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Invertebrate Microbiology

The Introduction into Bacillus sphaericus of the Bacillus thuringiensis subsp. medellin cyt1Ab1 Gene Results in Higher Susceptibility of Resistant Mosquito Larva Populations to B. sphaericus

I. Thiéry, S. Hamon, A. Delécluse, S. Orduz
I. Thiéry
Unité des Bactéries Entomopathogènes, Institut Pasteur, 75724 Paris Cedex 15, France and
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S. Hamon
Unité des Bactéries Entomopathogènes, Institut Pasteur, 75724 Paris Cedex 15, France and
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A. Delécluse
Unité des Bactéries Entomopathogènes, Institut Pasteur, 75724 Paris Cedex 15, France and
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S. Orduz
CIB Biological Control Unit, Medellin, Columbia
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DOI: 10.1128/AEM.64.10.3910-3916.1998
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  • Fig. 1.
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    Fig. 1.

    Protein analysis in wild-type and recombinantB. sphaericus strains. Fifteen micrograms of protein of the washed FWC was subjected to electrophoresis on an SDS-PAGE gel (12% polyacrylamide) followed by staining with Coomassie brilliant blue. Lanes: A, purified inclusion bodies of Cyt1Ab1 from B. thuringiensisSPL407(pcyt1Ab1); B, wild-type strain 2297; C, recombinant nonsporulating mutant strain 2297(pcyt1Ab1); D, recombinant strain 2297(pMK3); E, mutant asporulated strain 2297; F, recombinant strain 2297(pcyt1Ab1); G, recombinant strain Iab872(pcyt1Ab1); H, recombinant strain Iab872(pMK3); I, recombinant strain 2362(pcyt1Ab1); MW, low-molecular-mass kit standard protein markers from Pharmacia.

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    Fig. 2.

    Western blot of 15 μg of protein from FWC or inclusion bodies of recombinant and wild-type B. sphaericus strains and of 5 μg of purified Cyt1Ab1 inclusion bodies from B. thuringiensisSPL407(pcyt1Ab1). The filter was incubated with antiserum (dilution 1/2,000) raised against Cyt1Ab1 protein purified from B. thuringiensisSPL407(pcyt1Ab1). Lanes: 1, 2297(pcyt1Ab1); 2, wild-type strain 2297; 3, nonsporulating mutant 2297(pcyt1Ab1); 4, nonsporulating mutant 2297(pMK3); 5, purified inclusion bodies of 2297(pcyt1Ab1); 6 and 7, purified inclusion bodies of nonsporulating mutant 2297(pcyt1Ab1) with 15 and 30 μg of protein, respectively; 8, Iab872(pcyt1Ab1); 9, 2362(pMK3); 10, 2362(pcyt1Ab1); 11, purified Cyt1Ab1 inclusion bodies; 12, wild-type strain Iab872; 13, Iab872(pMK3); 14, Iab872(pcyt1Ab1).

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    Fig. 3.

    Protein analysis after sonication of FWC, inclusion body, and spore-pellet (after ultracentrifugation) suspensions from wild-type and recombinant B. sphaericus 2297 strains. Fifteen micrograms of protein per well was subjected to SDS-PAGE (12% polyacrylamide). Lanes: 1 to 3, wild-type strain 2297 (lane 1, FWC; lane 2, inclusion body suspension; lane 3, spore pellet); 4 and 5, mutant nonsporulating strain 2297(pMK3) (lane 4, FWC; lane 5, inclusion body suspension); 6 to 8, recombinant 2297(pcyt1Ab1) (lane 6, FWC; lane 7, inclusion body suspension; lane 8, spore pellet); 9 and 10, mutant nonsporulating strain 2297(pcyt1Ab1) (lane 9, FWC; lane 10, inclusion body suspension); MW, standard protein markers from Pharmacia low-molecular-mass kit.

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    Fig. 4.

    Electron micrographs of ultrathin sections of two crystalline inclusions of purified Cyt1Ab1 crystals from B. thuringiensis SPL407(pcyt1Ab1) (A) and inclusion bodies with various dense areas (B). (C) Cuboid crystalline inclusion body. Bar, 200 nm; Cry, crystal. The arrows indicate the crystalline lattice.

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    Fig. 5.

    Electron micrographs of ultrathin sections ofB. sphaericus 2297. (A) Spore crystal of recombinant B. sphaericus2297(pcyt1Ab1) after cell lysis. (B) Spore crystal of wild-type strain 2297. (C) Purified inclusion bodies from recombinant strain 2297(pMK3). Bar, 200 nm. S, spore; Cry, crystal.

Tables

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  • Table 1.

    Larvicidal activity of B. sphaericus recombinant strains against C. pipiens subsp. pipiens larvae

    B. sphaericus strainSuspension (all sonicated)aLarvicidal activityb
    LC50cLC90
    2297 (parental)Spore pelletd2.1 ± 0.412.6 ± 8.8
    Purified inclusion bodies8 ± 0.0515.5 ± 1.3
    2297(pMK3)Washed FWC31 ± 2.559.7 ± 7.3
    2297(pcyt1Ab1)Washed FWC73.3 ± 17.3181.3 ± 75.3
    Spore pellet7.1 ± 0.918.6 ± 2.8
    Purified inclusion bodies72.4 ± 4.8306 ± 222
    2362Washed FWC15.9 ± 1.928.6 ± 4.4
    2362(pcyt1Ab1)Washed FWC28.5 ± 4.564.5 ± 0.4
    Iab872(pcyt1Ab1)Washed FWC27.9e194  
    Iab872(pcyt1Ab1)Spore pellet5.8 ± 3.527.4 ± 19
    Iab872(pMK3)Spore pellet 2.6e 7.1
    2297 asporulated(pMK3)Washed FWC95.8 ± 45 μg/ml526.9 ± 299 μg/ml
    2297 asporulated(pcyt1Ab1)Washed FWC109 ± 29.4 μg/ml360.0 ± 180 μg/ml
    B. thuringiensis SPL 407(pcyt1Ab1)Purified crystals5.7 ± 3.1 μg/ml29.7 ± 17.3 μg/ml
    • ↵a All B. sphaericus suspensions were subjected to sonication two times for 10 min each before the experiments.

    • ↵b Unless otherwise noted, values are in nanograms per milliliter.

    • ↵c Lethal concentrations after 48 h of larval exposure.

    • ↵d Spores pelleted after ultracentrifugation.

    • ↵e The result of one experiment.

  • Table 2.

    Larvicidal activity of B. sphaericus recombinant strains against C. pipiens subsp. pipiens (SPHAE) and C. quinquefasciatus (GeoR) larval populations resistant toB. sphaericus

    Mosquito strainStrain (FWC)aLarvicidal activity (μg/ml)
    LC50bLC90
    C. quinquefasciatus2297 (parental)134.0 ± 6.7c383.6 ± 28.4
    2297(pcyt1Ab1)6.0 ± 2.816.4 ± 8.8
    C. pipiens subsp. pipiens2297 (parental)193.2 ± 46611.4 ± 42
    2297(pcyt1Ab1)14.1 ± 7.438.8 ± 18
    2362 (parental)66.8 ± 24.9115.2 ± 65
    2362(pcyt1Ab1)45.8 ± 7.8122.3 ± 67
    2297 asporulated(pMK3)595d7,806
    2297 asporulated(pcyt1Ab1)215 ± 781,346 ± 944
    B. thuringiensis SPL 407 (pcyt1Ab1)e43.2 ± 21416 ± 219
    • ↵a All strains are B. sphaericus, except as noted.

    • ↵b LC after 48 h of larval exposure ± standard error; mean of three experiments.

    • ↵c Result of two experiments.

    • ↵d Result of one experiment.

    • ↵e Purified crystals of Cyt1Ab1 protein.

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The Introduction into Bacillus sphaericus of the Bacillus thuringiensis subsp. medellin cyt1Ab1 Gene Results in Higher Susceptibility of Resistant Mosquito Larva Populations to B. sphaericus
I. Thiéry, S. Hamon, A. Delécluse, S. Orduz
Applied and Environmental Microbiology Oct 1998, 64 (10) 3910-3916; DOI: 10.1128/AEM.64.10.3910-3916.1998

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The Introduction into Bacillus sphaericus of the Bacillus thuringiensis subsp. medellin cyt1Ab1 Gene Results in Higher Susceptibility of Resistant Mosquito Larva Populations to B. sphaericus
I. Thiéry, S. Hamon, A. Delécluse, S. Orduz
Applied and Environmental Microbiology Oct 1998, 64 (10) 3910-3916; DOI: 10.1128/AEM.64.10.3910-3916.1998
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KEYWORDS

Aedes
Bacillus
Bacillus thuringiensis
Bacterial Proteins
Bacterial Toxins
Culex
Endotoxins

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