Activity of free and clay-bound insecticidal proteins from Bacillus thuringiensis subsp. israelensis against the mosquito Culex pipiens

Abstract

Bacillus thuringiensis subsp. israelensis produces parasporal insecticidal crystal proteins (ICPs) that have larvicidal activity against some members of the order Diptera, such as blackflies and mosquitoes. Hydrolysis of the ICPs in the larval gut results in four major proteins with a molecular mass of 27, 65, 128, and 135 kDa. Toxicity is caused by synergistic interaction between the 25-kDa protein (proteolytic product of the 27-kDa protein) and one or more of the higher-molecular-mass proteins. Equilibrium adsorption of the proteins on the clay minerals montmorillonite and kaolinite, which are homoionic to various cations, was rapid (<30 min for maximal adsorption), increased with protein concentration and then reached a plateau (68 to 96% of the proteins was adsorbed), was significantly lower on kaolinite than on montmorillonite, and was not significantly affected by the valence of the cation to which the clays were homoionic. Binding of the toxins decreased as the pH was increased from 6 to 11, and there was 35 to 66% more binding in phosphate buffer at pH 6 than in distilled water at pH 6 or 7.2. Only 2 to 12% of the adsorbed proteins was desorbed by two washes with water; additional washings desorbed no more toxins, indicating that they were tightly bound. Formation of clay-toxin complexes did not alter the structure of the proteins, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the equilibrium supernatants and desorption washes and by dot blot enzyme-linked immunosorbent assay of the complexes, which was confirmed by enhanced chemiluminescence Western blot analysis. Free and clay-bound toxins resulted in 85 to 100% mortality of the mosquito Culex pipiens. Persistence of the bound toxins in nonsterile water after 45 days was significantly greater (mortality of 63% +/- 12.7%) than that of the free toxins (mortality of 25% +/- 12.5%).

FIG. 1.

ECL Western blots of B. thuringiensis subsp. israelensis proteins subjected to different treatments. Free B. thuringiensis subsp. israelensis proteins (lanes 1 and 8), Ca-montmorillonite-protein complex (lanes 2, 4, and 6), and kaolinite-Ca-protein complex (lanes 3, 5, and 7) were used. Equilibrium adsorption supernatant of the protein complexes before (lanes 2 and 3) and after first (lanes 4 and 5) and second (lanes 6 and 7) washes of the equilibrium adsorption complexes were examined. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the blot.

FIG. 2.

Rate of adsorption of the toxins from B. thuringiensis subsp. israelensis on montmorillonite (M) and kaolinite (K) homoionic to calcium (Ca), magnesium (Mg), and aluminum (Al). The adsorption mixture contained 100 μg of clay and 250 μg of solubilized toxins in phosphate buffer (pH 6.0) in a total volume of 1 ml. Data are expressed as the means ± standard errors of the means (error bars).

FIG. 3.

Adsorption of the toxins from B. thuringiensis subsp. israelensis on montmorillonite (M) and kaolinite (K) homoionic to calcium (Ca), magnesium (Mg), or aluminum (Al) as a function of protein concentration. There was 100 μg of clay per ml of adsorption mixture. The contact time was 30 min. Data are expressed as the means ± standard errors of the means (error bars).

FIG. 4.

Adsorption of the toxins from B. thuringiensis subsp. israelensis on different concentrations of montmorillonite (M) homoionic to calcium (Ca) or magnesium (Mg). There was 250 μg of toxin per ml of adsorption mixture. The contact time was 30 min. Data are expressed as the means ± standard errors of the means.