Mechanism Of Action Among Plant, Diamoneback Moth, (Plutella Xylostella (L.)) And Insecticide

Diamond-back moth (Plutella xylostella, Lepidoptera: Plutellidae) was one of themost important and destructive pest of crucifer. We studied the relationships andmechanism of action among plant, diamond-back moth and insecticide, using different typeinsecticides (fenvalerate,spinosyns,azadirachtin), different host (hobby and non-hobbyhost) and different resistant strains (spinosad-resistant strain, fenvalerate-resistant strain andazadirachtin-resistant strain).1. Risistance selection to different type pesticide by P. xylostella and their cross-resistanceThe resistance was selected by feeding diamond back moth larvae with leaf have beendipped in insecticide water solution. The resistance developed up to 24.31, 3.66 and 8.42folds, respectively, to fenvalerate, spinosyns and azadirachtin after 12 generations. Thedevelopment of resistance to fenvalerate and spinosyns were exponential type, thedevelopment of resistance to spinosad was smooth, and the development of resistance toazadirachtin was discrete, which have a distinctness period of increase every other 3-4generation. Azadirachtin-resistant strain was positive cross-resistant to spinosad andfenvalerate, the resistance to spinosad was 2.57-fold, to fenvalerate was 7.71-fold.Spinosad-resistant strain was positive cross-resistant to azadirachtin and fenvalerate, theresistances were 5.18 and 7.03 folds, respectively. Fenvalerate-resistant strain has lightcross-resistance to spinosad, the resistance index were 1.18, and has positivecross-resistance to azadirachtin, the resistance index was 5.44.2. Electroantennogramre responses of diamond-back moth strains, P. xylostella to the ethanol extract of hostsThe electroantennogram responses of unmated female of resistance strains to Chinesecabbage,flowering Chinese cabbage,radish and rape were stronger than that of susceptiblestrains. For mated female, the responses of resistant strains to all hosts were stronger thanthat of susceptible strains. The responses of unmated males of different strains to theethanol extract of hosts were similar, and were all weaker than that of the susceptible strains. After mated, the responses of resistance strains were different from that ofsusceptible strain, the response of fenvalerate-resistant strain to cabbage extract wasstronger than that of susceptible strains.3. The relative fitness of different P. xylostella strainsThe periods of development were different among azadirachtin-resistant strain,fenvalerate-resistant strain and spinosad-resistant strain when larvae were fed with rape.The developmental time of larva of azadirachtin-resistant strain and spinosad-resistantstrain were prolonged, and that of fenvalerate-resistant strain was shortened. Thedevelopmental time of pupa of all resistant strains were longer than that of susceptiblestrain. The fecundities of all resistant strains were less than that of susceptible strain, thedifference ranged from 16.27 to 21.33. Relative fitness of fenvalerate-resistant,azadirachtin-resistant and spinosad-resistant strains were 0.825, 0.689 and 0.61,respectively.There were no differences among hosts in developmental time of egg and pupa ofsusceptivity strains, the former ranged from 4.24 to 4.47d and later ranged from 4.28d to4.74d. The fecundities ranged from 108.07 to 124.73, and the higher with cabbage, thelower with rape and radish. The relative fitness were 1.31, 1.25, 1.31, and 1.45, when fedwith flowering Chinese cabbage,Chinese cabbage,radish and cabbage, respectively.4. Feeding and ovipositing preference of P. xylostellaThe feeding experiment with different Brassica vegetables showed that P. xylostellalarva preferred seedlings of Chinese cabbage(Brassica campestris L), radish(Raphamussativus L), and flowering Chinese cabbage(B, campestris L), to seedlings of rape(B. napusL) and cabbage(B. olereacea L), with 93.33% preferred to Chinese cabbage with 6.67% torape, and 16.67% preferred to cabbage with 83.33% to flowering Chinese cabbage. Thefeeding preference of P. xylostella was affected by hosts on which it was reared. The larvapreferred to feed the plant damaged by other diamond back moth larvae.Chinese cabbage was the most preferred host for the moth ovipositing, following wereflowering Chinese cabbage, radish, cabbage and rape. The order of ovipositing preferencewas the same as the content of glucosinolates in hosts. The hosts affect the survival rate, thesurvival of 1st instars was higher when fed on the seedling of cabbage than that of larvae fedon other hosts, the survival of 2nd instars were the lowest in all treatments, it ranged from3.33% to 6.67%. No death happened to 3rd and 4th instars.5. Change of Juvenile hormone titer of P. xylostella treated by different pesticidesThe juvenile hormone titer (JH titer) of diamond back moth varied after larvae weretreated with pesticides. The results were different according to different pesticides anddosage used. The juvenile hormone titer of 3rd instars changed with a pattern ofincrease-decrease-increase after treated with LC₂₀ dosage of spinosad, and that wasdecrease-increase-decrease after treated with LC₂₀ of fenvalerate, and the titer increasedafter larvae were treated with LC₂₀ of azadirachtin. Treated with LC₅₀ dosage, spinosadimproves the degradation rate of JH ahead, then descending slowing the degradation ratebelow that of the controlling sample. The degradation rate of JH sufferred by fenvalerateand azadirachtin varies all below that of the controlling sample. The LC₈₀ dosage ofspinosad can reduce the JH titer in vivo, the JH titer change varies flectional, the same asfenvalerate. LC₈₀ dosage of azadirachtin can improve the JH titer, and would be durative.6. Change of activity of detoxifcation enzyme in P. xylostella treated with different pesticidesAfter larvae were treated with LC₂₀ dosage insecticides, O-demethylation activityincreased in the earlier stage of 3rd instars. Change pattern of activity of O-demethylationlike letter "M" after larvae were treated with fenvalerate. The changes of activity ofO-demethylation were smooth if larvae were treated with azadirachtin and spinosad.Overall, the activity of O-demethylation was restrained. Activity of N-demethylation wasrestrained in 24h after molting when larvae were treated with LC₂₀ dosage azadirachtin, theother insecticides restrained the activity of the enzyme. The LC₅₀, LC₈₀ dosages of allinsecticide restrained the activity of N-demethylation.The activity of detoxifcation enzyme varied with the increasing of resistance, itappears that O-demethylation activity increase with the resistance, and N-demethylationactivity was reverse. Host affected the O-demethylation activity, and has a little effect onN-demethylation activity.7. Study on the proteomics of P. xylostellaBy using the method of two-dimensional gal electrophoresis, proteins of P. xylostella ofdifferent resistant strains were analyzed. The results showed that there were 847 proteinspots in 2-DE maps of fenvalerate-resistant strain, and 885 protein spots in 2-DE maps ofazadirachtin-resistant strain, 1732 protein spots in 2-DE maps of spinosad-resistant strain,748 protein spots in 2-DE maps of susceptible strain. The position and quantity weredifferent in different maps. These mean that the mechanism of resistance to different insecticides were different.8. Clone and analysis of cytoehrome P450 gene from P. xylostellaSome new cDNA fragment about 240 bp of cytoohrome P450 were amplified by usingRT-PCR technique. Results of sequence analysis showed that there were high homologousamong the fragments, cyp6 family, cyp9 family and cyp4 family. The fragment fromsusceptible strain may belong to cyp6 family, and fragment from azadirachtin-resistantstrain may belong to cyp4 family.By using RT-PCR and RACE technique, the cytochrome P450 gene(CYP6BF1v1/v2) were cloned and determined, GenBank access number were AY971374and DQ088989. The CYP6BF1v1 includes 1661bp, and the opening reading frame (ORF)is 1545bp, the melting point is 102℃, anneal temperature is 87℃, molecule numerator is539.83 kDa(single) and 1079. 65kDa(duble). The protein is formed by 514 amino acids,and the molecular formula C₂₆₈₂H₄₁₅₄N₇₀₂O₇₄₇S₃₀, molecular weight 59146.5. Higherstructure is similar to the Cytochrome P450 Bm-3.9. Repellent and antifeedant effect of the ethanol extracts from not hobby host on P. xylostellaBased on antifeeding rate, inhibitory rate, oviposition deterrent effects and IIPCvalue, the repellent and antifeedant effect of the ethanol extracts from thetomato(Lycopersicon escu lentum) and pumpkin( Cucurbita moschata) on P. xylostella wasstudied. Oviposition deterrent effect of the ethanol extracts from the tomato is over 60%,which was higher than that of ethanol extracts from pumpkin. Antifeeding rate andinhibitory rate were not significantly different between tomato and pumpkin.Results of our research indicated that the relationships among plant, diamond-backmoth and insecticide were complex. In the whole population, the results was the reaction ofbehavior and biological characteristics owing to the communication transferring betweenplant/insecticide and insect, such as preference, avoiding, change of fitness. In thephysiological level, the results were varieties of metabolism responded to the externalstimulate (hypo-substance, insecticide), such as the variety of JH titer and MFOs activity.In the molecular biology level, it was the diversity of the quantity and quality of geneinduced by external stimulates (hypo-substance, insecticide).