Endocrinology of Reproduction in Crustaceans

Crustaceans have become the most popular proteinacious foods to meet the food demand of ever growing human population in the World. But, the culturing of crustacean species has many problems, including limited availability of quality seed. Out of all conventional methods practiced to increase seed of good quality and quantity, manipulation of the endocrine system of brood stock is found to be one of the best methods. Regulation of crustacean reproduction is under the control of many hormones and factors. The eyestalk hormones, namely gonad/ vitellogenin-inhibiting hormone (VIH) and mandibular organ inhibiting hormone (MOIH) show negative effects on maturation, whereas the other eyestalk hormones show mixed effects on maturation. The non-eyestalk hormones namely gonad stimulating hormone (GSH), methyl farnesoate (MF) and ecdysteroids are ovarian maturation inducers in crustaceans. The pros and cons of endocrine manipulation in crustaceans are discussed in this chapter.


Introduction
Crustaceans, a major group of animals which serve as food for humans and animals come under phylum Arthropoda. There are about 45,000 crustacean species distributed throughout the World. The crab, marine shrimps, crayfishes, lobsters and freshwater prawns are edible and they belong to crustacea. This group of animals is free-living and the habitat of most of them is freshwater or marine, where few of them are semi terrestrial. Edible crustaceans have lots of importance because of its role in acting as rich protein food, sustainability in culturing and trading. They possess significant economic value in Nations of developing which undoubtedly provide food security in both the production and transportation to within and to other Nations. The acceptance of crustacean food in World has also increased due to its softness, flavor, easy digestion and numerous health benefits due to the presence of protein, minerals and vitamins which are known to prevent a range of diseases. The seafood, especially crustacean proteinacious food is famous in many countries and has its own demand. Most of crustacean food is produced in China and other Asian countries. The fresh, the frozen and the snacks are different forms of crustacean food available throughout the World, supplied from the food industry. Besides food industry, other major industries that use crustaceans are pharmaceutical and cosmetic industries. The crustacean pigments and natural compounds of shell are holding high value in the cosmetic industry. Globally, the edible crustacean production is about 10 million tons per year through fisheries and aquaculture farming. Due to its significance, the crustacean World market has reached to US $ 147 in the year 2017 and is anticipated to grow at a reasonable rate in the years to follow. Though the crustacean production is increasing steadily in the global market, it is not at a great rate to meet the demands of the ever growing human population on the globe. Due to several reasons, crustacean aquaculture industry is facing numerous troubles in obtaining expected productivity. One of such reasons is limited availability of good quality seed, a potential startup for high yield of protein.
Crustaceans are mostly unisexual and can be easily distinguished into the male and female by morphology. Male and female reproductive organs are developed to produce and reproduce young ones in the brood sack of the female. The usage of quality seed is a key to get increased productivity of the quality protein in aquaculture. Naturally captured crustacean seed is found the most promising one to get more protein through culture. But, the availability of seed in the nature is limited and tedious to obtain. To overcome this, a classical eyestalk ablation method is introduced to induce spawning in the brood stock [1]. Removal of eyestalk eliminates the synthesis and release of eyestalk gonad inhibitory principle from neurohemal X-organ sinus gland complex, which promotes maturation [2]. Because of cautery of eyestalk, this method causes the highest mortality in the brood stock and loss of hemolymph very often leads to production of inferior quality seed. In search of an alternative, the researchers are working in multiple directions to get quality seed. Among all, the endocrine manipulation of crustacean hormones is one of the best methods for quality seed production.

Maturation and hormones
The proper maturation of gonads in crustacea provides the quality seed production. The maturation in crustaceans happens mainly due to activation of biosynthesis of female reproductive egg protein called vitellogenin. Vitellogenin is a lipoprotein used for the nourishment (energy requirement) of developing embryo. The site of vitellogenesis in crustaceans is various tissues, including the ovary. The major sites for vitellogenesis are ovary, hepatopancreas and epidermis. Most of the crabs are holding hepatopancreas as vitellogenin synthesizing site whereas in other crustaceans, it is epidermis or hepatopancreas along with the ovary. In natural maturation vitellogenin is produced as pre-vitellogenin and it undergoes cleavage to produce the mature vitellogenin/vitellin molecules of bearing different molecular sizes. The size and number of vitellins varies from one species to other. These vitellins via hemolymph are transported to ovary and they deposit in the developing oocytes. The accumulation of vitellin in the developing ovary is continued until the ovary reaches to fully matured stage [3]. The female reproductive protein vitellin/vitellogenin is a good indicator of reproductive activity in crustaceans and is frequently used to determine the regulatory functions of endocrine hormones [4].
There are many factors that regulate the maturation of ovary in crustacean brood stock. The eyestalk neural tissue in crustacea secrets a number of neuroendocrine hormones and is analogous to vertebrate pituitary-hypothalamus. The vitellogenesis/gonad-inhibiting hormone (VIH/GIH), mandibular organ-inhibiting hormone (MOIH), crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), biogenic amines and opioids are the major secretory products of eyestalk. These eyestalk products play a crucial role in regulating the maturation in crustaceans. The products of Y-organs and mandibular organs called ecdysteroids and methyl farnesoate (MF) respectively and gonad stimulating hormone (GSH) are non-eyestalk hormones involved in the regulation of ovarian maturation [5, 6]. Besides these, the ovarian maturation is also regulated by the external factors such as temperature, salinity, pH, food availability in the pond and heavy metals present in the water through endocrine regulation. However, the regulation of reproduction is a complex process in crustacea and its understanding requires the detailed description of action of internal and external factors.

Factors involved in the regulation of reproduction
Endocrine hormones such as eyestalk neuropeptides, GSH, opioids, biogenic amines, ecdysteroids and MF are endogenous factors that regulate reproduction in crustaceans [6]. The internal factors are of two types based on their action in the regulation of reproduction. Hormones which promote reproduction come under "positive regulators", whereas those that suppress the reproduction are called "negative regulators". The positive regulators are MF, ecdysteroids and GSH, and the negative regulators are VIH/GIH and MOIH. Some factors like CHH, MIH, biogenic amines and opioids are found to be having both the actions. The Y-organs, mandibular organs, brain, thoracic ganglia and ovary are responsible for the release of a variety of reproductive regulatory hormones and factors beside the major endocrine centers of the eyestalks. The role of these hormones and factors in the regulation of crustacean maturation, especially in the ovarian growth is discussed here.

Factors of X-organ sinus gland complex
The eyestalk neural tissue is divided into medulla extern interna and terminalis. Neural clusters located in these three parts of neural tissue are responsible for the secretion of all eyestalk peptide hormones and other factors. These secretions are initially stored in a neurohemal organ sinus gland located in the eyestalk and it finally secretes into the stream of hemolymph to show its action at the target tissue. The eyestalk neural tissue, cluster of neuronal cells and sinus gland are collectively called "neurohemal X-organ sinus gland complex". The hormones of eyestalk such as CHH, VIH, MIH, MOIH and other peptides are collectively called as CHH-family peptides. These peptide hormones are classified as Type I and II based on the presence or absence of Glycine residue at 12th position of their mature peptides [7]. The secretions of eyestalk are as follows and are discussed in Section 3.1.1.

Vitellogenesis/gonad-inhibiting hormone
The prime role of eyestalk GIH/VIH in crustacea is inhibition of ovarian/gonad maturation. Panouse [1] first identified the presence of GIH in Palaemon (Leander) serratus (P. serratus). He founded the speedy rise in size of ovary and precocious egg deposition in the ovaries by the removal of eyestalk gonad inhibitory factor (GIH) from immature P. serratus. Later the same was reported in other crustacean species (See review [3,8]). Since the lack of exact bioassay, identification and characterization of VIH neuropeptides the literature available is limited. However, VIH was identified and characterized in a variety of crustaceans. The ovarian fragments of Procambarus bouvieri were incubated in vitro with isolated VIH from the same species and inhibition of vitellogenin m-RNA levels were found in ovarian fragments [9]. Soyez et al.
In the recent past, recombinant proteins were identified as potential molecules for altering the biological functions. The recombinant GIH/VIH protein was synthesized for a variety of crustaceans. The r-GIH was synthesized for lobster Nephrops norvegicus and using this identified the GIH synthesizing cells in eyestalk neural tissue [12]. The VIH activity of secondary structure of H. americanus recombinant VIH (rHoa-VIH) which was assured by bacterial expression system was incubated with Marsupenaeus japonicus (M. japonicus) ovarian fragments in vitro [13]. Ohira et al. [13] was tested the reduced vitellogenin m-RNA levels in the ovary of M. japonicus incubated with c-terminus amidated rHoa-VIH. Advancement of technology reveals the gene function and is not restricted to characterize the crustacean genes. The gene -silencing using RNAi methodology is in use to silence the inhibitory CHH-family peptides, to activate reproduction and growth in crustaceans. VIH gene silencing was demonstrated in Penaeus monodon (P. monodon). The in vivo functional gene silencing of P. monodon VIH (Pem-VIH) with increased vitellogenin m-RNA levels was reported by administration of Pem-VIH ds m-RNA [14].
Though the mechanism of VIH/GIH induced inhibition of reproduction is not clear, from the available literature, it is identified that VIH shows its action in two intracellular signaling pathways in the ovary such as a) cyclic nucleotide signaling and b) calcium ion and protein kinase C signaling [15]. In one way, VIH induces its inhibitory action by activating the cyclic nucleotides adenylyl cyclase or guanylyl cyclase or both (Figure 1). Binding of VIH to G protein receptor activates the G protein which activates adenylyl cyclase that produce cAMP from ATP. On the other hand VIH may also stimulate the guanylyl cyclase through its receptors (GCR)  which leads to conversion of GTP into cGMP. Both the cyclic nucleotides by the action of phosphodiesterase activate protein kinase A and G respectively for cAMP and cGMP. Increased protein phosphorylation induced by protein kinase A or G or both suppresses the gene expression of vitellogenin in the nucleus, thereby reduces the vitellogenin protein levels in the ovarian follicles [16]. Figure 2 explains the second mechanistic action of VIH inhibitory pathway through activation of protein kinase C by diacylglycerol, inosital and calcium ions (calmodulin). The high titer of circulatory VIH, binds to G protein receptors (GpR) in the developing ovarian follicles. Upon binding of GpR with VIH, activates the G protein, which initiates the conversion of phosphotidyl bisphosphate into diacylglycerol and inositol 1, 4, 5 -triphosphate upon activation of phoshpolipase C. The diacylglycerol activates the protein phosphorylation through protein kinase C. On the other hand inositol 1, 4, 5 -triphosphate induces the increase of intracellular free calcium (Ca 2+ ) from the endoplasmic reticulum in addition to the calcium available through calcium transported from the extracellular space to cytosol. Calmodulin bound or free Ca 2+ activates the synthesis of cAMP, which directly inhibits the gene expression of vitellogenin gene in the nucleus along with high levels of protein phosphorylation [16].

Mandibular organ-inhibiting hormone
The MOIH, an eyestalk neuropeptide known negative regulator of reproduction is identified in many crustaceans [17]. More specifically, mandibular organs (MOs) involved in maturation of crustaceans through its secretory product methyl farnesoate are under negative control of MOIH. The elevated MF levels of 54.1 and 106.9 ng/gland in MO are reported respectively, after unilateral and bilateral eyestalk ablation respectively in H. americanus [18]. In the same study the suppressed MF levels are recorded in MOs after injection of sinus gland extracts to bilateral eyestalk ablated H. americanus. Chaves [19] demonstrated the negative effect of MOIH on MF synthesis from MO in crayfish Procambarus clarkii (P. clarkii) by measuring the activity of farnesoic acid O-methyl transferase an enzyme involved in MF synthesis and also found 20-100 fold rise in hemolymph MF levels during 8th-12th day after eyestalk ablation. An increase in MO size is also recorded in H. americanus during ovarian maturation [20]. Premature vitellogenesis is observed in juvenile Libinia emarginata after MO implantation [21]. During the ovarian maturation process an increase in the size of MO along with an increase in ovarian index and oocyte diameter is reported in the natural reproductive cycle of crab Oziothelphusa senex senex (O. senex senex) [22]. Direct studies showing the MOIH action against the release of MF from MO are demonstrated in many studies. The above mentioned studies are evidenced in the stimulatory role of MO and MF in crustacean reproduction. However, the role of MOIH in the regulation of reproduction by inhibiting the synthesis and secretion of MF from MO is clear from the limited literature, but the mechanistic action of MOIH is not clear and further studies are needed to establish it.

Crustacean hyperglycemic hormone
The major CHH-family peptide synthesized and released from the eyestalk neural tissue is a crustacean hyperglycemic hormone (CHH). The onset of vitellogenesis is regulated by CHH besides its principal activity in carbohydrate metabolism [23]. Studies on CHH regulated reproduction are limited. At first, the role of CHH-A and CHH-B on reproduction is demonstrated in H. americanus [9,23]. De Kleijn et al. [24] has found that the onset of vitellogenin synthesis is triggered by Hoa-CHH-A and oocyte development in late stages of maturation is by Hoa-CHH-B. Similarly, in Metapenaeus ensis (M. ensis) isolated two CHH-family peptides involved in ovarian development, especially in early stage (CHH-A) and, middle and later stage (CHH-B) [25]. In contrast, in vitro inhibition of vitellogenin and its expression in Penaeus semisulcatus ovarian fragments by Penaeus japonicus CHH are reported [26].
The isoforms of CHH may be received by different types of receptors responsible for a variety of physiological functions including reproduction. It is evidenced through occurrence of CHH receptors on the membrane of Y-organs the principle glands synthesize ecdysteroids in Carcinus maenas (C. maenas) and Gecarcinus lateralis [27,28]. The recombinant CHH reduced 51% of ecdysteroid levels from Y-organs of crab C. maenas [27]. Since CHH isoforms are showing similarity with other CHH-family peptides and its pleiotropic nature, they might display functions of important depends on the tissue they reach. Due to the CHH importance in crustacean reproduction and endocrine manipulation, further mechanistic actions are necessary to elucidate.

Molt-inhibiting hormone
Molt inhibition is the fundamental role of eyestalk MIH. Through inhibiting the synthesis and secretion of molting hormone (ecdysteroids) from Y-organs (molting glands) MIH regulates molting. Due to its pleiotropic nature, MIH is reported for its involvement in the onset of vitellogenesis in crustaceans. The MIH mediated reproduction is not very clear and reports are showing both positive and negative effects on maturation. The concealed onset of vitellogenesis by MIH is identified in shrimp  [29]. In the same species also reported recombinant Me-MIH-B induced vitellogenin expression in ovary and hepatopancreas in a dose dependent manner in vitro. Recent studies identified and characterized the hepatopancreatic MIH binding proteins in maturing female crab Callinectes sapidus (C. sapidus) [30]. Zmora et al. [30] Identified high cAMP (a secondary messenger) levels in hepatopancreatic tissue incubated with C. sapidus MIH and predicted the mechanistic action of MIH in the onset of reproduction. Zmora et al. [30] reported the dynamics of MIH during different stages of ovarian maturation in C. sapidus. They reported the antagonistic action of MIH on growth (inhibition) and reproduction (induction) by comparing the MIH titers at premature (low) and mature stages (four fold higher) of ovary. Moreover, the gonad stimulatory effect of MIH-B isolated from M. ensis is tested and its pattern of expression in eyestalk neural tissue is correlated with ovarian maturation cycle [31]. Tiu and Chan [29] found elevated hemolymph and ovarian vitellogenin protein levels along with hepatopancreatic and ovarian vitellogenin m-RNA levels upon administration with Me-MIH-B in vivo. The ds-RNA of Me-MIH-B injection concealed the vitellogenin m-RNA levels in hepatopancreas and ovary along with lowered vitellogenin protein levels in hemolymph and ovary in M. ensis [29]. However, the growth and reproduction are antagonistic in crabs and sequential in prawns and shrimps. Furthermore, studies are immensely needed on MIH for manipulation of crustacean reproduction in order to uphold sustainable aquaculture.

Opioid peptides
Small peptides containing five amino acid residues released from eyestalks are called opioids. Their presence is well defined in vertebrates [32] and is also reported in a few invertebrates including crustaceans using modern techniques like radioimmunoassay (RIA), high performance liquid chromatography (HPLC) and immunohistochemistry (IHC). In crustaceans, opioids are identified in the eyestalk neural tissue and are released from sinus glands into the hemolymph. The endogenous opioids found in crustaceans are named as leucine-enkephalin and methionineenkephalin. Limited reports are available for opioids induced reproduction in crustaceans [33]. The in vivo experiment was conducted to investigate the role of endogenous opioids on reproduction in crab Uca pugilator. Whereas, observed dose dependent inhibition of ovarian development by injecting methionine-enkephalin. In P. clarkii, it is proved that incubation of thoracic ganglion alone with ovarian fragments in vitro does not cause any change in the ovary, whereas incubation along with methionine-enkephalin reduced the ovarian maturation in a dose dependent manner [34]. Out of two enkephalins identified, only leucin-enkehpalin is found to be involved in the regulation of maturation in prawn Penaeus indicus (P. indicus) [35]. The induction of maturation by leucine-enkephalin is tested in P. indicus and O. senex senex [34,35]. However, the role of opioids in crustaceans is controversial and is required to conduct more studies to determine its specific mechanistic action and use in endocrine manipulation of reproduction.

Biogenic amines
In decapods, biogenic amines are well reported as regulators of reproduction. Initially these amines act as neuroregulators and later as neurohormones. They play an important role in synthesis and releasing of endocrine hormones involved in the regulation of reproduction [36]. The hormones like CHH-family peptides (GIH, CHH, MIH, RPDH and BPDH) and GSH are influenced by biogenic amines [37]. The identified biogenic amines in crustaceans are serotonin (5-hydroxy tryptamine; 5-HT) and dopamine, and they influence the ovarian maturation (Table 1).
Serotonin induces the release of GSH from thoracic ganglion, whereas dopamine promotes release of eyestalk GIH and reduces thoracic ganglion GSH [47]. The GSH triggered ovarian development by 5-HT in both in vitro and in vivo is reported in Penaeus vannamei [41]. Recently Sainath et al. [38] well demonstrated the reproductive function of serotonin in freshwater rice field crab O. senex senex. Since its importance in maturation, biogenic amines are potential candidate molecules for endocrine manipulation of reproduction in crustaceans.

Gonad stimulating hormone
The gonad stimulating hormone (GSH) is found to be synthesized and released from the crustacean brain and thoracic ganglia. Many in vivo and in vitro studies have explained the ovarian stimulating effects of brain and thoracic ganglia [48] without isolation and characterization of GSH principle. At first, induced ovarian growth is reported by implanting the brain in Paratelphusa hydrodromous [49], later in Macrobrachium kistnesis, Parapenaeopsis hardwickii [50,51]. The stimulatory role of implanted thoracic ganglia is reported for the first time in Potamon dehaani [52] and later in Cherax quadricarinatus [53]. The combined effect of brain and thoracic ganglia on ovarian development is demonstrated in P. clarkii [42]. The role of copper and cadmium on ovarian development through GSH is tested in Chasmagnathus granulata, where they incubated ovarian explants and thoracic ganglia along with heavy metals and found inhibition of ovarian maturation [54]. Though the GSH has lots of importance in regulating crustacean reproduction, it could not be used in endocrine manipulation until its structural characterization done in crustaceans.

S.no.
Crustacean species Biogenic amine and its function References 1

Ecdysteroids
In crustaceans, ecdysteroids are polyhydroxylated ketosteoroids synthesized and released from the molting gland called Y-organ with a primary role in regulation of growth. Y-organs are homologous to prothoracic glands of insects and are non-neural paired endocrine glands. Besides molting, ecdysteroids regulate crustacean reproduction and embroyogenesis [55]. The mechanistic action of crustecdysone is explained after the discovery of its receptors called ecdysone receptors (EcR) [56]. EcR binds with retinoid X receptor (RXR) to form heterodimer and stimulates DNA regulatory elements of ecdysteroid signaling pathway [56,57]. Various physiological functions of ecdysteroids are reported with the EcR and its domain activation, which decides to exert the physiological function by activation or suppression of gene function [58,59].
At first Charniaux-Cotton and Touir [60] has identified the role of ecdysteroids in the maturation of oocyte of Lysmata seticaudata by Y-organectomy, later in Orchestia gammarella [32]. The levels of ecdysteroids at the initial stages of spermatogonial mitoses and ovarian maturation are found along with expression of vitellogenin gene in crustaceans [61]. The ecdysteroids and its role is well established in crustaceans. Furthermore, the endocrine manipulation can be executed by supplementing the ecdysteroids through feed to the brood stock to induce reproduction. As a limitation, this method takes longer time to induce ovarian maturation and the animal may enter molt instead of the reproduction.

Methyl farnesoate
The terpenoid hormone identified through reverse endocrinology is called the methyl farneosate (MF) an analog of insect juvenile hormone III confined only to decapoda and is a secretory product of mandibular organ [62]. MF synthesis and secretion from MO is repressed by eyestalk neuropeptide MOIH [22]. The hemolymph titers of MF are founded high during ovarian maturation and it has a direct relation to promoting maturation in crustaceans [63]. The mechanism of MF induced maturation is found through ecdysteroid synthesis in Y-organs. The mandibular organ disruption due to exposure of environmental pollutants decreases the hemolymph levels of 20-hydroxyecdysone, which is a stimulator product of MF [64]. MF acts as a ligand for retinoid-X-receptor (RXR) and synergize with ecdysteroids to stimulate RXR-EcR heterodimer complex for expression of combined regulatory genes of these two hormones [56,57]. In C. maenas administration of MF elevated the levels of RXR m-RNA in both in vitro and in vivo [65]. MF is the best suited hormone for endocrine manipulation of reproduction in crustaceans.

Conclusion
Seed production is purely done in hatcheries and is supplied to the farmers. Though the reproductive manipulation is not directly influencing the high amount of quality protein production, it is an initial key factor to produce quality feed. The quality seed usage in culture pond is one of the key for high amount of quality protein production. Healthy seed is always superior to maintain and is not easily affected by bacterial, viral and fungal pathogens. More promisingly, superior seed get adjusted easily to new environment and is not affected by fluctuations in the pond management. Moreover, decreased mortality rate in seed, increases the population which reflects the amount of protein/yield at the harvesting time. It is most important that, crustacean hatchery industry should improve the seed quality by following the new methodologies developed from time to time. The systematic pond management and adequate food supply increases protein yield. The high yield influences (increases) the socioeconomic status of farmers ultimately improve the economy of country besides supplementing quality protein forever increasing population. The waste generated (other than protein) in aqua industry is used as feed for poultry, pig, fish and other farm animals, and also in cosmetic industry.
The factors such as pH, temperature, photoperiod, availability of food, salinity of water and heavy metal contamination influences the synthesis and release of CHH-family and other eyestalk secretions in cultured crustaceans.
Regulation of reproduction in crustaceans is a complex process, where a number of internal and external factors are involved. The endocrine hormones and other internal factors act accordingly along with external factors and regulate maturation. The role of well-known internal and external factors is depicted in Figure 3. Established endocrine authoritarian mechanisms of crustacean maturation were interpreted in the present chapter. The best method to manipulate endocrine hormones in crustaceans is through supplementation of reproduction positive regulators by means of the feed and is a best alternative for eyestalk ablation technique to improve good quality and quantity of seed from brood stock. Though the administration of positive regulators yielding quality seed, it may injure the animal and increases stress and sometimes it may lead to mortality. The promising method emerging in recent years is gene knockout studies, where the functional genes (negative regulatory genes of reproduction) made inactive. However, knockout studies are not initiated yet in crustacean aquaculture. The identified and reported method for temporary knockdown of gene function in crustaceans is RNAi silencing technology. Silencing of VIH and other genes inhibit reproduction may help to induce maturation thereby quality seed [23]. Controlled external factors with manipulation of internal factors (neither positive nor negative) provide fruitful results without any stress in the brood stock.