Enduring toxicity of transgenic Anabaena PCC 7120 expressing mosquito larvicidal genes from Bacillus thuringiensis ssp. israelensis

Persistence of biological control agents against mosquito larvae was tested under simulated field conditions. Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing cry4Aa, cry11Aa and p20 from Bacillus thuringiensis ssp. israelensis was greater than B. thuringiensis ssp. israelensis primary powder (fun 89C06D) or wettable powder (WP) (Bactimos products) when either mixed with silt or exposed to sunlight outdoors. Reduction of Bactimos primary powder toxicity was at least 10-fold higher than Anabaena's after mixing with silt. In outdoors experiments, Bactimos WP remained toxic (over 30% mortality of 3rd instar Aedes aegypti larvae) for 2-4 days only, while transgenic Anabaena's toxicity endured 8-21 days.     

Factors affecting the efficacy of three commercial formulations of Bacillus thuringiensis var. israelensis against species of European black flies

The efficacy of three commercial formulations of Bacillus thuringiensis var. israelensis against two European black fly species was determined. The effect of seston concentration, temperature and larval size on the efficacy of the formulations was also determined. Teknar‐WDC was found to be almost four times as effective as Bactimos‐WP though only slightly more efficient than Vectobac‐AS against Simulium variegatum gp. larvae. Small larvae ( < 2.5 mm) were more susceptible to both Teknar‐WDC and Vectobac‐AS than large larvae ( > 5 mm) though no difference was seen with Bactimos‐WP. High seston concentrations both before and during bioassays significantly decreased the efficacy of all formulations. Increased temperatures increased the efficacy of all formulations.     

Optimization of spray-drying conditions for the large-scale preparation of Bacillus thuringiensis var. israelensis after downstream processing

Reduction of water activity in the formulations of mosquito biocontrol agent, Bacillus thuringiensis var. israelensis is very important for long term and successful storage. A protocol for spray drying of B. thuringiensis var. israelensis was developed through optimizing parameters such as inlet temperature and atomization type. A indigenous isolate of B. thuringiensis var. israelensis (VCRC B-17) was dried by freeze and spray drying methods and the moisture content and mosquito larvicidal activity of materials produced by the two methods were compared. The larvicidal activity was checked against early fourth instars Aedes aegypti larvae. Results showed that the freeze-dried powders retained the larvicidal activity fairly well. The spray-dried powder moderately lost its larvicidal activity at different inlet temperatures. Between the two types of atomization, centrifugal atomization retained more activity than the nozzle type atomization. Optimum inlet temperature for both centrifugal and nozzle atomization was 160 degrees C. Keeping the outlet temperature constant at 70 degrees C the moisture contents for the spray-dried powders through centrifugal atomization and freeze-dried powders were 10.23% and 11.80%, respectively. The LC(50) values for the spray-dried and freeze-dried powders were 17.42 and 16.18 ng/mL, respectively. Spore count of materials before drying was 3 x 10(10) cfu/mL and after spray drying through nozzle and centrifugal atomization at inlet and outlet temperature of 160 degrees C/70 degrees C were 2.6 x 10(9) and 5.0 x 10(9) cfu/mL, respectively.     

Evaluation of a briquet formulation of Bacillus thuringiensis var. israelensis (H‐14) against aedes spp. and culex spp. larvae in dambos in Kenya

Bacillus thuringiensis Berliner var. israelensis serotype H‐14 (Bti) in briquet formulation (Bactimos) was tested in a field trial against ground‐pool breeding mosquitoes in a dambo located in a Rift Valley fever virus‐enzootic/epizootic area in central Kenya. Bactimos (10% Bti; 7000 AA International Toxic Units/mg) was tested for 30 days in 3 separate treatment areas at the rate of 1 briquet/9 m2, later increased to 1 briquet/1.5 m2 on day 13 postflood; 1 briquet/9 m2; and 1 briquet(4.6 m2). An estimate of the daily survival rate of larvae at different periods during the study revealed that mosquitoes in the area treated with 1 briquet/9 m2 had a significantly lower (64%) survival rate than those in the control site (92%) against Aedes spp. and was not significantly different from the site treated with 1 briquet/4.6 m². Mosquitoes in the site treated with I briquet/1.5 m² on day 13 postflood had a much‐reduced survival rate (25%;) when compared to those in the control site (67%) during the 3rd Culex spp. generation (22‐ 26 days postflood). This five‐fold increase above standard label dosage still failed to prevent Culex spp. emergence. There was no significant difference in survival rates between the control and any of the three treatment sites during the first or second Culex generation (9–20 days postflood). Emerged adults collected as pupae from control and treatment sites indicated that Aedes mcintoshi Huang and Culex antennatus (Becker) were the most predominant Aedes and Culex species.     

Bacillus thuringiensis serovar. israelensis (ABG-6108) against chironomids and some nontarget aquatic invertebrates

A wettable powder formulation (ABG-6108) of Bacillus thuringiensis serovar. israelensis was tested against chironomid larvae in experimental ponds at 1, 2, 3, 4 and 10 kg/ha (0.25–2.5 ppm), and in a golf pond at 3 kg/ha (0.5 ppm). Significant reductions (18–88%) in populations of Chrinomini and Tanytarsini experimental ponds, and 27–65% reduction of midges in the golf pond occurred during the 4-week evaluation period. The highest dose in experimental ponds had no adverse effects on rotifers, Cyclops spp., Daphnia sp., ostracods, Baetis sp., corixids and notonectids, and Coleoptera. The nontarget invertebrates in the golf pond also remained unaffected.     

Laboratory and field evaluations of industrial formulations of Bacillus thuringiensis serovar. israelensis against some mosquito species of central Italy

Two wettable powder formulations (Bactimos and Vectobac) and a flowable concentrate (Teknar) formulation of Bacillus thuringiensis serovar. israelensis were evaluated as larvicides of Culex pipiens, Aedes caspius, and Aedes detritus. In the laboratory, the levels of susceptibility of these species to the test formulations were essentially similar and corresponded to their relative potencies; the LC₉₀ values ranged from 0.042 to 0.37 ppm. C. pipiens, A. caspius, and A. detritus, in that order, were susceptible to the biocide. Under field conditions in central Italy. Bactimos at 0.5 kg/ha gave 98% control of C. pipiens in an irrigation canal. Teknar at 1.25 liters/ha gave 86–100%, and at 2.5 liters/ha gave 90–100% control of C. pipiens in two natural ponds. Against A. caspius in salt marsh habitats, Bactimos at 0.5 kg/ha and Teknar at 2.5 liters/ha yielded complete control of the larvae, while a lower rate (0.2 kg/ha) of Bactimos, and Vectobac at 0.5 kg/ha resulted in 82–94% and 67–91% control, respectively. Higher rates (0.75 and 1.0 kg/ha) of Vectobac gave 76–100% and 98–100% control of A. caspius. Bactimos at 0.15 kg/ha gave 93–98% control of A. detritus in two salt marsh ponds. B. thuringiensis serovar. israelensis is practically economical for the control of C. pipiens, A. caspius, and A. detritus in the various biotopes in central Italy.     

Biochemical and immunological characterization of the cloned crystal toxin of Bacillus thuringiensis var. israelensis

The protein components of the cloned crystal toxin of Bacillus thuringiensis var. israelensis were separated by polyacrylamide gel electrophoresis under denaturing conditions. Using an antiserum to the solubilized B. thuringiensis var. israelensis crystal protein as a probe, immunological homology between the crystal protein components of B. thuringiensis var. israelensis and those of the recombinant B. megaterium strain VB131 was tested. The results from this study indicate that the crystal inclusion of the recombinant strain contains only the 130 kilodalton protein and not the 68 or the 28 kilodalton proteins of the crystal toxin of B. thuringiensis var. israelensis and that the 130 kilodalton protein is primarily responsible for the mosquitocidal activity of this organism.     

Digestion of Bacillus thuringiensis var. israelensis spores by larvae of Aedes aegypti.

The larvicidal activity of Bacillus thuringiensis var. israelensis against mosquitoes and the blackfly is included in parasporal crystalline bodies which are produced during sporulation. Following ingestion, the crystals are solubilized in the larval midgut and induce death within a short time; the spores germinate in the dead larvae and complete a growth cycle. The fate of the spores in surviving live larvae was elucidated by using a nonlarvicidal B. thuringiensis var. israelensis mutant. When introduced as the only food source, spores of this mutant support development to the adult stage of newly hatched Aedes aegypti larvae at a rate directly related to spore concentration. The conclusion that spores of B. thuringiensis var. israelensis are digested in the larval gut was substantiated by following the incorporation of [35S]methionine-labeled spores into larval tissues.     

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.     

Efficacy of Czechoslovak and Soviet Bacillus thuringiensis (serotype H-14) formulations against mosquito larvae

Laboratory and field comparisons were made with two wettable powder formulations of Bacillus thuringiensis serotype H-14 (B. t. H-14) prepared in Czechoslovakia ("Moskitur") and the USSR ("Baktokulicid"). Expressed in the international Aedes aegypti toxic units (TU X mg-1) the potency of these two test formulations was greater than that of the Institute Pasteur Standard IPS-78 (= 1,000 TU X mg-1), i.e. Moskitur had a potency of about 1,500 TU X mg-1 and the Soviet Baktokulicid 2,000 TU X mg-1. The Baktokulicid and Moskitur LC 90 values for laboratory-reared Aedes aegypti larvae were, respectively, 0.11 and 0.16 mg X l-1. The range of LC 90 values for the Czechoslovak wild-caught mosquito species of the genera Aedes and Culex was 0.14-0.31 mg X liter-1 with Moskitur, 0.11-0.41 mg X l-1 with Baktokulicid, and 0.16-0.48 mg X l-1 with IPS-78. The susceptibility of laboratory Anopheles stephensi larvae was close to that of Aedes aegypti, larvae of An. messeae required many times as much Baktokulicid (1.6 mg X l-1) and Moskitur (more than 6.4 mg X l-1) for 90% mortality as did other mosquito species. The aim of outdoor assays was to establish the minimum Moskitur and Baktokulicid rates giving a 100% control of mosquito larvae. For Ae. cantans breeding habitat in flood plain forest areas these rates ranged between 0.1-0.5 mg X l-1 (0.2-1.0 kg X ha-1), for Ae. vexans control on artificially irrigated meadows between 0.8-2.0 mg X l-1 (1.2-3.0 kg X ha-1). Consistently with laboratory bioassays, Baktokulicid gave 100% control of An. messeae 4th instar larvae at a dose as high as 3.2 mg X l-1, Moskitur gave 23.1% kill at 6.4 mg X l-1. The effect of Moskitur and Baktokulicid formulations was immediate, larvae that hatched 7-14 days posttreatment survived. The efficacy of B. t. H-14 outdoor treatments tended to markedly decrease with the larval densities exceeding 100 larvae per 1 dm2. Species of nontarget aquatic organisms, including the Diptera Chaoborus crystallinus, Mychlonyx sp. and Dixidae, were not noticeably affected by treatments with B. t. H-14 formulations used.     

Immobilization in alginate as a technique for the preservation of Bacillus thuringiensis var. israelensis for long-term preservation

Technique for immobilization using sodium alginate as the matrix to preserve Bacillus thuringiensis var. israelensis isolates for long time storage was developed. Two strains of B. thuringiensis var. israelensis viz., VCRC B-17 and WHO standard strain IPS-82 were immobilized in alginate matrix and preserved at 4 degrees C and when tested both were found to have maintained excellent viability and mosquito larvicidal activity for 10 years. Mosquito larvicidal activity of B-17 and IPS-82 alginate beads, in term of LC(50) values before storage was 72.07 ng/ml and 47.07 ng/ml, respectively and after storage at 4 degrees C for a period of 1 to 10 years the values ranged from 69.88 to 73.86 ng/ml with a mean of 72.38 ng/ml and 45.32 to 48.60 ng/ml with a mean of 47.49 ng/ml, respectively. Similarly spore count of the beads of the respective strains was 4.37 x 10(8) and 3.33 x 10(10) CFU/mg before storage. After storage at 4 degrees C for a period of 1 to 10 years the counts of the beads of the respective strains ranged from 4.23 x 10(8) to 4.83 x 10(8) CFU/mg (mean of 4.49 x 10(8) CFU/mg) and 3.2 x 10(10) to 3.87 x 10(10) CFU/mg (mean of 3.54 x 10(10) CFU/mg). The alginate matrix immobilization technique has many advantages over free cells are that they enhance the stability of both spores and toxin against several physicochemical conditions and confer reduced susceptibility to contamination.     

Bacillus spp. toxicity against Haemonchus contortus larvae in sheep fecal cultures

The gastrointestinal nematode Haemonchus contortus is a major productivity constraint in sheep. In this study, the nematicidal effects of Bacillus circulans, Bacillus cereus, Bacillus thuringiensis var. israelensis, Bt. var. osvaldocruzi, Bt. var. morrisoni, and Bt. var. kurstaki were assessed in free-living larval stages of H. contortus. A spore-crystal suspension containing approximately 2×10(8)UFC/mL of each strain was added to sheep feces that were naturally infected with H. contortus eggs, and the presence of larvae was then evaluated. We observed a significant (p>0.05) reduction in larval development when using B. circulans, B. thuringiensis var. israelensis, Bt. var. osvaldocruzi and Bt. var. kurstaki, and these effects were proportional with the amount of bacteria added to the feces. However, no effect was observed when Bt. var. morrisoni or B. cereus was added. These observations suggest that these bacteria might be effective as nematicides and may allow for the development of integrated biological control of zooparasitic nematodes.     

Transformation of Bacillus thuringiensis by electroporation

Plasmids were transformed by electroporation into various strains of Bacillus thuringiensis with frequencies of up to 10(5) transformants/micrograms. pC 194 transformed all strains tested at a high frequency and cells could be stably transformed with pC194 and pUB110 simultaneously by electroporation with a frequency of 10(2) pC194+ pUB110 transformants/micrograms DNA. Low transformation frequencies observed with some plasmids, especially those grown initially in Escherichia coli, could be increased by passage through B. thuringiensis, B. thuringiensis var. israelensis and in acrystalliferous mutant of the same strain transformed at frequencies of 10(4)-10(5)/micrograms DNA with most of the plasmids tested. A cloned israelensis 27-kDa delta-endotoxin gene was introduced into the israelensis acrystalliferous mutant and a kurstaki acrystalliferous mutant by electroporation. Both transformants were shown to express the endotoxin gene and to be toxic to Aedes aegypti larvae.     

Influence of media composition on the production of delta-endotoxin by Bacillus thuringiensis var. thuringiensis

Powders of edible leguminous seeds, greengram (Vigna radiata) or soybean (Glycine max), were used as the major protein source with different combinations of soluble starch and/or cane sugar molasses as the major carbohydrate source for the production of delta-endotoxin by Bacillus thuringiensis var. thuringiensis serotype 1 in submerged fermentation. The primary product (lyophilized with 6 g of lactose) yield was 8.7 to 9.1 g/liter from media with dehusked greengram powder and 9.7 to 10.3 g/liter from media with defatted soybean powder in basal medium. The toxicity of primary products was assayed against fifth-instar Bombyx mori larvae by force-feeding. The primary product from the medium containing defatted soybean powder and soluble starch gave a maximum viable spore count of 91.3 x 10(6)/mg, with a corresponding potency of 35,800 IU/mg, whereas the medium containing dehusked greengram powder and cane sugar molasses gave a spore count of 49.5 x 10(6)/mg, with a highest potency of 38,300 IU/mg. Either legume protein in combination with cane sugar molasses yielded primary product 2.1 to 2.4 times more potent than the U.S. standard. The combined carbohydrate source consisting of soluble starch and cane sugar molasses, irrespective of the source of protein in the media, drastically reduced delta-endotoxin production, thereby reducing the potency of the primary products compared to the U.S. standard.     

Micro-lipid-droplet encapsulation of Bacillus thuringiensis subsp. israelensis delta-endotoxin for control of mosquito larvae.

The crystal delta-endotoxin of Bacillus thuringiensis subsp. israelensis is less toxic to larvae of Anopheles freeborni than to larvae of Aedes aegypti. However, when solubilized crystal was used, larvae from both species showed similar sensitivities. This effect presumably was due to the differences in feeding behavior between the two mosquito larvae when crystal preparations are used. A procedure is described whereby both crystal and solubilized B. thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity. The use of Freund incomplete adjuvant also allowed one to assay the solubilized toxin at a low nanogram level. Furthermore, coating the toxin with lipophilic material altered the buoyancy of the toxin and reversed the sensitivities of the two mosquito larvae toward the B. thuringiensis subsp. israelensis toxin. This difference in buoyancy was determined by using an enzyme-linked immunosorbent assay that was specific for the toxic peptides. These data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp. israelensis crystal can be developed.     

Micro-lipid-droplet encapsulation of Bacillus thuringiensis subsp. israelensis delta-endotoxin for control of mosquito larvae

The crystal delta-endotoxin of Bacillus thuringiensis subsp. israelensis is less toxic to larvae of Anopheles freeborni than to larvae of Aedes aegypti. However, when solubilized crystal was used, larvae from both species showed similar sensitivities. This effect presumably was due to the differences in feeding behavior between the two mosquito larvae when crystal preparations are used. A procedure is described whereby both crystal and solubilized B. thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity. The use of Freund incomplete adjuvant also allowed one to assay the solubilized toxin at a low nanogram level. Furthermore, coating the toxin with lipophilic material altered the buoyancy of the toxin and reversed the sensitivities of the two mosquito larvae toward the B. thuringiensis subsp. israelensis toxin. This difference in buoyancy was determined by using an enzyme-linked immunosorbent assay that was specific for the toxic peptides. These data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp. israelensis crystal can be developed.     

Influence of molting on efficacy of two functionally different larvicides: Bti and temephos

The sensitivity of the second, third (L3), and fourth (L4) instars of Culex pipiens L. and Ochlerotatus sticticus (Meigen) to two larvicides, temephos and Bacillus thuringiensis variety israelensis (Bti) was tested with and without molting occurring in the experiments. In the first experiment for both tested mosquito species, LC50 values increased from the second to the fourth instar when tested against temephos, whereas for Bti the opposite trend was observed. The highest LC50 value was found for fourth instars of Cx. pipens (0.00405 mg/liter) against temephos and for second instars of Oc. sticticus (0.267 mg/liter) against Bti. The determined LC50 values for the second and third instars of both species decreased with an increased number of molted larvae in the experiments with temephos. For the experiments with Bti molting did not have any significant influence on LC50 values, except a small increase in toxicity during the L3/L4 molt of Oc. sticticus. These findings could help assess and define larviciding, as well as influence the quantity of larvicides needed for an efficient treatment.     

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.     

Immobilization of Alginate-Encapsulated Bacillus thuringiensis var. israelensis Containing Different Multivalent Counterions for Mosquito Control

Immobilized techniques have been used widely for the controlled release formulation of mosquitoes. Among the microbial formulations, polymeric matrices play an important role in the controlled release of microbial pesticide at rates sufficiently effective to kill mosquitoes in the field. The advantage of these matrices is that they enhance the stability of both spores and toxin against pH, temperature variations, and UV irradiation. The disadvantage of using calcium alginate beads is that they are unstable upon contact with phosphate of potassium or sodium ions rich in the mosquito habitats. To overcome these problems, attempts were made to encapsulate Bacillus thuringiensis var. israelensis within alginate by using different multivalent counterions, namely, calcium chloride, zinc sulfate, copper sulfate, cobalt chloride, and ferric chloride, and the beads formed were tested for its mosquito larvicidal activity. Among all the beads tested, zinc alginate beads resulted in maximum larvicidal activity of 98% (+/-1.40 SE) against Culex quinquefasciatus IIIrd instar larvae and maximum spore count of 3.36 x 10(5) (+/-5291.50 SE) CFU/ml. Zinc alginate beads maintained their structure for up to 48 h when shaken vigorously on a rotary shaker at 180 rpm in the presence of 10 mM potassium phosphate buffer (pH 6.8 +/- 0.1). In conclusion, our results suggest that the use of zinc sulfate as counterions to encapsulate B. thuringiensis var. israelensis within alginate may be a potent mosquito control program in the habitats where more phosphate ions are present.