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Macrobrachium rosenbergii

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Intro

Development of the aquaculture industryrequires a reliable and adequate supply ofhigh quality eggs from broodstock.

The ability to control production of viableeggs and their successful rearing throughthe larval stages are necessary for the

reliable supply of the seedstock on whichintensive culture systems depend.

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The understanding of the reproductive biology,

especially the regulatory mechanisms involved ingonadal maturation of cultivable species ofcrustaceans, is needed for intensive culture.

In recent years procedures for stimulating

ovarian maturation in penaeid shrimps and otheraquaculture species under controlled conditionshave been developed.

Environmental control of photoperiod and

temperature were found to result in successfulreproduction in Penaeus stylirostrisand P.

japonicus.

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Female reproduction1. Vitellogenesis

In crustaceans, oocytes grow during oogenesis throughthe process of vitellogenesis.

During vitellogenesis, vitellogenin, the precursor of the

major yolk protein, vitellin, is synthesized and is taken inby the oocytes. In the oocytes, vitellogenin is processed and

accumulated as vitellin. Vitellin is utilized as a nutritional source during

embryogenesis. Vitellin and vitellogenin have been purified in several

shrimps and determined to be large lipoproteinmolecules (molecular weight, 280-700 kDa).

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Oogonia start meiotic division I and become

oocytes. While the oocytes remain arrested atprophase of meiotic division I, they accumulateRNA at the previtellogenic stage, oil globulesand PAS (periodic acid-Schiff)-positive vesicles at

the endogenous vitellogenic stage, and yolkglobules at the exogenous vitellogenic stage.

At the exogenous vitellogenic stage, oocytesgrow rapidly by yolk accumulation.

After the completion of yolk accumulation,oocytes recommence meiosis, and the germinalvesicle at the cell center disintegrates (germinalvesicle breakdown, GVBD).

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Stage I, underdeveloped and/orspent stage

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Stage II, developing stage

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Stage III, nearly ripe stage

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Stage IV, ripe stage

The complete ovary extends

from the head to the tail. The

majority of the ovarian mass is

within the cephalothorax

region which cannot be

observed by torchlight.

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OVARIAN DEVELOPMENT 4

Schematic diagram of the major endocrine organs in P. monodon. The sinus gland is composed of the

terminals from neurons which have their cell bodies in the X-organ and brain. (b) Electron microscopy

section of the sinus gland demonstrating hormone filled vesicles (dark circles) which fuse and release

their contents into the blood. Magnified 8500x.

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SECTION II

DEVELOPMENT OF THE EGG:Post-Fertilization to Hatching

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TIME AFTER SPAWNING0-30 MINUTES

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TIME AFTER SPAWNING1 HOUR

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TIME AFTER SPAWNING1 HOUR 30 MINUTES

Cell division is occurringrapidly. Within a sampleof eggs a range ofdevelopmental stages willbe seen (Fig. 2.3a, b, c).

The majority will be inthe 4-cell stage but willrange between the 8-celland 16-cell stage. At thistime it is still possible to

distinguish betweenfertilised and non-fertilised eggs due to thecleavage pattern on theoutside edge of the egg.

2.3a

2.3b

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2.3c

2.3d

Fertilised eggshave an evenregular patternof cell division

whereasunfertilised eggsare uneven andnonsymmetrical(Fig. 2.3d).

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TIME AFTER SPAWNING2 HOURS

The egg is well intoembryogenesis at this time.This figure shows a number ofstages of embryodevelopment. The latest stage

of development at this time isapproximately the 6th (64-cellstage) and 7th (128-cell stage)cleavage. By this time a layerof cells has formed at theperiphery of the egg, called

the blastoderm. Differentiationof cells to form various tissuesrequired for development ofthe nauplius has begun.

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TIME AFTER SPAWNING3-4 HOURS

The embryoscontinue toundergo rapid celldivision, thenumber ofcleavages isdifficult to estimateby this stage. It isincreasingly

difficult todistinguishbetween afertilised andunfertilised egg.The edges of theeggs have acorrugatedappearance and it

is difficult tovisualise individualcells.

Time 4 hoursTime 3 hours

There appears to bevery little difference in

external appearance of

the embryo even though

cell division continues at

a rapid rate. At this time

a secondary envelope,

within the external

hatching envelope, canbe seen. An invagination

can be seen on the

surface of the egg that is

due a process called

gastrulation. It involves a

dynamic morphological

change from amonolayered to a

multilayered embryo,

accompanied by tissue

differentiation.
http://www.aims.gov.au/pages/research/mdef/mdef-03a-4.htmlhttp://www.aims.gov.au/pages/research/mdef/mdef-00.html

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Time 5 hours

Time 6 hours

A wide range of

embryonic

development may

be observed. Limb

buds may be

distinguishablewhich will form the

major appendages

of the nauplii.

Depending on the

orientation of the

individual egg the

limb buds may or

may not be visible.

Further

embryonic

development

has occurred.

Some embryoswill appear

similar to those

at 5 hours after

spawning.

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The appendages

are more defined

as the naupliar

body develops

into a more

distinguishableform. Depending

on orientation, it

may be possible

to distinguish the

formation of

setae on the tips

of the developing

appendages.

Major

developmental

changes are

distinguishable.

The naupliar

body hasthickened and

further

development,

including setae,

may be

observed.

Time 8 hours

Time 7 hours

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Time 9 hours : Embryos progressively

develop with little change in

observable form

Time 10 hours :

Further differentiation

of appendages are

observable on the

naupliar body.

Depending on

orientation, the pairs

of major naupliar

appendages are

clearly

distinguishable.

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Time 11 hours : The

naupliar form is

recognisable. A single

reddish pigmented

spot can be seenwhich is the naupliar

eye. Appendages are

well developed.

Time 12 hours : The nauplii begin to hatch, typically around

12.5 hours after spawning. In this figure the bottom of thehatching envelop has been broken and the nauplii tears their

way out using their appendages. Commercial hatcheries have

reported that occasionally vigorous active nauplii can be seen

within the hatching envelope but are unable to hatch and die

within the shell. The reason for this is unknown but may be due

to poor quality and weak nauplii that are unable to tear open

the hatching envelope.

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TIME AFTER SPAWNING13 HOURS

Time 13 hours : Not all eggs will have hatched by this time. Hatching does not occur synchronouslyand its timing is temperature dependent. However, if the eggs do not hatch out within a few hours ofeach other then the nauplii will be of questionable quality. In addition, if hatching has not occurred by15 hours post-spawning the nauplii may be of inferior quality. This figure (2.15b) shows the 1stnaupliar stage with appendages fully extended.

2.15a 2.15b

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2. Eyestalk hormones

It is well-known that eyestalk ablation inducesovarian development and oviposition. This is

because the source (X-organ-sinus glandcomplex) of the vitellogenesis- inhibitinghormone (VIH) is removed by the ablation.

VIH has been purified as a peptide in theAmerican lobster, Homarus americanusand inthe isopod,Armadillidium vulgare.

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The regulatory mechanism of VIH ispartially understood.

Previous study that purified eyestalkpeptides, including VIH, inhibited protein-synthesis activity of ovaries, VIH may acton the vitellogenin synthesis sites directly.

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3. Ecdysteroids

Ecdysteroids are knownas a molting hormone incrustaceans and insects.

In crustaceans, the Y-

organ produces andsecretes ecdysone and 3-dehydroecdysone, andthey are converted to 20-

hydroxyecdysone, thebiologically activeecdysteroid.

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4. Vertebrate-type steroidhormones

Several vertebrate-type steroidhormones such as estradiol-17 and progesterone havebeen identified in crustaceans.

The administration of thesteroids has been attempted,but the results are varied andsometimes inconsistent.

Previous studies suggestingthat vertebrate-type steroidhormones are not involved inovarian development.

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Male reproduction1. Spermatogenesis

In crustaceans, as in vertebrates,spermatozoa are produced throughspermatogenesis.

Spermatogonia start meiotic division afterproliferation and become spermatocytes,and spermatocytes differentiate into

spermatozoa through spermatids.

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Eyestalk ablation has been used to maturefemale shrimp in captivity in conjunction withmanagement of water temperature,

photoperiod, light intensity, density, sex ratio,and nutrition.

The recent research has focused mostly onorgans (brain, thoracic ganglion and ovary) and

their functions which are closely related to therelease of vitellogenesis-stimulating hormonesand ovarian hormone.

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The crustacean endocrine system consists of classical ephitelial-type endocrine glands, and

endocrine structures of neural origin, the neurosecretory cells,and neurohemal organs.

This neuroendocrine component is of majorsignificance with respect to both the number ofhormones (neurohormones) and their broadarray of roles.

There are two types of crustacean reproductiveneurohormones, gonad inhibiting hormone (GIH), and

gonad stimulating hormone (GSH).

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The decapod sinus gland is the source of agonad-inhibiting hormone (GIH); eyestalkremoval results in precocious gonadaldevelopment, this inhibitory hormone is also

found in male prawn.A second decapod reproductive neurohormone,

found in the brain and thoracic ganglia, is thegonad-stimulating hormone (GSH).

GSH is present in the brain and thoracic gangliaof females with maturing ovaries, but absent (orin low quantity) in the brain and thoracic gangliaof immature females.

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This suggest that thoracic ganglia from maturing

female shrimp could be used to induce ovarianmaturation. GIH has a molecular weight of 9135 Da, and its

structurally related to the crustaceanhyperglycemic hormone (CHH) and the molt-

inhibiting hormone (MIH). The three forming a family of neuropeptides

unique to crustaceans. GSH has a molecularweight of 1000-2000 Da. GSH activity is present

in the same fraction as CHH and raised thepossibility that CHH may have, in addition to itshyperglycemic activity, a stimulatory action onthe reproductive system.

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Male crustaceans have a pair of androgenicglands, and one gland is attached to eachsperm duct.

The androgenic gland hormone controls: differentiation of the male reproductive system,

functioning and the development of the malesecondary sexual characteristics.

In females GSH and GIH act directly on theovaries.

But in males, these hormone appear to exerttheir effect on the testes only undirectly bydirectly affecting the androgenic glands.

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In penaeid shrimp, reproductive maturationinvolves two main processes,

Vitellogenesis, and

final maturation

germinal vesicle breakdown (GVBD)

ovulation in oocytes.

Vitellogenesis occurs in hardshelled shrimp atthe intermolt stage C4 , shortly after moltingand continues until immediately beforefinal

maturation. Oil globule stage is an initial stage of primary

vitellogenesis.

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Follicle cells on the oil globule stage oocytes expandrapidly and reach maximum size during oogenesis.

The follicle cells are possibly responsible for ovarianvitellogenin (Vg) synthesis in the kuruma prawn Penaeusjaponicus.

Vg appears to be secreted only from enlarged follicle

cells surrounding oil globule stage oocytes and not fromshrunken follicle cells related to yolk granule stage.

Furthermore, the surface of the shrunken follicle cellswas relatively smooth compared to the irregular surfaceof the oocyte, which had numerous well-developed

microvilli. This suggests that follicle cells on yolk granule stage

oocytes are not related to the route of egg yolk proteinuptake in this prawn.

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Final maturation of ovarian oocytesimmediately precedes spawning, which in

P.japonicusoccurs after dark. Two phases are, involved: the appearance

of ripe ova, and germinal vesiclebreakdown (GVBD) in preparation forfertilization after spawning.

Ovulation occurs when nuclei, shrunkenduring the late prematuration phase, have

migrated to the peripheral cytoplasm ofthe oocytes.

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In the late phase of the maturation cycle, meioticmetaphase is arrested and remains visible just beneaththe cytoplasmic membrane of the ovarian oocyte,indicating that GVBD is completed after ovulation.

Rapid shrinkage of the nucleus can be observed in the

ovary after sunset and meiotic metaphase followsbetween 21.00 and 03.00 in P.japonicus, implying thatGVBD is initiated in the evening and completed duringthe night, over a period of several hours.

Immediately after release from the female gonopore, the

mature eggs, still in metaphase, are fertilized by spermreleased into the seawater from the spermatophore heldin the thelycum.

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It is well known that in penaeid shrimp manysmall clusters of immature eggs often becomeattached to the walls of the spawning tank,together with separated fertilized eggs,immediately after spawning.

Histological studies of the maturation cycle

revealed eggs surrounded by many immatureoocytes.

These immature oocytes, which are at early andlate perinucleolus stages, are interconnected,

forming a coating around the egg.

The coating of immature eggs is not yet formedin the early phase of prematuration in the ovary.

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This suggests that immature oocytes may increaserapidly after the late phase of prematuration and thenquickly surround the mature oocytes immediatley priorto spawning.

Probably, the coating of immature eggs lubricates the

mature oocyte during release from the ovary. Once begun, spawning is continuous, females releasing

batches of eggs from the ripe ovary and sperm from thespermatophore into the seawater, where fertilizationtakes place.

Therefore, female shrimp have to repeat the process ofmolting, mating, and sexual maturation in order toachieve several spawnings during their life.

Th i li h

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Thoracic ganglion hormoneeffects on vitellogenesis

Smaller size P. vannameidid not initiatevitellogenesis even after eyestalk ablation.

Thus, vitellogenesis is controlled by twofactors, one which inhibits and the otherwhich stimulates.

In previtellogenic (immature) females, thevitellogenesis-stimulating principle isabsent or not yet functioning.

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The accumulation of yolk granules inoocytes was stimulated by repeatedimplantation of pieces of thoracic ganglionin the immature female crab Potamon

dehaani.Vitellogenesis in shrimp can be stimulated

by implantation of pieces of thoracicganglion tissue prepared from the femalelobster, Homarus americanus, withvitellogenic ovaries.

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This result indicated that vitellogenesiscould be stimulated by a vitellogenesis-stimulating hormone (VSH), also known as

the gonad-stimulating hormone. Injection of thoracic ganglion extract

prepared from vitellogenic females is

effective in increasing serum Vg in thekuruma prawn, P.japonicus.

E t lk h di tl

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Eyestalk hormone directlyinhibits vitellogenin synthesis

Eyestalk ablation stimulates ovarianmaturation in penaeid shrimp.

This treatment reduces the production of

a vitellogenesis-inhibiting hormone (VIH),also known as the gonad-inhibitinghormone (GIH), and thus permits

maturation of the ovaries in femalepenaeid shrimp.

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Reproductive maturation in penaeid shrimp isregulated by a VIH from the X-organ-sinus glandcomplex in the eyestalks.

In this complex, the acidophilic sinus gland isconnected with axons from the neurosecretorycell of the X-organ, which is located in themedulla terminalis of penaeid shrimp and theother crustaceans.

In general, the sinus gland is easily recognizableon the neurilemma, surrounding the opticganglia.

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In the kuruma prawn, P.japonicus,Vg

synthesis in previtellogenic ovary can beinitiated when the ovary is not affecteddirectly by VIH from eyestalks.

These results suggest that Vg synthesis inthe ovary may be inhibited directly by VIHsecreted by the X-organ-sinus glandcomplex of eyestalks.

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Unilateral eyestalk ablation is not effective inincreasing serum Vg in vitellogenic femalekuruma prawn.

This suggests that the VIH level decreasesquickly immediately before the initiation ofvitellogenesis and stays at a low level until aftervitellogenesis is completed.

Therefore, eyestalk ablation may no longer beeffective in regulating the production of VIHafter vitellogenesis has been initiated.

I d i f i ll i h i i

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Induction of vitellogenin synthesis inovary by estradiol-17

Vg synthesis in incubated ovarian piecescan be stimulated by estradiol-17in thekuruma prawn, P.japonicus.

In addition, oil globule stage oocytes werefound in incubated previtellogenic ovarianpieces after treatment with estradiol-17in vitro.

Evidence has been presented to show that17-hydroxy-progesterone is effective inincreasing serum Vg in the kuruma prawn.

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The hormone estradiol-17 or 17-hydroxy-progesterone is generally distributed in theovary of crustaceans.

In crustaceans, 17-hydroxy-progesterone can

be converted intro the estradiol-17. 17-hydroxy-progesterone may be worked into

the Vg synthesis mechanism in kuruma prawn asa precusor of estradiol-17.

Probably, that estradiol-17 secreted fromovarian follicle cells, induces Vg synthesis in theovary as a Vg-stimulating ovarian hormone inpenaeid shrimp.

P th f t id h i

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Pathway of steroid hormone indecapod crustacean animals

Cholesterol

Pregnenolone

Progesterone

17-hydroxy-progesteroneAndrost-4-ene-3, 17-dione

Testosterone

Estradiol-17

Serotonin stimulates the release of

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Serotonin stimulates the release ofvitellogenesis-stimulating hormone

(VSH)

It is well known that biogenic amines releasepeptide neurohormones from neuroendocrinestructures in several crustaceans.

Serotonin (5-hydroxytryptamine) stimulatesgonadal maturation in male and female sandfiddler crabs, and red swamp crayfish.

This action of serotonin is indirect, serotonin

apparently stimulating release of VSH that ispresent in the brain or thoracic ganglia.

Serotonin is found widely distributed throughoutthe nervous system in decapod crustaceans.

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Facts suggest that the release of VSHfrom thoracic ganglion may be induced byserotonin present in the central nervous

system in crustaceans. Therefore, neurohormonal serotonin is

nominated as a vitellogenesis-stimulatinghormone-releasing hormone (VSH-RH)

responsible for ovarian Vg systhesis inpenaeid shrimp.

Prostaglandine stimulates final

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Prostaglandine stimulates finalmaturation

Final maturation may be induced bycertain nutrients obtainable only from a

combination of foods.

A formulated, pelleted diet rich in vitaminE and containing fish oil, induced and

accelerated final maturation in P.monodon.

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Vitamin C-rich, pelleted diet containing fish oilalso induced and accelerated final maturation.

A high level of vitamin E or C is thought to

protect the eicosapentaenoic acid (EPA), aprecusor of prostaglandin which is essential forreproduction in crustacean or otherinvertebrates, from oxidation during digestion or

peroxidation in the shrimp body.

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EPA is one of the essential fatty acids incrustaceans and so spawners must take this upas a precusor of the prostaglandin required forfinal maturation.

Probably, under high levels of vitamin E or C,active EPA could be converted to prostaglandin.Secretion of prostaglandin, which is essential forthe induction of final maturation in crustaceans,

may therefore be stimulated by matingbehaviour and subsequent spermatophoretransfer in open-thelycum species.

Reproductive maturation is affected by

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Reproductive maturation is affected bywater temperature and photoperiod

It is well known that ovarian maturation is affected bywater temperature and photoperiod in female P.japonicus, a photoperiod of 14 to 16 h (light) andtemperature of 24 to 26 oC stimulate ovarian maturation.

Even in eyestalk ablated P. japonicus, full sexualmaturation did not occur below 17 oC. Hormonal controlof reproductive maturation is affected by the watertemperature and photoperiod in penaeid shrimp.

The release of serotonin (VSH-RH) from central nervous

system, VSH from the thoracic ganglia, VIH from the X-organ-sinus gland complex and estradiol-17B from theovary may be be induced directly or indirectly by thewater temperature and light stimuli via the antennuleand eye, respectively, in female penaeid shrimp.

Perspectives on hormonal

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Perspectives on hormonalmanipulation

At present, only eyestalk ablation for the induction of female maturation ispractically used in shrimp farming. This method is not repeatable andsometimes causes high mortality.

If the mechanism of VIH function is fully understood, some methods forblocking VIH activity, such as anti-VIH antibody and antagonists, can beanticipated.

Methyl farnesoate may be used to stimulate female maturation in the nearfuture, however, further studies are necessary. In addition, additionalstimulating factors may be newly characterized in the future.

To characterize additional hormones, a suitable bioassay is required.Recently, vitellogenin was purified and its cDNA was cloned. Subsequently,enzyme-immunoassay of vitellogenin and quantitative reverse-transcription(RT)-polymerase chain reaction (PCR) of vitellogenin have been developed.These assay methods are very sensitive, and will be used for bioassay of

the characterization process. After characterization, the factors may be synthesized by molecular

biological techniques as recombinant molt-inhibiting hormone andrecombinant androgenic gland hormone.

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Conclusion

To develop hormonal manipulationtechniques, much needs to be done. Onlya few hormones have been discovered so

far. Many hormones are still unidentified.

Characterizing new hormones should be

given priority.

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Control of reproductive maturation is a majorproblem in the development of commercialaquaculture programs for penaeid shrimp.

Controlling reproductive maturation in captivitycould help to provide a reliable year-roundsupply of juveniles, serve in developing selectivebreeding programs, and be generally useful for

obtaining disease-free spawners.

Th k

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