Pergamon 0305-1978(95)00030-5 Biochemical ~tematics and Ecology, Vol. 23, No. 5, pp. 461-468.1995 Copyright 0 1995 Elsetier Science Ltd Printed in Great Britain. All tights reserved 03051978/95 $9.50+0.00 Highly Mosquitocidal Isolates of Bacillus thuringiensis subspecies kenyae and entomocidus from Mexico JOEL LbPEZ-MEZA,” BRIAN A. FEDERICl,t WILLIAM J. POEHNER,t ANA MARTINEZ-CASTILLO” and JORGE E. IBARRAY l Departamento de Biotecnologla y Bioquimica, Centro de Investigaci6n y de Estudios Avanzados del I. P. N., Apdo. Postal 629, Irapuato, Gto., Mexico; tDepartment of Entomology and Interdepartmental Graduate Program in Genetics, University of California, Riverside, CA 92521, U.S.A. Key Word Index--Bacillus thuringiensis; mosquitocidal isolates; biochemical systematics; serotype/sub- species; ecological distribution, in grain, in soil; geographical occurrence, in Mexico. Abstract-Isolates of Bacillus thuringiensis ssp. kenyae and B. thuringiensis ssp. entomocidus that produce highly mosquitocidal parasporal bodies similar to those produced by B. tburingiensis ssp. israelensis have been obtained from, respectively, sorghum dust and soil samples in Mexico. The parasporal bodies of both isolates are spherical, contain several inclusions enveloped in a fibrous matrix, and are composed of pro- teins of 27, 65, 128 and 134 kDa which cross-react with polyclonal antisera made against the parasporal body of B. thudngiensis ssp. israelensis. Southern hybridization analyses of the plasmid complements of these new isolates using the cry/VD gene as a probe show that this gene is located on a plasmid of about 110 kb, the same size as the plasmid that bears the CM, cry/VA, cty/VB and cry/\/D genes in B. thur- ingiensis ssp. israelensis. These results demonstrate that the 110 kb plasmid bearing these insecticidal genes occurs in ecologically diverse habitats as well as in different subspecies of B. tburingiensis. More- over, the latter finding in conjunction with previous studies shows further that the serotype/subspecies designation used to classify isolates of this bacterium is not a definitive indicator of the insecticidal spec- trum of activity. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Introduction The first isolate Bad/us thuringiensis reported with significant toxicity to the larvae of mosquitoes, blackflies and other nematocerous dipterans was the ONR 60A isolate discovered in 1976 at a mosquito breeding site in the Negev Desert of Israel (Goldberg and Margalit, 1977). This isolate was subsequently shown to belong to a new subspecies, B. thuringiensis ssp. israelensis (serotype H14), by de Barjac (1978). Several years later, another mosquitocidal isolate, the PG-14 isolate of B. thuringiensis ssp. momsoni (serotype H8a8b), was discovered at a mosquito breeding site in the Philippines (Padua et d, 1980). Subsequent studies of these isolates showed that their insecticidal properties are due to a spherical parasporal body produced during sporulation which is similar but not identical in structure and protein composition in both isolates. The parasporal body produced by B. thuringensis ssp. israelensis contains three inclusion types and four proteins, the CytA (27 kDa), CrylVA (128 kDa), CrylVB (134 kDa), and CrylVD (72 kDa) proteins (Ibarra and Federici, 1986a), whereas the isolate of B. thuringiensis ssp. morrisoni contains four inclusion types and the same proteins as B. thuringiensis ssp. israelensis as well as an additional protein of 144 kDa (Ibarra and Federici, 1986b). In plasmid curing studies and gene mapping experiments, it was shown that the genes encoding the CytA and CrylV proteins in both isolates are located on a large plasmid of about 110 kb, whereas the gene for the 144-kDa protein in the B. *Author to whom correspondence should be addressed. (Received 20 December 1994; accepted 15 May 1995) 461