Evaluation of Bacillus thuringiensis serovar israelensis produced from a new culture medium for the control of the filariasis vector,Culex quinquefasciatus

The efficacy of Bacillus thuringiensis serovar israelensis (Bti) produced from a culture medium based on chicken feather extract was found to effectively control the filariasis vector, Culex quinquefasciatus for 3 weeks (>90% mortality). The results were on par with Bti produced from the conventional medium.     

Phenotypic and genotypic features of new autoagglutinating Bacillus thuringiensis strains

A total of 28 autoagglutinating strains of Bacillus thuringiensis were isolated from different ecologic niches and distinct sites. Twenty-six strains demonstrated toxicity to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. The electrophoretic protein profiles of the crystal components were studied. Twenty-three out of the 28 strains showed the same larvicidal activity and the same protein profiles as B. thuringiensis serovar israelensis. Using isoenzyme analysis (MLEE), it was observed the presence of three electrophoretic types (ETs). The mosquitocidal strains grouped into one ET. The random amplified polymorphic DNA analysis (RAPD) was evaluated using six primers, which demonstrated three different patterns for the 28 autoagglutinating strains, allowing correlation of the profiles obtained with the toxicity observed in the bioassays. The RAPD patterns for mosquitocidal strains were identical to the one of serovar israelensis. However, to strains of low toxicity, each primer generated distinctive RAPD patterns, which demonstrated that these strains belong to different serovars. Although the antigenic classification the 26 autoagglutinating strains of B. thuringiensis could not be determined by classical flagellar serotyping, MLEE and RAPD profiles proved these strains to be compatible with B. thuringiensis serovar israelensis.     

Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis

Summary The crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28 kDa, 68 kDa and 130 kDa. The genes encoding the 28 kDa protein and the 130 kDa protein have been cloned from a large plasmid of Bti. Escherichiacoli recombinant clones containing the 130 kDa protein gene were highly active against larvae of Aedes aegypti and Culex pipiens, while B. subtilis recombinant cells containing the 28 kDa protein gene were haemolytic for sheep red blood cells. A fragment of the Bti plasmid which is partially homologous to the 130 kDa protein gene was also isolated; it probably corresponds to part of a second type of mosquitocidal toxin gene. Furthermore, restriction enzyme analysis suggested that the 130 kDa protein gene is located on the same Bti EcoRI fragment as another kind of Bti mosquitocidal protein gene cloned by Thorne et al. (1986). Hybridization experiments conducted with the 28 kDa protein gene and the 230 kDa protein gene showed that these two Bti genes are probably present in the plasmid DNA of B. thuringiensis subsp. morrisoni (PG14), which is also highly active against mosquito larvae.     

An α‐amylase is a novel receptor for Bacillus thuringiensis ssp. israelensis Cry4Ba and Cry11Aa toxins in the malaria vector mosquito Anopheles albimanus (Diptera: Culicidae)

Bacillus thuringiensis ssp. israelensis (Bti) produces four Cry toxins (Cry4Aa, Cry4Ba, Cry10Aa and Cry11Aa), and two Cyt proteins (Cyt1Aa and Cyt2Ba), toxic to mosquito‐larvae of the genus Aedes, Anopheles and Culex, important human disease vectors that transmit dengue virus, malaria and filarial parasites respectively. Previous work showed that Bti is highly toxic to Anopheles albimanus, the main vector for transmission of malaria in Mexico. In this work, we analysed the toxicity of isolated Cry proteins of Bti and identified an An. albimanus midgut protein as a putative Cry4Ba and Cry11Aa receptor molecule. Biossays showed that Cry4Ba and Cry11Aa of Bti are toxic to An. albimanus larvae. Ligand blot assays indicated that a 70 kDa glycosylphosphatidylinositol‐anchored protein present in midgut brush border membrane vesicles of An. albimanus interacts with Cry4Ba and Cry11Aa toxins. This protein was identified as an α‐amylase by mass spectrometry and enzymatic activity assays. The cDNA that codes for the α‐amylase was cloned by means of 5′‐ and 3′‐RACE experiments. Recombinant α‐amylase expressed in Escherichia coli specifically binds Cry4Ba and Cry11Aa toxins.     

Development of mutants of the mosquitocidal bacterium Bacillus thuringiensis subspecies morrisoni (PG-14) toxic to lepidopterous or dipterous insects

The parasporal body of the mosquitocidal isolate (PG-14) of Bacillus thuringiensis subsp. morrisoni (BTM) contains five major proteins with molecular masses of, respectively, 27.3, 65, 128, 135, and 144 kDa. Proteins corresponding in mass to the first four of these also occur in the mosquitocidal strain, B. thuringiensis subsp. israelensis (BTI), and it is thought therefore that the mosquitocidal activity of both strains is due to these four proteins. In other studies it has been shown that each of these proteins exhibits from moderate to high toxicity to mosquitoes, though the specific toxicity of the 144 kDa protein in PG-14 to mosquitoes remains unknown. In the present study, two parasporal body mutants (M146 and M242) of PG-14 were developed growing the wild-type strain at 42 degrees C. The parasporal body of M146 contained less of the 65-kDa protein and was less toxic (LC50 = 108 ng/ml) to mosquitoes than the wild-type strain (LC50 = 8.3 ng/ml). The parasporal body of M242 consisted of a bipyramidal crystal composed of a 144-kDa protein that was not toxic to the mosquito, Aedes aegypti, but exhibited substantial toxicity (LC50 = 2.5 micrograms/ml) to the lepidopteran. Trichoplusia ni. Because the parasporal bodies of BTI and BTM PG-14 are similar in mosquitocidal toxicity on a weight basis, the latter results suggest the 144-kDa protein, though not mosquitocidal alone, can contribute to mosquitocidal, activity when in the presence of other mosquitocidal proteins.     

Investigation of Mosquito-Specific Larvicidal Activity of a Soil Isolate of Bacillus thuringiensis serovar canadensis

The LC₅₀ value of alkali-solubilized parasporal inclusion proteins of a Diptera-specific strain, belonging to Bacillus thuringiensis serovar canadensis, was 2.4 μg/ml for larvae of the mosquito, Aedes aegypti. A significant loss in larvicidal activity occurred when solubilized inclusion proteins were treated with A. aegypti larval gut extract, silkworm (Bombyx mori) larval gut juice, and the proteinase K. Approximately 90% of the larvicidal activity was destroyed upon treatment with proteases in 30 min. The parasporal inclusion was composed of major proteins of 65, 53, and 28 kDa and some other minor proteins. Proteolysis profiles showed that the 65-kDa major protein is highly sensitive to proteases. Purification experiments with DEAE-Toyopearl column chromatography revealed that the 65-kDa protein is responsible for the mosquitocidal activity of this strain. The LC₅₀ value of the purified protein was 5.4 μg/ml. Received: 2 December 1996 / Accepted: 7 January 1997     

DNA homology between the crystal toxin genes of several mosquito pathogenicBacillus thuringiensis strains

With the recombinant pVB131 plasmid, which encodes the mosquitocidal 130 kilodalton peptide ofBacillus thuringiensis var.israelensis as a probe, DNA homology between crystal toxin genes of several dipteran-toxic strains was tested. Results from this study indicate that, while the crystal toxin genes ofB. thuringiensis var.kyushuensis and var.morrisoni isolate PG-14 share homology to the crystal toxin gene of var.israelensis, the -endotoxin genes of other dipteran-active strains tested (i.e., var.colmeri and var.kurstaki) do not exhibit any homology. The crystal toxin genes of vars.kyushuensis andmorrisoni isolate PG-14 were found to be located on plasmids of 60 and 94 megadaltons, respectively.     

Identification of a mosquitocidal toxin from Bacillus thuringiensis using mass spectrometry

The Bacillus thuringiensis strain S2160-1 has previously been identified as being highly toxic to mosquito larvae and a viable alternative to strains currently used commercially to control these insects. A PCR approach had identified the presence of four putative insecticidal toxin genes (cry30Ea, cry30 Ga, cry50Ba and cry54Ba) in this strain, but did not identify the genes that encoding three of the main crystal toxin proteins of size 140 and 130 and 30 kDa. In this study we used mass spectrometry to identify the 130 kDa toxin as a rare Cry4 toxin (Cry4Cb3). The gene encoding this toxin was cloned and expressed and the toxin shown to have mosquitocidal activity against Culex quinquefasciatus.     

Cloning and Characterization of Two Novel Genes, cry24B and s1orf2, from a Mosquitocidal Strain of Bacillus thuringiensis serovar sotto

Two new crystal protein genes, cry24B and s1orf2, were cloned from a mosquitocidal Bacillus thuringiensis serovar sotto strain. The cry24B and s1orf2 genes encoded a 76-kDa and 62-kDa protein, respectively. The Cry24B protein retained five conserved regions commonly found in the existing Cry proteins. The amino acid sequence of the S1ORF2 had a high homology to that of the ORF2 protein of B. thuringiensis serovar jegathesan. Southern hybridization experiments with a cry24B gene-specific probe revealed that these genes are located on two large plasmids of > 100 kb. When the two genes, cry24B and s1orf2, were expressed in an acrystalliferous B. thuringiensis host, the proteins were synthesized and accumulated as inclusions. These inclusions exhibited no larvicidal activities against three mosquito species: Aedes aegypti, Anopheles stephensi, and Culex pipiens molestus. Likewise, the inclusions contained no cytocidal activity against HeLa cells.     

Co-Expression of the Mosquitocidal Toxins Cyt1Aa and Cry11Aa from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Subsp. <Emphasis Type="Italic">israelensis</Emphasis> in <Emphasis Type="Italic">Asticcacaulis excentricus</Emphasis>

The cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti), whose product synergizes other mosquitocidal toxins, and functions as a repressor of resistance developed by mosquitoes against Bacilli insecticides, was introduced into the aquatic Gram-negative bacterium Asticcacaulis excentricus alongside the cry11Aa gene. The genes were introduced as an operon, but although mRNA was detected for both genes, no Cyt1Aa toxin was detected. Both proteins were expressed using a construct in which a promoter was inserted upstream of each gene. Recombinant A. excentricus expressing both toxins was found to be approximately twice as toxic to third instar larvae of Culex quinquefasciatus as transformants expressing just Cry11Aa.     

Coexpression of cyt1Aa of Bacillus thuringiensis subsp. israelensis with Bacillus sphaericus Binary Toxin Gene in Acrystalliferous Strain of B. thuringiensis

The cyt1Aa gene of Bacillus thuringiensis subsp. israelensis and binary toxin gene of Bacillus sphaericus C3-41 were introduced into an acrystalliferous strain of B. thuringiensis independently and in combination by using shuttle vector pBU4. SDS-PAGE and Western blot analysis proved that cyt1Aa and binary toxin genes coexpressed during the sporulation of the recombinant. Transformant strain expressing the Cyt1Aa and binary toxin proteins in combination was more toxic to susceptible and resistant Culex pipiens quinquefasciatus than the transformants expressing Cyt1Aa protein or binary toxin proteins independently. It was suggested that large amount of production of Cyt1Aa protein and binary toxin proteins possibly interacted synergistically, thereby increasing its mosquitocidal toxicity significantly. Received: 22 October 1999 / Accepted: 22 November 1999     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

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.     

Characterization of Cyt2Bc Toxin from Bacillus thuringiensis subsp. medellin

We cloned and sequenced a new cytolysin gene from Bacillus thuringiensis subsp. medellin. Three IS240-like insertion sequence elements and the previously cloned cyt1Ab and p21 genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from Bacillus thuringiensis subsp. israelensis and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of B. thuringiensis. Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, including strains resistant to the Bacillus sphaericus binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant C. quinquefasciatus.     

The dual-activity insecticidal protein,Cry2Aa,does not enhance the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis

Cry2Aa, one of the major insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki HD1, is known to be active against both lepidopteran and dipteran larvae. In order to determine whether Cry2Aa could enhance or synergize the mosquitocidal activity of B. thuringiensis subsp. israelensis, we constructed a plasmid vector that harbored the cry2Aa operon and transformed crystalliferous and acrystalliferous strains of this bacterium. The wild-type B. thuringiensis subsp. israelensis, a recombinant B. thuringiensis subsp. israelensis producing Cry2A along with its native major mosquitocidal proteins, and a recombinant B. thuringiensis subsp. israelensis producing Cry2Aa alone were tested against three major mosquito species — Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Our results demonstrated that Cry2Aa does not synergize or enhance the mosquitocidal activity of B. thuringiensis subsp. israelensis against these important vectors of disease.     

Fingerprinting of Bacillus thuringiensis Type Strains and Isolates by Using Bacillus cereus Group-Specific Repetitive Extragenic Palindromic Sequence-Based PCR Analysis

A total of 119 Bacillus thuringiensis strains (83 type strains and 26 native isolates), as well as five B. cereus group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six B. cereus group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive B. cereus (Bc)-Rep-PCR pattern, except for B. thuringiensis serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native β-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the B. thuringiensis type strain. The B. cereus group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including nonflagellar and self-agglutinated strains.     

Production of the bioinsecticide Bacillus thuringiensis subsp. israelensis with deltamethrin increases toxicity towards mosquito larvae

Bacillus thuringiensis subsp. israelensis is a bioinsecticide used for larval mosquito control and it represents a safe alternative to chemical insecticides. Despite its environmental safety, it is less efficient and persistent than chemical insecticides. To bypass these limitations, we propose to combine the advantages of chemical and biological insecticides by producing Bti in a medium supplemented with a chemical insecticide (DDT, deltamethrin, permethrin, propoxur or temephos). Among the investigated insecticides, the addition of deltamethrin in the medium induced a higher toxicity (over 6·72‐fold) of the composite deltamethrin‐Bti towards mosquito larvae as compared to Bti alone. This was mainly due to the insertion of deltamethrin into the membranes of Bti spores, as evidenced by a quantification of membrane‐extracted deltamethrin by HPLC. This composite larvicide is a promising tool to decrease the quantity of chemicals dispersed in the environment, to increase the efficacy of Bti and to facilitate its widespread use as a transition between chemical and biological insecticides. Further experiments are required to characterize the mechanisms that underline the incorporation of deltamethrin into Bti to optimize the production and the toxicity of this composite larvicide.

Significance and Impact of the Study

This study is the first report of an increased efficacy of the mosquitocidal bioinsecticide Bacillus thuringiensis subsp. israelensis (Bti) when produced with a chemical insecticide. The results clearly demonstrate that deltamethrin is able to synergize the insecticidal activity of Bti through inclusion into spore membranes, reducing off‐target and nonspecific toxicity occurring when the chemical is used alone as sprays. This new composite chemical–biological insecticide can become an invaluable tool as an intermediate between single chemical usage and the widespread use of Bti, notably in developing countries with limited financial resources for intensive mosquito control campaigns.     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

Importance of the Central Region of 130-kDa Insecticidal Proteins of Bacillus thuringiensis var. israelensis for Their Activity in Vivo and in Vitro

To delineate the mosquitocidal regions of the ISRH3 (CryIVB) and ISRH4 (CryIVA) proteins, which are two of the mosquitocidal 130-kDa proteins contained in the crystalline protein bodies (CPBs) of Bacillus thuringiensis var. israelensis (BTI), a deletion analysis of these protein genes has been done. Based on the evidence that each 130-kDa protein had two mosquitocidal regions, N-terminal and C-terminal ones, and these two regions shared a common part in the center of the 130-kDa proteins, deleted genes on this region were constructed. As the protein products which lacked the central region had reduced activities, the central region could be important for the mosquitocidal activity. The mosquitocidal and non-mosquitocidal truncated gene products of 130-kDa protein genes were also applied to a cultured lepidopteran cell line, TN-368. The mosquitocidal proteins caused the swelling and disruption of the cells in spite of the insecticidal specificity of CPBs of BTI, but the non-mosquitocidal proteins did not. Therefore, TN-368 cells were sensitive to the mosquitocidal fragments of 130-kDa proteins of BTI under the assay conditions used.