Molecular cloning of the 130-kilodalton mosquitocidal delta-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus

A 3.7-kilobase (kb) XbaI fragment harboring the cryIVB gene (L. Thorne, F. Garduno, T. Thompson, D. Decker, M. A. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock, J. Bacteriol. 166:801-811, 1986) which encoded a 130-kilodalton (kDa) mosquitocidal toxin from a 110-kb plasmid of Bacillus thuringiensis subsp. israelensis 4Q2-72 was cloned into pUC12 and transformed into Escherichia coli. The clone with a recombinant plasmid (designated pBT8) was toxic to Aedes aegypti larvae. The fragment (3.7 kb) was ligated into pBC16 (tetracycline resistant [Tcr]) and transformed by the method of protoplast transformation into Bacillus sphaericus 1593 and 2362, which were highly toxic to Anopheles and Culex mosquito larvae but less toxic to Aedes larvae. After cell regeneration on regeneration medium, the Tcr plasmids from transformants (pBTC1) of both strains of B. sphaericus were prepared and analyzed. The 3.7-kb XbaI fragment from the B. thuringiensis subsp. israelensis plasmid was shown to be present by agarose gel electrophoresis and Southern blot hybridization. In addition, B. sphaericus transformants produced a 130-kDa mosquitocidal toxin which was detected by Western (immuno-) blot analysis with antibody prepared against B. thuringiensis subsp. israelensis 130-kDa mosquitocidal toxin. The 50% lethal concentrations of the transformants of strains 1593 and 2362 against A. aegypti larvae were 2.7 X 10(2) and 5.7 X 10(2) cells per ml, respectively. This level of toxicity was comparable to the 50% lethal concentration of B. thuringiensis subsp. israelensis but much higher than that of B. sphaericus 1593 and 2362 (4.7 X 10(4) cells per ml) against A. aegypti larvae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of the 130-kilodalton mosquitocidal delta-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus.

A 3.7-kilobase (kb) XbaI fragment harboring the cryIVB gene (L. Thorne, F. Garduno, T. Thompson, D. Decker, M. A. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock, J. Bacteriol. 166:801-811, 1986) which encoded a 130-kilodalton (kDa) mosquitocidal toxin from a 110-kb plasmid of Bacillus thuringiensis subsp. israelensis 4Q2-72 was cloned into pUC12 and transformed into Escherichia coli. The clone with a recombinant plasmid (designated pBT8) was toxic to Aedes aegypti larvae. The fragment (3.7 kb) was ligated into pBC16 (tetracycline resistant [Tcr]) and transformed by the method of protoplast transformation into Bacillus sphaericus 1593 and 2362, which were highly toxic to Anopheles and Culex mosquito larvae but less toxic to Aedes larvae. After cell regeneration on regeneration medium, the Tcr plasmids from transformants (pBTC1) of both strains of B. sphaericus were prepared and analyzed. The 3.7-kb XbaI fragment from the B. thuringiensis subsp. israelensis plasmid was shown to be present by agarose gel electrophoresis and Southern blot hybridization. In addition, B. sphaericus transformants produced a 130-kDa mosquitocidal toxin which was detected by Western (immuno-) blot analysis with antibody prepared against B. thuringiensis subsp. israelensis 130-kDa mosquitocidal toxin. The 50% lethal concentrations of the transformants of strains 1593 and 2362 against A. aegypti larvae were 2.7 X 10(2) and 5.7 X 10(2) cells per ml, respectively. This level of toxicity was comparable to the 50% lethal concentration of B. thuringiensis subsp. israelensis but much higher than that of B. sphaericus 1593 and 2362 (4.7 X 10(4) cells per ml) against A. aegypti larvae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant strain of Bacillus thuringiensis producing Cyt1A,Cry11B,and the Bacillus sphaericus binary toxin

A novel recombinant Bacillus thuringiensis subsp. israelensis strain that produces the B. sphaericus binary toxin, Cyt1Aa, and Cry11Ba is described. The toxicity of this strain (50% lethal concentration [LC(50)] = 1.7 ng/ml) against fourth-instar Culex quinquefasciatus was higher than that of B. thuringiensis subsp. israelensis IPS-82 (LC(50) = 7.9 ng/ml) or B. sphaericus 2362 (LC(50) = 12.6 ng/ml).     

Clearance of Bacillus sphaericus and Bacillus thuringiensis ssp. israelensis from mammals

The maximum recovery period following topical ocular instillation and intraperitoneal injection of two preparations of Bacillus thuringiensis ssp. israelensis de Barjac and two preparations of Bacillus sphaericus 2362 was evaluated in rabbits and mice. B. sphaericus 2362 persisted for 8 wk after administration to the conjunctival cul-de-sac of rabbits; B. thuringiensis ssp. israelensis persisted for 1 wk. Infection was not evident, but both entomopathogens were recovered from flushed and unflushed eyes. High doses of B. sphaericus 2362 (greater than or equal to 10(8) colony-forming units) were toxic to CD-1 mice, and the toxic factor was heat stable. Injection of 10(7) colony-forming units of B. sphaericus 2362 resulted in clearance from the spleens of euthymic and athymic mice. Recovery occurred up to 67 d after injection. Mice failed to remove one preparation of B. thuringiensis ssp. israelensis from their spleen, and a constant number of colony-forming units were recovered for 80 d. B. sphaericus 2362 and B. thuringiensis ssp. israelensis were recovered from heart blood; their disappearance from heart blood coincided with their clearance from the spleen. There was no evidence that either organism was infectious. We conclude that these organisms can be used safely in environments where human exposure might occur.     

Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression

The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.     

Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression.

The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.     

Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes.

Toxicity of Bacillus thuringiensis subsp. israelensis (ONR-60A/WHO 1897) parasporal crystals to three medically important mosquito larvae is described. The numbers of larvae killed are in relation to crystal dry weight. The crystals are lethally toxic to Aedes aegypti Linnaeus (mean 50% lethal concentration [LC50] = 1.9 x 10(-4) micrograms/ml), Culex pipiens var. quinquefasciatus Say (LC50 = 3.7 x 10(-4) micrograms/ml), and Anopheles albimanus Wiedemann (LC50 = 8.0 x 10(-3) micrograms/ml). Purfied crystals of B. thuringiensis subsp. kurstaki, which are toxic to lepidopteran insects, are ineffective against the mosquito larvae. Likewise, B. thuringiensis subsp. israelensis parasporal crystals are not efficacious for larvae of the lepidopteran, Manduca sexta.     

Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes.

Toxicity of Bacillus thuringiensis subsp. israelensis (ONR-60A/WHO 1897) parasporal crystals to three medically important mosquito larvae is described. The numbers of larvae killed are in relation to crystal dry weight. The crystals are lethally toxic to Aedes aegypti Linnaeus (mean 50% lethal concentration [LC50] = 1.9 x 10(-4) micrograms/ml), Culex pipiens var. quinquefasciatus Say (LC50 = 3.7 x 10(-4) micrograms/ml), and Anopheles albimanus Wiedemann (LC50 = 8.0 x 10(-3) micrograms/ml). Purfied crystals of B. thuringiensis subsp. kurstaki, which are toxic to lepidopteran insects, are ineffective against the mosquito larvae. Likewise, B. thuringiensis subsp. israelensis parasporal crystals are not efficacious for larvae of the lepidopteran, Manduca sexta.     

Co-expression of Bacillus thuringiensis Cry4Ba and Cyt2Aa2 in Escherichia coli revealed high synergism against Aedes aegypti and Culex quinquefasciatus larvae

Cry4Ba is a delta-endotoxin produced by Bacillus thuringiensis subsp. israelensis and Cyt2Aa2 is a cytolytic delta-endotoxin produced by B. thuringiensis subsp. darmstadiensis. Cry4Ba produced in Escherichia coli was toxic to Aedes aegypti larvae (LC(50)=140 ng ml(-1)) but virtually inactive to Culex quinquefasciatus larvae. Cyt2Aa2 expressed in E. coli exhibited moderate activity against A. aegypti and C. quinquefasciatus larvae with LC(50) values of 350 and 250 ng ml(-1), respectively. Co-expression of both toxins in E. coli dramatically increased toxicity to both A. aegypti andC. quinquefasciatus larvae (LC(50)=7 and 20 ng ml(-1), respectively). This is the first report to demonstrate that Cry4Ba and Cyt2Aa2 have high synergistic activity against C. quinquefasciatus larvae.     

A new black fly isolate of Bacillus thuringiensis autoagglutinating strain highly toxic to Simulium pertinax (Kollar) (Diptera, Simuliidae) larvae

Formulations containing the entomopathogenic Bacillus thuringiensis serovar israelensis strain IPS-82 has been widely applied for mosquito control around the world. Strain IPS-82 is highly active against Aedes aegypti but less active against other well-known vectors such as Culex quinquefasciatus and Simulium spp. larvae. Eighteen strains of B. thuringiensis were isolated from Simulium pertinax larvae naturally occurring in rivers of Southeast Brazil with one demonstrating special toxic effects. Simulated field tests against S. pertinax larvae showed that the native Brazilian autoagglutinating B. thuringiensis (LFB-FIOCRUZ 1035) has an LC50 at least 25 times lower than the standard IPS-82 strain. The same bacterial preparation was also tested against Ae. aegypti larvae in laboratory trials and the LC50 values obtained with LFB-FIOCRUZ 1035 were at least three times lower than the one for the IPS 82 strain. The results indicate that this strain is more toxic than the standard B. thuringiensis serovar israelensis (H14) in the two Dipteran species tested. It is noteworthy that differences between LC50 values were more pronounced in S. pertinax larvae, the source of the original isolation.     

Transfer and expression of the cryIVB and cryIVD genes of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus 2297

Abstract The genes encoding the CryIVB and CryIVD crystal polypeptides of B. thuringiensis subsp. israelensis were cloned indepently on a stable shuttle vector, and transfered into B. sphaericus 2297. Recombinant cells expressed the B. thuringiensis toxins during sporulation and were shown to be toxic to Aedes aegypti fourth instar larvae, whereas the parental strain was not.     

Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae)

Bacillus sphaericus is a mosquitocidal bacterium recently developed as a commercial larvicide that is used worldwide to control pestiferous and vector mosquitoes. Whereas B. sphaericus is highly active against larvae of Culex and Anopheles mosquitoes, it is virtually nontoxic to Aedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A from Bacillus thuringiensis subsp. israelensis to enhance the toxicity of B. sphaericus toward A. aegypti. Various combinations of these two materials were evaluated, and all were highly toxic. A ratio of 10:1 of B. sphaericus to Cyt1A was 3, 600-fold more toxic to A. aegypti than B. sphaericus alone. Statistical analysis showed this high activity was due to synergism between the Cyt1A toxin and B. sphaericus. These results suggest that Cyt1A could be useful in expanding the host range of B. sphaericus.     

The fate of Bacillus thuringiensis var. israelensis in B. thuringiensis var. israelensis-killed pupae of Aedes aegypti

Carcasses of mosquito larvae killed by Bacillus thuringiensis var. israelensis allow its complete growth cycle (germination, vegetative growth, and sporulation), thus becoming toxic themselves to scavenging larvae. In this study, we demonstrate that the bacterium is capable of inducing death of Aedes aegypti pupae and of recycling in the resulting carcasses. B. thuringiensis var. israelensis-killed pupae were obtained by treating 40-hr-old synchronized fourth instar larvae with a low dose of spores (8000/ml). The fraction of dead pupae was reduced by higher or lower spore concentrations as well as by treating younger or older larval populations (both fourth instar): Increased proportions of dead larvae were obtained at higher concentration or by earlier treatment, whereas lower concentrations or later treatment resulted in more living pupae. Multiplication of B. thuringiensis var. israelensis is shown to occur in the carcasses of dead pupae. The number of spores in each pupal carcass followed a similar kinetic as in larval carcasses, but the final yield was about 10-fold higher, apparently reflecting the difference in dry weight between the two mosquito developmental stages (426 micrograms vs 83 micrograms, respectively). The specific larvicidal activity in a homogenized dead pupa was similar to that of B. thuringiensis var. israelensis powder, LC50 of about 600 spores/ml.     

Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis.

Integrative plasmids were constructed to enable integration of foreign DNA into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Integration of the aphA3 kanamycin resistance gene by a two-step procedure demonstrated that this strategy was applicable with antibiotic resistance selection. Hybridization experiments evidenced two copies of the operon encoding the binary toxin from B. sphaericus in the recipient strain. The Bacillus thuringiensis subsp. israelensis cry11Aal gene (referred to as cry11A), encoding a delta-endotoxin with toxicity against Culex, Aedes, and Anopheles larvae, was integrated either by a single crossover event [strain 2297 (::pHT5601), harboring the entire recombinant plasmid] or by two successive crossover events [strain 2297 (::cry11A)]. The level of the Cry11A production in B. sphaericus was high; two crystalline inclusions were produced in strain 2297 (::pHT5601). Synthesis of the Cry11A toxin conferred toxicity to the recombinant strains against Aedes aegypti larvae, for which the parental strain was not toxic. Interestingly, the level of larvicidal activity of strain 2297 (::pHT5601) against Anopheles stephensi was as high as that of B. thuringiensis subsp. israelensis and suggested synergy between the B. thuringiensis and B. sphaericus toxins. The toxicities of parental and recombinant B. sphaericus strains against Culex quinquefasciatus were similar, but the recombinant strains killed the larvae more rapidly. The production of the Cry11A toxin in B. sphaericus also partially restored toxicity for C. quinquefasciatus larvae from a population resistant to B. sphaericus 1593. In vivo recombination therefore appears to be a promising approach to the creation of new B. sphaericus strains for vector control.     

Mtx toxins synergize Bacillus sphaericus and Cry11Aa against susceptible and insecticide-resistant Culex quinquefasciatus larvae

Two mosquitocidal toxins (Mtx) of Bacillus sphaericus, which are produced during vegetative growth, were investigated for their potential to increase toxicity and reduce the expression of insecticide resistance through their interactions with other mosquitocidal proteins. Mtx-1 and Mtx-2 were fused with glutathione S-transferase and produced in Escherichia coli, after which lyophilized powders of these fusions were assayed against Culex quinquefasciatus larvae. Both Mtx proteins showed a high level of activity against susceptible C. quinquefasciatus mosquitoes, with 50% lethal concentrations (LC(50)) of Mtx-1 and Mtx-2 of 0.246 and 4.13 microg/ml, respectively. The LC(50)s were 0.406 to 0.430 microg/ml when Mtx-1 or Mtx-2 was mixed with B. sphaericus, and synergy improved activity and reduced resistance levels. When the proteins were combined with a recombinant Bacillus thuringiensis strain that produces Cry11Aa, the mixtures were highly active against Cry11A-resistant larvae and resistance was also reduced. The mixture of two Mtx toxins and B. sphaericus was 10 times more active against susceptible mosquitoes than B. sphaericus alone, demonstrating the influence of relatively low concentrations of these toxins. These results show that, similar to Cyt toxins from B. thuringiensis subsp. israelensis, Mtx toxins can increase the toxicity of other mosquitocidal proteins and may be useful for both increasing the activity of commercial bacterial larvicides and managing potential resistance to these substances among mosquito populations.     

Proteolysis in the gut of mosquito larvae results in further activation of the Bacillus sphaericus toxin.

Gut proteases from the larvae of the mosquito Culex pipiens convert the 43-kilodalton (kDa) toxin from Bacillus sphaericus 2362 to a 40-kDa peptide. The 50% lethal concentration of this peptide for tissue culture-grown cells of Culex quinquefasciatus was 1.0 microgram/ml (as determined by the intracellular ATP assay), 54-fold less than that of the 43-kDa peptide. Gut proteases from Anopheles gambiae and Aedes aegypti, as well as bovine pancreatic trypsin, also converted the 43-kDa protein to a 40-kDa peptide which was indistinguishable from the peptide formed by the proteases from C. pipiens with respect to its toxicity to tissue culture-grown cells of C. quinquefasciatus. Evidence for the in vivo conversion of the 43-kDa protein to the 40-kDa peptide was also obtained from experiments in which larvae of C. pipiens, Anopheles gambiae, and Aedes aegypti were fed crystals from B. sphaericus 2362. By using the exclusion of trypan blue as an indication of cell viability, it was shown that chitobiose, chitotriose, N-acetylmuramic acid, and N-acetylneuraminic acid decreased the toxicity of the 40-kDa peptide (from 100 to 50% mortality at about 10 mM concentrations of these sugars). Muramic acid, N-acetylgalactosamine, and N-acetylglucosamine were less effective, while several sugars had no effect, suggesting that the 40-kDa toxin binds to specific receptors on the cell membrane. The 40-kDa protein was less toxic to tissue culture-grown cells of Anopheles gambiae and Aedes dorsalis, and the same sugars which reduced the toxicity for cells of C. quinquefasciatus were also effective in reduction of toxicity for these cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolysis in the gut of mosquito larvae results in further activation of the Bacillus sphaericus toxin

Gut proteases from the larvae of the mosquito Culex pipiens convert the 43-kilodalton (kDa) toxin from Bacillus sphaericus 2362 to a 40-kDa peptide. The 50% lethal concentration of this peptide for tissue culture-grown cells of Culex quinquefasciatus was 1.0 microgram/ml (as determined by the intracellular ATP assay), 54-fold less than that of the 43-kDa peptide. Gut proteases from Anopheles gambiae and Aedes aegypti, as well as bovine pancreatic trypsin, also converted the 43-kDa protein to a 40-kDa peptide which was indistinguishable from the peptide formed by the proteases from C. pipiens with respect to its toxicity to tissue culture-grown cells of C. quinquefasciatus. Evidence for the in vivo conversion of the 43-kDa protein to the 40-kDa peptide was also obtained from experiments in which larvae of C. pipiens, Anopheles gambiae, and Aedes aegypti were fed crystals from B. sphaericus 2362. By using the exclusion of trypan blue as an indication of cell viability, it was shown that chitobiose, chitotriose, N-acetylmuramic acid, and N-acetylneuraminic acid decreased the toxicity of the 40-kDa peptide (from 100 to 50% mortality at about 10 mM concentrations of these sugars). Muramic acid, N-acetylgalactosamine, and N-acetylglucosamine were less effective, while several sugars had no effect, suggesting that the 40-kDa toxin binds to specific receptors on the cell membrane. The 40-kDa protein was less toxic to tissue culture-grown cells of Anopheles gambiae and Aedes dorsalis, and the same sugars which reduced the toxicity for cells of C. quinquefasciatus were also effective in reduction of toxicity for these cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of Cry11A and Cry11Ba toxins in Bacillus sphaericus confers toxicity towards Aedes aegypti and resistant Culex populations.

Cry11A from Bacillus thuringiensis subsp. israelensis and Cry11Ba from Bacillus thuringiensis subsp. jegathesan were introduced, separately and in combination, into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Two loci on the B. sphaericus chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against Aedes aegypti and Culex pipiens. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to A. aegypti larvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible C. pipiens larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of C. pipiens SPHAE and Culex quinquefasciatus GeoR to B. sphaericus strain 2297.     

High-Level Field Resistance to Bacillus sphaericus C3-41 in Culex quinquefasciatus from Southern China

A flowable mosquito-larvicidal formulation of Bacillus sphaericus strain C3-41 has been continuously used for 8 years to control Culex quinquefasciatus larvae in Dongguan, Guangdong Province, China. This formulation had high efficacy against the target larvae during the first 6 years of treatment. However, under this high selection pressure, C. quinquefasciatus showed a significant level of resistance to C3-41 from years seven to eight. The resistance ratio of field-collected larvae at LC 50 was calculated to be 22 672-fold in comparison with the susceptible laboratory colony. Interestingly, no cross-resistance was observed to B. sphaericus strain LP1-G which had the same toxicity against both susceptible and resistant larvae, and B. thuringiensis subsp. israelensis was found to be more active to the latter than the former. After six months treatment with the B. thuringiensis subsp. israelensis formulation in the B. sphaericus resistant population area, the mosquito population recovered its susceptibility to B. sphaericus C3-41, with the resistance ratio of field-collected larvae dropping from 22 672- to 5.67-fold.     

Baseline susceptibility to bacterial insecticides in populations of Culex pipiens complex (Diptera: Culicidae) from California and from the Mediterranean Island of Cyprus

Bacterial insecticides play an increasingly important role in mosquito control. To establish guidelines for detecting resistance at an early stage, information on natural variation in susceptibility of insect populations to these insecticides is needed. Between 1990 and 1993, the susceptibility of Culex pipiens L. complex to Bacillus thuringiensis subsp. israelensis de Barjac and/or Bacillus sphaericus Neide was determined in 31 collections from California. These collections were undertaken before the widespread use of B. thuringiensis subsp. israelensis and before the registration of B. sphaericus in California. Seven collections from the Mediterranean island of Cyprus, where no microbial insecticides have been used, also were tested. The 1990-1991 California collections exhibited limited variation in susceptibility to B. thuringiensis subsp. israelensis. LC50 and LC95 values spanned about a three-fold and four-fold range, respectively. The 1993 Cyprus collections exhibited both higher mean LC values, and greater variability in those values, than the California collections. The LC50s for the Cyprus collections varied over a 10-fold range, whereas the LC50s varied over a 12.5-fold range. Variation in susceptibility to B. sphaericus among the 1991 California collections was about five-fold at the LC50 and LC95. No significant geographic variation in susceptibility to B. thuringiensis subsp. israelensis was observed among regions within California. Although variation in susceptibility was limited among California collections, the greater variability observed among the Cyprus collections and between the Cyprus and California collections illustrates the importance of establishing regional baselines to monitor accurately for changes in susceptibility.