Susceptibility of Spodoptera frugiperda and S. exigua to Bacillus thuringiensis Vip3Aa insecticidal protein

The Vip3Aa protein is an insecticidal protein secreted by Bacillus thuringiensis during the vegetative stage of growth. The activity of this protein has been tested after different steps/protocols of purification using Spodoptera frugiperda as a control insect. The results showed that the Vip3Aa protoxin was stable and retained full toxicity after being subjected to common biochemical steps used in protein purification. Bioassays with the protoxin in S. frugiperda and S. exigua showed pronounced differences in LC(50) values when mortality was measured at 7 vs. 10d. At 7d most live larvae were arrested in their development. LC(50) values of "functional mortality" (dead larvae plus larvae remaining in the first instar), measured at 7d, were similar or even lower than the LC(50) values of mortality at 10d. This strong growth inhibition was not observed when testing the trypsin-activated protein (62 kDa) in either species. S. exigua was less susceptible than S. frugiperda to the protoxin form, with LC(50) values around 10-fold higher. However, both species were equally susceptible to the trypsin-activated form. Processing of Vip3Aa protoxin to the activated form was faster with S. frugiperda midgut juice than with S. exigua midgut juice. The results strongly suggest that the differences in the rate of activation of the Vip3Aa protoxin between both species are the basis for the differences in susceptibility towards the protoxin form.     

Cloning and characterization of a cytolytic and mosquito larvicidal delta-endotoxin from Bacillus thuringiensis subsp. darmstadiensis

The cytolytic delta-endotoxin gene from Bacillus thuringiensis subsp. darmstadiensis was amplified from genomic DNA by PCR by using primers designed from the sequence of cyt2Aa1 gene of B. thuringiensis subsp. kyushuensis. DNA sequence analysis revealed an open reading frame translating to a 259-amino acid sequence. The cloned gene was designated cyt2Aa2. This gene was highly expressed in Escherichia coli as inclusion bodies that could be solubilized in 50 m M Na(2)CO(3), pH 10.5. Activation of the solubilized protoxin by proteinase K (1% wt/wt, proteinase K/protoxin) yielded the active fragment of about 23 kDa. Cyt2Aa2 showed high hemolytic activity against sheep erythrocytes (hemolytic end- point 0.25 microgram/ml) and was toxic to Culex quinquefasciatus and Aedes aegypti larvae (LC(50) 0.5-1.0 microgram/ml).     

Binding and toxicity of Bacillus thuringiensis protein Cry1C to susceptible and resistant diamondback moth (Lepidoptera: Plutellidae)

We studied mechanisms of resistance to Bacillus thuringiensis insecticidal crystal protein Cry1C in the diamondback moth, Plutella xylostella (L.). Binding assays with midgut brush border membrane vesicles prepared from whole larvae showed no significant difference between resistant and susceptible strains in binding of radioactively-labeled Cry1C. These results indicate that reduced binding of Cry1C to midgut membrane target sites did not cause resistance to Cry1C. Thus, the mechanism of resistance to Cry1C differs from that observed in several previously reported cases of resistance to Cry1A toxins in diamondback moth. We tested Cry1C toxin and Cry1C crystalline protoxin against resistant and susceptible larvae using leaf disk bioassays. After adjusting for the size difference between Cry1C toxin and protoxin, we found that with resistant larvae, toxin was significantly more toxic than protoxin. In contrast, with susceptible larvae, no significant difference in toxicity occurred between Cry1C toxin and protoxin. The resistance ratios for Cry1C were 19 for toxin and 48 for protoxin. These results suggest that reduced conversion of Cry1C protoxin to toxin is a minor mechanism of resistance to Cry1C. Because neither reduced binding nor reduced conversion of protoxin to toxin appear to be major mechanisms, one or more other mechanisms are important in diamondback moth resistance to Cry1C.     

Only part of the protoxin gene of Bacillus thuringiensis subsp. berliner 1715 is necessary for insecticidal activity.

Escherichia coli strains harboring deletion mutations of the insecticidal protoxin gene of Bacillus thuringiensis subsp. berliner 1715 were constructed. Although these strains did not produce intact protoxin, cell extracts from one of the mutants were extremely toxic to tobacco hornworm (Manduca sexta) larvae, indicating that only a part of the protoxin gene is required for insecticidal activity.     

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.     

Only part of the protoxin gene of Bacillus thuringiensis subsp. berliner 1715 is necessary for insecticidal activity

Escherichia coli strains harboring deletion mutations of the insecticidal protoxin gene of Bacillus thuringiensis subsp. berliner 1715 were constructed. Although these strains did not produce intact protoxin, cell extracts from one of the mutants were extremely toxic to tobacco hornworm (Manduca sexta) larvae, indicating that only a part of the protoxin gene is required for insecticidal activity.     

Adsorption and anti-ultraviolet light characteristics of the protoxin from Bacillus thuringiensis on montmorillonite,kaolinite, zinc oxide and rectorite

The adsorption, desorption and anti-ultraviolet light characteristics of the protoxin from Bacillus thuringiensis strain WG-001 on montmorillonite, kaolinite, zinc oxide and rectorite were studied. The protoxin was easily adsorbed onto minerals and the adsorption reached equilibrium within 0.5–1.0 h (except for rectorite). The adsorption isotherms of protoxin at different concentrations in sodium carbonate buffer (pH 9) followed the Langmuir (R ² >0.97) and Freundlich (R ² >0.95) equations. The maximum amounts of protoxin adsorbed were in the order: montmorillonite>rectorite>znic oxide>kaolinite. In the range of pH from 9 to 11 (carbonate buffer), the protoxin adsorbed decreased with increasing pH. The adsorption was not significantly affected by the temperature between 5 and 45°C. Both free and adsorbed protoxin were toxic to larvae of Heliothis armigera. The LC₅₀ value of free and adsorbed protoxin on montmorillonite, rectorite, zinc oxide and kaolinite were 14±1.16, 1.76±0.31, 2.94±0.71, 4.78±2.08 and 1.91±0.91 µg mL^?1, respectively. After 1 h of ultraviolet irradiation, the LC₅₀ of the above samples increased by 41.4, 19.3, 16.3, 125.9 and 62.3%, respectively. The desorption of adsorbed protoxin in water ranged from 30.1 to 64.9% and from 18.5 to 48.7% in carbonate buffer.     

Use of silkworm larvae to study pathogenic bacterial toxins

Injection of stationary phase culture-supernatants of Staphylococcus aureus and Pseudomonas aeruginosa into the hemolymph of silkworm larvae caused their death, whereas a culture-supernatant of a non-pathogenic strain of Escherichia coli did not. A culture-supernatant of a mutant of agr, a global virulence regulator of S. aureus that is required for exotoxin production, was much less toxic to silkworm larvae. A culture-supernatant of a disruption mutant of the S. aureus beta-toxin gene did not kill larvae, whereas one of a deletion mutant of alpha-toxin, gamma-toxin, or aureolysin killed larvae, indicating that the beta-toxin gene is required for staphylococcal supernatant-mediated killing of silkworm larvae. The 50% lethal doses (LD50) of staphylococcal alpha-toxin and beta-toxin, Pseudomonas exotoxin A and diphtheria toxin were 12 microg/g, 9 microg/g, 0.14 microg/g and 1.1 microg/g, respectively. As the purified toxins killed the larvae, silkworm larvae could be used as a model to study the actions of pathogenic bacterial toxins in animal bodies.     

Bacillus thuringiensis crystal protein: effect of chemical modification of the cysteine and lysine residues.

The 16 cysteine residues of reduced protoxin from Bacillus thuringiensis subsp. kurstaki HD-73 can be quantitatively reacted with: (a) iodoacetic acid, to give carboxymethyl protoxin; (b) iodoacetamide, giving carbaminomethyl protoxin and (c) N-(beta-iodoethyl)trifluoroacetamide to give aminoethyl protoxin. The carboxymethyl derivative was found to be significantly more soluble at neutral pH values where both the native protoxin and the carbaminomethyl derivative exhibit low solubilities. At the alkaline pH values (pH 9.5-10.5) normally used to solubilize the crystal protein, the native protein was slightly more soluble than either the carboxymethyl or the carbaminomethyl derivatives. The aminoethyl derivative had an extremely low solubility at all pH values. Succinic anhydride reacted with only 35% of the lysine residues in both the carboxymethyl and the carbaminomethyl protoxin derivatives. Nonetheless, these succinylated protoxins exhibited significantly increased solubilities at neutral pH values. All the derivatives were found to retain full insecticidal activity toward spruce budworm (Choristeneura fufimerana) larvae. It is concluded that all the cysteine residues and modified lysine residues are on the surface of the protein and that derivatization does not alter the conformation of the solubilized protoxin.     

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC₅₀ of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.     

Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae.

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.     

Prey-mediated effects of Cry1Ab toxin and protoxin and Cry2A protoxin on the predator Chrysoperla carnea

Laboratory feeding experiments were carried out to study prey-mediated effects of artificial diet containing Bacillus thuringiensis proteins on immature Chrysoperla carnea. Activated Cry1Ab toxin and the protoxins of Cry1Ab and Cry2A were mixed into standard meridic diet for Spodoptera littoralis (Boisduval) larvae at the following concentrations; for Cry1Ab toxin, 25, 50, 100 g g^–1 diet were used; for Cry1Ab protoxin, the concentration was doubled (50 g g^–1 diet, 100 g g^–1 diet and 200 g g^–1 diet) to give relative comparable levels of toxin concentration. Cry2A protoxin was incorporated into the meridic diet at one concentration only (100 g g^–1 diet). For the untreated control, the equivalent amount of double distilled water was added to the meridic diet. Individual C. carnea larvae were raised on S. littoralis larvae fed with one of the respective treated meridic diets described above. The objectives were to quantify and compare the resulting effects on mortality and development time of C. carnea with those observed in two previous studies investigating prey-mediated effects of transgenic Cry1Ab toxin-producing corn plants and the other studying effects of Cry1Ab toxin fed directly to C. carnea larvae. Mean total immature mortality for chrysopid larvae reared on B. thuringiensis-fed prey was always significantly higher than in the control (26%). Total immature mortality of C. carnea reared on Cry1Ab toxin 100 g g^–1 diet-fed prey was highest (78%) and declined with decreasing toxin concentration. Cry1Ab protoxin-exposed C. carnea larvae did not exhibit a dose response. Prey-mediated total mortality of Cry1Ab protoxin-exposed chrysopid larvae was intermediate (46–62%) to Cry1Ab toxin exposed (55–78%) and Cry2A protoxin (47%) exposed C. carnea. In agreement with the previous studies, total development time of C. carnea was not consistently, significantly affected by the Bt-treatments except at the highest Cry1Ab toxin concentration. However, both highest mortality and delayed development of immature C. carnea raised on Cry1Ab toxin 100 g g^–1 diet – fed prey may have been confounded with an increased intoxication of S. littoralis larvae that was observed at that concentration. At all other B. thuringiensis protein concentrations S. littoralis was not lethally affected. Comparative analysis of the results of this study with those of the two previous studies revealed that in addition to prey/herbivore by B. thuringiensis interactions, also prey/herbivore by plant interactions exist that contribute to the observed toxicity of B. thuringiensis – fed S. littoralis larvae for C. carnea. These findings demonstrate that tritrophic level studies are necessary to assess the long-term compatibility of insecticidal plants with important natural enemies.     

Characterization and comparison of midgut proteases of Bacillus thuringiensis susceptible and resistant diamondback moth (Plutellidae: Lepidoptera)

The midgut proteases of the Bacillus thuringiensis resistant and susceptible populations of the diamondback moth, Plutella xylostella L. were characterized by using protease specific substrates and inhibitors. The midgut contained trypsin-like proteases of molecular weights of 97, 32, 29.5, 27.5, and 25 kDa. Of these five proteases, 29.5 kDa trypsin-like protease was the most predominant in activation of protoxins of Cry1Aa and Cry1Ab. The activation of Cry1Ab protoxin by midgut protease was fast (T(1/2) of 23-24 min) even at a protoxin:protease ratio of 250:1. The protoxin activation appeared to be multi-step process, and at least seven intermediates were observed before formation of a stable toxin of about 57.4 kDa from protoxin of about 133 kDa. Activation of Cry1Aa was faster than that of Cry1Ab on incubation of protoxins with midgut proteases and bovine trypsin. The protoxin and toxin forms of Cry proteins did not differ in toxicity towards larvae of P. xylostella. The differences in susceptibility of two populations to B. thuringiensis Cry1Ab were not due to midgut proteolytic activity. Further, the proteolytic patterns of Cry1A protoxins were similar in the resistant as well as susceptible populations of P. xylostella.     

Delineation of a toxin-encoding segment of a Bacillus thuringiensis crystal protein gene

Crystals of Bacillus thuringiensis subsp. kurstaki HD-1-Dipel contain a Mr 134,000 protoxin which can be cleaved by proteolysis to a peptide of Mr approximately 70,000; this peptide is lethal to lepidopteran larvae. We have analyzed the peptides produced by recombinant Escherichia coli strains bearing deletions and fusions of the protoxin gene in order to delineate the portion of the gene which encodes the toxic peptide. The recombinant strains produced the toxic peptide as well as larger peptides whose size was related to the length of the deleted gene. The results indicate that the amino-terminal 55% of the protoxin protein is sufficient for toxicity. While two different gene fusions to the 10th codon allowed the synthesis of toxic polypeptides, fusions to the 50th codon did not. 3' end deletions up to the 645th codon allowed synthesis of the toxic peptide whereas a deletion to the 603rd codon yielded a non-toxic peptide. Some of the 5' and 3' end alterations to the gene caused changes in the proteolytic cleavage patterns of the polypeptides synthesized by E. coli, suggesting that the alterations led to conformational changes in the proteins. The presence of different 3' end segments affected the levels of synthesis of the altered crystal proteins.     

Comparative study of Bacillus thuringiensis Cry1Ia and Cry1Aa delta-endotoxins: Activation process and toxicity against Prays oleae

Cry1Ia and Cry1Aa proteins exhibited toxicities against Prays oleae with LC₅₀ of 189 and 116 ng/cm², respectively. The ability to process Cry1Ia11 protoxin by trypsin, chymotrypsin and P. oleae larvae proteases was studied and compared to that of Cry1Aa11. After solubilization under high alkaline condition (50 mM NaOH), Cry1Aa11 was converted into a major fragment of 65 kDa, whereas Cry1Ia11 protoxin was completely degraded by P. oleae larvae proteases and trypsin and converted into a major fragment of 70 kDa by chymotrypsin. Using less proteases of P. oleae juice, the degradation of Cry1Ia11 was attenuated. When the solubilization (in 50 mM Na₂CO₃ pH 10.5 buffer) and activation were combined, Cry1Ia11 was converted into a proteolytic product of 70 kDa after 3 h of incubation with trypsin, chymotrypsin and P. oleae juice. These results suggest that the in vivo solubilization of Cry1Ia11 was assured by larval proteases after a swelling of the corresponding inclusion due to the alkalinity of the larval midgut.     

The solubility of inclusion proteins from Bacillus thuringiensis is dependent upon protoxin composition and is a factor in toxicity to insects.

Bacillus thuringiensis subsp. aizawai HD133 is one of several strains particularly effective against Plodia interpunctella selected for resistance to B. thuringiensis subsp. kurstaki HD1 (Dipel). B. thuringiensis subsp. aizawai HD133 produces inclusions containing three protoxins, CryIA(b), CryIC, and CryID, and the CryIC protoxin has been shown to be active on resistant P. interpunctella as well as on Spodoptera larvae. The CryIA(b) protoxin is very similar to the major one in B. thuringiensis subsp. kurstaki HD1, and as expected, this protoxin was inactive on resistant P. interpunctella. A derivative of B. thuringiensis subsp. aizawai HD133 which had been cured of a 68-kb plasmid containing the cryIA(b) gene produced inclusions comprising only the CryIC and CryID protoxins. Surprisingly, these inclusions were much less toxic for resistant P. interpunctella and two other Lepidoptera than those produced by the parental strain, whereas the soluble protoxins from these strains were equally effective. In contrast, inclusions from the two strains were about as active as soluble protoxins for Spodoptera frugiperda larvae, so toxicity differences between inclusions may be due to the solubilizing conditions within particular larval guts. Consistent with this hypothesis, it was found that a higher pH was required to solubilize protoxins from inclusions from the plasmid-cured strain than from B. thuringiensis subsp. aizawai HD133, a difference which is probably attributable to the absence of the CryIA(b) protoxin in the former. The interactions of structurally related protoxins within an inclusion are probably important for solubility and are thus another factor in the effectiveness of B. thuringiensis isolates for particular insect larvae.     

Hemolymph factor in armyworm larvae infected with a nuclear-polyhedrosis virus toxic to Apanteles militaris

The parasitoid, Apanteles militaris, was killed or failed to pupate when its armyworm host was infected with the hypertrophic strain of a nuclear-polyhedrosis virus. Virion-free plasma from diseased larvae killed the parasitoids when injected into uninfected larvae. The toxic factor was partially precipitated from plasma that was 50% saturated with ammonium sulfate and was completely precipitated in 50% ethanol or methanol. Parasitoids in larvae infected with the typical strain of nuclear-polyhedrosis virus seemed unaffected.     

Identification of Henosepilachna vigintioctomaculata (Coleoptera: Coccinellidae) midgut putative receptor for Bacillus thuringiensis insecticidal Cry7Ab3 toxin

Bt WZ-9 strain, containing a single Cry7Ab3 toxin, had effective insecticidal activity against larvae of Henosepilachna vigintioctomaculata. By incubation with larvae midgut homogenate and trypsin in vitro, 130 kDa Cry7Ab3 protoxin was degraded into the ～75 kDa proteinase-resistant fragments. In vivo analysis, 130 kDa Cry7Ab3 protoxin was also processed into ～75 kDa fragment. Histopathological observations indicated that Cry7Ab3 ingestion by H. vigintioctomaculata larvae causes acceleration in the blebbing of the midgut epithelium cells into the gut lumen and eventual lysis of the epithelium cells resulting in larval death. A ligand blotting experiment demonstrated that Cry7Ab3 toxin bound a 220 kDa BBMV protein. This receptor protein was identified as cadherin by matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS). The cadherin protein may be the receptor of Cry7Ab3. The data obtained may contribute to a better understanding of the mechanism of Cry7Ab3 toxin against H. vigintioctomaculata larvae.