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

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.
Author Summary Molting is a crucial process in the growth and development in insects. Disturbing the molting process represents an attractive strategy for developing safe and effective insecticides. The migratory locust is a hemimetabolous pest that undergoes five molting stages in its life cycle. Similar molting defects can be observed in expression silencing of the key genes both in miRNA processing and in chitin metabolism. However, any link between a specific miRNA to chitin metabolism has not yet been established. In this study, we elucidated a mechanism by which two miRNAs regulate chitin metabolism related to the molting process. We found that miR-71 and miR-263 directly repress two genes, chitin synthase1 (CHS1) and chitinase10 (CHT10), which are required for chitin biosynthesis and degradation in chitin metabolism. Manipulation of miR-71 and miR-263 expression blocked molting and resulted in abnormal molting by negatively regulating the expression of LmCHS1 and LmCHT10. Furthermore, both up-regulation and down-regulation of LmCHS1 and LmCHT10 by miRNA manipulation altered the chitin content of the new cuticle and old cuticles, leading to a similar aberrant molting phenotype. Our results demonstrate a balancing modulation pattern of two miRNAs in chitin biosynthesis and degradation that controlled the precise molting process in locusts.