Mutation Effect Of Target Genes On Ethiprole Resistance In Laodelphax Striatelus(Fallén)

The small brown planthopper(SBPH),Laodelphax striatellus(Fallen),belongs to Delphacidae,Hemiptera.It is not only infests important cereal crops including rice,wheat,maize and sorghum but also feeding gramineous weeds,not only affect rice production by sucking plant juice but also transmising a variety of plant virus diseases.A large number of chemical pesticides have been used to control this pest,and serious resistance has emerged.Currently,the use of chemical insecticides is still the main way to control SBPH,while the development of resistance in L.striatellus makes chemical control facing more difficulties.Therefore,insecticide resistance management strategies must be developed to prevent further increase in resistance and obtain higher control efficacy of SBPH.Lacking of the knowledge of resistance mechanism is the barrier for implementation of resistance management and efficient control.In this paper,the strain resistant to ethiprole was selected in laboratory and a corresponding susceptible strain with similar heredity background was developed,the genes for ethiprole acting target and ATP-binding cassette(ABC)transporters were cloned,the resistant and susceptible strains were compared for their splicing types and mutations associated with resistance.Thus,the target resistance mechanism was studied to provide a scientific basis for management of the resistance in L.striatellus.1.Selection of ethiprole resistance in L.striatellus and related toxicity bioassayIn order to reveal the tendence of ethiprole resistance development and the cross-resistance between ethiprole and other kinds of insecticides,a resistance strain that had been selected for 8 generations with ethiprole in laboratory and suspended for a period was used as the original strain(GO),and subjected to further selection with ethiprole for another 6 generations.Meanwhile,a susceptible strain with similar heredity background was developed by breeding the hoppers collected in the same area in laboratory without contacting any insecticides.Then,toxicity bioassay was performed to evaluate the toxicity of imidacloprid,chlorpyrifos and deltamethrin on both the resistant and susceptible strains.The results showed that previous 8 generation selection had enhanced the resistant ratio 107 times in the original field population to 180 times,and the suspendence to the start of this study led it decreased to 71 times(in GO generation).Re-selection to G2 generation made the resistance ration increased to 136 times,and to G4 it became 325 times.The resistance of G6 generation reached the extremely high resistance level.Dosing 200ng/insect caused only 37.5%mortality,the resistance ratio was more than 725-fold and the resistance increase ratio during the 6 generation selection was more than 10-fold.It is indicated that if the planthopper developed resistance,suspendence could make resistance decrease,but reinstation of ethiprole will make the resistance developed more rapidly.Bioassay demonstrated that the resistant strain showed only 4.2-fold,4.4-fold and 4.3-fold resistance to imidacloprid,chlorpyrifos and deltamethrin,respectively.This implied that there was no significant cross-resistance between ethiprole and these three insecticides in L.striatellus.2.Cloning of the genes related to the resistance of ethiprole in L.striatellusFor ethiprole resistance in L.striatellus,well study has been performed on the role of metabolic enzymes.However,the important target resistance and transport excretion is not clear yet.In this study,the ABC transporter gene fragment was searched and cloned by using the transcriptome data of L.striatellus,and the terminal clones were obtained by using RACE technique.After deduplication,6 ABC transporter genes with full length were obtained.Meanwhile,GABA receptor gene for ethiprole target were cloned according to previous reports.As the results,we not only obtained the well-known RDL1 gene but also discovered a new RDL2 gene that has not been reported previously.The full-length of the RDL1 gene was 1830 bp,with a 1464 bp open reading frame encoding 487 amino acids.Its 5' UTR was 55 bp,and 3' UTR was 311 bp.The full-length of the RDL2 gene was 2129 bp,with a 1452 bp open reading frame encoding 483 amino acids,and its 5' UTR and 3' UTR were 55 bp and 622 bp,respectively.The amino acid sequence similarity of RDL1 and RDL2 was 91%.The difference between two genes appeared at their 3'-ward sequence area after the third transmembrane region,especially for their 3'UTR.3.Variation analysis of different target genes in resistance and susceptible strains of L.striatellusBased on the discovery above,this study tried to clone the two RDL genes from both resistant and susceptible strains of L.striatellus and compared the sequences in order to find the resistance associated mutations.The results showed similar splicing in the two RDL genes,including 3a,3b and 3c of exon 3,6a and 6b of exon 6,and two varried types between the third and the fourth transmembrane region.The distribution of different splice types was found similar in both strains.The majority of the theorial splicing types were detected in the resistance and susceptible strain with only a few exceptance.Although the distribution frequency of different splicing types has slightly different between the resistant and susceptible strains,the integral trend is consistent.Thus,no splice types related to resistance could be determined yet.In addition,by comparing the two RDL gene sequences of two strans,we not only found the A2'N mutations related to resistance reported in many insects,but also found another two A337V and G376 deletion.All the two mutations and one deletion appeared simultaneously like linkage.The results showed the mutation frequency of RDL1 was 46%in the resistant strain,and 3%in the susceptible strain.The RDL2 was 72%and 11%.It can be seen that the mutation frequencies of both genes in the resistant strain were significantly higher than those in the susceptible strain,and the mutation frequency of RDL2 in the two strains was higher than that of RDL1.Thus,the linkage mutations were thought related to ethiprole resistance of the planthopper,and both genes played roles.In view of the fact that most region of the two gene sequences are identical,further studies need to clarify the origin of the two genes and provide direct evidence that mutations contribute to the ethiprole resistance.