Study On The Resistance Mechanism Of Leptinotarsa Decemlineata To Thiamethoxam

Leptinotarsa decemlineata(Say)is destruct quarantine insect of potato worldwide and also one of main invasive species in China.Since it invaded in Xinjiang in early 1990s,the beetle has spread from west to east continuously and distributed in the main areas of northern Xinjiang,which poses potent threate to potato planting in China.The beetle had developed different level resistance to all classes of insecticides because of long-time and excessive use.Neonicotinoid insecticides is commonly used for controlling L.decemlineata at present,which are efficient for the beetle resistant to the other insecticides.Many studies abroad reported resistance to imidacloprid of the beetle.The previous investigation of our research group showed that L.decemlineata in Xinjiang had increasing resistance to thianethoxam.However,the relevant study on the resistance of L.decemlineata in Xinjiang is little.The elucidation of resistance mechanism is important premise for establishing resistance management strategy and has important implication to delay use period of neonicotinoids.In this study,the resistance levels to thiamethoxam and imidacloprid of four field populations of L.decemlineata in Xinjiang were assayed in 2018,and the resistance mechanism was preliminarily studied.The results of the study are as follows:1.Monitoring on resistance to neonicotinoids of Leptinotarsa decemlineata in XinjiangTo understand the risks and determine possible mechanisms to neonicotinoid insecticides resistance of L.decemlineata,the resistance level to thiamethoxam and imidacloprid of five field populations of L.decemlineata collected from Qapqal,Yining,Tacheng and Urumqi in Xinjiang were assayed using topical application in 2018.The results showed that the adults had developed low to moderate levels resistance to thiamethoxam with the highest resistance ratio in Qapqal population with RR ranging from 5.8 to 19.7 fold,and only Qapqal adults had low level of resistance to imidacloprid with RR of 9.85 fold.The 4th instar larvae from most of populations remained sensitivie to the two insecticides.The adults in Qapqal and Urumqi had extremely high resistance to lambda-cyhalothrin(RR of 745.13-10938.88 fold),which further indicated that there might exist cross-resistance between thiamethoxam and lambda-cyhalothrin.2.The expression difference of nAChR in L.decemlineata form different populations and response to thiamethoxamIn order to screen the optimal internal reference genes for real-time quantitative PCR(qPCR)of L.decemlineata under thiamethoxam stress,and clarify the effect of thiamethoxam on the expression of nAChR in L.decemlineata.GeNorm and Normfinder software were used to evaluate the stability of eight candidate internal reference genes,and the selected reference genes were used to analyze the expression levels of nAChR subunit genes in L.decemlineata under treatment with thiamethoxam.The results showed that RPL4 and Ef1α were the most suitable reference genes in the 4th instar larvae and adults of L.decemlineata.Using RPL4 and Ef1α as internal reference genes,quantitative PCR results showed that the expression of nAChR subunit genes were different in L.decemlineata after thiamethoxam exposure.The expression levels of Ldα3,Ldα4 in adults and Ldα9,Ldα10 in 4th instar larvae were significantly decreased,and Ldα1 was significantly up-regulated in 4th instar larvae and adults.Similar changes were observed in the expression of the above genes in different field populations.The expression levels of Ldα9 and Ldα10 in Urumqi,Qapqal and Yining adults were significantly decreased,while the expression of Ldαl in Urumqi population was significantly increased.From the above results,it is speculated that Ldαl and Ldα9 or Ldα10 may be copolymerized with the same receptor.These genes may play an important role in the resistance of L.decemlineata to thiamethoxam,which provides a basis for further investigation of the molecular mechanisms of target resistance to thiamethoxam in L.decemlineata.3.Study on the activity of the detoxification enzymes and expression analysis of detoxification enzyme genes in L.decemlineataThe activities of detoxification enzyme were tested in field populations of L.decemlineata and the expression of detoxifying enzyme genes was evaluated by real-time quantitative PCR.Assays of enzyme activity in vitro demonstrated that the Qapqal adults had higher activity of Cytochrome P450 monooxygenase than the susceptible strain(2.20-fold).Glutathione-S-transferase activity were found higher in adults and larvae from Qapqal and Yining than the susceptible strain when CDNB was used as substrate.The results by qPCR revealed that CYP9Ya,CYP9Yc and CYP9Z26 were over-expressed in adults and larvae from resistant populations.Thiamethoxam showed a time effect on the induction of P450 in beetles.The relative expression levels of CYP9Yc induced by 0.0048μg/larva and 0/0097 μg/beetle thiamethoxam were 4.67 and 2.39-fold as high as the control,respectively.Based on the above results,it was speculated that the overexpression of CYP9Yc might be related to the resistance to thiamethoxam of the beetles in Xinjiang.4.The heterologous expression and pharmacological characteristics of nAChR α3 and nAChR α8 from L.decemlineataRecombinant receptors of L.decemlineata nAChR α3,α8 and rat nAChR β2 were constructed and expressed in Xenopus laevis oocytes,and the functional properties of recombinant receptors were studied by two-electrode voltage clamp technique.The cRNAs of L.decemlineata nAChR α3,α8 and rat nAChR β2 were successfully synthesized.However,in Xenopus laevis oocytes,the recombinant receptor Ldα3/rβ2,Ldα8/rβ2 and Ldα3/Ldα8/rβ2 were not form functional receptors.