RNAi-mediated nuclear receptor HR96 gene knockdown inhibits feeding and gut development in Locusta migratoria

Nuclear receptors (NRs) play key roles in many biological processes such as cell differentiation, metabolic regulation and insect development. Despite the significant roles of HR96 plays in holometabolous insects, its molecular characteristics and functions in hemimetabolous insects remain poorly understood. We first identified a HR96 gene in a orthopteran insect, L. migratoria. The classical domains of NRs were present in LmHR96, which include a DNA-binding domain (DBD) and a ligand-binding domain (LBD). RT-qPCR results showed that LmHR96 mRNA exhibited a higher level of expression in the integument, foregut and hindgut. And LmHR96 expressed in the examined stages, which were from the early 3rd through the terminal 5th instar. We further demonstrated that LmHR96 expression significantly increased by 20E supplement, and was suppressed after silencing LmEcR than those in the control group. The injection of double-stranded RNAs against LmHR96 resulted in the feeding cessation, with body weight reduced. Hematoxylin-eosin staining and TEM analysis further showed that gut cells in dsLmHR96 treatments were ruptured and fragmented. Accordingly a thinner old cuticle of foregut and hindgut were found, and the structural integrity of the brush border (microvilli) of midgut were destroyed. RNA-seq and RT-qPCR analysis showed that the expression of digestion-related genes, trehalase genes, wingless and notch signaling pathway genes were dramatically declined in dsLmHR96-injected groups. These results indicate that LmHR96 is essential for the morphogenesis of guts in L. migratoria and targeting LmHR96 via RNAi in nymphal locusts inhibits locust feeding and induces high mortality rates, therefore LmHR96 could be a key target for RNAi-based pest management strategies.