Genotoxic evaluation in Oreochromis niloticus (Fish: Characidae) of recombinant spore–crystal complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis

Abstract

Bioinsecticides from Bacillus thuringiensis (Bt) are widely used around the world in biological control against larval stages of many insect species. Bt has been considered a biopesticide that is highly specific to different orders of insects, non-polluting and harmless to humans and other vertebrates, thus becoming a viable alternative for combating agricultural pests and insect vectors of diseases. The family of Bt δ-endotoxins are crystal-protein inclusions showing toxicity to insects' midgut, causing cell lysis leading to starvation, septicemia and death. The aim of this study is to evaluate the genotoxic potential of recombinant Bt spore-crystals expressing Cry1Ia, Cry10Aa and Cry1Ba6 on peripheral erythrocyte cells of Oreochromis niloticus, through comet assay, micronucleus (MN) test and nuclear abnormalities (NA) analysis. Fish (n = 10/group) were exposed for 96 h at 10(7) spores 30 l(-1), 10(8) spores 30 l(-1) or 10(9) spores 30 l(-1) of Bt spore-crystals. Cry1Ia showed a significant increase in comet cells at levels 1 and 2, but not at levels 3 and 4, so it was not mutagenic nor did it induce MN or NA. These three spore-crystals showed some fish toxicity at only the highest exposure level, which normally does not occur in the field.

Evaluation of Cytotoxicity, Genotoxicity and Hematotoxicity of the Recombinant Spore-Crystal Complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis in Swiss Mice

“…When analyzing hematologic data from mice, not only concurrent controls should be used as the primary comparison for interpretation of treatment-related changes, but also comparisons with reference intervals, which are desirable to put hematologic changes in perspective [ 15 ]. Thus, this study has demonstrated that the levels of toxicity depend on the strain of spore-crystal and its exposure levels, as we have also shown in our previous study with these same spore-crystals [ 19 ]. Considering that more than 50 Cry proteins have already been well-characterized, and that their structure shows three domains responsible for forming pores in the membranes of the susceptible cells, causing lysis, variations in these domains could be responsible for the variations in toxicity because of their requirement to bind to specific receptors for activity [ 29 ].…”

“…Aquatic exposure of spore-crystals Cry1Aa, Cry1Ab, Cry1Ac e Cry2Aa to zebrafish at different exposure levels showed low toxicity and no genotoxicity, except for Cry1Aa which increased the micronucleus levels in peripheral erythrocyte cells [ 18 ]. In addition, in our previous study of Oreochromis niloticus exposed to Bt spore-crystals Cry1Ia, Cry10Aa and CyrBa6 for 96 h no mutagenicity was observed through micronucleus test or DNA damage by the comet assay in peripheral erythrocyte cells [ 19 ]. Additionally, mice orally treated with Bt spore-crystals genetically modified to express individually Cry1Aa, Cry1Ab, Cry1Ac or Cry2A showed no genotoxic effect [ 20 ], corroborating our results.…”

Dissecting the Environmental Consequences of Bacillus thuringiensis Application for Natural Ecosystems

“…Non-target aquatic vertebrates can be exposed to Bt as a result of biological treatments aimed against insect pests or mosquitos. Most researchers report that Bt spores parasporal bodies are not toxic for fishes [ 152 , 154 , 155 , 156 , 157 ]. Snarski [ 157 ] observed no adverse effects of exposure of Bt var.…”

“…Moreover, upon the transfer to Bt -free water, fishes effectively excreted the toxins from their organisms [ 157 , 158 ]. No genotoxic or mutagenic effects of Bt toxins on peripheral erythrocyte cells of Oreochromis niloticus were observed [ 154 ]. Cry1Ba6 and Cry10Aa toxins did not show adverse effects to O .…”

“…Cry1Ba6 and Cry10Aa toxins did not show adverse effects to O . niloticus, although Cry1Ia induced slight DNA damage when applied at extraordinarily high concentrations [ 154 ]. In order to provide a more precise assessment of Bt toxicity to fishes, several studies utilized hematological parameters as indicators of total fitness.…”