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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (CYPs) play essential physiological functions in insects. CYP303A1 is highly conserved in insect species studied to date, and shows an indispensable role for adult eclosion in both Locusta migratoria and Drosophila melanogaster. However, how CYP303A1 is regulated to control insect developmental processes remains uninvestigated. In this study, we discovered functional binding sites for miR‐184 in the coding sequence of LmCYP303A1. The luciferase reporter assay showed that miR‐184 could target LmCYP303A1 and regulate its expression in vitro. Furthermore, overexpression of miR‐184 through microinjection of agomir to locusts reduced the transcripts of LmCYP303A1 and led to abnormal molting, which is similar to the phenotype of silencing LmCYP303A1 by direct injection of dsLmCYP303A1 to locusts. Meanwhile, down‐regulation of miR‐184 by injection of antagomir increased the LmCYP303A1 transcript and caused molting defects. These findings suggested that miR‐184 could target LmCYP303A1 to regulate the molting process in L. migratoria, which might be considered as a novel target for pest control. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSPs) are known as chaperones that help with folding of other proteins when cells are under environmental stresses. The upregulation of HSPs is essential for cold survival during insect diapause. The ectoparasitoid Habrobracon hebetor, a potential biological control agent, can enter reproductive diapause when reared at low temperature and short photoperiod. However, the expression of HSPs during diapause of H. hebetor has not been studied. In this study, we sequenced and characterized the full-length complementary DNAs of three Hsp70 genes ( HhHsp70I, HhHsp70II and HhHsp70III) from H. hebetor. Their deduced amino acid sequences showed more than 80% identities to their counterparts from other insect species. However, the multiple sequence alignment among the three deduced amino acid sequences of HhHsp70s showed only 46% identities. A phylogenetic analysis of the three HhHsp70s and all other known Hsp70 sequences from Hymenoptera clustered all the Hsp70s into four groups, and the three HhHsp70s were distributed into three different groups. Real-time quantitative polymerase chain reaction analysis showed that the expression of the three HhHsp70 genes in H. hebetor reared at different conditions was quite different. HhHsp70I showed higher relative expression when H. hebetor were reared at 27.5°C than at two lower temperatures (17.5°C and 20°C) regardless of the photoperiod, whereas HhHsp70II showed higher expression when H. hebetor were reared at 20°C and 10 : 14 L : D than when reared at 17.5°C and either 16 : 8 L : D or 10 : 14 L : D. In contrast, HhHSP70III was expressed at similar levels regardless of the rearing conditions. These results may suggest functional differences among the three HhHsp70 genes in H. hebetor. [ABSTRACT FROM AUTHOR]

Chitin synthase (CHS) is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control. However, our understanding of biochemical properties of insect CHSs has been very limited. We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito, Anopheles gambiae. Our study, which represents the first time to use a nonradioactive method to assay CHS activity in an insect species, determined the optimal conditions for measuring the enzyme activity, including pH, temperature, and concentrations of the substrate uridine diphosphate N-acetyl- d-glucosamine (UDP-GlcNAc) and Mg++. The optimal pH was about 6.5-7.0, and the highest activity was detected at temperatures between 37°C and 44°C. Dithithreitol is required to prevent melanization of the enzyme extract. CHS activity was enhanced at low concentration of GlcNAc, but inhibited at high concentrations. Proteolytic activation of the activity is significant both in the 500 × g supernatant and the 40 000 × g pellet. Our study revealed only slight in vitro inhibition of A. gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration (2.5 μmol/L) examined. There was no in vitro inhibition by polyoxin D at any concentration examined. Furthermore, we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined. Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A. gambiae. [ABSTRACT FROM AUTHOR]

β-N-acetylglucosaminidases are crucial enzymes involved in chitin degradation in insects. We identified a β-N-acetylglucosaminidase gene ( LmNAG1) from Locusta migratoria. The full-length complementary DNA (cDNA) of LmNAG1 consists of 2 667 nucleotides, including an open reading frame (ORF) of 1 845 nucleotides encoding 614 amino acid residues, and 233- and 589-nucleotide non-coding regions at the 5′- and 3′-ends, respectively. Phylogenetic analysis grouped the cDNA-deduced LmNAG1 protein with the enzymatically characterized β-N-acetylglucosaminidases in group I. Analyses of stage- and tissue-dependent expression patterns of LmNAG1 were carried out by real-time quantitative polymerase chain reaction. Our results showed that LmNAG1 transcript level in the integument was significantly high in the last 2 days of the fourth and fifth instar nymphs. LmNAG1 was highly expressed in foregut and hindgut. RNA interference of LmNAG1 resulted in an effective silence of the gene and a significantly reduced total LmNAG enzyme activity at 48 and 72 h after the injection of LmNAG1 double-stranded RNA (dsRNA). As compared with the control nymphs injected with GFP dsRNA, 50% of the ds LmNAG1-injected nymphs were not able to molt successfully and eventually died. Our results suggest that LmNAG1 plays an essential role in molting process of L. migratoria. [ABSTRACT FROM AUTHOR]

The cytochrome P450 monooxygenase (cytochrome P450) gene superfamily comprises many genes that may be involved in the biotransformations of pesticides and other xenobiotics. To date, very little is known about cytochrome P450 genes in the oriental migratory locust, Locusta migratoria manilensis. In this study, we carried out a genome-wide analysis of cytochrome P450 genes of the locust to identify putative cytochrome P450 genes and characterize their expression responses to insecticide exposures. We identified 15 cytochrome P450-like genes from a locust expressed sequence tag database (LocustDB). Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that most cytochrome P450-like genes displayed different tissue and developmental stage expression patterns. However, most of them were predominantly expressed in the midgut, gastric caeca, fatbodies, and/or hindgut. Biochemical analysis showed that cytochrome P450 was differentially affected by three different insecticides. Deltamethrin caused significant inductions in 12 h at LD30 (dose to kill 30% of the tested individuals) in the nymphs, whereas malathion and carbaryl did not have significant effect on cytochrome P450 enzyme activity. Further RT-PCR analysis showed significant increases of transcriptions of several cytochrome P450 genes in deltamethrin-treated locusts. Thus, the increased cytochrome P450 enzyme activity is likely due to increased transcriptions of multiple cytochrome P450 genes in response to deltamethrin exposure. These results are expected to help us better understand the interactions between insecticides and major detoxification enzymes, and possible changes of the susceptibility to other insecticides in deltamethrin-treated insects at various molecular levels. [ABSTRACT FROM AUTHOR]