Interactions between the tobacco budworm,Heliothis virescens,and the δ-endotoxin produced by the HD-1 isolate of Bacillus thuringiensis var. kurstaki: Relationship between length of exposure to the toxin and survival

Larvae of the tobacco budworm, Heliothis virescens, which were fed ad libitum for 24, 48, and 72 hr on a diet treated with various levels of the δ-endotoxin produced by the HD-1 isolate of Bacillus thuringiensis var. kurstaki and then transferred to an untreated diet, showed an unexpected capacity to recover from the effects of the toxin, although, as the length of exposure increased, the capacity decreased. Observations on larvae held to emergence indicated that recovery from the toxin was complete. X-ray studies using Ba²⁺ incorporated into the diet showed that, although the toxin paralyzed the midgut of the treated animals, many animals recovered after the toxin was removed, with food once again passing through the gut.     

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).     

Incidence of insect cell cytolytic activity among Bacillus thuringiensis serotypes

Fourteen serotypes of Bacillus thuringiensis were surveyed for cytolytic activity toward established cell lines of two lepidopteran and one dipteran insect species. Toxic protein extracted from spore-crystal combinations from each serotype was examined by an in vitro cell bioassay for cytotoxicity. In general, a cell line from the tobacco hornworm (Manduca sexta) was more responsive to entomocidal protein than cells from the spruce budworm (Choristoneura fumiferana). Significant cytotoxicity toward either or both lepidopteran cell lines was present among all of the serotypes tested with the single exception of serotype 14 (B. thuringiensis subsp. israelensis). This serotype exhibited the only significant cytotoxicity towards dipteran cells (Anopheles gambiae). The degree of cytotoxicity among the soluble but unactivated preparations varied widely and may have resulted from endogenous protease activity. Activation by α-chymotrypsin digestion improved cytotoxicity by as much as 500-fold in some instances, but failed to significantly improve the activity of those serotypes which contained high initial cytotoxicity.     

Specificity of cultured insect tissue cells for bioassay of entomocidal protein fromBacillus thuringiensis

Summary Cultured tissue cells from lepidopteran and dipteran sources displayed an order-specific response to entomocidal protein from crystals ofBacillus thuringiensis. Protein isolated from crystals ofB. thuringiensis subsp.kurstaki was effective against cells of the spruce budworm (Choristoneura fumiferana) and the tobacco hornworm (Manduca sexta), but was inactive against both mosquito cell lines tested (Aedes aegypti andAnopheles gambiae). Conversely, protein from inclusion bodies ofB. thuringiensis subsp.israelensis was fully active only against the mosquito cell lines but displayed reduced (four- to seven-fold) toxicity for the lepidopteran cell lines. One exception to this pattern of specificity was observed with aPlodia interpunctella cell line, which failed to respond to either crystal protein preparation. The moth toxin was stable at 4° C for months, whereas the mosquito toxin was susceptible to proteolytic degradation and was unstable for periods longer than 2 wk.     

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.     

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.     

The crystal δ-endotoxins of Bacillus thuringiensis: Models for their mechanism of action on the insect gut

The crystal δ-endotoxins of Bacillus thuringiensis (Bt) are a family of insecticidal proteins which have been known for some time to kill insects by lysing their gut epithelial cells, but the precise molecular mechanism of toxicity has remained elusive. The recent publication of the crystal structure of a Bt δ-endotoxin has made it possible for us to model the molecular events that occur as the toxin binds to its receptor and inserts into the membrane to form a pore. Using our knowledge of insect gut physiology, we can also predict the effect on the insect of the formation of a toxic pore. We present a new model to explain the events that occur in the insect gut during toxin action.     

Mode of action of Bacillus thuringiensis δ-endotoxin: Effect on TN-368 cells

TN-368 cells swelled and burst upon treatment with the dissolved δ-endotoxin of Bacillus thuringiensis. However, the cytotoxic response was greatly affected by the ionic conditions of the solutions employed for the toxin tests. Ions, in addition to K⁺, seemed to participate in the cytotoxic expression of δ-endotoxin, if their concentration was sufficient (>100 mm). Although similar swollen cells were observed with valinomycin treatment, some differences appeared on the ultrastructural level, especially in the mitochondria. Those of the toxin-treated cells were transformed into the “condensed” form, while those of valinomycin-treated cells were transformed into the “swollen” form. Therefore, the cytotoxic effect of δ-endotoxin, unlike that of valinomycin, seemed to be a general breakdown of ion regulation on the cell level.     

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.     

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.     

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.     

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.     

A protein complex from Choristoneura fumiferana gut-juice involved in the precipitation of δ-endotoxin from Bacillus thuringiensis subsp, sotto

A 75 kDa protein from spruce budworm (Choristoneura fumiferana) gut-juice has been isolated and shown to cause a specific precipitation of the δ-endotoxin from Bacillus thuringiensis subsp. sotto. This 75 kDa protein, separated by either column chromatography or SDS-PAGE, caused precipitation of the sotto toxin in both agarose diffusion gels and the PAGE gels. The precipitation event leads to limited proteolysis of the toxin and loss of larval toxicity. SDSPAGE analysis of the precipitated toxin indicates that proteolysis of the toxin is not a prerequisite for precipitation. The protein responsible for precipitation, exhibits elastase-like activity and appears to be a complex which partially dissociates during boiling in SDS-PAGE sample buffer. Gut-juice from gypsy moth, forest tent caterpillar and white mark tussock moth also precipitated δ-endotoxin, but silkworm gut-juice gave a much weaker response. These results provide further evidence that, in the larval gut, differential processing of δ-endotoxin may play a role in the expression of activity towards various insect larvae.     

Susceptibility of the spruce budworm, Choristoneura fumiferana, and the white-marked tussock moth, Orgyia leucostigmata, to Bacillus thuringiensis: Chemical insecticide combinations

Bacillus thuringiensis mixed with the organophosphate insecticides, fenitrothion (Sumithion), Gardona^®, and Orthene^®, or the synthetic pyrethroid, SBP 1382, was incorporated into synthetic diet and fed larvae of the spruce budworm, Choristoneura fumiferana, and the white-marked tussock moth, Orgyia leucostigma. Mortality was highest when larvae were fed combinations of low concentrations of the insecticides and low to moderate concentrations of the pathogen. The data indicated that applications of a B. thuringiensis dosage expected to produce about 45% mortality of third and fourth instar larvae of the spruce budworm combined with a dosage of fenitrothion causing about 40% mortality or a dosage of Orthene causing from 5 to 25% mortality should result in low budworm survival. With a B. thuringiensis dosage causing 20–60% mortality combined with a fenitrothion dosage causing 15–50% mortality or a sublethal dosage of Gardona, a low survival rate of young white-marked tussock moth larvae may be expected.     

Three years of aerial field experiments with Bacillus thuringiensis plus chitinase formulation against the spruce budworm

From 1971 to 1973 several Bacillus thuringiensis formulations were tested in the field against larvae of the spruce budworm under various conditions of population and tree defoliation. The results showed B. thuringiensis treatments can be a weapon in the control of spruce budworm outbreaks and the beneficial effect of B. thuringiensis treatments appear to be prolonged over 1 or 2 years. A new compact formulation was developed making B. thuringiensis treatments more economical and competitive with chemical insecticides.     

The effects of continuous exposure to low concentrations of the δ-endotoxin of Bacillus thuringiensis on the development of the tobacco budworm,Heliothis virescens

The tobacco budworm, Heliothis virescens, was reared on diets containing various low concentrations of the spore-δ-endotoxin complex of Bacillus thuringiensis var. kurstaki. As the concentration of the complex was increased, the development time increased, pupal weights of the surviving larvae decreased, and the numbers of larvae able to complete the cycle and reach adulthood was reduced. In all these cases, the changes were directly proportional to the log of the concentration of the complex in the diet. Fertility and fecundity were reduced in adult tobacco budworms emerging from larvae reared in the presence of the toxin, but these effects seemed to result indirectly from the general debilitation produced by the toxin, since their occurrence was not related to the concentration of the toxin in the diet.     

Inhibitors of Eicosanoid Biosynthesis and Their Effect upon Bacillus thuringiensisδ-Endotoxin Response in Cultured Insect Cells and Developing Larvae

Twelve inhibitors of eicosanoid biosynthesis were examined for their ability to affect the response of insect cells in vitro and developing larvae to δ-endotoxin from Bacillus thuringiensis. The response of cultured insect cells from Manduca sexta, Choristoneura fumiferana, and Plodia interpunctella to CryIA(c) and CryIC protein from Bacillus thuringiensis was measured while exposed to various concentrations of specific cyclooxygenase and/or lipoxygenase inhibitors. Five of the inhibitors (curcumin, baicalein, nordihydroguaiaretic acid, indomethacin, and eicosatetraynoic acid) were toxic to the cells at high concentrations (>20 μM). Surprisingly, the same inhibitors had no significant effect upon normal larval development, except for nordihydroguaiaretic acid. No true, consistent difference was detected with either lipoxygenase or cyclooxygenase inhibitors for cells or larvae treated with δ-endotoxin. However, the δ-endotoxin response of insect cells in vitro and developing larvae in the presence of nordihydroguaiaretic acid was strong evidence of an involvement with P₄₅₀ cytochromes in the B. thuringiensis toxic response.     

Partial Purification and Characterization of Bacillus thuringiensis Cry1A Toxin Receptor A from Heliothis virescens and Cloning of the Corresponding cDNA

Although extensively studied, the mechanism of action of insecticidal Bacillus thuringiensis Cry toxins remains elusive and requires further elucidation. Toxin receptors in the brush border membrane demand particular attention as they presumably initiate the cascade of events leading to insect mortality after toxin activation. The 170-kDa Cry1Ac toxin-binding aminopeptidase from the tobacco budworm (Heliothis virescens) was partially purified, and its corresponding cDNA was cloned. The cDNA encodes a protein with a putative glycosyl phosphatidylinositol anchor and a polythreonine stretch clustered near the C terminus with predicted O-glycosylation. Partial purification of the 170-kDa aminopeptidase also resulted in isolation of a 130-kDa protein that was immunologically identical to the 170-kDa protein, and the two proteins had identical N termini. These proteins were glycosylated, as suggested by soybean agglutinin lectin blot results. Cry1Ac toxin affinity data for the two proteins indicated that the 130-kDa protein had a higher affinity than the 170-kDa protein. The data suggest that posttranslational modifications can have a significant effect on Cry1A toxin interactions with specific insect midgut proteins.     

A method of visualizing and assessing deposits of aerially sprayed insect microbes

A technique is described for visualizing and assessing deposits of various types of insect microbes on membrane filter surface. It is proposed as an accurate method of determining coverage of entomopathogenic microorganisms sprayed in a tower or from an aircraft in microbial control operations. The actual numbers of spores and/or crystals and number of International Units deposited per unit area, spore: crystal ratio, microbial impact droplet size, and spore viability of Bacillus thuringiensis have been determined with simulated aerial applications. The morphological integrity of different types of insect pathogens is easily distinguishable with the technique.Field trials with nuclear polyhedrosis virus of the spruce budworm, Choristoneura fumiferana, demonstrate the utility of the method in insect pest control by aerially applied microbial agents.     

Cytotoxicity Analysis of Three Bacillus thuringiensis Subsp. israelensis δ-Endotoxins towards Insect and Mammalian Cells

Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ), resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7) when tested at 20 µg/mL.