Objective 2: Using the same approach as objective one, we will use Coho salmon embryos to identify if we can accurately use the same RiboTag for a different species while also running same assays described above for ROS production and hair cell impairment and death. This will give us a better understanding of species differences and, if the RiboTag is effective, we can make predictions as to what the downstream effects of B(a)P may have on lateral line development and function when exposed to PAHs.
H01: Due to species differences, Ribotagging for lateral line development cannot be used.
H02: When exposing B(a)P to Coho salmon, there will be no effect on lateral line cells nor their development.
Rational: From embryo to juvenile stages, Coho salmon inhabit coastal freshwaters where exposure to stormwater containing high levels of PAHs such as B(a)P is relevant. After modeling with zebrafish, it is critical to observe species differences comparing fish naturally found in areas of contamination that are economic and ecologically relevant to the area of concern.
Experimental methods and techniques: A Tg(myo6b:GFP-2A-rpl10a-3xHA) Coho salmon transgenic line will be developed in collaboration with Matern et al., and the University of Maryland School of Medicine. This model will show expression of an HA-tagged 60S ribosomal protein (Rpl10a) and green fluorescence protein (GFP) in hair cells similar to the zebrafish model. Using same protocols and statistical analyses from objective 1, wildtype Coho embryos 4hpf will be exposed to the same environmentally relevant concentrations of B(a)P until 2dph.
Due to species differences the developmental timeline is different in Coho and that of zebrafish so exposure time will be different in order to target embryogenesis. We will observe hair cell death, ROS, and mechanotransduction channel closure. With same concentrations, number of embryos and days post hatch as previously described we will expose the transgenic Coho embryos and then examine fluorescence, immunoprecipitation and RT-qPCR to analyze hair cell gene expression when exposed to environmentally relevant concentrations of B(a)P.
Expected results: Similar to that of objective 1, if B(a)P does impact development of hair cells due to downregulation of specific hair cell genes, we will see reduced fluorescence and down regulation of gene expression when running RT-qPCR. ROS production, hair cell impairment and death will be visualized via dye assays mentioned above.
Objective 3: Within holding tanks in the University of Saskatchewan’s ATRF, we will observe behavioral impacts of Coho salmon when exposed to B(a)P testing specifically for rheotaxis and predator-prey responses.
H01: We will not observe rheotaxis impairment due to lateral line perturbations of Coho salmon when exposed to B(a)P.
H02: We will not observe impairment to predator prey responses due to lateral line perturbations of Coho salmon when exposed to B(a)P.
Rational: If there is an impact on the development of lateral line hair cells due to exposure to B(a)P, it is important to assess what this means behaviorally. Assessing behavior of Coho after treatment to B(a)P can help us understand if exposure and possible lateral line impairments can potentiate population decline.