIAN
Central Institute of Brackishwater Aquaculture, Chennai

introduction

Artemia cysts are widely used as source of live feed to

shrimp and fish larvae. In the dormant cyst, the embryo occurs in gastrula stage.
It further develops and comes out as Arfernia nauplius when cyst is subjected to
hatching. When Artemia nauplii are offered as feed to fishlprawn larvae, they
have to be free from the unhatched and empty cysts. Consumption of empty
cysts blocks the gut when fish larvae are fed with uncleaned Artemia nauplii.
Further, unhatched and empty cysts have a very high bacterial load. Hence,
separation of hatched nauplii from debris gains importance. However, complete
separation of Arfemia nauplii from their cyst-shells, after hatching, is not always
possible. Short exposure of the hydrated cysts to hypochlorite solution removes
the hard shell without affecting the viability of the Artemia cysts. This process is
known as decapsulation and it can be followed before subjecting the cysts for
hatching. Decaps'ulation eliminates the process of separating hatched nauplii
from debris after hatching. Further, it disinfects the cysts thereby ensuring to get
Artemia nauplii of desirable quality. Cyst quality can be assessed on the basis
of its hatching efficiency (HR), hatching percentage (H%) and hatching rate (HR).

Artemia cyst hatching

BLACK COLOliRED PORTION

TRANSPARENT PORTION

VALVE

Fig.1. Artemia cyst hatching container.

Container for hatching Artemia cysts

should be

funnel shaped. Its cone portion is to be transparent while its cylindrical portion
should be black in colour. Filtered seawater is to be filled in the container to
required level. Maximum 5 gm cysts can be kept for hatching in 1 litre of water.
At least five conditions are essential for restarting
the embryological development in the cysts leading to the hatching of the nauplii.
They are (i) hydration of the cysts in seawater, (ii) oxygenation of the medium,
(iii) illumination of the hydrated cysts, (iv) pH above 8.0 and (v) temperature of

26 - 30C.

Hatching can be carried out in salinities ranging from

5 to 70 ppt. Oxygen content of the medium plays a vital role in hatching. It is

reported that hatching rate in the cysts of California strain is constant in the range
of 2 to 8 ppm dissolved oxygen; decreases below this range and is completely
inhibited at oxygen level 0.6 to 0.8 pprn. Aeration, at the rate of 10 to 20 litre of
air

per minute, is to be supplied. Continuous and moderate aeration, which

keeps the cysts in suspension, is beneficial to hatching as it prevents the

accumulation of cysts in the bottom, where cysts will be forced to anaerobic
condition. Light triggers the development of the embryo in the hatching process.
Brief illumination of the cysts after hydration is sufficient to assure good hatching.
Minimum 1000 lux light is to be provided or hatching should be started at
daytime. After 12 - 15 hours, regular samples have to be taken and observed
under microscope.

lh
sw'

cyst

breaking

start metabolism

hatching

Fig. 2. Arfemia cyst hatching process

Observation under microscope reveals that breaking stage in
the hatching process occurs after 12 - 24 hours of incubation. Time taken for
breaking stage varies in different strains.

Subsequent to breaking stage,

umbrella stage follows. Embryo in the umbrella stage is observed to assume the
shape of the nauplius within the hatching membrane and it hangs from the shell
just like an umbrella. After umbrella stage, the nauplius comes out by breaking
the hatching membrane, Generally, all the nauplii hatch out within 48 hours.

SEPARATION OF HATCHED ARTEMIA NAUPLII FROM HATCHING DEBRIS

After completion of hatching, the conical container has to be

covered with a dark cloth. Aeration has to be stopped and light source has to
be provided at the cone portion. Positive phototactic behaviour of the Arfernia
nauplii is exploited for separating the hatched nauplii from empty and unhatched
cysts.

Nauplii will swim towards the lighted cone portion of the hatching

container and accumulate from where they have to be collected by

siphoningldraining

through the provision in the bottom.

This separation

technique has a few disadvantages. it is a time consuming process. It requires

skill to remove the nauplii without eliminating the debris which are accumulated
in the bottom and surface. Many empty shells have exactly the same density and
consequently will be siphoned off or drained with the nauplii. These problems
can be solved by using nauplii separator.

Nauplii separator has two compartments. Mixture of

nauplii and debris is introduced in the inner dark compartment of the separator.
It can be observed that nauplii alone move through the slits because of the light
stimulus and reach the lighted outer compartment when the connection is made
by opening the slits. The debris is retained in the central dark compartment,
thereby ensuring complete separation. Once separation is completed, the
interconnection can be closed and the nauplii are to be collected from the outer
compartment.

Imer dark comoartment

Slit closed

--------------

7- - - - - - -7\ - -- - -b- - - I - - - -

---I
I

Outer-lighted compartment

inner dark compartment

b Retention of hatching debris only
Nauplii movement towards light
from inner compartment through slit
Slit open

Outer-lighted compartment

Fig. 3. Separation of Nauplii with Nauplii separator

DEGAPSULATON OF ARTEMlA CYSTS

Procedure for decapsulation

a

Hydrate the cysts, taken in a conical container, for a period of one to two
hours in freshwaterlsea water.

Provide moderate aeration.

Prepare the decapsulation solution with NaOCI, 40% NaOH and

sea water. For Igm of cysts, 14 ml of decapsulation solution with 0.5

gm active product is required. 0.33 ml 40% NaOH is needed for "I gm

of cysts.

After complete hydration, transfer the cysts in decapsulation solution

and stir it.
Note the rise in temperature and keep the temperature well below

40% by adding ice, if necessary and never allow it to exceed 40C.

Periodically observe the colour change in the cyst, under microscope.
Don't keep the cysts in decapsulation solution for more than 15 minutes.
Filter the cysts on 100 micron sieve.
Wash thoroughly in tap water.

Dip the cysts in 0.1 N HCI twice.

Subsequent to 0.1 N HCI treatment, wash in tap water.
For temporary storage of the decapsulated cysts, dehydrate them in
saturated brine.

Tap water
Decapsulation bath

Acid hath

Dehydration treatment

Hydration bath
Cooling Systeln

Water bath

Fig. 4. Decapsulaltion treatment

Complete decapsulation can only be ensured when

the cysts are spherical in shape. To obtain this desired stage, the cysts are
allowed to swell by hydration.

it can be observed that full hydration is reached

within 1 - 2 hours of introduction to freshwaterlsea water. Prolonged hydration is

to be avoided as it induces embryological development. Within a few minutes
after the transfer of the cysts to the decapsulation solution, exothermic oxidation
reaction starts.

Because of this reaction, a rise in temperature is resulted.

Further, dissolution of shells start. Foam formation can be noticed. A gradual

colour change in the cysts will be observed from dark brown to grey and then to
orange.

Attaining orange colour indicates the completion of the decapsulation

process. Afterward, there won't be any further increase in temperature. 40C is
lethal to cysts and hence care should be taken to retain the temperature well
below 40C during decapsulation.

Prolonged immersion in decapsulation

solution will kill the embryo. Hence, the cysts have to be removed from the
decapsulation solution as soon as the process is over. In any case, it should not
be more than 10 minutes. After washing in tap water, decapsulated cysts have
to be dehydrated by immersing them in saturated brine. They can be stored in
freshly prepared saturated brine. It can be noticed that decapsulated cysts will
sink in saturated brine.

Cyst quality analysis

Cyst quality can be assessed on the basis of its

moisture content, hatching efficiency, hatching percentage and hatching rate
besides its nutritional value. The quality of Arfemia cysts mainly depends on the
way the cysts are processed and stored.

During prolonged period of cyst

storage, periodical analysis is necessary to make sure that the cysts are retaining
their viability.

Matching efficiency (HE)

Hatching efficiency (HE) is the number of Arfemia
nauplii hatched out of 1 gm of cysts under normal hatching conditions (i.e., 48
hours incubation in 35 ppt seawater with 8.0

- 8.5

pH at 25-2gC). It can be

calculated by applying the following formula.

HE

N x 4 x I 0 0 x 4 where N is the average number of Arfemia nauplii

present in 0.25 mi sample.

Procedure to find out hatching efficiency (HE)

Take 80 ml of sea water in 100 ml measuring cylinder.

Aerate and add 250 mg Artemia cysts, to be anaiysed.
After one hour, adjust to 100 ml by adding sea water.
Provide continuous aeration and illumination.
After 48 hours, take 5 subsamples of 0.25 mi each in petridishes with 1 rnl
graduated pipette.
Add 1 drop of Lugol's solution.
Count the nauplii per petridish and make an average (N).
Number of naupli

------------------------- =
One gram of cyst

HE =

Nx4xIOOx4.

- 250 mg sample
- 80 ml seawater

100 ml

Adjust volume to

mi with seawater

- 25 O C

- Continuous

r)

1 hr,

Automatic
pipette

Take 5 sub samples of 2501.d

iml nlastic tube

Clnse with can

Incubation in continuous
light at 25 "C for 24,36
or 48 hours

Rotator

Fixatinn with 2 dmns of 1,11~nl

indine snlut'on

1
.b

Count Nauplii per tube

Average nauplii (N)

HATCHING EFFICIENCY (HE) = N x 4 x 100 x 4

Fig. 5. Determination of Hatching efficiency of Artemia cyst

42

Hatching percentage (H%)

Hatching percentage (H%) reveals the number of
nauplii, obtained out of 200 full (embryo containing) cysts. Treatment with
sodium hypochlorite after 48 hours, dissolves the shell thereby exposing the
unhatched embryos from the unhatched cysts. Hatching percentage can be
calculated by making use of the following formula.

Number of nauplii
H o/o = --------------------------------------------------------------------- x

100

Number of nauplii + Number of unhatched embryos

Properly processed cysts will have more than 90%

hatching as hatching percentage depends mainly on the nature of processing
and storage.
Procedure to find out hatching percentage (H%)

Take 80 ml of sea water in 100 ml measuring cylinder.

Aerate and add 250 mg Artemia cysts, to be analysed.

After one hour, adjust to 100 ml by adding sea water.

Provide continuous aeration and illumination.

After 48 hours, take 5 subsamples of 0.25 ml each in petridishes with I ml

graduated pipette.

Add few drops of sodium hypochlorite solution.

Count the nauplii and unhatched embryos in each petridish and make
an average.

Hatching rate (HR)

Hatching rate (HR) refers to the time period from the

start of incubation (hydration of the cysts), till the completion of the release of all
nauplii (completion of hatching). The foilowing time intervals can be worked out.
To is the incubation time till the appearance of the first free swimming

nauplii.
e

Tjo is the incubation time, taken for the appearance of 10% of the total
hatchable nauplii.
TgO is considered as the incubation time, needed for

the appearance

of 90% of the total hatchable nauplii.

To find out the hatching rate (HR), hatching efficiency
(HE) is to be calculated in the interval of three hours during the whole hatching
period from the initial 9 hours. From this, TO,Tlo and TgOfor the cysts, to be
analysed, can be worked out. It can be observed that they vary from strain to
strain and even batch to batch of the same strain.
Procedure to find out hatching rate (HR)

Take 80 ml of sea water in 100 ml measuring cylinder.

Aerate and add 250 mg Arfemia cysts, to be analysed.
After one hour, adjust to 100 mi by adding sea water.
Provide continuous aeration and illumination.
Starting from 9 hours incubation, take 5 subsamples of 0.25 ml each, in
five petridishes and add Idrop of Lugol's solution in each petridish.
Repeat the sampling at every three hours i.e., 12, 15, 18,21, 24, 27, 30,
33, 36, 39, 42, 45 and 48 hours incubation (totally 70 petridishes).
Count the nauplii and make an average for every 3 hours incubation after
initial 9 hours.
Plot the average nauplii number and time in hours as hatching rate curve.
From the curve, To, Tlo, TsOand TgOcan be extrapolated.

STUDENTS XEROX
Adyar