CYP4G7 and CYP4G14 mediate cuticle formation and pigmentation in Tribolium confusum

Cytochrome P450 genes compose a superfamily in insects, acting as the terminal oxidase in the monooxygenase system coupled with an electron donor such as NADPH-cytochrome P450 reductase (CPR). Recent studies have shown that CYP4G subfamily genes have a considerable role in cuticle formation and hydrocarbon synthesis in insects. However, CYP4Gs-related phenotypes and functions need to be further explored in beetles. In this study, CPR and two CYP4G genes, CYP4G7 and CYP4G14, were identified in Tribolium confusum. CPR, CYP4G7 and CYP4G14 were detected during different developmental stages and highly expressed at a late stage of pupae, with the levels of CYP4G14 much higher than those of CPR and CYP4G7 in T. confusum. Microinjection-based RNAi was applied to T. confusum and all three genes obtained over 50% reduce in expression levels in adults. RNAi of three genes performed in sixth-instar larvae resulted in higher than 60% mortalities in T. confusum during eclosion and cuticular pigmentation was inhibited in adults, leading to light coloration in adults. dsRNA injection in pupae led to defects in T. confusum eclosion and cuticular pigmentation. Abnormal adults had deformed epicuticle confirmed with scanning electron microscope and died within two weeks after eclosion. The results illustrated that CYP4G7 and CYP4G14, coupled with CPR, mediated cuticle formation and pigmentation in adults. The results provided new insights into the function of CYP4G subfamily genes in insects.