Crustacean Hyperglycemic Hormone Family Research Articles

Molecular characterization, localization, and physiological roles of ITP and ITP-L in the mosquito, Aedes aegypti.

The insect ion transport peptide (ITP) and its alternatively spliced variant, ITP-like peptide (ITP-L), belong to the crustacean hyperglycemic hormone family of peptides and are widely conserved among insect species. While limited, studies have characterized the ITP/ITP-L signaling system within insects, and putative functions including regulation of ion and fluid transport, ovarian maturation, and thirst/excretion have been proposed. Herein, we aimed to molecularly investigate Itp and Itp-l expression profiles in the mosquito, Aedes aegypti, examine peptide immunolocalization and distribution within the adult central nervous system, and elucidate physiological roles for these neuropeptides. Transcript expression profiles of both AedaeItp and AedaeItp-l revealed distinct enrichment patterns in adults, with AedaeItp expressed in the brain and AedaeItp-l expression predominantly within the abdominal ganglia. Immunohistochemical analysis within the central nervous system revealed expression of AedaeITP peptide in a number of cells in the brain and in the terminal ganglion. Comparatively, AedaeITP-L peptide was localized solely within the pre-terminal abdominal ganglia of the central nervous system. Interestingly, prolonged desiccation stress caused upregulation of AedaeItp and AedaeItp-l levels in adult mosquitoes, suggesting possible functional roles in water conservation and feeding-related activities. RNAi-mediated knockdown of AedaeItp caused an increase in urine excretion, while knockdown of both AedaeItp and AedaeItp-l reduced blood feeding and egg-laying in females as well as hindered egg viability, suggesting roles in reproductive physiology and behavior. Altogether, this study identifies AedaeITP and AedaeITP-L as key pleiotropic hormones, regulating various critical physiological processes in the disease vector, A. aegypti.

In silico analysis of crustacean hyperglycemic hormone family G protein-coupled receptor candidates.

Ecdysteroid molting hormone synthesis is directed by a pair of molting glands or Y-organs (YOs), and this synthesis is inhibited by molt-inhibiting hormone (MIH). MIH is a member of the crustacean hyperglycemic hormone (CHH) neuropeptide superfamily, which includes CHH and insect ion transport peptide (ITP). It is hypothesized that the MIH receptor is a Class A (Rhodopsin-like) G protein-coupled receptor (GPCR). The YO of the blackback land crab, Gecarcinus lateralis, expresses 49 Class A GPCRs, three of which (Gl-CHHR-A9, -A10, and -A12) were provisionally assigned as CHH-like receptors. CrusTome, a transcriptome database assembled from 189 crustaceans and 12 ecdysozoan outgroups, was used to deorphanize candidate MIH/CHH GPCRs, relying on sequence homology to three functionally characterized ITP receptors (BNGR-A2, BNGR-A24, and BNGR-A34) in the silk moth, Bombyx mori. Phylogenetic analysis and multiple sequence alignments across major taxonomic groups revealed extensive expansion and diversification of crustacean A2, A24, and A34 receptors, designated CHH Family Receptor Candidates (CFRCs). The A2 clade was divided into three subclades; A24 clade was divided into five subclades; and A34 was divided into six subclades. The subclades were distinguished by conserved motifs in extracellular loop (ECL) 2 and ECL3 in the ligand-binding region. Eleven of the 14 subclades occurred in decapod crustaceans. In G. lateralis, seven CFRC sequences, designated Gl-CFRC-A2α1, -A24α, -A24β1, -A24β2, -A34α2, -A34β1, and -A34β2, were identified; the three A34 sequences corresponded to Gl-GPCR-A12, -A9, and A10, respectively. ECL2 in all the CFRC sequences had a two-stranded β-sheet structure similar to human Class A GPCRs, whereas the ECL2 of decapod CFRC-A34β1/β2 had an additional two-stranded β-sheet. We hypothesize that this second β-sheet on ECL2 plays a role in MIH/CHH binding and activation, which will be investigated further with functional assays.

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.

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 ∼ 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.

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.

Examination of the potential role of CHH in regulating the expression of IAGBP gene through the eyestalk-testis pathway

Crustacean hyperglycemic hormone (CHH) is a peptide hormone of the CHH family produced by the X-organ/sinus gland complex and plays role in metabolism, osmoregulation, molting, and reproduction. However, the mechanism by which CHH regulates crustacean sexual behavior remains unclear. To elucidate the mechanism of CHH-mediated sexual regulation in this study, the giant freshwater prawn (Macrobrachium rosenbergii) CHH gene (MrCHH) was cloned and characterized. The MrCHH open reading frame was 408 bp in length and was found to encode a 135–amino acid putative protein of 15.42 kDa. In normal prawns, MrCHH mRNA transcripts were abundant in the examined tissues, as follows: eyestalk > intestine > androgenic gland > testis > ventral nerve cord > stomach > gills > heart > brain. In prawns injected with dsCHH RNA, MrCHH mRNA and protein expression were down-regulated at 24 h in the eyestalk and testis compared with prawns injected with negative control dsGFP. Moreover, MrCHH dsRNA increased mRNA and protein expression of M. rosenbergii insulin-like androgenic gland hormone–binding protein (MrIAGBP), indicating that MrCHH inhibits MrIAGBP expression. Additionally, after unilateral or bilateral eyestalk ablation, expression of both insulin-like androgenic gland hormone (IAG) and IAGBP in the prawn testis was significantly up-regulated. Immunohistochemical analyses showed that after unilateral or bilateral eyestalk ablation, MrIAGBP protein levels also increased in the testis, further suggesting that MrCHH is an inhibitor that negatively regulates the expression of genes that play roles in male gonadal development. Our findings thus suggest that MrCHH serves to enrich the “eyestalk-androgenic gland-testis” endocrine axis involved in IAG signaling, providing significant information for further explorations of the mechanism underlying sex regulation in other decapod crustaceans.

Regulation of VIH by miR-277 in the eyestalk of mud crab Scylla paramamosain

The regulatory role of microRNAs in gonad development and reproduction is attracting more and more attention in recent years.Vitellogenesis−/Gonadal-inhibiting hormone (VIH/GIH) is a member of the crustacean hyperglycemic hormone (CHH) family. It plays a key role in regulating ovarian development and gonadal maturation in crustaceans. In this study, based on our preliminary studies of Spvih (Scylla paramamosain vih, Spvih), the regulation of expression of Spvih gene was further investigated. Predicted by bioinformatics software, we found that miR-34/miR-277 may play a regulatory role in Spvih expression. Analysis of luciferase activity of HEK293T cells transfected with the synthetic miR-34/miR-277 mimics and miR-34/miR-277 inhibitor revealed that miR-277 mimics significantly reduced the activity and miR-277 inhibitor significantly increased the activity compared to the control group. However, there was no significant difference in activity between miR-34 mimics and miR-34 inhibitor. The relative expression of Spvih and miR-277 was detected in the eyestalk, which had been injected with stroke-physiological saline solution(SPSS)and chemically synthesized miR-277 agomir and miR-277 antagomir, respectively. The results showed that compared with the control group SPSS, the expression of miR-277 in the eyestalk was increased significantly after miR-277 agomir injection, while the expression of Spvih was decreased significantly. After the injection of miR-277 antagomir, the expression of miR-277 in the eyestalk decreased significantly, while the expression of Spvih increased significantly. The inhibitory effect of miR-277 on Spvih expression was further verified. Based on miR-277 agomir and miR-277 antagomir injected into the base of the crab, the relative expression of the Spvtg gene in the ovarian and hepatopancreas was detected. The results showed that compared with the control group SPSS, the expression of Spvtg in the ovary and hepatopancreas were significantly increased after injection of miR-277 agomir, while the expression of Spvtg was significantly decreased after injection of miR-277 antagomir. Our study may provide an evidence of how miRNA works on the regulation of Spvih, which indirectly affects the ovarian development of mud crab, and suggest a new strategy for controlling crab reproduction.

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.

The thirsty fly: Ion transport peptide (ITP) is a novel endocrine regulator of water homeostasis in Drosophila.

Animals need to continuously adjust their water metabolism to the internal and external conditions. Homeostasis of body fluids thus requires tight regulation of water intake and excretion, and a balance between ingestion of water and solid food. Here, we investigated how these processes are coordinated in Drosophila melanogaster. We identified the first thirst-promoting and anti-diuretic hormone of Drosophila, encoded by the gene Ion transport peptide (ITP). This endocrine regulator belongs to the CHH (crustacean hyperglycemic hormone) family of peptide hormones. Using genetic gain- and loss-of-function experiments, we show that ITP signaling acts analogous to the human vasopressin and renin-angiotensin systems; expression of ITP is elevated by dehydration of the fly, and the peptide increases thirst while repressing excretion, promoting thus conservation of water resources. ITP responds to both osmotic and desiccation stress, and dysregulation of ITP signaling compromises the fly’s ability to cope with these stressors. In addition to the regulation of thirst and excretion, ITP also suppresses food intake. Altogether, our work identifies ITP as an important endocrine regulator of thirst and excretion, which integrates water homeostasis with feeding of Drosophila.

Tissue distribution and biochemical characteristics of receptors for sinus gland peptide VII as a crustacean hyperglycemic hormone and vitellogenesis-inhibiting hormone of the kuruma prawn, Marsupenaeus japonicus

Crustacean hyperglycemic hormone (CHH) and vitellogenesis-inhibiting hormone (VIH) belong to the CHH family, a neuropeptide superfamily conserved in ecdysozoans. To date, no receptor for the CHH family peptides has been identified in crustaceans. Here, we used a CHH family isoform, Mj-sinus gland peptide (SGP)-VII, as a representative of CHH and VIH in order to determine its target tissues and obtain biochemical information regarding its receptor in the kuruma prawn Marsupenaeus japonicus (Crustacea, Decapoda). An in vitro binding assay using a radiolabeled recombinant Mj-SGP-VII and tissue membranes showed that ligand-receptor binding was specific and dissociable. Six tissues, including the hepatopancreas, gill, heart, skeletal muscle, hindgut, and ovary, were identified as the main targets for Mj-SGP-VII. Scatchard analysis of these six tissues determined the dissociation constant and maximum binding capacity values as Kd = 0.86–3.6 nM and Bmax = 102–915 fmol/mg protein, respectively. Of these six tissues, the hepatopancreas, heart, and ovary showed changes in the levels of ligand-binding after the elimination of endogenous ligands by eyestalk ablation. In the hepatopancreas, an increase in the amount of ligand-binding was observed after eyestalk ablation, independent of gender, which appears to be associated with hypoglycemia caused by the treatment. The change observed in the hepatopancreas was due to the increase in the ligand-binding capacity, but not in the ligand-binding affinity, of the receptors. Furthermore, chemical cross-linking analysis demonstrated the presence of target tissue-specific receptors for Mj-SGP-VII with molecular masses of 34–62 kDa. Collectively, the present data provided important information on tissue distribution, temporal changes in expression level, and molecular mass, for the identification and characterization of receptors for CHH family peptides in crustaceans.

A molting-inhibiting hormone-like protein from Pacific white shrimp Litopenaeus vannamei is involved in immune responses

The molting-inhibiting hormones (MIHs) from the crustacean hyperglycemic hormone (CHH) family are a group of neuropeptides that are implicated in regulation of molting and reproduction in crustaceans. In this study, a novel protein containing a typical crustacean neuropeptide domain was identified from Litopenaeus vannamei. The protein showed high homology with other shrimp MIHs and was then designated as a MIH-like protein (MIHL). Among the detected tissues, the heart expressed the highest level of MIHL. The expression of MIHL could be significantly up-regulated after infection with white spot syndrome virus (WSSV), gram-negative bacterium Vibro parahaemolyticus and gram-positive bacterium Staphylococcus aureus, indicating that MIHL could be involved in immune responses. The promoter of MIHL was predicted to contain two NF-κB binding sites and could be regulated by the NF-κB family protein Relish but not Dorsal, suggesting that MIHL could be an effector gene of the IMD/Relish pathway. Silencing of MIHL in vivo by RNAi strategy significantly down-regulated the expression of many immune effector genes and increased the mortalities of shrimp infected by V. parahaemolyticus and WSSV and their copy numbers in tissues. These confirmed that MIHL could play a role in antiviral and antibacterial immune responses in shrimp.

VIH from the mud crab is specifically expressed in the eyestalk and potentially regulated by transactivator of Sox9/Oct4/Oct1

Vitellogenesis-inhibiting hormone (VIH) is known to regulate ovarian maturation by suppressing the synthesis of vitellogenin (Vtg) in crustaceans, which belongs to a member of crustacean hyperglycemic hormone (CHH) family synthesized and secreted from the X-organ/sinus gland complex of eyestalks. In this study, the cDNA, genomic DNA (gDNA) and the 5′-upstream regulatory (promoter region) sequences of VIH gene were obtained by conventional PCR, genome walker and tail-PCR techniques according to our transcriptomic database of Scylla paramamosain. The full-length cDNA of SpVIH is 634bp including 105bp 5′UTR, 151bp 3′UTR and 378bp ORF that encodes a peptide of 125 amino acids. The full length gDNA of SpVIH is 790bp containing two exons and one intron. The 5′-flanking promoter regions of SpVIH we isolated are 3070bp from the translation initiation (ATG) and 2398bp from the predicted transcription initiation (A), which consists of putative core promoter region and multiple potential transcription factor binding sites. SpVIH was only expressed in eyestalk. The expression level of SpVIH in eyestalk of female crab decreased gradually along with the development of ovary. As there is not cell line of crabs available, we chose the mature transfection system HEK293FT cell lines to explore the mechanism of transcription regulation of SpVIH in crabs. Sequential deletion assays using luciferase reporter gene in HEK293FT cells revealed that the possible promoter activity regions (including positive and negative transcription factors binding sites simultaneously) presented between pSpVIH-4 and pSpVIH-6. In order to further identify the crucial transcription factors binding site in this region, the site-directed mutagenesis of Sox9/Oct4/Oct1 binding site of pSpVIH-4 was created. The results demonstrated that the transcriptional activity of pSpVIH-4△ decreased significantly (p<0.05). Thus, it is reasonable to deduce that the Sox9/Oct4/Oct1 may be the essential positive transcription factors which regulate the expression of SpVIH.

Crustacean hyperglycemic hormones directly modulate the immune response of hemocytes in shrimp Litopenaeus vannamei

A robust immune response against invading pathogens is crucial for host to survive, which depends greatly on the well balance of metabolism. Increasing evidence has indicated that some metabolic hormones, such as insulin, could modulate immune responses directly. Crustacean hyperglycemic hormone (CHH) family is a group of ecdysozoans-specific peptide hormone involved in glucose metabolism and other biological events. In the present study, two members of CHH family (designated as LvCHH I and LvCHH II) in shrimp Litopenaeus vannamei with one and two crustacean neurohormone domains respectively were chosen to investigate their putative modulatory roles in both glucose metabolism and immune response. LvCHH I and LvCHH II were both expressed in the sinus gland and lamina ganglionalis of eyestalks and were significantly induced after white spot syndrome virus (WSSV) infection. Meanwhile, significant increases of hemolymph glucose levels were observed in shrimp at 12 and 24 h after WSSV infection while the glucose inside the hemocytes decreased at 6 h and then increased at 12 h. Gain-of-function of rLvCHHs was subsequently conducted in vivo by injecting the recombinant proteins (rLvCHH I and rLvCHH II). The hemolymph glucose increased significantly from 0.5 h to 3 h after the shrimps received an injection of rLvCHH I, while it decreased at 0.5 h and increased afterward at 3 h post rLvCHH II injection. At the meantime, significant decreases of reactive oxygen species level in hemocytes were observed at 3 h and 6 h post rLvCHH I injection, while it remained unchanged in rLvCHH II injection group. rLvCHH I and rLvCHH II could bind to the cytomembrane of primary shrimp hemocytes in vitro, and the expressions of superoxide dismutase and LvRelish increased when the hemocytes were incubated with rLvCHH I for 3 h. Meanwhile, the expression of antimicrobial peptides, crustin and penaeidin-4, were also induced by rLvCHH I and rLvCHH II. These results demonstrated that host immune response, in addition to glucose metabolism, could be directly modulated by LvCHH family, and the present study provided new insights into the immunomodulation role of metabolic hormones in invertebrate.

A novel CHH gene from the Pacific white shrimp Litopenaeus vannamei was characterized and found highly expressed in gut and less in eyestalk and other extra-eyestalk tissues

The crustacean hyperglycemic hormone (CHH) family is an important group of neuropeptides involved in controlling growth, reproduction, and stress response in decapod species. In this study, a new gene containing 4 exons-3 introns flanked by canonical 5′-GT-AG-3′ intron splice-site junctions was isolated from Litopenaeus vannamei. Two full length transcripts of this CHH were isolated from eyestalk and pericardial tissue of males and females using rapid amplification of cDNA ends (RACE). Transcripts sequences were 1578bp in length in males pericardial tissues and in males and females eyestalk with 100% identity, but the transcript isolated from females pericardial tissues was shorter (974bp). The differences in transcripts length is a result of two polyadenylation sites present in the 3'UTR resulting in two transcription termination signals. Transcript sequences encoded one unique protein that can be classified as type I CHH subfamily because of the 4 exons and 3 introns structure, although the CPRP region is not-well conserved and there is no amidation in the C-terminal of the deduced amino acid sequence. Furthermore, there is a glycine inserted in the mature peptide not at position 12 as in type II CHHs but after amino acid 31 and the phylogenetic analysis did not group the peptide within type I, but closer to type II CHHs. We demonstrated by endpoint-PCR, qPCR, and in situ hybridization (ISH), that this gene is expressed in neuroendocrine organs known to express CHHs in penaeid shrimp, including X-organ and optic nerve in eyestalk, supraesophageal ganglion (SoG), but it is also expressed in other organs as gill, gut, pericardial cavity, as well as in terminal ampoule or spermatophore and vas deferens of males.

Functional Assessment of Residues in the Amino- and Carboxyl-Termini of Crustacean Hyperglycemic Hormone (CHH) in the Mud Crab Scylla olivacea Using Point-Mutated Peptides.

To assess functional importance of the residues in the amino- and carboxyl-termini of crustacean hyperglycemic hormone in the mud crab Scylla olivacea (Sco-CHH), both wild-type and point-mutated CHH peptides were produced with an amidated C-terminal end. Spectral analyses of circular dichroism, chromatographic retention time, and mass spectrometric analysis of the recombinant peptides indicate that they were close in conformation to native CHH and were produced with the intended substitutions. The recombinant peptides were subsequently used for an in vivo hyperglycemic assay. Two mutants (R13A and I69A rSco-CHH) completely lacked hyperglycemic activity, with temporal profiles similar to that of vehicle control. Temporal profiles of hyperglycemic responses elicited by 4 mutants (I2A, F3A, D12A, and D60A Sco-CHH) were different from that elicited by wild-type Sco-CHH; I2A was unique in that it exhibited significantly higher hyperglycemic activity, whereas the remaining 3 mutants showed lower activity. Four mutants (D4A, Q51A, E54A, and V72A rSco-CHH) elicited hyperglycemic responses with temporal profiles similar to those evoked by wild-type Sco-CHH. In contrast, the glycine-extended version of V72A rSco-CHH (V72A rSco-CHH-Gly) completely lost hyperglycemic activity. By comparing our study with previous ones of ion-transport peptide (ITP) and molt-inhibiting hormone (MIH) using deleted or point-mutated mutants, detail discussion is made regarding functionally important residues that are shared by both CHH and ITP (members of Group I of the CHH family), and those that discriminate CHH from ITP, and Group-I from Group-II peptides. Conclusions summarized in the present study provide insights into understanding of how functional diversification occurred within a peptide family of multifunctional members.

Transcriptome profiling of the eyestalk of precocious juvenile Chinese mitten crab reveals putative neuropeptides and differentially expressed genes

Chinese mitten crabs that reach maturity 1year earlier than normal crabs are known as precocious juvenile crabs. The molecular mechanisms underlying the precocity of the Chinese mitten crab are poorly understood. To identify the genes that may be involved in the control of precocity in Chinese mitten crab, we measured the expression profile of eyestalk genes in precocious and normally developed juvenile crabs using high-throughput sequencing on an Illumina HiSeq 2500 platform. We obtained 56,446,284 raw reads from the precocious crabs and 58,029,476 raw reads from the normally developed juvenile crabs. Reads from the two libraries were combined into a single data set. De novo assembly of the combined read set yielded 78,777 unigenes with an average length of 1563bp. A total of 41,405 unigenes with predicted ORFs were selected for functional annotation. Among these genes, we identified three neuropeptide genes belonging to the crustacean hyperglycemic hormone family and two neuropeptide genes encoding the chromatophorotropic hormones. Transcriptome comparison between the two libraries revealed 42 genes that exhibited significant differential expression, of which 29 genes were up-regulated and 13 genes were down-regulated in the precocious crabs. To confirm the sequencing data, six differentially expressed genes with functional annotations were selected and validated by qRT-PCR. In conclusion, we obtained the comprehensive transcriptome of the eyestalk tissues of precocious juvenile crabs. The sequencing results may provide new insights into the biomolecular basis of precocity in the Chinese mitten crab.

Characterization, expression, and function analysis of gonad-inhibiting hormone in Oriental River prawn, Macrobrachium nipponense and its induced expression by temperature

Gonad-inhibiting hormone (GIH) is a member of crustacean hyperglycemic hormone family and plays a major role in regulating reproduction in crustaceans. In this study, a full-length cDNA of GIH of Oriental River prawn, Macrobrachium nipponense (Mn-GIH) was cloned from the eyestalk. A 1350bp full-length Mn-GIH cDNA harbored 336bp of an open reading frame encoding signal peptide of 112 amino acid residues. Sequence analysis revealed that the overall cDNA sequence and specific functional sites of Mn-GIH were highly conserved with those in other crustacean species. Expression analysis by quantitative real-time PCR demonstrated its tissue-specific, larval developmental stage-specific, and ovary developmental stage-specific expression pattern, respectively. The RNAi by GIH-ds-RNA in vivo injection was effective in this study and resulted a 50% (day 1), 83% (day 5) and 63% (day 9) down-regulation compared to control. The obvious changes of gonad somatic index (GSI) rate also provided strong evidence to the inhibition effects of GIH on ovary maturation and spawning. Four temperature gradients (12°C±1°C, 17°C±1°C, 22°C±1°C, 27°C±1°C) were set to imitate the temperature in breeding and non-breeding seasons. The observed expression profiles suggest that Mn-GIH did not display a high level expression as supposed to maintain an immature ovary state under low temperature (12°C). The results indicated that GIH was probably activated to concentrating and working by a proper temperature before reaching to breeding season.

Differential regulation of hepatopancreatic vitellogenin (VTG) gene expression by two putative molt-inhibiting hormones (MIH1/2) in Pacific white shrimp (Litopenaeus vannamei)

Molt-inhibiting hormone (MIH), a peptide member of the crustacean hyperglycemic hormone (CHH) family, is commonly considered as a negative regulator during the molt cycle in crustaceans. Phylogenetic analysis of CHH family peptides in penaeidae shrimps suggested that there is no significant differentiation between MIH and vitellogenesis-inhibiting hormone (VIH, another peptide member of CHH family), by far the most potent negative regulator of crustacean vitellogenesis known. Thus, MIH may also play a role in regulating vitellogenesis. In this study, two previously reported putative MIHs (LivMIH1 and LivMIH2) in the Pacific white shrimp (Litopenaeus vannamei) were expressed in Escherichia coli, purified by immobilized metal ion affinity chromatography (IMAC) and further confirmed by western blot. Regulation of vitellogenin (VTG) mRNA expression by recombinant LivMIH1 and LivMIH2 challenge was performed by both in vitro hepatopancreatic primary cells culture and in vivo injection approaches. In in vitro primary culture of shrimp hepatopancreatic cells, only LivMIH2 but not LivMIH1 administration could improve the mRNA expression of VTG. In in vivo injection experiments, similarly, only LivMIH2 but not LivMIH1 could stimulate hepatopancreatic VTG gene expression and induce ovary maturation. Our study may provide evidence for one isoform of MIH (MIH2 in L. vannamei) may serve as one of the mediators of the physiological progress of molting and vitellogenesis. Our study may also give new insight in CHH family peptides regulating reproduction in crustaceans, in particular penaeidae shrimps.

Identification and Characterization of Receptors for Ion Transport Peptide (ITP) and ITP-like (ITPL) in the Silkworm Bombyx mori

Ion transport peptide (ITP) and its alternatively spliced variant, ITP-like (ITPL), are insect peptides that belong to the crustacean hyperglycemic hormone family. These peptides modulate the homeostatic mechanisms for regulating energy metabolism, molting, and reproduction and are specifically conserved in ecdysozoans. Many of the details of the molecular mechanisms by which crustacean hyperglycemic hormone family peptides exert pleiotropy remain to be elucidated, including characterization of their receptors. Here we identified three Bombyx mori orphan neuropeptide G protein-coupled receptors (BNGRs), BNGR-A2, -A24, and -A34, as receptors for ITP and ITPL (collectively referred to as ITPs). BNGR-A2 and -A34 and BNGR-A24 respond to recombinant ITPs, respectively, with EC50 values of 1.1-2.6 × 10(-8) M, when expressed in a heterologous expression system. These three candidate BNGRs are expressed at larval B. mori tissues targeted by ITPs, with cGMP elevation observed after exposure to recombinant ITPs. ITPs also increased the cGMP level in B. mori ovary-derived BmN cells via membrane-bound and soluble guanylyl cyclases. The simultaneous knockdown of bngr-A2 and -A34 significantly decreased the response of BmN cells to ITP, whereas knockdown of bngr-A24 led to decreased responses to ITPL. Conversely, transient expression of bngr-A24 potentiated the response of BmN cells to ITPL. An in vitro binding assay showed direct interaction between ITPs and heterologously expressed BNGRs in a ligand-receptor-specific manner. Taken together, these data demonstrate that BNGR-A2 and -A34 are ITP receptors and that BNGR-A24 is an ITPL receptor in B. mori.

Pacific White Shrimp (Litopenaeus vannamei) Vitellogenesis-Inhibiting Hormone (VIH) Is Predominantly Expressed in the Brain and Negatively Regulates Hepatopancreatic Vitellogenin (VTG) Gene Expression1

Ovarian maturation in crustaceans is temporally orchestrated by two processes: oogenesis and vitellogenesis. The peptide hormone vitellogenesis-inhibiting hormone (VIH), by far the most potent negative regulator of crustacean reproduction known, critically modulates crustacean ovarian maturation by suppressing vitellogenin (VTG) synthesis. In this study, cDNA encoding VIH was cloned from the eyestalk of Pacific white shrimp, Litopenaeus vannamei, a highly significant commercial culture species. Phylogenetic analysis suggests that L. vannamei VIH (lvVIH) can be classified as a member of the type II crustacean hyperglycemic hormone family. Northern blot and RT-PCR results reveal that both the brain and eyestalk were the major sources for lvVIH mRNA expression. In in vitro experiments on primary culture of shrimp hepatopancreatic cells, it was confirmed that some endogenous inhibitory factors existed in L. vannamei hemolymph, brain, and eyestalk that suppressed hepatopancreatic VTG gene expression. Purified recombinant lvVIH protein was effective in inhibiting VTG mRNA expression in both in vitro primary hepatopancreatic cell culture and in vivo injection experiments. Injection of recombinant VIH could also reverse ovarian growth induced by eyestalk ablation. Furthermore, unilateral eyestalk ablation reduced the mRNA level of lvVIH in the brain but not in the remaining contralateral eyestalk. Our study, as a whole, provides new insights on VIH regulation of shrimp reproduction: 1) the brain and eyestalk are both important sites of VIH expression and therefore possible coregulators of hepatopancreatic VTG mRNA expression and 2) eyestalk ablation could increase hepatopancreatic VTG expression by transcriptionally abolishing eyestalk-derived VIH and diminishing brain-derived VIH.