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.     

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.     

Purification and properties of soluble cytoplasmic toxin from the mosquito pathogen Bacillus sphaericus strain 1593

Soluble cytoplasmic toxin from broken Bacillus sphaericus 1593 sporulating cells was partially purified by ammonium sulfate precipitation, ion exchange, and gel filtration chromatography. Purification was monitored by electrophoresis. The toxin remained active after incubation in the presence of several enzymes and in buffers from pH 6 to 10, but was destroyed by Pronase and subtilisin, and by heating to 80°C for 30 min. Results indicate that the B. sphaericus 1593 cytoplasm contains a single proteinaceous toxin with a molecular weight of 100,000 daltons.     

Bioassay of three strains of Bacillus sphaericus on field-collected mosquito larvae.

Bacillus sphaericus strains 1593, 1404, and SSII-1 were assayed for infectivity against field-collected larvae of Psorophora columbiae, Culex nigripalpus, and Aedes taeniorhynchus in southwest Florida. Results indicate that all three strains are highly active against the Psorophora and Culex species. A. taeniorhynchus is also susceptible but requires higher dosages to achieve lethal responses. Tests were also conducted on the rate of infection and the differences in susceptibility of different instars to B. sphaericus. These tests indicate that nearly 75% of the mortality that occurs in the course of exposure to B. sphaericus occurs within 48 hr post-incubation with the bacteria. Furthermore, our tests indicate P. columbiae larvae decrease in susceptibility to the Bacillus with increase in larval age (instar). This investigation shows B. sphaericus to be a feasible biological control agent that warrants further study.     

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.     

Insecticidal Activity of Bacillus laterosporus

The Bacillus laterosporus strains 921 and 615 were shown to have toxicity for larvae of the mosquitoes Aedes aegypti, Anopheles stephensi, and Culex pipiens. The larvicidal activity of B. laterosporus was associated with spores and crystalline inclusions. Purified B. laterosporus 615 crystals were highly toxic for Aedes aegypti and Anopheles stephensi.     

Studies on the culicine mosquito host range of Bacillus sphaericus and Bacillus thuringiensis var. israelensis with notes on the effects of temperature and instar on bacterial efficacy

Toxicity tests of three strains of Bacillus sphaericus against late instars of 12 culicine mosquito species indicated a wide range of susceptibility. Culex pipiens and C. salinarius were highly susceptible (LC₅₀s < 10⁴ spores/ml) to strain 1593, and C. pipiens and C. restuans were highly susceptible to strain 2013-4. The potency of strain SSII-1 was approximately one-tenth that of strains 1593 and 2013-4 against C. pipiens. Susceptibility of Aedes species to strain 1593 was highly variable. At temperatures ≥ 20°C, A. fitchii, A. intrudens, A. stimulans, and A. vexans were moderately to highly susceptible (LC₅₀s 6 × 10³−4 × 10⁴ spores/ml), A. triseriatus was only slightly susceptible (LC₅₀ > 10⁶ spores/ml), and A. aegypti was refractory. Susceptibility of Aedes mosquitoes to strain SSII-1 was less variable, with LC₅₀s against A. aegypti, A. canadensis, A. stimulans, and A. triseriatus all being between 10⁴ and 10⁶ vegetative cells + spores/ml. All species of mosquitoes tested were, in general, highly susceptible to B. thuringiensis var. israelensis (LC₅₀s 2.3 × 10³−2.5 × 10⁴ spores/ml). In B. sphaericus toxicity tests, decreased temperatures resulted in up to a 16-fold increase in LC₅₀ and a substantial reduction in probit line slope. First-instar A. aegypti larvae were more susceptible to B. sphaericus strain SSII-1 than the three later instars, which were approximately equally susceptible; however, no significant difference was observed in the susceptibility of the four instars of A. triseriatus.     

Selection of the most potent Bacillus sphaericus strains based on activity ratios determined on three mosquito species

Summary The larvicidal power of more than 180 Bacillus sphaericus strains belonging to six H serotypes has been assayed on Culex pipiens, Anopheles stephensi and Aedes aegypti under standardized conditions. The most potent strains are distributed into serotype H5a5b, generally toxic to the three mosquito species, and serotypes H6 and H25, toxic to C. pipiens and A. stephensi. Strains of serotypes 26a26b and H2a2b are much less toxic and most often only on C. pipiens. The relative potency of each strain can be expressed by specific titres on the different mosquito species and by activity ratios derived from such titres.     

Evaluation of the toxicity of different phytoextracts of Ocimum basilicum against Anopheles stephensi and Culex quinquefasciatus

The larvicidal effect of the crude carbon tetrachloride, methanol and petroleum ether leaf extracts of a widely grown medicinal plant, Ocimum basilicum, against Anopheles stephensi and Culex quinquefasciatus was evaluated. Petroleum ether extract was found to be the most effective against the larvae of both mosquitoes, with LC₅₀ values of 8.29, 4.57; 87.68, 47.25 ppm and LC₉₀ values of 10.06, 6.06; 129.32, 65.58 ppm against A. stephensi and C. quinquefasciatus being observed after 24 and 48 h of treatment, respectively. The efficacy of petroleum ether was followed by that of the carbon tetrachloride and methanol extracts, which had LC₅₀ values of 268.61, 143.85; 446.61, 384.84 ppm and LC₉₀ values of 641.23, 507.80; 923.60, 887.00 ppm against A. stephensi after 24 and 48 h, respectively, and LC₅₀ values of 24.14, 17.02; 63.48, 53.77 ppm and LC₉₀ values of 295.38, 204.23; 689.71, 388.87 ppm against C. quinquefasciatus after 24 and 48 h of treatment, respectively. These extracts are highly toxic against mosquito larvae from a range of species; therefore, they may be useful for the management of mosquito larvae to control vector borne diseases.     

Insecticidal activity of the crystalline parasporal inclusions and other components of the Bacillus sphaericus 1593 spore complex

Bacillus sphaericus 1593 spore complexes were disrupted by French pressure cell. Fractions recovered from centrifugation of these complexes on 10–50% NaBr gradients were assayed against mosquito larvae and examined using the electron microscope. Crystalline parasporal inclusions were concentrated in the fraction of highest insecticidal activity. The fractions containing sporangium, exosporium, and spores also were insecticidal at a lower level. These results indicate that the crystals are the major source of insecticidal toxin in strains of B sphaericus which produce them.     

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.     

Comparative studies of eleven isolates of the fungal entomopathogen Tolypocladium cylindrosporum and two isolates of Tolypocladium extinguens

The virulence of 11 isolates of Tolypocladium cylindrosporum and 2 isolates of Tolypocladium extinguens was evaluated against mosquito larvae. Second-instar Aedes aegypti larvae were more susceptible to infection by blastospores than were second-instar Anopheles stephensi larvae. Only the T. extinguens isolates were not infectious for A. aegypti or A. stephensi. Nine of the remaining strains killed 100% of larvae at the highest dose tested (10⁶ blastospores/ml). In vitro sporulation and conidiospore size were also studied. Significant differences were observed for both characteristics. Characterization of strains according to in vitro sporulation and virulence made it possible to select the most promising strains for future laboratory and field tests against mosquitoes.     

Plasmid deoxyribonucleic acid in strains of Bacillus sphaericus and in Bacillus moritai

Bacillus moritai and six strains of Bacillus sphaericus pathogenic to dipteran larvae were examined for the presence of covalently closed circular (CCC) DNA. The plasmid profiles of the bacteria were analyzed using a cleared lysate electrophoresis technique. Four of the six strains of B. sphaericus examined contained CCC DNA. Strain SSII-1 contained two plasmids (pKA1, pKA2) having molecular weights of about 8.4 and 2.0 megadaltons (MDa). Strains 1404 and 1881 each contained one plasmid, pKA3 and pKA4, respectively. pKA3 had a molecular weight of about 8.2 MDa. pKA4 had a relatively large plasmid with a molecular weight of about 33.5 MDa. Strain K contained five size classes of CCC DNA. The plasmids pKA5, pKA6, pKA7, pKA8, and pKA9 had molecular weights of about 11.4, 10.9, 7.4, 7.0, and 6.4 MDa, respectively. Strains 1593-4 and 1691 were plasmidless and could not be distinguished from each other based on their plasmid profiles. B. moritai ATCC 21042 contained two size classes of CCC duplex DNA; pRF100 had a molecular weight of about 4.6 MDa and pRF101 had a molecular weight of about 2.1 MDa. No phenotype association with any of the isolated plasmids has been determined.     

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.     

The effect of the microsporidan, Nosema algerae, on Anopheles stephensi

A bioassay method was established to examine infectivity differences between different batches of Nosema algerae spores. The IC₅₀ of N. algerae spores produced in one unusual host, Heliothis zea, was the same as for spores from the normal mosquito host, Anopheles stephensi. Soil and sand bottoms caused an approximate 200–400 fold increase in the IC₅₀. Nosematosis had little effect on the survival of larvae and pupae but the adult life span was reduced to the extent that malaria transmission would be doubtful.     

Evaluation of larvicidal potential of certain insect pathogenic fungi extracts against Anopheles stephensi and Culex quinquefasciatus

Fungal metabolites are attracting attention as potential microbial insecticides, and they are anticipated to overcome the problems of pesticide resistance and environmental pollution that are associated with the indiscriminate use of conventional synthetic insecticides. The relative bioefficacies of selected fungal pathogens, Aspergillus flavus, A. niger, A. parasiticus, Fusarium sporotrichoides and Penicillium verrucosum were observed against Anopheles stephensi and Culex quinquefasciatus larvae. A. flavus demonstrated the greatest bioefficacy with 50% lethal concentration (LC₅₀) values of 9.54 and 10.98 ppm against Anopheles stephensi and Culex quinquefasciatus larvae, respectively, after 24‐h exposure. The bioefficacy of A. flavus increased in both species with an exposure time of 48 h, with LC₅₀ values of 7.26 and 8.55 ppm, respectively.     

Virulence of natural and insect-passaged strains of Metarhizium anisopliae to mosquito larvae

Forty-seven isolates of Metarhizium anisopliae var. anisopliae (small-spored form) and five isolates of M. anisopliae var. major (large-spored form) obtained from widely separated geographical regions from various insect hosts were screened for virulence against Culex pipiens pipiens larvae. Pathogenesis was variable with mortalities ranging from 0 to 100%. However, much of the variation in mortality among small-spored isolates was due to lowered natural viabilities. The most virulent isolates were from Austria, Australia, and Brazil from insect species in three different orders. Isolates from the major strain were generally avirulent. There was no correlation of strain morphology, geographical region of isolation, or original host species with strain virulence. The strains most virulent to C. pipiens larvae were also highly infective to Aedes aegypti and Anopheles stephensi larvae. Virulence of two strains (E₆ and E₉) to C. pipiens larvae was significantly enhanced by one passage through a C. pipiens larval siphon. Relative potencies increased approximately 1.63 to 2.45 times. A smaller increase in virulence, depending upon the isolate, was also shown when these same strains were tested against A. aegypti and A. stephensi. Virulence of strain E₉ was also increased significantly by passage through an alternate host, Nilaparvata lugens.     

Isolation and identification of entomopathogenic fungus from Eastern Ghats of South Indian forest soil and their efficacy as biopesticide for mosquito control

The repeated usage of chemical insecticides, responsible for insecticide resistance in mosquitoes and environmental toxicity. Currently effective and environmental-safe control strategies are needed for the control disease-vector mosquitoes. Entomopathogens can be an effective alternative to chemical insecticide. Herein we isolated and tested 46 soil-borne entomopathogenic fungi belonging to six genera, namely Beauveria sp., Metarhizium sp., Fusarium sp., Aspergillus sp., Trichoderma sp., and Verticillium sp., fungi conidia were tested on Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus larvae. Bioassays results show that M. anisopliae fungal isolate causes a 100%, 98.6% and 92% mortality within six days, on Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus, respectively. M. anisopliae treated three mosquito larvae have lower lifetime with LT₅₀ values in A. stephensi, 2.931 days; A. aegypti, 2.676 days and C. quinquefasciatus, 3.254 days. 18 s rDNA sequence analysis confirmed that the isolated fungus are belonging to the genus of M. anisopliae-VKKH3, B. bassiana-VKBb03, and V. lecanii-VKPH1. Our results clearly show that M. anisopliae has good potential, as a low-cost, environmentally safe tool for the control of A. aegypti, A. stephensi, and C. quinquefasciatus mosquitoes.     

Fitness cost of malaria vector Anopheles stephensi induced with larvicidal and nutritional stress

Our study focused on how externally applied single or multiple stressors alter the fitness of early IV instar Anopheles stephensi larvae by inducing various larval stressors such as starvation and sublethal doses (LC₁₀, LC₂₅ and LC₅₀ for 24, 48 and 72 h) of various conventional and biorational larvicides. Larval stress specific response was observed in terms of their nutritional (glycogen, sugar, lipid and protein) and biochemical (DNA) status compared with respective control group which were found to be significantly (P < 0.05) altered. Nutrition depletion index was found to be concentration dependent depicting maximum reduction at LC₅₀ concentration with all applied larvicides. Significant (P < 0.05) reduction in DNA level was observed only with neem oil (10–23%) and Bti (21–23%) treatments. DNA damage was further evidenced by generating RAPD profiles that revealed variations in band intensities along with addition or deletion of few band in stress induced larvae. Overall, our results depicted that An. stephensi larvae may possibly tolerate the induced stress within certain limits by modifying their nutritional and biochemical levels, which may occur at a significant fitness cost.     

Enhanced expression of a second mosquito larvicidal gene fromB.sphaericus 1593M inE.coli

Summary Enhanced expression of a second mosquito larvicidal gene fromB.sphaericus 1593M inE.coli has been achieved by the recloning of the DNA fragment encoding for larvicidal activity previously reported by us, in a pMal vector system. The potency of this recombinant strain was only 10 fold lower than the parentalB.sphaericus 1593M strain. The protein encoded was different from the previously reported larvicidal gene products ofB.sphaericus. Neverthelesss, this protein is recognized by the antiserum raised against crystal proteins. This result has indicated the presence of multiple mosquito larvicidal genes inB.sphaericus, a situation similar to that encountered withB.thuringiensis toxins.