Effect of temperature on proteinase activities of enteral microbiota and intestinal mucosa of fish of different ecological group

Effect of temperature on proteinases activities of enteral microbiota and of intestinal mucosa was studied in five fish species (roach Rutilus rutilus, crucian carp Carassius carassius, common perch Perca fluviatilis, pike-perch Zander lucioperca, and pike Esox lucius) belonging by the nutrition type to different ecological groups. Essential differences of temperature characteristics of proteinases of intestinal mucosa and of enteral microbiota are revealed in fish belonging by the nutrition type to different ecologic groups. The character of the t0-function of proteinases of intestinal mucosa and enteral microbiota by casein and hemoglobin as a rule is different. The highest values of relative proteinases activities for casein in the zone of low temperatures (38 and 45.3 % of the maximal activity) are found at study of proteinases of enteral microbiota in common perch and crucian carp. The latter indicates a significant adaptability of the enteral microbiota proteinases of common perch and crucial carp to functioning at low temperatures.     

Effect of temperature on proteinase activites of enteral microbiota and intestinal mucosa of fish of different ecological groups

Effect of temperature on proteinases activities of enteral microbiota and of intestinal mucosa was studied in five fish species (roach Rutilus rutilus, crucian carp Carassius carassius, common perch Perca fluviatilis, pike-perch Zander lucioperca, and pike Esox lucius) belonging by the nutrition type to different ecological groups. Essential differences of temperature characteristics of proteinases of intestinal mucosa and of enteral microbiota are revealed in fish belonging to different ecological groups. The character of the t-function of proteinases of intestinal mucosa and enteral microbiota for casein and hemoglobin as a rule is different. The values of the apparent E _act proteinases of intestinal mucosa for casein in most cases are higher than those of enteral microbiota, while those for hemoglobin, on the contrary, are lower. The lowest values of relative proteinase activities for casein in the zone of low temperatures (38 and 45.3% of the maximal activity) and the E_act value (less than 2.0 kcal/mol) are found at study of proteinases of enteral microbiota in common perch and crucian carp. The latter indicates a significant adaptability of the enteral microbiota proteinases of common perch and crucian carp to functioning at low temperatures.     

Effect of temperature on proteinase activities in intestinal chyme and mucosa of fish of different ecological groups

Effect of temperature on activities of proteinases in intestinal chyme and mucosa was studied in three fish species (pike-perch, zope, roach) belonging to different ecological groups by their nutrition type. There was revealed a significant difference of dependence of enzyme activities in chyme on temperature in the benthophage, roach (a higher level of relative activity in the range of lower temperatures and a wider zone of temperature optimum) as well as of values of apparent energy of activation of the protein hydrolysis process as compared with that in planktoand ichtyophages, zope and pike-perch, which indicates a significant effect of the enteral microbiota proteinases and of nutrition objects on characteristics of hydrolases functioning in fish intestine.     

Effect of temperature on proteinase activities in the intestinal chyme and the mucosa of various ecological fish groups

Effect of temperature on activities of proteinases in intestinal chyme and mucosa was studied in three fish species (pike-perch, zope, roach) belonging to different ecological groups by their type of feeding. There was revealed a significant difference of dependence of enzyme activities in chyme on temperature in the benthophage roach (a higher level of relative activity in the zone of lower temperatures and a larger zone of temperature optimum) as well as of values of apparent energy of activation of the protein hydrolysis process as compared with that in plankto- and ichthyophages--zope and pike-perch, which indicates a significant effect of the enteral microbiota proteinases and of nutrition objects on characteristics of hydrolases functioning in fish intestine.     

Effect of pH on the proteinase activities in the intestine mucosa,chyme, and enteral microbiota in the piscivorous fish differing in their ecological traits

The effect of pH on the activity of proteinases of intestine mucosa, chyme, and enteral microbiota was studied in three piscivorous fish species of the Rybinsk Reservoir differing in their ecological traits: pike Esox lucius, burbot Lota lota, and zander Sander lucioperca. In all fish species, studied pH of ten is optimal for the functioning of proteinases of intestine mucosa; in chyme proteinases, optimal pH values vary from six to ten. Optimal pH for functioning of proteinases of enteral microbiota is seven for zander and pike; pH of nine, for burbot. High activity of chyme and microbiota proteinases within the diapason of pH values of six to nine is a characteristic of burbot. Relative activity of proteinases of intestine mucosa in all fish species is not more than 15% of the maximal activity; that of chyme and enteral microbiota is lower than 40% in zander, close to 50% in burbot, and 80 and 50%, respectively, in pike. It is suggested that diversity of the patterns of pH-dependence of enteral microbiota proteinases relates to the specificity of microbiota in various ecological zones of the reservoir (littoral, sublittoral, and bathyal).     

Effect of pH on the activity of proteinases in intestinal mucosa,chyme, and microbiota of fish from the Cuciurgan reservoir

Proteolytic activities of the intestinal mucosa, chyme, and enteral microbiota have been studied in a wide range of pH values in five fish species from the Cuciurgan reservoir (Moldova). Differences in pH dependence of the intestinal proteinase activity of fish are determined by their feeding type. The maximum activity of proteinases is found in the pumpkinseed Lepomis gibbosus. The minimum activity of proteinases has been demonstrated by the zander Zander lucioperca. The pH optimum of the mucosa and chyme in all fish species (except for the European perch Perca fluviatilis) is 10. The pH optimum of the intestinal microflora varies from 6.0 (in the common carp Cyprinus carpio) to 10 (in the crucian carp Carassius carassius), whereas that in the perch from the Cuciurgan and Rybinsk reservoirs is 7. The majority of fish species, mostly Zander lucioperca and Lepomis gibbosus, are characterized by high proteniase activity of the microbiota, in the pH ranging from 6 to 9. It is assumed that proteinases in the enteral microbiota of fish are able to make up for the relatively low activity of those synthesized by their digestive system in the range of low pH values.     

Changes in microbiota of rainbow trout caused by sediments contamination

The abundance, composition and hydrocarbon-degrading bacteria, as possible biomarkers of contamination with oil hydrocarbons, of autochthonous and alochtonous microbiota of the digestive tract of rainbow trout have been estimated. The samples of the bottom sediments for microbiological tests have been collected and a response of natural bacterial communities in the digestive tract of rainbow trout and nutritional changes has been investigated. Experimental fish have been fed with a mixture of three substances with the aim to assess the influence of hydrocarbon-degrading bacteria contained in the sediments on the microbiota of rainbow trout’s digestive tracts. The abundance values of rainbow trout intestinal heterotrophic bacteria were found to change depending on alochtonous microbiota of different bottom sediments given to the experimental fish with food in vitro. According to the results of our research, it is likely that the changes in the abundance values of the microbiota of the digestive tract of fish and in the proportions of functional groups of the bacteria allow us to determine changes in the functional activity of bacteria depending on food composition. Any relative increase or decrease of abundance or activity of alochtonous microbiota allows the prediction of toxic effects of the contaminants on animals and the environment.     

Evening rather than morning increased physical activity alters the microbiota in mice and is associated with increased body temperature and sympathetic nervous system activation

Voluntary training and food modulate the fecal microbiota in humans and mice. Although there are some reports of the timing effects of voluntary training and feeding on metabolism, the timing effects of these factors on microbiota have not been investigated. Therefore, we investigated the effects of the timing of voluntary training and feeding on the gut microbiota.The ICR mice were housed under conditions with an early (in the morning) or late (evening) active phase of increased physical activity. Furthermore, to investigate why voluntary training affects the gut microbiota, mice were housed in a cold environment and received propranolol administration with increased physical activity. After that, we collected cecal contents and feces and measured cecal pH. Short-chain fatty acids (SCFA) were measured from cecal contents. Microbiota was determined using sequencing of the V3-V4 region of the 16S rDNA gene.This study found that increased evening physical activity rather than morning activity decreases cecal pH, increases SCFA, and changes the microbiota. It is especially important that increased evening physical activity is induced under the post-prandial voluntary training condition. Also, we found that cold room housing, sympathetic blockade, or both suppressed the increased physical activity-induced changes in cecal pH, SCFA, and microbiota. Allobaculum responded to increased physical activity through body temperature increases and sympathetic activation.Post-prandial increased physical activity, rather than pre-prandial increased physical activity by evening voluntary wheel training, altered the microbiota composition, which may be related to the increase in body temperature and sympathetic nervous system activation.     

Aquacultured Rainbow Trout (Oncorhynchus mykiss) Possess a Large Core Intestinal Microbiota That Is Resistant to Variation in Diet and Rearing Density

As global aquaculture fish production continues to expand, an improved understanding of how environmental factors interact in fish health and production is needed. Significant advances have been made toward economical alternatives to costly fishmeal-based diets, such as grain-based formulations, and toward defining the effect of rearing density on fish health and production. Little research, however, has examined the effects of fishmeal- and grain-based diets in combination with alterations in rearing density. Moreover, it is unknown whether interactions between rearing density and diet impact the composition of the fish intestinal microbiota, which might in turn impact fish health and production. We fed aquacultured adult rainbow trout (Oncorhynchus mykiss) fishmeal- or grain-based diets, reared them under high- or low-density conditions for 10 months in a single aquaculture facility, and evaluated individual fish growth, production, fin indices, and intestinal microbiota composition using 16S rRNA gene sequencing. We found that the intestinal microbiotas were dominated by a shared core microbiota consisting of 52 bacterial lineages observed across all individuals, diets, and rearing densities. Variations in diet and rearing density resulted in only minor changes in intestinal microbiota composition despite significant effects of these variables on fish growth, performance, fillet quality, and welfare. Significant interactions between diet and rearing density were observed only in evaluations of fin indices and the relative abundance of the bacterial genus Staphylococcus. These results demonstrate that aquacultured rainbow trout can achieve remarkable consistency in intestinal microbiota composition and suggest the possibility of developing novel aquaculture strategies without overtly altering intestinal microbiota composition.     

Seasonal habitat drives intestinal microbiome composition in anadromous Arctic char (Salvelinus alpinus)

Intestinal microbial communities from 362 anadromous Arctic char (Salvelinus alpinus) from the high Arctic Kitikmeot region, Nunavut, Canada, were characterized using high-throughput 16S rRNA gene sequencing. The resulting bacterial communities were compared across four seasonal habitats that correspond to different stages of annual migration. Arctic char intestinal communities differed by sampling site, salinity and stages of freshwater residence. Although microbiota from fish sampled in brackish water were broadly consistent with taxa seen in other anadromous salmonids, they were enriched with putative psychrophiles, including the nonluminous gut symbiont Photobacterium iliopiscarium that was detected in >90% of intestinal samples from these waters. Microbiota from freshwater-associated fish were less consistent with results reported for other salmonids, and highly variable, possibly reflecting winter fasting behaviour of these char. We identified microbiota links to age for those fish sampled during the autumn upriver migration, but little impact of the intestinal content and water microbiota on the intestinal community. The strongest driver of intestinal community composition was seasonal habitat, and this finding combined with identification of psychrophiles suggested that water temperature and migratory behaviour are key to understanding the relationship between Arctic char and their symbionts.     

Bovine serum eliminates rapid nonspecific toxic reactions during bioassay of stored fish for Clostridium botulinum toxin

When stored fish or some fish products were tested for the presence of Clostridium botulinum toxin, nonspecific toxic reactions in mice often occurred, rendering the bioassay inconclusive. The nonspecific toxic reactions were mediated by the gram-negative microbiota, inherent to the fish, which were the source of lethal, heat-stable endotoxins. The treatment of assay samples with bovine serum eliminated nonspecific reactions through the interaction of constituent serum immunoglobulin M (IgM) with endotoxic material. Removal of IgM from bovine serum through treatment with protein A or concanavalin A resulted in a loss of protective activity.     

Bovine serum eliminates rapid nonspecific toxic reactions during bioassay of stored fish for Clostridium botulinum toxin.

When stored fish or some fish products were tested for the presence of Clostridium botulinum toxin, nonspecific toxic reactions in mice often occurred, rendering the bioassay inconclusive. The nonspecific toxic reactions were mediated by the gram-negative microbiota, inherent to the fish, which were the source of lethal, heat-stable endotoxins. The treatment of assay samples with bovine serum eliminated nonspecific reactions through the interaction of constituent serum immunoglobulin M (IgM) with endotoxic material. Removal of IgM from bovine serum through treatment with protein A or concanavalin A resulted in a loss of protective activity.     

Characterization of the gut microbiota in early life stages of pikeperch Sander lucioperca

This study characterized the gastrointestinal microbiome of nine juvenile farmed pikeperch Sander lucioperca using a metagenomics approach based on bacterial 16S rRNA gene sequencing. Potential changes in the gut microbiota during 2 months of S. lucioperca juvenile life were investigated. Results revealed that gut microbiota was dominated by Proteobacteria (95–92%), while other phyla Firmicutes (1–1·5%) and Actinobacteria (0·9–1·5%) were less abundant. At the family level, fish‐gut microbiota were dominated by Enterobacteriaceae, which constituted c. 83% of all DNA sequence reads. Such a situation was present in all of the examined fish except one, which showed a different proportion of particular microbial taxa than the other fish. In this fish, a higher relative abundance (%) of Fusobacteria (21·0%), Bacteroidetes (9·5%) and Firmicutes (7·5%) was observed. There were no significant differences in the gut microbiome structure at different stages of development in the examined fish. This may indicate that Proteobacteria inhabiting the gut microbiota at an early stage of life are a necessary component of the pikeperch microbiome that may support proper nutrition of the fish. The information obtained on the gut microbiome could be useful in determining juvenile S. lucioperca health and improving rearing conditions by welfare monitoring in aquaculture.     

Inter Individual Variations of the Fish Skin Microbiota: Host Genetics Basis of Mutualism?

The commensal microbiota of fish skin is suspected to provide a protection against opportunist infections. The skin of fish harbors a complex and diverse microbiota that closely interacts with the surrounding water microbial communities. Up to now there is no clear evidence as to whether the host regulates the recruitment of environmental bacteria to build a specific skin microbiota. To address this question, we detected Quantitative Trait Loci (QTL) associated with the abundance of specific skin microbiota bacterial strains in brook charr (Salvelinus fontinalis), combining 16S RNA tagged-amplicon 454 pyrosequencing with genetic linkage analysis. Skin microbiota analysis revealed high inter-individual variation among 86 F2 fish progeny based upon the relative abundance of bacterial operational taxonomic units (OTUs). Out of those OTUs, the pathogenic strain Flavobacterium psychrophilum and the non-pathogenic strain Methylobacterium rhodesianum explained the majority of inter-individual distances. Furthermore, a strong negative correlation was found between Flavobacterium and Methylobacterium, suggesting a mutually competitive relationship. Finally, after considering a total of 266 markers, genetic linkage analysis highlighted three major QTL associated with the abundance of Lysobacter, Rheinheimera and Methylobacterium. All these three genera are known for their beneficial antibacterial activity. Overall, our results provide evidence that host genotype may regulate the abundance of specific genera among their surface microbiota. They also indicate that Lysobacter, Rheinheimera and Methylobacterium are potentially important genera in providing protection against pathogens.     

Intestinal microbiota composition in fishes is influenced by host ecology and environment

The digestive tracts of vertebrates are colonized by complex assemblages of micro-organisms, collectively called the gut microbiota. Recent studies have revealed important contributions of gut microbiota to vertebrate health and disease, stimulating intense interest in understanding how gut microbial communities are assembled and how they impact host fitness (Sekirov et al. 2010). Although all vertebrates harbour a gut microbiota, current information on microbiota composition and function has been derived primarily from mammals. Comparisons of different mammalian species have revealed intriguing associations between gut microbiota composition and host diet, anatomy and phylogeny (Ley et al. 2008b). However, mammals constitute <10% of all vertebrate species, and it remains unclear whether similar associations exist in more diverse and ancient vertebrate lineages such as fish. In this issue, Sullam et al. (2012) make an important contribution toward identifying factors determining gut microbiota composition in fishes. The authors conducted a detailed meta-analysis of 25 bacterial 16S rRNA gene sequence libraries derived from the intestines of different fish species. To provide a broader context for their analysis, they compared these data sets to a large collection of 16S rRNA gene sequence data sets from diverse free-living and host-associated bacterial communities. Their results suggest that variation in gut microbiota composition in fishes is strongly correlated with species habitat salinity, trophic level and possibly taxonomy. Comparison of data sets from fish intestines and other environments revealed that fish gut microbiota compositions are often similar to those of other animals and contain relatively few free-living environmental bacteria. These results suggest that the gut microbiota composition of fishes is not a simple reflection of the micro-organisms in their local habitat but may result from host-specific selective pressures within the gut (Bevins & Salzman 2011).     

Intestinal epigenotype of Atlantic salmon (Salmo salar) associates with tenacibaculosis and gut microbiota composition

It remains a challenge to obtain the desired phenotypic traits in aquacultural production of Atlantic salmon, and part of the challenge might come from the effect that host-associated microorganisms have on the fish phenotype. To manipulate the microbiota towards the desired host traits, it is critical to understand the factors that shape it. The bacterial gut microbiota composition can vary greatly among fish, even when reared in the same closed system. While such microbiota differences can be linked to diseases, the molecular effect of disease on host-microbiota interactions and the potential involvement of epigenetic factors remain largely unknown.The aim of this study was to investigate the DNA methylation differences associated with a tenacibaculosis outbreak and microbiota displacement in the gut of Atlantic salmon. Using Whole Genome Bisulfite Sequencing (WGBS) of distal gut tissue from 20 salmon, we compared the genome-wide DNA methylation levels between uninfected individuals and sick fish suffering from tenacibaculosis and microbiota displacement. We discovered >19,000 differentially methylated cytosine sites, often located in differentially methylated regions, and aggregated around genes.The 68 genes connected to the most significant regions had functions related to the ulcerous disease such as epor and slc48a1a but also included prkcda and LOC106590732 whose orthologs are linked to microbiota changes in other species. Although the expression level was not analysed, our epigenetic analysis suggests specific genes potentially involved in host-microbiota interactions and more broadly it highlights the value of considering epigenetic factors in efforts to manipulate the microbiota of farmed fish.     

Altered gut microbiota associated with intestinal disease in grass carp (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>)

Gut microbiota plays a crucial importance in their host. Disturbance of the microbial structure and function is known to be associated with inflammatory intestinal disorders. Enteritis is a significant cause of high mortality in fish species, including grass carp (Ctenopharyngodon idellus). Study regarding the association between microbial alternations and enteritis in grass carp is still absent. In this study, changes in the gut microbiota of grass carp suffering from enteritis were investigated using NGS-based 16S rRNA sequencing. Six healthy and ten abnormal fish (showing reddening anus, red odiferous fluid accumulating in the abdominal capacity, and flatulence and haemorrhage in the intestine) were collected from a fish farm in Huanggang Fisheries Institute (Hubei, China). Our results revealed that the diversity, structure, and function of gut microbiota were significantly different between diseased and healthy fish (P?<?0.05). Particularly, members of the genera Dechloromonas, Methylocaldum, Planctomyces, Rhodobacter, Caulobacter, Flavobacterium, and Pseudomonas were significantly increased in diseased fish compared with that in healthy fish (P?<?0.05). Predicted function indicated that microbiota significantly changed the specific metabolic pathways (related to amino acid metabolism, xenobiotics biodegradation and metabolism, and carbohydrate metabolism) in diseased fish (P?<?0.05). Taken together, our findings point out the association between changes of the gut microbiota and enteritis in grass carp, which provide basic information useful for diagnoses, prevention, and treatment of intestinal diseases occurring in cultured fish.     

Genetic and Physiological Characterization of the Intestinal Bacterial Microbiota of Bluegill (Lepomis macrochirus) with Three Different Feeding Habits

Bluegill (Lepomis macrochirus) in Lake Biwa, Japan, feed on benthic invertebrates (benthivorous type), aquatic plants (herbivorous type), and zooplankton (planktivorous type). To evaluate the effect of food on intestinal bacterial microbiota, we characterized and compared the intestinal microbiota of these three types of bluegill in terms of community-level physiological profile (CLPP) and genetic structure. The CLPP was analyzed using Biolog MicroPlates (Biolog, Inc., Hayward, CA, USA), and multivariate analysis of variance revealed that the CLPP of intestinal microbiota differed significantly between any pairs of the three types of bluegill. The genetic profiles were analyzed by temperature gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rDNA fragments, and multidimensional scaling indicated the existence of specific intestinal bacterial structures for both the benthivorous and the planktivorous types. These results suggest that the host's feeding habit can be one factor controlling the intestinal microbiota of fish in the natural environment.     

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.