GREAT LAKES FISHERY COMMISSION 2003 Project Completion Report1 Effects of Low Level Aquatic Contaminants on Lake Trout Reproduction: Implication in Lake Trout Rehabilitation by:

We have completed this project and anticipate submitting in total of three manuscripts. Briefly, we developed several novel methods to further understand and characterize the induction and function of this gene in lake trout and other salmonids species. First, we developed the measurement of gill CYP1A using quantitative PCR with the sampling of non-lethal gill biopsies in both wild and cultured Atlantic salmon. This study demonstrated that gill biopsies coupled with quantitative PCR analysis was a potentially valuable tool in environmental assessment of wild fish populations. Second, advances in the quantitative PCR method and instruments led us to develop a real-time quantitative PCR assay useful for measuring CYP1A mRNA in four salmonid species; lake trout, brook trout, rainbow trout, and Atlantic salmon. In order to obtain necessary information for the design of a cRNA standard, full-length CYP1A cDNA sequences were determined for two Salvelinus species, lake trout (Salvelinus namaycush) and brook trout (Salvelinus fontinalis). Each cDNA was found to share the same characteristics with known CYP1A sequences of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss, and shared greater than 97% coding region sequence. The developed a CYP1A-specific real-time quantitative PCR assay indicated that BNF treated fish showed 1.8 to 3.0 orders of magnitude higher CYP1A than control fish in all four species studied. Finally, we examined the effects BNF exposure had on juvenile lake trout brain tissue using a multidisciplinary approach. Over a 32 day time-course, CYP1A mRNA induction in response to BNF exposure occurs rapidly and continued to rise in the BNF-treated lake trout after 4 hours, 8 hours, and 24 hours with a peak in CYP1A mRNA expression after 2 days. At each of these time periods, significantly higher levels of CYP1A expression were found in each induced group over their respective control groups (Tukey-Kramer, p < 0.0001). In situ hybridization study supports the Q-RT-PCR results in that CYP1A mRNA level was universally induced in the brain of BNF-exposed fish, and that CYP1A mRNA were mainly expressed in the endothelia and occasionally in the neurons or glial cells. CYP1A immunoreactivity was induced in the olfactory bulb and valvula cerebelli of BNF-treated fish. Notably, some BNF-treated fish contained multifocal hemorrhages in the brain tissue. These fish had overall depressed CYP1A immunoreactivity in the brain. These results show the relationship between transcriptional and translational effects of contaminant exposure in the brain of juvenile lake trout and provide knowledge to the potential physiological effects sublethal levels of contaminants have on fish from the population level.