RNA interference (RNAi) is a sequence-specific gene silencing mechanism that holds great promise for effective management of agricultural pests. Previous studies have shown that the efficacy of RNAi varies among different insect species, which limits its wide spread application in the field of crop protection. In this study, we identified and characterized six core RNAi pathway genes including OfDicer1, OfDicer2, OfR2D2, OfAgo1, OfAgo2, and OfAgo3 from the transcriptomic database of the Asian corn borer (Ostrinia furnacalis). Domain analysis showed that the six deduced proteins contained the necessary functional domains. Insect developmental stage- and tissue-specific expression analysis showed that five genes were expressed in all the stages and tissues examined except OfAgo3, which showed low expression in larvae, and high expression in pupae and adults and in the midgut. RT-qPCR was performed to examine the response of these six genes to exogenous double-stranded RNA (dsRNA). Interestingly, the transcript levels of OfDicer2 and OfAgo2 were significantly enhanced after the injection of dsEGFP at different time points and tissues investigated. Consequently, the RNAi efficiency in targeting the insect endogenous genes can be greatly enhanced in the hemolymph or midgut. Taken together, our investigations suggest that RNAi efficiency can be enhanced by pre-injection of dsRNA to induce the RNAi core machinery genes, which could be a useful strategy to improving RNAi efficiency for studying gene functions under laboratory conditions.

Small interfering RNAs (siRNAs) are non-coding RNAs with a length of 21~23 nucleotides (nt) and present in almost all eukaryotes. The formation of siRNA is a highly conserved post-transcriptional gene-silencing mechanism mediated by key proteins, including Dicer2, Argonaute2 (Ago2) and R2D2. R2D2 has been identified as a double-stranded RNA (dsRNA)-binding protein and reported as an integral component of the siRNA pathway in Drosophila. However, the involvement of R2D2 in the siRNA pathway of Locusta migratoria is still unknown. In the present study, we identified an LmR2D2 gene from the transcriptome of L. migratoria. It consists of a 954-bp open reading frame that encodes a protein of 318 amino acid residues. Further sequence analysis revealed that LmR2D2 possesses two tandem dsRNA-binding domains (dsRBD) at the N-terminus. Analysis of the developmental expression profile of LmR2D2 indicated that its transcript level was stable in third-instar nymphs of L. migratoria, whereas the tissue-dependent expression profile exhibited high levels of expression of LmR2D2 in the testis and ovary. When LmR2D2 was silenced by RNAi, the RNAi efficiency against Lmβ-tubulin as a marker gene was significantly diminished, as indicated by the 37.7% increased Lmβ-tubulin transcript level. Additionally, the prokaryotic expression system was used to obtain the LmR2D2 supernatant protein. By incubating the LmR2D2 protein with biotin-dsRNA, we found that LmR2D2 can bind to dsRNA in vitro, which supports our conclusion that LmR2D2 plays an essential role in the siRNA pathway of L. migratoria.

Fushi-tarazu factor 1 (FTZ-F1) is a class of transcription factors belonging to the nuclear receptor superfamily and an important molting regulator in insects; however, its detailed function in the molting process of Locusta migratoria is still unclear. This study identified two FTZ-F1 transcripts (LmFTZ-F1-X1 and LmFTZ-F1-X2) in L. migratoria. The classical domains of FTZ-F1 were present in their protein sequences and distinguished based on their variable N-terminal domains. Reverse-transcription quantitative polymerase chain reaction analysis revealed that LmFTZ-F1-X1 and LmFTZ-F1-X2 were highly expressed in the integument. RNA interference (RNAi) was used to explore the function of LmFTZ-F1s in the molting of the third-instar nymph. Separate LmFTZ-F1-X1 or LmFTZ-F1-X2 silencing did not affect the normal development of third-instar nymphs; however, the simultaneous RNAi of LmFTZ-F1-X1 and LmFTZ-F1-X2 caused the nymphs to be trapped in the third instar stage and finally die. Furthermore, the hematoxylin-eosin and chitin staining of the cuticle showed that the new cuticles were thickened after silencing the LmFTZ-F1s compared to the controls. RNA-seq analysis showed that genes encoding four cuticle proteins, two chitin synthesis enzymes, and cytochrome P450 303a1 were differentially expressed between dsGFP- and dsLmFTZ-F1s-injected groups. Taken together, LmFTZ-F1-X1 and LmFTZ-F1-X2 are involved in the ecdysis of locusts, possibly by regulating the expression of genes involved in cuticle formation, chitin synthesis, and other key molting processes.

The epidermal cells of insects are polarized epithelial cells that play a pivotal role in the insect’s molting process. Sinuous, a pivotal structural protein involved in the formation of septate junctions among epithelial cells, is essential for its physiological function. In this study, to determine whether sinuous participates in the regulation of insect molting, we identified the sinuous gene, Lmsinu, in Locusta migratoria, which encodes a protein belonging to the claudin family and shares 62.6% identity with Drosophila’s sinuous protein. Lmsinu is expressed in multiple tissues, and its expression level in the integument significantly increases prior to molting. Knockdown of Lmsinu in L. migratoria results in larval mortality during molting. Furthermore, hematoxylin and eosin and chitin staining demonstrate that the downregulation of Lmsinu led to a prolonged degradation process of the old cuticle during the molting process. Electron microscopy analysis further revealed that knockdown of Lmsinu disrupts the formation of septate junctions among epidermal cells, which are a monolayer of polarized epithelial cells, which may hinder the functionality of epidermal cells during the process of molting. In summary, these findings suggest that Lmsinu plays a role in nymph molting by regulating the formation of septate junctions among epidermal cells.