Bursicon receptor gene HLGR2 as a potential RNA interference target for control of the fall webworm Hyphantria cunea.

BACKGROUND: Insect G protein-coupled receptors (GPCRs) have been identified as a new generation of attractive targets for RNA interference (RNAi)-based pest control. A functional study of the leucine-rich repeat-containing (LGR2) gene in Hyphantria cunea (HLGR2) was performed to examine whether it can be used in the molecular control of this notorious pest. RESULTS: The complementary DNA (cDNA) sequence and deduced amino acids of HLGR2 were obtained and analyzed in the present study. HLGR2 is a typical GPCR and shows high structural and sequence similarity with other insect LGR2 proteins. The spatiotemporal expression profiles of HLGR2 showed that HLGR2 was highly expressed at the egg stage and tissues of head and silk gland. After RNAi of HLGR2, distinct phenotypes were observed when HLGR2 expression was suppressed, indicating that HLGR2 is essential in pupation and eclosion. HLGR2 RNAi led to a low pupation rate (45.00%), body malformation, abnormal wing expansion, failed cuticle melanization (63.33%), and high mortality rate (48.33%). Furthermore, we identified eight genes that are regulated by HLGR2. The expression of these eight genes was induced by the HLGR2 signaling pathway and correlated well with cuticle sclerotization. Unlike LGR2 in other insect species, HLGR2 was found to play a crucial role in the control of H. cunea during ecdysis and postecdysial stages. CONCLUSION: HLGR2 is essential for the growth and development and wing expansion and maturation in H. cunea, suggesting HLGR2 is a promising candidate for application in RNAi-based control of this notorious agriculture-forest pest. © 2021 Society of Chemical Industry.

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.

Targeted Top-Down Mass Spectrometry for the Characterization and Tissue-Specific Functional Discovery of Crustacean Hyperglycemic Hormones (CHH) and CHH Precursor-Related Peptides in Response to Low pH Stress.

Crustacean hyperglycemic hormones (CHHs) are a family of neuropeptides that were discovered in multiple tissues in crustaceans, but the function of most isoforms remains unclear. Functional discovery often requires comprehensive qualitative profiling and quantitative analysis. The conventional enzymatic digestion method has several limitations, such as missing post-translational modification (PTM) information, homology interference, and incomplete sequence coverage. Herein, by using a targeted top-down method, facilitated by higher sensitivity instruments and hybrid fragmentation modes, we achieved the characterization of two CHH isoforms from the sinus glands (SG-CHH) and the pericardial organs (PO-CHH) from the Atlantic blue crab, Callinectes sapidus, with improved sequence coverage compared to earlier studies. In this study, both label-free and isotopic labeling approaches were adopted to monitor the response of CHHs and CHH precursor-related peptide (CPRP) under low pH stress. The identical trends of CPRP and CHH expression indicated that CPRP could serve as an ideal probe in tracking the CHH expression level changes, which would greatly simplify the quantitative analysis of large peptides. Furthermore, the distinct patterns of changes in the expression of CHHs in the SG and the PO suggested their tissue-specific functions in the regulation of low pH stress. Ion mobility-mass spectrometry (IM-MS) was also employed in this study to provide conformation analysis of both CHHs and CPRPs from different tissues.

A relaxin-like gonad-stimulating peptide identified from the starfish Astropecten scoparius.

A relaxin-like gonad-stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B-chain (21 aa), C-peptide (47 aa), and A-chain (24 aa). There are three putative processing sites (Lys-Arg) between the B-chain and C-peptide, between the C-peptide and A-chain, and within the C-peptide. This structural organization revealed that the mature AscRGP is composed of A- and B-chains with two interchain disulfide bonds and one intrachain disulfide bond. The C-terminal residues of the B-chain are Gln-Gly-Arg, which is a potential substrate for formation of an amidated C-terminal Gln residue. Non-amidated (AscRGP-GR) and amidated (AscRGP-NH2 ) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP-GR and AscRGP-NH2 induced oocyte maturation and ovulation in similar dose-dependent manners. This is the first report on a C-terminally amidated functional RGP. Collectively, these results suggest that AscRGP-GR and AscRGP-NH2 act as a natural gonadotropic hormone in A. scoparius.

D-Amino Acids in Peptides from Animals, Including Human: Occurrence, Structure, Bioactivity and Pharmacology.

All life forms typically possess homochirality, with rare exceptions. In the case of peptides and proteins, only L-amino acids are known to be encoded by genes. Nevertheless, D-amino acids have been identified in a variety of peptides, synthesized by animal cells. They include neuroexcitatory and neuroprotective peptides, cardioexcitatory peptides, hyperglycemic hormones, opioid peptides, antimicrobial peptides, natriuretic and defensin-like peptides, and fibrinopeptides. This article is a review of their occurrence, structure and bioactivity. It further explores the pharmacology and potential medical applications of some of the peptides.

Effect of chimeric relaxin-like gonad-stimulating peptides on oocyte maturation and ovulation in the starfish Asterias rubens and Aphelasterias japonica.

A relaxin-like gonad-stimulating peptide (RGP), comprising two peptide chains (A- and B-chains) linked by two interchain bonds and one intrachain disulfide bond, acts as a gonadotropin in starfish. RGP orthologs have been identified in several starfish species, including Patiria pectinifera (PpeRGP), Asterias rubens (AruRGP) and Aphelasterias japonica (AjaRGP). To analyze species-specificity, this study examined the effects on oocyte maturation and ovulation in ovaries of A. rubens and A. japonica of nine RGP derivatives comprising different combinations of A- and B-chains from the three species. All nine RGP derivatives induced spawning in A. rubens and A. japonica ovaries. However, AruRGP, AjaRGP and their chimeric derivatives were more potent than peptides containing the A- or B-chain of PpeRGP. Three-dimensional models of the structures of the RGP derivatives revealed that residues in the B-chains, such as AspB6, MetB10 and PheB13 in PpeRGP and GluB7, MetB11, and TyrB14 in AruRGP and AjaRGP, respectively, are likely to be involved in receptor binding. Conversely, it is likely that ArgA18 in the A-chain of AruRGP and AjaRGP impairs binding of these peptides to the PpeRGP receptor in P. pectinifera. In conclusion, this study provides new insights into the structural basis of RGP bioactivity and RGP receptor activation in starfish.

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.

CRISPR/Cas9-mediated deletion of EcMIH shortens metamorphosis time from mysis larva to postlarva of Exopalaemon carinicauda.

The recently emerged CRISPR/Cas9 technology is the most flexible means to produce targeted mutations at the genomic loci in a variety of organisms. In Crustaceans, molt-inhibiting hormone (MIH) is an important negative-regulatory factor and plays a key role in suppressing the molting process. However, whether precise disruption of MIH in crustacean can be achieved and successfully used to improve the development and growth has not been proved. In this research, the complementary DNA (cDNA) and genomic DNA, including flanking regions of the MIH gene (EcMIH) of ridgetail white prawn Exopalaemon carinicauda, were cloned and sequenced. Sequence analysis revealed that EcMIH was composed of three exons and two introns. Analysis by RT-PCR showed that EcMIH mainly expressed in eyestalks. During different development periods, EcMIH was highest in juvenile stage and extremely low in others but adult prawns eyestalks. In addition, we applied CRISPR/Cas9 technology to generate EcMIH knock-out (KO) prawns and then analyzed the changes in their phenotypes. We efficiently generated 12 EcMIH-KO prawns out of 250 injected one-cell stage embryos and the mutant rate reached 4.8% after embryo injection with one sgRNA targeting the second exon of EcMIH. The EcMIH-KO prawns exhibited increased the body length and shortened the metamorphosis time of larvae from mysis larva to postlarva. Meanwhile, EcMIH-KO did not cause the health problems such as early stage death or deformity. In conclusion, we successfully obtained EcMIH gene and generated EcMIH-KO prawns using CRISPR/Cas9 technology. This study will certainly lead to a wide application prospect of MIH gene in prawns breeding.

Functional and evolutionary implications from the molecular characterization of five spermatophore CHH/MIH/GIH genes in the shrimp Fenneropenaeus merguiensis.

The recent use of RNA-Seq to study the transcriptomes of different species has helped identify a large number of new genes from different non-model organisms. In this study, five distinctive transcripts encoding for neuropeptide members of the CHH/MIH/GIH family have been identified from the spermatophore transcriptome of the shrimp Fenneropenaeus merguiensis. The size of these transcripts ranged from 531 bp to 1771 bp. Four transcripts encoded different CHH-family subtype I members, and one transcript encoded a subtype II member. RT-PCR and RACE approaches have confirmed the expression of these genes in males. The low degree of amino acid sequence identity among these neuropeptides suggests that they may have different specific function(s). Results from a phylogenetic tree analysis indicated that these neuropeptides were likely derived from a common ancestor gene resulting from mutation and gene duplication. These CHH-family members could be grouped into distinct clusters, indicating a strong structural/functional relationship among these neuropeptides. Eyestalk removal caused a significant increase in the expression of transcript 32710 but decreases in expression for transcript 28020. These findings suggest the possible regulation of these genes by eyestalk factor(s). In summary, the results of this study would justify a re-evaluation of the more generalized and pleiotropic functions of these neuropeptides. This study also represents the first report on the cloning/identification of five CHH family neuropeptides in a non-neuronal tissue from a single crustacean species.

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.

Regulating gonad inhibition and vitellogenin/vitellin induction in Penaeus monodon using mature GIH fusion protein and polyclonal antisera.

Gonad inhibiting hormone (GIH), type II class of the CHH family neuropeptides, is released by the neurohaemal XO-SG complex of the eyestalk. The inhibitory function of GIH has a pivotal role in gonad development and reproduction. In this study, we report the expression and production of a thioredoxin-fused mature GIH protein (mf-PmGIH) of Penaeus monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmGIH). The mature GIH gene of 237bp that codes for 79 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The expression vector construct (mf-PmGIH+pEt32a+) upon induction produced 32.16kDa mature GIH fusion protein (mf-PmGIH)·The purified fusion protein was used as exogenous GIH and as antigen to raise polyclonal antisera. The fusion protein when injected into juvenile shrimp significantly reduced vitellogenin/vitellin levels by 31.55% within 72h in comparison to the controls showing the gonad inhibiting property. Vitellogenin/vitellin levels were significantly induced by 74.10% within 6h when polyclonal antiserum (anti-mf-PmGIH - 1:500) was injected in P. monodon. Anti-mf-PmGIH immunolocalized GIH producing neurosecretory cells in the eyestalk of P. monodon. The present manuscript reports an innovative means of gonad inhibition and vitellogenin/vitellin induction with thioredoxin fused GIH and antisera developed.

Identification and functional characterization of a novel antistasin/WAP-like serine protease inhibitor from the tropical sea cucumber, Stichopus monotuberculatus.

A novel antistasin/WAP-like serine protease inhibitor, named as StmAW-SPI, was identified from sea cucumber (Stichopus monotuberculatus) and functionally characterized in this study. The full-length cDNA of StmAW-SPI is 1917 bp in length with a 72 bp 5'-untranslated region (UTR), a 294 bp 3'-UTR and a 1551 bp open reading frame (ORF) encoding a protein of 516 amino acids with a deduced molecular weight of 54.56 kDa. The StmAW-SPI protein has 5-fold internal repeats (IRs) of antistasin domain and 6-fold IRs of WAP domain. For the gene structure, StmAW-SPI contains 10 exons separated by 9 introns. The StmAW-SPI mRNA expression pattern was determined using quantitative real-time PCR. The highest level of StmAW-SPI was found in the intestine, followed by coelomocytes, gonad, body wall and respiratory tree. The StmAW-SPI expressions were significantly up-regulated after polyriboinosinic polyribocytidylic acid [Poly (I:C)] or lipopolysaccharides (LPS) challenge in in vitro experiments performed in primary coelomocytes. In addition, the serine protease inhibitory activity and bacterial protease inhibitory activity of StmAW-SPI were examined, and the antibacterial activity was also demonstrated in this study. Our study, as a whole, suggested that StmAW-SPI might play a critical role in the innate immune defense of sea cucumber against microbial infections, by not only inactivating the serine protease but also inhibiting the growth of pathogens.

Crystal structure of a crustacean hyperglycemic hormone (CHH) precursor suggests structural variety in the C-terminal regions of CHH superfamily members.

The crustacean hyperglycemic hormone (CHH) is one of the major hormones in crustaceans, and peptides belonging to the CHH superfamily have been found in diverse ecdysozoans. Although the basic function of CHH is to control energy metabolism, it also plays various roles in crustacean species, such as in molting and vitellogenesis. Here, we present the crystal structure of Pej-SGP-I-Gly, a partially active precursor of CHH from the kuruma prawn Marsupenaeus japonicus, which has an additional Gly residue in place of the C-terminal amide group of the mature Pej-SGP-I. The 1.6-angstrom crystal structure showed not only the common CHH superfamily scaffold comprising three α-helices, three disulfide bridges, and a hydrophobic core but also revealed that the C-terminal part has a variant backbone fold that is specific to Pej-SGP-I-Gly. The α-helix 4 of Pej-SGP-I-Gly was much longer than that of molt-inhibiting hormone (Pej-MIH) from the same species, and as a result, the following C-terminal helix, corresponding to α-helix 5 in MIH, was not formed. Unlike monomeric Pej-MIH, Pej-SGP-I-Gly forms a homodimer in the crystal structure via its unique α-helix 4. The unexpected dissimilar folds between Pej-SGP-I-Gly and Pej-MIH appear to be the result of their distinct C-terminal amino acid sequences. Variations in amino acid sequences and lengths and the resulting variety of backbone folds allow the C-terminal and sterically adjoining regions to confer different hormonal activities in diverse CHH superfamily members. DATABASE: Structural data are available in the PDB under the accession number 5B5I.

Localization and identification of crustacean hyperglycemic hormone producing neurosecretory cells in the eyestalk of blue swimmer crab, Portunus pelagicus.

This study intensely focuses on to the localization and identification of crustacean hyperglycemic hormone (CHH) producing neurosecretory cells in the eyestalk of the blue swimmer crab Portunus pelagicus. Anti-Carcinus maenas-CHH was used to identify the location of CHH neurosecretory cells by immunohistochemistry. Ten pairs of eyestalks were collected from intact adult intermoult female crab and fixed in Bouin's fixative. Eyestalks were serially sectioned and stained with chrome-hematoxylin-phloxine stain. Histological studies show the presence of different types of neurosecretory cells namely A (multipolar), B (tripolar), C (bipolar), D (unipolar), E (oval), and F (spherical) in the medulla interna, externa, and terminalis regions based on their size, shape, and tinctorial properties. The neurohemal organ, sinus gland (SG) was observed laterally between medulla interna and terminalis regions. Immunohistochemical studies showed the presence of distinct CHH-like immunoreactivity in the optic ganglia. Divergent group of neurosecretory cells with varying degree of immunoreactivity with Anti-Carcinus maenas-CHH (low, moderate, and intense reactivity) were identified in medulla terminalis, medulla interna, medulla externa, and sinus gland. The present study maps the various types of neurosecretory cells in the optic ganglia and also shows the presence of CHH-like immunoreactivity in various regions of optic ganglia in P. pelagicus. The presence of these unique neurosecretory cell types with larger cell diameter in medulla terminalis, a region that bears the neurosecretory cell bodies, suggest high secretory activity.

Characterization, localization and temporal expression of crustacean hyperglycemic hormone (CHH) in the behaviorally rhythmic peracarid crustaceans, Eurydice pulchra (Leach) and Talitrus saltator (Montagu).

Crustacean hyperglycemic hormone (CHH) has been extensively studied in decapod crustaceans where it is known to exert pleiotropic effects, including regulation of blood glucose levels. Hyperglycemia in decapods seems to be temporally gated to coincide with periods of activity, under circadian clock control. Here, we used gene cloning, in situ hybridization and immunohistochemistry to describe the characterization and localization of CHH in two peracarid crustaceans, Eurydice pulchra and Talitrus saltator. We also exploited the robust behavioral rhythmicity of these species to test the hypothesis that CHH mRNA expression would resonate with their circatidal (12.4h) and circadian (24h) behavioral phenotypes. We show that both species express a single CHH transcript in the cerebral ganglia, encoding peptides featuring all expected, conserved characteristics of other CHHs. E. pulchra preproCHH is an amidated 73 amino acid peptide N-terminally flanked by a short, 18 amino acid precursor related peptide (CPRP) whilst the T. saltator prohormone is also amidated but 72 amino acids in length and has a 56 residue CPRP. The localization of both was mapped by immunohistochemistry to the protocerebrum with axon tracts leading to the sinus gland and into the tritocerebrum, with striking similarities to terrestrial isopod species. We substantiated the cellular position of CHH immunoreactive cells by in situ hybridization. Although both species showed robust activity rhythms, neither exhibited rhythmic transcriptional activity indicating that CHH transcription is not likely to be under clock control. These data make a contribution to the inventory of CHHs that is currently lacking for non-decapod species.

Prediction of Scylla olivacea (Crustacea; Brachyura) peptide hormones using publicly accessible transcriptome shotgun assembly (TSA) sequences.

The aquaculture of crabs from the genus Scylla is of increasing economic importance for many Southeast Asian countries. Expansion of Scylla farming has led to increased efforts to understand the physiology and behavior of these crabs, and as such, there are growing molecular resources for them. Here, publicly accessible Scylla olivacea transcriptomic data were mined for putative peptide-encoding transcripts; the proteins deduced from the identified sequences were then used to predict the structures of mature peptide hormones. Forty-nine pre/preprohormone-encoding transcripts were identified, allowing for the prediction of 187 distinct mature peptides. The identified peptides included isoforms of adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, bursicon ß, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, diuretic hormone 31, eclosion hormone, FMRFamide-like peptide, HIGSLYRamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide and tachykinin-related peptide, all well-known neuropeptide families. Surprisingly, the tissue used to generate the transcriptome mined here is reported to be testis. Whether or not the testis samples had neural contamination is unknown. However, if the peptides are truly produced by this reproductive organ, it could have far reaching consequences for the study of crustacean endocrinology, particularly in the area of reproductive control. Regardless, this peptidome is the largest thus far predicted for any brachyuran (true crab) species, and will serve as a foundation for future studies of peptidergic control in members of the commercially important genus Scylla.

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.

Neuropeptidergic Signaling in the American Lobster Homarus americanus: New Insights from High-Throughput Nucleotide Sequencing.

Peptides are the largest and most diverse class of molecules used for neurochemical communication, playing key roles in the control of essentially all aspects of physiology and behavior. The American lobster, Homarus americanus, is a crustacean of commercial and biomedical importance; lobster growth and reproduction are under neuropeptidergic control, and portions of the lobster nervous system serve as models for understanding the general principles underlying rhythmic motor behavior (including peptidergic neuromodulation). While a number of neuropeptides have been identified from H. americanus, and the effects of some have been investigated at the cellular/systems levels, little is currently known about the molecular components of neuropeptidergic signaling in the lobster. Here, a H. americanus neural transcriptome was generated and mined for sequences encoding putative peptide precursors and receptors; 35 precursor- and 41 receptor-encoding transcripts were identified. We predicted 194 distinct neuropeptides from the deduced precursor proteins, including members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin C, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, FLRFamide, GSEFLamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, proctolin, pyrokinin, SIFamide, sulfakinin and tachykinin-related peptide families. While some of the predicted peptides are known H. americanus isoforms, most are novel identifications, more than doubling the extant lobster neuropeptidome. The deduced receptor proteins are the first descriptions of H. americanus neuropeptide receptors, and include ones for most of the peptide groups mentioned earlier, as well as those for ecdysis-triggering hormone, red pigment concentrating hormone and short neuropeptide F. Multiple receptors were identified for most peptide families. These data represent the most complete description of the molecular underpinnings of peptidergic signaling in H. americanus, and will serve as a foundation for future gene-based studies of neuropeptidergic control in the lobster.

Defining the Neuropeptidome of the Spiny Lobster Panulirus interruptus Brain Using a Multidimensional Mass Spectrometry-Based Platform.

Decapod crustaceans are important animal models for neurobiologists due to their relatively simple nervous systems with well-defined neural circuits and extensive neuromodulation by a diverse set of signaling peptides. However, biochemical characterization of these endogenous neuropeptides is often challenging due to limited sequence information about these neuropeptide genes and the encoded preprohormones. By taking advantage of sequence homology in neuropeptides observed in related species using a home-built crustacean neuropeptide database, we developed a semi-automated sequencing strategy to characterize the neuropeptidome of Panulirus interruptus, an important aquaculture species, with few known neuropeptide preprohormone sequences. Our streamlined process searched the high mass accuracy and high-resolution data acquired on a LTQ-Orbitrap with a flexible algorithm in ProSight that allows for sequence discrepancy from reported sequences in our database, resulting in the detection of 32 neuropeptides, including 19 novel ones. We further improved the overall coverage to 51 neuropeptides with our multidimensional platform that employed multiple analytical techniques including dimethylation-assisted fragmentation, de novo sequencing using nanoliquid chromatography-electrospray ionization-quadrupole-time-of-flight (nanoLC-ESI-Q-TOF), direct tissue analysis, and mass spectrometry imaging on matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF. The high discovery rate from this unsequenced model organism demonstrated the utility of our neuropeptide discovery pipeline and highlighted the advantage of utilizing multiple sequencing strategies. Collectively, our study expands the catalog of crustacean neuropeptides and more importantly presents an approach that can be adapted to exploring neuropeptidome from species that possess limited sequence information.