Trematodes coupled with neonicotinoids: effects on blood cell profiles of a model amphibian

Habitat loss, climate change, environmental contaminants, and parasites and pathogens are among the main factors thought to act singly or together in causing amphibian declines. We tested for combined effects of neonicotinoid pesticides and parasites (versus parasites-only) on mortality, growth, and white blood cell profiles of a model amphibian: the northern leopard frog (Rana pipiens). We first exposed infectious stages of frog trematodes (cercariae of Echinostoma spp.) to low and high concentrations of thiamethoxam or clothianidin versus water-only controls. There were no differences in survival of trematode cercariae between treatments. For the main experiment, we exposed tadpoles to clean water versus high concentrations of clothianidin or thiamethoxam for 2 weeks and added trematode cercariae to all tanks after 1 week. Exposure of tadpoles and parasites to high concentrations of thiamethoxam or clothianidin did not affect parasite infection success. Tadpole survival was not different between treatments before or after parasite addition and there were no significant differences in tadpole snout-to-vent lengths or developmental stages between treatments. Tadpoles exposed to thiamethoxam + parasites had smaller widths than parasite-only tadpoles, whereas tadpoles exposed to clothianidin + parasites had higher eosinophil to leukocyte ratios compared to parasite-only tadpoles. Tadpoles of both neonicotinoid + parasite treatments had significantly lower monocyte to leukocyte ratios relative to parasite-only tadpoles. High concentrations of neonicotinoid combined with parasites appear to influence tadpole immune function important for further defense against parasites and pathogens. This work highlights the need for more holistic approaches to ecotoxicity studies, using multiple stressors.