High-throughput sequencing reveals the gut and lung prokaryotic community profiles of the Chinese giant salamander (Andrias davidianus).

Increasing attention has been attracted to host microbiota, due to their vital impact on host health. Little is known about the microbiota of the Chinese giant salamander (Andrias davidianus), in spite of the high economic and scientific value of this endangered species. This study was designed to characterise and compare the gut and lung prokaryotic communities of the Chinese giant salamander by high-throughput sequencing. Our study showed that the giant salamander had a lung prokaryotic community that clustered separately from its intestinal microbiota. Statistical analysis (LEfSe) revealed that the bacterial populations were dominated by Geobacter, Sulfurimonas, and Dechloromonas from Proteobacteria phylum, and Corynebacterium from Actinobacteria phylum in the lung, while Parabacteroides, Bacteroides, and PW3 from Bacteroidetes phylum, and Oscillospira from Firmicutes phylum were predominant in the intestine. A particularly innovative finding was the fairly high abundance of Archaea, especially methanogenic Euryarchaeota. The gut dominant Archaea were Methanocorpusculum and Thermoplasmata vadinCA11, while Methanosaeta and Methanoculleus were the main Archaea in the lung. PICRUSt analysis revealed differentiated functional profiles between the intestinal miacrobiota and the lung microbiota. Specially, some microbial metabolic functions were significantly more active in the intestinal microbiota, while the functional genes involved in infectious diseases were much richer in the lung microbiota. This study characterized the prokaryotic microbial community profiles in the gut and lung of the Chinese giant salamander, providing foundational support for future study seeking to understand microbiota of the giant salamander and the role of its microbiota on infectious diseases.

Adaptation to Fasting in Crucian Carp (Carassius auratus): Gut Microbiota and Its Correlative Relationship with Immune Function.

Fasting influences the overall physiology of fish, and the knowledge how the gut microbiota, growth performances, and immune function in response to intermittent and long-term fasting is still insufficient. Here, we characterized the effects of fasting on the host-gut microbiota in crucian carp, which would enhance our insight into physiological adaptation to fasting. To achieve this, we investigated the gut microbial communities of crucian carp with different fasting stress, and corresponding immune and growth parameters. The gut microbial communities were structured into four clusters according to different fasting stress, namely one control group (feed regularly), two intermittent fasting groups (fasting period and re-feeding period, respectively), and one long-term fasting group. Intermittent fasting significantly improved the activity of superoxide dismutase (SOD) and lysozyme (LZM) (ANOVA, p < 0.05) and significantly increased alpha diversity and ecosystem stability of gut microbiota (ANOVA, p < 0.05). Gut length (GL) and condition factor (CF) showed no significant difference between the control group (CG) and intermittent fasting group under re-feeding period (RIF) (ANOVA, p = 0.11), but relative gut length (RGL) in group RIF was higher than that in the CG (ANOVA, p = 0.00). The bacterial genera Bacteroides, Akkermansia, and Erysipelotrichaceae were enriched in fishes under intermittent fasting. Two Bacteroides OTUs (OTU50 and OTU1292) correlated positively with immune (SOD, complement, and LZM) and growth (GL and RGL) parameters. These results highlight the possible interplay between growth performances, immune function, and gut microbiota in response to fasting.

Managing the Microbial Community of Marine Fish Larvae: A Holistic Perspective for Larviculture.

The availability of high-quality juveniles is a bottleneck in the farming of many marine fish species. Detrimental larvae-microbe interactions are a main reason for poor viability and quality in larval rearing. In this review, we explore the microbial community of fish larvae from an ecological and eco-physiological perspective, with the aim to develop the knowledge basis for microbial management. The larvae are exposed to a huge number of microbes from external and internal sources in intensive aquaculture, but their relative importance depend on the rearing technology used (especially flow-through vs. recirculating systems) and the retention time of the water in the fish tanks. Generally, focus has been on microbes entering the system, but microbes from growth within the system is normally a substantial part of the microbes encountered by larvae. Culture independent methods have revealed an unexpected high richness of bacterial species associated with larvae, with 100-250 operational taxonomic units associated with one individual. The microbiota of larvae changes rapidly until metamorphosis, most likely due to changes in the selection pressure in the digestive tract caused by changes in host-microbe and microbe-microbe interactions. Even though the microbiota of larvae is distinctly different from the microbiota of the water and the live food, the microbiota of the water strongly affects the microbiota of the larvae. We are in the early phase of understanding larvae-microbe interactions in vivo, but some studies with other animals than fish emphasize that we so far have underestimated the complexity of these interactions. We present examples demonstrating the diversity of these interactions. A large variety of microbial management methods exist, focusing on non-selective reduction of microbes, selective enhancement of microbes, and on improvement of the resistance of larvae against microbes. However, relatively few methods have been studied extensively. We believe that there is a lot to gain by increasing the diversity of approaches for microbial management. As many microbial management methods are perturbations of the microbial community, we argue that ecological theory is needed to foresee and test for longer term consequences in microbe-microbe and microbe-larvae interactions. We finally make some recommendations for future research and development.

Multi-Omics Analysis Reveals a Correlation between the Host Phylogeny, Gut Microbiota and Metabolite Profiles in Cyprinid Fishes.

Gut microbiota play key roles in host nutrition and metabolism. However, little is known about the relationship between host genetics, gut microbiota and metabolic profiles. Here, we used high-throughput sequencing and gas chromatography/mass spectrometry approaches to characterize the microbiota composition and the metabolite profiles in the gut of five cyprinid fish species with three different feeding habits raised under identical husbandry conditions. Our results showed that host species and feeding habits significantly affect not only gut microbiota composition but also metabolite profiles (ANOSIM, p ≤ 0.05). Mantel test demonstrated that host phylogeny, gut microbiota, and metabolite profiles were significantly related to each other (p ≤ 0.05). Additionally, the carps with the same feeding habits had more similarity in gut microbiota composition and metabolite profiles. Various metabolites were correlated positively with bacterial taxa involved in food degradation. Our results shed new light on the microbiome and metabolite profiles in the gut content of cyprinid fishes, and highlighted the correlations between host genotype, fish gut microbiome and putative functions, and gut metabolite profiles.

Bacterial Signatures of "Red-Operculum" Disease in the Gut of Crucian Carp (Carassius auratus).

Fish gut microbiota play important roles in fish immunity, nutrition, and the adaptation to environmental changes. To date, few studies have focused on the interactions among environmental factors, fish diseases, and gut microbiota compositions. We compared the gut bacterial communities of healthy crucian carps (Carassius auratus) with those of individuals affected by "red-operculum" disease and corresponding water and sediment microbiota in four fish farm ponds. Distinct gut bacterial communities were observed in healthy and diseased fish. The bacterial communities of diseased fish were less diverse and stable than those of healthy individuals. The differences in bacterial community compositions between diseased and healthy fish were explained by the changes in the relative abundances of some specific bacterial OTUs, which belonged to the genera such as Vibrio, Aeromonas, and Shewanella, and they were prevalent in diseased fish, but rare or even absent in environmental samples. Water temperature and ammonia concentration were the two most important environmental factors that impacted gut microbiota in diseased fish. These results highlighted the surge of some potential pathogens as bacterial signatures that were associated with "red-operculum" disease in crucian carps.

The highly variable microbiota associated to intestinal mucosa correlates with growth and hypoxia resistance of sea bass, Dicentrarchus labrax, submitted to different nutritional histories.

BACKGROUND: The better understanding of how intestinal microbiota interacts with fish health is one of the key to sustainable aquaculture development. The present experiment aimed at correlating active microbiota associated to intestinal mucosa with Specific Growth Rate (SGR) and Hypoxia Resistance Time (HRT) in European sea bass individuals submitted to different nutritional histories: the fish were fed either standard or unbalanced diets at first feeding, and then mixed before repeating the dietary challenge in a common garden approach at the juvenile stage. RESULTS: A diet deficient in essential fatty acids (LH) lowered both SGR and HRT in sea bass, especially when the deficiency was already applied at first feeding. A protein-deficient diet with high starch supply (HG) reduced SGR to a lesser extent than LH, but it did not affect HRT. In overall average, 94 % of pyrosequencing reads corresponded to Proteobacteria, and the differences in Operational Taxonomy Units (OTUs) composition were mildly significant between experimental groups, mainly due to high individual variability. The highest and the lowest Bray-Curtis indices of intra-group similarity were observed in the two groups fed standard starter diet, and then mixed before the final dietary challenge with fish already exposed to the nutritional deficiency at first feeding (0.60 and 0.42 with diets HG and LH, respectively). Most noticeably, the median percentage of Escherichia-Shigella OTU_1 was less in the group LH with standard starter diet. Disregarding the nutritional history of each individual, strong correlation appeared between (1) OTU richness and SGR, and (2) dominance index and HRT. The two physiological traits correlated also with the relative abundance of distinct OTUs (positive correlations: Pseudomonas sp. OTU_3 and Herbaspirillum sp. OTU_10 with SGR, Paracoccus sp. OTU_4 and Vibrio sp. OTU_7 with HRT; negative correlation: Rhizobium sp. OTU_9 with HRT). CONCLUSIONS: In sea bass, gut microbiota characteristics and physiological traits of individuals are linked together, interfering with nutritional history, and resulting in high variability among individual microbiota. Many samples and tank replicates seem necessary to further investigate the effect of experimental treatments on gut microbiota composition, and to test the hypothesis whether microbiotypes may be delineated in fish.

Alterations of the gut microbiome of largemouth bronze gudgeon (Coreius guichenoti) suffering from furunculosis.

High-throughput sequencing was applied to compare the intestinal microbiota in largemouth bronze gudgeon either healthy or affected by furunculosis. Proteobacteria, Actinobacteria, Tenericutes, Firmicutes and Bacteroidetes were detected as the predominant bacterial phyla in the gut of both diseased and healthy fish. The abundance of Proteobacteria differed significantly between the two groups of fish, mainly due to the overwhelming prevalence of Aeromonas in the diseased fish (81% ± 17%), while the genus was unevenly spread among the apparently healthy fish (33% ± 33%). The bacterial diversity in the intestine of diseased fish was markedly lower than in healthy fish. Analysis revealed the significant dissimilarity between the gut microbiota of diseased and healthy fish. The bacterial profiles in the gut were further characterized with the 28 phylotypes that were shared by the two groups. In diseased fish, two shared OTUs (OTU0001 and OTU0013) were closely related to Aeromonas salmonicida, their total proportion exceeding 70% of the sequences in diseased fish, while averaging 5.2% ± 4.6% in the healthy fish. This result suggested the presence of healthy carriers of pathogenic A. salmonicida among the farmed fish, and the gut appeared as a probable infection source for furunculosis in largemouth bronze gudgeon.

Comparative analysis of the intestinal bacterial communities in different species of carp by pyrosequencing.

Gut microbiota is increasingly regarded as an integral component of the host, due to important roles in the modulation of the immune system, the proliferation of the intestinal epithelium and the regulation of the dietary energy intake. Understanding the factors that influence the composition of these microbial communities is essential to health management, and the application to aquatic animals still requires basic investigation. In this study, we compared the bacterial communities harboured in the intestines and in the rearing water of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius cuvieri), and bighead carp (Hypophthalmichthys nobilis), by using 454-pyrosequencing with barcoded primers targeting the V4 to V5 regions of the bacterial 16S rRNA gene. The specimens of the three species were cohabiting in the same pond. Between 6,218 and 10,220 effective sequences were read from each sample, resulting in a total of 110,398 sequences for 13 samples from gut microbiota and pond water. In general, the microbial communities of the three carps were dominated by Fusobacteria, Firmicutes, Proteobacteria and Bacteroidetes, but the abundance of each phylum was significantly different between species. At the genus level, the overwhelming group was Cetobacterium (97.29 ± 0.46 %) in crucian carp, while its abundance averaged c. 40 and 60 % of the sequences read in the other two species. There was higher microbial diversity in the gut of filter-feeding bighead carp than the gut of the two other species, with grazing feeding habits. The composition of intestine microbiota of grass carp and crucian carp shared higher similarity when compared with bighead carp. The principal coordinates analysis (PCoA) with the weighted UniFrac distance and the heatmap analysis suggested that gut microbiota was not a simple reflection of the microbial community in the local habitat but resulted from species-specific selective pressures, possibly dependent on behavioural, immune and metabolic characteristics.

Intestinal microbiota of gibel carp (Carassius auratus gibelio) and its origin as revealed by 454 pyrosequencing.

The intestinal microbiota has received increasing attention, as it influences growth, feed conversion, epithelial development, immunity as well as the intrusion of pathogenic microorganisms in the intestinal tract. In this study, pyrosequencing was used to explore the bacterial community of the intestine in gibel carp (Carassius auratus gibelio), and the origin of these microorganisms. The results disclosed great bacterial diversities in the carp intestines and cultured environments. The gibel carp harbored characteristic intestinal microbiota, where Proteobacteria were predominant, followed by Firmicutes. The analysis on the 10 most abundant bacterial operational taxonomic units (OTUs) revealed a majority of Firmicutes in the intestinal content (by decreasing order: Veilonella sp., Lachnospiraceae, Lactobacillales, Streptococcus sp., and Lactobacillus sp.). The second most abundant OTU was Rothia sp. (Actinobacteria). The most likely potential probiotics (Lactobacillus sp., and Bacillus sp.) and opportunists (Aeromonas sp., and Acinetobacter sp.) were not much abundant. Bacterial community comparisons showed that the intestinal community was closely related to that of the sediment, indicating the importance of sediment as source of gut bacteria in gibel carp. However, 37.95 % of the OTUs detected in feed were retrieved in the intestine, suggesting that food may influence markedly the microbiota of gibel carp, and therefore may be exploited for oral administration of probiotics.

Influence of the diet on the microbial diversity of faecal and gastrointestinal contents in gilthead sea bream (Sparus aurata) and intestinal contents in goldfish (Carassius auratus).

Fish intestinal microbiota changes with the diet and this effect is of particular interest considering the increasing substitution of fish meal by plant protein sources. The objective of this work was to study the effects of partial substitution of fish meal with lupin and rapeseed meals on gut microbiota of the gilthead sea bream (Sparus aurata) and in goldfish (Carassius auratus). Faecal, gastrointestinal and intestinal contents were characterized using culture-based and molecular methods. Vibrionaceae was high in faeces and in the intestine of sea bream, while a more diverse microbiota was retrieved from the stomach, where Bacillales and Flavobacteriaceae appeared to be influenced by the diet. PCR-denaturing gradient gel electrophoresis profiles revealed a high diversity of the microbiota transiting in the sea bream digestive tract, with a shift between gastric and intestinal communities, especially in the group fed with lupin meal. The goldfish was different, with a predominance of Aeromonas spp., Shewanella putrefaciens and Staphylococcus spp. among the aerotolerant-cultivable bacteria. The culture-independent methods revealed the presence of anaerobes like Cetobacterium somerae, and that of Vibrio spp., likely in a viable, but noncultivable state. There was a trend towards decreasing diversity in goldfish microbiota with the partial substitution by lupin, which seemed to inhibit some taxa.

Updating the importance of lactic acid bacteria in fish farming: natural occurrence and probiotic treatments.

Many recent papers have deepened the state of knowledge about lactic acid bacteria (LAB) in fish gut. In spite of high variability in fish microbiota, LAB are sometimes abundant in the intestine, notably in freshwater fish. Several strains of Streptococcus are pathogenic to fish. Streptococcus iniae and Lactococcus garvieae are major fish pathogens, against which commercial vaccines are available. Fortunately, most LAB are harmless, and some strains have been reported for beneficial effects on fish health. A major step forward in recent years was the converging evidence that LAB can stimulate the immune system in fish. An open question is whether viability can affect immunostimulation. The issue is crucial to commercialize live probiotics rather than inactivated preparations or extracts. There has been a regain of interest in allochthonous strains used as probiotics for terrestrial animals or humans, due to economical and regulatory constraints, but the short survival in sea water may limit application to marine fish. If viability is required, alternative treatments may include the incorporation of prebiotics in feed, and other dietary manipulations that could promote intestinal LAB. Antagonism to pathogens is the other main feature of candidate probiotics, and there are many reports concerning mainly carnobacteria and Enterococcus. Some bacteriocins were characterized which may be of interest not only for aquaculture, but also for food preservation.