Article Figures & Data
Figures
- 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.
- 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).
- 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.
- 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.
- 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
- Table 1.
Larvicidal activity of B. sphaericus recombinant strains against C. pipiens subsp. pipiens larvae
B. sphaericus strain Suspension (all sonicated)a Larvicidal activityb LC50c LC90 2297 (parental) Spore pelletd 2.1 ± 0.4 12.6 ± 8.8 Purified inclusion bodies 8 ± 0.05 15.5 ± 1.3 2297(pMK3) Washed FWC 31 ± 2.5 59.7 ± 7.3 2297(pcyt1Ab1) Washed FWC 73.3 ± 17.3 181.3 ± 75.3 Spore pellet 7.1 ± 0.9 18.6 ± 2.8 Purified inclusion bodies 72.4 ± 4.8 306 ± 222 2362 Washed FWC 15.9 ± 1.9 28.6 ± 4.4 2362(pcyt1Ab1) Washed FWC 28.5 ± 4.5 64.5 ± 0.4 Iab872(pcyt1Ab1) Washed FWC 27.9e 194 Iab872(pcyt1Ab1) Spore pellet 5.8 ± 3.5 27.4 ± 19 Iab872(pMK3) Spore pellet 2.6e 7.1 2297 asporulated(pMK3) Washed FWC 95.8 ± 45 μg/ml 526.9 ± 299 μg/ml 2297 asporulated(pcyt1Ab1) Washed FWC 109 ± 29.4 μg/ml 360.0 ± 180 μg/ml B. thuringiensis SPL 407(pcyt1Ab1) Purified crystals 5.7 ± 3.1 μg/ml 29.7 ± 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 strain Strain (FWC)a Larvicidal activity (μg/ml) LC50b LC90 C. quinquefasciatus 2297 (parental) 134.0 ± 6.7c 383.6 ± 28.4 2297(pcyt1Ab1) 6.0 ± 2.8 16.4 ± 8.8 C. pipiens subsp. pipiens 2297 (parental) 193.2 ± 46 611.4 ± 42 2297(pcyt1Ab1) 14.1 ± 7.4 38.8 ± 18 2362 (parental) 66.8 ± 24.9 115.2 ± 65 2362(pcyt1Ab1) 45.8 ± 7.8 122.3 ± 67 2297 asporulated(pMK3) 595d 7,806 2297 asporulated(pcyt1Ab1) 215 ± 78 1,346 ± 944 B. thuringiensis SPL 407 (pcyt1Ab1)e 43.2 ± 21 416 ± 219
