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 (LC₅₀) of Mtx-1 and Mtx-2 of 0.246 and 4.13 μg/ml, respectively. The LC₅₀s were 0.406 to 0.430 μg/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.     

Isolation of novel Bacillus species showing high mosquitocidal activity against several mosquito species

Two novel mosquitocidal bacteria, VB17 and VB24, identified as new Bacillus species were isolated from dead mosquito larvae obtained in Florida aquatic habitats. Gas chromatographic analysis of fatty acid methyl esters (GC-FAME) and 16S rRNA sequencing indicated that VB24 is closely related to Bacillus sphaericus whereas VB17 does not have a close relationship with either Bacillus thuringiensis or B. sphaericus. Both isolates were significantly more active than B. sphaericus 2362 against Aedes taeniorhynchus, Anopheles quadrimaculatus, Culex quinquefasciatus larvae, and as active as B. sphaericus 2362 against Anopheles gambiae. Interestingly, however, both were not active against Aedes aegypti larvae, indicating some level of insecticidal specificity.     

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 [LC₅₀] = 1.7 ng/ml) against fourth-instar Culex quinquefasciatus was higher than that of B. thuringiensis subsp. israelensis IPS-82 (LC₅₀ = 7.9 ng/ml) or B. sphaericus 2362 (LC₅₀ = 12.6 ng/ml).     

Effects of temperature and instar on the efficacy of Bacillus thuringiensis var. israelensis and Bacillus sphaericus strain 1593 against Aedes stimulans larvae

Laboratory trials of Bacillus thuringiensis var. israelensis (serotype 14) and B. sphaericus strain 1593 against field-collected Aedes stimulans showed that susceptibility declined with increasing instar and decreasing temperature. Test results with B. sphaericus were more erratic than with B. thuringiensis, and the efficacy of the former declined more rapidly with decreasing temperature. B. thuringiensis was significantly more active than B. sphaericus under all treatment conditions. These results indicate that the effective use of this strain of B. sphaericus as a mosquito biological control agent may be limited to warm water situations against more susceptible species.     

Production of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus strain 1593 on media using a byproduct from a monosodium glutamate factory

Two newly developed media, H4 and H7, were found to be highly suitable for culturing Bacillus thuringiensis subsp. israelensis and B. sphaericus, respectively. These media contained 0.05% K₂HPO₄ and 4% HDL (H4 medium) or 0.05% K₂HPO₄ and 7% HDL (H7 medium); HDL is the by-product from a monosodium glutamate factory. Tests to compare endospore formation and toxicity values of B. thuringiensis subsp. israelensis in H4 medium and nutrient broth supplemented with salts and glucose (NBSG) medium were carried out in a 3-liter fermentor. The viable cell count and LC₅₀ value of B. thuringiensis subsp. israelensis in H4 medium at 48 hr were 2.5 × 10⁸ cells/ml and 10^?7.2 (dilution), respectively, while those in NBSG medium were 1.6 × 10⁸ cells/ml and 10^?6.5, respectively. In the case of B. sphaericus grown in H7 medium, the number of cells and LC₅₀ value were found to be 1.4 × 10⁹ cells/ml and 10^?7.8, respectively. B. sphaericus grown in nutrient broth supplemented with salt and yeast extract (NBSY) were found to produce 6.4 × 10⁸ cells/ml and an LC₅₀ value of 10^?6.8. The toxicity of B. thuringiensis subsp. israelensis was tested against Aedes aegypti larvae, while that of B. sphaericus was tested against Culex quinquefasciatus. The cost of 10 liters of medium for production of B. thuringiensis subsp. israelensis and in B. sphaericus and H4 and H7 was $0.02 and $0.03, respectively. The cost of these newly developed media was much less than that of NBSG medium ($7.05 per 10 liters) for cultivation of B. thuringiensis subsp. israelensis and NBSY medium ($11.67 per 10 liters) for cultivation of B. sphaericus.     

Conjugal Transfer of a Toxin-Coding Megaplasmid from Bacillus thuringiensis subsp. israelensis to Mosquitocidal Strains of Bacillus sphaericus

Both Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis produce mosquitocidal toxins during sporulation and are extensively used in the field for control of mosquito populations. All the known toxins of the latter organism are known to be encoded on a large plasmid, pBtoxis. In an attempt to combine the best properties of the two bacteria, an erythromycin resistance-marked pBtoxis plasmid was transferred to B. sphaericus by a mating technique. The resulting transconjugant bacteria were significantly more toxic to Aedes aegypti mosquitoes and were able to overcome resistance to B. sphaericus in a resistant colony of Culex quinquefasciatus, apparently due to the production of Cry11A but not Cry4A or Cry4B. The stability of the plasmid in the B. sphaericus host was moderate during vegetative growth, but segregational instability was observed, which led to substantial rates of plasmid loss during sporulation.     

Bacillus sphaericus Mtx1 and Mtx2 toxins co-expressed in Escherichia coli are synergistic against Aedes aegypti larvae

Mtx1 and Mtx2 are mosquitocidal toxins produced by some strains of Bacillus sphaericus during vegetative phase of growth. Mtx1 from B. sphaericus 2297 shows higher toxicity against Culex quinquefasciatus larvae than to Aedes aegypti larvae whereas Mtx2 from B. sphaericus 2297 shows lower toxicity against C. quinquefasciatus than to A. aegypti larvae. To test synergism of these toxins against A. aegypti larvae, mtx1 and mtx2 genes were cloned into a single plasmid and expressed in Escherichia coli. Cells producing both Mtx1 and Mtx2 toxins exhibited high synergistic activity against A. aegypti larvae approximately 10 times compared to cells expressing only a single toxin. Co-expression of both toxins offers an alternative to improve efficacy of recombinant bacterial insecticides. There is a high possibility to develop these toxins to be used as an environmentally friendly mosquito control agent.     

Mosquito larvicidal activity of Aloe vera (Family: Liliaceae) leaf extract and Bacillus sphaericus,against Chikungunya vector,Aedes aegypti

The bio-efficacy of Aloe vera leaf extract and bacterial insecticide, Bacillus sphaericus larvicidal activity was assessed against the first to fourth instars larvae of Aedes aegypti, under the laboratory conditions. The plant material was shade dried at room temperature and powdered coarsely. A. vera and B. sphaericus show varied degrees of larvicidal activity against various instars larvae of A. aegypti. The LC₅₀ of A. vera against the first to fourth instars larvae were 162.74, 201.43, 253.30 and 300.05 ppm and the LC₉₀ 442.98, 518.86, 563.18 and 612.96 ppm, respectively. B. sphaericus against the first to fourth instars larvae the LC₅₀ values were 68.21, 79.13, 93.48, and 107.05 ppm and the LC₉₀ values 149.15, 164.67, 183.84, and 201.09 ppm, respectively. However, the combined treatment of A. vera + B. sphaericus (1:2) material shows highest larvicidal activity of the LC₅₀ values 54.80, 63.11, 74.66 and 95.10 ppm; The LC₉₀ values of 145.29, 160.14, 179.74 and 209.98 ppm, against A. aegypti in all the tested concentrations than the individuals and clearly established that there is a substantial amount of synergist act. The present investigation clearly exhibits that both A. vera and B. sphaericus materials could serve as a potential larvicidal agent. Since, A. aegypti is a container breeder vector mosquito this user and eco-friendly and low-cost vector control strategy could be a viable solution to the existing dengue disease burden. Therefore, this study provides first report on the mosquito larvicidal activity the combined effect of A. vera leaf extract and B. sphaericus against as target species of A. aegypti.     

Efficacy of dry powders from Bacillus sphaericus: RB 80, a potent reference preparation for biological titration

Larvicidal potency of three primary powders based on Bacillus sphaericus strains 1593 and 1881 was studied on mosquito larvae. Two acetone powders, P 1593 and P 1881, were very toxic for Anopheles stephensi larvae. The potency of a third lyophilized powder RB 80 made from 1593 strain compared even better when tested against Anopheles stephensi and Culex pipiens pipiens larvae. LC₅₀'s after 48 hr were 0.15 and 0.003 mg/ml, respectively. After storage of RB 80 aqueous suspensions over 2 years or after heat exposure of RB 80 powder, larvicidal potency was still high, indicating an excellent stability. The use of RB 80, because of all its qualities, is suggested as a first experimental standard for titration of B. sphaericus preparations.     

Interpreting the SDS-PAGE protein patterns with self-organizing maps: application for the characterization of mosquito-pathogenic Bacillus strains

Aims: To present the pairwise comparison of potential mosquito‐pathogenic Bacillus strains based on their SDS‐PAGE protein patterns and to evaluate their characteristic toxicity patterns. Methods and Results: In this work, 20 Bacillus strains were subjected to qualitative toxicity tests against Aedes aegypti and Culex quinquefasciatus larvae. The selected strains were then characterized by SDS‐PAGE protein profiles. The highly heterogeneous multiple protein components of protein patterns were analysed using self‐organizing map (SOM), a ‘visualization and clustering’ tool. Members of mosquitocidal Bacillus species were classified in four distinct clusters, and then toxicity patterns were examined. Cluster (1, 1) comprised of three highly toxic strains of Bacillus sphaericus: SPH88, 1593 and KSD‐4; cluster (1, 2) consisted of two B. sphaericus strains: SSII‐1 and Bsp‐R that showed weak larvicidal activity; cluster (2, 1) constituted two B. sphaericus strains: WHO2297 and ISPC‐5 that possessed moderate toxicity; and cluster (2, 2) contained four B. thuringiensis ssp. israelensis strains: ONR‐60A, HD500, IPS70 and IPS82 belonging to serotype H14 but exhibited moderate to high mosquito larvicidal toxicity. Conclusions: SOM served as a colour‐coded alternate for easy visualization of similarities or dissimilarities between the strains even at the infra subspecies level. Furthermore, characteristic toxicity patterns of Bacillus strains of different clusters were determined. Significance and Impact of the Study: Analysis of electrophoretic protein patterns using SOM provides a better insight into the inter‐relationships of bacterial strains through similarity‐based clustering and pairwise comparison of two strains.     

Evolution of resistance to the Bacillus sphaericus Bin toxin is phenotypically masked by combination with the mosquitocidal proteins of Bacillus thuringiensis subspecies israelensis

Two insecticidal bacteria are used as larvicides to control larvae of nuisance and vector mosquitoes in many countries, Bacillus thuringiensis ssp. israelensis and B. sphaericus. Field studies show both are effective, but serious resistance, as high as 50 000‐fold, has evolved where B. sphaericus is used against Culex mosquitoes. To improve efficacy and deal with even greater potential problems of resistance, we previously developed several recombinant larvicidal bacteria that combine the best mosquitocidal proteins of these bacteria. In the present study, we report laboratory selection studies using our best recombinant strain against larvae of Culex quinquefasciatus. This recombinant, Bti/BsBin, is a strain of B. thuringiensis ssp. israelensis engineered to produce a large amount of the B. sphaericus binary (Bin) toxin, which makes it more than 10‐fold as mosquitocidal as the its parental strains. Here we show that larvae exposed to Bti/BsBin failed to develop significant resistance after 30 successive generations of heavy selection pressure. The highest level of resistance obtained at the LC₉₅ level was 5.2‐fold, but declined to less than two‐fold at the 35th generation. Testing the selected populations against B. sphaericus alone showed resistance to Bin evolved, but was masked by combination with B. thuringiensis ssp. israelensis. These results suggest that recombinant bacterial strains have improved mosquito and vector management properties compared with the wild‐type strains used in current commercial formulations, and should prove useful in controlling important human diseases such as malaria and filariasis on a long‐term basis, even when used intensively under field conditions.     

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.     

Cyt1Ab1 and Cyt2Ba1 from Bacillus thuringiensis subsp. medellin and B. thuringiensis subsp. israelensis Synergize Bacillus sphaericus against Aedes aegypti and Resistant Culex quinquefasciatus (Diptera: Culicidae)

The interaction of two cytolytic toxins, Cyt1Ab from Bacillus thuringiensis subsp. medellin and Cyt2Ba from Bacillus thuringiensis subsp. israelensis, with Bacillus sphaericus was evaluated against susceptible and resistant Culex quinquefasciatus and the nonsensitive species Aedes aegypti. Mixtures of B. sphaericus with either cytolytic toxin were synergistic, and B. sphaericus resistance in C. quinquefasciatus was suppressed from >17,000- to 2-fold with a 3:1 mixture of B. sphaericus and Cyt1Ab. This trait may prove useful for combating insecticide resistance and for improving the activity of microbial insecticides.     

球形芽孢杆菌对致倦库蚊的后致死作用

研究了球形芽孢杆菌Bacillus sphaericus C3-41菌株对致倦库蚊Culex quinquefasciatus幼虫的毒力及其后致死作用。生物测定表明,该菌株对目标蚊幼虫具有很高的毒力,其丙酮粉剂对3～4龄幼虫48 h的半致死浓度(LC₅₀)为(6.92±0.22) μg/L。用不同亚致死浓度处理2～3龄致倦库蚊幼虫,存活幼虫在后期发育中存在明显的延续死亡和损伤现象,经LC₃₀、LC₅₀、LC₇₀、LC₉₀和LC₉₈剂量的C3.41粉剂处理的致倦库蚊羽化前的总死亡率分别为84%、91%、95%、97%和100%,同时存活的幼虫、蛹和成蚊的发育和行为也受到一定的影响。这种后致死作用随处理浓度的升高而增强,可能同球形芽孢杆菌毒素蛋白对处理期间蚊幼虫中肠上皮细胞造成的损伤相关。     

Purification of the toxic protein from Bacillus thuringiensis serotype 10 isolate demonstrating a preferential larvicidal activity to the mosquito

The protein demonstrating larvicidal activity to the mosquito Aedes aegypti was purified from the alkali extract of the spore-parasporal inclusion complex of the isolate, 73-E-10-2, belonging to Bacillus thuringiensis serotype 10. By Sepharose CL-4B gel filtration and DEAE-cellulose column chromatography, a toxic protein was obtained, and its homogeneity was confirmed by Sephadex G-150 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the toxic protein was 67,000, when estimated by SDS-PAGE. The LC₅₀ of the toxic protein against 4-day-old larvae of A. aegypti was 16.8 μg/ml. There was no serological relationship between the toxic protein from the isolate 73-E-10-2 and that (M_r 67,000) from the type strain of B. thuringiensis subsp. israelensis.     

Improving the Insecticidal Activity against Resistant Culex quinquefasciatus Mosquitoes by Expression of Chitinase Gene chiAC in Bacillus sphaericus

Expression of a chitinase gene, chiAC, from Bacillus thuringiensis in B. sphaericus 2297 using the binary toxin promoter yielded a recombinant strain that was 4,297-fold more toxic than strain 2297 against resistant Culex quinquefasciatus. These results show that this chitinase can synergize the toxicity of the binary toxin against mosquitoes and thus may be useful in managing mosquito resistance to B. sphaericus.     

Characterization of Cyt2Bc Toxin from Bacillus thuringiensis subsp. medellin

We cloned and sequenced a new cytolysin gene from Bacillus thuringiensis subsp. medellin. Three IS240-like insertion sequence elements and the previously cloned cyt1Ab and p21 genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from Bacillus thuringiensis subsp. israelensis and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of B. thuringiensis. Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, including strains resistant to the Bacillus sphaericus binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant C. quinquefasciatus.     

Introduction of Culex Toxicity into Bacillus thuringiensis Cry4Ba by Protein Engineering

Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC₅₀], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba. The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C. quinquefasciatus, and C. pipiens. Putative loop 1 and 2 exchanges eliminated activity towards A. aegypti and A. quadrimaculatus. Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C. quinquefasciatus (LC₅₀ 114 ng/ml) and C. pipiens (LC₅₀ 37 ng/ml), respectively. The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or Aedes. These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B. thuringiensis Cry toxins.     

Development of low-cost media for the culture of mosquito larvicides, Bacillus sphaericus and Bacillus thuringiensis serovar. israelensis

Mosquito larvicides like Bacillus sphaericus and Bacillus thuringiensis serovar. israelensis have been widely and effectively used in mosquito control programs, but the industrial production of these bacilli is expensive. Here we have attempted to develop three cost-effective media, based on cheap sources, potato, common sugar and bengalgram. Growth and production of the insecticidal proteins from these bacteria were satisfactory. Bioassay studies with different mosquito larvae showed considerable toxicity. Therefore the investigation suggests that potato-based culture media are more economical for the industrial production of B. sphaericus and B. thuringiensis serovar. israelensis.     

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.