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.     

Laboratory and simulated-field bioassays for assessing mixed cultures of <Emphasis Type="Italic">Lysinibacillus sphaericus</Emphasis> against <Emphasis Type="Italic">Aedes aegypti</Emphasis> (Diptera: Culicidae) larvae resistant to temephos

Aedes aegypti (L.) is the main vector of tropical diseases such as dengue, chikungunya and Zika. Due to the overuse of insecticides, Ae. aegypti resistant populations have increased. Biological control with Lysinibacillus sphaericus (Ahmed) has been used against Culex sp. and Anopheles sp. Although Ae. aegypti is refractory to the binary toxin of L. sphaericus spores, vegetative cells have been shown to be effective against Ae. aegypti larvae. In this work, the effect of L. sphaericus vegetative cells on Ae. aegypti temephos-resistant larvae was assessed under lab and simulated field conditions. L. sphaericus caused about 90% mortality of insecticide-resistant Ae. aegypti larvae under simulated field conditions. Likewise, Ae. aegypti larvae were more sensitive to mixed cultures of L. sphaericus than to individual strains; then, the most effective mixed culture exhibited an LC₅₀ of 1.21 × 10⁵ CFU/mL with Rockefeller larvae and 8.04 × 10⁴ CFU/mL with field-collected larvae. Additionally, we found that mixed cultures composed of two L. sphaericus strains were more effective than a culture formed by the three strains. Our results suggest that mixed cultures comprising L. sphaericus vegetative cells could be useful for controlling temephos-resistant populations of Ae. aegypti, as evidenced by the effectiveness demonstrated under laboratory and simulated field conditions.     

Metal tolerance and larvicidal activity of Lysinibacillus sphaericus

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

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.     

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.     

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.     

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.     

Enzymatic synthesis of l-cysteine by O-acetylserine sulfhydrylase of 3-chloro-l-alanine resistant Bacillus sphaericus L-118

The regulatory properties of serine-O-transacetylase and O-acetylserine sulfhydrylase have been investigated with 3-chloro-l-alanine resistant Bacillus sphaericus L-118. The enhancement of O-acetylserine sulfhydrylase formation by 3-chloro-l-alanine was observed and this effect was counteracted by corepressor l-cysteine. O-Acetylserine sulfhydrylase occurring in B. sphaericus L-118 can catalyse β-replacement reaction of 3-chloro-l-alanine in the presence of a high concentration of sodium hydrosulfide to form l-cysteine. The optimal reaction conditions for l-cysteine production were studied using resting cells. Under optimal conditions, about 80% of the added 3-chloro-l-alanine could be converted to l-cysteine. The highest yield achieved was 70 mg of l-cysteine per 1.0 ml of the reaction mixture.     

Cyt1A from Bacillus thuringiensis Synergizes Activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae)

Bacillus sphaericus is a mosquitocidal bacterium recently developed as a commercial larvicide that is used worldwide to control pestiferous and vector mosquitoes. Whereas B. sphaericus is highly active against larvae of Culex and Anopheles mosquitoes, it is virtually nontoxic to Aedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A from Bacillus thuringiensis subsp. israelensis to enhance the toxicity of B. sphaericus toward A. aegypti. Various combinations of these two materials were evaluated, and all were highly toxic. A ratio of 10:1 of B. sphaericus to Cyt1A was 3,600-fold more toxic to A. aegypti than B. sphaericus alone. Statistical analysis showed this high activity was due to synergism between the Cyt1A toxin and B. sphaericus. These results suggest that Cyt1A could be useful in expanding the host range of B. sphaericus.     

Conjugal Transfer of a Toxin-Coding Megaplasmid from Bacillus thuringiensis subsp. israelensis to Mosquitocidal Strains of Bacillus sphaericus

Both Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis produce mosquitocidal toxins during sporulation and are extensively used in the field for control of mosquito populations. All the known toxins of the latter organism are known to be encoded on a large plasmid, pBtoxis. In an attempt to combine the best properties of the two bacteria, an erythromycin resistance-marked pBtoxis plasmid was transferred to B. sphaericus by a mating technique. The resulting transconjugant bacteria were significantly more toxic to Aedes aegypti mosquitoes and were able to overcome resistance to B. sphaericus in a resistant colony of Culex quinquefasciatus, apparently due to the production of Cry11A but not Cry4A or Cry4B. The stability of the plasmid in the B. sphaericus host was moderate during vegetative growth, but segregational instability was observed, which led to substantial rates of plasmid loss during sporulation.     

Production of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus strain 1593 on media using a byproduct from a monosodium glutamate factory

Two newly developed media, H4 and H7, were found to be highly suitable for culturing Bacillus thuringiensis subsp. israelensis and B. sphaericus, respectively. These media contained 0.05% K₂HPO₄ and 4% HDL (H4 medium) or 0.05% K₂HPO₄ and 7% HDL (H7 medium); HDL is the by-product from a monosodium glutamate factory. Tests to compare endospore formation and toxicity values of B. thuringiensis subsp. israelensis in H4 medium and nutrient broth supplemented with salts and glucose (NBSG) medium were carried out in a 3-liter fermentor. The viable cell count and LC₅₀ value of B. thuringiensis subsp. israelensis in H4 medium at 48 hr were 2.5 × 10⁸ cells/ml and 10^?7.2 (dilution), respectively, while those in NBSG medium were 1.6 × 10⁸ cells/ml and 10^?6.5, respectively. In the case of B. sphaericus grown in H7 medium, the number of cells and LC₅₀ value were found to be 1.4 × 10⁹ cells/ml and 10^?7.8, respectively. B. sphaericus grown in nutrient broth supplemented with salt and yeast extract (NBSY) were found to produce 6.4 × 10⁸ cells/ml and an LC₅₀ value of 10^?6.8. The toxicity of B. thuringiensis subsp. israelensis was tested against Aedes aegypti larvae, while that of B. sphaericus was tested against Culex quinquefasciatus. The cost of 10 liters of medium for production of B. thuringiensis subsp. israelensis and in B. sphaericus and H4 and H7 was $0.02 and $0.03, respectively. The cost of these newly developed media was much less than that of NBSG medium ($7.05 per 10 liters) for cultivation of B. thuringiensis subsp. israelensis and NBSY medium ($11.67 per 10 liters) for cultivation of B. sphaericus.     

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.     

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.     

Cyt1Ab1 and Cyt2Ba1 from Bacillus thuringiensis subsp. medellin and B. thuringiensis subsp. israelensis Synergize Bacillus sphaericus against Aedes aegypti and Resistant Culex quinquefasciatus (Diptera: Culicidae)

The interaction of two cytolytic toxins, Cyt1Ab from Bacillus thuringiensis subsp. medellin and Cyt2Ba from Bacillus thuringiensis subsp. israelensis, with Bacillus sphaericus was evaluated against susceptible and resistant Culex quinquefasciatus and the nonsensitive species Aedes aegypti. Mixtures of B. sphaericus with either cytolytic toxin were synergistic, and B. sphaericus resistance in C. quinquefasciatus was suppressed from >17,000- to 2-fold with a 3:1 mixture of B. sphaericus and Cyt1Ab. This trait may prove useful for combating insecticide resistance and for improving the activity of microbial insecticides.     

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.     

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.     

Recycling potential and selective retrieval of Bacillus sphaericus from soil in a mosquito habitat

A selective medium containing a combination of starch, milk proteins, and streptomycin was used as a reliable indicator for the presence of Bacillus sphaericus in soil samples collected from a mosquito habitat where this pathogen was previously applied. The medium can also be used as an indicator substrate to retrieve B. sphaericus from infected mosquito larvae. Results show that B. sphaericus remains viable and infective 9 months after application as a larvacidal agent of mosquitoes in a roadside ditch.     

Genome comparison provides molecular insights into the phylogeny of the reassigned new genus Lysinibacillus

Background

Lysinibacillus sphaericus (formerly named Bacillus sphaericus) is incapable of polysaccharide utilization and some isolates produce active insecticidal proteins against mosquito larvae. Its taxonomic status was changed to the genus Lysinibacillus in 2007 with some other organisms previously regarded as members of Bacillus. However, this classification is mainly based on physiology and phenotype and there is limited genomic information to support it.

Results

In this study, four genomes of L. sphaericus were sequenced and compared with those of 24 representative strains belonging to Lysinibacillus and Bacillus. The results show that Lysinibacillus strains are phylogenetically related based on the genome sequences and composition of core genes. Comparison of gene function indicates the major difference between Lysinibacillus and the two Bacillus species is related to metabolism and cell wall/membrane biogenesis. Although L. sphaericus mosquitocidal isolates are highly conserved, other Lysinibacillus strains display a large heterogeneity. It was observed that mosquitocidal toxin genes in L. sphaericus were in close proximity to genome islands (GIs) and mobile genetic elements (MGEs). Furthermore, different copies and varying genomic location of the GIs containing binA/binB was observed amongst the different isolates. In addition, a plasmid highly similar to pBsph, but lacking the GI containing binA/binB, was found in L. sphaericus SSII-1.

Conclusions

Our results confirm the taxonomy of the new genus Lysinibacillus at the genome level and suggest a new species for mosquito-toxic L. sphaericus. Based on our findings, we hypothesize that (1) Lysinibacillus strains evolved from a common ancestor and the mosquitocidal L. sphaericus toxin genes were acquired by horizontal gene transfer (HGT), and (2) capture and loss of plasmids occurs in the population, which plays an important role in the transmission of binA/binB.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1359-x) contains supplementary material, which is available to authorized users.Keyword: Lysinibacillus, Bacillus, Lysinibacillus sphaericus, Genome, Phylogeny     

Molecular Characterization of a Glucokinase with Broad Hexose Specificity from Bacillus sphaericus Strain C3-41

Bacillus sphaericus cannot metabolize sugar since it lacks several of the enzymes necessary for glycolysis. Our results confirmed the presence of a glucokinase-encoding gene, glcK, and a phosphofructokinase-encoding gene, pfk, on the bacterial chromosome and expression of glucokinase during vegetative growth of B. sphaericus strains. However, no phosphoglucose isomerase gene (pgi) or phosphoglucose isomerase enzyme activity was detected in these strains. Furthermore, one glcK open reading frame was cloned from B. sphaericus strain C3-41 and then expressed in Escherichia coli. Biochemical analysis revealed that this gene encoded a protein with a molecular mass of 33 kDa and that the purified recombinant glucokinase had K_m values of 0.52 and 0.31 mM for ATP and glucose, respectively. It has been proved that this ATP-dependent glucokinase can also phosphorylate fructose and mannose, and sequence alignment of the glcK gene indicated that it belongs to the ROK protein family. It is postulated that the absence of the phosphoglucose isomerase-encoding gene pgi in B. sphaericus might be one of the reasons for the inability of this bacterium to metabolize carbohydrates. Our findings provide additional data that further elucidate the specific metabolic pathway and could be used for genetic improvement of B. sphaericus.