Abstract
Insect resistance to the Cry toxins of Bacillus thuringiensis (Bt) has been examined previously using a number of traditional biochemical and molecular techniques. In this study, we utilized a proteomic approach involving two-dimensional differential gel electrophoresis, mass spectrometry, and function-based activity profiling to examine changes in the gut proteins from the larvae of an Indianmeal moth (IMM, Plodia interpunctella) colony exhibiting resistance to Bt. We found a number of changes in the levels of certain specific midgut proteins that indicate increased glutathione utilization, elevation in oxidative metabolism, and differential maintenance of energy balance within the midgut epithelial cells of the Bt-resistant IMM larva. Additionally, the electrophoretic migration pattern of a low molecular mass acidic protein, which apparently is an ortholog of F(1)F(0)-ATPase, was considerably altered in the Bt-resistant insect indicating that variations in amino acid content or modifications of certain proteins also are important components of the resistance phenomenon in the IMM. Furthermore, there was a dramatic decrease in the level of chymotrypsin-like proteinase in the midgut of the Bt-resistant larva, signifying that reduction of chymotrypsin activity, and subsequently decreased activation of Cry toxin in the insect midgut, is an important factor in the resistant state of the IMM. The proteomic analysis of larval gut proteins utilized in this study provides a useful approach for consolidating protein changes and physiological events associated with insect resistance to Bt. Our results support the hypothesis that physiological adaptation of insects and resistance to Bt is multifaceted, including protein modification and changes in the synthesis of specific larval gut proteins. We believe that increased oxidative metabolism may be an adaptive response of insects that undergo survival challenge and that it could mediate detoxification as well as higher rates of generalized and localized mutations that enhance their resistance and provide survival advantage.
Highlights
Insect resistance to the Cry toxins of Bacillus thuringiensis (Bt) has been examined previously using a number of traditional biochemical and molecular techniques
1 The abbreviations used are: Bt, Bacillus thuringiensis; DIGE, differential gel electrophoresis; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; Isoelectric Focusing (IEF), isoelectric focusing; IMM, Indianmeal moth; IPG, immobilized pH gradient; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; NL, nonlinear; P, standard consisting of S and R proteins; R, Bt-resistant; SAAPFpNA, N-succinyl-Ala-AlaPro-Phe p-nitroanilide; S, Bt-susceptible; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; TEMED, N,N,NЈ,NЈtetramethylethylenediamine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; V-ATPase, vacuolar ATPase
We found a number of changes in the levels of certain specific midgut proteins that indicate increased glutathione utilization, elevation in oxidative metabolism, and differential maintenance of energy balance within the midgut epithelial cells of the Bt-resistant IMM larva
Summary
Bacillus thuringiensis; DIGE, differential gel electrophoresis; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; IEF, isoelectric focusing; IMM, Indianmeal moth; IPG, immobilized pH gradient; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; NL, nonlinear; P, standard consisting of S and R proteins; R, Bt-resistant; SAAPFpNA, N-succinyl-Ala-AlaPro-Phe p-nitroanilide; S, Bt-susceptible; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; TEMED, N,N,NЈ,NЈtetramethylethylenediamine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; V-ATPase, vacuolar ATPase. Insect Resistance to B. thuringiensis ive responses such as changes in protein expression and the physiological state of the larval midgut can interrupt toxin action, rendering the gut tissue refractory to Cry toxins [8]. We examined alterations in the larval midgut proteome of such an insect, the Indianmeal moth (IMM), to determine which proteins may be involved mechanistically in sustaining a resistant state. The results of this study support our view that resistance to Bt is a complex response that can be displayed by proteomic expression profiles of midgut proteins from resistant and susceptible strains of an insect
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