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.

Revealing CO2-Fixing SAR11 Bacteria in the Ocean by Raman-Based Single-Cell Metabolic Profiling and Genomics.

The majority of marine microbes remain uncultured, which hinders the identification and mining of CO2-fixing genes, pathways, and chassis from the oceans. Here, we investigated CO2-fixing microbes in seawater from the euphotic zone of the Yellow Sea of China by detecting and tracking their 13C-bicarbonate (13C-HCO3-) intake via single-cell Raman spectra (SCRS) analysis. The target cells were then isolated by Raman-activated Gravity-driven Encapsulation (RAGE), and their genomes were amplified and sequenced at one-cell resolution. The single-cell metabolism, phenotype and genome are consistent. We identified a not-yet-cultured Pelagibacter spp., which actively assimilates 13C-HCO3-, and also possesses most of the genes encoding enzymes of the Calvin-Benson cycle for CO2 fixation, a complete gene set for a rhodopsin-based light-harvesting system, and the full genes necessary for carotenoid synthesis. The four proteorhodopsin (PR) genes identified in the Pelagibacter spp. were confirmed by heterologous expression in E. coli. These results suggest that hitherto uncultured Pelagibacter spp. uses light-powered metabolism to contribute to global carbon cycling.

Complete Genome Sequence of Macrobrachium rosenbergii Golda Virus (MrGV) from China.

In a meta-transcriptome study of the giant freshwater prawn Macrobrachium rosenbergii sampled in 2018 from a hatchery, we identified a variant of Macrobrachium rosenbergii golda virus (MrGV) in postlarvae without clinical signs. The virus belongs to the family Roniviridae, and the genome of this MrGV variant, Mr-18, consisted of 28,957 nucleotides, including 4 open reading frames (ORFs): (1) ORF1a, encoding a 3C-like protein (3CLP) (4933 aa); (2) ORF1b, encoding a replicase polyprotein (2877 aa); (3) ORF2, encoding a hypothetical nucleocapsid protein (125 aa); and (4) ORF3, encoding a glycoprotein (1503 aa). ORF1a overlaps with ORF1b with 40 nucleotides, where a -1 ribosomal frameshift with slippage sequence 5'-G14925GGUUUU14931-3' produces the pp1ab polyprotein. The genomic sequence of Mr-18 shared 97.80% identity with MrGV LH1-2018 discovered in Bangladesh. The amino acid sequence identities between them were 99.30% (ORF1a), 99.60% (ORF1b), 100.00% (ORF2), and 99.80% (ORF3), respectively. Phylogenetic analysis of the RNA-dependent RNA polymerase (RdRp) proteins revealed that they clustered together and formed a separate cluster from the genus Okavirus. The finding of MrGV in China warrants further studies to determine its pathogenicity and prevalence within the region.

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.

Seasonal dynamics of the coastal bacterioplankton at intensive fish-farming areas of the Yellow Sea, China revealed by high-throughput sequencing.

Marine aquaculture areas are facing stressed environmental challenges, especially the degradation of coastal ecosystems. Here a coordinated time-series study was used to investigate the coastal bacterioplankton biodiversity dynamics of the Yellow Sea, China. Bacterial 16S rRNA gene sequencing revealed a temporal pattern of decreasing of diversity in summer. Functional prediction indicated that metabolic pathways related to the adenosine triphosphate (ATP)-binding cassette transporters and other membrane transporters were significantly enriched in May, while the genetic information processing category was most abundant in March. The May microbiomes showed most significant positive correlation with phosphate concentration, while the August and November microbiomes correlated with temperature and chemical oxygen demand (COD) most, and the March microbiomes showed significant correlation with Cu2+ level, pH and salinity. The correlations between representative bacteria and environmental parameters revealed in this study may provide insights into the potential influences of human aquaculture activities, on the biodiversity of coastal bacterioplankton.

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.

Raman-activated cell sorting and metagenomic sequencing revealing carbon-fixing bacteria in the ocean.

It is of great significance to understand CO2 fixation in the oceans. Using single cell Raman spectra (SCRS) as biochemical profiles, Raman activated cell ejection (RACE) was able to link phenotypes and genotypes of cells. Here, we show that mini-metagenomic sequences from RACE can be used as a reference to reconstruct nearly complete genomes of key functional bacteria by binning shotgun metagenomic sequencing data. By applying this approach to 13 C bicarbonate spiked seawater from euphotic zone of the Yellow Sea of China, the dominant bacteria Synechococcus spp. and Pelagibacter spp. were revealed and both of them contain carotenoid and were able to incorporate 13 C into the cells at the same time. Genetic analysis of the reconstructed genomes suggests that both Synechococcus spp. and Pelagibacter spp. contained all genes necessary for carotenoid synthesis, light energy harvesting and CO2 fixation. Interestingly, the reconstructed genome indicates that Pelagibacter spp. harbored intact sets of genes for ß-carotene (precursor of retional), proteorhodopsin synthesis and anaplerotic CO2 fixation. This novel approach shines light on the role of marine 'microbial dark matter' in global carbon cycling, by linking yet-to-be-cultured Synechococcus spp. and Pelagibacter spp. to carbon fixation and flow activities in situ.

Adjuvant and immunostimulatory effects of LPS and ß-glucan on immune response in Japanese flounder, Paralichthys olivaceus.

In order to investigate the adjuvant and immunostimulatory effects of LPS and ß-glucan, a highly effective vaccine candidate for Edwardsiella tarda known as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was recombinantly expressed in Escherichia coli, which was intramuscularly injected into Japanese flounder alone or containing LPS or ß-glucan. ELISA analysis of sera showed that the fish immunized with rGAPDH containing LPS or ß-glucan had significant higher antibody titers than rGAPDH immunization group at day 14 and 30 (P<0.05). Flow cytometric analysis showed that the surface immunoglobulin positive (sIg+) lymphocytes percentages in head kidney of the immunized fish exhibited significant higher than the fish of control group at day 14 and 30, and immunization with rGAPDH containing LPS or ß-glucan could induce significant higher sIg+ percentages than immunization with rGAPDH alone. Simultaneously, the expressions of genes coding for immunoregulatory molecules involved in innate and acquired responses, including IFN-γ, IL-1ß, IL-6, TNF-α, TCRα, IgM, C-type lysozyme and MHC-IIα were analyzed by quantitative PCR. The transcription levels of all selected genes except TNF-α displayed up-regulation after immunization, and immunization with rGAPDH containing LPS or ß-glucan could induce significant higher levels than that induced only by rGAPDH. Moreover, to investigate the protection against infection, challenge with E. tarda was performed at day 30 after immunization. Fish immunized with rGAPDH containing ß-glucan exhibited the highest relative percentage survival (RPS) of 63% compared to the other groups. These results indicated that LPS and ß-glucan employed as co-immunostimulatory molecules could significantly trigger the innate and acquired immune responses in Japanese flounder.

Tumour gene expression predicts response to cetuximab in patients with KRAS wild-type metastatic colorectal cancer.

BACKGROUND: Although it is accepted that metastatic colorectal cancers (mCRCs) that carry activating mutations in KRAS are unresponsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, a significant fraction of KRAS wild-type (wt) mCRCs are also unresponsive to anti-EGFR therapy. Genes encoding EGFR ligands amphiregulin (AREG) and epiregulin (EREG) are promising gene expression-based markers but have not been incorporated into a test to dichotomise KRAS wt mCRC patients with respect to sensitivity to anti-EGFR treatment. METHODS: We used RT-PCR to test 110 candidate gene expression markers in primary tumours from 144 KRAS wt mCRC patients who received monotherapy with the anti-EGFR antibody cetuximab. Results were correlated with multiple clinical endpoints: disease control, objective response, and progression-free survival (PFS). RESULTS: Expression of many of the tested candidate genes, including EREG and AREG, strongly associate with all clinical endpoints. Using multivariate analysis with two-layer five-fold cross-validation, we constructed a four-gene predictive classifier. Strikingly, patients below the classifier cutpoint had PFS and disease control rates similar to those of patients with KRAS mutant mCRC. CONCLUSION: Gene expression appears to identify KRAS wt mCRC patients who receive little benefit from cetuximab. It will be important to test this model in an independent validation study.

[Study on the volatile oil by SFE-CO, from crude and processed Rhizoma Atractylodis by GC-MS].

OBJECTIVE: To analyze the chemical constituents of the volatile oil by SFE-CO2 from Rhizoma Atractylodis and its roasted products. METHODS: The chemical constituents of the volatile oil extracted by SFE-CO2 were separated and identified by GC-MS. RESULTS: 33 kinds of constituents were identified from Atractylodes lancea (Thunb.) DC. and its roasted products. The relative contents of 12 kinds of constituents from the processing products were descended and 7 kinds were ascended; 30 kinds of constituents were identified from Atractylodes chinensis (DC.) Koidz. and its roasted products. The relative contents of 10 kinds of constituents from the roasted products were descended and 11 kinds were ascended. CONCLUSION: In the processed products, the relative contents of some low boiling point constituents are descended but some high are ascended.