Crab Hemocytes Research Articles

The cellobiose and α-ketoglutarate as metabolic modulators in Eriocheir sinensis to resist Spiroplasma eriocheiris infection

As one of the most devastating diseases in Eriocheir sinensis, tremor disease (TD) is caused by Spiroplasma eriocheiris and dramatically increases the economic losses of crab aquaculture. With the development of biotechnology, metabolomics plays an important role in the screening of metabolites related to physiological and pathological changes. In this study, the different metabolites induced by S. eriocheiris were investigated in hemocytes of crab E. sinensis using metabolomics based on GC/MS among diverse phenotypes groups (tremor group, no-symptom group, and control group). Compared with the tremor group, 42 differential metabolites (26 up-regulated and 16 down-regulated) were significantly different in the no-symptom group. The KEGG pathway enrichment analysis showed that the differential metabolites were enriched in “ABC transporters”, “Vitamin B6 metabolism”, “Ascorbate and aldarate metabolism” and “Starch and sucrose metabolism”. The cellobiose and α-ketoglutarate (AKG) were chosen to verify the function of these two metabolites. The crabs were fed with dietary cellobiose (1, 5, and 10 mg/g diet weight) or AKG (1, 5, and 10 mg/g diet weight) before S. eriocheiris injection. The results show that all three concentrations of cellobiose and AKG could reduce the mortality of crabs and decrease the copies of S. eriocheiris in the hemocytes. The histopathological sections of the hindgut of E. sinensis show that cellobiose and AKG affect the connective tissue of the outer layer of muscle. Overall, these results provide insight into TD control and the sustainable development of aquaculture.

A truncated peptide Sp-NPFin from the neuropeptide FII SpNPFII of Scylla paramamosain exhibiting potent antimicrobial activity

Neuropeptide (NP) F is a homolog of vertebrate neuropeptide Y, and mounting evidence suggests that it may play a role in innate immunity in addition to its function in invertebrate neurobiology. However, so far, there have been few reports on the immunological properties of crustacean NPs. Here, we cloned a neuropeptide FII gene, naming it SpNPFII, from the mud crab Scylla paramamosain. The full-length cDNA sequence of SpNPFII was 551 bp, encoding 124 amino acids. Structural prediction suggested that the SpNPFII mature peptide mainly formed an α-helix. SpNPFII was predominately expressed in male crab gonadal tissues, with the highest expression in the anterior vas deferens and seminal vesicle. The expression level of SpNPFII changed significantly under challenge by lipopolysaccharide (LPS) or Vibrio alginolyticus. After bioinformatic analysis, a truncated 45-amino acid peptide derived from SpNPFII and having potent antimicrobial activity was identified and named Sp-NPFin. Sp-NPFin exerted strong, broad-spectrum antibacterial (minimum inhibitory concentration [MIC] ranged from 1.5 to 12 μM) and antimildew activity (MIC ranged from 1.5 to 48 μM), a low fungicidal concentration (e.g., minimum bactericidal concentration [MBC] for Fusarium oxysporum was 1.5 to 3 μM) and rapid bactericidal kinetic (killed all bacteria within 1 to 6 h). Sp-NPFin could bind to several microbial surface components (e.g. LPS, lipoteichoic acid, peptidoglycan and glucan), and induce significant damage to microbial membranes and cause leakage of cell contents in a concentration-dependent manner. Confocal microscopy indicated that Sp-NPFin was localized at microbial surfaces. In addition, Sp-NPFin exhibited no cytotoxicity to primary cultured crab hemocytes and mammalian cells, and in vivo study demonstrated that it could significantly improve the survival rate of S. paramamosain under the challenge of V. alginolyticus. Taken together, SpNPFII might play a role in the immune system of S. paramamosain, and its truncated peptide, Sp-NPFin, would be a promising antibacterial and antimildew agent with broad application prospects in the fields of aquaculture, veterinary practices and medicine.

The role of myosin-9 in Scylla paramamosain against Vibrio alginolyticus and white spot syndrome virus infection

Myosin-9 is an important component of myofibrillar filament which is widely distributed in the various tissues and organs of animals. In this research we studied the role of the Scylla paramamosain myosin-9 gene in the immune process against white spot syndrome virus (WSSV) or Vibrio alginolyticus infection. The myosin-9 gene with a total length of 2735 bp, including a 2145 bp ORF encoding a 715aa protein, was cloned from S. paramamosain. The expression of myosin-9 in muscle was the highest and decreased in crab hemocytes after challenges with WSSV or V. alginolyticus. After myosin-9 knockdown by double-stranded RNA (myosin-9-dsRNA), the expression of c-type-lectin, Toll-like-receptor, and crustin antimicrobial peptide was increased significantly, and the expression of JAK, STAT, and prophenoloxidase was decreased significantly. An immune activity test showed that the total number of hemocytes decreased significantly and the activity of phenoloxidase (PO) and superoxide dismutase (SOD) were decreased after myosin-9 knockdown. After myosin-9 knockdown and WSSV infection, the phagocytosis rate of hemocytes was significantly decreased but the apoptosis rate of hemocytes was increased, and the copy number of virus and the mortality rate of crabs were significantly increased compared with that of control. These results indicated that myosin-9 may participate in the antiviral immune process of S. paramamosain against WSSV infection. After myosin-9 knockdown and V. alginolyticus infection, the phagocytosis rate of hemocytes was significantly reduced, the apoptosis rate of hemocytes was significantly increased, and the mortality rate of crabs was significantly increased compared with that of control. It was revealed that myosin-9 may play a positive role in the immune response to V. alginolyticus through increasing the phagocytosis rate of hemocytes. This study interpreted the role of myosin-9 in the innate immunity of S. paramamosain, and provided strong support for the prevention and treatment of diseases in S. paramamosain culture.

Elucidation of metabolic responses in mud crab Scylla paramamosain challenged to WSSV infection by integration of metabolomics and transcriptomics

White spot syndrome virus (WSSV) is a severe pathogen of mud crab Scylla paramamosain (S. paramamosain). Hemolymph, containing three types of hemocytes, is the key immunoregulatory tool of mud crab in response to pathogens. Herein, the metabonomics and transcriptomics analysis of hemocytes were adopted to investigate the immune response of S. paramamosain challenged to WSSV. We established the metabolic and transcriptional profiles of mud crab hemocytes with different treatments, including the control group (WT), WSSV early infected group (WSSV-6 h) and WSSV later infected group (WSSV-72 h). The results showed that 68 metabolites were dysregulated both in WSSV-infected mud crab of early stage and later stage, while 4452 genes were up-regulated and 9746 genes were down-regulated in WSSV-6 h, and 2016 genes were up-regulated and 6229 genes were down-regulated compared in WSSV-72 h. We found that several pathways were dysregulated at both metabolic and transcriptional levels, including ABC transporters, purine metabolism, taurine and hypotaurine metabolism in the WSSV early infected group, cysteine metabolism, methionine metabolism and biosynthesis of unsaturated fatty acids in the WSSV later infected group. In this context, through the integration of metabolomics and transcriptomics, our study provided a more comprehensive understanding of the biological process in mud crab against viral invasion.

Transcriptome and metabolome integration analysis of mud crab Scylla paramamosain challenged to Vibrio parahaemolyticus infection

Vibrio parahaemolyticus (V. parahaemolyticus) is a common pathogen for marine crustacean, which causes severe illnesses in aquatic animals. Therefore, it is meaningful to explore the mechanism during V. parahaemolyticus infection. In this study, to investigate the immune responses of mud crab Scylla paramamosain (S. paramamosain) to V. parahaemolyticus, we established the metabolic and transcriptional profiles of mud crab hemocytes challenged with V. parahaemolyticus. The results indicated that V. parahaemolyticus infection could induce a series of metabolism alterations at both metabolome and transcriptome levels, including biosynthesis of amino acids and Aminoacyl-tRNA, Purine and pyrimidine metabolism, TCA cycle and glutamine metabolism. In this context, through the integration of metabolomics and transcriptomics, our study provided a more comprehensive understanding of the biological process in mud crab against pathogen infection.

The miRNAs profiling revealed by high-throughput sequencing upon WSSV infection in mud crab Scylla paramamosain

microRNAs (miRNAs) are known to regulate various immune functions by silencing the target genes in both vertebrates and invertebrates. However, in mud crab Scylla paramamosain, the role of miRNAs during the response to virus invasion remains unclear. To investigate the roles of miRNAs in S. paramamosain during virus infection, the mud crab was challenged with white spot syndrome virus (WSSV) and then subjected to the transcriptional analysis at different conditions. The results of high-throughput sequencing revealed that 940,379 and 1,306,023 high-quality mappable reads were detected in the hemocyte of normal and WSSV-infected mud crabs, respectively. Besides, the total number of 261 unique miRNAs were identified. Among them, 131 miRNAs were specifically expressed in the hemocytes of normal mud crabs, 46 miRNAs were specifically transcribed in those of WSSV-infected individuals, the other 84 miRNAs were expressed in both normal and WSSV-infected individuals. Furthermore, a number of 152 (89 down-regulated and 63 up-regulated) miRNAs were found to be differentially expressed in the WSSV-infected hemocytes, normalized to the controls. The identified miRNAs were subjected to GO analysis and target gene prediction and the results suggested that the differentially regulated miRNAs were mainly correlated with the changes of the immune responses of the hemocytes, including phagocytosis, melanism, and apoptosis as well. Taken together, the results demonstrated that the expressed miRNAs during the virus infection were mainly involved in the regulation of immunological pathways in mud crabs. Our findings not only enrich the understanding of the functions of miRNAs in the innate immune system but also provide some novel potential targets for the prevention of WSSV infection in crustaceans.

Sp-CBL inhibits white spot syndrome virus replication by enhancing apoptosis in mud crab (Scylla paramamosain)

In mammals, casitas B-lineage lymphoma (CBL) family proteins, a RING-type E3 ubiquitin ligase, are involved in many signal transduction pathways. However, the functions of CBL in invertebrates are not well elucidated. In this study, Sp-CBL containing CBL-N, CBL-2, CBL-3 and RING domains was identified in mud crab Scylla paramamosain. Sp-CBL was widely expressed in all tissues tested and found to be significantly up-regulated in the hemocytes of mud crab challenged by white spot syndrome virus (WSSV). The RNA interference of Sp-CBL increased the copy number of WSSV and declined the apoptosis rate of hemocytes. In addition, Sp-CBL could affect the activities of caspase 3 and the mitochondrial membrane potential. Taken together, the results of this study revealed that Sp-CBL could restrict WSSV proliferation through enhancing the apoptosis of the hemocytes, which would provide a novel insight into the anti-viral response in the innate immunity system of mud crab.

TMT-based quantitative proteomic analysis of Eriocheir sinensis hemocytes and thoracic ganglion during Spiroplasma eriocheiris infection

Spiroplasma eriocheiris, a novel pathogen of Chinese mitten crab Eriocheir sinensis tremor disease, has led into catastrophic economic losses in aquaculture. S. eriocheiris invaded the hemocytes in the early stage, then invaded nerve tissue and caused typically paroxysmal tremors of pereiopod in the late stage of infection. The purpose of this study was to detect the infection mechanism of hemocytes in the early stage and thoracic ganglion in the late stage of S. eriocheiris infection at the protein level. Hemocytes and thoracic ganglion were collected at 24 h and 10 d after injection (the crabs with typical paroxysmal tremors of the pereiopod), respectively. TMT was performed with isobaric markers, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). In hemocytes, 127 proteins were up-regulated and 85 proteins were down-regulated in 2747 quantified proteins. Many proteins and process including proPO system proteins, hemolymph coagulation system proteins and lectins were differently expressed in hemocytes and involved in the early immune process of E. sinensis against S. eriocheiris infection. Meanwhile, 545 significantly different expression proteins (292 down-regulated and 253 up-regulated protein including a number of immune-associated, nervous system development and signal transmission related proteins) were identified in thoracic ganglion in the late stage of S. eriocheiris infection. The qRT-PCR analysis results shown that the selected significantly changed proteins in hemocytes and thoracic ganglion were consistent with the TMT proteomics. This paper reported for the first time to study the responses of crab hemocyte and thoracic ganglion against the S. eriocheiris infection at different stages. These findings help us understand the infection mechanism of S. eriocheiris at different stage with the different tissue.

Alternatively spliced down syndrome cell adhesion molecule (Dscam) controls innate immunity in crab

Alternatively-spliced hypervariable immunoglobulin domain-encoding molecules, called Down syndrome cell adhesion molecule (Dscam), have been widely detected as components of the arthropod immune system. Although its ability to specifically bind pathogens and enable phagocytosis of bacteria has been elucidated, the signal transduction mechanisms or effectors that activate post-Dscam-binding pathogens remain poorly characterized. Here, we reveal the alternative splicing exons of Dscam's cytoplasmic tail and its isoforms in the hemocytes of crab (Eriocheir sinensis), showing that the expression of Dscam was acutely induced after an immune challenge, which suggested its functioning for innate immunity. Significantly decreased expression levels of antimicrobial molecular peptides (AMPs) were detected in Dscam-silenced crab hemocytes in vitro, which coincided with their vulnerability to infection by Staphylococcus aureus and higher bacterial concentrations occurring in Dscam-silenced crabs in vivo Further experimental investigation demonstrated that Dscam-regulated AMP expression via the Src homology (SH)3-binding domain in the first constant exon translated protein of the cytoplasmic tail bound with the SH3 domain of the Dock, an SH3/SH2 adaptor protein required for axon guidance. Dock promoted extracellular signal-regulated kinase (ERK) phosphorylation via indirect binding and then regulated dorsal phosphorylation and translocation from the cytoplasm to the nucleus, subsequently promoting AMP expression for the effective removal of bacteria. To the best of our knowledge, this comprehensive study is the first to highlight the critical role of the alternatively-spliced Dscam cytoplasmic tail in antimicrobial control activity. It also suggests possible cross-talk occurring between Dscam and other pattern recognition receptors.

Characterization, subcellular localization and function analysis of myeloid differentiation factor 88 (Pt-MyD88) in swimming crab, Portunus trituberculatus

Myeloid differentiation factor 88 (MyD88) is a universal and essential adaptor protein required for the Toll-like receptors (TLRs) pathway activation in invertebrates as well as in vertebrates. Herein, we characterized a MyD88 (Pt-MyD88) cDNA sequence in the swimming crab (Portunus trituberculatus). The Pt-MyD88 ORF is predicted to encode 469 peptides with an N-terminal death domain and a typical C-terminal TIR domain. Real-Time quantitative PCR analysis showed that the Pt-MyD88 transcriptions were constitutively expressed in hemocytes, gill, intestine, heart and muscle in normal crab. The expressions of Pt-MyD88 would be down-regulated by V. alginolyticus or LPS challenge, and be up-regulated by WSSV infection in hemocytes. Intracellular localization showed Pt-MyD88 was distributed mainly in the cytoplasm when it was over-expressed in human cell HEK293T or in Drosophila Schneider 2 (S2). Functionally, over-expression of Pt-MyD88 could either activate the NF-κB in HEK293T cells or activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. In primary cultured hemocytes of swimming crab, after Pt-MyD88 was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), hyastatin3, crustin1 and crustin3 have been significantly inhibited, while the expression of other AMPs is normal compared to non-specific dsRNA treated cells.

SpBAG1 promotes the WSSV infection by inhibiting apoptosis in mud crab (Scylla paramamosain)

Bcl-2 associated athanogene-1 (BAG1) is involved in various signalling pathways including apoptosis, cell proliferation, gene transcriptional regulation and signal transduction in animals. However the functions of BAG1 during the antiviral response of mud crab Scylla paramamosain is still unclear. In this study, the mud crab BAG1 (SpBAG1) was characterized to consist of 1761 nucleotides, containing an opening frame of 630bp encoding 209 amino acids with an ubiquitin domain and a BAG1 domain. SpBAG1 was found to be significantly up-regulated at 6 h–24 h, but down-regulated from 48 h–72 h in the hemocytes of mud crab after challenge with white spot syndrome virus (WSSV). RNAi knock-down of SpBAG1 significantly reduced the copies of WSSV and increased the apoptotic rate in mud crabs. The finding from this study suggested that SpBAG1 could promote the WSSV infection by inhibiting apoptosis in mud crab. Therefore, to the best of our knowledge, this is the first study demonstrating the role of SpBAG1 as a novel apoptosis inhibitor to promote virus infection in mud crab.

Application of a Recombinant Three-Factor Chromogenic Reagent, PyroSmart, for Bacterial Endotoxins Test Filed in the Pharmacopeias.

Assays using lysate reagents prepared from horseshoe crab hemocyte extract (limulus amoebocyte lysate, LAL) are commonly and widely used to detect and measure endotoxin in parenteral drugs and medical devices. However, lysate reagents suffer from lot-to-lot variations leading to possible fluctuations in testing. Also, this continued usage of lysate reagents leads to the possible decline of the horseshoe crab population. Recently, a new recombinant chromogenic reagent, PyroSmart, consisting of three recombinant factors was introduced to the market. There are now three recombinant products; two with recombinant factor C reagents and PyroSmart with the complete recombinant LAL system. We evaluated the applicability of the reagent to the harmonized bacterial endotoxins test in the United States, European and Japanese pharmacopeias. The recombinant product showed equivalent potency of thirteen endotoxins from different bacterial strains to conventional chromogenic lysate reagents as long as their assay modes are identical. All analytical characteristics or assay parameters of the reagent satisfied the acceptance criteria which are set for the use for the bacterial endotoxins test filed in the pharmacopeias. All of 109 parenteral drugs tested can be measured with PyroSmart within respective maximum allowable dilutions. The lot-to-lot variation in recovery of endotoxin added in the parenteral drugs for PyroSmart was equal to or less than those of six limulus lysate reagents. In conclusion, the present study suggests that the recombinant reagent, PyroSmart, provide a good alternative to the LAL reagents with better lot-to-lot variation.

Molecular characterization of carboxypeptidase B-like (CPB) in Scylla paramamosain and its role in white spot syndrome virus and Vibrio alginolyticus infection

Carboxypeptidase plays an important physiological role in the tissues and organs of animals. In this study, we cloned an entire 2316 bp carboxypeptidase B-like (CPB) sequence with a 1302 bp open reading frame encoding a 434 amino acid peptide from Scylla paramamosain. The CPB gene was expressed highly in hepatopancreas and decreased in crab hemocytes after challenges with white spot syndrome virus (WSSV) or Vibrio alginolyticus. After CPB gene knockdown using double-stranded RNA (CPB-dsRNA), the expression of JAK, STAT, C-type lectin, crustin antimicrobial peptide, Toll-like receptors, prophenoloxidase, and myosin II essential light chain-like protein were down-regulated in hemocytes at 24 h post dsRNA treatment. CPB knockdown decreases total hemocyte count in crabs indicated that CPB may negatively regulate crab hemocyte proliferation in crabs. CPB showed an inhibitory effect on hemocyte apoptosis in crabs infected with WSSV or V. alginolyticus. The phagocytosis rate of WSSV by hemocytes was increased after CPB-dsRNA treatment. After WSSV challenge, the mortality and WSSV copy number were both decreased but the rate of hemocyte apoptosis was increased in CPB-dsRNA-treated crabs. The results indicate that the antiviral activity of the crabs was enhanced when CPB was knocked down, indicating WSSV may take advantage of CPB to benefit its replication. In contrast, the absence of CPB in crabs increased mortality following the V. alginolyticus challenge. The phagocytosis rate of V. alginolyticus by hemocytes was increased after CPB-dsRNA treatment. It was revealed that CPB may play a positive role in the immune response to V. alginolyticus through increasing the phagocytosis rate of V. alginolyticus. This research further adds to our understanding of the CPB and identifies its potential role in the innate immunity of crabs.

The Development and Characterization of a Cell Culture System from Indian Mud Crabs Scylla serrata

Commercially available culture media and supplements were tested for their potential to produce primary cell cultures from tissues of Indian mud crabs Scylla serrata. Eight commercially available culture media from Sigma-Aldrich (Leibovitz's L-15, Medium 199, Grace's InsectMedium, Minimal Essential Medium, Dulbecco's Modified Eagle Medium, TC-100 Insect Medium, IPL-41 Insect Medium, and Roswell Park Memorial Institute) were examined. Three different supplements (amino acid and sugar [AS], crab muscle extract [CME], and natural seawater [NSW]) were also examined. The hemocyte culture appeared to grow well for a maximum period of 21d in 2×L-15 medium supplemented with AS and 15% fetal bovine serum (FBS). Partial amplification and sequencing of the cytochrome oxidase subunit I (COI) gene confirmed that the primary hemocytes originated from Indian mud crabs. The effects of four metals on hemocyte viability were evaluated using the MTT assay. Of the four metals examined (arsenic, lead, cobalt, and nickel), cobalt and nickel were more toxic to the crab cells than the other metals. Both acridine orange/ethidium bromide and Hoechst staining showed the presence of apoptosis and necrosis in metal-treated groups, which suggests that metals in an aquatic environment induce death of the Indian mud crab's hemocytes. The hemocyte primary cell culture was also used to study the cytotoxicity effect of bacterial extracellular products from Vibrio harveyi and white spot syndrome virus. This study demonstrates that hemocyte primary cell culture can be used as a tool to study viral and bacterial pathogenesis and to assess the cytotoxicity of pollutants present in aquatic environments.

Modulation of oxidative damage by lipopolysaccharides and antioxidant responses in the freshwater crab Sinopotamon henanense Bott 1967 (Decapoda: Brachyura: Potamonidae) during cadmium exposure

AbstractCadmium (Cd) is a toxic pollutant that exerts adverse effects on organisms. We examined oxidative damage and antioxidative parameters in hemocytes of the freshwater crab Sinopotamon henanense Bott 1967 exposed to sublethal levels of Cd for 21 d. We also examined the immunomodulation of lipopolysaccharides (LPS) on oxidation caused by Cd in the crab. Cd induced higher reactive oxygen species (ROS) levels but ROS production in crab hemocytes was down-regulated after LPS injection. The contents of malondialdehyde (MDA) and protein carbonyl derivatives (PCO), DNA-protein crosslinks (DPCs) coefficient in the hemocytes of crabs exposed to Cd increased. LPS injection could decrease these oxidative parameters. The total antioxidant capacity (T-AOC) was significantly inhibited by Cd and LPS enhanced the activity of T-AOC in crab hemocytes. Cd treatment also up-regulated activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Only CAT activity decreased when crabs were exposed to Cd followed by LPS stimulation. Peroxidase (POD) activities showed little change after Cd or LPS treatments. The oxidative damage caused by Cd exposure may be reduced by LPS, suggesting its possible use in aquaculture as an immunostimulant for crustaceans.

Proteomic analysis and white spot syndrome virus interaction of mud crab (Scylla olivacea) revealed responsive roles of the hemocytes

White spot disease (WSD) is a highly virulent viral disease in shrimps. Clinical signs and high mortality of WSD is generally observed after a few days of infection by White Spot Syndrome virus (WSSV). Mud crabs are the major carrier and persistent host for the WSSV. However, an elucidation of viral interaction and persistent mode of WSSV infection in mud crab is still limited. We investigated the defensive role of mud crab (Scylla olivacea) hemocytes against WSSV infection by using comparative proteomic analysis coupled with electrospray ionization liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS). The proteomic maps of expressed proteins obtained from WSSV infected hemocytes revealed differential proteins related to various biological functions, including immune response, anti-apoptosis, endocytosis, phosphorylation signaling, stress response, oxygen transport, molting, metabolism, and biosynthesis. Four distinctive cell types of crab hemocytes: hyaline cells (HC), small granular cells (SGC), large granular cells (LGC) and mixed granular cells (MGC) were found susceptible to WSSV. However, immunohistochemistry analysis demonstrated a complete replication of WSSV only in SGC and LGC. WSSV induced apoptosis was also observed in HC, SGC and MGC except for LGC. These results suggested that HC and MGC may undergo apoptosis prior to a complete assembly of virion, while SGC is more susceptible showing higher amplification and releasing of virion. In contrast, WSSV may inhibit apoptosis in infected LGC to stay in latency. This present finding provides an insight for the responsive roles of crustacean hemocyte cells involved in molecular interaction and defense mechanism against WSSV.

Beclin-1 is involved in the regulation of antimicrobial peptides expression in Chinese mitten crab Eriocheir sinensis

Beclin-1, the mammalian ortholog of yeast Atg6, plays essential roles in the regulation of various processes, including autophagy, apoptosis, embryonic development and immune responses in vertebrates. However, the information about Beclin-1 in invertebrates especially in crustaceans is still very limited. In the present study, a novel Beclin-1 (designated as EsBeclin-1) was identified from Chinese mitten crab Eriocheir sinensis. The open reading frame of EsBeclin-1 cDNA was of 1,275 bp, encoding a typical APG6 domain. The deduced amino acid sequence of EsBeclin-1 shared high similarity ranging from 42.9% to 63.6% with the previously identified Beclins. In the phylogenetic tree, EsBeclin-1 was firstly clustered with Drosophila melanogaster Atg6 and then assigned into the branch of invertebrate Beclin-1. The mRNA transcripts of EsBeclin-1 were highly expressed in hepatopancreas, hemocytes and gill. After lipopolysaccharide (LPS) and Aeromonas hydrophila stimulations, the relative mRNA expression of EsBeclin-1 in hemocytes was significantly increased from 3 to 24 h with the peak level of 4.70-fold (p < 0.01) and 2.91-fold (p < 0.01) at 6 h, respectively. EsBeclin-1 protein was diffusely distributed in the cytoplasm of crab hemocytes under normal conditions, whereas it displayed predominantly punctuate distribution after LPS stimulation. After EsBeclin-1 was interfered with specific EsBeclin-1-dsRNA, the mRNA transcripts of some antimicrobial peptides, including EsALF2, EsLYZ, EsCrus and EsCrus2 in crab hemocytes were significantly decreased at 6 h post LPS stimulation. These results implicated that EsBeclin-1 played a role in regulating the antimicrobial peptides expressions in the immune responses of E. sinensis.

Dopamine and Serotonin Modulate Free Amino Acids Production and Na+/K+ Pump Activity in Chinese Mitten Crab Eriocheir sinensis Under Acute Salinity Stress.

The Chinese mitten crab Eriocheir sinensis lives in saline or fresh water during different life stages and exhibits a complex life history, making it an ideal model to study the salinity adaptation of euryhaline animals. In this study, RNA-seq techniques, and determinations of free amino acids (FAAs), monoamine neurotransmitters, and Na+/K+ pump activity, were employed to understand the osmoregulatory mechanism in Chinese mitten crab. A total of 15,138 differentially expressed genes were obtained from 12 transcriptome libraries. GO enrichment analysis revealed that the mRNA expression profiles were completely remodeled from 12 to 24 h after salinity stress. The neuroendocrine system was activated under stimulation, and the monoamine neurotransmitters including dopamine (DA) and serotonin (5-HT) were released to modulate osmoregulation. Furthermore, the Na+/K+ pump in crab hemocytes was significantly inhibited post salinity stress, resulting in increased intracellular ion concentrations and osmotic pressure to sustain the osmotic balance. Moreover, six key FAAs, including alanine (Ala), proline (Pro), glycine (Gly), glutamate (Glu), arginine (Arg), and aspartate (Asp), were overexpressed to modulate the extracellular osmotic balance during salinity adaptation. Interestingly, the immune genes were not enriched in the GO analysis, implying that the immune system might not contribute fundamentally to the tolerance upon fluctuating ambient salinity in the Chinese mitten crab. These results collectively demonstrated that the Chinese mitten crab had evolved an efficient regulation mechanism by modulating the FAAs production and Na+/K+ pump activity to sustain the osmotic balance independent of the immune system, in which the neuroendocrine modulation, especially generated by the monoamine neurotransmitter, played an indispensable role.

SpToll1 and SpToll2 modulate the expression of antimicrobial peptides in Scylla paramamosain

Tolls and Toll-like receptors (TLRs) were the first pattern recognition receptors (PRRs) identified to play key roles in host innate immunity. However, relatively little is known about other types of Toll-like receptors in Scylla paramamosain, although a Toll-like receptor (SpToll1) has recently been cloned. In this study, we cloned and characterized another novel Toll-like receptor 2 (SpToll2) from S. paramamosain. The full-length cDNA of SpToll2 is 3391 bp with a 2646 bp open reading frame (ORF) encoding a putative protein of 881 amino acids, and predicted to contain six extracellular leucine-rich repeat (LRR) domains, a transmembrane domain and an intracellular Toll/IL-1 receptor (TIR) domain. Phylogenetic analysis revealed that SpToll2 clustered with Drosophila Toll1, and shared high homology with PtToll4. Real-time qPCR analysis showed that SpToll2 was widely expressed in all tissues tested, with the highest level found in hemocytes and hepatopancreas while the lowest in heart and muscle. The transcript levels of both SpToll1 and SpToll2 in mud crabs hemocytes was induced following challenge with Vibrio parahaemolyticus, Staphylococcus aureus, Polyinosinic: polycytidylic acid (Poly I:C) and white spot syndrome virus (WSSV). In addition, recombinant SpToll1-LRR and SpToll2-LRR proteins could bind to V. parahaemolyticus, S. aureus, Escherichia coli, and Beta Streptococcus. In order to study the signaling pathway of AMPs' expression in mud crab, RNA interference were used to test the expression of SpAMPs after the challenges with V. parahaemolyticus or S. aureus. The data suggested that SpToll1and SpToll2 could regulate the transcripts of several AMPs and four immune related mediators (SpMyD88, SpTube, SpPelle and SpTRAF6) at different scale. While silencing of SpToll1 post pathogens challenge attenuated the expression of SpHistin, SpALF1 and SpALF5 in mud crab's hemocytes, depletion of SpToll2 post pathogens challenge inhibited the expression of SpALF1-6, SpGRP, SpArasin and SpHyastastin. Furthermore, the results of overexpression assay also showed SpToll1 and SpToll2 could enhance the promoter activities of SpALFs in mud crab. Taken together, these results indicated that SpToll1 and SpToll2 might play important roles in host defense against pathogen invasions in S. paramamosain.

Comparative transcriptomic analysis of crab hemocytes in response to white spot syndrome virus or Vibrio alginolyticus infection

To assess the immune response of hemocytes to white spot syndrome virus (WSSV) or Vibrio alginolyticus infection in the mud crab Scylla paramamosain, a transcriptome analysis was performed. We report the analysis of 45131 transcripts from S. paramamosain hemocytes by de novo assembly. A comparison with GenBank protein and nucleotide sequences identified 33699 genes as previously known. The length distribution of the genes was 8147 genes ≥200 bp, 4714 genes ≥300 bp, and 3517 genes ≥2000 bp. A total of 21579 simple sequence repeats (SSRs) were found in the transcriptomic dataset, including 9% monomers, 53.34% dimers and 32.55% trimers. A total of 13172 and 5087 differentially expressed transcripts were found in the V. alginolyticus-infected group and WSSV-infected group, respectively. Of these, 5920 transcripts were up-regulated and 7252 were down-regulated in the V. alginolyticus-infected crabs and 2302 transcripts were up-regulated and 2785 were down-regulated in the WSSV-infected crabs. Additionally, 3096 transcripts were differentially expressed simultaneously in the V. alginolyticus-infected crabs and the WSSV-infected crabs. Several known immune-related genes such as heat shock protein, Janus kinase, STAT, relish, caspase, Ca2+-transporting ATPase and lysosomal alpha-mannosidase were found among the differentially expressed transcripts. Transcription and its regulation were significant biological processes, and ATP binding and zinc ion binding were significant molecular functions. This is the first report of comparative transcriptomic analysis of crab hemocytes in response to WSSV or V. alginolyticus infection. These findings will contribute to our understanding of the immune response to WSSV and V. alginolyticus infection in crustaceans.