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First published online September 23, 2003

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Recombinant bacteria for mosquito control

B. A. Federici^*, H.-W. Park, D. K. Bideshi, M. C. Wirth and J. J. Johnson

Department of Entomology, University of California Riverside, Riverside, CA 92521, USA

View larger version (14K): [in a new window]   Fig. 1. Toxicity of Bacillus sphaericus (strain 2362) alone and in combination with a recombinant strain of Bacillus thuringiensis that only produces Cyt1A against fourth instars of Aedes aegypti. The figure illustrates dose-response regression lines of technical powder combinations (B. sphaericus: B. thuringiensis) of different ratios of the two bacterial strains.

View larger version (45K): [in a new window]   Fig. 2. (A) Sporulating cell of Bacillus thuringiensis subsp. israelensis and (B) a typical parasporal body of this species showing individual toxin inclusions and their toxin composition. Sp, spore; E, exosporium; PB, parasporal body.

View larger version (81K): [in a new window]   Fig. 3. Binding of Bin and Cyt1A to midgut epithelial cells of Bin-resistant fourth instars of Culex quinquefasciatus five hours after treatment. (A) Phase-contrast micrograph of a posterior midgut epithelial cell of a Bin-resistant larva fed only Bin labeled with Oregon Green. (B) The same cell as in A viewed with an Oregon Green filter, showing that little or no Bin bound to the microvilli or entered the cell. (C-F) Micrographs of a single posterior midgut epithelial cell from a Bin-resistant larva fed a 1:1 mixture of Bin and Cyt1A. Bin was labeled with Oregon Green, and Cyt1A with Rhodamine Red-X. (C) Phase contrast without filter; (D) superimposed micrographs taken with Oregon Green and Rhodamine Red-X filters; (E) micrograph of the cell using Oregon Green filter showing Bin throughout the cytoplasm, with no Bin detectable bound to the microvilli. (F) Micrograph of the cell taken using Rhodamine Red-X filter showing Bin bound primarily to the microvilli. N, nucleus; bb, microvilli of the brush border.

View larger version (27K): [in a new window]   Fig. 4. Maps of recombinant plasmids and strategy for constructing a strain of Bacillus thuringiensis that produces Cyt1A, Cry11B and the Bacillus sphaericus 2362 binary toxin. (A) p45S1 containing cyt1A from B. thuringiensis subsp. israelensis and binary toxin gene from B. sphaericus 2362. (B) pPFT11Bs-CRP containing cry11B from B. thuringiensis subsp. jegathesan. Amp, ampicillin-resistant gene; Erm, erythromycin-resistant gene; Cm, chloramphenicol-resistant gene; cyt1A-p, cyt1A promoters; cry1Ac-p, cry1Ac promoters; E. c. ori, E. coli replication origin; B. t. ori, B. thuringiensis replication origin.

View larger version (61K): [in a new window]   Fig. 5. Phase-contrast micrograph of Bacillus thuringiensis subsp. israelensis strain 4Q7/p45S1-11B, which produces crystals of Cry11B, Cyt1A and the Bacillus sphaericus binary toxin.

View larger version (89K): [in a new window]   Fig. 6. Analysis of endotoxin content in recombinant strains of Bacillus thuringiensis. M, molecular mass marker; lane 1, B. thuringiensis subsp. israelensis 4Q7 producing Bs binary toxin and Cyt1A (4Q7/p45S1); lane 2, B. thuringiensis subsp. israelensis 4Q7 producing Cry11B (4Q7/pPFT11Bs-CRP); lane 3, B. thuringiensis subsp. israelensis 4Q7 producing Cry11B, Cyt1A and Bs binary toxin (4Q7/p45S1-11B).

View larger version (60K): [in a new window]   Fig. 7. Recombinant strains of Bacillus thuringiensis subsp. israelensis (Bti) that produce the Bacillus sphaericus (Bs) 2362 binary toxin. (A) Acrystalliferous strain transformed with a plasmid that produces the Bs 2362 binary toxin using cyt1A promoters and the STAB/SD mRNA stabilizing sequence. (B) IPS-82 transformed with the same plasmid. A large crystal of the Bs binary toxin and a typical Bti crystal are obvious in the sporulated cell. (C) SDS-PAGE analysis of IPS-82 (lane Bti) and IPS-82 producing the Bti proteins and the Bs binary toxin (lane Bti/BsB).