Combined effects of three crystalline toxins from Bacillus thuringiensis with seven proteinase inhibitors on beet armyworm, Spodoptera exigua Hubner (Lepidoptera: Noctuidae)

Spodoptera exigua is a highly polyphagous pest and causes extensive damage to many field and truck crops. Since the larvae are not or only sublethally affected by Cry1Ac, the fused Cry1Ac/Cry1Ab, or the stacked Cry1Ac + CpTI transgenic cottons that are widely planted in China, S. exigua has become a major economic pest of cotton across a wide distribution since the commercialization of Bt cottons in 1997 in China. Proteinase inhibitors are potential candidates for enhancing Bt toxicity against, and for expanding control spectrum for insect pests. In the present paper, we first found that S. exigua larval midgut fluids could remarkably degrade activated Cry1Ca, and slightly hydrolyze Cry1Ac and Cry1Ab. Subsequently, we investigated interactions between the 3 Cry toxins and 7 proteinase inhibitors, i.e., phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, tannic acid, N-alpha-tosyl-L-phenylalanine chloromethyl ketone, N-alpha-tosyl-L-lysine chloromethyl ketone, elastatinal and ethylenediaminetetraacetic acid (EDTA), by monitoring larval growth and mortality rate. A 6-day dietary exposure of the newly molted 2nd instars to either the inhibitors (3 test concentrations) or the toxins (Cry1Ac, 31.30 ng/cm(2); Cry1Ab, 3.2 ng/cm(2); Cry1Ca, 0.6 ng/cm(2)) alone only slightly affected larval growth. In contrast, exposure to the mixtures containing an inhibitor and a toxin, with the exception of those containing EDTA, synergistically reduced larval weight. In general, the synergisms were more obvious at higher inhibitor concentrations. Regarding larval mortality, the inhibitors except EDTA at the highest test concentration showed significant synergism to both Cry1Ab (0.2 mu g/cm(2)) and Cry1Ca (0.04 mu g/cm(2)). As the inhibitor concentration reduced, however, the synergistic effects decreased. These results indicated that trypsin-, chymotrypsin-, and elastase-like proteinases in S. exigua larval midgut were involved in proteolytical hydrolyzation of the 3 activated Cry toxins, and protection of Bt Cry toxins from proteinase degradation in the midgut by inhibitors may greatly enhance toxicity against S. exigua larvae. (C) 2013 Elsevier Inc. All rights reserved.