Identification and Functional Characterization of Chitinase Genes During Larva–Pupa–Adult Transitions in Tuta absoluta

Chitinases are crucial in the molting process of insects and represent potential targets for the development of RNA interference (RNAi)-based insecticidal strategies. In this study, we identified and characterized 11 chitinase genes (<i>TaChts</i>) in <i>T. absoluta</i>, each harboring at least one GH18 catalytic domain. Phylogenetic analysis placed these proteins into 11 established groups (I-X and h), revealing conserved lineage-specific patterns. Spatiotemporal expression profiling showed that most <i>TaChts</i> peak during key developmental transitions. <i>TaCht1</i>, <i>TaCht2</i>, <i>TaCht3</i>, <i>TaCht5</i>, <i>TaCht6</i>, <i>TaCht7</i>, <i>TaCht10</i>, <i>TaCht-h</i>, and <i>TaIDGF</i> were predominantly expressed in the integument, whereas <i>TaCht8</i> and <i>TaCht11</i> showed gut-enriched expression, indicating functional specialization. Stage-tailored RNAi assays demonstrated that silencing <i>TaCht5</i>, <i>TaCht7</i>, <i>TaCht10</i>, and <i>TaIDGF</i> caused pronounced mortality in both larvae and pupae. In larvae, delivery of dsRNA using carbon quantum dots (CQDs) disrupted molting: knockdown of <i>TaCht7</i>, <i>TaCht10</i>, and <i>TaIDGF</i> prevented shedding of the old cuticle, whereas <i>TaCht5</i> silencing produced severe cuticular shrinkage and blackening. Hematoxylin and eosin staining revealed detachment of epidermal cells from the cuticle, providing cytological evidence of disrupted cuticle-epidermis remodeling. In pupae, microinjection of dsRNA induced cuticular collapse, melanization, and eclosion failure due to persistent adhesion of the pupal case, accompanied by marked reductions in chitinase activity. Together, our findings highlight the critical roles of four key chitinases in endocuticular turnover and metamorphic progression in <i>T. absoluta</i>, offering mechanistic insight into chitin-mediated developmental processes. The identified genes represent strong candidates for RNAi-based, species-specific pest management strategies against this globally significant insect.