Endocuticle structural glycoproteins are involved in bursicon signaling pathway: insights into their roles in regulating wing expansion in the oriental fruit fly, <i>Bactrocera dorsalis</i>

Bursicon is indispensable for wing expansion in Bactrocera dorsalis, acting through PKA-mediated regulation of the wing-development gene Bdhh. Although cuticular proteins are recognized as critical regulators of insect wing development, it remains unclear whether they directly respond to the bursicon signaling pathway. Here, we conducted a functional dissection of cuticular protein genes during wing expansion. Transmission-electron-microscopy (TEM) analysis revealed that bursicon controls endocuticle development in the wing cuticle. Genome-wide mining uncovered sixteen CPR-type endocuticle structural glycoprotein genes, of which three cuticular protein genes (BdABD-4a, BdABD-4b, and BdABD-4c) were selected for RNAi on the basis of their expression profiles. Silencing of each gene via dsRNA injection at the 5-d-old pupal stage produced wing malformations in 37.5%, 32.5%, and 40% of adults, respectively, and reduced cuticle thickness by 40.4%, 42.4%, and 44.2% relative to controls. TEM confirmed the presence of thinner endocuticle in malformed wings. Furthermore, expression levels of all three endocuticle structural glycoprotein genes were markedly altered following knock-down of Bursicon genes, Bdhh and after PKA inhibition, indicating that these proteins operate downstream of the bursicon signaling cascade. The results in this study demonstrated that the functional role of BdABD-4a, BdABD-4b, and BdABD-4c in wing expansion, and provide new insights into the molecular mechanism underlying bursicon regulated wing expansion in B. dorsalis.