Recombinant Strain of Bacillus thuringiensis Producing Cyt1A, Cry11B, and the Bacillus sphaericus Binary Toxin

A novel recombinant Bacillus thuringiensis subsp. israelensis strain that produces the B. sphaericus binary toxin, Cyt1Aa, and Cry11Ba is described. The toxicity of this strain (50% lethal concentration [LC₅₀] = 1.7 ng/ml) against fourth-instar Culex quinquefasciatus was higher than that of B. thuringiensis subsp. israelensis IPS-82 (LC₅₀ = 7.9 ng/ml) or B. sphaericus 2362 (LC₅₀ = 12.6 ng/ml).     

Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Variable Cross-Resistance to Cry11B from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) Resistant to Single or Multiple Toxins of Bacillus thuringienisis subsp. israelensis

A novel mosquitocidal bacterium, Bacillus thuringiensis subsp. jegathesan, and one of its toxins, Cry11B, in a recombinant B. thuringiensis strain were evaluated for cross-resistance with strains of the mosquito Culex quinquefasciatus that are resistant to single and multiple toxins of Bacillus thuringiensis subsp. israelensis. The levels of cross-resistance (resistance ratios [RR]) at concentrations which caused 95% mortality (LC₉₅) between B. thuringiensis subsp. jegathesan and the different B. thuringiensis subsp. israelensis-resistant mosquito strains were low, ranging from 2.3 to 5.1. However, the levels of cross-resistance to Cry11B were much higher and were directly related to the complexity of the B. thuringiensis subsp. israelensis Cry toxin mixtures used to select the resistant mosquito strains. The LC₉₅ RR obtained with the mosquito strains were as follows: 53.1 against Cq4D, which was resistant to Cry11A; 80.7 against Cq4AB, which was resistant to Cry4A plus Cry4B; and 347 against Cq4ABD, which was resistant to Cry4A plus Cry4B plus Cry11A. Combining Cyt1A with Cry11B at a 1:3 ratio had little effect on suppressing Cry11A resistance in Cq4D but resulted in synergism factors of 4.8 and 11.2 against strains Cq4AB and Cq4ABD, respectively; this procedure eliminated cross-resistance in the former mosquito strain and reduced it markedly in the latter strain. The high levels of activity of B. thuringiensis subsp. jegathesan and B. thuringiensis subsp. israelensis, both of which contain a complex mixture of Cry and Cyt proteins, against Cry4- and Cry11-resistant mosquitoes suggest that novel bacterial strains with multiple Cry and Cyt proteins may be useful in managing resistance to bacterial insecticides in mosquito populations.     

Activity of a Brazilian Strain of Bacillus thuringiensis israelensis Against the Cotton Boll Weevil Anthonomus grandis Boheman (Coleoptera: Tenebrionidae)

A Brazilian Bacillus thuringiensis subspecies israelensis, toxic to Diptera, including mosquitoes, was found also to show toxicity to the coleopteran boll weevil Anthonomus grandis Boheman at an equivalent level to that of the standard coleopteran-active B. thuringiensis subspecies tenebrionis T08017. Recombinant B. thuringiensis strains expressing the individual Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa toxins from this strain were assessed to evaluate their potential contribution to the activity against A. grandis, either alone or in combination. Whilst individual toxins produced mortality, none was sufficiently potent to allow calculation of LC₅₀ values. Combinations of toxins were unable to attain the same potency as the parental B. thuringiensis subsp. israelensis, suggesting a major role for other factors produced by this strain.     

Genetic and Biochemical Approach for Characterization of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Field Population of the Diamondback Moth, Plutella xylostella

Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F₄ to F₈) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F₉ found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F₉. The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F₉ to F₁₃) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with ¹²⁵I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F₁₅). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F₂ progeny from a backcross of F₁ progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.     

Genetic and Biochemical Characterization of Field-Evolved Resistance to Bacillus thuringiensis Toxin Cry1Ac in the Diamondback Moth, Plutella xylostella

The long-term usefulness of Bacillus thuringiensis Cry toxins, either in sprays or in transgenic crops, may be compromised by the evolution of resistance in target insects. Managing the evolution of resistance to B. thuringiensis toxins requires extensive knowledge about the mechanisms, genetics, and ecology of resistance genes. To date, laboratory-selected populations have provided information on the diverse genetics and mechanisms of resistance to B. thuringiensis, highly resistant field populations being rare. However, the selection pressures on field and laboratory populations are very different and may produce resistance genes with distinct characteristics. In order to better understand the genetics, biochemical mechanisms, and ecology of field-evolved resistance, a diamondback moth (Plutella xylostella) field population (Karak) which had been exposed to intensive spraying with B. thuringiensis subsp. kurstaki was collected from Malaysia. We detected a very high level of resistance to Cry1Ac; high levels of resistance to B. thuringiensis subsp. kurstaki Cry1Aa, Cry1Ab, and Cry1Fa; and a moderate level of resistance to Cry1Ca. The toxicity of Cry1Ja to the Karak population was not significantly different from that to a standard laboratory population (LAB-UK). Notable features of the Karak population were that field-selected resistance to B. thuringiensis subsp. kurstaki did not decline at all in unselected populations over 11 generations in laboratory microcosm experiments and that resistance to Cry1Ac declined only threefold over the same period. This finding may be due to a lack of fitness costs expressed by resistance strains, since such costs can be environmentally dependent and may not occur under ordinary laboratory culture conditions. Alternatively, resistance in the Karak population may have been near fixation, leading to a very slow increase in heterozygosity. Reciprocal genetic crosses between Karak and LAB-UK populations indicated that resistance was autosomal and recessive. At the highest dose of Cry1Ac tested, resistance was completely recessive, while at the lowest dose, it was incompletely dominant. A direct test of monogenic inheritance based on a backcross of F₁ progeny with the Karak population suggested that resistance to Cry1Ac was controlled by a single locus. Binding studies with ¹²⁵I-labeled Cry1Ab and Cry1Ac revealed greatly reduced binding to brush border membrane vesicles prepared from this field population.     

Binding of Cyt1Aa and Cry11Aa Toxins of Bacillus thuringiensis Serovar israelensis to Brush Border Membrane Vesicles of Tipula paludosa (Diptera: Nematocera) and Subsequent Pore Formation

Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.     

Single concentration tests show synergism among Bacillus thuringiensis subsp. israelensis toxins against the malaria vector mosquito Anophelesalbimanus

Bioassays of insecticidal proteins from Bacillus thuringiensis subsp. israelensis with larvae of the malaria vector mosquito Anophelesalbimanus showed that the cytolytic protein Cyt1Aa was not toxic alone, but it increased the toxicity of the crystalline proteins Cry4Ba and Cry11Aa. Synergism also occurred between Cry4Ba and Cry11Aa toxins. Whereas many previous analyses of synergism have been based on a series of toxin concentrations leading to comparisons between expected and observed values for the concentration killing 50% of insects tested (LC₅₀), we describe and apply a method here that enables testing for synergism based on single concentrations of toxins.     

Cyt1Aa from Bacillus thuringiensis subsp. israelensis Is Toxic to the Diamondback Moth, Plutella xylostella, and Synergizes the Activity of Cry1Ac towards a Resistant Strain

The Bacillus thuringiensis subsp. israelensis cytolytic protein Cyt1Aa was found to be toxic to an insecticide-susceptible laboratory population of Plutella xylostella. Cry1Ac-resistant populations of P. xylostella showed various degrees of resistance to Cyt1Aa. Cyt1Aa/Cry1Ac mixtures showed a marked level of synergism in the Cry1Ac-resistant populations.     

Fate of Bacillus thuringiensis subsp. israelensis in the Field: Evidence for Spore Recycling and Differential Persistence of Toxins in Leaf Litter

Bacillus thuringiensis subsp. israelensis is a bioinsecticide increasingly used worldwide for mosquito control. Despite its apparent low level of persistence in the field due to the rapid loss of its insecticidal activity, an increasing number of studies suggested that the recycling of B. thuringiensis subsp. israelensis can occur under specific, unknown conditions. Decaying leaf litters sampled in mosquito breeding sites in the French Rhône-Alpes region several months after a treatment were shown to exhibit a high level of larval toxicity and contained large amounts of spores. In the present article, we show that the high concentration of toxins found in these litters is consistent with spore recycling in the field, which gave rise to the production of new crystal toxins. Furthermore, in these toxic leaf litter samples, Cry4Aa and Cry4Ba toxins became the major toxins instead of Cyt1Aa in the commercial mixture. In a microcosm experiment performed in the laboratory, we also demonstrated that the toxins, when added in their crystal form to nontoxic leaf litter, exhibited patterns of differential persistence consistent with the proportions of toxins observed in the field-collected toxic leaf litter samples (Cry4 > Cry11 > Cyt). These results give strong evidence that B. thuringiensis subsp. israelensis recycled in specific breeding sites containing leaf litters, and one would be justified in asking whether mosquitoes can become resistant when exposed to field-persistent B. thuringiensis subsp. israelensis for several generations.     

Assessment of protoxin composition of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by use of polyacrylamide gel block and mass spectrometry

Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Zujiao Fu and Yunjun Sun contributed equally to this work.     

Synergism between Bacillus thuringiensis Spores and Toxins against Resistant and Susceptible Diamondback Moths (Plutella xylostella)

We studied the effects of combinations of Bacillus thuringiensis spores and toxins on the mortality of diamondback moth (Plutella xylostella) larvae in leaf residue bioassays. Spores of B. thuringiensis subsp. kurstaki increased the toxicity of crystals of B. thuringiensis subsp. kurstaki to both resistant and susceptible larvae. For B. thuringiensis subsp. kurstaki, resistance ratios were 1,200 for a spore-crystal mixture and 56,000 for crystals without spores. Treatment of a spore-crystal formulation of B. thuringiensis subsp. kurstaki with the antibiotic streptomycin to inhibit spore germination reduced toxicity to resistant larvae but not to susceptible larvae. In contrast, analogous experiments with B. thuringiensis subsp. aizawai revealed no significant effects of adding spores to crystals or of treating a spore-crystal formulation with streptomycin. Synergism occurred between Cry2A and B. thuringiensis subsp. kurstaki spores against susceptible larvae and between Cry1C and B. thuringiensis subsp. aizawai spores against resistant and susceptible larvae. The results show that B. thuringiensis toxins combined with spores can be toxic even though the toxins and spores have little or no independent toxicity. Results reported here and previously suggest that, for diamondback moth larvae, the extent of synergism between spores and toxins of B. thuringiensis depends on the strain of insect, the type of spore, the set of toxins, the presence of other materials such as formulation ingredients, and the concentrations of spores and toxins.     

Towards novel Cry toxins with enhanced toxicity/broader: a new chimeric Cry4Ba / Cry1Ac toxin

Attempts have been made to express or to merge different Cry proteins in order to enhance toxic effects against various insects. Cry1A proteins of Bacillus thuringiensis form a typical bipyramidal parasporal crystal and their protoxins contain a highly conserved C-terminal region. A chimerical gene, called cry(4Ba-1Ac), formed by a fusion of the N-terminus part of cry4Ba and the C-terminus part of cry1Ac, was constructed. Its transformation to an acrystalliferous B. thuringiensis strain showed that it was expressed as a chimerical protein of 116 kDa, assembled in spherical to amorphous parasporal crystals. The chimerical gene cry(4Ba-1Ac) was introduced in a B. thuringiensis kurstaki strain. In the generated crystals of the recombinant strain, the presence of Cry(4Ba-1Ac) was evidenced by MALDI-TOF. The recombinant strain showed an important increase of the toxicity against Culex pipiens larvae (LC₅₀ = 0.84 mg l^?1 ± 0.08) compared to the wild type strain through the synergistic activity of Cry2Aa with Cry(4Ba-1Ac). The enhancement of toxicity of B. thuringiensis kurstaki expressing Cry(4Ba-1Ac) compared to that expressing the native toxin Cry4Ba, might be related to its a typical crystallization properties. The developed fusion protein could serve as a potent toxin against different pests of mosquitoes and major crop plants.     

Cyt1A of Bacillus thuringiensis Delays Evolution of Resistance to Cry11A in the Mosquito Culex quinquefasciatus

Insecticides based on Bacillus thuringiensis subsp. israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported. Moreover, in contrast to B. thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B. thuringiensis subsp. israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations. Selection of Culex quinquefasciatus with mutants of B. thuringiensis subsp. israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance. This hypothesis, however, has not been tested experimentally. Here we report experiments in which separate C. quinquefasciatus populations were selected for 20 generations to recombinant strains of B. thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population. The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains. When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold. This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent. These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins.     

Marginal cross-resistance to mosquitocidal Bacillus thuringiensis strains in Cry11A-resistant larvae: presence of Cry11A-like toxins in these strains

Culex quinquefasciatus mosquito larvae resistant to the Cry11A toxin showed marginal cross-resistance to the multiple toxin crystals from B. thuringiensis subsp. israelensis and also to toxin crystals from three other mosquitocidal strains, i.e. B. thuringiensis subsp. fukuokaensis, subsp. jegathesan, and subsp. kyushuensis. Cross-resistance patterns of the Cry11A-resistant larvae to mosquitocidal strains of B. thuringiensis together with the immunological screening using antisera raised against Cry11A indicated the presence of Cry11A-like toxins in these strains and could be used as a screening tool for the identification of novel toxins. The Cry11A-resistant larvae had significantly less resistance to the Cry11B toxin from B. thuringiensis subsp. jegathesan. The occurrence of cytolytic toxins in all of these mosquitocidal strains partially explains the marginal cross-resistance observed with multiple toxin crystals since each of these crystals also contains cytolytic toxins.     

Synergism of mosquitocidal toxicity between CytA and CrylVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis

The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp. israelensis and the PG-14 isolate of B. thuringiensis subsp. morrisoni is at least 20-fold greater than any of the four mosquitocidal proteins of which It is composed (CytA, CrylVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CrylVD toxins were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis cells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bio-assays against first instars of Aedes aegypti. The LC₅₀ for the CytA inclusion was 60 ng ml⁻¹, whereas the LC₅₀ for the CryIVD was 85ng ml⁻¹ In comparison, the LC₅₀s for different combinations of CytA and CrylVD inclusions ranged from 12–15 ng ml⁻¹, 4–5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild-type parasporal bodies of B. thuringiensis subspp. israelensis and morrisoni Is due to synergism among three or four of their major proteins.     

Mtx Toxins Synergize Bacillus sphaericus and Cry11Aa against Susceptible and Insecticide-Resistant Culex quinquefasciatus Larvae

Two mosquitocidal toxins (Mtx) of Bacillus sphaericus, which are produced during vegetative growth, were investigated for their potential to increase toxicity and reduce the expression of insecticide resistance through their interactions with other mosquitocidal proteins. Mtx-1 and Mtx-2 were fused with glutathione S-transferase and produced in Escherichia coli, after which lyophilized powders of these fusions were assayed against Culex quinquefasciatus larvae. Both Mtx proteins showed a high level of activity against susceptible C. quinquefasciatus mosquitoes, with 50% lethal concentrations (LC₅₀) of Mtx-1 and Mtx-2 of 0.246 and 4.13 μg/ml, respectively. The LC₅₀s were 0.406 to 0.430 μg/ml when Mtx-1 or Mtx-2 was mixed with B. sphaericus, and synergy improved activity and reduced resistance levels. When the proteins were combined with a recombinant Bacillus thuringiensis strain that produces Cry11Aa, the mixtures were highly active against Cry11A-resistant larvae and resistance was also reduced. The mixture of two Mtx toxins and B. sphaericus was 10 times more active against susceptible mosquitoes than B. sphaericus alone, demonstrating the influence of relatively low concentrations of these toxins. These results show that, similar to Cyt toxins from B. thuringiensis subsp. israelensis, Mtx toxins can increase the toxicity of other mosquitocidal proteins and may be useful for both increasing the activity of commercial bacterial larvicides and managing potential resistance to these substances among mosquito populations.     

Effects of the 20-Kilodalton Helper Protein on Cry1Ac Production and Spore Formation in Bacillus thuringiensis

Bacillus thuringiensis produces large amounts of various pesticidal proteins during the stationary phase. In order to achieve a high yield and form crystals, some pesticidal proteins require the presence of other proteins. Helper protein P20 is required for efficient production of both the Cyt1A and Cry11A crystal proteins in B. thuringiensis subsp. israelensis. Although full-length Cry1 protoxins are usually independent in terms of expression and crystallization in B. thuringiensis, in this study P20 significantly enhanced production of Cry1Ac protoxin (133 kDa) in an acrystalliferous and plasmid-negative strain. In the presence of P20, the yield of Cry1Ac protoxin increased 2.5-fold, and on average the resulting crystals were 1.85 μm long and 0.85 μm wide, three times the size of the crystals formed in the control lacking P20. Correspondingly, the recombinant strain that coexpressed P20 and Cry1Ac exhibited higher toxicity against Heliothis armigera larvae than the control. Furthermore, serious degradation of Cry1Ac in vivo was observed, which has seldom been reported previously. Actually, most protein was completely degraded during synthesis, and after synthesis about one-third of the expressed protoxins were degraded further before crystallization. In this process, P20 protected only nascent Cry1Ac from degradation, indicating that it acted as a molecular chaperon. In addition, spores were smaller and rounder and had a thinner exosporium layer when they were produced in the presence of P20. In summary, Cry1Ac was severely degraded during synthesis; this degradation was effectively relieved by P20, which resulted in enhanced production. Our results indicated that P20 is an effective tool for optimizing protein production in vivo.     

Cross-Resistance and Stability of Resistance to Bacillus thuringiensis Toxin Cry1C in Diamondback Moth

We tested toxins of Bacillus thuringiensis against larvae from susceptible, Cry1C-resistant, and Cry1A-resistant strains of diamondback moth (Plutella xylostella). The Cry1C-resistant strain, which was derived from a field population that had evolved resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai, was selected repeatedly with Cry1C in the laboratory. The Cry1C-resistant strain had strong cross-resistance to Cry1Ab, Cry1Ac, and Cry1F, low to moderate cross-resistance to Cry1Aa and Cry9Ca, and no cross-resistance to Cry1Bb, Cry1Ja, and Cry2A. Resistance to Cry1C declined when selection was relaxed. Together with previously reported data, the new data on the cross-resistance of a Cry1C-resistant strain reported here suggest that resistance to Cry1A and Cry1C toxins confers little or no cross-resistance to Cry1Bb, Cry2Aa, or Cry9Ca. Therefore, these toxins might be useful in rotations or combinations with Cry1A and Cry1C toxins. Cry9Ca was much more potent than Cry1Bb or Cry2Aa and thus might be especially useful against diamondback moth.