Monitoring Of Insecticide Resistance In Field Populations And Role Of GABA Receptor α3 Subunit In Fipronil Sensitivity Of Helicoverpa Armigera

The cotton bollworm,Helicoverpa armigera(Hübner),is a worldwide agricultural pest.It has more than 200 hosts and could harm many important crops.It has always been the main control target in cotton fields.Before 1997,many chemical insecticides were heavily used to control H.armigera.The consequence of large-scale misuse was that it had developed serious resistance to insecticides in the early 1990s.After 1997,with the planting of Bt insect-resistant cotton,the amount of H.armigera was effectively controlled,and the amount of insecticides in cotton fields was significantly reduced.Since 2010,with adjustment of cropping structure in our country,the planting area of cotton has been decreasing,otherwise the planting area of corn,peanuts and other crops has been increasing,and the control of Bt cotton to H.armigera has declined.As a result,the cotton bollworm has recovered in northern China in recent years,especially the amount in other crops except cotton.So chemical control is still an important means to prevent H.armigera.Green prevention and control technology is encouraged to control crop pests.The scientific and reasonable use of chemical insecticides is more important,therefore,the monitoring of insecticide resistance in field populations of H.armigera will be a long-term fundamental and important task.Insect GABA receptors are the targets of cyclopentadienes(dieldrin)and benzopyrazoles(fipronil).The Rdl gene is the first insect GABA receptor subunit cloned from Drosophila melanogaster.There are contains three Rdl homologous genes HaGABAα1,HaGABAΛ2 and HaGABAα3 in H.armigera.In our laboratory,these three gene knockout lines were established by CRISPR/Cas9 technology,and the toxicity of fipronil and other insecticides varies greatly in the three lines.The strains with or without HaGABAal and HaGABAα2 had no significant difference in toxicity to fipronil,but the sensitivity of HaGABAα3 knockout strain SCD-GABAα3-KO to fipronil increased by 100-fold.In this paper,in order to clarify the difference in the sensitivity of the three Rdl homologous genes to fipronil in H.armigera,several areas of work have been carried out.First,a genetic linkage analysis of HaGABAα3 gene and fipronil tolerance was performed.Secondly,the types of alternative splicing of 3 Rdl homologous genes in H.armigera were analyzed.And finally using the Xenopus oocyte expression system to express these three genes,combined with the two-electrode voltage clamp technique,the effects of GABA and fipronil on the three GABA receptor subunit channel proteins of bollworm were compared,this results could to further clarify the role of GABA receptor 3 subunit in fipronil sensitivity.1.Monitoring of insecticides resistance in H.armigera field populationsFrom 2017 to 2019,field populations of H.armigera were collected from 10 sites in 6 provinces of the three major cotton regions of China for the resistance monitoring,compared with the SCD strain,the resistance levels to 7 insecticides in the field populations are 3-10 fold to phoxim,3-29 fold to lambda-cyhalothrin,0.1-5 fold to emamectin benzoate,0.9-2 fold to chlorfenapyr,0.6-3 fold to indoxacarb,0.1-4 fold to chlorantraniliprole,1-13 fold to spinosad,respectively.The monitoring result indicates that many field populations from cotton region of the Yellow River Basin and the Yangtze River Basin have developed varying resistance to phoxim,lambda-cyhalothrin and spinosad,but all populations are still sensitive to emamectin benzoate,chlorfenapyr,indoxacarb,and chlorantraniliprole.Combined with the previous monitoring data of our laboratory,it was found that the resistance of H.armigera to phoxim,lambda-cyhalothrin,and emamectin benzoate in cotton areas of the Yellow River Basin and the Yangtze River Basin has declined,however,the resistance level of many field populations to spinosad.has increased significantly.2.Genetic linkage analysis of fipronil sensitivity with HaGABAα3 of H.armigeraThe sensitivity to fipronil of HaGABAα3 gene knockout strain SCD-GABAα3-KO increased by 100-fold.In this paper,SCD and SCD-GABAα3-KO were subjected to forward and reverse cross and the sensitivity of the orthogonal F1 and the inverse F1’ to fipronil were determine.Compared with SCD-GABAα3-KO strain,it was found that F1 had 41-fold tolerance to fipronil and F1’ had 32-fold tolerance to fipronil.The dominant D value of F1 is 0.59 and F1’ is 0.49,both are between 0-1.These results indicate that the tolerance to fipronil of SCD relative to SCD-GABAα3-KO is autosomal,incomplete dominant.The backcross population of F1 and SCD-GABAα3-KO strains was constructed.The progeny individuals were divided into two groups(BC1 and BC2),BC2 was treated with fipronil.Genotype of the offspring was determined by molecular detection method.The genotype of the surviving individuals in BC2 were all heterozygous,and the ratio of heterozygous and homozygous with HaGABAα3 knockout in BC1 is 1:1.Based the above results,the tolerance to fipronil in SCD with the knockout of HaGABAa3 gene is tightly genetic linkage.3.Pharmacological responses of three GABA receptor subunits to GABA and fipronil in H.armigeraThere are three GABA receptor subunits homologous to Rdl in H.armigera.In this study,the alternative splicing types of three genes compared.The results showed that there were 3 transcript types,3a6b,3b6b and 3a with deletion of exons 6 in HaGABAα1,of which 3a6b accounted for 84.6%;there were 3 transcript types,3a6b and 3a6b with deletion of exon 2 and 6b with exon 3 deletion in HaGABAa2,of which 3a6b accounts for 68.2%;there were 5 transcript types 3a6b,3b6b,3b6a,3b6b with insertion of intron 3 and 3a with deletion of exons 6,7,8,9 and insertion of intron 5 in HaGABAα3,of which 3a6b accounts for 40%.In order to further study the pharmacological properties of different GABA receptor subunits of H.armigera and explore the effect of fipronil on the currents of three different GABA receptor subunit channels.Xenopus oocyte system was used to express each receptor subunit(They are the 3 a6b transcript types with the highest relative content among the three GABA receptor subunits H.armigera),the current detection of the expressed channel protein was performed by the two-electrode voltage clamp technique.The results show that the EC50 of HaGABAal,HaGABAa2 and HaGABAα3 receptor channels in response to GABA were 28.38 μM,12.90 μM and 153.70 μM,respectively,and HaGABAα3 receptor had the lowest sensitivity to GABA.Fipronil can obviously block the GABA(EC50)induced current of HaGABAal and HaGABAa2 subunit cRNA expressed in oocytes,IC50 values are 114 nM and 41.27 nM.When the oocyte expresses HaGABAα3 subunit,IC50>10000,it indicated that fipronil could not effectively block the GABA(EC50)induced current of HaGABAα3 subunit receptor expressed in oocytes.This indicates thatα3-type GABA receptors play a unique role different from α1 and α2-type GABA receptors in the sensitivity to fipronil in H.armigera.