Fig. 2.

Methods to determine the relationships among atrazine exposure, corticosterone levels, host-associated gut microbiota, and Bd infection risk in Cuban tree frogs. Superscript letters represent the timing of treatment exposure and sampling effort: ^aOn day one, tadpoles were placed in tanks with water that was treated experimentally with atrazine and/or metryapone using a 2×2 factorial design (8 tadpoles per tank). ^bOn day six, tadpoles from each tank: 1) were euthanized and their gut bacterial community was characterized (n = 1 tadpole per tank), 2) remained in their respective tanks after the pond water was changed to remove residual chemicals from the water treatment (n = ≤ 3 tadpoles per tank), or 3) were used to quantify their corticosterone levels in response to water treatment and then exposed to Bd or a solvent control; four tadpoles from each original tank were either exposed to Bd (n = 2) or the solvent control (n = 2) in new tanks. ^cOn day 27, Bd load was quantified from the skin of tadpoles that were exposed to Bd or the solvent control. ^dTadpoles that remained in their respective tanks, after the experimental water treatment of atrazine and/or metyrapone, were allowed to metamorphose, which occurred, on average, on day 50. ^eOn day 84, post-metamorphic (adult) frogs were exposed to Bd or the solvent control and then ^fBd was quantified on approximately day 100 from the skin of adults (n = ≤ 2 adults per treatment per tank). ^gOn day 119, adult frogs were euthanized and their gut bacterial community was characterized. Photos by Mark Yokoyama.