Gene misexpression system for functional studies of genes involved in cuticle development in the cricket <i>Gryllus bimaculatus</i>

The insect epidermis possesses the potential to generate remarkable diversity in exoskeletal cuticle traits, including colour, thickness and mechanical properties. Genetic manipulation is essential for investigating the molecular mechanisms; however, spatiotemporally controlled gene overexpression remains technically challenging despite the widespread use of loss-of-function approaches in many insects. In this study, we established an epidermal gene misexpression system in the model hemimetabolous insect Gryllus bimaculatus by inserting a gene expression cassette into the evolutionarily conserved yellow gene, which is involved in melanin synthesis, using genome editing. Insertion of the EGFP expression cassette into yellow gene resulted in fluorescence in the epidermis of pre-hatching embryos and post-moult individuals across developmental stages, corresponding to melanin pigmentation. Using this system, we induced the misexpression of Arylalkylamine N-acetyltransferase (aaNAT), an enzyme involved in the synthesis of N-acetyldopamine (NADA)-sclerotin, which serves as both a white pigment and a crosslinker in the cuticle. Compared to the background strain, the aaNAT misexpression strain exhibited a brighter body coloration, indicating that Gryllus aaNAT has the function of producing white NADA sclerotin and suppressing melanin pigment production. Wing thickness exhibited no substantial alteration, whereas a significant reduction in puncture resistance was observed. Collectively, these results suggest that an imbalance in the molecular components essential for cuticular crosslinking leads to alterations in the mechanical properties of the cuticle. This system serves as a gain-of-function analysis tool for cuticle research and has the potential to be applicable across a wide range of insect species, thereby helping to elucidate cuticle diversity.