Abstract
Susceptibility of Tribolium castaneum (Tc) larvae was determined against spore-crystal mixtures of five coleopteran specific and one lepidopteran specific Bacillus thuringiensis Cry toxin producing strains and those containing the structurally unrelated Cry3Ba and Cry23Aa/Cry37Aa proteins were found toxic (LC50 values 13.53 and 6.30 µg spore-crystal mixture/µL flour disc, respectively). Using iTRAQ combined with LC-MS/MS allowed the discovery of seven novel differentially expressed proteins in early response of Tc larvae to the two active spore-crystal mixtures. Proteins showing a statistically significant change in treated larvae compared to non-intoxicated larvae fell into two major categories; up-regulated proteins were involved in host defense (odorant binding protein C12, apolipophorin-III and chemosensory protein 18) and down-regulated proteins were linked to metabolic pathways affecting larval metabolism and development (pyruvate dehydrogenase Eα subunit, cuticular protein, ribosomal protein L13a and apolipoprotein LI-II). Among increased proteins, Odorant binding protein C12 showed the highest change, 4-fold increase in both toxin treatments. The protein displayed amino acid sequence and structural homology to Tenebrio molitor 12 kDa hemolymph protein b precursor, a non-olfactory odorant binding protein. Analysis of mRNA expression and mortality assays in Odorant binding protein C12 silenced larvae were consistent with a general immune defense function of non-olfactory odorant binding proteins. Regarding down-regulated proteins, at the transcriptional level, pyruvate dehydrogenase and cuticular genes were decreased in Tc larvae exposed to the Cry3Ba producing strain compared to the Cry23Aa/Cry37Aa producing strain, which may contribute to the developmental arrest that we observed with larvae fed the Cry3Ba producing strain. Results demonstrated a distinct host transcriptional regulation depending upon the Cry toxin treatment. Knowledge on how insects respond to Bt intoxication will allow designing more effective management strategies for pest control.
Highlights
The entomophatogenic bacterium Bacillus thuringiensis (Bt) represents an environmentally safe alternative for pest control producing parasporal inclusions, which contain one or several insecticidal proteins
Bt Cry toxin producing strains display toxicity against Tribolium castaneum (Tc) To determine the susceptibility of Tc larvae to five coleopteran specific Bt toxin producing strains (Cry3Aa, Cry3Ba, Cry3Ca, Cry23Aa/Cry37Aa and Cry34Ab/Cry35Ab) and one lepidopteran specific Bt toxin producing strain (Cry1Ac) a single dose of 3 mg of spore-crystal mixture of each Bt toxin expressing strain per microliter of flour disc were initially assayed
The profiles of Tc dose-response curves to both Bt active spore-toxin treatments were similar with a 70% maximum mortality percentage achieved after seven days of exposure, supporting that in the assay conditions, surviving larvae might be able to mount a defensive response that counteracted the toxic action
Summary
The entomophatogenic bacterium Bacillus thuringiensis (Bt) represents an environmentally safe alternative for pest control producing parasporal inclusions, which contain one or several insecticidal proteins. The red flour beetle, Tribolium castaneum (Tc), is a major global pest of stored grain, cereal products, and peanuts for human consumption [4]. This coleopteran insect, readily adaptable to all classes of insecticides, is an ideal subject for the identification of new pesticide targets for which many genetic and genomics tools have been developed and it has become the genetic model for agriculturally important coleopteran species [5].
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