Toxin resistance mechanisms span biological scales in the Royal Ground Snake (Colubridae: Erythrolamprus reginae)

Exposure to multiple toxic compounds imposes diverse selective pressures, potentially leading to a toxin-resistant phenotype that operates across biological levels. There are several known toxin resistance mechanisms–such as behavioral avoidance, metabolic detoxification, and target-site insensitivity. However, most studies have been conducted with exposure to a single toxin or have focused on only one of these mechanisms. More integrative approaches are necessary to capture the complexity of the toxin-resistant phenotype in a single organism. Predators of amphibians, for example, must counteract multiple chemicals secreted by different species or even by the same individual prey. The pan-Amazonian snake Erythrolamprus reginae (Squamata: Colubridae) preys on multiple species of poisonous frogs, including members of the Dendrobatidae family, and is therefore exposed to a chemically diverse diet. We aimed to evaluate the process of consuming a toxic prey, from behavioral decisions to a suite of possible resistance mechanisms. We tested interrelated hypotheses to understand the complexity of toxin resistance in E. reginae. First, feeding assays revealed that E. reginae exhibited longer handling times and aversive behaviors toward the highly toxic Ameerega trivittata, suggesting that prey toxicity imposes searching and handling costs that influence foraging strategies. Second, we developed a novel assay to screen liver extracts for toxin neutralization and showed that soluble proteins in the liver partially restored sodium channel activity inhibited by A. trivittata alkaloids and neosaxitoxin, indicating the presence of toxin-binding proteins that mediate detoxification. Third, transcriptomic profiling across tissues revealed liver-specific upregulation of transporters, such as the solute carrier protein family, and stress-response genes following exposure to A. trivittata, supporting a complementary detoxification mechanism. Finally, using two-electrode voltage-clamp recordings, we showed that one variant of the E. reginae muscle-expressed voltage-gated sodium channel NaV1.4 is highly resistant to tetrodotoxin, saxitoxin, and neosaxitoxin. However this same NaV1.4 channel variant did not prevent inhibition by A. trivittata alkaloids, suggesting that resistance to these compounds relies on alternative mechanisms such as the putative liver binding proteins. These findings demonstrate that E. reginae populations may be adapting to a chemically diverse diet by evolving multiple, overlapping forms of resistance, highlighting the complexity of resistance where selection favors multiple mechanisms acting at different physiological levels to mitigate the effects of prey toxins. This study provides unparalleled insight into whole-organismal resistance to toxin ingestion, advancing our understanding of the genetic architecture underlying toxin adaptation and its broader physiological and evolutionary implications.