Toxicity of bacillus thuringiensis subsp. kurstaki strain HD-1 against insect larvae

Abstract

B. thuringiensis subsp. kurstaki HD-1 contains two types of proteinaceous crystals, that is a bipyramidal crystal (M(,r) = 134 kDa), which is toxic to lepidopteran larvae, and a cuboidal crystal (M(r) = 65 kDa), which is toxic to dipteran larvae. In order to study the toxicities of these toxin, the purified crytals must be obtained. Vigorous shaking in 0.2 M NaCl was used to produce purified crystals containing impurities of 1.3% spores from cultures of B. thuringiensis subsp kurstaki HD-1. Bioassays of these crystals were done against Spodoptera exigua and Aedes aegypti larvae in parallel with the sample derived from cultures washing in distilled water and/or the sample derived from cultures washing in 1 M. NaCl. When S. exigua larvae were used, the sample derived from cultures washing in 1 M NaCl gave highest toxicity than others. This sample gave LC(,50) = 62 crystals/mm(,2) diet surface in first instar larvae to 1291 crystals/mm(2) diet surface in fifth instar larvae. While the purified crystals gave the least toxicity, it gave LC(,50) = 207 crystals/mm(2) diet surface against first instar larvae to 32,734 crystals/mm(2) diet surface in fifth instar larvae. When mosquito larvae were used to test all of these specimens the purified crystals was the most effective one when it was compared with the crude preparations. The LC(,50) was 3,682 crystals/ml against early instar larvae and 23,550 crystals/ml against late instar larvae. When the intact crystals were solubilized in 2% 2-mercaptoethanol at pH 10, the 134-kDa protein was recovered. After this protein was removed, the residue was solubilized in 0.1 M NaOH and the 65-kDa protein was obtained. The toxicity of these proteins were tested against third instar larvae of S. exigua and second instar larvae of Ae. aegypti. The 139-kDa protein could kill S. exigua larvae with 2 folds higher activity than that of the intact crystal. The 65-kDa protein could kill Ae. aegypti larvae with 10 folds higher activity than those of the intact crystals. However, this protei