How Does Reference Site Selection Influence Interpretation of Omics Data?: Evaluating Liver Transcriptome Responses in Male Rainbow Darter (Etheostoma caeruleum) across an Urban Environment.

Studies quantifying the influence of reference site selection on transcriptomic profiles in aquatic organisms exposed to complex mixtures are lacking in the literature, despite the significant implications of such research for the interpretation of omics data sets. We measured hepatic transcriptomic responses in fish across an urban environment in the central Grand River watershed (Ontario, Canada). Adult male rainbow darter (RBD) (Etheostoma caeruleum) were collected from nine sites at varying distances from two major municipal wastewater treatment plants (MWWTPs) (Waterloo, Kitchener), including three upstream reference sites. The transcriptomic response in RBD was independently compared with that of fish from each of the three reference sites. Data collected in fish downstream of the Waterloo MWWTP (poorest effluent quality) suggested that ∼15.5% of the transcriptome response was influenced by reference site selection. In contrast, at sites where the impact of MWWTPs was less-pronounced and fish showed less of a transcriptome response, reference site selection had a greater influence (i.e., ∼56.9% of transcripts were different depending on the site used). This study highlights the importance of conducting transcriptomics studies that leverage more than one reference site, and it broadens our understanding of the molecular responses in fish in dynamic natural environments.