Analysis of the molecular basis of insecticidal specificity of Bacillus thuringiensis crystal delta-endotoxin.

The mechanism of action and receptor binding of a dual-specificity Bacillus thuringiensis var. aizawai ICl delta-endotoxin was studied using insect cell culture. The native protoxin was labelled with 125I, proteolytically activated and the affinity of the resulting preparations for insect cell-membrane proteins was studied by blotting. The active preparations obtained by various treatments had characteristic specificity associated with unique polypeptides, and showed affinity for different membrane proteins. The lepidopteran-specific preparation (trypsin-treated protoxin containing 58 and 55 kDa polypeptides) bound to two membrane proteins in the lepidopteran cells but none in the dipteran cells. The dipteran-specific preparation (protoxin treated sequentially with trypsin and Aedes aegypti gut proteases, containing a 53 kDa polypeptide) bound to a 90 kDa membrane protein in the dipteran (A. aegypti) cells but bound to none in the lepidopteran cells or Drosophila melanogaster cells. The toxicity of trypsin-activated delta-endotoxin was completely inhibited by preincubation with D-glucose, suggesting a role for this carbohydrate in toxin-receptor interaction. The toxicity was also decreased by osmotic protectants to an extent proportional to their viscometric radius. These results support a proposal that initial interaction of toxin with a unique receptor determines the specificity of the toxin, following which cell death occurs by a mechanism of colloid osmotic lysis.     

Comparison of the in vivo and in vitro activity of the delta-endotoxin of Bacillus thuringiensis var. morrisoni (HD-12) and two of its constituent proteins after cloning and expression in Escherichia coli

The insecticidal crystal delta-endotoxin of Bacillus thuringiensis var. morrisoni HD-12 contains at least five polypeptides in the range 126-140 kDa. Immune blotting revealed that individual proteins in this complex share homology with a range of other B. thuringiensis delta-endotoxins. In vivo the native HD-12 crystal killed a lepidopteran larva (Pieris brassicae) and a dipteran larva (Anopheles gambiae), but not the related dipteran Aedes aegypti. In vitro the solubilized activated crystal lysed Choristoneura fumiferana cells (lepidopteran) and dipteran cells derived from Anopheles gambiae and Culex quinquefasciatus but not those from Aedes aegypti. An intragenic probe derived from a B. thuringiensis var. sotto lepidoptera-specific delta-endotoxin gene hybridized with one of six plasmids extracted from HD-12. When cloned into pUC18 two HindIII fragments from this plasmid (pEG1 and pEG2) were shown to encode polypeptides cross-reacting with HD-12 antiserum. Escherichia coli lysates containing pEG2 were toxic in vivo to lepidoptera and diptera larvae and in vitro to a broader range of insect cell lines than the native crystal. E. coli cells containing pEG3, a subclone derived from pEG1, synthesised large amounts of a 140-kDa protein in the cytoplasm as inclusion bodies. The cytotoxicity of the protein encoded by pEG3 was restricted to C. fumiferana and A. gambiae cell lines.     

Cytotoxicity of a cloned Bacillus thuringiensis subsp. israelensis CryIVB toxin to an Aedes aegypti cell line

A cloned CryIVB toxin was purified from a cured strain of Bacillus thuringiensis (BT) containing the cryIVB gene on the recombinant plasmid Cam135. Solubilized protoxin was treated with Aedes gut extract or trypsin for varying times and tested for toxicity in vitro on three dipteran and one lepidopteran cell line. Treatment with the Aedes extract but not trypsin, produced an active toxin which lysed only Aedes aegypti cells out of those tested. This activation was time-dependent reaching a maximum after 6 h. Both the Aedes extract-treated and trypsin-treated toxin killed A. aegypti larvae, but this toxicity declined rapidly with increasing time of exposure to the proteolytic preparations.     

Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture

An insecticidal protein gene from Bacillus thuringiensis var. aizawai was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteolytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resistant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CF1 cells but not dipteran (Aedes albopictus) cells. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawai toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran cell membranes, which may be the receptor for this toxin.     

Mosquitocidal activity of the CryIC delta-endotoxin from Bacillus thuringiensis subsp. aizawai.

The cloned 135-kDa CryIC delta-endotoxin from Bacillus thuringiensis is a lepidopteran-active toxin, displaying high activity in vivo against Spodoptera litoralis and Spodoptera frugiperda larvae and in vitro against the S. frugiperda Sf9 cell line. Here, we report that the CryIC delta-endotoxin cloned from B. thuringienesis subsp. aizawai HD-229 and expressed in an acrystalliferous B. thuringiensis strain is also toxic to Aedes aegypti, Anophles gambiae, and Culex quinquefasciatus mosquito larvae. Furthermore, when solubilized and proteolytically activated by insect gut extracts, CryIC is cytotoxic to cell lines derived from the first two of these dipteran insects. This activity was not observed for two other lepidopteran-active delta-endotoxins, CryIA(a) and CryIA(c). However, in contrast to the case with a lepidopteran and dipteran delta-endotoxin cloned from B. thuringiensis subsp. aizawai IC1 (M.Z. Haider, B. H. Knowles, and D. J. Ellar, Eur. J. Biochem. 156:531-540, 1986), no differences in the in vitro specificity or processing of CryIC were found when it was activated by lepidopteran or dipteran gut extract. The recombinant CryIC delta-endotoxin expressed in Escherichia coli was also toxic to A. aegypti larvae. By contrast, a second cryIC gene cloned from B. thuringiensis subsp. aizawai 7.29 (V. Sanchis, D. Lereclus, G. Menou, J. Chaufaux, S. Guo, and M. M. Lecadet, Mol. Microbiol. 3:229-238, 1989) was nontoxic. DNA sequencing showed that the two genes were identical. However, CryIC from B. thuringiensis subsp. aizawai 7.29 had been cloned with a truncated C terminus, and when it was compared with the full-length CryIC delta-endotoxin, it was found to be insoluble under alkaline reducing conditions. These results show that CryIC from B. thuringiensis subsp. aizawai is a dually active delta-endotoxin.     

Distribution, Frequency, and Diversity of Bacillus thuringiensis in an Animal Feed Mill

Bacillus thuringiensis was isolated from 36 of 50 residue samples obtained from an animal feed mill (a stored-product environment). Of 710 selected colonies having Bacillus cereus-B. thuringiensis morphology isolated from the samples, 477 were classified as B. thuringiensis because of production of parasporal delta-endotoxin crystals. There was a diverse population of B. thuringiensis, as revealed by differentiation of the isolates into 36 subgroups by using (i) their spectra of toxicity to the lepidopterans Heliothis virescens, Pieris brassicae, and Spodoptera littoralis and the dipteran Aedes aegypti and (ii) their parasporal crystal morphology. A total of 55% of the isolates were not toxic to any of these insects at the concentrations used in the bioassays; 40% of all isolates were toxic to one or more of the Lepidoptera; and 20, 1, and 1% of the isolates were toxic to only P. brassicae, H. virescens, and S. littoralis, respectively. The most frequent toxicity was toxicity to P. brassicae (36% of all isolates); 18% of the isolates were toxic to A. aegypti (5% exclusively), 10% were toxic to H. virescens, and 4% were toxic to S. littoralis. Toxicity to P. brassicae was more often linked with toxicity to H. virescens than with toxicity to S. littoralis. The frequency of toxicity was significantly greater in isolates that produced bipyramidal crystals than in isolates that produced irregular pointed, irregular spherical, rectangular, or spherical crystals.     

Purification and properties of a 28-kilodalton hemolytic and mosquitocidal protein toxin of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2.

The mosquitocidal crystal of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2 was purified, bioassayed against third-instar Aedes aegypti larvae (50% lethal concentration, 7.5 micrograms/ml), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealing polypeptides of 125, 50, 47, and 28 kilodaltons (kDa). When solubilized and proteolytically activated by insect gut proteases or proteinase K, the crystal was cytotoxic to insect and mammalian cells in vitro and was hemolytic. By using nondenaturing polyacrylamide gel electrophoresis, a polypeptide of 23 kDa, derived from the 28-kDa protoxin, was identified which was hemolytic and cytotoxic to Aedes albopictus, A. aegypti, and Choristoneura fumiferana CF1 insect cell lines. The 23-kDa polypeptide was purified by ion-exchange chromatography and gave 50% lethal dose values of 3.8, 3.3, and 6.9 micrograms/ml against A. albopictus, A. aegypti, and C. fumiferana CF1 cells lines, respectively. Cytotoxicity in vitro was both dose and temperature dependent, with a sigmoidal dose-response curve. The cytotoxicity of the 23-kDa toxin and the solubilized and proteolytically activated delta-endotoxin was inhibited by a range of phospholipids containing unsaturated fatty acids and by triglyceride and diglyceride dispersions. An interaction with membrane phospholipids appears important for toxicity. Polyclonal antisera prepared against the 23-kDa polypeptide did not cross-react with polypeptides in the native crystals of four other mosquitocidal strains.     

Monoclonal Antibody Analysis and Insecticidal Spectrum of Three Types of Lepidopteran-Specific Insecticidal Crystal Proteins of Bacillus thuringiensis

We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pieris brassicae and had no detectable activity against Spodoptera littoralis. However, the 4.5-, 5.3-, and 6.6-kilobase subtypes differed in their toxicity against Heliothis virescens and Mamestra brassicae. Type B crystal proteins consisted of 140-kDa protoxins with a 55-kDa tryptic core fragment. These were only active against one of the five insect species tested (P. brassicae). The protoxin and the trypsin-activated toxin of type C were 135- and 63-kDa proteins, respectively. Proteins of this type were associated with high toxicity against S. littoralis and M. brassicae. A panel of 35 monoclonal antibodies was used to compare the structural characteristics of crystal proteins of the three different types and subtypes. Each type of protein could be associated with a typical epitope structure, indicating an unambiguous correlation between antigenic structure and insect specificity.     

Identification and purification of the 69-kDa intracellular protease involved in the proteolytic processing of the crystal delta-endotoxin of Bacillus thuringiensis subsp. tenebrionis

The dynamics of appearance of intracellular proteases in relation to the synthesis of crystal delta-endotoxin was studied to identify the native intracellular protease(s) involved in the proteolytic processing of the 73-kDa protoxin of Bacillus thuringiensis subsp. tenebrionis. In vitro proteolytic activation of the 73-kDa protoxin indicated the possible role of 69-kDa protease in the proteolytic processing of 73-kDa protoxin. The purified 69-kDa protease was able to cause the proteolytic activation of the 73-kDa protoxin to 68-kDa toxin and this conversion was inhibited by ethylenediamine tetraacetic acid and 1,10-phenanthroline.     

Nucleotide sequence and deduced amino acid sequence of a coleopteran-active delta-endotoxin gene from Bacillus thuringiensis subsp. san diego

A gene from Bacillus thuringiensis subsp. san diego that is responsible for a delta-endotoxin active against Colorado potato beetle and some other Coleoptera was sequenced and shown to have surprising regional homology with both lepidopteran and dipteran active delta-endotoxins from other strains of B. thuringiensis. Unlike the lepidopteran active toxins from B. thuringiensis subsp. kurstaki that exist as approx. 130-kDa protoxins and form bipyramidal crystalline inclusions, the coleopteran toxic protein forms a square-shaped crystal composed of an approx. 65-kDa protein. Comparisons of the gene sequences encoding the active portions of these protoxins indicate conservation of N-terminal hydrophilic and hydrophobic regions, and suggest a distant ancestral origin for these insecticidal proteins.     

Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture

An insecticidal protein gene from Bacillus thuringiensis var. aizawal was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteloytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resist-ant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CFI ceils but not dipteran (Aedes albopictus) calls. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawal toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran ceil membranes, which may be the receptor for this toxin.     

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo

When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.     

Folding and unfolding of the protoxin from Bacillus thuringiensis: evidence that the toxic moiety is present in an active conformation

The action of trypsin or papain on the 130-kDa crystal protein (protoxin) from Bacillus thuringiensis subsp. kurstaki HD-73 yields a 67-kDa proteinase-resistant toxic fragment (toxin) which is derived from the N-terminal half of the molecule. Sensitivity to proteolysis and fluorescence emission spectroscopy showed that the toxin unfolded to a much greater extent in 6 M guanidinium chloride (GuHCl) than in 8 M urea. Protoxin also unfolded extensively in 6 M GuHCl, whereas in 8 M urea only the C-terminal half of the molecule had unfolded extensively. Both unfolded protoxin and unfolded toxin refolded to their native and biologically active conformations. The biphasic unfolding observed for protoxin suggests that the C-terminal half of the molecule unfolded rapidly, whereas the N-terminal toxic moiety unfolded at a much slower rate, similar to that of the free 67-kDa toxin. A 67-kDa fragment, derived from the N-terminal half of the molecule, could be generated from the protoxin in the presence of either urea or GuHCl by treatment with proteinases. Compared to toxin in denaturants, this fragment was found to be more sensitive to proteolysis. However, on removal of the denaturants the fragment had the same proteinase resistance and cytolytic activity as native toxin. The increased proteinase sensitivity of the fragment generated in the presence of denaturants appears to be due to a perturbation in the conformation of the N-terminal toxic moiety. This perturbation is attributed to the unfolding of the C-terminal region of the protoxin prior to its proteolysis to yield the 67-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Micro-lipid-droplet encapsulation of Bacillus thuringiensis subsp. israelensis delta-endotoxin for control of mosquito larvae

The crystal delta-endotoxin of Bacillus thuringiensis subsp. israelensis is less toxic to larvae of Anopheles freeborni than to larvae of Aedes aegypti. However, when solubilized crystal was used, larvae from both species showed similar sensitivities. This effect presumably was due to the differences in feeding behavior between the two mosquito larvae when crystal preparations are used. A procedure is described whereby both crystal and solubilized B. thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity. The use of Freund incomplete adjuvant also allowed one to assay the solubilized toxin at a low nanogram level. Furthermore, coating the toxin with lipophilic material altered the buoyancy of the toxin and reversed the sensitivities of the two mosquito larvae toward the B. thuringiensis subsp. israelensis toxin. This difference in buoyancy was determined by using an enzyme-linked immunosorbent assay that was specific for the toxic peptides. These data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp. israelensis crystal can be developed.     

Micro-lipid-droplet encapsulation of Bacillus thuringiensis subsp. israelensis delta-endotoxin for control of mosquito larvae.

The crystal delta-endotoxin of Bacillus thuringiensis subsp. israelensis is less toxic to larvae of Anopheles freeborni than to larvae of Aedes aegypti. However, when solubilized crystal was used, larvae from both species showed similar sensitivities. This effect presumably was due to the differences in feeding behavior between the two mosquito larvae when crystal preparations are used. A procedure is described whereby both crystal and solubilized B. thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity. The use of Freund incomplete adjuvant also allowed one to assay the solubilized toxin at a low nanogram level. Furthermore, coating the toxin with lipophilic material altered the buoyancy of the toxin and reversed the sensitivities of the two mosquito larvae toward the B. thuringiensis subsp. israelensis toxin. This difference in buoyancy was determined by using an enzyme-linked immunosorbent assay that was specific for the toxic peptides. These data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp. israelensis crystal can be developed.     

Purification of a protein from Bacillus thuringiensis toxic to larvae of lepidoptera

The protein toxin of the parasporal body or crystal of Bacillus thuringiensis (Mattés isolate) has been purified severalfold by a combination of Sephadex G-200 gel filtration and ammonium sulphate precipitation. It has been shown that the use of highly alkaline conditions for dissolution of the crystals does not lead to serious artifacts. The crystal toxin has been shown to be quantitatively related to the crystal antigen. It is possible that there is a second distinct toxin present in the crystal and this too can be detected by its antigenic reaction. Purified toxic protein has been hydrolysed in vitro by regurgitated Pieris brassicae gut enzymes, chymotrypsin, trypsin and subtilisin. In each case the digest contained a product that was still antigenic, had mol.wt. about 40000 and was toxic to P. brassicae larvae. Smaller toxic molecules (mol.wt. approx. 10000) that did not react as antigens were also produced by proteolysis. It is possible that these smaller molecules were hydrolytic products of the larger digestion product.     

Common features of Bacillus thuringiensis toxins specific for Diptera and Lepidoptera

The complete nucleotide sequence of a cloned gene encoding a 130-kDa crystal protein of Bacillus thuringiensis (B.t.) subspecies israelensis has been determined. The recombinant protein (Bt8) was purified and shown to be a mosquito-specific toxin with a LC50 value of 43 ng/ml to third-instar larvae of Aedes aegypti. Bt8 is processed by proteases or midgut extracts of mosquito larvae into toxic fragments of 68-78 kDa. Deletion mapping indicated that the active fragment of Bt8 is localized in the N-terminal half of the protoxin molecule. The deduced amino acid sequence of Bt8 has been compared with that of Bt2, a Lepidoptera-specific toxin, previously cloned from Bacillus thuringiensis berliner. Highly homologous amino acid stretches are present in the C-terminal half of the proteins. The N-terminal parts show much less sequence homology but they display a strikingly similar distribution of hydrophilic and hydrophobic amino acids. In addition, Bt8 and Bt2 show a significant immunological cross-reaction. The data indicate that although these B.t. delta endotoxins exhibit a different insect-host specificity, they are structurally related and might use a similar mechanism to interact with insect cell membranes.     

Ex vivo cytotoxicity of the Bacillus thuringiensis Cry4B delta-endotoxin to isolated midguts of Aedes aegypti larvae

The pathological effect of the Bacillus thuringiensis Cry delta- endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protoxin. However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.     

Optimised expression in Escherichia coli and purification of the functional form of the Bacillus thuringiensis Cry4Aa delta-endotoxin

Achieving high-level expression of the Bacillus thuringiensis Cry4Aa mosquito-larvicidal protein was demonstrated. The 130-kDa Cry4Aa protoxin was overexpressed as an inclusion body in Escherichia coli under the control of the tac promoter together with the cry4Ba promoter. The solubility of the toxin inclusions in carbonate buffer, pH 10.0, was markedly enhanced at a cultivation temperature of 30 degrees C. Elimination of the tryptic cleavage site at Arg-235 in the loop between helices 5 and 6 still retained the high-level toxicity of E. coli cells expressing the Cry4Aa mutant against Aedes aegypti larvae. Trypsin digestion of the R235Q mutant protoxin produced a protease-resistant fragment of ca. 65kDa. A homogeneous product of the 65-kDa trypsin-treated R203Q protein was obtained after size-exclusion chromatography that would pave the way for the further crystallisation and X-ray crystallographic studies.     

Comparative toxicity of Bacillus thuringiensis var. israelensis crystal proteins in vivo and in vitro

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.