Study On Metabolic Resistance Mechanism Of Laodelphax Striatellus (Fallén) To Triflumezopyrim

The small brown planthopper（SBPH）Laodelphax striatellus（Fallén）,is one of the most important pest insect of gramineous crops including rice,corn and wheat.It can directly damage crops by sucking plant juice and spread many plant viruses,resulting in seriously damage to crop production and yield in China.The control of L.striatellus is mainly based on using insecticides.However,L.striatellus has developed resistance with different resistance level to many insecticides.Triflumezopyrim is a new type of mesoionic insecticide,which has high insecticidal activity against L.striatellus and has important value in the integrated control and resistance management of SBPH in field.The laboratory has obtained a triflumezopyrim-resistant strain(RS₂₁)（RR=26.29 times）previously.To obtain a more higher triflumezopyrim-resistant strain of SBPH,the RS₂₁strain was continuously screened by triflumezopyrim for more ten generations in laboratory.Then,the activities of detoxification metabolic enzymes and the relative expression levels of detoxification metabolic genes in the susceptible and resistant strains were compared.RNA interference technology was used to further explore the role of overexpressed metabolic genes in resistance,and the metabolic mechanism of L.striatellus resistance to triflumezopyrim was preliminarily discussed.The RS₃₁strain was obtained from the RS₂₁strain of SBPH with continuous triflumezopyrim selection of ten generations by rice seedling spraying method in the laboratory.The toxicity results showed that the LC₅₀value increased from 14.45 mg·L^-1(RS₂₁)to 26.22 mg·L^-1(RS₃₁),and the resistance ratio increased from 26.29 times to 47.67 times.The resistance development showed a rapid upward trend.In order to clarify the correlation between detoxification enzymes and the metabolic resistance mechanisms of L.striatellus to triflumezopyrim,the differences of three detoxification enzymes activities between resistant strain(RS₃₁)and susceptible strain（SS）were compared.The results showed that the activities of cytochrome P450 monooxygenase（P450s）and carboxylesterase（Car E）in the resistant strain RS₃₁were significantly higher than those in the susceptible strain,which were 1.85 times and 1.30 times higher than those in the susceptible strain.However,there was no significant difference in glutathione S-transferase（GST）activity between the two strains.To further clarify the association between detoxification enzymes genes and the metabolic resistance mechanisms of SBPH,the expression levels of 46 P450 genes,15 Car E genes,16 GST genes and 7 ABC transporter genes was determined and compared between the SS and RS₃₁strain.The results showed that 24 of the 46 P450 genes were differentially expressed,among which,CYP303A1,CYP4CE2 and CYP419A1v2 genes were up-regulated by 7.6 times,4.4 times and 15.8 times,respectively in RS₃₁strain compared with SS strain.Among the 15 Car E genes,9 genes were significantly differentially expressed.Compared with SS strain,two Car E genes（Ls Car E6 and Ls Car E12）were significantly up-regulated by8.3 and 16.8 times,respectively.Among the 16 GST genes and 7 ABC transporter genes screened,10 genes and 2 genes were significantly differentially expressed,but no high-fold up-regulated genes were found.Two P450 genes and two Car E genes with high expression levels were selected for next RNAi experiment.The artificial diet feeding method was used to silence the gene by RNAi technology,and its role in anti-triflumezopyrim was detected.The results of silencing efficiency showed that the artificial diet containing 50 ng/μL ds RNA for 5 days had a good silencing effect on the four metabolic genes.The gene silencing efficiency of CYP303A1,CYP419A1v2,Ls Car E6 and Ls Car E12 was 83.2%,54.7%,62.5%and 72.5%,respectively.The activity of triflumezopyrim（20 mg/L）on the resistant strain of L.striatellus after silencing single gene and co-silencing two P450 genes and two Car E genes was determined by rice seedling dipping method.The results showed that the mortality rates of single gene silencing L.striatellus were 51.00%,61.33%,56.67%and 57.00%,respectively,which were significantly higher than that of the control group（26.67%）.The mortality of L.striatellus with co-silencing P450 gene and Car E gene was 90.00%and 85.00%,respectively,which were significantly higher than that of single gene silencing.The results showed that the increased expression levels of P450 genes CYP303A1 and CYP419A1v2,Car E genes Ls Car E6 and Ls Car E12 in L.striatellus could lead to resistance to triflumezopyrim,and multiple genes could cooperate to increase the resistance level.