Positive selection analysis

Many butterfly and moth larvae have evolved a wide range of adaptations to feed on plants that produce toxic secondary metabolites. <i>Crotalaria </i>species are mostly known for containing Pyrrolizidine Alkaloids (PAs) that produce harmful effects on humans, wildlife and livestock. However, numerous tiger moths, including the Bella moth (<i>Utetheisa ornatrix</i>), can metabolize, sequester and use PAs for predator defense by both caterpillars and adults. Here, we used male Bella moth caterpillars as model systems to investigate adaptations to PAs through an integrative morphological, genetic and evolutionary framework. We used micro-CT scanning to describe the morphology of organs associated with secondary metabolite metabolism and used qPCR to quantify differences in detoxification gene expression in these organs by comparing caterpillars fed on a PA and PA-devoid diet. Furthermore, we performed positive selection analyses on two PA detoxification genes across twenty-eight tiger moth species to determine if they have undergone adaptative evolution in response to their PA diet. Our results characterized the morphology and spatial organization of the fat body, gut, integument, Malpighian tubules and testes and revealed that detoxification gene expression varied across organs and diets. In particular, the mRNA levels of <i>UGT</i> and <i>SOD2</i> differed significantly among diets. Additionally, we identified specific lineages and amino acid residues under positive selection. Collectively, these findings provide new insights into the morphological specialization of organs, the diet-dependent regulation of detoxification genes and the evolutionary pressures shaping adaptations to secondary metabolites.