A General Approach to Test for Interaction Among Mixtures of Insecticidal Proteins Which Target Different Orders of Insect Pests

Abstract

A shift toward transgenic crops which produce combinations of insecticidal proteins has increased the interest (Syngenta Seeds, Inc., Minnetonka, MN) in studying the potential for interactions amongst those proteins. We present a general testing method which accommodates proteins with nonoverlapping spectrums of activity. Our sequential testing approach first investigates groups of the proteins with overlapping activity; e.g., proteins active against Lepidoptera or Coleoptera, respectively. The Colby method is used to test for interactions within each respective group. Subsequently, the mixture of proteins within each group is regarded as a single entity and tests for interactions between the groups (when combined) is conducted using analysis of variance. We illustrate the method using Cry1Ab, Vip3Aa20, and Cry1F (a mixture of proteins active against Lepidoptera), and mCry3A and eCry3.1Ab (a mixture of proteins active against Coleoptera). These insecticidal proteins are produced by Bt11 × MIR162 × TC1507 × MIR604 × 5307 maize. We detected no interactions between Cry1Ab, Vip3Aa20, and Cry1F in tests using larvae of two different lepidopteran species, and possible slight antagonism between mCry3A and eCry3.1Ab with a coleopteran test species. We detected no effect of (eCry3.1Ab + mCry3A) on the potency of (Cry1Ab + Vip3Aa20 + Cry1F) to lepidopteran larvae, and no effect of (Cry1Ab + Vip3Aa20 + Cry1F) on the potency of (mCry3A + eCry3.1Ab) to coleopteran larvae. We discuss implications of these results for characterization of Bt11 × MIR162 × TC1507 × MIR604 × 5307 maize, and the value of the method for characterizing other transgenic crops that produce several insecticidal proteins.

Keywords: Bacillus thuringiensis; Cry; stacked; synergism; trait.

Fig. 1.

Testing for the interaction of insecticidal protein mixtures in both Lepidoptera and Coleoptera when the mixtures target susceptible pests across both orders.

Fig. 2.

Difference between the observed and expected mortality for ECB or FAW on diets containing mixtures of Cry1Ab + Vip3Aa20 + Cry1F 120 hours after treatment. X refers to the A or B undiluted mixture as described in the Methods section for ECB or FAW, respectively. X/2 to X/16 refers to serial dilutions for each respective mixture.

Fig. 3.

Difference between observed and expected mortality of CPB on diet containing a mixture of eCry3.1Ab and mCry3A after 120 h. X refers to the undiluted mixture as described in the Methods. X/2 to X/16 refers to serial dilutions for the mixture.

Fig. 4.

Percent mortality of ECB exposed to the lepidopteran-active protein mixture treatments. Treatment X contains 400 ng Cry1Ab + 200 ng Vip3Aa20 + 800 ng Cry1F per ml diet as described in the Methods section.

Fig. 5.

Percent mortality of CPB exposed to the coleopteran-active protein mixture treatments. Treatment X contains 8 µg mCry3A + 8 µg eCry3.1Ab per ml diet as described in the Methods section.