MOSQUITO BIOCONTROL VIA CLONING THE BTI TOXIN GENE

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Control of many human diseases is based on control of their mosquito vectors. Biological control programs have been made possible by the recent discovery of Bacillus thuringiensis var. israelensis (Bti) which produces a protein crystal toxic to mosquito larvae. The goals of this project are to: 1) identify which component of the crystal constitutes the actual toxin; 2) characterize the plasmid coding for the toxin; 3) clone the toxin gene into bacteria more suitable for long term mosquito control. Bti has a mammalian and a mosquito toxin within the nine proteins found in purified crystals. No information is yet available regarding the identity of these toxins or indeed whether they are separate toxins. The 9 proteins will be separated by nondenaturing gel electrophoresis and tested for larval toxicity and red blood cell lysis. If the 2 toxins are distinct, it should be possible to obtain mutants or recombinants that produce only the mosquito toxin, thus eliminating potential health hazard incurred by the widespread use of Bti. Both the 4.5 and 72 Md plasmids of Bti have been reported to be the site of the toxin gene. Plasmids will be separated electrophoretically, eluted, and transformed into protoplasts of a plasmid-free Bti. Transformants will be detected by hybridization and tested for toxin gene expression by immunoassay and larval toxicity. If the toxin plasmid is large, it will be digested with restriction enzymes and then ligated until it has been reduced to a more manageable size. The Bti toxin plasmid will then be cloned into 2 more suitable bacteria: 1) Baccillus sphaericus (Bs). Bti does not survive well in nature while the ecological persistence of Bs, also a mosquito pathogen, has been well documented. Accordingly, the Bti toxin gene will be ligated to the pUB 110 plasmid which stably transforms Bs. Hopefully, the recombinant will produce the Bti toxin and maintain the ecological fitness of Bs. 2) The cyanobacterium Anacystis nidulans (An). Bti is less effective against the Anopheles malaria mosquitoes because their larvae are surface feeders while the Bti toxin particles rapidly settle to the bottom. Accordingly, the Bti toxin gene will be ligated to the pUC 104 plasmid which stably transforms An. Gas vacuole-containing strains of An will float at the surface and remain available for Anopheles larvae. Additionally, An, unlike Bti, is adapted for survival in the brackish waters where 75-80% of the U.S. mosquito abatement programs are located.