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

## TL;DR

This study identifies key chitinase genes in Tuta absoluta and shows their essential roles in insect molting and metamorphosis, offering new targets for pest control.

## Contribution

The study identifies and functionally characterizes chitinase genes in Tuta absoluta, revealing four key genes critical for molting and metamorphosis.

## Key findings

- Four chitinase genes (TaCht5, TaCht7, TaCht10, and TaIDGF) are essential for molting and metamorphosis in Tuta absoluta.
- RNAi silencing of these genes caused high mortality and developmental defects in larvae and pupae.
- Functional specialization of chitinases was observed, with some predominantly expressed in the integument and others in the gut.

## Abstract

The South American tomato leaf miner, Tuta absoluta, is a highly destructive invasive pest of solanaceous crops worldwide. Intensive reliance on chemical insecticides has led to widespread resistance and heightened environmental concerns, underscoring the need for species-specific and physiologically informed alternatives. Chitinases, which mediate chitin turnover during molting and metamorphosis, represent promising molecular targets for RNA interference (RNAi)-based control strategies. Therefore, this study aims to systematically characterize the chitinase family genes in T. absoluta and investigate their roles in larva–pupa–adult transitions through molecular characterization, spatiotemporal expression, and RNAi analyses. These findings reveal that four key chitinases (TaCht5, TaCht7, TaCht10, and TaIDGF) are indispensable for the molting physiology and metamorphic progression of T. absoluta, thereby providing potential molecular targets for leaf miner pest management.

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 (TaChts) in T. absoluta, 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 TaChts peak during key developmental transitions. TaCht1, TaCht2, TaCht3, TaCht5, TaCht6, TaCht7, TaCht10, TaCht-h, and TaIDGF were predominantly expressed in the integument, whereas TaCht8 and TaCht11 showed gut-enriched expression, indicating functional specialization. Stage-tailored RNAi assays demonstrated that silencing TaCht5, TaCht7, TaCht10, and TaIDGF caused pronounced mortality in both larvae and pupae. In larvae, delivery of dsRNA using carbon quantum dots (CQDs) disrupted molting: knockdown of TaCht7, TaCht10, and TaIDGF prevented shedding of the old cuticle, whereas TaCht5 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 T. absoluta, 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.