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Chitin synthase and chitinase play crucial roles in chitin biosynthesis and degradation during insect molting. Silencing of Dicer-1 results in reduced levels of mature miRNAs and severely blocks molting in the migratory locust. However, the regulatory mechanism of miRNAs in the molting process of locusts has remained elusive. In this study, we found that in chitin metabolism, two crucial enzymes, chitin synthase (CHS) and chitinase (CHT) were regulated by miR-71 and miR-263 during nymph molting. The coding sequence of  CHS1  and the 3’-untranslated region of  CHT10  contain functional binding sites for miR-71 and miR-263, respectively. miR-71/miR-263 displayed cellular co-localization with their target genes in epidermal cells and directly interacted with  CHS1  and  CHT10  in the locust integument, respectively. Injections of miR-71 and miR-263 agomirs suppressed the expression of  CHS1  and  CHT10 , which consequently altered chitin production of new and old cuticles and resulted in a molting-defective phenotype in locusts. Unexpectedly, reduced expression of miR-71 and miR-263 increased  CHS1  and  CHT10  mRNA expression and led to molting defects similar to those induced by miRNA delivery. This study reveals a novel function and balancing modulation pattern of two miRNAs in chitin biosynthesis and degradation, and it provides insight into the underlying molecular mechanisms of the molting process in locusts.

miR-71 and miR-263 Jointly Regulate Target Genes Chitin synthase and Chitinase to Control Locust Molting