Insecticidal activity and cytotoxicity of Bacillus thuringiensis Cry1Ia7 protein
Abstract: Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces insecticidal proteins whether accumulated in parasporal crystal inclusions in the stationary phase, or secreted in the vegetative phase. Cry1I proteins belong to this second group of Bt proteins, although their structure is similar to the Cry proteins which are generally accumulated in the parasporal body. Moreover, Cry1I proteins have attracted attention due to their dual activity to Lepidoptera and Coleoptera. The aim of the present work was to explore additional insect species potentially susceptible to Cry1Ia7 by two different methods. First, insecticidal activity was tested in bioassays against Grapholita molesta, Mamestra brassicae and Ostrinia nubilalis neonates, with trypsin activated Cry1Ia7 at a concentration of 1000 ng/cm2. Second, for the first time, the cytotoxicity of Cry1Ia7 was analyzed in different insect cell lines by the method of tetrazolium salt reduction (MTT). Lepidopteran cell lines from Spodoptera frugiperda (Sf21 cells) and Trichoplusia ni (BTI-Tn-5B1-4, Hi5 cells) were challenged with trypsin-activated Cry1Ia7 at several toxin concentrations and exposure times. We found that trypsin-digested Cry1Ia7 was almost not toxic for M. brassicae and O. nubilalis. Our results agree with published data about low toxicity of other Cry1I proteins to M. brassicae and are in disagreement with the Cry1Ie toxicity reported for O. nubilalis. In addition, the treatment of G. molesta (a lepidopteran pest that is very susceptible to Cry1 proteins) with Cry1Ia7, showed low mortality and moderate growth inhibition. We observed a 40% loss of cell viability in both insect cell lines, Sf21 and Hi5, after 48 hours of exposure to the highest concentration of protein (20 μg/ml). The results of this study show that none of the studied lepidopteran species revealed high susceptibility to Cry1Ia7 protein, and that the use of insect cell lines could be a good tool to study the mode of action of this protein.