The toxins of Bacillus thuringiensis (Bt) have shown great potential in the control of harmful insects affecting human health and agriculture, used as the main biological agent for the formulation of bioinsecticides due to its specificity to target different insects' orders. This has led Bt-based products to become the best-selling biological insecticides in the world since the genes encoding insecticidal proteins have been successfully used in novel insecticidal formulation, genetically engineered (GE) crops, and development of transgenic rice that produce insecticidal toxins derived from Bacillus thuringiensis. It has been proven that insecticidal activity of Bt protein crystals can prolong their toxicity in shelf life or field under specific conditions, and this can improve the use of special strains and formulations to control insect vectors of diseases. Bt toxins have shown well-documented toxicity against lepidopterans, coleopterans, hemipterans, dipterans, nematodes, Rhabditida and human cancer cells of various origins. These crystal toxins may be responsible for other novel biological properties suggesting a pluripotential nature with different specificities.

This study was carried out to determine the persistence of toxicity of fermentation extracts of Bacillus thuringiensis var. israelensis after more than three decades of storage. For this purpose, a population of Aedes aegypti was established. The mortality rate of 20 spore-crystal extracts purified using the acetone-lactose coprecipitation method was measured and evaluated by bioassays according to a modified WHO protocol. The extracts with the highest mortality rate were determined in triplicate by their LD50 and LD98. All extracts showed toxicity at the highest tested dose (1000 ppm) and some, such as strains 3260 and 3501, still killed larvae at doses as low as 0.01 ppm. These data are surprising because no study on the activity of B. thuringiensis toxic proteins after such a long storage time has been reported.

Background Bacillus thuringiensis subsp. israelensis (Bti) produces insecticidal endotoxins known as Cry and Cyt. Its efficiency and specificity make it the most widely used substance as a biopesticide for controlling disease from vector insects, such as mosquitoes, responsible for important human diseases such as malaria, filariasis, dengue, and yellow fevers. To date, it is proven difficult to develop a commercial product that has more than 2 years of shelf life, and there is little information on the viability of these commercial proteins under prolonged storage conditions. Results This study aimed to evaluate biological activity of reconstituted Bti endotoxins after 40 years of storage against the mosquito Aedes aegypti larvae. Five concentrations of Bti extracts were used for bioassays against 3rd and 4th instars of A. aegypti larvae. All reconstituted endotoxins from stored extracts showed a potency increase. The strain HD-500 from extract 3260 was the most effective insecticide (LC50 = 0.0014 mg/l), followed by 3756 (LC50 = 0.0037 mg/l). These strains were particularly notable, increasing their larvicidal potency one hundredfold and one thousandfold, respectively. Protein profiles in polyacrylamide gels revealed a greater presence of Cyt toxins compared to the stored Bti extracts, which maintained their activity at high concentrations. Conclusion The reconstituted Bti strains presented a great biological activity against A. aegypti larvae, specially extract 3260 (median lethal concentration (LC50) value = 0.0014 mg/l). This considerable larvicidal activity after 40 years under storage was an encouraging signal for the development of future formulation strategies regarding their useful life. The stability of extracts of stored endotoxins produced by Bti decreased significantly, particularly Cyt1A protein, which is responsible for their synergistic activity.