Indian Journal of Experimental Biology Vol. 43, March 2005, pp. 209-223

Review Article

Current progress in shrimp endocrinology-A review

ADDiwan Indian Council of Agricultural Research (ICAR), Krishi Anusandhan Bhavan-II, Pusa, New Delhi 110 012, India

One problem in aquaculture is obtaining brood because many commercially important species are incapable of spontaneous maturation under artificial conditions. Commercial shrimp hatcheries commonly use eyestalk ablation to stimulate gonadal maturation in shrimps. Research has been conducted on the inhibition of reproductive maturation by hormones originating in the eyestalk glands and on other endocrine sources (e.g.,brain, thoracic ganglion, ovary, mandibular organ, androgenic gland and Y-organs) to determine their roles. Alternate techniques for acceleration of gonad maturation through the use of synthetic hormones or neurotransmitters may benefit aquaculture. Neurohormones and neuroregulators have been shown to accelerate gonadal maturation but an effective delivery technique must be developed for use in a large­scale aquaculture operation.

Keywords: Shrimp, Hormones, Neurotransmitters, X-organ, Y-organ, Sinus gland

Shrimps are common to worldwide capture fisheries and to aquaculture. Demand for shrimp in the international markets has increased greatly over time. The high demand has led to over-exploitation of the natural shrimp stocks in many parts of the world. A decline in the capture fisheries led to increased aquaculture production as an alternative source for sustaining and expanding the seafood industry. Due to overexploitation of the wild stock of marine shrimp and the decrease in total natural productivity, shrimp farming activity has developed rapidly in two decades, with an the annual global production of 700,000 mtl. Shrimp culture has many challenges. One major problem is the lack of genetic selection of brood. Zeleny2 and Panouse3 showed the effect of eyestalk ablation on moult and ovary development in some crustaceans. Reviews by Keller4 on crustacean neuropeptides, Fingerman5

.8 and Waddy and Aiken9

on crustacean endocrine mechanisms, and Huberman 10 on shrimp endocrinology have been published. The objective of this review is to

. summarise endocrinological developments in shrimps which may benefit aquaculture.

Hormonal regulation of growth Growth pattern in shrimp

All crustaceans have an exoskeleton and ecdysis is necessary for growth. During early stages of the life

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cycle, shrimps undergo several ecdysis before they attain juvenile size. As size increases, frequency of ecdysis is reduced. Female shrimp moult as a requirement for matingll . Thus, the control of growth is dependent on the control of moulting. The moulting process in crustaceans has been demonstratedI2-ls; the process is regulated through ecdysteroid honnones from the Y -organs during proecdysis and moult­inhibiting honnone (MIH) produced by a group of neurosecretory cells (NSC) in the X-or~an sinus gland complex of the eyestalks of shrimpI3.16- 3.

Role of Y -organs Gabe24 observed a Y -organ in 58 species of

malacostracan crustaceans. Since then, Y -organs have been identified and described in numerous species of crustaceans including shrimps2s-28. The Y -organ is generally located in the musculature of the anterior branchial chamber and appears as a compact mass in crabs or a less compact mass in crayfish and lobsters 19. The size of the Y-organ is found to be 60-300 11m in isopods and amphipods and 1-3 mm in decapods. Histological studies revealed that the cells of the Y -organ show secretory activity in relation to the moult cycle28.

The endocrine function of the Y -organ in control of moulting was suggested by Gabe24-29and subsequently this was demonstrated through surgical experiments by Echalie~0.31 in Carcinus maenas, and by Passano and Jyssum32, Maissiae3

-34 and Blanchees in several

other species of decapods, isopods and amphipods respectively. The first evidence of Y -organs

210 INDIAN] EXP BIOL, MARCH 2005

controlling the moulting process through production of ecdysteroid hormones was demonstrated by injecting exogenous ecdysteroid (20-hydro­xyecdysone) 17. In vitro culture of the Y -organ provided direct evidence that it produces ecdysteroids in Procambarus clarkU36 and Penaeus vannamei37

The major secretory product of the Y -organs is an ecdysteroid which is synthesized from cholesterol19

and converted to 20-hydroxyecdysone by several tissues in crustacea. Kaeuser et al.38 reported lack of evidence on identification of the target organs of ecdysteroids and the hormone receptor complex, which results in increased RNA synthesis through certain genes. However, recently Durica et al. 39 while studying the characterization of crab (Uca pugilator) homologs of ecdysteroid (ECR) and retenoid-X (RXR) receptors during limb regeneration and oocyte maturation found that ovarian tissue is a potential target for hormonal control in crustacea. High levels of ecdysteroid in blood and tissue during premoult and reduced levels of same just prior to ecdysis have been reported by Snyder and Chang40 while working on the American lobster, Homarus americanus. Blais et al. 38 reported that in P. vannamei at late premoult ecdysteroid concentrations increase in the haemolymph following activation of the Y-organ while the concentration remains low during postmoult and intermoult. The rate of ecdysteroid synthesis is generally controlled by MIH from the X-organ sinus gland complex. Experiments with eyestalk ablated shrimp and with Y -organs incubated in vitro in presence of sinus gland extract indicated that the X -organ regulates the synthesis of ecdysone in the Y-organ. It has been reported Palaemon serratus and Sicyonia ingentis have low levels of ecdysteroids in eggs at extrusion and the levels increase towards hatching41 . Alpheus heterochaelis exposed to a micromolar concentration of 20-0H-ecdysone for 5 days showed that the winter moult cycle was shortened by 18 days or by 65%42. Vijayan et al. 28

demonstrated that the size and tinctorial affinity of Y -organ cells in F. indicus change during the moult cycle. Further, they have shown that the Y -organ removal in this shrimp inhibits the onset of premoult. Chan43 cloned a eDNA from the shrimp Metapenaeus ensis which encodes a nuclear receptor, homologous to the insect ecdysone-inducible E 75 gene. The amino acid sequence has all five domains of a nuclear receptor and it is expressed in the epidermis, eyestalk and nervous tissue of premoult shrimp.

Role of the eyestalk X-organ sinus gland complex The eyestalks and their involvement in moult

control were initially discovered by Zelen/. Hanstrom44 provided a detailed structural description of the X-organ. With the advancement of science in neuroendocrine aspects of crustaceans, many workers have described the X-org~n-sinus gland complex that contains neurosecretory cells, in details8,12,17.45,46, Several types of neurosecretory cells in the X -organs containing neurosecretory granules in their cell bodies, axons and terminals have been mentioned by a number of workers using light and electronmicroscopl7,48,17.49.46,50. The distribution of neurosecretory cells in different parts of the optic ganglia has also been observed and depending on the location, the terminology adopted was the medulla terminalis ganglionic X-organ (MTGX), medulla externa ganglionic X-organ (MEGX) and medulla interna ganglionic X-organ (MIGX). The sinus gland is actually formed of axon endings of neurosecretory cells and glial cells lining haemolymph sinuses51 . Exocytosis appears to be the dominant mechanism for the release of granules into the haemolymph52,17,53,51. In recent years, Huberman 10 reviewed the chemical and functional aspects of various ' crustacean hormones and emphasized their importance in shrimp aquaculture.

Functional aspects The role of the optic ganglia of the eyestalk in the

control of various physiological processes including growth and moulting in crustaceans has been described54,55,46.4,23. It was demonstrated that eyestalk ablation in decapods leads to precocious moult, and moulting occurs several times following ablation, particularly in young animals. Reimplantation or injection of eyes talk extract in several crustaceans has been shown to reverse or inhibit the process of moulting13. The impact of eyes talk removal on acceleration of the moulting/growth process is due to loss of MIH, which is predominantly present in the optic ganglia, particularly in younger stages of the life cycles. The presence of MIH inhibits the moult promoting effect of Y -organs 12,52,23.

Once the inhibitory effect of the eyestalk (MIH) on the moulting process was confirmed in several decapods, research was conducted to determine the chemical nature and other physiological functions of this hormone. MIH was then isolated and its peptidic nature established in several brachyuran and macruran

DIW AN: CURRENT PROGRESS IN SHRIMP ENDOCRINOLOGY 211

species56. In Carcinus maenas MIH was characterised57, purified58 and also sequenced59. MIH has also been sequenced in Homarus americanus60

and in Callinectes sapidui l• Aguilar et al. 62 isolated

MIH from the eyestalk of the crayfish Procambarus bouvieri and compared its sequence with four other known peptides from H. americanus, C. maenas, C.sapidus and P. vannamei. The lengths of these peptides vary between 72 and 78 residues and their molecular masses between 8 and 9 KDa. The first cloning of cDNA encoding a MIH like neuropeptide from P. vannamei was accomplished by Sun63. Further, it has been reported that MIH-like mRNA is found exclusively in the medulla terminalis ganglionic X-organ MTGXO of the eyestalk and in the brain the MIH-like gene transcript was detected in the neurosecretory cells64

• This could mean that a MIH-like neuropeptide could have specific functions in the nervous system in addition to its hormonal function. This neuropeptide has similarities in its amino acid sequence with other neuropeptides synthesized in the X-organ, i.e., the crustacean hyperglycemic hormone (CHH) and the vitellogenesis inhibiting hormone (VIH). A smaller neuropeptide (53-55 amino acids) with MIH activity was isolated from P. bouvien-65. This peptide was later sequenced and bioassayed62. Yang et al. 66 isolated and sequenced a peptide with MIH activity from sinus glands of the shrimp Marsupenaeus japonicus. In vitro experiments demonstrated that this peptide inhibited the synthesis of ecdysone in Y -organs of the crayfish P. clarkii. Four peptides with both MIH and CHH activity were isolated from the sinus gland of P. vannamei by Sefiani et al. 67. It was hypothesized that different types of peptides that are detected in sinus glands display different activities, including the site responsible for MIH activity; but this hypothesis must be confirmed experimentally 10. Mattson and Spaziani68 have shown that MIH is able to inhibit ecdysteroid secretion by Y -organs in vitro in a dose dependent manner. The injection of serotonin (5-0H­tryptamine) lowers the haemolymph ecdysteroid levels and these effects are revoked by eyes talk ablation. In this experimental evidence Spaziani et al. 69 proposed a cycle of MIH regulation sensory input via 5-HT that would release MIH and inhibit ecdysteroid synthesis and release by the Y -organs, and that MIH release would be subjected to a negative feedback by elevated ecdysteroid titers. Increase in the mitotic activity in the Y -organ cells after eyestalk

ablation in Pachygrapsus mannoratus70 clearly indicated the possibility of enhanced steroidogenic activity. Mattson and Spaziani68

•71 found that sinus

gland extracts suppress Y -organ steroidogenesis. A conceptual model defining the mode of action of sinus gland extracts on steroidogenesis by the Y -organ was proposed by Mattson and Spaziani72 and the same was later reviewed by many other workers73.19. According to this model MIH increases intracellular cAMP, which activates a protein kinase that leads to inhibition of protein synthesis and steroidogenesis. Gu et al. 74 while working on MIH in the shrimp Metapenaeus ensis showed that specific cells in three different clusters of the X-organ, the sinus gland and axonal tract of the eyestalk contain MIH. To test the moult inhibiting activity of MIH, shrimp of intermoult stage were injected with r-MIH (recombinant protein) and there was a significant increase in the moult cycle duration for the shrimp74.

Mandibular organs The histology and ultrastructure of mandibular

organs (MO) were described in a number of species of crustaceans25.9. The main characteristics of cells of MO are the presence of a centrally located nucleus with abundant cytoplasm, a large number of mitochondria and smooth endoplasmic reticulum. Three types of cells have been identified in MO of H. americanus75 . The detailed structure of MO of F. indicus has been described by Vijayan and Diwan76.

There is sufficient evidence that terpenoids such as methyl farnesoate (MF) and farnesoic acid (FA), both secreted by the MO, have stimulatory effects on Y-organs and may influence reproductive activity. Methyl famesoate has been detected in the MOs of Libinia emarginata77-79 and several other crustacean species8o. The possible implications of MO and MF in the control of moult and growth have also been suggestedI5-23. The nature of the MO inhibiting factor has been explored81 -83. Eyestalk ablation causes hypertrophy of the gland and a higher density of smooth endoplasmic reticulum84,85. Tsukimura and Borst86 observed increased levels of MF following eyes talk ablation in H. americanus. Ablation of MO is difficult but transplantation of the MO reduces the length of the moult cycle in Penaeus setiferui7. While studying regulation of the crustacean MO Borst et al. 88 described the role of MO in controlling reproduction and development. Further, they

212 INDIAN J EXP BIOL. MARCH 2005

mentioned89 that methyl farnesoate (MF) production by MO is negatively regulated by neuropeptides from the sinus gland in the eyes talk. , Two neuropeptides (MO-IH-I and 2) have been isolated from the SG of the crab, Cancer pagurus that inhibit MF synthesis by MO of female crabs in vitro. It was revealed that the regulation of MO is complex and may involve several SG compounds89

• Further studies are needed to determine the exact role of MO in relation to growth and reproduction.

Hormonal control of reproduction Reproductive processes of crustaceans are

controlled by hormones. Hormones that influence gonadal development and spawning are produced in the brain and thoracic ganglia. The ovaries also produce hormones. Y-organs, MO and androgenic glands also produce hormones which promote gonadal development directly or indirectly. In recent years, the roles of neuroregulators have been appraised in enhancing maturation and the spawning process23,90

Reproduction pattern in shrimps Shrimps are bisexual and development of gonads is

a slow process, which takes place along with the moulting process during a large part of the adult life. During reproductive cycles the gonads undergo a sequence of major morphological and physiological transformations which require a large amount of energy. In females, development of the oocyte starts with the process of oogenesis and in males with spermatogonial cells. The various stages in the development of the oocytes as well as spermatozoa h b d 'b d & I' 91-93,46.50 ave een escn e lor severa speCIes Determination of sexual maturity of live shrimps in early stages is difficult. However, as a shrimp advances towards the ripening phases of gonadal development, identitification of the maturation phase is possible. For example, the developing ovary and developed thalycum of female shrimp for storing spermatozoa can be seen through the cephalothoracic region. In males, the development of white glitterhening spermatheca or sperm boxes are visible on the bases of the fifth per~iopods. Many have developed immunochemical techniques for determing the scale of maturity. Derelle et al. 94 developed an ELISA titration assay for measuring vitellogenin synthesis in the freshwater prawn Macrobrachium rosenberg ii, using a monoclonal antibody. Similar studies have been done with the lobster

H. americanus95 and crab Callinectes sapidus96• The

most accurate method now available is based on a micro-anatomical survey of the reproductive organs using histological procedures.

Many shrimps are seasonal breeders and in tropical countries generally the breeding season lasts from October to February. Environmental factors particularly temperature, photoperiod and salinity play an important role in enhancing breeding and spawning activitl7

. All the marine shrimps breed and spawn in deep sea waters. The larvae migrate to shallow coastal waters for their growth to attain the adult size. Shrimps undergo breeding and spawning several times and produce millions of larvae during their lifetime98

• Currently, technologies are available for domestication of shrimps in hatcheries and laboratory conditions so that induction of breeding and spawning at a desired time is possible. The role of the central nervous system and its coordination with neuroendocrine centres, along with environmental cues, are now well understood so far as regulating the reproductive organs is concerned7

.

Regulation of reproduction The reproductive activity of crustaceans is partially

under the control of neuroendocrine factors. Panouse3

showed the presence of gonad inhibiting hormone(GIH) in the eyes talk of a female prawn, P. serratus. The increase in ovarian growth after eyes talk removal was primarily due to removal of the inhibitory effect on vitellogenesis46

. Otsu99 showed the existence of the gonad stimulating hormone (GSH) in the brain and thoracic ganglion. Based on these findings it was deduced that gonadal maturation in shrimp was regulated by two antagonistic neurohormones, GIH and GSH. This model concept has been well reviewed I 00,87,97,46,7,23,8. Khalaila et al. 101

while studying the role of eyes talk-borne hormones on spermatogenic activity in the testis and androgenic gland of the crayfish, Cherax quadricarinatus, found that the sinus gland directly controls the activity of the androgenic gland which suggests an endocrine axis­like relationship between the sinus gland and AG and the male reproductive system in decapod crustaceans.

Eyestalk X-organ sinus gland complex The X-organ-sinus gland complex, which is the

prime neuroendocrine centre in the eyestalks of shrimps, produces hormonal factors (neuropeptides / neurohormones) that control the physiological process of gonads7

• GIH was localized by specific antibodies

DIW AN: CURRENT PROGRESS IN SHRIMP ENDOCRINOLOGY 213

in the X-organ SG complex of the lobster H.americanus,102 prominently at the metanauplius stagelO3 . By using the technique of nonradioactively labeled cDNA probes it was possible to detect neuropeptides in X-organ SG complex of this lobster and its larvae, which has the property of GIH I04

•105.

This study revealed that GIH is also present in males. In the prawn P. varians and the crayfish P. bouvieri neuropeptides extracted from the eyes talks have a negative effect on the growth of vitellogenic oocytes l06.107 Now the eyestalk ablation technique is being practiced commercially for inducement of

. d . f h ' 10846109 Th maturatlon an spawnlllg 0 s nmps ". e presence of GIH in embryos and larvae \05 may be an indication of an inhibitory role before adolescence. Chang et al. I \0 isolated and purified the peptide from sinus glands of shrimp S. ingentis which is responsible for inhibition of ovarian development and spawning. Quackenbush and Keeley III have also isolated a factor from eyestalks of the shrimp P. setiferus, which has inhibitory effect on vitellin synthesis. Similar, studies have been carried out by Quackenbushl12 in Penaeus vannamei. Huberman lo

recently reviewed the biomolecular aspects regarding the work carried out on GIH in crustaceans in general and shrimps in particular. Edomi et at. 113 while studying GIH of the Norway lobster (Nephros norvegicus) reported that the GlH is actively involved in gonad maturation process and plays a more compex role in control of reproduction and mOUlting. With a combination of the reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends approaches, they determined the cON A sequence of N. no rvegicus prepro-GIH. The possibility of the involvement of neurohumoral agents in relation to control of reproduction was debated for quite some time, but in recent years experimental evidences are available that 5-HT that is present in the nervous system including the X-organ sinus gland complex has a stimulatory effect on reproductive activity.

Cerebral and thoracic ganglia A considerable amount of research has been

conducted in crustaceans that indicates the presence of gonad stimulating factors in cerebral and thoracic ganglia. Implantation experiments carried out by Otsu99 and Gomez and Nayar l14 indicated that the thoracic and supraoesophageal ganglia can induce' ovarian growth. Injections of aqueous extracts

prepared from thoracic ganglia and brain as well as in vitro incubation experiments indicated vitellogenesis .. 4511511646117.50 can be stlmulated III several crustaceans . . . .

Induction of ovarian maturation in P. vannamei has been accomplished by injecting lobster brain extract I 18. By injecting thoracic ganglion extract of P. semisulcatus, Shoji50 maturated and spawned P. monodon. Yano1l9.120 modeled the mode of action of hormones related to maturation. According to him GnRH is produced in the brain and in response to this the thoracic ganglion produces gonadotropins (GnH) and under the influence of GnH maturation is effected. However, the molecular structure of GSH is yet to be elucidated.

Role of endocrine organs Mandibular organ(MO)

The possible role of the MO in reproductive activity of crustaceans was reviewed by Laufer et al. 121 and Waddy and Aiken9. Terpenoids like methyl farnesoate (MF) and farnesoic acid (FA), both secreted by MO, influence reproduction of both in male and female crustaceans. Liu and Lauferl22 found that the activity of MO is regulated by sinus gland neuropeptides. It was mentioned that MO-inhibiting hormonal compounds have the similar type molecular masses and amino acid composition as noticed with other sinus gland neuropeptides. Chang et at. 110 by using a radiolabeled photoaffinity analog of MF found in S. ingentis the presence of MF binding proteins in the ovaries, testis and accessory glands in addition to the haemolymph. The culturing in vitro of ovarian tissue of the shrimp, P. vannamei in presence of MF resulted in a significant increase in the size of the oocytes. This can be interpreted as the involvement of MF in the early events related to secondary vitellogenesis. MF was reported to increase fecundity . I d h ' P ·123 124 G d III cu ture s nmp . vannamel . . unawar ene et al. 74 while working on functions and cellular localization of farnesoic acid O-methyl transferase (FAMeT) in the shrimp, Metapenaeus ensis reported that FAMeT directly or indirectly through methyl farnesoate (MF) modulates the reproduction and growth of shrimp by interacting with the eyes talk neuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland.

Androgenic gland In decapod crustaceans, the androgenic glands are

generally found associated with the terminal portion

214 INDIAN 1 EXP BIOL, MARCH 2005

of the male gamete duct. Earlier investigators have noted the differences and / or uniformities in the function of the androgenic gland among various group of crustaceans in general and hermaphroditic and non­hermaphroditic decapods in particular. Mohamed and Diwan l25 described the structure of the androgenic gland in F. indicus and also demonstrated the impact of bilateral andrectomy on sex reversal of the shrimp. It was reported that andrectomized male shrimps have lost their secondary sexual characters and exhibited absence of sperm in the lumen of their testicular acini. Decapod androgenic glands are not necessary for completion of spermatogenesis and their absence results only in a reduction of spermatogenesis intensity 125. Mohamed and Diwanl25 reported that the lack of androgenic glands in F. indicus appeared to inhibit spermatogonial differentiation. Many studies have been devoted to determine the chemical nature

. of the androgenic hormone. Laufer and Landau 126 have mentioned that these glands are capable of producing several compounds including proteins and the terpenes, hexahydroxy farnesylacetone and farnesy lacetone, and the exact role of these compounds is not yet known. Charmantier et at. 22

reported that the androgenic gland hormone regulates the spermatogenic activity in the testis and is responsible for the development and maintenance of the secondary sexual characteristics in male crustaceans.

V-organs The role of the Y -organs in crustaceans in the

regulation of the moulting process and growth is well known30,127,32,128,33,34,27,23 H 't I . . owever, I s ro e m reproduction has been reported by Chang2o. As stated earlier, the Y-organ is the source of ecdysteroids and correlations between vitellogenesis and ecdysteroids levels in haemolymph have been reported in some

. 129,87 Th I f d 'd' hi' speCIes . e ro e 0 ec ysteroI s m t e regu attOn of vitellogenesis in female crustaceans is not known. Ecdysteroids may directly or indirectly participate in the regulation of spermatogenesis 130 and induction of gonadal growth in males 121 •

Ovaries The ovarian tissue in most crustaceans, particularly

in decapods produces vitellogenin-stimulating­ovarian-hormone (VSOH) and under the influence of this, the growth of oocytes takes place I31 ,132,10I,92,97. Besides VSOH, many ecdysteroids have been identified in the follicle cells and oocytes of the

ovarian tissue21 . Yanol20 described in detail the control of the process of vitellogenesis by the endocrine system in penaeid shrimp. He reported that estradiol-17~ is effective in increasing serum V g in the Kuruma prawn, M. japonicus. The hormone estradiol-17~ is generally distributed in the ovary of crustaceans and it was suggested that estradiol-17{3 secreted from ovarian follicle cells induces V g synthesis in the ovary as a V g-stimulating ovarian hormone in penaeid shrimpl20.

Chemistry of hormones

Ecdysteroids Ecdysteroid or ecdysone, a moulting hormone,

similar to the hormone found in insect prothoracic glands is produced by the Y -organs in crustaceans 133. It was also demonstrated that 3-dehydroecdysone is produced by the Y -organ in several crustaceans including the shrimp P. vannameP7. Besides, 3-dehydroecdysone, the Y -organ also produces 20-hydroxyecdysone, 25 deoxyecdysone l34 and several other ecdysteroids 135

• However, the biosynthetic pathway for the production of ecdysone is not known, Cholesterol is tr p ~recusor for ecdysone production, Cholesterol can not be synthesized by

t 136· b b' d f . crus aceans , It must e 0 tame rom dIetary sources. A number of studies indicated the involvement of ecdysteroids not only in the moulting process but also in the reproduction of crustaceans 137.

Neuropeptides Considerable research on chemical aspects of

crustacean neuropeptides has been conducted. Chang et at. 60 reported an amino acid sequence of lobster MIH. It has been observed that the amino acid sequence of lobster MIH was almost identical to that of crustacean hyperglycemic hormone A (CHHA) discovered by Tensen et at138

• Aguilar et at. 62 isolated MIH from crayfish and compared its sequence with other four known peptides of crustaceans. Their lengths vary between 72 and 78 residues and their molecular masses between 8 and 9 kDa. All have six cysteines that farm three disulfide bonds. Sun63 by using molecular techniques investigated the chemistry of MIH like neuropeptides of the shrimp P. vannamei. She reported that this MIH consists of a 72 residue mature peptide and a 30-residue region of a propeptide, By using similar techniques Aguilar et at. 139 isolated the MIH and found that it consists of a 72-residue peptide with amino and carboxyl termini

DIWAN: CURRENT PROGRESS IN SHRIMP ENDOCRINOLOGY 215

and six cysteines forming three disulfide bonds. Similar investigations have been made by Yang et al. 66 and it was reported that M. japonicus MIH consists of a 77-residue peptide with both free amino and carboxyl termini. Sefiani et al. 67 isolated an MIH­like peptide from P. vannamei and by mass spectrometry its molecular mass was estimated to be 8627 Da and consists of only 38 residues. Gu and Chan43 have isolated MIH from M. ensis and detected the amino acid sequence of 77 residues preceded by a signal peptide of 28 residues. Kawakami et al. 140

chemically synthesized a moult inhibiting hormone (pre-MIH) from the American crayfish, Procambarus clarkii, which consist of 75 amino acid residues. This product they found was almost similar to natural pre­MIH chemically.

Chang141 reported that activity of growth hormone peptide has not been reported in either CHH or MIH assays. Further it is described that the CHHlMIHlVIH peptide family appears to be a novel group of crustacean neuropeptides l41

• Much amount of work has been done on the expression of the CHH gene(s). Soyez et al. 142 isolated a 7500 Da peptide from the sinus gland of H. americanus and assayed its GIH activity in vitro in a shrimp by measurement of oocyte diameter. They 106 further investigated the structure of GIH and found that this peptide consists of 77 residues and its molecular weight · is 9135kDa. Dircksen et al. 143 while studying the crustacean hyperglycemic hormone (CHH) and CHH-precusor­related peptides from pericardial organ (PO) neurosecretory cells in the shore crab, Carcinus maenas, observed PO-CHH is a 73 amino acid peptide with a free C-terminus. PO-CHH and sinus gland CHH have been found to share an identical N-terminal sequence at positions 1-40 but the remaining sequence, positions 41-73 or 41-72, differs considerably. They have also reported that PO-CHH may have different precursors and CHH genes coding for precusor products are presumably modified at the post-transcriptional or post-translational level. It has been also mentioned that PO-CHH unlike SG-CHH, has neither a hyperglycemic effect nor it is active in inhibition of ecdysteroid production by crab Y-organs.

Katayama et al. 144 studied the structure of moult inhibiting hormone (MIH) from the Kuruma prawn, Marsupenaeus japonicus. It has been reported that the amino acid sequence of MIH is similar to that of crustacean hyperglycemic hormone (CHH). Further, it

was shown that the homology-modeh!d struCture of M. japonicus CHH was similar to the structure of MIH with the exception of the absence of the N-terminal a-helix and the C-terminal tail. This is the first report on the tertiary structure analysis of a crustacean neuropeptide. This kind of elucidation is expected to provide new insights not only on structure-activity relationship but on the molecular evolution of the CHH family peptides.

MF is a secretory product of the MO in crustaceans and is related to insect juvenile hormone a sesquiterpene that has a significant effect on growth

d d ··· L & I 78 79· I d an repro uctlOn III msects. aUier et a. . ISO ate MF from the haemolymph of crabs. Later a number of workers have isolated MF from a number of other crustaceans l41

• Wainwright et al. 82 and Liu et al. 83

have characterised mandibular organ inhibiting hormones (MOIH) from crabs and found that the hormones have the molecular masses of approximately 8400 Da and the amino acid composition of this neuropeptide is similar to those of MIH and CHH. There is another compound called farnesoic acid secreted by cultured MO from P. clarkU145 and Cancer magister l46

• Chang141 has mentioned that MF is converted rapidly to farnesoic acid by esterases that are present in tissues.

Androgenic hormone Once the physiological function of androgenic

hormone was known efforts were made to purify and characterise the active factors present in this hormone. Hasegawa et al. 147,148 purified two proteins, androgenic gland hormone I (AGHI) and AGH II consisting of 157 and 160 amino acids respectively with molecular weights of 17.0 and 18.3 kDa. The other biological active factors isolated were identified as the terpenes farnesylacetone and hexhydroxy farnesylacetone l49

• However, the relative role of these proteins and terpenes is unknown.

Roles of neuropeptides I neurotransmitters in reproduction

The known neurotransmitters in crustaceans are acetylcholine (Ach), gamma-aminobutyric acid (GABA), glutamate, octopamine (OA), dopamine (DA), and 5-hydroxytryptamine/serotonine (5-HT). Among the neurotransmitters tested for possible roles in crustacean reproduction and molting, 5-HT, OA, and DA seem to be attracting more attention than the others. Each of these is present in the crustacean nervous systems.

216 INDIAN J EXP BIOL, MARCH 200S

S-Hydroxytryptamine (S-HT, Serotonin) 5-HT is an ubiquitous substance found in plants

and animals. The Fa1ck-Hillarp fluorescence histochemical technique for monoamines was used by several investigators to study the distribution of 5-HT in crustacean tissues. With this method, 5-HT emits yellow light. Osborne and Dando '50 detected fluorescence characteristic of 5-HT in some cell bodies and in most of the neuropile in the stomatogastric ganglion of the lobster Homarus vulgaris.

In recent years, immunocytochemical techniques have largely supplanted the FaIck-Hillarp fluorescence technique for localization studies of biogenic amines '51 . In the eyestalk, immunoreactive neurons are present in three of the four optic ganglia (medulla externa, medulla interna, and medulla terminalis). Some of the immunoreactive cell bodies in the medulla terminalis send out fibers through the optic peduncle that terminate in the protocerebrum. Immunoreactive cell bodies are also present in the brain. The brain and every ganglion in the ventral nerve cord displayed at least one immunoreactive cell body. In addition, several axons in the pericardial organs are immunoreactive, but the origin and termination of these axons could not be determined.

In addition to identification and localization of 5-HT by histochemical methods, precise measurements of tissue concentration of biogenic amines are now possible. With the HPCL technique, 5-HT was detected in the central nervous system and

I 152 A . f hemolymph of P. leniuscu us. senes 0

experiments done elsewhere revealed that 5-HT stimulates ovarian development when injected into the fiddler crab and red swamp crayfish. These crabs showed increased, dose dependent ovarian

8,1 51 development . Supporting evidence for this hypothesis was

obtained by determining the effects of two 5-HT agonists on ovarian development in the crab. For this purpose, the 5-HT releaser fenfluramine and the 5-HT potentiator fluoxetine were used. The ovaries of crabs that received fenfluramine, fluoxetine, 5-HT alone, 5-HT plus fenfluramine, or 5-HT plus f1uoxetine exhibited significant increase in ovarian index and oocyte size compared to the ovaries of untreated initial control crabs and saline-injected concurrent control specimens 151. Supporti ng evidence for a neurotransmitter role of 5-HT in stimulating GSH

b S .. . 1153 release in P. clarkii was reported y aroJIDI et. a

In M. japonicus, V g synthesis in ovarian pieces incubated with thoracic ganglion pieces prepared from vitellogenic females can be stimulated by 5_HTI20.

In addition, 5-HT stimulates testicular maturation in P. clarkii as shown by the use of a series of 5-HT agonists and antagonists. 5-HT and its agonists induce testicular maturation and help in the development of the androgenic glands. In contrast, 5-HT antagonists had no stimulatory effects on the testes or androgenic glands. This stimulatory action of 5-HT on the testes and androgenic glands was hypothesized to be indirect, i.e. 5-HT stimulates the release of GSH, which in tum activates the androgenic glands to synthesize and release androgenic gland hormone, and the androgenic gland hormone then triggers testicular maturation90. While discussing 5-HT modulation of crustacean hyperglycerine hormone (CHH) secretion by isolated cells of the crayfish retina and optic lobe, Escamilla-Chemal et al. 154 found that retinal CHH secreting cells correspond to a population of retinal tapetal cells and optic lobe CHH-secreting cells correspond to two sUbpopulations of CHH of medulla terminalis-X organ cells. Further, they mentioned that CHH secretion generally increases as a function of 5-HT concentration. Gu et al. 74 reported the cloning and characterization of the cDNA and the gene encoding the hyperglycemic hormone (CHH-B) of the shrimp Metapaneus ensis. It was shown that the amino acid sequence of the hyperglycemic hormone of M. ensis is identical to that of the CHH-like neuropeptide (CHH-A) of M. ens is. They further observed the presence of CHH-B in the eyestalk of vitellogenic females. At the middle stage of gonadal maturation,a minimum level of CHH-B transcript and a maximum level of CHH-A transcript were detected which indicated that both CHH-related neuropeptides may play an important role during the female gonad maturation cycle in shrimp.

Dopamine (DA) Dopamine (DA) is present in crustacean nervous

systems. Histochemical studies employing the fluorescence method of Falck and Hillarp indicated that catecholamines, including DA, are present and

h'b' f1 151 catecholamines ex 1 It a green uorescence . However, both DA and norephinephrine (NE) evoke this green fluorescence, both being catecholamines. Much green fluorescence was apparent in the medulla

DIWAN: CURRENT PROGRESS IN SHRIMP ENDOCRINOLOGY 217

externa and medulla interna of the eyes talk, and in the brain and ventral nerve cord of Astacus astacus l55

•

As with 5-HT, immunocytochemical procedures have been used to demonstrate the presence of DA in crustaceans. By use of an anti-DA antibody, the presence of DA-like neurons in the terminal abdominal ganglion, intestinal nerve, and axons in the hindgut musculature of Orconectes limos us was demonstrated. Mercierl56 identified two immuno­reactive neurons in the abdominal nerve cord of P. clarkii that contribute axons through the intestinal nerve to the plexus that surrounds the hindgut. HPLC analysis confirmed the presence of DA in this nerve8

.

Role of dopamine in reproduction DA, when injected into female P. clarkii, inhibited

ovarian maturation. The DA-injected individuals had a smaller mean ovarian index than the control. The crayfish given DA plus 5-HT had a significantly smaller mean ovarian index than the crayfish that received 5-HT alone, but larger than the crayfish that received DA alone. 5-HT exerts its effect on the ovary indirectly, but stimulating GSH release, the inhibitory action of DA on ovarian maturation induced by 5-HT could have been due to (1) inhibition of GSH release, thereby directly counteracting the action of 5-HT; (2) stimulation of release of the GSH antagonist, GIH; or (3) both (1) and (2)153.151.

Octopamine (OA) The presence of OA in central nervous organs of

H. american us was shown by using thin layer chromatography. With the use of radiolabeled compounds and HPLC it was shown that OA is synthesized in the brain of Orconectes. By HPLC, OA is detected in the eyestalks, brain and hemolymph of P. leniusculus. Additionally, by HPLC, Butler and Fingerman l57 found OA in the central nervous system of U. panacea and Luschen et al. 158 found OA in Y-organs of C. maenas. Wallace et al. 159 by using various staining techniques such as treatment with neutral red and osmium tetroxide and performing OA analysis by use of a radiolabeled OA precursor reported the presece of OA in the specific neurons of the lobster nervous system. Many chromatographic and electrophoretic techniques were able to identify neurons in the second thoracic ganglion of H. americanus that appeared to be the ones that contain this biogenic amine. The role of OA in reproduction appears to be at least in part stimulation

of contraction of the ovarian walls, liberating the oocytes.

Red pigment-concentrating hormone (RPCH) RPCH was isolated from eyestalks of the prawn,

Pandalus borealis. It was later found to be an octapeptide. Extracts of all portions of the central nervous system, including the brain and thoracic ganglia of the crayfishes Cambarellus shufeldi and Faxonella clypeata have concentrated red pigment hormone in the integumentary chromatophores, thereby revealing that RPCH is widely distributed throughout the central nervous system of the crayfish l61

.

Role of RPCH in reproduction RPCH, in addition to its hormonal role in

regulating pigmentation, appears to have another hormonal role , stimulation of MF synthesis in the mandibular organ of P. clarkii. MF may function as a hormone involved in the regulation of crustacean reproduction to complement the two peptidergic neurohormones, GIH and GSH, that have well documented roles in controlling gonadal maturation in crustaceans 136.

Opioid peptides The first report of the presence of an opioid-like

substance in crustaceans was published by Mancillas et al. 161. Through immunocytochemistry, they found Leu-Enk-like immunoreactivity in all the retinular cells of the spiny lobster, Panulirus interruptus, and P. clarkii. In addition, such immunoreactivity was also apparent in nerve fibers in chiasma 3 that run from the medulla interna to the medulla terminalis. Later, other crustaceans were similarly studied. Immunoreactivity was apparent in the retinular cells, lamina 'ganglionaris, sinus gland, optic peduncle, the three chiasma, and medulla terminalis. Immunoreac­tivity was present in ovarian follicle cells and avitellogenic and early vitellogenic oocytes but not in fully ripe oocytes. In the testes, the immunoreactivity was present in spermatogonia, spermatocytes and spermatids but not in mature spermatozoa. This was the first demonstration of opioid-like Immunoreac­tivity in a crustacean gonad.

Role of opioids in reproduction The potential involvement of an endogenous opioid

system in the regulation of ovarian development in

218 INDIAN J EXP BIOL, MARCH 2005

U. pugilator has been investigated in viV0162• Injection

of synthetic Met-Enk into female crabs significantly slowed ovarian maturation. The inhibition was dose­dependent. The mean ovarian index and mean oocyte diameter of the crabs that received the opioid were significantly smaller than the corresponding values for the saline-injected concurrent control specimens. In contrast, injection of the opioid antagonist, naloxone, produced dose-dependent ovarian matura­tion. It was hypothsized that the opioid (1) stimulates GIH release, (2) inhibits GSH release or (3) does both (1) and (2).

The role of an opioid in testicular maturation of the fiddler crab was also investigated. Injection of synthetic Met-Enk into male crabs significantly slowed maturation of the testes. The inhibition was dose-dependent. The mean testicular index, testicular weight, testicular lobe diameter and the number of mature spermatocytes in each testicular lobe were significantly less than the corresponding values for the saline-injected concurrent control crabs. Injection of naloxone produced dose-related testicular maturation 162.

Conclusion The shrimp industry today all over the world is

developing rapidly, particularly in Southeast Asian countries due to the high demand of shrimp in the international market and also due to its high export value. The success of the industry often depends on the availability of the shrimp resource on a continuous basis. Today the shrimp industry is mostly dependent for this resource from the wild and to meet the demand, shrimps are being exploited continuously and in the process overexploitation takes place, and as a result shrimp catches have dwindled and in some places even the fishery has declined to a large extent. In order to reverse this trend and manage this resource on a sustainable basis the FAO has recently introduced the Code of Conduct For Responsible Fisheries (CCRF) for strict adoption by the all the maritime countries. Even if this code becomes effective, it will take a long time to rejuvenate the depleted stock. Therefore, in order to fulfill the demand of shrimps by the industries on a continuous basis, one of the alternate ways is to produce them in captive conditions.

In the recent past several efforts have been made to evolve methods for the induction of maturation and spawning of shrimps in captive conditions. Though

eyes talk ablation has been accepted as the most successful method for induction of maturation and spawning, this method does not work for many shrimps of commercial value. Moreover, there are other inherent and social problems in adopting this technique. Hence, the need of the hour is to explore / devise methods involving other possible neuro­endocrine manipulations. The use of neurohumours or neurotransmitters, or extracts from the brain and thoracic ganglia containing reproductive hormones, has to be explored to initiate gonadal maturation.

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