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.

The crustacean hyperglycemic hormone (CHH) is a multifaceted neuropeptide instrumental in regulating carbohydrate and lipid metabolism, reproduction, osmoregulation, molting, and metamorphosis. Despite its significance, there is a dearth of research on its metabolic impact on the gills and epidermis-key organs in osmoregulation and molting processes. This study employed CHH dsRNA injections to silence CHH gene expression in Procambarus clarkii, followed by a metabolomic analysis of the gills and epidermis using nuclear magnetic resonance spectroscopy. Metabolic profiling through principal component analysis revealed the most pronounced changes at 24 h post-injection (hpi) in the epidermis and at 48 hpi in the gills. At 24 hpi, the epidermis exhibited significant modulation in 25 enrichment sets and 20 KEGG pathways, while at 48 hpi, 5 metabolite sets and 6 KEGG pathways were prominently regulated. Notably, pathways associated with amino acid metabolism, carbohydrate metabolism, and cofactor and vitamin metabolism were affected. A marked decrease in glucose and other carbohydrates suggested a compromised carbohydrate supply, whereas increased levels of citrate cycle intermediates implied a potential boost in energy provision. The silencing of CHH gene expression hampered the carbohydrate supply, which was possibly the main energy derived substrates. Conversely, the gills displayed significant alterations in 15 metabolite sets and 16 KEGG pathways at 48 hpi, with no significant changes at 24 hpi. These changes encompassed amino acid, carbohydrate, and lipid metabolism pathways. The decline in TCA cycle intermediates pointed to a potential downregulation of the cycle, whereas a decrease in ketone bodies indicated a shift towards lipid metabolism for energy production. Additionally, increased levels of nicotinate, nicotinamide, and quinolinate were observed in both organs. Overall, CHH\'s impact on the epidermis was prominent at 24 hpi and diminished thereafter, whereas its influence on metabolism in gills was delayed but intensified at 48 hpi. This differential CHH effect between gills and epidermis in P. clarkii provides new insights into the organ-specific regulatory mechanisms of CHH on energy metabolism and osmoregulation, warranting further comparative studies to elucidate the distinct roles of CHH in these organs.

Salinity and temperature influence growth, survival, and reproduction of crustacean species such as Penaeus vannamei where Na +/K+-ATPase plays a key role in maintaining osmotic homeostasis in different salinity conditions. This ability is suggested to be mediated by other proteins including neuropeptides such as the crustacean hyperglycemic hormones (CHHs), and heat shock proteins (HSPs). The mRNA expression of Na+/K+-ATPase, HSP60, HSP70, CHH-A, and CHH-B1, was analyzed by qPCR in shrimp acclimated to different salinities (10, 26, and 40 PSU) and temperature conditions (20, 23, 26, 29, and 32 °C) to evaluate their uses as molecular stress biomarkers. The results showed that the hemolymph osmoregulatory capacity in shrimp changed with exposure to the different salinities. From 26 to 32 °C the Na+/K+-ATPase expression increased significantly at 10 PSU relative to shrimp acclimated at 26 PSU and at 20 °C increased at similar values independently of salinity. The highest HSP expression levels were obtained by HSP70 at 20 °C, suggesting a role in protecting proteins such as Na+/K+ -ATPase under low-temperature and salinity conditions. CHH-A was not expressed in the gill under any condition, but CHH-B1 showed the highest expression at the lowest temperatures and salinities, suggesting its participation in the Na+/K+-ATPase induction. Since Na+/K+-ATPase, HSPs, and CHHs seem to participate in maintaining the osmo-ionic balance and homeostasis in P. vannamei, their expression levels may be used as a stress biomarkers to monitor marine crustacean health status when acclimated in low salinity and temperature conditions.

Peptide hormones and neuropeptides form a diverse class of bioactive secreted molecules that control essential processes in animals. Despite breakthroughs in peptide discovery, many signaling peptides remain undiscovered. Recently, we demonstrated the use of somatostatin-mimicking toxins from cone snails to identify the invertebrate ortholog of somatostatin. Here, we show that this toxin-based approach can be systematically applied to discover other unknown secretory peptides that are likely to have signaling function. Using large sequencing datasets, we searched for homologies between cone snail toxins and secreted proteins from the snails\' prey. We identified and confirmed expression of five toxin families that share strong similarities with unknown secretory peptides from mollusks and annelids and in one case also from ecdysozoans. Based on several lines of evidence we propose that these peptides likely act as signaling peptides that serve important physiological functions. Indeed, we confirmed that one of the identified peptides belongs to the family of crustacean hyperglycemic hormone, a peptide not previously observed in Spiralia. We propose that this discovery pipeline can be broadly applied to other systems in which one organism has evolved molecules to manipulate the physiology of another.

In mammals, energy homeostasis is regulated by the antagonistic action of hormones insulin and glucagon. However, in contrast to the highly conserved insulin, glucagon is absent in most invertebrates. Although there are several endocrine regulators of energy expenditure and catabolism (such as the adipokinetic hormone), no single invertebrate hormone with all of the functions of glucagon has been described so far. Here, we used genetic gain- and loss-of-function experiments to show that the Drosophila gene Ion transport peptide (ITP) codes for a novel catabolic regulator that increases energy expenditure, lowers fat and glycogen reserves, and increases glucose and trehalose. Intriguingly, Ion transport peptide has additional functions reminiscent of glucagon, such as inhibition of feeding and transit of the meal throughout the digestive tract. Furthermore, Ion transport peptide interacts with the well-known signaling via the Adipokinetic hormone; Ion transport peptide promotes the pathway by stimulating Adipokinetic hormone secretion and transcription of the receptor AkhR. The genetic manipulations of Ion transport peptide on standard and Adipokinetic hormone-deficient backgrounds showed that the Adipokinetic hormone peptide mediates the hyperglycemic and hypertrehalosemic effects of Ion transport peptide, while the other metabolic functions of Ion transport peptide seem to be Adipokinetic hormone independent. In addition, Ion transport peptide is necessary for critical processes such as development, starvation-induced foraging, reproduction, and average lifespan. Altogether, our work describes a novel master regulator of fly physiology with functions closely resembling mammalian glucagon.

Viruses are obligate intracellular parasites relying on host cells to obtain biosynthetic precursors and energy to successfully infect the host. The metabolic profile of the host cell is known to be altered in response to viral infection to satisfy the resources demanded during viral replication. Previous data of ours showed that white spot syndrome virus (WSSV) elicited in a crustacean host (Procambarus clarkii) a rapid and long-lasting release of crustacean hyperglycemic hormone (CHH), a well-known carbohydrate-regulating and stress response-mediating endocrine hormone. Therefore, the WSSV-enhanced release of CHH could be responsible at least in part for the metabolic alterations in the WSSV-challenged host. To investigate the possible metabolic roles of CHH in the host-parasite interaction, we studied whether silencing CHH gene expression could inhibit WSSV propagation in tissues and reduce the mortality of the WSSV-infected animals. Data presented in this study showed that CHH gene silencing indeed resists the WSSV infection. Injection of CHH dsRNA at the dosage of 140 μg/g BW caused significant decreases of viral copy number in tissues of WSSV-infected host, particularly showing a pronounced effect in the endodermal tissues (including hepatopancreas and gastrolith disk). Furthermore, results from the cumulative mortality showed that the treatment of CHH dsRNA delayed death from WSSV. Injection of CHH dsRNA at the dosages of 70, 17, and 10 μg/ g BW significantly extended the mean survival time. Together, this study concludes that the silencing of the CHH gene does have an inhibitory effect on the replication of the white spot syndrome virus and can assist the host to mitigate the invasion of WSSV, through attenuating CHH-mediated stress responses.

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

Hyperglycemia is a stress responsible mechanism induced in crustaceans through the secretion of Crustacean Hyperglycemic Hormone (CHH). The effect of thermal shock on the hemolymph CHH levels was studied in P. pelagicus. Crabs were exposed to varying temperatures for 3 h and were then transferred to ambient temperature (28 °C). A higher CHH level of 47.30 ± 2.26 fmol/ml was observed on exposure of crabs to 24 °C, over a recovery period of 3 h. This was reflected with increase in hemolymph glucose causing hyperglycemia and subsequent decrease in hepatopancreas glycogen levels. The results suggest the modulatory role of CHH in producing the energy required for the physiological reparation faced by the crabs during thermal stress.

To unveil the neuroendocrine-immune (NEI) mechanism of crustaceans under high ambient ammonia-N, crustacean hyperglycemic hormone (CHH) in L. vannamei was knocked down under 20 mg/L ammonia-N exposure. The results showed that the expression of CHH in the eyestalks decreased significantly when CHH was silenced. After CHH was knocked down, the levels of CHH, ACh, DA, NE, and 5-HT in the haemolymph decreased significantly. Correspondingly, the expressions of GC, ACh7R, DM1, DA1R, and 5-HT7R in haemocytes down-regulated significantly, while DA4R and α2AR up-regulated significantly. Besides, the expression of Toll3 reduced significantly. And significantly changes occurred in the levels of G protein effectors (AC and PLC), second messengers (cAMP, cGMP, CaM, and DAG), protein kinases (PKA, PKC and PKG), and nuclear transcription factors (CREB, Dorsal, Relish and NKRF). Furthermore, immune defense proteins (BGBP and PPO3, Crustin A, ALF, LYC, TNFα, and IL-16), phagocytosis-related proteins (Cubilin, Integrin, Peroxinectin, Mas-like protein, and Dynamin-1) and exocytosis-related proteins (SNAP-25, VAMP-2 and Syntaxin) changed significantly. Eventually, a significant decrease in the levels of THC, haemocytes phagocytosis rate, plasma PO, antibacterial and bacteriolytic activities was detected. Therefore, these results indicate that under ammonia-N stress, the combination of CHH and GC mainly affects exocytosis of shrimp through the cGMP-PKG-CREB pathway. Simultaneously, CHH stimulates the release of biogenic amines, and then activate G protein effectors after binding to their specific receptors, to regulate exocytosis mainly via the cAMP-PKA-CREB pathway and influence phagocytosis primarily by the cAMP-PKA-NF-κB pathway. CHH can enhance ACh, and then activate G protein effectors after binding to the receptors, and finally regulate exocytosis mainly through the cAMP-PKA-CREB pathway and regulate phagocytosis by the cAMP-PKA-NF-κB pathway. CHH can also promote Toll3-NF-κB pathway, thereby affecting the expressions of immune defense factors. This study contributes to a further understanding of the NEI mechanism of crustacean in response to environmental 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.