Drinkall, Emma
(2022)
A comparative investigation of the effect of ploidy on rainbow trout gastrointestinal health.
PhD thesis, University of Nottingham.
Abstract
Aquaculture is the fastest growing food producing industry and demand is expected to increase with the growing global population. Thus, there is also a growing demand for farmed high value salmonids such as Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Production of triploid trout is preferred by commercial fish farms, primarily because they grow larger and quicker than their diploid counterparts and their sterility ensures the prevention of breeding with wild populations in the event of escape.
The epithelial surface of the intestine is a primary site of nutrient absorption, and the mucosal barrier provides protection from mechanical insult as well as forming the interface between the host microbiota, host immune system and potential pathogens. However, little is known about the effect of ploidy on the immunocompetence, function and resident microbiota of the gastrointestinal tract.
This study examined the intestinal tract of a cohort of healthy juvenile triploid and diploid rainbow trout, reared under identical conditions, and studied the histo-morphological of the GI tract as well as teleost-specific TLR and mucin gene identification and expression, to better understand the role of ploidy in intestinal function and health.
Through a multimodal bioinformatics investigation putative sequences of TLR18 and TLR21 were identified for the first time in the rainbow trout genome as well as a complete sequence of putative TLR19. Expression of TLR2, TLR18, TLR19 and TLR21 in both diploid and triploid intestinal tissues was confirmed by qPCR and suggested that ploidy state had little impact on relative gene expression. Appropriate reference genes were determined as part of this comparison. Existing algorithms for identification of TLRs through Leucine Rich Repeat (LRR) domains, were found to be inadequate for complete identification in teleost sequences, likely due to the presence of atypical amino acids in the conserved regions. These atypical LRRs were often categorised into the previously identified sub group of LxxLxLxx(L/I/M)xL or the newly suggested LxxLx(M/A)xxNxL sub group and indicated that current dentification tools require adaption or alternative teleost-specific algorithms are needed to facilitate more efficient identification of teleost TLRs.
Examination of the intestinal morphology demonstrated that the histo-morphological structure of both populations was consistent with that reported in other salmonid species, with a general trend of thicker individual intestinal layers seen in the triploid fish, although this was not statistically significant. Goblet cell distribution was similar for both populations, with greater density of cells found in the proximal rather than distal tissues. Although goblet cell distribution was higher in triploid fish, the differences between ploidy were not statistically significant, indicating very similar anatomy between fish of differing ploidy. A multimodal approach identified putative sequences mucin sequences belonging to the Muc5 family were identified for the first time in the rainbow trout genome and presence confirmed using PCR in samples from both ploidy.
Finally, investigation using 16S rRNA analysis, identified a core bacterial microbiota of the intestinal mucosa, which comprised of a number of classified and unclassified genera including Mycoplasma the Ruminococcaceae Group, RFN20, Pseudomonas, Clostridium, Sphingomonas, Citrobacter and Fusicatenibacter. Mucosal microbial communities were shown to differ between the proximal and distal parts of the intestinal tract with Mycoplasmataceae predominant in proximal intestine and Erysipelotrichaceae predominant in the distal populations for both ploidy. Mycoplasma were found to be of higher abundance in diploid fish in both intestinal sites, while Adlercreutzia, Sandaracinaceae and Tenacibaculum were of higher abundance in triploid samples. In addition, a number of novel genera were identified for the first time in the trout gastrointestinal including Fusicatenibacter, Desulfatigans, Aldercreutzia sp., the Sandaracinaceae Group and Leptolyngbya.
Overall, the identification and confirmation of expression of trout TLRs within the gastrointestinal tract may contribute to the adaption of existing vaccines for enhanced protection against infectious disease or to the development of new vaccine adjuvants for oral vaccinations, leading to improved preventative health care strategies for rainbow trout production. The lack of statistically significant differences between the expression of TLR and morphology of the intestinal tract between populations indicates that ploidy may have little impact of intestinal structure and function. The findings regarding the core microbiota of rainbow trout will inform future studies examining dysbiosis and potential impact of pre and probiotic supplements and may lead to the development of ploidy specific nutritional management to optimise the gut microbiota.
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