Crustacean Hyperglycemic Hormone Research Articles

Impacts of a novel live shrimp (Litopenaeus vannamei) water-free transportation strategy on flesh quality: Insights through stress response and oxidation in lipids and proteins

A novel strategy of water-free transportation (WFT) for live shrimp was recently developed. In this study, survival rate, hormone response, free radical content, oxidation in lipids and proteins, and flesh quality were evaluated after WFT. Meanwhile water transportation (WT) was employed as a reference for comparison. Results showed only 5.1% mortality after 10 h of WFT, while crustacean hyperglycemic hormone (CHH), reactive nitrogen species (RNS), free fatty acid content (FFA), peroxide value (PV), carbonyl concentration, intrinsic fluorescence intensity (IFI), and surface hydrophobicity significantly increased. Myosin heavy chain (MHC) and actin degradation were also detected after WFT. Although oxidation occurred in lipids and proteins, no significant changes in texture and water-holding capacity (WHC) were detected after WFT or WT (p > 0.05). The 10 h of WFT showed little influence on flesh quality of shrimp even though lipids and proteins oxidation occurred during the mimicked water-free transportation.

Insights on Molecular Mechanisms of Ovarian Development in Decapod Crustacea: Focus on Vitellogenesis-Stimulating Factors and Pathways.

Vitellogenesis in crustaceans is an energy-consuming process. Though the underlying mechanisms of ovarian maturation in decapod Crustacea are still unclear, evidence indicates the process to be regulated by antagonistically-acting inhibitory and stimulating factors specifically originating from X-organ/sinus gland (XO/SG) complex. Among the reported neuromediators, neuropeptides belonging to the crustacean hyperglycemic hormone (CHH)-family have been studied extensively. The structure and dynamics of inhibitory action of vitellogenesis-inhibiting hormone (VIH) on vitellogenesis have been demonstrated in several species. Similarly, the stimulatory effects of other neuropeptides of the CHH-family on crustacean vitellogenesis have also been validated. Advancement in transcriptomic sequencing and comparative genome analysis has led to the discovery of a large number of neuromediators, peptides, and putative peptide receptors having pleiotropic and novel functions in decapod reproduction. Furthermore, differing research strategies have indicated that neurotransmitters and steroid hormones play an integrative role by stimulating neuropeptide secretion, thus demonstrating the complex intertwining of regulatory factors in reproduction. However, the molecular mechanisms by which the combinatorial effect of eyestalk hormones, neuromediators and other factors coordinate to regulate ovarian maturation remain elusive. These multifunctional substances are speculated to control ovarian maturation possibly via the autocrine/paracrine pathway by acting directly on the gonads or by indirectly exerting their stimulatory effects by triggering the release of a putative gonad stimulating factor from the thoracic ganglion. Acting through receptors, they possibly affect levels of cyclic nucleotides (cAMP and cGMP) and Ca2+ in target tissues leading to the regulation of vitellogenesis. The “stimulatory paradox” effect of eyestalk ablation on ovarian maturation continues to be exploited in commercial aquaculture operations, and is outweighed by the detrimental physiological effects of this procedure. In this regard, the development of efficient alternatives to eyestalk ablation based on scientific knowledge is a necessity. In this article, we focus principally on the signaling pathways of positive neuromediators and other factors regulating crustacean reproduction, providing an overview of their proposed receptor-mediated stimulatory mechanisms, intracellular signaling, and probable interaction with other hormonal signals. Finally, we provide insight into future research directions on crustacean reproduction as well as potential applications of such research to aquaculture technology development.

Do plasticity in gene expression and physiological responses in Palaemonid prawns facilitate adaptive response to different osmotic challenges?

Integrating physiological and genomic approaches in a comparative framework offers excellent opportunity to investigate the underlying mechanisms for acclimation to specific challenges. The present study was conducted on three different prawn species (inhabitants of different salinity environments) of the genus Macrobrachium (M. australiense, M. tolmerum and M. novaehollandiae) to investigate the salinity induced changes in expression patterns of 10 candidate genes in the gill tissue (that previously had been inferred to play important functional roles in acclimation and adaptation to freshwater environments), and hemolymph osmolality. The prawn individuals were maintained in laboratory condition under three different salinity levels (0‰, 6‰ and 12‰) for 28 days using 6‰ as the control. All of the genes studied, showed salinity induced differential expression patterns. Genes with more important functional roles under low ionic conditions (i.e. Claudin, Na+/H+exchanger, V-type H+-ATPase and UNT2) showed 2.5 to 6 fold higher expression at 0‰ compared with at higher salinities (6‰ and 12‰) but no significant differences (p > 0.05) were observed between 6‰ and 12‰ for the same genes. In contrast, 1.5 to 4 fold higher expression levels were observed at 6‰ and 12‰ for genes that have important roles in mediating salinity tolerance (i.e., Na+/K+-ATPase, Na+/K+/2Cl−Co-transporter, Diuretic Hormone, Crustacean Hyperglycaemic Hormone and UNT1). The osmotic stress response gene, Calreticulin, showed significant differences (p < 0.05) in expression between different salinity comparisons. Hemolymph osmolality also was impacted in all three species with a strong correlation evident between hemolymph osmolality and expression of genes influencing this trait. Findings indicate an important role of plasticity that facilitates rapid acclimation to changing salinity levels.

The Crustacean Hyperglycemic Hormone Superfamily: Progress Made in the Past Decade.

Early studies recognizing the importance of the decapod eyestalk in the endocrine regulation of crustacean physiology—molting, metabolism, reproduction, osmotic balance, etc.—helped found the field of crustacean endocrinology. Characterization of putative factors in the eyestalk using distinct functional bioassays ultimately led to the discovery of a group of structurally related and functionally diverse neuropeptides, crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH). These peptides, along with the first insect member (ion transport peptide, ITP), constitute the original arthropod members of the crustacean hyperglycemic hormone (CHH) superfamily. The presence of genes encoding the CHH-superfamily peptides across representative ecdysozoan taxa has been established. The objective of this review is to, aside from providing a general framework, highlight the progress made during the past decade or so. The progress includes the widespread identification of the CHH-superfamily peptides, in particular in non-crustaceans, which has reshaped the phylogenetic profile of the superfamily. Novel functions have been attributed to some of the newly identified members, providing exceptional opportunities for understanding the structure-function relationships of these peptides. Functional studies are challenging, especially for the peptides of crustacean and insect species, where they are widely expressed in various tissues and usually pleiotropic. Progress has been made in deciphering the roles of CHH, ITP, and their alternatively spliced counterparts (CHH-L, ITP-L) in the regulation of metabolism and ionic/osmotic hemostasis under (eco)physiological, developmental, or pathological contexts, and of MIH in the stimulation of ovarian maturation, which implicates it as a regulator for coordinating growth (molt) and reproduction. In addition, experimental elucidation of the steric structure and structure-function relationships have given better understanding of the structural basis of the functional diversification and overlapping among these peptides. Finally, an important finding was the first-ever identification of the receptors for this superfamily of peptides, specifically the receptors for ITPs of the silkworm, which will surely give great impetus to the functional study of these peptides for years to come. Studies regarding recent progress are presented and synthesized, and prospective developments remarked upon.

Effects of ZnO nanoparticles on the Giant freshwater prawn (Macrobrachium rosenbergii, de Man, 1879): Reproductive performance, larvae development, CHH concentrations and anti-oxidative enzymes activity

The Giant freshwater prawn (Macrobrachium rosenbergii) breeds when in captive conditions. The eggs of a clutch are attached to the abdomen of berried females. Zinc oxide (ZnO) is one of the most important metal oxide-nanoparticles (NPs) that is widely used in various industries and is released into aquatic environments from wastewater management facilities. The present study was conducted to evaluate effects of ZnO on values for the reproductive variables: larvae development, crustacean hyperglycemic hormone (CHH) release from the X-organ into the hemolymph and anti-oxidative enzymes activity of M. rosenbergii. There were five groups including a group not treated (control), and groups treated with10, 20, 50, 100 mg/L ZnO in triplicate during a 90-day period. Results indicated that ZnO-NPs have marked effects on reproductive performance, offspring development, CHH release from the X-organ into the hemolymph and anti-oxidant enzymes activities with there being no spawning of brood-stock in the 100 mg/L ZnO group and in the prawns treated with 50 mg/L there was spawning but there was larvae mortality immediately subsequent to hatching. Also, values for viability rate of eggs, dry weight of eggs, brood-stock inter-spawn period and egg clutch somatic index (ESI) reproductive variables were affected by the NP. This NP was found to have a dose-dependent effect on CHH release from the X-organ into the hemolymph and also superoxide dismutase (SOD) and catalase activities in M. rosenbergii. The results indicate that M. rosenbergii, a freshwater decapod crustacean, is an appropriate species to study nano-material effects on reproduction in freshwater ecosystems.

Transcriptomic Analysis of the Kuruma Prawn Marsupenaeus japonicus Reveals Possible Peripheral Regulation of the Ovary.

Crustacean reproduction has been hypothesized to be under complex endocrinological regulation by peptide hormones. To further improve our understanding of the mechanisms underlying this complex regulation, knowledge is needed regarding the hormones not only of the central nervous system (CNS) such as the X-organ/sinus gland (XOSG), brain, and thoracic ganglia, but also the peripheral gonadal tissues. For example, in vertebrates, some gonadal peptide hormones including activin, inhibin, follistatin, and relaxin are known to be involved in the reproductive physiology. Therefore, it is highly likely that some peptide factors from the ovary are serving as the signals among peripheral tissues and central nervous tissues in crustaceans. In this work, we sought to find gonadal peptide hormones and peptide hormone receptors by analyzing the transcriptome of the ovary of the kuruma prawn Marsupenaeus japonicus. The generated ovarian transcriptome data led to the identification of five possible peptide hormones, including bursicon-α and -β, the crustacean hyperglycemic hormone (CHH)-like peptide, insulin-like peptide (ILP), and neuroparsin-like peptide (NPLP). Dominant gene expressions for the bursicons were observed in the thoracic ganglia and the ovary, in the CNS for the CHH-like peptide, in the heart for NPLP, and in the ovary for ILP. Since the gene expressions of CHH-like peptide and NPLP were affected by a CHH (Penaeus japonicus sinus gland peptide-I) from XOSG, we produced recombinant peptides for CHH-like peptide and NPLP using Escherichia coli expression system to examine their possible peripheral regulation. As a result, we found that the recombinant NPLP increased vitellogenin gene expression in incubated ovarian tissue fragments. Moreover, contigs encoding putative receptors for insulin-like androgenic gland factor, insulin, neuroparsin, and neuropeptide Y/F, as well as several contigs encoding orphan G-protein coupled receptors and receptor-type guanylyl cyclases were also identified in the ovarian transcriptome. These results suggest that reproductive physiology in crustaceans is regulated by various gonadal peptide hormones, akin to vertebrates.

Effect of recombinant crustacean hyperglycemic hormones rCHH‐B1 and rCHH‐B2 on lipid metabolism in the Pacific white shrimp Litopenaeus vannamei

The crustacean hyperglycemic hormones (CHH) are pleiotropic neuropeptides controlling diverse processes in the life cycle of crustaceans. In the Pacific white shrimp Litopenaeus vannamei, CHH-B1 and CHH-B2 are generated by alternative splicing of the same gene, and the recombinant versions of these peptides have shown differential effects on glucose metabolism and osmoregulation in this species. In this work, we explored the role of both recombinant variants, expressed in yeast Pichia pastoris, on lipid metabolism. The results showed that rCHH-B1 and rCHH-B2 injections into eyestalk-ablated shrimp significantly increased the phospholipid and triglyceride levels in haemolymph and restored free fatty acid levels, suggesting that these CHH variants mobilize lipid reserves in shrimp. The effects on lipids were concomitant to its hyperglycemic activity, suggesting that both variants, in addition to their hyperglycemic activity, have hyperlipidemic functions.

Molecular cloning of crustacean hyperglycemic hormone (CHH) family members (CHH, molt-inhibiting hormone and mandibular organ-inhibiting hormone) and their expression levels in the Jonah crab, Cancer borealis

The crustacean hyperglycemic hormone (CHH) neuropeptide family has multiple functions in the regulation of hemolymph glucose levels, molting, ion, and water balance and reproduction. In crab species, three neuroendocrine tissues: the eyestalk ganglia (medulla terminalis X-organ and -sinus gland = ES), the pericardial organ (PO), and guts synthesize a tissue-specific isoforms of CHH neuropeptides. Recently the presence of the mandibular organ-inhibiting hormone (MOIH) was reported in the stomatogastric nervous system (STNS) that regulates the rhythmic muscle movements in esophagus, cardiac sac, gastric and pyloric ports of the foregut. In this study, we aimed to determine the presence of a tissue-specific CHH isoform in the Jonah crab, Cancer borealis using PCR with degenerate primers and 5′, 3′ rapid amplification of cDNA ends (RACE) in the ES. PO, and STNS. The analysis of CHH sequences shows that C. borealis has one type of CHH isoform, unlike other crab species. We also isolated the cDNA sequence of molt-inhibiting hormone (MIH) in the ES and MOIH in the ES and STNS. The presence of CHH, MOIH and MIH in the sinus gland of adult females and males is confirmed by using a dot-blot assay with the putative peaks collected from RP-HPLC and anti-Cancer sera for CHH, MIH, and MOIH. The present of crustacean female sex hormone (CFSH) in the sinus gland of adult females was examined with a dot-blot assay with anti-Callinectes CFSH serum. Levels of CHH, MOIH, and MIH in the sinus gland and their expressions in the eyestalk ganglia are estimated in the adult males, where CHH is the predominant form among these neuropeptides.

Diet composition and long-term starvation do not affect crustacean hyperglycemic hormone (CHH) transcription in the burrowing crab Neohelice granulata (Dana, 1851)

The burrowing crab Neohelice granulata is a key omnivorous species in intertidal areas along the southwestern Atlantic from southern Brazil to northern Argentinean Patagonia. This crab is adapted to starvation and can endure natural periods of food deprivation. The metabolic adjustments during starvation depend on the type of diet the crabs were fed previously. Since eyestalk-crustacean hyperglycemic hormone (CHH) is the principal regulator of glucose homeostasis in decapods, we investigated whether CHH transcription was affected by diet composition and starvation. Crabs were maintained in the laboratory for two weeks and subsequently divided in two groups. One received a high carbohydrate (HC) diet, and the other was fed a high protein (HP) diet. After this period, they were starved for four weeks. The full-length cDNA sequence of N. granulata CHH was determined and aligned with CHH sequences of other crabs. Levels of circulating glucose and glycogen were higher in the hepatopancreas and muscle of the HC-fed group and decreased after starvation. Glucose and glycogen concentrations were not altered by starvation in the HP group. Triglyceride levels within the hemolymph were not altered by diet or starvation. However, triglycerides concentration was higher in the hepatopancreas of HC compared to HP-fed group. Long-term starvation and diet composition did not affect CHH transcription.

Crustacean hyperglycemic hormone (CHH) regulates the ammonia excretion and metabolism in white shrimp, Litopenaeus vannamei under ammonia-N stress

To understand the adaptation of Litopenaeus vannamei to high environmental ammonia-N, RNA interference was used to investigate the function of crustacean hyperglycemic hormone (CHH) in the physiological process of neuroendocrine signaling transduction, and ammonia excretion and metabolism. The shrimp were exposed to 25 mg/L NH4Cl and injected with 20 μg/shrimp CHH dsRNA for 72 h. The results showed that hemolymph ammonia content increased under ammonia-N stress and further increased after CHH knockdown, suggesting that CHH can promote ammonia excretion. Moreover, after CHH knockdown, the levels of CHH, DA, and Wnts decreased significantly, the expression of receptor GC, DA1R, Frizzled and LRP 5/6 also decreased, while DA4R increased remarkably. Moreover, PKA and PKG decreased, while PKC markedly increased, and nuclear transcription factors (CREB and TCF) as well as effector proteins (β-catenin, FXYD2, and 14–3-3) were significantly downregulated. Furthermore, ammonia transporters Na+/K+-ATPase (NKA), K+channel, Rh protein, AQP, V-ATPase, and VAMP decreased significantly, while Na+/H+ exchangers (NHE) and Na+/K+/2Cl− cotransporter (NKCC) increased significantly. These results suggest that CHH regulates ammonia excretion in three ways: 1) by mainly regulating ion channels via PKA, PKC, and PKG signaling pathways; 2) by activating related proteins primarily through Wnt signaling pathway; and 3) by exocytosis, mostly induced by the PKA signaling pathway. In addition, the levels of Gln, uric acid, and urea increased in accordance with the activities of GDH/GS, XDH, and arginase, respectively, suggesting that ammonia excretion was inhibited but ammonia metabolism was slightly enhanced. This study deepens our understanding of the mechanism by which crustaceans respond to high environmental ammonia-N.

The Complete Genome of an Endogenous Nimavirus (Nimav-1_LVa) From the Pacific Whiteleg Shrimp Penaeus (Litopenaeus) Vannamei.

White spot syndrome virus (WSSV), the lone virus of the genus Whispovirus under the family Nimaviridae, is one of the most devastating viruses affecting the shrimp farming industry. Knowledge about this virus, in particular, its evolution history, has been limited, partly due to its large genome and the lack of other closely related free-living viruses for comparative studies. In this study, we reconstructed a full-length endogenous nimavirus consensus genome, Nimav-1_LVa (279,905 bp), in the genome sequence of Penaeus (Litopenaeus) vannamei breed Kehai No. 1 (ASM378908v1). This endogenous virus seemed to insert exclusively into the telomeric pentanucleotide microsatellite (TAACC/GGTTA)n. It encoded 117 putative genes, with some containing introns, such as g012 (inhibitor of apoptosis, IAP), g046 (crustacean hyperglycemic hormone, CHH), g155 (innexin), g158 (Bax inhibitor 1 like). More than a dozen Nimav-1_LVa genes are involved in the pathogen-host interactions. We hypothesized that g046, g155, g158, and g227 (semaphorin 1A like) were recruited host genes for their roles in immune regulation. Sequence analysis indicated that a total of 43 WSSV genes belonged to the ancestral/core nimavirus gene set, including four genes reported in this study: wsv112 (dUTPase), wsv206, wsv226, and wsv308 (nucleocapsid protein). The availability of the Nimav-1_LVa sequence would help understand the genetic diversity, epidemiology, evolution, and virulence of WSSV.

Regulation of amino acid and nucleotide metabolism by crustacean hyperglycemic hormone in the muscle and hepatopancreas of the crayfish Procambarus clarkia.

To comprehensively characterize the metabolic roles of crustacean hyperglycemic hormone (CHH), metabolites in two CHH target tissues of the crayfish Procambarus clarkii, whose levels were significantly different between CHH knockdown and control (saline-treated) animals, were analyzed using bioinformatics tools provided by an on-line analysis suite (MetaboAnalyst). Analysis with Metabolic Pathway Analysis (MetPA) indicated that in the muscle Glyoxylate and dicarboxylate metabolism, Nicotinate and nicotinamide metabolism, Alanine, aspartate and glutamate metabolism, Pyruvate metabolism, and Nitrogen metabolism were significantly affected by silencing of CHH gene expression at 24 hours post injection (hpi), while only Nicotinate and nicotinamide metabolism remained significantly affected at 48 hpi. In the hepatopancreas, silencing of CHH gene expression significantly impacted, at 24 hpi, Pyruvate metabolism and Glycolysis or gluconeogenesis, and at 48 hpi, Glycine, serine and threonine metabolism. Moreover, analysis using Metabolite Set Enrichment Analysis (MSEA) showed that many metabolite sets were significantly affected in the muscle at 24hpi, including Ammonia recycling, Nicotinate and nicotinamide metabolism, Pyruvate metabolism, Purine metabolism, Warburg effect, Citric acid cycle, and metabolism of several amino acids, and at 48 hpi only Nicotinate and nicotinamide metabolism, Glycine and serine metabolism, and Ammonia recycling remained significantly affected. In the hepatopancreas, MSEA analysis showed that Fatty acid biosynthesis was significantly impacted at 24 hpi. Finally, in the muscle, levels of several amino acids decreased significantly, while those of 5 other amino acids or related compounds significantly increased in response to CHH gene silencing. Levels of metabolites related to nucleotide metabolism significantly decreased across the board at both time points. In the hepatopancreas, the effects were comparatively minor with only levels of thymine and urea being significantly decreased at 24 hpi. The combined results showed that the metabolic effects of silencing CHH gene expression were far more diverse than suggested by previous studies that emphasized on carbohydrate and energy metabolism. Based on the results, metabolic roles of CHH on the muscle and hepatopancreas are suggested: CHH promotes carbohydrate utilization in the hepatopancreas via stimulating glycolysis and lipolysis, while its stimulatory effect on nicotinate and nicotinamide metabolism plays a central role in coordinating metabolic activity in the muscle with diverse and wide-ranging consequences, including enhancing the fluxes of glycolysis, TCA cycle, and pentose phosphate pathway, leading to increased ATP supply and elevated protein and nucleic acid turnovers.

Sex-Biased CHHs and Their Putative Receptor Regulate the Expression of IAG Gene in the Shrimp Litopenaeus vannamei.

The “eyestalk-androgenic gland (AG)-testis” endocrine axis is involved in male sexual differentiation of crustaceans. The insulin-like androgenic gland hormone (IAG), secreted from the AG, plays a central role in this axis, however key factors upstream the IAG are still poorly understood. Here, two crustacean hyperglycemic hormone (CHH) genes (LvCHH1 and LvCHH2) and their putative receptor guanylate cyclase (LvGC) were identified in Litopenaeus vannamei. LvCHH1 and LvCHH2 belonged to CHH subfamily I members and LvGC was a membrane-bound guanylate cyclase. They were all differentially expressed in eyestalks and gonads of males and females. RNA interference (RNAi) of either LvCHH1 or LvCHH2 increased LvIAG expression, while injection of their recombinant protein decreased LvIAG expression, indicating that LvCHH1 and LvCHH2 are inhibitory factors of LvIAG expression. Yeast two-hybrid assay showed that both LvCHH1 and LvCHH2 interacted with LvGC and their RNAi and recombinant protein injection exerted opposite regulatory effects on the transcriptional expression of LvGC. Meanwhile, knockdown of LvGC increased LvIAG expression. These results suggest that LvGC is the receptor of LvCHH1 and LvCHH2 and they are all involved in male sexual development by regulating LvIAG expression. The present study unveils missing upstream elements in the “eyestalk-AG-testis” endocrine axis in crustacean.

Comparative transcriptome reveals the potential modulation mechanisms of estradiol affecting ovarian development of female Portunus trituberculatus.

Estradiol is an important sex steroid hormone that is involved in the regulation of crustacean ovarian development. However, the molecular regulatory mechanisms of estradiol on ovarian development are largely unknown. This study performed transcriptome sequencing of ovary, hepatopancreas, brain ganglion, eyestalk, and mandibular organ of crabs after estradiol treatment (0.1μg g-1 crab weight). A total of 23, 806 genes were annotated, and 316, 1300, 669, 142, 383 genes were expressed differently in ovary, hepatopancreas, brain ganglion, eyestalk, and mandibular organ respectively. Differentially expressed gene enrichment analysis revealed several crucial pathways including protein digestion and absorption, pancreatic secretion, insect hormone biosynthesis, drug metabolism-cytochrome P450 and signal transduction pathway. Through this study, some key genes in correlation with the ovarian development and nutrition metabolism were significantly affected by estradiol, such as vitelline membrane outer layer 1-like protein, heat shock protein 70, Wnt5, JHE-like carboxylesterase 1, cytochrome P302a1, crustacean hyperglycemic hormone, neuropeptide F2, trypsin, carboxypeptidase B, pancreatic triacylglycerol lipase-like, and lipid storage droplet protein. Moreover, RT-qPCR validation demonstrated that expression of transcripts related to ovarian development (vitelline membrane outer layer 1-like protein and cytochrome P302a1) and nutrition metabolism (trypsin, glucose dehydrogenase and lipid storage droplet protein) were significantly affected by estradiol treatment. This study not only has identified relevant genes and several pathways that are involved in estradiol regulation on ovarian development of P. trituberculatus, but also provided new insight into the understanding of the molecular function mechanisms of estradiol in crustacean.

Advances in the Science of Crustacean Reproductive Physiology and Potential Applications to New Seed Production Technology

Wilder, M.N., 2019. Advances in the science of crustacean reproductive physiology and potential applications to new seed production technology. In: Jithendran, K.P.; Saraswathy, R.; Balasubramanian, C.P.; Kumaraguru Vasagam, K.P.; Jayasankar, V.; Raghavan, R.; Alavandi, S.V., and Vijayan, K.K. (eds.), BRAQCON 2019: World Brackishwater Aquaculture Conference. Journal of Coastal Research, Special Issue No. 86, pp. 6–10. Coconut Creek (Florida), ISSN 0749-0208.The shrimp culture industry continues to show expansion world-wide, and a large number of crustacean species having commercial importance are found within the order Decapoda. At present, eyestalk ablation is frequently used in hatcheries for marine shrimp in order to induce maturation under captivity, but the method is considered undesirable from the viewpoint of animal welfare. In this regard, this review details the current status of knowledge regarding crustacean reproductive mechanisms, especially in relation to research covering vitellogenin structure, structure and function of vitellogenesis-inhibiting hormone (VIH), identity of putative factors stimulating vitellogenesis, and applications of such research to developing new seed production technology. Regarding vitellogenin, full structure is now known for nearly 20 decapod species, not only for prawns/shrimps, but also for certain crab and crayfish species; vitellogenin molecules in these species have a primary structure consisting of more than 2,500 amino acid residues. VIH is a member of the crustacean hyperglycemic hormone (CHH)-family. Mature CHH-family peptides are typically comprised of 72-78 amino acids having six conserved cysteine residues. The complete identity of vitellogenesis-stimulating hormone (VSH) remains elusive, but eyestalk hormones such as red pigment-concentrating hormone (RPCH) may play a role in promoting ovarian maturation in certain species. The challenge for the future is to seek a viable alternative to eyestalk ablation based on hormonal manipulation and application of the above knowledge.

Effects of 17β-estradiol injection and red-spectrum light on eyestalk hormones and vitellogenesis of the ornamental cleaner shrimp Lysmata amboinensis (De Man, 1888) (Decapoda: Caridea: Lysmatidae)

Abstract We analyzed the effects of 17β-estradiol (E2) injection and red light on the sexual maturation and vitellogenesis of the cleaner shrimp Lysmata amboinensis (De Man, 1888) by measuring the change of mRNA expression levels of eyestalk and vitellogenesis-related hormones induced by both factors. We examined the eyestalk crustacean hyperglycemic hormone (CHH) and vitellogenesis-inhibiting hormone (VIH) genes. The E2 injection did not affect changes of eyestalk hormones. The red light, however, caused a significant increase (P &amp;lt; 0.05) of CHH promoting maturity and a significant decrease of VIH, which suppressed vitellogenesis. The vitellogenin receptor (VTG-R) and mRNA expression (in situ hybridization) also caused the E2 injection and red-wavelength irradiation to induce synthesis of vitellogenin (VTG). Red-light-induced gonadal maturation, promoting hormone and VTG synthesis, but E2 did not change the eyestalk hormone. Red-light irradiation could thus be used as a novel, maturity-promoting alternative method in the culture of the species, which is different from the eyestalk-ablation method that has so far been used.

Effects of crustacean hyperglycemic hormone (CHH) on regulation of hemocyte intracellular signaling pathways and phagocytosis in white shrimp Litopenaeus vannamei

Shrimps like other arthropods rely on innate immune system, and may have some form of adaptive immunity in defending against pathogens. Phagocytosis is one of the oldest cellular processes, serving as a development process, a feeding mechanism and especially as a key defense reaction in innate immunity of all multicellular organisms. It is confirmed that crustacean hyperglycemic hormone (CHH) is one of the most important neuropeptides produced by Neuro-endocrine Immune (NEI) regulatory network, which undertakes important roles in various biological processes, especially in immune function and stress response. In this study, the recombinant Litopenaeus vannamei CHH (rLvCHH) was obtained from a bacterial expression system and the intracellular signaling pathways involved in the mechanism of phagocytosis after rLvCHH injection was investigated. The results showed that the contents of adenylyl cyclase (AC), phospholipase C (PLC) and calmodulin (CaM) in hemocytes were increased significantly after rLvCHH injection. Furthermore, the mRNA expression levels of NF-kB family members (relish and dorsal) and phagocytosis-related proteins in hemocytes were basically overexpressed after rLvCHH stimulation, while the expression level of NF-kB repressing factor (NKRF) gene was down-regulated significantly. Eventually, the total hemocyte count and phagocytic activity of hemocyte were dramatically enhanced within 3 h. Collectively, these results indicate that shrimps L. vannamei could carry out a simple but ‘smart’ NEI regulation through the action of neuroendocrine factors, which could couple with their receptors and trigger the downstream signaling pathways during the phagocytic responses of hemocytes.

The serotonin or dopamine by cyclic adenosine monophosphate-protein kinase A pathway involved in the agonistic behaviour of Chinese mitten crab, Eriocheir sinensis

Agonistic behaviour is common in an encounter between two crustaceans. It often causes limb disability and consumes a lot of energy, which is harmful for the growth and survival of commercially important crustaceans. In the present study, we mainly focused on the agonistic behaviour of the Chinese mitten crab, Eriocheir sinensis, which is an important species of the aquaculture industry in China. We recorded agnostic behaviour with a high-definition camera and preliminarily evaluated the role of serotonin (5-HT) or dopamine (DA)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway and eyestalk in the behaviour. The results showed that agonistic behaviour in E. sinensis consisted of three stages: approach, contact and fight. We found that the number of fights and cumulative time of fight were significantly higher in the male vs. male group than in the female vs. female and female vs. male groups (P < 0.05). After 1 h of agonistic behaviour, 5-HT concentration showed a significant increase and DA concentration showed a significant decrease when compared with the control group (no encounter; P < 0.05). 5-HT1B and 5-HT2B mRNA levels showed a significant increase in the eyestalk (P < 0.05). 5-HT7 mRNA levels showed significant downregulation in the thoracic ganglia and DA1A mRNA levels showed upregulation in the intestine (P < 0.05). DA2 mRNA levels showed a significant decrease in the eyestalk (P < 0.05). These changes were accompanied by a significant increase in cAMP level and significant decrease in PKA level in the haemolymph (P < 0.05). In addition, a significant decrease in glucose levels was detected after the agonistic behaviour. Crustacean hyperglycemic hormone (CHH) mRNA levels showed significant upregulation in the eyestalk and significant downregulation in the intestine (P < 0.05). The number of fights and cumulative time of fight in the left eyestalk ablation (L-X vs. L-X) group were more and longer than those in the intact eyestalk (C vs. C), right eyestalk ablation (R-X vs. R-X) and bilateral eyestalk ablation (D-X vs. D-X) groups. In short, E. sinensis shows special agonistic behaviour modulated by 5-HT or DA-cAMP-PKA pathway and eyestalk, especially the left eyestalk.

Molecular characterization of a vitellogenesis-inhibiting hormone (VIH) in the mud crab (Scylla olivacea) and temporal changes in abundances of VIH mRNA transcripts during ovarian maturation and following neurotransmitter administration

The vitellogenesis-inhibiting hormone (VIH), also known as gonad-inhibiting hormone, is a neuropeptide hormone in crustaceans that belongs to the crustacean hyperglycemic hormone (CHH)-family peptide. There is regulation vitellogenesis by VIH during gonad maturation in crustaceans. A full-length Scylla olivacea VIH (Scyol-VIH) was identified through reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. The open reading frame consists of 378 nucleotides, which encodes a 126-amino acid precursor protein, including a 22-residue signal peptide and a 103-amino acid mature peptide in which 6 highly conserved cysteine residues are present. There was expression of the Scyol-VIH gene in immature female Scylla olivacea in the eyestalk, brain and ventral nerve cord. The Scyol-VIH gene expression was localized to the eyestalk X-organ, brain neuronal clusters 6 and 11, and in multiple neuronal clusters of the ventral nerve cord. The relative abundance of Scyol-VIH mRNA transcript in the eyestalk was relatively greater in immature stage females, then decreased as ovarian maturation progressed. Furthermore, eyestalk Scyol-VIH increased after dopamine (5 μg/g BW) injection. The present research provides fundamental information about Scyol-VIH and its potential effect in controlling reproduction.

Crustacean hyperglycemic hormone (CHH) affects hemocyte intracellular signaling pathways to regulate exocytosis and immune response in white shrimp Litopenaeus vannamei

Recombinant Litopenaeus vannamei CHH (rLvCHH) was obtained from a bacterial expression system and the intracellular signaling pathways involved in exocytosis and immune response after rLvCHH injection (0.2 and 2 μg/shrimp) was investigated in this study. The results showed that CHH contents increased 51.4%–110.2% (0.2 μg/shrimp) and 65.0%–211.3% (2 μg/shrimp) of the control level. And the contents of three biogenic amines in hemolymph presented a similar variation pattern after rLvCHH injection, but reached the highest level at different time points. Furthermore, the mRNA expression levels of membrane-bound guanylyl cyclase (mGC) (1.20–1.93 fold) and biogenic amine receptors, including type 2 dopamine receptor (DA2R) (0.72-0.89 fold), α2 adrenergic receptor (α2-AR) (0.72-0.91 fold) and 5-HT7 receptor (5-HT7R) (1.37–3.49 fold) in hemocytes were changed consistently with their ligands. In addition, the second messenger and protein kinases shared a similar trend and reached the maximum at the same time respectively. The expression levels of nuclear transcription factor (cAMP response element-binding protein, CREB) and exocytosis-related proteins transcripts were basically overexpressed after rLvCHH stimulation, which reached the peaks at 1 h or 3 h. Eventually, the phenoloxidase (PO) activity (37.4%–158.5%) and antibacterial activity (31.8%–122.3%) in hemolymph were dramatically enhanced within 6 h, while the proPO activity in hemocytes significantly decreased (11.2%–62.6%). Collectively, these results indicate that shrimps L. vannamei could carry out a simple but ‘smart’ NEI regulation by releasing different neuroendocrine factors at different stages after rLvCHH stimulation, which could couple with their receptors and trigger the downstream signaling pathways during the immune responses in hemocytes.