Vitellogenesis-inhibiting Hormone Research Articles

Activation and characterization of G protein-coupled receptors for CHHs in the mud crab, Scylla paramamosain

Crustacean hyperglycemic hormone (CHH) superfamily peptides constitute a group of neurohormones, including the crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH), which reportedly play an essential role in regulating various biological activities by binding to their receptors in crustaceans. Although bioinformatics analyses have identified G protein-coupled receptors (GPCRs) as potential CHH receptors, no validation through binding experiments has been carried out. This study employed a eukaryotic expression system, HEK293T cell transient transfection, and ligand-receptor interaction tests to identify the GPCRs of CHHs in the mud crab Scylla paramamosain. We found that four GPCRs (Sp-GPCR-A34-A37) were activated by their corresponding CHHs (Sp-CHH1-v1, Sp-MIH, Sp-VIH) in a dose-dependent manner. Of these, Sp-GPCR-A34 was exclusively activated by Sp-VIH; Sp-GPCR-A35 was activated by Sp-CHH1-v1 and Sp-VIH, respectively; Sp-GPCR-A36 was activated by Sp-CHH1-v1 and Sp-MIH; Sp-GPCR-A37 was exclusively activated by Sp-MIH. The half-maximal effective concentration (EC50) values for all CHHs/GPCRs pairs (both Ca2+ and cAMP signaling) were in the nanomolar range. Overall, our study provided hitherto undocumented evidence of the presence of G protein-coupled receptors of CHH in crustaceans, providing the foothold for further studies on the signaling pathways of CHHs and their corresponding GPCRs.

Potent ovarian development as being stimulated by cocktail hormone in the female Scylla olivacea

The mud crab Scylla olivacea is widely cultured for its economic value, but reproduction issues limit its production. Vitellogenesis-inhibiting hormone (VIH), serotonin (5-HT), and gonadotropin-releasing hormone (GnRH) are important neurohormones that control reproduction in crustaceans. Mimicking crab hormone stimulation during reproduction has scarcely been reported. Comparison of the single hormone and multiple hormone approaches to crab hormonal control in S. olivacea is limited. In situ hybridization showed that injection of dsRNA-VIH could abolish its gene expression in neuronal clusters of female S. olivacea eyestalks, potentially reducing its inhibitory effects on ovarian maturation. This was confirmed by assessing the ovarian gonadosomatic index (GSI), hemolymph vitellogenin (Vg), an indicator of vitellogenesis, and gonad histology using dsRNA-VIH and 5-HT/GnRH combinations. Based on our findings, we demonstrated that administration of dsRNA-VIH significantly increased the gonadosomatic index (GSI) on days 14 and 28 post-treatment. The combination cocktail, however, consisting of 5-HT + GnRH + dsRNA-VIH on days 14 and 28, and GnRH + dsRNA-VIH on day 28, was the most efficacious in increasing GSI and enhancing crab ovarian maturation. Upregulation of hemolymph Vg levels was observed solely on the 28th day following treatment with dsRNA-VIH, 5-HT + GnRH + dsRNA-VIH, and GnRH + dsRNA-VIH. Differential gene expression analysis using quantitative RNA-sequencing of the neural tissues (brain and ventral nerve cord), revealed a significant upregulation of certain receptors (5-HTR, GnRHR, LHR, and FSHR), neuropeptides (sNPF, NPF1, NPF2, SIFamide, AKH/Crz, CHH, and RPCH), downstream reproductive-related genes (FAMeT, ESULT, progesterone-like protein), and prostanoid-related genes (phospholipase A and C, COX, Thromboxane A synthase, prostaglandin D, E, and F synthases) following treatment, particularly dsRNA-VIH + GnRH and/or 5-HT-injected individuals. Upregulation of prostaglandin E synthase and estrogen sulfotransferase genes was confirmed by real-time PCR. Since the construction and propagation of dsRNA-VIH is costly, its lower dose application supplemented with synthetic GnRH and/or 5-HT may be an alternative approach to ensure that female S. olivacea attain sufficient reproductive fecundity in aquaculture. Furthermore, we propose that the administration of multiple hormones in crabs may better emulate the physiological conditions of crustaceans in their natural habitat.

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

The vitellogenesis-inhibiting hormone (SpVIH) of the crab Scylla paramamosain is not likely to have a vitellogenesis-inhibiting function in the ovary

The function and molecular characterization of the previously reported crab (Scylla paramamosain) vitellogenesis inhibiting hormone SpVIH is still unclear. In this experiment, SpVIH’s bioinformatic and functional characterizations were analyzed. Sequence analysis showed SpVIH was clustered with several type II MIH/GIHs of the Brachyura. The expression of SpVIH in the eyestalk was low in the early ovarian developmental stage I and increased with the advancement of ovary maturation. Interestingly, the SpVIH transcript was still at a high level in the ovary of females at the middle to late maturation stage. SpVIH’s function was studied according to the expression of vitellogenin (Vg) and vitellogenin receptor (VgR) in the ovary and hepatopancreas after incubation with SpVIH-dsRNA and recombinant SpVIH protein (rSpVIH). In vitro assay results indicated that rSpVIH has a significant stimulative effect on the expression of Vg in the hepatopancreas in the middle stage of vitellogenesis and in the ovary of each vitellogenesis stage. However, SpVIH-dsRNA has no significant effect on the expression of Vg when SpVIH-dsRNA is introduced to the ovary and hepatopancreas at the early, middle, and late stages of vitellogenesis. Furthermore, the co-incubation experiment result indicated that the eyestalk consists of factor(s) that can significantly up-regulate the expression of Vg in the ovary, with a further combination of dsVIH and eyestalk under incubation, the significant increase of Vg-mRNA expression levels were also found in the ovary of four ovarian development stages of S. paramamosain. Contrary to the previous reports, SpVIH does not possess a vitellogenesis-inhibiting function but can promote vitellogenesis in the ovary and hepatopancreas.

Regulation of CHH family in eyestalk by A-type allatostatin in the mud crab Scylla paramamosain

The eyestalk ganglion is a principal neuroendocrine organ in crustaceans for the synthesis, storage and release of the crustacean hyperglycemic hormone neuropeptide (CHH) family. A-type allatostatin (AST-A) is involved in a variety of physiological functions, although its function in regulating the CHH family in the eyestalk ganglion has not been reported. The present study identified AST-A in the mud crab Scylla paramamosain. AST-A was highly expressed in the ganglia as shown by reverse transcription PCR, while AST-A receptor (AST-AR) was expressed in multiple tissues, such as the eyestalk ganglion. Both in vitro and in vivo experiments revealed that AST-A mature peptide significantly increased mRNA expression of AST-AR, vitellogenesis-inhibiting hormone (VIH), molt-inhibiting hormone (MIH), and crustacean hyperglycemic hormone (CHH). In contrast, RNA interference by injection of AST-AR-dsRNA into crabs significantly reduced the expressions of VIH, MIH, and CHH in the eyestalk ganglion. This study reports for the first time that AST-A might play roles in ovarian development, molting and glucose metabolism in S. paramamosain via regulating CHH family neuropeptides in the eyestalk ganglion.

Role of short neuropeptide F in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain

The eyestalk ganglion serves as an essential endocrine organ in Crustacea. Although the short neuropeptide F (sNPF) is involved in a broad range of physiological functions via its counterpart receptor (sNPFR), its roles in regulating the crustacean hyperglycemic hormone (CHH) family in the eyestalk ganglion is still unclear. In this study, reverse transcription PCR indicates that the sNPF was most highly expressed in the eyestalk ganglion, whereas sNPFR was expressed in 11 tissues of the mud crab Scylla paramamosain, including eyestalk ganglion. Immunohistochemistry analyses further display that sNPF immunoreactive neurons were localized in the X-organ of eyestalk ganglion. Moreover, the stimulatory effect of sNPF on the gene expression of vitellogenesis-inhibiting hormone (VIH), molt-inhibiting hormone (MIH), and crustacean hyperglycemic hormone (CHH) in the eyestalk ganglion was confirmed by in vivo experiment. In contrast, RNA interference by injection of sNPF dsRNA or sNPFR dsRNA into crabs significantly reduced VIH, MIH, and CHH expressions in the eyestalk ganglion, respectively. This study first reported that sNPF might play roles in the reproduction, molting and glucose metabolism in S. paramamosain via regulating CHH family neuropeptides in the eyestalk ganglion.

Acceleration of Ovarian Maturation in the Female Mud Crab With RNA Interference of the Vitellogenesis-Inhibiting Hormone (VIH)

In the present study, double strand RNA technology (dsRNA) was used to inhibit transcripts of vitellogenesis-inhibiting hormone (VIH) that mainly synthesized and secreted from the central nervous system in Scylla olivacea females. The results presented in this study clearly demonstrate the potential dsRNA-VIH was highly effective to inhibit VIH in the eyestalks of females injected with dsRNA-VIH on the 3rd, 7th and 14th day, respectively. The dsRNA-VIH injections were performed at 14-day intervals, a single dsRNA dose of 0.6 µg/gram body weight was enough to suppress VIH expression until 14th day after injection. The dsRNA-VIH injection significantly increased gonad-somatic index (GSI) and hemolymph vitellin level at day 14 and 28 when compared with control groups. The histological observation found that the number of oocyte step 4 in dsRNA-VIH group was significantly higher than that of the control group. Also, dsRNA-VIH has stimulatory function on other reproduction-related genes such as the Scyol-PGES and Scyol-ESULT that both genes gradually increased their expressions in brain and ventral nerve cord. In conclusion, the silence of VIH gene could reduce the production of VIH from eyestalk and brain that affected other downstream genes related to ovarian maturation in the mud crab.

Suppression of a Novel Vitellogenesis-Inhibiting Hormone Significantly Increases Ovarian Vitellogenesis in the Black Tiger Shrimp, Penaeus monodon.

In this study, a novel Crustacean Hyperglycemic Hormone-type II gene (CHH-type II) was identified and biologically characterized in a shrimp, Penaeus monodon. Based on its structure and function, this gene was named P. monodon vitellogenesis-inhibiting hormone (PemVIH). The complete cDNA sequence of PemVIH consisted of 1,022 nt with an open reading frame (ORF) of 339 nt encoding a polypeptide of 112 amino acids. It was classified as a member of the CHH-type II family based on conserved cysteine residues, a characteristically positioned glycine residue, and the absence of CHH precursor-related peptide (CPRP) domain. The deduced mature PemVIH shared the highest sequence similarities with giant river prawn sinus gland peptide A. Unlike P. monodon gonad-inhibiting hormone (PemGIH), PemVIH was expressed only in the brain and ventral nerve cord, but not the eyestalks. Whole mount immunofluorescence using a newly generated PemVIH antiserum detected positive signals in neuronal cluster 9/11 and 17 of the brain, commissural ganglion (CoG), and neuronal clusters of ventral nerve cord. The presence of PemVIH-positive neurons in CoG, a part of stomatogastric nervous system, suggested a potential mechanism for crosstalk between nutritional and reproductive signaling. The role of PemVIH in vitellogenesis was evaluated using RNA interference technique. Temporal knockdown of PemVIH in female subadults resulted in a 3-fold increase in ovarian vitellogenin expression, suggesting an inhibitory role of PemVIH in vitellogenesis. This study provided novel insight into the control of vitellogenesis and additional strategies for improving ovarian maturation in P. monodon without the current harmful practice of eyestalk ablation.

Progress and challenges in the comprehensive management of chronic viral hepatitis: Key ways to achieve the elimination.

Chronic viral hepatitis is a significant health problem throughout the world, which already represents high annual mortality. By 2040, chronic viral hepatitis due to virus B and virus C and their complications cirrhosis and hepatocellular carcinoma will be more deadly than malaria, vitellogenesis-inhibiting hormone, and tuberculosis altogether. In this review, we analyze the global impact of chronic viral hepatitis with a focus on the most vulnerable groups, the goals set by the World Health Organization for the year 2030, and the key points to achieve them, such as timely access to antiviral treatment of direct-acting antiviral, which represents the key to achieving hepatitis C virus elimination. Likewise, we review the strategies to prevent transmission and achieve control of hepatitis B virus. Finally, we address the impact that the coronavirus disease 2019 pandemic has had on implementing elimination strategies and the advantages of implementing telemedicine programs.

Comparison of the effect of red light exposure and eyestalk ablation for inducing sexual maturation and improving egg quality in the cleaner shrimp Lysmata amboinensis

Abstract Female cleaner shrimps Lysmata amboinensis either underwent eyestalk ablations (EA), or were treated with two different wavelength intensities of red light (peak at 630 nm), and then their sex maturation and the effects on their egg quality parameters were compared. Vitellogenesis-inhibiting hormone (VIH), vitellogenin (VTG), VTG-receptor (VTG-R), and crustacean hyperglycemic hormone (CHH) expressions in the hepatopancreas were analysed. VTG-R expression (in situ) levels that caused the EA and red irradiation to induce the synthesis of VTG-R were assessed, and the triglycerides (TG) and glucose levels in the eggs were analysed. VIH expression was decreased in all experimental groups, but VTG and VTG-R expressions were increased. CHH expression and TG and glucose concentrations were higher in the red irradiation group, compared to the EA group. These results confirmed that the red light not only had a positive effect on the promotion of sex maturation, but it also improved the quality of the eggs, when compared to the EA method.

Assessment of the Effects of Double-Stranded RNAs Corresponding to Multiple Vitellogenesis-Inhibiting Hormone Subtype I Peptides in Subadult Female Whiteleg Shrimp, Litopenaeus vannamei.

Vitellogenesis-inhibiting hormone (VIH) negatively regulates reproduction in shrimp and other decapod crustaceans. In order to assess the effects of transcriptional silencing by multiple VIH subtype I sinus gland peptides (SGPs) on ovarian maturation in female whiteleg shrimp, Litopenaeus vannamei, we synthesized five dsRNAs targeting Liv-SGP-A, −B, −C, −F, and −G and injected them into subadults. The following treatments were employed: sgpG-dsRNA (targeting Liv-SGP-G), sgpC-dsRNA (targeting Liv-SGP-C), and mixed-dsRNA (targeting Liv-SGP-A, −B, and −F). The expression of Liv-SGP-G in eyestalks was significantly decreased at 10, 20, and 30 days after the injection of sgpG-dsRNA In addition, it was significantly decreased at 10 and 30 days after the injection of mixed-dsRNA. The expression of vitellogenin (Vg) gene expression in the ovaries, and concentrations of Vg protein in the hemolymph, were not changed by the administration of any dsRNA treatment (the ovaries remained immature in all treated individuals and contained mostly oogonia and previtellogenic oocytes). Although the administration of dsRNAs corresponding to multiple VIHs did not promote ovarian maturation, this is the first report of the co-transcriptional repression of Liv-SGP-G by the injection of dsRNA for homologous genes (Liv-SGP-A, −B, and −F). These results indicate that subadults can respond to the techniques of transcriptional silencing.

Transcriptional silencing of vitellogenesis-inhibiting and molt-inhibiting hormones in the giant freshwater prawn, Macrobrachium rosenbergii, and evaluation of the associated effects on ovarian development

In crustaceans, vitellogenesis-inhibiting hormone (VIH) plays a major regulatory role in vitellogenesis and the reproductive cycle, and molt-inhibiting hormone (MIH) controls both the molt cycle and the conditional pre-mating molt of mature Macrobrachium rosenbergii females occurring before every egg spawning event. In the current study, we investigated the effect of silencing the VIH gene alone or in combination with silencing the MIH gene on vitellogenesis and ovarian maturation in immature and mature M. rosenbergii females with the aim of determining the associated effects on ovarian development. The first step involved the construction of a dsRNA based on sequence alignment. Importantly, the sequence alignment and phylogenetic relationship between M. rosenbergii (Mr) VIH and Mr-MIH revealed that nucleotides 251–508 of VIH are almost identical to a sequence in Mr-MIH. However, nucleotides 9–250 – including the untranslated region (UTR; nucleotides 9–172) and the first 78 nucleotides of the open reading frame (ORF) - of VIH are completely different from the comparable MIH sequence. As a strategy to induce vitellogenesis and hence ovarian maturation, we used a single dsRNA retrieved from the above-mentioned conserved ORF (dsORF) sequence to silence VIH and MIH simultaneously. Another dsRNA retrieved from the distinct (dsDIS) sequence was used to silence VIH alone. Our results indicated that both dsRNAs are capable of inducing VIH silencing, as shown by a decline in mRNA levels. The results also indicated that VIH silencing is more effective in mature adults than in immature animals, leading to ovary maturation and vitellogenesis. Mature females injected with dsORF showed a decline in MIH levels. In addition, dsDIS also affected the expression of MIH, but the effect lagged behind that of dsORF by one week. We therefore concluded that dsDIS might be as effective as dsORF in silencing VIH, but without a marked influence on MIH and its mutual effects on reproduction and growth. The study thus lays down the foundation for the development of a unique novel tool for molecular manipulation of ovarian maturation and induction of the reproductive cycle in mature M. rosenbergii females. The suggested strategy might prove valuable for sustainable culture protocols not only in M. rosenbergii but also in other crustacean species. Further investigation is, however, needed to elucidate the regulation of the expression of VIH and MIH and how the neuroendocrine system controls growth molt, reproductive molt and reproduction in crustaceans.

Transcriptional Regulation of Vih by Oct4 and Sox9 in Scylla paramamosain.

Mud crab (Scylla paramamosain) is one of the most economically-important marine crabs in China. However, research on mechanisms of reproductive regulation is not sufficient. Vitellogenesis-inhibiting hormone (VIH) is a member of the crustacean hyperglycemia hormones (CHH) family, which plays an essential role in the regulation of gonadal development and maturation in crustaceans, and current studies on the regulation of Vih transcription in crabs are relatively rare. Our previous studies on the transcriptional regulation of mud crab Vih (SpVih) have proved that the binding site of Oct4/Sox9 transcription factor may be the key region for positively regulating the expression of SpVih. In this study, the electrophoretic mobility shift assay (EMSA) experiment confirmed that the nuclear protein extracted from the eyestalk could bind to the key region of SpVih promoter, and these specific bindings were dependent on the presence of Oct4/Sox9 binding sites. Two specific binding complex bands were detected in the supershift group of EMSA supershift experiments by Oct4 and Sox9 antibodies, further confirming the specific recognition of these two transcription factors on the key regulatory region of SpVih. In vitro, Oct4 and Sox9 gene overexpression vectors and SpVih core promoter fragment vector were constructed and co-transfected into HEK293T cells. As a result, SpVih activity increased with the concentration of transcription factors. In vivo, when Oct4 and Sox9 dsRNA were injected into the eyestalks of mud crab, respectively, the expression level of SpVih decreased significantly after interference with Oct4 or Sox9, and the expression level of SpVtg in the ovary and hepatopancreatic increased. Both in vitro and in vivo experiments showed that Oct4 and Sox9 had a positive regulatory effect on SpVih. The GST pull-down experiment was carried out by purified Oct4 and Sox9 proteins, and the results showed that there was an interaction between them. It was speculated that they regulated the expression of SpVih through the interaction.

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.

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.

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 < 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.

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.

Transcriptional silencing of vitellogenesis-inhibiting hormone (VIH) subtype-I in the whiteleg shrimp, Litopenaeus vannamei

Eyestalk ablation, which is a method for the removal of the source of vitellogenesis-inhibiting hormone (VIH), is frequently used in Litopenaeus vannamei in order to stimulate ovarian maturation and spawning in captivity. In this study, we assessed the effects of vih silencing using double-stranded RNA (dsRNA) in L. vannamei females. The relative expression levels of vitellogenin (vg) and vih were analyzed in the ovary and eyestalks, respectively, and levels of Vg and VIH in hemolymph were quantified at 10 and 20 days following injection with VIH-dsRNA, green fluorescent protein (GFP)-dsRNA (a non–VIH-related dsRNA), or Tris-EDTA (TE) buffer. Expression of vih in the eyestalks was significantly suppressed at 10 and 20 days after VIH-dsRNA injection. Hemolymph VIH levels were significantly increased at 10 days after VIH-dsRNA injection and then decreased to initial levels. However, there were no significant differences in the levels of Vg in hemolymph or vg mRNA in the ovary.

Modulation of complex coordinated molecular signaling by 5HT and a cocktail of inhibitors leads to ovarian maturation of Penaeus monodon in captivity.

In aquaculture practices, prawn cultivation holds the major share and Penaeus monodon is the main species cultured. The decline in production of P. monodon is mainly due to the limited availability of domesticated broodstock, which is attributed to its reproductive cycle, controlled by complex coordinated signaling mechanisms. Unilateral eyestalk ablation of domesticated females held in captivity is done to induce ovary development, which has certain disadvantages, including a high rate of mortality. Thus, developing alternative techniques for eyestalk ablation in captive broodstock is necessary to induce maturation of ovary. This study exemplifies the role of 5HT along with a cocktail of inhibitors (U0126, Rp-cAMP, and LY294002) in inducing ovarian maturation. In this study, inhibition of pERK by U0126 inhibited vitellogenesis-inhibiting hormone (VIH), which in turn led to the overexpression of vitellogenin. 5HT induces steroidogenesis (estradiol-17β) through induction of the gonadotropin-releasing hormone by activating calcium-calmodulin signaling. Steroidogenesis is also aided by synthesis of StAR protein. Estradiol-17β stimulates the formation of the maturation-promoting factor (MPF) complex by cdc25 activation and Myt1 inactivation. LY294002 aids in keeping cdc25 activated by inhibiting calcium-calmodulin induced phosphorylation of Akt which is a negative regulator of mitogen-activated protein kinases. VIH induced activation of Myt1, through protein kinase A (PKA), was inhibited by Rp-cAMP which inhibits adenylate cyclase, thus stabilizing the activated MPF complex. To conclude, the coordinated effect of inhibitors and 5HT accelerates the development of ovary from previtellogenic to matured oocytes, yielding high quality and quantity larvae compared with eyestalk-ablated P. monodon.