Detection And Modeling Of Cry1Ac Resistance Evolution In Field Populations Of Helicoverpa Armigera

Cotton bollworm,Helicoverpa armigera is a worldwide important lepidopteran pest.The methods for suppressing this pest all most relayed on chemical control before 1996.However,severe resistance evolved due to the large-scale and intensive use of chemical insecticides,and then caused control failure in field.Since 1996,Bt crops were adopted not only suppressed some major pest including H.armigera,but also reduced the use of toxic chemical pesticides,improved the abundance of natural enemies,reduced environment pollution,and improved profits for farmers.However,such benefits will be impaired by Bt resistance that evolved by target pests.Therefore,series of resistance management strategies are designed for delaying development of resistance.In the United States and Australia,two resistance management strategies are successively adopted:(1)refuge strategy,which require farmers to plant a part of non-Bt crops for susceptible insects;(2)pramid strategy,which replaced Bt cotton that only producing single Cry1Ac toxin with Bt cottons that producing two or three toxins(i.e.Cry2Ab or Vip3Aa).In Australia,where two strategies were strictly used,insects are keeping susceptible to Bt crops.In China,Bt cotton was planted for controlling some major pests such as H.armigera and Pectinophora gossypiella since 1997,but without requirement of any resistance management strategy by government.However,other non-Bt host plants than cotton(i.e.soybean,corn and peanut)act as natural refuges for H.armigera,with corresponding equivalent effectiveness of up to 56%non-Bt cotton,thereby contributing to the delay in resistance evolution.Moreover,in Yangtze river valley,hybrid Bt cottons that unconscious planted by farmers were reported served as refuges in bag and effectively delayed resistance in P.gossypiella.Though high proportion of refuge were planted in northern China,Cry1Ac resistant individuals still increased from 0.93%in 2010 to 5.5%in 2013,and nonrecessive resistance increased from 37%in 2010 to 84%in 2013 due to the long-term monoculture of Cry1Ac Bt cotton.Here,we monitored and modeling the evolution of Cry1Ac resistance in northern China,as well as developed and used a BSA-seq detection for uncover the diversity of dominant Cry1Ac resistance in field H.armigera populations.The present study in order to illustrate the evolutionary mechanism in molecular level that providing suggestion for revising and imporing resistant management strategy in northern China.1.Detection and modeling of Cry1Ac resistance evolution in field populations of H.armigera based on the frequency of resistant individuals.The monitoring data shows that the frequency of Cry1Ac resistant individuals increased 8-fold from 0.93%in 2010 to 7.59%in 2016(Mann-Whitney U test,two-tailed,P=0.0023),and 84.4%(95%CI:80.5-88.4%)of resistant individuals harboring at least one dominant resistance genes,which indicating that refuge strategies are not suffient for delay dominant Cry1Ac resistance.Previous study demonstrated that natural refuge effectively delayed Cry1Ac resistance in H.armigera in northern China from 2010 to 2013.However,we did not observe the same tendency when we conduct the simulation from 2013 to 2016 under the same assumptions.A potential reason is some other conditions evolved in resistance evolution in field,such as part of hybrid Bt cotton acting as refuges in bag.Here we conducted a simulation under the hypothesis that combination of refuge in bag and natural refuge delayed resistance in H.armigera.The result of simulation corresponding to the observed data demonstrate this assumption.The sensitive analysis of modeling indicate that dominance of resistance is the key factor for success use of refuge strategy.Therefore,resistance management strategies other than using refuge are urgently needed to delay dominant resistance in northern China.2.Detection and modeling of Cry1Ac resistance evolution in field populations of H.armigera based on the T92C mutation frequency of HaTSPAN1.The key assumption for refuge delaying resistance in field is that resistance inherence recessive.However,dominant resistance emerged and development severely threaten the successful use of Bt cotton in northern China.Here we developed an amplicon-seq detection based on high throughput sequencing in order to conducting a large-scale testing for frequency of a dominant resistance alleles(T92C on HaTSPAN1 gene)in field.The result shows the corresponding between observed data and theoretical value(R2=0.9992,P<0.0001)),indicating that amplicon-seq detection is appropriate for detecting allele frequency.Then we conducting a monitoring for T92C frequency using amplicon-seq detection,and the result shows the T92C frequency increased 100-fold from 0.1%in 2006 to 10%in 2016(Mann-Whitney U test,two-tailed,P<0.0001).The corresponding between the observed data and the tendency of computer simulation,which modeling under the assumption that refuges delayed resistance in northern China,indicated that refuge strategy is powerless for delaying the development of dominant resistance.3.Genetic diversity of dominant Cry1Ac resistance in field populations of H.armigera.The dominant resistance evolved by H.armigera is threatening the successful use of Bt cotton in northern China.Therefore,illustrating the diversity of dominant resistance is helpful for making scientific resistance management strategy.However,little is known about the mechanism of dominant resistance except for T92C substitution on HaTSPANl gene.Here,we developed and used a BSA-seq detection based on gene mapping and high throughput sequencing that can rapidly uncover the genes conferring dominant resistance in field.We detected 23 field-derived populations harboring dominant resistance gene.The results indicate a diverse of dominant resistance genes in northern China,and at least 5 genes present at high frequency in resistant individuals.The most frequent locus is 10.2-11.5 Mbp on chromosome 10,where harboring T92C mutation on HaTSPAN1 gene.The present study uncovered the diverse dominant Cry1Ac resistance genes present in field H.armigera populations.The monitoring and modeling of Cry1Ac resistance demonstrated that refuge strategy can no longer counter the dominant Cry1Ac resistance evolved by H.armigera in Yellow river and Yangtze river vallies.The new resistance management strategies other than refuge(i.e.using pyramid cotton produce Cry1Ac+Cry2Ab+Vip3Aa Bt toxins)are urgently needed to be adopted in these regions.