A novel defensin-like peptide C-13326 possesses protective effect against multidrug-resistant Aeromonas schubertii in hybrid snakehead (Channa maculate ♀ × Channa argus ♂).

The purpose of this study was to investigate whether a defensin-like antimicrobial peptide (C-13326 peptide) identified in Hermetia illucens could possess protective effect against multidrug-resistant Aeromonas schubertii in hybrid snakehead (Channa maculate ♀ × Channa argus ♂). The cDNA of C-13326 peptide comprised 243 nucleotides encoding 80 amino acids, with six conserved cysteine residues and the classical CSαß structure. The recombinant expression plasmid pPIC9K-C-13326 was constructed and transformed into GS115 Pichia pastoris, and the C-13326 peptide was expressed by induction with 1% methanol. The crude extract of C-13326 peptide was precipitated by ammonium sulfate, assayed by Braford method, detected by tricine-SDS-PAGE, evaluated by BandScan software and identified by liquid chromatography-mass spectrometry. The C-13326 peptide was shown to have inhibitory activity against the growth of multidrug-resistant A. schubertii DM210910 by using the minimum growth inhibitory concentration and Oxford cup method. In addition, scanning electron microscopy analysis suggested that C-13326 peptide inhibited the growth of A. schubertii DM210910 by damaging the bacterial cell membrane. To explore the role of peptide C-13326 in vivo, hybrid snakehead was fed with peptide C-13326 as feed additives for 7 days. The results revealed that C-13326 peptide could significantly down-regulate the expression levels of IL-1ß, IL-8, IL-12 and TNF-α (p < .05), and significantly improved the survival rate of hybrid snakehead after challenging with A. schubertii DM210910. Therefore, the C-13326 peptide is a promising antimicrobial agent for A. schubertii treatment in aquaculture.

The pathway of Edwardsiella piscicida infecting Lateolabrax maculatus via the immersion bath.

Edwardsiella piscicida, an infectious bacterium, causes great economic losses to the aquaculture industry. Immersion bath which is the closest way to how the fish infect bacterial pathogens in the natural environment is an effective route of artificial infection. In this study, the dynamic process of E. piscicida infection, in the spotted sea bass (Lateolabrax maculatus) was evaluated via the immersion bath. The results showed that soaking the spotted sea bass with 3 × 106 CFU mL-1 E. piscicida for 30 min could artificially induce edwardsiellosis. The higher culture temperature (28.5 ± 0.5°C) or the longer bath time (30 min) would lead to higher mortality of fish. E.piscicida first invaded the gill, then entered the blood circulation to infect the spleen and kidney, where it is colonized, and gradually multiplied in the liver and brain. Meanwhile, the fluorescence in situ hybridization showed that the localization of E. piscicida in the gill and foregut after the immersion challenge proceeded from the exterior to the interior. The invasion of pathogens triggers the immune response of fish and causes tissue damage to the host. The quantitative real-time PCR results displayed an increase in the relative expression level of immune genes (NK-lysin, LZM, IgM and IgD). Otherwise, the most notable histopathological changes of the infected spotted sea bass were multifocal necrosis. Findings in this study broaden our understanding of the infection conditions of E. piscicida and its pathogenicity to the spotted sea bass.

Identification and Characterization of Klebsiella pneumoniae from Farmed American Bullfrogs (Rana catesbeiana).

Klebsiella pneumoniae is a major cause of nosocomial infection and is considered a clinically important bacterium with antibiotic-resistant strains. There are few reports of K. pneumoniae infections in cultured aquatic animals, and no natural infection has been reported in amphibians. From September to October 2021, a high-mortality disease outbreak occurred in a pond-raised American bullfrog farm in Guangzhou, China. The infected bullfrogs were characterized by multiple organ congestive enlargement and inflammation. A pathogenic bacterium was isolated from the viscera of infected bullfrogs and confirmed to be K. pneumoniae by morphological, biochemical, and phylogenetic analyses. Infection experiments confirmed the virulence of the pathogenic strain against bullfrogs and tadpoles. A histopathological examination showed that the strain was harmful to multiple organs. Antibiotic resistance experiments indicated the isolate was a carbapenemase-producing multidrug-resistant K. pneumoniae (MDR-KP) strain. This study is the first report of K. pneumoniae infected American bullfrogs (Rana catesbeiana) and amphibians. These results will shed light on the pathogenicity of K. pneumoniae and help prevent and control K. pneumoniae infections in bullfrogs. IMPORTANCE Klebsiella pneumoniae is recognized as the most common multidrug-resistant bacterial pathogen in humans, and little is known about its pathogenicity in aquatic animals. Recently, K. pneumoniae was found to cause substantial mortality and morbidity in American farm frogs. This was the first report of K. pneumoniae infecting amphibians. In this study, we analyzed the biochemical, growth, and phylogenetic characteristics of the K. pneumoniae strain and described the symptoms and pathological features of infected bullfrogs and tadpoles; this will provide useful data for the prevention and control of infectious diseases, which has been suggested to decrease economic losses in bullfrog farming and reduce the potential threat to public health posed by K. pneumoniae.

Development of TaqMan quantitative PCR assay for detection of Nocardia seriolae in fish and the environment.

Nocardia seriolae is a gram-positive bacterium that causes nocardiosis, threatening fish farming. Advanced nocardiosis is challenging to control; thus, accurate detection methods of the causal agent in the early disease stage are required. In this study, we developed a TaqMan fluorescence quantitative PCR (qPCR) assay for quantitative detection of N. seriolae in fish tissues and water samples. A pair of highly specific primers and a TaqMan probe were designed based on the N. seriolae 16S23S rRNA internal transcribed spacer (ITS) region. A high correlation coefficient (R2 = 0.998) of the standard curve with a 99.5% efficiency was obtained. The qPCR detection limit of the method was as low as 19.8 copies/µL, 1000 times more sensitive than conventional PCR, and has a good performance in the detection of cultured bacteria (y = -3.750× + 48.075, R2 = 0.974). Even 1.42 CFU/mL N. seriolae collected from 500 mL of natural pond water can be detected. Furthermore, a linear model for the relationship between the log of bacteria load and Cq values in water was established (y = -3.239× + 40.978), and the R2 value was 0.979. This assay was used for accurate N. seriolae detection in fish tissues, water samples, feeds and soils. This study provides a valuable tool for the early detection and control of nocardiosis in aquaculture.

A novel chemerin receptor 1 (Chemerin1) takes part in the immune response of cobia (Rachycentron canadum).

Chemerin receptor 1 (Chemerin1) plays a critical role in innate and adaptive immune systems. In this study, a cobia (Rachycentron canadum) Chemerin1 was identified, and its molecular characterization and expression patterns were analyzed. Multiple sequence alignment revealed that the RcChemerin1 possessed a typical dynein regulatory complex (DRC) motif. There was also a potential N-glycosylation site in the extracellular regions of the N-terminus and intracellular loops (ICL) 1 region. Phylogenetic analysis demonstrated that the RcChemerin1 was clustered together with homologous proteins from other fish species. RcChemerin1 was constitutively expressed in a wide range of tissues (especially in immune-related tissues) with different expression levels, which suggests that the RcChemerin1 plays different roles in un-stimulated tissues. RcChemerin1 expression showed up-regulation in the head kidney after Vibrio harveyi challenge. Up-regulation in the head kidney and spleen was also observed after polyinosinic-polycytidylic acid (poly I: C) challenge, which suggests that RcChemerin1 may play vital roles during bacterial and viral infection. The differential responses of immune organs to bacteria and poly I: C imply the differences in defense mechanisms against viruses and bacteria.

Acute septicemia and immune response of spotted sea bass (Lateolabrax maculatus) to Aeromonas veronii infection.

A previous study confirmed that spotted sea bass (Lateolabrax maculatus), an economically important cultured species in East Asia, is a new host of Aeromonas veronii, which can cause acute death in these fish, but there is little in-depth understanding of this disease. In the present study, the virulence of 10 isolates of A. veronii derived from spotted sea bass was determined. It was found that the 18BJ181 isolate was a virulent strain and led to the fastest death of spotted sea bass. Death was determined to be within in 2-12 h, and resulted in abdominal effusion and varying degrees of hemorrhage in internal organs. Bacterial colonization analysis showed that the bacterial load in the spleen was highest, and was up to 3.1 × 105 cfu g-1. In addition, the bacteria proliferated massively in the blood and reached 2.4 × 107 cfu mL-1 at 12 h after 18BJ181 strain infection, which was also a typical feature of acute septicemia. Histopathology of the spleen revealed edema in interstitial tissue, degeneration, and necrosis in lymphoid tissue, and hemorrhage in the capillary network. Transcriptome analysis of the spleen showed that the expression level of HSP70, CCL19, and IL-1ß was extremely significantly up-regulated at 8 h after infection (P < 0.01), and the expression of these genes was normal at 24 h. These results revealed that A. veronii infection could rapidly activate the chemokine signal pathway and stimulate the acute inflammatory response in the host. The bacterial colonization, pathological features, and gene expression patterns in immune pathways will help us to better understand acute septicemia in spotted sea bass caused by A. veronii.

Effects of dietary black soldier fly (Hermetia illucens Linnaeus) on the disease resistance of juvenile grouper (Epinephelus coioides).

An experiment was performed to study the effects of dietary levels of black soldier fly larva meal (BSFLM) on the growth performance, immunity and disease resistance of juvenile grouper (Epinephelus coioides). Four isoproteic and isoenergetic diets were formulated with dietary BSFLM levels of 0 g/kg (T0), 25 g/kg (T2.5), 50 g/kg (T5) and 100 g/kg (T10). Each diet was randomly fed to triplicate groups, each containing 40 fish. The results of the 30-day study indicated that fish growth performance was not affected in the T2.5 and T5 groups compared with the T0 group. In the group with a dietary BSFLM level of 100 g/kg, the feed coefficient was significantly higher than that in the other three groups. The superoxide dismutase, catalase, glutathione peroxidase, lysozyme activity, and malondialdehyde content in the liver, and the interleukin-1 beta (IL-1ß), gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α) and heat shock protein 70 (HSP70) expression in the gills, head kidney, liver and spleen remained consistent in all groups. In addition, no significant differences in the cumulative mortality or parasite abundance in groupers after Vibrio harveyi and Cryptocaryon irritans infection were observed. These results suggested that BSFLM supplemented diets did not inhibit disease resistance in groupers.

Microbial communities in swamps of four mangrove reserves driven by interactions between physicochemical properties and microbe in the North Beibu Gulf, China.

Mangroves are distributed in coastal and estuarine regions and are characterized as a sink for terrestrial pollution. It is believed that complex interactions between environmental factors and microbial communities exist in mangrove swamps. However, little is known about environment-microbe interactions. There is a need to clarify some important environmental factors shaping microbial communities and how environmental factors interact with microbial assemblages in mangrove swamps. In the present study, physicochemical and microbial characteristics in four mangrove reserves (named ZZW, Qin, Bei, and GQ) in the North Beibu Gulf were determined. The interactions between environmental factors and microbial assemblages were analyzed with statistical methods in addition to CCA and RDA. Higher concentrations of sulfate (SO42--S) and Fe but lower concentrations of total phosphorus (TP) and NO3--N were detected in ZZW and Qin. Nutrient elements (NO3--N, NH4+-N, organic matter (OM), SO42--S, Fe, and TP) were more important than heavy metals for determining the microbial assemblages, and NO3--N was the most important factor. NO3--N, SO42--S, TP, and Fe formed a significant co-occurrence network in conjunction with some bacterial taxa, most of which were Proteobacteria. Notably, comparatively elevated amounts of sulfate-reducing bacteria (Desulfatibacillum, Desulfomonile, and Desulfatiglans) and sulfur-oxidizing bacteria (Thioprofundum and Thiohalophilus) were found in ZZW and Qin. The co-occurrence network suggested that some bacteria involved in sulfate reduction and sulfur oxidation drive the transformation of P and N, resulting in the reduction of P and N in mangrove swamps. Through the additional utilization of multivariate regression tree (MRT) and co-occurrence network analysis, our research provides a new perspective for understanding the interactions between environmental factors and microbial communities in mangroves.

Nutrients, temperature, and oxygen mediate microbial antibiotic resistance in sea bass (Lateolabrax maculatus) ponds.

Antibiotic resistance genes (ARGs) have drawn increasing attention as novel environmental pollutants because of the threat they impose on human and animal health. The sea bass (Lateolabrax maculatus) is the third most cultured marine fish in China. Therefore, a study of ARG pollution in the sea bass culture environment is of great significance for the healthy and sustainable development of the sea bass industry. Here, we systematic investigated the contents of 23 antibiotic resistance-related genes (ARRGs), including 19 ARGs and four mobile genetic elements, and analyzed bacterial community composition and environmental parameters in sea bass ponds. The relative abundance (ARRG copies/16S ribosomal RNA gene copies) of ARRGs was up to 3.83 × 10-2. Sul1 was the most abundant ARRG, followed by ereA, intI-1, sul2, dfrA1, and aadA. Both the ARRG changes and aquatic microbiota succession were mainly driven by water temperature (WT), dissolved oxygen (DO), and NO3-. WT is positively correlated with the most ARGs and some of the top 38 Operational Taxonomic Units (OTUs) belonging to the orders of Frankiales, Micrococcales, Chitinophagales, and Sphingomonadales. Furthermore, WT is negatively related with some other OTUs of the orders Frankiales, Xanthomonadales, Micrococcales, and Rhizobiales. However, DO and NO3- have the opposite function with WT on specific taxa and ARGs. These results indicate that sea bass ponds are reservoirs of ARGs, and are driven mainly by the nutrient, temperature, and oxygen with inducing specific microbial taxa. The regulation of environmental factors (increasing DO and NO3-) can be conducted to reduce drug resistance risk in aquaculture ponds. Therefore, environmental factors and specific taxa could be the indicators of ARG contamination and can be used to establish an antibiotic elimination system and consequently realize a sustainable aquaculture industry.

LuxS modulates motility and secretion of extracellular protease in fish pathogen Vibrio harveyi.

Vibrio harveyi can cause infections and diseases in a variety of marine vertebrates and invertebrates, which are harmful to the aquaculture industry. The LuxS quorum-sensing system regulates the expression of virulence factors in a wide variety of pathogenic bacteria. In this study, an in-frame deletion of the luxS gene was constructed to reveal the role of LuxS in the physiology and virulence of V. harveyi. Statistical analysis showed no significant differences in the growth ability, biofilm formation, antibiotic susceptibility, virulence by intraperitoneal injection, and ability of V. harveyi to colonize the spleen and liver of the pearl gentian grouper between the wild-type (WT) and luxS mutant. However, deletion of luxS decreased the secretion of extracellular protease, while increasing swimming and swarming abilities. Simultaneously, a luxS-deleted mutant showed overproduction of lateral flagella, and an intact luxS complemented this defect. Since motility is flagella dependent, 16 V. harveyi flagella biogenesis related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR (qRT-PCR), the expression levels of these genes, including the polar flagella genes flaB, flhA, flhF, flhB, flhF, fliS, and flrA and the lateral flagella genes flgA, flgB, fliE, fliF, lafA, lafK, and motY, were significantly upregulated in the ΔluxS: pMMB207 (ΔluxS+) strain as compared with the V. harveyi 345: pMMB207 (WT+) and C-ΔluxS strains during the early, mid-exponential, and stationary growth phases. Our results indicate that LuxS plays an important role in controlling motility, flagella biogenesis, and extracellular protease secretion in V. harveyi.

Immune Response and Apoptosis-Related Pathways Induced by Aeromonas schubertii Infection of Hybrid Snakehead (Channa maculata♀ × Channa argus♂).

Aeromonas schubertii is the etiological pathogen of internal organ nodules in snakehead fish. Infections with A. schubertii produce a significant economic loss in aquaculture. Therefore, it is important to examine the immune mechanisms by which snakeheads defend against A. schubertii infection. In this study, we established a hybrid snakehead infection model by intraperitoneal injection of A. schubertii that produced internal organ nodules. The splenic immune response of infected fish was examined at the transcriptome level by Illumina-seq analysis. Results showed 14,796 differentially expressed genes (DEGs) following A. schubertii infection, including 4441 up-regulated unigenes and 10,355 down-regulated unigenes. KEGG analysis showed 2084 DEGs to be involved in 192 pathways, 14 of which were immune-related. Twelve DEGs were used to validate quantitative real-time PCR results with RNA-seq data. Time-course expression analysis of six genes demonstrated modulation of the snakehead immune response by A. schubertii. Furthermore, transcriptome analysis identified a substantial number of DEGs that were involved in the apoptosis signaling pathway. TUNEL analysis of infected spleens confirmed the presence of apoptotic cells. This study provided new information for a further understanding of the pathogenesis of A. schubertii in snakeheads, which can be used to prevent and possibly treat A. schubertii infections.

Functional characterization of VscCD, an important component of the type â ¢ secretion system of Vibrio harveyi.

Vibrio harveyi is a Gram-negative bacterium that occurs widely in the ocean and a kind of pathogenic bacteria associated with vibriosis in grouper. We investigated whether the VscCD protein of the type Ⅲ secretion system (T3SS) was important for pathogenicity of V. harveyi. Mutations to the vscC and vscD genes (ΔvscCD) and complementation of the ΔvscCD mutant (C-ΔvscCD) were created. Moreover, the biological characteristics of the wild-type (WT) and mutant strains of V. harveyi 345 were compared. The results showed that deletion of the vscCD genes had no effect on bacterial growth, swimming/swarming ability, secretion of extracellular protease, or biofilm formation. However, as compared with the V. harveyi 345: pMMB207 (WT+) and complementary (C-ΔvscCD) strains, the ΔvscCD: pMMB207 (ΔvscCD+) mutant displayed decreased resistance to acid stress, H2O2, and antibiotics. In addition, infection of the pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatu) showed that as compared with the WT+ and C-ΔvscCD strains, the ΔvscCD+ strain significantly reduced cumulative mortality of the host. The colonization ability of the ΔvscCD+ mutant in the spleen and liver tissues of the pearl gentian grouper was significantly lower than that of the WT+ and C-ΔvscCD strains. In the early stage of infection with the ΔvscCD+ strain, the expression levels of IL-1ß, IL-16, TLR3, TNF-α, MHC-Iα, and CD8α were up-regulated to varying degrees. As compared with the WT+ and C-ΔvscCD strains, luxR expression was significantly up-regulated in the ΔvscCD+ strain, while the expression of vcrH and vp1668 was significantly down-regulated. As an important component of the T3SS, VscCD seemed to play a significant role in the pathogenesis of V. harveyi.

Snakehead vesiculovirus (SHVV) infection alters striped snakehead (Ophicephalus striatus) cells (SSN-1) glutamine metabolism and apoptosis pathways.

Snakehead vesiculovirus (SHVV) causes enormous economic losses in snakehead fish (Ophicephalus striatus) culture. Understanding replication mechanisms of virus is considerable significance in preventing and treating viral disease. In our previous studies, we have reported that glutamine starvation could significant inhibit the replication of SHVV. Furthermore, we also showed that SHVV infection could cause apoptosis of striped snakehead fish cells (SSN-1). However, the underlying mechanisms remain enigmatic. To decipher the relationships among the viral infection, glutamine starvation and apoptosis, SSN-1 cells transcriptomic profilings of SSN-1 cells infected with or without SHVV under glutamine deprived condition were analyzed. RNA-seq was used to identify differentially expressed genes (DEGs). Our data revealed that 1215 up-regulated and 226 down-regulated genes at 24 h post-infection were involved in MAPK, apoptosis, RIG-1-like and toll-like receptors pathways and glutamine metabolism. Subsequently, DEGs of glutamine metabolism and apoptosis pathways were selected to validate the sequencing data by quantitative real-time PCR (qRT-PCR). The expression patterns of both transcriptomic data and qRT-PCR were consistent. We observed that lack of glutamine alone could cause mild cellular apoptosis. However, lack of glutamine together with SHVV infection could synergistically enhance cellular apoptosis. When the cells were cultured in complete medium with glutamine, overexpression of glutaminase (GLS), an essential enzyme for glutamine metabolism, could significantly enhance the SHVV replication. While, SHVV replication was decreased in cells when GLS was knocked down by specific siRNA, indicating that glutamine metabolism was essential for viral replication. Furthermore, the expression level of caspase-3 and Bax was significantly decreased in SHVV infected cells with GLS overexpression. By contrast, they were significantly increased in SHVV infected cells with GLS silence by SiRNA, indicating that SHVV infection activated the Bax and caspase-3 pathways to induce apoptosis independent of glutamine. Our results reveal that SHVV replication and starvation of glutamine could synergistically promote the cellular apoptosis, which will pave a new way for developing strategies against the vial infection.

Effect of nitrite exposure on oxidative stress, DNA damage and apoptosis in mud crab (Scylla paramamosain).

Nitrite is one of major environmental pollutants that can impact immunological parameters in aquatic organisms. In the present study, we investigated the effects of nitrite exposure on oxidative stress, DNA damage and apoptosis in mud crab (Scylla paramamosain). Mud crab were exposed to 0, 5, 10 and 15 mg L-1 nitrite for 72 h. These data showed that acid phosphatase (ACP) and alkaline phosphatase (ALP) activity significantly decreased in treatments with various concentrations of nitrite (5, 10 and 15 mg L-1) after 24 and 48 h, while the levels of nitric oxide (NO) significantly increased in these treatments. Nitrite exposure could suppress superoxide dismutase (SOD) and catalase (CAT) activity, and increase the formation of malondialdehyde (MDA) after 48 and 72 h of exposure. In addition, nitrite exposure decreased total haemocyte counts after 48 and 72 h of exposure. Cytological damage, DNA damage and apoptosis was observed obviously at 72 h after nitrite exposure. Moreover, nitrite exposure significantly induced the mRNA levels of phosphorylated Jun N-terminal kinases (JNK), and eventually activated p53 signaling and caspase-3. These results indicated that nitrite exposure could induce oxidative stress, which further caused DNA damage and apoptosis in mud crab. Our results will be helpful to understand the mechanism of nitrite toxicity on crustaceans.

A First Report of Aeromonas veronii Infection of the Sea Bass, Lateolabrax maculatus in China.

The sea bass, Lateolabrax maculatus is commercially farmed in Zhuhai, located in the Guangdong Province of China. L. maculatus in aquaculture have suffered acute death, characterized by ulcerations on the body surface, congestion, and hemorrhage in internal organs such as liver, kidney, and spleen. The dominant infecting strain of bacteria isolated from the kidneys of diseased fish was identified as Aeromonas veronii (strain 18BJ181). This identification was based on analysis of morphological, physiological, and biochemical features, as well as 16S rRNA and gyrB gene sequences. Drug sensitivity testing showed that the strain 18BJ181 isolate was resistant to four antibacterial drugs, including amoxicillin, madinomycin, penicillin and sulfamethoxazole, while moderately sensitive to erythromycin and rifampicin. The detection of growth characteristics showed that the strain 18BJ181 exhibited adaptability to the environment. In addition, some virulence genes, such as aer, act, gcaT, tapA and fla, were detected in the strain 18BJ181. The median lethal dosage of the strain 18BJ181 isolate in L. maculatus was 8.5 × 105 and 4.2 × 105 cfu/g under the conditions of intraperitoneal injection and intramuscular injection, respectively. The experimentally induced infection showed that the 18BJ181 isolate caused considerable histological lesions in L. maculatus, including tissue degeneration, necrosis, and different degrees of hemorrhage. These results provided evidence for a more comprehensive understanding of A. veronii strain 18BJ181 infection in L. maculatus.

Horizontal gene transfer contributes to virulence and antibiotic resistance of Vibrio harveyi 345 based on complete genome sequence analysis.

BACKGROUND: Horizontal gene transfer (HGT), which is affected by environmental pollution and climate change, promotes genetic communication, changing bacterial pathogenicity and drug resistance. However, few studies have been conducted on the effect of HGT on the high pathogenicity and drug resistance of the opportunistic pathogen Vibrio harveyi. RESULTS: V. harveyi 345 that was multidrug resistant and infected Epinephelus oanceolutus was isolated from a diseased organism in Shenzhen, Southern China, an important and contaminated aquaculture area. Analysis of the entire genome sequence predicted 5678 genes including 487 virulence genes contributing to bacterial pathogenesis and 25 antibiotic-resistance genes (ARGs) contributing to antimicrobial resistance. Five ARGs (tetm, tetb, qnrs, dfra17, and sul2) and one virulence gene (CU052_28670) on the pAQU-type plasmid p345-185, provided direct evidence for HGT. Comparative genome analysis of 31 V. harveyi strains indicated that 217 genes and 7 gene families, including a class C beta-lactamase gene, a virulence-associated protein D gene, and an OmpA family protein gene were specific to strain V. harveyi 345. These genes could contribute to HGT or be horizontally transferred from other bacteria to enhance the virulence or antibiotic resistance of 345. Mobile genetic elements in 71 genomic islands encoding virulence factors for three type III secretion proteins and 13 type VI secretion system proteins, and two incomplete prophage sequences were detected that could be HGT transfer tools. Evaluation of the complete genome of V. harveyi 345 and comparative genomics indicated genomic exchange, especially exchange of pathogenic genes and drug-resistance genes by HGT contributing to pathogenicity and drug resistance. Climate change and continued environmental deterioration are expected to accelerate the HGT of V. harveyi, increasing its pathogenicity and drug resistance. CONCLUSION: This study provides timely information for further analysis of V. harveyi pathogenesis and antimicrobial resistance and developing pollution control measurements for coastal areas.

A novel sRNA srvg17985 identified in Vibrio alginolyticus involving into metabolism and stress response.

Vibrio alginolyticus is an opportunistic pathogen that is a threat to the aquaculture industry. Evidence has revealed critical roles for small RNAs (sRNAs) in bacterial physiology and pathology by modulating gene expression post transcription. However, little information about sRNA-mediated regulation in V. alginolyticus is available. We experimentally verified the existence and characterized the function of sRNA srvg17985 in V. alginolyticus ZJ-T. We identified a 179 nt and growth-phase-dependent transcript with a σ70 promoter and a ρ-independent terminator. The transcript consisted of five stem-loops and was conserved in Vibrio spp. Phenotype microarray assays showed that deletion of srvg17985 led to less use of Gly-Glu as a carbon source but a gain in ability to use l-phenylalanine as a nitrogen source. Srvg17985 regulated the osmotic stress response with stronger tolerance to NaCl but weaker tolerance to urea. In addition, srvg17985 inhibited the deamination of l-serine at pH 9.5 and promoted the hydrolysis of X-beta-d-glucuronide, thus affecting the pH stress response. Bioinformatics by IntaRNA and TargetRNA2 identified 45 common target mRNAs, some of which probably contributed to the observed phenotypes. These results indicated that srvg17985 regulated environmental adaptation. The results provide valuable information for in-depth studies of sRNA-mediated regulation mechanisms of the complex physiological processes of V alginolyticus and provide new targets for antibacterial therapeutics or attenuated vaccines for Vibrio spp.

Expression of lactate dehydrogenase is induced during hypoxia via HIF-1 in the mud crab Scylla paramamosain.

Lactate dehydrogenase (LDH) is a key enzyme involved in anaerobic metabolism in most organisms. In the present study, we determined the structure and function of LDH sequence in Scylla paramamosain (SpLDH) by gene cloning, expression and RNA interference techniques in order to explore the genetic characteristics of LDH and its relationship with HIF-1 during hypoxia. The full-length cDNA was 1453 bp with an open reading frame (ORF) of 996 bp, and encoded a polypeptide of 332 amino acids. Homology analysis showed that the SpLDH gene is highly similar to arthropods. The SpLDH transcript increased after hypoxia in all tested tissues. The silencing of HIF-1 blocked the increase in LDH mRNA and activity, which were induced by hypoxia in gill and muscle tissues. Our results indicated that SpLDH expression was regulated transcriptionally by HIF-1.

Molecular typing of Streptococcus agalactiae isolates of serotype Ia from tilapia in southern China.

Streptococcus agalactiae is an important pathogen of tilapia causing enormous economic losses worldwide. In this study, multilocus sequence typing indicated that 75 S. agalactiae isolates from tilapia in southern China belonged to sequence type-7, as well as belonging to serotype Ia, as confirmed by multiplex PCR assay. The putative-virulence gene profiles and genetic variation of these strains were determined by three sets of multiplex PCR and multi-virulence locus sequencing typing (MVLST), respectively. Analysis of putative-virulence gene profiles showed that each strain harbored 18 putative-virulence genes but lacked lmb and scpB. Three putative-virulence genes (srr-1, bibA and fbsA) were further selected for MVLST analysis. Our data showed that the strains had 14 MVLST types (1-14) and clustered in three groups (Groups I-Ⅲ). The period of time during 2013 and 2014 was an important turning point for the differentiation of the putative-virulence genes of S. agalactiae, as type 1 within Group Ⅱ became the predominant MVLST type. There were significant differences in MVLST types of S. agalactiae isolated from different tilapia farming regions. MVLST assay may improve the discriminatory power and is suitable for understanding the epidemiology of S. agalactiae serotype Ia and screening multivalent vaccine candidate strains.

First report of trypanosomiasis in farmed largemouth bass (Micropterus salmoides) from China: pathological evaluation and taxonomic status.

The aim of this study was to evaluate the effect of trypanosomes on cultured largemouth bass (Micropterus salmoides) and describe the taxonomic identification of the parasite. The effects of the parasite on M. salmoides were examined based on clinical symptoms, hemograms, histopathology, and serum biochemistry. Diseased fish showed typical clinical symptoms of trypanosomiasis, which included lethargy, anorexia, and histopathological lesions in the liver, head kidney, and spleen. The serum of diseased fish had significantly lower concentrations of glucose, triglyceride, and low-density lipoprotein, and significantly higher alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) activities. The morphology of the trypanosomes was also analyzed using light microscopy, and their 18S rDNA sequence was analyzed to establish genetic relationships with other known strains. We found that the trypomastigote form of the trypanosomes from M. salmoides was similar to those isolated from Pelteobagrus fulvidraco. The trypanosomes had a slender and narrow body with a relatively long free flagellum, not well-developed undulating membrane, and an oval kinetoplast located near the subterminal posterior end of the body. The 18S rDNA sequences of the trypanosome from M. salmoides had the highest similarity (99.8%) with that of P. fulvidraco, suggesting they are identical species. Based on the differences in morphological characteristics and 18S rDNA sequence compared to trypanosomes isolated from other freshwater fish, it is considered as a new species and we propose the name Trypanosoma micropteri n. sp.