Outdoor mesocosm study evaluating how mass, NaCl tolerance, and pesticide tolerance affect oxidative stress biomarkers (CAT, SOD, GR, GPx, TBARS) in larval wood frogs (Rana sylvatica) exposed to baseline and NaCl-contaminated conditions, 2019

Biomarkers of oxidative stress can aid in wildlife monitoring by allowing conservationists to detect sublethal environmental shifts. However, interpretation of stress responses can be complicated by multiple interacting factors (e.g., individual development, evolved physiological tolerance to stressors) which alter biomarker expression. Here, we investigated how individual ontogenetic traits and population-level tolerance traits influence oxidative stress responses under baseline and contaminated environmental conditions.

For our model contaminant, we used NaCl (common freshwater contaminant due to factors such as coastal flooding, irrigation, airborne salt circulation, drought, runoff from road deicing salts). For our model wildlife populations, we used larval wood frogs (Rana sylvatica) from six noninteracting populations known to vary in two population-level tolerance traits: NaCl tolerance (calculated as average time to death from lethal NaCl exposure) and pesticide tolerance (determined by proxy of distance to agriculture - a consistent and highly repeatable relationship).

At an outdoor research facility, R. sylvatica tadpoles were exposed to either baseline conditions (0 g/L NaCl added) or NaCl-contaminated conditions (1 g/L NaCl added for 21 days, then reduced to 0.5 g/L NaCl). Exposures were conducted in individual units with 40 replicates per population for each treatment. The experiment was terminated per individual to capture the full term of larval development (Developmental stage: Gosner stage 36), lasting between 33-48 days. For each individual, we measured mass, Snout-Vent-Length, and developmental stage before processing for biomarker expression. Individual homogenates were assayed for oxidative stress biomarkers superoxide dismutase (SOD; responsible for Reactive Oxygen Species capture and peroxide production), glutathione peroxidase (GPx; responsible for high-affinity peroxide reduction), catalase (CAT; responsible for low-affinity peroxide reduction), glutathione reductase (GR; responsible for glutathione reduction), and thiobarbituric acid reactive substances (TBARS; an indicator of lipid peroxidation).