Bacillus thuringiensis var. israelensis strains
Five Bti fermentation extracts with the code: 3260, 3501, 3691, 3696, and 3756, respectively, were produced by coprecipitation lactose-acetone method by Dulmage et al. (1970) at the US Department of Agriculture, Agricultural Research Service (USDA-ARS) from the strains HD-500 and HD-567, chosen from the previous work of Galán et al. (2017). During the storage period, all extracts were stored in dark and dry conditions exclusively for this purpose at 25 ± 3 °C in sterile and hermetic bottles.
Reconstitution of toxic complex: spore ∂-endotoxin
For each Bti extract, 20 mg of powder was dissolved and homogenized on a vortexer in Eppendorf tubes with 1 ml of sterile ionized water, reactivated in Petri dishes and incubated in BD Bioxon nutrient agar for 48 h at 30 °C. Subsequently, the colonies were inoculated into 50-ml flasks containing 10 ml nutrient broth (NB, Difco) as a culture medium for 12 h at 30 °C in an incubator shaker (New Brunswick Scientific Co., Inc., Edison, NJ) running at 150 rpm. Then, 1 ml from each of the cultures in the mid-logarithmic growth phase was transferred into 500-ml Erlenmeyer flasks containing 100 ml of the same medium for 72 h at 30 °C in the same shaker running at 150 rpm until 80% sporulation had been achieved and most of the spores and crystals had been released. The spore-crystal complex was then extracted using the lactose-acetone co-precipitation method (Dulmage et al. 1970).
Aedes aegypti bioassays
Toxicity against A. aegypti larvae was determined by multiple bioassays under laboratory conditions. The larvae of the 3rd and 4th instars of A. aegypti, used in these experiments, were obtained from the insectary of the Institute of Biotechnology of the School of Biology of the UANL. This mosquito colony has been permanently maintained under pathogen-free conditions at 28 to 30 °C, with 60–80% relative humidity and light/dark cycles of 12 h. The larvae were fed daily on finely ground presterilized dog food (Pedigree brand).
For each Bti extract tested, 4 replicates per concentration were performed on 25 individuals of the 3rd instar larvae, which contained 150 ml of tap water with the bioinsecticide, according to the standard bioassay procedure described by the World Health Organization (2005). Five negative controls consisting of larvae exposed to tap water only were performed, and as a positive control (100% mortality), a primary standard was prepared from VectoBac® 3000 UTI/mg (Valent BioSciences Corp. Libertyville, IL). All bioassays were performed at room temperature (25–28 °C). Suspensions of each fermentation extract were prepared at 200 ppm in Erlenmeyer flasks. Several dilutions were then made to obtain final concentrations of each treatment (5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, and 0.001 mg/l).
Protein profile analysis
The protein profiles of Bt spore-crystal toxins were analyzed by SDS-PAGE. A total of 1 mg of crystal and spores from each Bti strain was solubilized in 100 μl of Nanopure water. They were fractionated by protein buffer (2-mercaptoethanol 5%, Tris-HCL 60 mM pH 6.8, glycerol 25%, SDS 2%, bromophenol blue 0.1%), heated (95 °C for 5 min), and then analyzed by sodium dodecyl sulfate 12% polyacrylamide gel electrophoresis (SDS-PAGE). Finally, proteins were visualized by Coomassie blue staining (Green and Sambrook 2012).
Results were tabulated according to concentrations, considering the number of live and dead larvae with 4 replications per treatment, performed in triplicate at 24 and 48 h. Values of 50% lethal concentration (LC50) as well as confidence limits were obtained by probit analysis (Finney 1971) Repeated measures ANOVA and Tukey-Kramer post hoc measurements were performed to compare the means among the different extract treatments under different conditions, using the NCSS statistical software (2019).