Rhodobacter sphaeroides supplementation improves defense ability of Chinese mitten crab Eriocheir sinensis against Shewanella putrefaciens infection via intestinal flora and metabolism regulation.

Shewanella putrefaciens is a vital bacterial pathogen implicated in serious diseases in Chinese mitten crab Eriocheir sinensis. Yet the use of probiotics to improve the defense ability of E. sinensis against S. putrefaciens infection remains poorly understood. In the present study, the protective effect of dietary R. sphaeroides against S. putrefaciens infection in E. sinensis was evaluated through antioxidant capability, immune response, and survival under bacterial challenge assays, and its protective mechanism was further explored using a combination of intestinal flora and metabolome assays. Our results indicated that dietary R. sphaeroides could significantly improve immunity and antioxidant ability of Chinese mitten crabs, thereby strengthening their disease resistance with the relative percentage survival of 81.09% against S. putrefaciens. In addition, dietary R. sphaeroides could significantly alter the intestinal microbial composition and intestinal metabolism of crabs, causing not only the reduction of potential threatening pathogen load but also the increase of differential metabolites in tryptophan metabolism, pyrimidine metabolism, and glycerophospholipid metabolism. Furthermore, the regulation of differential metabolites such as N-Acetylserotonin positively correlated with beneficial Rhodobacter could be a potential protection strategy for Shewanella infection. To the best of our knowledge, this is the first study to illustrate the protective effect and mechanism of R. sphaeroides supplementation to protect E. sinensis against S. putrefaciens infection.

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.

Overcoming research challenges: In vitro cultivation of Ameson portunus (Phylum Microsporidia).

Ameson portunus is an intracellular pathogen that infects marine crabs Portunus trituberculatus and Scylla paramamosain, causing significant economic losses. However, research into this important parasite has been limited due to the absence of an in vitro culture system. To address this challenge, we developed an in vitro cultivation model of A. portunus using RK13 cell line in this study. The fluorescent labeling assay indicated a high infection rate (∼60 %) on the first day post-infection and quantitative PCR (qPCR) detection demonstrated successful infection as early as six hours post-inoculation. Fluorescence in situ hybridization (FISH) and qPCR were used for the detection of A. portunus infected cells. The FISH probe we designed allowed detection of A. portunus in infected cells and qPCR assay provided accurate quantification of A. portunus in the samples. Transmission electron microscopy (TEM) images revealed that A. portunus could complete its entire life cycle and produce mature spores in RK13 cells. Additionally, we have identified novel life cycle characteristics during the development of A. portunus in RK 13 cells using TEM. These findings contribute to our understanding of new life cycle pathways of A. portunus. The establishment of an in vitro culture model for A. portunus is critical as it provides a valuable tool for understanding the molecular and immunological events that occur during infection. Furthermore, it will facilitate the development of effective treatment strategies for this intracellular pathogen.

Identification and functional characterization of activating transcription factor 6 (ATF6) from the mud crab (Scylla paramamosain) in response to hydrogen peroxide and bacterial challenge.

Activating transcription factor 6 (ATF6) is critical for regulation of unfolded protein response (UPR), which is involved in the endoplasmic reticulum (ER) proteostasis maintenance and cellular redox regulation. In the present study, a ATF6 gene from the mud crab (designated as Sp-ATF6) has been cloned and identified. The open reading frame of Sp-ATF6 was 1917 bp, encoding a protein of 638 amino acids. The deduced amino acid sequences of Sp-ATF6 contained a typical basic leucine zipper (BZIP domain). Sp-ATF6 was widely expressed in all tested tissues, with the highest expression levels in the hemocytes and the lowest in the muscle. Subcellular localization showed that Sp-ATF6 was expressed in both nucleus and cytoplasm of S2 cells. The expression level of Sp-ATF6 was induced by hydrogen peroxide and V. parahaemolyticus challenge, indicating that the ATF6 pathway was activated in response to ER stress. In order to know more about the regulation mechanism of the Sp-ATF6, RNA interference experiment was investigated. Knocking down Sp-ATF6 in vivo can decrease the expression of antioxidant-related genes (CAT and SOD) and heat shock proteins (HSP90 and HSP70) after V. parahaemolyticus infection. All these results suggested that Sp-ATF6 played a crucial role in the defense against environmental stress and pathogen infection in crustaceans.

iTRAQ-based differential proteomic analysis of high- and low-virulence strains of Spiroplasma eriocheiris.

Spiroplasma eriocheiris is one of the major pathogenic bacteria in crustaceans, featuring high infectivity, rapid transmission, and an absence of effective control strategies, resulting in significant economic losses to the aquaculture industry. Research into virulence-related factors provides an important perspective to clarify how Spiroplasma eriocheiris is pathogenic to shrimps and crabs. Therefore, in this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology was utilized to undertake a differential proteomic analysis of high- and low-virulence Spiroplasma eriocheiris strains at different growth phases. A total of 868 differentially expressed proteins (DEPs) were obtained, of which 31 novel proteins were identified by proteogenomic analysis. There were 62, 61, 175, and 235 DEPs between the log phase (YD) and non-log phase (YFD) of the high-virulence strain, between the log phase (CD) and non-log phase (CFD) of the low-virulence strain, between YD and CD, and between CFD and YFD, respectively. All the DEPs were compared with virulence protein databases (MvirDB and VFDB), and 68 virulence proteins of Spiroplasma eriocheiris were identified, of which 12 were involved in a total of 21 metabolic pathways, including motility, chemotaxis, growth, metabolism and virulence of the bacteria. The results of this study form the basis for further research into the molecular mechanism of virulence and physiological differences between high- and low-virulence strains of Spiroplasma eriocheiris, and provide a scientific basis for a detailed understanding of its pathogenesis.

Identification and Characterization of a Potential Probiotic, Clostridium butyricum G13, Isolated from the Intestine of the Mud Crab (Scylla paramamosain).

The butyrate-producing bacterium Clostridium butyricum has been proven to be important in improving the growth and health benefits of aquatic animals. In this study, C. butyricum G13 was isolated for the first time from the gut of the mud crab (Scylla paramamosain). The results of this study showed that C. butyricum G13 could produce a high concentration of butyric acid and grow well in a wide range of pHs (4 to 9) and NaCl (1 to 2.5%) and bile salt (0.2 to 1.0%) concentrations. In vitro characterization revealed that C. butyricum G13 is a Gram-positive and gamma-hemolytic bacterium sensitive to most antibiotics and shows hydrophobicity and the capacity to degrade starch. In vitro fermentation using mud crab gut contents showed that C. butyricum G13 alone or in combination with galactooligosaccharides (GOS) and/or resistant starch (RS) significantly increased butyric acid production and beneficially affected the abundance and diversity of intestinal microbiota. In addition, C. butyricum G13 can improve the survival rate of mud crabs and effectively maintain the normal structure of gut morphology after infection with Vibrio parahaemolyticus. In conclusion, C. butyricum G13 can be considered a potential probiotic that improves the immune capacity and confers health benefits on mud crabs. IMPORTANCE With the development of society, more and more aquatic animals are demanded. Intensification in the aquaculture scale is facing problems, such as disease outbreaks, eutrophication of water bodies, and misuse of antibiotics. Among these challenges, disease outbreak is the most important factor directly affecting aquaculture production. It is crucial to explore new approaches effective for the prevention and control of diseases. Probiotics have been widely used in aquaculture and have shown beneficial effects on the host. In this study, the butyrate-producing bacterium Clostridium butyricum G13 was isolated for the first time from the intestine of the mud crab through in vitro fermentation. The bacterium has probiotic properties and changes the gut microbiota to be beneficial to hosts in vitro as well as protecting hosts from Vibrio parahaemolyticus infection in vivo. The outcomes of this study indicate that C. butyricum G13 can be used as a potential probiotic in mud crab aquaculture.

Integration of microbiome and Koch's postulates to reveal multiple bacterial pathogens of whitish muscle syndrome in mud crab, Scylla paramamosain.

BACKGROUND: For more than a century, the Koch's postulates have been the golden rule for determining the causative agents in diseases. However, in cases of multiple pathogens-one disease, in which different pathogens can cause the same disease, the selection of microorganisms that regress infection is hard when Koch's postulates are applied. Microbiome approaches can obtain relatively complete information about disease-related microorganisms and can guide the selection of target microorganisms for regression infection. In the present study, whitish muscle syndrome (WMS) of Scylla paramamosain, which has typical symptoms with whitish muscle and blackened hemolymph was used as an example to establish a new research strategy that integrates microbiome approaches and Koch's postulates to determinate causative agents of multiple pathogens-one disease. RESULTS: Microbiome results revealed that Aeromonas, Acinetobacter, Shewanella, Chryseomicrobium, Exiguobacterium, Vibrio and Flavobacterium, and Kurtzmaniella in hemolymph were bacterial and fungal indicators for WMS. A total of 23 bacteria and 14 fungi were isolated from hemolymph and muscle tissues, and among the bacteria, Shewanella chilikensis, S. xiamenensis, Vibrio alginolyticus, S. putrefaciens, V. fluvialis, and V. parahaemolyticus were present in hemolymph and/or muscle tissues in each WMS crab, and the last three species were also present in three Healthy crabs. The target bacteria and fungi were further screened to regression infections based on two criteria: whether they belonged to the indicator genera for WMS, whether they were isolated from both hemolymph and muscle tissues in most WMS crabs. Only S. chilikensis, S. putrefaciens, S. xiamenensis, V. alginolyticus, V. fluvialis, and V. parahaemolyticus met both two criteria. The six bacteria that met both two criteria and six fungi and another bacterium that unmatched any of two criteria were used to perform regression infection experiments based on Koch's postulates. S. chilikensis, S. putrefaciens, S. xiamenensis, V. alginolyticus, V. fluvialis, and V. parahaemolyticus met both two criteria, and the results indicate that they cause WMS in crabs independently. CONCLUSIONS: This study fully demonstrated that our research strategy that integrates the microbiome and Koch's postulates can maximize the ability to catch pathogens in one net for the situation of multiple pathogens-one disease. Video Abstract.

Glutaredoxin 2 in the mud crab Scylla paramamosain: Identification and functional characterization under hypoxia and pathogen challenge.

Glutaredoxin (Grx) is a glutathione-dependent oxidoreductase that plays a key role in antioxidant defense. In this study, a novel Grx2 gene (SpGrx2) was identified from the mud crab Scylla paramamosain, which consists of a 196 bp 5' untranslated region, a 357 bp open reading frame, and a 964 bp 3' untranslated region. The putative SpGrx2 protein has a typical single Grx domain with the active center sequence C-P-Y-C. The expression analysis revealed that the SpGrx2 mRNA was most abundant in the gill, followed by the stomach and hemocytes. Both mud crab dicistrovirus-1 and Vibrioparahaemolyticus infection as well as hypoxia could differentially induce the expression of SpGrx2. Furthermore, silencing SpGrx2 in vivo affected the expression of a series of antioxidant-related genes after hypoxia treatment. Additionally, SpGrx2 overexpression significantly increased the total antioxidant capacity of Drosophila Schneider 2 cells after hypoxia, resulting in a reduction of reactive oxygen species and malondialdehyde content. The subcellular localization results indicated that SpGrx2 was localized in both the cytoplasm and the nucleus of Drosophila Schneider 2 cells. These results indicate that SpGrx2 plays a crucial role as an antioxidant enzyme in the defense system of mud crabs against hypoxia and pathogen challenge.

New insight of variable lymphocyte receptor-likes in anti-bacteria activity from Eriocheir sinensis.

The Chinese mitten crab, Eriocheir sinensis, is a vital freshwater aquaculture species in China, however, is also facing various crab disease threats. In the present study, we identify three novel variable lymphocyte receptor-like (VLR-like) genes-VLR-like1, VLR-like3 and VLR-like4-from E. sinensis, which play vital roles in adaptive immune system of agnathan vertebrates. The bacterial challenge, bacterial binding and antibacterial-activity experiments were applied to study immune functions of VLR-likes, and the transcriptomic data from previous E. sinensis bacterial challenge experiments were analyzed to speculate the possible signaling pathway. VLR-like1 and VLR-like4 can respond to Staphylococcus aureus challenge and inhibit S. aureus specifically. VLR-like1 and VLR-like4 possess broad-spectrum bacteria-binding ability whereas VLR-like3 do not. VLR-likes in E. sinensis could associate with the Toll-like receptor (TLR) signaling pathway. The above results suggest that VLR-likes defend against bacteria invasion though exerting anti-bacteria activity, and probably connect with the TLR signaling pathway. Furthermore, studying the immune functions of these VLR-likes will provide a new insight into the disease control strategy of crustacean culture.

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.

Hematodinium sp. infection does not drive collateral disease contraction in a crustacean host.

Host, pathogen, and environment are determinants of the disease triangle, the latter being a key driver of disease outcomes and persistence within a community. The dinoflagellate genus Hematodinium is detrimental to crustaceans globally - considered to suppress the innate defences of hosts, making them more susceptible to co-infections. Evidence supporting immune suppression is largely anecdotal and sourced from diffuse accounts of compromised decapods. We used a population of shore crabs (Carcinus maenas), where Hematodinium sp. is endemic, to determine the extent of collateral infections across two distinct environments (open-water, semi-closed dock). Using a multi-resource approach (PCR, histology, haematology, population genetics, eDNA), we identified 162 Hematodinium-positive crabs and size/sex-matched these to 162 Hematodinium-free crabs out of 1191 analysed. Crabs were interrogated for known additional disease-causing agents; haplosporidians, microsporidians, mikrocytids, Vibrio spp., fungi, Sacculina, trematodes, and haemolymph bacterial loads. We found no significant differences in occurrence, severity, or composition of collateral infections between Hematodinium-positive and Hematodinium-free crabs at either site, but crucially, we recorded site-restricted blends of pathogens. We found no gross signs of host cell immune reactivity towards Hematodinium in the presence or absence of other pathogens. We contend Hematodinium sp. is not the proximal driver of co-infections in shore crabs, which suggests an evolutionary drive towards latency in this environmentally plastic host.

The Eriocheir sinensis calcium/calmodulin-dependent protein kinase II activates apoptosis to resist Spiroplasma eriocheiris infection.

Calcium/calmodulin-dependent protein kinase II is a downstream mediator of calcium signalling and participates in the regulation of various cellular physiological functions. In previous studies, the expression of Eriocheir sinensis CaMKII (EsCaMKII) was significantly decreased in the thoracic ganglion after Spiroplasma eriocheiris infection, as shown using TMT-based quantitative proteomic analysis; however, the specific functions of EsCaMKII are still unclear. In this study, the full-length cDNA of EsCaMKII was 3314 bp long, consisting of a 1605 bp open reading frame encoding a protein of 535 amino acids, including a 258 aa serine/threonine protein kinase catalytic domain (EsCaMKII-CD). EsCaMKII is highly transcribed in haemocytes, nerves (thoracic ganglion), gills, and muscles, but lowly transcribed in the hepatopancreas, heart, and intestines. The transcription levels of EsCaMKII were altered in E. sinensis haemocytes after S. eriocheiris infection. After the over-expression of EsCaMKII-CD in RAW264.7 cells, the apoptosis rate of RAW264.7 cells was significantly increased. After the over-expression of EsCaMKII-CD, the morphology of RAW264.7 cells became worse after being infected with S. eriocheiris. Meanwhile, the copy number of S. eriocheiris in RAW264.7 cells was significantly decreased. From 48 h to 96 h after EsCaMKII RNA interference, the transcription levels of EsCaMKII decreased significantly. The transcription of apoptosis genes and cell apoptosis were also inhibited in haemocytes after EsCaMKII RNAi. The knockdown of EsCaMKII by RNAi resulted in significant increases in the copy number of S. eriocheiris and in the mortality of crabs during S. eriocheiris infection. These results indicate that EsCaMKII could promote the apoptosis of E. sinensis and enhance its ability to resist S. eriocheiris infection.

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 Elevated Expressions of Anti-lipopolysaccharide Factors After Priming Stimulation Confer Lastingly Humoral Protection in Crab Eriocheir sinensis.

Evidence of immune memory in invertebrates (immune priming) has accumulated in various organisms, and both cellular and humoral immune reactions are speculated to be involved in immune priming. However, there is a lack of understanding of the molecular mechanisms involved. In the present study, the protective effect of primed haemolymph was further validated by the increased survival rate of naïve crabs receiving a transfusion of primed haemolymph. By proteomic analysis, there were 474 proteins identified from the primed haemolymph, and most of them were functionally annotated in transport and metabolism classes. A total of 70 proteins were found to be differentially expressed in haemolymph at 12 hours and 7 days after priming stimulation with Aeromonas hydrophila, among which anti-lipopolysaccharide factor 1 (EsALF-1) and 3 (EsALF-3) were identified as the most significant (p < 0.05). After being challenged with A. hydrophila, EsALF-1 and EsALF-3 were highly expressed at both mRNA (in haemocytes) and protein (in haemolymph) levels compared with blank crabs, and the mRNA expressions of components in the EsTLR1-EsMyd88-EsPelle-EsALF pathway also increased significantly (p < 0.05). The EsALF-3 and EsMyd88 were even significantly higher expressed in response to the second A. hydrophila challenge, but their expressions all decreased (p < 0.05) when EsTLR1 was knocked down by RNAi. After the naïve crabs received an injection with the recombinant protein of EsALF-1 (rEsALF-1) or EsALF-3 (rEsALF-3), their survival rate increased significantly (p < 0.05) upon A. hydrophila stimulation. In contrast, the survival rate of the primed crabs reduced significantly (p < 0.05) after they received an injection with the antibody of EsALF-1 or EsALF-3. The enhanced expressions of EsALF-1 and EsALF-3 after A. hydrophilap riming stimulation could sustain for four weeks. All the results suggested that the EsTLR1-mediated productions of EsALF-1 and EsALF-3 in haemolymph played an indispensable role in the month-long humoral immune protection induced by A. hydrophila, which provides solid evidence of immune priming in crabs and a valuable reference for further understanding immune memory in invertebrates.

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.

Exosomal miR-224 contributes to hemolymph microbiota homeostasis during bacterial infection in crustacean.

It is well known that exosomes could serve as anti-microbial immune factors in animals. However, despite growing evidences have shown that the homeostasis of the hemolymph microbiota was vital for immune regulation in crustaceans, the relationship between exosomes and hemolymph microbiota homeostasis during pathogenic bacteria infection has not been addressed. Here, we reported that exosomes released from Vibrio parahaemolyticus-infected mud crabs (Scylla paramamosain) could help to maintain the homeostasis of hemolymph microbiota and have a protective effect on the mortality of the host during the infection process. We further confirmed that miR-224 was densely packaged in these exosomes, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex, then the released TRAF6 further interacted with Ecsit to regulate the production of mitochondrial ROS (mROS) and the expression of Anti-lipopolysaccharide factors (ALFs) in recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the pathogenic bacteria infection in mud crab. To the best of our knowledge, this is the first document that reports the role of exosome in the hemolymph microbiota homeostasis modulation during pathogen infection, which reveals the crosstalk between exosomal miRNAs and innate immune response in crustaceans.

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.

Novel recombinant keratin degrading subtilisin like serine alkaline protease from Bacillus cereus isolated from marine hydrothermal vent crabs.

Microbial secondary metabolites from extreme environments like hydrothermal vents are a promising source for industrial applications. In our study the protease gene from Bacillus cereus obtained from shallow marine hydrothermal vents in the East China Sea was cloned, expressed and purified. The protein sequence of 38 kDa protease SLSP-k was retrieved from mass spectrometry and identified as a subtilisin serine proteinase. The novel SLSP-k is a monomeric protein with 38 amino acid signal peptides being active over wide pH (7-11) and temperature (40-80 °C) ranges, with maximal hydrolytic activities at pH 10 and at 50 °C temperature. The hydrolytic activity is stimulated by Ca2+, Co2+, Mn2+, and DTT. It is inhibited by Fe2+, Cd2+, Cu2+, EDTA, and PMSF. The SLSP-k is stable in anionic, non-anionic detergents, and solvents. The ability to degrade keratin in chicken feather and hair indicates that this enzyme is suitable for the degradation of poultry waste without the loss of nutritionally essential amino acids which otherwise are lost in hydrothermal processing. Therefore, the proteinase is efficient in environmental friendly bioconversion of animal waste into fertilizers or value added products such as secondary animal feedstuffs.

Comparative analysis of the intestinal bacterial communities in mud crab Scylla serrata in South India.

Little is known about the functions of the crustacean gut microbiome, but environmental parameters and habitat are known to affect the composition of the intestinal microbiome, which may in turn affect the physiological status of the host. The mud crab Scylla serrata is an economically important species, and is wild-caught, and farmed across the Indo-Pacific region. In this study, we compared the composition of the gut microbiome (in terms of gut microbial species richness and abundance) of S. serrata collected from wild sites, and farms, from the east and west coast of India, and also tested the effects of the environment on the composition. The water temperature had a statistically significant effect on gut microbiome composition, with microbial biodiversity decreasing with increasing water temperature. This could have negative effects on both wild and farmed mud crabs under future climate change conditions, although further research into the effects of temperature on gut microbiomes is required. By comparison, salinity, crab mass and carapace width, geographical location as well as whether they were farmed or wild-caught crabs did not have a significant impact on gut microbiome composition. The results indicate that farming does not significantly alter the composition of the gut microbiome when compared to wild-caught crabs.