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EVALUATING EXPERIMENTALLY INDUCED THIAMINE DEFICIENCY IN ATLANTIC SALMON USING IN SITU HYBRIDIZATION
Clifford Kraft1 and Andrew D. Miller2
1 Department of Natural Resources, Cornell University, Ithaca, NY 14850
2 College of Veterinary Medicine, Cornell University, Ithaca, NY 14850
Despite the longstanding recognition of fisheries mortality from thiamine deficiency, methods to evaluate the thiamine status of wild or hatchery fishes are inadequate. Pilot project funds from the Great Lakes Fishery Commission provided an opportunity to take steps to develop an in situ hybridization method to identify intermediate or incipient levels of thiamine deficiency in fishes. The underlying premise of this project was that thiamine dependent genes would be downregulated in Atlantic salmon raised in a hatchery for more than three years under thiamine-deficient conditions by a research group at the University of Western Ontario led by Bryan Neff. Brain, skin and skeletal muscle tissue sections from 20 hatchery-reared thiamine-deficient Atlantic salmon (Salmo salar) – and 20 control fish reared under similar conditions – were obtained, prepared and stained for in situ hybridization (ISH) using five RNA probes targeting thiamine-dependent enzymes, receptors, and transporters or enzymes previously identified as associated with thiamine deficiency. No differences in brain histology were observed in a comparison of Atlantic salmon raised in thiamine-deficient conditions versus thiamine-replete conditions. All five probes were successful in identifying the target enzymes in these three tissues, confirming their ability to quantify levels of thiamine-dependent enzymes in fish tissues. An initial quantitative comparison of expression levels of two of the thiamine-dependent enzymes in the thiamine-deficient and non-thiamine-deficient fish did not show a statistically significant difference, therefore expression levels of three other enzymes were not quantified using that same approach. Instead, these initial results pointed to the need to employ more sophisticated quantification approaches to examine expression levels of the target enzymes. Although this study met two proposed study objectives demonstrating the potential use of ISH probes to identify thiamine deficiency in fish, the study objectives associated with quantifying levels of these enzymes were not achieved. In hindsight, these last two objectives were unrealistic given the resources available for this pilot project. Fortunately, the initial success using these probes to identify thiamine-dependent enzymes provides an opportunity to do future work with stained histological sections prepared during this project to evaluate quantitative comparisons of expression of target enzymes that could provide an important tool for assessing thiamine deficiency in Great Lakes fishes.