SYNERGISM OF MOSQUITOCIDAL TOXICITY BETWEEN CYTA AND CRYIVD PROTEINS USING INCLUSIONS PRODUCED FROM CLONED GENES OF BACILLUS-THURINGIENSIS

The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp. israelensis and the PG-14 isolate of B. thuringiensis subsp. morrisoni is at least 20-fold greater than any of the four mosquitocidal proteins of which it is composed (CytA, CryIVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CryIVD toxins were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis cells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bioassays against first instars of Aedes aegypti. The LC(50) for the CytA inclusion was 60 ng ml(-1), whereas the LC(50) for the CryIVD was 85 ng ml(-1). In comparison, the LC(50)s for different combinations of CytA and CryIVD inclusions ranged from 12-15 ng ml(-1), 4-5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild-type parasporal bodies of B. thuringiensis subspp. israelensis and morrisoni is due to synergism among three or four of their major proteins.