Abstract
Bacillus thuringiensis (Bt) Cry toxins from the Cry1A family demonstrate significantly different toxicities against members of the family Noctuidae for unknown reasons. In this study, membrane potential was measured and analyzed in freshly isolated midgut samples from Mythimna separata and Agrotis ipsilon larvae under oral administration and in vitro incubation with Bt toxin Cry1Ab to elucidate the mechanism of action for further control of these pests. Bioassay results showed that the larvae of M. separata achieved a LD50 of 258.84 ng/larva at 24 h after ingestion; M. separata larvae were at least eightfold more sensitive than A. ipsilon larvae to Cry1Ab. Force-feeding showed that the observed midgut apical-membrane potential (Vam) of M. separata larvae was significantly depolarized from −82.9 ± 6.6 mV to −19.9 ± 7.2 mV at 8 h after ingestion of 1 μg activated Cry1Ab, whereas no obvious changes were detected in A. ipsilon larvae with dosage of 5 μg Cry1Ab. The activated Cry1Ab caused a distinct concentration-dependent depolarization of the apical membrane; Vam was reduced by 50% after 14.7 ± 0.2, 9.8 ± 0.4, and 7.6 ± 0.6 min of treatment with 1, 5, and 10 μg/mL Cry1Ab, respectively. Cry1Ab showed a minimal effect on A. ipsilon larvae even at 20 μg/mL, and Vam decreased by 26.3% ± 2.3% after 15 min. The concentrations of Cry1Ab displayed no significant effect on the basolateral side of the epithelium. The Vam of A. ipsilon (−33.19 ± 6.29 mV, n = 51) was only half that of M. separata (−80.94 ± 6.95 mV, n = 75). The different degrees of sensitivity to Cry1Ab were speculatively associated with various habits, as well as the diverse physiological or biochemical characteristics of the midgut cell membranes.
Highlights
Bacillus thuringiensis (Bt) Cry toxins are the active ingredients in most widely used biological insecticides and insect-resistant transgenic crops; pests can develop resistance, thereby reducing the effectiveness of these toxins [1,2,3]
A. ipsilon is described to be a group of more Bt-tolerant insects, which are difficult to control with sprayable Bt biopesticides or transgenic Bt plants [1,10]
Bioassay results demonstrated that M. separata was more susceptible to Cry1Ab than A. ipsilon
Summary
Bacillus thuringiensis (Bt) Cry toxins are the active ingredients in most widely used biological insecticides and insect-resistant transgenic crops; pests can develop resistance, thereby reducing the effectiveness of these toxins [1,2,3]. Crystal proteins are first ingested as protoxins, which are solubilized and proteolytically converted into smaller and protease-stable polypeptides in the insect midgut These activated toxins bind to specific receptors on the surface of midgut epithelial cells, thereby allowing them to enter the membrane and form poorly selective pores permeable to small molecules, such as inorganic ions, amino acids, and sugars [13,14]. A simple technique for intracellular recording with a standard microelectrode was developed to measure the electrical membrane potential of epithelial cells in freshly isolated lepidopteran larval midgut samples by Peyronnet et al [23]. Bt toxin Cry1Ab to elucidate the mechanism of action and verify whether the membrane potential depolarization is correlated with the different susceptibilities to Bt toxins of both larvae for further control of these pests
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