Comparative analysis of cytochrome P450-like genes from Locusta migratoria manilensis： expression profiling and response to insecticide exposure

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 genomewide analysis ofcytochrome P450 genes of the locust to identify putative cytochrome P450 genes and characterize their expression responses to insecticide exposures. We identified 15 cytochrome P450-1ike genes from a locust expressed sequence tag database （LocustDB）. Reverse transcription polymerase chain reaction （RT-PCR） analysis showed that most cytochrome P450-1ike genes displayed different tissue and developmental stage expression patterns. However, most of them were predominantly expressed in the midgnt, 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.

RNA interference to reveal roles of β-N-acetylglucosaminidase gene during molting process in Locusta migratoria

β-N-acetylglucosaminidases are crucial enzymes involved in chitin degrada- tion 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 dsLmNAGl-injected nymphs were not able to molt successfully and eventually died. Our results suggest that LmNAG1 plays an essential role in molting process ofL. migratoria.

Heterologous expression and characterization of two chitinase 5 enzymes from the migratory locust Locusta migratoria

Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix ofmidgut. In Locusta migratoria, two duplicated Cht5s （LmCht5-1 and LmCht5-2） have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate （4MU-（GlcNAe）3）, and higher Km value for the longer substrate （CM-Chitin- RBV） compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain （CBD） tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5- I and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-（GlcNAc）3, whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBE These findings are helpful for further research to clarit}g their different roles in insect growth and development.

The microRNA miR-184 regulates the CYP303A1 transcript level to control molting of Locusta migratoria

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.

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(OfdsRNaseL Ofd-sRNase2,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,OfHexl,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.

Characteristics of Halloween genes and RNA interference-mediated functional analysis of LmCYP307a2 in Locusta migratoria

Halloween genes are involved in the biosynthesis of the molting hormone, which plays a key role in insect ecdysis, development, metamorphosis, and reproduction. Our previous work identified five Halloween genes from Locusta migratoria, but their functions are currently unknown. In this study, the sequences of these five Halloween genes were analyzed and characterized. LmCYP307a2, LmCYP306a1, LmCYP302a1, and LmCYP315a1 were primarily expressed in the prothoracic glands, while LmCYP314a1 was universally expressed in peripheral tissues, especially in the ovaries and Malpighian tubules. All five Halloween genes were mainly expressed from the 5th to the 7th d in 5th-instar nymphs. RNA interference (RNAi) silencing of LmCYP307a2 resulted in severe molting delays and molting failure, which could be rescued by supplementary 20-hydroxyecdysone. A hematoxylin and eosin staining analysis suggested that the RNAi of LmCYP307a2 inhibited the ecdysis process by inhibiting the apolysis and degradation of the old cuticle, and by promoting the synthesis of a new cuticle. Quantitative reverse transcription polymerase chain reaction results showed that the expressions of LmE74, LmCht5, and LmCht10 were dramatically down-regulated, while that of LmChsI was substantially up-regulated, after knockdown of LmCYP307a2. The results suggest that LmCYP307a2 is related to the molt process via regulation of chitin synthesis and degradation.