Mechanisms of field-evolved Cry1Ac resistance in Helicoverpa zea

Abstract

Global large-scale adoption of Bt transgenic crops has provided effective management of key insect pests and have greatly reduced insecticide use. However, some field populations of several insect species have evolved resistance to Bt crops in the field, which threatens the continuing success of Bt crops. The cotton bollworm (Helicoverpa zea) is among the first pest reported to have field-evolved resistance to Cry1Ac and Cry2Ab, but the underlying mechanisms remain largely unknown. To determine the current resistance status of the field populations of H. zea and to elucidate the mechanisms of Bt resistance in this pest, I conducted a series of experiments including bioassays of field populations as well as biochemical and molecular comparisons of midgut proteases and putative Cry1Ac receptors between Cry1Ac-susceptible and -resistant strains. Diet incorporation bioassays of six field populations of H. zea collected from Tifton, Georgia USA in 2008 and 2009 indicated that, comparing to LAB-S, a susceptible laboratory strain, all six field populations were significantly resistant to Cry1Ac toxin and one of three field strains was significantly resistant to Cry2Ab toxin. Across the five populations, survival on leaf-discs producing Cry1Ac was positively correlated to the lethal concentration that kills 50% of the population (LC₅₀) for Cry1Ac from diet bioassays. These results support previous findings of field-evolved resistance to Bt crops in H. zea and suggest an overall increase in resistance to Cry1Ac from 2002 to 2009.One of the six field population, which was designed as GA and had 55-fold resistance to Cry1Ac, was further selected with Cry1Ac in the laboratory to generate a more resistant strain, which was designated as GA-R and had 560-fold resistance to Cry1Ac. Total protease activity of the midgut extracts from GA-R and GA strains is significantly lower than that from the susceptible laboratory strain LAB-S. Among the proteases contributing to the total activity, trypsin-like and chymotrypsin-like activities of GA-R and GA midgut extracts are significantly lower than that from the susceptible strain, while no difference in elastase-like activity is evident. Decreased proteolytic activity was correlated to the decreased Cry1Ac activation rate of midgut extracts of the GA-R and GA strains. Cytotoxicity assays with H. zea midgut cells show that the product of Cry1Ac protoxin digested with GA-R and GA midgut extracts has significantly lower cytotoxicity when compared with that digested with the susceptible strain midgut extracts. Transcriptional analysis of a limited number of protease genes did not identify specific proteases involved in the decline in Cry1Ac activation in GA-R and GA. These results indicate that the decreased Cry1Ac activation rate by midgut proteases is involved in the field originated Cry1Ac resistance in the H. zea GA-R and GA strains. I also compared the cDNA sequences and expression levels of the putative Cry1Ac receptors cadherin, aminopeptidase 1 (APN1), alkaline phosphatase 2 (ALP2) and ATP-binding cassette subfamily C member 2 (ABCC2) in LAB-S and GA-R. No indels (insertions and deletions) were found in the cDNA sequences of the resistant alleles of the four receptors, relative to those of the susceptible alleles. While there were no amino acid point mutations in the resistant alleles of ALP2 and ABCC2, we found 2 and 14 consistent amino acid point mutations in the resistant alleles of cadherin and APN1, respectively. However, neither cadherin nor APN1 point mutations were genetically linked to Cry1Ac resistance in GA-R. Quantitative RT-PCR analysis revealed no differences in the transcripts of the four receptors between the two strains. Taken together, these results indicate that the four receptors are not involved in Cry1Ac resistance in the GA-R strain of H. zea.