Effects of Bacillus thuringiensis var. kurstaki δ-endotoxin on isolated lepidopteran mitochondria

An investigation was undertaken to determine the effects of Bacillus thuringiensis var. kurstaki δ-endotoxin on mitochondria isolated from Bombyx mori midgut epithelium. Using manometric and colorimetric techniques, the investigation revealed that toxic polypeptides had stimulatory effects on mitochondria oxygen uptake and inhibitory effects on ATP production. These results indicated that B. thuringiensis δ-endotoxin could act as an uncoupler of oxidative phosphorylation. Loss of ATP production caused by the action of the δ-endotoxin would lead to metabolic imbalance and possible cell death.     

Bacillus thuringiensis isolates from Korean forest environments

We investigated the distribution, toxicity, morphology, and protein profiles of Bacillus thuringiensis isolates from forests in Korea to isolate naturally occurring novel B. thuringiensis. A total of 170 B. thuringiensis isolates were obtained from 832 samples producing spore and parasporal inclusion bodies. In toxicity tests for lepidopteran, dipteran, and coleopteran insects, 57.6% isolates were toxic only to Lepidoptera, 5.3% were toxic only to Diptera, and 24.1% were toxic to both Diptera and Lepidoptera. The remaining collections (13.0%) were not toxic to the tested insects. The shapes of the parasporal crystals produced in B. thuringiensis isolates were bipyramidal, spherical, ovoid, or irregular. As their toxicities varied with parasporal crystal shape, B. thuringiensis isolates possessing bipyramidal or irregular parasporal crystals were largely toxic to lepidopteran species whereas those producing spherical parasporal crystals were mainly toxic to dipteran species. B. thuringiensis toxic to both dipteran and lepidopteran insects contained 130- and 70-kDa parasporal crystals, whereas B. thuringiensis toxic to lepidopteran insects expressed 130-kDa parasporal crystals. The results suggest that forest areas in Korea are a rich source of B. thuringiensis and need to be further explored to discover novel B. thuringiensis isolates.     

Mode of Action of Bipyramidal δ-Endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1

The mode of action of the toxic fragment (P-59) derived from bipyramidal-shaped δ-endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 on the silkworm Bombyx mori was investigated. An enzyme-linked immunosorbent assay showed that there was no translocation of P-59 from the gut lumen to the hemocoel. When membrane vesicles prepared from silkworm midgut were incubated with P-59, normally smooth surface of vesicles became rough, and patch formation was observed on the surface. Vesicles treated with P-59 tended to agglutinate. The vesicle-denaturing activity of a 130,000-dalton subunit protein of bipyramidal toxin was enhanced by treatment with a gut juice protease of the silkworm. P-59 did not cause any uncoupling effect on mitochondria of the silkworm midgut. These results suggest that the attacking site of this toxin is not the mitochondrion but the cell membrane of the susceptible cell.     

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.     

Neither barium nor calcium prevents the inhibition by Bacillus thuringiensis δ-endotoxin of sodium- or potassium gradient-dependent amino acid accumulation by tobacco hornworm midgut brush border membrane vesicles

Rapid filtration assays were used to determine the effects of barium, calcium and an insecticidal δ-endotoxin from Bacillus thuringiensis on sodium and potassium ion gradient dependent phenylalanine accumulation by brush border membrane vescles from the larval midgut of the tobacco hornworm (Manduca sexta). Neither barium nor calcium had a significant effect on sodium ion gradient dependent phenylalanine accumulation by the membrane vesicles. Both barium and calcium inhibited potassium ion gradient dependent phenylalanine accumulation by the membrane vesicles. B. thuringiensis δ-endotoxin inhibited both sodium and potassium ion gradient dependent phenylalanine accumulation by the vesicles. Inhibition of both sodium and potassium ion gradient dependent phenylalanine accumulation increased similarly with increasing δ-endotoxin inhibition of either sodium or potassium dependent phenylalanine accumulation by the vesicles.     

Mode of action of Bacillus thuringiensis δ-endotoxin: General characteristics of intoxicated Bombyx larvae

The general pathology induced by δ-endotoxin in terms of larval behavior and hemolymph chemistry has been widely studied in the so-called Type I insect, Bombyx mori. The succession of symptoms is divided into four arbitrary stages: Stage 0, appearance and locomotion normal, no feeding; Stage 1, slightly sluggish; Stage 2, extremely sluggish; and Stage 3, complete paralysis. The action of δ-endotoxin is highly specific to the midgut since contractile movement of both foregut and hindgut continues long after all locomotor activity and heartbeat have stopped. Immediately after the silkworm stops feeding and blood pH sharply rises, there is an associated abrupt rise in the K⁺ concentration of hemolymph. Thereafter, the rise in K⁺ is linear while the rise in pH is not. In vivo measurements have not yielded the same simple linear dependence of pH on K⁺ concentrations that is found in in vitro mixtures of hemolymph and midgut juice. Ligation experiments showed that the same pathological sequence (rise of pH and K⁺ concentration, and general paralysis) follows whether the toxin has unrestricted access to the entire midgut or only part of it (anterior or posterior). From the results of injections of midgut juice or various salt solutions into hemocoel, we came to the conclusions that the blood pH and the symptoms are not necessarily parallel and the intact midgut and Malpighian tubules have strong functions for ion regulation.     

The delta-endotoxin of Bacillus thuringiensis. V. On the occurrence of endotoxin fragments in hemolymph

Leucine-³H labeled crystals of Bacillus thuringiensis δ-endotoxin were fed to last-instar larvae of spruce budworm, Choristoneura fumiferana, eastern forest tent caterpillar, Malacosoma disstria, and silkworm, Bombyx mori. Radioactivity was detected in hemolymph 1 min after feeding in the first two species, but not until 3–5 min after feeding in silkworm larvae. Most of the radioactivity from hemolymph of all three species eluted from gel filtration columns at the same elution volume indicating similar moleculear weights (<1800 daltons).     

The delta-endotoxin of Bacillus thuringiensis. II. On the mode of action

Uptake of glucose-¹⁴C by guts of the silkworm Bombyx mori was stimulated within 1 min after administration of Bacillus thuringiensis δ-endotoxin. Uptake of amino acids or carbonate ions was not similarly stimulated. General metabolic breakdown of the gut epithelium appears to occur between 10 and 20 min after administration of the toxin.     

Toxicity of Bacillus thuringiensis entomocidal protein toward cultured insect tissue

The entomocidal protein from crystalline inclusion bodies of Bacillus thuringiensis can be bioassayed in vitro using cultured insect tissue. Larval cells of the spruce budworm, Choristoneura fumiferana, are damaged by enzyme-digested (activated) protein isolated from B. thuringiensis crystals. Measurement of toxicity is accomplished by detection of adenosine triphosphate (ATP) in treated cultures using firefly bioluminescence. The ATP content of toxin-treated tissue is inversely proportional to the amount of toxin added. Tissue cells from the spruce budworm exhibited maximum susceptibility to activated δ-endotoxin after 120 hr incubation. Probit analysis of tissue ATP response to toxin dose indicated 50% of the cells were damaged by 14.6 μg or less of toxin protein per 2 × 10⁵ insect tissue cells. Activated δ-endotoxin was entomocidal to insects as well, as detemined by mortality studies with second-instar larvae of the European corn borer. Electron microscopic observations of insect tissue treated with activated δ-endotoxin protein for 60 min revealed massive outer membrane disruption and subsequent loss of cytoplasmic constituents, accompanied by swelling of the nuclear membrane.     

Immunological properties of entomocidal protein of Bacillus thuringiensis and its insecticidal activity

The immunological properties of the proteinaceous component of the parasporal crystal (δ-endotoxin) of Bacillus thuringiensis var. kurstaki were analyzed by rocket immunoelectrophoresis. Two antisera, one against the k-l-type crystal containing two components, and the other against the k-73-type crystal containing one component, were made in rabbits. The antigens consisting of purified and dissociated crystals were run in electrophoresis with these two antisera. The ratio between the two peak heights of precipitin lines, which were formed by the dissociated crystal of one B. thuringiensis isolate in two antisera, was compared with the ratios of other isolates under identical conditions. The difference in the ratio reflected a difference in the structure of the crystal component and correlated closely with the insecticidal activity spectrum. This method can be used to evaluate a newly isolated B. thuringiensis, and it can further differentiate the isolates which have been classified as one serotype.     

苏云金芽孢杆菌δ—内毒素的杀虫机理及其增效途径

苏云金芽孢杆菌（Ｂａｃｉｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ,Ｂｔ）制剂是当前应用最广、最有效的微生物杀虫剂。Ｂｔ属于革兰氏阳性细菌,在形成芽孢的同时,产生伴孢晶体。伴孢晶体是Ｂｔ杀虫活性的主要来源,它可能由几种晶体蛋白即δ－内毒素组成。δ－内毒素的专一...     

Bacillus thuringiensis subsp. sichuansis strain MC28 produces a novel crystal protein with activity against Culex quinquefasciatus larvae

The Bacillus thuringiensis subsp. sichuansis MC28 strain produces spherical parasporal crystals during sporulation and exhibits remarkable insecticidal activity against dipteran and lepidopteran pests. We characterized a novel cry gene (cry69Aa1), which was found in the pMC95 plasmid of the MC28 strain. The cry69Aa1 gene was inserted into a shuttle vector (pSTK) and expressed in an acrystalliferous mutant B. thuringiensis HD73^?. In this transformant, a large number of spherical parasporal crystals, which were toxic to Culex quinquefasciatus (Diptera), were formed.     

Larvicidal activity of Bacillus thuringiensis subsp. israelensis, serovar H14 in Aedes aegypti: Histopathological studies

Bacillus thuringiensis subsp. israelensis, serovar H14, when applied as a primary commercial powder, caused the rapid death of Aedes aegypti larvae. Mortality started 6 min after application of 4 μg/ml of the pathogen and reached a maximum 27 min later. When the LC₅₀ (10 ng/ml) was applied, mortality began after 37 min and reached a maximum 120 min later. Histopathological changes in B. thuringiensis israelensis-treated larvae could be observed only in the midgut and caeca. In B. thuringiensis israelensis-treated “dead larvae”, the epithelial layer is disorganized, most of the cells have disappeared and the peritrophic membrane is broken. The epithelium in the B. thuringiensis israelensis-treated “living larvae” still maintains its monolayer structure, but with marked cellular hypertrophy and vacuolized cytoplasm. Also, the “brush border” is thinner and disrupted. Based on the fact that mortality of A. aegypti is a quick process, and because the histopathological changes caused by B. thuringiensis israelensis are similar to those found in lepidopterous larvae treated with pure δ-endotoxin of other B. thuringiensis variants, it is suggested that larvicidal activity of B. thuringiensis israelensis in A. aegypti is due to its δ-endotoxin.     

Study of the irreversible binding of Bacillus thuringiensis Cry1Aa to brush border membrane vesicles from Bombyx mori midgut

The binding of Bacillus thuringiensis δ-endotoxin to brush border membrane vesicles (BBMVs) from the target insect larval midgut comprises with not only a reversible but also an irreversible component. The irreversible binding of δ-endotoxin is thought to be a pathologically important factor. Here, we studied the irreversible binding of Cry1Aa to the BBMVs of Bombyx mori. The ¹²⁵I-labeled Cry1Aa bound to the solubilized brush border membrane (BBM) through rapid dissociation only, unlike the binding to BBMVs, indicating that the toxin bound to the solubilized BBM through only a reversible process. Low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the toxin bound irreversibly to BBMVs formed an oligomer of 220 kDa, whereas that bound reversibly to the solubilized BBM did not oligomeraize. When the ¹²⁵I-labeled Cry1Aa bound irreversibly to the BBMVs was digested by proteinase K, approximately 40% of the toxin observed to be resistant to proteinase K. The molecular mass of the toxin resistant to proteinase K was 60 kDa, suggesting that the irreversible binding comprise two forms. These results support the notion that the irreversible binding of the toxin to BBMVs is due to the insertion of the toxin into the lipid bilayers and oligomerization to form channels.     

EFFECT OF DISSOLUTION AND DEGRADATION OF BAClLUlS THURlNGIENSlS PARASPORAL CRYSTALS ON TOXICITY TO COTTON BOLLWORM*

Abstract The effect of dissolution and buffer pH values on the proteolysis of Bacillus thuringiensis subvar. kurstaki HD-1 parasporal crystals were studied by SDS-PAGE. Dissolution made crystal much easier to be digested by larval Helicoverpa armigera midgut proteases; the rate of proteolysis of protoxin was faster than that of the crystal. Incubated with larval midgut juice, bovine trypsin and chymotrypsin for 17 h at 30°C, the proteolytic process of crystals deepened as the pH of buffer rose from 6. 9 to 10.5; the complete degradation of crystals occurred at pH 10.5, and the active domains were similar, with molecular weight of 60.5±1.26Kd, 61.6±1.16Kd, and 61.7±2.0Kd respectively. Bioassay results suggested that incubation in alkaline buffer could improve the toxicity of crystals to H. armigera, proteolysis gave no further modification to the toxicity and the toxicity was not significantly different among the products from proteolysis by larval midgut juice, bovine trypsin and chymotrypsin, Whether diets with different pH would affect the toxicity of crystals to H. armiger were also tested. Diets made by 0. 01mol.L^-1 Tris-HC1 buffer, pH 8.0 and 0.01mol*L^-1 glycine-NaOH buffer, pH 10.0, all significantly increased the toxicity of crystals and no difference between them was observed.     

Specific activity of a Bacillus thuringiensis strain against Locusta migratoria manilensis

Bacillus thuringiensis (Bt) has played an important role in biocontrol of pests. However, insecticidal activity of B. thuringiensis against locusts has been rarely reported. Bt strain BTH-13 exhibiting specific activity to locusts was isolated from a soil sample in China and characterized. Its bipyramidal parasporal crystal is mainly composed of a protein of 129 kDa, and produces a mature toxin of 64 kDa after activation. The pattern of total DNA from BTH-13 showed a large and three small plasmid bands. Known δ-endotoxin genes, cry1Aa, cry1Ab, cry1Ac, cry1C, cry3, cry4 and cry7Aa were not found from strain BTH-13 by PCR amplification. The sequence analysis of a DNA fragment produced by PCR amplification with degenerate cry-selective primers revealed that the fragment encoded a δ-endotoxin segment, which exhibited some similarity to several Cry proteins (41% of the highest similarity to Cry7Ba1). Toxicity tests were performed against Locusta migratoria manilensis, and the results demonstrated that trypsin-treated sporulated cultures and crystal proteins had high toxicity to larval and adult locusts. Cry toxin of BTH-13 was detected on the midguts of treated locusts using immunofluorescent technology, which confirmed the site of action of the crystal proteins in their toxicity for locusts.     

Invertebrate pathogenicity and toxin-producing potential of strains of Bacillus thuringiensis endemic to Antarctica

Several strains of Bacillus thuringiensis were previously isolated from soil in Antarctica and appeared to have physiological adaptations to this cold, nutrient-poor environment. In spite of this they could produce abnormally large, parasporal crystals under laboratory conditions. Here, they have been further characterised for toxin genes and invertebrate pathogenicity. All of the strains were positive in PCR assays for the cry1Aa and cry2 genes. This was confirmed by sequence analysis and the parasporal crystals of all strains contained polypeptides of about 130 kDa. This potential for lepidopteran toxicity was borne out in bioassays of purified δ-endotoxins against larvae of Pieris brassicae: the LD₅₀ values of B2408 (288 μg) were comparable to that of the reference strain, HD-12 (201 μg). There was no activity against the nematode Caenorhabditis elegans in spite of the fact that all strains appeared to possess the cry6 gene. PCR screening for genes encoding other nematode-toxic classes of toxins (Cry5, 4 and 21) was negative. B. thuringiensis has never previously been shown to be toxic to Collembola (springtails) but the purified δ-endotoxins of one of the Antarctic strains showed some activity against Folsomia candida and Seira domestica (224 μg and 238 μg, respectively). It seems unlikely that the level of toxicity demonstrated against springtails would support a pathogenic life-style in nature. All of the strains were positive for genes encoding Bacillus cereus-type enterotoxins. In the absence of higher insects and mammals the ecological value of retaining the toxic capability demonstrated here is uncertain.     

Bacillus thuringiensis: laboratory tests against four species of biting lice (Mallophaga: Trichodectidae)

Four species of mammalian biting lice, Bovicola bovis, B. crassipes, B. limbata, and B. ovis, were more susceptible in the laboratory to the spore-δ-endotoxin complex of Bacillus thuringiensis than to the β-exotoxin. The relative potency of these active principles was thus reversed from that for avian biting lice. The activity of the preparations tested was not homologous with that of E-61, the international standard for δ-endotoxin activity.     

Morphology of a spectrum of parasporal endotoxin crystals from cultures of Bacillus thuringiensis ssp. kurstaki isolate A3-4

Morphology of the parasporal -endotoxin crystals of Bacillus thuringiensis subsp. kurstaki isolate A3-4, a native (Taiwan) strain was studied by scanning (SEM) and transmission (TEM) electron microscopy. The typical bipyramidal crystals and an unusual parasporal inclusion with an embedded body were observed. The parasporal -endotoxin crystal with an embedded body, which was in different size and shape was well demonstrated in thin sections by transmission electron microscopy. The sporulated cells had multiple individually separated inclusions up to four crystals in one cell, which was unique to this isolate, and has not been reported before. The -endotoxin crystals included in the cell or released from the cell after batch fermentation or fed-batch fermentation did not show any altered morphological characteristics. However, judging from thin-sections of TEM, cells and the included parasporal crystals from fed-batch fermentation appeared larger than those from batch fermentation. It was observed that release of spore and parasporal crystals from the bacterial cell produced from batch cultures was earlier than that of fed-batch cultures. Preliminary bioassay results showed that the isolate cultures from both types of culture were equally effective against Plutella xylostella larvae. Based on the morphological observations, this strain may have a multiple insecticidal activities toward different insect species.     

Dissolution and Degradation of Bacillus thuringiensis δ-Endotoxin by Gut Juice Protease of the Silkworm Bombyx mori

The dissolution and degradation of †-endotoxin (crystal) of Bacillus thuringiensis subsp. kurstaki strain HD-1 were investigated. Crystals were dissolved in 0.1 M phosphate-carbonate-NaOH buffer at pH > 12. Swelling of crystals occurred in the buffer between pH 10 and 11, and crystals dissolved in the same buffer supplemented with gut juice protease of the silkworm Bombyx mori. The proteolytic dissolution of crystals occurred after a time lag of several minutes in 0.1 M carbonate-NaOH buffer, pH 10.2. The time lag was not observed when crystals were suspended in the buffer for 30 min before the addition of protease. After the dissolution of the crystals and further degradation of the solubilized protein, the appearance of a toxic protein with a molecular weight of 59,000, designated P-59, was observed. Lower-molecular-weight peptides (less than 40,000) showed no toxicity to the silkworm larvae on feeding. Digestion of the 120,000-dalton subunit of the crystal by gut juice protease also produced P-59. These observations suggest the occurrence of a similar process in vivo, i.e., the swelling of crystals due to the alkalinity of gut juice and the production of P-59, dependent on the hydrolysis of swollen crystals by gut juice protease.