Dietary lauric acid promoted antioxidant and immune capacity by improving intestinal structure and microbial population of swimming crab (Portunus trituberculatus).

Lauric acid (LA), a saturated fatty acid with 12 carbon atoms, is widely regarded as a healthy fatty acid that plays an important role in disease resistance and improving immune physiological function. The objective of this study was to determine the effects of dietary lauric acid on the growth performance, antioxidant capacity, non-specific immunity and intestinal microbiology, and evaluate the potential of lauric acids an environmentally friendly additive in swimming crab (Portunus trituberculatus) culture. A total of 192 swimming crabs with an initial body weight of 11.68 ± 0.02 g were fed six different dietary lauric acid levels, the analytical values of lauric acid were 0.09, 0.44, 0.80, 1.00, 1.53, 2.91 mg/g, respectively. There were four replicates per treatment and 8 juvenile swimming crabs per replicate. The results indicated that final weight, percent weight gain, specific growth rate, survival and feed intake were not significantly affected by dietary lauric acid levels; however, crabs fed diets with 0.80 and 1.00 mg/g lauric acid showed the lowest feed efficiency among all treatments. Proximate composition in hepatopancreas and muscle were not significantly affected by dietary lauric acid levels. The highest activities of amylase and lipase in hepatopancreas and intestine were found at crabs fed diet with 0.80 mg/g lauric acid (P < 0.05), the activity of carnitine palmityl transferase (CPT) in hepatopancreas and intestine significantly decreased with dietary lauric acid levels increasing from 0.09 to 2.91 mg/g (P < 0.05). The lowest concentration of glucose and total protein and the activity of alkaline phosphatase in hemolymph were observed at crabs fed diets with 0.80 and 1.00 mg/g lauric acid among all treatments. The activity of GSH-Px in hepatopancreas significantly increased with dietary lauric acid increasing from 0.09 to 1.53 mg/g, MDA in hepatopancreas and hemolymph was not significantly influenced by dietary lauric acid levels. The highest expression of cat and gpx in hepatopancreas were exhibited in crabs fed diet with 1.00 mg/g lauric acid, however, the expression of genes related to the inflammatory signaling pathway (relish, myd88, traf6, nf-κB) were up-regulated in the hepatopancreas with dietary lauric acid levels increasing from 0.09 to 1.00 mg/g, moreover, the expression of genes related to intestinal inflammatory, immune and antioxidant were significantly affected by dietary lauric acid levels (P < 0.05). Crabs fed diet without lauric acid supplementation exhibited higher lipid drop area in hepatopancreas than those fed the other diets (P < 0.05). The expression of genes related to lipid catabolism was up-regulated, however, and the expression of genes related to lipid synthesis was down-regulated in the hepatopancreas of crabs fed with 0.80 mg/g lauric acid. Lauric acid improved hepatic tubular integrity, and enhanced intestinal barrier function by increasing peritrophic membrane (PM) thickness and upregulating the expression of structural factors (per44, zo-1) and intestinal immunity-related genes. In addition, dietary 1.00 mg/g lauric acid significantly improved the microbiota composition of the intestinal, increased the abundance of Actinobacteria and Rhodobacteraceae, and decreased the abundance of Vibrio, thus maintaining the microbiota balance of the intestine. The correlation analysis showed that there was a relationship between intestinal microbiota and immune-antioxidant function. In conclusion, the dietary 1.00 mg/g lauric acid is beneficial to improve the antioxidant capacity and intestinal health of swimming crab.

The involvement of VEGF and VEGFR in bacterial recognition and regulation of antimicrobial peptides in Eriocheir sinensis.

Vascular endothelial growth factor (VEGF) and VEGF reporter (VEGFR) are essential molecules in VEGF signalling pathway. Although the functions of VEGF and VEGFR have been well reported in vertebrates, their functions are still poorly understood in invertebrates. In this study, the open reading frame sequences of EsVEGF1 and EsVEGFR4 were cloned from Eriocheir sinensis, and their corresponding proteins shared typical structure characteristics with their counterparts in other species. EsVEGF1 were predominantly expressed in hepatopancreas and muscle while EsVEGFR4 mainly expressed in hemocytes and intestine. The expression levels of EsVEGF1 in hemocytes were rapidly induced by Staphylococcus aureus and Vibrio parahaemolyticus, and it also increased rapidly in hepatopancreas after being challenged with V. parahaemolyticus. The expression levels of EsVEGFR4 only increased in hepatopancreas of crabs injected with S. aureus. The extracellular immunoglobulin domain of EsVEGFR4 could bind with Gram-negative and Gram-positive bacteria as well as lipopolysaccharide and peptidoglycan. EsVEGF1 could act as the ligand for EsVEGFR4 and Toll-like receptor and regulate the expression of crustins and lysozyme with a tissue-specific manner, while have no regulatory function on that of anti-lipopolysaccharide factors. This study will provide new insights into the immune defense mechanisms mediated by VEGF and VEGFR in crustaceans.

Microencapsulation of ginger essential oil using mung bean protein isolate-chitosan complex coacervates: Application in the preservation of crab meatballs and the prediction of shelf life.

Crab meatballs with more unsaturated fat tend to spoil. Ginger essential oil (GEO) with oxidation resistance was encapsulated into microcapsules (GM) by complex cohesion of mung bean protein isolate (MBPI) and chitosan (CS) in a ratio of 8:1 at pH = 6.4, encapsulation efficiency (EE) and payload (PL) of GM (D50 = 26.16 ± 0.45 µm) with high thermal stability were 78.35 ± 1.02% and 55.43 ± 0.64%. GM (0.6%, w/w) did not interfere with the original flavor of crab meatballs, and lowered values of pH, thiobarbituric acid reactive substances (TBARS) and total bacteria counts (TBC) of the products than those spiked with GEO and the control. The prediction accuracy of the logistic first-order growth kinetic equation in line with TBC (2.84%) was better than that of zero-order and Arrhenius coupled equation based on pH (7.48%) and TBARS (5.94%), but all of them could predict the shelf life of crab meatballs containing GM stored at 4-25 °C.

Deubiquitinase cylindromatosis (CYLD) regulates antibacterial immunity and apoptosis in Chinese mitten crab (Eriocheir sinensis).

Ubiquitination and deubiquitination of target proteins is an important mechanism for cells to rapidly respond to changes in the external environment. The deubiquitinase, cylindromatosis (CYLD), is a tumor suppressor protein. CYLD from Drosophila melanogaster participates in the antimicrobial immune response. In vertebrates, CYLD also regulates bacterial-induced apoptosis. However, whether CYLD can regulate the bacterial-induced innate immune response in crustaceans is unknown. In the present study, we reported the identification and cloning of CYLD in Chinese mitten crab, Eriocheir sinensis. Quantitative real-time reverse transcription polymerase chain reaction analysis showed that EsCYLD was widely expressed in all the examined tissues and was upregulated in the hemolymph after Vibrio parahaemolyticus challenge. Knockdown of EsCYLD in hemocytes promoted the cytoplasm-to-nucleus translocation of transcription factor Relish under V. parahaemolyticus stimulation and increased the expression of corresponding antimicrobial peptides. In vivo, silencing of EsCYLD promoted the removal of bacteria from the crabs and enhanced their survival. In addition, interfering with EsCYLD expression inhibited apoptosis of crab hemocytes caused by V. parahaemolyticus stimulation. In summary, our findings revealed that EsCYLD negatively regulates the nuclear translocation of Relish to affect the expression of corresponding antimicrobial peptides and regulates the apoptosis of crab hemocytes, thus indirectly participating in the innate immunity of E. sinensis.

Shell Disease Syndrome Is Associated with Reduced and Shifted Epibacterial Diversity on the Carapace of the Crustacean Cancer pagurus.

Cancer pagurus is highly susceptible to shell disease syndrome. However, little is known about concomitant changes in the epibacterial community. We compared the bacterial communities of black spot affected and nonaffected areas of the carapace by amplicon sequencing of 16S rRNA genes and 16S rRNA. Within each spot, bacterial communities of affected areas were less diverse compared to communities from nonaffected areas. Communities of different affected spots were, however, more divergent from each other, compared to those of different nonaffected areas. This indicates a reduced and shifted microbial community composition caused by the black spot disease. Different communities found in black spots likely indicate different stages of the disease. In affected areas, Flavobacteriaceae rose to one of the most abundant and active families due to the increase of Aquimarina spp., suggesting a significant role in shell disease syndrome. We isolated 75 bacterial strains from diseased and healthy areas, which are primarily affiliated with Proteobacteria and Bacteroidetes, reflecting the dominant phyla detected by amplicon sequencing. The ability to degrade chitin was mainly found for Gammaproteobacteria and Aquimarina spp. within the Flavobacteriia, while the ability to use N-acetylglucosamine, the monomer of the polysaccharide chitin, was observed for most isolates, including many Alphaproteobacteria. One-third of the isolates, including most Aquimarina spp., showed antagonistic properties, indicating a high potential for interactions between the bacterial populations. The combination of bacterial community analysis and the physiological properties of the isolates provided insights into a functional complex epibacterial community on the carapace of C. pagurus. IMPORTANCE In recent years, shell disease syndrome has been detected for several ecologically and economically important crustacean species. Large proportions of populations are affected, e.g., >60% of the widely distributed species Cancer pagurus in different North Sea areas. Bacteria play a significant role in the development of different forms of shell disease, all characterized by microbial chitinolytic degradation of the outer shell. By comparing the bacterial communities of healthy and diseased areas of the shell of C. pagurus, we demonstrated that the disease causes a reduced bacterial diversity within affected areas, a phenomenon co-occurring also with many other diseases. Furthermore, the community composition dramatically changed with some taxa rising to high relative abundances and showing increased activity, indicating strong participation in shell disease. Characterization of bacterial isolates obtained from affected and nonaffected spots provided deeper insights into their physiological properties and thus the possible role within the microbiome.

Effect of infection with Aeromonas hydrophila on antioxidant capacity, inflammation response, and apoptosis proteins in Chinese mitten crab (Eriocheir sinensis).

Aeromonas hydrophila (A. hydrophila) as a serious bacterial disease endangering aquaculture and the Chinese mitten crabs (Eriocheir sinensis) industry. The present study was conducted to investigate the effects of A. hydrophila on the antioxidant, inflammation, immunity and apoptosis of the E. sinensis. The E. sinensis (female: 150 crabs and male: 150 crabs; 67.11 ± 0.76 g) were randomly divided into the control group (Foot injection with 200 µl PBS) and infection group (Foot injection with 200 µl A. hydrophila of 106 cfu/mL). The hepatopancreas and serum was collected to detect the related indicators after injection 24 h. The results showed that A. hydrophila significantly reduced the malondialdehyde (MDA) level and gamma-glutamyl-cysteine synthetase (γ-GCS) activity in the hepatopancreas of male and female crabs (P < 0.05). A.hydrophila also significantly decreased the total-superoxide dismutase (T-SOD) activity while the levels of total antioxidant capacity (T-AOC) and total glutathione (T-GSH) were significantly increased in the hepatopancreas and serum of male crabs (P < 0.05). At the transcriptional level, the expression of catalase (CAT) and glutathione peroxidases (GPx), Glutathione S-transferase (GST) in the hepatopancreas of male and female crabs was significantly reduced compared to the control group (P < 0.05). However, A. hydrophila could not significantly change the Kelch-like ECH-associated protein 1 (Keap1) gene expression level in both of male and female carbs. A. hydrophila injection for 24 h, the lysozyme (LZM) and phenoloxidase (PO) activity was significantly increased in the hepatopancreas and serum of the male and female crabs (P < 0.05). Simultaneous increase of immune-related enzyme activity (acid phosphatase and alkaline phosphatase) was found in the serum of male and female crabs (P < 0.05). However, the acid phosphatase (ACP) and alkaline phosphatase (ALP) activity was significantly decreased in the hepatopancreas of male and female crabs (P < 0.05). Meanwhile, the LZM mRNA level was significantly decreased in the hepatopancreas of E. sinensis (P < 0.05). Furthermore, A. hydrophila significantly inhibited the mRNA expression of immune regulated factors (Interleukin enhancer binding factor 2: ILF2, interleukin-16: IL-16, Toll-like receptor: TLR) in the male and female crabs. The levels of inflammatory cytokines (interleukin-1ß: IL-1ß, interleukin-6: IL-6, interleukin-8: IL-8, interleukin-10: IL-10) were significantly increased in the hepatopancreas of male and female crabs. Moreover, A.hydrophila increased the mRNA expression of apoptosis - related genes in male crabs (p38 mitogen-activated protein kinase: p38, adamalysin 17: ADAM17, Cysteine-aspartic acid protease 3: Caspase 3, and Bcl-2-associated X: BAX), but reduced the expression of p38, ADAM17, Caspase 3 and BAX genes in female crabs. In conclusion, A. hydrophila could induce oxidative stress and the response of inflammation and immunity, and also trigger the mRNA expression changes of apoptosis related-genes in E. sinensis. This study provides a theoretical basis for the study of E. sinensis diseases.

The role of caspase 3 in the mud crab (Scylla paramamosain) after Vibrio parahaemolyticus infection.

Apoptosis plays essential roles in the immune defense mechanism against pathogen infection. Caspase 3 is a family of cysteine proteases involved in apoptosis and the immune response. In this study, the full-length of mud crab (Scylla paramamosain) caspase 3 (designated as Sp-caspase 3) was cloned and characterized. The open reading frame of Sp-caspase 3 was comprised a 1035 bp, which encoded a putative protein of 344 amino acids. Sp-caspase 3 was ubiquitously expressed in various tissues with a high-level expression in hemocytes. Cellular localization analysis revealed that Sp-caspase 3 was located in the cytoplasm and nucleus. Over-expression of Sp-caspase 3 could induce cell apoptosis. In addition, V. Parahaemolyticus infection induced the relative expression of caspase-3 mRNA and increased caspase-3 activity. Knocking down Sp-caspase 3 in vivo significantly reduced cell apoptosis and increased mortality of mud crab after V. parahaemolyticus infection. These results indicated that Sp-caspase 3 played important roles in the immune response and apoptosis against bacterial infection.

The effects of ammonia-N stress on immune parameters, antioxidant capacity, digestive function, and intestinal microflora of Chinese mitten crab, Eriocheir sinensis, and the protective effect of dietary supplement of melatonin.

Ammonia nitrogen pollution seriously affects the economic benefits of Chinese mitten crab (Eriocheir sinensis) farming. In this study, we first evaluated the protective effects of melatonin (MT) on immune parameters, antioxidant capacity, and digestive enzymes of E. sinensis under acute ammonia nitrogen stress. The results showed that ammonia-N stress significantly decreased the antibacterial ability of crabs, nevertheless MT could significantly improve it under ammonia-N stress (P < 0.05). Ammonia-N group hemolymph antioxidant capacity indicators (T-AOC, T-SOD, GSH-Px) were significantly decreased than control (p < 0.05), while the MT ammonia-N group hemolymph T-SOD activity significantly increased than ammonia-N group (p < 0.05). For hepatopancreas, ammonia-N group GSH-PX activity significantly decreased than control group, but MT ammonia-N group was significant increased than ammonia-N (p < 0.05). Ammonia-N stress has significantly increased the content of MDA in hemolymph and hepatopancreas (p < 0.05), but MT ammonia-N treatment significantly decreased than ammonia-N group (p < 0.05). Compared with the control group, ammonia-N significantly reduced the activities of Trypsin in the intestine and hepatopancreas (p < 0.05), while MT ammonia-N group can significantly improve the intestinal trypsin activity than ammonia-N (p < 0.05). The intestinal microbiota of E. sinensis results showed that ammonia-N stress significantly decreased the relative abundance of Bacteroidetes (p < 0.05). Ammonia-N stress significantly decreased the Dysgonomonas and Rubellimicrobium, and the Citrobacter significantly increased. In summary, melatonin has a protective effect on E. sinensis under ammonia-N stress. Acute ammonia-N stress may lead to the decrease of probiotics and the increase of pathogenic bacteria, which may be closely related to the impairment of digestive function and immune function.

A New C-Type Lectin Homolog SpCTL6 Exerting Immunoprotective Effect and Regulatory Role in Mud Crab Scylla paramamosain.

C-type lectin (CTL), a well-known immune-related molecule, has received more and more attention due to its diverse functions, especially its important role in development and host defense of vertebrate and invertebrate. Since the research on crab CTLs is still lack, we screened a new CTL homolog, named SpCTL6 from mud crab Scylla paramamosain. The full-length cDNA sequence of SpCTL6 was 738 bp with a 486 bp of ORF, and the deduced amino acids were 161 aa. SpCTL6 was predicted to have a 17 aa signal peptide and its mature peptide was 144 aa (MW 16.7 kDa) with pI value of 5.22. It had typical CTL structural characteristics, such as a single C-type lectin-like domain, 4 conserved cysteines, similar tertiary structure to that of vertebrate CTLs and a mutated Ca2+ binding motif Gln-Pro-Thr (QPT), clustering into the same branch as the crustacean CTLs. SpCTL6 was highly expressed in the entire zoeal larval stages and widely distributed in adult crab tissues with the highest transcription level in testis. During the molting process of juvenile crabs, the expression level of SpCTL6 was remarkably increased after molting. SpCTL6 could be significantly upregulated in two larval stages (Z1 and megalopa) and adult crab testis under immune challenges. Recombinant SpCTL6 (rSpCTL6) was successfully obtained from eukaryotic expression system. rSpCTL6 exhibited binding activity with PAMPs (LPS, lipoteichoic acid, peptidoglycan, and glucan) and had a broad spectrum bacterial agglutination activity in a Ca2+-dependent manner. In addition, rSpCTL6 could enhance the encapsulation activity of hemocytes and has no cytotoxic effect on hemocytes. Although rSpCTL6 had no bactericidal activity on Vibrio alginolyticus, rSpCTL6 treatment could significantly reduce the bacterial endotoxin level in vitro and greatly improved the survival of S. paramamosain under V. alginolyticus infection in vivo. The immunoprotective effect of rSpCTL6 might be due to the regulatory role of rSpCTL6 in immune-related genes and immunological parameters. Our study provides new information for understanding the immune defense of mud crabs and would facilitate the development of effective strategies for mud crab aquaculture disease control.

The role of tumor suppressor protein p53 in the mud crab (Scylla paramamosain) after Vibrio parahaemolyticus infection.

The tumor suppressor protein p53 plays important roles in DNA repair, cell cycle and genetic stability. In the present study, a p53 gene in the mud crab (Scylla paramamosain) (designated as Sp-53) was identified and characterized. The open reading frame of Sp-53 was comprised a 1383 bp, which encoded a putative protein of 460 amino acids. Sp-53 is expressed in all examined tissues, with the highest expression in hepatopancreas and hemocytes. Vibrio parahaemolyticus infection induced oxidative stress, and led to DNA damage. The Sp-53 transcriptions in hepatopancreas were significantly up-regulated after V. parahaemolyticus infection. RNA interference (RNAi) experiment was used to understand the roles of Sp-53 in response to V. parahaemolyticus infection. Knocking down Sp-53 in vivo significantly reduced the expression of the Mn-SOD, Gpx3 and caspase 3 after V. parahaemolyticus infection. Moreover, the mortality of mud crabs and DNA damage in Sp-53-silenced mud crab challenged with V. parahaemolyticus were significantly higher than those in the control group. All these results suggested that Sp-53 played an important role in responses to V. parahaemolyticus infection through its participation in regulation of antioxidant defense, DNA repair and apoptosis.

An insulin-like peptide serves as a regulator of glucose metabolism in the immune response of Chinese mitten crab Eriocheir sinensis.

A robust immune response against invading pathogens greatly depends on the balance of metabolism, which could be vigorously modulated by insulin/IGF signaling (IIS) pathway in vertebrates. However, knowledge on the IIS pathway, especially the function of insulin-like peptides (ILPs) in invertebrates remained largely unknown. In the present study, a novel ILP was identified from Eriocheir sinensisis (designated EsILP). The coding sequence of EsILP was of 216 bp, which encoded a polypeptide of 71 amino acids containing an IlGF-like domain with four conserved cysteine residues. The mRNA transcripts of EsILP were found to be expressed dominantly in eyestalks and hepatopancreas, and EsILP protein was found to be distributed in the anterior median area of thoracic ganglion mass and the edges of hepatic tubules correspondingly. After Aeromonas hydrophila stimulation, EsILP transcripts were significantly increased at 3, 12 and 24 h post-stimulation in eyestalks and 6 and 48 h in hemocytes, respectively. In contrast, the expression level of EsILP decreased significantly in hepatopancreas from 6 h to 12 h after the stimulation. The glucose level in the hemolymph of crabs was significantly decreased from 6 to 12 h after the injection of recombinant EsILP. These results collectively demonstrated that the ancient ILP protein in E. sinensisis could negatively regulate glucose metabolism and participate in the immune response of the crabs against pathogen infection, which provided clues for the further investigation about the evolution and function of the IIS pathway in invertebrates.

The role of Astakine in Scylla paramamosain against Vibrio alginolyticus and white spot syndrome virus infection.

Astakine is a crucial factor in the proliferation and differentiation of hematopoietic stem cells and is directly involved in hematopoiesis in crustaceans. To assess the role of Astakine in the innate immune system of Scylla paramamosain, the immune responses in healthy and Astakine-inhibited S. paramamosain were investigated in the present study. The RNA transcripts of Astakine were widely distributed in all examined tissues, with significantly higher levels of expression in hemocytes of both healthy and challenged S. paramamosain with Vibrio alginolyticus and WSSV. When Astakine was knocked down by RNA interference technology, immune-related genes, including Janus kinase, prophenoloxidase, hemocyanin, ß-actin, myosin II essential light chain-like protein, signal transducer and activator of transcription, Relish, and C-type-lectin, were significantly down-regulated in hemocytes. The levels of phenoloxidaseactivity (PO), total hemocyte counts (THC) and hemocyte proliferation decreased significantly in hemocytes of Astakine-dsRNA treated S. paramamosain. After being challenged with V. alginolyticus and WSSV, the THC decreased significantly and the levels of hemocyte apoptosis increased significantly in Astakine-dsRNA treated S. paramamosain in comparison with those in infected groups without Astakine-dsRNA treatment. After being challenged with WSSV, the WSSV copies were significantly lower in Astakine-dsRNA treated groups than those in the WSSV infection group, which suggested that knockdown of Astakine was not conductive to WSSV replication and this might be associated with the decreasing THC. The results of survival analysis showed that the survival rate of V. alginolyticus or WSSV infected S. paramamosain decreased significantly following Astakine knockdown. These results suggested that RNA interference of Astakine might weaken the resistance of S. paramamosain to V. alginolyticus or WSSV infection. The weaken resistivity after knockdown Astakine might be related to the changes of important immune-related gene expression, THC, PO activity, proliferation and apoptosis of hemocytes.

Clone, identification and functional characterization of a novel toll (Shtoll1) from the freshwater crab Sinopotamon henanense in response to cadmium exposure and Aeromonas hydrophila infection.

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

Identification, functional characterization and the potential role of variable lymphocyte receptor EsVLRA from Eriocheir sinensis in response to secondary challenge after Vibrio parahaemolyticus vaccine.

Variable lymphocyte receptors (VLRs) play an important role via their antigen-special reorganization in jawless vertebrates (agnathans) adaptive immune response. In the present study, the open reading frame (ORF) of Eriocheir sinensis VLRA (designated as EsVLRA) was identified. EsVLRA comprised a 799-amino-acid polypeptide with one LRR_NT domain, thirteen LRR domains and one LRR_CT domain, which showed a high domain consistency of the VLR genes in lamprey (Petromyzon marinus). The transcript of EsVLRA was detected in all examined tissues with the highest level detected in hepatopancreas. Notably, the expression of EsVLRA in hepatopancreas, gonads, gill and intestine of male crabs was significantly higher than that in females. The recombinant EsVLRA exhibited strong bacteria-binding activity rather than antibacterial activity, suggesting its crucial role in immune recognition. Furthermore, 6 h earlier response and a significantly higher peak of EsVLRA mRNA expression was observed after challenge with live Vibrio parahaemolyticus (240.6-fold, P < 0.01, crabs receive secondary challenge after V. parahaemolyticus vaccine to the carbs only receive twice PBS injection, N = 6), compared with those only received first injection with formalin-inactivated V. parahaemolyticus (39.7-fold, P < 0.01, challenge 6 h to vaccination 12 h). The findings of this study together demonstrated that EsVLRA plays an important role in the immune system of E. sinensis, serving as a pattern recognition receptor and involving in the immune priming.

A class B scavenger receptor mediates antimicrobial peptide secretion and phagocytosis in Chinese mitten crab (Eriocheir sinensis).

Scavenger receptors (SRs) are pattern recognition receptors (PRRs) vital for innate immunity. As well as their importance in immune recognition, microbe phagocytosis, and the clearance of modified endogenous molecules, they also activate downstream immune responses as co-receptors. In the current study, we identified a class B scavenger receptor in Eriocheir sinensis (EsSR-B2). The full-length gene is 2,517 bp and encodes a 517 amino acid polypeptide. EsSR-B2 is expressed widely in all tested tissues and is induced by microbial stimulation. Recombinant EsSR-B2 binds to bacteria and pathogen-associated molecular patterns in vitro. Upon knockdown of EsSR-B2 and bacterial challenge with Staphylococcus aureus or Vibrio parahaemolyticus, phagocytosis rates in hemocytes are decreased. Moreover, the expression of several antimicrobial peptides (AMPs) in response to distinct microorganism stimulation is decreased following EsSR-B2 silencing. Thus, EsSR-B2 is a PRR that protects E. sinensis against invading pathogens by promoting phagocytosis and enhancing AMP expression.

Molecular characterization, expression and antimicrobial activities of a c-type lysozyme from the mud crab, Scylla paramamosain.

Lysozyme is an important immune protein involved in the first line of defense for crustaceans. In the present study, a c-type lysozyme gene (SpLyzC) was cloned and characterized from the mud crab, Scylla paramamosain. The full-length cDNA was 849 bp with an open reading frame of 669 bp, and encoded a polypeptide of 223 amino acids with a calculated molecular mass of 23.7 kDa and an isoelectric point of 8.90. SpLyzC shared conserved active sites with c-type lysozymes from other species, detected in all tested tissues and had higher expression levels in hepatopancreas and gill tissues. The expression of SpLyzC was up-regulated in hepatopancreas and gill after infection with Vibrio parahaemolyticus and Staphylococcus aureus. The density of bacteria in the hemolymph and the mortality of crabs increased following infection with V. parahaemolyticus after SpLyzC expression was silenced by injecting double-strand RNA of SpLyzC. The recombinant protein of the S. paramamosain c-type lysozyme (rSpLyzC) exhibited antibacterial activities against Micrococcus lysodeikticus, S. aureus, Vibrio harveyi and V. parahaemolyticus. These results indicate that SpLyzC could help eliminate bacteria in S. paramamosain and may play an important role in resistance to bacterial infection.

Crab-meat-isolated psychrophilic spore forming bacteria inactivation by electron beam ionizing radiation.

The present work was performed to evaluate the potential of electron beam ionizing radiation for the inactivation of three psychrophilic spore forming bacteria (Bacillus mycoides, Bacillus weihenstephanensis and Psychrobacillus psychrodurans) isolated from ready-to-eat brown crab (Cancer pagurus). Inactivation curves for the three spores were performed in both types of crab meat, brown and white. Also the effect of pH and water activity (aw) on the lethal efficacy of ionizing radiation, for the three different psychrophilic spore forming bacteria, was evaluated. The effects of pH, aw and their possible interactions were assessed in citrate-phosphate buffers of different pH, ranging between 7 and 4, and aw, ranging from <0.99 to 0.80. A reduction of aw increased the spores resistance between >0.99 and 0.90, while an aw reduction from 0.90 to 0.80 had a minor impact on their resistance. In contrast to aw, the effect of pH showed a greater variability depending on the spore species. While pH did not affect the resistance of B. weihenstephanensis at any aw, B. mycoides showed slightly higher resistance at pH 5.5 at aw of 0.90 and 0.80. pH showed a significant effect on the resistance of P. psychrodurans. For the two types of crab meat, slightly differences were observed in 6D values. B. weihenstephanensis was the most resistant, requiring 7.3-7.6 kGy to inactivate 6 Log10-cycles of this spore forming bacterium, while for B. mycoides and P. psychrodurans 6.1-6.3 and 5.4-5.3 kGy respectively were necessary to reach the same inactivation level in crab meat. An agreement between spore resistance in crab meats and lab media, with similar characteristics in pH and aw, was also observed. The results obtained in this research demonstrated the potential for ionizing radiation to achieve an appropriate inactivation level of spores naturally present in brown crab with the application of doses lower than 10 kGy.

Is Marine Dispersion of the Lethargic Crab Disease Possible? Assessing the Tolerance of Exophiala cancerae to a Broad Combination of Salinities, Temperatures, and Exposure Times.

Since 1997, an emergent fungal disease named lethargic crab disease (LCD) has decimated stocks of the edible mangrove land crab Ucides cordatus (Linnaeus, 1763) (Brachyura: Ocypodidae) along the Brazilian coast, threatening the mangrove ecosystem and causing socioeconomic impacts. Evidence from a variety of sources suggests that the black yeast Exophiala cancerae (Herpotrichiellaceae, Chaetothyriales) has been responsible for such epizootic events. Based on the spatiotemporal patterns of the LCD outbreaks, the well-established surface ocean currents, and the range of ecological traits of Exophiala spp., a marine dispersal hypothesis may be proposed. Using in vitro experiments, we tested the survival and growth of E. cancerae CBS 120420 in a broad combination of salinities, temperatures, and exposure times. While variation in salinity did not significantly affect the growth of colony-forming units (CFUs) (P > 0.05), long exposure times visibly influenced an increase in CFUs growth (P < 0.05). However, higher temperature (30 °C) caused a reduction of about 1.2-fold in CFUs growth (P < 0.05). This result suggests that sea surface temperatures either above or below the optimum growth range of E. cancerae could play a key role in the apparent north-south limits in the geographical distribution of LCD outbreaks. In light of our results, we conclude that a fundamental step toward the understanding of LCD epidemiological dynamics should comprise a systematic screening of E. cancerae in estuarine and coastal waters.

Characterization and functional analysis of a novel C-type lectin from the swimming crab Portunus trituberculatus.

C-type lectins (CTLs) are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a novel C-type lectin (designated as PtCTL1) was identified and characterized from Portunus trituberculatus. The full-length cDNA of PtCTL1 was of 702 bp, containing a 5' untranslated region (UTR) of 91 bp, a 3' UTR of 110 bp with a poly (A) tail, and an open reading frame (ORF) of 501 bp encoding a polypeptide of 166 amino acids with a putative signaling peptide of 21 amino acids. A C-type lectin carbohydrate-recognition domain (CRD) containing four conserved cysteines was identified in the amino acid sequence of PtCTL1. The cDNA fragment encoding the mature peptide of PtCTL1 was recombined into pET-21a(+) with a C-terminal hexa-histidine tag fused in-frame and expressed in Escherichia coli Origami (DE3). The recombinant PtCTL1 (rPtCTL1) can agglutinate all the tested bacteria, including three Gram-positive bacterial strains and three Gram-negative bacterial strains. In addition, erythrocyte agglutination and LPS-binding activity were observed in a Ca2+-dependent manner. The erythrocyte agglutination was inhibited by EDTA, indicating that PtCTL1 was Ca2+-dependent. The mRNA transcripts of PtCTL1 were detected mainly in the tissues of hepatopancreas and hemocytes and its levels were significantly up-regulated in hemocytes following Vibrio alginolyticus challenge. These results indicate that PtCTL1 may function as a pattern recognition receptor (PRR) for protecting P. trituberculatus from bacterial infection. Moreover, such findings also provide evidence for further understanding the innate immunology of invertebrate.

Lacinutrix chionocetis sp. nov., isolated from gut of a red snow crab.

A Gram-negative, aerobic, non-motile, rod-shaped bacterial strain, designated MAB-07T, was isolated from the gut of a red snow crab. The novel strain grew optimally at 20 °C, pH 7.0-8.0, and in the presence of 3% (w/v) NaCl. A phylogenetic analysis based on the 16S rRNA gene sequence indicated that the strain MAB-07T belongs to the type strains of species of the genus Lacinutrix. Strain MAB-07T exhibited 16S rRNA gene sequence similarity values of 95.5-97.8% with the type strains of species of the genus Lacinutrix. The predominant cellular fatty acids of strain MAB-07T were iso-C15:1 G (27.5%) and iso-C15:0 (21.7%). The major respiratory quinine was identified as MK-6. The polar lipids consisted of phosphatidylethanolamine, four unidentified aminolipids, and two unidentified lipids. The genomic DNA G + C content was determined to be 33.3%, and its DNA-DNA relatedness values with the type strains of L. venerupis, L. mariniflava, L. jangbogonensis, L. algicola, and Olleya aquimaris were 28-32%. Based on the data from this polyphasic taxonomic study, strain MAB-07T is considered to represent a novel species of the genus Lacinutrix, for which the name L. chionocetis sp. nov. is proposed. The type strain is MAB-07T (=KCTC 42767T = JCM 30988T).