SHELL FISH BREEDING & HATCHERY MANAGEMENT-FAQ-

312(2+1)

Learning Objectives

This Unit has been designed to provide the following to the students:

1. General introduction and history of shell fish seed production in the world
2. The seed resources (natural) and the need for the captive seed production in shellfishes
3. A basic and general understanding on the shellfish seed production

1.1.1 Shrimps

4. The controlled spawning and larval rearing of shrimps was initiated by Hudinaga (1942)
with wild spawners of P. japonicus caught from fishing grounds. He successfully bred
and reared under controlled conditions in the laboratory. In course of time, series of
developments have taken place in different parts of the world as a follow up of the
above which helped in the commercial production of shrimp seed for farming. The
hatchery systems for shrimps are primarily of two types:

a) Japanese system / community culture or fertilized system. (Green water culture system) -
originally developed and followed by Hudinaga and others for Japan wWhite shrimps.

b) Galveston system – or clear water system - This system was developed in Europe for the
shrimps, in which the green water culture system was modified to overcome the troubles in the
green water culture. Since it was developed and followed in Galveston laboratory it is named as
Galveston system.

1
Following the successful seed prodcution with [Link], seed production was standardised
for P. monodon in Taiwan and later on this technology was transferred to many parts of the
world during early 70s. The seed production of [Link] was worked out in many Asian
countries including India in early 1960s.

1.1.2 Japanese System of shrimp seed production

This was the first developed system for the seed production of shrimps. Hudinaga adopted this
system for the seed production of Penaeus japonicus. This system involved spawning, hatching
and larval rearing in the same pond or tank with same conditions. This is based on the initial
attempt to produce the seed of Penaeid shrimp and hence is vogue at present. However, this
was the first designed system for the larval rearing and hence regarded as the first developed
technology for closing the larval cycle in captivity.

In this system, the larval rearing was done along with the culture of live feed (i.e., microalgae).
Therefore the water in the rearing tank will always remain green due to the enrichment of
algae. Due to uncontrolled aquatic conditions, the survival in this system was low compared to
clear water culture system.

1.1.3 Galveston system for seed production shrimp

This system was developed in the Galveston laboratory, USA (Cook and Murphy, 1966). In this
system, rearing of brood stock, spawning hatching, larval rearing and live feed culture are done
separately in separate tanks. Since live feeds were not cultured in the larval rearing tank (LRT),
the water in the LRTs remained clear. Therefore this system is also referred as clear water
rearing system.

In this clear water system further developments took place leading to the development of larval
feeds and advanced larval rearing conditions. Thus resulting in higher survival rate, better
economical seed production, etc. Hence, this system is widely followed in many countries for
the shrimp seed production and has been successful for many other species of crustaceans.

Therefore further refinement of technologies were developed keeping with the clear water
system as the base.

1.1.4 Breeding of freshwater prawns

The first successful attempt of captive breeding of freshwater prawn was done by S.W. Ling in a
Malaysian Laboratory for Macrobrachium rosenbergii in the year 1969. He completed his
experimental larval cycle in captivity for M. rosenbergii. Later the larval rearing under
laboratory conditions was perfected by Fujimura in the year 1970 in Hawaii. This was further
picked up by countries like France, Thailand, Taiwan and India for controlled seed production of
M. rosenbergii. Further research was done on different aspects of rearing such as feeds and

2
feeding, health management, etc in Taiwan, Hawaii, French Guyana , and India for M.
rosenbergii and M. malcolmsonii simultaneously.

Similar to M. rosenbergii, controlled breeding and seed production was developed for many
native Macrobrchium spp in other countries like China for M. nipponense. This has led to the
expanded farming of Macrobrchium spp in the world.

1.1.5 Mussel

Mussel hatchery technology has been developed in countries like China, Tahiti and Thailand. In
India, CMFRI at Madras and N.I.O. Goa have developed technology for the production of spat of
green mussel(Perna viridis) and Vizhingam centre of CMFRI has developed the spat production
for brown mussel (Perna indica).

1.1.6 Status of shellfish seed production in India

Indian shellfish seed production dates back to 1960s as earlier works indicate that the seed
collection had been a serious occupation for the coastal folk in many states. Presently, India has
about 400 registered hatcheries in which about 120 were for freshwater prawn seed
production. The major emphasis is on Tiger shrimp (Penaeus monodon) while a few have
concentrated on Indian white shrimp (P. indicus) too. After 2005, the thrust is slowly shifting
towards P. vannamei while in the freshwater prawn sector, it was only for Macrobrachium
rosenbergii.

Due to biological complexity, lobster hatcheries are not operated by entrepreneurs in India. A
few experimental hatcheries are present. A private entrepreneur has his crab hatchery in
Chennai that has been supplying juvenile crabs for farming.

Molluscan hatcheries do not have much appeal for commercial start-up and therefore, only
government run oyster, clam and holothurian hatcheries are present in India.

It is worth to note that there are many hatcheries in coastal Tamil Nadu and Andhra Pradesh
working on shrimp, prawn and fishes all in one facility. This type of multi functional hatcheries
are known for their year round operation and business.

1.1.7 Shrimp and prawn seed production

Shrimps and Prawns being the thrust among all the shellfishes, it is necessary to know the
status of their seed production in the country. Among the shrimp species available in India,
Tiger shrimp and White shrimp are the species of preference due to their international
[Link] Indian hatheries concentrate more on the above two species.

Tiger shrimp is the most preferred among the two shrimp species. Quality brooders for the
hatcheries is remaining as a major problem for the production of quality seeds. Captive

3
broodstock development has not become a popular reality for P. monodon while, some
attempts on P. indicus yielded good results. But in general all the shrimp hatcheries can work on
both the species for captive seed production.

 Chapter 2: Seed Resources

1.2.1 Natural seed resources of crustaceans

The seeds of cultivable crustaceans were originally collected from the natural wild waters as
they are their breeding grounds. Most of the crustaceans have preferences for particular
breeding ground or micro climate and environmental stimulii to complete their spawning. To
perform the spawning they undertake migration also (spawning migration) and the obstruction
in their migratory path affects their spawning drastically (E.g., Macrobrachium malcolmsonii
and few other species of shrimps). These factors are known to affect the natural seed
prodcution and seed resources in natural waters.

In general Indian estuaries, river mouths, coastal waters and marine ecosystems are known as
rich sources for natural seed collection. A wide variety of species are known to inhabit and
spawn in these waters and their seeds are available either in specified seasons or through the
year (based on species). Usually estuaries are considered to be nursery grounds for the
crustaceans due to their rich feed and varied ecological parameters.

1.2.1 Natural seed resources of crustaceans

The seeds of cultivable crustaceans were originally collected from the natural wild waters as
they are their breeding grounds. Most of the crustaceans have preferences for particular
breeding ground or micro climate and environmental stimulii to complete their spawning. To
perform the spawning they undertake migration also (spawning migration) and the obstruction
in their migratory path affects their spawning drastically (E.g., Macrobrachium malcolmsonii
and few other species of shrimps). These factors are known to affect the natural seed
prodcution and seed resources in natural waters.

In general Indian estuaries, river mouths, coastal waters and marine ecosystems are known as
rich sources for natural seed collection. A wide variety of species are known to inhabit and
spawn in these waters and their seeds are available either in specified seasons or through the
year (based on species). Usually estuaries are considered to be nursery grounds for the
crustaceans due to their rich feed and varied ecological parameters.

Unit 2: Hormonal interaction in the reproduction of crustaceans

Learning Objectives

This unit will educate the students on the following:

4
 1. The role of hormones in the reproduction of crustaceans.
2. Different hormones and their physiological importance.
3. The glands and their location for clear understanding of hormonal manipulation.
4. The effects of gonadal amputation in the maturation of crustaceans.

Chapter 1: Glands and hormones in crustaceans

2.1.1 Introduction

Next to fin fishes, crustaceans such as shrimps, prawns crabs, lobsters, etc are of much
biological and economical importance due to their high value and ready market acceptance.
This has motivated the scientists to work out the breeding programme of these species which
has come a long way as on date. The neuroendocrime system and mechanisms controlling
reproduction in crustaceans are similar to that of fishes with some deviations. Being
invertebrates, their reproduction involves more external and internal stimulii.

Reproduction in crustaceans is basically controlled by the hormones secreted by the glands

present in different parts of the body.

2.1.2 X-organ

This is a specialised neurosecretory organ located in both the eye stalks. The cluster of
neurosecretory neurons (X-organ) are located in the medulla terminalis. The axons of these
secretory neurons and neurohaemal organ are together called sinus gland, which serves as a
storage – release centre for these hormones. As the sinus gland serves for the storage and
release of neurohormones secreted by secretary neurons of x-organ it is called “X- organ-sinus-
gland complex”.

2.1.3 Functions

The X-organ secretes several neurohormones such as

a. Ovary (gonad) Inhibiting Hormones (OIH/GIH)

b. Moult Inhibiting Hormone (MIH)
c. Light adapting hormone or distal retinal pigment hormone
d. Hyper Glycemic Factor (CHH- Crustacean Hyperglycaemic Hormone)
e. Erythrophore concentrating hormone &
f. Neuro depressing hormone.

Out of these, OIH and MIH are directly involved in reproduction. This hormone acts on the molt
stimulating hormone and inhibits its action. The action of MIH over MSH is called bihormonal
action.

2.1.4 Growth Stimulating Hormone

5
Growth Stimulating Hormone(GSH) is also known as Moult or Gonad Stimulating Hormone. It is
secreted by the Thoracic ganglia.

Neurosecretory neurons of supra oesophageal ganglia believed to secrete a gonad stimulatory

hormone releasing factor (GSHRF) which stimulates the secretion of GSH (Gonad Stimulating
Hormone) from thoracic ganglia(But in Mexican cray fish, Procambarus borverii, GSH has been
found in sinus gland).

Major functions of this GSH include stimualting the development of gonads, gametogenesis,
energy conservation for spawning and prepartion of the animal for breeding. This hormone is
believed to be a complex with more fractions responsible for the ion regulation and
physiological control of the animal for breeding. The modular - activator - inhibitor mechanism
present in this complex will lead to the release of GIH or MIH from X-organ sinus gland.

2.1.5 Y-organ

Located in the antennary / maxillary segment of the body anteriorly. This secretes a moulting
hormone (ecdysone) which is a steroid hormone (Crust ecdysteroid). The secretary activity of
this gland is controlled by moult inhibiting hormone secreted by X-organ and secretion of
mandibular organ. MIH inhibits Y-organ whereas secretion of mandibular organ will stimulate
the gland.

2.1.6 Mandibular organ

The mandibular organ in the crustaceans is similar to that of an endocrine gland in higher
vertebrates. It secretes hormones or hormone like substances, that will have the influence on
the reporducitve organs or reproductive process.

It has been involved in ovarian growth and vitellogenesis. Mandibular organ synthesizes
asubstance called methyl farnesoate (MF) and the same has been reported from lobsters,
crayfish and shrimps. MF has been shown to have stimulatory effect on Y-organ to secrete
ecdysone.

2.1.7 Effects

Testes are said to have no endocrine role whereas the androgeneic gland is shown to be
endocrine in function. The secretion of this gland (androgenic hormone) controls male
secondary sexual characters and spermatogenesis. If androgenic gland is removed from a
genetic male, it may develop ovary and change into a female. So, a genetic female can be
changed to male by the transplantation of androgenic glands into females.

OIH or GIH of X-organ-sinus gland complex is reported to control the ovarian development.
Ovary develops rapidly and undergoes vitellogenesis and maturation if MIH is inhibited.
Because, of its specific role in vitelleogenesis it is called VIH (Vitellogenesis Inhibiting Hormone).
6
It is for this reason that eye stalk, ablation is practiced to induce ovarian maturation in shrimps,
prawns / crabs in hatcheries. During eye-stalk ablation X-organ sinus gland complex is removed
along with the eye-stalk thereby the ovary is free from the inhibiting effect of OIH and MIH.

Unit 3: Reproductive Biology of Penaeid Shrimps and Prawns

Learning Objectives

This unit has been designed to achieve the following objectives:

1. To provide biological information on the reproduction of penaeid shrimps.

2. To give a detailed information on the reproductive organs of penaeid shrimps
3. To educate the students in mating and spawning behaviour of penaeid shrimps.

Chapter 1: General description of the reproductive systems

3.1.1 Age & Size

Age at first breeding of tiger shrimp is not clearly established. It is reported that a combination
of age and body size along with feed availabe and favourable environmental conditions
influence the onset of sexual maturity. Nevertheless, the size is a matter for the high fecundity
and yield of good young ones.

The males of body weight between 40 and 70g and females with body weight of 60 to 80g are
found sexually matured among wild stock, whereas among farm reared shrimps, male weighing
35g and females weighing 40g are found sexually matured. Other contributing factors might be
resposnisble for differnce in maturity.

3.1.2 Sexual dimorphism

Male Female
1. Presence of petasma in the 1st pleopod. Petasma is absent in the 1st pleopod in
2. Presence of appendix masculina in the female.

7
 2nd pleopod.
Appendix masculina is absent.
3. Thelycum is absent.
Thelycum is present between pereopods
th
4. Gonopore is located at the base of 5 IV & V.
walking log. Gonopore is located at the base of 3rd
walking leg.
5. Smaller in size. Larger than male of the same age group.

Penaeus monodon

Penaeus indicus

3.1.3 Male reproductive system

The male reproductive system consists of a pair of testes located in the cephalothorax (CT)
above the hepatopancreas (HP). The testes are translucent, composed of 6 lobes and all the
lobes are connected on the inner margin leading to vas deferens (vd). Vas deferens has 4
regions such as a proximal region, which is short and narrow, a medium portion which is thick

8
and larger, long narrow distal part and a muscular portion called terminal ampoule which opens
at the base of coxa of 5th perieopod. The gametes produced by the lobules of testis are
aggregated and stored in a bag and are called spermatophores, which are stored in terminal
ampoule.

Spermatozoa of shrimps are small in size and lacks flagellum. Sperms are immotile and attach to
the egg at spike end.

3.1.4 Male reproductive organ and the process of gamete delivery

Male reproductive system externally consists of a pair of pleopods as accessory reproductive

organ, present on the inner side of the abdominal flap. The ejaculatory ducts of the testes open
into a small gential papilla at the base of the 3rd pair of the walking legs.

The first pair of pleopod of the shrimp is made up of two segments, the basal one is broad,
rectangular and flattened and positioned close to the sternal wall and the terminal one is long
tube-like and tapering towards the tip. During mating with the female shrimp, this tip is
inserted into the seminal receptacle of the female. The second pleopod helps in passing the
spermatophores from the ejaculatory ducts into the funnel-like portion of the first pleopod.

The ejaculate from the gonad (testes) consists of non-motile sperms and seminal plasma.
Sperms are enclosed in numerous small spermatophores and stored in the anterior vas
deferens (vd), while the seminal plasma is produced and stored in the posterior vas deferens.

3.1.5 Maturity stages of testes

In shrimps, the testes show marked variations for different maturity stages. As the time
advances, the maturation progresses. Different identifiable maturity stages of testes are
described below:

Immature: Transparent / creamy in colour; occupying less than 1/6th of body cavity; without a
prominent vas deferens.

Maturing: Creamy white; occupying 1/4th of body cavity.

Matured: Milky white with thick vas deferens; occupying full body cavity.

3.1.6 Female reproductive system

There are a pair of ovaries which are partly fused extending almost the entire length of a
mature specimen. Each ovary consists of an anterior lobe, lateral lobes located dorsal to the
hepatopancreas and an abdominal lobe lying dorso-lateral to the intestine. The oviduct
originates from sixth lateral lobe, leading to the external genital papilla located at the base of
3rd periopod.

9
 3.1.7 Female reproductive organ (External)

The female reproductive organ consists of a sperm receiving organ called Thelycum, which is
present in the middle portion of the last pair of the walking legs. There are two types of
Thelycum, namely closed thelycum and open thelycum based on the presence and absence of
the lateral plates in the thelycum, respectively.

3.1.8 Maturity stages of ovary

In general the ovaries can be classified based on the maturity stages of the eggs. Broadly the
maturity stages are:

Immature: The ovaries in immatured female shrimps will be transparent / light yellowish to
ivory in colour; occupying 1/6th of body cavity; or like fili form. The Gonado Somatic Index (GSI)
at this stage will be strikingly low.

Maturing: Ovaries in maturing shrimps will be light to dark yellowish or orange in colour;
ovaries are filled with egg mass and therefore they will be occupying 1/3 rd of body cavity.

10
Matured: Ovaries in matured females will be orange-red in colour; filled with matured eggs
with or without yolk granules. prominent seminal receptacle will be present; occupying full
body cavity and showing a high GSI. The females with matured ovary can be identified by the
presence of fully widened ovary in the body extending from cephalothorax to the abdomen
with prominent bulged portion in the ovary.

3.1.9 Mating characteristics in open vs closed species

Penaeus species are grouped into two broad categories based on differences in morphology of
the female genital organ or thelycum which occupies the area from the third to the fifth
periopods. The “open thelycum” species include the members of the Litopenaeus subgenera.
They receive spermatophore or sperm packet from the male and retain it externally for a few
hours perior to spawning.

In contrast, the “closed thelycum” species are those which mate each time the female molts.
The closed thelycum female receives the spermatophore into her thelycum and then her new
exoskeleton gets hardened over it. She retains the spermatophore until she utilizes the sperm
in one or several spawning. The subgenera Penaeus, Fenneropenaues, Marsupenaeus and
Melicrotus have the closed or internal type thelycum.

This open thelycum females follow a sequence of molt-mature- mate and spawn while closed
thelycum species follow a sequence of molt- mate-mature and spawn. In both groups, males
with hard exoskeleton deposit spermatophores into females. Multiple spawns may occur within
one intermolt period for both open and closed thelycum speices.

The stimulus for courtship behaviour in Penaeus sp. is believed to be motivated by the sex
pheromone secreted in the females and received by the males through their antennules or
antennular flagella. In open thelycum species, mating occurs mainly in the evening and in the
night spawning will occur.

3.1.10. Male and female reproductive systems in prawns

The male reproductive system of freshwater prawn consists of a pair of testes united at the
anterior end. It is located within the cephalothorax region of the male.

The female reproducitve system consists of a massive ovary situated in the cepahlothorax
region. The ovary can be clearly seen from the dorsal side of the body in the CT region as a
yellow coloured mass. The oviduct arising from the ovary opens outside in the base of the 3rd
walking leg. The seminal receptacles are present in the exterior of the female genital openings
situated in the left and right sternites of sixth thoracic segment.

Externally four pairs of pleopods serve as accessory reproductive organs present on the inner
side of the second to fifth segments of the abdominal flap. Each pleopod consists of a basal
propodite from which arise the medial endopodite and lateral exopodite. The exopodite bears a

11
large number of pinnate setae. A cluster of long and very smooth setae are on the endopodite,
to which the extruded eggs are attached at the time of spawning .

Unit 4: Larval stages of shrimps

Learning Objectives

This unit has been designed to provide the following to the students:

 Details of larval stages and their morphological features (shrimps and prawns).

 Details of post larval behaviour of shrimps and prawns.

Introduction

Hatching of eggs in the decapod crustaceans release only a larva that has entirely a different life
and habits. Larvae of shrimps are either planktonic or free swimming and not crawlers. The
feed preference differs from one larval stage to another making the larval life distinctively clear
and well defined. There are many characters like, eye, rostrum length and teeth, body
segments and appearence of appendages, etc help in the identification of larval stages. The
process of metamorpohosis helps the larvae to move on to the next stage and therefore feed
and water quality are important parameters for the development of larvae, particulary passing
from one stage to another.

Chapter 1: Description of identification features

4.1.1. Nauplius

Newly hatched nauplius is dark yellow in color. They are attracted to light. When at rest, it
keeps its dorsal side down, remaining in water in a perpendicular position. The nauplius is a
non-feeding stage as it utilizes the reserved nutrients of the egg. As it grows, it moults once in
4-6h. There are 6 nauplii stages. At each moult the size of the body increases along with change
in body structure. The duration of nauplii stage is 36-48 h.

4.1.2 Protozoea

At the completion of Nauplii VI, the larva transforms to Protozoea (PZ). As the nauplius grows to
protozoea, it starts feeding. It moults three times, thereby distinguishing three stages such as
protozoa I, II & III. Each stage lasts for 36-48 h.

12
 4.1.3 Mysis

Mysis swims with their head down, telson up keeping the body in a slanting position. They are
not attracted to light. Clear and distinguishable morphological differences do not exist between
3 sub-stages of Mysis except tin the size of the body, length of pleopod and number of setae in
the antenal scale, maxillary exopod and uropod. At the end of 3rd stage, Mysis attains a size of
3.1mm to 4.5 mm.

4.1.4 Post Larvae

The Mysis after III stages, metamorphoses into Post Larvae(PL). PL are the miniature form of
shrimps resembling the adult in all morphological and functional features. They will change
their life from the swimming to crawling and settle down to the bottom.

They will be omnivore in feeding habit and can feed on variety of items. The body is complete in
development and thier growth will be depending on the feed intake.

4.1.5 Identification of larval stages of shrimps

Nauplius stage (N) :

 Pear shaped with 3 pairs of appendages.

 Moults every 3-5 hours and passes 6 substages (N1-N6).
 Swims actively towards weak source of light.
 No feeding due to absence of mouth and alimentary canal.
 After 2 days, the nauplius converts to protozoea by metamorphosis.

Protozoa stages (PZ) :

It has 3 sub stages:
PZ I - Broad head and narrow tail with forked end. Eyes sessile.
PZ II - Eyes stalked. Rostrum developed.
PZ III - Abdominal segments develop dorsomedian spines. Uropod bud
appears near forked end of tail.
The stages lasts for 3-4 days and is succeeded by mysis stage.
Mysis stage (M) :
It also has 3 sub stages:
M I - No pleopod bud
M II - Pleopod bud appears

13
M III - Pleopod bud with 2 segments
This stage lasts for 3-4 days and then pass to I post larval stage.
First post larval stage (PL1) :
· Superficially resembles 3rd mysis except for the development of setae on the pleopods.
· First 3 walking legs lose the swimming setae and their claws become functional.
Metamorphosis:
XII POST-LARVAE:

 Teeth on upper and lower margin of rostrum.

 Behavioral changes in swimming.
 Body transparent with slightly brown coloured.
 Chromatophores on the head.

4.1.6. Identification of Post Larvae of Penaeus Spp

Characteristic features of early postlarvae of Penaeus spp.

Postlarvae I of [Link], [Link], [Link] are more or less morphologically similar.

Following characters can be easily observed .

 Long and slender body with total length of 7-13 mm.

 Absence of antennal spine on carapace;
 Rostrum reaching end of eye and with 4-6 dorsal spines.
 Supraorbital and hepatic spines present on carapace;
 Telson with 8 pairs of setae.

Characters of postlarvae of Penaeus indicus :

 Body white and transparent, except telson and tip of rostrum.

 Rostrum with 1-2 dorsal spine and single dorsal tooth extending beyond eye.
 Total length 5-10 mm. Yellowish eye stalk.
 Few mid abdominal segments.
 Telson with few branched brownish-yellow chromatophores.
 Eye stalk yellowish.

Characters of post larvae of P. merguiensis :

 Rostrum slender and elongated as in [Link]

 Basal region of rostrum not elevated.
 4-5 dorsal rostral spines.

14
  Telson and half the distal end of the uropod with chromatophores.

Unit 5: Larval stages of freshwater prawn

Learning Objectives

This unit will help the students to acquire knowledge on the following subjects:

1. Identification features of larvae of shrimps and prawns.

2. Understanding the characteristics of post larvae of Penaeus spp.
3. Behaviour of larvae and post larvae of Penaeus spp.

Chapter 1: Description of life cycle and larval stages

[Link] Larval Stages

The newly hatched Zoea larva is about 1.92 mm in the length. It has the zoeal characteristics
such as a body distinguishable into cephalothorax (CT) and abdomen (AB). The cephalothorax is
covered by a carapace having the dorsomedian rostrum. All thoracic and cephalic appendages
are present. The larvae pass through 11 zoeal stages within a period of 23-32 days based on the
availability of feed, light and appropriate water quality.

15
 5.1.2 Identification of larvae and post-larvae of Macrobrachium rosenbergii

Macrobrachium rosenbergii has 11 larval and one post larval stage (i.e. 12 stages) in its life
cycle. The larva passes through 11 zoea stages within a period of 23-35 days depending upon
the temperature and availability of feed.

After these 11 zoeal stage, the post larvae stage is reached, which exhibits the features of adult
prawn.

Key to the stages of larvae of Macrobrachium rosenbergii:

Stage Prominent characters Days after Size (mm)

hatching

16
 I Sessile eyes 1-2 2.0 – 2.1
II Eyes stalked 2-4 2.2 - 2.4
III Uropods appear 4-7 2.5-2.7
IV Two dorsal epigastric teeth at base 7-12 2.8-3.1
of rostrum
V Telson narrower and elongated 11-16 3.2-3.4
VI Pleopod buds appear 15-21 3.5-3.6
VII Pleopod biramous and bare 18-24 3.7-4.2
VIII Pleopods with setae 22-28 4.3-4.6
IX Endopods of pleopods with 25-31 4.7 – 5.2
appendices
X 3-4 dorsal teeth on rostrum 28-33 5.3 – 5.8
XI Teeth on half of upper dorsal margin 31-35 5.8 – 6.2
XII (PL) Teeth on upper and lower margin of
rostrum; behavioural changes in
swimming. They are generally
transparent at this stage and have
slightly brown coloured
chromatophores on the head.

17
Unit 6: Reproductive biology of crabs

Learning Objectives

This unit has been designed to provide the following to the students:

1. General introduction to identification of sexes in crab and their breeding behaviour.

2. Sizes at first maturity for different crabs.

Chapter 1: Sexual dimorphism

6.1.1 Identification of sexes

Sexes can be morphologically distinguished based on the shape of the abdominal flap. Though
the shape of this flap is similar in both immature and mature male, it is different in mature and
immature female. In mature female the shape of abdominal flap is half-round, while in
immature female it is broad and triangular.

6.1.2. Sexual dimorphism

The sexes are separate and there is distinct sexual dimorphism in crabs:

Distinction between male & female based on morphological characters.

18
 Male Female
Abdominal flap which is folded Abdominal flap folded against the
firmly against the ventral side is ventral side of body is broad
narrow (triangle shape) of (Round shape) and berried females.
cephalothorax is slender and
triangular. There are 4 pairs of abdominal
appendages present from 2nd to 5th
st
Appendages are present only on 1 segment and the same are used for
and 2nd abdominal segments and carrying the eggs.
the same are modified to
copulatory organs. The claws are smaller.

The claws are comparatively larger.

6.1.3. Size at maturity
The size range at first maturity in males and females of S. tranquebarica and S. serrata is
presented in the Table below
Size range at first maturity in S. tranquebarica and S. serrata

Size range at first maturity

Species Sex
(Carapace width in mm)
Male 125-133
[Link] Female 129-135
[Link] Male 80-89
Female 85-96
Chapter 2: Mating and spawning

6.2.1. Mating

The size at first sexual maturity is 120 mm (carapace width) for larger species ([Link])
and 83 mm (cw) for S. serrata. The early maturing ovary is bright orange in colour where as, in
matured – ready to spawn female, it is deep yellow in colour. Copulation takes place between a
hard shelled male and a freshly moulted, soft bodied female. The courtship is initiated by a
“premating embrace” between hard shelled male and hard shelled female which lasts for 2-3
days.

19
 6.2.2. Spawning

The ova are extruded by the female and the same are fertilized by the sperm stored in the
spematophores. The fertilized eggs are attached to the ovigerous setae of the abdominal
appendages. In S. tranquebarica, the nos. of eggs may be 2-3 million per female of body weight
whereas in S. serrata it is 0.5-2.5 million such as females with eggs attached to the abdomen
are called berried.

6.2.3. Incubation & hatching

The berried females carry their eggs for 2 weeks, during which period the embryos develop. The
eggs undergo change of colour from orange to grey / brown. Just before releasing the larvae,
the eggs become grey black. After the incubation period, the larvae hatch out. The hatched out
larvae need feed for their growth.

Unit 7: Life cycle of crabs an

Learning Objectives

This unit has been designed to provide the following to the students:

1. Life cycle of crabs and lobsters

20
 2. Breeding biology and larval development in lobsters
3. Identification features of larvae of crabs

Chapter 1: Life cycle of Crabs

7.1.1 Introduction

In general, the crustacean eggs hatch to produce nauplius larva which is free swimming. It is not
always true as in some cases the nauplius stage is passed within the eggs so that there is no
free swimming nauplius larva produced after hatching, E.g. freshwater prawns, cray fishes,
crabs, etc. In such animals, the freshly hatched out larvae are called Zoea and these larvae will
be taking food immediately after hatching and their development is faster than other stages.
But the risk is that in the absence of feed, they may become canibals or die of starvation.

7.1.2 Larval development

The zoea larva undergoes 5 successive moults to become the megalopa larva. Thus there are 5
zoeal stages. For each moult, it takes 3-4 days. Thus to develop a megalopa from a zoea it takes
15-20 days. Megalopa develops into juvenile crab after 8-11 days.

A newly hatched zoea larva of crab measures 1.2 mm and it consists of a cephalothorax and a 5
segmented abdomen and telson. The carapace bears 4 spines, one dorsal spine bent
backwards, rostral spine bent forwards and 2 lateral spine closely pressed against the sides of
the body. The eyes are not stalked. The first maxilla consists of coax, basis and endopodite
(each with 4,5 and 6 setae respectively), a second maxilla has coax and basis bilobed, with 1-1
and 2-3 setae respectively, the endopodite is having 3 rudimentary setae at its tip where as
exopodite is with 4 rudimentary setae. The first maxilliped also has coax, basis and exopodite
and endopodite, but the endopodite is 3 segmented, and it has only 3 rudimentary setae.
Second and third segment of abdomen has a pair of knobs. Telson is rudimentary and forks are
not well defined.

As it passes through 5 zoel stages, growth in body size, appendages etc take place. Zoea is 3.5
mm in length. From zoea II onwards the eyes are stalked (for distinguishing characteristics of
larva stages of crab).

The megalopa larva has a crab like appearance. The cheliped is well developed. 5 pairs of
pleopods are present which becomes locomotory in function. Chelipeds are used to catch prey.

21
 7.1.4. Identification of larval stages of crabs (Scylla serrata)
Larval stages Important morphological characters
Zoea I Eyes sessile ; 5 abdominal segments; telson with 3 + 3
spines.
Zoea II Eyes stalked ; 5 abdominal segments; telson with 4 + 4
spines.
Zoea III 6 abdominal segments
Zoea IV Rudiments of remaining thoracic appendages,
abdominal segments with buds of pleopod.
Zoea V Remaining thoracic appendages developed; pleopods
on abdominal segments with setae; telson with 5 + 5
spines.
Megalopa Carapace longer than wider; abdomen with 5 pairs of
pleopods ; a pair of cheliped; 4 pairs of legs.
First crab instar Carapace with 9 anterolateral teeth on either side; 3
pairs of walking legs; last pair of legs with paddle
shaped dactylus; resembles parent.

22
23
Chapter 2: Reproductive biology & physiology of lobsters

7.2.1 Introduction

Spiny lobsters are abundant in Gulf of Kutch, Maharastra,Gulf of Mannar and Andaman
Nicobar islands. It is a valuable fishery resource for the country as it brings in huge foreign
exchange and a much demanded sea food in the world. In recent years there has been a
decline in the catch due to over- exploitation of juveniles and spawning females. It is necessary
to protect this valuable resource by artificial seed production and ranching. This has been
emphasized by many natural and biological researchers in the world.

There are many bottle necks in the hatchery seed production of lobsters. However, constantly
efforts are being made to overcome the bottle necks and make the seed production possible.
Clear understanding of the basic reproductive biology and physiology of lobsters is essential to
make success in the seed production.

7.2.2. Reproductive biology

Sexual dimorphism

Sexes are separate in lobsters and usually males are larger than females. In the male, the 5 th
periopod or walking leg ends in single claws but in the female the tips of these legs are provided
with three points formed by spurs used for carrying eggs attched to the under side of the tail.

24
Identification of males and females

The males differ by the presence of swollen genital opening at the base of last pair of periopod,
whereas in the females it opens at the base of 3rd pair of periopod or walking legs.

The female lobsters have two branches of swimming legs whereas the male is having a single
branch. The animal has a prolonged breeding periopod and the peak season is October-
December under natural condition but it is observed that in confined conditions, it breeds
throughout the year in India.

Mating and spawning

Male lobster deposits spermatophores on the sternum of the female between the last three
pairs of appendages. The spermatophores turn black and are called as tar spots. Development
of eggs will take 24-72 hrs, during this periopod eggs will develop and remain attached with
pleopods of female lobster or oveigerous setae. The female carries the eggs until the hatch out.

7.2.3 Larval development

Metamorphosis and growth of larvae

The eggs hatch into transparent, flat and leafy shaped larvae quite different from the adult and
are called as Phyllosoma larvae. The Phyllosoma larvae have antennules, antennae, paired eyes
and very long jointed setose appendages for swimming. Phyllosoma larvae are planktonic and
are dispersed far and wide by currents. Their dispersal is further aided by the lengthy larval life,
during which the larvae undergo about a dozen moults. In this process, lobster undergoes
indirect development, involving a wide variety of Phyllosomal larval forms. In this indirect
developmental process there is a final larval stage, which differs from the adult in many
features. It acquires adult features to Puerulus larval stage. From this stage there is direct
development to adult hood through progressive growth during which differentiation takes
place.

7.2.4 Larvae of Lobster

The size of this larvae is very large, having head, throax, abdominal regions, and is transparent
as glass. A large carapace covers the first two thoracic segments and head. Abdomen is very
much reduced and may be indistinctly segmented, but without appendages, the antennae and
maxillae are degenerated before hatching. The seventh and eight thoracic appendages and
abdominal somites are not clearly segmented in the larvae, even though they are well defined
in the embryo. The first thoracic appendages are rudimentary and second is uniramous.

The Phyllosoma undergoes several moults before becoming an young lobster(juvenile) and
duration of this stage may extend upto 250 days (90-250 days). There are 7-13 Phyllosoma
stages(some times a pre-Phyllosoma, and pro Phyllosoma). The larval periods of palinurids

25
ranging from few months to almost 2years before last Phyllosoma stage at about 35 mm in
length.

Puerulus stage (7-56 days,I - stage)

After 6-10 months in the planktonic larval stage, the Phylllosoma larva metamorphoses into the
Puerulus stage.

It is transparent, free swimming, non feeding post larval stage that moves inshore where it
settles to the bottom. Subsequently through ecdysis, Puerulus becomes juvenile lobster which
leads a nectonic swimming life with well developed appendages.

Un

Learning Objectives

The following are the objectives that can be achieved through this unit:

1. Acquiring knowledge on the secondary sexual characters of shrimps and prawns.

2. Importance of understanding the secondary sexual characters.
3. Ability to asses the bioeconomical values of the brooders.

it 8: Brood stock of crustaceans and their management

Learning Objectives

The following are the objectives that can be achieved through this unit:

1. Acquiring knowledge on the secondary sexual characters of shrimps and prawns.

2. Importance of understanding the secondary sexual characters.
3. Ability to asses the bioeconomical values of the brooders.

 Chapter 1: Secondary sexual characters

8.1.1 Significance of learning secondary sexual characters

Study of secondary sexual characters is of much importance, as it not only helps to differentiate
between the sexes of a species; it also helps to differentiate mature ones from immature or at
different stages of maturity.
According to some evolutionary biologists, the secondary sexual characters may help in
understanding evolution and gradual changes in these characters, that may help in
understanding the relationship between species.

26
The secondary sexual characters are important to the animals as they assist in reproduction and
help in identification or recognition of useful matter in natural or hatchery conditions.

Observations of the secondary sexual character is important for the clear understanding of
natural reproduction so that captive reproduction technology can be developed.
8.1.2 Secondary sexual characters of shrimps (Penaues spp.)
Male :

 Modified abdominal appendages (1st and 2nd pairs) and abdominal segments (the
petasma and appendix masculina) that deliver sperm to the female’s sperm receptacle
(the thelycum). The petasma and appendix masculina are located on the ventral surface.
 The petasma is formed by the fusion of the endopodites of the first pair of pleopods
which are modified as interlocking structures for spermatophore transfer.
 The appendix masculina are on the endopodites of the second pair of pleopods and
serve to open the petasma.
 The gonopore is located at the base of 5th walking leg.

Female :

 A thelycum is present in the thoracic sterna between peropods IV and V. It is meant for
storing the spermatophore.
 Thelycum consists of a median plate and 2 lateral plates which enclose a seminal
receptacle for storing spermatophores.
 Female gonopore is located at the base of 3rd walking legs.

8.1.3 Secondary sexual characters of prawns ([Link])

Male :

 Second pair of swimmerets are modified to form spinous projection known as appendix
masulina, which helps in copulation
 Genital pores are found at the base of 5th walking legs.
 The endopodite of 2nd pleopod has appendix interna alongwith appendix masculina.
 Reproductive setae are absent on the ventral side of the thorax and pleopods of mature
male.
 Ventral side of the first abdominal segment has a hard lump at the centre, which can be
felt with finger.

Female :

27
  First 5 pairs of softer swimmrets are the attachment sites for cluster of eggs within the
brood chamber.
 Genital pores at base of 3rd walking legs.
 Extended pleura of 1st, 2nd and 3rd tail segments to form brood chamber to carry eggs
after spawning, fertilization and before hatching.
 Reproductive (ovipositing and ovigerous) setae on the ventral side of thorax and
pleopods of mature females.
 Ovipositing setae appear on the coxa of last 3rd pair of pereopods and posterior margin
of sperm receptacle area and the pleopods. It helps in guiding and propelling eggs
during spawning.
 Ovigerous setae serve to anchor the eggs to pleopods.
 Ovaries can be seen as large orange-coloured mass occupying a large portion of dorsal
and lateral cephalothorax in a ripe or ovigerous female.

8.1.4 Bioeconomic values of the brooders

The brooders have a tremendous biological as well as economical value. In general a healthy
brooder is the base for the population proliferation and establishment. The following
parameters are considered while fixing the bioeconomic value of the brooders.

1. The size of the female brooders

2. The healthiness of the brooders
3. The natural habitat of the brooder
4. The stress bearing ability (tolerence)
5. The age of the brooders
6. The known fecundity of the brooder

Based on the above, the value of the brooder is ascertained and fixed.

Unit 9: Selection, collection and transportation of shellfish brood stock

Learning Objectives

This unit has been designed to provide the following:

1. Parameters to evaluate the breeders available from wild.

2. Methods of collection, criteria for selection and transportation of the brooders.
3. Safe handling methods of brooders.
4. Quarantine and prophylactic measures to be adopted in hatchery.

Chapter 1: Qualities of brood stock

9.1.1 Qualities of brood stock – Parameters for evaluation

28
Generally shrimp or prawn brooders must possess some qualities in order to get quality seeds
and economical hatchery operation.

Parameters of brooder or mother shrimp quality are,

1. Bigger in size without any damages in their body

2. Good pigmentation

3. Clear eyes and open telson (broad)

4. Complete antenna without any damages or cuts

5. Full stomach - no white patches

6. Ovary seen occupying the full abdomen portion

7. Thelycum loaded with spermatophores

8. If non-impregnated, they must be fully matured and ready for mating

9. If males, bigger size and active.

Brooders with such parameters are considered useful for the seed production.

9.1.2 broodstock quality

The basic requirement for healthy and quality seed production is healthy broodstock of desired
size so that it will yield maximum number of viable eggs. There are two sources for the
broodstock. One being the wild natural waters, the other from the culture ponds.

For shrimp hatcheries, mostly broodstock is obtained form wild habitat. Captive broodstock for
[Link] is still under experimentation, while for P. indicus α P. japanicus, captive broodstock
development has become possible. However, the brooders from wild are found to be superior
to captive brooders which are usually smaller in size. Males of a minimum size of 200 mm total
length weighing over 90-150g (9-12 months) are reported to give good results in tiger shrimp
seed production. Other criteria of brooder or mother shrimp quality are,

1. Big size without any damages

2. Pigmentation

3. Clear eyes and open telson (broad)

29
4. Complete antenna

5. Full stomach - no white patches

6. Ovary occupying the full abdomen portion

7. Thelycum loaded with sperm mass

8. If non-impregnated, there must be readiness for mating

9. If males, bigger size and active.

Transportation of brood stock

Usually the adults are collected from deep sea and from the collection site to the hatchery site,
the breeders are transported in bag containing 40-50 litres of sea water @ 4 - 6 nos per bag.
After introduction of the breeders, the bag is filled with oxygen and sealed. In this condition the
brooders can be transported without much stress. In order to avoid any casuality, the
temperature of the bag may be reduced with ice cubes packed polythene bags kept
surrounding.

Spawner transportation, particularly by air, by adopting chilled saw dust method is not
advisable to any of the currently cultured species in India. P. monodon can not tolerate a
temperature below 120C and P. semisulcalus can not withstand exposure for long.

Acclimatization of brood stock

After the brooders are brought to the hatchery, they are to be acclimated to the hatchery
conditions by transferring the brooders to tubs with seawater used in hatchery. Care must be
taken to do this slowly in case there is considerable difference in salinity, pH and temperature.

After acclimation, brooders are treated with 100 ppm formalin for about ½ hr for disinfection.
In case brooders exhibit some sign of disease such as lying on one side/ abnormal
pigmentation/ lesions in the exosleleton, a dip treatment of antibiotic (50 ppm oxytetracycline/
erythromycin/ perfuran) for 10 minutes can be given. Such disinfected and healthy brooders
are then transferred to maturation tanks.

Chapter 2: Handling of the brooders and transport

9.2.1 Freshwater prawn brooder transport

Transport of freshwater prawn brooders

30
Berried females collected from the natural water bodies can be transported from the collection
site by keeping them individually in perforated plastic or PVC pipes or cylinders capped on both
sides with pieces of netting. The pipes/ cylinder carrying brooders should be kept in the water
in the polythene bags. During transit, the polythene bags are oxygenated by oxygen. For shrot
distance, tanks or bins are well suited for this purpose.

During long distance transportation tips or restrum and telson may be banded or capped with
protective rubber tubes and transported in plactic bags filled with water and oxygen (1:1). This
is to avoid physical damage to spawners and bag.

Temperature can be controlled by keeping small ice bags alongside the plastic containers &
keeping inside the bins/ tanks. Metabolite accumulation in the medium can be delayed by
starving the animals for few hours before packing for transport.

9.2.2 Quarantine

Immediately on arrival in the hatchery, the spawners may be given a dilute formalin dip
treatment (200-400 ppm for 1-2 hours and then rinse). This prevents introduction of epifauna
into the hatchery.

9.2.3 Mud crab brood stock - collection, transportation, acclimatization and management

Live mature male and female S. tranquebarica and S. serrata can be obtained from the inshore
sea and brackish water regions where these mud crabs form a commercial fishery.

Transport of broodstock

Generally live mud crabs are tied with jute / nylon thread to arrest the movement of chelipeds
for easy handling. Live adult crabs obtained from the fishermen / merchants are dipped in fresh

31
and clean sea / brackishwater in order to moisten the gills and packed in bamboo baskets with
wet sea grass / seaweeds and are transported either by road or rail or air. By this method of
packing, mud crabs are known to survive for a maximum period of 3 - 4 days.

If the duration of transport is longer, those crabs with all limbs intact are given another dip in
sea / brackishwater and packed in new bamboo baskets en route. This will ensure their survival
for another 3-4 days.

In another method, adult crabs weighing 350-700g are packed at the rate of one crab per
packet in polythene bags each containing six litres of oxygenated seawater after placing plastic
sticking tape over the chelipeds and a tight plastic tube over the tip (dactylus) of walking legs
and transported by road, rail or air for a duration of 24 hours.

Prophylactic treatment of broodstock

Mud crabs obtained either from the wild or from the culture ponds should be disinfected
individually in 100ppm Formalin for 30 minutes before transferring them to the broodstock
holding thanks.

Acclimatization of broodstock

After disinfection, the adult mud crabs should be held in tanks containing sea water (30 to 35
ppt), for a week, in order to acclimatize them to the hatchery conditions, before the initiation of
trials on induced maturation and breeding.

Unit 10: Captive brood stock development techniques (Crustaceans)

Learning Objectives

This unit has been designed to achieve the following objectives:

1. To provide information about the broodstock handling and transport

2. Maintenance of brood stock in the hatchery

3. Basic factors to be remembered in the handling of brooders

4. To provide a base for the collection and transportation of brooders to the hatchery

Chapter 1: General care for the brooders

10.1.1 Source of broodstock

32
Broodstock of shrimps and prawns can be obtained either from wild waters of captive ponds.
For Penaeids like P. monodon, the source happens to be wild waters due to their complexity in
the captive maturation. For P. indicus, P. japonicus and P. vannamei, the brooders can be
developed in the captive conditions. This helps in the development of SPF and SPR brooders for
the quality seed production.

Macrobrachium brooders can be easily collected from the captive ponds as they mature and
mate in captivity. Live mature male and female S. tranquebarica and S. serrata can be obtained
from the inshore sea and brackish water regions where these mud crabs form a commercial
fishery. For lobsters the brooders have to be collected from the wild waters. But the hatchery
seed prodcution is yet to be commercialised for the lobsters in India.

10.1.2 Transport of broodstock

Transportation of brood stock will become essential if the hatchery is located far away from the
brooder collection site. The following points are to be kept in mind before taking up the
transportation of brood stock:

1. The brooders must be healthy and fit for transportation.

2. The transportation duration must be as less as possible

3. There should not be any un due dealy in the transportation once packed.

4. Over stocking must be avoided

5. If infected or carrying pathogens or parasites the brooders must be transported only after
they are fully recovered or healed.

6. There should be low metabolic activity in the brooders during transport to avoid
accumulation of metabolites in the water.

Care must be taken for the safer transport.

33
 10.1.3 Stocking and maintenance of the brooders

Tanks with cement / RCC either circular or rectangular in shape tanks (five or ten ton capacity)
with facility for draining water are used for holding the brooders of crustaceans, (both induced
or ablated females and unablated males). The stocking rate should be 1-2 brooders per square
metre to facilitate easy movement and less infighting. Adult unablated males and unilaterally
eye-stalk ablated females should be stocked either in the ratio of [Link] or 1:1.5 in the case of
shrimps and crustaceans.

In the case of freshwater prawns (Macrobrachium spp) the brooders can be stocked in the
tanks in the hatchery or in net cages or haps in the ponds. Their helath should be maintained
with good feed and water quality in both the places.

Chapter 2: Crab brooder development and maintenance

10.2.1 Marking of reared crabs

Marking is essential for the correct identification of the brooder for breeding purposes.

Both male and female crabs that are stocked in the brooder tanks should be marked on their
carapace with white colour epoxy paint. Since the crabs undergo moulting there is a possibility
of losing the mark. Therefore, when a crab moults, the same marking as present or made in
their old exoskeleton may be painted on the newly formed carapace after hardening of the
exoskeleton of moulted crab. Thus, the identity of an individual crab in the stock is continuously
monitored. Re-marking of crabs may be done, whenever the old marking fades away.

10.2.2 Refuge system for crabs

To facilitate the burying habit of mud crabs, a refuge system is constructed in one part of the
holding tank. For this purpose, one-third floor area of the tank should be provided with a clean
sandy substratum up to a height of 10cm. A brick-wall of 15 cm height may be erected to hold
the sand in position. Otherwise, while burying, mud crabs may scatter the sand. To reduce
cannibalism among the brood crabs, hollow-brick blocks may be placed as hide-outs for the
crabs.

34
 10.2.3 Size of broodstock
The size range of male and female brood crabs to be maintained under captive conditions is
given below
Size range of brood crabs
[Link] S. serrata
Sex
( Carapace width in mm / Total weight in g)
Males 125-165 mm / 330-685 g 80-135 mm / 90-590 g
Females 129-175 mm / 300-690 g 85-137 mm / 98-340 g
Filtered seawater drawn from a bore-well or from the open sea, with a salinity of 30-35 ppt
should be used for rearing crab broodstock. After removing the left-over feed, 60 to 70% of
water should be changed daily in the morning. The water level should be maintained at 60 cm if
the tank height is 100 cm. Otherwise, if the water level is maintained at 80 cm, the brood crabs
may climb out. Water temperature should be 25 to 290C.
10.2.4 Feeds and feeding schedules

Animal flesh such as bivalve meat and squid meat which contain high protein levels may be
used as maturation diets for crabs. The rate of feeding should be 8-10% of stocked biomass.
The daily ration may be divided into two equal quantities and offered twice a day, one in the
morning and another in the evening with 12hrs time interval. The crabs should be fed to their
satiation.

Studies on the food and feeding habits of wild S. serrata have shown that the main food items
included the remains of crustacean (44.3%) molluscs (14.3%) and others (19.1%). Therefore a
feed dominating with animal matter is essential for the health and breeding performance of the
crabs.

Pellet feeds are also found to be useful with limited success.

10.2.5 Mating

Male and female crabs are found paired together continuously for days. During this period, the
male would climb over the female and clasp her with his chelipeds and walking legs. The pair
separated on the verge of precopulatory moult.

After the copulatory moult of the female, the male gently turn the female over on her back
using chelipeds. The female unfolded her abdominal flap and held the male in position for the
deposition of the spermatophores. The whole process will last for two days.

10.2.6 Spawning

The eggs are yellow in colour at the time of spawning and turn to grey or brownish grey or black
before they hatch and release the larvae in 14 days.

35
The eggs will measure from 280 to 380 micro meter in diameter, while the early and late zoea
larvae measure at 1.0 and 1.4 mm in length, respectively. A berried female measuring 140mm
will relase 2 million larvae. The size of berried females from the wild ranged from 93-175 mm
(400-1000g) and that of ablated females will be 135-140 mm (340-700g).

Unit 11: Breeding biology and reproductive physiology of Bivalves

Learning Objectives

This unit has been designed to provide the following to the students:

1. General introduction of the reproductive biology of bivalves.

2. Maturity stages of bivalves.

3. Factors influencing the breeding of bivalves.

4. The importance of bivalve seed production.

Introduction

Oysters are the economically important bivalves along with mussels and clams. Their economic
importance is due to the quality of their meat and shell utilities. While edible oysters of
different species are world wide preferred for their nutritious meal, pearl oysters are of
immense value as they yield natural pearls.

In general bivalves are sedentary found in the ocean at a depth of 18-22m and found attached
to rocky ridges, usually 19 Kms offshore and also in freshwaters.

About 28 species of pearl oysters are reported so far. The common species found along the
Indian coasts are Pinctada fucata, P. chemmitzi, P. margaratifera, P. anomioides and P.
atropurpurea.

Chapter 1: Factors affecting the breeding of bivalves

11.1.1 Temperature
Temperature plays an important role in the biological activities of pearl oyster in the temperate
waters (The optimum temperature for the growth of oyster in Japan is found to be 20-25 oC).
Above 28oC, the pearl oysters show signs of exhaustion. Spawning is effected by the heating the
water temp. from 25-30oC. The calcification is stopped when the water temperature goes below
13oC. The oyster perishes at 6oC.

36
Oysters are very common in tropical waters than in temperate waters due to their preference
of higher temperature for their growth and reproduction.
11.1.2 Salinity

Generally pearl oysters seem to prefer high salinities. They can tolerate a wide range of salinity
(24-50ppt) for short duration of 2-3 days. Salinities; below 14ppt and above 55ppt are known to
be fatal for pearl oysters. Normally they prefer to live in waters with narrow changes in salinity.

11.1.3 Primary productivity

The plankton are the main source of amino acids and proteins for the pearl oysters. But the
blooms of Trichodesmium had caused mortality to the oysters. Suitable phyto plankton if
available in sufficient quantity can promote the growth and gonadal development in bivalves.
Therefore areas where the primary is high are prefered for the erection of bivalves culture
cages and poles and racks.

Chapter 2: Maturity stages in bivalves

You are here

 [Link]
 / ► FAQ 312
 / ► Resources
 / ► 11.2.1 General classification of maturity stages in P. fucata

31552 true D7zPBKndcn Update this Resource

11.2.1 General classification of maturity stages in P. fucata

Male and female cannot be distinguished externally. There is a pair of gonads present over the
intestine and hepatopancreas. Duct arising from the gonads opens as gonophores located at
the anterior end of the gills.

STAGES CHARACTERISTICS
Inactive /
Gonads translucent, sex is in indeterminate form.
Spent resting
Developing / Transparent gonad, Oocyte size is 60 x 47.5 µ
Maturing Gametogonic materials begin to appear.
Gonads yellowish cream in females. Oocyte size is 68
Mature
x 50µ. Average nucleus size is 25 µ.
Partially spawned Gonad becomes loose. Oocyte size is 51.7 µ.

37
 Spent Immature Oocytes

Chapter 3: Other bivalves

11.3.1 Mussel

The sexes are separate green mussel is faster growing species than brown mussel. It has been
also found that cultured species grow faster than natural ones, and in open sea it grows faster
in bays or lagoon. P. viridis attain sexual maturity at a very small size such as 15.5-28.0 mm
length i.e., 2-3 months old.

11.3.2 Clams

Anadora granosa males attain first maturity at 20 mm length and females at 24 mm length. It is
an year round spawner having 2-4 reproductive cycles a year. Fecundity is about 50,0000 eggs
per spawner. Brood stock may be of 3-4 cm length and 1-1.5 year old.

P. malabarica - Size at first maturity is 11-20mm. It spawns from September to January in the
year.

K. opima - Size at first maturity is 11-20mm; main spawning season is October – November.

Chapter 4: Bivalve larval development under natural conditions

11.4.1 Comparison of larval development among the bivalves

In pearl oyster, pediveliger develops to plantigrade having a size of 200 µm. All along the
margin except umbo region rapid shell growth takes place. Labial palps, additional gill filament
and byssus threads develop. Plantigrade develops into spat (300µm). It is a juvenile having left
valve larger than right valve.

In mussles, Veliger grows to umbo stage approximately 20h after fertilization. It develops a
pedal organ and it is called pediveliger larvae. Spat settlement takes place from 20th day
onwards and continious for 5-9 days.

In clams, the different stages in the development is mostly similar to that of green mussles. The
differences being that D- hinge veliger starts consuming food 1-3 days after fertilization in clam,
Umbo becomes clearly visible 7-15 days after fertilization and settlement stage is 21-35 days
after the date of fertilization.

38
 11.4.2 Spat collection from natural spawning ground

During the spawning season, when there is an onset of spat fall, spats can be collected by
providing spat collecting materials in the ground. In Thailand and The Phillipines, bamboo
poles / wooden sticks are used by the farmers. The spat attches to those objects easily.

Another popular type of spat collectors are ropes made of either coir or synthetic material such
as polypropylene or polyethylene. Ropes made of natural fibre like coir is very successfully used
for collecting the mussel spat because of their hairy and crevised nature offering much
advantages in attachment. There is poorer settlement of spat on smooth ropes. But the
disadvantage with coir rope is that it has lesser longivity in water whereas synthetic fibre can be
used for several years. Several modifications of the rope system have been deviced for better
efficiency of spat collection.

11.4.3 Spat collectors

Tiles suspended from rafts, used tyres suspended in water, or polyethylene nets are used as the
spat collectors. It is necessary to season the spat collecting material by immersing in sea water
for atleast 2 weeks. Settling of young spat is successful only if it is seasoned. It is due to the fact
that during seasoning other marine organisms attach to the substrate prior to spat fall. The pre
coating of similar organisms is considered useful in the spat settlement.

Unit 12: Life cycle of cray fish and commercially important molluscs (other than bivalves)

Learning Objectives

The study will help understanding the following

1. Breeding biology of molluscs other than bivalves.

2. Life cycles of squid, cuttle fish, cray fish, etc.,

3. The captive breeding of the cray fish, squid and cuttle fish.

Chapter 1: Life cycle and breeding biology of cray fish

12.1.1 Importance of cray fish

Cray fish or craw fish are popular freshwater crustaceans in Europe and Australia. Their
commercial exploitation and culture are very popular in the above continents due to high
demand and consumers' preference. They are being cultured in ponds and marketed.
Worldwide cray fishes are the freshwater crustaceans contributing more to the aquaculture
production. Procambarus clarkii is the most widely cultured species among the cray fishes and
Cherax spp are the next ones.

39
Craw fish are temperate in distribution and can live and grow only in temperatures below 20
deg C. Therefore they are not distributed in tropical countries. Their breeding biology is well
known and therefore their seed production is also popular in the countries where they are
cultured.

Chapter 2: Breeding biology of cephalopods

[Link] cycles of Cuttle fish and Squids

Squids and Cuttle fishes are Cephalopods (having appendages in their head) contributing more
for the revenue from seafood. They are much preferred delicacies in many countries. Although
many of them are tropical in distribution there are some species that are found in temperate
waters too. They are not populary cultured in the ponds or any other aquaculture systems. But
they are widely fished out from natural waters and exploited.

Understanding their biology and reproduction will help in conserving this species for
sustainable yield from the wild waters.

Unit 13: Shellfish hatcheries - Design and construction of hatcheries for different shellfishes

Learning Objectives

This unit has been designed to provide the following:

1. Guidelines for the selection of sites for the construction of hatcheries

2. Basic designs and layout for shrimp and prawn hatcheries
3. Implications of different factors in the designing and construction of the hatcheries
4. .[Link]
5. / ► FAQ 312
6. / ► Resources
7. / ► Introduction

31574 true D7zPBKndcn Update this Resource

Introduction

Successful hatchery seed production largely depends on the perfect location of the hatchery.
Before locating the hacthery it is essential to study the physical, chemical, biological and social
parameters of the site so as to minimise the risks.

A site identified for the establishment of the hatchery should be thoroughly screened for
various parameters in order to assess the usefulness of the site for the particular species. The
supply of various raw materials needed for the hatchery opreation needs to be looked into

40
 along with their quality and quantity aspects. The social problems that can influence the
hatchery process also need to be identified.

The following chapters deal with the criteria for the site selection and their limiting factors for
the shellfish hatcheries

Chapter 1: General guidelines

13.1.1 Site selection for a hatchery - Basic parameters

[Link]. Factor Desired Undesired Implication Corrective measure
character character
1. Site level Moderately high Very high or very Very high level will Pumping facility should
( 1 to 1.5 m from low (>1.5 m & make pumping cost be erected for high
normal GL) <0.1m from high and fast seeping if level site. The bundh
normal GL) ground is not should be made
impervious. impervious.

Very low level willFor low level ground, a

make drainage separate drain canal
impossible or digging
below the pond bottom
will lead to further
level should be
downward move. planned. Mechanical
drain should be made.
2. Soil Clay mixed with High clay or high High clay will interfere Sand to be mixed if clay
composition sand (Loamy soil sand with the nutrient is more. Clay should be
– clay 60 – 65%) trapping and high imported into the pond
sandy area will not site if sand is more.
hold water.

Involves high cost for

the construction of the
water holding pond.
3. Soil pH Must be between >8.5 – highly High alkaline or acidic Correction can be done
8 and 8.5 alkaline soil will make the with the application of
culture difficult in alam or peaty soil or
<7.5 – acidic terms of nutrient lime based on pH.
release and growth
promotion.
4. Extend of the Must be between Less than 1 acre Lesser extend will not Only the required area
site 1 – 25 acres) and more than 25 be economically should be developed
acres feasible. for the farming.

More extend will

41
 enhance cost of
production as it
involves more
overheads.
5. Cost of the Should be Too high price Will make the project Site with the affordable
land affordable for the unviable. Will increase cost alone should be
project (Project the cost of production selected.
dependent) thru indirect costing
6. Accessibility to Must be easily Remotely located. Visiting the site will Minimum accessibility
the site accessible by Inaccessible. make cumbersome. level should be
road The farmer will lose satisfied. Site with the
interest in going to the accessibility should be
farm. Cost will be high selected. Accessible
for going and visiting mode must be
the site. designed.
7. Influence of Free of any Frequently Unexpected damage Site should not be
natural natural calamity affected by and loss. Farm may be selected.
calamities like flooding, natural calamity a severe loss
heat waves, (easily flooded, enterprise.
cyclone, etc highly drought
prone, more heat,
windy or cyclone
hit areas)
8. Water Must be plenty Low availability of Difficult to manage the Suitable water sources
availability water crop in times of should be located. Site
emergencies. with abundant water
alone should be
selected.
9. Water pH must be >7.5 Deviating from No growth or fish kill, Corrections can be
suitability & <8.5; the desired No productivity done with water
characters treatment, but involves
Hardness < 100 cost and will affect the
ppm economics.

No ions, no Iron,
no pollutants,
Less BOD & COD,
No
contamination
with sewage
10. Nearness of Must be so near Far away from the High cost for Open conduits or closed
good water so that expenses ponds transportation of pipes can be used for

42
 source could be water. Water loss transportation.
minimized on during transportation
water
transportation
11. Accessibility to Accessible by Inaccessible. No Reaching the site for Road access has to be
the site road. road connection construction as well as made for easy approach
to the site during crop operation
will be very difficult
12. Labour Enough labour No labour near Cost for bringing the Low cost labour force
availability must be available the site labour for work will be should be identified
near to the site near the site. high near to the site
13. Cost of labour Must be High cost for the Construction cost will Labour cost must be
reasonable labour be too high and project checked and kept in
cost will be unbearable control
14. Availability of Technical No technical Difficulty in the regular High cost has to be paid
technical managerial level manpower operation of the farm for bringing in
manpower personnel should available near the manpower for the
be available near site project
the site
15. Availability of Power must be No power in the Use of diesel genset Alternative power
power available near or vicinity of the site will increase the cost of source should be
in the site production identified and
employed.
16. Availability of All the inputs Inputs availability Cost of production will Inputs have to be
inputs needed for the is not assured or increase on the brought from faraway
farm should be uncommon transportation of the places will affect the
available near inputs economics of the
the site farming
17. Availability of Seeds must be Seeds not It will be difficult to Seeds must be located
seeds for the available easily available near the stock the farm with the
farm near the farm site seeds and cost of
site for stocking transportation will
or brood stock affect economics
must be available
nearby.
18. Availability of Must be available No technical Trouble shooting in the Suitable place should be
Institutional near the site support for the farming will be very identified for the
technical farming difficult technical support.
support
19. Poaching or No poaching or Site with anti Trouble in running the Sites with such troubles
theft pilferage in the social elements farm should be avoided
area.

43
20. Social No social Area with social More difficulties in Site with social unrest
problems problems in the unrest running the farms. should be avoided.
area. No social More issues to be
issues for the settled
farming
21. Market Market for the No marketing More difficult to Suitable market should
farm produce facility market the farm be identified before the
must be available produces. selection of the site.
near the site
22. Construction More suitable for Difficulty in civil
More cost will be Suitable designs should
feasibility constructing the work execution involved in the be prepared for the
ponds and other construction of the construction.
structures ponds and other civil
structures
Unit 14: Broodstock maintenance and different inducing methods for shrimps

Learning Objectives

This unit has been designed to achieve the following objectives:

1. Imparting knowledge on the natural spawning behaviour of the shrimps

2. How induced maturation will help in achieving the targets in hatchery seed production
3. What is the safe and best method of inducement for shrimps in captivity.

 Chapter 1: Maturation and spawning in shrimps

14.1.1 Natural spawning

All the shrimp species will naturally spawn in their preferred spawning ground and expand their
[Link] is a natural phenomenon wherein the animals choose the pattern for the
expansion of their poupulation.

Shrimps have different preferences to perform their natural spawning and in the absence of
their preferred parameters, their breeding or spawning is delayed or sometimes do not occur.

In general shrimps can attain maturity in near shore or in shore or deep sea waters. On
attaining maturity, the mating occurs and the female shrimps for spawning migrate to deep sea
where they will spawn. The young nauplii will be drifted by the currents and water movement
and brought to near shore waters. They will undergo metamorphosis and reach next larval
stages such as Protozoea and Mysis. By this time they will enter into the estuaries where they
will settle as Post Larvae.

44
Although this is the broad life process of the shrimps, there can be variations based on the
species. This might be due to the type of thelycum the females have. Breeding migration and
spawning process will be more or less similar for majority of the cultivable shrimps.

The minimum age of spawning females varies between species and according to the
environmental conditions. The minimum age requirements for the cultivable shrimps are given
below:

Shrimp species Min. age of spawning

Penaeus indicus 4-8 months (weight 6-8 g)
P. japonicus 7-12 months
P. merguiensis 4-8 months (weight 6-8 g)
P. monodon 9-15 months (32-45 g)
P. vannamei 8-9 months
14.1.2 Need for inducement

Under captive conditions, the maturity and spawning do not take place for some of the shrimp
species, eg, Tiger shrimp. Therefore, suitable inducement has become essential for the female
and / or male shrimp brooders. The type of inducement varies from animal to animal based on
their maturity condition.

Basically the inducements are needed for the shrimps, for the following reasons:

1. They depend on a specific ground for maturation

2. Specific micro climatic conditions are required for maturation
3. Specific feed is required for maturation
4. Dependence on the endocrine system for the maturation
5. Presence of bihormonal action in the animals

14.1.3 Induced Maturation

Eye-stalk ablation

The X-organ-sinus gland complex in crustaceans plays an important role in the regulation of
metabolism (Discussed in Unit 7 - Hormonal interaction). Two hormones responsible for the
development and control of breeding are Moulting or Growth or Gonad or Ovarian Stimulating
Hormone (MSH / GSH / OSH) and Moulting or Growth Inhibiting Hormone (MIH / GIH). While
the MSH / GSH/ OSH will stimulate the growth and ovarian development of the crustaceans,
the MIH /GIH will inhibit the development of the gonads.

Eye-stalk ablation in shrimps and crustaceans results in increased food intake, faster growth of
ovary irrespective of developmental stage or season, significant increase in weight and size of
oocytes and advancement of extrusion of eggs. Thus, the female shrimps (crustaceans) will be

45
ready for spawning. Therefore they are subjected to unilateral eye-stalk ablation for the
inducement of growth of ovary at any required time.

Unit 15: Shrimp and prawn breeding and embryonic developments

Learning objectives

This unit has been designed to achieve the following objectives:

1. To provide a broad idea on the breeding of Tiger shrimp with details of their ability in
the hatchery seed production
2. To know about the development process in freshwater prawns
3. To gain the ability to identify different larval stages in M. rosenbergii

Chapter 1: Seed production protocol for shrimp and prawn

15.1.1 Shrimp (P. monodon) seed production

Performance of Penaeus monodon for hatchery seed production

Weight range of mother shrimp - 70-150g

Average weight for best breeding - 120g

Average fecundity of P. monodon - 8-10 lakhs per female shrimp of 100g

Hatchability _________________ - 95 –100% (sometimes fractional)

Breeding programme:

Breeder stocking density : 1per 2 m2

Breeder type :Impregnated in wild waters (females)

(tobe collected for every breeding)

Hatching time : Early morning hours – induced by EDTA

Total number of larval stage : 12 (Twelve stages)

:Nauplii 1 – 6

46
Proto Zoea 1 -3

Mysis 1 – 3

Feed to be given :First feeding Micro algae – Skeletonema or

Tetraselmis

After PZ 3 – Artemia and Spirulina

Duration of larval rearing : 13 – 15 days (depending onwater temperature)

Average survival of larvae : 20 %

Average yield per cycle :1.0 to 2.5 lakhs per cycle

(For a breeder of 100g)

Embryonic development:

Development
Day
1st day First division takes place at about four hours after
fertilization and subsequent division takes place at 90
minutes intervals. Cleavage is completed in about 24
hours.
2nd day Germinal disc appears on the Ventral side of embryo.
Gastrulation takes place followed by the formation of
blastomere. A, V-Shaped germinal fends is differentiated
from the blastoderm on the ventral surface & assuming
the position of the developing embryo.
3rd day The rudiments of embryonic regions are formed and the
embryo develops into the embryonic nauplius stage
4th day Development of appendages of the nauplius takes place.
5th day past nauplius segment begin to the formed followed by
the appearance of rudiments of other cephalic
appendages
6th day The caudal papilla appears with rudiments of thoracic legs
and telson

47
 7th day The optic rudiments develop & rudiments of appendages
and caudal papilla elongate to becomes the telson
th th
8 & 9 day Eye pigment develops, the carapace rudiments to formed
th th
10 & 11 day The heart begins to beat and get is formed.
12th & 16th day The embryo lives parallel to the long axis of the egg & the
larva steadily grows.
th
17 day Appendages elongate & organs are full
th th
19 & 20 day The larva hatches out.
15.1.2 Larval development process in Macrobrachium rosenbergii

15.1.3 Larval stages

Larval stages No. of days Characters
after hatching
I 1-2 Sessile eyes
II 2-5 Stalked eyes
III 6-8 Uropods appear
IV 8-13 Dorsal teeth on rostrum
V 12-19 Telson narrower & elongated
VI 15-24 Pleopod buds appear;Chromatopore mass
present on mid- ventral
VIII 24-28 Pleopods with setae
IX 26-30 Endopods of pleopods with appendix
internae
X 29-33 3 - 4 dorsal teeth on rostrum
XI 32-35 Teeth on half of the upper dorsal margin of
rostrum
Post Larvae 34-45 Teeth on upper & lower margin of rostrum
and setae appear in telson. Starts settling at
(PL) the bottom of tank and swim & crawl like
adults

48
Unit 16: Breeding, larval rearing and post larvae production in Crabs & Lobsters

Learning Objectives

This unit has been designed to provide the following to the students:

1. Hatchery seed production technology for crabs and lobster (General)

2. Constraints in the seed production and ways to overcome them

Chapter 1: Hatchery seed production of crab

16.1.1 Broodstock rearing

The berried females (females carrying eggs) can be either collected from the natural ground or
raised in a pond so that breeders shall be available as and when required. Berried females with

49
yellow eggs mature to grey eggs within 5-7 days and it can be fed on squid meat twice daily.
Feeding can be done with trash fish at @ 5% body weight which can be increased to 10%
towards the latter part of rearing period of 4 months, when chopped trash fish along with
bivalve or gastropod meat is provided. From the culture ponds either berried females are
collected or to induce gonadal maturation unilateral eyestalk ablation of females can also be
done. Within 15 days from the time of eyestalk ablation, females can be obtained for hatchery
use. In some attempts eye stalk ablation just after moulting was also tried, but with limited
success.

16.1.2 Crab hatchery

For crab seed production, following are the components required:

a) Brood stock development unit

b) Hatching unit

c) Larval rearing unit

d) Live feed culture unit

To obtain mature and berried females for breeding, adult crabs of both sexes can be stocked in
earthen ponds and indoor tanks. The sizes of the specimens stocked must be 150-175 mm for
males and 150-170 mm for females. The male and female ratio at stocking can be 2:3. The
female crabs can be subjected to unilateral eyestalk ablation. The duration of broodstock
rearing should be from 120 to 135 days.

In the indoor tanks pre mating embrace copulatory moult and the mating process will last for
48 hours. A month after mating, berried crabs can be obtained.

The same female crab can become berried three times with an interval of one month between
spawning.

16.1.3 Hatching unit and constraints

Hatching unit

The berried females are dipped in 10 ppm malachite green / methylene blue / formalin for 5
minutes as a prophylactic measure and released into FRP tanks/ cement tanks of 500 litres
capacity covered with black cloth to prevent the light. Providing darkness is found to enhance
the reproductive activity of the crustaceans in general. Incubation period will last for 8-15 days.

50
Hatching usually takes place during early morning hours which lasts for 3-4 hours. Before the
larvae hatch out, the abdominal flap of female makes jerking movements in quick succession,
whereas the 2nd & 4th walking legs tightly tab the egg mass.

The zoea are positively phototropic and therefore soon after they are hatched out they will be
attracted towards light. The freshly hatched zoea have to be stocked @ 200-400 nos per litre
glass / cement rearing tanks of 2 ton capacity. Filtered seawater having salinity 30-35 ppt and
temperature of 27-290C can be used for rearing the larvae. Each zoea stage is of 3-4 days
duration. After 5th moult, the larva becomes Megalopa. Thus it takes 27-30 days for the first
zoea to become first crab instar. Megalopa grows to juvenile crab within 8-11 days.

Constraints

During the transition period from zoea I to zoea V, heavy mortality (50-70%) may be observed.
During the development of zoea V to Megalopa, less mortality (10-20%) is observed. During the
growth of Megalopa to crab I stage, cannibalism is a serious threat. Cannibalism can be as high
as 60% leading to a reduction of the desnity. The cannibalism will be severe if the stocking
density is more (10 nos/ l). Cannibalism could be controlled by lowering the stocking density to
5 pieces per liter.

16.1.4 Feeding

Feeding regime for larval rearing

Larval Stage Morning Afternoon Supplementary

feed
Z1 Rotifer 5-10 Rotifer 5-10/ ml -
Z2 Rotifer 5-10 Frozen Artemia 6 5a
Z3 Rotifer 10-15 Frozen Artemia 7b
10
Z4 Rotifer 20-30 Frozen Artemia 9b
15
Z5 Frozen Artemia 1 Frozen Artemia 12 b
20
M 2 days lod 2 day Artemia 40 15b
Artemia 10
C1 2 days old 2 day Artemia 10 15-20b
Artemia 10
16.1.5 Rearing of juvenile crab

Once they metamorphose into post larvae, they can be transferred to rearing tanks. Rearing of
postlarvae (juvenile crab) is done in tank having 5 cm deep sand in order to facilitate the
burrowing of crabs. The PL are stocked @5-10 baby crabs/ m2. They are fed on boiled molluscan

51
meat @ 1-2 %. In this rearing tank they are grown to 4 g and 30 mm carapace width. The
growth of these baby crabs depends on temperature and food available to them.

Chapter 2: Lobster seed production

16.2.1 Larval development of lobster

Egg: The eggs are adhesive in nature. (Brick red colour)

Embryonic development:

It takes place inside the egg.

In the embryo 16 development stages have been observed during early embryonic
development. The egg passed through all these stages before hatching into phyllosoma larvae.
The total time taken for embryonic development and organ formation depend on temperature.
It took 8-20 days at temperature of 310C & 25-260C. The brick red colour of egg changes into
orange red, light yellow, and finally egg colour changed to light brown colour.

Stage I – Undeveloped and appeared brick red

Stage II – Cleavage of eggs

Stage III – Yolk utilization

Stage IV – IX – Developing egg passed through blastula, gastrula stage. During this stage much
of the yolk will be utilized

Stage X – non- rhythmic heart beat can be seen in the dorsal part of the egg. 60% yolk
consumed indicating the development of different organells. Egg mass become brown.

Stage XIII – Full differentiation of eyes.

Stage XVI – Fully differentiated larvae can be seen.

Egg mass appear dark green black in this stage. After complete development, the phyllsoma
larvae hatched out of the egg in the surrounding water.

Phyllosoma larvae:

The size of this larva is very large, having head, throax, abdominal regions and is transparent as
glass. A large carapace covers the first two thoracic segments and head. Abdomen is very much
reduced and may be indistinctly segmented, but without appendages. The antennae and
maxillae are degenerating before hatching. The seventh and eight thoracic appendages and

52
abdominal somites are indistinct in the larvae. even through they are well defined in the
embryo. The first thoracic appendage is rudimentary second is uniramous. The phyllosoma
undergoes several moults before becoming an young adult. Duration of this stage is 90-250
days. There are 7-13 phyllosoma stages (some times a pre-phyllosoma, & pro phyllosoma).

These stages last for not more than few hours. The larval periods of palinurids ranging from few
months to almost 2 years before it reaches last phyllosoma stage (about 35 mm in length).

Puerulus stage (7-56 days, I - stage)

After 6-24 months, the planktonic larval stage metamorphoses into the puerulus stage. It is
transparent, free swimming, non feeding post larval stage that moves inshore is where it settles
to the benthos.

Subsequently through ecdysis, puerulus becomes adult which leads a nectonic swimming life
with well developed appendages.

Unit 17: Eye stalk ablation in crustaceans

Learning Objectives

This unit has been designed to provide the following to the students:

1. General introduction on the induced maturation through eye stalk ablation in shrimps
2. Precautions to be taken for the eye stalk ablation
3. Methods of eye stalk ablation

Introduction

4. Shrimp, prawn and crab brood stock are collected either from the culture ponds or
natural water bodies. The brooders may be at different stages of maturity and usually
either matured or berried or ready to spawn brooders are preferred for the hatchery
seed production. However, some times the brooders may be in their early maturing or
late maturing stages that may require some more time for the release of their gametes.
Under such circumstances, the speeding of maturity is necessary to make them spawn.
Therefore their maturity is induced through biological interference in crustaceans.
5. There are many inducement methods, some may be external and some may be internal.
Some species like freshwater prawns respond well to the feed given and may mature
fast if right feed is given at right quantity. In penaeids, the maturation is well controlled
by hormones and therefore the manipulations are required at their hormone production
sites to make them mature and breed. This applies to crabs too.

Chapter 1: Importance of eye stalk ablation

53
 17.1.1 Eye stalk ablation

It was earlier understood that the endocrine glad present in the eyestalk of the crustaceans is
responsible for the blocking of certain activities connected with the gonaldal maturation. It has
been named as X organ sinus glad and sinus complex, that secretes Moulting Inhibiting
Hormone (MIH). It is also termed as Growth Inhibiting Hormone (GIH) since it inhibits growth in
general. Therefore removal or blocking of this hormone from its activitiy is believed to help in
the speedy maturation of the animals.

Panouse (1943) observed that removal of eyestalk of shrimp Palaemon serratus led to ovarian
development. Ever since this discovery several workers have tried to induce gonadal
maturation in prawn through ablation of eyestalk and also the breeding of different crustacean.

17.1.2 Eye stalk ablation and its role in gonadal development

Introduction :

Shrimp seeds were collected from natural wild waters in the past. Following the development
of hatchery technology and larval production in captivity, seeds have been produced in the
hatcheries and supplied to the farms. However, there has been a difficulty in getting the mother
shrimps for the seed production. Matured mother shrimps have been collected from wild
waters for copious seeds and production of good quality seeds.

It is a fact that all species of shrimps do not attain sexual maturity in confined waters. It has
been found that hormones secreted by the X-organ sinus gland complex are responsible for the
inhibition of the gonad maturation and breeding. Therefore, removal of eyestalk in the matured
shrimps is believed to result in the inducement for the maturation and spawning. Ablation or
removal of the eyestalk for induction of maturation in crustacean is an approved practice in the
crustacean seed production as an essential prerequisite for the maturation of the shrimps in
captivity. In female penaeid shrimp and female crabs the eyestalk has the gland (a complex) for
the production and storage of the gonad inhibiting hormone (GIH) that inhibits the maturation
of the ovary.

Eye stalk ablation

It is the removal or extirpation or elimination of one or both eyestalks in order to at least

reduce the level of inhibitory hormone or to fully prevent their secretion in the body for full and
accelerated maturation of the ovaries.

The eye stalk ablation can be either unilateral or bilateral ablation based on the removal of
single or both eye stalks. If both eye stalks are removed, then it is called as bilateral eye stalk
ablation.

Background of eye stalk ablation

54
The stimulating effect of eyestalk ablation on reproduction of decapod crustacean was first
evaluated for Penaeid culture in the early 1970’s when French researchers attempted bilateral
eyestalk ablation (both eyes). Ablated females suffered high mortality (Probably due to
hormone non-synchronization) and ova were typically reabsorbed with out subsequent
spawning.

These problems were alleviated by the ablation of only one eye stalk (unilateral eyestalk
ablation) which provided moderate hormonal stimulus without re-absorption of ova or
excessive mortality. Consequently, unilateral eyestalk ablation rapidly emerged worldwide as a
simple procedure for inducing reproduction of numerous species of Penaeid shrimp reared in
captivity.

The ablation process

It is more essential to perform the ablation at appropriate stage in order to avoid loss of mother
shrimps. For all practical reasons, it is emphasized that only inter molt shrimp is to be used for
ablation. Post-molt (stage) female shrimps are not recommended for ablation, due to increased
risk of handling mortality associated with softened exoskeleton and weakness of the animal
Pre-molt (Stage IV) individuals are also not recommended since there may be immediate
molting during recovery from the ablation process leading to loss or delay in spawning.

It was observed that ablation under taken between 8-20 days post-molt resulted in significantly
greater egg production than that of ablation at 13-15 days post-molt indicating the inter moult
period is the best one for the ablation process.

Methods of eyestalk ablation:

Several methods are available for the removal of eyestalk. They are,

(i) Cutting the eye stalk near the base with a pair of scissors followed by sealing with a pencil
type soldering iron. This method is called electrocautrization.

(ii) Pinching of eyestalk and squeezing of eye ball contents out

(iii) Incision of eyeball followed by eunucleation of contents.

Enucleation, cauterization and ligation are the methods that have been effective in removing
(or) destroying the X organ / sinus gland complex. Never the less, the stress that will be arising
due to the ablation has its own ill effects on the animals.

Stress can be reduced and losses minimized if shrimps are held in chilled water before and after
ablation. To minimize stress, the ablation should be performed as quickly as possible under
chilled water. Further the stress to the mother shrimps can be reduced by doing the ablation in

55
the early morning hours, when the temperatures is the lowest. Care should be taken to see that
female mortality due to ablation is very low.

a) Enucleation

Grasp the eyestalk just behind the eyeball using the thump and index finger. Squeeze hard and
roll the thump and finger outwards away from the body, thus crushing the eyestalk and
squeezing out the contents of the eye. The objective is to squeeze the contents outwards and
not let them follow the eyestalk back in to the head region. Enucleation has the advantages of
simplicity and rapid clotting of hemolymph within the empty eyestalk.

b) Ligation

A string is tied around the base of the eyestalk as close to the carapace as possible. The string
should be drawn fairly tight causing the eyestalk to fall off in a few days. The procedure does
not leave the shrimp with open wound. However, successful ablation is often unpredictable and
this process will give the result only after two or three days.

c) Cautery: (Electrocauterisation)

This method is the severing eyestalk followed by sealing of the wound through the process
called electrocauterization. Heated forceps, or the application of a silver nitrate bar on the
wound will prevent the secondary infection at the wound site.

Ø Pinching method of ablation required one person and it is without application of antibiotics.

Ø Ligation requires two persons, one to hold the shrimp and the other to tie the eyestalk.

Ø Cautery requires either a cautrizer (or) silver nitrate bar.

Effects of eye stalk ablation

The effects of eyestalk ablation vary with season and stage in the molt cycle. Ablation is
performed on either left or right compound [Link] damaged eye should be ablated in
order to leave the shrimp with one unablated functional eye.

Fecundity and possibilities for spawning:

Ovarian development in sexually matured female can commence within 3 days of ablation,
followed by a first spawn within one week. If ablated during the inter molt stage, the females
will mature and spawn immediately. If ablated during early molt, they will molt before
maturing. There have been varied observations and results due to ablation.

56
- The fecundity and viability of larvae that are obtained from abated females are observed to be
inferior to larvae from females matured in the wild indicating the superiority of the larvae from
the natural spawning.

- In the captive maturation there might be poor or low embryonic development that may
influence juvenile’s survival and growth.

- If the females are smaller, there will be higher lipid in the body and variations in the
distribution of yolk among oocytes. These were identified from the crab (Paratelphusa
hydrodromus) during the pre-spawning.

- Those differences presumably are consequences of hormonal imbalances due to ablation.

They are physiological (or) embryological limitation such as improper oocyte differentiation,
nutrient storage food supply (or) temperature.

17.1.3 Role of eyestalk in the reproduction of crustaceans

In prawns, the neutrosecretory centers located in the ganglia of eyestalk (X organ sinus gland
complex), brain and thoracic ganglia mainly influence the egg production (vitellogenesis). While
the X organ sinus gland complex of the eyestalk inhibits vitellogenesis under the influence of its
hormone, viz., Gonad Inhibiting Hormone (GIH), the brain and thoracic ganglia promote
vitellogenesis by their hormone, viz., Gonad Stimulating Hormone (GSH).

Under natural conditions, when the physiological parameters of the prawns and the
environmental factors are favorable, the GSH promotes vitellogenesis. On the basis of this
principle, the ovarian development and maturation of gonads are obtained in prawns through
the unilateral eyestalk ablation (removal of one of the eyestalk). It is also worth mentioning
here, that the removal of both the eyestalks though lead to rapid ovarian growth, spawning
does not result due to physiological stress. It has been observed that the ova are reabsorbed in
the ovary. The eye stalks apart from GIH, produce other neutrosecretory hormones which
regulate lipid metabolism and protein synthesis in hepatopancreas; induce hyperglycemia in
blood to combat stress; regulate calcium metabolism during cuticle formation; effect water
balance during moulting; inhibit production of moulting hormone by ‘Y’ organ and influence
movement of pigments in chromatophores.

- Removal of both eyestalk is called as bilateral eye stalk ablation.

- The behavioral pattern of the eye stalk ablated females especially their feeding and matting
are also not affected.

Points in eyestalk ablation in crustaceans:

57
1. For the procurement of broodstock of crustaceans for inducement, some of the shrimps /
prawns / crabs are reared in ponds to harvestable size and later they may be allowed to grow
for further period of one year before subjecting them for eyestalk ablation.

2. The eyestalk ablation is carried out only in female and not in male.

3. For eyestalk ablation, P. indicus with a carapace length of 30 mm (total length, 150 mm
weight > 20 g) and P. monodon with the carapace length of 50-80 mm (total length > 200mm
and weight >100g) are normally selected.

4. Before removing eyestalks, the brood stocks are kept in large plastic pools containing filtered
sea water of 33 ± 20%.

5. After about 24 hrs, the eyestalks are removed in the females using electrocauterization
technique.

6. The ablated female and unablated males are then kept at 2:1 in larger circular plastic lined
pools, in which the sea water is made to circulate through sub-gravel filter by air lifts.

7. The breeders are fed ad libitum using chopped fish or calm meat.

8. The temperature, salinity and pH of the medium are maintained at 28 ± 2, 32 ± 2% and 8-8.2
respectively.

Generally the mating and impregnation are ensured in the shrimps if the males and females are
kept separately in two tanks and the females are introduced into the male tank when they are
about to molt. The separation of sexes seems to enhance the attraction between them and
facilities early spawning.

Chapter 2: Eye stalk ablation methods

17.2.1. Methods of eye stalk ablation

Eye stalk abalation can be done in two types.

They are, unilateral abalation and bilateral abalation.

In unilateral abalation, only one eye will be removed and the animal will be allowed to live one
eye, while in bilateral abalation method, both the eyes will be removed with the stalks of the
eye. Although the bilateral abalation results in faster maturation adn yielding desirable results,
there are some demerits in this method.

Unit 18: Broodstock and Larval Nutrition

58
 Learning Objectives

Learning of this unit will help the students to get knowledge on the following:

1. Importance of brood stock diet

2. Ingredients and their usefulness in the formulation of feeds for broodstock and larvae
3. Artemia and their importance in shell fish seed production
4. Larval nutrition and feeds

5. Introduction
6. Nutrition plays an important role in the life of any organism. It is practically true in the
case of aquatic organisms, in which the development and population sustainability is
largely depending on the availability of right quality feed at right qunatity.
7. In the life of the shellfishes, their growth and reproduction depend on extrinsic factors
such as water quality, environment and feed. Understanding their nutritional
requirements is essential to provide the required feed for their development and living.
8. Shellfish brooders demand special feed items in order to go thorugh tough maturation
and spawning process. In many species, migration is also an integral part of the
reproduction and therefore the energy becomes an inevitable parameter in deciding
their sucessful breeding and survival.

Chapter 1: Feed ingredients and feed formulation

18.1.1 Feed ingredients for feed preparation

a) Fish meal

Fish meal should contain atleast 60% of crude protein and not more than 1% of sand. In case,
commercial fish meal is not available, dried anchovies and silver bellies can be powdered and
used in place of fish meal.

b) Meat of shrimp:

It is collected in fresh condition, washed and dried in electrical dryer at 70 ° c until the moisture
content is reduced to less than 10%. Fresh meat of shrimp contains 40-45% crude protein, 5-7%
of lipid & 20-23%

c) Prawn Head waste:

It is collected from prawn processing units and dried in a dryer at 70 ° c to reduce the moisture
to less than 10%. Prawn head meal contains 20-35% crude protein of which 10-15% is due to
chitin. The prawn head waste has 5-7% lipid rich in PUFA & pigments.

d) Squid meal:

59
Squid meal, prepared from whole squid is a good source of high quality (60-70%) rich in
essential amino acid. It is also having lipid content of 13e-19% richin PUFA. Squid meal contains
short chain polypeptides which are found to be good feed and growth promoter for prawn.

e) Soybean cake

It has 50% of protein

f) Tapioca

g) Alfalfa

h) Lecithin

i) Vitamin Mixture

j) Minerd mixture BHA

18.1.2 Proximate composition of feeds materials (% on dry basis)

Moistur
Ingredient Protein Lipid CHO Fibre Ash
e
Fishmeal 6.8 64.4 4.7 0.9 - 19.3
Shrimp meat 8.0 44.1 7.6 1.3 8.2 23.6
Prawn waste 81.5 35 7.5 0.9 14.2 23.9
Squid 80.5 67.8 19.5 2.3 - 10.4
Soy bean cake 6 50.3 2.5 24.8 6.6 9.8
Tapioca 4.8 2 0.5 72.0 19.2 1.5
Alf alfa 5.6 19.9 1.8 31.5 29.5 11.7

Proximate Composition of microparticulate feed

Moisture – 5.2%

Crude protein – 39.5%

Lipid - 8%

CHO - 26.8%

Crude fibre - 6.7%

Ash - 13.8%

60
Chapter 2: Artemia as feed

18.2.1 Feeding rate of Artemia nauplii to freshwater prawn larvae

No. of Artemia nauplii/
Age of larvae (d) Larval stage
larvae/ day
3 II-III 5
4 II-III 10
5-6 III-IV 15
7-8 IV-V 20
9 IV-VI 30
10-11 V-VII 35
12 VI-VII 40
13-14 VI-VIII 45
15-24 VII-PL 50
25-30 VIII-PL 40
31-35 IX-PL 30

Feeding starts from 2nd day of hatching. Prepared feed are usually given in the day time,
Artemia nauplii in the night time. Different stages require 50 to 150 µg prepared feed/ larvae/
day and Artemia nauplii from 5 to 50 per larva/ day.

Brine shrimp nauplii are given in the night to ensure the presence of food in the larval rearing
medium throughout night. While inadequate feeding leads to started growth due to
strarvation, cannibalism and prolonged metamorphosis over feeding leads to water pollution.
The larval rearing tank is to be cleaned every day, by siphoning out feed residues, metabolic
waste and faecal matter from the bottom of tank. This is to be done daily after stopping
aeration before water exchange and feeding of Artemia nauplii.

PL-3 to PL-6 stage Artemia nauplii given @ 2 nauplii/ml 3 times daily. PL-7 to PL-12 – 3
nauplii/ml, 3 times daily. PL-13 to PL-20 – 3 nauplii/ml once daily. While adding Artemia nauplii
care must be taken to avoid cyst shell as it may bring bacteria into the rearing medium.

In addition to Artemia nauplii, artificial feed is also given such as microencapsulated feed and
egg custard. Microencapsulated feed is dissolved in water, stirred and allowed to stand for 5-10
minutes. The feeding is done @ 0.5 g/ton.

18.2.2 Feeding the larvae of P. monodon

Day Substage Water Mesh size Feed management
management of screen Algal Artemia
(µ) feeding([Link]) (no./ml)
1. N6/Z1 Fill 60% of tank 100 1,00,000 -
2. Z1 Make to 80% 100 1,00,000 -

61
 3. Z2 Make to 100% 100 1,00,000 -
4. Z3 30% exchange 250 1,00,000 -
5. Z3 50% 250 1,00,000 -
6. M1 70% 350 1,00,000 0.25
7. M1 70% 350 1,00,000 0.25
8. M2 70% 350 1,00,000 0.25
9. M3 70% 350 1,00,000 0.5
10. M3 70% 350 1,00,000 0.5
11. PL1 100% 350 60,000 1
12. PL2 100% 350 60,000 1
13. PL3 100% 350 60,000 1
18.2.3 Artemia and Shell Free Artemia for larvae

1. What is Artemia?

It is a micro crustacean or small crustacean just like shrimp or prawn (but very small in size) so
that they can be used as the feed for the larvae of prawns,shrimps, crabs and any fishes.

2. Why it is to be used as larval feed?

Because it is very rich in protein, which is 100% digestible for any aquatic organism (fish or
shrimp or prawn or crab). The biological value of the protein is similar to that of fish or prawn
or shrimp and therefore this stands as the first preferred feed for the larvae.

It contains more vitamins and minerals in bio form that can be used by the larvae.

The first feed of the larvae is very critical. It must be good enough to sustain the life of the
larvae. Therefore it is considered as the best suitable feed for the aquatic organisms.

3. For which this is used as the live feed?

For all aquatic organisms – like, shrimp, prawn, fish, crabs, lobsters, etc particularly the aquatic
organisms that prefer or need only animal matter for its feed. Or we can say that all aquatic
animals having carnivorous feeding habit can be fed with this

4. What are the advantages of Artemia?

1. It is a live feed – that is available as cysts which can be stored for a long period (may be
100 years if not hydrated)
2. The hatched out cysts will release nauplii, which care tiny and suitable for the mouth
size of the larvae.
3. More of protein and other nutrients –except vitamin C
4. Available in more safe form – free from any bacterial load

62
 5. They are readily available as cysts, which can be used for producing the nauplii in the
hatchery.

5. What is shell free Artemia?

The Artemia is available as cysts – i.e. eggs produced under unfavorable conditions from which
the organism can come to life once the situations are very favorable to them. These cysts are
very hard materials that they have a strong epi coat over their cysts. Thiepi coat can resist to
any intolerable conditions even to acidic [Link] they can absorb water and be hydrated.
The real Artemia is inside this epi coats developing nauplii but in inactive form until it touches
the water of favorable quality. Most of the aquatic organisms can not digest this epi coat and
therefore they can not use this Artemia nauplii unless until the epi coais removed or hatched
out. Therefore forgetting the Artemia nauplii the cysts must be hatched out through the
Artemia hatching process, which is cumbersome and will take about 24 hrs.

Considering the cumbersome process of hatching, which requires seawater, light, aeration and
correct pH to make them hatching up to 85% now many hatchery operators are using the
Artemia cysts as such after decapsulating them or removing the epi coat over the nauplii. This
process is called Decapsulation of the cysts there by the epi coat is digested and removed from
the cysts. This will make the cysts readily usable by the larvae or fry.

Until very recently the cysts are available as cysts and used for hatching and nauplii production.
But after careful thought and bio processing, now the epi coat is removed and available as Shell
Free Artemia which do not have the shell or epi coat over them. This is called Shell Free
Artemia.

6. What are the advantages of Shell Free Artemia?

1. The shell free Artemia is a ready to use product – that means it does not require any
hatching
2. During hatching not all cysts will hatch out. The maximum possible hatching percentage
is only 90% - that means – out of 100 cysts only 90 cysts alone can be used in the case of
normal cysts. But in Shell Free Artemia all the 100 cysts (i.e. 100%) can be used.
3. There is no requirement for hatching – that means no need for electricity expenses or
aeration or seawater.
4. On hatching the nauplii will start using the stored up energy (i.e – yolk) so that
ultimately there is an energy loss as per the energy pyramid. In shell free Artemia there
is no loss in energy and the total energy stored up in the cysts will be delivered to the
target organisms.
5. There will be chances for the infection through the nauplii once they are hatched out in
the seawater. In the Shell Free Artemia, no such infection as the cysts will not touch the
seawater.

63
 6. More importantly the Shell Free Artemia can be used as a ready to serve feed in the
hatchery thereby it reduces the risk ok non-fed conditions arising due to non-availability
of nauplii.

Unit 19: Compounded (prepared ) larval feeds for shrimp hatcheries

Learning Objectives

This unit will provide the following to the students:

1. A broad knowledge on the different larval feeds.

2. Making of larval feeds with different feed items.

3. Criteria for the selection of suitable larval feeds.

Chapter 1: Larval feeds- their importance and use

19.1.1 Artificial larval feeds for shrimp hatcheries

There are different larval stages in shrimp viz., nauplii, protozoea, mysis and post larvae.

In nature, the nauplii do not feed as it utilizes the yolk content in yolk sac. At protozoea stage
the larvae normally feed phytoplankton of 3-10μ in size , while at mysis stage the larvae feed
food of 30-100 μ in size, while at post larvae stage the larvae feed, food of till bigger in size.

Mostly shrimp larvae take live feeds and it is believed that the live feeds help in getting more
survival and growth. Recently the live feeds are substituted with the prepared feeds. Hatcheries
continue to make their own larval feeds, because commercially available feeds are expensive
and their quality and texture are varying a lot. Larval feeds are made for both freshwater
prawns and marine shrimps. There are different types of feeds available for differet stages of
larvae.

Hatcheries use egg custard and micro encapsulated diets as a substitute for live feed.

Chapter 2: Preparation of artificial larval feeds

19.2.1 Egg custard

Egg custard are the wet type of microfeed commonly used in hatcheries. The preparation of egg
custard may vary in hatcheries.

64
 Preparation of farm made crustacean larval feed
60 egg cracked into bucket

Add powdered milk at rate of 2 teaspoon / egg

whisk the mixture until it thickens and rises.

pour into trays and steam for 30 min.

(Trays layered above boiling water)

Resultant egg custard is then forced through a series of sieves using high pressure water
and retained in plankton net (sieves sizes vary according to size of larvae to be fed)

Egg custard then added to larval tank.

19.2.2 Common method for preparation of egg custard

Ingredients: Egg – 3 - 6 no.; Beef liver – 130g;Polycheate worm – 150g; Vitamin – 20 drop; Flesh
of squid / prawn – 200g ; Milk powder – 75g.

Mix all ingredients

(except vitamin)

Cook in cooker (1hr)

65
 Add vitamins

Take required quantity and sieve it through screen (500 μ)

Wash granules

Apply to larvae.

Feeding rate : Feeding rates for egg custa rd are about 2-2.5 times as great as those
recommended for commercial encapsulated larval diets because they are moist.

15g to 40g feed depending on stage.

PL8 – PL10 - 15g /feed x 2times

PL11 – PL15 - 25g /feed x 3times

PL16 - PL20 - 40g /feed x 5times

However feeding rate with egg custard done on demand and care should be taken to siphon
out daily, the left over [Link] case of small hatcheries, simple egg custards are still popular.

Freshwater prawn, Macrobrachium rosenbergii, larvae is carnivorous in habit, so the

preparation of egg custard for freshwater prawn hatcheries varies.

* Egg custard are fed along with Artemia nauplii.

19.2.3 Egg/Mussel Mixture

Blend 0.5kg of shelled mussel

Strain chopped mussel through coarse cloth & discard

connective tissue, retain only material passes strainer

66
Using whole of mussel which passed strainer, add 3 or 4

whole egg and stirr thoroughly in blender

steam the mixture over until it solidifies into custard

Preparation method.

Fillet fish, discard head, bones and viscera

Blend 0.5kg of shelled mussel

Strain chopped mussel through coarse cloth&discard

connective tissue, retain only material passes strainer

Using whole of mussel which passed strainer, add 3 or 4

whole egg and stirr thoroughly in blender

steam the mixture over until it solidifies into custard

Grind and liquidize flesh in a blender

Blend 0.5kg of shelled mussel

Strain chopped mussel through coarse cloth&discard

connective tissue, retain only material passes strainer

Using whole of mussel which passed strainer, add3 or 4

67
whole egg and stirr thoroughly in blender

steam the mixture over until it solidifies into custard

Force flesh through stainless steel sieves with a

strong jet of fresh water. This grades particles and

washes flesh free of blood and excess oil

Mesh size chosen to produce particles of a size relevant to age of prawn larvae.

prawn larvae.

Fish : Skipjack tuna, Mackeral

3 days after hatching, prepared feed can be introduced in increasing weaning quantities. By 5 th

day, BSN given only at evening, feeding to ensure presence of food at all times. Use of artificial

feed for night feeding not recommended because quantity necessary to supply requirement

through out night in one feeding would foul the water. On 5 th day, prepared feed gives as 4-5

meals spread through out day-light hours at 1.5-2 hours interval.

Feeding rate :

12-16kg of prepared feed for each larval cycle/10m 3 tanks initial quantity at 5th day is

30-60g/tank and rise to around 200g/tank/feed.

68
* 0.3mm size till 10th day

* 0.3-1mm size till metamorphosis

Particle of prepared feed must be kept close to larvae.

The egg/mussel mixture can also be replaced by fish.

Unit 20: Seed packing and transportation

Learning Objectives

This unit has been designed with the following objectives:

1. To make the students to get an idea of the packing of seeds

2. To know the prerequisites for packing and transportation of seeds
3. To practically transport the seeds with oxygen packing

Chapter 1: General Guidelines

20.1.1 Prerequisites for seed packing

Seed packing is essentially done in the hatcheries as the seeds are to be transferred from the
hatcheries to the ponds. In order to minimise the loss during such transfer seeds are packed
with more care to withstand the transportation.

The following factors are to be considered before going for seed packing:

1. Type of seeds to be packed

2. Size of the seeds
3. Distance to be travelled
4. Duration of travel
5. Time of packing
6. External temperature
7. Salinity and other water quality parameters in the hatchery and also in the farm

69
 8. Special requirements for the seeds like feeds and chemicals
9. Handling methods
10. Nature of the seeds.

After ascertaing the above, the right method of packing will be decided.

20.1.2. Seed packing methods and transportation

Normally seeds can be packed in two methods, viz.,

1. Oxygen inflated bags

2. Drums with oxygen supplied from the cylinder

Among the above two methods, oxygen inflated bags with sufficient water is considered good
due to their advantages like reduced damages to the seeds and easy handling. Further the
seeds are in batches in bags and therefore even if mass mortality occurs that will affect only
lesser number of seeds in the whole lot. In the drum it may affect the whole lot of seeds that
are packed in the drum.

The seeds can be transported in a carrier either in inflated bags or in oxygen supplied drums for
distances to be determined based on the following:

1. External temperature
2. Number of seeds in the bag
3. Size of the seeds
4. Metabolic wastes that are released by the seeds
5. Nature of the seeds (cannibals or compatible)
6. The possibility for repacking en route

Unit 21: Breeding, larval rearing and post larvae (spat) production in oysters

Learning Objectives

Following are the objectives of learning:

1. To understand the development and larval production in oysters.

2. To study the technology of pearl oyster seed production and spat collection

Introduction

3. Oyster seed production although a standardized technology, is not widey practised in

India for may reasons. However, scope still exists for the adoption of the technology in
many parts of the coastal states in the country.

70
 4. Among the oysters, seed production technology is available for Crossastrea madrasensis
and Pinctada fucata.

Chapter 1: Development stages of edible oyster and Mussels

21.1.1. Interaction of different factors in the development process of bivalves

The developmental biology of bivalves is strongly dependent on many factors that are
prevailing in their natural environment. Life and their growth in the natural environment are
greatly influenced by the physical and chemical parameters of the living water and even minor
changes will affect them.

In general bivalves have many larval stages in their life cycle starting from Umbo stage to
Plantigrade stage which will end with the settlement as spat. During this process they are
depending on the following important factors:

1. Right quality of feed

2. Appropriate quantity

3. Salinity (35 - 37ppt)

4. Absence of pollutants

5. Absence of predators

6. Temperature (28 to 30 deg. C)

7. Fishing disturbances

8. Turbidity and water current

9. Availability of suitable substrates

10. No human activities

By controlling the above in the breeding grounds the population of bivalves can be protected.

21.1.2 Development stages of Crassostrea madrasensis

After about 20 hrs of fertilization, ‘D’ larval stage (Straight hinge stage of 70 mm size) with a
semi transparent velum appears. These larvae are also known for their vigorous swimming and
circular movements. On the third day, the “D” shell larva measures 100 mm and becomes
"early umbo” stage, getting a slightly oval shape which in turn becomes late umbo stage in 12th

71
–15th day and measures 250-300 mm. At this stage, they are transferred to eyed stage (on 13th
to 17th day) which measures 300 mm. This is characterized by an irregular eye spot, gets
transformed into a pediveliger stage (350 mm) between 14th & 18th day, when the functional
foot develops to facilitate crawling at the bottom.

Depending on the availability of favourable substrates for attachment, the pediveliger takes 1-6
days for settlement as spat (450 mm) in which the velum, eye spot and foot disappear and
develop gills.

21.1.3 Developmental stages of Perna indica (Mussel)

The fecundity of mussels is 0.5-10 million. Following external fertilization through cleavage &
gastrulation, embryo becomes first larval stage, viz, trochophore which is then transformed to
veliger. All embryonic developments take 4 hours after fertilization (Morula stage – 58-60 um).

The shell gland of veliger secretes larval shell, i.e., Prodissconch I which subsequently attains
straight hinge or ‘D” shaped stage. The straight to hinge larva measures 70-76 um. This stage is
reached in 17-10 hours.

The development of straight hinge larvae to umbo stage is reached on 7th day. At this stage,
larva measures 120-140 um. Eye spot stage is reached on13th to 14th day when the larval
second larval shell, Prodissoconch II with the help of mantle becomes “Veliconcha’. In
pediveliger stage the foot is developed on 16th day at size of 280-320 mm. At this stage the larva
is in crawling stage and sinks down & attaches a suitable substratum in a bottom on 20 th day
after fertilization by developing Byssus threads.

After complete metamorphosis, larva acquires adult characteristics and becomes ‘plantigrade’
(or) “spat”. The spat attains a maximum length of 2.7 mm by 32nd day. It look line a true mussel.

Chapter 2: Pearl oyster seed production

21.2.1 Development of Pearl oyster (Pinctada fucata)

The Indian pearl oyster. Pinctada fucata inhabits in the sea bottom. The male and female
oysters spawn and discharge the gametes. The eggs are fertilized and assume spherical shape .
The fertilized egg measures few microns and the first & second polar bodies will be released.

The first cell division lakes place 45 min after fertilization. Repeated cell divisions result in the
morular stage in 3-4 hrs. The morula looks like a ball with cells.

The morula, after development will attain the plastula and gatrula stages. The trochophore
stage is reached in 10 hrs. The trochophore larva has preoral and postoral cilia with a single
filagellum. In 20-22 hrs after fertilization, the trochophore develops into veriger larva with the
formation of shell, stright hinge, mantle and velum. It is also called strigh hinge larva or 'D'

72
shape larva and measures 67.5 um in anteroposterior axis and 52.2 um in dorsoventral axis.
From D - shape stage onwards algal food is given.

On 10th to 12th day the veliger reaches the umbo stage with the formation of Umbo. It measures
135x130um. The umbo larva transforms into eyed larva with the development of eye spot at
the base. At this stage, the larva measures 180x170um.

On 18th day, pediveliger stage is reached with the development of functional foot. The foot
helps in movement and also in temporary adherence to objects. At this stage, the velum
disappears. The plantigrade stage is reached on 20th day and it measures 220x200 um. Byssus
threads are produced for attachment. The plantigrade metamorphoses by the formation of
stright hinge line and anterior and posterior ears. Byssus notch is well developed. It measures
300um and resembles the adult pearl oyster in appearance.

21.2.2 Pearly oyster spat collection

Collection of pearl oyster spat from the natural beds is not always dependaple, owing to their
irregular production. Therefore setting up of artificial spat collectors is recommended in the
farm area for the spats to attach in large numbers, especially during the peak spawning season,
Keeping the spat collectors only during peak season of their breeding will prevent the spat
collectors becoming fouled with barnacles and other organisms.

There are several practices for spat collection of different species of oysters in different parts of
the world. In the eest coast of India lime coated- semi cylindrical tiles are used to collect spat.
Strings of scallop or other oyster shells can also be used for the spat collection.

In Japan, long lines with leaves and twigs are used as spat collectors. Shells of abalones, oysters
and scallops in strings are suspended from rafts just below the surface to about 3 m depth for
collecting the spat of pearl oyster. Nylon ropes are used for collecting spat of pearl oysters at
Vizhingam.

21.2.3 Materials used in the spat collection

Although, spawning of tropical oysters occurs throughout the year, the peak is invariably during
the onset of rains or at the end of the dry season. In the case of Pinctada fucata, the peak
spawning has been recorded during April – May & August - September.

After a brief period of planktonic life, the larvae begin to settle on a hard substratum and at this
stage they are called "spat". In the natural environment these spats can be collected with the
help of spat collectors. The abundance of the larvae before their settlement can be confirmed
by periodic plankton collections in the seasons mentioned before. In order to avoid biofouling
in spat collectors, the man made collectors should be suspended at the appropriate times
otherwise the surface of the spat collectors will be preoccupied either by silt deposits or by

73
foulers like seaweeds and barnacles. Suspension of spat collectors below racks and trays where
oysters are grown may help in effective utilization of the natural seed supply.

The following materials are used for the spat collection:

Lime-coated tiles:

Semicylindrical roofing lites (24x15cm size) are first dipped in lime solution (12.5kg of lime
dissolved in 25 l of water would be sufficient for 500 tiles) and are dried under shade. After
drying, the tiles are given a second coating with lime-mortar (30kg of lime + 50kg of find sand
and 40l of water). These dried tiles are placed in rectangular trays (90x60x15 cm) in the rack at
50no/tray in such away that the concave sides of the tiles face dewnwards. These tiles are
periodically examined for the settlement of oyster spats. The spats that are settled in the tiles
are scraped off after they reach a size of about 2 cm for the tranfer to trays set on racks.

Oyster shell strings:

About 20 oyster shells with a central opening in each (made with a punching hammer) tied in a
1.5 m long No.10 galvanized wire are used for the collection of spats. Each unit is called as ’ren’
and 100 such rens may be hung horizontally from a rack,

Coconut shell string:

Tar coated and centre punched 10 coconut shells are held together by a 3 mm synthetic rope of
1.5m length. Between two shells, an interspace of 10-15 cm may be allowed. The strings are
held horizantally on a rack and kept in the spat settlement area.

Strips of corrugated roofing asbestos:

Lime coated 5-7 corrugated asbestos strips of 30x20 cm size may be alternately arranged one
above the other and are tied with a 3 mm synthetic rope in such a way that every unit can be
hung easily from a rack.

Asbestos sheet:

Strips of asbestos sheet of size 128 cm are given lime coating. After drying, the strips are placed
in grooves of palmyra reepers which are tied to 2m long poles installed in shallow areas.

Among the various types of spat collectors, the strips of corrugated roofing asbestos have been
proved to be useful in the collection of large number of spats.

Rope collectors:

74
The rope collectors consist of a 9mm main rope made of synthetic material in which bunches of
untwisted nylon filaments are inserted at intervals of 25cm. One end of the main rope is firmly
tied on the wooden pole of the raft and the other end is tied to the granite stone weighing
around 5kg. When the rope is suspended from the raft the filaments from the bunch spread in
the entire water column from surface to bottom. The synthetic ropes and flamentous bunches
are of three colours, viz., blue, green and yellow.

Synthetic filamentous spindle:

Bunches of synthetic monofilaments of 6 m length secured at both ends in the form of spindle
was suspended vertically into the water column. The spindle was supported by main rope in the
middle, one end was tied on the wooden pole of the raft and the other end in a 5kg of granite
stone. A good spread of monofilament was noticed in the entire water column commencity
from surface to bottom.

Facundity, Incubation & Hatching

Fecundity is generally estimated as the number of eggs carried externally. Facundity varies
greatly to between species and within species among size classes. The range of clutch size are
as follows: metapnephrops andamanicus 6000-1400, N norvegcus, 900-6000, H. gammarus,
5000-17,000 H. americanus,

10% per month in [Link] and a small egg loss over the incubation period in [Link]. The
number of eggs in a brood is not as important to the overall biology of the animal as is the
valative fecundity. This is developed from data concerning brood size, egg loss, number of
broods per year, and proportion of the population represented by each size class of funales.
Beory (1971) estimated that by far the highest fecunclity was achieved by medium size P-
homarus rubellus. The relative reproductive contribution of the very large fanales was
diminished by the small proportion that they comprise of the population, although they breed
upto four times per season and carry many more eggs than chair smaller counterparts.

The incubation period of the eggs is temperature dependent, but at least for [Link], it is
also dependents, it is also dependent on the age of embryos at the time of exposure to a given
temperature. The early embryos develop faster at a given temperature may synchronize
hatching to a limited period, even when egg extrusion is staggered in different individuals.

Hatching in all species studied occurs at night, & may occur over several nights. In fact,
paterson (1969) reported days. In homarus, hatching lasts from 2-3 days to 2-6 weeks and
nephrops relcases larwae over a period of several days. The process of hatching in all the
Nephropidal seems to be similar. Internal water pressure is thought to burst the outer egg
membrnes independently of any action by the female. Larwal release occurs when the female
assumes the hatching posture described by Templeman (1937) for [Link]. The female
assumes a tail-up posture, standing on the tips of her walking legs, and beats the pleopds
rapidly, releasing several hundred larval at a time. The violent pleopod action may initiate

75
ecdysis while the prelarvae are still enveloped in the embryonic cuticle. larval release appears
to be under endogenous control, since shaking of the female does not induce it, although such
motion appears to be similar to female hotching behaviour. In H. gammarus, larwal release
occurs almost entirely in the first few hours of darkness, and there appears to be an
endogenous rhythm associated with the release, since the temporal darkness. H. americanus is
not as closely lied to the light dark cycle, although the kanority of its larval release also occurs in
short bursts rather than continuously, Ennis()1975 pointed out the surviva rate, relative to
preclation, of rleasing larwae in small batches, usually at night, over a long period of time.

Unit 22: Breeding, larval rearing and post larvae production in Holothurians

Learning Objectives

This unit has been designed to provide the following to the students:

1. General introduction and background of seed production in sea cucumber (Holothuria

spp)
2. Life cycle of holothuirians and their importance in seed production
3. Broodstock management techniques in holothurian seed production
4. Spawning and captive seed productio

Chapter 1: Introduction and background

22.1.1 Introduction to Holothurian seed production

Holothurians are wonderful natural resources in India and they are found aggregated in
selected maritime states and Tamilnadu is one among them. Sandy shores together with
conducive climate for their breeding make them habituate in the particular location restricting
its distribution.
Hornell (1917) reported that the beche-de-mer industry in India was an ancient one probably
introduced by the Chinese (might be about 1000 years ago). Since sea cucumbers fall easy prey
and easily captured, their population is over exploited in many parts of the world. (Example,
beche-de-mer industry in the Maldives, which developed in 1985 with a modest export of 31
kg, now the resource was in danger of over exploitation (Joseph and Shakeel, 1991; Joseph,
1992). James and James (1994)wrote on the needs for conservation and management of sea
cucumbers in India.
A new resource of sea cucumber, Actinopyga echinites, was exported for the first time from
India in 1989 and by 1992 this resource had become scarce (James and Badrudeen, 1995).
Another species, A. miliaris, was collected for the first time in 1992 and, within 2 months, more
than 0.6 million specimens were caught.

James and James (1994) published a handbook on Indian sea cucumbers to facilitate
identification of commercially important sea cucumbers from India. James and Ruparani (1999)
gave an account of new resources of beche-de-mer and the management of these resources in

76
India. In order to replenish the natural populations, hatchery technology for juvenile production
and sea ranching was developed by the Central Marine Fisheries Research Institute in India.

James et al. (1988) produced juveniles of H. scabra for the first time in 1988 at Tuticorin in
India. Following this same technology, juveniles of this species have been produced in Australia,
Indonesia, New Caledonia, Maldives, Solomon Islands and Viet Nam in recent years.

The fertilized eggs develop into larvae which may be planktonic for 65 -70 before settling on the
bottom and changing into juvenile sea cucumbers. Juveniles are very secretive and hide among
sea weed, under stones, and in crevices. Growth is slow, requiring at least four to five years to
attain adult size of 500g bw. Their life span may be more than eight years.

22.1.2 History of seed production in India

Although the seed production is not a big commercial activity in India, it is worth to note that
more attempts were done in this line only in India.

During late 80s, the first attempt was made by James and his research team in CMFRI, Tuticorin.
They produced Holothuria scabra juveniles for the first time in India in 1988. Since 1988 this
species has been bred in captivity on a number of occasions (James 1993, 1997, 1998; James
and James, 1993; James et al. 1994). Chen and Chan (1990) reported the larval development of
Actinopyga echinites and James et al. (1993) reported the spawning technique of A. mauritiana,
while Asha and Rodrigo (2001) and Asha and Muthiah (2002) reported on spawning and larval
rearing of [Link] in India.

Chapter 2: Brood stock management

22.2.1 Collection and selection of broodstock

Brooders for the seed production needs to be collected from the natural habitat as there is no
captive production of broodstock. Generally, large and healthy adults measuring more than
150mm are collected from the commercial catch. Most individuals can be collected by divers in
shallow waters up to a depth of 10 m. The diving season for sea cucumbers is from October to
March in the Gulf of Mannar and from March to October in Palk Bay. The brooders should not
have any injuries or infestations with parasites. From the captured group only animals suitable
for breeding based on their morphology and condition will be selected for the breeding. The
selected brooders will be transported in filtered seawater with aeration to the hatchery.

22.2.2 Maintenance of broodstock

After collection, individuals are to be brought to the hatchery and stocked in one ton
tanks. The tanks should be cleaned, disinfected and dried before stocking of the
brooders. Usually 8 -10 animals will be stocked in 1 sq.m area. The bottom of the tank
should be covered with mud from the natural habitat to 15 cm to allow the brooders to
77
 bury themselves. The water in the holding tanks is to be changed every morning and the
mud at the bottom of the tanks every fortnight.

Chapter 3: Induced spawning and larval rearing

22.3.1 Spawning and early development in holothurian lifecycle

Spawning

Spawning occurs mostly from June to August by releasing of ova and sperm in the water where
fertilization takes place. During spawning, sea cucumbers adopt a "cobralike" posture, with the
front end lifted off the bottom.

Fertilization

The fertilization is external and takes place in the water column. The oocytes are fertilized
quickly as they make contact with the spermatozoa. After the oocytes and sperm are released
in the water, the sea cucumbers are removed from the tank. The eggs are washed in fresh
seawater several times to remove the excess spermatozoa. Excess spermatozoa can reduce the
rate of fertilization and cause the development of deformed embryos.

Early development

Large females can release about one million oocytes. About 0.7 million eggs were stocked in
750 litres of water. The eggs were spherical, white and visible to the naked eye and were found
floating in the water. The diameter of the oocytes ranged from 180 to 200 µm. After
fertilization the eggs underwent cleavage and developed into the dipleurula stage with eggs
ranging in length from 190 to 250 µm. The dipleurula transformed into early auricularia larva
after 24 hours. They are 430 µm long and 280 µm wide at this stage.
The early auricularia larvae have a buccal cavity, ciliary bands, a cloaca and an anus and they
actively feed. They were fed on the microalgae, Isochrysis galbana, and a mixed culture
dominated by species of Chaetoceros spp. and Skeletonema spp. As days passed the auricularia
became more and more transparent and the lateral projections also became prominent. On
each side of the late auricularia larva, four lateral projections were seen and at the end of each
projection there was a hyaline sphere. The oesophagus and the pear-shaped stomach were well
demarcated. Right and left stomatocoel were clearly seen. The ciliary bands showed a number
of pigment spots. The length of the late auricularia larvae varied from 660 to 1050 µm (with an
average of 860 µm) and the width was 240-690 µm (with an average of 500 µm). Some of the
auricularia larvae remained small.
A few of the late auriculariae transformed into doliolariae on the tenth day. The doliolariae
were barrel-shaped with five hyaline spheres on each side. Later, the first two tentacles
developed at the anterior end. Their length varied from 420 to 570 µm (with an average of 485
µm) and 240-390 µm in breadth (average of 295 µm).
On the thirteenth day, some of the doliolariae transformed into pentactula larvae . The body of

78
the pentactula was tubular with five tentacles at the anterior end and with one short stumpy
tube foot at the posterior end which helps in locomotion. The anal opening was distinct. The
length varied from 330 to 750 µm (average of 307 µm). By the eighteenth day the tube feet and
tentacles became distinct. Two long tube feet developed at the posterior end.

At this stage the length of the juveniles was 550-720 µm (average of 656 µm) and the width
varied from 210 to 320 µm (average of 262 µm). The pentactulae had the habit of moving to
the edge of the tank, remaining just below the surface of the water. They soon settle down on
the bottom of the tank.

22.3.2 Induced Spawning & Fertilization

Spawning Inducement
Thermal stimulation
Best results for thermal stimulation were obtained only during the breeding peaks (March-May)
of the sea cucumbers. The temperature of the water was raised by 3-5 °C by slowly adding hot
seawater and stirring uniformly. Usually 20 specimens were introduced into the tanks at 10:00
hrs. By 13:00 hrs the males had released sperm by raising the anterior end . One hour or so
after the males had released sperm, the females started releasing the oocytes. Simultaneously,
several males in the tank were still releasing sperm. It is better to keep only one male spawning
in the tank, otherwise the over-abundance of spermatozoa clouds the water.
Spawning behaviour
In sea cucumbers the sexes are separate but it is not possible to separate the males and
females from an external examination in most species. In the case of H. scabra, only a
microscopic examination of the gonads reveals whether the specimen is a male or a female.
However, it is possible to distinguish them at the time of spawning since the spawning
behaviour of the males and females is different. Typically, males spawn first, followed by
females. The male first lifts the anterior end and exhibits swaying movements just like a snake.
After exhibiting such movements for some time the males start releasing the sperm in a fine
white stream from the gonopore situated at the anterior end and in the mid-dorsal position.
When a male starts releasing the sperms it continues for nearly 2 hours. Meanwhile the ripe
females start reacting, possibly to the presence of sperm released in the water. The anterior
ends of females become bulged due to the pressure created inside the gonopore by the
accumulation of oocytes. The female releases the oocytes which settle down on the bottom of
the tank. Sometimes the same females spawn for a second or even third time; this is mainly
observed in large specimens. The oocytes are ejected out through the single gonopore. They
are ejected out in a powerful jet reaching a distance of about one metre, helping in the
dispersal over a wide area. The gametes are released as a light yellow mucus-like substance.

22.3.3 Larval rearing & collection of juveniles

Rearing of the post larvae

Preparation of the rearing tanks
All rearing tanks used in breeding, especially new tanks must be scrubbed clean and filled with
79
seawater for 20 days. During this period the water in the tanks is repeatedly changed. Before
the tanks are used, they are then scrubbed and filled with seawater containing 40 ppm
bleaching powder and then washed clean with filtered seawater.
Rearing density
Strict control over the rearing density of the larvae (i.e. the number of larvae per ml of water) is
maintained. At present there are two methods used to rear the larvae: still water rearing and
flowing water rearing. Auricularia larvae during their early and middle stages concentrate at the
surface of the water. If the density of the larvae is high, they will form agglomerations and sink
to the bottom of the tank resulting in their death. Rearing density, therefore, should be
controlled to ensure better survival rates. The desirable density of auricularia is 300-700 per
litre. In a one tonne tank filled with 750 litres of water, 0.3 million auriculariae can be reared.
Selecting and counting of larvae
After the embryos are transferred to rearing tanks, they develop into auricularia larvae in about
30 hours. Healthy larvae occupy the surface layer of the water while deformed larvae and dead
embryos are found in the lower layer of the water column or on the bottom of the tank. A
sample can be removed for counting the larvae. Samples can be taken separately from the two
ends and the middle of the tank using 250 ml beakers. The sample is stirred and a 1 ml aliquot
is taken with a pipette and placed on a plankton counting chamber to assess the number of
larvae. Two more samples are taken and the average of three counts is taken as an indication of
the density of the larvae. When the auricularia larvae are in the early stage of development,
they should be reared at a density of about 500 larvae per litre. The development of the
auricularia can be divided into three stages: early, middle and late.
Water management
During their development, the larvae eject faeces and consume dissolved oxygen constantly.
Some of the larvae will die. These, together with excess food, will produce harmful substances
such as hydrogen sulphide and ammonia. In addition, bacteria reproduce rapidly with the rise
of temperature. Poor water quality directly affects the normal development of the larvae.
Therefore proper water management and sanitation is essential, including regular cleaning of
the tanks and frequent changing of water. Sediment and deformed larvae at the bottom of the
tank have to be siphoned out preferably on a daily basis.
During water changes a sieve (80 µm mesh size) is used to prevent loss of eggs and/or larvae.
While the water is being changed, it is advisable to constantly stir the water lightly around the
tank. This will prevent damage to the larvae during the water change as without stirring the
larvae would be forced into the sieve causing mechanical injury.
Larval feeding and feeding rates
High quality microalgae and proper feeding schedules are key factors in the successful rearing
of sea cucumber larvae. Early auriculariae possess a well formed alimentary tract and must be
fed. Ingestion by these larvae consists of conveying the suspended particles of food into the
alimentary canal through the mouth parts by the swaying of peristomial cilia. The effectiveness
of Isochrysis galbana, Dunaliella salina, Dicrateria spp. and mixed diets consisting of all the
above mentioned microalgae was tested. The best growth rates and lower mortality were
observed when larvae were first fed I. galbana and supplemented with mixed cultures, chiefly
consisting of Chaetoceros spp., four or five days later. Unicellular algae were given twice a day
but the amount given depended on the particular stage of the larvae. In general, a

80
concentration of 20 000 to 30 000 cells per ml was maintained in the rearing tank. The amount
of food given should be increased or decreased depending on the abundance of food observed
in the stomach of the larvae. This can be visually assessed everyday before feeding.

Unit 23: Water quality management in shellfish hatcheries

Learning Objectives

This unit has been designed to provide the following to the students:

1. General introduction on the water quality and its importance in the hatcheries
2. The parameters to be managed in the hatchery
3. Water quality needed for different species of shellfishes

Chapter 1: Water quality in shell fish hatcheries

23.1.1 Introduction

Generally water is considered and referred as universal solvent, which has diverse properties.
The nature of the water is highly influenced by the components it has with it. By and large
water contains gases, solids (both dissolved and suspended), life (micro and macro organisms)
and chemicals or salts or minerals. It can be said that all the non-living things that are present in
the water will strongly influence the living things present in the same water.

Since water is the basic medium for the shellfishes, its quality will greatly infulence the life and
their regular activities like feeding, breeding and growth. Every organism prefers to stay in
waters with desirable qualities for them. E.g,, lobsters preferring the rocky or sandy areas,
shrimps adn prawns prefer muddy waters, etc. The preference is for certain chemical and
biological qualities and also the movement of water (currents).

When the water quality changes, the organisms try to move away from the locaality and find
another suitable place for their living. Such migration is not possible in the pond or in hatchery.
Therefore it is necessary to maintain the water quality as per the requirements of the species
under captivity.

23.1.2 Shellfishes and their environment

Management of the tank water has become more inevitable in the case of all aquatic
organisms. The confined environment makes the shellfishes to suffer if the water quality is bad
and the parameters are not at preferred level. It exerts much pressure on the brooders and also
larvae.

The following can be considered as the general recommendation for the good water in
hatcheries.

81
 Shellfish
S.N Parameter Fish Hatcheries
hatcheries
1. Alkalinity 10 – 400 75 – 150
2. Total Hardnes (ppm of CaCO3) 10 – 400 20 – 80
3. Ammonia (ppm) <0.02 <0.001
4. Ammonia (Total Ammonia <1.0 <0.01
Nitrogen)
5. Calcium (ppm) 4 – 160 20 – 80
6. Dissolved oxygen (ppm) 3 - Saturation 4 to Saturation
7. Carbon dioxide (ppm) 0 – 10 0 –1
8. Chlorine (ml/l) <0.003 <0.001
9. Hydrogen sulphide (ppm) <0.003 <0.001
10. pH 6.5 – 8 7.5 – 8.0
11. Total Dissolved solids (TDS) < 400 < 200
(ppm)
12. Total suspended solids (TSS) < 80 < 20
(ppm)
13. Micro elements (Zn, Cd, Se, Fe, < 0.01 < 0.01
Mn, Cu, Ni, Cr.) (ppm)
14. Macro elements (Na, K, Kg, S, < 1.0 < 1.0
etc)
15. Turbidity (Light penetration 30 – 50 cm 30 – 80 cm
depth)

There are many minor components in the water other than the items listed above, which can
also influence the shellfishes. However, managing the above parameters will control all the
other quality parameters in the culture water.

The following table shows the effect of major water quality parameters on the crustaceans:

S. Nature of the Influence on the organism Nature of the

No. parameter effect (negative)
1. Dissolved oxygen - Gas bubbles will be May be lethal
super saturation trapped in the eye, gills,
etc..
Low concentration - System will become Fishers will be
3ppm anaerobic in the bottom: affected badly
Decomposition will result is
the production of
unwanted gaser.
Nitrification process will

82
 be stripped
2. Carbon di oxide pH Variation in water will Lethal
High concentration affect the fish growth
> 5ppm Will alter the alkalinity, so
that the fish growth will be
reduced
3. High Minerals Osmorgulation will be Lethal
affected laner conversion of
High Handers feed to flesh
Calcification on the body
surface. Skin diseases -
growth of unwanted
organism in the tank
4. High or low pH Affects the growth. Lead to Lethal
death of the fish
23.1.3 Water quality management

The congenial or suitable qualities of the water required for different shellfishes are given in the
previous sub heading in this unit.

The tank water will not have the stable qualities due to varied biological activities and it is
necessary to restore the qualities to their optimum level by proper management techniques.

Water quality management involves the following steps :

1. Removal of suspended solid

2. Removal of dissolved gases & solids

3. Reduction or elimination of microbial load

4. Adjusting the basic gudities and kiss if water.

Unit 24: Different water treatment techniques for shellfish hatcheries

Learning Objectives

Following are the learning objectives of this unit:

 To know about the water treatment methods

 To decide the best suitable water treatment method needed for the particular hatchery
operation

83
 Chapter 1: Water treatment methods

24.1.1 Removal of suspended solids

This is more essential for further treatment of water. Therefore it is referred as primary
treatment. In this process, all the suspended matter will be removed.

In the hatchery tank water, the suspended particles are mainly the unused feed and fecal
matter. They might be having a particle size ranging from 0.001 – 0.01mm depending on the
feed used. These particles can be separated by the following tools:

a) Seives – appropriate size to remove the particles

b) Sedimentation chamber - make the particles to settle through the process called
sedimentation.

c) cartridge filter - Cartridges are used for filtering the water. The cartridges will have filter
media or cloth with micro pores.

d) Granular media filter - Filter containing granules for the filtration of water. The milli pores
offered by the mass of granules will filter the suspended solids from the water.

In the above cartridge filter and granular media filter, the solids are removed while the water is
passed through with or without pressure through a media with holes or pores smaller than the
size of the particles. These filters are commercially available in different models to suit the flow
characteristian of the water.

Sedimentation filter is to be designed based on the volume of the water that is to be filtered.
This is working oo the principle of separation based as the mass or weight of the particles as
against the density of the water. This has to be designed for every hatchery.

24.1.2 Removal of dissolved solids and gases

The culture tank water has dissolved solids arising due to the excretion of the fishes and
dissociation of various compounds from the organic matter due to bacterial decomposition. The
particles may vary in size from 10-6 to 10-4mm which can not be filtered by the mechanical filter.
Therefore classical or biological treatment is recommended.

Chemical treatment is done through precipitation method, which is not recommended for the
shellfish hatchery tank water. Biological treatment in bio filter is the only option to remove the
dissolved gases and compounds in the water.

Water in the hatchery tanks will have high amount of nitrogenous wastes and ammonia in the
diet and gaseous compounds in the water. Carbon-di-oxide and hydrogen sulphide are other

84
major gases present in the water and nitrogen and sulphur along with other minerals may be
other significant matter present in the water.

Biological filtration will help in removing the gases by the process called nitrification with the
help of nitrifiers. The bio media used inside the biofilters will provide the space for the living of
the Nitrosomonas and Nitrobacter. These microbes will use the nitrogenous wastes in the
water and convert them as harmless Nitrates. This process is highly useful for the hatcheries in
maintaining the water quality in the hatchery tanks.

24.1.3 Bio filtration

This is no filtration process making as the biological principles. The unwanted materials are
either reduced or removed viz.

Bio filter can either be designed micro organisms constructed bio filter cab also be fitted in the
flow line for filtration. Whether it is pryamicated or designed for a system, bio filter with correct
specification will be more useful is giving the best results.

The bio filter consists of the following materials for the following purpose:

1. Wood/cotton/silk cloth filter - to reuse any suspended solids

material or parlider of large size.
2. Activated carbon / charcoal / - to remove gases
burnt shells / ceramic rings
3. Bio balls / Bio media / Bio beads - to convert ammonia into
nitrate through nitrification
process.

Process :

The water from the screen or any mechanical filter will be allowed to pass through the biofilter.
The biofilter is packed with the above materials through which water is to pass through. There
are two methods of water flow in a biofilter.

Unit 25: Health management & sanitation in shellfish hatcheries

Learning Objectives

Following are the objectives of learning of this unit:

1. To gain a basic knowledge on the larval diseases

2. To know about physical and chemical methods of disinfection

85
 3. To understand the bacterial diseases in the hatcheries

Chapter 1: Larval diseases

25.1.1 Bacterial disease

It affects zoea, mysis and post larvae. It may choke the gills affecting respiration. Luminous
bacterial disease. It is caused by Vibrio harveyi / V. alginolyticus. Infected larvae exhibit
contimuous greenish luminescence when observed on total darkness.

Vibriosis- It affects haemolymph and mid gut gland.

Fungal disease Mysis stages are most vulnerable to fungal attacks. The common fungae that
attack the shrimp larvae are Lagenidium.

Viral disease: They appear mainly during post larval and juvenile stages. This may cause high
mortality in post larvae. Beculovirus group affect mid gut and hepatopancreas whereas
parvovirus group (IHHNV) affect epidermal, hypodermis & mesoderm.

Larval fouling – Large scale infectation of Zoothamnium, Vorticella etc on the gills, eyes of larvae
lead to hypoxia and death. Though they are not directly pathogenic, they can cause mortality in
larvae.

25.1.2 Bacterial Necrosis

Necrosis – It starts on the antennae or uropod in protozoa III or pleopods in mysis. The affected
areas get melanised. Hepatopancreas and stomach become greyish and hence called grey
stomach disease

Chapter 2. Disinfection in hatcheries

25.2.1 Need for Disinfection in Hatcheries

During the routine operation of hatchery, many pathogenic microbes may accumulate in the
equipment and components of hatchery system. When there is any drastic change in water
quality favouring the microbes, it will enhance in number and prone to cause disease to culture
organisms. The best method to avoid the infection is the breaking the life cycle or continuity of
such pathogens in the hatchery. For this the hatchery should be disinfected and shutdown
between production cycles so that the pathogens will have no host to infect and will get
eliminated from the system. This is to ensure non-settlement of microbes in the system.

Disinfection has widespread application as a method of sanitation, to eliminate or suppress the

presence of the initial infective unit that is the pathogens. Therefore disinfection has to be done
in all possible ways to completely avoid the infection. In this process a variety of hatchery

86
system components are to be subject to disinfection, like equipment such as pipes, tanks, bio-
filters, nets, floor, boots, culture water, input water, food, stock and air, output of effluent,
debris and dead fish, etc.

25.2.2 Methods of Disinfection

Disinfection is broadly classified into two methods.

i) Physical Method

ii) Chemical Method

[Link] Physical Methods of Disinfection

i) Cleaning
The cleaning removes sessile organisms. It is mainly used for equipment disinfection. The tanks
and other equipments are scrubbed with brushes or scrub pads of various designs. This can be
enhanced by using standard detergent powders to inactivate the organism even if they cannot

87
be removed. Use of detergents will also help in the removal of nutrient base that will encourage
the growth of microbes.
ii) Heating
This is very common method of disinfection that will help in reducing the microbial load in any
system or component. The basic idea is that the temperature above certain level would kill the
microbes by dehydration of the cells. Heat exchangers of some hatchery systems raise the
water temperature to a level that is intolerable to some microbial pathogens. Heating of water
is suitable only for smaller systems.
iii) Filtration
This is the physical removal of debris and other solid material that is present in suspension and
is mainly used to remove solids from supply water. This solid removal also helps in the
reduction of microbial load associated with those solids in suspension. This filtration is effected
by designed filters and filter materials that are non-toxic and non-corrosive. There are physical
filters and biological filters with the application of biological principles such as Nitrification for
removal of Ammonia in the rearing water. It can be said that a perfectly designed biological
filter will keep the water free from ammonia and mortality due to ammonia.
iv) Dehydration
Dehydration or drying and reducing water contact of equipment or entire system provides a
degree of disinfection. Drying easily destroys fragile stages of many parasites and dry surface
have greater exposure to the effects of ultra violet light.
v) Radiation
Disinfection by radiation is especially used in hatcheries. The ultraviolet light of approximately
2500-2650 Ǻ wave length effectively destroys vegetative stages of many pathogens such as
virus, bacteria and fungi. During water disinfection by radiation, pretreatment such as
sedimentation and filtration make the water suitable for UV radiation treatment, by removing
the hurdles for penetration of UV light. The ultraviolet rays are used to disinfect the organisms
in limited dose. The dosage depends on intensity of light, duration of UV-light on organisms.
[Link] Chemical Methods of Disinfection
i) Chlorination

88
It is commonly used method for disinfection of water in shellfish hatcheries. The water is
treated with 0.2-0.5 mg Cl 2/litre of water for 20 to 30 minutes. The removal of residual chlorine
prior to introducing the disinfected water into shellfish hatchery is inevitable. Vigorous aeration
was given to remove the residual Chlorine present in water or it is passed over charcoal for
removal of chlorine. If any chlorine residue is present in treated water, it is toxic to shellfishes.
Chlorine in the form of calcium and sodium hypochlorites and chloramines are commonly used.
Nowadays chlorine-dioxide is also used as disinfectant, which is considered as safe as it seldom
leaves any residual chlorine in harmful form.
ii) Ozonation
Electric discharge and UV radiation of a suitable wavelength produce a 3-atom form of Oxygen
(Ozone). The contact concentration of Ozone is around 3 mg/ liter of water with residuals of
below 0.15 mg/ liter. The residual ozone may also be removed with charcoal prior to use.
However, the cost of equipment and use is relatively high.
iii) Formalin and Formaldehyde
Formalin water containing dissolved Formaldehyde is a readily available industrial Chemical. A
1:100 mixture of formalin and water is used to disinfect the water. Formaldehyde [HCHO] gas
has been used for the disinfection of sealed building, usually generated by heating Para
formaldehyde and the polymerized solid of Formaldehyde. It is effective against many viruses
and bacteria.
iv) Quaternary Ammonium Compounds [QAC]
QAC are widely available in numerous brands and strengths. The active concentration is
approximately 1200 mg/ liter (1200 ppm) of water. It is effective against gram +ve bacteria,
some viruses but less effective on fungi and gram -ve bacteria.
v) Hydrogen Peroxide:
It is clear colorless, water like in appearance, has pungent odour, and do not highly corrosive or
dangerous toxic effect.
vi) Iodophores

89
Iodophores are generally available at 1% solution. They are organic compounds that release
free iodine. The active concentration for disinfection of water is 50 mg/ liter and for
equipments 250 mg/ liter. It effectively disinfects bacteria and some viruses.
During unfavourable and lean demand periods have to be advantageously utilized for the
purpose of disinfecting the entire shellfish hatchery.
Unit 26: Disease prevention in hatcheries (Guidelines)
Learning Objectives

This unit has been designed to provide the following to the students:

 Need for disease prevention in the hatcheries

 Basic information on the disinfection protocols and application
 How to make the hatcheries free from disease outbreak.

Chapter 1:Prevention methods

26.1.1 Disinfection and Disease Prevention

Generally there are lot of doubts on the need for disinfection and the process of disinfection.
The following text will help in getting a fair understanding on this topic

What is Disinfection?

The technique or method of selectively controlling or removing organisms that enter into
aquaculture system is called disinfection. In the process of disinfection, the unwanted
microorganisms are removed from the water.

Why it is required in the hatchereis?

In hatcheries, the major input is the formulated and prepared feed for the breeders and larvae,
which is purely organic in nature. This feed results in the production of fecal matter by the
fishes. The uneaten feed is another fraction to add with the fecal matter offer good scope for
the growth of microbes, as they are the principal feed for the heterotrophic bacteria. The
nutrients released or leeched from the feed and fecal matter also encourage the growth of
diverse microorganisms. In many instances these microbes turn to be pathogens causing or
leading to disease outbreak in the population. Therefore it is very much essential to disinfect to
reduce or remove the bacterial load in the culture water.

In practice, disinfection is commonly adopted in hatcheries as a separate treatment measure.

Disinfection can be accomplished by using one or a combination of readily accepted methods.
To minimize the outbreak and transmission of disease causing organisms and parasites,

90
disinfection is becoming one of the promising methods in the super intensive aquaculture
system.

Disinfection methods

Basically, 4 methods can disinfect the water. They are,

a. Heat disinfection,

b. Chlorination,

c. Ultraviolet irradiation &

d. Ozone treatment

The agent or chemical used for the disinfection process is referred as disinfectant. The
effectiveness of a given disinfectant depends on some of the basic factors, viz.,

i. Contact time

ii. Temperature

iii. Turbidity

iv. Particulate concentration

v. Characteristics or type of the of the target organisms

Unit 27: Culture of different live –feeds (Skletonema, Chaetoceros, Spirulina,

Dunaliella, Isochrysis, Cyclotella, Azolla, Cladocerans, Artemia, Streptocephalus, etc)

Learning Objectives

Following are the objectives of learning this unit:

1. To get a basic knowledge on the micro algae and their production methods
2. To understand the different media that are used for the live feed culture
3. To carry out the algal culture in the hatcheries
4. To understand the advantages of the live feeds for the hatcheries

Chapter 1: Natural feed for shellfish larvae

27.1.1 Microalgae

91
Micro-algae:

Micro-algae are plants consisting either of single cells or small filaments. Microalgae serve as
live-food for commercially important mollurscs, fish & crustaceans

Important Micro-algae in aquahatcheries:

1. Chaetoaros sp.

2. Skeletonema sp.

3. Isochrysis sp.

4. Tetraselmis sp.

5. Chlorella sp.

(Chlorella has thick cell wall and therefore is not suitable for bivalves)

Size of micro-algae

Chaetoceros 4-6 m m

Isochrysis 3-5 m m

27.1.2 Role of Micro-algae in shrimp hatcheries

Successful operation of shrimp hatcheries largely depends on how effectively one handles the
“Critical phase” of the delicate larval stages. This is why researchers all over the world are
concentrating their efforts towards developing a right type of food, to be provided to the larvae
at a right time, in a right manner Larvae of shell fishes such as shrimp or bivalves have
restrictions in their feeds and the digestive ability.

The first and foremost problem in the feeding of the shellfish larvae is their mouth size and
digetive processes. Most of the larvae will have very tiny mouth opening and therefore the feed
must be lesser than the opening of the mouth.

The next issue will be their digestive enzymes in their alimentary canal. It is understood that
their development is primitive and therefore with less complext enzymes secreted by the
alimentary canal.

Considering above restrictions, the micro algae with their small and tiny size (<10 micron) and
easily digestible composition are the most accepatable first feed for many shell fish hatcheries.

92
 Chapter 2: Mass production of algae

27.2.1 Culture media

The following are the media that are used for the culture of microalgae:

Chu-10 medium – For myxophyceae, chlorophyceae,Bacillariophyceae,

and Euglenophyceae,

Fogg’S medium – For blue green algae

Zarrouk’s medium – For spirulina

Modified “F” medium – For chlorella

Although the above can be used for individual group of micro algae, usually Walne’s medium is
used for microalgae culture.

27.2.2 Algal culture techniques

Algal culture techniques are classified as below:

1. Indoor and Outdoor culture method

2. Open and Closed culture method

3. Batch culture

4. Continuous culture method

5. Semicontinuous culture method

1. Indoor and Outdoor:

Indoor culture allows control over illumination, temperature, nutrient level, contamination with
predators & competing algae, where as outdoor algal culture systems make it very difficult to
grow specific algae culture for extended periods.

2. Open/Closed:

Open culture such as uncovered ponds & tanks (indoor or outdoors) are mostly readily
contaminated than closed culture vessels such as tubes, flakes, Carboys, bags etc.

93
3. Axenic (=sterile)/xenic:

Axenic cultures are free of any foreign organisms such as bacteria & require strict sterilization
of all glass ware, culture media & vessels to avoid contamination. The latter makes it impractical
for commercial operation.

4. Batch continuous & Semi continuous

Batch culture technique:

The batch culture consists of a single inoculation of cells into a container of fertilized seawater
followed by a growing period of several days & Finally harvesting when the algal population
reaches its maximum or near maximum density.

Inoculation 2-10%

Continuous culture:

Two categories

1. Turbidostat culture

2. Chemostat culture

Turbidostat culture:

In which the algal concentrationis kept at a preset level by diluting the culture with fresh
medium by means of an automatic system.

Chemostat culture:

In which a flow of fresh medium is introduced into culture at a steady, predetermined rate. The
latter adds a limiting vital nutrient (eg nitrate) at a fixed rate and in this way the growth rate &
not the cell density is kept constant.

Semi-continuous culture:

The semi-continuous techniques prolongs the use of large tank culture by partial periodic
harvesting followed immediately by topping upto the original volume & supplementing with
nutrients to achieve the original level of enrichment.

27.2.3 Feeding regimes of penaeid larvae: ( cells/ml)

Substage Chaetoceros sp. Tetraselmis sp.

94
 N5 or N6 60,000 0-15,000
P1 1,00,000-1,20,000 30,000
P2 1,20,000 35,000
P3 1,20,000 35,000
M1 1,00,000 30,000
M2 75,000 20,000
PL1 to PL5 50,000-75,000 20,000
20,000-75,000 5,000-20,000

27.2.4 Advantage of algae in Shrimp hatchery

1. Natural occurrence
2. Alternative systems
3. High production with minimum inputs
4. Rich in nurrient value
5. Supplemental value to conventional food

27.2.5 Disadvantages

 Predation – contamination
 Upscaling & collapsing of pure culture
 Species control in induced blooms of natural phytoplankton
 Toxicity & digestibility
 Variation in nutritional value
 High production cost

Unit 28: Spirulina as a feed for the shellfish larvae

Learning Objectives

This unit will provide the following to the student:

 General introduction of Spirulina and its use in aquaculture.

 Advantages of Spirulina as a larval feed.
 Production methods of Spirulina.

Chapter 1: Introduction and significance of Spirulina

28.1.1 Spirulina in Aquaculture

95
Breeding of fish has emerged as a major industry in India over they past decade, so much so
that today prasons and shuimps are major export items Spirulina is a good protein diet with
adequate minualsand intamins.

The single cell protein spirulina has been iroghiyed as one of the potential

Ingredients for use in aquaculture and successful attempts have been made in the mass culture
of this algar. The better digestibility of protein and fat observed in spirulina incorporated diets
must be due to better absartron. Spirulina flabes are being used in shrimps hatcheries to obtain
better growth and scusval

In Taiwan fishuy experimental station. Spirulina was pound to are an excellent source of food
for shrimps lowae and milh fry and yearlings. Its role in augmentader the pigmentatron of
anamental fish and prawn larvae has commercial significance.

28.1.2 Spirulina and its importance in aqua hatcheries

Spiulina is a blue green algae with very high nututional profile. It is rich in vitamin B-12 and has
a good protein anturt with excellent profile 70% aminoacid above intains natural carotene and
ranthophyll phytoprotains. Spirulina has a soft cell wall made of complex sugars and protein. It
is digestible too .

Spirulina is known to possess an amazing ability to thrive in conditions considered too harsh for
other algae. With such ability it is not surprising that it grows naturally in rakes with extremely
high alkaline levels. Todays, however, soirulina is hawested. In large-scale commercially ponds
in acologically controlled conditions. The worlds largest scientifically designed algae farm is the
Earth riese farms located in the sunny desest of californis. Blue-green algae of the genus
spirulina have found world wide interest for having unmatched nutrient composition and its
uses invarious other felds. Spirulina is extremely popular for complete nutrition and instant
energy synthesis.

28.1.3 Ecology

Spirulina are cyanobacteria that grow as multicellular filaments that are helical wiled hence its
name. Spirulina are found in soil, thermal springs and waters that range in salinity from to full
sea H2O. It has been recorded from waterlogged rice fields. Pards, lakes, watatec channels by
spirulina, the water generally has a PH of atleast 10. Being a typical organism, Spirulina is
thermophilic, the optima temperature for more strains is 36+(or) – 2 c. Though many strain
have been expecimentally cultured, probably the most commonly cultured species are S .
platensis & S. maxima.

28.1.4 Taxonomy

Class: Myxophyceae
96
Order: Hormogonales

Family: Oscillatoriaceae

Genus: Spirulina

Culture Systems:

Spirulina is being cultural successfully in many countries. There are two types of culture ponds.

(1) The air-lift type culture pond, developed by IFP(Institute Francais due petrole)

(2) Receway-type culture ponds driven by paddle wheels.

For outdoor culture, cement raceway orvat-having capacity is culture is sunlight. The average
light intensity in typical countries is usually 60,000 – 70,000 lux during mid aternoon. However,
at this intensity the algae culture may get bleached. To avoid this, a shade is mounted left
above the tank level, which cut 60-70 per cent of the light intensity.

Chapter 2: Culture and production of Spirulina

28.2.1 Nutrient Requirement for spirulina culture

Nutrients

Sodium bicarbonate: 4.50(g/l)

Dipotassium phosphate: 0.50 (g/l)

Sodium carbonate: 1.5 (g/l)

Sodium nitrate: 1.5 (g/l)

Magnesium sulphate: 1 (g/l)

Ferrous sulphate: 0.01 (g/l)

Potassium sulphate: 1 (g/l)

Calcium sulphate: 0.04 (g/l)

The pH is maintained between 9.5 & 10.5 . Spirulina is reported to use bicarbonate as the
carbon source for photosynthesis. Laboratary studies have shown the difference in growth of
spirulina at the level of 4.5 g/l of bicarbonate.

97
Mass Culture:

For mass culture of spirulina, a simplified culture is recommended by adding chemicals like
sodium carbonate 4.00, Magnesium sulphate 0.20, N:P:K([Link]) 100 zincsulphate 0.01 EDTA
(Ethylene diamine fetre acctic acid) 0.08 (allg/litre).

28.2.2 Harvesting and processing

Harvesting can be done by

(i) Curasity cloth filtratron

(ii) Plate and frame filter press.

(iii) Centrifugation Method

For gravity cloth filtration method, 50 mesh. Cloth is used. Whereas in plate and frame filter
press method. 140mesh cloth used. Centrifugation at 8400rpm using a self-opening bowl type
centrifugal separate is used in centrifugation method.

Spirulina is usually dired by steam heated table drum deying vaccum shelf deying and
sundeying. Among these, sun deying holds food promise both in terms of cost and flowover.

Bio-chemical Composition (%) of spirulina

Protein : 55-70%

Hipid : 5-7%

Minual : 3-7%

RNA : 3-5%

Nitugen from

Nucleic acids : 1.99%

DNA : 1%

Crude fibre : 0.90%

Nutritional Value

Protein Efficiency rate (PER) - 2.2-2.6 (77-87% that of caesin)

98
Net Protein Utiliz (NPU) – 53-61% (85-92% that of caesin)

Digestability - 83-84%

Available lysine - average 85%

Physical Properties:

Appearance : Fine powder

Colour : Dark green

Odocuand Taste : Mills resembling sea weeds.

Bulk density : 0.5 g/l

Practical size : 9.25 miuons

28.2.4 Conclusion

The culture and use of Spirulina is gaining popularity. Recently many Indian companies have
developed Spirulina for human cousumptron and also to use in aquaculture practice. There is
much scope for improving the culture and harvestry technology of this super food in view of its
goods export value.

Use of spirulina showed excellent economic feasibility.

Unit 29: Genetic improvement and biotechnological application in shellfish hatcheries

Learning Objectives

Following are the objectives of this unit:

1. To provide information on the biotechnological application in shrimp hatcheries

2. To understand and assess the benefits of biotechnological application for the shrimp seed
production.

3. To know the hybridization attempts in shrimp seed production.

Chapter 1: Biotechniques in shrimp seed production

29.1.1 Steroids in Sex Differentiation & Reproduction of Crustacean

99
The genetic composition of the individual decides whether or not the androgenic gland will be
differentiated from this primordial gonadal tissue. In the male, the androgenic gland develops
and its hormonal activity results in the production of remaining gonadial tissue into a testis. In
the female the androgenic gland does not develop and gonad becomes the ovary.

There are many experiments in which the androgenic glands are removed from the male and
the male will either become sterile or functional female due to the dominance of the estrogens

29.1.2 Male Sex determination

The androgenic gland is meut is male and responsible for producing androgenic hormone (AH),
which determins all the primary and secondary sexual characters of the male. Although it was
first discovered in the males of the amphipod orchestia, the gland is now known to present in
all super – order of higher crustaue. Usually it consists at a strand of cells lying along the var
differences near it termination, and so between the coxopodite muscles at the last thorak leg;
but in some isopods it lies close to the testicular tubues. It is absent in females.

Whenever, the androgenic gland is implemented into a young female it prevents the
development of female secondary sexual characters, such as oostegits, and result in
masculnization, including a change in the apendages as that of males male condition.

The effect of the hormone is clearly a direct one, since the ovary can be removed prior to
implantation without altering the result. If the ovary is not removed, it ceases to produce eggs
and instead forms sperm which are fertile and can be used artificially to fertilize normal eggs.
Females with implanted andriogenic glands display normal male behaviour and will mate the
normal female. They cannot, however, fertilize the females as, although a var deferences with a
lumen is developed, it is not functional. Injection of tests without androgenic gland into females
has no mascularizing effect.

Reciprocal transplants of ovaries into males give further proof that control of male
characteristics is governed slowly by the androgenic gland. Thus, when an ovary is transplanted
into a normal male, it is converted into a testis. When an ovary is put into a male which the
androgenic gland has been remove, however it remains as an ovary. Finally, removal of the
androgenic glands from a male can result in a progressive loss of male secondary characters
and in at least one case. Orchestia montag, the tests may become converted into an ovary
about a month after the operation.

Injection of steroid, which in mammals have androgenic effects, have no such action in
crustacea and, since the histological appearance of the gland resembles that of vertebrate
protein – producing cells, it seems possible that the effective hormone is a protein or peptide.

100
The determination of sex is also influenced by the environmental factors. In the female the
androgenic gland does not develop and gonad becomes the ovary.

Reproduction:

One of the obvious features of crustaceans is a thick chitin cuticle (or) exoskeleton. The
replacement of the old exoskeleton is accomplished by moulting. In addition moulting is also for
the purpose of mating which is usually occur during the reproductive cycle. Whether the
moulting is for growth or for purpose of reproduction.

29.1.3 Ecdysteroids (Arthropod molting Hormones)

The Y organ (Molting gland) secrete the moulting hormone, ecdysone. Ecdysone initiate the
molting.

Ecdysone structure was determined from insects, horn and his colleagues isolated and
determined the structure of 20 – hydroxyecdysone (20 HE) from the lobster Jasus Lalandei.
They demonstrated the identify of the arthropod molting hormone from both insects &
crustaceans.

Ecdysteroids and Reproduction:

Some evidence exists that ecdysteroids are involved in crustaecan eggs and embryonic
development with RIA detection after HPLC separation, ecdysone, 20HE, ecdysteroid
conjugater, and (in some cases) ponasterone A have been identified in follicle cells oocyter, and
embryos of various species including shrimp palalmon serratus. Embryonic ecdysteroid
concentrations were low after fertilization, but increased rapidly following the appearance of
the embryonic Y organ.

The actual function of these ovarian ecdysteroide is uncertain. There are correlation between
vitellogenis and increasing hemolymph ecdysteroid is some species but not in stimulatory or
inhibitory effects on vitellogenis. The conflicting result are difficult to interpret due to special
differences, animal age, and stage in the molt and reproductive cycle, Adiyodi (1985) gives
more through discussion.

Ecdysteroids concentration changes have been observed in developing embryos of various

species.

These observations support the hypothesis that they are at least partially of embryonic origin,
and not entirly sequestered from the mother during vitllogenesis . These embryos, which were
spawned at fertilization and in some cases incubated on the female’s obdomen, were without
and cytoplasmic connections to the parent. Ecdysteroid concentration were must likely due to

101
endogenous synthesis. A low concentration of ecdysteroids initially deposited in the eggs, and
synthesis and secretion by the embryo begins soon after fertilization.

Further evidence for larval ecdysteroid synthesis is the Y organ description in larval crob cancer
anthonyi The function of these embryonic ecdysteroids are not know, It is plusible, However,
that they are involved in the deposition of embryonic envelops and in embryonic molting.

Female reproduction:

One of the major contraints still facing shrimp culture is control of female reproduction.
Reproduction molting are often antagonistic events since both require large energy expenditure
and are often mechanically incompatible.

Gonad – Inhibiting Hormone (GIH) :

The primary action of GIH in females apporetly occures during secondary vitellogenesis, the
time when the ovary increases dramatically in size due to syntesis and uptake of yolk produced
in either tollicle cells or at extraovarian sites.

The stuidy by quackenbush and keeley (1968) sguggests that the target of GIH may be synthesis
of golk protein. there are other data, however, that suggest that GIH may inhibit binding and
uptake of yolk by the oocyte. Whether the same or different factors are responsible for these
activities is unclear. purification and amino acid sequencing of GIH is one of the most active
areas of research in crustacean endocrinology.

Mythyl Farnesoate:

Based on endocrine regulation of moliting analogie between crustaean & insects It is

responsible to hypothesize that an analog to the insect juvenile hormone (JH)is present in
crustuan. The insect JH plays important roles in regulating morphological development &
Reproduction.

There are correlations between secondary activity of the MO and vitellogenesis. In the sipider
crab, MOs from junvenile females secreted small amount of MF in culture. During the later
stages of ovanion development, however, when vitellgeonesis was host pernounced, secretion
of MF by the Mo increused to 3.2 ng/gland/hr. At the completion of vitellogenesis, Mo
secretion dropped to 1.6 ng/gland /hr. The precise role of Mf in vitellogenesis remains unclear.

THE END

102