Site-specific impacts on gene expression and behavior in fathead minnows exposed to streams adjacent to STPs

Environmental monitoring for pharmaceuticals and endocrine disruptors in the aquatic environment traditionally employs a variety of methods including analytical chemistry, as well as a variety of histological and biochemical endpoints that correlate with the fish fitness. It is now clear that analytical chemistry alone is insufficient to identify aquatic environments that are compromised because these measurements do not identify the biologically available dose. The biological endpoints that are measured are important because they relate to known impairments, however, they are not specific to the contaminants and often focus on only a few known endpoints. These studies can be enhanced by looking more broadly at changes in gene expression, especially if the analysis focuses on biochemical pathways. The present study was designed to obtain additional information on well-characterized sites adjacent to sewage treatment plants in MN that are thought to be impacted by endocrine disruptors. Here we examine five sites that have been previously characterized and examine changes in gene expression in fathead minnows (Pimephales promelas) that have been caged for 48 h in each of the aquatic environments. We find that the gene expression changes are characteristic and unique at each of the five sites. Also, fish exposed to two of the sites, 7 and 12, display a more aggressive behavior compared to control fish. Our results show that short-term exposure to sewage treatment plant effluents was able to induce a site-specific gene expression pattern in the fathead minnow gonad and liver. The short-term exposure was also sufficient to affect fish sexual behavior. Our results also show that microarray analysis can be very useful for determining potential exposure to chemicals, and could be used routinely as a tool for environmental monitoring. To conduct the field exposures at each field site, FHM were transported from the laboratory to the field site in aerated, insulated tanks. At each site, 25 males and 25 females were placed in separate wire mesh minnow traps with the entrance funnel plugged. The traps were anchored to the bottom in the stream current with the top of the traps submerged. Fish were removed from the traps 48 h later and transported back to the laboratory in aerated, insulated tanks containing the stream water. Immediately upon arrival at the laboratory, four males and four females were sacrificed as described for the laboratory exposures. Liver and gonads were removed and stored in liquid nitrogen until processed for arrays. All procedures involving live fish were reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC). Fathead minnow 22,000 gene arrays were designed by EcoArray (Alachua, FL) and purchased from Agilent. Array hybridizations were performed using a reference design, where each sample was compared to a reference sample. The reference sample consisted of equal amounts of RNA from control female and male tissues (liver, brain and gonad). Four replicates, each consisting of a different individual, were analyzed for each of the treatment sites (sites 7, 11, 12, 13 and 21). cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturer’s kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit and Agilent 60-mer oligo microarray processing protocol; Agilent, Palo Alto, CA).