| Woolly apple aphid(Eriosoma lanigerum Hausmann)(Hemiptera:Pemphigidae),is an important pest of applesand one kind of important quarantine pests in the world,which caused hypertrophic gall formation on the roots and limbs of the tree.The insect has distributed many areas in China posing potaintial threat to fruit industry in our country.The chemicals of choice have always been main means to control E.lanigerum.High selection pressure eventually resulted in a large number of insecticide-resistant E.lanigerum populations,but there seem no related study reports so far.In this study,the insecticides resistance survey,biochemical mechanism and cloning of the two nicotinic acetylcholine receptor subunits in E.lanigerum were carried out.The results may provide important informations for resistance management strategies and understanding of the role of nAChR in neonicotinoid resistance in E.lanigerum.1.Comparison on the bioassay methods and field resistance monitoring of three insecticides in Eriosoma lanigerumTo determine the resistance level to conventional insecticides of E.lanigerumin China,resistance to chlorpyrifos,imidachloprid and lambda-cyhalothrinin the populations collected from Jinan,Feng county,Qapqal and Qian county provinces were tested using leaf dipping method.Firstly,the sensitivity of the insecticides against E.lanigerum were determined and compared by leaf dipping method,residual film and topical application method,and the results showed that the toxicities of the three methods were consistent.The toxicity of chlorpyrif'os to E.lanigerum was the highest and imidacloprid exhibited the lowest toxicities among the three insecticides.Because Leaf dipping method showed the stable results and was convenience,it was recommended to determine insecticides resistance.Secondly,the current situation and development of insecticide resistance of E.lanigerum were investigated by the leaf dipping method during 2014-2015.The Qapqal population in 2014 was the most susceptible and selected as a reference strain.Compared with Qapqal 2014 population,all the populations developed high levels of resistance to imidacloprid with the highest level in Qapqal and Feng populations in 2015(RR>258 folds).For lambda-cyhalothrin,Qian,Jinan 2014 and Jinan,Feng in 2015 exhibited moderate to high level resistance and Qapqal in 2015 exhibited highest level of resistance.For chlorpyrifos,Jinan 2014 and 2015 populations,Qapqal 2015 and Feng populations displayed moderate,low level resistance and low susceptibility respectively.The study demonstrated that most of the tested populations developed different levels of resistance to three tested insecticides with the tendency of increase yearly.The resistance level of E.lanigerum to imidacloprid was the highest,it should be paid attention to using the insecticides in reasonable rotation for control of E.lanigerum.2.Detoxifying enzyme activity test of different populations in Eriosoma lanigerumThe biochemical mechanisms of imidacloprid,chlorpyrifos and lambda-cyhalothrin resistance in E.lanigerum populations were studied by detoxifying enzyme activity test.The results demonstrated that Feng population had lower activity of esterase than the Qapqal and Jinan populations.For glutathione S-transferase activity,Feng and Qapqal populations had higher activity than Jinan population when DCNB was used as substrate.There was significant correlation between resistance levels of imidacloprid and activity of glutathione S-transferase respectively not including esterase.The study suggests that glutathione S-transferase may be associated with the lambda-cyhalothrin and imidacloprid resistance of E.lanigerum.3.Cloning of nAChR genes from Eriosoma lanigerumNicotinic acetylcholine receptors(nAChR)play major roles in the fast excitatory neurotransmitter at cholinergic synapses in the central nervous system of insects and are also the target site for neonicotinoids.With general primers designed for nAChR genes,RT-PCR was performed and two target cDNA fragments were cloned from E.lanigerum.Then,the two different genes were successfully cloned to their full-length using RACE.The two genes are 2064 and 2072 bp,containing an open reading frame of 1758 and 1638 bp encoding 586 and 546 amino acidresidues respectively.Homology analysis indicated that the cloned genes had the typical characteristics of the nAChR a-subunits and shared high amino acid sequence similarities with nAChR ? subunits from other insect,especially those from the Coleoptera insect,Tribolium castaneum.So,the two genes were determined as nAChR a-subunits of E.lanigerum,named as Ela2 and Ela5 respectively.In addition,the sequence of El?2 and El?5 from Jinan,Qapqal and Feng county E.lanigerum populations were analyzed and compared.The results showed that some amino acid polymorphism were detected specifically in the tested populations.These findings may provide basis for further study on target resistance mechanism of imidacloprid in E.lanigerum. |
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