The success of the degradation of the extraembryonic serosal cuticle and the second embryonic cuticle (pro-nymphal cuticle) is essential for the development and molting of nymph from egg in Orthoptera Locusta migratoria. Chitinase 5 is an important gene for chitin degradation in nymphs and in the egg stage. In this study, we investigated the important roles of chitinase 5-1 (LmCht5-1) and chitinase 5-2 (LmCht5-2) in the degradation of the serosal and pro-nymphal cuticles during locust embryonic development. The serosal cuticle degrades from 7-day-old embryos (E7) to E13, along with the degradation of the pro-nymphal cuticle, which begins at E12 to E14. The mRNA and protein of LmCht5-1 and LmCht5-2 are expressed during the degradation process of the serosal cuticle and the pro-nymphal cuticle. RNAi experiments at the embryonic stage show that both dsLmCht5-1 and dsLmCht5-2 contribute to the failure of development in early and late embryogenesis. Further, during the serosal cuticle molting process, ultra-structure analysis indicated that dsLmCht5-1 prevented the loss of the coarse chitin layer in the upper part in both early and late embryogenesis. Meanwhile, dsLmCht5-2 blocked the degradation of the lower fine chitin layer at the early stage and blocked the chitin degradation of loose coarse chitin in the late molting process. During the degradation of the pro-nymphal cuticle, dsLmCht5-1 suppresses chitin degradation between layers in the procuticle, while dsLmCht5-2 suppresses chitin degradation into filaments inside of the layer. In summary, our results suggest that both LmCht5-1 and LmCht5-2 contribute to the degradation of the serosal and pro-nymphal cuticles during the locust embryonic stage.

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.