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 characterization of mosquitocidal Bacillus sphaericus BinA protein

Certain strains of Bacillus sphaericus produce a highly toxic mosquito-larvicidal binary toxin during sporulation. The binary toxin is composed of toxic BinA (41.9 kDa) and receptor binding BinB (51.4 kDa) polypeptides and is active against vectors of filariasis, encephalitis and malaria. The toxin has been tested with limited use for the control of vector mosquitoes for more than two decades. The binA gene from a local ISPC-8 strain of B. sphaericus that is highly toxic to Culex and Anopheles mosquito species was cloned into pET16b and expressed in Escherichia coli. The purified BinA protein differs by one amino acid (R197 M) from BinA of the highest toxicity strains 1593/2362/C3-41. Majority of the expressed protein was observed in inclusion bodies. BinA inclusions alone from E. coli did not show toxic activity, like reported previously. However, the active form of BinA could be purified to homogeneity from the soluble fraction of E. coli cell lysate, grown at reduced temperature after isopropyl β-d-thiogalactopyranoside induction. The purified BinA protein with and without poly-histidine tag showed LC₅₀ dose of 82.3 and 66.9 ng ml⁻¹, respectively, at 48 h against Culex quinquefasciatus larvae. The secondary structure of BinA is expected to be mainly β strands as estimated using far-UV circular dichroism. The estimates matched well with the secondary structure predictions using amino acid sequence. This is the first report of large-scale purification and accurate toxicity estimation of soluble B. sphaericus BinA. This can help in design and synthesis of improved bacterial insecticide.     

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.     

Increased toxicity of modified mosquitocidal binary toxins of Bacillus sphaericus expressed in Escherichia coli

The binary mosquitocidal genes of 51-kDa and 42-kDa proteins isolated from Bacillus sphaericus 1593 have been expressed at moderate levels in Escherichia coli employing the pQE expression system. The expressed proteins are readily visible in Coomassie-blue-stained protein gels. The recombinant E. coli cells expressing toxic proteins were toxic towards Culex larvae. During the assembly of crystals in B. sphaericus, the 42-kDa toxin is first cleaved at the N-terminal end by a specific B. sphaericus protease. To express the toxins in E. coli the B.sphaericus specific protease-recognition site was deleted at the N-terminal end of the 42-kDa toxin, thereby mimicking the structure of the toxin as present in the crystal. This modification resulted in a twofold increase in the toxicity of the E. coli cells expressing the modified 42-kDa toxin as a constituent of the binary toxin. Our results demonstrate the utility of this modification for heterologous expression of the binary toxin genes from B. sphaericus. Received: 18 July 1997 / Received revision: 6 October 1997 / Accepted: 14 October 1997     

Effect of UV Light on Spore Viability and Mosquito Larvicidal Activity of Bacillus sphaericus 1593

UV light from a germicidal lamp rapidly reduced the viability of Bacillus sphaericus 1593 spores, but insecticidal activity was resistant to inactivation by continuous exposure to UV light for 4 h.     

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.     

Functional Complementation of Nontoxic Mutant Binary Toxins of Bacillus sphaericus 1593M Generated by Site-Directed Mutagenesis

Alanine residues were substituted by site-directed mutagenesis at selected sites of the N- and C-terminal regions of the binary toxin (51- and 42-kDa peptides) of B. sphaericus 1593M, and the mutant toxins were cloned and expressed in Escherichia coli. Bioassays with mosquito larvae, using binary toxins derived from individual mutants, showed that the substitution of alanine at some sites in both the 51-kDa and the 42-kDa peptides resulted in a total loss of activity. Surprisingly, after mixing two nontoxic derivatives of the same peptide, i.e., one mutated at the N-terminal end and the other mutated at the C-terminal end of either the 51-kDa or the 42-kDa peptide, the toxicity was restored. This result indicates that the altered binary toxins can functionally complement each other by forming oligomers.     

Efficient mosquitocidal toxin production by Bacillus sphaericus using cheese whey permeate under both submerged and solid state fermentations

Whey permeate (WP) was used efficiently for production of mosquitocidal toxin by Bacillus sphaericus 2362 (B. sphaericus 2362) and the Egyptian isolate, B. sphaericus 14N1 (B. sphaericus 14N1) under both submerged and solid state fermentation conditions. Under submerged fermentation, high mosquitocidal activity was produced by B. sphaericus 2362 and B. sphaericus 14N1 at 50-100% and 25-70% WP, respectively. Initial pH of WP was a critical factor for toxin production by both tested organisms. The highest toxicity was obtained at initial pH 7. Egyptian isolate, B. sphaericus 14N1 was tested for growth and toxin production under solid state fermentation conditions (SSF) by using WP as moistening agent instead of distilled water. The optimum conditions for production of B. sphaericus 14N1 on wheat bran-WP medium were 10 g wheat bran/250 ml flask moistened with 10-70% WP at 50% moisture content, inoculum size ranged between 17.2 × 10⁷ and 34.4 × 10⁷ and 6 days incubation under static conditions at 30 °C. Preliminary pilot-scale production of B. sphaericus 14N1 under SSF conditions in trays proved that wheat bran-WP medium was efficient and economic for industrial production of mosquitocidal toxin by B. sphaericus.     

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.     

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.     

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.     

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.     

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.     

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.     

Identification of Cry48Aa/Cry49Aa toxin ligands in the midgut of Culex quinquefasciatus larvae

A binary mosquitocidal toxin composed of a three-domain Cry-like toxin (Cry48Aa) and a binary-like toxin (Cry49Aa) was identified in Lysinibacillus sphaericus. Cry48Aa/Cry49Aa has action on Culex quinquefasciatus larvae, in particular, to those that are resistant to the Bin Binary toxin, which is the major insecticidal factor from L. sphaericus-based biolarvicides, indicating that Cry48Aa/Cry49Aa interacts with distinct target sites in the midgut and can overcome Bin toxin resistance. This study aimed to identify Cry48Aa/Cry49Aa ligands in C. quinquefasciatus midgut through binding assays and mass spectrometry. Several proteins, mostly from 50 to 120 kDa, bound to the Cry48Aa/Cry49Aa toxin were revealed by toxin overlay and pull-down assays. These proteins were identified against the C. quinquefasciatus genome and after analysis a set of 49 proteins were selected which includes midgut bound proteins such as aminopeptidases, amylases, alkaline phosphatases in addition to molecules from other classes that can be potentially involved in this toxin's mode of action. Among these, some proteins are orthologs of Cry receptors previously identified in mosquito larvae, as candidate receptors for Cry48Aa/Cry49Aa toxin. Further investigation is needed to evaluate the specificity of their interactions and their possible role as receptors.     

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     

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.     

Tightly Bound Binary Toxin in the Cell Wall of Bacillus sphaericus

We have shown that urea-extracted cell wall of entomopathogenic Bacillus sphaericus 2297 and some other strains is a potent larvicide against Culex pipiens mosquitoes, with 50% lethal concentrations comparable to that of the well-known B. sphaericus binary toxin, with which it acts synergistically. The wall toxicity develops in B. sphaericus 2297 cultures during the late logarithmic stage, earlier than the appearance of the binary toxin crystal. It disappears with sporulation when the binary toxin activity reaches its peak. Disruption of the gene for the 42-kDa protein (P42) of the binary toxin abolishes both cell wall toxicity and crystal formation. However, the cell wall of B. sphaericus 2297, lacking P42, kills C. pipiens larvae when mixed with Escherichia coli cells expressing P42. Thus, the cell wall toxicity in strongly toxic B. sphaericus strains must be attributed to the presence in the cell wall of tightly bound 51-kDa (P51) and P42 binary toxin proteins. The synergism between binary toxin crystals and urea-treated cell wall preparations reflects suboptimal distribution of binary toxin subunits in both compartments. Binary toxin crystal is slightly deficient in P51, while cell wall is lacking in P42.