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.     

Growth,sporulation and larvicidal activity of Bacillus sphaericus

Summary A new medium (MBS) for optimal sporulation of Bacillus sphaericus was defined. With the two main mosquito pathogenic strains grown in this medium, 1593-4 and 2297, highest cell and spore yields were obtained, concomitantly with an highest larvicidal activity against Culex pipiens. Study of both strains asporulated mutants showed a decrease in larvicidal power. After plasmid curing treatments, toxicity of strain 1593-4 did not decrease, neither toxic parasporal inclusion bodies of strain 2297 disappear.     

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.     

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.     

Cloning and expression in Escherichia coli of two DNA fragments from Bacillus sphaericus encoding mosquito-larvicidal activity

A library of Bacillus sphaericus 1593 DNA was constructed in Escherichia coli using pBR322 as vector and screened for clones expressing larvicidal activity against Culex mosquito larvae. Two larvicidal clones were identified and their plasmids characterized by restriction mapping. pAS233 and pAS377 contained inserts of 8.6 and 15 kb which were reduced by subcloning to 3.6 and 4.3 kb, respectively. A peptide of 29 kDa was the single product detected by maxicell expression of pAS377PT, a plasmid subcloned from pAS377. No insert-encoded peptide could be detected for pAS233HA, a subclone of pAS233, although maxicells containing this plasmid encoded larvicidal activity. The insert of pAS377PT was transcribed from a vector promoter whereas the insert of pAS233HA was transcribed from its own promoter and hence its expression in B. subtilis was possible. The insert was ligated to a shuttle vector yielding pSVI which was then used to transform B. subtilis. Recombinant E. coli and B. subtilis clones showed equivalent larvicidal activity of 1–10 μg cell protein per ml. Larvicidal activity was observed during vegetative growth for recombinant B. subtilis even though B. sphaericus 1593 synthesizes its mosquito-toxin only during sporulation.     

Growth,sporulation and toxin production byBacillus thuringiensis subsp.israelensis andB. sphaericus in media based on mustard-seed meal

Bacillus thuringiensis subsp.israelensis andB. sphaericus strains 2362 and 1593 were grown in media based on defatted mustard-seed meal (MSM). The meal contains 40% (w/w) protein, with glutamic acid and arginine as the major amino acids. The toxic potencies of the final bacterial powders towardsCulex pipens quinquefasciatus Say, compared with those of the respective international reference standards, were 46% forB. thuringiensis subsp.israelensis, 62% forB. sphaericus 2362 and 88% forB. sphaericus 1593 when 2% (w/v) MSM was used for growth. With 4% (w/v) MSM,B. thuringiensis subsp.israelensis grew better but had undetectable larvicidal activity, whereas theB. sphaericus strains not only grew better but gave a higher degree of sporulation and toxicity. The potencies ofB. sphaericus in medium with 4% MSM were comparable with those of international reference standards.The authors are with the Department of Life Sciences, University of Bombay, Bombay 400 098, India.     

Various Levels of Cross-Resistance to Bacillus sphaericus Strains in Culex pipiens (Diptera: Culicidae) Colonies Resistant to B. sphaericus Strain 2362

We studied the cross-resistance to three highly toxic Bacillus sphaericus strains, IAB-59 (serotype H6), IAB-881 (serotype H3), and IAB-872 (serotype H48), of four colonies of the Culex pipiens complex resistant to B. sphaericus 2362 and 1593, both of which are serotype H5a5b strains. Two field-selected highly resistant colonies originating from India (KOCHI, 17,000-fold resistance) and France (SPHAE, 23,000-fold resistance) and a highly resistant laboratory-selected colony from California (GeoR, 36,000-fold resistance) showed strong cross-resistance to strains IAB-881 and IAB-872 but significantly weaker cross-resistance to IAB-59 (3- to 43-fold resistance). In contrast, a laboratory-selected California colony with low-level resistance (JRMM-R, 5-fold resistance) displayed similar levels of resistance (5- to 10-fold) to all of the B. sphaericus strains tested. Thus, among the mosquitocidal strains of B. sphaericus we identified a strain, IAB-59, which was toxic to several Culex colonies that were highly resistant to commercial strains 2362 and 1593. Our analysis also indicated that strain IAB-59 may possess other larvicidal factors. These results could have important implications for the development of resistance management strategies for area-wide mosquito control programs based on the use of B. sphaericus preparations.     

Biocide gene(s) and biocidal activity in different strains of Bacillus sphaericus. Expression of the gene(s) in E. coli maxicells

Summary The recently cloned biocidal determinant of the highly toxic strain of B. sphaericus 1593M (Ganesan et al. 1983) was used as probe to investigate homologous sequences in different toxic and non-toxic strains of B. sphaericus. It was found that the potent strains we have analysed are characterised by the presence of DNA sequences (6.6, 6.4, 5.8, 1.6, 1.3 and 0.6 Kb) not found in the non-toxic strains. These results further show that one of the two weakly toxic strains analysed presents a hybridisation pattern completely different from that observed with the highly potent strains of B. sphaericus. When the DNA of the two non-toxic strains was analysed, SSII-I failed to hybridise to the probe and Rem4 exhibited mainly one hybridisable sequence of 2.3 Kb not detectable in the toxic strains.No region of homology to the probe was found in the DNA of two strains of B. thuringiensis (var. berliner and var. israeliensis) analysed.By dot blot hybridisation experiments it was estimated that the larvicidal determinant might be present in about one to three copies per genome.With the use of E. coli maxicells we have shown further that the toxin gene(s) encoded four polypeptides with molecular weights of 21, 19, 15, and 12 Kd. The significance of these findings is discussed.     

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.     

Bacillus sphaericus for the control of mosquitoes

From 1972 to 1977 a large laboratory effort was devoted to determining data on efficacy, safety, environmental impact (on nontarget organisms), and some preliminary field work using several isolates of Bacillus sphaericus. The B. sphaericus strains were found to be specific in their mosquito larvicidal activity, not causing mammalian toxicity nor apparent perturbation of the environment. During this period several fermentation and industrialization problems were investigated so that by 1978, using new strains and cultures, it was possible to have prepared kilogram amounts of an active dry stable powder, of strain 1593, for field evaluation. These field evolution. These field evaluations are presently still in progress. Control has been seen particularly against Culex, Anopheles, and Psorophora species, with some what less control aganst Aedes species. Unlike the agriculturally oriented Bacillus thuringiensis candidates, B. sphaericus bacterial cell, which is digested in the larval midgut (within a peritrophic membrane), releasing a toxin as early as 15 min after ingestion. Subsequent death of the larva ensues. Recent evidence suggests that applied B. sphaericus powder will survive in aquatic situations (ditches, ponds, and tree holes) for at least nine month. Comparisons of the B. sphaeicus strains with recently isolated strains of B. thuringiensis (var. israelensis), the latter being particularly active against Aedes species, indicates that they may be useful complements of each other in overall mosquito control strategies. The recent isolation of several new strains of B. thuringiensis, from WHO-CCBC accessions from Roumania, indicate that although the B. thuringiensis isolate is a rare event when compared to the occurrence of B. sphaericus isolates (they usually occur together in accessions from which B. thuringiensis is isolated), several new useful strains of B. thuringiensis should be anticipated. The longevity of the B. thuringiensis strains in the wild has not yet been investigated.     

The Introduction into Bacillus sphaericus of the Bacillus thuringiensis subsp. medellin cyt1Ab1 Gene Results in Higher Susceptibility of Resistant Mosquito Larva Populations to B. sphaericus

The fragment containing the gene encoding the cytolytic Cyt1Ab1 protein from Bacillus thuringiensis subsp. medellin and its flanking sequences (I. Thiery, A. Delécluse, M. C. Tamayo, and S. Orduz, Appl. Environ. Microbiol. 63:468–473, 1997) was introduced into Bacillus sphaericus toxic strains 2362, 2297, and Iab872 by electroporation with the shuttle vector pMK3. Only small amounts of the protein were produced in recombinant strains 2362 and Iab872. The protein was detected in these strains only by Western blotting and immunodetection with antibody raised against Cyt1Ab1 protein. Large amounts of Cyt1Ab1 protein were produced in B. sphaericus recombinant strain 2297, and there was an additional crystal, other than that of the binary toxin, within the exosporium. The production of the Cyt1Ab1 protein in addition to the binary toxin did not increase the larvicidal activity of the B. sphaericus recombinant strain against susceptible mosquito populations of Culex pipiens or Aedes aegypti. However, it partially restored (10 to 20 times) susceptibility of the resistant mosquito populations of C. pipiens (SPHAE) and Culex quinquefasciatus (GeoR) to the binary toxin. The Cyt1Ab1 protein produced in recombinant B. thuringiensis SPL407(pcyt1Ab1) was synthesized in two types of crystal—one round and with various dense areas, surrounded by an envelope, and the other a regular cuboid crystal, very similar to that found in the B. sphaericus recombinant strain.     

Effect of oxygen on growth,sporulation, and mosquito larval toxin formation byBacillus sphaericus 1593

The effect of oxygen on growth, sporulation, and mosquito larval toxin synthesis byBacillus sphaericus 1593 grown in a small fermentor was investigated. With air as the source of oxygen, about one-half of the cells sporulated and 1022 units of toxicity/mg of cell dry weight were formed. A shift to 100% oxygen in the gas stream maintained a higher level of dissolved oxygen in the medium, but this produced a late block in sporulation; however, toxin synthesis was normal. The mechanism of oxygen inhibition of sporulation byB. sphaericus is unknown, but the same effect was observed inB. subtilis 168. Stopping of the air flow at 8 h, after forespores were completed in about one-half the cells, inhibited the completion of sporulation, but did not decrease toxin production.     

Cloning and Characterization of a Mosquito Larvicidal Toxin Produced During Vegetative Stage of <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> 2297

The mosquitocidal toxin 1 (mtx1) gene from genomic DNA of B. sphaericus strain 2297 was cloned and expressed in E. coli. DNA sequencing analysis of the cloned gene revealed a single open reading frame encoding an 870-amino acid polypeptide. Expression level of the full-length gene in E. coli was very low even though strong promoter was used or the gene was expressed as a fusion protein. Expression level was highly improved after the putative leader sequence was deleted, and the truncated gene was expressed as a fusion protein with glutathione S-transferase (GST-tMtx1). E. coli cells expressing GST-tMtx1 was highly toxic to Culex quinquefasciatus larvae and showed lower toxicity against Anopheles dirus and Aedes aegypti larvae. Enterobacter amnigenus An11, a mosquito larval gut colonizable bacteria, transformed with the cloned gene exhibited mosquito larvicidal activity. Result suggested that there is a potential to develop this protein to be used as an alternative mosquito control agent.     

Metabolic response of Bacillus sphaericus 1593M for dual-substrate limitation in continuous and total-cell-retention cultures

A chemically defined medium has been developed to support the growth and the production of mosquito larvicidal factor(s) (MLF) of Bacillus sphaericus 1593M. On the basis of the data of steady-state continuous cultures, it has been understood that acetate can serve as a sole carbon and energy source for B. sphaericus 1593M. Utilization of acetate by B. sphaer-icus 1593M and the production of MLF are further enhanced by the addition of glutamate at low concentrations, both in steady-state continuous as well as in total-cell-retention cultures (TCRC). A two-step TCRC procedure resulted in better biomass and MLF production by B. sphaericus 1593M. It was also found that glutamate can serve as a carbon source as well as a growth factor in the presence of acetate and hence is a partially substitutable carbon source. Received: 3 January 1997 / Accepted: 31 January 1997     

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.     

A Sandwich Enzyme-Linked Immunosorbent Assay for the Bacillus sphaericus Binary Toxin

Bacillus sphaericus (Bs) binary toxin was purified from recombinant E. coli DH5α harboring the recombinant plasmid pAR5, which carries a 3.6-kb DNA fragment of Bs 1593M encoding mosquito larvicidal activity. The binary toxin preparation, designated BsEcAg, contained mainly 51- and 42-kDa toxin proteins and was toxic to 50% of Culex quinquefasciatus larvae at a concentration of 9.22 ng toxin protein/ml. This preparation was used to raise antibodies in sheep and mice. The sandwich ELISA used sheep antitoxin antibody as primary antibody (coating antibody), mouse antitoxin antibody as second antibody, and goat antimouse antibody as an alkaline phosphatase-conjugated detecting antibody. The assay sensitivity was 200 ng/ml for both BsEcAg and binary toxin antigen (BsAg) from Bs 2362 cells. There is a significant correlation between toxin level determined by ELISA and bioassay. This procedure has also been used to monitor toxin levels in batch fermentations of Bs 2362. Received: 2 July 1997 / Accepted: 12 August 1997     

Construction of direct selection vectors for use inBacillus sphaericus

Genetic manipulation in the mosquito larvicidal organismBacillus sphaericus has been hampered by a lack of effective molecular biological techniques and tools. Currently available vectors forBacillus subtilis are inappropriate as they are selected with antibiotics to whichB. sphaericus is naturally resistant. Construction of chimeras between theStaphylococcus aureus plasmid pE194 and theB. subtilis direct selection vector pBD214 permitted selection of plasmid transformants with erythromycin. As with pBD214, direct selection of recombinants is accomplished by insertional inactivation of the plasmid-borne thymidylate synthetase gene and subsequent selection with trimethoprim.     

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.     

Expression of the larvicidal gene of Bacillus sphaericus 1593M in the cyanobacterium Anacystis nidulans R2

Summary A 3.6 kb HindIII DNA fragement from Bacillus sphaericus 1593M was cloned and expressed in Escherichia coli and B. subtilis using pHV33 as shuttle vector and in the cyanobacterium Anacystis nidulans R2 with pUC303 as shuttle vector. The level of toxin activity of the respective recombinant plasmids pGsp04 and pGsp12 against Culex mosquito larvae was found to be the same in Escherichia coli and in the cyanobacterium.