Crustacean Hyperglycemic Hormone Research Articles

Comparative Genomic and Transcriptomic Analyses of CHHs and Their Putative Receptors in Scylla paramamosain, Portunus trituberculatus, and Eriocheir sinensis

Crustacean hyperglycemic hormone superfamily neuropeptides (CHHs) are typical crustacean eyestalk hormones that include the crustacean hyperglycemic hormone (CHH), moult-inhibiting hormone (MIH), vitellogenesis/gonad-inhibiting hormone (VIH/GIH) and mandibular organ-inhibiting hormone (MOIH), which are divided into two subfamilies: type I CHH (included CHH) and type II CHH (consisting of MIH, VIH/GIH, and MOIH). They are involved in various biological activities, such as metabolism, molting, reproduction, and osmotic regulation. Discovery of the ion transport peptide (ITP) in insects expanded the members of CHHs and revealed that CHHs are not restricted to crustaceans. In this study, we focused on three economically important crabs: the mud crab, Scylla paramamosain, the swimming crab, Portunus trituberculatus, and the Chinese mitten crab, Eriocheir sinensis. Their genomes, Pacbio full-length transcriptomic data as well as comparative RNA-seq data were obtained and used to analyze the genomic structures and expression patterns of CHHs and their putative receptors through bioinformatic methods. Two type I CHH members (CHH1 and CHH2) were identified, of which CHH1 had two splice variants, CHH1-v1 and CHH1-v2. One copy of type II CHH (MIH) was found in P. trituberculatus and E. sinensis. While most decapods, including S. paramamosain, have two copies of type II CHHs (MIH/VIH), these MIH/VIHs are adjacent to each other on the same chromosome. Besides type I and II CHH, ITP-like peptides have also been found in the three crabs, and they are mainly expressed in the eyestalk. Four, five, and three G protein-coupled receptors (GPCRs) were identified in S. paramamosain, P. trituberculatus, and E. sinensis, respectively, which might be putative CHH receptors. These GPCRs were divided into three groups. One group was composed of two contiguous genomic position GPCRs, and they were mainly expressed in the hepatopancreas. These findings provide a basis for further studies on CHHs receptor binding tests and on CHHs/GPCRs signaling pathways.

Dietary chromium modulates glucose homeostasis and induces oxidative stress in Pacific white shrimp (Litopenaeus vannamei).

While chromium (Cr) has been recognized as an essential nutrient for all animals, and dietary supplementation can be beneficial, it can also be toxic. The present study aimed to investigate the contrasting effects of dietary chromium in Pacific white shrimp Litopenaeus vannamei. Five experimental diets were formulated to contain Cr at levels of 0.82 (Cr0.82, unsupplemented diet), 1.01 (Cr1.01), 1.22 (Cu1.22), 1.43 (Cr1.43) and 1.63 (Cr1.63) mg/kg and were fed to shrimp for 8 weeks. Highest weight gain was recorded in shrimp fed the diet containing 1.22 mg/kg Cr. Shrimp fed the diet containing the highest level of Cr (1.63 mg/kg) showed the lowest weight gain and clear signs of oxidative stress and apoptosis as evidenced by higher levels of H2O2, malondialdehyde and 8-hydroxydeoxyguanosine, and expression of caspase 2, 3, 5, and lower contents of total and oxidized glutathione, and expression of Cu/Zn sod, cat, gpx, mt, bcl2. Chromium supplementation promoted glycolysis and inhibited gluconeogenesis as shown by increased activities of hexokinase, phosphofructokinase and pyruvate kinase, and reduced activity of phosphoenolpyruvate carboxykinase in shrimp fed the diet containing 1.43 mg/kg Cr. Shrimp fed the diet with 1.63 mg/kg Cr had lowest contents of crustacean hyperglycemic hormone and insulin like peptide in hemolymph. Expression of genes involved in insulin signaling pathway and glycose metabolism including insr, irs1, pik3ca, pdpk1, akt, acc1, gys, glut1, pk, hk were up-regulated, and foxO1, gsk-3β, g6pc, pepck were down-regulated in shrimp fed the diets supplemented with Cr. This study demonstrated that optimum dietary supplementation of Cr had beneficial effects on glucose homeostasis and growth, whereas excess caused oxidative damage and impaired growth. The results contribute to our understanding of the biological functions of chromium in shrimp.

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.

Experimental effects of cadmium on physiological response of Callinectes danae (Crustacea, Portunidae) from environments with different levels of Cd contamination

Cadmium (Cd) can adversely affect aquatic life, altering reproductive and molting processes in crustaceans. The objective of this study was to evaluate the influence of Cd on reproduction and molting in the crab Callinectes danae. Adult females were obtained from environments with different levels of pollution: low (LC), medium (MC), and high contaminated (HC) areas. Animals from LC, MC, and HC areas were exposed to 0, 0.5, and 2 mg L−1 of CdCl2 for 3 h. Cd bioaccumulation, oxidative stress (evaluated by antioxidant enzymes activity), and lipid peroxidation (LPX) were analyzed in mature ovaries (stage II), gills, and hepatopancreas. The expression levels of crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) genes were quantified in the eyestalks, while 17β-estradiol (E2) and melatonin concentration were measured in the hemolymph. Cd bioaccumulated mainly in the hepatopancreas and gills, with increased E2, LPX, and antioxidant enzymes in HC compared to the LC region. Decreased CHH and MIH transcripts were observed in the animals from HC regions compared to LC and MC areas. Physiological differences were recorded, especially for bioaccumulation, oxidative stress, and hormone levels, in animals sampled in HC areas compared to LC and MC regions. In conclusion, the physiological damage triggered by Cd could be reduced due to higher levels of melatonin and antioxidant enzymes in HC areas.

Dose-dependent effect of recombinant CHH-B1 on osmoregulatory capacity and Na+/K+-ATPase expression in bilaterally eyestalk-ablated shrimp Litopenaeus vannamei

Osmoregulation in crustaceans is under neuroendocrine control by the crustacean hyperglycemic hormones (CHHs), which modify water and ion concentrations in diverse species. Previous studies done by our group suggested that CHH variant B1 (CHH-B1) has effects on the osmoregulatory responses of the Pacific white shrimp Litopenaeus vannamei. For a better understanding of the molecular action mechanisms of CHH-B1 in osmoregulation, in this work, we established the dose-dependent effect of recombinant CHH-B1 on the gene expression of Na+/K+-ATPase (NKA) in gills, in contrast to changes in the osmoregulatory capacity (OC) of bilaterally eyestalk-ablated shrimp under hyper-osmotic conditions. The results indicate that CHH-B1 regulates the OC of shrimp during hypo-regulation by modulating Na+/K+-ATPase at a transcriptional level. Our results suggest that CHH has a direct participation in the control of osmo-ionic regulation mechanisms, not only in L. vannamei but in crustaceans in general.

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.

Understanding molt control switches: Transcriptomic and expression analysis of the genes involved in ecdysteroidogenesis and cholesterol uptake pathways in the Y-organ of the blue crab, Callinectes sapidus.

The crustacean molting process is regulated by an interplay of hormones produced by the eyestalk ganglia and Y-organs (YO). Molt-inhibiting hormone and crustacean hyperglycemic hormone released by the sinus gland of the eyestalk ganglia (EG) inhibit the synthesis and secretion of ecdysteroid by the YO, hence regulating hemolymph levels during the molt cycle. The purpose of this study is to investigate the ecdysteroidogenesis pathway, specifically genes linked to changes in ecdysteroid levels occurring at early premolt (ePM). To this end, a reference transcriptome based on YO, EG, and hepatopancreas was de novo assembled. Two genes (cholesterol 7-desaturase Neverland and cytochrome p450 307a1-like Spook) involved in ecdysteroidogenesis were identified from the YO transcriptome using sequence comparisons and transcript abundance. Two other candidates, Hormone receptor 4 and probable cytochrome p450 49a1 potentially involved in ecdysteroidogenesis were also identified. Since cholesterol is the ecdysteroid precursor, a putative cholesterol carrier (Apolipoprotein D-like) was also examined to understand if cholesterol uptake coincided with the increase in the ecdysteroid levels at the ePM stage. The expression level changes of the five candidate genes in the YO were compared between intermolt (IM) and induced ePM (iePM) stages using transcriptomic analysis. Expression analysis using qPCR were carried out at IM, iePM, and normal ePM. The increase in Spook and Neverland expression in the YO at the ePM was accompanied by a concomitant rise in ecdysteroid levels. The data obtained from iePM stage were congruent with those obtained from the normal ePM stage of intact control animals. The present findings support the role of Halloween genes in the ecdysteroidogenesis and molt cycle in the blue crab, Callinectes sapidus.

The exploration of neuroendocrine regulation of crustacean hyperglycemic hormone (CHH) on innate immunity of Litopenaeus vannamei under ammonia-N 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.

Thermal stress induced hyperglycemia in the blue swimmer crab, Portunus pelagicus

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 3h and were then transferred to ambient temperature (28°C). A higher CHH level of 47.30±2.26fmol/ml was observed on exposure of crabs to 24°C, over a recovery period of 3h. 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.

A new insight to characterize immunomodulation based on hepatopancreatic transcriptome and humoral immune factor analysis of the Cherax quadricarinatus infected with Aeromonas veronii

Cherax quadricarinatus is a type of large freshwater crayfish that is characterized by rapid growth and formidable adaptability. It has also been widely cultured and studied as a model organism. Aeromonas veronii is the dominant pathogen in aquatic environments and the primary threat to aquaculture’s economic stability. To better understand the interactions between C. quadricarinatus and A. veronii, high-throughput RNA sequencing of the C. quadricarinatus hepatopancreas was carried out on a control group, susceptible group (6 h after infection), and resistant group (48 h after infection). A total of 65,850,929 genes were obtained. Compared with the control group, 2616 genes were up-regulated and 1551 genes were down-regulated in the susceptible group; while 1488 genes were up-regulated and 1712 genes were down-regulated in the resistant group. GO and KEGG analysis showed that these differentially expressed genes (DEGs) were associated with multiple immune pathways, including Toll-like receptors (TLRs), antigen processing and presentation, NOD-like receptor signaling pathway, phagosome, lysosome, JAK-STAT signaling pathway. qRT-PCR showed that infection by A. veronii changed the expression pattern of the serine proteinase inhibitor (SPI), crustacean hyperglycemic hormone (CHH), anti-lipopolysaccharide factor (ALF), and extracellular copper/zinc superoxide dismutase (SOD1), all of which were significantly higher than in the control group up to 48 h after infection. In addition, detection of superoxide dismutase (SOD), catalase (CAT), lysozyme (LZM), and phenoloxidase (PO) activity, as well as ceruloplasmin (CP) concentration at different times after infection showed diverse trends. Furthermore, pathological sections obtained 24 h after infection show lesions on the hepatopancreas and intestinal tissues caused by A. veronii. The results of this study provide a foundation for analyzing the immune mechanism of C. quadricarinatus infected with A. veronii at the transcriptional level and a theoretical basis for screening disease-resistant individuals to ensure healthy economic development of the aquatic industry.

Effects of crustacean hyperglycaemic hormone RNA interference on regulation of glucose metabolism in Litopenaeus vannamei after ammonia-nitrogen exposure.

To unveil the adaptation of Litopenaeus vannamei to elevated ambient ammonia-N, crustacean hyperglycaemic hormone (CHH) was knocked down to investigate its function in glucose metabolism pathway under ammonia-N exposure. When CHH was silenced, haemolymph glucose increased significantly during 3-6 h, decreased significantly during 12-48 h and recovered to the control groups' level at 72 h. After CHH knock-down, dopamine (DA) contents reduced significantly during 3-24 h, which recovered after 48 h. Besides, the expressions of guanylyl cyclase (GC) and DA1R in the hepatopancreas decreased significantly, while DA4R increased significantly. Correspondingly, the contents of cyclic AMP (cAMP), cyclic GMP (cGMP) and diacylglycerol (DAG) and the expressions of protein kinase A (PKA), protein kinase G (PKG), AMP active protein kinase α (AMPKα) and AMPKγ were significantly down-regulated, while the levels of protein kinase C (PKC) and AMPKβ were significantly up-regulated. The expressions of cyclic AMP response element-binding protein (CREB) and GLUT2 decreased significantly, while GLUT1 increased significantly. Moreover, glycogen content, glycogen synthase and glycogen phosphorylase activities in hepatopancreas and muscle were significantly increased. Furthermore, the levels of key enzymes hexokinase, pyruvate kinase and phosphofructokinase in glycolysis (GLY), rate-limiting enzymes citrate synthase in tricarboxylic acid and critical enzymes phosphoenolpyruvate carboxykinase, fructose diphosphate and glucose-6-phosphatase in gluconeogenesis (GNG) were significantly decreased in hepatopancreas. These results suggest that CHH affects DA and then they affect their receptors to transmit glucose metabolism signals into the hepatopancreas of L. vannamei under ammonia-N stress. CHH acts on the cGMP-PKG-AMPKα-CREB pathway through GC, and CHH affects DA to influence cAMP-PKA-AMPKγ-CREB and DAG-PKC-AMPKβ-CREB pathways, thereby regulating GLUT, inhibiting glycogen metabolism and promoting GLY and GNG. This study contributes to further understand glucose metabolism mechanism of crustacean in response to environmental stress.

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

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

Analysis of transcriptome difference between rapid-growing and slow-growing in Penaeus vannamei.

Penaeus vannamei is the principle cultured shrimp species in China. However, with the increase of culture density, the growth difference between individuals is also expanding. Here, we make use of RNA-seq to study the growth mechanisms of P. vannamei. After 120days, we examined the transcriptomes of rapid-growing individuals (RG) and slow-growing individuals (SG). A total of 2116 and 176 differentially expressed genes (DEGs) were found in SG and RG, respectively. Moreover, the main DEGs are opsin, heat shock protein (HSP), actin, myosin, superoxide dismutase (SOD), cuticle protein, and chitinase. GO analysis further revealed that the DEGs were enriched in biological processes significantly, such as "sensory perception," "sensory perception of light stimulus," "response to stimulus," and "response to stress." Additionally, KEGG enrichment analysis showed that the DEGs were mainly enriched in "pentose and glucuronate interconversions," "amino sugar and nucleotide sugar metabolism," "glycophospholipid biosynthesis," and "glutathione metabolism." Interestingly, the upstream genes in the ecdysone signaling pathway, including molting inhibition hormone (MIH) and crustacean hyperglycemic hormone (CHH), did not differ significantly between RG and SG, which suggests that the cause for the inconsistent growth performance is due to the stress levels rather than the ecdysone signal pathway. In summary, this work provides data that will be useful for future studies on shrimp growth and development.

RNAi Reveals Role of Insulin-Like Androgenic Gland Hormone 2 (IAG2) in Sexual Differentiation and Growth in Hermaphrodite Shrimp

Insulin-like androgenic gland hormone (IAG) is the most widely known hormone that regulates sexual differentiation in crustaceans. Previously, a transcriptome study described two transcripts of IAGs (Lvit-IAG1 and Lvit-IAG2) in the peppermint shrimp Lysmata vittata, a species characterized by a rare reproductive system of protandric simultaneous hermaphroditism (PSH). Herein, we explored the function of Lvit-IAG2 via RNA interference (RNAi) experiments, and then compared the functional differences between the two IAGs. We demonstrated that Lvit-IAG2 positively regulated the expression of crustacean hyperglycemic hormone (Lvit-CHH) in eyestalk ganglion but exhibited no significant effect on the expression of gonad-inhibiting hormone (Lvit-GIHs) and crustacean female sex hormone (Lvit-CFSHs). Besides, Lvit-IAG2 gene knockdown caused degeneration in appendices masculinae (AM) and suppressed germ cells at the secondary spermatocyte stage. Moreover, silencing the Lvit-IAG2 gene impeded ovarian development, including smaller previtellogenic oocytes, and lower expression of vitellogenin (Lvit-Vg) gene in hepatopancreas and vitellogenin receptor (Lvit-VgR) gene in the ovarian region. Notably, silencing the Lvit-IAG2 gene impeded individual growth of the species. Collectively, findings from this study demonstrate that Lvit-IAG2 and Lvit-IAG1 coordinatively function to modulate sexual differentiation in L. vittata; meanwhile, Lvit-IAG2 stimulates the growth of the PSH species.

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.

Identification and characterisation of crustacean hyperglycaemic hormone (CHH) from Mediterranean shore crab Carcinusaestuarii

Identification and characterisation of crustacean hyperglycaemic hormone (CHH) from Mediterranean shore crab Carcinusaestuarii

Gamma-aminobutyric acid regulates glucose homeostasis and enhances the hepatopancreas health of juvenile Chinese mitten crab (Eriocheir sinensis) under fasting stress

The ability of immune defense and resistance to physiological stress is crucial to animal health and survival. This study investigated the regulation of γ-aminobutyric acid (GABA) on metabolic homeostasis and its enhancement of hepatopancreas health in juvenile Chinese mitten crab (Eriocheir sinensis) under food deprivation. Juvenile crabs of 400 individuals were divided into four treatment groups: a control group without injection, and injections with a phosphate-buffered saline solution, 100 μmol GABA/mL and 1000 μmol GABA/mL, respectively. Hypoglycemia was induced by fasting, whereas the GABA treatment regulated hemolymph glucose homeostasis. The quantitative real-time PCR (qRT-PCR) results showed that the GABA treatment significantly up-regulated the mRNA expression levels of crustacean hyperglycemic hormone (CHH) and pyruvate kinase (PK). In contrast, the expression of E. sinensis insulin-like peptide (EsILP) was significantly down-regulated in the cranial ganglia, thoracic ganglia and hepatopancreas. Moreover, acid phosphatase (ACP), alkaline phosphatase (AKP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were significantly increased in the hepatopancreas by the GABA treatment. Furthermore, the hemocyanin content in serum was significantly increased with the GABA injection, and the glutathione (GSH) content, total superoxide dismutase (T-SOD) activity and catalase (CAT) activity in the hepatopancreas showed a similar increasing trend with the dose elevation of GABA. Therefore, these results indicate that GABA can effectively maintain the hemolymph glucose homeostasis by regulating the levels of glucose metabolism-related hormones and key enzymes to promote the degradation and utilization of hepatopancreas glycogen. Meanwhile, GABA can improve the hepatopancreas function and immune status of juvenile E. sinensis under fasting stress. The treatment with GABA may provide a clue to guide health management in crab farming.

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.

Three different C: N ratios for Pacific white shrimp, Penaeus vannamei under practical conditions: Evaluation of growth performance, immune and metabolic pathways

A 28-day experiment was carried out to evaluate different C/N ratios (10:1, 15:1 and 20:1) on growth performance, water quality, survival rate and gene expression related to metabolic pathways of Penaeus vannamei in a biofloc system. To maintain the C/N ratio of the feed input, different carbon sources (molasses, rice bran and wheat flour) in combination were applied after fermentation for a day with biofloc consortium. Results showed that shrimps reared in C/N 10 (630 mg) and C/N 15 (646 mg) biofloc systems showed significantly (p = .0229 and .0263) higher growth when compared to C/N 20 (528 mg) and control (374 mg). With regard to the production of extracellular enzymes, protease, lipase and xylanase were found predominantly in colonized bacteria isolated from biofloc treatments, whereas amylase was commonly found in all the treatments. The proPO activity was considerably increased in C/N 10 and 15 treatments than C/N 20 and control. The ranges of relative expression levels of potential genes entailed in metabolite pathway like hexokinase (HK- 6.57–8.17), pyruvate kinase (PK-5.79–7.96), crustacean hyperglycaemic hormone (CHH- 6.99–7.66) and fatty acid synthase (FAS- 6.57–8.86), triacylglycerol lipase (TGL-6.99–8.66) and immune gene (Toll receptor- 4.57–4.86) fold up-regulated expression were observed in the biofloc treatments than that of the control. The overall findings indicate that the rearing of P. vannamei shrimp in C/N 10 and 15 biofloc systems could improve the growth performance, water quality and development of immune and metabolic responses.

Impacts of a novel live shrimp (Litopenaeus vannamei) water-free transportation strategy on flesh quality: Insights through stress response and oxidation in lipids and proteins

A novel strategy of water-free transportation (WFT) for live shrimp was recently developed. In this study, survival rate, hormone response, free radical content, oxidation in lipids and proteins, and flesh quality were evaluated after WFT. Meanwhile water transportation (WT) was employed as a reference for comparison. Results showed only 5.1% mortality after 10 h of WFT, while crustacean hyperglycemic hormone (CHH), reactive nitrogen species (RNS), free fatty acid content (FFA), peroxide value (PV), carbonyl concentration, intrinsic fluorescence intensity (IFI), and surface hydrophobicity significantly increased. Myosin heavy chain (MHC) and actin degradation were also detected after WFT. Although oxidation occurred in lipids and proteins, no significant changes in texture and water-holding capacity (WHC) were detected after WFT or WT (p > 0.05). The 10 h of WFT showed little influence on flesh quality of shrimp even though lipids and proteins oxidation occurred during the mimicked water-free transportation.