Scanning electron microscopy of the disruption of tobacco hornworm, Manduca sexta, midgut by Bacillus thuringiensis endotoxin

The ingestion of Bacillus thuringiensis crystal endotoxin by Manduca sexta causes the destruction of both goblet and columnar cells of the midgut. One hour after ingestion, the microvilli show pathological effects. Nearly complete destruction of the goblet and columnar cells has taken place after 4 hr exposure to the toxin.     

Intravital imaging of Bacillus thuringiensis Cry1A toxin binding sites in the midgut of silkworm

Identification of the resistance mechanism of insects against Bacillus thuringiensis Cry1A toxin is becoming an increasingly challenging task. This fact highlights the need for establishing new methods to further explore the molecular interactions of Cry1A toxin with insects and the receptor-binding region of Cry1A toxins for their wider application as biopesticides and a gene source for gene-modified crops. In this contribution, a quantum dot-based near-infrared fluorescence imaging method has been applied for direct dynamic tracking of the specific binding of Cry1A toxins, CrylAa and CrylAc, to the midgut tissue of silkworm. The in vitro fluorescence imaging displayed the higher binding specificity of CrylAa–QD probes compared to CrylAc–QD to the brush border membrane vesicles of midgut from silkworm. The in vivo imaging demonstrated that more CrylAa–QDs binding to silkworm midgut could be effectively and distinctly monitored in living silkworms. Furthermore, frozen section analysis clearly indicated the broader receptor-binding region of Cry1Aa compared to that of Cry1Ac in the midgut part. These observations suggest that the insecticidal activity of Cry toxins may depend on the receptor-binding sites, and this scatheless and visual near-infrared fluorescence imaging could provide a new avenue to study the resistance mechanism to maintain the insecticidal activity of B. thuringiensis toxins.     

Histopathological effects of Bacillus thuringiensis on the midgut of tobacco hornworm larvae (Manduca sexta): Low doses compared with fasting

The cytology and ultrastructure of the midgut cells of Manduca sexta larvae are described for untreated controls, larvae which fed on a spore preparation of Bacillus thuringiensis, and larvae which were fasted for either 24 or 48 hr. New observations on the ultrastructure of midgut cells in Manduca larvae included the finding of specialized Golgi vesicles in anteriormost columnar cells and of regular arrays of expanded rough endoplasmic reticulum in goblet cells of the posterior midgut region. The present observations reveal that the columnar cells of the midgut responded cytologically in the same way to fasting as they did to exposure to the toxic spores of B. thuringiensis. The goblet cells, however, appeared unaffected by fasting but became swollen in response to feeding of B. thuringiensis spore preparation.     

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.     

Persistence of Bacillus thuringiensis parasporal crystal insecticidal activity in soil

Spores and parasporal crystals of a Bacillus thuringiensis var. aizawai (H-serotype 7), strain HD137, streptomycin-resistant mutant were added to acidic (pH 5.0) natural and autoclaved soil and incubated at ?0.10 MPa, 25°C. Populations of B. thuringiensis in both soil treatments showed exponential rates of mortality which were represented by linear regression, the loss of viability being greater in natural than autoclaved soil. In natural soil, parasporal crystal insecticidal activity was lost at a complex, nonexponential rate. The initial, rapid decrease of activity gradually slowed, and the level of activity stabilized at 10% of the original inoculum level after 250 days incubation, until the cessation of sampling at >2 years. In autoclaved soil no significant (P > 0.2) loss of parasporal crystal insecticidal activity was detected over the same period, which suggested that soil microorganisms were responsible for the loss of crystal insecticidal activity in the natural, nonsterilized soil. The rate of loss of crystal activity in natural soil correlated well with assay data reported in the literature using Galleria mellonella, which measures the combined activity of spore and crystal. In autoclaved soil correlation was poor, probably due to variability in the bioassay data.     

Bacillus thuringiensis parasporal crystal toxin: Dissociation into toxic low molecular weight peptides

Parasporal crystals of Bacillus thuringiensis can be dissociated into low molecular weight peptides (< 5000 daltons) by dissolving them in 0.1 M N-morpholinopropane sulfonic acid buffer pH 7.8 containing 0.05 M dithiothreitol and 2M–4M KSCN, or by performic acid oxidation. The peptides obtained by dissolving in KSCN were still toxic to silkworm larvae.     

Insecticidal activity of Bacillus thuringiensis crystal proteins

Published data on insecticidal activity of crystal proteins from Bacillus thuringiensis are incorporated into the Bt toxin specificity relational database. To date, 125 of the 174 holotype known toxins have been tested in ∼1700 bioassays against 163 test species; 49 toxins have not been tested at all; 59 were tested against 71 Lepidoptera species in 1182 bioassays; 53 toxins were tested against 23 Diptera species in 233 bioassays; and 47 were tested against 39 Coleoptera species in 190 bioassays. Activity spectra of the tested toxins were summarized for each order. Comparisons of LC₅₀ values are confounded by high variability of the estimates, mostly due to within-species variation in susceptibility, and errors associated with estimation of toxin protein content. Limited analyses suggest that crystal protein toxicity is not affected by quarternary toxin rank or host used for gene expression, but that pre-ingestion treatment by solubilization or enzymatic processing has a large effect. There is an increasing number of toxin families with cross-order activity, as 15 of the 87 families (secondary rank) that are pesticidal are active against more than one order. Cross-order activity does not threaten environmental safety of B. thuringiensis-based pest control because toxins tend to be much less toxic to taxa outside the family’s primary specificity range.     

Kinetics of pore formation by the Bacillus thuringiensis toxin Cry1Ac

After binding to specific receptors, Cry toxins form pores in the midgut apical membrane of susceptible insects. The receptors could form part of the pore structure or simply catalyze pore formation and consequently be recycled. To discriminate between these possibilities, the kinetics of pore formation in brush border membrane vesicles isolated from Manduca sexta was studied with an osmotic swelling assay. Pore formation, as deduced from changes in membrane permeability induced by Cry1Ac during a 60-min incubation period, was strongly dose-dependent, but rapidly reached a maximum as toxin concentration was increased. Following exposure of the vesicles to the toxin, the osmotic swelling rate reached a maximum shortly after a delay period. Under these conditions, at relatively high toxin concentrations, the maximal osmotic swelling rate increased linearly with toxin concentration. When vesicles were incubated for a short time with the toxin and then rapidly cooled to prevent the formation of new pores before and during the osmotic swelling experiment, a plateau in the rate of pore formation was observed as toxin concentration was increased. Taken together, these results suggest that the receptors do not act as simple catalysts of pore formation, but remain associated with the pores once they are formed.     

Characterization of crystal prototoxin and an activated toxin from Bacillus thuringiensis var. entomocidus

Toxin crystals from Bacillus thuringiensis var. entomocidus were lysed by proteases present in gut juice from larval Philosamia ricini (Lepidoptera) with the release of a prototoxin and an activated toxin. Some of the toxic activity of the lysate was complexed with a pheophytinlike pigment and this complex was retarded on filtration through Sephadex gels. A method is described for the removal of the pheophytin from larval protease preparations. The prototoxin has a molecular weight greater than 200,000, determined by its exclusion from Sephadex G-200 and on activation produces a toxin of molecular weight about 50,000. Isoelectric focusing of crystal lysates gave pI values of 4.5 and 6.4 for the prototoxin and toxin, respectively. The antigenic composition of the prototoxin and of the toxin are compared and the significance of antigen h as an indicator of activation is discussed.     

Distribution of phenotypes among Bacillus thuringiensis strains

An extensive collection of Bacillus thuringiensis isolates from around the world were phenotypically profiled using standard biochemical tests. Six phenotypic traits occurred in 20–86% of the isolates and were useful in distinguishing isolates: production of urease (U; 20.5% of isolates), hydrolysis of esculin (E; 32.3% of isolates), acid production from salicin (A; 37.4% of isolates), acid production from sucrose (S; 34.0% of isolates), production of phospholipase C or lecithinase (L; 79.7% of isolates), and hydrolysis of starch (T; 85.8% of isolates). With the exception of acid production from salicin and hydrolysis of esculin, which were associated, the traits assorted independently. Of the 64 possible combinations of these six phenotypic characteristics, 15 combinations accounted for ca. 80% of all isolates, with the most common phenotype being TL (23.6% of isolates). Surprisingly, while the biochemical traits generally assorted independently, certain phenotypic traits associated with the parasporal crystal were correlated with certain combinations of biochemical traits. Crystals that remained attached to spores (which tended to be non-toxic to insects) were highly correlated with the phenotypes that included both L and S. Among the 15 most abundant phenotypes characterizing B. thuringiensis strains, amorphous crystals were associated with TLE, TL, T, and Ø (the absence of positive tested biochemical traits). Amorphous crystal types displayed a distinct bias toward toxicity to dipteran insects. Although all common phenotypes included B. thuringiensis isolates producing bipyramidal crystals toxic to lepidopteran insects, those with the highest abundance of these toxic crystals displayed phenotypes TLU, TLUA, TLUAE, and TLAE.     

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.     

Pore-forming properties of the Bacillus thuringiensis toxin Cry9Ca in Manduca sexta brush border membrane vesicles

The toxicity and pore-forming ability of the Bacillus thuringiensis Cry9Ca insecticidal toxin, its single-site mutants, R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using Manduca sexta neonate larvae and fifth-instar larval midgut brush border membrane vesicles, respectively. Neither the mutations nor the proteolytic cleavage altered Cry9Ca toxicity. Compared with Cry1Ac, Cry9Ca and its mutants formed large poorly selective pores in the vesicles. Pore formation was highly dependent on pH, however, especially for wild-type Cry9Ca and both mutants. Increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization that was not related to a change in the ionic selectivity of the pores. Pore formation was much slower with Cry9Ca and its derivatives, including the 55-kDa fragment, than with Cry1Ac and its rate was not influenced by the presence of protease inhibitors or a reducing agent.     

Pathogenesis and midgut histopathology of Bacillus thuringiensis in Simulium vittatum (Diptera: Simuliidae)

The pathogenesis and midgut histopathology which resulted when larvae of the blackfly, Simulium vittatum, were exposed to Bacillus thuringiensis at various temperatures and periods of exposure were investigated. The onset of mortality was studied at 10°, 15°, 19°, and 24°C. For each 4–5°C increase in temperature above 15°C, the onset of mortality was shortened by 24 hr. Exposures as brief as 15 min to 10 ppm of a whole spore preparation resulted in an average mortality of 29% in late-instar larvae. Mortality increased sharply for exposures up to 3 hr, approaching a maximum of 80%.The gross signs of disease included cessation of feeding and tetany with brachytosis. The tissue most affected was the midgut epithelium in the regions of the gastric caeca and posterior stomach. The formation of cytoplasmic vacuoles followed by cell lysis and/or sloughing were very apparent in moribund larvae. Death resulted without bacteremia.     

Detection of Bacillus thuringiensis in soil by immunofluorescence

Persistence of viable and heat-killed vegetative cells, parasporal crystals, and spores of Bacillus thuringiensis in soil was monitored by immunofluorescence. The rates of disappearance of the different bacterial components decreased in the following order: viable cells, heat-killed cells, parasporal crystals, and spores. Vegetative cells disappeared at rapid, exponential rates; viable cells autolysed, whereas heat-killed cells were digested by an actinomycete-like, soil microorganism. Parasporal crystals disappeared at a slower, nonexponential rate. Numbers of spores remained unaltered throughout 91 days incubation at 25°C and no germination was detected in this period.     

Midgut juice components affect pore formation by the Bacillus thuringiensis insecticidal toxin Cry9Ca

The pore-forming ability of the Bacillus thuringiensis toxin Cry9Ca, its two single-site mutants R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at R164 was evaluated under a variety of experimental conditions using an electrophysiological assay. All four toxin preparations depolarized the apical membrane of freshly isolated third-instar Manduca sexta midguts bathing in a solution containing 122 mM KCl at pH 10.5, but the 55-kDa fragment was considerably more active than Cry9Ca and its mutants. The activity of the latter toxins was greatly enhanced, however, when the experiments were conducted in the presence of fifth-instar M. sexta midgut juice. This effect was also observed after midgut juice proteins had been denatured by heating at 95 °C or after inorganic ions and small molecules had been removed from the midgut juice by extensive dialysis. A similar stimulation of toxin activity was also observed when the experiments were carried out in the presence of the lipids extracted from an equivalent volume of midgut juice. Depolarization of the cell membrane was also greatly enhanced, in the absence of midgut juice, by the addition of a cocktail of water-soluble protease inhibitors. These results indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.     

Ultrastructural studies of midgut epithelium formation in Lepisma saccharina L. (Insecta, Zygentoma)

At the end of embryogenesis of Lepisma saccharina L. (Insecta, Zygentoma), when the stomodaeum and proctodaeum are completely formed, the midgut epithelium is replaced by the primary midgut, a yolk mass is surrounded by a cell membrane. Midgut epithelium formation begins in the 1st larval stage. Energids migrate toward the yolk periphery and aggregate just beneath the cell membrane. They are gradually enclosed by cell membrane folds of the primary midgut. Single cells are formed. Succeeding energids join just formed cells. Thus, groups of cells, regenerative cell groups, are formed. Their number gradually increases. The external cells of the regenerative cell groups transform into epithelial cells and their basal regions spread toward the next regenerative cell groups. Epithelial cells of neighboring regenerative cell groups join each other to form the epithelium. At the end of the 2nd larval stage, just before molting, degeneration of newly the formed epithelium begins. Remains of organelles and basal membrane occur between the regenerative cell groups. The new epithelium is formed from the regenerative cell groups, which are now termed stem cells of the midgut epithelium.     

Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

Toxic impact of ingested Jatropherol-I on selected enzymatic activities and the ultrastructure of midgut cells in silkworm, Bombyx mori L.

Abstract:  Jatropherol-I, a phorbol-type diterpene from Jatropha curcas seeds was found highly toxic to third instars silkworm larvae after ingestion with LC₅₀ values 0.5793, 0.2197 and 0.1578 mg/ml at 48, 72 and 120 h respectively. The acute toxicity was associated with changes in activities of several midgut enzymes and pathological changes in midgut epithelial cells. Jatropherol-I caused various fluctuations in activities of different midgut enzymes in third instars larvae of silkworm. Compared with controls, both esterase and carboxyliesterase showed significantly depressed activities at 3 h ( t -test, P < 0.05) and increased activities at 24 h and rapidly decreased activities at 48 h after ingestion of 0.125 and 0.25 mg/ml of Jatropherol-I. Jatropherol-I induced two (E4 and E5) and depressed one (E2) of midgut esterase isozymes analysed 3 h after ingestion. There was no significantly different glutathione S-transferase activity between most of treated silkworm and control ( t -test, P > 0.05). Activities of acetylcholinesterase fluctuated weakly in treated silkworm in 48 h. Jatropherol-I induced a gradual decline in midgut protease activities in silkworm with an obvious dose- and time-dependent effect. Jatropherol-I also caused pathological changes in midgut cells, especially in their endoplasmic reticulum. They were noted slight dilatation on 12 h exposure, while extreme dilatation, vesiculations and shedding of ribosome from membrane were observed in endoplasmic reticulum after a longer time exposure. Other pathological changes in microvilli, lysosome, mitochondria and chromatin were also observed in midgut cell of treated silkworm.     

Loop residues of the receptor binding domain of Bacillus thuringiensis Cry11Ba toxin are important for mosquitocidal activity

Using a Cry11Ba toxin model, predicted loops in domain II were analyzed for their role in receptor binding and toxicity. Peptides corresponding to loops α8, 1 and 3, but not loop 2, competed with toxin binding to Aedes midgut membranes. Mutagenesis data reveal loops α8, 1 and 3 are involved in toxicity. Loops 1 and 3 are of greater significance in toxicity to Aedes and Culex larvae than to Anopheles. Cry11Ba binds the apical membrane of larval caecae and posterior midgut, and binding can be competed by loop 1 but not by loop 2 peptides. Cry11Ba binds the same regions to which anti-cadherin antibody binds, and this antibody competes with Cry11Ba binding suggesting a possible role of cadherin in toxication.     

Proteolytic processing of Bacillus thuringiensis toxin Cry1Ab in rice brown planthopper, Nilaparvata lugens (Stål)

To understand the low toxicity of Cry toxins in planthoppers, proteolytic activation of Cry1Ab in Nilaparvata lugens was studied. The proteolytic processing of Cry1Ab protoxin by N. lugens midgut proteases was similar to that by trypsin activated Cry1Ab. The Cry1Ab processed with N. lugens midgut proteases was highly insecticidal against Plutella xylostella. However, Cry1Ab activated either by trypsin or the gut proteases of the brown planthopper showed low toxicity in N. lugens. Binding analysis showed that activated Cry1Ab bound to brush border membrane vesicles (BBMV) from N. lugens at a significantly lower level than to BBMV from P. xylostella.