Comparative transcriptomic characterization of the ovary in the spawning process of the mud crab Scylla paramamosain.

Oviposition is induced upon mating in most insects. Spawning is a physiological process that is fundamental for the reproduction of Scylla paramamosain. However, the molecular mechanisms underlying the spawning process in this species are poorly understood. Herein, comprehensive ovary transcriptomic analysis was conducted at the germinal vesicle breakdown stage (GVBD), spawning stage, 0.5 h post-spawning stage, and 24 h post-spawning stage of S. paramamosain for gene discovery. A total of 67,230 unigenes were generated, and 27,975 (41.61%) unigenes were annotated. Meanwhile, the differentially expressed genes (DEGs) between the different groups were identified, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was subsequently conducted. These results suggested that octopamine (OA) and tyramine (TA) could induce oviposition, while dopamine (DA) and serotonin (5-hydroxytryptamine [5-HT]) inhibit oviposition. The 20-hydroxyecdysone (20E) and methyl farnesoate (MF) signal pathways might be positively associated with oviposition. Furthermore, numerous transcripts that encode neuropeptides and their G-protein-coupled receptors (GPCRs), such as CNMamide, RYamide, ecdysis-triggering hormone (ETH), GPA2/GPB5 receptor, and Moody receptor, appear to be differentially expressed during the spawning process. Eleven unigenes were selected for qRT-PCR and the pattern was found to be consistent with the transcriptome expression pattern. Our work is the first spawning-related investigation of S. paramamosain focusing on the ovary at the whole transcriptome level. These findings assist in improving our understanding of spawning regulation in S. paramamosain and provide information for oviposition studies in other crustaceans.

Effects of geniposide on innate immunity and antiviral activity of Scyllaparamamosain.

In this study, we examined the impact of geniposide on the innate immunity of the mud crab Scylla paramamosain, specifically in relation to WSSV infection. Through the use of in vitro cell culture experiments, we assessed the effects of geniposide on various parameters of hemocyte activity in S. paramamosain. Our findings revealed that high doses of geniposide inhibited hemocyte growth, with an optimal dose of 100 mg/kg determined. Additionally, we observed that geniposide increased the total hemocyte counts in S. paramamosain following WSSV infection. Geniposide also enhanced the enzymatic activities in hemolymph following treatment. The enzymes affected by geniposide encompassed ACP (acid phosphatase), POD (phenol oxidase catalase), PO (phenoloxidase), SOD (superoxide dismutase), CAT (catalase), and LZM (lysozyme). Furthermore, the activities of ACP, POD, PO, and LZM were also observed to increase subsequent to infection with WSSV. Notably, geniposide was found to enhance the phagocytosis of V. alginolyticus within the hemocytes. Geniposide can reduce hemocyte apoptosis rates after treatment, as well as hemocytes infected with WSSV. Furthermore, geniposide treatment significantly up-regulated the expression level of Myosin, but expression levels of Astakine, C-type lectin (CTL), STAT, JAK, proPO, minichromosome maintenance protein (MCM7), caspase-3 and crustin were down-regulated in the hemocytes. Additionally, geniposide treatment inhibited WSSV replication in hemocytes of S. paramamosain, and enhanced the survival rates of mud crabs following WSSV infection. These experimental results provide evidence that geniposide can improve the immune response by regulating humoral immunity and cellular immunity, and enhance pathogen resistance in S. paramamosain.

Identification and functional characterization of laminin receptor in the mud crab, Scylla paramamosain, in response to MCDV-1 challenge.

Laminin receptor (LR), which mediating cell adhesion to the extracellular matrix, plays a crucial role in cell signaling and regulatory functions. In the present study, a laminin receptor gene (SpLR) was cloned and characterized from the mud crab (Scylla paramamosain). The full length of SpLR contained an open reading frame (ORF) of 960 bp encoding 319 amino acids, a 5' untranslated region (UTR) of 66 bp and a 3' UTR of 49 bp. The predicted protein comprised two Ribosomal-S2 domains and a 40S-SA-C domain. The mRNA of SpLR was highly expressed in the gill, followed by the hepatopancreas. The expression of SpLR was up-regulated after mud crab dicistrovirus-1(MCDV-1) infection. Knocking down SpLR in vivo by RNA interference significantly down-regulated the expression of the immune genes SpJAK, SpSTAT, SpToll1, SpALF1 and SpALF5. This study shown that the expression level of SpToll1 and SpCAM in SpLR-interfered group significantly increased after MCDV-1 infection. Moreover, silencing of SpLR in vivo decreased the MCDV-1 replication and increased the survival rate of mud crabs after MCDV-1 infection. These findings collectively suggest a pivotal role for SpLR in the mud crab's response to MCDV-1 infection. By influencing the expression of critical innate immune factors and impacting viral replication dynamics, SpLR emerges as a key player in the intricate host-pathogen interaction, providing valuable insights into the molecular mechanisms underlying MCDV-1 pathogenesis in mud crabs.

Transcriptome analysis of Scylla paramamosain hepatopancreas response to mud crab dicistrovirus-1 infection.

Scylla paramamosain, an economically significant crab, is widely cultivated worldwide. In recent years, S. paramamosain has faced a serious threat from viral diseases due to the expansion of culture scale and increased culture density. Among these, mud crab dicistrovirus-1 (MCDV-1) stands out as highly pathogenic, presenting substantial challenges to the healthy development of mud crab aquaculture. Therefore, a comprehensive understanding of the mud crab immune response to MCDV-1 infection is imperative for devising effective disease prevention strategies. In this study, transcriptomic analyses were conducted on the hepatopancreas of mud crabs infected with MCDV-1. The findings revealed a total of 5139 differentially expressed genes (DEGs) between healthy and MCDV-1 infected mud crabs, including 3327 upregulated and 1812 downregulated DEGs. Further analysis showed that mud crabs resist MCDV-1 infection by activating humoral immune-related pathways, including the MAPK signaling pathway, MAPK signaling pathway-fly, and Toll and Imd signaling pathway. In contrast, MCDV-1 infection triggers host metabolic disorders. Several immune-related vitamin metabolism pathways (ascorbate and aldarate metabolism, retinol metabolism, and nicotinate and nicotinamide metabolism) were significantly inhibited, which may create favorable conditions for the virus's self-replication. Notably, endocytosis emerged as significantly upregulated both in GO terms and KEGG pathways, with several viral endocytosis-related pathways showing significant activation. PPI network analysis identified 9 hub genes associated with viral endocytosis within the endocytosis. Subsequent GeneMANIA analysis confirmed the association of these hub genes with viral endocytosis. Both transcriptome data and qPCR analysis revealed a significant upregulation of these hub genes post MCDV-1 infection, suggesting MCDV-1 may use viral endocytosis to enter cells and facilitate replication. This study represents the first comprehensive report on the transcriptomic profile of mud crab hepatopancreas response to MCDV-1 infection. Future investigations should focus on elucidating the mechanisms through which MCDV-1 enters cells via endocytosis, as this may holds critical implications for the development of vaccine targets.

Vitamin C as a functional enhancer in the non-specific immune defense, antioxidant capacity and resistance to low-temperature stress of juvenile mud crab, Scylla paramamosain.

This experiment was conducted to explore the effects of dietary vitamin C supplementation on non-specific immune defense, antioxidant capacity and resistance to low-temperature stress of juvenile mud crab (Scylla paramamosain). Mud crabs with an initial weight of 14.67 ± 0.13 g were randomly divided into 6 treatments and fed diets with 0.86 (control), 44.79, 98.45, 133.94, 186.36 and 364.28 mg/kg vitamin C, respectively. The experiment consisted of 6 treatments, each treatment was designed with 4 replicates and each replicate was stocked with 8 crabs. After 42 days of feeding experiment, 2 crabs were randomly selected from each replicate, and a total of 8 crabs in each treatment were carried out 72 h low-temperature challenge experiment. The results showed that crabs fed diets with 186.36 and 364.28 mg/kg vitamin C significantly improved the activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in hemolymph and hepatopancreas (P < 0.05). Crabs fed diet with 133.94 mg/kg vitamin C significantly decreased the concentration of nitric oxide (NO) and the activity of nitric oxide synthase (NOS) in hemolymph (P < 0.05). Diet with 133.94 mg/kg vitamin C was improved the activity of polyphenol oxidase (PPO) and the concentration of albumin (ALB) in hemolymph. Crabs fed diet with 133.94 mg/kg vitamin C showed lower concentration of malondialdehyde (MDA) in hemolymph and hepatopancreas than those fed the other diets. Meanwhile, crabs fed diet with 98.45 mg/kg vitamin C showed higher activity of total superoxide dismutase (T-SOD) in hemolymph, and crabs fed diet with 133.94 mg/kg vitamin C showed higher activity of T-SOD in hepatopancreas. Crabs fed diet with 186.36 mg/kg vitamin C significantly decreased the concentration of reduced glutathione (GSH) and the activity of glutathione peroxidase (GSH-PX) in hepatopancreas (P < 0.05). In normal temperature, crabs fed diets with 133.94 mg/kg vitamin C significantly up-regulated the expression levels of gpx (glutathione peroxidase) and trx (thioredoxin) in hepatopancreas compared with the control treatment (P < 0.05). The highest expression levels of relish, il16 (interleukin 16), caspase 2 (caspase 2), p38 mapk (p38 mitogen-activated protein kinases) and bax (bcl-2 associated x protein) in hepatopancreas were found at crabs fed control diet (P < 0.05). Moreover, crabs fed diet with 133.94 mg/kg vitamin C showed higher expression levels of alf-3 (anti-lipopolysaccharide factor 3) and bcl-2 (B-cell lymphoma 2) in hepatopancreas than those fed the other diets (P < 0.05). Under low-temperature stress, crabs fed diet with 133.94 mg/kg vitamin C significantly improved the expression levels of hsp90 (heat shock protein 90), cat (catalase), gpx, prx (thioredoxin peroxidase) and trx in hepatopancreas (P < 0.05). In addition, dietary with 133.94 vitamin C significantly up-regulated the expression levels of alf-3 and bcl-2 (P < 0.05). Based on two slope broken-line regression analysis of activity of PPO against the dietary vitamin C level, the optimal dietary vitamin C requirement was estimated to be 144.81 mg/kg for juvenile mud crab. In conclusion, dietary 133.94-144.81 mg/kg vitamin C significantly improved the non-specific immune defense, antioxidant capacity and resistance to low-temperature stress of juvenile mud crab.

Identification of toll-like receptor family and the immune function of new Sptlr-6 gene of Scylla paramamosain.

As a crucial member of pattern-recognition receptors (PRRs), the Tolls/Toll-like receptors (TLRs) gene family has been proven to be involved in innate immunity in crustaceans. In this study, nine members of TLR gene family were identified from the mud crab (Scylla paramamosain) transcriptome, and the structure and phylogeny of different SpTLRs were analyzed. It was found that different SpTLRs possessed three conserved structures in the TIR domain. Meanwhile, the expression patterns of different Sptlr genes in examined tissues detected by qRT-PCR had wide differences. Compared with other Sptlr genes, Sptlr-6 gene was significantly highly expressed in the hepatopancreas and less expressed in other tissues. Therefore, the function of Sptlr-6 was further investigated. The expression of the Sptlr-6 gene was up-regulated by Poly I: C, PGN stimulation and Vibrio parahaemolyticus infection. In addition, the silencing of Sptlr-6 in hepatopancreas mediated by RNAi technology resulted in the significant decrease of several conserved genes involved in innate immunity in mud crab after V. parahaemolyticus infection, including relish, myd88, dorsal, anti-lipopolysaccharide factor (ALF), anti-lipopolysaccharide factor 2 (ALF-2) and glycine-rich antimicrobial peptide (glyamp). This study provided new knowledge for the role of the Sptlr-6 gene in defense against V. parahaemolyticus infection in S. paramamosain.

Myeloid differentiation protein 2 regulates the innate immunity and the disease resistant against Vibrio alginolyticus in Scylla paramamosain.

Myeloid differentiation protein 2 (MD2), generally functions as a coreceptor of Toll-like receptor 4 (TLR4), facilitating the activation of TLR4 and the recognition of lipopolysaccharides (LPS) in host organisms. While the role of MD2 in immune activation is well-documented across various species, the specific role of the MD2 homolog in Scylla paramamosain (SpMD2) remains unidentified. In this study, we applied RNA interference to reduce SpMD2 expression, aiming to elucidate its role in immune system of mud crabs. Notably, SpMD2 interference leded to decrease in the hemocyte counts and phagocytic activity, along with increase in apoptosis rates and level of reactive oxygen species (ROS). Furthermore, the activities of key enzymes related to immune, such as superoxide dismutase (SOD), catalase (CAT), phenoloxidase (PO), peroxidase (POD), lysozyme (LZM), and acid phosphatase (ACP), were reduced by SpMD2 knockdown. Following infection with Vibrio alginolyticus, increase of SpMD2 expression level was observed. This was accompanied by alterations in the expression levels of genes related to immune in mud crabs. Challenge experiment with Vibrio alginolyticus showed a higher mortality rate after SpMD2 interference. Our study underscore the critical role of SpMD2 in enhancing the innate immunity and disease resistant in S. paramamosain, advancing our understanding of the innate immune regulatory mechanisms in crustaceans.

Transcriptional regulation of IAG by dsx and foxl-2 in mud crab (Scylla paramamosain).

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.

Temporal and Spatial Signatures of Scylla paramamosain Transcriptome Reveal Mechanistic Insights into Endogenous Ovarian Maturation under Risk of Starvation.

Variability in food availability leads to condition-dependent investments in reproduction. This study is aimed at understanding the metabolic response and regulatory mechanism of female Scylla paramamosain in response to starvation in a temporal- and tissue-specific manner. The mud crabs were starved for 7 (control), 14, 28, and 40 days for histological and biochemical analysis in the hepatopancreas, ovary, and serum, as well as for RNA sequencing on the hepatopancreas and ovary. We further highlighted candidate gene modules highly linked to physiological traits. Collectively, our observations suggested that starvation triggered endogenous ovarian maturation at the expense of hepatopancreas mass, with both metabolic adjustments to optimize energy and fatty acid supply from hepatopancreas to ovary in the early phase, followed by the activation of autophagy-related pathways in both organs over prolonged starvation. These specific adaptive responses might be considered efficient strategies to stimulate ovarian maturation of Scylla paramamosain under fasting stress, which improves the nutritional value of female mud crabs and other economically important crustaceans.

p53 Ubiquitination Comediated by HUWE1 and TRAF6 Contributes to White Spot Syndrome Virus Infection in Crustacean.

p53, the guardian of the genome, is a short-lived protein that is tightly controlled at low levels by constant ubiquitination and proteasomal degradation in higher organisms. p53 stabilization and activation are early crucial events to cope with external stimuli in cells. However, the role of p53 ubiquitination and its relevant molecular mechanisms have not been addressed in invertebrates. In this study, our findings revealed that both HUWE1 (HECT, UBA, and WWE domain-containing E3 ubiquitin-protein ligase 1) and TRAF6 (tumor necrosis factor receptor-associated factor 6) could serve as E3 ubiquitin ligases for p53 in mud crabs (Scylla paramamosain). Moreover, the expression of HUWE1 and TRAF6 was significantly downregulated during white spot syndrome virus (WSSV) infection, and therefore the ubiquitination of p53 was interrupted, leading to the activation of apoptosis and reactive oxygen species (ROS) signals through p53 accumulation, which eventually suppressed viral invasion in the mud crabs. To the best of our knowledge, this is the first study to reveal the p53 ubiquitination simultaneously induced by two E3 ligases in arthropods, which provides a novel molecular mechanism of invertebrates for resistance to viral infection. IMPORTANCE p53, which is a well-known tumor suppressor that has been widely studied in higher animals, has been reported to be tightly controlled at low levels by ubiquitin-dependent proteasomal degradation. However, recent p53 ubiquitination-relevant research mainly involved an individual E3 ubiquitin ligase, but not whether there exist other mechanisms that need to be explored. The results of this study show that HUWE1 and TRAF6 could serve as p53 E3 ubiquitin ligases and synchronously mediate p53 ubiquitination in mud crabs (Scylla paramamosain), which confirmed the diversity of the p53 ubiquitination regulatory pathway. In addition, the effects of p53 ubiquitination are mainly focused on tumorigenesis, but a few are focused on the host immune defense in invertebrates. Our findings reveal that p53 ubiquitination could affect ROS and apoptosis signals to cope with WSSV infection in mud crabs, which is the first clarification of the immunologic functions and mechanisms of p53 ubiquitination in invertebrates.

KCMF1-like suppresses white spot syndrome virus infection by promoting apoptosis in mud crab (Scylla paramamosain).

Potassium channel modulatory factor 1 (KCMF1), an E3 ubiquitin ligase, plays a vital role in renal tubulogenesis, preeclampsia, and tumor development in mammals. Nevertheless, the function of KCMF1 in invertebrates remains to be investigated. Here, we identified KCMF1-like from Scylla paramamosian, encoding 242 amino acids with two zinc finger domains at the N-terminal. Real-time quantitative PCR analysis revealed that KCMF1-like was expressed in all tested tissues, including hemocytes, brain, mid-intestine, subcuticular epidermis, gills, muscle, heart, and stomach, with higher levels in muscle and mid-intestine. KCMF1-like was up-regulated in the hemocytes of mud crabs challenged with white spot syndrome virus (WSSV). RNA interference (RNAi) was performed to investigate the impact of KCMF1-like on the proliferation of WSSV in mud crabs. Knock-down of KCMF1-like resulted in an increase of the WSSV copy number and an impairment of the hemocytes apoptosis rate in vivo. In addition, KCMF1-like could also affect the mitochondrial membrane potential. Collectively, these results revealed that KCMF1-like might play a crucial role in the defense against virus infection in mud crab. This study contributes a novel insight into the role of KCMF1-like in the antiviral immune defense mechanism in crustaceans.

Integration of transcriptomic and metabolomic analyses reveal the molecular responses of the mud crab Scylla paramamosain to infection by an undescribed endoparasite Portunion sp.

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.

SpTNF regulates apoptosis and antimicrobial peptide synthesis in mud crab (Scylla paramamosain) during white spot syndrome virus infection.

Tumor necrosis factor (TNF) is an inflammatory cytokine that is important in cell survival, proliferation, differentiation, and death. However, the functions of TNF in the innate immune responses of invertebrates have been less studied. In this study, SpTNF was cloned and characterized from mud crab (Scylla paramamosain) for the first time. SpTNF contains an open reading frame of 354 bp encoding 117 deduced amino acids, with a conserved C-terminal TNF homology domain (THD) domain. RNAi knockdown of SpTNF reduced hemocyte apoptosis and antimicrobial peptide (AMP) synthesis. Expression of SpTNF was initially down-regulated but subsequently up-regulated after 48 h in hemocytes of mud crabs after WSSV infection. Results of RNAi knockdown and overexpression showed that SpTNF inhibits the WSSV infection through activating apoptosis, NF-κB pathway, and AMP synthesis. Furthermore, the lipopolysaccharide-induced TNF-α factor (SpLITAF) can regulate the expression of SpTNF, induction of apoptosis, and activation of the NF-κB pathway and AMP synthesis. The expression and nuclear translocation of SpLITAF were found to be regulated by WSSV infection. Knocking down of SpLITAF increased the WSSV copy number and expression of VP28 gene. Taken together, these results proved the protective function of SpTNF, which is regulated by SpLITAF, in the immune response of mud crabs against WSSV through the regulation of apoptosis and activation of AMP synthesis.

Essential role of the HSC70 in the mud crab Scylla paramamosain in response to Vibrio parahaemolyticus infection.

Heat shock proteins play an important role in host defense, and modulate immune responses against pathogen infection. In this study, a novel HSC70 from the mud crab (designated as SpHSC70) was cloned and characterized. The full length of SpHSC70 contained a 58 bp 5'untranslated region (UTR), an open reading frame (ORF) of 2,046 bp and a 3'UTR of 341 bp. The SpHSC70 protein included the conserved DnaK motif. The mRNA of SpHSC70 was highly expressed in the hemocytes, heart and hepatopancreas, and lowly expressed in the intestine. The subcellular localization results indicated that SpHSC70 was localized in both the cytoplasm and the nucleus. Moreover, SpHSC70 was significantly responsive to bacterial challenge. RNA interference experiment was designed to investigate the roles of SpHSC70 in response to bacterial challenge. V. parahaemolyticus infection induced the expression levels of SpPO, SpHSP70, SpSOD and SpCAT. Knocking down SpHSC70 in vivo can decrease the expression of these genes after V. parahaemolyticus infection. These results suggested that SpHSC70 could play a vital role in defense against V. parahaemolyticus infection via activating the immune response and antioxidant defense signaling pathways in the mud crab.

Essential role of the Cytochrome P450 2 (CYP2) in the mud crab Scylla paramamosain antioxidant defense and immune responses.

Cytochrome P450 (CYPs) enzymes are one of the critical detoxification enzymes, playing a key role in antioxidant defense. However, the information of CYPs cDNA sequences and their functions are lacked in crustaceans. In this study, a novel full-length of CYP2 from the mud crab (designated as Sp-CYP2) was cloned and characterized. The coding sequence of Sp-CYP2 was 1479 bp in length and encoded a protein containing 492 amino acids. The amino acid sequence of Sp-CYP2 comprised a conserved heme binding site and chemical substrate binding site. Quantitative real-time PCR analysis revealed that Sp-CYP2 was ubiquitously expressed in various tissues, and it was highest in the heart followed by the hepatopancreas. Subcellular localization showed that Sp-CYP2 was prominently located in the cytoplasm and nucleus. The expression of Sp-CYP2 was induced by Vibrio parahaemolyticus infection and ammonia exposure. During ammonia exposure, ammonia exposure can induce oxidative stress and cause severely tissue damage. Knocking down Sp-CYP2 in vivo can increase malondialdehyde content and the mortality of mud crabs after ammonia exposure. All these results suggested that Sp-CYP2 played a crucial role in the defense against environmental stress and pathogen infection in crustaceans.

Effects of environmental pollutant benzop[α]yrene on the innate immunity of Scylla paramamosain and its mechanism.

Benzo[α]pyrene (BaP), a polycyclic aromatic hydrocarbon, is present in the aquatic environment and may be harmful to aquatic animals. We exposed the mud crab Scylla paramamosain to BaP for 7 days, the of superoxide dismutase (SOD), catalase (CAT), phenoloxidase (PO), lysozyme (LZM), glutathione (GSH), glutathione-S-transferase (GST), and acid phosphatase (ACP) activities in the hemolymph of mud crab were reduced. Additionally, the reactive oxygen species content was increased in mud crabs after exposed to BaP. When BaP concentration was increased, the total hemocyte count (THC), the survival rate of hemocytes and their proliferation were decreased. Histopathology analysis revealed damaged hepatopancreas cells, which indicating that BaP exposure is cytotoxic to crab hemocytes. However, the degree of DNA damage did not worsen with increasing BaP concentration. The expression levels of p53, MCM7, Caspase-3, and Myosin were changed with increasing concentration of BaP, which indicated that BaP exposure may affect apoptosis and phagocytosis in mud crabs. As BaP concentration was increased, the apoptosis rate of hemocytes was increased and the phagocytosis was decreased. These results confirmed that BaP exposure inhibited the innate immune response of mud crabs. A possible explanation for this effect is that BaP reduces the antioxidant enzyme activity and increases the reactive oxygen species content in mud crabs, thereby oxidizing and damaging hemocytes, which stimulates phagocytosis and apoptosis and negatively affects the innate immunity of S. paramamosain.

The first identification and functional analysis of two drosophila mothers against decapentaplegic protein genes (SpSmad1 and SpSmad2/3) and their involvement in the innate immune response in Scylla paramamosain.

Smad，a member of the TGF-ß superfamily，controls cell proliferation，growth and guiding cell differentiation, thus playing a crucial role in diseases. However, the presence as well as specific function of Smad in crabs is still unknown. In this study, two Smads (Smad1 and Smad2/3) were identified for the first time from the mud crab Scylla paramamosain. The complete open reading frames of SpSmad1 and SpSmad2/3 were 1,497bp and 1,338bp, encoding deduced proteins of 498 and 445 amino acids respectively. Moreover, under the administration of Vibrio alginolyticus and WSSV, the relative expression levels of SpSmad1 and SpSmad2/3 were significantly increased, indicating their involvement in the innate immune response of mud crabs. Knockdown of SpSmad1 and SpSmad2/3 in vivo not only led to the increasement of the expressions of NF-κB signaling genes and antimicrobial peptides genes, but also significantly affected the bacterial clearance process of mud crabs. Additionally, overexpression of SpSmad1 and SpSmad2/3 in HEK293T cells could markedly activate NF-κB signaling. These results indicated that Smad1 and Smad2/3 participated in the innate immunity of Scylla paramamosain, and might provide a better understanding of the presence and immune regulatory functions of Smad1 and Smad2/3 in crabs and even invertebrates.

Identification and functional analysis of endoplasmic reticulum oxidoreductase 1 (ERO1) from the green mud crab Scylla paramamosain: The first evidence of ERO1 involved in invertebrate immune response.

Endoplasmic reticulum oxidoreductase 1 (ERO1) is an important mediator in regulating disulfide bond formation and maintaining endoplasmic reticulum homeostasis. Its activity is transcriptionally regulated by the unfolded protein response (UPR) in the endoplasmic reticulum, which is known to be essential in immunity. However, whether ERO1 is involved in innate immunity in invertebrates remains unclear. In the present study, two subtypes of ERO1 from Scylla paramamosain were first identified and characterized. Sequence analysis revealed the conserved ERO1 domain and the oxidative capacity assay verified the oxidative capacity of SpERO1 recombinant protein. Moreover, SpERO1s were found to be ubiquitously expressed in all the tested tissues, with the highest expression observed in hemocytes. Two SpERO1s exhibited distinct expression patterns in response to Vibrio alginolyticus and White Spot Syndrome Virus (WSSV). Importantly, the downregulation of the expression of immune factors upon bacterial challenge in SpERO1-silenced crabs was observed. These results provided an initial foundation for further investigations into the role of ERO1 in the innate immunity of invertebrates.

The effect of reactive oxygen species (ROS) in immunity and WSSV infection of Scylla paramamosain.

Reactive oxygen species (ROS) are typically regarded as being generated by the cellular respiratory chain or by cells under pathological damage, which play a crucial role as signaling molecules in promoting hemocytes circulation and normal cellular physiological functions. In this study, the antioxidant N-acetylcysteine (NAC) was used to reduce ROS in vivo and in vitro, which to analyze the effect of ROS on innate immunity and viral infection of mud crab. The total hemocyte count (THC), phenoloxidase (PO), superoxide dismutase (SOD) activity, immune-relative genes were analyzed, respectively. Moreover, the effect of ROS on WSSV infection was analyzed by THC and hemocytes apoptosis. The data showed that NAC could effectively remove and inhibit intracellular ROS. The THC of NAC group was reduced at 12 h and 24 h compared with that of control. And the inhibition of ROS by NAC could increase the SOD activity with control group, while increased the PO activity caused by early WSSV infection. And NAC could up-regulate the expression of MCM7, JAK, TLR and proPO significantly, while down-regulate the expression of Astakine, proPO, caspase and p53. Similarly, NAC could inhibit WSSV-induced apoptosis of S. paramamosain hemocytes. The data illustrated that ROS participates in the interaction between hemocytes and virus infection by regulating innate immunity. Especially, after NAC inhibited ROS, the expression of hemocytes proliferation gene Astakine was also inhibited, which may indicate that ROS is related to the process of hemocytes proliferation. The data will show a preliminary exploration on the regulatory role of ROS in crustacean immune system.

A novel C-type lectin (SpccCTL) suppresses MCRV replication by binding viral protein and regulating antiviral peptides in Scylla paramamosain.

Pattern recognition receptors (PRRs) play a crucial role in the recognition and activation of innate immune responses against invading microorganisms. This study characterizes a novel C-type lectin (CTL), SpccCTL. The cDNA sequence of SpccCTL has a full length of 1744 bp encoding a 338-amino acid protein. The predicted protein contains a signal peptide, a coiled-coil (CC) domain, and a CLECT domain. It shares more than 50 % similarity with a few CTLs with a CC domain in crustaceans. SpccCTL is highly expressed in gills and hemocytes and upregulated after MCRV challenge, suggesting that it may be involved in antiviral immunity. Recombinant SpccCTL (rSpccCTL) as well as two capsid proteins of MCRV (VP11 and VP12) were prepared. Pre-incubating MCRV virions with rSpccCTL significantly suppresses the proliferation of MCRV in mud crabs, compared with the control (treatment with GST protein), and the survival rate of mud crabs is also significantly decreased. Knockdown of SpccCTL significantly facilitates the proliferation of MCRV in mud crabs. These results reveal that SpccCTL plays an important role in antiviral immune response. GST pull-down assay result shows that rSpccCTL interacts specifically with VP11, but not to VP12. This result is further confirmed by a Co-IP assay. In addition, we found that silencing SpccCTL significantly inhibits the expression of four antimicrobial peptides (AMPs). Considering that these AMPs are members of anti-lipopolysaccharide factor family with potential antiviral activity, they are likely involved in immune defense against MCRV. Taken together, these findings clearly demonstrate that SpccCTL can recognize MCRV by binding viral capsid protein VP11 and regulate the expression of certain AMPs, suggesting that SpccCTL may function as a potential PRR playing an essential role in anti-MCRV immunity of mud crab. This study provides new insights into the antiviral immunity of crustaceans and the multifunctional characteristics of CTLs.