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Environmental cues such as temperature induce macroscopic changes in the molting cycle of crustaceans, however, the physiological mechanisms behind these changes remain unclearWe aimed to investigate the regulatory mechanisms in the intermolt and premolt stages of the Callinectes sapidus molt cycle in response to thermal stimuli. The concentration of ecdysteroids and lipids in the hemolymph, and the expression of heat shock proteins (HSPs) and molt key genes were assessed at 19 °C, 24 °C and 29 °C. The premolt animals exhibited a much larger response to the colder temperature than intermolt animals. Ecdysteroids decreased drastically in premolt animals, whereas the expression of their hepatopancreas receptor (CasEcR) increased, possibly compensating for the low hemolymphatic levels at 19 °C. This decrease might be due to increased HSPs and inhibited ecdysteroidogenesis in the Y-organ. In addition, the molting-inhibiting hormone expression in the X-organ/sinus gland (XO/SG) remained constant between temperatures and stages, suggesting it is constitutive in this species. Lipid concentration in the hemolymph, and the expression of CasEcR and CasHSP90 in the XO/SG were influenced by the molting stage, not temperature. On the other hand, the expression of HSPs in the hepatopancreas is the result of the interaction between the two factors evaluated in the study. Our results demonstrated that temperature is an effective modulator of responses related to the molting cycle at the endocrine level and that temperature below the control condition caused a greater effect on the evaluated responses compared to the thermostable condition, especially when the animal was in the premolt stage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Sinopotamon Henanense expresses two metal‒induced metallothioneins (MTs), Cd‒induced MT and Cu‒induced MT (ShCuMT). The Cd‒induced MT has been characterized as a Cd‒thiolate MT. However, it is unknown whether ShCuMT is a Cu‒thiolate MT. In the present study, ShCuMT was expressed heterologously in Escherichia coli and purified by Ni‒NTA column and superdex‒75 column. And its metal‒binding feature was evaluated by DTNB reaction, circular dichroism spectroscopy (CD), isothermal microtitration (ITC), electrospray flight mass spectrometry (ESI‒TOF‒MS), and matrix‒assisted laser desorption ionization flight mass spectrometry (MALDI‒TOF‒MS). Bioinformatics analysis demonstrated that ShCuMT possessed the cysteine‒triplet motif of a Cu‒specific MT. Expression and purification of ShCuMT illustrated that SUMO tag used as the production system for ShCuMT resulted in a high production yield. The stability order of ShCuMT binding metal ions were Cu (Ⅰ) > Cd (Ⅱ) > Zn (Ⅱ). The CD spectrum indicated that ShCuMT binding with Cu (I) exhibited a compact thiol metal clusters structure. Besides, there emerged no a visible nickel‒thiol absorption after Ni‒NTA column affinity chromatography. The ITC results implied that Cu‒ShCuMT possessed the optimal thermodynamic conformation and the highest stoichiometric number of Cu (Ⅰ). Overall, the results suggested that SUMO fusion system is a robust and inexpensive approach for ShCuMT expression and Ni‒NTA column had no influence on metal binding of ShCuMT and Cu(Ⅰ) was considered its cognate metal ion, and ShCuMT possessed canonical Cu‒thiolate characteristics. The metal binding feature of ShCuMT reported here contributes to elucidating the structure‒function relationship of ShCuMT in S. Henanense., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ecdysteroid molting hormones coordinate arthropod growth and development. Binding of 20-hydroxyecdysone (20E) to ecdysteroid receptor EcR/RXR activates a cascade of nuclear receptor transcription factors that mediate tissue responses to hormone. Insect ecdysteroid responsive and Forkhead box class O (FOXO) transcription factor gene sequences were used to extract orthologs from blackback land crab (Gecarcinus lateralis) Y-organ (YO) transcriptome: Gl-Ecdysone Receptor (EcR), Gl-Broad Complex (Br-C), Gl-E74, Gl-Hormone Receptor 3 (HR3), Gl-Hormone Receptor 4 (HR4), Gl-FOXO, and Gl-Fushi tarazu factor-1 (Ftz-f1). Quantitative polymerase chain reaction quantified mRNA levels in tissues from intermolt animals and in YO of animals induced to molt by multiple limb autotomy (MLA) or eyestalk ablation (ESA). Gl-EcR, Gl-Retinoid X Receptor (RXR), Gl-Br-C, Gl-HR3, Gl-HR4, Gl-E74, Gl-E75, Gl-Ftz-f1, and Gl-FOXO were expressed in all 10 tissues, with Gl-Br-C, Gl-E74, Gl-E75, and Gl-HR4 mRNA levels in the YO lower than those in most of the other tissues. In MLA animals, molting had no effect on Gl-Br-C, Gl-E74, and Gl-Ftz-f1 mRNA levels and little effect on Gl-EcR, Gl-E75, and Gl-HR4 mRNA levels. Gl-HR3 and Gl-FOXO mRNA levels were increased during premolt stages, while Gl-RXR mRNA level was highest during intermolt and premolt stages and lowest at postmolt stage. In ESA animals, YO mRNA levels were not correlated with hemolymph ecdysteroid titers. ESA had no effect on Gl-EcR, Gl-E74, Gl-HR3, Gl-HR4, Gl-Ftz-f1, and Gl-FOXO mRNA levels, while Gl-RXR, Gl-Br-C, and Gl-E75 mRNA levels were decreased at 3 days post-ESA. These data suggest that transcriptional up-regulation of Gl-FOXO and Gl-HR3 contributes to increased YO ecdysteroidogenesis during premolt. By contrast, transcriptional regulation of ecdysteroid responsive genes and ecdysteroidogenesis were uncoupled in the YO of ESA animals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study investigated the effects of nanoplastics (NPs) of varying particle sizes (75, 500, and 1000 nm) and concentrations (2.5 and 10 mg/L) on the gut health of Chiromantes dehaani. The experimental groups included a control (Cg0), and varying combinations of particle size and concentration. Our results showed that 75 nm NPs were more likely to enhance pathogenic bacterial growth than other sized NPs. Compared with CK, Low NPs concentrations (2.5 mg/L) raised total cholesterol (T-CHO) levels in the gut, while high concentrations significantly decreased both triglyceride (TG) and T-CHO levels (p < 0.05). The enzymatic activities of intestinal lipase and amylase were inhibited by NPs exposure, with greater inhibition at higher NPs concentrations. The 500 nm NPs exhibited a notably higher inhibitory effect than the 75 and 1000 nm NPs (P < 0.05). In terms of apoptosis, NPs exposure led to reduced mRNA expression of Bcl2 and increased expression of Caspase-3, Caspase-8, and Caspase-9, indicating an induction of apoptosis. This effect was more pronounced at higher NPs concentrations, with 75 nm NPs more likely to induce apoptosis in intestinal cells than 500 nm and 1000 nm NPs. Moreover, NPs triggered intestinal inflammatory responses, evidenced by the increased mRNA expression of TNF-β, TNF-α, IL1β, IL6, and IL8, and the decreased expression of IL10. High NPs concentrations were more likely to induce intestinal inflammation, with 500 nm NPs imparting the strongest effect. In summary, the study demonstrated that NPs, and particularly those at higher concentrations, disrupted the gut environment of C. dehaani by altering the microflora, reducing microbial diversity, inhibiting digestion and metabolism, inducing apoptosis, and triggering inflammation. Among the sizes of NPs tested, 500 nm NPs had the most significant adverse impact on digestion, metabolism, and inflammation, while 75 nm NPs most strongly induced apoptosis in C. dehaani's intestinal cells., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As a fat-soluble vitamin, vitamin D 3 relies on fat to perform its biological function, affecting lipid metabolism and innate immunity. This study used different percentages of lipid and vitamin D 3 diets to evaluate the synergistic effects on the growth, lipid metabolism and immunity of juvenile Eriocheir sinensis (5.83 ± 0.01 g) for 56 days, including low lipid (LL, 1.5%) and normal lipid (NL, 7.5%) and three levels of vitamin D 3 : low (LVD, 0 IU/kg), medium (MVD, 9000 IU/kg) and high (HVD, 27,000, IU/kg). The synergistic effect of lipid and vitamin D 3 was not significant on growth but significant on ash content, total protein, hepatopancreas lipid content, hemolymph 1α,25-hydroxy vitamin D 3 [1α,25(OH) 2 D 3 ] content, hepatopancreas lipolysis and synthesis genes. Crabs fed normal lipid (7.5%) and medium vitamin D 3 (9000 IU/kg) had the highest hepatopancreas index, hemolymph 1α,25(OH) 2 D 3 content, antibacterial ability, immune-related genes and hepatopancreatic lipid synthesis genes expression, but down-regulated the lipolysis genes expression. In contrast, crabs fed diets with low lipid percentage (1.5%) had low growth performance, hemolymph 1α,25(OH) 2 D 3 , mRNA levels of lipid synthesis genes, antibacterial ability and immune-related gene expression. At the 1.5% lipid level, excessive or insufficient vitamin D 3 supplementation led to the obstruction of ash and protein deposition, reduced growth and molting, aggravated the reduction in antioxidant capacity, hindered antimicrobial peptide gene expression and reduced innate immunity, and resulted in abnormal lipid accumulation and the risk of oxidative stress. This study suggests that diets' lipid and vitamin D 3 percentage can enhance antioxidant capacity, lipid metabolism and innate immunity in E. sinensis. A low lipid diet can cause growth retardation, reduce antioxidant capacity and innate immunity, and enhance lipid metabolism disorder., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The leucine-rich repeat (LRR) domain is a crucial structure in a variety of immune related proteins and displays multiple immune functions. In this study, the open reading frame (ORF) of an LRR-only protein was cloned from the Chinese mitten crab, Eriocheir sinensis (EsLRRop1). The protein sequence of EsLRRop1 contained seven LRR motifs, three LRR-TYP motifs and an LRRCT motif. Tissue distribution exhibited that EsLRRop1 mainly expressed in nervous tissues including thoracic ganglion, eyestalk and brain while showed relatively lower transcriptional level in hemocyte. Based on the above expression characteristics, the responses of EsLRRop1 to the challenge of Vibrio parahaemolyticus and Staphylococcus aureus were tested. The result showed that the transcript of EsLRRop1 in thoracic ganglion and eyestalk up-regulated after being challenged with S. aureus, while it decreased post injection with V. parahaemolyticus. The transcript of EsLRRop1 in hemocytes up-regulated sharply at 3 h and decreased at 12 h and 24 h after being challenged with V. parahaemolyticus, while it decreased at 12 h and 24 h post injection with S. aureus. The recombinant protein of EsLRRop1 (His-EsLRRop1) displayed binding activities to V. alginolyticus, V. harveyi, V. parahaemolyticus, S. aureus, Corynebacterium glutamicum and Micrococcus lysodeikticus as well as lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, the His-EsLRRop1 exhibited inhibitory activity against V. parahaemolyticus and V. harveyi with minimum inhibitory concentration (MIC) of 3.57-7.14 μM and 7.14-14.28 μM, respectively. These results provide theoretical basis for the application of EsLRRop1 in inhibiting bacteria in aquaculture practice., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Scylla paramamosain is an important cultured crab species on the southeast coast of China. However, the molecular regulation mechanism of its gonadal development still has not been thoroughly studied. Dsx (doublesex) and foxl-2 (forkhead transcription factor gene 2) are important transcription factors involved in gonadal development. So far, studies on the functions of dsx and foxl-2 in crustaceans are very limited. Insulin-like androgenic gland hormone (IAG) is an effector molecule that regulates the differentiation, development and sex maintenance of testes in crustaceans. In this study, the promoter region of Sp-IAG was predicted, and several potential binding sites of dsx and foxl-2 were found. Site-directed mutagenesis was performed on the predicted potential binding sites, and their promoter activity was analyzed. The results showed that there was a dsx and a foxl-2 binding site, respectively, that could regulate the expression of Sp-IAG. In order to verify the regulatory effect of these two transcription factors on Sp-IAG, we constructed the expression plasmids of dsx and foxl-2 and co-transfected them into HEK293T cell lines with the promoter of Sp-IAG, respectively. The results showed that dsx could significantly promote the expression of Sp-IAG, while foxl-2 could inhibit its expression substantially. Then we carried out in vivo RNA interference experiment on mud crabs. The expression of dsx and foxl-2 in crabs was interfered respectively. The results of qRT-PCR showed that the expression of Sp-IAG was significantly inhibited after interfering with dsx, while significantly increased after interfering with foxl-2, which was consistent with the cell experiment. In conclusion, dsx and foxl-2 transcription factors play opposite roles in regulating the expression of Sp-IAG., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dimethoate is a broad-spectrum organophosphate insecticide and acaricide. Through various pathways, such as runoff and drift, dimethoate can reach marine environment, and easily impact common organisms in coastal areas, close to agriculture lands, namely crustaceans. The purpose of this study was to investigate the potential effects of dimethoate exposure (50, 100, and 200 μg/l), for 1 day, on a wide range of markers of oxidative stress and neurotransmission impairment, as well as fatty acids composition and histopathological aspect in the gills of the green crab Carcinus aestuarii. A significant increase in n-3 polyunsaturated fatty acids series, namely the eicosapentaenoic acid (C20: 5n3) and its precursor alpha-linolenic acid (C 18: 3n3) in dimethoate-treated crabs was recorded. Concerning n-6 polyunsaturated fatty acids, we noted a high reduction in arachidonic acid (C20:4n-6) levels. Dimethoate exposure increased the levels of hydrogen peroxide, malondialdehyde, lipid hydroperoxides, protein carbonyl, and caused the advanced oxidation of protein products along with enzymatic and non-enzymatic antioxidant-related markers. Acetylcholinesterase activity was highly inhibited following exposure to dimethoate in a concentration-dependent manner. Finally, deleterious histopathological changes with several abnormalities were noted in exposed animals confirming our biochemical findings. The present study offered unique insights to establish a relationship between redox status and alterations in fatty acid composition, allowing a better understanding of dimethoate-triggered toxicity., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Tumor necrosis factor (TNF) is an important cytokine that can regulate a variety of cellular responses by binding tumor necrosis factor receptor (TNFR). We studied whether the TNF of Eriocheir sinensis can regulate hemocyte proliferation. The results showed that the EsTNF and EsTNFR were constitutively expressed in all tested tissues, including the heart, hepatopancreas, muscles, gills, stomachs, intestines, and hemocytes. We found that low levels of EsTNF and EsTNFR transcripts were present in hemocytes. The gene expression levels were significantly increased in the hemocytes after being stimulated by Staphylococcus aureus or Vibrio parahaemolyticus. We also found some genes related to cell proliferation were expressed at a higher level in pulsing rTNF-stimulated hemocytes compared with the control group. We also knocked down the EsTNFR gene with RNAi technology. The results showed that the expression level of these genes related to cell proliferation was significantly down-regulated compared with the control group when the TNF does not bind TNFR. We used Edu technology to repeat the above experiments and the results were similar. Compared with the control group, the hemocytes stimulated by rTNF showed more significant proliferation, and the proliferation rate was significantly down-regulated after knocking down the EsTNFR gene. Therefore, we indicate that TNF binding TNFR can affect the proliferation of E. sinensis hemocytes, which might be manifested by affecting the expression of some proliferation-related genes., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Protein kinases of the MAPK cascade family (MAPKKK-MAPKK-MAPK) play an important role in the growth and development of organisms and their response to environmental stress. The MAPKK gene families in the Chinese mitten crab Eriocheir sinensis have never been systematically analyzed. We identified four MAPKK family genes, EsMEK, EsMAPKK4, EsMAPKK6, and EsMAPKK7, in E. sinensis and analyzed their molecular features and expression patterns. All four MAPKK genes are composed of multiple exons and introns, all have a conserved domain, and all have 10 conserved motifs (except EsMEK and EsMAPKK7 which are missing motif 10). The four MAPKK genes are on four different chromosomes and have no gene duplications, and the results of phylogenetic tree analysis indicate that the ESMAPKK gene family is highly conserved evolutionarily. The EsMAPKK genes were widely expressed in all the examined tissues with higher expression in hemocytes, hepatopancreas, and gills. Notably, EsMAPKK6 was also highly expressed in the ovary. Vibrio parahaemolyticus infection significantly increased the mRNA levels of the EsMAPKK genes in hemocytes. Further disruption of the EsMAPKK gene family expression affects the expression levels of multiple antimicrobial peptides in hemocytes. Our experimental results provide a starting point for a more in-depth study of the innate immunity functional roles of members of the MAPKK gene families in E. sinensis., Competing Interests: Declaration of competing interest None., (Copyright © 2023. Published by Elsevier Ltd.)

Menippe mercenaria, the Florida stone crab, supports an unconventional fishery across the southern USA and Caribbean that involves claw-removal and the return of de-clawed animals to the sea. We provide pathological, ultrastructural, and genomic detail for a novel hepatopancreatic, nucleus-specific virus - Menippe mercenaria nudivirus (MmNV) - isolated from M. mercenaria, captured during fisheries-independent monitoring. The virus has a genome of 99,336 bp and encodes 84 predicted protein coding genes and shows greatest similarity to Aratus pisonii nudivirus (ApNV) (<60% protein similarity and 31 shared genes of greatest similarity), collected from the Florida Keys, USA. MmNV is a member of the Gammanudivirus genus (Naldaviricetes: Lefavirales: Nudiviridae). Comparisons of virus genome size, preferred host environment, and gene number revealed no clear associations between the viral traits and phylogenetic position. Evolution of the virus alongside the diversification of host taxa, with the potential for host-switching, remain more likely evolutionary pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lectins are non-immune glycoproteins or proteins having a unique capacity to interact with carbohydrate ligands found on the surface of their host cells. In the present investigation, the lectin was purified from the hemolymph of freshwater crab, Oziotelphusa naga and its antimicrobial, anti-inflammatory and anti-arthritic activity was analysed. The preliminary characterization of the hemagglutinin was carried out to identify the erythrocyte and sugar specificity, optimum pH and temperature and cation dependency. The agglutinin was found to be highly specific to rabbit erythrocyte and inhibited by fetuin and α-lactose. Maximum hemagglutination activity was noted at pH 7.5-8 and temperature 20-40 °C. An O-acetyl sialic acid specific 75 kDa hemolymph lectin, designated as NagLec was isolated from the freshwater crab, Oziotelphusa naga by affinity chromatography on fetuin coupled Sepharose 4 B, with a purification fold of 185. The bacteria Staphylococcus aureus, Proteus mirabilis and fungus Candida albicans had the greatest zone of inhibition when treated with NagLec. The results of the Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) assays showed that the purified lectin inhibited the growth of Staphylococcus aureus at 0.031 and 0.065 μg/ml, which proved the bactericidal property of NagLec. NagLec generated alterations on the bacterial cells and led to protein leakage, which was dosage (24 and 48 μg/ml) and time dependent (10-40 min). COX and LOX enzyme was inhibited to 49.43% and 61.81% with 100 μg/ml concentration of NagLec respectively, demonstrating NagLec's ability to reduce inflammation. Furthermore, NagLec (500 μg) suppressed protein denaturation up to 77.12% whereas diclofenac sodium (a standard drug) was inhibited by 89.36%. The results indicate that NagLec, a sialic acid specific lectin isolated from the freshwater crab O. naga could be formulated as a nano drug in future owing to its antimicrobial, anti-inflammatory and anti-arthritic potential that could be targeted to specific pathogenic microbes and treat arthritis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Portunion is a rare endoparasitic isopod genus, recently observed inhabiting the hemocoel of the commercially important mud crab, Scylla paramamosain. For better understanding of the host-parasite interaction between S. paramamosain and Portunion sp., the metabolomic and transcriptomic changes in the hemolymph of the S. paramamosain were analyzed. We detected a total of 143 and 126 differentially accumulated metabolites in the positive and negative modes, respectively. Pathways related to amino acids and vitamin synthesis, such as Aminoacyl-tRNA biosynthesis, Tyrosine metabolism, Cysteine and methionine metabolism, Vitamin B6 metabolism, and Biotin metabolism were significantly enriched. Based on the transcriptomic data, a total of 942 differentially expressed genes were identified, of which 25 and 36 were significantly related to the immune system and metabolic pathways, respectively. Based on the metabolomic and transcriptomic data, 90 correlated metabolite-gene pairs were selected to build a regulatory network. Common significantly enriched pathways, including Starch and sucrose metabolism, Metabolism of xenobiotics by cytochrome P450, Aminoacyl-tRNA biosynthesis, Nitrogen metabolism, and Galactose metabolism were detected. On the basis of our analysis, the endoparasite Portunion sp. places a heavy metabolic burden on the host, particularly with respect to fundamental resources, such as amino acids, vitamins, carbohydrates, and lipids. In summary, these data provide an overview of the global metabolic and transcriptomic changes of the S. paramamosain resulting from Portunion sp. infection., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A pair of Y-organs (YOs) synthesize ecdysteroids that initiate and coordinate molting processes in decapod crustaceans. The YO converts cholesterol to secreted products through a biosynthetic pathway involving a Rieske oxygenase encoded by Neverland (Nvd) and cytochrome P450 monooxygenases encoded by Halloween genes Spook (Spo; Cyp307a1), Phantom (Phm; Cyp306a1), Disembodied (Dib; Cyp302a1), and Shadow (Sad; Cyp315a1). NAD kinase (NADK) and 5-aminolevulinic acid synthase (ALAS) support ecdysteroid synthesis in insects. A 20-hydroxylase, encoded by Shed in decapods and Shade in insects, converts ecdysone to the active hormone 20-hydroxyecdysone (20E). 20E is inactivated by cytochrome P450 26-hydroxylase (Cyp18a1). Contigs encoding these eight proteins were extracted from a Gecarcinus lateralis YO transcriptome and their expression was quantified by quantitative polymerase chain reaction. mRNA levels of Gl-Spo and Gl-Phm were four orders of magnitude higher in YO than those in nine other tissues, while mRNA levels of Gl-NADK and Gl-ALAS were similar in all ten tissues. In G. lateralis induced to molt by multiple leg autotomy, YO mRNA levels of Gl-Nvd, Gl-Spo, Gl-Phm, Gl-NADK, and Gl-ALAS were highest in intermolt and premolt stages and lower in postmolt. Gl-Dib mRNA level was not affected by molt stage. mRNA level of Gl-Sad, which converts 2-deoxyecdysone to ecdysone, was higher in mid- and late premolt stages, when YO ecdysteroidogenic capacity is greatest. Gl-Cyp18a1 mRNA level was highest in intermolt, decreased in premolt stages, and was lowest in postmolt. In animals induced to molt by eyestalk ablation, YO mRNA levels of all eight genes were not correlated with increased hemolymph 20E titers. These results suggest that YO ecdysteroidogenic genes are differentially regulated at transcriptional and translational levels., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

QM protein was previously discovered as a tumor suppressor, and numerous studies have shown that QM protein also played important roles in the immune responses. To investigate the potential roles of the QM protein gene in Eriocheir sinensis, the QM protein gene (designated as EsQM) has been cloned from E. sinensis using the rapid amplification of cDNA ends (RACE) technique. The cDNA of EsQM is 781 bp in length, consisting of a 654 bp open reading frame (ORF), encoding 219 amino acids, a 27 bp 5' untranslated region (UTR) and a 94 bp 3' UTR. The EsQM protein has a calculated molecular weight of 25.4 kDa and a theoretical isoelectric point of 10.10. The deduced protein sequence of EsQM contains a Ribosomal&#95;L16 domain, an SH3-binding motif, an N-acylation site, two putative antibiotic binding sites, two putative protein kinase C phosphorylation sites, and two amidation sites. EsQM is extremely conserved and exhibits more than 85% similarities to previously identified arthropod QM protein genes. By real-time quantitative PCR (qPCR) analysis, we found that EsQM mRNA transcripts were detectable in all the examined tissues, with the highest expression in hemocytes. The mRNA expression of EsQM in hemocytes was significantly upregulated after the stimulation of Aeromonas hydrophila or polybrominated diphenyl ether-47 (BDE-47). Moreover, EsQM mRNA expression in hemocytes responded more quickly and lasted longer when stimulated by A.hydrophila than BDE-47. Thus, EsQM can respond to bacterial infection and environmental pollution, and might be involved in the defense mechanism to both biological and non-biological stimulation of arthropods., Competing Interests: Declaration of competing interest The authors declared no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The occurrence of hepatopancreatic necrosis syndrome (HPNS) has seriously affected the sustainable development of Chinese mitten crab (Eriocheir sinensis) farming industry. Limited studies have focused on the immune responses in crabs with HPNS. Serine proteases (SPs) and SP homologs (SPHs) play important roles in the innate immunity of crustaceans. This study investigated the effects of HPNS on the expression levels of genes related to prophenoloxidase (proPO) activation system, and the relationship between Runt transcription factor and the transcriptions of these genes. Eight SPs and five SPHs (SPH1-4, Mas) were identified from E. sinensis. SPs contain a catalytic triad of "HDS", while SPHs lack a catalytic residue. SPs and SPHs all contain a conservative Tryp&#95;SPc domain. Evolutionary analysis showed that EsSPs, EsSPHs, EsPO, and EsRunt were clustered with SPs, SPHs, POs, and Runts of other arthropods, respectively. In crabs with HPNS, the expression levels of six SPs (1, 3, 4, 6, 7, and 8), five SPHs, and PO were significantly upregulated in the hepatopancreas. The knockdown of EsRunt could evidently decrease the expression levels of four SPs (3, 4, 5 and 8), five SPHs (SPH1-4, Mas), and PO. Therefore, the occurrence of HPNS activates the proPO system. Furthermore, the expression levels of partial genes related to proPO system were regulated by Runt. The activation of innate immune system may be a strategy for crabs with HPNS to improve immunity and fight diseases. Our study provides a new understanding of the relationship between HPNS and innate immunity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Because invertebrates lack acquired immunity, they rely primarily on the innate immune system to defend themselves against viral and bacterial infections. SVWC, also called Vago, is a class of small-molecule proteins characterized by a single von Willebrand factor C-domain and appears to be restricted to arthropods. It has been reported that SVWC is involved in antiviral immunity in invertebrates, but whether it is involved in antimicrobial immunity and the mechanism of its involvement in antimicrobial immunity remains unclear. In this study, we identified a novel SVWC gene in Eriocheir sinensis and named it EsSVWC. EsSVWC was found to respond positively to bacterial stimulation and to regulate the expression of related antimicrobial peptides (AMPs). The EsSVWC protein recognized and bound to a variety of pathogen-associated molecular patterns (PAMPs) but did not exhibit direct bactericidal effects. Thus, the EsSVWC protein in crabs helps resist bacterial infection and improve survival rates. In summary, EsSVWC may regulate the innate immune system of crabs in response to microbial invasion in an indirect manner., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Rice crab co-culture is a new integrated farming model in China. The application of triazole plant growth regulators (PRGs) is often used as an advantageous option to combat rice lodging. However, there is still a gap regarding the toxicity of these PRGs on the growth and development of the Chinese mitten crab (Eriocheir sinensis, E. sinensis). Here the effect of triazoles (paclobutrazol and uniconazole) on the molting mechanism of E. sinensis was investigated. Monitoring of regulatory genes associated with molting showed that the two PRGs were found to inhibit the expression of ecdysteroid hormone (EH), ecdysteroid receptors gene (EcR), and retinoid X receptors gene (RXR) and induce secretion of molt-inhibiting hormone (MIH) gene. In addition, the activities of chitinase (CHIA) and N-acetyl-β-d-aminoglucosidase (β-NAGase) were also inhibited by exposure to PRGs. Exposure to PRGs also elevated the mRNA expression of the growth-related myostatin gene (MSTN). These results revealed that there is a long-term risk of exposure to triazoles PRGs that may inhibit molting and affect normal development and immune system of E. sinensis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ficolin, a member of the fibrinogen-related proteins family (FREPs), functions as a pattern recognition receptor (PRR) in vertebrates and in invertebrates as a novel lectin. In this study, we discovered the Ficolin homolog of Chinese mitten crab (Eriocheir sinensis), which we named EsFicolin. The obtained sequence showed that it has a highly conserved C-terminal fibrinogen-related domain (FReD) and a coiled-coil structure for trimer formation. EsFicolin was up-regulated in hemocytes after being stimulated by bacteria. Recombinant EsFicolin protein binds to gram-negative and gram-positive bacteria and agglutinates bacteria through pathogen-associated molecular patterns. In-depth study found that recombinant EsFicolin could effectively remove bacteria and showed direct antibacterial activity. EsFicolin could also promote the phagocytosis of hemocytes to enhance bacterial clearance. These findings suggest that EsFicolin plays an important role in the crab antibacterial immune response., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

To study the effects of dietary methionine on growth performance, immunity, antioxidant capacity, protein metabolism, inflammatory response and apoptosis factors in Chinese mitten crabs (Eriocheir sinensis). Five diets with different methionine levels (0.63%, 0.85%, 1.06%, 1.25% and 1.47%) were fed to E. sinensis for 8 weeks. Results showed that in the 1.25% Met group, both growth performance and feed utilization were significantly increased. The crude protein content of crab muscle in the 1.06% and 1.25% Met groups was significantly higher than that in the control group. The immune and antioxidant enzyme activities, as well as gene expression levels of anti-lipopolysaccharide factor 1 (ALF1), Crustin-1, prophenoloxidase (proPO), cap 'n' collar isoform C (CncC) in 1.25% Met group were significantly higher than other groups. The activities of adenosine deaminase (ADA) and glutamate transaminase (GPT) in serum decreased first and then increased with the increase of methionine content, while the changes of ADA and GPT in hepatopancreas increased first and then decreased. 1.25% Met group exhibited significantly increased levels of GOT, GPT, and ADA compared to the control group. 1.25% Met diet group significantly up-regulated protein synthesis and anti-apoptotic factors, and significantly down-regulated inflammatory and pro-apoptotic factors in hepatopancreas. At 1.25% in the diet, methionine was found to boost E. sinensis growth, muscle protein deposition and immunity, as well as its antioxidant capacity. Combined with the above results, based on the expression of factors involved in the mammalian target of rapamycin (mTOR) signaling pathway and the mitogen-activated protein kinase (MAPK) signaling pathway, it is proved that methionine can not only promote protein metabolism, improve feed utilization, but also alleviate the inflammatory response and apoptosis caused by oxidative stress in the body., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mud crab reovirus (MCRV) is a serious pathogen that leads to large economic losses in the mud crab farming. However, the molecular mechanism of the immune response after MCRV infection is unclear. In the present study, physiological, transcriptomic, and metabolomic responses after MCRV infection were investigated. The results showed that MCRV infection could increase lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase activities. MCRV infection decreased antioxidant enzyme activity levels, induced oxidative stress, and caused severe histological damage. Transcriptome analysis identified 416 differentially expressed genes, including 354 up-regulated and 62 down-regulated genes. The detoxification, immune response, and metabolic processes-related genes were found. The results showed that two key pathways including phagocytosis and apoptosis played important roles in response to MCRV infection. The combination of transcriptomic and metabolomic analyses showed that related metabolic pathways, such as glycolysis, citrate cycle, lipid, and amino acid metabolism were also significantly disrupted. Moreover, the biosynthesis of unsaturated fatty acids was activated in response to MCRV infection. This study provided a novel insight into the understanding of cellular mechanisms in crustaceans against viral invasion., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

This study aims to investigate the effects of glycerol monolaurate (GML) on growth performance, non-specific immunity, antioxidant capacity and intestinal microflora in Chinese mitten crabs. The crabs were randomly arranged to three experimental diets groups containing 0 (control group), 1000 mg/kg GML (GML1000 group), and 2000 mg/kg GML (GML2000 group), respectively. After 8 weeks of breeding, results showed a better growth performance in GML2000 group, with a higher PWG, SGR and lower FCR (P < 0.05). Meanwhile, in GML2000 group the activities of phenoloxidase, alkaline phosphatase, acid phosphatase and lysozyme in hemolymph were increased (P < 0.05), also the activities of hemolymph and hepatopancreas superoxide dismutase (SOD) and glutathione peroxidase (GPx) were increased in hepatopancreas (P < 0.05). While malondialdehyde (MDA) concentrations were lower significantly (P < 0.05) both in GML1000 and GML2000 groups. Furthermore, the mRNA expression of TLR1, TLR2, which related to the Toll pathway were increased (P < 0.05). Supplementation of 2000 mg/kg GML up-regulated the expression of ALF and LZM (P < 0.05), and down-regulated the expression of caspase-3 (P < 0.05). The abundance of Firmicutes increased in GML2000 group (P < 0.05), and Shewanella was significantly increased (P < 0.05) in both GML1000 and GML2000 groups. In conclusion, dietary supplemented with GML enhanced the growth performance and antioxidant capacity, enhanced hemolymph immune enzymes activities and antimicrobial peptides expression through regulating the proPO system and Toll pathway, and improved gut microflora in Chinese mitten crabs., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Phosphofructokinase (PFK), the key enzyme of glycolysis, can catalyze the irreversible transphosphorylation of fructose-6-phosphate forming fructose-1, 6-biphosphate. In the present study, a PFK gene from the mud crab Scylla paramamosain, named SpPFK, was cloned and characterized. The full length of SpPFK contained a 5'untranslated region (UTR) of 249 bp, an open reading frame of 2,859 bp, and a 3'UTR of 1,248 bp. The mRNA of SpPFK was highly expressed in the gill, followed by the hemocytes and muscle. The expression of SpPFK was significantly up-regulated after mud crab dicistrovirus-1 (MCDV-1) infection. Knocking down SpPFK in vivo by RNA interference significantly reduced the expression of lactate dehydrogenase after MCDV-1 infection. Furthermore, silencing of SpPFK in vivo increased the survival rate of mud crabs and decreased the MCDV-1 copies in the gill and hepatopancreas after MCDV-1 infection. All these results suggested that SpPFK could play an important role in the process of MCDV-1 proliferation in mud crab., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is an immunomodulator to inhibit immune-mediated pro-inflammatory response and has been used to treat various immune-related diseases in mammals. However, the immunoregulatory effect of GABA in crustaceans has not been reported. This study evaluates the regulatory effect of dietary GABA supplementation on the innate immune status and immunoregulatory potential in lipopolysaccharide (LPS)-induced immune response in juvenile Eriocheir sinensis. Juvenile crabs were fed with six diets supplemented with graded GABA levels (0, 40, 80, 160, 320 and 640 mg/kg dry matter) for 8 weeks and then 24 h LPS challenge test was carried out. The results showed that dietary GABA supplementation significantly decreased mortality at 4 and 8 weeks. Moreover, the hemocyanin content, acid phosphatase, and alkaline phosphatase activities significantly increased in the crabs fed GABA supplementation compared with the control. On the contrary, the alanine aminotransferase and alanine aminotransferase activities in serum decreased significantly in the GABA supplementation groups compared with the control. Similarly, superoxide dismutase activity, glutathione content, and the transcriptional expression of the antioxidant-related genes and immune-related genes were significantly higher in the GABA supplementation groups than in the control. In addition, the mRNA expressions of anti-lipopolysaccharide factors (ALF 1, ALF 2, ALF 3) and inflammatory signaling pathways related genes (TLR, Myd88, Relish, LITAF, P38-MAPK, ADAM17) were significantly up-regulated in LPS stimulation groups compared with PBS treatment. Meanwhile, pro-apoptosis-related genes' mRNA expressions were significantly up-regulated, and anti-apoptosis-related genes were significantly down-regulated under LPS stimulation compared with PBS treatment. However, GABA pretreatment effectively alleviated LPS-induced immune overresponse and apoptosis. Therefore, this study demonstrates that dietary GABA supplementation could be used as an immunomodulator to improve the non-specific immunity and antioxidant capacity and alleviate the immune-mediated immune overresponse of juvenile E. sinensis., (Copyright © 2022. Published by Elsevier Ltd.)

Endocrine-disrupting chemicals (EDCs), distributed at various concentrations in freshwater and marine ecosystems, affect the survival, reproduction, and behavior of wide ranges organisms. Most toxicology studies on EDCs have focused on the endocrine system of invertebrates, and research on invertebrate neurotransmitters is limited. In the present study, we investigated the expression of Macrophthalmus japonicus genes encoding γ-aminobutyric acid transporter subtype 2 (GAT-2) and glutamine synthetase (GS), which play important roles as neurotransmitters at synapses. We observed differences in the mRNA expression levels of GAT-2 and GS as well as histological changes in various tissues after exposure to bisphenol-A (BPA) and di-(2-ethylhexyl) phthalate (DEHP). The amino acid sequences of M. japonicus GAT-2 and GS formed separate branches in crustaceans, fish, insects, and mammals. M. japonicus GAT-2 and GS expression levels were highest in the gills, hepatopancreas, and stomach, and showed different between DEHP or BPA treatments. In particular, hepatopancreas GS expression on Day 1, the first step in the presynaptic process, was upregulated after BPA and DEHP exposure, while GAT-2, sequential step in the presynaptic process, was significantly elevated only in DEHP. After BPA treatments, gill GS expression was increased at all concentrations, whereas GAT-2 expression was overall down regulations. In contrast, in DEHP treatment groups hepatopancreatic GS and GAT-2 expression at Day 1 was only significantly higher and all groups including gill GS and GAT-2 expression were downregulation. Histological changes in the gills and hepatopancreas were observed in a concentration-dependent manner. Accordingly, BPA and DEHP exposure in crabs could be stimulate neurotransmitter gene expression and alter the morphological structure of gill and hepatopancreas., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Crustacean Y-organs secrete ecdysteroid molting hormones. Ecdysteroids are released in increased amount during premolt, circulate in hemolymph, and stimulate the events in target cells that lead to molting. During much of the molting cycle, ecdysteroid production is suppressed by molt-inhibiting hormone (MIH), a peptide neurohormone produced in the eyestalks. The suppressive effect of MIH is mediated by a cyclic nucleotide second messenger. A decrease in circulating MIH is associated with an increase in the hemolymphatic ecdysteroid titer during pre-molt. Nevertheless, it has long been hypothesized that a positive regulatory signal or stimulus is also involved in promoting ecdysteroidogenensis during premolt. Data reviewed here are consistent with the hypothesis that an intracellular Ca 2+ signal provides that stimulus. Pharmacological agents that increase intracellular Ca 2+ in Y-organs promote ecdysteroidogenesis, while agents that lower intracellular Ca 2+ or disrupt Ca 2+ signaling suppress ecdysteroidogenesis. Further, an increase in the hemolymphatic ecdysteroid titer after eyestalk ablation or during natural premolt is associated with an increase in intracellular free Ca 2+ in Y-organ cells. Several lines of evidence suggest elevated intracellular calcium is linked to enhanced ecdysteroidogenesis through activation of Ca 2+ /calmodulin dependent cyclic nucleotide phosphodiesterase, thereby lowering intracellular cyclic nucleotide second messenger levels and promoting ecdysteroidogenesis. Results of transcriptomic studies show genes involved in Ca 2+ signaling are well represented in Y-organs. Several recent studies have focused on Ca 2+ transport proteins in Y-organs. Complementary DNAs encoding a plasma membrane Ca 2+ ATPase (PMCA) and a sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase (SERCA) have been cloned from crab Y-organs. The relative abundance of PMCA and SERCA transcripts in Y-organs is elevated during premolt, a time when Ca 2+ levels in Y-organs are likewise elevated. The results are consistent with the notion that these transport proteins act to maintain the Ca 2+ gradient across the cell membrane and re-set the cell for future Ca 2+ signals., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Invertebrate insulin-like peptide-binding proteins (ILPBPs) are structurally homologous to vertebrate insulin-like growth factor binding protein 7 (IGFBP7). One of the invertebrate ILPBPs is considered as a potential binding protein of insulin-like androgenic gland factor (IAG) in males of crayfish, Cherax quadricarinatus. However, the ILPBP expression is not limited in the androgenic gland and found in most examined tissues, implicating that ILPBP may have additional functions in crustaceans. Here, the full-length cDNA sequence of ILPBP (termed ChqILPBP) is isolated from the ovary of the red deep-sea crab, Chaceon quinquedens. ChqILPBP transcripts are present in the various tissues, as similar to other crab species. The crustacean ILPBPs have their putative amino acid sequences conserved much less than vertebrate IGFBP7s. To understand if ChqILPBP is involved in ovarian development, examined are levels of ChqILPBP, together with vitellogenin (ChqVTG) in the same ovary and hepatopancreas of adult females at the different ovarian stages: 2, 3, and 5. Chaceon hepatopancreas exhibits as the primary VTG synthesis site, while VTG transcript levels do not differ by the ovarian stages. The ovary contains ChqILPBP transcripts ~10-fold higher than hepatopancreas that changes significantly from stage 2 to 3. Such an expression pattern mirrors that of ovarian ChqVTG. In hepatopancreas, ChqILPBP transcripts are similar at stages 2 and 3 and increase significantly at stage 5. The data indicate that ovarian ILPBP may function differently from that of the hepatopancreas and may play a role in ovarian development. ChqAK transcripts are ~six folds higher in the ovary than the hepatopancreas. While they do not differ by ovarian stages, suggesting that AK may not be involved in vitellogenesis of the cold water crustacean species., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The epidermal growth factor receptor (EGFR) is a pleiotropic glycoprotein which plays a role in regulating cell proliferation, migration and differentiation. However, to date little is known about its functions in crustaceans. In this study, we successfully identified SpEGFR from mud crab Scylla paramamosain. RT-PCR result showed that SpEGFR was widely distributed in all tested tissues and highly expressed in ovary. In situ hybridization revealed that SpEGFR mainly localized in oocyte perinuclear region with notably obvious signals. In vitro experiments showed that the expression of SpVgR and SpCyclin B in ovary explants from late vitellogenic stage crabs (summer) were significantly increased when treated with 1 nM human EGF (hEGF) for 1 h, while there was no obvious change towards SpEGFR. Interestingly, as for winter crab at the same vitellogenic stage, the expression of SpVgR and SpCyclin B in ovary explants did not show significant increase until treated with higher concentration of 10 nM hEGF and longer incubation time of 12 h. In addition, the hEGF-induced effect could be suppressed by pre-treated with EGFR inhibitor AG1478 and PD153035, respectively, which further indicated that EGF-EGFR pathway played a vital role in ovarian development in mud crab. In conclusion, SpEGFR might promote ovarian development by stimulating the expression of SpVgR and SpCyclin B under hEGF-induced treatment. The different physiological response to hEGF in the same vitellogenic stage crabs between summer and winter might be attributed to the changes in metabolism and physiological sensitivity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Eriocheir sinensis is an important euryhaline catadromous crustacean of the Yangtze River and an important commercial species for breeding in China. However, wild E. sinensis have suffered serious damage attributed to overfishing, climate change, etc. The Ministry of Agriculture of China issued a notice banning the commercial fishing of wild E. sinensis. E. sinensis megalopa migrates upriver into fresh water for growth and fattening, which creates optimal conditions to experimentally explore its hyposaline osmoregulation mechanism. We performed comparative transcriptome analyses of E. sinensis megalopae under hyposaline stress. The results suggest that KEGG pathways and genes related to genetic information processing, developmental regulation, immune and anti-stress responses were differentially expressed. The present study reveals the most significantly enriched pathways and functional gene groups, and explores the hyposaline osmoregulation mode of E. sinensis megalopae. This study lays a theoretical foundation for further studies on the osmoregulation and developmental mechanisms of E. sinensis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Till date numerous microRNAs (miRNAs) have been discovered from various organisms, including fish, shellfish and crustaceans. The miRNAs are known to regulate immune functions in crustaceans, but little is known about the role of miRNAs against viral infection in crab. We performed small RNA sequencing to characterize the differentially expressed miRNAs in WSSV infected Scylla paramamosain, in comparison to that in uninfected crab, at 2 h and 12 h post infection. In total, 24 host miRNAs were up-regulated and 25 host miRNAs were down-regulated in response to WSSV infection at 2 h post infection. And 27 host miRNAs were up-regulated and 30 host miRNAs were down-regulated in response to WSSV infection at 12 h post infection. Further, the gene ontology analysis revealed that many signaling pathways were mediated by these miRNAs. The integral component of membrane is the most important biological process and endocytosis pathway is the most important pathway, which indicates that endocytosis is very important for WSSV infection. This study is one important attempt at characterizing crab miRNAs that response to WSSV infection, and will help unravel the miRNA pathways involved in antiviral immunity of crab., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Infection by the rhizocephalan parasite Sacculina beauforti can have detrimental effects on mud crab Scylla olivacea. However, the molecular changes that occur during rhizocephalan infection are poorly understood. Due to the disruption in the reproductive system after infection, the gonadal transcriptomic profiles of non-infected and infected Scylla olivacea were compared. A total of 686 and 843 unigenes were differentially expressed between non-infected and infected males, and females, respectively. The number of DEGs increased after infection. By comparing shared DEGs of non-infected and infected individuals, potential immune- and reproduction-related of host, and immune- and metabolism-related genes of parasite are highlighted. The only shared KEGG pathway between non-infected and infected individuals was the ribosome pathway. In summary, findings in this study provide new insights into the host-parasite relationship of rhizocephalan parasites and their crustacean hosts., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Endocrine control of molting in decapod crustaceans involves the eyestalk neurosecretory center (X-organ/sinus gland complex), regenerating limbs, and a pair of Y-organs (YOs), as molting is induced by eyestalk ablation or multiple leg autotomy and suspended in early premolt by limb bud autotomy. Molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), produced in the X-organ/sinus gland complex, inhibit the YO. The YO transitions through four physiological states over the molt cycle: basal in intermolt; activated in early premolt; committed in mid- and late premolt; and repressed in postmolt. We assembled the first comprehensive YO transcriptome over the molt cycle in the land crab, Gecarcinus lateralis, showing that as many as 23 signaling pathways may interact in controlling ecdysteroidogenesis. A proposed model of the MIH/cyclic nucleotide pathway, which maintains the basal YO, consists of cAMP/Ca 2+ triggering and nitric oxide (NO)/cGMP summation phases. Mechanistic target of rapamycin (mTOR) signaling is required for YO activation in early premolt and affects the mRNA levels of thousands of genes. Transforming Growth Factor-β (TGFβ)/Activin signaling is required for YO commitment in mid-premolt and high ecdysteroid titers at the end of premolt may trigger YO repression. The G. lateralis YO expresses 99 G protein-coupled receptors, three of which are putative receptors for MIH/CHH. Proteomic analysis shows the importance of radical oxygen species scavenging, cytoskeleton, vesicular secretion, immune response, and protein homeostasis and turnover proteins associated with YO function over the molt cycle. In addition to eyestalk ganglia, MIH mRNA and protein are present in brain, optic nerve, ventral nerve cord, and thoracic ganglion, suggesting that they are secondary sources of MIH. Down-regulation of mTOR signaling genes, in particular Ras homolog enriched in brain or Rheb, compensates for the effects of elevated temperature in the YO, heart, and eyestalk ganglia in juvenile Metacarcinus magister. Rheb expression increases in the activated and committed YO. These data suggest that mTOR plays a central role in mediating molt regulation by physiological and environmental factors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Matrix metalloproteinases (MMPs) are a cluster of enzymes that degrade the extracellular matrix (ECM) and some intracellular proteins; as such, they play an important role in tissue regeneration, infant growth, animal reproduction, and immunity. Most research into MMPs focuses mainly on their effects on the mammalian immune system. However, it is not clear how MMPs affect immune processes in crustaceans. Here, we cloned the open reading frame (ORF) of Eriocheir sinensis (Chinese mitten crab) MMP-14 (EsMMP-14) to explore the role of MMPs in crustacean innate immune responses. RT-PCR results showed that stimulation of crab with LPS and poly I:C upregulated expression of EsMMP-14 markedly. Besides, following the stimulation of 20-Hydroxyecdysone, the expression level of EsMMP-14 increased robustly, suggesting that EsMMP-14 involved in the molt process of E. sinensis. Hematoxylin and eosin staining of hepatopancreas and intestine revealed that knocking down EsMMP-14 maintained morphology following infection by Bacillus thuringiensis. Moreover, downregulated expression of EsMMP-14 increased the survival rate of infected E. sinensis. These results show that EsMMP-14 plays a role in innate immune responses of E. sinensis and fills a gap in our knowledge about the function of MMPs in crustaceans., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Troponin C (TnC) is one member of the EF-hand superfamily. In many species, this gene had been identified and related functions had been elucidated. The TnC gene was still blank in the Scylla paramamosain. We obtained the TnC gene for the first time in the S. paramamosain. And we systematically analyzed the possible role of this gene in the innate immunity of S. paramamosain while infected with white spot syndrome virus (WSSV) or Vibrio alginolyticus. The full-length 1427 bp sequence of TnC contains a 453 bp open reading frame (ORF) for encoding a 151 amino acid protein. Detection of tissue specificity of gene expression showed that the TnC was primarily expressed in muscle tissue. The expression of TnC was successfully inhibited by RNA interference technology, and several immune genes were affected. The activity of phenoloxidase and superoxide dismutase increased, and the total hemocytes counts increased after RNAi of TnC. It was found that after infection with V. alginolyticus and WSSV, the expression of TnC in hemocytes decreased. Infected with V. alginolyticus and WSSV, the cumulative mortality and apoptotic rate of hemocytes increased after silencing the TnC gene. Our results indicate that TnC takes participate in the innate immunity of S. paramamosain and may plays a different role in the antiviral and antibacterial immune response., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Toll is essential in innate immune system which is important for defense against bacterial, fungal and viral infections in invertebrates. Our previous study showed that cadmium (Cd) could change the expression pattern of ShToll3 in the epithelium (gills and midgut from the freshwater crab Sinopotamon henanense) infected by Aeromonas hydrophila. To investigate the diverse innate immune roles of crustacean homolog Tolls, in this study, we cloned Shtoll1 from S. henanense. The full-length cDNA of Shtoll1 was 4746 bp, with an ORF of 3033 bp encoding a putative protein of 111 amino acids, a 5'-untranslated region of 255 bp and a 3'-untranslated region of 1713 bp. Phylogenetic analysis showed that ShToll1 was clustered into the group of DmToll1, DmToll 4 and DmToll 5. In addition, the tissue distribution results showed that Shtoll1 was expressed widely in different tissues, with the highest expression in heamocytes. Besides, Shtoll1 expressions were upregulated in heamocytes and hepatopancreas after A. hydrophila infection. At the same time, the increase of Shtoll1 expressions were examined in heamocytes in response to Cd exposure and A. hydrophila infection in combination. Through western blotting and immunohistochemical analysis, the ShToll1 expressions in heamocytes were increased in response to A. hydrophila and Cd independently as well as in combination. Moreover, the mRNA level of three antimicrobial peptides (AMPs) alf5, alf6, and c-lys, which possibly responded to Cd and A. hydrophila stimulation through Shtoll1, were analyzed. Thus, we conclude that Cd expand the susceptibility of ShToll1 to A. hydrophila infection in heamocytes. This suggest that ShToll1 may contribute to the innate immune defense of S. henanense against A. hydrophila and Cd in heamocytes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chinese mitten crab Eriocheir sinensis is probably the most important freshwater cultured crab in China. A tiny minority of brownish-orange individuals have been discovered in the long period of artificial breeding history of E. sinensiss. Those mutants are usually accompanied with slow growth rate, low molting frequency and poor survival rate, which may be the results of growth defects and immunodeficiency. To better understand the relationship between body color determination and the immune system as well as the related genes expression in E. sinensiss, we performed the whole-body transcriptome analysis in different color of first stage zoea (ZI) larvae using next-generation sequencing (NGS) technology. We randomly assembled 175.40 and 177.52million clean reads from the wild and mutant ZIs, respectively. Finally, we identified 7153 differentially expressed genes (DEGs) (p < 0.05), with 5194 up-regulated and 1959 down-regulated. A total of 13 KEGG pathways related to immune system were detected among 248 pathways. Except the first whole-body RNA sequencing of color-specific transcriptomes for E. sinensis, this study will offer a better understanding of the underlying molecular mechanisms of interaction between color determination and the immune system., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Neuroparsin (NP) is an important neuropeptide in invertebrates. It is well-known that NP displays multiple biological activities, including antidiuretic and inhibition of vitellogenesis in insects. However, the information about its effect in crustaceans is scarce. In this study, the sequence of Sp-NP1 was selected from the transcriptome database from the mud crab, Scylla paramamosain. Sequence analyses indicate that the Sp-NP1 amino acid (AA) sequences consist of a 27 AA signal peptide and a 74 AA mature peptide, which contains 12 cysteine residues. qRT-PCR analysis has revealed that the expressions of Sp-NP1 gene are high in the nervous tissues and extremely low in the ovary and hepatopancreas. In situ hybridization has shown that the positive signals are localized in cell cluster 6 of protocerebrum and cell clusters 10 and 11 of deutocerebrum. The presence of Sp-NP1 in the haemolymph has been detected in S. paramamosain through western blot, which indicates that Sp-NP1 serves as an endocrine factor in the regulation of physiological activities. In vitro experiments have further shown that the mRNA level of vitellogenin in the hepatopancreas notably decreases following administration of recombinant Sp-NP1, while the mRNA level of vitellogenin receptor and cyclin B in the ovary shows no significant differences. Collectively, Sp-NP1 possibly can inhibit the production of vitellogenin in the hepatopancreas and has no direct effect on the ovary in S. paramamosain., (Copyright © 2019 Elsevier Inc. All rights reserved.)

PIWI-interacting RNAs (piRNAs) are abundantly found in germ cells and involved in gametogenesis and gonadal development. Information on the regulatory roles of piRNAs in crustacean reproduction, however, is scarce. Thus, we identified gonadal piRNAs of mud crab Scylla paramamosain. Of the 115,491 novel piRNAs, 596 were differentially expressed. Subsequently, 389,887 potential piRNA-target genes were predicted. The expression of 4 piRNAs and 9 genes with high piRNA interactions were validated with the inclusion of additional immature specimens, including LRP2 that is involved in growth and reproduction, MDN1 in ribosome biogenesis pathway and gametogenesis, and PRKDC, a DNA repair gene involved in gonadal differentiation and maturation. KEGG analysis further revealed the involvement of predicted piRNA target genes in gametogenesis- and reproduction-related pathways. Our findings provide baseline information of mud crab piRNAs and their differential expression between testes and ovaries suggests that piRNAs play an essential role in regulating gametogenesis and gonadal development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The Chinese mitten crab (Eriocheir sinensis), an economically valuable crustacean that is popular for its flavor, exhibits catadromous spawning migration. Overfishing and environmental pollution have inflicted serious damage on wild E. sinensis populations, and the Chinese government has banned the commercial fishing of this species in the Yangtze River. Studies have examined the sexual dimorphism in the body size and morphology of crabs, but there are few reports on the molecular regulatory mechanisms that occur during the reproduction of E. sinensis. In this study, we performed the first comparative transcriptome analyses of the cerebral ganglion and hepatopancreas of E. sinensis during reproduction. The results indicate that E. sinensis has significant sexual dimorphism in signal transduction, metabolism, substance transportation, and cellular protection. This study aims to provide information that can be used as a basis for further research on the molecular mechanisms that underlie sexual dimorphism in E. sinensis during reproduction. Furthermore, the results can be used to support the development of the E. sinensis breeding industry and the restoration of wild E. sinensis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The SIFamides are a broadly conserved arthropod peptide family characterized by the C-terminal motif -SIFamide. In decapod crustaceans, two isoforms of SIFamide are known, GYRKPPFNGSIFamide (Gly 1 -SIFamide), which is nearly ubiquitously conserved in the order, and VYRKPPFNGSIFamide (Val 1 -SIFamide), known only from members of the astacidean genus Homarus. While much work has focused on the identification of SIFamide isoforms in decapods, there are few direct demonstrations of physiological function for members of the peptide family in this taxon. Here, we assessed the effects of Gly 1 - and Val 1 -SIFamide on the cardiac neuromuscular system of two closely related species of Cancer crab, Cancer borealis and Cancer irroratus. In each species, both peptides were cardioactive, with identical, dose-dependent effects elicited by both isoforms in a given species. Threshold concentrations for bioactivity are in the range typically associated with hormonal delivery, i.e., 10 -9 to 10 -8  M. Interestingly, and quite surprisingly, while the predicted effects of SIFamide on cardiac output are similar in both C. borealis and C. irroratus, frequency effects predominate in C. borealis, while amplitude effects predominate in C. irroratus. These findings suggest that, while SIFamide is likely to increase cardiac output in both crabs, the mechanism through which this is achieved is different in the two species. Immunohistochemical/mass spectrometric data suggest that SIFamide is delivered to the heart hormonally rather than locally, with the source of hormonal release being midgut epithelial endocrine cells in both Cancer species. If so, midgut-derived SIFamide may function as a regulator of cardiac output during the process of digestion., (Copyright © 2019. Published by Elsevier Inc.)

To investigate the effects of arginine (Arg) on the growth, antioxidant capacity, immunity and disease resistance of juvenile Chinese mitten crab, three diets containing Arg levels at 1.72% (control), 2.73% and 3.72% were formulated and fed to Chinese mitten crab (0.22 ± 0.03 g) for eight weeks. The weight gain, ecdysterone and growth hormone in the serum, relative expression of insulin-like growth factor 2 in the hepatopancreas significantly increased in crabs fed the 2.73% and 3.72% Arg diets. The protein and lipid contents significantly increased in crabs fed the 3.72% Arg diet. The feed conversion ratios in crabs fed the diets with Arg additions were lower than in the control. Arg supplementation also enhanced the antioxidative capacity by increasing the activities of superoxide dismutase, catalase and the relative expression of Kelch-like ECH-associated protein 1 gene in the hepatopancreas, which subsequently decreased malondialdehyde content in the hepatopancreas. Besides, Arg also decreased nitric oxide content in the serum and the activity of nitric oxide synthetase in the hepatopancreas. The relative mRNA levels of crustin, relish, lysozyme and cryptocyanin genes were significantly upregulated by Arg supplementation. The activities of acid phosphatase and alkaline phosphatase in the serum significantly increased in crabs fed the 3.72% Arg diet than those in the control. Similarly, the relative mRNA levels of crustin, cryptocyanin and proPO genes were significantly upregulated in crabs fed the 2.73% Arg diet after lipopolysaccharide challenge, and in crabs fed the 3.72% Arg diet after the Poly (I:C) challenge. The crabs fed the 2.73% and 3.72% Arg diets had higher survival rate after bacterial infection than those fed the control diet. This study indicates that the addition of Arg to the diet at 2.7-3.7% can improve the growth, survival, antioxidant capacity, immunity and disease resistance in juvenile Chinese mitten crab., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study evaluates the effect of dietary supplementation of immunostimulants on the Chinese mitten crab (Eriocheir sinensis) with a single administration of mannan oligosaccharide (MOS), or its combination with either β-glucan or with inulin for 8 weeks. Four diets included an untreated control diet (C), MOS alone (3 g kg -1 , M), MOS with β-glucan (3 g kg -1 MOS + 1.5 g kg -1 β-glucan, MB), and MOS with inulin (3 g kg -1 MOS + 10 g kg -1 inulin, MI). The weight gain and specific growth rate of the crabs fed M, MB, and MI diets were improved by lowing feed conversion ratio. The growth and feed utilization of the crabs fed the MB diet were improved compared with the other three groups. The crabs fed the M, MB and MI diets showed a higher intestinal trypsin activity than that in the M and control groups. The highest trypsin activity in the hepatopancreas was observed in the MB group. Crabs fed M, MB and MI diets increased antioxidant system-related enzyme activities, but reduced malondialdehyde. The highest activities of alkaline phosphatase, acid phosphatase, lysozyme and phenol oxidase in the gut and the respiratory burst of the crabs were found in the MB group. The MB diet promoted the mRNA expression of E. sinensis immune genes (ES-PT, ES-Relish, ES-LITAF, p38MAPK and Crustin) compared with the control. After 3 days of infection with Aeromonas hydrophila, the highest survival of crabs was also found in the MB group. This study indicates that the combination of MOS with β-glucan or with inulin can improve growth, antioxidant capacity, non-specific immunity and disease resistance in E. sinensis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Estradiol is an important sex steroid hormone that involved in regulation of animal lipid metabolism. However, the effect of estradiol on lipid metabolism in swimming crab (Portunus trituberculatus) is unclear. The present study investigated the effect of four concentrations of exogenous estradiol (0, 0.01, 0.1 and 1 μg g -1 crab weight) on the expression levels of lipid metabolism-related genes, lipid composition and histology of hepatopancreas in the P. trituberculatus. The results showed that the mRNA levels of carnitine palmitoyltransferase I and II (CPT-I and CPT-II) increased significantly at the low concentrations (0.01 μg g -1 and 0.1 μg g -1 ), while decreased significantly in the highest concentration (1 μg g -1 ). The mRNA levels of acyl-CoA oxidase (ACOX), fatty acid transport protein (FATP), fatty acid-binding protein (FABP), diacylglycerol acyltransferase 1 (DGAT1) and acetyl-CoA carboxylase (ACC) were significantly down-regulated. The transcripts of fatty acid synthase (FAS) and fatty acyl desaturase (FAD) decreased significantly only in 1 μg g -1 treatment. All estradiol treatments (0.01, 0.1 and 1 μg g -1 ) had significantly higher percentages of 20:4n6, 20:5n3 and 22:6n3, but lower percentages of total monounsaturated fatty acids and polar lipids than the control treatment (0 μg g -1 ). Histological observations indicated the size of B cell became larger under estradiol treatment. The results indicated that estradiol promoted lipid catabolism in the hepatopancreas of P. trituberculatus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We investigated the effects of icariin (ICA) on growth performance, antioxidant capacity and non-specific immunity in Chinese mitten crab (Eriocheir sinensis). A total of 200 healthy crabs (average weight: 33.58 ± 0.05 g) were randomly assigned to four treatments with five replicates, each with ten individuals per pool. There were four dietary treatments: the control group (fed with the basal diet), the ICA 50 group, the ICA100 group, and the ICA 200 group (fed with the basal diet supplemented with 50, 100, and 200 mg/kg ICA, respectively). These diets were provided for 8 weeks. Results indicated that ICA100 crabs had higher weight gain (WG), specific growth rate (SGR) and survival rate (SR) than the controls. Protein carbonyl content (PCC) and malondialdehyde (MDA) concentrations in the haemolymph and hepatopancreas of ICA100 crabs were significantly lower than in the control group, while the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were significantly higher. The activities of PO, LZM, ACP and AKP were significantly enhanced with ICA supplementation at 50 and 100 mg/kg, yet decreased subsequently at 200 mg/kg. Furthermore, supplementation of 100 mg/kg ICA up-regulated the mRNA expression of prophenoloxidase (proPO), catalase (CAT), mitochondrial manganese superoxide dismutase (mtMnSOD), thioredoxin-1 (Trx1) and peroxiredoxin 6 (Prx6), while the mRNA expression of toll like receptors (TLRs), NF-κB-like transcription factor Relish and lipopolysaccharide-induced TNF-α factor (LITAF) were down-regulated in the hepatopancreas (P < 0.05). These findings indicate that dietary ICA supplementation at an optimum dose of 100 mg/kg may be effective in improving growth performance, antioxidant capability and non-specific immunity of Chinese mitten crab., (Copyright © 2019. Published by Elsevier Ltd.)

Vibrio parahaemolyticus is one of the major pathogens caused diseases in cultured mud crab (Scylla paramamosain). Mud crabs lack an adaptive immune system, their defenses depend almost on innate immunity. Evaluation of the molecular responses of mud crabs to pathogens is essential for control of disease occurrence in farmed animals. In this study, the impacts of V. parahaemolyticus on immunity-related genes and metabolites in mud crabs of different groups (PG, SG and MG refer to controlled, survival and moribund groups, respectively) were investigated. Our results revealed that V. parahaemolyticus infection stimulated significant expressions of immune-related genes (prophenoloxidase, alpha 2-macroglobulin, lysosomal-associated membrane protein, Rab5, C-type lectin B and anti-lipopolysaccharide factor 5) in the MG within 72 h post-infection. The ATP content was significantly reduced in all tissues except muscle of moribund mud crabs. A total of 668 metabolites (including 190 down-regulated and 145 up-regulated) were identified and assigned to 77 pathways in both SG and MG. Metabolites involved in the saturated fatty acid are up-regulated, whereas unsaturated fatty acid and amino acid metabolisms are down-regulated in the immune system of mud crabs during the bacterial infection in MG. Furthermore, a reduction of hemocyte number and an increase of microbial abundance was found in MG. Our results demonstrated that V. parahaemolyticus induced death of mud crabs through reducing the metabolites associate with energy biosynthesis and innate immune system (i.e. proliferation of hemocyte and melanization), resulting in decrease of ATP in different tissues and failed to clearance of pathogens, respectively. The findings of this study provide a basic information of the responses of mud crab on bacterial infection, which is essential for prevention and control of diseases in mud crab aquaculture., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As a broad-spectrum herbicide, glyphosate was extensively utilised in China for several decades. The contradiction between glyphosate spraying and crab breeding in the rice-crab co-culture system has become more obvious. In this study, the antioxidative status and immunological responses of Chinese mitten crab, Eriocheir sinensis, under sublethal exposure of glyphosate were investigated by detecting the antioxidative and immune-related enzyme activity, acetylcholinesterase (AChE) activity and relative mRNA expression of heat shock proteins (HSPs) in hepatopancreas. The results showed that high concentrations of glyphosate (44 and 98 mg/L) could induce significant alteration of superoxide dismutase (SOD), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP), and phenoloxidase (PO) activities by first rising then falling during the exposure. However, AChE activity in all treatments including 4.4 mg/L was inhibited markedly after 6 h of exposure. In addition, the relative mRNA expression of HSP 60, HSP 70, and HSP 90 was significantly upregulated at both 48 h and 96 h. These results revealed that glyphosate has a prominent toxic effect on E. sinensis based on antioxidative and immunological response inhibition and AChE activity reduction even at the lowest concentration of 4.4 mg/L, and a protective response by upregulation of HSPs was carried out by the species to ease the environmental stress., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Neuropeptides, ubiquitous signaling molecules, commonly achieve their signaling function via interaction with cell membrane-spanning G-protein coupled receptors (GPCRs). In recent years, in the midst of the rapid development of next-generation sequencing technology, the amount of available information on encoded neuropeptides and their GPCRs sequences have increased dramatically. The repertoire of neuropeptides has been determined in many crustaceans, including the commercially important mud crab, Scylla paramamosain; however, determination of GPCRs is known to be more difficult and usually requires in vitro binding tests. In this study, we adopted a combinatorial bioinformatics analysis to identify S. paramamosain neuropeptide GPCRs. A total of 65 assembled GPCR sequences were collected from the transcriptome database. Subsequently these GPCRs were identified by comparison to known neuropeptide GPCRs based on the sequence-similarity-based clustering and phylogenetic analysis, which showed that many of them are closely related to insect GPCR families. Of these GPCRs, most of them were detected in various tissues of the mud crab and some of them showed differential expression by gender, suggesting they are involved in different physiological processes, such as sex differentiation. By employing ligand-receptor binding tests, we demonstrated that the predicted crustacean cardioactive peptide (CCAP) receptor was activated by CCAP peptide in a dose-dependent manner. This is the first CCAP receptor that has been functionally defined in crustaceans. In summary, the present study shortlists candidate neuropeptide GPCRs for ligand-receptor binding tests, and provides information for subsequent future research on the neuropeptide/GPCR signaling pathway in S. paramamosain., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A bone morphogenetic protein ligand (BMP7) and its two receptors (BMPRIB and BMPRII) were recently cloned and characterized in the mud crab, Scylla paramamosain. However specific functions of BMP7 and the mechanistic pathways regulating its function are largely unidentified. In the present study, we separated oocytes and follicle cells from the ovarian explants of S. paramamosain. Subsequent analysis using semi-quantitative PCR demonstrated that the mRNA of Sp-BMP7 was exclusively expressed in follicle cells while Sp-BMPRs were expressed in both oocytes and follicle cells. In vitro experiments further showed that the mRNA and protein levels of Cyclin B increased but Sp-BMP7 declined in 17α, 20β-Dihydroxyprogesterone (DHP)-induced oocytes. Furthermore, the inhibitory effects of Sp-BMP7 were not affected by the elimination of the contact/gap junction-mediated communication between oocytes and follicle cells. Our data indicate that BMP7 may play a role in the suppression of DHP-induced oocyte maturation by affecting autocrine/paracrine pathways in S. paramamosain., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The present study determined the effect of osmotic stress on the insulin-like receptor binding characteristics and on glucose metabolism in the anterior (AG) and posterior (PG) gills of the crab Neohelice granulata. Bovine insulin increased the capacity of the PG cell membrane to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and the glucose uptake in the control crab group. The crabs were submitted to three periods of hyperosmotic (HR) and hyposmotic (HO) stress, for 24, 72 and 144 h, to investigate the insulin-like receptor phosphorylation capacity of gills. Acclimation to HO for 24 h or HR for 144 h of stress inhibited the effects of insulin in the PG, decreasing the capacity of insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and decreasing the glucose uptake. Hyperosmotic stress for the same period of 144 h significantly affected 125 I-insulin binding in the AG and PG. However, HO stress for 24 h significantly reduced 125 I-insulin-specific uptake only in the PG. Therefore, osmotic stress induces alterations in the gill insulin-like receptors that decrease insulin binding in the PG. These findings indicate that osmotic stress induced a pattern of insulin resistance in the PG. The free-glucose concentration in the PG decreased during acclimation to 144 h of HR stress and 24 h of HO stress. This decrease in the cell free-glucose concentration was not accompanied by a significant change in hemolymph glucose levels. In AG from the control group, neither the capacity of bovine insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) nor the glucose uptake changed; however, genistein decreased tyrosine-kinase activity, confirming that this receptor belongs to the tyrosine-kinase family. Acclimation to HO (24 h) or HR (144 h) stress decreased tyrosine-kinase activity in the AG. This study provided new information on the mechanisms involved in the osmoregulation process in crustaceans, demonstrating for the first time in an estuarine crab that osmotic challenge inhibited insulin-like signaling and the effect of insulin on glucose uptake in the PG., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Serpin families classified serine protease inhibitors regulate various physiological processes. However, there is not study on the role of serpin in immune responses against Spiroplasma eriocheiris as a novel causative pathogen in the Chinese mitten crab, Eriocheir sinensis. In our study, quantitative real-time PCR (qRT-PCR) revealed that the mRNA transcripts of Esserpin-2 were ubiquitous in every tissue, relative higher expression in hepatopancreas, gill and hemocytes, while the intestine, muscle, heart and nerve showed relative lower expression. Followed by infection with S. eriocheiris, the transcripts of Esserpin-2 were significantly down-regulated from 1 d to 7 d. After double-stranded RNA injection, the transcripts of Esserpin-2 dramatically declined from 48 h to 96 h. The transcripts of proPO were found to be obviously increased after Esserpin-2 silenced, meanwhile, LGBP with no significant difference. The copy number of S. eriocheiris and subsequently the mortality of crabs in a silencing Esserpin-2 group were significantly less than control groups during infection. The subcellular localization experiment suggested that recombinant Esserpin-2 was mainly located in the cytoplasm. Finally, over-expression assay in Drosophila S2 cells indicated that Esserpin-2 could increase copies of S. eriocheiris and result in cell death. These findings demonstrated that Esserpin-2 involved in the innate immune mechanism of E. sinensis in response to S. eriocheiris infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)