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[Effect of the pC194 plasmid on the development of Bacillus thuringiensis phages].

Bacillus thuringiensis var. galleriae strains were transformed by plasmid pC194, coding for chloramphenicol resistance (CmR). Efficiency of plating and the yields of bacteriophages Tg13 and Tg27 maturating in CmR transformant cells were decreased for 2-3 orders as compared with the ones in parental strains. The CmR transformants are characterized by the increased level of spontaneous induction of bacteriophage Tg22.

Bacillus thuringiensis↗

Larvicidal activity of chimeric Bacillus thuringiensis protoxins.

Bacillus thuringiensis subspecies kurstaki (Btk) and subspecies berliner (Btb) both produce lepidopteran-specific larvicidal protoxins with different activities against the same insect species. Toxic activity resides in the amino-terminal half of both protoxins, whereas the carboxy-terminal half of the molecules is not required for toxicity. The protoxins are 90% homologous, with a major cluster of differences in the amino-terminal half, and a 26 consecutive amino-acid insertion within the carboxy-terminal half of the Btk protoxin. Protoxin chimeras composed of the amino-terminal half of one subspecies and the carboxy-terminal half of the other were generated. Wild-type and chimeric protoxins were compared in bioassays against tobacco hornworm larvae. The amino-terminal half, the toxin itself, dictates specific larvicidal activity.

Animals↗

Recovery of commercially produced Bacillus thuringiensis var. israelensis and Bacillus sphaericus from tires and prevalence of bacilli in artificial and natural containers.

We conducted surveys to identify the species of spore-forming bacteria present in natural and artificial containers. Most of our samples came from Illinois. Identification was based on the cellular fatty acid composition of the bacterial cell wall. In addition, we utilized a custom database for commercially produced strains of Bacillus thuringiensis var. israelensis (Bti) and B. sphaericus, to differentiate between larvicidal isolates with commercial or native origin. Native Bti was present at low levels in almost all habitats but was not recovered from bromeliads and metal containers. In temporary woodland pools, 27.9% of the colonies recovered were native Bti. We did not recover larvicidal B. sphaericus in untreated habitats. VectoBac and VectoLex were applied to tires containing water and the tires were sampled 3 months and 9 months after treatment. Isolates of Bti and B. sphaericus with commercial origin were recovered as long as 9 months after application. We noticed numerous cadavers of Aedes triseriatus in several tires 9 months after treatment with VectoBac. We could not determine if this mortality resulted from recycling of Bti in these tires or whether insecticidal crystal proteins from the original treatment were resuspended. Bacillus thuringiensis var. israelensis isolates with commercial ancestry were recovered from untreated tires 9 months after application. Isolates of larvicidal B. sphaericus that differed from the bacteria in VectoLex were also recovered from untreated tires.

Animals↗

Comparative sensitivity to UV-B radiation of two Bacillus thuringiensis subspecies and other Bacillus sp.

Susceptibility of Bacillus thuringiensis spores and toxins to the UV-B range (280--330 nm) of the solar spectrum reaching Earth's surface may be responsible for its inactivation and low persistence in nature. Spores of the mosquito larvicidal B. thuringiensis subsp. israelensis were significantly more resistant to UV-B than spores of the lepidopteran-active subsp. kurstaki. Spores of subsp. israelensis were as resistant to UV-B as spores of B. subtilis and more resistant than spores of the closely related B. cereus and another mosquito larvicidal species B. sphaericus. Sensitivity of B. thuringiensis subsp. israelensis spores to UV-B radiation depended upon their culture age; 24-h cultures, approaching maximal larvicidal activity, were still sensitive. Maximal resistance to UV-B was achieved only at 48 h.

Bacillus↗

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.

Aedes↗

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai in Bacillus subtilis and in the thermophile Bacillus stearothermophilus by using the alpha-amylase promoter of the thermophile.

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai IPL7 in B. subtilis MI113 and B. stearothermophilus SIC1 was examined. Production of the protein (130 kilodaltons [KDa]) was analyzed by its reaction with antibody against the insecticidal proteins of the parental B. thuringiensis. When the original gene containing its own promoter was subcloned in B. subtilis, only a small amount of the protein was produced. Therefore, both the promoter for the B. stearothermophilus alpha-amylase gene and the insecticidal protein gene were inserted in a repA (low-copy-number) plasmid to yield the recombinant plasmid pTBT-Pamy. B. subtilis MI113 carrying pTBT-Pamy produced more of the 130-kDa protein (about 10(4) molecules per cell) at 37 degrees C. In contrast, B. stearothermophilus SIC1 carrying pTBT-Pamy produced a small amount of 130-kDa protein (10(2) to 10(3) molecules per cell) at 55 degrees C.

Amylases↗

Cyt1A from Bacillus thuringiensis restores toxicity of Bacillus sphaericus against resistant Culex quinquefasciatus (Diptera: Culicidae).

The 2362 strain of Bacillus sphaericus, which produces a binary toxin highly active against Culex mosquitoes, has been developed recently as a commercial larvicide. It is being used currently in operational mosquito control programs in several countries including Brazil, France, India, and the United States. Laboratory studies have shown that mosquitoes can develop resistance to B. sphaericus, and low levels of resistance have already been reported in field populations in Brazil, France, and India. To develop tools for resistance management, the Cyt1A protein of Bacillus thuringiensis subsp. israelensis De Barjac was evaluated for its ability to suppress resistance to B. sphaericus in a highly resistant population of Culex quinquefasciatus Say. A combination of B. sphaericus 2362 in a 10:1 ratio with a strain of B. thuringiensis subsp. israelensis that only produces Cyt1A reduced resistance by >30,000-fold. Resistance was suppressed completely when B. sphaericus was combined with purified Cyt1A crystals in a 10:1 ratio. Synergism was observed between the Cyt1A toxin and B. sphaericus against the resistant mosquito population and accounted for the marked reduction in resistance. However, no synergism was observed between the toxins against a nonresistant mosquito population. These results indicate that Cyt1A could be useful for managing resistance to B. sphaericus 2362 in Culex populations, and also provide additional evidence that Cyt1A may synergize toxicity by enhancing the binding to and insertion of toxins into the mosquito microvillar membrane.

Animals↗

Gene dosage effect on the expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis and Bacillus megaterium.

Significant differences in expression of the delta-endotoxin genes cryA1 and cryA2 of Bacillus thuringiensis subsp. kurstaki were observed in B. subtilis and B. megaterium. The cryA1 gene was expressed when present on a high-copy-number (hcn) vector in B. megaterium but not in B. subtilis. The cryA2 gene was expressed in both hosts, but at a higher level in B. megaterium. Expression of the cryA2 gene in B. megaterium was better from a hcn vector than from a low copy number vector. Inhibition of sporulation was observed when the toxin genes were present on hcn plasmids in B. subtilis while no such effect was evident in B. megaterium. In addition, there was a significant reduction in copy numbers in both B. subtilis and B. megaterium when delta-endotoxin genes or a spoVG promoter-containing fragment of DNA were cloned into hcn plasmids.

Bacillus↗

Bacillus thuringiensis var. israelensis delta-endotoxin. Nucleotide sequence and characterization of the transcripts in Bacillus thuringiensis and Escherichia coli.

The nucleotide sequence of a 1408 base-pair DNA fragment encoding the insecticidal 27,340 Mr delta-endotoxin of Bacillus thuringiensis var. israelensis has been determined by analysis of a recombinant plasmid from Escherichia coli. The hydropathy plot of the protein shows it to be highly hydrophobic, consistent with a postulated cytolytic mechanism of action for the toxin. In addition, the delta-endotoxin transcriptional start points that are used in B. thuringiensis and an E. coli recombinant have been determined. In B. thuringiensis var. israelensis, transcription initiates from a single start point, and gene-specific transcripts are not observed before stage II of sporulation. This is the stage at which delta-endotoxin antigen is first detected, indicating that control of expression is primarily at the transcriptional level for this protein. Analysis of gene-specific transcription in E. coli indicates that at least three start points are utilized in this organism. Interestingly, the highest level of delta-endotoxin mRNA is seen during mid-exponential growth of E. coli and the level appears to decrease as the cells enter the stationary phase of growth.

Amino Acid Sequence↗

Bacterial control of mosquito larvae: investigation of stability of Bacillus thuringiensis var. israelensis and Bacillus sphaericus standard powders.

Bacillus thuringiensis var. israelensis and Bacillus sphaericus products were assayed against their respective reference powders IPS82 and SPH88. Since their production in 1982 and 1988, the potency and larvicidal activity of these standard powders have been regularly checked on their test insects Aedes aegypti (for IPS82) or Culex pipiens (for SPH88). Over the 16-year evaluation period of IPS82 and 10-year evaluation period of SPH88, their potencies were considered stable. The global mean of each year's mean showed a coefficient of variation of less than 20%. Larval rearing was the most important factor in the reproducibility of the bioassay, although some variation also originated from the person performing the bioassay. This study demonstrated that the SPH88 standard could be kept in a stock suspension at 4 degrees C for 3 years without loss of potency. Moreover, after 9 years of storage in suspension, only a 2-fold decrease in the potency of SPH88 was detected.

Aedes↗

Factors affecting the toxicity of Bacillus thuringiensis var. israelensis and Bacillus sphaericus to fourth instar larvae of Chironomus tepperi (Diptera: Chironomidae).

Laboratory bioassays (48h duration, 25+/-1 degrees C) were used to determine the toxicity of Bacillus thuringiensis var. israelensis (B.t.i.) and Bacillus sphaericus to fourth instar larvae of Chironomus tepperi, a major pest of rice in southern Australia. Bioassays were conducted using different combinations of larval ages and densities to determine if these factors affected toxicity. The effects of temperature and substrate type on B.t.i. toxicity were also investigated. Tests were conducted using a commercial B.t.i. formulation (VectoBac WDG, 3000ITU/mg), a spore/crystal mixture derived from the VectoBac WDG strain, and VectoLex WDG, a commercial B. sphaericus formulation (650ITU/mg). VectoBac WDG was highly toxic to fourth instar C. tepperi in bioassays using a sand substrate (LC(50) 0.46mg/L, older larvae); younger fourth instar larvae were more susceptible (LC(50) 0.20mg/L). Increasing larval densities (from 10 to 30 per bioassay cup) increased LC(50) values for both age groups, significantly so in the case of older larvae (higher density LC(50) 0.80mg/L). Use of a soil substrate increased the LC(50) value (older larvae, 10 per cup) to 0.99mg/L. Similar differences in toxicity relative to larval age and substrate type were found in bioassays using the B.t.i. spore/crystal mixture. VectoBac WDG and the spore/crystal mixture both showed similar (approximately 6-fold) declines in activity between 30 and 17.5 degrees C. At lower temperatures (between 17.5 and 15 degrees C), activity of the spore/crystal mixture declined much more rapidly than that of VectoBac WDG. VectoLex WDG showed very low toxicity to C. tepperi larvae, and the overall impact of larval age and density was relatively minor (LC(50) values 1062-1340mg/L). Autoclaving VectoLex WDG did not substantially reduce its toxicity (LC(50) 1426mg/L), suggesting that formulation additives (i.e., surfactants and other adjuvants) are responsible for much of the toxicity occurring at the high product concentrations required to cause C. tepperi mortality. Whilst VectoLex WDG was ineffective against C. tepperi, VectoBac WDG has the potential to provide selective control of this rice pest at economically viable application rates.

Animals↗

Mosquito control and bacterial flora in water enriched with organic matter and treated with Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus formulations.

Three tests were conducted during July 17 to October 30, 1998 to study the impact of two mosquitocidal microbial agents on mosquito larvae and their contribution to bacterial flora in aquatic microcosms. Formulations of Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus strain 2362 (Bsph) were applied at various rates to outdoor tubs enriched with rabbit pellets and filled with irrigation water from a reservoir. Mosquito larvae were effectively controlled by all treatments; the magnitude of initial and persistent control depended on materials and dosages applied. Bacterial flora were assessed in the irrigation water as well as water in the enriched tubs before and after treatment with the microbial agents. The irrigation water contained 800-1000 total bacterial cells/ml. The populations of total bacteria and spore formers peaked on day 3 after enriching and filling the tubs, then declined progressively to the low levels at the end of the tests. After treatment, the numbers of Bti and Bsph spores in treated tubs prevailed at a dosage-dependent manner, their populations peaked at three hours after treatment, and declined progressively thereafter. The contribution of Bti and Bsph spores to the total bacterial flora was negligible but significant to the counts of spore-forming bacteria. The gram-negative bacteria made up more than 80% of the total bacterial flora during the test periods; and, of these, gram-negative rods constituted the greatest proportion, gradually increasing from the time of flooding to the end of the tests. Gram-negative cocci also occurred in relatively great proportion, but showed a reverse trend as compared with the gram-negative rods, declining gradually from pretreatment to the end of the tests. Gram-positive rods (spore formers), including Bti and Bsph, occurred in low numbers in all the tests but increased slightly in treated tubs due to the addition of Bti and Bsph spores. Gram-positive cocci occurred occasionally in some water samples.

Animals↗

[Duration of the larvicidal effect of spore crystalline mass of bacteria Bacillus thuringiensis spp. israelensis and Bacillus sphaericus in the laboratory setting].

The duration of action of sporocrystalline mass of Bacillus thuringiensis spp. Israelensis (Bti) and Bacillus sphaericus (Bsph) against three-age larvae of An. stephensi mosquitoes was studied in the laboratory setting. The duration of action depends on its initial concentration. The optimal concentration should be at least 10(5) spores/ml for An. stephensi larvae. Decreasing the concentration reduces the duration of larvicidal action. Increasing the number of the larvae placed also diminishes the duration of action shown by Bti. This has no significant influence on the duration of action by Bsph. The presence of dead larvae in the test vessel increases the duration of action by Bti only by 4 times whereas Bsph acts rather long. The follow-ups lasted 60 weeks with full death of the larvae placed again. A significant water resistance of Bti sporocrystalline mixture was found. The mixture retained its larvicidal activity 6 months following its presence in the dechlorinated water without larval placement. Bsph was less resistant and its complete inactivity was noted under the same conditions just following 2 months.

Animals↗

Comparison of development of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus in mosquito larvae.

Immunofluorescent staining was used with thin sections of paraffin-embedded specimens to detect the development of Bacillus thuringiensis var. israelensis and Bacillus sphaericus in the gut of mosquito larvae. The third- and fourth-instar larvae of Aedes aegypti, Anopheles maculatus, and Culex quinquefasciatus were fed either vegetative cells or spores of the bacteria. Spore germination, multiplication, and sporulation were studied in the larvae of each species. The spores of B. thuringiensis var. israelensis and B. sphaericus strain 2297 could germinate and cells could sporulate in the larval body. The vegetative cells of B. sphaericus strain 810428 were also able to produce spores in the mosquito larval gut, but the germination of spores could not be detected in the larvae. Multiplication of all bacterial species was observed after the larvae died. Growth of the bacteria in distilled water containing crude extracts of larvae made from each species was compared with that in synthetic medium (nutrient broth). They could produce spores and toxins in all the media used and the toxins had larvicidal activity against the target mosquitos Ae. aegypti, An. maculatus, and C. quinquefasciatus.

Animals↗

Vegetative expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis.

Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars. A HincII-SmaI double digest of pHBHE was then ligated to BglII-cut plasmid pBD64 and introduced into Bacillus subtilis by transformation. The transformants were identified by colony hybridization and confirmed by Southern blot hybridization. A 135,000-dalton protein which bound to an antibody specific for the crystal protein of B. thuringiensis was detected from the B. subtilis clones containing the toxin gene insert in either orientation. A toxin gene insert cloned into a PvuII site distal from the two drug resistance genes of the pBD64 vector also expressed a 135,000-dalton protein. These results suggest that the toxin gene is transcribed from its own promoter. Western blotting of proteins expressed at various stages of growth revealed that the crystal protein expression in B. subtilis begins early in the vegetative phase, while in B. thuringiensis it is concomitant with the onset of sporulation. The cloned genes when transferred to a nonsporulating strain of B. subtilis also expressed a 135,000-dalton protein. These results suggest that toxin gene expression in B. subtilis is independent of sporulation. Another toxin gene encoding a 130,000- to 135,000-dalton protein was cloned in E. coli from a library of B. thuringiensis genes established in lambda 1059. This gene was then subcloned in B. subtilis. The cell extracts from both clones were toxic to caterpillars. Electron microscope studies revealed the presence of an irregular crystal inclusion in E. coli and a well-formed bipyramidal crystal in B. subtilis clones similar to the crystals found in B. thuringiensis.

Bacillus subtilis↗

Field evaluation of new water-dispersible granular formulations of Bacillus thuringiensis ssp. israelensis and Bacillus sphaericus against Culex mosquitoes in microcosms.

A variety of formulations of Bacillus thuringiensis var. israelensis de Barjac (B.t.i.) and Bacillus sphaericus Neide (B.s.) have been studied for mosquito control under laboratory and field conditions. High efficacy, specificity, low risk of development of resistance, long shelf-life, and transportability, as well as the safety to nontarget organisms of these 2 microbial agents have been well documented. Some of the currently available formulations of B.t.i. and B.s. have low potency per unit mass. Research and development efforts are focusing on commercializing formulations with high potency and low minimum effective dosage that are suitable for long-distance shipment. To achieve this goal, new water-dispersible granule (WDG) formulations of both microbial agents were prepared and made available by Abbott Laboratories for evaluation. The newly developed WDGs of B.t.i. and B.s. with high potency dispersed readily in water with gentle agitation. These WDGs were evaluated and the minimum effective dosages were determined in microcosms against natural populations of Culex mosquitoes. The minimum effective dosage for B.t.i. WDGs with 4,000 International Toxic Units (ITU)/mg was 0.27-0.53 lb/acre which yielded significant control for up to 7-12 days. The minimum effective dosage for B.s. WDGs with 350-630 ITU/mg was 0.05-0.10 lb/acre, which yielded significant control of immature mosquitoes for up to 14-20 days.

Animals↗

Stable integration and expression of mosquito-larvicidal genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus into the chromosome of Enterobacter amnigenus: a potential breakthrough in mosquito biocontrol.

Previously, we have successfully integrated a spectinomycin/streptomycin resistance gene into Enterobacter amnigenus strain An11, a potential host for mosquito control, using in vivo recombination via homologous recombination (An11S4::Omega). We now report the successful transfer of two mosquito-larvicidal genes, cry4B from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus, into the host genome. To facilitate the screening procedure, the E. amnigenus derivative, An11S4::Omega, was used as a host. The integration of both toxin genes by two successive crossover events interrupted the Omega region yielding two integrants designated An11S4::cry4B and An11S4::Omega::bin, respectively. Differences in the integration efficiency of these toxin genes were observed. The presence of both genes in the target sites of the host genome was verified by PCR. Cry4B was expressed weakly from An11S4::cry4B, but no expression of the binary toxin gene could be detected from An11S4::Omega::bin. Nevertheless, these two integrants exhibited mosquito-larvicidal activity against Aedes and Culex, suggesting that both proteins were expressed, but at very low levels.

Animals↗

Laboratory and field efficacy of Bacillus thuringiensis var. Israelensis and Bacillus sphaericus against Anopheles gambiae s.l. and Culex quinquefasciatus in Ouagadougou, Burkina Faso.

Two wettable powders (Bactimos and Vectobac) and one flowable concentrate (Teknar) of Bacillus thuringiensis var. israelensis (B.t.i.) and primary powders of Bacillus sphaericus isolates 1593 and 2362 were evaluated (laboratory) against field-collected larvae of Anopheles gambiae s.l. and Culex quinquefasciatus in Ouagadougou, Burkina Faso. Bactimos, Vectobac and a Corn-cob B.t.i. formulation (ABG-6138G) were field tested against Cx. quinquefasciatus and An. gambiae s.l. The isolates of B. sphaericus were also tested against An. gambiae s.l. in artificial ponds. Both wettable powders of B.t.i. showed superior activity than the flowable concentrate formulation against An. gambiae s.l. in the laboratory. Culex quinquefasciatus was more susceptible (3-4X) to B.t.i. (Bactimos) than An. gambiae s.l. The isolates of B. sphaericus were more effective (2-3X) against both mosquito species than Bactimos. In a ditch and two channels, Bactimos, Vectobac and ABG-6138G at 0.65, 1.5 and 5.6 kg/ha, respectively, gave 91-100% control of Cx. quinquefasciatus within 3 days of treatment. The same formulations at rates ranging from 0.25 to 5.6 kg/ha, produced 82-97% control of An. gambiae s.l. in rainwater pools 24 h after treatment. Isolates 1593 and 2362 at 0.12 and 0.24 kg/ha gave excellent control of An. gambiae s.l. in artificial ponds.

Animals↗