| In insects, chitin plays a significant role in the physical progress of growth and development.Pest control by interfering and disrupting chitin biosynthesis have a show potential role inresearch on novel pesticides and integrated pest management. Insect trehalases and chitinsynthases are the first and last enzymes in the pathway of chitin biosynthesis, respectively, andare ideal targets for designing eco-friendly insecticides. Benzoylphenylureas (BUPs), belongingto the insect growth regulators, can specifically affect various physiological process, such asinsect chitin biosynthesis and molting, thereby providing toxicity against the pest. The cottonbollworm, Helicoverpa armigera (Lepidoptera: Noctuidae) is a worldwide pest in agriculture.In this research,using selected H. armigera as the object of study, the genes for trehalaseand chitin synthase were cloned, and the expression levels of those genes in differentdevelopmental stages and tissues were analyzed by quantitative real-time PCR. Furthermore, westudied the toxicities of benzoylphenylureas insecticides, chlorfluazuron and hexaflumuron atdifferent concentration, and their primary effects of these two insecticides against H. armigera,including physiological and biochemical, and even molecular aspects. The results aresummarized as follows:1. Cloning and expression analysis of trehalase genes in H. armigeraBy RT-PCR and RACE techniques, the two genes of trehalase, the soluble trehalase gene(HaTreh-1) and the membrane-bound trehalase gene (HaTreh-2), were cloned and characterizedin H. armigera. HaTre-1contains an open reading frame of1716bp that encoded for a572amino acid residue protein with a predicted mass of approximately65.38kDa and an isoelectricpoint of5.02. HaTreh-2has an open reading frame of1938bp, encoding a protein of646aminoacids, a mass of approximately73.85kDa, and an isoelectric point of6.04. Sequence alignmentand phylogenetic analysis of the two putative proteins revealed that HaTreh1and HaTreh2 belong to soluble and membrane-bound trehalase groups respectively. The expression pattern indifferent developmental stages and tissues of HaTrehs in H. armigera was measured byquantitative real-time PCR (qPCR), and the results revealed that the HaTreh-1gene wasexpressed mainly in the midgut, followed by the integument and head, and was very weaklyexpressed in the Malpighian tubules, trachea, and fat body. The expression levels of theHaTreh-2gene were detected in all6tissues, with HaTreh-2mainly expressed in the midgut andhead. Expression of HaTreh-1was higher throughout larvae stage, and lower on days1and2ofthe pupal stage, but the expression level of HaTreh-2was higher during the4th-and5th-instarlarval stages. Taken together, these results demonstrated that HaTreh-1and HaTreh-2havedifferent functions in various stages and tissues.2. Cloning and expression analysis of chitin synthase1gene in H. armigeraIn this research, chitin synthase1gene (HaCHS-1) was cloned from H. armigera. Thefull-length cDNA of HaCHS-1gene was5,285bp, containing an open reading frame of4,698bp,5’-UTR of206bp and3’-UTR of381bp, which encoded for a1,565amino acid residues protein，with a predicted mass of approximately180.7kDa and an isoelectric point of6.02. Sequencealignment and phylogenetic analysis of the putative proteins revealed that HaCHS-1belong tochitin synthase1group. The expression patterns in different developmental stages and tissues ofHaCHS-1in H. armigera were measured by quantitative real-time PCR (qPCR), and the resultsrevealed that the HaCHS-1gene expressed mainly in the integument and head, while it wasseldom expressed in other tissues. The expression level of HaCHS-1was higher at the primary orlast periods of4th and5th larval stages, while it increased significantly after pupal stage,showing the highest expression, then decreased gradually at the day-1and day-2pupal stage.3. Toxic bioassay of insect growth regulators on3rd-instar larvae of H. armigeraAfter exposure of chorfluazuron and hexaflumuron to3rd-instar larvae of cotton bollworm,the toxicity effects were expressed as slow response, abnormal molting and abnormal cuticulardevelopment on cuticle with malformation finally, leading to death. Both chlorfluazuron andhexaflumuron caused tangible contact toxicities to H. armigera, and the toxicity regressionequation of chlorfluazuron is: y=2.409+1.027x (r=0.996)，LC50=333.67mg/L；hexaflumuronis：y=3.6891+0.5566x (r=0.995), LC50=226.43mg/L.4. Effects of insect growth regulators on chitin content, chitinase activity and the geneexpression levels of chitin synthase and chitinase in H.armigeraWhen the chlorfluazuron was at120and240mg/L concentrations, the chitin contentdecreased significantly at96h post-exposure in3rd-instar larvae of cotton bollworm. At96hpost-exposure to chlorfluazuron, the chitinase activity was significantly activated at the lower sublethal concentration (60mg/L), and showed inhibition at the two higher concentrations (120and240mg/L). At a sublethal concentration (60mg/L), the expression levels of HaCHS-1showed changes of “inhibition-activation-re-inhibition” and “from activation to inhibition”respectively, in larvae treated by chlorfluazuron.At72h post-exposure to hexaflumuron, the chitin content decreased significantly in larvaetreated with120mg/L and240mg/L concentrations, in accordance with a significant activationof chitinase activities. At the concentration of60mg/L, the expression levels of HaCHS-1andHaCHA showed consistent inhibition in larvae post-exposure to hexaflumuron, and the inhibitionbecame dramatical with the increasing exposure-time.5. Effects of insect growth regulators on trehalose content, trehalase activity and their geneexpression in H. armigeraAfter exposure to chlorfluazuron with3rd-instar larvae, the content of trehalose and glucoseincreased, then decreased significantly than control at72h and96h post-exposure in theconcentration of60mg/L, while both were significantly lower than the control at96h aftertreatment at concentrations of120mg/L and240mg/L. After exposure to60mg/Lchlorfluazuron, the soluble trehalase activity in treated larvae showed activation to some extent,and the degree of activation decreased with the increasing exposure-time. But themembrane-bound trehalase activity showed fluctuation with no significant difference whencompared to the control. At96h after exposure to240mg/L hexaflumuron with3rd-instar larvae,the content of trehalose and glucose increased significantly compared to control. The resultsshowed the changing expression levels of HaTreh-1and HaTreh-2, from activation to inhibitionin H. armigera post-exposure to chlorfluazuron and hexaflumuron with concentration of60mg/L,while up-regulation of HaTreh-2expression lagging behind that of HaTreh-1. |
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