Genomic and physiological responses to strong selective pressure during late organogenesis: how natural Fundulus heteroclitus populations cope with persistent environmental pollution Superfund Site

This study explores the synergistic effects of two model PAHs, an aryl hydrocarbon receptor (AHR) agonist (β-naphthoflavone) and a cytochrome P4501A (CYP1A) inhibitor (α-naphthoflavone), on gene expression in stage 31 embryos from two different population. One population (Elizabeth River population) is relatively resistant to the pollutants in its environment. A loop design (Figure 5) was used for the microarray hybridizations where each sample is hybridized to 2 arrays using both Cy3 and Cy5 labeled fluorophores (Kerr and Churchill, 2001). The loop consisted of Cy3 and Cy5 labeled embryo aRNAs from 4 biological samples and six different treatments (T1-T6: control, 1 μg/L BNF, 50 μg/L ANF, 100 μg/L ANF, 1 μg/L BNF + 50 μg/L ANF, 1 μg/L BNF + 100 μg/L ANF). In total, 48 biological samples were hybridized to 24 microarrays. Each array had different combinations of biological samples, so that the most direct comparisons (i.e., 50 µg/L ANF resistant embryo and 50 µg/L sensitive embryo) are hybridized to the same array. The loop formed was T1S→ T1R → T2S→ T2R →T3S→ T3R→ T4S→ T4R→ T5S→ T5R → T6S→T6R→ T1S→ T2S → T3S → T4S → T5S → T6S → T1S → T1R → T2R → T3R → T4R → T5R → T6R, where each arrow represents a separate hybridization (array) with the biological sample at the base of the arrow labeled with Cy3 and the biological pool at the head of the arrow labeled with Cy5. T1-6 is treatment, and S and R represent sensitive and resistant embryos.