Rnai-based functional analysis of bursicon genes related to cuticle pigmentation in a ladybird beetle.

In arthropods, the binding of a bursicon (encoded by burs and pburs) heterodimer or homodimer to a leucine-rich repeat-containing G protein coupled receptor LGR2 (encoded by rk) can activate many physiological processes, especially cuticle pigmentation during insect ecdysis. In the current paper, we intended to ascertain whether bursicon signaling mediates body coloration in the 28-spotted larger potato ladybird, Henosepilachna vigintioctomaculata, and if so, by which way bursicon signal governs the pigmentation. The high expression of Hvburs, Hvpburs and Hvrk occurred in the young larvae, pupae and adults, especially in the head and ventral nerve cord. RNA interference (RNAi) aided knockdown of Hvburs, Hvpburs or Hvrk in the prepupae caused similar phenotypic defects. The pigmentation of the resultant adults was affected, with significantly reduced dark areas on the sternums. Moreover, the accumulated mRNA levels of two sclerotin biosynthesis genes, aspartate 1-decarboxylase gene Hvadc and N-ß-alanyldopamine synthase gene Hvebony, were significantly increased in the Hvburs, Hvpburs or Hvrk RNAi beetles. Furthermore, depletion of either Hvadc or Hvebony could completely rescue the impaired coloration on the sternums of Hvpburs RNAi adult. Our results supported that bursicon heterodimer-mediated signal regulate cuticle pigmentation. The bursicon signaling may tune the ratio of melanins (dark/black, brown) to sclerotins (light yellow, colorless) exerting its regulative role in the pigmentation of H. vigintioctomaculata sternums.

A new type II CHH neuropeptide involves ovarian development in the peppermint shrimp, Lysmata vittata.

Type II crustacean hyperglycemic hormone (CHH) neuropeptides play diverse roles in crustaceans. In the hermaphrodite shrimp Lysmata vittata, two transcripts of type II CHHs (molt-inhibiting hormone/gonad-inhibiting hormone, MIH/GIH1 and MIH/GIH2) were identified by transcriptome sequencing, and MIH/GIH1 was later named Lvit-GIH1 for its inhibitory effect on ovarian development. Based on the high similarity of MIH/GIH2 to Lvit-GIH1, we named tentatively MIH/GIH2 as Lvit-GIH2 and explored the role of Lvit-GIH2 in ovarian development. The open reading frame (ORF) of Lvit-GIH2 was 333 bp in length, encoding a precursor consisted of a 32-aa signal peptide and a 78-aa mature peptide, which shared high sequence similarity with the type II subfamily peptides in crustaceans. Notably, Lvit-GIH2 was widely expressed in multiple tissues. The qRT-PCR findings indicated a rising trend in the expression of Lvit-GIH2 from the male phase to the euhermaphrodite phase. Both RNA interference and addition of GIH2 recombinant proteins (rGIH2) experiments showed that Lvit-GIH2 suppressed Lvit-Vg expression in hepatopancreas and Lvit-VgR expression in ovary. To further investigate the role of Lvit-GIH2 in ovarian development, the RNA-sequence analysis was performed to examine the changes in ovary after addition of rGIH2. The results showed that the pathways (Cysteine and methionine metabolism, Apoptosis-multiple species, etc.) and the genes (17bHSD8, IGFR, CHH, etc.) related to ovarian development were negatively regulated by rGIH2. In brief, Lvit-GIH2 might inhibit the ovarian development in L. vittata.

Identification of ETH receptor and its possible roles in the mud crab Scylla paramamosain.

Ecdysis-triggering hormone (ETH) is a neuropeptide hormone characterized by a conserved KxxKxxPRx amide structure widely identified in arthropods. While its involvement in the regulation of molting and reproduction in insects is well-established, its role in crustaceans has been overlooked. This study aimed to de-orphanise a receptor for ETH in the mud crab Scylla paramamosain and explore its potential impact on ovarian development. A 513-amino-acid G protein-coupled receptor for ETH (SpETHR) was identified in S. paramamosain, exhibiting a dose-dependent activation by SpETH with an EC50 value of 75.18 nM. Tissue distribution analysis revealed SpETH was in the cerebral ganglion and thoracic ganglion, while SpETHR was specifically expressed in the ovary, hepatopancreas, and Y-organ of female crabs. In vitro experiments demonstrated that synthetic SpETH (at a concentration of 10-8 M) significantly increased the expression of SpVgR in the ovary and induced ecdysone biosynthesis in the Y-organ. In vivo experiments showed a significant upregulation of SpEcR in the ovary and Disembodied and Shadow in the Y-organ after 12 h of SpETH injection. Furthermore, a 16-day administration of SpETH significantly increased 20E titers in hemolymph, gonadosomatic index (GSI) and oocyte size of S. paramamosain. In conclusion, our findings suggest that SpETH may play stimulatory roles in ovarian development and ecdysone biosynthesis by the Y-organ.

Egg-laying hormone expression in identified neurons across developmental stages and reproductive states of the nudibranch Berghia stephanieae.

Neuropeptides play essential roles in coordinating reproduction. Egg-laying hormone (ELH) is conserved in genetic sequence and behavioral function across molluscs, where neuronal clusters secrete ELH to modulate and induce egg-laying. Here we investigated ELH in the nudibranch mollusc, Berghia stephanieae. ELH preprohormone gene orthologs, which showed clade-specific differences at the C-terminus of the predicted bioactive peptide, were identified in brain transcriptomes across several nudipleuran species, including B. stephanieae. ELH shares deep homology with the corticotropin-releasing hormone gene family, which has roles broadly in stress response. Injection of synthesized B. stephanieae ELH peptide into mature individuals induced egg-laying. ELH gene expression in the brain and body was mapped using in-situ hybridization chain reaction. Across the adult brain, 300-400 neurons expressed ELH. Twenty-one different cell types were identified in adults, three of which were located unilaterally on the right side, which corresponds to the location of the reproductive organs. Ten cell types were present in pre-reproductive juvenile stages. An asymmetric cluster of approximately 100 small neurons appeared in the right pedal ganglion of late-stage juveniles. Additional neurons in the pleural and pedal ganglia expressed ELH only in adults that were actively laying eggs and sub-adults that were on the verge of doing so, implicating their direct role in reproduction. Outside the brain, ELH was expressed on sensory appendages, including in presumptive sensory neurons. Its widespread expression in the nudibranch B. stephanieae suggests that ELH plays a role beyond reproduction in gastropod molluscs.

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.

Neuropeptide hormone bursicon mediates female reproduction in the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae).

Bursicon, a neuropeptide hormone comprising two subunits-bursicon (burs) and partner of burs (pburs), belongs to the cystine-knot protein family. Bursicon heterodimers and homodimers bind to the lucine-rich G-protein coupled receptor (LGR) encoded by rickets to regulate multiple physiological processes in arthropods. Notably, these processes encompass the regulation of female reproduction, a recent revelation in Tribolium castaneum. In this study we investigated the role of burs/pburs/rickets in mediating female vitellogenesis and reproduction in a hemipteran insect, the whitefly, Bemisia tabaci. Our investigation unveiled a synchronized expression of burs, pburs and rickets, with their transcripts persisting detectable in the days following eclosion. RNAi-mediated knockdown of burs, pburs or rickets significantly suppressed the transcript levels of vitellogenin (Vg) and Vg receptor in the female whiteflies. These effects also impaired ovarian maturation and female fecundity, as evidenced by a reduction in the number of eggs laid per female, a decrease in egg size and a decline in egg hatching rate. Furthermore, knockdown of burs, pburs or rickets led to diminished juvenile hormone (JH) titers and reduced transcript level of Kruppel homolog-1. However, this impact did not extend to genes in the insulin pathway or target of rapamycin pathway, deviating from the results observed in T. castaneum. Taken together, we conclude that burs/pburs/rickets regulates the vitellogenesis and reproduction in the whiteflies by coordinating with the JH signaling pathway.

Eyestalk neuropeptide identification in the female red deep-sea crab, Chaceon quinquedens.

Eyestalk-derived neuropeptides, primarily the crustacean hyperglycemic hormone (CHH) neuropeptide family, regulate vitellogenesis in decapod crustaceans. The red deep-sea crab, Chaceon quinquedens, a cold-water species inhabiting depths between 200 and 1800 m, has supported a small fishery, mainly harvesting adult males in the eastern US for over 40 years. This study aimed to understand the role of eyestalk-neuropeptides in vitellogenesis in C. quinquedens with an extended intermolt stage. Chromatography shows two CHH and one MIH peak in the sinus gland, with a CHH2 peak area four times larger than CHH1. The cDNA sequence of MIH and CHH of C. quinquedens is isolated from the eyestalk ganglia, and the qPCR assay shows MIH is significantly higher only at ovarian stages 3 than 4 and 5. However, MIH transcript and its neuropeptides do differ between stages 1 and 3. While CHH transcripts remain constant, its neuropeptide levels are higher at stages 3 than 1. Additionally, transcriptomic analysis of the de novo eyestalk ganglia assembly at ovarian stages 1 and 3 found 28 eyestalk neuropeptides. A GIH/VIH or GSH/VSH belonging to the CHH family is absent in the transcriptome. Transcripts per million (TPM) values of ten neuropeptides increase by 1.3 to 2.0-fold at stage 3 compared to stage 1: twofold for Bursicon α, followed by CHH, AKH/corazonin-like, Pyrokinin, CCAP, Glycoprotein B, PDH1, and IDLSRF-like peptide, and 1.3-fold of allatostatin A and short NP-F. WXXXRamide, the only downregulated neuropeptide, decreases TPM by âˆ¼ 2-fold at stage 3, compared to stage 1. Interestingly, neuroparsin with the highest TPM values remains the same in stages 1 and 3. The mandibular organ-inhibiting hormone is not found in de novo assembly. We report that CHH, MIH, and eight other neuropeptides may play a role in vitellogenesis in this species.

RNAi-based functional analysis of bursicon genes related to wing expansion in gypsy moths.

Bursicon is a heterodimeric neuropeptide composed of Burs-α and Burs-ß subunits that plays an important role in cuticle tanning and wing expansion in insects. In this study, full-length cDNAs of Burs-α (LdBurs-α) and Burs-ß (LdBurs-ß) genes were identified in gypsy moth (Lymantria dispar) and cloned. The 480 bp and 420 bp open reading frames (ORFs) encode 159 and 129 amino acid polypeptides, respectively. LdBurs-α and LdBurs-ß have 11 conserved cysteine residues, and LdBurs-α and LdBurs-ß genes were expressed during all developmental stages according to quantitative reverse transcription PCR (qRT-PCR), with highest expression in the egg stage. High expression levels were also detected in the haemolymph, cuticle and head. To explore the physiological functions of LdBurs-α and LdBurs-ß, the genes were knocked down in larvae and pupae using RNA interference (RNAi), and expression levels of LdBurs-α and LdBurs-ß were decreased by 42.26-80.09%. Wing defects were observed in L. dispar pupae following Ldbursion silencing, with a phenotypic percentage ranging from 10.17% to 15.00%. RNAi-mediated knockdown of Ldbursicon prevented the expansion of male and female L. dispar adult wings, with malformation rates ranging from 6.38% and 30.00% to 57.69% and 69.23%, but no cuticle tanning defects were observed in pupae or adults. The results indicate that bursicon plays a key role in wing expansion in L. dispar adults, making it a potentially novel molecular target for insecticide-based control of this pest species.

Structure-Based Functional Analysis of a Hormone Belonging to an Ecdysozoan Peptide Superfamily: Revelation of a Common Molecular Architecture and Residues Possibly for Receptor Interaction.

A neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs, and increased significantly after the animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, the solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy, revealing that it has an N-terminal tail, three α-helices (α2, Gly9-Asn28; α3, His34-Gly38; and α5, Glu62-Arg72), and a π-helix (π4, Cys43-Tyr54), and is structurally constrained by a pattern of disulfide bonds (Cys7-Cys43, Cys23-Cys39, and Cys26-Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substitution, and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, and G73) are located at either end of the sequence, which are sterically close to each other and presumably constitute the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily, revealing a folding pattern, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand-receptor binding specificity.

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.

Insulin-like androgenic gland hormone from the shrimp Fenneropenaeus merguiensis: Expression, gene organization and transcript variants.

Male sex differentiation in the crustacean is best known to be controlled by the insulin-like androgenic gland hormone (IAG). In this report, the cDNA and gene of the shrimp Fenneropenaeus merguiensis FmIAG were studied and characterized. FmIAG gene shares a high sequence identity in the coding region as well as the promoter region with that of F. chinensis. FmIAG gene is most likely consists of 5 exons and 4 introns. The cDNA reported here is the most abundant transcript that retained cryptic intron 4. The use of different splicing acceptor sites in exon 2 can produce a long-form FmIAG transcript variant with 6 additional amino acids inserted. Splicing of cryptic intron 4 would produce a transcript variant with a different C-terminal end. Therefore 4 different FmIAG transcripts can be produced from the FmIAG gene. During the molt cycle, the expression level of FmIAG was low in the early intermolt, increase steadily towards the late premolt and decreased rapidly in the early postmolt. In addition to the androgenic gland, FmIAG is also expressed in the hepatopancreas and ovary of adult females. Unilateral eyestalk ablation caused a significant increase in FmIAG transcript suggesting that the eyestalk consists of inhibiting factor(s) that suppressesFmIAGexpression. To explore the function of FmIAG in males, injection of FmIAG dsRNA knock-down the expression of FmIAG and up-regulated the expression of the vitellogenin gene in the testis and hepatopancreas. Interestingly a CHH-like gene identified in the androgenic gland was down-regulated. CHH-like gene knock-down resulted in altered expression of FmIAG in males suggesting that the CHH-like may be involved in FmIAG's regulation. RT-PCR with specific primers to the different transcript variant were used to determine if there is an association of different sizes of male and the type of IAG transcript. Results indicated that a high percentage of the large male shrimp expressed the long-form of FmIAG. The results suggested that FmIAG may be useful as a size marker for male shrimp aquaculture. In summary, the results of this study have expanded our knowledge of shrimp insulin-like androgenic gland hormone in male sex development and its potential role as a marker gene for growth regulation in shrimp.

The Origin and Evolution of Antistasin-like Proteins in Leeches (Hirudinida, Clitellata).

Bloodfeeding is employed by many parasitic animals and requires specific innovations for efficient feeding. Some of these innovations are molecular features that are related to the inhibition of hemostasis. For example, bloodfeeding insects, bats, and leeches release proteins with anticoagulatory activity through their salivary secretions. The antistasin-like protein family, composed of serine protease inhibitors with one or more antistasin-like domains, is tightly linked to inhibition of hemostasis in leeches. However, this protein family has been recorded also in non-bloodfeeding invertebrates, such as cnidarians, mollusks, polychaetes, and oligochaetes. The present study aims to 1) root the antistasin-like gene tree and delimit the major orthologous groups, 2) identify potential independent origins of salivary proteins secreted by leeches, and 3) identify major changes in domain and/or motif structure within each orthologous group. Five clades containing leech antistasin-like proteins are distinguishable through rigorous phylogenetic analyses based on nine new transcriptomes and a diverse set of comparative data: the trypsin + leukocyte elastase inhibitors clade, the antistasin clade, the therostasin clade, and two additional, unnamed clades. The antistasin-like gene tree supports multiple origins of leech antistasin-like proteins due to the presence of both leech and non-leech sequences in one of the unnamed clades, but a single origin of factor Xa and trypsin + leukocyte elastase inhibitors. This is further supported by three sequence motifs that are exclusive to antistasins, the trypsin + leukocyte elastase inhibitor clade, and the therostasin clade, respectively. We discuss the implications of our findings for the evolution of this diverse family of leech anticoagulants.

Bursicon mediates antimicrobial peptide gene expression to enhance crowded larval prophylactic immunity in the oriental armyworm, Mythimna separata.

It has been reported that a high population density alters insect prophylactic immunity. Bursicon plays a key role in the prophylactic immunity of newly emerged adults. In this paper, full-length cDNAs encoding the alpha and beta subunits of bursicon in Mythimna separata larvae (Msburs α and Msburs ß) were identified. The cDNAs of Msburs α and Msburs ß contain open reading frames (ORFs) encoding 145- and 139-amino acid residue proteins, respectively. Multiple alignment sequences and phylogenetic analysis indicated that Msbursicons (Msburs α and Msburs ß) are orthologous to bursicons in other lepidopterans. The Msbursicons were expressed throughout all developmental states with higher relative expression during the egg, pupae, and adult stages. Msbursicons (Msburs α and Msburs ß) were highly expressed in the ventral nerve cord and brain relative to other tested tissues. Msbursicon expression of larvae subject to high-density treatment (10 larvae per jar) was significantly increased compared with that of the larvae subject to low-density treatment (1 larva per jar) in the whole fourth and fifth instar stages. The trend in the expression of the antimicrobial peptide (AMP) genes cecropin C and defensin in the test stage was accorded and delayed with increased expression of bursicons. Silencing Msburs α (or Msburs ß) expression by dsRNA injection in larvae subject to high-density treatment significantly decreased the expression levels of the cecropin C and defensin genes. Recombinant Msbursicon homodimers significantly induced the expression of the cecropin C and defensin genes. There was a notable decrease in the survival rate of the Msburs α (or Msburs ß or Mscecropin C or Msdefensin) knockdown larvae infected by Beauveria thuringiensis. Our findings provide the first insights into how larval density mediates AMP gene expression, which subsequently affects the prophylactic immunity of insects under high-density conditions.

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.

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.

Transcriptional Inhibition of Sp-IAG by Crustacean Female Sex Hormone in the Mud Crab, Scylla paramamosain.

In crustaceans, the regulation of sex differentiation is mediated by insulin-like androgenic hormone (IAG) and crustacean female sex hormone (CFSH). CFSH is reported to inhibit IAG gene (Sp-IAG) expression in the mud crab Scylla paramamosain, but the regulatory mechanism is not well understood. A 2674 bp 5' flanking Sp-IAG contains many potential transcription factor binding sites. In this study, analysis of serially deleted 5' flanking Sp-IAG and site-directed mutation (SDM) of transcription factor binding sites of the same gene showed that the promoter activity of reporter vectors with Sox-5-binding site, signal transducers and activators of transcription (STAT)-binding site and activator protein 1 (AP-1)-binding site were significantly higher than that of vectors without these regions, suggesting that they were involved in transcriptional regulation of Sp-IAG expression. The expression analysis of these transcription factor showed that there was no difference in the level of mRNA in Sox-5 and AP-1 in androgenic gland treated with recombinant CFSH, but expression of Sp-STAT was significantly reduced, suggesting that CFSH regulates the expression of Sp-STAT, inhibiting its function to regulate Sp-IAG. Further experiment revealed that RNAi mediated Sp-STAT gene knockdown reduced the expression of Sp-IAG. These results suggested that Sp-CFSH regulates Sp-IAG by inhibiting STAT. This is a pioneering finding on the transcriptional mechanism of IAG gene in crustaceans.

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.

Genomic organization of the molt-inhibiting hormone gene in the red swamp crayfish Procambarus clarkii and characterization of single-nucleotide polymorphisms associated with growth.

Molt-inhibiting hormone (MIH), a neuropeptide synthesized in the eyestalk in crustaceans, is mainly responsible for the molting by negatively controlling the ecdysteroids secretion. Although there are several reports of the isolation and protein sequencing of MIH in the red swamp crayfish, little is known about the nucleotide sequence and gene organization of this neuropeptide, even less about the association of MIH polymorphisms and growth traits. Here, a 1237 bp full-length MIH cDNA was obtained from the crayfish eyestalk, which encodes a putative protein of 106 amino acids, with a 191 bp 5'-UTR and a 728 bp 3'-UTR. The MIH genomic DNA sequence is 4205 bp in length, which includes three exons interrupted by two introns, and a 929 bp 5'-flanking region. Potential transcription initiation site and transcription factor binding sites were identified in the 5'-flanking region, implying a potential role in transcriptional regulation. Seventeen SNPs in the 5'-flanking region and 3'-UTR were identified, and the associations between these SNPs and growth traits were evaluated with a two-stage design. A SNPs g. -12C > G that showed a significant association with body weight was identified. Individuals with GG genotype had a significantly higher body weight than those with CC genotype (43.98 ±â€¯9.82 g vs. 34.27 ±â€¯6.87 g; P ﹤ 0.001), indicating a beneficial effect of the G allele on the growth of red swamp crayfish. The obtained MIH gene, as well as the identified SNPs, may serve as targets for molecular marker-aided selection in growth improvement of the red swamp crayfish in future studies.

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.