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.     

Effects of temperature, pH and salinity on the infection of Leptolegnia chapmanii Seymour (Peronosporomycetes) in mosquito larvae

The effects of temperature, pH, and NaCl concentrations on the infectivity of zoospores of Leptolegnia chapmanii (Argentine isolate) were determined for Aedes aegypti and Culex pipiens under laboratory conditions. Zoospores of L. chapmanii were infectious at temperatures between 10 and 35 degrees C but not at 5 or 40 degrees C. At the permissive temperatures, mortality rates in young instars were much higher than in older instars and larvae of Ae. aegypti were more susceptible to L. chapmanii than larvae of Cx. pipiens. At 25 degrees C, Ae. aegypti larvae challenged with L. chapmanii zoospores resulted in 100% infection at pH levels ranging from 4 to 10. Larvae of Cx. pipiens exposed to similar pH and zoospore concentrations resulted in increasing mortality rates from 62% to 99% at pH 4 to 7, respectively, and then decreased to 71% at pH 10. Aedes aegypti larvae exposed to L. chapmanii zoospores in NaCl concentrations ranging from 0 to 7 parts per thousand (ppt) at 25 degrees C resulted in 100% mortality while mortality rates for Cx. pipiens decreases from 96% in distilled water to 31.5% in water with 6 ppt NaCl. Control Cx. pipiens larvae died when exposed at a NaCl concentration of 7 ppt. Vegetative growth of L. chapmanii was negatively affected by NaCl concentrations. These results have demonstrated that the Argentinean isolate of L. chapmanii tolerated a wide range of temperatures, pH, and salinity, suggesting that it has the potential to adapt to a wide variety of mosquito habitats.     

Deletion by in vivo recombination shows that the 28-kilodalton cytolytic polypeptide from Bacillus thuringiensis subsp. israelensis is not essential for mosquitocidal activity.

The cytA gene encoding the 28-kDa polypeptide of Bacillus thuringiensis subsp. israelensis crystals was disrupted in the 72-MDa resident plasmid by in vivo recombination, thus indicating that homologous recombination occurs in B. thuringiensis. The absence of the 28-kDa protein in B. thuringiensis did not affect the crystallization of the other toxic components of the parasporal body (68-, 125-, and 135-kDa polypeptides). The absence of the 28-kDa protein abolished the hemolytic activity of B. thuringiensis subsp. israelensis crystals. However, the mosquitocidal activity of the 28-kDa protein-free crystals did not differ significantly from that of the wild-type crystals when tested on Aedes aegypti and Culex pipiens larvae. The 28-kDa protein contributed slightly to the toxicity to Anopheles stephensi larvae. This indicates that the 28-kDa protein is not essential for mosquitocidal activity, at least against the three species tested.     

Role of the CryIVD polypeptide in the overall toxicity of Bacillus thuringiensis subsp. israelensis.

The gene encoding the CryIVD protein of B. thuringiensis subsp. israelensis crystals was disrupted by in vivo recombination. The toxicity of the CryIVD protein-free inclusions was similar to that of the wild-type crystals on Anopheles stephensi larvae but was half the wild-type toxicity on Culex pipiens and Aedes aegypti larvae.     

Medium for the production of primary powder of Bacillus thuringiensis subsp. israelensis

Five media, formulated from the seeds of five legume varieties, dried cow blood, and mineral salts, were assessed for the growth and production of insecticidal properties of Bacillus thuringiensis subsp. israelensis. Bacterial powders prepared from the broth cultures were assayed against the larvae of Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae. A standard primary powder of B. thuringiensis subsp. israelensis (IPS78) was included in the assay for comparison. Good growth was obtained in all the media, and all powders were effective against the three types of mosquito larvae. The powder containing ground seeds of Voandzeia subterranean was the most effective and compared favorably with the standard (IPS78). The concentrations required to kill 50% of the larvae of Aedes aegypti, C. quinquefasciatus, and Anopheles gambiae were 1.13 X 10(-2) +/- 1.79 X 10(-3), 1.83 X 10(-2) +/- 2.55 X 10(-3), and 2.25 X 10(-2) +/- 1.88 X 10(-3) micrograms/ml, respectively. This investigation shows that the medium containing V. subterranean can be used for the production of B. thuringiensis subsp. israelensis primary powder.     

Medium for the production of primary powder of Bacillus thuringiensis subsp. israelensis.

Five media, formulated from the seeds of five legume varieties, dried cow blood, and mineral salts, were assessed for the growth and production of insecticidal properties of Bacillus thuringiensis subsp. israelensis. Bacterial powders prepared from the broth cultures were assayed against the larvae of Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae. A standard primary powder of B. thuringiensis subsp. israelensis (IPS78) was included in the assay for comparison. Good growth was obtained in all the media, and all powders were effective against the three types of mosquito larvae. The powder containing ground seeds of Voandzeia subterranean was the most effective and compared favorably with the standard (IPS78). The concentrations required to kill 50% of the larvae of Aedes aegypti, C. quinquefasciatus, and Anopheles gambiae were 1.13 X 10(-2) +/- 1.79 X 10(-3), 1.83 X 10(-2) +/- 2.55 X 10(-3), and 2.25 X 10(-2) +/- 1.88 X 10(-3) micrograms/ml, respectively. This investigation shows that the medium containing V. subterranean can be used for the production of B. thuringiensis subsp. israelensis primary powder.     

Cloning and expression of two crystal protein genes, cry30Ba1 and cry44Aa1, obtained from a highly mosquitocidal strain, Bacillus thuringiensis subsp. entomocidus INA288

Two novel crystal protein genes, cry30Ba and cry44Aa, were cloned from Bacillus thuringiensis subsp. entomocidus INA288 and expressed in an acrystalliferous strain. Cry44Aa crystals were highly toxic to second-instar Culex pipiens pallens (50% mortality concentration [LC50] = 6 ng/ml) and Aedes aegypti (LC50 = 12 ng/ml); however, Cry30Ba crystals were not toxic.     

Laboratory evaluation of Mesocyclops annulatus (Wierzejski, 1892) (Copepoda: Cyclopidea) as a predator of container-breeding mosquitoes in Argentina

In laboratory bioassays we tested the predatory capacity of the copepod Mesocyclops annulatus on Aedes aegypti and Culex pipiens larvae. A single adult female of M. annulatus caused 51.6% and 52.3% mortality of 50 first instar larvae of Ae. aegypti and Cx. pipiens respectively, in a 72 h test period. When alternative food was added to the containers, mortality rates declined to 16% and 10.3% for Ae. aegypti and Cx. pipiens respectively. When 50 first instar larvae of each of the two mosquito species tested were placed together with a single adult female of M. annulatus, mortality rates were 75.5% for Ae. aegypti larvae and 23.5% for Cx. pipiens larvae in a three day test period. Different density of adult females of M. annulatus ranged from 5 to 25 females produced mortality rates of Ae. aegypti first instar larvae from 50% to 100% respectively. When a single adult female of M. annulatus was exposed to an increasing number of first-instar Ae. aegypti larvae ranging from 10 to 100, 100% mortality was recorded from 1 to 25 larvae, then mortality declined to 30% with 100 larvae. The average larvae killed per 24 h period by a single copepod were 29.     

Cloning and expression of a novel toxin gene from Bacillus thuringiensis subsp. jegathesan encoding a highly mosquitocidal protein.

A gene, designated cry11B, encoding a 81,293-Da crystal protein of Bacillus thuringiensis subsp. jegathesan was cloned by using a gene-specific oligonucleotide probe. The sequence of the Cry11B protein, as deduced from the sequence of the cry11B gene, contains large regions of similarity with the Cry11A toxin (previously CryIVD) from B. thuringiensis subsp. israelensis. The Cry11B protein was immunologically related to both Cry11A and Cry4A proteins. The cry11B gene was expressed in a nontoxic strain of B. thuringiensis, in which Cry11B was produced in large amounts during sporulation and accumulated as inclusions. Purified Cry11B inclusions were highly toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi. The activity of Cry11B toxin was higher than that of Cry11A and similar to that of the native crystals from B. thuringiensis subsp. jegathesan, which contain at least seven polypeptides.     

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.     

Identification of a gene for Cyt1A-like hemolysin from Bacillus thuringiensis subsp. medellin and expression in a crystal-negative B. thuringiensis strain.

A gene designated cyt1Ab1, encoding a 27,490-Da protein, was isolated from Bacillus thuringiensis subsp. medellin (H30 serotype) by using an oligonucleotide probe corresponding to the cyt1Aa1 gene. The sequence of the Cyt1Ab1 protein, as deduced from the sequence of the cyt1Ab1 gene, was 86% identical to that of the Cyt1Aa1 protein and 32% identical to that of the Cyt2Aa1 protein from B. thuringiensis subsp. kyushuensis. The cyt1Ab1 gene was flanked upstream by a p21 gene, in the same orientation, encoding a 21,370-Da protein that showed 84% similarity to the putative chaperone P20 protein from B. thuringiensis subsp. israelensis and downstream, on the opposite strand, by a sequence showing 85% identity to the IS240A insertion sequence. The cyt1Ab1 gene was expressed at a high level in a nontoxic strain of B. thuringiensis subsp. israelensis in which large inclusions of the Cyt1Ab1 protein were produced. Purified Cyt1Ab1 crystals were as hemolytic as those of the Cyt1Aa1 protein and were twice as hemolytic as those from the wild-type strain. Mosquitocidal activity toward Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae was assayed. The toxicity of the Cyt1Ab1 protein was slightly lower than that of the Cyt1Aa1 protein for all three mosquito species, and Cyt1Ab1 was 150, 300, and 800 times less active toward Culex, Anopheles, and Aedes larvae, respectively, than were the native crystals from B. thuringiensis subsp. medellin.     

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.     

Toxic activity of Bacillus Thuringiensis isolates to Aedes Aegypti (L.) (Diptera: Culicidae) larvae

Aedes aegypti (L.), the main vector of dengue fever in Brazil, has been controlled with the use of massive chemical products, contributing to the development of resistance and decreasing the insect control efficiency. The control of dipterans with bioinsecticides based on Bacillus thuringiensis has been satisfactory, due to the production of insecticidal proteins denominated Cry (crystal), Cyt (cytolytic) toxins and Chi (chitinase), and to the synergistic effects among them. The present work aimed to select B. thuringiensis isolates efficient against A. aegypti larvae. A bacterial collection containing 1,073 isolates of B. thuringiensis, obtained from different locations of Brazilian territory, had the DNA isolated and submitted to PCR amplifications using specific primers for cry4Aa, cry4Ba, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa and chi genes. For the LC50 and LC90 determination, the entomopathogenic isolates were evaluated by selective and quantitative bioassays. Only 45 isolates (4.2%) presented amplicons for the cry and cyt genes. The chi gene sequence was detected in 25 (54.3%) of those isolates. From the 45 isolates submitted to the selective bioassays, 13 caused 100% mortality of A. aegypti larvae. The identification of cry, cyt and chi genes of B. thuringiensis and the toxicity analysis on A. aegypti led to the selection of a set of isolates that have the potential to be used in the formulation of new bioinsecticides.     

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.     

Analysis of mosquito larvicidal potential exhibited by vegetative cells of Bacillus thuringiensis subsp. israelensis

Vegetative Bacillus thuringiensis subsp. israelensis cells (6 X 10(5)/ml) achieved 100% mortality of Aedes aegypti larvae within 24 h. This larvicidal potential was localized within the cells; the cell-free supernatants did not kill mosquito larvae. However, they did contain a heat-labile hemolysin which was immunologically distinct from the general cytolytic (hemolytic) factor released during solubilization of B. thuringiensis subsp. israelensis crystals. The larvicidal potential of the vegetative cells was not due to poly-beta-hydroxybutyrate. Instead, it correlated with the ability of vegetative cells to sporulate during the bioassays. No toxicity was observed when bioassays were conducted in the presence of chloramphenicol or streptomycin. It is unlikely that the vegetative cells sporulate in the alkaline (pH 9.5 to 10.5) larval guts after ingestion. B. thuringiensis subsp. israelensis is not an alkalophile; we have been unable to grow it in culture at pH values of greater than or equal to 9.5. Moreover, we have been unable to demonstrate formation of a protective capsule. However, bacteria may replicate in the gut fluids of dead or dying mosquito larvae because their alkaline gut pH values drop markedly after exposure to the B. thuringiensis subsp. israelensis crystal toxins.     

Analysis of mosquito larvicidal potential exhibited by vegetative cells of Bacillus thuringiensis subsp. israelensis.

Vegetative Bacillus thuringiensis subsp. israelensis cells (6 X 10(5)/ml) achieved 100% mortality of Aedes aegypti larvae within 24 h. This larvicidal potential was localized within the cells; the cell-free supernatants did not kill mosquito larvae. However, they did contain a heat-labile hemolysin which was immunologically distinct from the general cytolytic (hemolytic) factor released during solubilization of B. thuringiensis subsp. israelensis crystals. The larvicidal potential of the vegetative cells was not due to poly-beta-hydroxybutyrate. Instead, it correlated with the ability of vegetative cells to sporulate during the bioassays. No toxicity was observed when bioassays were conducted in the presence of chloramphenicol or streptomycin. It is unlikely that the vegetative cells sporulate in the alkaline (pH 9.5 to 10.5) larval guts after ingestion. B. thuringiensis subsp. israelensis is not an alkalophile; we have been unable to grow it in culture at pH values of greater than or equal to 9.5. Moreover, we have been unable to demonstrate formation of a protective capsule. However, bacteria may replicate in the gut fluids of dead or dying mosquito larvae because their alkaline gut pH values drop markedly after exposure to the B. thuringiensis subsp. israelensis crystal toxins.     

The glycoprotein toxin of Bacillus thuringiensis subsp. israelensis indicates a lectinlike receptor in the larval mosquito gut.

The mosquito-active protein crystals produced by Bacillus thuringiensis subsp. israelensis contain covalently attached aminosugars which are critical for their larvicidal activity. The 50% lethal concentrations toward Aedes aegypti larvae were increased up to 10-fold by mild periodate treatment, up to 40-fold by forming the protein crystals in the presence of tunicamycin, and up to 7-fold by the presence during the mosquito bioassays of N-acetylglucosamine or its trimer, triacetylchitotriose. Periodate-treated crystals and crystals formed in the presence of tunicamycin had greatly reduced binding capacities for wheat germ agglutinin, an N-acetylglucosamine-specific lectin. These results suggest that the B. thuringiensis subsp. israelensis glycoprotein toxin binds to a lectinlike receptor in the larval mosquito gut. Furthermore, the distinct lectin-binding patterns exhibited by diptera-active versus lepidoptera-active B. thuringiensis crystals suggest that host specificity for the microbial insecticides is determined, in part, by the carbohydrate portion of their glycoprotein crystals.     

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)