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.     

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.     

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.     

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.     

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.     

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).     

Properties and applied use of the mosquitocidal bacterium,Bacillus sphaericus

Strains of Bacillus sphaericus exhibit varying levels of virulence against mosquito larvae. The most potent strain, B. sphaericus 2362, which is the active ingredient in the commercial product VectoLex^®, together with another well-known larvicide Bacillus thuringiensis subsp. israelensis, is used to control vector and nuisance mosquito larvae in many regions of the world. Although not all strains of B. sphaericus are mosquitocidal, lethal strains produce one or two combinations of three different types of toxins. These are (1) the binary toxin (Bin) composed of two proteins of 42 kDa (BinA) and 51 kDa (BinB), which are synthesized during sporulation and co-crystallize, (2) the soluble mosquitocidal toxins (Mtx1, Mtx2 and Mtx3) produced during vegetative growth, and (3) the two-component crystal toxin (Cry48Aa1/Cry49Aa1). Non-mosquitocidal toxins are also produced by certain strains of B. sphaericus, for example sphaericolysin, a novel insecticidal protein toxic to cockroaches. Larvicides based on B. sphaericus-based have the advantage of longer persistence in treated habitats compared to B. thuringiensis subsp. israelensis. However, resistance is a much greater threat, and has already emerged at significant levels in field populations in China and Thailand treated with B. sphaericus. This likely occurred because toxicity depends principally on Bin rather than various combinations of crystal (Cry) and cytolytic (Cyt) toxins present in B. thuringiensis subsp. israelensis. Here we review both the general characteristics of B. sphaericus, particularly as they relate to larvicidal isolates, and strategies or considerations for engineering more potent strains of this bacterium that contain built-in mechanisms that delay or overcome resistance to Bin in natural mosquito populations.     

Horizontal transfer of large plasmid with type IV secretion system and mosquitocidal genomic island with excision and integration capabilities in Lysinibacillus sphaericus

We identified a ~30-kb genomic island (named GI8) carrying the binary toxin gene operon binA/binB on both the chromosome and large pBsph plasmid in the mosquitocidal Lysinibacillus sphaericus C3-41 strain. We found that GI8 is related to the occurrence of binA/binB within L. sphaericus and displays excision and integration capability by recognizing the attB region, which consists of a 2-nt target site (AT) flanked by an 11-nt imperfect inverted repeat. pBsph and two pBsph-like plasmids (p2362 and p1593) were found to carry a type IV secretion system (T4SS) and displayed transmissibility within a narrow host range specific to L. sphaericus. GI8 can be co-transferred with pBsph as a composite element by integration into its attB site, then excised from pBsph and re-integrated into the chromosomal attB site in the new host. The potential hosts of GI8, regardless of whether they are toxic or non-toxic to mosquito larvae, share good collinearity at the chromosomal level. Data indicated that the appearance of the mosquitocidal L. sphaericus lineage was driven by horizontal transfer of the T4SS-type conjugative plasmid and GI8 with excision and specific integration capability.     

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.     

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.     

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.     

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.     

Contribution of S-Layer Proteins to the Mosquitocidal Activity of Lysinibacillus sphaericus

Lysinibacillus sphaericus strains belonging the antigenic group H5a5b produce spores with larvicidal activity against larvae of Culex mosquitoes. C7, a new isolated strain, which presents similar biochemical characteristics and Bin toxins in their spores as the reference strain 2362, was, however, more active against larvae of Culex mosquitoes. The contribution of the surface layer protein (S-layer) to this behaviour was envisaged since this envelope protein has been implicated in the pathogenicity of several bacilli, and we had previously reported its association to spores. Microscopic observation by immunofluorescence detection with anti S-layer antibody in the spores confirms their attachment. S-layers and BinA and BinB toxins formed high molecular weight multimers in spores as shown by SDS-PAGE and western blot detection. Purified S-layer from both L. sphaericus C7 and 2362 strain cultures was by itself toxic against Culex sp larvae, however, that from C7 strain was also toxic against Aedes aegypti. Synergistic effect between purified S-layer and spore-crystal preparations was observed against Culex sp. and Aedes aegypti larvae. This effect was more evident with the C7 strain. In silico analyses of the S-layer sequence suggest the presence of chitin-binding and hemolytic domains. Both biochemical characteristics were detected for both S-layers strains that must justify their contribution to pathogenicity.     

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.     

Efficient Expression of the Mosquito Larvicidal Binary Toxin Gene from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The binary toxin gene encoding BinA (42 kDa) and BinB (51 kDa) from Bacillus sphaericus strain 2297 was cloned and expressed in E. coli. Low expression level was found when both proteins were expressed from a single operon. High expression was observed when the gene encoding an individual protein was placed downstream of the T7 promoter. The expression level of BinB was not different when expressed alone (non-fusion) or as a fusion form with T7 peptide (T7-BinB). Both forms of BinB were equally stable. Unlike BinB, the non-fusion form of BinA was less stable than T7-BinA. The mosquito larvicidal test showed that BinA or BinB alone was not toxic to mosquito larvae, but high toxicity was found when both BinA and BinB were applied. The results suggest that a short peptide of T7 linked to the N-terminus of either BinA or BinB does not affect their toxicity, but may make the toxin, especially BinA, more stable.     

Recombinant Enterobacter amnigenus Highly Toxic to Anopheles dirus Mosquito Larvae

The mosquito-larvicidal binary toxin of Bacillus sphaericus 2297 was expressed in Enterobacter amnigenus, a Gram-negative bacterium isolated from Anopheles dirus larvae gut. The toxin was placed under the regulation of various promoters in order to improve the expression level of the toxin. Amongst the recombinants obtained, E. amnigenus harboring pBS373, a plasmid which contains the toxin genes under the control of the native B. sphaericus promoter, expressed a significant amount of protein, comparable to that found in B. sphaericus 2297. In addition, this recombinant provided approximately twenty times higher toxicity against second-instar Anopheles dirus larvae when compared to B. sphaericus 2297. The procedure of obtaining this environmentally isolated bacterium from larvae gut and introducing the system for mosquito-larvicidal toxin synthesis is noteworthy. The promising result presented here provides a substantial degree of confidence for further field studies.     

Cytopathological Effects of Bacillus sphaericus Cry48Aa/Cry49Aa Toxin on Binary Toxin-Susceptible and -Resistant Culex quinquefasciatus Larvae

The Cry48Aa/Cry49Aa mosquitocidal two-component toxin was recently characterized from Bacillus sphaericus strain IAB59 and is uniquely composed of a three-domain Cry protein toxin (Cry48Aa) and a binary (Bin) toxin-like protein (Cry49Aa). Its mode of action has not been elucidated, but a remarkable feature of this protein is the high toxicity against species from the Culex complex, besides its capacity to overcome Culex resistance to the Bin toxin, the major insecticidal factor in B. sphaericus-based larvicides. The goal of this work was to investigate the ultrastructural effects of Cry48Aa/Cry49Aa on midgut cells of Bin-toxin-susceptible and -resistant Culex quinquefasciatus larvae. The major cytopathological effects observed after Cry48Aa/Cry49Aa treatment were intense mitochondrial vacuolation, breakdown of endoplasmic reticulum, production of cytoplasmic vacuoles, and microvillus disruption. These effects were similar in Bin-toxin-susceptible and -resistant larvae and demonstrated that Cry48Aa/Cry49Aa toxin interacts with and displays toxic effects on cells lacking receptors for the Bin toxin, while B. sphaericus IAB59-resistant larvae did not show mortality after treatment with Cry48Aa/Cry49Aa toxin. The cytopathological alterations in Bin-toxin-resistant larvae provoked by Cry48Aa/Cry49Aa treatment were similar to those observed when larvae were exposed to a synergistic mixture of Bin/Cry11Aa toxins. Such effects seemed to result from a combined action of Cry-like and Bin-like toxins. The complex effects caused by Cry48Aa/Cry49Aa provide evidence for the potential of these toxins as active ingredients of a new generation of biolarvicides that conjugate insecticidal factors with distinct sites of action, in order to manage mosquito resistance.Bacillus sphaericus is considered an important entomopathogen due to its capacity to produce insecticidal proteins with specific action against mosquitoes (Diptera: Culicidae). The binary (Bin) toxin, which is produced during bacterial sporulation and deposited in parasporal crystalline inclusions, is the most important larvicidal factor. Other proteins characterized, such as mosquitocidal toxins (Mtx proteins), can be produced during vegetative growth, and although these proteins may have larvicidal potential, they play a minor role in the toxicity of the native strains since they are produced by vegetative cells and are degraded by B. sphaericus proteinases (20, 30), and do not form components of the spore-crystal preparations that are used in control programs. Recently, a new two-component toxin was characterized from B. sphaericus strain IAB59. This is formed by the proteins Cry48Aa (135 kDa) and Cry49Aa (53 kDa), which are produced as crystalline inclusions (13). The toxin has a unique composition since the Cry48Aa component belongs to the three-domain family of Cry proteins with 30% similarity to the mosquitocidal Cry4Aa protein from Bacillus thuringiensis serovar israelensis, while Cry49Aa is one of the Bin-toxin-like proteins, a family that comprises the Bin toxin from B. sphaericus, in addition to the Cry36 and Cry35 proteins from B. thuringiensis (9, 13).Cry48Aa/Cry49Aa is considered a two-component toxin because neither component shows toxicity alone, whereas both can act in synergy and the optimum level of toxicity to Culex species is achieved when the two are present at an equimolar ratio. The 50% lethal concentration for third-instar larvae equates to 15.9 ng/ml Cry48Aa and 6.3 ng/ml Cry49Aa of purified toxins, which is a level of toxicity comparable to that of the Bin toxin (13). However, in contrast to the Bin toxin, which is naturally produced in an equimolar ratio, Cry48Aa production is low in native strains and does not confer high toxicity (13). The initial steps of the mode of action of Bin and Cry48Aa/Cry49Aa crystals are similar and comprise the ingestion of crystals, solubilization under alkaline pH, and activation of protoxins into toxins by midgut proteases. After processing, Bin toxin recognizes and binds to specific receptors in the midgut of Bin-toxin-susceptible species through its subunit BinB (51 kDa), while the component BinA (42 kDa) confers toxicity and is likely to form pores in the cell membrane (7, 25). The membrane-bound receptors of Bin toxin on the midgut of Culex quinquefasciatus larvae, Cqm1, were characterized as 60-kDa α-glucosidases (24). The mode of action of Cry48Aa/Cry49Aa is still unknown, but a remarkable feature of this new two-component toxin is the capacity to overcome C. quinquefasciatus resistance to the Bin toxin (13, 19, 21). Resistance of Culex larvae to the Bin-toxin-based larvicides often relies on the absence of functional Cqm1 receptors in the midgut (19, 24, 26). As a consequence, toxins with a distinct mode of action, such as Cry48Aa/Cry49Aa as well as B. thuringiensis serovar israelensis toxins (Cry11Aa, Cry4Aa, Cry4Ba, and Cyt1Aa), do not experience cross-resistance in the Bin-toxin-resistant larvae (12, 21, 32). Such toxins can play a strategic role in the management of resistance, and the major goal of this study was to investigate the ultrastructural effects of the Cry48Aa/Cry49Aa toxin on Bin-toxin-susceptible and -resistant C. quinquefasciatus larvae and to compare these with the effects of a synergistic mixture of Bin/Cry11Aa used to overcome Bin toxin resistance.     

Economic production of Lysinibacillus sphaericus under solid state fermentation

A highly active mosquitocidal Lysinibacillus sphaericus namely Ls 9B24 was isolated from soil of Alexandria governorate in Egypt. It was more active than the standard strain, L. sphaericus 2362. The sporulation and toxin formation of both cultures grown on different leguminous seeds and by-products under solid state fermentation (SSF) were studied. Among the tested substrates, 6% cotton seed meal enhanced sporulation and the mosquitocidal activity of L. sphaericus 2362, while 6% fodder yeast enhanced sporulation and the mosquitocidal activity of Ls 9B24. The optimum SSF growth conditions for maximum mosquitocidal activity by both cultures were using coarse wheat bran as a carrier material, 50% initial moisture content, 4–64 × 10⁶ colony forming units (CFU)/g solid medium inoculum and 6 days’ incubation period at 30°C. Addition of 0.5% yeast extract enhanced toxicity about 2.2 and 1.8 fold for L. sphaericus 2362 and Ls 9B24, respectively.     

Proteolytic Stability of Insecticidal Toxins Expressed in Recombinant Bacilli

The production of the vegetative mosquitocidal toxin Mtx1 from Bacillus sphaericus was redirected to the sporulation phase by replacement of its weak, native promoter with the strong sporulation promoter of the bin genes. Recombinant bacilli developed toxicity during early sporulation, but this declined rapidly in later stages, indicating the proteolytic instability of the toxin. Inhibition studies indicated the action of a serine proteinase, and similar degradation was also seen with the purified B. sphaericus enzyme sphericase. Following the identification of the initial cleavage site involved in this degradation, mutant Mtx1 proteins were expressed in an attempt to overcome destructive cleavage while remaining capable of proteolytic activation. However, the apparently broad specificity of sphericase seems to make this impossible. The stability of a further vegetative toxin, Mtx2, was also found to be low when it was exposed to sphericase or conditioned medium. Random mutation of the receptor binding loops of the Bacillus thuringiensis Cry1Aa toxin did, in contrast, allow production of significant levels of spore-associated protein in the form of parasporal crystals. The exploitation of vegetative toxins may, therefore, be greatly limited by their susceptibility to proteinases produced by the host bacteria, whereas the sequestration of sporulation-associated toxins into crystals may make them more amenable to use in strain improvement.     

Overproduction of a mosquitocidal chloramphenicol-resistant Lysinibacillus sphaericus mutant obtained through UV irradiation

A highly active mosquitocidal mutant UV-chloramphenicol-resistant mutant (UCR-146) of Lysinibacillus sphaericus 2362 was isolated by UV irradiation and selected through resistance to chloramphenicol which was inhibitory for growth of the parent strain. The growth of UCR-146 in NYSMCL at different concentrations of chloramphenicol (20–100 µg/ml) showed high mosquitocidal activity against Culex pipiens larvae with the optimum concentration of 35µg/ml. At this level, LC₅₀ of UCR-146 was decreased about five times from that of L. sphaericus 2362. Comparative efficiency of mosquitocidal activity of UCR-146 and L. sphaericus 2362 within 28 days of consecutive growth exhibited notable stability of both cultures through seven cycles of growth in their optimal media. UCR-146 was grown in industrial by-products media for production of the binary toxin under economic conditions. Offal medium at 2% showed the highest mosquitocidal activity of UCR-146 with LC₅₀ of 0.53 PPM which was 16.6, 13, 12.3 and 3.4 times less than that produced with L. sphaericus 2362 grown in NYSM, L. sphaericus 2362 grown in offal, UCR-146 grown in NYSM and UCR-146 grown in NYSMCL, respectively. Hence, the mosquitocidal activity of L. sphaericus 2362 increased several times through ultraviolet (UV) irradiation followed by chloramphenicol resistance selection and then growth in 2% offal medium. Finally, this procedure for selecting UV-chloramphenicol-resistant mutant and the medium used could potentially be a simple and cost effective approach for obtaining and producing a highly active mosquitocidal mutant.