Structural and Functional Insights into Methuselah Genes of Plutella xylostella (Lepidoptera: Plutellidae): Evolutionary Adaptations and Their Responses to Chlorantraniliprole

G protein-coupled receptors (GPCRs) are considered the largest and most variable family of transmembrane receptors regulating physiological processes such as toxicological responses and insecticide resistance development. The present study investigated the responses of Methuselah (<i>Mth</i>), belonging to GPCR family B in the Diamondback Moth (DBM), <i>Plutella xylostella</i>, to chlorantraniliprole (CAP). Genome-wide identification and phylogenetic analysis of <i>Pxmth</i> genes revealed their evolutionary relationships and functional classifications. Expression profiling demonstrated significant overexpression of <i>Pxmth2</i> in the CAP-resistant strain. Additionally, the tertiary and secondary structures of <i>Pxmth2</i> were characterized, providing insights into its functional role. Silencing <i>Pxmth2</i> via RNA interference (RNAi) reduced resistance of DBM to CAP and suppressed downstream stress-associated genes (<i>CYP6B6</i>, <i>CYP6B7</i>, <i>CYP6BF1</i>), increasing susceptibility to the insecticide. The function of <i>Pxmth2</i> was further explored using a transgenic line of <i>Drosophila melanogaster</i> engineered to overexpress the gene; flies overexpressing <i>Pxmth2</i> exhibited a significantly increased resistance to CAP compared to controls. These findings indicate that <i>Pxmth2</i> contributes to CAP resistance in DBM and highlights potential molecular targets for improving pest management strategies.