Effect of Hypoxia on the Gut Microflora of a Facultative Air-Breathing Loach Lepidocephalichthys guntea.

One of the main risks to fish health in an aquatic environment is hypoxia, which can either lead to respiratory failure or the emergence of various diseases in a fish population. This investigation examined the impact of hypoxia on the gut bacteria of a loach, Lepidocephalichthys guntea, which under the dissolve oxygen stress can gulp air from surface and breathe using its posterior intestine. High-throughput sequencing was used to examine the anterior and posterior parts of the gut of L. guntea during both normoxia and hypoxia. According to the community profiling of the gut bacteria, prolonged exposure to hypoxia increased the diversity and abundance of bacteria in the posterior part while decreasing both in the anterior part of the gut. Additionally, for both parts of the gut, the core microbiota showed a significant alteration during hypoxia. In correlation network analysis, a more interactive and intricate network was developed at normoxia. According to the comparative analyses of the gut bacteria, hypoxia causes more pronounced alterations in the posterior gut than the anterior gut at various taxonomic levels. As a consequence of hypoxia, several genera like Aeromonas, Pseudomonas, Plesiomonas, Acinetobacter, and Enterobacter were replaced by Streptococcus, Escherichia-Shigella, Janthinobacterium, and Clostridia. A surge in probiotic genera, including Bifidobacterium, Lactobacillus, Blautia, and Cetobacterium, was also seen. The fatty acid biosynthesis pathway was induced only in hypoxia, although all other metabolic pathways were present in both situations, albeit with fewer hits in hypoxia.

TAK1 of blunt snout bream promotes NF-κB activation via interaction with TAB1 in response to pathogenic bacteria.

Transforming growth factor-ß activated kinase-1 (TAK1) is an important upstream signaling molecules involved in the NF-κB signaling pathway. TAK1 interacts with TAB1 to form the TAK1-TAB1 complex, which elicits NF-κB activation through a series of cascade reactions in mammals. However, the function of TAK1 in blunt snout bream (Megalobrama amblycephala ( maTak1) and the effects of their interaction between TAK1 and TAB1 on the NF-κB activation still remains largely unknown. In the present study, maTak1 was cloned and characterized successfully based on transcriptome data. Its open reading frame is composed of 1626 nucleotides and the predicted maTAK1 protein contains 541 amino acids, which includes an N-terminal Serine/Threonine protein kinases (S/TKc) and a C-terminal coiled-coil region. Phylogenetic analysis showed that maTAK1 were clustered with those of other teleosts. MaTak1 displayed ubiquitous transcriptional expression in all the examined tissues of healthy blunt snout bream but with varied expression levels. And maTrak1 expression was dramatically enhanced in different tissues and MAF cells after LPS stimulation and A. hydrophila challenge. The result from subcellular localization analysis indicated that both maTAK1 and maTAB1 were cytoplasmic protein. The activity of NF-κB promoter could not be induced by overexpression of maTak1 or maTab1 alone, however, it could be enhanced by co-expression of maTak1 and maTab1. Co-immunoprecipitation and subcellular co-localization assay revealed that maTAK1 can combine with maTAB1 directly. The transcriptional expression level of pro-inflammatory cytokines (IL-1ß, IL-6 and IL-8) increased distinctly after the overexpression of maTak1 and maTab1. Taken together, the data obtained in this study demonstrated that the direct interaction between maTAK1 and maTAB1 might play a pivotal role in mediating host innate immune response to pathogen invasion by the production of pro-inflammatory cytokines via NF-κB signaling pathway, which might lay a solid foundation for the establishment of novel therapeutic approach to combat bacterial infection in fish.

Isolation, identification and characterisation of an emerging fish pathogen, Acinetobacter pittii, from diseased loach (Misgurnus anguillicaudatus) in China.

Although members of the genus Acinetobacter have emerged as important nosocomial pathogens causing severe human infections, there are few reports about their occurrence as fish pathogens. In this study, five bacterial strains were isolated from diseased loach (Misgurnus anguillicaudatus) cultured in a farm in China. The diseased loach displayed shedding of skin mucus and many petechial haemorrhages all over the body. Based on sequence analyses of 16S rRNA and rpoB genes, the isolates were identified as Acinetobacter pittii. An experimental infection assay confirmed their pathogenicity to loach. The results of artificial infection in zebrafish (Barchydanio rerio) and nematode (Caenorhabditis elegans) suggested that, as well as loach, these A. pittii isolates are pathogenic and highly virulent to these organisms. Multilocus sequence typing analysis revealed that all the isolates belong to sequence type (ST) 839, which may be the dominant clone causing fish disease and exhibits a close phylogenetic relationship with ST396 from human clinical samples in Korea or Taiwan China. This is the first report demonstrating that A. pittii is an emerging causal agent of mass mortality in loach and poses significant risks to fish culturing besides causing human clinical infection worldwide.

Assessment of bacterial contributions to the biochemical changes of chill-stored blunt snout bream (Megalobrama amblycephala) fillets: Protein degradation and volatile organic compounds accumulation.

In this study, we evaluated the contributions of three bacteria (Pseudomonas versuta, Shewanella putrefaciens, and Aeromonas sobria) to the proteolysis, biogenic amines formation, volatile organic compounds accumulation, lipid oxidation, nucleotide catabolism, discoloration, and water migration of bream flesh during chilled storage. The results showed that P. versuta exhibited hydrolyzing activity against sarcoplasmic proteins, and all three strains could degrade myofibrillar proteins, specifically actin. The highest producer of putrescine was S. putrefaciens, which reached a maximum level 5.05 mg/kg after 14 days. Compared with the A. sobria group, hypoxanthine riboside degraded faster in samples inoculated with P. versuta or S. putrefaciens, A. sobria, P. versuta, and S. putrefaciens were responsible for the production of alcohol and aldehydes, whereas only S. putrefaciens produced thiophene and partial esters. Fish flesh inoculated with P. versuta, S. putrefaciens, and A. sobria presented slight green, yellow, and pink discoloration, respectively.

Differences in the digestive enzyme activity, intestinal mucosa and microbial community in loach cultivated in two separate environments.

BACKGROUND: Fish culture in rice paddies can contribute to increasing yields of rice and surplus fish products. Environmental impacts and food-safety issues have become important topics in aquaculture, and organic foods currently were paid attention by researchers and industry practitioners. But the mechanism of differences in quality of Loach (Paramisgurnus dabryanus) reared in rice fields and ponds remains largely uncharacterized. In this study,digestive enzyme activity, intestinal mucosa cells and the gut microbial community of loach were determined under the two separate cultivation modes. RESULTS: The levels of intestinal digestive enzyme activity of fish reared in the paddy-cultivated mode (PACM) were higher (P < 0.05) than those in the pond-cultivated mode (POCM). It was extremely significant (P < 0.01) for the activity of lipase in the liver, foregut and midgut, and for the activities of amylase and trypsin in the hindgut. Acid mucous cells in the loach foregut in PACM were fewer than in POCM (P < 0.01). In summer, the abundance of the Firmicutes, Lactobacillus spp., Aeromonas hydrophila, Enterobacteriaceae and Streptococcus spp. in loach intestinal mucosa in PACM was higher than in POCM. In fall, the abundance of total bacteria, the Bacteroidetes, Bifidobacterium and Enterobacteriaceae in the intestinal mucosa in PACM was likewise higher than in POCM. These differences were significant (P < 0.05 or P < 0.01) between loach in the two separate culture modes for all microorganisms except for A. hydrophila and Streptococcus spp. In addition, quantitative PCR assays showed that some microorganisms presented consistently similar abundances in the gut as in the culture water. CONCLUSIONS: These results showed some enzymatic activities involved in digestion in liver and intestine of loach in PACM were higher than those in POCM, as using digestive enzyme analysis and histological observation of intestinal sections. These findings suggest most of the microorganisms examined in the gut mucosa of loach in the two culture modes significantly differed in abundance between summer and fall. However, some pathogenic bacteria in the gut, particularly A. hydrophila, presented lower abundance in PACM in fall, yet did not differ in abundance between loach in the two cultivation modes.

Effects of fulvic acid on growth performance and intestinal health of juvenile loach Paramisgurnus dabryanus (Sauvage).

A 60-day feeding trial was conducted to determine the effect of dietary fulvic acid supplements on intestinal digestive activity (enzymatic analysis), antioxidant activity, immune enzyme activity and microflora composition of juvenile loach (initial weight of 6.2 ± 0.1 g) reared in experimental aquaria. Five test diets containing 0, 0.5, 1.0, 1.5, and 2% fulvic acid were randomly assigned to three aquaria, respectively. Elevated growth performance including final weight, weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR) was observed in loaches that were fed fulvic acid. Maximal weight gain rates and specific growth rates occurred at the 1.5% additive level. The optimal dietary fulvic requirement for maximal growth of juvenile loach is 16.4 g per kg of the diet based on the quadratic regression analysis of specific growth rate against dietary fulvic acid levels. Furthermore, intestinal protease activity, antioxidant activity, lysozyme activity (LZM), complement 3 (C3) content, immunoglobulin M (IgM) content, acid phosphatase activity (ACP) and alkaline phosphatase activity (AKP) were significantly elevated with concomitant increasing levels of dietary fulvic acid. Following a deep sequencing analysis, a total of 42,058 valid reads and 609 OTUs (operational taxonomic units) obtained from the control group and the group displaying the most optimal growth rate were analyzed. Fulvic acid supplementation resulted in an abundance of Firmicute and Actinobacteria sequences, with a concomitant reduction in the abundance of Proteobacteria. Results indicated that fulvic acid supplementation resulted in a reduction in the relative abundance of Serratia, Acinetobacter, Aeromonas and Edwardsiella, and a relative increase in the abundance of Lactobacillus in the intestine. In conclusion, these results suggest that fulvic acid improves growth performance and intestinal health condition of loach, indicates that fulvic acid could be used as an immunoenhancer in loach culture.

Observational comparisons of intestinal microbiota characterizations, immune enzyme activities, and muscle amino acid compositions of loach in paddy fields and ponds in Sichuan Province.

A balanced intestinal microbial ecosystem is crucial for the growth and health of animals because it can influence the digestion and absorption of nutrients in the intestine. Different culture conditions may change the ecology of microbial in intestine and thus affect the overall growth performance of an animal. In this study, we compared intestinal morphologies, microbiota characterizations, immune enzyme activities, and muscle amino acid compositions of loach cultured in paddy fields and ponds. The fish were fed with the same diets from May 5 to November 5 (2015) in three paddy or ponds. Fish samples were collected for analysis in the August (summer season) and November (fall season) during the feeding trial. In both culture conditions, results based on microscopy observation showed that the intestinal perimeter, fold height, fold radical, and total absorption of the gut were significantly higher in the foregut than that found in the midgut and hindgut (P < 0.01). The average final body weight of fish was similar between the two culture conditions (P > 0.05). The percentage of carcass weight to whole loach weight for samples collected from paddy field (91.6 ± 1.1) was significantly higher than the index measured for loach from pond (87.3 ± 3.4, P < 0.05). Results based on denaturing gradient gel electrophoresis demonstrated that the Shannon-diversity index, evenness, and richness of intestinal flora were increased from summer to fall in paddy cultivation. In pond culture condition, however, the above indexes obtained from mucosa and intestinal contents decreased in fish from summer to fall. The sequencing results of bands indicated that the predominant microorganisms are Proteobacteria, Firmicutes, and Actinobacteria in the intestine of fish being cultured in both cultures. Activities of alkaline phosphatase (AKP, in two culture conditions) and superoxide dismutase (SOD, in paddy field) presented a gradual decrease trend from foregut to hindgut of fish. The activities of acid phosphatase (ACP, in midgut), AKP (in midgut and hindgut), SOD (in foregut), and lysozyme (LZM, in midgut) were significantly higher in fish cultured in paddy than those in pond (P < 0.01). In addition, the percentage of some essential amino acids (valine, methionine, and phenylalanine) based on total amino acids in muscle was significantly higher in fish cultured in paddies than in ponds. In summary, the fish cultured in paddy or pond was not significantly different in growth but the two culture conditions seems to generate different carcass yield and changed the amino acid profiles of fish muscle. The similar predominance microorganisms were identified in the intestine of fish from two conditions, and the quantification of microbial in the intestine will be determined in the future, but part activities involved in immune protection were higher for fish cultured in paddy fields.

Protocol to establish a disease model in Lepidocephalichthys guntea using Aeromonas hydrophila.

The fish disease model facilitates our understanding of disease dynamics, risk assessment for disease outbreaks, the response of the gut immune system, and the maintenance of ecosystem health. Here, we present a protocol for studying gut immunity modulation by infecting Lepidocephalichthys guntea, a loach fish, with Aeromonas hydrophila. We describe steps for performing intra-peritoneal injection on fish and a bath challenge. We detail procedures for conducting periodic population calculations during the infection phase to corroborate Aeromonas hydrophila invasion in the gut. For complete details on the use and execution of this protocol, please refer to Basak and Chakraborty.1.

Identification, expression and bioactivity of Paramisgurnus dabryanus ß-defensin that might be involved in immune defense against bacterial infection.

ß-defensins are a large family of multi-disulfide-bonded peptides with broad-spectrum antimicrobial activities that contribute to innate host defense in many organisms, but little information is available about ß-defensins produced by freshwater fish lacking scales. We therefore cloned and identified a ß-defensin gene from Chinese loach (Paramisgurnus dabryanus) by designing degenerate primers and using thermal asymmetric interlaced PCR. This gene is the first defensin gene ever identified in a non-scaled freshwater fish. Annotation of the protein domain architecture showed that the putative Chinese loach ß-defensin contains the signature motif of six conserved cysteines within the mature peptide, an aspect similar to ß-defensins of other marine fish. We also used quantitative real-time PCR to investigate the expression pattern of the Chinese loach ß-defensin gene, mRNA of which could be observed in various tissues. After challenge with the pathogenic bacterium Aeromonas hydrophila, ß-defensin expression was induced in the eye, gill, skin, and spleen of the adult loach. The bioactivity of the recombinant P. dabryanus ß-defensin was examined against pathogenic bacteria, and the results suggest that this class 2 ß-defensin has potential applications for treatment of bacterial infections.

Identification and characterization of circRNAs in the liver of blunt snout bream (Megalobrama amblycephala) infected with Aeromonas hydrophila.

Circular RNAs (circRNAs), a class of non-coding RNAs, play an important role in regulating various biological processes. In the present study, circRNAs from the Megalobrama amblycephala liver were identified at five different time points post Aeromonas hydrophila using RNA-seq technology. A total of 250 circRNAs were identified, of which 106 were differentially expressed (DE) in ten pairwise comparisons. GO and KEGG analyses showed that the parental genes of DE circRNAs were enriched in phagocytosis, complement and coagulation cascades, and Fc gamma R-mediated phagocytosis pathways. According to ceRNA hypothesis, the interaction network of circRNAs, miRNAs and mRNAs was constructed. Moreover, WGCNA was conducted, and five specific modules significantly related to bacterial infection were identified. All the above results reveal the important role of circRNAs in immune response, which enriches the information of circRNAs in teleost, and helps to understand the immune response mechanism of M. amblycephala to A. hydrophila.

De novo whole transcriptome profiling of Edwardsiella tarda isolated from infected fish (Labeo catla).

Edwardsiella tarda belongs to the genera of Gram negative bacterium mainly associated with edwardsiellosis, the most commonly found infectious fish disease throughout the globe. E. tarda is also a widespread pathogen which cause infections such as cellulitis or gas gangrene and generalized infections in humans. To control the escalating infection of E. trada on various species, it is essential to decoded the mysterious mechanism behind the bacterial infection at transcript level. In this present study, we carry out a de novo E. tarda Whole transcriptome sequencing, isolated from infected fish intestine using SOLiD sequencing platform. RNA-Seq data analysis was performed using various bioinformatics pipelines. Protein-protein interaction study for pathway enrichment and gene ontology study were executed for further investigation. Assembly statistics for E. tarda dataset showed that the number of transcript contigs was 9657 out of which 6749 were GO annotated whereas 1528 were not assigned any GO terms. GO analysis showed that the expressed genes were enhanced with molecular function, cellular component and biological process. A KEGG enrichment study showed that pathway's that are directly linked with immune diseases like Rheumatoid arthritis (0.2%), Tuberculosis (0.3%) Endocytosis (0.6%) was considerably enriched. Protein-protein interaction study showed that most of the expressed proteins were involved in metabolic pathways, flagellar assembly, Propanoate metabolism, Microbial metabolism in diverse environments, Butanoate metabolism and Carbon. The present study provides novel E. tarda transcriptome sequence data, allowing us to identify biologically significant genes and their functional relationship with fish diseases, and will be useful in recognize the reliable therapeutic targets in near feature.

Molecular characterization and expression analysis of interleukin 15 (IL15) and interleukin-15 receptor subunit alpha (IL15Rα) in dojo loach (Misgurnus anguillicaudatus): Their salient roles during bacterial, parasitic and fungal infection.

Interleukin 15 (IL15) is a pleiotropic cytokine that participates in innate and adaptive immunity along with its receptor α-chain (IL15Rα). In order to investigate the potential roles of IL15 and IL15Rα in dojo loach (Misgurnus anguillicaudatus), we firstly cloned the cDNA sequence of Ma-IL15 and Ma-IL15Rα, which contain 1096bp and 1236bp and code proteins of 193 amino acids and 210 amino acids, respectively. A short signal peptide and Pfam IL15 domain were found in Ma-IL15, while a highly conserved sushi domain existed in Ma-IL15Rα. Ontogeny analysis indicated that significantly increased expression of Ma-IL15 and Ma- IL15Rα mRNA were detected in larvae from 1d to 7d post hatching, while relative high expression levels were detected in both systematic and mucosal immune-related tissues of adult dojo loach. Then three dojo loach infection models with F. columnare G4, I. multifiliis and Saprolegnia parasitica were constructed, which resulted in increased skin goblet cells and serious lesions in gill. Ma-IL15 and Ma-IL15Rα showed different expression patterns in different tissues during three infection models. Ma-IL15Rα mRNA was found to be more significantly elevated than Ma-IL15 after infection with F. columnare G4 in all examined tissues including kidney, spleen, gill and skin. I. multifiliis infection induced higher expression of Ma-IL15 in mucosal tissues including skin and gill, while it mainly increased Ma-IL15Rα expression in kidney. Moreover, our study firstly evaluated the influence of fungal infection on IL15 and IL15Rα expression in teleost, and it is interesting to find that both Ma-IL15 and Ma-IL15Rα expression showed consistent up-regulation after Saprolegnia parasitica infection compared to two other infection models. Therefore, our results suggest that Ma-IL15 and Ma-IL15Rα possess important defensive roles in systematic and mucosal tissues of dojo loach during bacterial, fungal and parasitic infection.

[The symbiotic microflora associated with the tegument of proteocephalidean cestodes and the intestines of their fish hosts].

The indigenous symbiotic microflora associated with the tegument of proteocephalidean cestodes and the intestines of their fish hosts has been investigated in morphological and ecological aspects. The indigenous microflora associated with the cestode tegument consists of the nannobacteria population, which was present obligatorily on the surface of tegument, and the "deep microflora". The deep microflora associates with some few species of parasites only. Each individual host-parasite micro-biocenosis includes specific indigenous symbiotic microorganisms, with the differing microfloras of host intestine and parasite. Physiology, biochemistry and/or diet of hosts apparently influence on the symbiotic microflora's structure of parasites. The least bacteria abundance and diversity of their morphotypes were observed in the parasites from baby fishes. The diversity and abundance of bacteria were increased with the fish host ageing and the formation of the definitive structure of its intestine. It is an evidence of the gradual invading of the intestinal parasites (cestodes) tegument by bacterial cells. The invading is realized on the base of the microflora that was present in the food of fish host. The symbiotic microflora has specific morphological features, can regulate the homeostasis of the cestodes and fish hosts and also can maintain equilibrium of alimentary and immune interrelations in the host-parasite system.

Epizootic haematopoietic necrosis virus (EHNV): improved ELISA for detection in fish tissues and cell cultures and an efficient method for release of antigen from tissues.

An improved antigen capture ELISA for the detection of epizootic haematopoietic necrosis virus (EHNV) is described. Affinity purified rabbit anti-EHNV immunoglobulin was used as capture antibody, enabling the detection of EHNV at a concentration of 10(3.5) TCID50/ml cell culture supernatant. The sensitivity and specificity of the ELISA, determined by examination of clarified tissue homogenates from 55 infected fish and 348 uninfected fish in relation to virus isolation, were 81.2% and 98.9%, respectively. Specificity for redfin perch (Perca fluviatilis) tissue samples was 100%, however, high background optical density (OD) in samples from uninfected rainbow trout (Oncorhynchus [corrected] mykiss) resulted in lower specificity (98.5%). Specificity for rainbow trout tissues was increased to 100% by testing clarified tissue homogenates that had been diluted 1:10. Sensitivity and specificity estimates for cell culture supernatants were 96.2% and 99.9% based on the results obtained for 158 infected and 355 uninfected samples. Incubation steps in the ELISA can be shortened to enable its completion in less than 4 h if a rapid qualitative diagnosis is required. Of eight methods of releasing EHNV from infected tissue that were evaluated, manual grinding of tissue with a fitted pestle in a 1.5-ml plastic disposable tube, followed by vortexing in the same tube with 3 mm diameter glass beads and clarification in a microcentrifuge was the most efficient. Samples from 150-200 individual fish could be prepared for ELISA and cell culture by a single operator in a day. Antigen capture ELISA can replace virus isolation as the method of choice for diagnosis of EHNV.