Risk Assessment And Mechanisms Of Resistance To Cry2Ab Toxin In Helicoverpa Armigera From China

Cotton bollworm,Helicoverpa armigera,is one of the most serious agricultural pests around the world.In 2016,the total plant area of Bt cotton had reached 15 million ha since genetically modified cotton expressing the Bt toxin was first introduced to Australia and the US in 1996 to control cotton bollworm,which had developed wide-spread resistance to synthetic insecticides.The planting of Cry1Ac cotton(Bollgard I)could reduce the damage of H.armigera and has brought a significant economic,social,and environmental benefits.The high-does/refuge strategy has been executed strictly as a resistance management tactic since the Bt cotton was used in the field in 1996.Afterwards,for the expanding of insecticidal spectrum,reducing the refuge area and suspending resistance to the target pest,the second generation of Bt cotton expressed Cry1Ac and Cry2Ab toxins(Bollgard ?)was first planted in Austrlia and the US in 2003,that means the application of a new resistance management strategy of gene pyramiding or gene stacking.From 1997 Cry1Ac cotton has been used in China and the natural refuge strategy for resistance management has been adopted since then.Monitoring data in 2010 indicated that the early resistance to Cry1Ac of H.armigera from northern China presented,but H.armigera from these areas were still susceptible to Cry2Ab.Although the natural refuge still works in China,the frequency of resistance to Cry1Ac,especially the non-recessive resistance,is increasing regularly and significantly.How about the influence on the resistance to Cry2Ab under the increasing resistance to Cry1Ac of H.armigera in northern China?In this study,the susceptibility detection to Cry2Ab in field populations of H.armigera from 2013 to 2016 was conducted and the inheritance mode,cross-resistance pattern and fitness costs of a selected resistance strain An2Ab were investigated.Based these results,which are the condition underlying success of the refuge strategy for delaying resistance to the pyramided Bt crops,the risk of resistance to CrylAc+Cry2Ab cotton in China is assessed.It is different with the understanding about the resistance mechanisms to Cry1Ac of H.armigera.The resistance mechanism to Cry2Ab is far from clear except that those mutations in ABCA2 were found in Austrilia H.argmigera?In this study,Cry2A toxin binding proteins in BBMV prepared from midguts of H.armigera were captured under non-denaturing conditions using modified Pull-down method and bio-tinylated Cry2A toxin and then identified by liquid chromatography coupled with tandem mass spectrometry(LC-MS/MS).The resistance to Cry2Ab was not linked with any receptor mutations and reduction of binding.Furthermore,the genetic mapping and transcriptomic analysis were employed to screen candidate resistance genes broadly.Bioassay with the inhibitors targeting difference signal pathway proteins and CRISPR/dCas9 system were utilized to find the possible resistance genes.These results in this paper could be helpful to design an effective resistance management stategy after 20 years planting of Cry1Ac cotton in China and to understand the multiple possibilitise of resistance mechanism to Cry2Ab of H.armigera.1.Detection of susceptibility to Cry2Ab in field populations of H.armigeraThe susceptibility to Cry2Ab in 12 field populations(Shawan,Anyang,Kaifeng,Anci,Gaoyang,Nanpi,Qiuxian,Huimin,Xiajin,Jingzhou,Wangjiang,Yancheng)of H.armigera from different areas of China in 2013 to 2016 was detected utilizing LC-P line.The difference of LC50 values in these populations are less than 10-fold variations.Further,compared with the population from Shawan where fewer Cry1Ac cotton was planted and the laboratory susceptible strain An,the variations were 2.0-fold to 2.7-fold in 2013-2016.The frequency of Cry2Ab resistance individuals was measured in 2016 using the diagnostic concentration bioassay.The survival rates were between 1.4%and 5.2%in these 11 populations and the mean survival rate was 3.3%(106/3168)which was higher than the rate of Shawan population(3/288,1%)and An strain(0/288,0).This means the frequency of Cry2Ab resistance individuals is related with the planting of Cry1Ac cotton(?2 = 13.7,df = 1,P = 0.025).Spearman test indicates that the frequency of Cry2Ab resistant individuals is linear correlated with LC50 for each test population.The results indicate that intensive planting of Cry1Ac cotton has resulted in reduction of susceptibility to Cry2Ab in some field populations of H.armigera in northern China.Switching to Cry1Ac + Cry2Ab cotton from Cry1Ac cotton would not be the best option in China.2.Selection and fitness costs of resistance to Cry2Ab of H.armigeraIn 2009,several resistant female lines were isolated by F2 screen,then these lines were mixed and the progeny has been selected with Cry2Ab for more than 37 generations to generate a resistance strain An2Ab.An2Ab has 130-fold resistance to Cry2Ab.Strong cross-resistance occurs between Cry2Ab and Cry2Aa(81-fold)and weaker cross-resistance(18-to 22-fold)between Cry2Ab and CrylA toxins also present on An2Ab stain.The mode of inheritance is and significantly increased survival of An2Ab relative to An(both were 0)on cotton cultivars producing the fusion protein CrylAc/CrylAb(6.7%±0.7%)or CrylAc(5.3%± 1.3%).Survival on CrylAc+Cry2Ab cotton was also significantly higher in An2Ab(4.0%±2.0%)than in An,showing that redundant killing on this pyramid was incomplete.Survival on non-Bt cotton did not differ significantly between An2Ab(23.3%±3.3%)and An(30%±5.8%),indicating an absence of fitness costs affecting this trait.These results indicate that a switch to three-toxin pyramided cotton could be valuable for increasing durability of Bt cotton in China.The non-recessive resistance to Cry2Ab,cross-resistance to CrylA toxins,survive on Bt cotton and the lack of fitness cost in An2Ab strain incated that the redundant killing of dual Bt toxins cotton can not control resistance to Cry2Ab effectively.So,the introduction of CrylAc+ Cry2Ab cotton may not have very satisfied effect in China.A novel factor was demanded for the preferably management of rensistance to Bt crops.3.Identification of Cry2A binding proteins in the midgut BBMV of H.armigeraThe binding proteins of Cry2A toxin in BBMV of H.armigera were captured under non-denaturing conditions using modified Pull-down method and bio-tinylated Cry2A toxin and identified by LC-MS/MS.Several binding proteins were be identified,including Cadherin,APN1,APN2,APN3,ADP/ATP transferase and V-ATPase et al.Cadherin,APN1,APN2,APN3 and ABCA2 were cloned and no deletion and insertion were found in these genes.Binding quantity with Cry2A toxin shows no difference between An and An2Ab,by contrast,there was no binding of BBMV from ABCA2 konckout strain SCD-A2-KO.Genetic linkage analysis indicated that Cry2Ab resistance in An2Ab was not linked with the above receptor genes.In conclusion,resistance to Cry2Ab in An2Ab strain was not caused by the mutations in binding proteins and the reduction of binding to Cry2Ab.Our study shows that there were no changes in the proteolytic activity of digestive enzymes between An and An2Ab,so it could be a new mechanism of resistance to cry2Ab in An2Ab.4.Genetic mapping and identification of Cry2Ab resistance genes.Genetic mapping results revealed that there are four genetic loci groups linked with resistance to Cry2Ab:Chr09 3.65-4.59M,7.05-8.10M;Chr10 0.78-3.98M,11.86-12.82M.Transcriptome sequencing was employed to screen the candidate genes broadly.Most of those genes in the four genetic loci groups are related with different signal pathway.The synergisms of different signal pathway inhibitors to Cry toxins were detected.BQU57 targeting Ral protein in Chr09 linked area and LY404039 targeting GPCRs in Chr10 linked area can recover the susceptibility to Cry toxins in An2Ab strain.After knockdown the expression of Ral gene via CRISPR/dCas system,the mortality under the diagnostic concentration of Cry2Ab was increased from 33%to 42%with the increase of concentration of dCas9+ sgRNA from 200 ng/?l to 600 ng/?l and the control mortality was 7%.The mortality under the diagnostic concentration of Cry1Ac was also increased.All results indicated that resistance to Cry2Ab was linked with increased expression of Ras-related protein.GPCR/G protein signal pathway may be involved in the non-recessive resistance to Cry2Ab.