Resistance to the Cry1Ac δ-Endotoxin of Bacillus thuringiensis in the Cotton Bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract

Three laboratory strains of Helicoverpa armigera (Hübner) were established by mating of field-collected insects with an existing insecticide-susceptible laboratory strain. These strains were cultured on artificial diet containing the Cry1Ac protoxin of Bacillus thuringiensis using three different protocols. When no response to selection was detected after 7-11 generations of selection, the three strains were combined by controlled mating to preserve genetic diversity. The composite strain (BX) was selected on the basis of growth rate on artificial diet containing Cry1Ac crystals. Resistance to Cry1Ac was first detected after 16 generations of continuous selection. The resistance ratio (RR) peaked approximately 300-fold at generation 21, after which it declined to oscillate between 57- and 111-fold. First-instar H. armigera from generation 25 (RR = 63) were able to complete their larval development on transgenic cotton expressing Cry1Ac and produce fertile adults. There appeared to be a fitness cost associated with resistance on cotton and on artificial diet. The BX strain was not resistant to the commercial Bt spray formulations DiPel and XenTari, which contain multiple insecticidal crystal proteins, but was resistant to the MVP formulation, which only contains Cry1Ac. The strain was also resistant to Cry1Ab but not to Cry2Aa or Cry2Ab. Toxin binding assays showed that the resistant insects lacked the high affinity binding site that was detected in early generations of the strain. Genetic analysis confirmed that resistance in the BX strain of H. armigera is incompletely recessive.

Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: Identification of proteins binding the δ-endotoxin, Cry1Ac of Bacillus thuringiensis

“…While five Cry1Ac-binding proteins were isolated from H. armigera in this study, in situ studies predicted only two binding sites (Akhurst et al, 2003;Liao et al, 1996). These in situ studies were conducted with BBMVs, which, without the membrane solubilisation step, might better reflect in vivo binding whereas the treatment of BBMVs with CHAPS as part of the affinity purification process could have exposed binding epitopes that are concealed in vivo.…”

“…All the proteins isolated in this study have predicted GPI anchor sites but only some have the mucin-like domain. These data might be reconciled by the suggestion that the two classes of binding sites identified kinetically (Liao et al, 1996;Akhurst et al, 2003) might correspond with GalNAc contained in mucin-like domains and GalNAcmodified GPI-anchors rather than two particular proteins. If this is correct then either of these features might be sufficient to enable an APN to bind Cry1Ac under the conditions reported here.…”

“…The propensity of field populations of H. armigera to develop resistance to chemical insecticides (Forrester et al, 1993), its capacity for resistance to Cry1A toxin (Akhurst et al, 2003), and the already evident field-developed resistance of Plutella xylostella (Tabashnik et al, 1990), has highlighted the need for resistance management when controlling field populations of H. armigera. Such precautions require detecting resistant insects in field populations, which can be handled efficiently by using resistance markers.…”

“…In the majority of Cry-resistant insect species investigated, resistance has been linked to an alteration in the binding of Bt toxins to midgut epithelia, often correlated with reduced expression of a protein ligand (reviewed by Ferré and Van Rie, 2002). In H. armigera, Cry1Ac resistance was linked to an alteration in Cry1Ac binding to a cadherin-like protein (Akhurst et al, 2003;Xu and Wu, 2008). However, two isoenzymes of aminopeptidase (APN) from H. armigera interacted with Cry1Ac after expression in Trichoplusia cells.…”

Genetic contributions to agricultural sustainability

“…Previous research had reported resistance to Cry proteins in the Indian meal moth (Plodia interpunctella) and the diamondback moth (Plutella xylostella). Akhurst et al (2003) were able, under laboratory conditions, to select a strain of H. armigera that was resistant to the toxicity of Cry1Ac proteins, so it was clear that target pests could possibly develop resistance to the active ingredient of Ingard cotton. The cotton industry had for years grappled with the problem of chemical insecticide resistance and was reluctant to see Ingard technology wasted.…”