Gut microbiota communities of reciprocal hybrids from koi (Cyprinus carpio) and goldfish (Carassius auratus) are more similar to koi than goldfish.

AIMS: To investigate the gut microbiota communities of reciprocal hybrids and inbred lines of koi (Cyprinus carpio) and goldfish (Carassius auratus), as well as the genetic effect of intestinal microbiota between hybrids and parents. METHODS AND RESULTS: The reciprocal hybrids and inbred lines derived from the parents, koi and goldfish, were established. Then, the bacterial 16S rRNA gene of intestinal contents was sequenced using Illumina Miseq PE300. Alpha diversity in the two types of hybrids was lower than inbred lines of koi or goldfish and was highest in goldfish, followed by koi. For beta diversity, microbial samples presented clear clusters and the two types of hybrids were more similar to koi than goldfish, indicating the gut microbiota of the reciprocal hybrids was more affected by koi. The dominant phyla were Proteobacteria, Actinobacteria and Firmicutes in koi, and Proteobacteria, Fusobacteria and Actinobacteria in goldfish, and Proteobacteria, Fusobacteria and Firmicutes in the reciprocal hybrids. In the case of Proteobacteria, the dominant classes were Alphaproteobacteria and Gammaproteobacteria in four fish. The dominant genera were norank_f_Rhizobiales_Incertae_Sedis and Plesiomonas in koi, Cetobacterium in goldfish, and Cetobacterium and ZOR0006 in the reciprocal hybrids. PICRUSt1 predictive function analysis showed that the reciprocal hybrids had lower abundance in the most functional categories than koi and goldfish. CONCLUSIONS: The gut microbiota of reciprocal hybrids was more affected by koi. Two types of hybrids possessed the same dominated phyla and were different from the inbred lines of koi and goldfish. SIGNIFICANCE AND IMPACT OF THE STUDY: It enhanced our understanding of gut microbiota of hybrid lines of goldfish and koi and provided a new perspective for the selective breeding of gut microbiota traits.

Long-term effects of three compound probiotics on water quality, growth performances, microbiota distributions and resistance to Aeromonas veronii in crucian carp Carassius auratus gibelio.

Probiotics could promote the healthy growth of aquatic animals and have been widely used in aquaculture. However, the influence of high concentration compound probiotics on the aquatic animals has not been reported. In the present study, a compound probiotics was used in high-density culture of crucian carps under the condition of micro-water exchange. During nearly 7-weeks feeding experiment, the aquaculture water quality, growth performances, disease resistance and microbiota distributions of crucian carps were tested. Under the high concentrations of compound probiotics, the content of total ammonia nitrogen and nitrite were finally in a state of dynamic equilibrium. The body length and weight of crucian carps in the experimental group (E) was significantly higher than that in the recirculating group (R). The antioxidant enzymes in the intestines and gills of the E group including SOD, CAT, GSH and MDA, were significantly higher than those in R group. The mortality of crucian carps in E group was significantly lower after the immersion infection of Aeromonas veronii. The addition of compound probiotics significantly increased the number of microorganisms detected in the intestines and gills of crucian carps in E group. The bacteria including Firmicutes, Planctomycetes, Verrucomicrobiota at the phylum level in E group were higher than those in R group. At the genus level, these bacteria (Pirellula, Roseimicrobium, Malikia) were not only higher in E group water, but also significantly higher in the intestines and gills than R group. The results of present study systematically analyzed the impact of high-concentration probiotics on crucian carps breeding, and speculated genus Pirellula, Roseimicrobium, Malikia may be used as aquatic probiotics. The present study will provide a new idea for the green and sustainable development of aquaculture.

A novel application of bubble-eye strain of Carassius auratus for ex vivo fish immunological studies.

In this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at elevated temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and elevated rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

Molecular characterization and biological effect of a C-type lectin receptor in Qihe crucian carp, Carassius auratus.

C-type lectin receptors, as the important members of pattern-recognition receptors, play the crucial roles in the innate immune system, which discriminate self and non-self by recognizing and binding the carbohydrates on the surface of microorganism. In this study, we identified a C-type lectin receptor gene in Qihe crucian carp Carassius auratus (named as CaCLR). The full-length cDNA of CaCLR was composed of 1130 bp, with a 226 bp 5'-untranslated region (UTR), a 792 bp ORF encoding a 263aa protein, and a 112 bp 3'-UTR with a polyadenylation signal sequence AATAAA and a poly (A) tail. The predicted amino acid sequence of CaCLR is a single transmembrane receptor with a typical carbohydrate recognition domain (CRD) at its C-terminus. With regard to the mRNA transcript of CaCLR, it was ubiquitously detected in the tested tissues, among which it was the most abundant in head kidney. The temporal expressions of CaCLR were obviously up-regulated in liver, spleen, kidney, and head kidney after Aeromonas hydrophila and poly I: C challenge, respectively, and the patterns of expression changes were in a time-depended manner. The recombinant CaCLR (rCaCLR) purified from Escherichia coli BL21 (DE3), exhibited strong binding ability with lipopolysaccharide (LPS), peptidoglycan (PGN), ß-Glucan, and Mannan, as well as five microorganisms including fungus (Saccharomyces cerevisiae), Gram-negative bacteria (A. hydrophila, E. coli and Vibrio anguillarum), and Gram-positive bacteria (Micrococcus lysodeikticus). In the presence of rCaCLR, the eliminating capacity against A. hydrophila could be enhanced in C. auratus. Taken together, CaCLR is involved in the antibacterial defense in C. auratus.

Antibiotic-resistant Enterobacteriaceae from diseased freshwater goldfish.

Goldfish farming gained more attention among the ornamental fishes in aquaculture industry. The occurrence of bacterial infections and further antimicrobial treatment lead to the major crisis of antibiotic resistance in aquaculture. We have isolated diverse enterobacteriaceae groups which affect the goldfish and identified their response towards 46 antimicrobials of 15 different classes. Thirteen significant bacterial isolates such as Edwardsiella tarda, Serratia marcescens, Klebsiella aerogenes, Proteus penneri, P. hauseri, Enterobacter cloacae, E. cancerogenus, E. ludwigii, Citrobacter freundii, E. coli, Kluyvera cryocrescens, Plesiomonas shigelloides and Providencia vermicola were recovered from the infected fish with the Shannon-wiener diversity index of 2.556. Multiple antibiotic resistance (MAR) index was found to be maximum for P. penneri (0.87) and minimum for C. freundii and E. cloacae (0.22), highlighting the hyper antibiotic selection pressure in the farm. The minimum concentration of antibiotics required to inhibit most of the resistant isolates was found to be > 256 mcg/ml. All the isolates were susceptible towards ciprofloxacin. Plasmid curing and further AMR tests could reveal the location of antibiotic resistance genes mainly as plasmids which determine the large extent of AMR spread through horizontal gene transfer. This study is the first of its kind to investigate the antimicrobial resistance profile of enterobacteriaceae recovered from goldfish, before and after plasmid curing.

Effect on intestinal microbiota, bioaccumulation, and oxidative stress of Carassius auratus gibelio under waterborne cadmium exposure.

Cadmium (Cd) is a hazardous pollutant known to exert various toxic effects and other sublethal effects on aquatic organisms, and induce a variety of adverse effects on human health, and can be commonly found in environment. The aim of this study was to explore the effects of waterborne Cd exposure on the intestinal microbiota, and Cd accumulation and oxidative response in Carassius auratus gibelio (C. gibelio). The fish were exposed to waterborne Cd at 0, 1, 2, and 4 mg/L for 30 days. Waterborne Cd exposure resulted marked alterations in the composition of microbiota. At the genus level, Bacteroides, Aeromonas, Akkermansia, Acinetobacter, Chryseobacterium, Shinella, Cetobacterium and Bacillus were significantly changed in Cd groups. The results obtained indicate that Cd exposure significantly increased the bioaccumulation level of Cd and profoundly affected antioxidant enzyme including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), total antioxidant (T-AOC), malonaldehyde (MDA), and glutathione-S-transferase (GST). The present findings may provide a new framework for the role of gut microbiota in the response to environmental chemical contamination and in the pathogenesis of body disorders.

Characterization of tetracycline effects on microbial community, antibiotic resistance genes and antibiotic resistance of Aeromonas spp. in gut of goldfish Carassius auratus Linnaeus.

The gut of aquatic animals was a significant niche for dissemination of antibiotic resistance genes (ARGs) and direct response of living conditions. In this study, the gut microbiota of goldfish Carassius auratus Linnaeus was sampled at 7 days and 21 days after treatment with tetracycline at 0.285 and 2.85 µg L-1 to investigate the influences on the microbial structure and antibiotic resistance. The proportion of tetracycline resistance bacteria was 1.02% in the control group, while increased to 23.00%, 38.43%, 62.05% in groups of high concentration for 7 days (H7), low concentration for 21 days (L21) and high concentration for 21 days (H21), respectively. Compared to the control group, the diversity of isolated Aeromonas spp. was decreased in the treatment groups and the minimal inhibitory concentration (MIC) of resistant isolates was enhanced from 32 to 256 µg mL-1 with the treatment of tetracycline in time- and dose-dependent manners. Furthermore, the abundance of most genes was increased in treatment groups and efflux genes mainly responded to the stress of tetracycline with an average level of 1.0 × 10-2. After treatment with tetracycline, the predominant species were changed both at phylum and genus levels. The present study explored the impact of tetracycline on gut microbiota of goldfish at environmentally realistic concentrations for the first time and our findings will provide a reference for characterizing the microbiome of fish in the natural environment.

Quorum quenching potential of Enterococcus faecium QQ12 isolated from gastrointestinal tract of Oreochromis niloticus and its application as a probiotic for the control of Aeromonas hydrophila infection in goldfish Carassius auratus (Linnaeus 1758).

Quorum quenching (QQ), the obstruction of quorum sensing, is the most attractive way to break down the N-acyl-homoserine lactones (AHL) molecules. This work was focused at isolating AHL degrading bacteria from gastrointestinal tract of Oreochromis niloticus, with abilities appropriate for use as probiotic in aquaculture. The presence of an autoinducer inactivation (aiiA) homolog gene and AHL inactivation assay showed that Enterococcus faecium QQ12, which was one among the 20 isolates, could rapidly degrade synthetic C6-HSL in vitro and hampered violacein production by Chromobacterium violaceum. It had excellent biodegrading ability of natural N-AHL produced by Aeromonas hydrophila, suggesting that it can be used as a potential quencher bacterium for disrupting the virulence of A. hydrophila. It was susceptible to all the five antibiotics tried out. The isolate grew well at pH 3.0-7.0, was resistant to high level of bile salts (0-0.9%) and 0.5% of phenol. QQ12 also exhibited high degree of auto-aggregation and co-aggregation, confirming that it possessed good probiotic attributes. Goldfish fed diet incorporated with 108 and 1010 CFU g-1 of the QQ12 for 30 days showed 76.66-86.66% survival when challenged with A. hydrophila. The study indicates that Enterococcus faecium QQ12 could be used as a non-antibiotic feed additive in aquaculture to control bacterial diseases.

Study on Immune Response of Organs of Epinephelus coioides and Carassius auratus After Immersion Vaccination With Inactivated Vibrio harveyi Vaccine.

Immersion vaccination relies on the response of fish mucosa-associated lymphoid tissues, the Crucian carp (Carassius auratus) and Grouper (Epinephelus coioides) were researched in this paper to examine local mucosal immune responses and associated humoral system responses following immersion vaccination. We administered 1.5 × 107 CFU/ml formalin-inactivated Vibrio harveyi cells and measured mucus and serum antibody titers as well as IgM, MHC II mRNA levels in immune organs. The mucosal antibody response preceded the serum response indicating a role for local mucosal immunity in immersion vaccination. IgM and MHC II mRNA levels were relatively greater for the spleen and head kidney indicating the importance and central position of systemic immunity. Expression levels were also high for the gills while skin levels were the lowest. IgM and MHC II mRNA levels were altered over time following vaccination and the hindgut, liver and spleen were similar indicating a close relationship, so the absolute value of r is used to analyze the correlation among different organs immunized. It can be inferred the existence of an internal immune molecular mechanism for Immune synergy hindgut-liver-spleen, from the peak time (14th day), the relative ratio of genes expression in the same tissues between the immunized grouper and the control group (26 times), and Pearson correlation coefficient (0.8<|r|<1). Injection challenges with live V. harveyi indicated that the relative protection rates for the crucian carp and Grouper was basically the same at 44.4% and 47.4%, respectively. It is believe that crucian carp may be used as a substitute for the valuable grouper in immunity experiment, just from aspect of the relative percent survival (RPS) and how it changes with time. But they were not consistent about the IgM mRNA expression between that of crucian carp and grouper after immersion the Vibrio vaccine.

Amelioration of Cd-induced bioaccumulation, oxidative stress and intestinal microbiota by Bacillus cereus in Carassius auratus gibelio.

The heavy metal cadmium (Cd) is a hazardous pollutant known to exert various toxic effects and other sublethal to lethal effects on aquatic organisms, and can be commonly found in environment. The genus Bacillus was one of dominant probiotics, which was commonly used in aquaculture. The aim of this study was to evaluate the effects of dietary administration of Bacillus cereus (B. cereus) on bioaccumulation, oxidative stress and intestinal microbiota of Carassius auratus gibelio (C. gibelio) after Cd exposure. Fish were exposed for 4 weeks to waterborne Cd at 0, 1 and 2 mg/L and/or dietary B. cereus at 108 cfu/g. At 2 and 4 weeks, the fish were sampled and bioaccumulation, antioxidant activity and intestinal microbiota were assessed. Waterborne Cd exposure caused marked alterations in the composition of the microbiota. Dietary supplementation with B. cereus can reverse the changes in the composition of intestinal microbiota in Cd exposure and increase the abundance of Bacteroides, Akkermansia, Cetobacterium in the 0 and 1 mg/L Cd. The results obtained indicate that B. cereus can provide a significant protective effect on the toxicity of cadmium by inhibiting alterations in the levels of bioaccumulation and antioxidant enzyme including Superoxide dismutase (SOD), catalase (CAT), total antioxidant (T-AOC), glutathione (GSH), malonaldehyde (MDA) and Glutathione-S-transferase (GST). The present findings imply that dietary supplement of B. cereus can effectively protect C.gibelio to combat cadmium toxicity.

Diversity of antimicrobial-resistant pathogens from a freshwater ornamental fish farm.

The indiscriminate use of antimicrobials in aquaculture results in antibiotic selection pressure and proliferation of antimicrobial resistant (AMR) bacteria. Frequent assessment of antimicrobial resistance in aquaculture environment is inevitable so as to reduce the passage of clinically important AMR from aquatic to other environment. The present study analysed the antimicrobial resistance of pathogens associated with diseased koi carp and goldfish from an ornamental fish farm. Phenotypic and genotypic characterization of the recovered isolates from both fishes revealed significant pathogens in aquaculture such as Aeromonas, Edwardsiella tarda, Acinetobacter, Lactococcus, Citrobacter, Enterobacter and Comamonas. Shannon-Wiener diversity of koi isolates (2·359) was found to be higher than that of goldfish (1·864). Antibiotic susceptibility testing using disc diffusion with 47 antibiotics revealed significant resistance pattern of Acinetobacter, Comamonas, Klebsiella and Enterobacter from goldfish and Edwardsiella, Aeromonas, Lactococcus, Enterobacter and Acinetobacter from koi with higher multiple antibiotic resistance indexes (>0·3). The minimum inhibitory concentration of antibiotics for the major resistant isolates was found to be very high with >256 µg. All the isolates were susceptible to amoxicillin, kanamycin, cefepime, cefexime, cefotaxime, ceftazidime, doripenem, ciprofloxacin and norfloxacin, recommending their successful application in the farm. SIGNIFICANCE AND IMPACT OF THE STUDY: Antimicrobial resistance is a major threat faced in aquaculture industry. The current study provides baseline information regarding the antibiotic resistance patterns of diverse pathogens recovered from ornamental koi carp and goldfish. The higher MAR index of pathogens and greater MIC of antibiotics for the resistant isolates highlighted the intense use of antibiotics in aquaculture farm. The potential of the pathogens to exhibit resistance even towards the new generation antibiotics remind the need of prudent use of antibiotics and continuous monitoring and surveillance programmes.

Microorganism's adaptation of Crucian carp may closely relate to its living environments.

The relationship between the internal microbiome of an individual organism and that of its external environment has been little investigated in freshwater ecosystems. Thus, this is an area of interest in freshwater fish biology. Along with the genotype of the fish host, external environment plays an important role in determining the composition of the internal microbiome. Here, we characterized the variability of the microbiome of wild Crucian carp (Carassius auratus), along with those of their surrounding environments (water and mud). We found that each environment had distinct bacterial communities, with varying composition and structure. The primary bacterial phyla identified in the Crucian carp gut were Fusobacteria and Proteobacteria (90% of all bacterial phyla identified); the primary genera identified were Cetobacterium, Aeromonas, and Plesiomonas (85% of all bacterial phyla identified). We identified 1,739 operational taxonomic units (OTUs) in the Crucian carp gut, 1,703 in water, and 5,322 in mud. Each environment had unique OTUs, but the fewest unique OTUs (97) were found in the Crucian carp gut. There were significant differences in the relative abundances of different bacterial phyla in the different environments. It may be that only bacterial phyla vital for efficient fish function (e.g., immune response or metabolism), such as Fusobacteria and Proteobacteria, are retained in the Crucian carp gut.

Effect on Serum Parameters and Immune Responses of Carassius auratus gibelio Exposed to Dietary Lead and Bacillus subtilis.

Lead (Pb), a heavy metal and an environmental stressor, may affect many physiological processes, including the serum index and the immune response. The aim of this study was to explore the toxic effects of Pb on the serum index and the immune response of Carassius auratus gibelio (C. gibelio) fed 0, 120, or 240 mg/kg Pb, and 109 cfu/g Bacillus subtilis (B. subtilis). After 15 and 30 days of dietary exposure, the serum indices and the immune responses of the fish were assessed. Dietary Pb exposure significantly affected various components of the serum index, including calcium, magnesium, glucose, cholesterol, total protein, glutamic-pyruvic transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH). However, sIgA activity in the gut increased significantly following B. subtilis supplementation. Notable changes were also observed in the expression levels of immune-related genes, including HSP70, IgM, HSP90, IL-1ß, IL-6, and TNF-α. B. subtilis supplementation effectively attenuated the effects of dietary Pb exposure.

Study of Bioaccumulation, Hematological Parameters, and Antioxidant Responses of Carassius auratus gibelio Exposed to Dietary Lead and Bacillus subtilis.

Lead (Pb) is one of the most ubiquitous and toxic elements in the aquatic environment. Bacillus subtilis (B. subtilis) is a widely used probiotic in aquaculture. The aim of this study was to explore the toxic effects on bioaccumulation, hematological parameters, and antioxidant responses of Carassius auratus gibelio (C. gibelio) exposed to dietary lead at 0, 120, and 240 mg/kg and/or B. subtilis at 109 cfu/g. At 15 and 30 days, the fish were sampled and bioaccumulation, hematological parameters, and antioxidant responses were assessed. The result showed that B. subtilis administration can provide a significant protection against lead toxicity by reducing lead bioaccumulation in tissues, increasing the antioxidant enzymes activity, recovering δ-aminolevulinic acid dehydratase activity and optimizing the hematological parameters. Our results suggested that administration of B. subtilis (109 cfu/g) has the potential to combat dietary lead toxicity in C. gibelio.

Influences of dietary selenomethionine exposure on tissue accumulation, blood biochemical profiles, gene expression and intestinal microbiota of Carassius auratus.

A 30-days feeding trail was conducted to determine the sensitivity of Carassius auratus to the toxicological effects of elevated dietary Selenomethionine (Se-Met). C. auratus averaging 23.56 ±â€¯1.82 g were exposed to four Se-Met concentrations (mg Se/kg): 0 (Se-Met0), 5 (Se-Met5), 10 (Se-Met10) and 20 (Se-Met20) to estimate the effects on tissue selenium (Se) accumulation, blood biochemical profiles, transcript expression and intestinal microbiota. Se accumulated in the kidney, liver and muscle in a dose-dependent manner and followed this order: kidney > liver > muscle, the highest accumulation were obtained in kidney of Se-Met20 diet after 30 days of feeding. Serum contents of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) in fish exposed to Se-Met20 group was significantly highest among Se-Met exposure groups. Hydrogen peroxide (H2O2) concentrations in liver were affected by dietary Se-Met exposures. Liver contents of total antioxidant capacity (TAC), catalase (CAT), glutathione peroxidase (GPx) and malondialdehyde (MDA) in fish exposure to Se-Met5 group was significantly highest among Se-Met exposure groups. Growth hormone receptor (GHR), insulin-like growth factor 1 (IGF-1) and antioxidant enzyme related genes including glutathione peroxidase (GPx), catalase (CAT) and glutathione S-transferase (GST) expression in liver were down-regulated with the concentration of Se-Met exposure groups. The results of high-throughput sequencing showed that gut microbial communities and hierarchy cluster heatmap analysis were significantly affected by Se-Met exposure. The abundances of Cetobacterium and Vibrio increased while fish exposed to Se-Met20 group. The abundance of Ralstonia increased when the Se-Met exposure dose reached 10 mg Se kg-1. The results suggested that the exposure to elevated dietary Se-Met may result toxic effects in C. auratus.

Distribution of Antimicrobial Resistance Genes and Class 1 Integron Gene Cassette Arrays in Motile Aeromonas spp. Isolated from Goldfish (Carassius auratus).

Aeromonas spp. are opportunistic pathogens related to multiple infectious diseases in ornamental fishes. In the present study, the antimicrobial susceptibility, resistance genes, and integrons of 65 goldfish-borne Aeromonas spp. were evaluated. The isolates were identified as A. hydrophila (n = 30), A. veronii (n = 32), and A. punctata (n = 3) by gyrB sequencing. The antimicrobial susceptibility testing of the isolates designated that most of the isolates were resistant to amoxicillin (100.00%), nalidixic acid (100.00%), ampicillin (98.46%), tetracycline (92.31%), rifampicin (86.15%), and cephalothin (61.54%) and each of the isolates showed multiple antimicrobial resistance phenotype (resistant to ≥3 classes of antimicrobials). PCR amplification of antimicrobial resistance genes revealed that the plasmid-mediated quinolone resistance gene, qnrS, was the most prevalent (73.85%) among the isolates. The other antimicrobial resistance genes were detected in the following proportions: qnrB (26.15%), aac(6')-Ib-cr (4.60%), tetA (16.92%), tetE (21.54%), aac(6')-Ib (29.23%), and aphAI-IAB (7.69%). The IntI gene was found in 64.62% isolates, and four class 1 integron gene cassette profiles (incomplete dfrA1, catB3-aadA1, dfrA1-orfC, and qacE2-orfD) were identified. These data suggest that goldfish-borne Aeromonas spp. serve as a reservoir of antimicrobial resistance genes and class 1 integrons.

Significant improvement of intestinal microbiota of gibel carp (Carassius auratus gibelio) after traditional Chinese medicine feeding.

AIMS: Increasing attention has been attracted to intestinal microbiota, due to interactions with nutrition, metabolism and immune defence of the host. Traditional Chinese medicine (TCM) feed additives have been applied in aquaculture to improve fish health, but the interaction with fish gut microbiota is still poorly understood. This study aimed to explore the effect of adding TCM in feed on the intestinal microbiota of gibel carp (Carassius auratus gibelio). METHODS AND RESULTS: Bacterial communities of 16 fish intestinal contents and one water sample were characterized by high-throughput sequencing and analysis of the V4-V5 region of the 16S rRNA gene. The results showed that the composition and structure of the bacterial community were significantly altered by the TCM feeding. Some phyla increased markedly (Proteobacteria, Actinobacteria, Acidobacteria, etc.), while Fusobacteria were significantly reduced. Concurrently, the richness and diversity of the taxonomic units increased, and the microbiota composition of TCM-treated fish was more homogeneous among individuals. At the genus level, the addition of TCM tended to reduce the incidence of potential pathogens (Aeromonas, Acinetobacter and Shewanella), while stimulating the emergence of some potential probiotics (Lactobacillus, Lactococcus, Bacillus and Pseudomonas). CONCLUSIONS: These data suggested that the feed additive could regulate the fish intestinal microbiota by reinforcing the microbial balance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study may provide useful information for further application of TCM for diseases prevention and stress management in aquaculture.

Composition of Gut Microbiota in the Gibel Carp (Carassius auratus gibelio) Varies with Host Development.

To understand how a bacteria-free fish gut ecosystem develops microbiota as the fish ages, we performed a 1-year study on the gut microbiota of hatchling gibel carp (Carassius auratus gibelio). Our results indicate that the gut microbial diversity increases significantly as the fish develop. The gut microbial community composition showed significant shifts corresponding to host age and appeared to shift at two time points despite consistent diet and environmental conditions, suggesting that some features of the gut microbial community may be determined by the host's development. Dietary and environmental changes also seem to cause significant shifts in the fish gut microbial community. This study revealed that the gut microbiota of gibel carp assemble into distinct communities at different times during the host's development and that this process is less affected by the surrounding environment than by the host diet and development. Community phylogenetic analyses based on the net relatedness index further showed that environmental filtering (host selection) deterministically governs the gut microbial community composition. More importantly, the influence of host-associated deterministic filtering tends to weaken significantly over the course of the host's development. However, further studies are needed to assess whether this host development-dependent shift in gut microbiota will still exist under different rearing strategies.

Diversity of culturable bacterial communities in the intestinal tracts of goldfish (Carassius auratus) and their ability to produce N-acyl homoserine lactone.

Intestinal bacteria isolated from goldfish (Carassius auratus) were identified based on 16 ribosomal RNA (rRNA) gene sequences and screened for their ability to produce N-acyl homoserine lactone (AHL), an autoinducer of the quorum sensing (QS) system. The 230 aerobes/facultative anaerobes that were isolated comprised members of the genera Aeromonas (184 isolates), Citrobacter (11), Enterobacter (2), Shewanella (28), Vagococcus (1), and Vibrio (4). Among these genera, the two most abundant species were Aeromonas veronii (163 isolates) and Shewanella xiamenensis (27). In addition, 142 obligate anaerobes consisting of Cetobacterium somerae (139 isolates), Clostridium frigidicarnis (2), and Cetobacterium sp. (1) were also isolated. One hundred seventy isolates (74.2%) belonging to the genera Aeromonas, Citrobacter, Enterobacter, Shewanella, and Vibrio produced AHL, while 155 (67.7%) and 91 (39.7%) isolates possessed the luxR and luxI gene homologs, respectively. None of the obligate anaerobes produced AHL or possessed luxRI homologs. Total viable counts ranged from 1.2 × 107 to 2.2 × 109 CFU/g, which were accounted for 0.8 to 15.2% of direct counts. Aeromonas veronii, S. xiamenensis, and C. somerae were detected from five goldfish at densities ranging from 4.0 × 106 to 1.7 × 109 CFU/g, indicating that these bacteria are dominant components of the culturable gut flora in goldfish. In addition, members of the genera Aeromonas and Shewanella appeared to communicate with each other by using the QS system to some extent when the concentration of AHL reaches a certain threshold. It is therefore suggested that bacteria with the ability to disrupt AHL secretion in intestinal environments are potential candidates for probionts for preventing opportunistic infections in freshwater fish such as goldfish.

Bacillus sp. QSI-1 Modulate Quorum Sensing Signals Reduce Aeromonas hydrophila Level and Alter Gut Microbial Community Structure in Fish.

Quorum sensing (QS) is a cell density dependent process that enables bacteria to communicate with each other based on the production, secretion and sensing of the auto-inducer molecules and then subsequently regulate virulence associated gene expression. Interrupting quorum sensing may represent a novel alternative approach to combat bacterial pathogen. Several bacteria can produce quorum quenching (QQ) enzymes. However, the role of QQ bacteria in shaping the microbiota and the level of N-acyl-homoserine lactones (AHLs, a prevalent type of QS molecules) producing bacteria remains largely unknown. The objective of this study was to examine the presence of AHLs in the fish intestine and investigate the modulation of gut microbiota and its effect on Aeromonas hydrophila level by a QQ enzyme producing probiotic Bacillus sp. QSI-1. AHLs were found in fish gut content and were confirmed in Aeromonas species using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens AT 136 (pZLR4) as reporter strains. We demonstrated that the composition of fish gut microbiota was affected by quenching bacteria QSI-1, and the percentage of A. hydrophila was decreased significantly. Taken together, these results provide valuable insights into QQ enzyme producing probiotics can modulate the microbiota structure and decrease the percentage of AHL-producing pathogenic bacteria in the gut. These data strongly suggest that QQ probiotics may serve as non-antibiotic feed additive in aquaculture to control bacterial diseases.