Fundulus heteroclitus
In order to maximize the number of expressed genes for our transcriptome assemblky, F. heteroclitus tissues were isolated from 57 unique samples. These included gill, liver, or kidney isolated from male and female fish exposed to different environmental conditions (i.e., stabile salinity, variable salinity, metal, metal mixture), as well as, different developmental stages. Stabile salinity conditions included fish acclimated to seawater, 10‰ salinity, and freshwater. Seawater acclimated fish were collected from seawater acclimated animal stocks housed as previously detailed. Fish living in seawater were acclimated to 10‰ salinity following exposure to 10‰ salinity for four weeks. Fish were acclimated to freshwater (0.3ppt) prior to experiments by first transitioning them from seawater to 10% seawater for two weeks and then to soft freshwater (48mg/L NaHCO3, 30 mg/L CaSO4, 30 mg/L MgSO4, 2mg/L KCl, pH7.5-8.0) for an additional two weeks. Fish exposed to variable conditions were seawater or freshwater acclimated fish that were immediately transferred to opposing salinity gradients (e.g., seawater to freshwater or freshwater to seawater for 30 minutes, 1 hour, 3 hour). Metal treatments included seawater acclimated fish exposed for 48 hours to arsenic (8,000 and 12,000 μg/l as sodium arsenite), cadmium (500 and 5000 μg/l as cadmium chloride), lead (500 and 5000 μg/l as lead chloride), and copper (4.8 and 480 μg/l as copper chloride), as well as, binary combinations of lead (500 μg/l) and copper (4.8 μg/l), arsenic (12,000 μg/l) and copper (480 μg/l), and cadmium (5000 μg/l) and copper (480 μg/l).   