The comparative persistence of toxicity of Bacillus sphaericus strain 1593 and Bacillus thuringiensis serotype H-14 against mosquito larvae in different kinds of environments

Bacillus sphaericus strain 1593 and B. thuringiensis serotype H-14 were evaluated for persistence of toxicity against two species of mosquito larvae, Culex quinquefasciatus and Aedes aegypti, in a selected simulating plot in Bangkok. Both strains of bacteria demonstrated larvicidal activity towards both species of mosquito larvae. In tap water, the toxicity of B. sphaericus strain 1593 was found to be greater towards C. quinquefasciatus larvae than A. aegypti larvae, whereas the toxicity of B. thuringiensis serotype H-14 was found to be greater towards A. aegypti larvae than C. quinquefasciatus larvae. The persistence of toxicity of these two bacteria was found to be different. The lethal concentration of B. thuriengiensis H-14 against A. aegypti decreased from LC₉₀ to below LC₅₀ in about 15 weeks when tested in tap water. The decrease was faster in polluted water. The toxicity of B. sphaericus 1593 towards C. quinquefasciatus larvae persisted for at least 9 months in tap water and 6 months in polluted water. The multiplication of bacteria was indicated only in populations of B. sphaericus 1593 tested with C. quinquefasciatus larvae.     

Preparation of a flowable liquid bacterial insecticide based on Bacillus sphaericus

Fermenter-produced Bacillus sphaericus 2362 was formulated into a thick, dark flowable liquid concentrate containing 4.8×10⁹ c.f.u./ml and charcoal as protector against ultraviolet light. The potencies of the formulation against L₄ Culex pipiens quinquefasciatus before and after storage for 2 years were 5714 and 5862 International Toxic Units (ITU), respectively, when compared with a standardized B. sphaericus from the WHO at 1000 ITU. In field trials, treatment at 1.01/ha gave 96 to 100% control of mosquito larvae. B. sphaericus could be re-isolated in 5% of the samples 9 months after application.The authors are at the Department of Applied Microbiology & Brewing, Anambra State University of Technology, P.M.B. 5025, Awka, Nigeria.     

球形芽孢杆菌C3-41对致倦库蚊的毒效及在蚊体内的再循环

球形芽孢杆菌Csub3-41菌株(Bacillus sphaericus C3-41)对致倦库蚊(Culex puinquefa-seiatus)幼虫有很高毒效,对2龄和3-4龄幼虫的半致死剂量(LD₅₀)分别为63.1 和89.7芽孢/蚊幼虫。处理浓度越高,取食时间越长,蚊幼虫取食到的杀蚊活性物质量越多,死亡率越高。当蚊幼虫取食亚致死剂量杀蚊活性物质后,球形芽孢杆菌在感染的活幼虫体内不增殖;但当蚊幼虫取食致死剂量杀蚊活性物质后,蚊幼死亡,球形芽孢杆菌在死蚊幼虫体内增殖明显,6天内芽孢从感染初期的1.86X10²蚊幼虫增加到1.59X10⁶/蚊幼虫。芽孢在死蚊幼虫体内能正常萌发、生长、产孢和形成毒素。增殖的芽孢同样对致倦库蚊幼虫有较高毒力。     

Pathogenesis of Bacillus sphaericus strain SSII-1 infections in Culex pipiens quinquefasciatus ( = C. pipiens fatigans) larvae

Numbers of viable bacteria in second instar Culex pipiens quinquefasciatus larvae were determined following ingestion of pathogenic strain SSII-1 and nonpathogenic Bacillus sphaericus. Numbers of nonpathogenic B. sphaericus recovered from larvae declined rapidly after cessation of feeding, as did numbers of pathogenic SSII-1 cells fed at LD₂₀ dosage. When pathogenic cells were fed at LD₇₀ dosage, the number of B. sphaericus in larvae increased following initial decline. When chloroformtreated SSII-1 cultures, in which all bacteria except spores were dead, were fed at LD₁₀ and LD₉₈ dosages, no viable B. sphaericus were recovered from larvae. In all SSII-1 treatments, other bacterial flora multiplied rapidly in larvae following onset of mortality; the role of this multiplication in the pathogenesis was not determined. It is proposed that toxic material is released when SSII-1 cells are digested and that multiplication of B. sphaericus in the larval gut is not essential in the pathogenesis. There appears to be no difference in the pathogenesis when differing numbers of B. sphaericus. i.e., LD_10–20 or LD_70–98 dosages, are ingested. Possible nature of the toxic material is discussed.     

A cost-effective medium for the large-scale production of Bacillus sphaericus H5a5b (VCRC B42) for mosquito control

Bacillus sphaericus has been widely used in mosquito control programs, but the large-scale production of this bacterium is expensive because of the high cost of the medium. In this study, we attempted to develop a cost-effective medium, based on inexpensive, locally available raw materials including soybean flour (Glycine max) and peanut cake powder (Arachis hypogea) by using 100-l bioreactor. Sporulation, toxicity and biomass were satisfactory after B. sphaericus was produced on both media. Use of the soybean culture medium resulted in “maximum” toxicity (LC₅₀ 14.02 ng/ml against third instar Culex quinquefasciatus larvae), highest spore count (3.7 × 10⁹ spores/ml) and maximum biomass (4.6 g/l) within a short fermentation time of 21 h. Hence, this soybean-based culture medium was considered most economical for the large-scale industrial production of B. sphaericus.     

Efficient expression of mosquito-larvicidal proteins in a gram-negative bacterium capable of recolonization in the guts of Anopheles dirus larva

The gram-negative bacterium, An11/2 G1, isolated from the guts of Anopheles dirus mosquito larvae, was identified as Enterobacter amnigenus. The E. amnigenus was able to recolonize in the gut of An. dirus larva but not in those of Aedes aegypti and Culex quinquefasciatus larvae. It was able to float in water for a longer period than Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus. These are desirable characteristics for a delivery vehicle of mosquito-larvicidal toxins for the control of mosquito larvae, and E. amnigenus was therefore used as a host to express the cryIVB gene of B. thuringiensis subsp. israelensis and the binary toxin genes of B. sphaericus. The recombinant E. amnigenus produced a high level of CryIVB protein, which was toxic to larvae of Ae. aegypti and An. dirus. Another E. amnigenus producing the 51-kDa protein of B. sphaericus was toxic to larvae of An. dirus and Cx. quinquefasciatus. The recombinant plasmids were stable in E. amnigenus without the presence of selective pressure for at least 23 generations. The recombinant E. amnigenus should represent a desirable biological agent for controlling mosquito larvae. Received: 20 February 1998 / Received last revision: 5 October 1998 / Accepted: 11 October 1998     

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.     

Effects of Bacillus sphaericus 1593 and 2362 spore/crystal toxin on cultured mosquito cells

An in vitro assay system for the toxin of Bacillus sphaericus strains 1593 and 2362 has been developed utilizing cultured Culex quinquefasciatus cells. The cytotoxic activity of extracts of B. sphaericus strain 1593 did not necessarily correlate with insecticidal activity. Cytotoxicity and larvicidal activity were neutralized by immune rabbit serum prepared against crude toxin extracts as well as by serum prepared against purified toxin from strain 2362. This purified toxin was also found to be cytotoxic. Activation with mosquito larval gut homogenates enhanced cytotoxicity of both 1593 extracts and purified toxin from 2362. The activity of cytotoxic preparations against three mosquito cell lines paralleled the activity of B. sphaericus spores against larvae of these mosquito species. The results suggest the presence of a protoxin and one or more cytotoxic proteins derived from it.     

Detection of an Allele Conferring Resistance to Bacillus sphaericus Binary Toxin in Culex quinquefasciatus Populations by Molecular Screening

The activity of the Bacillus sphaericus binary (Bin) toxin on Culex quinquefasciatus larvae depends on its specific binding to the Cqm1 receptor, a midgut membrane-bound α-glucosidase. A 19-nucleotide deletion in the cqm1 gene (cqm1_REC) mediates high-level resistance to Bin toxin. Here, resistance in nontreated and B. sphaericus-treated field populations of C. quinquefasciatus was assessed through bioassays as well as a specific PCR assay designed to detect the cqm1_REC allele in individual larvae. Resistance ratios at 90% lethal concentration, gathered through bioassays, were close to 1 and indicate that the selected populations had similar levels of susceptibility to B. sphaericus, comparable to that of a laboratory colony. A diagnostic PCR assay detected the cqm1_REC allele in all populations investigated, and its frequency in two nontreated areas was 0.006 and 0.003, while the frequency in the B. sphaericus-treated population was significantly higher. Values of 0.053 and 0.055 were detected for two distinct sets of samples, and homozygote resistant larvae were found. Evaluation of Cqm1 expression in individual larvae through α-glucosidase assays corroborated the allelic frequency revealed by PCR. The data from this study indicate that the cqm1_REC allele was present at a detectable frequency in nontreated populations, while the higher frequency in samples from the treated area is, perhaps, correlated with the exposure to B. sphaericus. This is the first report of the molecular detection of a biolarvicide resistance allele in mosquito populations, and it confirms that the PCR-based approach is suitable to track such alleles in target populations.     

Metal tolerance and larvicidal activity of Lysinibacillus sphaericus

Lysinibacillus sphaericus is a spore-forming bacterium used in the biological control of mosquitoes and in bioremediation. Mosquito larvae exposed to heavy metals are tolerant to concentrations above the permissible limit for industrial residual waters. In this work, we characterize 51 L. sphaericus strains for metal tolerance and larvicidal activity against Culex quinquefasciatus. Lysinibacillus sphaericus OT4b.2, OT4b.20, OT4b.25, OT4b.26 and OT4b.58 were as toxic as the spores of the reference strain 2362 against C. quinquefasciatus larvae. 19 Mosquito-pathogenic L. sphaericus strains and 6 non-pathogenic strains were able to grow in arsenate, hexavalent chromium and/or lead. 16S rRNA gene sequences and phylogenetic analyses clustered 84 % of the metal-tolerant strains in L. sphaericus group 1, which encompasses the mosquitocidal strains. The larvicidal activity of vegetative and sporulated cells and its high tolerance to arsenate, hexavalent chromium and lead indicate that L. sphaericus OT4b.26 is a strong candidate for further studies examining its potential for biological control of mosquitoes in waters contaminated with metals.     

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.     

UV protectants for the biopesticide based on Bacillus sphaericus Neide and their role in protecting the binary toxins from UV radiation

The UV protectant properties of 26 natural and synthetic compounds were investigated for a biopesticide based on an indigenously isolated strain (ISPC-8) of Bacillus sphaericus Neide. In initial screening, spores of ISPC-8 with 0.1% (w/w for solid and v/w for liquid materials) concentration of different compounds were exposed to UV-B radiation (4.9 × 10⁵ J/m²) for 6 h and their spore viability and larvicidal activity were studied. The larvicidal activity was evaluated against third-instar larvae of Culex quinquefasciatus Say. There was a complete loss of spore viability (1.4% viable spores) and partial reduction in larvicidal activity (57.7% of original activity) after exposure of spores to UV-B for 6 h. However, spore viability as well as larvicidal activity protected significantly when spores were mixed with different compounds before exposing them to UV-B. Among the different compounds tested benzaldehyde, congo red, para-aminobenzoic acid (PABA) and cinnamaldehyde were found to be promising in protecting the spores from UV-B radiation. The presence of binary toxins (41.9 kDa and 51.4 kDa) in protected and unprotected samples were examined by SDS–PAGE. The binary toxin bands disappeared in unprotected spores after 24 h of exposure to UV-B, whereas toxin bands were distinctly visible when spores with benzaldehyde and cinnamaldehyde were exposed to UV-B for 96 h and 120 h, respectively. Congo red and PABA were found to be most effective in protecting binary toxins even after 168 h of exposure to UV-B. Incorporation of these promising UV protectant compounds in biopesticides would help in protecting the spores from the adverse effects of UV radiation and prolong the persistence of biopesticides under field conditions.     

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.     

Expression of Binary Toxin Genes in the Mosquito-Colonizable Bacteria, <Emphasis Type="Italic">Bacillus cereus</Emphasis>, Leads to High Toxicity Against <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> Larvae

Two B. cereus strains, Ae10 and Cx5, isolated from mosquito larval guts, were transformed with a recombinant plasmid, pBS373, harboring binary toxin genes from Bacillus sphaericus 2297. Immunoblotting analysis clearly revealed the production and presence of the 51-kDa toxin protein in both strains. Two recombinant B. cereus strains Ae10 and Cx5 showed very high toxicity against C. quinquefasciatus larvae. Since both strains have a close relationship with the mosquito larvae in the native environment and are capable of recolonizing in the guts of mosquito larvae, these strains can be considered promising new hosts for an effective delivery of mosquito-larvicidal toxins.     

Stored Bacillus popilliae spores and their infectivity against Popillia japonica larvae

Bacillus popilliae spores were stored for about 7 years under three separate conditions: frozen in sterile distilled water, smeared on glass microscope slides, and stored in loam soil at room temperature. In separate experiments, each of the 7-year-old preparations was fed to Popilla japonica larvae at concentrations of 10³, 10⁵, 10⁷, and 10⁹ spores/g of soil. A significant decrease in the percentage of larvae infected occurred in all of the aged spore tests. B. popilliae spores stored in soil, for the extended period, produced 3% larval infection only at the 10⁹ spores concentration; similar results were obtained from frozen spores. When P. japonica larvae were fed spores stored dried on slides, about 20% of the larvae developed milky disease. When aged frozen spores were artificially injected into larvae, 12% became infected at concentrations of 1 × 10⁶ spores/larvae; dried spores at the same concentration infected about 38% of the insect larvae. We conclude from these data that aged B. popilliae spores are significantly less infective against P. japonica larvae than young spores.     

Effects of components of theBacillus sphaericus toxin on mosquito larvae and mosquito-derived tissue culture-grown cells

Bacillus sphaericus 2362 produces a parasporal crystal containing 42 and 51 kilodalton (kDa) proteins. Both of these proteins are required for toxicity to mosquito larvae; neither is toxic alone. When overexpressed inB. subtilis, these two proteins accumulate as amorphous inclusions (AIs). Bioassays involving larvae ofCulex pipiens and different ratios of these AIs indicated that maximal toxicity was observed at a ratio of approximately one 42-kDa protein to one 51-kDa protein. Purified preparations of these proteins, as well as derivatives similar to those which accumulate in the gut of mosquito larvae, were also toxic when combined, but not toxic singly. Different results were obtained when the toxicity of these preparations was tested for tissue culture-grown cells ofC. quinquefasciatus. Under these conditions, the 39-kDa derivative of the 42-kDa protein was alone sufficient for toxicity, which was not increased by the addition of the 51-kDa protein or its derivatives. These results indicate that theB. sphaericus larvicide acts as a binary toxin in mosquitos, whereas only the 39-kDa protein is required for full toxicity to tissue culture-grown cells.     

Concentration of larvicidal Bacillus spores at the water surface by adherence to oil droplets

Adherence of two larvicidal spores, Bacillus sphaericus 2297 and Bacillus thuringiensis israelensis, to hexadecane and corn oil was determined. Adherence was greatly promoted in the presence of 2 M ammonium sulphate, reaching levels of up to 89%. The data suggest that larvicidal spores may be concentrated at their operational niche (the water surface) by flotation, adhering to the surface of oil droplets. The data suggest that adherence of the spores to oil droplets may enable their concentration at the water surface, where they can effectively kill mosquito larvae.     

A field study of potential ecological costs of resistance by ‘stem ducking’ in tall goldenrod,Solidago altissima

Culex quinquefasciatus Say (Diptera: Culicidae) is an abundant urban mosquito that is the vector of filariasis. Breeding in septic tanks, where there are very high levels of bacterial food, it is likely to have a different reaction to crowding compared with other mosquitoes. To test for the presence and type of crowding effects, four larval densities of C. quinquefasciatus varying from 0.4 to 3.2 larvae ml^?1 of water were reared in tubes. Mortality was found to greatly increase at densities above 0.8 larvae, whereas larval duration increased even above 0.4 larvae ml^?1. Changing the water in the tubes daily gave a small (but significant) response in reducing mortality and larval duration. However, when larvae kept at a low density shared the same water with larvae at high density, there was no chemical influence on their growth rate and mortality. The effect of crowding was primarily due to physical disturbances between larvae. When larvae were kept at a high density in the same volume of water, but in shallow trays with a large surface area and therefore much less contact between them, mortality was the same as for the lowest density. There was still, however, a significant increase in larval duration from 8.6 days at 0.4 larvae ml^?1 to 12.1 days at 3.2 larvae ml^?1. It is therefore concluded that the larvae respond to physical rather than chemical factors by prolonging larval development and having some increase in mortality.     

A Strain of Bacillus sphaericus Causes Slower Development of Resistance in Culex quinquefasciatus

Two field-collected Culex quinquefasciatus colonies were subjected to selection pressure by three strains of Bacillus sphaericus, C3-41, 2362, and IAB59, under laboratory conditions. After 13 and 18 generations of exposure to high concentrations of C3-41 and IAB59, a field-collected low-level-resistant colony developed >144,000- and 46.3-fold resistance to strains C3-41 and IAB59, respectively. A field-collected susceptible colony was selected with 2362 and IAB59 for 46 and 12 generations and attained >162,000- and 5.7-fold resistance to the two agents, respectively. The pattern of resistance evolution in mosquitoes depended on continuous selection pressure, and the stronger the selection pressure, the more quickly resistance developed. The resistant colonies obtained after selection with B. sphaericus C3-41 and 2362 showed very high levels of cross-resistance to B. sphaericus 2362 and C3-41, respectively, but they displayed only low-level cross-resistance to IAB59. On the other hand, the IAB59-selected colonies had high cross-resistance to both strains C3-41 and 2362. Additionally, the slower evolution of resistance against strain IAB59 may be explained by the presence of another larvicidal factor. This is in agreement with the nontoxicity of the cloned and purified binary toxin (Bin1) of IAB59 for 2362-resistant larvae. We also verified that all the B. sphaericus-selected colonies showed no cross-resistance to Bacillus thuringiensis subsp. israelensis, suggesting that it would be a promising alternative in managing resistance to B. sphaericus in C. quinquefasciatus larvae.