Effects of Bacillus thuringiensis δ-Endotoxins on the Pea Aphid (Acyrthosiphon pisum)

Four Bacillus thuringiensis δ-endotoxins, Cry3A, Cry4Aa, Cry11Aa, and Cyt1Aa, were found to exhibit low to moderate toxicity on the pea aphid, Acyrthosiphon pisum, in terms both of mortality and growth rate. Cry1Ab was essentially nontoxic except at high rates. To demonstrate these effects, we had to use exhaustive buffer-based controls.Many species of aphids are important sucking-insect pests that feed on plant vascular fluids. Their feeding mechanism makes these insects excellent vectors for many plant pathogens, especially viruses, yet less amenable to standard, nonsystemic chemical control by insecticides. Minor effects on the survival and fecundity of aphids reared on Bacillus thuringiensis (Bt) crops have been noted in some studies but not in others (1, 3, 6). However, the sensitivity of aphids to Bt toxins, or the lack thereof, has not been previously tested through artificial-diet bioassays with exhaustive buffer-based controls.Bt δ-endotoxins Cyt1A, Cry4A/Cry4B, and Cry11, obtained from three recombinant strains of B. thuringiensis subsp. israelensis, as well as Cry1Ab and Cry3A, obtained from recombinant Escherichia coli, were purified by ultracentrifugation in a discontinuous sucrose gradient as described previously (9). Cry proteins were solubilized in solubilization buffer (50 mM Na₂CO₃, 100 mM NaCl, pH 10) with dithiothreitol (10 mM) added before use. Cyt1A was first solubilized on 10 mM Na₂CO₃ (pH 11) buffer and then neutralized at pH 7.5 to 8 with 10 μl HCl (1 N). Both solubilized and trypsin-digested samples (1:30 over toxin weight) were used at different concentrations (32, 125, and 500 μg/ml; trypsin-activated toxin concentrations were calculated on the basis of the preactivation concentrations of the protoxins) to supplement the AP3 aphid synthetic diet (7) used to feed Acyrthosiphon pisum (LL01 green clone). Ampicillin (100 μg/ml), an ineffective antibiotic for A. pisum or its obligate symbiont Buchnera, was added to the medium to avoid bacterial growth. For each concentration, 30 nymphs (10 nymphs/box and three repetitions) were bioassayed at 20°C and under a 16:8 (light-dark) photoperiod. Survival time was calculated from aphid deposition on the test diet (day 0). Mortality was surveyed daily, and body weights of survivors were noted at day 7. ST₅₀ (median survival time after challenge) was calculated by using an actuarial survival analysis (Statview) with censoring values of survivors at the end of the experiments. The approximate concentrations resulting in a 50% decrease in mean body weight (IC₅₀) and killing of 50% of the insects tested (LC₅₀) were calculated at the end of the experiments from the growth reduction and mortality data, respectively, derived with the three doses by using Statview and the censoring values of survivors.All of the Cry δ-endotoxins tested were lethal to A. pisum and retarded the growth of survivors (Fig. ​(Fig.11 and ​and2).2). Mortalities ranged from only 25% (Cry1Ab) to 100% (Cry4 and Cry11) after 3 to 6 days of exposure to 500 μg/ml of solubilized protein (Fig. ​(Fig.1).1). When significant mortalities were achieved (Cry3A, Cry4, and Cry11), trypsin activation enhanced toxicity. Activation of Cry4 at the intermediate concentration tested (125 μg/ml) resulted in a twofold increase in mortality (Fig. ​(Fig.1D).1D). ST₅₀s were calculated for both solubilized protoxins and activated Cry3A, Cry4, and Cry11. The ST₅₀s (at 500 μg/ml) ranged from 1.8 ± 0.14 days for solubilized Cry4 and Cry11 to 3.7 ± 1.2 days for trypsin-activated Cry3A (Table ​(Table1).1). Control aphids fed buffer all survived for >8 days. The LC₅₀ of Cry1Ab was not calculated, since mortality associated with Cry1Ab reached a plateau at 500 μg/ml. The LC₅₀ of Cry4 was estimated to be 70 to 100 μg/ml (data not shown).Open in a separate windowFIG. 1.Mortality assays over the nymphal life stage of the pea aphid, A. pisum, upon ingestion of artificial diets containing purified Bt toxins after either solubilization (open symbols) or solubilization and trypsin activation (solid symbols). The toxins used were Cry1Ab (circles), Cry3A (squares), a mixture of Cry4A and Cry4B (diamonds), and Cry11A (triangles). The soluble-toxin doses used were low at 32 μg/ml (blue), intermediate at 125 μg/ml (violet), and high at 500 μg/ml (red). Assays were carried out with 30 initial neonate insects in three batches of 10 individuals.Open in a separate windowFIG. 2.Growth inhibition assays with purified Bt toxins Cry3A, Cry4, and Cry 11 (A) and Cry1Ab and Cyt1A (B) on the pea aphid, A. pisum. Toxins were added to the diet either after solubilization (open symbols) or after solubilization and trypsin activation (solid symbols). Error bars show the standard errors (SE) of individual weights at day 7 of experiments, standardized by the control group mean weight (toxin dose, 0; initial number, 30). Color coding of toxins: Cry3A, red squares; Cry4A and Cry4B mixture, violet diamonds; Cry11A, blue triangles; Cry1Ab, green circles; Cyt1A, yellow squares. In the experiment with Cry1Ab (B), the toxin was purified by high-performance liquid chromatography and activated toxin was provided as a salt-free lyophilisate by W. Moar (Auburn University, Auburn, AL).

TABLE 1.

ST₅₀s of pea aphids feeding on solubilized Cry toxins and solubilized Cry toxins activated with trypsin

Growth, Sporulation, δ-Endotoxins Synthesis, and Toxicity During Culture of Bacillus thuringiensis H14

Growth, sporulation, synthesis of δ-endotoxins, and toxicity against the larvae of Aedes aegypti and Culex pipiens were studied during fermentation of Bacillus thuringiensis H14 in a 20-L fermentor. Measurements of optical density and dielectric permittivity for biomass determination suggest a highly promising technique for on-line evaluation of sporulation. The synthesis of 65-, 25- and 130-kDa proteins started at 16, 18, and 23 h, respectively. These proteins were enriched in different ways until the end of culture (48 h). Toxicity in the course of sporulation was significantly different for the larvae of both mosquito species. Maximal activity against Ae. aegypti was obtained at the end of culture, whereas for Cx. pipiens, the sample at 38 h was the most active.     

Limited Proteolysis of Bacillus thuringiensis CryIG and CryIVB δ-Endotoxins Leads to Formation of Active Fragments That Do Not Coincide with the Structural Domains

Bacillus thuringiensis “true” toxins consist of three domains: the N-terminal, α-helical domain followed by two β-structural domains. Their limited proteolysis does not proceed at the domain boundaries, but is directed to the loops within the domains. There are at least two patterns of the limited proteolysis of “true” toxins. The first pattern, observed for CryIA and CryIVD δ-endotoxins, results in the proteolysis of the loops connecting β-strands of the second domain. The second pattern, detected for CryIG and CryIVB proteins, consists in the cleavage of the loop connecting the fifth and the sixth α-helixes of the first domain. The choice between the routes depends on the size, sequence, and dynamics of the loop that define its accessibility to a proteinase. Bioassay of CryIG and CryIVB δ-endotoxin fragments indicates that only two α-helixes, the sixth and the seventh within the first domain, followed by the two β-structural domains are sufficient for the insecticidal activity.     

The main features of Bacillus thuringiensis δ-endotoxin molecular structure

The crystal-forming proteins (-endotoxins) produced by various serotypes of Bacillus thuringiensis and toxic for Lepidoptera reveal the same pattern of molecular organisation. These proteins (M _r of ca. 145,000–130,000) contain an N-terminal domain (M _r of 85,000–65,000) resistant to proteolysis whereas their C-terminal moieties (M _r of 65,000) undergo an extensive degradation by trypsin that leads to stepwise cleavage off the fragments with M _r of 15,000–35,000.The N-terminal domain from serotype V -endotoxin is active when introduced into the hemocoel of Galleria mellonella larvae. Hence, it correponds to the true toxin normally formed by larva proteases action on the crystalforming protein (protoxin). Some differences were found in the properties of the N-terminal domains isolated from the crystal-forming proteins of III, V and IX serotypes, e.g., in their solubility, digestion by subtilisin, molecular weights and the distribution of methionine residues along the polypeptide chains.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacryl amide gel electrophoresis - CFP crystal-forming protein - DNS 5-dimethylamino-1-naphthalene-sulphonyl     

Relationship between Poly- β-hydroxybutyrate Production and δ-endotoxin for Bacillus thuringiensis var. kurstaki

A linear relationship between total solid concentration (TSC), δ-endotoxin production [Cry = 0.2795(TSC)−0.2472, R² = 0.8644] and poly-β-hydroxybutyrate (PHB) accumulation [PHB = 0.1327(TSC) + 0.3974, R² = 0.9877] in Bacillus thuringiensis var. kurstaki HD-73 was observed. A similar correlation between δ-endotoxin and PHB accumulation [Cry = 2.1573(PHB)−1.1248, R² = 0.9181] was found. A minimum PHB accumulation of 0.52 mg l⁻¹ was required before the onset of δ-endotoxin production. Revisions requested 28 September 2005 and 4 November 2005; Revisions received 28 October 2005 and 1 February 2006     

Improvement of Bacillus thuringiensis δ-endotoxins Synthesis Yields Through Acquisition of Erythromycin Resistance

The acquisition of the erythromycin resistance by Bacillus thuringiensis kurstaki improved yields of δ-endotoxins in sporulating cells ranging from 134 to 215%. Resistance to erythromycin decreased the final spore count by at least 50%. Consequently, erythromycin resistance is an efficient tool for the improvement of bioinsecticides yields with a high ratio of δ-endotoxins to spores. Revisions requested 31 October 2005; Revisions received 28 November 2005     

Host specificity of the Bacillus thuringiensis δ-endotoxin toward Lepidopteran species: Spodoptera littoralis Bdv. and Pieris brassicae L

Crystals from several strains of Bacillus thuringiensis among the first 10 serotypes were tested for their effectiveness (in terms of LC₅₀ or LD₅₀) in killing Spodoptera littoralis larvae. The δ-endotoxin obtained from the isolates aizawai 7–29 and entomocidus 601 was found to be the most active against S. littoralis; lethal doses were considerably lower than those obtained with the berliner 1715 strain but were nevertheless 10 times higher than that of this last strain against Pieris brassicae. Interestingly, entomocidus crystals were active toward both Lepidopteran species. Protein fractions with molecular mass ranging from 60 to 70 kDa, obtained by dissolving crystals with gut proteases from the two insect species, proved to be active when delivered by intrahemocoel injection. Based on the use of mild detergents such as Triton N-101, sodium cholate, or both, conditions for stabilizing the activity of the solubilized fractions are reported. Under these conditions only, the molecules of toxin thus obtained were as active against S. littoralis larvae as the native crystals, whatever the route of administration and whatever the enzymes used. The same fractions induced different responses in P. brassicae larvae, which manifested a much higher sensitivity to the proteolysis-activated molecules, particularly after intrahemocoel injection, thus suggesting differences between the two insect species as regards the nature of toxic effects. The results clearly illustrate two different patterns in activity spectrum among Lepidopteran species and they also indicate that structural characteristics of the protoxin are predominant factors in determining host specificity.     

The effect of oxygen on the sporulation, δ-endotoxin synthesis and toxicity of Bacillus thuringiensis H14

Summary The sporulation and toxicity of Bacillus thuringiensis H14 were studied as a function of aeration. The fed-batch cultures carried out in the similar aeration conditions were followed in four different oxygen transfer rates containing 0, 20, 100 and 250 mmol/l/h. The percentage of total cells which had formed refractile spores in these four oxygen transfer rates were 100, 93, 84 and 48%, respectively. The highest rate of sporulation was observed in the absence of oxygen and the mature spores were the only population present under this condition at the end of culture. Sporulation in a large portion of cells failed under saturated oxygenation and either mature spores or vegetative cells were present at the end of culture. In the intermediate conditions, cells in different physiological states could be observed at the end of culture. It was found that the optimal conditions for spore yield and for δ-endotoxin yield were not the same, even though sporulation and δ-endotoxin formation proceed simultaneously during the fermentation process. The 130-kDa δ-endotoxin seemed to be more sensitive to aeration conditions. The higher toxicity against Culex pipiens was obtained under the saturated condition.     

Hyper-Production of Insecticidal Crystal Protein (δ-Endotoxin)by Bacillus thuringiensis var. israelensis Is Not Related toSporulation-Specific Biochemical Functions

Hypertoxic mutant strains of Bacillus thuringiensis var. israelensis were isolated by mutagenesis of the parent strain. The correlation, if any, between hyper-production of insecticidal crystal protein (δ-endotoxin) by hypertoxic mutant strains of Bacillus thuringiensis var. israelensis and sporulation-specific biochemical functions was studied. No increase in sporulation-specific biochemical markers was observed in the hypertoxic mutant strains. Asporogenous mutants of hypertoxic mutant strains blocked at different stages of sporulation were isolated, and larvicidal activity was studied. The hypertoxic parent strains and the sporulation-deficient, hypertoxic mutant strains showed almost identical larvicidal activity. Therefore, the increased production of toxin is not related to sporulation-specific biochemical changes. Received: 14 February 2000 / Accepted: 21 March 2000     

Influence of some plant phenolics on the activity of δ-endotoxin of Bacillus thuringiensis var. galleriae on Heliothis armigera

Bioassays with Bacillus thuringiensis var. galleriae Berliner -endotoxin and plant phenolics on Heliothis armigera Hübner enhanced the activity of B.t. var. galleriae endotoxin. The presence of plant phenolics with B.t var. galleria endotoxin not only reduced feeding potential and weight gain by the larvae, but also enhanced the LC50 value of the toxin. Our study demonstrates the effect of phytochemicals from resistant crop plants on the biocidal activity of B. thuringiensis strains in laboratory conditions.     

Bacillus thuringiensis growth,sporulation and δ-endotoxin production in oxygen limited and non-limited cultures

The production of crystals and spores ofBacillus thuringiensis var.israelensis was studied under different aeration conditions. The results with 4 l batch cultures showed that for O₂ non-limited, cultures cell yield, toxin production and spore count were constant for all oxygen transfer rates (OTR). Under O₂ limitation, °-endotoxin concentrations and spore counts were lower than those obtained in non-limited cultures. In addition, -endotoxin yields diminished under O₂ limitation, suggesting that the toxin synthesis mechanism could have been affected.     

The 20-kDa chaperone-like protein of Bacillus thuringiensis ssp. israelensis enhances yield, crystal size and solubility of Cry3A

Aims: To determine whether the 20‐kDa chaperone‐like protein of Bacillus thuringiensis ssp. israelensis enhances synthesis, crystallization and solubility of the Cry3A coleopteran toxin and whether the crystalline inclusions produced are toxic to neonates of the Colorado potato beetle, Leptinotarsa decemlineata. Methods and Results: The cry3A gene was expressed in the 4Q7 strain of B. thuringiensis ssp. israelensis in the absence or presence of the 20‐kDa gene. The 20‐kDa protein enhanced Cry3A yield by 2·7‐fold per unit of fermentation medium. Crystal volumes averaged 2·123 and 0·964 μm³ when synthesized in, respectively, the presence or absence of the 20‐kDa protein. Both crystals were soluble at pH 5 and pH 6; however, the larger crystal was 1·7× and 1·5× more soluble at, respectively, pH 7 and pH 10. No significant difference in toxicity against L. decemlineata neonates was observed. Conclusions: This report demonstrated that the 20‐kDa chaperone‐like protein enhances yield, volume and solubility of the coleopteran Cry3A crystalline inclusions per unit crystal/spore mixture. Significance and Impact of the Study: This is the first report showing that an accessory protein (20‐kDa) could enhance synthesis and crystallization of Cry3A, a finding that could be beneficial for commercial production of this coleopteran‐specific insecticidal protein for microbial insecticides and possibly even for transgenic crops.     

Interaction of Bacillus thuringiensis δ-endotoxins with Midgut Brush Border Membrane Vesicles of Helicoverpa armigera

Pesticidal activity of different Bacillus thuringiensis (Bt) δ-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac and Cry2A, were investigated against Helicoverpa armigera infesting cotton crop worldwide. Cry1Ac toxin was found to be the most potent toxin towards H. armigera. All selected Bt toxins were found stable in vitro processing by midgut juice of H. armigera. Saturation and competition binding experiments were performed with iodine-125 labeled proteins and brush border membrane vesicles prepared from the midgut of H. armigera. The results show saturable, specific and high affinity of all toxins except for Cry2A. Both the toxins were bound with low binding affinity but with high binding site concentration. Heterologous competition experiments showed that Cry1Aa, Cry1Ab and Cry1Ac recognized or share the same binding site which is different from that of Cry2A. The data suggest that development of multiple toxin system in transgenic plants with toxin pyramiding, which recognize different binding sites, may be useful in the deployment strategies to decrease the rate of pest adaptation to Bt toxins in transgenic plants.     

Susceptibility of major lepidopterans to δ-endotoxins and a formulation of Bacillus thuringiensis (B.t.) on vegetable brassicas in Taiwan

Baseline susceptibility of Plutella xylostella, Crocidolomia binotalis and Hellula undalis to Bacillus thuringiensis (B.t.) δ-endotoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca) and a formulation (Xentari®) was assessed using a leaf-dip bioassay method. The toxins Cry1Ac, Cry1Aa and Cry1Ca were equally toxic to P. xylostella. Crocidolomia binotalis was highly susceptible to all Cry1A toxins and it was least sensitive to Cry1Ca. Conversely, H. undalis was highly sensitive to Cry1Ca, but less susceptible to Cry1A toxins. Hence, the susceptibility of H. undalis to Xentari®, a formulation containing Cry1C as a major toxin, was significantly higher than C. binotalis or P. xylostella.     

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Colorado potato beetle-active Bacillus thuringiensis δ-endotoxins

Many Bacillus thuringiensis crystal proteins, particularly those active against lepidopteran insects, have carboxy-terminal extensions that mediate bipyramidal crystal formation. These crystals are only soluble at high (>10.0) pH in reducing conditions such as generally found in the lepidopteran midgut. Most of the Colorado potato beetle (CPB)-active toxins lack such an extension, yet some toxins with a carboxy-terminal extension have cryptic activity against this insect, revealed only after in vitro solubilization. Crystal formation, morphology, protein content, and activity against CPB were compared for two sets of proteins, the Cry 1-hybrid SN19 and Cry3Aa, both with and without a carboxy-terminal extension. Cry3Aa, with or without extension, formed flat square or rectagular crystals. SN19 (with extension) and its derivative without extension formed irregular inclusion bodies. All Cry3Aa and SN19 crystals and inclusion bodies were almost equally active before and after in vitro presolubilization and could be solubilized in diluted CPB midgut extract. In contrast, bipyramidal crystals of Cry1Ba were insoluble under these conditions. Our results suggest that bipyramidal crystal formation typical for proteins with a carboxy-terminal extension may preclude activity against CPB, but that interfering with this crystal formation can increase the activity.     

Expression of insecticidal activity in Rhizobium containing the δ-endotoxin gene cloned from Bacillus thuringiensis subsp. tenebrionis

Bacillus thuringiensis subsp. tenebrionis produces a 65 kilodalton polypeptide toxin which is lethal to various coleopteran insect larvae. The gene encoding this toxin was cloned in E. coli in the broad host range vector pKT230 and subsequently transferred to Rhizobium leguminosarum by conjugation. Western blot analysis showed that the toxin gene was expressed in the free living state of Rhizobium producing two major polypeptides of 73 and 68 kilodalton in size. The level of expression of the toxin gene in Rhizobium varied from strain to strain. Cell extracts from toxin-producing rhizobia were toxic to larvae of Gasterophysa viridula. Bioassays also showed that the -endotoxin was toxic to larvae of the clover weevil Sitona lepidus. Furthermore, pea (Pisum sativum) and white clover (Trifolium repens) plants suffered less root and nodule damage by Sitona larvae when they were inoculated with Rhizobium strains containing the toxin gene. This suggests that such rhizobia could be useful in the biological control of this important legume pest.Abbreviations B.t.t. Bacillus thuringiensis subsp. tenebrionis - IPTG isopropyl--D-thiogalactoside     

Bacillus thuringiensis subsp. Aizawai δ-endotoxin inhibits the k+/amino acid cotransporters of lepidopteran larval midgut

1. The δ-endotoxin of Bacillus thuringiensis subsp. aizawai inhibits in a dose-dependent manner the K⁺-driven accumulation of histidine and lysine as well as their equilibrative uptake into brush border membrane vesicles from the midgut of Bombyx mori larvae.2. The decrease of uptake in the absence of a K⁺-gradient is neither due to a leakage of the labelled substrate from the vesicles nor to a reduction of the osmotically active space available, as a result of a detergent-like effect of the toxin.3. The toxin acts as a non competitive inhibitor of the K⁺/amino acid cotransporters of the larval midgut of Lepidoptera, with no preference for a specific transport system.     

Rifampicin Increases Expression of Plant Codon-Optimized Bacillus thuringiensis δ-Endotoxin Genes in Escherichia coli

The Protein Journal - Transgenic crops expressing Cry δ-endotoxins of Bacillus thuringiensis for insect resistance have been commercialized worldwide with increased crop productivity and...