Microbiote intestinal de la truite arc-en-ciel : déterminisme génétique et alimentaire

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Effets de l’alimentation végétale sur les capacités digestives de la truite arc-en-ciel et sur le microbiote associé à sa muqueuse digestive en fonction de son génotype

Over-fishing pressure and increasing aquaculture production led to an important substitution of fish oil and fish meal with oil and meal from plant origin in feed meant for farming fish. However this replacement has some deleterious incidence on fish. For rainbow trout, which are carnivorous fish, some growth delay often appears from the early life stages when they are fed with plant based diet. The aim of this work was to assess, at alevin and juvenile stages, the impact of a total replacement of fish meal and oil on rainbow trout gastrointestinal tract, and more particularly on the digestive capacity and the associate microbiota. The objective, in fine, being to determine if some digestive enzymes, intestinal transporters, or bacterial communities are impacted by the dietary replacement and if these biological factors can be related to the observed growth delay. Metagenomic approach using next generation sequencing was used to characterize the gut bacterial communities, while digestive capacity was assessed through quantitative PCR and enzymatic measurements in order to compare rainbow trout responses to a plant-based diet. In our investigations, rainbow trout isogenic lines that diverge in their response to this alternative diet (tolerant or rather reluctant) were adopted because they constitute a pertinent biological material for answering this question.In alevin rainbow trout, a plant-based diet led to an increase of pepsinogen, trypsinogen, and chymotrypsinogen genes which codes for proteolitic enzymes. Two main assumptions can explain this response, and their effectivness remains to investigate: wether this is a physiological response due to a lower weight of trout fed with the plant-based diet, or so it is due to an increase transcritpion of pancreatic enzymes to compensate for a reduction of protein digestibility. In the intestine, it appears that an increase transcription of IAP, SGLT1, CCK-t, and PEPT1 genes, and a decrease transcription of GLUT2 gene under a plant-based diet could reflect a disability to grow under a vegetable diet.In juvenile rainbow trout, a plant-based diet led to a decrease of lipid digestibility, and of triglycerides and total amino acid plasmatic levels. These perturbations could be explained in part by a decrease of the phosphatase alkaline activity, which suggest perturbancesof intestinal homeostasis, and by a decrease of phospholipase A2 activity. Transcriptional decrease of the triglycerides transporter MTP and of the prolidase, which is a peptidase from intestinal cell cytosol, has also been observed. Some modification of the microbiota associated to the intestinal mucosa could also contribute to the decrease of the intestinal homeostasis. The dietary replacement effectively led to reduce evenness of the bacterial communities in trout fed with a plant-based diet, which reflected a shift in the representativeness of some OTUs. Bacterial community from trout fed with a marine diet and trout fed with a plant-based diet were on average 70 % dissimilar. Dietary substitution led to the replacement of OTUs from the Firmicutes class, different bacterial species being observed according to the considered diet. The comparison of bacterial community between the isogenic lines showed that the genotype led to the replacement of β-Proteobacteria. Finally, abundance comparison suggested that Cetobacterium somerae, which is able to synthesise vitamin B12, and Shewanella, which has already been reported to stimulate pancreatic β cells, could be implicated in the trout response to vegetable.Modifications observed in this work constitute biological indicator that could be used to assess the response of the digestive tract to future feed formulations.La pression sur les quotas de pêche et l’augmentation de la production aquacole ont contribué à une substitution importante des farines et des huiles de poisson incorporées dans les aliments pour poissons carnivores, par des farines et des huiles végétales. La truite arc-en-ciel, qui est un poisson carnivore, est affectée par ce changement de régime. Ainsi un retard de croissance apparaît dès le plus jeune stade si certaines transformations et supplémentations ne sont pas apportées aux végétaux. L’objectif de ce travail a été d’évaluer, aux stades alevins et juvéniles, l’impact d’une substitution totale des huiles et farines de poisson sur le tractus digestif de la truite arc-en-ciel, et plus particulièrement sur ses capacités digestives et sur la composition de son microbiote intestinal. Le but in fine étant de déterminer si certaines enzymes de la digestion, transporteurs intestinaux, ou sous-communautés bactériennes sont impactés par le changement de régime et peuvent expliquer le retard de croissance observé. Chacun de ces facteurs ont été étudiés via une approche de métagénomique par séquençage de nouvelle génération NGS pour la caractérisation du microbiote, et via de la PCR quantitative et des mesures d’activités enzymatiques pour la comparaison des capacités digestives. Des lignées isogéniques de truites, identifiées comme divergentes dans leur réponse à l’alimentation végétale (capables d’adaptation ou réfractaires) ont permis de disposer d’un matériel biologique pertinent pour répondre à cette question.Chez la truite au stade alevin, une alimentation 100% végétale conduit à une plus forte transcription des gènes codant pour le pepsinogène, le trypsinogène, et le chymotrypsinogène qui sont des enzymes protéolytiques. Deux principales hypothèses peuvent expliquer cette réponse, et pourraient être étudiées : soit cette réponse est physiologique et s’explique par le plus faible poids des truites nourries avec un aliment végétal, soit cette réponse reflète une plus forte transcription d’enzymes digestives pancréatiques en compensation à une digestibilité protéique réduite. Au niveau de l’intestin, une augmentation de la transcription des gènes codant pour l’IAP, le SGLT1, la CCK-t, et PEPT1, et une diminution de la transcription du gène codant pour GLUT2 chez les truites nourries avec une alimentation végétale reflète une capacité réduite à grandir sous une alimentation végétale.Chez la truite au stade juvénile, l’alimentation végétale conduit à une baisse de la digestibilité des lipides et des niveaux plasmatiques des triglycérides et des acides aminés totaux. Ces perturbations pourraient en partie s’expliquer par une diminution de l’activité enzymatique de la phosphatase alcaline, qui témoigne de l’homéostasie intestinale, et de la phospholipase A2. Une baisse de la transcription du transporteur membranaire de triglycérides MTP et de la transcription de la prolidase, peptidase du cytosol des entérocytes, ont également était révélées. Une modification du microbiote intestinal associé à la muqueuse digestive pourrait également contribuer à la baisse de l’homéostasie intestinale. Le changement de régime conduit en effet à une équitabilité plus faible chez les truites ayant reçue un aliment végétal, ce qui reflète un changement dans la représentativité de certains OTUs. Ce changement de régime s’est également traduit par des communautés dissimilaires en moyenne à 70 %, d’après l’estimation de la β-diversité entre les communautés de truites nourries avec l’aliment marin et celles nourries avec l’aliment végétal. La sélection opérée par l’aliment a conduit à un remplacement des OTUs rencontrés au sein des Firmicutes, c’est-à-dire que différentes espèces bactériennes de Firmicutes sont rencontrées suivant le régime considéré. La comparaison de communautés bactériennes entre les différentes lignées isogéniques a montré que la sélection opérée par le génotype de l’hôte a davantage eu lieu sur le remplacement des β-Protéobactéries. Enfin, les comparaisons d’abondances en certaines espèces bactériennes particulières suggèrent que les bactéries Cetobacterium somerae, capables de synthétiser de la vitamine B12, et Shewanella, dont l’implication dans la stimulation des cellules β du pancréas endocrine a déjà été observée chez d’autres espèces, pourraient être impliquées dans la réponse métabolique des truites aux végétaux. Les modifications identifiées dans ce travail constituent des indicateurs biologiques qui pourront être mis à profit pour évaluer la réponse du tractus digestif des truites à de nouvelles formules alimentaires

Impact of plant-based diet nutrition on rainbow trout digestive capacity and on it associated mucosal microbiota

La pression sur les quotas de pêche et l’augmentation de la production aquacole ont contribué à une substitution importante des farines et des huiles de poisson incorporées dans les aliments pour poissons carnivores, par des farines et des huiles végétales. La truite arc-en-ciel, qui est un poisson carnivore, est affectée par ce changement de régime. Ainsi un retard de croissance apparaît dès le plus jeune stade si certaines transformations et supplémentations ne sont pas apportées aux végétaux. L’objectif de ce travail a été d’évaluer, aux stades alevins et juvéniles, l’impact d’une substitution totale des huiles et farines de poisson sur le tractus digestif de la truite arc-en-ciel, et plus particulièrement sur ses capacités digestives et sur la composition de son microbiote intestinal. Le but in fine étant de déterminer si certaines enzymes de la digestion, transporteurs intestinaux, ou sous-communautés bactériennes sont impactés par le changement de régime et peuvent expliquer le retard de croissance observé. Chacun de ces facteurs ont été étudiés via une approche de métagénomique par séquençage de nouvelle génération NGS pour la caractérisation du microbiote, et via de la PCR quantitative et des mesures d’activités enzymatiques pour la comparaison des capacités digestives. Des lignées isogéniques de truites, identifiées comme divergentes dans leur réponse à l’alimentation végétale (capables d’adaptation ou réfractaires) ont permis de disposer d’un matériel biologique pertinent pour répondre à cette question. Une modification du microbiote intestinal associé à la muqueuse digestive pourrait également contribuer à la baisse de l’homéostasie intestinale. Le changement de régime conduit en effet à une équitabilité plus faible chez les truites ayant reçue un aliment végétal, ce qui reflète un changement dans la représentativité de certains OTUs. Ce changement de régime s’est également traduit par des communautés dissimilaires en moyenne à 70 %, d’après l’estimation de la β-diversité entre les communautés de truites nourries avec l’aliment marin et celles nourries avec l’aliment végétal. La sélection opérée par l’aliment a conduit à un remplacement des OTUs rencontrés au sein des Firmicutes, c’est-à-dire que différentes espèces bactériennes de Firmicutes sont rencontrées suivant le régime considéré. La comparaison de communautés bactériennes entre les différentes lignées isogéniques a montré que la sélection opérée par le génotype de l’hôte a davantage eu lieu sur le remplacement des β-Protéobactéries. Enfin, les comparaisons d’abondances en certaines espèces bactériennes particulières suggèrent que les bactéries Cetobacterium somerae, capables de synthétiser de la vitamine B12, et Shewanella, dont l’implication dans la stimulation des cellules β du pancréas endocrine a déjà été observée chez d’autres espèces, pourraient être impliquées dans la réponse métabolique des truites aux végétaux. Les modifications identifiées dans ce travail constituent des indicateurs biologiques qui pourront être mis à profit pour évaluer la réponse du tractus digestif des truites à de nouvelles formules alimentaires.Over-fishing pressure and increasing aquaculture production led to an important substitution of fish oil and fish meal with oil and meal from plant origin in feed meant for farming fish. However this replacement has some deleterious incidence on fish. For rainbow trout, which are carnivorous fish, some growth delay often appears from the early life stages when they are fed with plant based diet. The aim of this work was to assess, at alevin and juvenile stages, the impact of a total replacement of fish meal and oil on rainbow trout gastrointestinal tract, and more particularly on the digestive capacity and the associate microbiota. The objective, in fine, being to determine if some digestive enzymes, intestinal transporters, or bacterial communities are impacted by the dietary replacement and if these biological factors can be related to the observed growth delay. Metagenomic approach using next generation sequencing was used to characterize the gut bacterial communities, while digestive capacity was assessed through quantitative PCR and enzymatic measurements in order to compare rainbow trout responses to a plant-based diet. In our investigations, rainbow trout isogenic lines that diverge in their response to this alternative diet (tolerant or rather reluctant) were adopted because they constitute a pertinent biological material for answering this question.In alevin rainbow trout, a plant-based diet led to an increase of pepsinogen, trypsinogen, and chymotrypsinogen genes which codes for proteolitic enzymes. Two main assumptions can explain this response, and their effectivness remains to investigate: wether this is a physiological response due to a lower weight of trout fed with the plant-based diet, or so it is due to an increase transcritpion of pancreatic enzymes to compensate for a reduction of protein digestibility. In the intestine, it appears that an increase transcription of IAP, SGLT1, CCK-t, and PEPT1 genes, and a decrease transcription of GLUT2 gene under a plant-based diet could reflect a disability to grow under a vegetable diet.In juvenile rainbow trout, a plant-based diet led to a decrease of lipid digestibility, and of triglycerides and total amino acid plasmatic levels. These perturbations could be explained in part by a decrease of the phosphatase alkaline activity, which suggest perturbancesof intestinal homeostasis, and by a decrease of phospholipase A2 activity. Transcriptional decrease of the triglycerides transporter MTP and of the prolidase, which is a peptidase from intestinal cell cytosol, has also been observed. Some modification of the microbiota associated to the intestinal mucosa could also contribute to the decrease of the intestinal homeostasis. The dietary replacement effectively led to reduce evenness of the bacterial communities in trout fed with a plant-based diet, which reflected a shift in the representativeness of some OTUs. Bacterial community from trout fed with a marine diet and trout fed with a plant-based diet were on average 70 % dissimilar. Dietary substitution led to the replacement of OTUs from the Firmicutes class, different bacterial species being observed according to the considered diet. The comparison of bacterial community between the isogenic lines showed that the genotype led to the replacement of β-Proteobacteria. Finally, abundance comparison suggested that Cetobacterium somerae, which is able to synthesise vitamin B12, and Shewanella, which has already been reported to stimulate pancreatic β cells, could be implicated in the trout response to vegetable.Modifications observed in this work constitute biological indicator that could be used to assess the response of the digestive tract to future feed formulations

Interplay between host genetics and the dynamics of the gut microbiota in pigs

International audiencePig gut microbiota displays high inter-individual variability and it remains an open question to determine to what extent its taxonomic composition relies on host genetic determinism and not only on environmental conditions. We will illustrate links between host genetics and gut microbiota composition by reporting results from different experiments carried out on Large White pig cohorts. Under similar controlled feeding and environmental conditions, we have shown that the gut microbiota after weaning can be consistently classified into two enterotypes by sequencing the V3-V4 variable region of the 16S rRNA gene from fecal DNA. The two enterotypes are characterized by either an overabundance of the genera Prevotella and Mitsuokella or Ruminococcus and Treponema. We explored the contribution of host genetics to the gut microbiota composition by estimating genetic parameters and by launching a directional selection based on the enterotype assignment of 60-day-old pigs. Calculated heritabilities of the relative abundance of fecal microbiota bacteria genera and the response to selection confirmed a significant contribution of host genetics to microbiota variability. The results on this ongoing selection experiment should help to investigate coevolution of the gut microbiota with its host

Response to selection on fecal microbiota composition in Large White piglets

International audiencePig gut microbiota displays high inter-individual variability and it remains an open question to determine to what extent its taxonomic composition relies on host genetic determinism and not only on environmental conditions. We carried out a study to demonstrate coevolution of the host and its gut microbiota established one month post-weaning, by directional selection over two generations. The gut microbiota was characterized by sequencing the V3-V4 variable region of the 16S rRNA gene from fecal samples collected on 60-day-old Large White piglets. Amplicon sequence variants were inferred from amplicon data and the microbial community was further studied at the genus level. Based on the stratification of the initial population (generation G0) according to the two major pig enterotypes, characterized by relative overabundance of either Prevotella and Mitsuokella or Ruminococcus and Treponema, we used the relative abundance of these four genera as selection criteria. From the G0 population of 317 piglets, we selected 6 males and 30 females per line and produced two successive generations (G1 and G2) of approximately 130 pigs per line. We consistently confirmed a moderate heritability for each of the selected genera (h²=0.3 to 0.4). We also estimated the heritability values of the relative abundances for 64 additional bacterial genera, which ranged from 0.1 to 0.5. We showed significant differences between the two lines in the relative abundance of the four bacterial genera at G1 (P<0.001, from 0.6 genetic standard deviation for Treponema to 1.3 for Prevotella). In the following generation G2, response to selection was maintained for Prevotella and was even increased for the three other genera. The observed contrasts were in the expected direction for the genera under direct selection, and we extended the analysis to the 64 other bacterial genera with estimated heritabilities higher than 0.1. All these results confirm a significant influence of host genetics on the composition of gut microbiota at 60 days of age in pigs, and a capacity of directional selection over generations that will be further explored together with early and late host traits

Broilers divergently selected for digestibility differ for their digestive microbial ecosystems

Improving the digestive efficiency of broiler chickens (Gallus gallus) could reduce organic waste, increase the use of alternative feed not used for human consumption and reduce the impact of feed in production costs. By selecting chicken lines divergently for their digestive efficiency, we showed previously that digestive efficiency is under genetic control and that the two resulting divergent lines, D+ (high digestive efficiency or "digestibility +") and D- (low digestive efficiency or "digestibility -"), also differ for the abundance of specific bacteria in their caeca. Here we perform a more extensive census of the bacteria present in the digestive microbiota of 60 chickens selected for their low apparent metabolizable energy corrected for nitrogen balance (AMEn-) or high (AMEn+) digestive efficiency in a [D+ x D-] F8 progeny of 200 individuals. We sequenced the 16S rRNA genes of the ileal, jejunal and caecal microbiotas, and compared the compositions and predicted functions of microbiotas from the different intestinal segments for 20 AMEn+ and 19 AMEn- birds. The intestinal segment of origin was the main factor structuring the samples. The caecal microbiota was the most impacted by the differences in digestive efficiency, with 41 bacterial species with abundances differing between highly and poorly efficient birds. Furthermore, we predicted that the caecal microbiota of efficient birds might be enriched in genes contributing to the degradation of short chain fatty acids (SCFA) from non-starch polysaccharides. These results confirm the impact of the genetic selection led on digestibility on the caecal microbiota taxonomic composition. They open the way toward the identification of specific, causal genes of the host controlling variations in the abundances of bacterial taxons

Caecal microbiota composition of experimental inbred MHC-B lines infected with IBV differs according to genetics and vaccination

International audienceAbstract Interactions between the gut microbiota and the immune system may be involved in vaccine and infection responses. In the present study, we studied the interactions between caecal microbiota composition and parameters describing the immune response in six experimental inbred chicken lines harboring different MHC haplotypes. Animals were challenge-infected with the infectious bronchitis virus (IBV), and half of them were previously vaccinated against this pathogen. We explored to what extent the gut microbiota composition and the genetic line could be related to the immune response, evaluated through flow cytometry. To do so, we characterized the caecal bacterial communities with a 16S rRNA gene amplicon sequencing approach performed one week after the IBV infectious challenge. We observed significant effects of both the vaccination and the genetic line on the microbiota after the challenge infection with IBV, with a lower bacterial richness in vaccinated chickens. We also observed dissimilar caecal community profiles among the different lines, and between the vaccinated and non-vaccinated animals. The effect of vaccination was similar in all the lines, with a reduced abundance of OTU from the Ruminococcacea UCG-014 and Faecalibacterium genera, and an increased abundance of OTU from the Eisenbergiella genus. The main association between the caecal microbiota and the immune phenotypes involved TCR ϒδ expression on TCR ϒδ + T cells. This phenotype was negatively associated with OTU from the Escherichia - Shigella genus that were also less abundant in the lines with the highest responses to the vaccine. We proved that the caecal microbiota composition is associated with the IBV vaccine response level in inbred chicken lines, and that the TCR ϒδ + T cells (judged by TCR ϒδ expression) may be an important component involved in this interaction, especially with bacteria from the Escherichia - Shigella genus. We hypothesized that bacteria from the Escherichia-Shigella genus increased the systemic level of bacterial lipid antigens, which subsequently mitigated poultry γδ T cells

Postprandial kinetics of gene expression of proteins involved in the digestive process in rainbow trout (O. mykiss) and impact of diet composition

International audienceThe impact of increased incorporation of plant ingredients on diets for rainbow trout was evaluated in terms of gene expression of gastric (gastric lipase, pepsinogen) and intestinal (prolidase, maltase, phospholipase A2) digestive enzymes and nutrient transporters (peptide and glucose transporters), as well as of postprandial levels of plasma glucose, triglycerides and total free amino acids. For that purpose, trout alevins were fed from the start of exogenous feeding one of three different experimental diets: a diet rich in fish meal and fish oil (FM–FO), a plant-based diet (noFM–noFO) totally free from fish meal and fish oil, but containing plant ingredients and a Mixed diet (Mixed) intermediate between the FM–FO and noFM–noFO diets. After 16 months of rearing, all fish were left unfed for 72 h and then given a single meal to satiation. Blood, stomach and anterior intestine were sampled before the meal and at 2, 6 and 12 h after this meal. The postprandial kinetics of gene expression of gastric and intestinal digestive enzymes and nutrient transporters were then followed in trout fed the FM–FO diet. The postprandial profiles showed that the expression of almost all genes studied was stimulated by the presence of nutrients in the digestive tract of trout, but the timing (appearance of peaks) varied between genes. Based on these data, we have focused on the molecular response to dietary factors in the stomach and the intestine at 6 and 12 h after feeding, respectively. The reduction in FM and FO levels of dietary incorporation induced a significant decrease in the gene expression of gastric lipase, GLUT2 and PEPT1. The plasma glucose and triglycerides levels were also reduced in trout fed the noFM–noFO diet. Consequently, the present study suggests a decrease in digestive capacities in trout fed a diet rich in plant ingredients

Searching for links between gut microbiota collected before vaccination and variabilities of vaccine response in pigs

International audienceUnderstanding and predicting why some animals respond better to vaccination than others is a main concern to strengthen vaccination efficiency. Our aim was to study whether the gut microbiota before vaccination presents composition patterns associated with individual variabilities of vaccine responses in pigs. Ninety-eight Large White piglets were vaccinated against the influenza A virus (IAV) at weaning at 28 days of age (D28) with a booster three weeks later. Stools were collected before the vaccination at D28, and were further processed to perform 16SRNA gene sequencing (Illumina MiSeq) and assess microbial taxonomic composition. The piglets’ humoral response was evaluated by ELISA of seric IAV-specific IgGs and by hemagglutination inhibition assays (HAI) at D49, D56, D63, and D146 to identify extreme animals with either high or low responses to vaccination. Piglets with a richer microbiota had higher levels of HAI at D63 (p<0.05) and had a tendency towards more IAV-specific IgGs. Extreme high and low responders for IAV-specific IgGs at D63 had also a dissimilar microbiota (p<0.01) and displayed differentially abundant operational taxonomic units (OTUs); bacteria from the Paludibacteraceae family and Prevotella genera were more abundant in high responders, while bacteria from Helicobacter and Escherichia-Shigella genera were more abundant in low responders (FDR<0.05). Thus, our results show that the faecal microbiota before vaccination could be further investigated to identify biomarkers predictive of vaccine response levels and analyse the underlying biology

Searching for links between gut microbiota collected before vaccination and variabilities of vaccine response in pigs

International audienceUnderstanding and predicting why some animals respond better to vaccination than others is a main concern to strengthen vaccination efficiency. Our aim was to study whether the gut microbiota before vaccination presents composition patterns associated with individual variabilities of vaccine responses in pigs. Ninety-eight Large White piglets were vaccinated against the influenza A virus (IAV) at weaning at 28 days of age (D28) with a booster three weeks later. Stools were collected before the vaccination at D28, and were further processed to perform 16SRNA gene sequencing (Illumina MiSeq) and assess microbial taxonomic composition. The piglets’ humoral response was evaluated by ELISA of seric IAV-specific IgGs and by hemagglutination inhibition assays (HAI) at D49, D56, D63, and D146 to identify extreme animals with either high or low responses to vaccination. Piglets with a richer microbiota had higher levels of HAI at D63 (p<0.05) and had a tendency towards more IAV-specific IgGs. Extreme high and low responders for IAV-specific IgGs at D63 had also a dissimilar microbiota (p<0.01) and displayed differentially abundant operational taxonomic units (OTUs); bacteria from the Paludibacteraceae family and Prevotella genera were more abundant in high responders, while bacteria from Helicobacter and Escherichia-Shigella genera were more abundant in low responders (FDR<0.05). Thus, our results show that the faecal microbiota before vaccination could be further investigated to identify biomarkers predictive of vaccine response levels and analyse the underlying biology