Studies On Adaptive Response Of Tetranychus Cinnabarinus To Abamectin And High Temperature Stress

Due to its strong reproductive ability,short generation time,small movement area, high inbreeding rate and frequent insecticide-contacted opportunity,the problem of mite's resistance to insecticide is more severe than other crop insect pests.Tetranychus cinnabarinus(Boisduva1) is widely distributed in china,and difficult to prevent or control,which has caused severe damage to cotton and vegetable crops.The insecticide-resistance of mite has been produced in the most regions of China. Especially in recent years,being the widespread planting of transgenic cotton,the bollworm incidence was greatly restrained,but the damage issues of aphid and red mite are even more prominent.The avermectin used in production has become the leading medicament to control pests and mite,because of its unique mechanisms and excellent control effect.However,the resistance to avermectin of pest(mite) is quickly produced with its large-area and high-reliance use.Different to the general results of the resistance biology,pests(mites) to abamectin resistance do not always show fitness disadvantage, instead,they can show fitness advantage in some times,which will make it more difficult to resistance management.The studies on the pesticide resistance,heat resistance and their relationship of Tetranychus cinnabarinus,and increase or decrease mechanism of resistance fitness clarified at physical,biochemical,as well as molecular level,has important biological significance for deeply understanding the the biological response to environmental stress,and also have important practical significance for better understanding and using the resistance fitness to make resistance management. The research was supported by the National Natural Science Foundation(No.30600059). Through three-year efforts,we have drawn thereinafter main conclusions.1 Insecticide-resistance,high temperature adaptability and cross-endurance detection of Tetranychus cinnabarinusThe susceptible populations(SS),the abamectin-resistance populations(RA) and the heat-resistance populations(RH) of Tetranychus cinnabarinus were selected to study drug- resistance,high temperature adaptability and cross-endurance,by using bioassay and life table method.Our results showed that,under high temperature(46℃), the 50%individual death time(Lt₅₀) was RH＞RA＞SS,the toxicity of abamectin(LC₅₀) to Tetranychus cinnabarinus was RA＞RH＞SS,which indicated the long-term heat stress could induce abamectin-resistance,conversely,abamectin-resistance also could induce heat-resistance.Analyzing the life table of Tetranychus cinnabarinus showed that at high temperature(40℃),fecundity and survival rates of RA and RH were higher than SS,the most of population parameters(except T) were superior to SS,the relative fitness(Compared with SS) had different degree of increase,indicated that high temperature adaptability of Tetranychus cinnabarinus was enhanced by long-term Avermectins and heat stress,and the fitness of RA and RH was superior to SS under high stress.2 Studies on heat shock response and heat shock proteins(HSPs) of Tetranychus cinnabarinus(Boisduva1)The abamectin-resistance populations(RA) and susceptible populations(SS) were selected to study survival rate of Tetranychus cinnabarinus treated by heat pre-exposure at Ultimate high temperature,and sorts and contents of heat shock proteins (HSPs) by using SDS-PAGE.Our results showed that Non-lethal heat pre-exposure significantly increased the ability of endurance to Ultimate high temperature.there was difference of sorts and contents of HSPs between two strains treated by heat shock. Compared with the RA,eight kinds proteins was lack in the SS in normal condition,but the SS had five more kinds of proteins by heat shock,their molecular mass was 97.2 KDa,74.3 KDa,62.4 KDa,53.0KDa and 30.3 KDa respectively.The RA had no Specific Proteins but some proteins's expression increased after heat shock.This result helped to explain T.cinnabarinus resistant strains of fitness advantages phenomena.3 Effects of abamectin and temperature stresses on free radicals and protective enzyme activities of Tetranychus cinnabarinusThe free radicals(O₂^?)contents and activities of superoxide dismutase(SOD), catalase(CAT),and peroxidase(POD) in the RA,the SS and RH of Tetranychus cinnabarinus treated by abamectin,high temperature and low temperature were determined by enzyme activity assay.The result showed that free radical contents of the SS,RA and RH,after 4h treated by abamectin,were 3.54,3.42 and 2.91 times higher than those of the control group,respectively.Meanwhile,the activities of SOD and CAT in treatment group were evidently higher than those in control group.In treatment group, the O₂^? content of the RAwas lower than that of SS,but the activities of SOD and CAT were opposite.The activities of O₂^?,SOD,CAT and POD under unsuitable temperature were higher than that under normal temperature.The maximum activities of SOD,CAT and POD in the SS of Tetranychus cinnabarinus were at 42℃heat-shock,which were 4.25,2.06,and 1.81 times higher than in the control group,respectively,and the difference was significant from other treatments(P＜0.05),then declined,but the enzyme activities in the RA and RH exhibited stably uprising tendency at experimental temperature.The analysis result of enzyme activity chang rates showed that SOD had the strongest irritability to heat stress,POD was the weakest,and CAT was between the two.therefore,the change rates of enzyme activities of SOD,CAT,POD in RA and RH were superior to that of SS.4 Molecular cloning,prokaryotic expression of heat shock proteins (HSP90) gene in Tetranychus cinnabarinusIn this study,we cloned the cDNA of Tetranychus cinnabarinus HSP90(registered number:EU851046),and sequenced by the combination of reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(RACE) approaches.The full-length cDNA of TcHSP90 was 2,595 bp,including a 5'-untranslated region(UTR) of 177 bp,3'-UTR of 249 bp,and an open reading frame (ORF) of 2,169bp.The ORF encoded a polypeptide of 722 amino acids with a predicted molecular weight of 83.45 kDa and a theoretical isoelectric point of 4.81.By the analyse of antheprot,five signature sequences of HSP90 family and C-terminal five amino acids of HSP90:MEEVD were detected in the predicted amino acid sequence. Through blast analysis,the ORF of the HSP90 gene sequence of Tetranychus cinnabarinus shared high similarity with other known HSP90s,especially with those from insects of arthropods.TcHSP90 gene was inserted into pET-43.1a(+) of prokaryotic expression vector,and recombinant expression vector was obtained, named pET43a-TcHSP90.The recombinant expression vector could express fusion proteins of recombinant TcHSP90 in E.coli BL21(origami).Specific strip appeared on gel in SDS-PAGE and on PVDF-Membran in Western blotting.Studying the separation of TcHSP90 from T.cinnabarinus had important theoretical and practical significance to study environmental stress response and furthermore to explore its function in the stress response.5 TcHSP90 gene express of Tetranychus cinnabarinus by fluorescent real time quantitative PCRwe used fluorescent real time quantitative PCR to examine the expression pattern of TcHSP90 mRNA relative to that ofβ-actin gene in T.cinnabarinus given the treatments of abamectin,high temperature and low temperature,respectively.Our results showed that under the normal condition,the mRNA level of TcHSP90 was 1.64 and 1.29-fold higher in the abamectin-resistance populations(RA) and the heat-resistance populations(RH) than that in the susceptible populations(SS), respectively.The TcHSP90 mRNA levels of SS,RA,and RH were also significantly increased after induced at 40℃,and they were 1.83,2.98,and 2.58-fold higher than that of their untreated controls,respectively,and the difference was significant from other treatments(P＜0.05).Unexpectedly,the mRNA level of TcHSP90 was also significantly increased after induced at 4℃,and they were 1.25,1.72,and 1.38-fold higher than that of their untreated controls,respectively,and the difference was also significant from other treatments(P＜0.05).After abamectin treatment of 8 h,the TcHSP90 mRNA levels of SS,RA,and RH were also significantly increased,and they were 1.38,2.36,and 2.06-fold higher than that of their untreated controls,respectively.