EFFECTS OF PLANT-BASED DIETS ON THE DISTAL GUT MICROBIOME OF RAINBOW TROUT (Oncorhynchus mykiss).

Abstract

Replacement of fish meal in aquaculture diets with plant proteins is economically desirable, but the effects of these alternative protein sources on fish intestinal microbiota are poorly understood. The fish intestine is colonized by a diverse population of micro-organisms of which bacteria form the biggest component. The intestinal microbiota plays important roles in digestion, pathogen exclusion, maintenance of intestinal homeostasis, and in the development of the immune system of the fish. The purpose of this thesis was to characterise the intestinal microbiota of rainbow trout (Oncorhynchus mykiss) fed with diets based on plant ingredients. Semi quantitative (clone library analysis and 454 pyrosequencing) and quantitative (qRT-PCR) techniques based on the cpn60 gene, and denaturing gradient gel electrophoresis (DGGE) based on the 16S rRNA were used to characterise the intestinal microbiota and evaluate the effects of plant based experimental diets on the structure of the intestinal microbiota. A study was also conducted to evaluate the extent to which individual fish in a recirculating aquaculture system share strains of bacteria, to understand the effect that a shared recirculating facility might have on our ability to resolve differences in the intestinal microbiota of fish fed with different diets. Regardless of the technique used, the intestinal microbiota of fish fed with plant based diets were always distinguishable from fish fed with fish meal diets. Intestinal microbiota of fish fed with plant based diets had higher Firmicutes:Proteobacteria ratios compared to their corresponding controls. This trend was seen with both cpn60 clone library analysis of pooled samples of intestinal contents, and cpn60 based pyrosequencing of intestinal microbiota from individual fish. Evaluation of the effects of plant based diets on the intestinal microbiota by DGGE and pyrosequencing showed that the microbiomes of fish fed with plant protein concentrate diets were less distinguishable from fish meal control diet associated microbiomes, than were microbiomes of fish fed with plant meal based diets. GTG5 rep PCR analysis of Carnobacterium maltaromaticum isolates showed that even though the fish were reared in a recirculating facility, individual fish hosted distinct C. maltaromaticum strains, suggesting a minimal level of sharing of bacterial strains between fish housed in a common environment. Quantitative real-time PCR was used to determine the abundance of specific bacterial targets in fish intestinal contents associated with particular plant based diets in two replicate trials, to determine if the diets had reproducible effects on the composition of intestinal microbiota. Even though the diets did not have a consistent, reproducible effect at the species level, it was observed that intestinal microbiota composition was always modified with change in diet. Results of these studies indicate that inclusion of plant ingredients to replace fish meal alters the structure of the intestinal microbiota of rainbow trout. Additionally, the protein concentrate forms of the experimental ingredients reduce the impact of plant ingredients on the intestinal microbiota structure making them favourable candidates for fish meal replacement.