Resistance to <i>Bacillus thuringiensis</i> Mediated by an ABC Transporter Mutation Increases Susceptibility to Toxins from Other Bacteria in an Invasive Insect

<div><p>Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium <i>Bacillus thuringiensis</i> (Bt) used widely in sprays and transgenic crops. Recent efforts to delay pest adaptation to Bt crops focus primarily on combinations of two or more Bt toxins that kill the same pest, but this approach is often compromised because resistance to one Bt toxin causes cross-resistance to others. Thus, integration of Bt toxins with alternative controls that do not exhibit such cross-resistance is urgently needed. The ideal scenario of negative cross-resistance, where selection for resistance to a Bt toxin increases susceptibility to alternative controls, has been elusive. Here we discovered that selection of the global crop pest, <i>Helicoverpa armigera</i>, for >1000-fold resistance to Bt toxin Cry1Ac increased susceptibility to abamectin and spineotram, insecticides derived from the soil bacteria <i>Streptomyces avermitilis</i> and <i>Saccharopolyspora spinosa</i>, respectively. Resistance to Cry1Ac did not affect susceptibility to the cyclodiene, organophospate, or pyrethroid insecticides tested. Whereas previous work demonstrated that the resistance to Cry1Ac in the strain analyzed here is conferred by a mutation disrupting an ATP-binding cassette protein named ABCC2, the new results show that increased susceptibility to abamectin is genetically linked with the same mutation. Moreover, RNAi silencing of <i>HaABCC2</i> not only decreased susceptibility to Cry1Ac, it also increased susceptibility to abamectin. The mutation disrupting ABCC2 reduced removal of abamectin in live larvae and in transfected Hi5 cells. The results imply that negative cross-resistance occurs because the wild type ABCC2 protein plays a key role in conferring susceptibility to Cry1Ac and in decreasing susceptibility to abamectin. The negative cross-resistance between a Bt toxin and other bacterial insecticides reported here may facilitate more sustainable pest control.</p></div

Responses to spineotram, endosulfan, phoxim, and cyhalothrin in the Cry1Ac-resistant strain (LF60) of <i>H</i>. <i>armigera</i> and its Cry1Ac-susceptible parent strain (LF).

<p>Responses to spineotram, endosulfan, phoxim, and cyhalothrin in the Cry1Ac-resistant strain (LF60) of <i>H</i>. <i>armigera</i> and its Cry1Ac-susceptible parent strain (LF).</p

Suppression of <i>HaABCC2</i> transcription by RNAi in the Cry1Ac-susceptible LF strain of <i>H</i>. <i>armigera</i>.

<p>Early third instar larvae were fed individually with water (control), dsRNA from <i>GFP</i> (control) or dsRNA from <i>HaABCC2</i>. <i>HaABCC2</i> transcription was monitored using qRT-pCR at 1, 3 and 5 days after treatment. The bars show mean transcript levels relative to reference genes (actin and GAPDH) and standard errors from three biological replicates (n = 5 larvae per replicate). For 1, 3 or 5 days after treatment, different letters indicate significantly different means (P < 0.05 by Duncan’s multiple range tests).</p

Concentration of abamectin in larval midgut for the Cry1Ac-resistant LF60 strain and the Cry1Ac-susceptible LF strain of <i>H</i>. <i>armigera</i>.

<p>Larvae from both strains fed on diet with 3 μg abamectin per ml. At 24 and 48 h after feeding on treated diet, the concentration of abamectin was significantly higher in LF60 than LF (t-tests, df = 4, 24 hours: t = 6.0, P = 0.004, 48 hours: t = 20.8, P < 0.0001).</p

Silencing <i>HaABCC2</i> with RNAi decreased susceptibility to Cry1Ac and increased susceptibility to abamectin.

<p>After treatment with water, dsGFP, or dsHaABCC2, larvae from the Cry1Ac-susceptible LF strain of <i>H</i>. <i>armigera</i> were given diet containing Cry1Ac or abamectin (three replicates of 24 larvae for each treatment for each diet, total n = 432 larvae). Bars show means and their standard errors. The asterisk indicates no larvae treated with dsHaABCC2 survived on diet with abamectin. For each diet type (Cry1Ac or abamectin), different letters indicate significantly different means (P < 0.05 by Duncan’s multiple range tests).</p

Transfection of Hi5 cells with wild type gene <i>HaABCC2-GFP</i> (brown) decreased residual abamectin concentration relative to Hi5 cells transfected with the mutant gene <i>mHaABCC2-GFP</i> (blue) or <i>GFP</i> (green).

<p>Different letters indicate significantly different means (P < 0.05 by Duncan’s multiple range tests).</p

Genetic linkage between resistance to Cry1Ac and increased susceptibility to abamectin.

<p>Previous work showed that the <i>r</i> allele of the <i>HaABCC2</i> gene confers resistance to Cry1Ac and the <i>s</i> allele confers susceptibility [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005450#ppat.1005450.ref027" target="_blank">27</a>]. The results here with F2 larvae from five single-pair families (total n = 200 larvae) show that on untreated diet (control), the genotype frequencies did not differ significantly from those expected under Mendelian inheritance (0.25 <i>ss</i>: 0.50 <i>rs</i>: 0.25 <i>rr)</i>, but on diet containing abamectin, the frequencies of <i>rr</i> and <i>rs</i> were significantly lower than expected (see text and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005450#ppat.1005450.s003" target="_blank">S3 Table</a> for details).</p

Transfection of Hi5 cells with the wild-type gene fused to GFP (<i>HaABCC2-GFP</i>), but not the mutant gene fused with GFP (<i>mHaABCC2-GFP</i>) or <i>GFP</i> alone, confers susceptibility to Cry1Ac.

<p>As the concentration increased from 0.0625 to 2 μg- Cry1Ac per ml, the percentage of aberrant cells increased from 2.4 to 91.9% for Hi5 cells transfected with <i>HaABCC2-GFP</i>, but remained ≤3% for cells transfected with <i>GFP</i> (control) or <i>mHaABCC2-GFP</i>. Different letters indicate significantly different means (P < 0.05 by Duncan’s multiple range tests).</p

Responses to abamectin in the Cry1Ac-resistant strain (LF60) of <i>H</i>. <i>armigera</i>, its Cry1Ac-susceptible parent strain (LF), and their F1 progeny; and in an independently derived susceptible strain (96S).

<p>Responses to abamectin in the Cry1Ac-resistant strain (LF60) of <i>H</i>. <i>armigera</i>, its Cry1Ac-susceptible parent strain (LF), and their F1 progeny; and in an independently derived susceptible strain (96S).</p