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.

Rhodobacter azotoformans supplementation improves defense ability of Chinese mitten crab Eriocheir sinensis against citrobacteriosis.

Rhodobacter probiotics are considered as good alternatives to antibiotics for aquaculture. Yet the beneficial effects of Rhodobacter on Chinese mitten crab Eriocheir sinensis are still unclear, and more functions of Rhodobacter supplementation need to be clarified. In this study, a 60-day feeding trial was performed to investigate the protective effects of R. azotoformans against citrobacteriosis in E. sinensis by growth performance, serum immunity, hepatopancreatic antioxidant capability, intestinal flora, and resistance to Citrobacter freundii challenge assays. The results showed that R. azotoformans supplementation significantly and dose-dependently increased weight gain and specific growth rate as well as activities of serum immune and hepatopancreatic antioxidant enzymes, leading to notable improvement in the growth performance, serum immunity and hepatopancreatic antioxidant status of E. sinensis. Besides, R. azotoformans supplementation significantly enhanced intestinal microbial abundance and diversity in E. sinensis, and conferred significant protection of the crabs against C. freundii challenge with seven-day survival rates of 70.0%-100.0%. To the best of our knowledge, this is the first study to reveal the protective effects of R. azotoformans against citrobacteriosis in E. sinensis.

Protective effects of Bacillus licheniformis against Citrobacter freundii infection in Chinese mitten crab Eriocheir sinensis.

Citrobacter freundii is an important bacterial pathogen that causes serious diseases in Chinese mitten crab Eriocheir sinensis. However, scarce information is available on the use of Bacillus licheniformis to protect E. sinensis from C. freundii infection by improving the immunity, antioxidant ability and intestinal flora. In the present study, a 60-day feeding trial was conducted to examine the potential effects of dietary supplementation with antagonistic B. licheniformis on the non-specific immunity, antioxidant capability, intestinal flora and resistance of E. sinensis to C. freundii infection. The results indicated that dietary supplementation of B. licheniformis could boost non-specific immunity and antioxidant capability of E. sinensis in a significant dose-dependent manner respectively by increasing serum lysozyme, superoxide dismutase, alkaline phosphatase, and catalase activities and hepatopancreatic superoxide dismutase, catalase, acid phosphatase activities. In addition, crabs fed diets with B. licheniformis displayed increased composition and diversity of the intestinal flora, and exhibited significant defense against C. freundii infection with the relative percentage survivals ranging from 61.9% to 100.0% for seven days. To our knowledge, this is the first report of antagonistic B. licheniformis as a supplement in Chinese mitten crab feed to effectively resist C. freundii infection by improving the non-specific immunity, antioxidant status and intestinal flora.

Phenotypic and genomic characterization of pathogenic Providencia rettgeri from kuruma shrimp Marsupenaeus japonicus.

Providencia rettgeri has been recognized as a zoonotic pathogen of humans and aquaculture animals and has become a global public health concern. However, scarce information is available on the characterization of pathogenic P. rettgeri from kuruma shrimp Marsupenaeus japonicus. In the present study, a P. rettgeri isolate (KM4) was confirmed as a causative agent of red leg disease in cultured M. japonicus, which showed a median lethal dose (LD50 ) value of 5.01 × 105 CFU·ml-1 and had multiple resistances to aminoglycosides, sulfonamides, and tetracycline antimicrobials used in aquaculture. In addition, the whole genome of isolate KM4 was sequenced and found to consist of a single circular chromosome of 4,378,712 bp and a circular plasmid of 171,394 bp. The genome sequence analysis further revealed the presence of potential virulence and antibiotic resistance genes in isolate KM4, which probably rendered this isolate particularly virulent. To our knowledge, this is the first study to characterize P. rettgeri pathogens from kuruma shrimp infected with red leg disease. The findings of this study can provide novel insights into the presence and distribution of pathogenicity-associated genes in shrimp-pathogenic P. rettgeri.

Identification of Vibrio ponticus as a bacterial pathogen of coral trout Plectropomus leopardus.

Vibrio ponticus is a vital pathogen with potential danger for aquaculture animals. Yet V. ponticus pathogenic to the coral trout Plectropomus leopardus is still unknown. In this study, a virulent bacterial strain, temporarily named DX2, was isolated from diseased coral trout suffering liver necrosis with cell vacuolar degeneration, and was identified molecularly and phenotypically as V. ponticus. Besides, the DX2 isolate showed an LD50 value of 6.64×105 CFU mL-1, developed multiple resistances to cephalosporins, macrolides, penicillins, peptides, and sulfonamides antimicrobials, and was highly susceptible to doxycycline and florfenicol in aquaculture use. To the best of our knowledge, this is the first report of the pathogenicity of V. ponticus to the coral trout, and the findings provide a reference for the control of pathogenic V. ponticus in the coral trout.

A convenient polyculture system that controls a shrimp viral disease with a high transmission rate.

Developing ecological approaches for disease control is critical for future sustainable aquaculture development. White spot syndrome (WSS), caused by white spot syndrome virus (WSSV), is the most severe disease in cultured shrimp production. Culturing specific pathogen-free (SPF) broodstock is an effective and widely used strategy for controlling WSS. However, most small-scale farmers, who predominate shrimp aquaculture in developing countries, cannot cultivate SPF shrimp, as they do not have the required infrastructure and skills. Thus, these producers are more vulnerable to WSS outbreaks than industrial farms. Here we developed a shrimp polyculture system that prevents WSS outbreaks by introducing specific fish species. The system is easy to implement and requires no special biosecurity measures. The promotion of this system in China demonstrated that it allowed small-scale farmers to improve their livelihood through shrimp cultivation by controlling WSS outbreaks and increasing the production of ponds.

Full-length transcriptome sequencing combined with RNA-seq analysis revealed the immune response of fat greenling (Hexagrammos otakii) to Vibrio harveyi in early infection.

Fat greenling (Hexagrammos otakii) is an important commercial marine fish species cultured in northeast Asia, but its available gene sequences are limited. Vibrio harveyi is a causative agent of vibriosis in fat greenling and also causes severe losses to the aquaculture industry in China. In order to obtain more high-quality transcript information and investigate the early immune response of fat greenling against V. harveyi, the fish were artificially infected with V. harveyi, and five sampling points were set within 48 h. Iso-Seq combined with RNA-Seq were applied in the comprehensive transcriptome analysis of V. harveyi-infected fat greenling. Total 42,225 consensus isoforms were successfully extracted from the result of Iso-Seq, and more than 19,000 ORFs were predicted. In addition, total three modules were identified by WGCNA which significantly positive correlated to the infection time, and the KEGG analysis showed that the immune-related genes in these modules mainly enriched in TLR signaling pathway, NF-κB signaling pathway and Endocytosis. The activation of inflammation and endocytosis was the most significant characteristics of fat greenling immune response during the early infection. Based on the WGCNA, a series of high-degree nodes in the networks were identified as hub genes. The protein structures of cold-inducible RNA-binding protein (CIRBP), poly [ADP-ribose] polymerase 1 (PARP1) and protein arginine N-methyl transferase 1 (PRMT1) were subsequently found to be highly conserved in vertebrate, and the gene expression pattern of CIRBP, PARP1, PRMT1 and a part of TLR/NF-κB pathway-related genes indicated that these proteins might have similar biological functions in regulation of inflammatory response in teleost fish. The results of this study provided the first systematical full-length transcriptome profile of fat greenling and characterized its immune responses in early infection of V. harvey, which will serve as the foundation for further exploring the molecular mechanism of immune defense against bacterial infection in fat greenling.

Recombinant outer membrane protein C of Aeromonas salmonicida subsp. masoucida, a potential vaccine candidate for rainbow trout (Oncorhynchus mykiss).

Aeromonas salmonicida subsp. masoucida (ASM) is an important bacterial pathogen of salmonid fish, which can cause huge economic losses to the fish farming industry. In order to screen effective vaccine candidate proteins, four outer membrane proteins of ASM, including OmpA, OmpC, OmpK and OmpW, were selected and recombinantly expressed in Escherichia coli. The result of western blotting showed that these four recombinant proteins could be recognized by rainbow trout anti-ASM antibodies. The immune protective effects of the four rOMPs were also investigated, and the relative percentage survival (RPS) of rOmpA, rOmpC, rOmpK and rOmpW were 71.1%, 81.6%, 55.3% and 42.1%, respectively. The RPS of rOmpC was significantly higher than the other three rOMPs, so the immune responses of rainbow trout induced by rOmpC were further investigated. The results showed that vaccination with rOmpC could significantly induced the production of specific serum antibodies and proliferation of sIg + lymphocytes in peripheral blood. Meanwhile, RT-qPCR analysis showed that rOmpC could significantly enhance the expression of the MHC-II, TCR, CD4, CD8, IL-8 and IgM genes compared with the BSA immunized group. These results demonstrated that rOmpC could induce strong humoral immune response in rainbow trout and provided effective immune protection against ASM challenge, which indicated that OmpC is a promising vaccine candidate against Aeromonas salmonicida infection.

Aeromonas hydrophila, an Emerging Causative Agent of Freshwater-Farmed Whiteleg shrimp Litopenaeus vannamei.

Aeromonas hydrophila is a well-known bacterial pathogen associated with mass mortalities in aquaculture. Yet, few reports are available on whiteleg shrimp-pathogenic A. hydrophila. In the present study, a virulent isolate WS05 was confirmed as a causative agent of diseased freshwater-cultured whiteleg shrimp and showed a mean lethal dose (LD50) value of 4.8 × 104 CFU mL-1. It was identified phenotypically and molecularly as an A. hydrophila strain, and exhibited susceptibility to several veterinary antibiotics extensively used in aquaculture, including cotrimoxazole, doxycycline, florfenicol, neomycin, and tetracycline. In view of the strongest inhibition zone of florfenicol against isolate WS05, the synergistic effect of the combinations of florfenicol and herb extracts was further evaluated, and the result indicated that Punica granatum extract was a potential synergist of florfenicol against isolate WS05 and the fractional inhibitory concentration index (FICI) for the florfenicol-P. granatum extract was calculated as 0.31. When combined with 7.81 mg mL-1 P. granatum extract, the minimum inhibitory concentration (MIC) of florfenicol against isolate WS05 was reduced from 0.50 to 0.03 mg L-1, and its activity against isolate WS05 was also enhanced with a significant reduction of ≥3.61 log in cell density after 24 h of treatment compared with that in the single drug treatment. In addition, the protective effect was potentiated by the combination of florfenicol and P. granatum extract, with a cumulative mortality of 36.66% (p < 0.05) and 33.33% (p < 0.05) lower than that in the single treatment with florfenicol and P. granatum extract after the challenge with isolate WS05 for seven days. As far as we know, this is the first study to describe whiteleg shrimp-pathogenic A. hydrophila and suggest P. granatum extract as a potential synergist of florfenicol against the A. hydrophila pathogen.

Transcriptome analysis of immune response in fat greenling (Hexagrammos otakii) against Vibrio harveyi infection.

Fat greenling (Hexagrammos otakii) is an important aquaculture fish species in northern China. Unfortunately, Vibrio infections have caused considerable losses to the fat greenling aquaculture industry. However, the study on immune response of fat greenling against Vibrio species has not been reported yet. In this paper, the immune response of fat greenling against V. harveyi at gene expression level was studied by transcriptome analysis. A total of 189753 high-quality unigenes with a N50 length of 672bp were obtained by transcriptome profiling, which provided abundant data for the future study of fat greenling. Comparative analysis showed that 5425 differentially expressed genes (DEGs) were identified on day 3 post-infection (3dpi), containing 1837 up-regulated and 3588 down-regulated genes. Further annotation and analysis revealed that the DEGs were enriched in complement and coagulation cascades, ribosome, oxidative phosphorylation, glycine, serine and threonine metabolism and peroxisome proliferator-activated receptor (PPAR) signaling pathway. These pathways were mainly associated with phagocytosis and pathogen clearance, rarely involved in bacteria adhesion and pathogen identification, which suggested that the host might begin to clear and kill the invading bacteria on 3dpi. The research might provide a valuable resource to further study immune response and suggest strategies against V. harveyi infection in fat greenling.