Study On Physiological,Biochemical And Molecular Mechanisms Of Insecticide-induced Resurgence Of Nilaparvata Lugens(Stal)(Hemiptera:Delphacidae)

The brown planthopper(BPH),Nilaparvata lugens(Stal)(Hemiptera:Delphacidae),is a serious pest of rice in the tropical regions of Asia.It is also a typical recurrent pest induced by pesticides.The previous investigations have focused more on ecological aspects of pest resurgence.However,physiological and molecular mechanisms effects of the insecticide-induced resurgence in BPH are not well understood.The present paper studied physiological and biochemical and molecular mechanisms of resurgence of N.lugens.Main results are showed as follows.1.Insecticide-induced changes in protein,RNA,and DNA contents in ovary and fat body of female Nilaparvata lugens(Stal)(Hemiptera:Delphacidae)The present study investigated insecticide-induced changes in protein,RNA and DNA levels in the ovary and fat body of female adults.The results showed that all sublethal dose of three insecticides caused a significant increase in protein and RNA contents in the ovary and fat body of female adults developed from nymphs fed on treated plants compared to those from on untreated plants,though there was no significant effect on DNA content.In general,protein and RNA contents in both ovary and fat body linearly increased with insecticide concentrations or days after emergence(DAEs).There was a significant correlation between protein content and RNA content in both ovary and fat body.In addition,the relationship between ratios of RNA to protein in ovary and fat body of adult female of N.lugens and insecticide concentrations showed a significant positive correlation for most DAEs of imidacloprid and several DAEs of triazophos and deltamethrin treatments.These findings suggest that RNA in both ovary and fat body was more sensitive to insecticides.The insecticide-induced changes in RNA content in turn influence protein synthesis in ovary and fat body.Consistently,RNA to protein ratios in ovary and fat body were 0.42 in all treatments and controls.2.Induction of Nlvg and suppression of Nljhe gene expression in Nilaparvata lugens(Stal)(Hemiptera:Delphacidae)adult females and males exposed to two insectidesJuvenile hormone(JH)plays a key role in regulation of growth,development,diapause and reproduction of insects.The fecundity of insects is mainly regulated by the synthesis of vitellin(Vt)and vitellogenin(Vg);vitellogenin synthesis,in turn,is regulated by JH.The present study investigated the effect of sublethal dose of two insecticides,deltamethrin and triazophos,on gene expression of vitellogenin(Nlvg)and juvenile hormone esterase(Nljhe)associated with reproduction.Our findings show that the Nlvg mRNA expression in adult females before and after mating is significantly induced by insecticides.Deltamethrin caused Nlvg mRNA expression to be 0.6,1.7 and 2.1 times the control levels at 1,2 and 3 days before mating(DBM)after adult emergence,respectively,and 2.5,4.1 and 5.2 times the control levels at 1,2 and 3 days after mating(DAM),respectively.Triazophos exposure induced Nlvg mRNA expression at levels that were 1.2,2.4 and 2.7 times the control levels at 1,2 and 3 DBM,respectively,and 4.1,5.4 and 6.0 times the control levels at 1,2 and 3 DAM,respectively.The transcript level of Nlvg mRNA was higher for triazophos than for deltamethrin before and after mating.However,expression of Nljhe mRNA in adult females before mating was significantly suppressed by insecticides.The two insecticides significantly induced Nljhe mRNA expression in females after mating,and the induction increased with increasing insecticide concentration,especially at 1 and 2 DAM.JHIII titer in N.lugens adult females both before and after mating increased with insecticide concentration.JHIII titer in N.lugens adult males increased before mating and decreased after mating compared to controls,indicating that JHIII in adult males is transferred to adult females via mating.JH titer and insecticide concentration could be fitted into a significant regression equation.3.Mating pair combinations of insecticide-treated male and female Nilaparvata lugens(Stal)(Hemiptera:Delphacidae)planthopper influence protein content in the male accessory glands(MAGs)and vitellin content in both fat bodies and ovaries of adult females The brown planthopper,Nilaparvata lugens(Stal)(Hemiptera:Delphacidae),is an insect pest in which offspring are produced by the mating of adult males with adult females.This species is a classic case in which pest resurgence is induced by insecticides.In the past,studies of resurgence mechanisms have focused on sublethal dose of insecticide-induced stimulation of reproduction in adult females.To date,however,the role that males play in the resurgence mechanisms of N.lugens has not been investigated.The aim of the present study is to examine changes in protein levels in male accessory glands(MAGs)induced by the insecticides triazophos and deltamethrin and to determine their relationship with vitellin content in the fat bodies and ovaries of adult females in the context of mating pairs.Our results show that protein content in MAGs is significantly affected by male mating status,insecticide type,and insecticide concentration.Insecticide application induced an increase of protein levels in MAGs.A greater quantity of MAG products was transferred to females via mating.Thus,protein levels in MAGs significantly decreased after mating.Experimental matings indicate that vitellin content in both fat bodies and ovaries of adult females in mating pairs consisting of a treated male and an untreated female((?)t ×(?)ck)is significantly greater than that of females in pairs consisting of an untreated male and an untreated female((?)ck ×(?)ck).Under various concentrations of the two insecticides,vitellin levels are highest in mating pairs consisting of a treated male and a treated female((?)t ×(?)t),followed by mating pairs consisting of an untreated male with a treated female((?)ck ×(?)t).These findings demonstrate that 1)insecticides have an effect on males;2)insecticide effect can be transferred to females;and 3)the reproductive effect of insecticides is strongest in mating pairs in which both the males and females are treated compared to pairs in which only one individual is treated.4.Proteomic analysis of insecticide triazophose-induced mating responsive proteins of Nilaparvata lugens Stal(Hemiptera:Delphacidae)It has been demonstrated that sublethal dose of triazophos treatment causes an increase in the of content of male accessory gland proteins(Acps)that can be transferred to females via mating,influencing female reproduction.However,the mechanism of this type of sublethal dose of insecticide-induced Acps in males and the subsequent stimulation of reproduction in females are not well understood.To identify changes in the types of Acps and reproductive proteins in mated females,we conducted a comparative proteomic analysis.Six samples were categorized into four different groups:(1)untreated unmated males compared to treated unmated males(UUM vs.TUM);(2)treated unmated males compared to treated mated males(TUM vs.TMM);(3)untreated unmated females compared to treated unmated females(UUF vs.TUF);(4)treated unmated females compared treated mated females(TUF vs.TMF).Protein expression changes between the four different groups were examined by two-dimensional gel electrophoresis(2-DE)and liquid chromatography tandem mass spectrometry(LC-MS/MS).Of the 500-600 reproducibly detected protein spots on each gel,107 protein spots were differentially expressed between the four different groups.Of the 63 proteins identified by LC-MS/MS analysis,38 were up-regulated and 25 were down-regulated in this process.Some novel proteins related to fecundity were observed including spermatogenesis-associated protein 5,PHD finger protein 7(NYD-SP6),arginine kinase,actin-5C,vitellogenin,and ovarian serine protease nudel.The function of these proteins need further study.5.The effects of triazophos on the trehalose content,trehalse activity and their gene expression in the brown planthopper Nilaparvata lugens(Stal)(Hemiptera:Delphacidae)A previous study demonstrated that the flight capacity of Nilaparvata lugens adults treated with sublethal dose of triazophos enhanced significantly.However,physiological and regulative mechanisms of the flight capacity enhancement are not well understood.Trehalose is a primary blood sugar in insects,and the enzyme trehalase is involved in energy metabolism.The present study investigated the effects of sublethal dose of triazophos on the trehalose content,trehalase activity(soluble trehalase and membrane-bound trehalase)and the mRNA transcript levels of their corresponding genes(NlTre-1 and NlTre-2)in fifth instar nymphs,as well as in the brachypterous and macropterous N.lugnes adult females.Our findings showed that the trehalose content in fifth instar nymphs as well as in the brachypterous and the macropterous adults significantly decreased following triazophos treatment.However,the glucose content,soluble trehalase activity and expression level of NlTre-1 mRNA increased significantly compared to the controls.No significant enhancement of NlTre-2 expression was found,indicating that the regulation of energy metabolism in N.lugens induced by triazophos was not associated with NlTre-2 expression.In addition,soluble trehalase activity and the expression level of NlTre-1 mRNA in the macropterous females was significantly higher than that in the brachypterous females.