Crab Hemocytes Research Articles

Ca2+-SpVAMP2 pathway promotes exosome secretion to resist the infection of Vibrio parahaemolyticus in mud crab (Scylla paramamosain)

Exosomes play an important role in the innate immune system. Many types of cells can secrete exosomes in both normal and abnormal status. However, the mechanism of exosome secretion in invertebrates has not been reported. In this study, the mechanism of the secretion of exosomes regulated by the Ca2+ signaling pathway in the hemocytes of mud crabs (Scylla paramamosain) under infection with Vibrio parahaemolyticus was investigated. The results showed that the increase of exosome secretion in mud crabs infected with V. parahaemolyticus was caused by the increase in intracellular Ca2+ level. In addition, transcriptome sequencing and differential expression analysis revealed that SpVAMP2, a vesicle-associated membrane protein 2, was found to be involved in the exosome secretion regulated by Ca2+ signaling. Further research revealed that SpVAMP2 can inhibit MVB from entering the lysosome for degradation and promote the release of exosomes. Additionally, the secretion of exosomes upregulated the expression of anti-lipopolysaccharide factors and enhanced the survival rate of mud crabs after V. parahaemolyticus infection. This study clarified the exosome secretion regulated by the Ca2+-SpVAMP2 pathway, which, in turn, increased the innate immune response in mud crabs to resist the invasion of V. parahaemolyticus.

Single-cell RNA-sequencing reveals Eriocheir sinensis hemocyte subpopulations and their molecular responses to Spiroplasma eriocheiris infection

The hemocytes is the central part of the Eriocheir sinensis innate immune system, however, their subclasses have not been completely defined and the single-cell gene transcriptional profiles and functions are still unknown. In the present study, the single-cell mRNA sequencing (scRNA-seq) was carried out to build a single-cell gene expression map of E. sinensis hemocytes and further analysis of the interaction molecular mechanism between the crab and novel aquatic pathogens Spiroplasma eriocheiris. By scRNA-seq, we successfully identified nine cell clusters (Hem1–9) and predicted their functions according to the specific marker genes. Pseudotime inference analysis revealed that all the identified cells undergo three stages, state 1 (the initial point), state 2 and state 3 (the other two branches). After being infected by S. eriocheiris, lots of different expression genes (DEGs) were identified in each cell cluster compared with the control group (with fold change ≥2 and p-value <0.05). The bioinformatics analysis found that the Hem1 and Hem5 were the main cell clusters synthesis and release of antimicrobial peptides (AMPs) for help the host against the pathogen infection. The Hem5 also could activate the proPO system to participate in the process of host eliminate the invading S. eriocheiris. Hem7, Hem8, and Hem9 were mainly responsible for killing the invading pathogen through phagocytosis. In particular, there were also many immune-related genes identified, including β-glucan binding protein (BGBP), Clip-domain serine proteases (CLIPs), phenoloxidase 1 (PO1), anti-lipopolysaccharide factors (ALFs), syndecan, and lysozyme C, which not only played critical roles in the crab immune system but also could serve as the marker genes for identifying cell subgroup. In conclusion, the results obtained in the present study could provide pivotal insights into the crab hemocytes cell-type-specific molecular mechanisms response S. eriocheiris infection at a single-cell level.

Cytoplasmic tail of transmembrane dscam controls antibacterial responses by regulating cell proliferation-related genes in hemocytes of Chinese mitten crab (Eriocheir sinensis)

In arthropods, the involvement of Dscam (Down syndrome cell adhesion molecule) in innate immunity has been extensively demonstrated. Its cytoplasmic tail contains multiple conserved functional sites, which indicates its involvement in different intracellular signaling pathways. In this study, we focused on the role of the cytoplasmic tail of Dscam in the Chinese mitten crab (Eriocheir sinensis) immune defense. In the group with cytoplasmic tail knockdown (the site was located on constant exons 37 and 38), 3885 differentially expressed genes (DEGs) were identified. The DEGs were enriched in small molecule binding, protein-containing complex binding, and immunity-related pathways. The expression of selected genes were validated using quantitative real-time reverse transcription PCR. We identified key Cell cycle, Janus kinase (JAK)-signal transducer, activator of transcription (STAT) and mitogen-activated protein kinase (MAPK) signaling pathway genes, the results indicated that the cytoplasmic tail of Dscam controls antibacterial responses by regulating cell proliferation-related genes in hemocytes.

Comparative putative metabolites profiling of Tachypleus gigas and Carcinoscorpius rotundicauda hemocytes stimulated with lipopolysaccharide

Horseshoe crabs are among the most studied invertebrates due to their unique, innate immune system and biological processes. The metabolomics study was conducted on lipopolysaccharide (LPS)-stimulated and non-stimulated hemocytes isolated from the Malaysian Tachypleus gigas and Carcinoscorpius rotundicauda. LC–TOF–MS, multivariate analyses, principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA) were included in this study to profile the metabolites. A total of 37 metabolites were identified to be differentially abundant and were selected based on VIP > 1. However, of the 37 putative metabolites, only 23 were found to be significant with ANOVA at p < 0.05. The metabolites were identified using several databases, and the literature review of the metabolites was reported in the manuscript. Thus, this study has provided further insights into the putative metabolites' presence in the hemocytes of horseshoe crabs that are stimulated and non-stimulated with LPS and their abundance in each species. Several putative metabolites showed they have medicinal values from previous studies.

Two autophagy-related proteins, ATG5 and ATG7, from Eriocheir sinensis involved into Spiroplasma eriocheiris infection

Autophagy-related proteins (ATGs) play an important role in the immune process by regulating autophagy. Eriocheir sinensis tremor disease (TD) is the most hazardous disease caused by Spiroplasma eriocheiris. However, it is unclear whether ATGs play a role in the development of TD. In this study, the function of autophagy and ATG (ATG5 and ATG7) was investigated. Both E. sinensis ATG5 and ATG7 (EsATG5 and EsATG7) were highly transcribed in hemocytes. After S. eriocheiris infection, the transcriptions of EsATG5 and EsATG7 were significantly up-regulated in crab hemocytes. After gene silencing and S. eriocheiris infection, the mortality rate of E. sinensis and the copy number of S. eriocheiris in hemocytes were remarkably increased. These results suggested that EsATG5 and EsATG7 played an important immune role in S. eriocheiris infection. According to the result of western blot using polyclonal antibody of EsATG7, EsATG7 protein expression was increased in S. eriocheiris infection. Meanwhile, EsATG7 protein expression was considerably changed after stimulation with autophagy activator (Rapamycin, RM) and autophagy inhibitor (3-methyladenine, 3-Ma). These results shown EsATG7 was involved in autophagy. After S. eriocheiris infection, the cell activity of the RM group was significantly increased, the copy number of S. eriocheiris in RM group was decreased, while the results in the 3-Ma group were completely opposite. In conclusion, the EsATG gene through regulated E. sinensis autophagy to improve the host's immune ability to resist S. eriocheiris infection.

Cellular localization and potential ligands of a novel scavenger receptor class B/CD36 protein homolog (Pt-SRB2) identified in the marine crab, Portunustrituberculatus

The scavenger receptor class B family proteins (SRB) are multiligand membrane receptor proteins. Herein, a novel SRB homolog (Pt-SRB2) was identified in Portunus trituberculatus. The open reading frame of Pt-SRB2 was predicted to encode 520 amino acid residues comprising a typical CD36 domain. Phylogenetic analysis showed that Pt-SRB2 distinctly clustered with the SRB homologs of most crustaceans and Drosophila but was separate from all vertebrate CD36/SRB. Semi-quantitative and Real-time quantitative PCR revealed that the abundance of Pt-SRB2 transcripts was the highest in hepatopancreas than in other tested tissues. Overexpressed Pt-SRB2 was distributed primarily in the cell membrane and cytoplasm of HEK293T or Drosophila Schneider 2 cells. In crab hemocytes, Pt-SRB2 was distributed primarily in the cell membrane by immunofluorescence staining. In addition, the immunofluorescence staining showed that green fluorescence signals were mainly located in the inner lumen membrane of the hepatopancreatic tubules. Moreover, solid-phase enzyme-linked immunosorbent assay revealed that rPt-SRB2-L exhibited relative high affinity with lipopolysaccharides, and relative moderate binding affinity with lipoteichoic acid or peptidoglycan. Of note, rPt-SRB2-L showed high binding affinity with eicosapentaenoic acid among a series of long-chain polyunsaturated fatty acids. Taken together, this study provided valuable data for understanding the functions of the crab CD36/SRB.

MicroRNA-125 modulates hemocyte proliferation through regulating Astakine expression and participate the disease resistant in Scylla paramamosain

The innate immunity is widely regulated by microRNAs (miRNAs) and that miRNA-125 (miR-125) is involved in many diseases and host immune response. This research was aimed to explore how miR-125 regulates innate immunity in Scylla paramamosain when response to pathogen infection. AMO-miR-125 (anti-miRNA-125 oligonucleotide) was applied to specifically inhibit miR-125 in mud crabs. We found that miR-125 induced hemocyte proliferation, enhanced total hemocyte count in the crabs and protected crabs from challenged with white spot syndrome virus (WSSV) and Vibrio parahaemolyticus. Astakine regulates hemocyte proliferation, and the expression of Astakine increased after miR-125 knockdown, the results indicated that miR-125 may regulate hemocyte proliferation and total hemocyte count by regulating the expression of Astakine. Moreover, miR-125 knockdown also reduced phagocytosis and apoptosis, showing that miR-125 could affect the two processes in crab hemocytes. And the cumulative mortality of WSSV or V. parahaemolyticus infected crabs increased after AMO-miR-125 treatment, which indicated that miR-125 could reduce the death of crab caused by the pathogens and significantly affect the crab's immune response to pathogen. In summary, our data confirmed that miR-125 participates in hemocyte proliferation, the innate immunity, and the resistance to microbial disease in Scylla paramamosain.

Host-parasite interactions: a study on the pathogenicity of different Mesanophrys sp. densities and hemocytes-mediated parasitic resistance of swimming crabs (Portunus trituberculatus).

Mesanophrys sp. is a parasitic ciliate that invades and destroys the hemocytes of the swimming crab (Portunus trituberculatus). In the present study, we employed an in vitro model to elucidate how Mesanophrys sp. destroys crab hemocytes. We also evaluated the relationship between the parasite's density, the destruction rate of the hemocytes, and the rapid proliferation pattern of parasites in host crabs. We found that the survival rate and cell integrity of crab hemocytes decreased with an increase in Mesanophrys sp. density, depicting a negative correlation between hemocyte viability and parasite density. Further analyses revealed that crab hemocytes could resist destruction by a low density (10 ind/mL) of Mesanophrys sp. for a long time (60h). Mesanophrys sp. and its culture medium (containing the ciliate secretions) destroy the host hemocytes. The natural population growth rate of Mesanophrys sp. decreased with an increase in the parasite density, but the Mesanophrys sp. density did not affect the generation time of the parasites. In summary, Mesanophrys sp. can destroy crab hemocytes, and the degree of destruction is directly proportional to the parasite density. The resistance of crab hemocytes to Mesanophrys sp. decreased gradually with an increase in the parasite density.

Effects of the aquatic pollutant sulfamethoxazole on the innate immunity and antioxidant capacity of the mud crab Scylla paramamosain

Sulfamethoxazole (SMZ) is commonly used in aquaculture to treat bacterial infections, but its long-term residual properties in natural water can pose a direct threat to aquatic animals. This study is to investigate the effects of continuous exposure to SMZ on mud crabs (Scylla paramamosain) at four different concentrations (0, 10, 100, and 1000 ng/L) that reflect the range found in natural aquatic environments. The results confirmed that SMZ exposure reduced the expression levels of genes related to the innate immunity in mud crabs, including JAK, Astakine, TLR, and Crustin. It also stimulated oxidative stress, caused the production of reactive oxygen species and lower activities of antioxidant enzymes such as peroxidase, superoxide dismutase, catalase, and glutathione. SMZ exposure damaged the DNA of crab hemocytes and hepatopancreas tissue, and reduced the phagocytosis, ultimately leading to a decreased survival rates of mud crabs infected with Vibrio alginolyticus. These findings demonstrate that SMZ exposure has immunotoxic effects on mud crabs' innate immunity and reduces the ability to resist pathogen infections.

Exosomal SpFlotillin-1 participates in resistance to Vibrio parahemolyticus infection in mud crab (Scylla paramamosain)

Exosomes have been considered anti-microbial immune factors in animals (including aquatic animals). However, the relationship between exosomal proteins and immune responses during bacterial infection has not been addressed. Flotillin-1 has previously been shown to enrich plasma membrane which implicates in cellular processes, including signal transduction, membrane trafficking, and molecular sorting. In this study, SpFlotillin-1 was cloned and characterized from mud crab (Scylla paramamosain). SpFlotillin-1 was found to be up-regulated in the hemocytes of mud crabs after infecting with V. parahaemolyticus. RNAi knockdown of SpFlotillin-1 showed capacity in suppressing phagocytosis and reducing the expression of antimicrobial peptides (AMPs) but increasing the ROS production in hemocytes. Furthermore, SpFlotillin-1 densely packaged in the exosomes can regulate the phagocytosis, expression of AMPs through MAPK and NF-κB pathway, and production of ROS, eventually activating the immune response to bacterial infection in mud crabs. Taken together, the results of this study provide a new finding on the mechanism that exosomal SpFlotillin-1 may participate in the V. parahaemolyticus infection through the regulation of phagocytosis and activation of AMP synthesis in mud crabs.

The impact of ocean acidification and cadmium toxicity in the marine crab Scylla serrata: Biological indices and oxidative stress responses

Ocean acidification (OA) and heavy metal pollution in marine environments are potentially threatening marine life. The interactive effect of OA and heavy metals could be more vulnerable to marine organisms than individual exposures. In the current study, the effect of OA on the toxicity of cadmium (Cd) in the crab Scylla serrata was evaluated. Crab instars (0.07 cm length and 0.1 g weight) were subjected to pH 8.2, 7.8, 7.6, 7.4, 7.2, and 7.0 with and without 0.01 mg l−1 of Cd for 60 days. We noticed a significant decrease in growth, molting, protein, carbohydrate, amino acid, lipid, alkaline phosphatase, and haemocytes of crabs under OA + Cd compared to OA treatment. In contrast, the growth, protein, amino acid, and haemocyte levels were significantly affected by OA, Cd, and its interactions (OA + Cd). However, superoxide dismutase, catalase, lipid peroxidation, glutamic oxaloacetate transaminase, glutamic pyruvate transaminase, and accumulation of Cd in crabs were considerably elevated in OA + Cd treatments compared to OA alone treatments. The present investigation showed that the effect of Cd toxicity might be raised under OA on S. serrata. Our study demonstrated that OA significantly affects the biological indices and oxidative stress responses of S. serrata exposed to Cd toxicity.

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.

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 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 regulated by WSSV infection. SpLITAF knockdown increased the WSSV copy number and VP28 gene expression. 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.

Large-scale lysine crotonylation analysis reveals the role of TRAF6-Ecsit complex in endoplasmic reticulum stress in mud crab (Scylla paramamosain)

Lysine crotonylation (Kcr) is a newly discovered type of post-translational modification. Although Kcr has been reported in several species, its role in crustaceans remains largely unknown. In this study, Kcr in hemocytes of mud crab (Scylla paramamosain) was characterized using pan anti-crotonyllysine antibody enrichment and high-resolution liquid chromatogram-mass spectrometry analysis after SpTRAF6 or SpEcsit silencing. Altogether, 1,800 Kcr sites with six conserved motifs were identified from 512 proteins. Subcellular localization analysis showed that the identified Kcr proteins were mainly localized to the cytoplasm, nucleus, and mitochondria. The cellular components analysis showed that the ‘chromosomal region’ was enriched in the hemocytes of SpTRAF6-or SpEcsit-silenced mud crabs. The KEGG and PPI analyses showed that the identified Kcr proteins in the hemocytes SpTRAF6-or SpEcsit-silenced mud crabs were related to the ‘protein processing in endoplasmic reticulum’; of which the marker of endoplasmic reticulum stress (Bip) was identified to be crotonylated. These datasets present the first comprehensive analysis of the crotonylome in mud crab hemocytes, providing invaluable insights into the regulatory functions of SpTRAF6 and SpEcsit in Kcr. Additionally, our findings shed light on the potential role of these proteins in activating marker proteins during endoplasmic reticulum stress in invertebrates.

Snow crab (Chionoecetes opilio) hemocytes and hepatopancreas transcriptomes: identification, validation, and application of immune-relevant biomarkers of exposure to noise

The snow crab (Chionoecetes opilio) fishery off the east coast of Newfoundland and Labrador, Canada, has been impacted by annual seismic oil and gas surveying in recent decades, and commercial crab harvesters are concerned that it could potentially affect this resource. A laboratory study was performed to investigate the effects of chronic noise exposure on the transcriptomes of snow crab hemocytes and hepatopancreas, which are important in immunity, metabolism and response to environmental stress. Snow crab were held in replicate control or experimental tanks; on alternating days, the experimental tanks were exposed to noise from an underwater speaker which played a recording of a seismic airgun firing every 10 sec in a continuous loop for ~ 22 weeks (chronic noise). RNA sequencing was used to identify candidate noise-responsive molecular biomarkers in both hemocytes and hepatopancreas. The quantified transcripts of individuals (i.e. n=10 from each of the four groups) were compared using DESeq2, identifying over 300 noise-responsive transcripts in each cell/tissue type. Real-time quantitative polymerase chain reaction (qPCR) assays were designed and run for 61 of these transcripts with significant BLASTx hits. One transcript (rgs2) was significantly (p &amp;lt; 0.05) higher expressed in both hemocytes and hepatopancreas in response to noise; five and seven other transcripts were either significant or showed a non-significant trend of differential expression (0.05 &amp;lt; p ≤ 0.1) in hemocytes (e.g. cfb upregulated, sacs and myof downregulated) and hepatopancreas (e.g. gpx3 and atf3 upregulated, sult1c4 downregulated), respectively. To further investigate the performance and utility of these candidate biomarkers in the field, expression levels of 25 selected transcripts were then examined in the hepatopancreas of snow crab that had been subjected to 2D and 3D seismic surveying using both qPCR and multivariate statistical analyses. Overall, few of these biomarkers showed consistent results in field-collected snow crab, highlighting the need to combine lab and field-based studies to fully evaluate the utility of biomarkers. Nonetheless, our study still identified noise-responsive biomarkers in both settings (including stress and immune relevant genes), providing valuable information for understanding the impact of ocean noise on snow crab physiology and health.

Studies on modulation of hemocyte surface antigen through agglutination reaction under arsenic toxicity in edible mudcrab (Scylla serrata)

Scylla serrata (Crustacea: Decapoda), which is widely spread on the intertidal mudflat of West Bengal, India's Sundarbans Biosphere Reserves, is a potential aqua crop and an economically significant edible species. One of the larger crab groups in the mangrove swamp of the Sundarbans is thought to be this one. The S. serrata's multifaceted immune response is directly tied to its diverse habitat and survival technique. It lives in dangerous surroundings and is constantly in danger of physiological stress brought on by various xenobiotics, such as arsenic. By producing a number of polyclonal antisera in rabbits (New Zealand White, albino), the study attempted to evaluate the surface antigen against crab hemocytes and murine lymphocytes. Control hemocytes and hemocytes treated to 1 ppm expressed very identical reactivity to antihemocyte sera for the agglutination reaction. The control results, however, shifted when exposed to 2 and 3 ppm of sodium arsenite, indicating arsenic-induced hemocyte surface modification. The agglutination reaction from the control sets of hemocytes that reacted with murine anti-lymphocyte sera gradually, shifted as the quantity of sodium arsenite in the medium of the treatment sets increased. The maximum equivalence zone of murine lymphocyte and hemocyte agglutination 98.6% and 99% respectively suggested a potential epitope sharing between two phylogenetically separate species. The situation may lead to a possible alteration of immune status and make opportunity for pathogenic foreign invaders within the mud crab body. Chronic arsenic exposure indicated a steady decline of edible and demandable S. serrata in the natural habitat of Sundarbans.

Subcellular Translocation of Yorkie through the PRP4K-CRM1 Axis Regulates Antimicrobial Peptides Transcription and Defense against Bacterial Infection in Crab.

The Hippo signaling pathway plays important roles in innate immunity. In the current study, we found that bacterial infection did not influence mRNA and protein levels of yorkie (Yki), which is an important terminal molecule of the Hippo signaling pathway. However, bacterial infection promoted the translocation of Yki from the nucleus to the cytoplasm in Chinese mitten crab (Eriocheir sinensis), thus attenuating Yki-suppressed transcription of antimicrobial peptides through Cactus. Chromosome region maintenance 1 (CRM1)-silenced crab hemocytes significantly suppressed Yki translocation from the nucleus to the cytoplasm upon bacterial infection, resulting in significantly increased expression of Cactus, decreased expression of antimicrobial peptides, and higher bacterial susceptibility, which demonstrated the regulatory role of CRM1 in subcellular localization of Yki. However, RNA interference of Scalloped (Sd) exhibited no effect on the subcellular localization of Yki and its regulation of Cactus/antimicrobial peptides. Moreover, we elucidated that both CRM1 and Sd could interact with Yki and that the PRP4K-mediated phosphorylation of a conserved serine amino acid residue in the nuclear export signal of Yki is essential for interaction between Yki and CRM1; however, the phosphorylation did not affect the binding of Yki with Sd. We also found that bacterial infection significantly promoted the expression of PRP4K in hemocytes; RNA interference of PRP4K and phosphatase inhibitor suppressed Yki translocation from the nucleus to the cytoplasm, promoting Cactus expression and inhibiting antimicrobial peptide expression. Thus, subcellular localization of Yki regulates antibacterial infection through both PRP4K and CRM1 in crabs.

Protective effect of chicken egg yolk immunoglobulin (IgY) against Spiroplasma eriocheiris infection in Chinese mitten crab

The aquaculture of the Chinese mitten crab Eriocheir sinensis has become the leading and pillar of aquaculture in China. The tremor disease (TD) caused by Spiroplasma eriocheiris is the most destructive disease of E. sinensis. At present, the treatment of TD in crabs mainly depends on antibiotics. To protect the environment and the health of aquatic products, it is urgent to establish an effective and pollution-free green prevention and control technology. This study aimed to investigate the prevention and control of the TD by immunization with chicken yolk antibody (IgY). Anti-S. eriocheiris IgY was obtained by intramuscular injection of ultrasonically inactivated S. eriocheiris into chickens. After successful isolation and purification of IgY from eggs, ELISA and sensitivity tests were performed. Specific IgY was able to specifically react at a titer of 1:10,000 to antigen at a minimum concentration of 20 μg/mL and to inhibit the growth of S. eriocheiris in vitro. The affection of IgY in vivo to resist S. eriocheiris infection was demonstrated by feeding or injection of specific IgY. The copy number of S. eriocheiris in crab hemocytes with specific IgY administration was significantly less than in crabs that received normal IgY or without IgY. Meanwhile, the survival rate of E. sinensis with anti-S.eriocheiris- specific IgY using both injection and feeding methods significantly increased during S. eriocheiris infection. The electron microscope results showed that the structure of S. eriocheiris in specific IgY group was completely irregular and broken pin vivo. Finally, this study provides a reference for clarifying the potential role of specific IgY to help E. sinensis resist S. eriocheiris infection.

Effects of Hizikia fusiforme polysaccharides on innate immunity and disease resistance of the mud crab Scylla paramamosain

In this study, we extracted the polysaccharides from Hizikia fusiforme (HFPs) and evaluated their effects on the immune response of the mud crab Scylla paramamosain. Compositional analysis revealed that HFPs were composed mainly of mannuronic acid (49.05%) and fucose (22.29%) as sulfated polysaccharides, and the sugar chain structure was β-type. These results indicated that HFPs have potential antioxidant and immunostimulation activity in vivo or in vitro assays. Through this research, we found that HFPs inhibited viral replication in white spot syndrome virus (WSSV)-infected crabs and promoted phagocytosis of Vibrio alginolyticus by hemocytes. Quantitative PCR results showed that HFPs up-regulated the expression levels of astakine, crustin, myosin, MCM7, STAT, TLR, JAK, CAP, and p53 in crab hemocytes. HFPs also promoted the activities of superoxide dismutase and acid phosphatase and the hemolymph antioxidant activities of crabs. HFPs maintained peroxidase activity after WSSV challenge, thereby providing protection against oxidative damage caused by the virus. HFPs also promoted apoptosis of hemocytes after WSSV infection. In addition, HFPs significantly enhanced the survival rate of WSSV-infected crabs. All results confirmed that HFPs improved the innate immunity of S. paramamosain by enhancing the expression of antimicrobial peptides, antioxidant enzyme activity, phagocytosis, and apoptosis. Therefore, HFPs have potential for use as therapeutic or preventive agents to regulate the innate immunity of mud crabs and protect them against microbial infection.

The first description of the blue swimming crab (Portunus pelagicus) transcriptome and immunological defense mechanism in response to white spot syndrome virus (WSSV)

In the global shellfish farming industry, white spot syndrome virus (WSSV) is a major cause of mortality and a significant factor in economic losses. However, information on molecular immune responses to WSSV in blue swimming crabs (Portunus pelagicus) has never been reported. First, viral loads were measured in the gills, hepatopancreas, intestines, subcuticular epithelium and hemocytes of blue swimming crabs (50 ± 10 g) (n = 4) after WSSV induction at 0, 24, 48 and 96 h post injection (hpi). A significant increase in WSSV particles was observed in gills at 48 and 96 hpi, as supported by histopathology. To further investigate the acute immune response to WSSV, total RNA from the same gill tissues at 0, 24, and 96 hpi was used to construct 16 high-quality RNA-seq cDNA libraries. In summary, 162,740 unigenes were discovered in these transcriptomic libraries analyzed with the GO, KO, KOG, NR, NT, PFAM and SwissProt databases. Intensive sequence analysis against control crabs using three major categories of gene oncology (GO) of DEGs, biological processes (BPs), molecular functions (MFs), and cellular components (CCs), indicated that induction of WSSV in blue swimming crabs strongly affected the immune responses of the target animals significantly during the early stages of infection from 24 to 96 hpi. Furthermore, KEGG identified approximately twenty biological pathways of gene expression that were both downregulated and upregulated. Interestingly, at 24 and 96 hpi, several immune-related genes involved in virus defense in the blue swimming crab, particularly crustin 2, chitinase, anti-lipopolysaccharide, proteinase inhibitor, and lysozyme, were highly expressed during the WSSV early infection stages. At the same time, viral mRNA transcripts, including WSV289, WSV343, WSV306, deoxyuridine 5’ triphosphate nucleohydrolase, RING finger containing E3 ubiquitin-protein ligase WSV403 and WSV404, were recorded in the top twenty upregulated genes. Moreover, some immune-responsive genes related to growth development, such as chitinase, tubulin alpha and beta chains, trypsin, and the cathepsin family, were also differentially expressed during these periods. Expression validation of 20 upregulated and 11 downregulated immune-related genes using qRT‒PCR showed similar patterns with transcriptome information. Overall, the data showed that during WSSV infection, a number of immune-, metabolism-, and growth-related pathways were activated, and several of the pathways involved differed depending on the stage of virus invasion. These findings could effectively help us better understand the impact of WSSV on the physiology of blue swimming crabs and serve as a valuable reference for future research on the immune system and disease control in this target species.