Ingestion, dissolution, and proteolysis of the Bacillus sphaericus toxin by mosquito larvae

Larvae of Culex quinquefasciatus are much more susceptible to the toxin of Bacillus sphaericus than are larvae of Aedes aegypti. In the present study, the rate of ingestion, dissolution, and the cleavage by midgut proteases of the B. sphaericus toxin were compared in larvae of these species to determine whether these factors account for the differences in susceptibility. During filter feeding, larvae of both species removed significant quantities of B. sphaericus toxin from suspensions. Filtration rates for 1 hr, the time at which C. quinquefasciatus exhibited marked intoxication, were higher for A. aegypti (576-713 microliters/larva/hr) than for C. quinquefasciatus (446-544 microliters/larva/hr). Within 24 hr of exposure, A. aegypti larvae ingested 97-99% of the toxin particulates and suffered not more than 10% mortality in suspensions which induced complete mortality in C. quinquefasciatus within 2 hr of exposure. Quantification of the particulate toxin present in larvae after exposure to B. sphaericus suspensions revealed that larvae of both species contained only minor amounts of the toxin, suggesting the larvae had been able to solubilize the toxin after ingestion. Proteases recovered from the feces of larvae cleaved at 43-kDa protein isolated from B. sphaericus toxin extract to 40 kDa in both species. Thus, differences in susceptibility to the B. sphaericus toxin between A. aegypti and C. quinquefasciatus are not due to differences in rates of ingestion, dissolution, or the specificity of proteases.     

Proteolysis in the gut of mosquito larvae results in further activation of the Bacillus sphaericus toxin.

Gut proteases from the larvae of the mosquito Culex pipiens convert the 43-kilodalton (kDa) toxin from Bacillus sphaericus 2362 to a 40-kDa peptide. The 50% lethal concentration of this peptide for tissue culture-grown cells of Culex quinquefasciatus was 1.0 microgram/ml (as determined by the intracellular ATP assay), 54-fold less than that of the 43-kDa peptide. Gut proteases from Anopheles gambiae and Aedes aegypti, as well as bovine pancreatic trypsin, also converted the 43-kDa protein to a 40-kDa peptide which was indistinguishable from the peptide formed by the proteases from C. pipiens with respect to its toxicity to tissue culture-grown cells of C. quinquefasciatus. Evidence for the in vivo conversion of the 43-kDa protein to the 40-kDa peptide was also obtained from experiments in which larvae of C. pipiens, Anopheles gambiae, and Aedes aegypti were fed crystals from B. sphaericus 2362. By using the exclusion of trypan blue as an indication of cell viability, it was shown that chitobiose, chitotriose, N-acetylmuramic acid, and N-acetylneuraminic acid decreased the toxicity of the 40-kDa peptide (from 100 to 50% mortality at about 10 mM concentrations of these sugars). Muramic acid, N-acetylgalactosamine, and N-acetylglucosamine were less effective, while several sugars had no effect, suggesting that the 40-kDa toxin binds to specific receptors on the cell membrane. The 40-kDa protein was less toxic to tissue culture-grown cells of Anopheles gambiae and Aedes dorsalis, and the same sugars which reduced the toxicity for cells of C. quinquefasciatus were also effective in reduction of toxicity for these cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolysis in the gut of mosquito larvae results in further activation of the Bacillus sphaericus toxin

Gut proteases from the larvae of the mosquito Culex pipiens convert the 43-kilodalton (kDa) toxin from Bacillus sphaericus 2362 to a 40-kDa peptide. The 50% lethal concentration of this peptide for tissue culture-grown cells of Culex quinquefasciatus was 1.0 microgram/ml (as determined by the intracellular ATP assay), 54-fold less than that of the 43-kDa peptide. Gut proteases from Anopheles gambiae and Aedes aegypti, as well as bovine pancreatic trypsin, also converted the 43-kDa protein to a 40-kDa peptide which was indistinguishable from the peptide formed by the proteases from C. pipiens with respect to its toxicity to tissue culture-grown cells of C. quinquefasciatus. Evidence for the in vivo conversion of the 43-kDa protein to the 40-kDa peptide was also obtained from experiments in which larvae of C. pipiens, Anopheles gambiae, and Aedes aegypti were fed crystals from B. sphaericus 2362. By using the exclusion of trypan blue as an indication of cell viability, it was shown that chitobiose, chitotriose, N-acetylmuramic acid, and N-acetylneuraminic acid decreased the toxicity of the 40-kDa peptide (from 100 to 50% mortality at about 10 mM concentrations of these sugars). Muramic acid, N-acetylgalactosamine, and N-acetylglucosamine were less effective, while several sugars had no effect, suggesting that the 40-kDa toxin binds to specific receptors on the cell membrane. The 40-kDa protein was less toxic to tissue culture-grown cells of Anopheles gambiae and Aedes dorsalis, and the same sugars which reduced the toxicity for cells of C. quinquefasciatus were also effective in reduction of toxicity for these cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variation in binding of Bacillus sphaericus toxin and wheat germ agglutinin to larval midgut cells of six species of mosquitoes

Bacillus sphaericus toxin labeled with fluorescein isothiocyanate was readily ingested by Culex pipiens, Aedes aegypti, Anopheles stephensi, Anopheles gambiae, Anopheles quadrimaculatus, and Anopheles albimanus larvae. Fluorescent toxin bound to the luminal cell surface in discrete regions of the posterior midgut and gastric caecum in C. pipiens. In Anopheles spp., toxin bound in a variable pattern to these structures and central and anterior midgut as well. The toxin did not bind to midgut cells of A. aegypti. The toxin was internalized in bright fluorescent vesicles in C. pipiens, but was not internalized in Anopheles spp. and appeared to be weakly bound in these larvae, leaking rapidly from the gut surface. The lectin, wheat germ agglutinin, which interferes with binding of the B. sphaericus toxin, bound to the posterior midgut and gastric caecum of all species, but was not internalized. These results suggest that the sugar moiety of the receptor is not solely responsible for specificity of this toxin, and that binding to Culex spp. midgut cells may be highly specific and of high affinity, whereas binding to Anopheles spp. cells may be nonspecific and/or of low affinity.     

Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush-border membranes from mosquito larvae.

The presence of specific receptors for Bacillus sphaericus binary toxin on brush-border membrane fractions (BBMF) from Culex pipiens larvae midgut cells was demonstrated by an in vitro binding assay. Both activated and radiolabelled polypeptides from the 51-kDa and 42-kDa binary toxin of B. sphaericus 1593 specifically bound to BBMF. Direct binding and homologous competition experiments indicated a single class of B. sphaericus toxin receptors, with a dissociation constant (Kd) of approximately 20 nM and a maximum binding capacity (Bmax) of approximately 7 pmol/mg BBMF protein. The sugars GalNAc, GlcNAc and N-acetyl neuraminic acid had no detectable inhibitory effect on toxin binding to C. pipiens BBMF. Binding experiments with the non-susceptible mosquito species Aedes aegypti failed to detect significant binding of B. sphaericus binary toxin to A. aegypti BBMF.     

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.     

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.     

Identification of the receptor for Bacillus sphaericus crystal toxin in the brush border membrane of the mosquito Culex pipiens (Diptera: Culicidae).

The binary toxin (Bin) from Bacillus sphaericus crystals specifically binds to soluble midgut brush border membrane proteins from Culex pipiens larvae. A single 60 kDa midgut membrane protein is identified as the binding protein. This protein is anchored in the mosquito midgut membrane via a glycosyl-phosphatidylinositol (GPI) anchor, and is partially released by phosphatidylinositol specific-phospholipase C (PI-PLC). Fractionation of soluble proteins by anion exchange chromatography indicates that the binding protein does not co-elute with leucine aminopeptidase activity. After partial purification, the sequences of internal amino acid fragments of the 60 kDa protein were determined. The peptide sequences were compared with data in GenBank, and showed a very high degree of similarity with enzymes belonging to the alpha-amylase family. Further enzymatic investigation showed that the receptor of the Bin toxin in C. pipiens larval midgut may be an alpha-glucosidase.     

Comparison of development of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus in mosquito larvae

Immunofluorescent staining was used with thin sections of paraffin-embedded specimens to detect the development of Bacillus thuringiensis var. israelensis and Bacillus sphaericus in the gut of mosquito larvae. The third- and fourth-instar larvae of Aedes aegypti, Anopheles maculatus, and Culex quinquefasciatus were fed either vegetative cells or spores of the bacteria. Spore germination, multiplication, and sporulation were studied in the larvae of each species. The spores of B. thuringiensis var. israelensis and B. sphaericus strain 2297 could germinate and cells could sporulate in the larval body. The vegetative cells of B. sphaericus strain 810428 were also able to produce spores in the mosquito larval gut, but the germination of spores could not be detected in the larvae. Multiplication of all bacterial species was observed after the larvae died. Growth of the bacteria in distilled water containing crude extracts of larvae made from each species was compared with that in synthetic medium (nutrient broth). They could produce spores and toxins in all the media used and the toxins had larvicidal activity against the target mosquitos Ae. aegypti, An. maculatus, and C. quinquefasciatus.     

Comparative studies of the mosquito-larval toxin of Bacillus sphaericus SSII-1 and 1593

Two strains of Bacillus sphaericus. SSII-1 and 1593, were bioassayed for toxic activity against second-instar larvae of the mosquito Culex pipiens quinquefasciatus. It was found that strain 1593 developed a level of toxicity 3000 times that of strain SSII-1. Although the toxic activity of B. sphaericus SSII-1 was relatively unchanged throughout growth, an increase in activity of strain 1593 occurred as the bacteria began to sporulate. Strain differences were examined by (i) growth cycle experiments, (ii) bioassays of the toxicity of oligosporogenous mutants, and (iii) manganese limitation experiments. The toxin of strain 1593 was shown to be more stable than that of strain SSII-1. Unlike the spores of strain SSII-1, the spores of B. sphaericus 1593 were found to be highly toxic. Thin sections of SSII-1 or 1593 cells did not reveal the presence of any inclusion body that might be related to toxicity.     

The receptor of Bacillus sphaericus binary toxin in Culex pipiens (Diptera: Culicidae) midgut: molecular cloning and expression

Culex pipiens larval midgut is the primary target of the binary toxin (Bin) present in parasporal inclusions of Bacillus sphaericus. Cpm1, a 60-kDa protein purified from brush border membranes, has been proposed as the receptor of the Bin toxin in the midgut epithelial cells of mosquitoes. We have cloned and characterized the corresponding cDNA from midgut of Culex pipiens larvae. The open reading frame predicted a 580 amino-acid protein with a putative signal peptide at the N-terminus and a putative GPI-anchoring signal at the C-terminus. The amino acid sequence of the cloned Cpm1 exhibited 39-43% identities with insect maltases (alpha-glucosidases and alpha-amylases). Recombinant Cpm1 expressed in E. coli specifically bound to the Bin toxin and had a significant alpha-glucosidase activity but no alpha-amylase activity. These results support the view that Cpm1 is an alpha-glucosidase expressed in Culex midgut where it constitutes the receptor for the Bin toxin. To date, this is the first component involved in the mosquitocidal activity of the Bacillus sphaericus Bin toxin to be characterized. Its identification provides a key step to elucidate the mode of action of the Bin toxin and the mechanisms of resistance developed against it by some mosquito strains.     

Ultrastructural effects of macrotetrolides of Streptomyces griseus LKS-1 in tissues of Culex pipiens larvae

The isolate of macrotetrolides produced by Streptomyces griseus strain LKS-1 was tested in its effect on the ultrastructure of larvae of Culex pipiens autogenicus. Changes were mainly in mitochondria where the cristae were destroyed and the outer membrane inflated. The endoplasmic reticulum was vacuolized and subsequently the nuclear membranes were seriously affected. Microvilli of the midgut epithelial cells and the surface membrane of these cells were unaltered and there were no changes in the arrangement of cells in the tissues. The effect of macrotetrolides on insect tissues is analogous to the effect of secondary metabolites of fungi such as beauvericin, destruxin E, cyclosporin or tolypin, and differs from the effects of bacterial endotoxins of Bacillus thuringiensis or B. sphaericus.     

Toxicity of Bacillus sphaericus crystal toxin to adult mosquitoes.

Adult Culex quinquefasciatus mosquitoes were killed by alkaline-solubilized Bacillus sphaericus toxin when it was introduced by enema into the midgut of the insect but not when it was administered orally. Adult Aedes aegypti mosquitoes were not affected by the toxin.     

Toxicity of Bacillus sphaericus crystal toxin to adult mosquitoes

Adult Culex quinquefasciatus mosquitoes were killed by alkaline-solubilized Bacillus sphaericus toxin when it was introduced by enema into the midgut of the insect but not when it was administered orally. Adult Aedes aegypti mosquitoes were not affected by the toxin.     

球形芽孢杆菌C3—41二元毒素基因在苏云金芽孢杆菌以色列亚种中的？…

球形芽孢杆菌Ｃ３－４１是我国分离的一株对蚊幼虫有毒杀作用的高毒力菌株,对库蚊、按蚊幼虫的毒性高于２３６２菌株,Ｓｏｕｔｈｅｒｎ杂交证明Ｃ３－４１总ＤＮＡ中３．５ＫｂＨｉｎｄＩＩＩ片段上带有４１．９和５１．４ｋＤ二元毒素基因。     

The 42- and 51-kilodalton mosquitocidal proteins of Bacillus sphaericus 2362: construction of recombinants with enhanced expression and in vivo studies of processing and toxicity.

After site-directed mutagenesis, the genes coding for the 42- and 51-kilodalton (kDa) mosquitocidal proteins of Bacillus sphaericus 2362 were placed under the regulation of the aprE (subtilisin) promoter of the Bacillus subtilis vector pUE (a derivative of pUB18). The levels of expression of the gene products in B. subtilis DB104 and B. sphaericus 718 were assessed by bioassays with larvae of Culex pipiens and by Western immunoblots. The results indicated that a higher amount of protein was produced in B. subtilis DB104. Electron microscopic examination of B. subtilis DB104 and B. sphaericus 718 containing the 42- and 51-kDa proteins indicated that amorphous inclusions accumulated in the former species and that crystals identical in appearance to that found in B. sphaericus 2362 were produced in the latter. Strains producing only the 42- or the 51-kDa protein were not toxic to larvae of C. pipiens. A mixture of both strains, a single strain producing both proteins, or a fusion of the 51- and the 42-kDa proteins was toxic. The amount of B. subtilis DB104 containing the 42- and the 51-kDa proteins necessary to kill 50% of the larvae of C. pipiens was 5.6 ng (dry weight) of cells per ml. This value was significantly lower than that for B. sphaericus 2362 (14 ng [dry weight] per ml). Larvae consuming purified amorphous inclusions containing the 42-kDa protein degraded this protein this protein to primarily 39- and 24-kDa peptides, whereas inclusions with the 51-kDa protein were primarily degraded to a protein of 44 kDa. Past studies involving purified proteins from B. sphaericus 2362 indicate an associate of toxicity with the 39-kDa peptide. The results presented here suggest that the 44-kDa degradation product of the 51-kDa protein may also be required for toxicity.     

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.     

球形芽孢杆菌C3-41二元毒素基因在苏云金芽孢杆菌以色列亚种中的克隆和表达

球形芽孢杆菌C3-41是我国分离的一株对蚊幼虫有毒杀作用的高毒力菌株,对库蚊、按蚊幼虫的毒性高于2362菌株,Southern杂交证明C\-3\|41总DNA中35Kb HindIII片段上带有419和514kD二元毒素基因,该片段由3479个核苷酸组成,核苷酸序列同2362菌株的二元毒素基因序列完全相同。含二元毒素基因的重组质粒pCW\|1和pCW\|2能在大肠杆菌中表达产生二元毒蛋白,但表达量低,重组子杀蚊毒性低。无晶体型苏云金芽孢杆菌以色列亚种重组子在其芽孢形成中能产生以晶体形式存在的二元毒素蛋白,其全发酵液和纯化晶体蛋白的杀蚊活性与C\-3\|41相近。     

Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxins.

In the course of sporulation, Bacillus sphaericus produces an inclusion body which is toxic to a variety of mosquito larvae. In this review we discuss the general biology of this species and concentrate on the genetics and physiology of toxin production and its processing in the midgut of the larval host. The larvicide of B. sphaericus is unique in that it consists of two proteins of 51 and 42 kDa, both of which are required for toxicity to mosquito larvae. There is a low level of sequence similarity between these two proteins, which differ in their sequences from all the other known insecticidal proteins of Bacillus thuringiensis. Within the midgut the 51- and 42-kDa proteins are processed to proteins of 43 and 39 kDa, respectively. The conversion of the 42-kDa protein to a 39-kDa protein results in a major increase in toxicity; the significance of the processing of the 51-kDa protein is not known. In contrast to the results with mosquito larvae, the 39-kDa protein is alone toxic for mosquito-derived tissue culture-grown cells, and this toxicity is not affected by the 51-kDa protein or its derivative, the 43-kDa protein. Comparisons of larvae from species which differ in their susceptibility to the B. sphaericus toxin indicate that the probable difference resides in the nature of the target sites of the epithelial midgut cells and not in uptake or processing of the toxin. A similar conclusion is derived from experiments involving tissue culture-grown cells from mosquito species which differ in their susceptibility to the B. sphaericus toxin.