Investigating Mechanisms of Environmental Chemical Tolerance and Toxicity in Brown Trout using RNA-seq
Uren Webster, Tamsyn
Date: 29 November 2013
University of Exeter
PhD Biological Sciences
Brown trout (Salmo trutta) are an ecologically and economically important native European species, known to be sensitive to environmental stressors. Compared to other model species, there is little information available on the toxicological responses of this species to environmental pollutants. High-throughput RNA-sequencing (RNA-seq) ...
Brown trout (Salmo trutta) are an ecologically and economically important native European species, known to be sensitive to environmental stressors. Compared to other model species, there is little information available on the toxicological responses of this species to environmental pollutants. High-throughput RNA-sequencing (RNA-seq) is emerging as a sensitive and accurate tool for conducting transcriptomics, but is yet to be widely used in ecotoxicology. A major advantage of RNA-seq is that it can be used to conduct non-biased, global gene expression analysis in species without existing genomic sequence information. Therefore, during this PhD I set out to investigate global mechanisms of toxicity for a selection of the most environmentally relevant chemicals likely to impact upon natural brown trout populations. By using RNA-seq, I also aimed to demonstrate the potential application of this technology as a valuable tool in ecotoxicology. To address these objectives, I conducted transcriptomic profiling, both on wild brown trout and on those exposed to agricultural pollutants in a laboratory setting. Using RNA-seq in combination with analysis of tissue metal concentration I found evidence of a high degree of metal tolerance in a chronically exposed wild population of brown trout from the river Hayle. The main molecular mechanisms responsible for this metal-tolerance included regulation of metal- and ion-homeostasis pathways. In the laboratory exposures, I found evidence of considerable transcriptomic changes in male brown trout exposed to 34.38 ng/L E2, including up-regulation of typical oestrogen-responsive transcripts (vitellogenins, zona pellucida proteins and estrogen receptor 1), as well as hepatic processes that can be associated with vitellogenesis such as lipid metabolism, cell proliferation and ribosome biogenesis. This concentration is within a range measured in sewage effluent and, more occasionally, in surface waters. I also exposed male brown trout to linuron, a widely used pesticide, and observed a striking down-regulation of enzymes involved in the cholesterol biosynthesis pathway and up-regulation of transcripts involved in cellular stress response following exposure to 250 µg linuron/L. There was also some evidence of similar responses occurring at the lower, environmentally relevant concentration (2.5 µg/L). I then compared the mechanisms of toxicity of glyphosate, the most widely used herbicide in the world, and its commercial formulation Roundup in juvenile brown trout. I found evidence of a cellular stress response consistent with generation of oxidative stress at concentrations of 10 µg/L and above which, importantly, is within the range of concentrations measured in the environment. To investigate the potential reproductive toxicity of these compounds I also conducted an exposure of breeding zebrafish to glyphosate and Roundup, and found evidence of reproductive toxicity, but only at a very high concentration (10 mg/L). This work therefore provides valuable information on the toxicological effects of these environmentally relevant chemicals in brown trout, which can potentially be used to assess the risk they pose to natural populations and therefore contribute to the sustainable management of this species. I have successfully employed RNA-seq to achieve the main objective of this PhD and, in so doing, have demonstrated the value of this technology in ecotoxicology. Specifically, we have demonstrated the ability of RNA-seq to identify conserved responses typically associated with oestrogen exposure. We also highlight the importance of optimising the experimental design and strategy for RNA-seq data analysis to improve the quality of transcript expression analysis. Throughout the course of this work we have benefited from improvements in sequencing technology and the tools available for data analysis. This technology is continuing to develop rapidly, and it is likely that RNA-seq will become the dominant tool for conducting transcriptomics in ecotoxicology in the future.
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