Genome sequencing analysis and validation of infestation-related functional genes of Vibrio parahaemolyticus LG2206 isolated from the hepatopancreas of diseased mud crab (Scylla paramamosain) in South China.

Vibrio parahaemolyticus (V. parahaemolyticus) is a major bacterial pathogen found in brackish environments, leading to disease outbreaks and great economic losses in the mud crab industry. This study investigated the molecular mechanism of V. parahaemolyticus infecting mud crabs through genome sequencing analysis, survival experiments, and the expression patterns of related functional genes. A strain of V. parahaemolyticus with high pathogenicity and lethality was isolated from diseased mud crab in South China. The genome sequencing results showed that the genome size of V. parahaemolyticus was a circular chromosome of 3,357,271 bp, with a GC content of 45 %, containing 2985 protein-coding genes, denoted as V. parahaemolyticus LG2206. Genome analysis data revealed that a total of 113 adherence coding genes were obtained, including 120 virulence factor coding genes, 37 type III secretion system (T3SS) coding genes, and 277 sequences of T3SS effectors. Survival experiments showed that the mortality was 20 % within 96 h in the 1 × 104 CFU/mL infection group, 90 % in the 3.2 × 105 CFU/mL treatment group, and 100 % in the 1 × 106 CFU/mL treatment group. The LD50 of V. parahaemolyticus LG2206 was determined as 4.6 × 104 CFU/mL. Six genes of znuA and fliD (flagellin encoding genes), yscE and yscR (T3SS encoding genes), and nfuA and htpX (virulence factor encoding genes) were selected and validated by quantitative real-time PCR analysis after infection with 4.6 × 104 CFU/mL of V. parahaemolyticus LG2206 for 96 h. The expression of the six genes exhibited a significant up-regulation trend at all tested time points. The results indicated that the infestation-related genes screened in the experiment play important roles in the infestation process. This study provides timely and effective information to further analyze the molecular mechanism of V. parahaemolyticus infection and develop comprehensive measures for disease prevention and control.

Identification of ETH receptor and its possible roles in the mud crab Scylla paramamosain.

Ecdysis-triggering hormone (ETH) is a neuropeptide hormone characterized by a conserved KxxKxxPRx amide structure widely identified in arthropods. While its involvement in the regulation of molting and reproduction in insects is well-established, its role in crustaceans has been overlooked. This study aimed to de-orphanise a receptor for ETH in the mud crab Scylla paramamosain and explore its potential impact on ovarian development. A 513-amino-acid G protein-coupled receptor for ETH (SpETHR) was identified in S. paramamosain, exhibiting a dose-dependent activation by SpETH with an EC50 value of 75.18 nM. Tissue distribution analysis revealed SpETH was in the cerebral ganglion and thoracic ganglion, while SpETHR was specifically expressed in the ovary, hepatopancreas, and Y-organ of female crabs. In vitro experiments demonstrated that synthetic SpETH (at a concentration of 10-8 M) significantly increased the expression of SpVgR in the ovary and induced ecdysone biosynthesis in the Y-organ. In vivo experiments showed a significant upregulation of SpEcR in the ovary and Disembodied and Shadow in the Y-organ after 12 h of SpETH injection. Furthermore, a 16-day administration of SpETH significantly increased 20E titers in hemolymph, gonadosomatic index (GSI) and oocyte size of S. paramamosain. In conclusion, our findings suggest that SpETH may play stimulatory roles in ovarian development and ecdysone biosynthesis by the Y-organ.

Comparative Analyses of Scylla olivacea Gut Microbiota Composition and Function Suggest the Capacity for Polyunsaturated Fatty Acid Biosynthesis.

Although numerous studies in aquatic organisms have linked lipid metabolism with intestinal bacterial structure, the possibility of the gut microbiota participating in the biosynthesis of beneficial long-chain polyunsaturated fatty acid (LC-PUFA) remains vague. We profiled the gut microbiota of the mud crab Scylla olivacea fed with either a LC-PUFA rich (FO) or a LC-PUFA-poor but C18-PUFA substrate-rich (LOCO) diet. Additionally, a diet with a similar profile as LOCO but with the inclusion of an antibiotic, oxolinic acid (LOCOAB), was also used to further demarcate the possibility of LC-PUFA biosynthesis in gut microbiota. Compared to diet FO treatment, crabs fed diet LOCO contained a higher proportion of Proteobacteria, notably two known taxonomy groups with PUFA biosynthesis capacity, Vibrio and Shewanella. Annotation of metagenomic datasets also revealed enrichment in the KEGG pathway of unsaturated fatty acid biosynthesis and polyketide synthase-like system sequences with this diet. Intriguingly, diet LOCOAB impeded the presence of Vibrio and Shewanella and with it, the function of unsaturated fatty acid biosynthesis. However, there was an increase in the function of short-chain fatty acid production, accompanied by a shift towards the abundance of phyla Bacteroidota and Spirochaetota. Collectively, these results exemplified bacterial communities and their corresponding PUFA biosynthesis pathways in the microbiota of an aquatic crustacean species.

The role of Nrf2 signaling pathway in the mud crab (Scylla paramamosain) in response to Vibrio parahaemolyticus infection.

The transcription factor Nrf2 plays vital roles in detoxification and antioxidant enzymes against oxidative stress. However, the function of Nrf2 in crustaceans is not well studied. In this study, a novel Nrf2 gene from the mud crab (Sp-Nrf2) was identified. It was encoded 245 amino acids. Sp-Nrf2 expression was ubiquitously expressed in all tested tissues, with the highest expression level in the gill. Sp-Nrf2 protein was mainly located in the nucleus. The expression levels of Sp-Nrf2, and antioxidant-related genes (HO-1 and NQO-1) were induced after Vibrio parahaemolyticus infection, indicating that Nrf2 signaling pathway was involved in the responses to bacterial infection. Over-expression of Sp-Nrf2 could improve cell viability after H2O2 exposure, indicating that Sp-Nrf2 might relieve oxidative stress. Silencing of Sp-Nrf2 in vivo decreased HO-1 and NQO-1 expression. Moreover, knocking down Sp-Nrf2 in vivo can increase malondialdehyde content and the mortality of mud crabs after V. parahaemolyticus infection. Our results indicated that Nrf2 signaling pathway played a significant role in immune response against bacterial infection.

Pathogenicity of white spot syndrome virus (WSSV) after multiple passages in mud crab, Scylla olivacea.

White spot syndrome virus (WSSV) is a highly virulent shrimp pathogen with a broad host range. Among the hosts, though mud crab, Scylla olivacea is reported to be more susceptible to WSSV than S. serrata and S. paramamosain, a detailed study on the pathogenicity and genome stability of the virus after multiple passages has yet to be reported. Firstly, to test the pathogenicity of the virus, WSSV was intramuscularly injected into healthy shrimp, Penaeus vannamei. Experimentally infected P. vannamei showed the first mortality at 36 h post-injection (hpi), followed by 100 % cumulative mortality in 7 days post-injection (dpi). However, S. olivacea injected with the WSSV inoculum derived from infected shrimp showed the first mortality at 48 hpi and a cumulative mortality of 70 % at the end of the ten days experiment. Subsequently, WSSV was sequentially passaged five times in Scylla olivacea to find out any change in the virulence of the virus in each passage. S. olivacea groups injected with 1st, second, third and fourth passages derived from the crab recorded the first mortality between 48 and 56 hpi and the cumulative mortality of 60 to 70 % at the end of the ten days experiment. Injection of WSSV inoculum in P. vannamei derived from multiple passages in S. olivaceae revealed the retention of the pathogenicity of the virus. Shrimp groups injected with WSSV derived from different passages showed first mortality between 24 and 36 hpi and cumulative mortality of 100 % between 6 and 7 dpi. The average viral load in the shrimp groups injected with WSSV inoculum derived from shrimp was 3.6 × 108, whereas in shrimp injected with the inoculum derived from 1st, third and fifth passages from crab showed 4.0 × 108, 4.7 × 108 and 4.3 × 108 copies per 100 ng DNA. Histological examination of the gill and stomach tissue of shrimp injected with inoculum prepared from shrimp as well as the inoculum derived from 1st, third and fifth passages in S. olivacea revealed characteristic pathological manifestations of the WSSV infection in gill and stomach tissues such as hypertrophied nuclei, Cowdry A-type inclusions as well as massive basophilic intranuclear inclusions. Further, to study the genome stability, the primers targeting highly variable regions of the WSSV genome (ORF94, ORF125, ORF75, variable region (VR) 14/15 and VR 23/24) were used to amplify WSSV derived from different passages and the amplified PCR products were sequenced. The sequence analysis revealed the WSSV genome stability after multiple passages in mud crab, S. olivacea.

Isolation of an Insulin-Like Receptor Involved in the Testicular Development of the Mud Crab Scylla paramamosain.

Insulin-like androgenic gland hormone (IAG) is a key regulator of male sexual differentiation in crustaceans that plays important roles in secondary sexual characteristics and testicular development. As a hormone, IAG interacts with its membrane receptor to initiate downstream signal pathways to exert its biological functions. In this study, we isolated a full-length cDNA of an insulin-like receptor (Sp-IR) from the mud crab Scylla paramamosain. Sequence analysis revealed that this receptor consists of a Fu domain, two L domains, three FN-III domains, a transmembrane domain, and a tyrosine kinase domain, classifying it as a member of the tyrosine kinase insulin-like receptors family. Our results also suggested that Sp-IR was highly expressed in the testis and AG in males. Its expression in the testis peaked in stage I but significantly decreased in stages II and III (p < 0.01). Next, both short- and long-term RNA interference (RNAi) experiments were performed on males in stage I to explore Sp-IR function in mud crabs. The results showed that Sp-vasa and Sp-Dsx expression levels in the testis were significantly down-regulated after the specific knockdown of Sp-IR by RNAi. Additionally, the long-term knockdown of Sp-IR led to a considerable decrease in the volume of seminiferous tubules, accompanied by large vacuoles and a reduced production of secondary spermatocytes and spermatids. In conclusion, our results indicated that Sp-IR is involved in testicular development and plays a crucial role in transitioning from primary to secondary spermatocytes. This study provided a molecular basis for the subsequent analysis of the mechanism on male sexual differentiation in Brachyuran crabs.

Biochemical, metabolic, and immune responses of mud crab (Scylla paramamosain) after mud crab reovirus infection.

Mud crab reovirus (MCRV) is a serious pathogen that leads to large economic losses in the mud crab farming. However, the molecular mechanism of the immune response after MCRV infection is unclear. In the present study, physiological, transcriptomic, and metabolomic responses after MCRV infection were investigated. The results showed that MCRV infection could increase lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase activities. MCRV infection decreased antioxidant enzyme activity levels, induced oxidative stress, and caused severe histological damage. Transcriptome analysis identified 416 differentially expressed genes, including 354 up-regulated and 62 down-regulated genes. The detoxification, immune response, and metabolic processes-related genes were found. The results showed that two key pathways including phagocytosis and apoptosis played important roles in response to MCRV infection. The combination of transcriptomic and metabolomic analyses showed that related metabolic pathways, such as glycolysis, citrate cycle, lipid, and amino acid metabolism were also significantly disrupted. Moreover, the biosynthesis of unsaturated fatty acids was activated in response to MCRV infection. This study provided a novel insight into the understanding of cellular mechanisms in crustaceans against viral invasion.

Monodon baculovirus (MBV) infects wild mud crab, Scylla serrata.

During a survey of farmed and wild crustaceans from India for viruses, spherical baculovirosis otherwise known as Penaeus monodon-type baculovirus (MBV) was detected in field-collected juvenile/sub-adult mud crab, Scylla serrata using a nested polymerase chain reaction (PCR)-based amplification of the hepatopancreatic DNA. Eight out of 115 mud crab (7.0%) examined during the study were found to be positive in the nested PCR resulting in a 361 nt amplicon. Mud crab, S. olivacea and other crustaceans such as marine crab, Portunus sanguinolentus and farmed penaeid shrimp, Penaeus vannamei and P. monodon were tested negative for the virus. Further, degenerate primers reported to amplify polyhedrin protein gene of MBV also showed PCR amplification in one of the MBV-positive crab samples resulting in a 250 nt amplicon. Sequencing of the two target amplicons (MBV- 361 nt and MBV polyhedrin - 216 nt) revealed more than 97.5 % and 92.8% sequence identity, respectively with the Penaeus monodon nudivirus and Penaeus monodon nucleopolyhedrovirus (MBV) reported from shrimp. Further, histological analysis of mud crab revealed nuclear hypertrophy, chromatin margination and intranuclear eosinophilic/basophilic inclusions in tubule epithelium of hepatopancreas. The hepatopancreatic tissue also showed unusually large, eosinophilic/basophilic inclusion-like structures. These inclusions resembled the viral inclusions reported from S. serrata from Australia. This is the first record of monodon-type baculovirus from a crab host and the second from a non-penaeid crustacean. Interestingly, some of the crab samples also showed deeply basophilic intranuclear inclusion-like bodies resembling hepatopancreatic parvovirus group of viruses (HPV). However, none of the crab samples subjected to PCR amplification using HPV-specific primers showed any amplification. The histological observations made in the present study indicate the possibility of the presence of two hepatopancreas-infecting viruses in S. serrata from India.

A Novel Antimicrobial Peptide Spampcin56-86 from Scylla paramamosain Exerting Rapid Bactericidal and Anti-Biofilm Activity In Vitro and Anti-Infection In Vivo.

The abuse of antibiotics leads to the increase of bacterial resistance, which seriously threatens human health. Therefore, there is an urgent need to find effective alternatives to antibiotics, and antimicrobial peptides (AMPs) are the most promising antibacterial agents and have received extensive attention. In this study, a novel potential AMP was identified from the marine invertebrate Scylla paramamosain and named Spampcin. After bioinformatics analysis and AMP database prediction, four truncated peptides (Spa31, Spa22, Spa20 and Spa14) derived from Spampcin were screened, all of which showed potent antimicrobial activity with different antibacterial spectrum. Among them, Spampcin56-86 (Spa31 for short) exhibited strong bactericidal activity against a variety of clinical pathogens and could rapidly kill the tested bacteria within minutes. Further analysis of the antibacterial mechanism revealed that Spa31 disrupted the integrity of the bacterial membrane (as confirmed by scanning electron microscopy observation, NPN, and PI staining assays), leading to bacterial rupture, leakage of cellular contents (such as elevated extracellular ATP), increased ROS production, and ultimately cell death. Furthermore, Spa31 was found to interact with LPS and effectively inhibit bacterial biofilms. The antibacterial activity of Spa31 had good thermal stability, certain ion tolerance, and no obvious cytotoxicity. It is worth noting that Spa31 could significantly improve the survival rate of zebrafish Danio rerio infected with Pseudomonas aeruginosa, indicating that Spa31 played an important role in anti-infection in vivo. This study will enrich the database of marine animal AMPs and provide theoretical reference and scientific basis for the application of marine AMPs in medical fields.

Comparative transcriptome profiling of heat stress response of the mangrove crab Scylla serrata across sites of varying climate profiles.

BACKGROUND: The fishery and aquaculture of the widely distributed mangrove crab Scylla serrata is a steadily growing, high-value, global industry. Climate change poses a risk to this industry as temperature elevations are expected to threaten the mangrove crab habitat and the supply of mangrove crab juveniles from the wild. It is therefore important to understand the genomic and molecular basis of how mangrove crab populations from sites with different climate profiles respond to heat stress. Towards this, we performed RNA-seq on the gill tissue of S. serrata individuals sampled from 3 sites (Cagayan, Bicol, and Bataan) in the Philippines, under normal and heat-stressed conditions. To compare the transcriptome expression profiles, we designed a 2-factor generalized linear model containing interaction terms, which allowed us to simultaneously analyze within-site response to heat-stress and across-site differences in the response. RESULTS: We present the first ever transcriptome assembly of S. serrata obtained from a data set containing 66 Gbases of cleaned RNA-seq reads. With lowly-expressed and short contigs excluded, the assembly contains roughly 17,000 genes with an N50 length of 2,366 bp. Our assembly contains many almost full-length transcripts - 5229 shrimp and 3049 fruit fly proteins have alignments that cover >80% of their sequence lengths to a contig. Differential expression analysis found population-specific differences in heat-stress response. Within-site analysis of heat-stress response showed 177, 755, and 221 differentially expressed (DE) genes in the Cagayan, Bataan, and Bicol group, respectively. Across-site analysis showed that between Cagayan and Bataan, there were 389 genes associated with 48 signaling and stress-response pathways, for which there was an effect of site in the response to heat; and between Cagayan and Bicol, there were 101 such genes affecting 8 pathways. CONCLUSION: In light of previous work on climate profiling and on population genetics of marine species in the Philippines, our findings suggest that the variation in thermal response among populations might be derived from acclimatory plasticity due to pre-exposure to extreme temperature variations or from population structure shaped by connectivity which leads to adaptive genetic differences among populations.

Molecular and allergenic characterization of recombinant tropomyosin from mud crab Scylla olivacea.

BACKGROUND: Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this study aimed to produce the recombinant tropomyosin protein from S. olivacea and subsequently investigate its IgE reactivity. METHODS AND RESULTS: The tropomyosin gene was cloned and expressed in the Escherichia coli system, followed by SDS-PAGE and immunoblotting test to identify the allergenic potential of the recombinant protein. The 855-base pair of tropomyosin gene produced was found to be 99.18% homologous to Scylla serrata. Its 284 amino acids matched the tropomyosin of crustaceans, arachnids, insects, and Klebsiella pneumoniae, ranging from 79.03 to 95.77%. The tropomyosin contained 89.44% alpha-helix folding with a tertiary structure of two-chain alpha-helical coiled-coil structures comprising a homodimer heptad chain. IPTG-induced histidine tagged-recombinant tropomyosin was purified at the size of 42 kDa and confirmed as tropomyosin using anti-tropomyosin monoclonal antibodies. The IgE binding of recombinant tropomyosin protein was reactive in 90.9% (20/22) of the sera from crab-allergic patients. CONCLUSIONS: This study has successfully produced an allergenic recombinant tropomyosin from S. olivacea. This recombinant tropomyosin may be used as a specific allergen for the diagnosis of allergy.

Panopeispora mellora n. gen. n. sp. (microsporidia) infecting Say's crab (Dyspanopeus sayi) from the Atlantic shoreline of Canada.

Say's mud crab, Dyspanopeus sayi (Brachyura: Panopeidae) is a native shallow subtidal and inter-tidal inhabitant of the Atlantic coastline of North America and an invasive species in the Mediterranean and Black Seas. Little is known about the microparasites of this host and the broader Panopeidae. We describe a novel microsporidian parasite infecting the musculature of D. sayi from Malagash, Nova Scotia (Canada), at a prevalence of 7%. Histopathology and molecular diagnostics were used to describe pathology and parasite phylogenetics, respectively. Based on SSU rDNA gene sequencing we propose that the microsporidian requires establishment of a new genus (Panopeispora n. gen.) and species (Panopeispora mellora n. sp.), due to significant differences to closest known taxa (e.g. Facilispora margolisi [81% similarity] and Thelohania butleri [80% similarity]), residing in Clade V of the Microsporidia. Archived, wax-embedded histological material was re-processed for transmission electron microscopy to obtain preliminary details of its intracellular development cycle and ultrastructure within the host musculature. The discovery of this pathogen is discussed with relevance to microsporidian taxonomy and the potential for achieving ultrastructural data from archived material.

Dietary soybean oil aggravates the adverse effects of low salinity on intestinal health in juvenile mud crab Scylla paramamosain.

Salinity is one of the important factors affecting the physiological state of crustaceans in marine environments. Lipid plays major roles in energy supply and is main sources of essential fatty acids for membrane integrity, which is critical in adaptations to changes in salinity. Here we evaluated the effects of salinity (medium, 23 ppt and low, 4 ppt) and dietary lipid source (fish oil, FO and soybean oil, SO) on intestinal health of the marine crustacean mud crab Scylla paramamosain. The results indicated that low salinity and dietary SO (LSO group) significantly affected intestinal histomorphology, with a significant decrease of intestinal fold height and width as well as down-regulation of intestinal mRNA levels of tight junction genes compared to crab reared at medium salinity and fed FO diets (MFO group). Crabs reared at low salinity and fed SO showed an increased inflammatory response in intestine, which stimulated a physiological detoxification response together with apoptosis compared to crab in the MFO group. Low salinity and SO diets also could be responsible for multiply the pathogenic bacteria of Photobacterium and inhibit the beneficial bacteria of Firmicutes and Rhodobacteraceae in intestine, and act on a crucial impact on the development of intestinal microbial barrier disorders. The results of microbial function predictive analysis also support these inferences. The findings of the present study demonstrated that soybean oil as the main dietary lipid source could exacerbate the adverse effects of low salinity on intestinal health of mud crab, and provided evidence suggesting that dietary lipid source and fatty acid composition may play vital roles in intestinal health and the process of adaptation to environmental salinity in marine crustaceans.

Gonadal transcriptomic analysis of the mud crab Scylla olivacea infected with rhizocephalan parasite Sacculina beauforti.

Infection by the rhizocephalan parasite Sacculina beauforti can have detrimental effects on mud crab Scylla olivacea. However, the molecular changes that occur during rhizocephalan infection are poorly understood. Due to the disruption in the reproductive system after infection, the gonadal transcriptomic profiles of non-infected and infected Scylla olivacea were compared. A total of 686 and 843 unigenes were differentially expressed between non-infected and infected males, and females, respectively. The number of DEGs increased after infection. By comparing shared DEGs of non-infected and infected individuals, potential immune- and reproduction-related of host, and immune- and metabolism-related genes of parasite are highlighted. The only shared KEGG pathway between non-infected and infected individuals was the ribosome pathway. In summary, findings in this study provide new insights into the host-parasite relationship of rhizocephalan parasites and their crustacean hosts.

Reference gene and tropomyosin expression in mud crab Scylla olivacea, Scylla paramamosain and Scylla tranquebarica.

Tropomyosin, a muscle tissue protein is a major allergen in most of shellfish including mud crab. Quantitative real time-PCR (qRT-PCR) using a stable reference gene is the most sensitive approach to produce accurate relative gene expression that has yet to be demonstrated for allergenic tropomyosin in mud crab species. This study was conducted to identify the suitable reference gene and tropomyosin expression in different body parts of local mud crabs, Scylla olivacea, Scylla paramamosain and Scylla tranquebarica. Myosin, 18S rRNA, GADPH and EF1α were selected as candidate reference genes and their expression was measured in the abdomen, walking leg and cheliped tissues of local Scylla spp. The expression stability was analyzed using the comparative delta-Ct method, BestKeeper, NormFinder and geNorm then comprehensively ranked by RefFinder algorithm. Findings showed that EF1α was the most suitable reference gene across three mud crab species. Meanwhile, the abdomen, walking leg and cheliped selected their own suitable reference gene either Myosin, 18S rRNA, EF1α or GADPH. Overall, tropomyosin was the highest in S. tranquebarica, whereas the least was in S. paramamosain. Interestingly, tropomyosin was the highest in the abdomen of all mud crab species. This is the first analysis on reference genes selection for qRT-PCR data normalization of tropomyosin expression in mud crab. These results will provide more accurate findings for further gene expression and allergen analysis in Scylla spp.

Role of crustacean female sex hormone (CFSH) in sex differentiation in early juvenile mud crabs, Scylla paramamosain.

Recent studies have shown that crustacean female sex hormone (CFSH) is involved in the development of reproductive phenotype. In the present study, observation of sexually dimorphic traits revealed that gender could be distinguished from the third stage juveniles onwards in the mud crab, Scylla paramamosain. Sp-cfsh expression levels were analyzed in early juveniles. The results showed that, Sp-cfsh expression levels differed among individuals at post-molt of the first stage and second stage, and significantly different between the two sexes at post-molt of the third stage, which suggested that Sp-cfsh might participate in the sex differentiation in early juveniles. The expression of Sp-cfsh was examined during the molting cycle at the third stage juveniles, and the results showed that it was highest at the pre-molt stage. Based on the results, the expression of Sp-cfsh at pre-molt stage was further analyzed between the sexes from the third stage to the fifth stage, and it was found that the expression of Sp-cfsh was similar between two sexes at the third stage and the fourth stage; whereas at the fifth stage, when the gonopores occurred, the expression of Sp-cfsh significantly increased in females but decreased in males; suggesting that the expression of Sp-cfsh could influence the formation of gonopores. Finally, the role of Sp-cfsh in the reproductive phenotypes was confirmed through RNA interference knockdown. The combined results suggest that CFSH is involved in the regulation of sex differentiation of early juvenile in S. paramamosain.

Characterization of Aquimarina hainanensis isolated from diseased mud crab Scylla serrata larvae in a hatchery.

Mass mortality due to necrosis signs occurred in hatchery-reared zoea stage larvae of the mud crab Scylla serrata in Okinawa, Japan, and a causative bacterium was isolated. In this study, we identified and characterized the bacterium by genome analysis, biochemical properties and pathogenicity. The bacterium was a Gram-negative, non-motile, long rod, forming yellow colonies on a marine agar plate. It grew at 20-33°C (not at 37°C) and degraded chitin and gelatin. Phylogenetic analysis of the 16S rRNA gene sequence identified the bacterium as Aquimarina hainanensis. Genome sequence data obtained from Illumina MiSeq generated 29 contigs with 3.56 Mbp in total length and a G + C content of 32.5%. The predicted 16 chitinase genes, as putative virulence factors, had certain homologies with those of genus Aquimarina. Experimental infection with the bacterium conducted on larvae of four crustacean species, brine shrimp Artemia franciscana, freshwater shrimp Caridina multidentata, swimming crab Portunus trituberculatus and mud crab S. serrata, revealed that this bacterium was highly virulent to these species. The present study suggests that the bacterium caused mass mortality in mud crab seed production was A. hainanensis and can be widely pathogenic to crustaceans.

Bioaccumulation of heavy metals (Hg, Cd and Ni) by sentinel crab (Macrophthalmus depressus) from sediments of Mousa Bay, Persian Gulf.

The presence of heavy metals (HMs) in the environment can increase their risk of transfer to the food chain. The present study was conducted to investigate the bioaccumulation of HMs including Hg, Cd and Ni in soft and hard tissues of sentinel crab (Macrophthalmus depressus Rüppell, 1830) in sediments of Mousa Bay in northwest of the Persian Gulf. The average (mean ± SD) amounts of Hg, Cd and Ni in the sediments were 6.27 ± 3.66, 3.8 ± 1.48 and 71.6 ± 5.09 µg/g, whereas the relevant amounts for soft tissues were 6.16 ± 4.64, 3.3 ± 1.7 and 19.7 ± 3.96 µg/g, and for hard tissues were, 2.9 ± 1.67, 3.5 ± 1.35 and 10.44 ± 3.1 µg/g, respectively. Accordingly, soft tissues of the crab could be suitable bioindicators of Hg and Cd, whereas the hard tissues could be used as Cd bioindicator. Since the bioaccumulation of Ni was the most, it might have a higher risk among the metals to enter the food chain in the region.

Role of Oxytocin/Vasopressin-Like Peptide and Its Receptor in Vitellogenesis of Mud Crab.

Oxytocin (OT)/vasopressin (VP) signaling system is important to the regulation of metabolism, osmoregulation, social behaviours, learning, and memory, while the regulatory mechanism on ovarian development is still unclear in invertebrates. In this study, Spot/vp-like and its receptor (Spot/vpr-like) were identified in the mud crab Scylla paramamosain. Spot/vp-like transcripts were mainly expressed in the nervous tissues, midgut, gill, hepatopancreas, and ovary, while Spot/vpr-like were widespread in various tissues including the hepatopancreas, ovary, and hemocytes. In situ hybridisation revealed that Spot/vp-like mRNA was mainly detected in 6-9th clusters in the cerebral ganglion, and oocytes and follicular cells in the ovary, while Spot/vpr-like was found to localise in F-cells in the hepatopancreas and oocytes in the ovary. In vitro experiment showed that the mRNA expression level of Spvg in the hepatopancreas, Spvgr in the ovary, and 17ß-estradiol (E2) content in culture medium were significantly declined with the administration of synthetic SpOT/VP-like peptide. Besides, after the injection of SpOT/VP-like peptide, it led to the significantly reduced expression of Spvg in the hepatopancreas and subduced E2 content in the haemolymph in the crabs. In brief, SpOT/VP signaling system might inhibit vitellogenesis through neuroendocrine and autocrine/paracrine modes, which may be realised by inhibiting the release of E2.

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

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