Dual roles of <i>LmCDA1</i> and <i>LmCDA2</i> in the assembly and integrity of the embryonic cuticle in <i>Locusta migratoria</i>

The embryonic pro-nymphal cuticle of hemimetabolous insects is a critical protective structure, yet the molecular mechanisms governing its formation, particularly the regulation of chitin organization, remain poorly understood. Chitin deacetylases (CDAs) are key enzymes that modify chitin physicochemical properties, prompting this study to investigate the functions of two Group I CDAs, LmCDA1 and LmCDA2, during the formation of the pro-nymphal cuticle in Locusta migratoria. We found that both LmCDA1 and LmCDA2 were highly expressed during the key period of pro-nymphal cuticle formation (E8-E11). Immunofluorescence localization revealed that LmCDA1 and LmCDA2 proteins were deposited in the apical region of the procuticle. RNA interference (RNAi)-mediated knockdown of LmCDA1 significantly reduced cuticle thickness but did not disrupt the helicoidal organization of chitin laminae. In contrast, knockdown of LmCDA2 completely abolished the laminar structure, resulting in disorganized chitin microfibrils and increased cuticle thickness. Together, these results demonstrate that LmCDA1 and LmCDA2 play distinct and complementary roles in regulating the biosynthesis and structural integrity of the pro-nymphal cuticle. Functional assays further revealed that suppression of either gene impaired the cuticular barrier, accelerated water loss under desiccating conditions, and significantly reduced egg tolerance to dryness. This study provides evidence that CDAs are essential for embryonic cuticle formation and desiccation resistance in a hemimetabolous insect. Our findings offer new insights into the evolutionary functional diversification of CDAs and suggest a potential novel strategy for locust control by targeting chitin metabolism during embryogenesis.