Imidacloprid Resistance In Sitobion Avenae (Fabricius) And The Mechanisms

Sitobion avenae(Fabricius) is one of the most important pests on wheat.Because of the resistance of S.avenae to conventionally used insecticide in the field,imidacloprid has become the key insecticide used for control of this pest.So,study on imidacloprid risistance is very important for sustainable control of S.avenae.In this paper,the resistance survey,resistance selection,relative fitness analysis,and cross-resistance test were all carded out to evaluate the risk for S.avenae to develop resistance to imidacloprid.With selected resistant strain and susceptible strain,the biochemical mechanisms for imidacloprid resistance were analyzed,and some genes for nicotinic acetylcholine receptors (nAChR) were cloned.Valuable breakthroughs have been achieved.1.Field survey for imidacloprid resistance in S.avenaeThe toxicity of imidacloprid and pirimicarb(another effective insecticide usually used to control wheat aphid) to field populations of S.avenae had been tested with the insects collected from Nanjing,Jianhu,Sihong,Dezhou,Zhoukou,Lanzhou and Tianshui in 2005 and 2006.Results showed that the sensitivity of S.avenae to imidacloprid varied obviously with the geographic populations.The aphids collected from Jianhu and Nanjiang had developed low level of resistance(RR was 11.31 and 6.29,respectively).But the aphids from other areas were still susceptible to imidacloprid(RR ranged 1.99-3.85).However, pirimicarb resistance varied not so much among different populations.The survey results showed that pirimicarb was much less toxic to S.avenae than imidacloprid.Comparing with the baseline data reported previously,S.avenae in different areas were found to be moderate resistant(RR:20-40).Furthermore,the data of different years showed that the resistance of S.avenae to imidacloprid and pirimicarb were increasing slowly.In conclusion,imidacloprid resistance was still in its early stage of development.Most field populations of S.avenae were still susceptible to imidacloprid,but a few populations had really developed resistance to this insecticides.Thus,it is necessary to study the development of imidecloprid resistance and complement reasonable resistance management.2.Imidacloprid resistance selected in S.avenae and the fitnessAn imidacloprid-resistant stain(25.22-fold) of S.avenae was developed by continuous selection for 32 generations with imidaclopfid in laboratory.During the selection, imidaclopfid resistance was found with a phased increase.From the 1^st to 18^th generation, the resistance developed very slowly.The rapid increase occurred from 18^th to 24^th generation,and leaving 24^th to 32^nd as a resistance developing plate.There should be a risk of resistance in S.avenae after long-time extensive use of imidaclopfid in the field.The effects of resistance on the fitness of S.avenae were evaluated in term of developmental and reproductive characteristics by constructing and comparing the life tables of imidaclopfid-resistant and susceptible strains.The results revealed that imidacloprid-resistant stain had no developmental disadvantage but obvious reproductive disadvantage.The fitness of irnidacloprid-resistant strain was determining by population number tendency index(I) and intrinsic rate of increase(r_m) with susceptible strain as standard.The relative fitness value for the imidalopfid-resistant strain was calculated to be 0.623 and 0.846 respectively.So,it was concluded that imidacloprid resistance in S.avenae costs lots of fitness,which could be one of the main factors for the slow development of imidacloprid resistance in the field population of S.avenae.3.Cross-resistance of the imidacloprid-resistant strain of S.avenaeThe cross-resistance of the imidacloprid-resistant strain of S.avenae to six conventionally used insecticides in the field was measure.It was found that selection with imidaclopfid could make a obvious increase in the resistance of S.avenae to imidaclopfid and acetamiprid(9.10 and 4.11 times,respectively),which means positive cross-resistance between imidacloprid and acetamiprid.However,no distinct cross-resistance to carbamate (pirimicarb and methomyl),organphosphates(omethoate and monocrotophos) and pyrethroid (deltaraethrin) was found. 4.Mechanism for imidacloprid resistance in Sitobion avenaeA strain(about 25.22 fold) of Sitobion avenae resistant to irnidacloprid was developed in the laboratory.Synergism experiments and detoxification enzyme analysis were carried out to reveal the resistance mechanisms.The results showed that piperonyl butoxide and triphenyl phosphite had significant synergism on imidaeloprid in both the resistant and susceptible strains,but diethyl maleate had not.The synergism ratio of piperonyl butoxide and triphenyl phosphite in the resistant strain were much higher(3.49-fold and 2.62-fold, respectively) than in the susceptible strain(1.59-fold and 1.29-fold,respectively).The resistant strain showed higher activity of carboxylesterases(about 1.53-fold) than the susceptible strain,but the activity of gluathione S-transferases was similar.These results indicated that the enhancement of the activity of carboxylesterase and Mixed function oxidase should contribute to the imidaeloprid-resistance in this S.avenae strain.5.Cloning of nAChR genes from S.avenaeNicotinic aeetylcholine receptor(nAChR) is one of the most important insecticide targets.With general primers designed for nAChR genes,RT-PCR was carded out and 6 cDNA fragment of nAChR genes were cloned from S.avenae.Of which,five had the typical characteristics of nAChR alpha subunits and one belonged to beta subunits.Further analysis demonstrated that this six fragments had high amino acid sequence identity with other insect nAChR subunits previously reported,especially those of Homoptera.Based on the five fragments of nAChRαsubunits,general primers were designed and five different genes were successfully cloned to their full-length by using the RACE strategy.They were named Saα1,Saα2,Saaα,Saα4 and Saα5,and their GenBank access number were EF363700,EF363701,EF211977,EF363702 and EF363703,respectively. These five genes had all the characteristics of nAChR gene family and were high homology with other nAChR genes from insects.This work facilitates the further studies on natural subunit composition of nAChR and the molecular mechanisms for target resistance to new nicotinic insecticides in this pest.Additionally,an unusual adenosine rich region were found in Saα3 and alternative splicing in Saα5.There were three forms of alternative splicing in N-coding regain in Saα5, causing absence,insert and change in the amino acid sequence.The role of these alternative splicing need further research.In conclusion,studies had demonstrated the imidaeloprid-resistance statue in S.avenae, the risk to develop resistance to imidacloprid,the reasons of the slow development of imidacloprid resistance in the field,and the main mechanisms for imieacloprid resistance. This work is significant in the management of S.avenae resistance.On the other hand,five nAChR subunits genes cloned from S.avenae facilitates the further studies on natural subunit composition of nAChR and the molecular mechanisms for target resistance to new nicotinic insecticides in this pest.