Predatory fireflies and their toxic firefly prey have evolved distinct toxin resistance strategies.

Toxic cardiotonic steroids (CTS) act as a defense mechanism in many firefly species (Lampyridae) by inhibiting a crucial enzyme called Na,K-ATPase (NKA). While most fireflies produce these toxins internally, species of the genus acquire them from a surprising source: predation on other fireflies. The contrasting physiology of toxin exposure and sequestration between and other firefly genera suggests that distinct strategies may be required to prevent self-intoxication. Our study demonstrates that both and their firefly prey have evolved highly-resistant NKAs. Using an evolutionary analysis of the specific target of CTS (ATPα) in fireflies, and gene-editing in , we find that the initial steps towards resistance were shared among and other firefly lineages. However, the lineage subsequently underwent multiple rounds of gene duplication and neofunctionalization, resulting in the development of ATPα paralogs that are differentially expressed and exhibit increasing resistance to CTS. In contrast, other firefly species have maintained a single copy. Our results implicate gene duplication as a facilitator in the transition of to its distinct ecological role as predator of toxic firefly prey.