Identification of Rab family genes and functional analyses of LmRab5 and LmRab11A in the development and RNA interference of Locusta migratoria.

Rab proteins constitute the largest family of small GTPases, which play pivotal roles in intracellular membrane trafficking in all eukaryotes. A number of Rab genes have been identified in eukaryotes; however, very little information about these genes has been reported in insects. In the current study, for the first time we identified and characterized 27 Rab family genes from Locusta migratoria. Phylogenetic analysis and comparison of domain architecture indicated that Rab family genes are highly conserved among insect species. Tissue-dependent expression profiles indicated that expression of Rab genes was highest in the ovary, except for LmRab3, which was most highly expressed in hemolymph. The biological function of each Rab gene was investigated using RNA interference (RNAi). Double-stranded RNA targeting each Rab gene was injected into the hemocoel of nymphs and revealed that suppression of two Rab genes (LmRab5 and LmRab11A) caused 100% mortality. In addition, nymphs injected with dsLmRab5 exhibited severe phenotypic defects in the gastric caeca and midgut, while dsLmRab11A arrested the molting process. We then applied the RNAi of RNAi technique to test if silencing either of these two genes would affect the suppression of the lethal giant larvae (LmLgl) reporter gene and found that suppression of LmRab5 diminished the RNAi efficiency of LmLgl, whereas suppression of LmRab11A enhanced RNAi efficiency of LmLgl. These results indicate that Rab genes contribute differently to RNAi efficiency in different tissues. Our study provides a foundation for further functional investigations of Rab genes and their contributions to RNAi efficiency in L. migratoria.

A dsRNA-degrading nuclease (dsRNase2) limits RNAi efficiency in the Asian corn borer (Ostrinia furnacalis).

The efficiency of RNA interference (RNAi) varies substantially among different insect species. Rapid degradation of double-stranded RNA (dsRNA) by dsRNA-degrading nucleases (dsRNases) has been implicated to cause low RNAi efficiency in several insect species. In this study, we identified four dsRNase genes (OfdsRNase1, OfdsRNase2, OfdsRNase3 and OfdsRNase4) from the Asian corn borer (Ostrinia furnacalis) transcriptome database. Bioinformatic analyses showed that each deduced protein sequence contained endonuclease NS domains and signal peptides. Gene expression analysis revealed that OfdsRNase2 was exclusively expressed in the midgut of larvae. RNAi efficiency was investigated in 2-d-old fifth-instar larvae (high expression of dsRNase2) and 2-d-old pupae (low expression of dsRNase2) by feeding or injecting dsRNA targeting a marker gene that encodes the lethal giant larvae protein (OfLgl). Our results showed that OfLgl only partially silenced the expression of OfLgl in pupae, but not in larvae, suggesting that OfdsRNase2 could contribute to lower RNAi efficiency in larval stages. This hypothesis was supported by our RNAi-of-RNAi experiment using a tissue culture technique where the silencing efficiency against the reporter gene, OfHex1, was significantly improved after knockdown of OfdsRNase2. When double luciferase assays were performed to evaluate the role of the four dsRNases in vitro, only OfdsRNase2 expressed in S2 cells significantly affected RNAi efficiency by degrading dsRNA. Taken together, our results suggested that the degradation of dsRNA by OfdsRNase2 in the midgut contributed to low RNAi efficiency in O. furnacalis larvae.