Synergism and Antagonism between Bacillus thuringiensis Vip3A and Cry1 Proteins in Heliothis virescens, Diatraea saccharalis and Spodoptera frugiperda

Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC50, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC90, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC90 level than at the LC50 level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Susceptibility of <i>H. virescens</i> neonate larvae to Cry1 and Vip3A protoxins.^a

a<p>Values represent the mean from three replicates of 16 larvae per replicate (n = 48).</p>b<p>Slope ± standard error.</p>c<p>Values are expressed as µg/cm² with 95% fiducial limits (at 5 days).</p><p>Susceptibility of <i>H. virescens</i> neonate larvae to Cry1 and Vip3A protoxins.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107196#nt101" target="_blank">a</a>}</p

SDS-PAGE of <i>Escherichia coli</i> lysates.

<p>MM, Molecular Mass Marker “Spectra TM Multicolor Broad Range Protein Ladder” (Fermentas); lane 1, Vip3A not induced with IPTG; lane 2, Vip3Aa; lane 3, Vip3Ae; lane 4, Vip3Af lane 5, Cry1Aa; lane 6, Cry1Ac; lane 7, Cry1Ca.</p

Evaluation of antagonism, at the LC₅₀ and LC₉₀ level, of combinations of Vip3A and Cry1 protoxins to <i>H. virescens</i> neonate larvae.

a<p>Each data point was obtained from three replicates of 16 larvae per replicate (n = 48).</p>b<p>Proportions of proteins were chosen approximately to match those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107196#pone-0107196-t002" target="_blank">Table 2</a>.</p>c<p>Slope ± standard error.</p>d<p>FL₉₅: 95% fiducial limits.</p>e<p>Expected mortality considering simple similar action.</p>f<p>AF: Antagonism factor, calculated as the ratio of the observed LC₅₀ over the expected LC₅₀. NS = not significant.</p><p>Evaluation of antagonism, at the LC₅₀ and LC₉₀ level, of combinations of Vip3A and Cry1 protoxins to <i>H. virescens</i> neonate larvae.</p

Susceptibility of <i>D. saccharalis</i> and <i>Spodoptera frugiperda</i> neonate larvae to Vip3Aa and Cry1Ca protoxins and their 1: 2 combination.

a<p>Slope ± standard error.</p>b<p>Each data point was obtained from three replicates of 16 larvae per replicate (n = 48).</p>c<p>FL₉₅: 95% fiducial limits.</p>d<p>Expected mortality considering simple similar action.</p>e<p>AF: Antagonism factor.</p><p>Susceptibility of <i>D. saccharalis</i> and <i>Spodoptera frugiperda</i> neonate larvae to Vip3Aa and Cry1Ca protoxins and their 1: 2 combination.</p

Susceptibility of <i>H. virescens</i> neonate larvae to combinations of Vip3A and Cry1 protoxins.

a<p>Concentrations of proteins were chosen such as to equal their respective LC₅₀ values. Values are expressed as µg/cm².</p>b<p>Each value represents the mean from three replicates of 16 larvae per replicate (n = 48).</p>c<p>Expected mortality considering simple independent action.</p>d<p>Asterisks indicate significant differences at P<0.05, and two asterisks at P<0.001.</p>e<p>Chi-square and P values.</p><p>Susceptibility of <i>H. virescens</i> neonate larvae to combinations of Vip3A and Cry1 protoxins.</p