Intracellular Ca2+ concentration was measured in single Cf1 cells (Choristoneura fumiferana, spruce budworm) loaded with Fura-2, a Ca2+-sensitive fluorescent probe. Cf1 cells displayed Ca2+ surges in response to Cry1Ac and Cry1C proteins, two Cf1-toxic Bacillus thuringiensis products, but not to Cry1Aa and Cry3A, which are not toxic to Cf1 cells. In the presence of extracellular Ca2+, the toxin-induced Ca2+ response was insensitive to methoxyverapamil, a voltage-dependent Ca2+ channel blocker, but was abolished by lanthanum, a general inhibitor of Ca2+ transport. In the absence of external Ca2+, Cry1Ac induced a small intracellular Ca2+ transient which was inhibited by TMB-8, a blocker of Ca2+ release from inositol-1,4,5-trisphosphate-sensitive pools. Under these conditions, thapsigargin, which inhibits intracellular Ca2+-ATPases, elicited a Ca2+ surge when applied alone. However, subsequent addition of Cry1Ac failed to induce a Ca2+ signal, indicating a depletion of intracellular Ca2+ pools. In Cf1 cells, therefore, bioactive B. thuringiensis toxins triggered intracellular Ca2+ surges which were mainly due to the influx of extracellular Ca2+ through toxin-made pores, as confirmed by planar lipid bilayer experiments. Furthermore, TMB-8- and thapsigargin-sensitive Ca2+ stores contributed to the Cry1Ac-induced Ca2+ signal.

The inclusion bodies produced during sporulation by Bacillus thuringiensis, a Gram-positive soil bacterium, are highly specific gut poisons causing insect death within a few hours of ingestion (Höfte and Whiteley, 1989; Gill et al. 1992). Several formulated products based on B. thuringiensis toxins are currently used as efficient tools for the control of agro-forestry insect pests (Cannon, 1996) and of the insect vectors of several human and animal diseases (Federici, 1995). The exact mechanism of action of B. thuringiensis toxins is not well understood (Gill et al. 1992; Knowles, 1994). Following ingestion and solubilisation by intestinal secretions in the insect midgut, the crystal proteins are cleaved by gut proteases. The resulting products are 60–65 kDa activated proteins which bind to specific sites of the brush-border membrane of the columnar cells lining the gut lumen. This triggers a cascade of poorly elucidated events leading to the death of the insect. It is believed that the pore-related increased permeabilisation of the target cells and the resulting cellular ionic and metabolite imbalance constitute the critical steps leading to cell disruption. With the recent elucidation of the atomic structures of Cry3A, a coleopteran-specific toxin (Li et al. 1991), and Cry1Aa, a lepidopteran-specific toxin (Grochulski et al. 1995), a better understanding of the molecular mode of action of these proteins should emerge. This will be essential to deal with insect resistance to B. thuringiensis insecticides, the most serious problem which these products will inevitably face (Tabashnik, 1994).

So far, only limited attention has been given to the interactions of B. thuringiensis toxin with physiological processes at the cell level, possibly because of the lack of appropriate cellular models. Only a few B. thuringiensis toxin-susceptible insect cell lines are available for physiological studies. While these cells are not the natural targets of the pathogens and their sensitivity to the crystal proteins is several orders of magnitude lower than that of the insects from which they originate, they allow appropriate detection of the entomocidal activity of activated B. thuringiensis products with reasonable species and interspecies selectivity (Johnson, 1994; McCarthy, 1994b). Several physiological mechanisms have been investigated in Sf9 cells from the fall armyworm (Spodoptera frugiperda, Lepidoptera) (Hu et al. 1994a,b), UCR-SE-1a cells from the beet armyworm (Spodoptera exigua, Lepidoptera) (Monette et al. 1994) and Cf1 cells from the spruce budworm (Choristoneura fumiferana, Lepidoptera)

(Gole et al. 1987; Orr et al. 1988; Gupta and Downer, 1993). These cells, because of their sensitivity to B. thuringiensis toxins, provide simple and convenient models for studies of the mechanism of action of the toxin at the cellular level. Recently, the cellular effects of Cry1 toxins on Sf9 cells have been the object of intense scrutiny in our laboratories (Schwartz et al. 1991; Vachon et al. 1995a,b; Monette et al. 1997; Villalon et al. 1997). Cry1C induced cell swelling and caused the rapid diffusion and equilibration of K+, Na+ and H+ across the plasma membrane of Sf9 cells (Vachon et al. 1995b; Villalon et al. 1997). The toxin triggered an intracellular Ca2+ surge within seconds of toxin exposure and thereafter activated anion-selective channels in the cell membrane (Schwartz et al. 1991). We observed a similar Cry1C-mediated Ca2+ response in UCR-SE-1a cells (Monette et al. 1994). Furthermore, it was established that Cry1C toxicity to Sf9 cells was substantially stimulated by extracellular Ca2+ in a dose-dependent manner and that this effect was related to an increased concentration of intracellular Ca2+ (Monette et al. 1997). These data suggested that cellular Ca2+ changes related to toxin exposure represent an early step in the activity of the toxin and may be a general response of susceptible insect cells to the detection of B. thuringiensis toxins. Furthermore, they supported the concept of a synergetic interaction between Ca2+ and B. thuringiensis toxins, as demonstrated in several lepidopteran pests in vivo (for a review, see Dent, 1993) and by a recent in vivo study on the interaction of caffeine with B. thuringiensis toxin activity against the bertha armyworm (Mamestra configurata, Lepidoptera), suggesting that the augmented toxicity was mediated by the deregulation of cellular Ca2+ transport processes (Morris et al. 1994).

In this report, we used Fura-2, a Ca2+ fluorophore, to examine the effects of B. thuringiensis toxins on the intracellular Ca2+ concentration of Cf1 cells, a B. thuringiensis toxin-sensitive cell line (for a review, see McCarthy, 1994a). We demonstrated that active toxins triggered Ca2+ surges which were produced largely by the influx of extracellular Ca2+ through toxin-made membrane pores but also by a small component originating from the release of Ca2+ from intracellular stores. Furthermore, planar lipid bilayer experiments conducted in the present study demonstrated that the pores induced by Cry1Ac, which are permeable to K+ (Slatin et al. 1990; Schwartz et al. 1997), also allowed the passage of Ca2+.

Cells

Cf1 cells are derived from trypsin-treated larval tissue of the spruce budworm Choristoneura fumiferana. They were obtained from S. Sohi (Natural Resources Canada, Sault Sainte Marie, Ontario, Canada) and grown in Grace’s medium supplemented with 0.25 % (w/v) tryptose and 10 % (v/v) heat-inactivated foetal bovine serum. Cultures were maintained in 25 cm2 plastic tissue culture flasks (Sarstedt Inc, Newton, North Carolina, USA) at 27 °C and were subcultured every 3–4 days to a final concentration of 1.5×106 to 2×106 cells ml−1. In preparation for the experiments, 500 μl of cells from cultures at 80–90 % confluence were deposited on glass coverslips (24 mm in diameter) in supplemented Grace’s medium at room temperature (20–22 °C). Attachment to the coverslips occurred within 2 h to a final confluence of 80–90 %.

Solutions

The normal bath solution (NBS) was a simplified Grace’s solution containing 50 mmol l−1 KCl, 21 mmol l−1 NaCl, 6.8 mmol l−1 CaCl2, 14 mmol l−1 MgCl2, 11 mmol l−1 MgSO4, 3.9 mmol l−1 D-glucose and 20 mmol l−1 Pipes. Osmolarity was adjusted to 380 mosmol l−1 with sucrose, and pH was set to 6.4 with NaOH. Ca2+-free NBS (0NBS) was obtained by replacing CaCl2 with 5 mmol l−1 EGTA. Concentrated stock solutions of test agents were prepared in NBS, 0NBS or, when required, dimethylsulphoxide (DMSO). Working solutions were obtained by dilution in NBS or 0NBS. Fura-2/AM (Fura-2 acetoxymethyl ester) stock solution (1 mmol l−1) was prepared in DMSO and was used at a final concentration of 2 μmol l−1 in NBS.

Cell Ca2+ measurements

Intracellular Ca2+ concentration in single cells or small group of cells was determined using Fura-2 as described previously (Schwartz et al. 1991). Briefly, cells were loaded with Fura-2/AM, the Ca2+-insensitive, membrane-permeant form of Fura-2, a fluorescent Ca2+ indicator, in NBS for 1 h at room temperature. Cells were washed three times and incubated for 10 min in NBS to achieve intracellular Fura-2/AM hydrolysis by cellular esterases into Fura-2, which remained trapped intracellularly. Upon binding to Ca2+ (135 nmol l−1 dissociation constant at 20 °C), the excitation spectrum of Fura-2 undergoes a dose-dependent shift towards lower wavelengths with no change in emission peak (505 nm). This spectral property of Fura-2 was used for high-sensitivity, largely artefact-free determination of cellular Ca2+ concentration, which can be derived from the ratio of Fura-2 fluorescence intensities measured at 340 nm and 380 nm excitation (Grynkiewicz et al. 1985). Coverslips were mounted in a custom-made experimental chamber containing 0.5 ml of NBS or 0NBS and located on the stage of an IMT-2 inverted fluorescence microscope (Olympus Optical Co, Tokyo, Japan) equipped with a 40×, 0.85 NA epifluorescence objective and attached to a dual-excitation photometric instrument (Photon Technology Instrument, Monmouth Junction, New Jersey, USA). Test compounds were added to the chamber after a 2 min control period. Diluted working solutions of B. thuringiensis toxins were prepared in NBS or 0NBS and added to the chamber to a final concentration of 0.35 μmol l−1. For calibration, the maximum and minimum fluorescence ratios were determined at the end of each experiment by the sequential addition of 20 μmol l−1 ionomycin and 20 mmol l−1 EGTA. All experiments were performed at room temperature. Autofluorescence of Cf1 cells at either wavelength was at least 10 times lower than that of Fura-2 loaded cells and, therefore, fluorescence levels were not corrected for background. The fluorophore was uniformly distributed in the cell cytosol, with no sign of compartmentalisation in the nucleus or cytoplasmic organelles. The basal level of intracellular Ca2+ in NBS was 82±14 nmol l−1 (mean ± S.E.M., N=23).

Planar lipid bilayer

Reconstitution of B. thuringiensis toxins in planar lipid bilayers has been described in detail elsewhere (Schwartz et al. 1993). Briefly, phospholipid membranes were formed from a 7:2:1 lipid mixture of phosphatidylethanolamine, phosphatidylcholine and cholesterol painted on a 250 μm circular aperture in a Delrin wall separating two low-volume chambers (4 ml trans, 3.5 ml cis). Under the experimental conditions used in the present study, membranes had a capacitance of approximately 150–200 pF and remained stable for hours. Channel activity, following injection of 0.3 μmol l−1 activated protein near the membrane in the cis chamber, was monitored by step changes in the current recorded when test voltages were applied to the planar lipid bilayer. All experiments were performed at room temperature in solutions containing either 50 or 450 mmol l−1 CaCl2 and buffered with 10 mmol l−1 Tris, pH 9.0. Single-channel currents were recorded with an Axopatch-1D patch-clamp amplifier (Axon Instruments, Foster City, California, USA). Analysis was performed on a personal computer using pClamp and Axotape software (Axon Instruments).

Toxins and chemicals

Toxins were produced, activated, purified and tested for purity as described previously (Masson et al. 1989). They were stored in lyophilised form and reconstituted to a final concentration of 1 mg ml−1 in high-purity water with 10 mmol l−1 Tris at pH 10.0. Grace’s insect cell culture medium was purchased from Gibco BRL (Life Technologies, Burlington, Ontario, Canada). Foetal bovine serum was obtained from PDI Bioscience (Aurora, Ontario, Canada). Tryptose was purchased from Oxoid, Unipath Ltd, Basingstoke, Hampshire, UK. TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride], thapsigargin (a naturally occurring sesquiterpene lactone) and EGTA were obtained from Sigma, St Louis, Missouri, USA. D600 (methoxyverapamil, a phenethylamine derivative) and ionomycin-free acid (from Streptomyces conglobatus) were purchased from Calbiochem Corp, La Jolla, California, USA. Anhydrous DMSO was obtained from Aldrich Chemicals, Milwaukee, Wisconsin, USA. Fura-2/AM was purchased from Molecular Probes, Eugene, Oregon, USA. Lipids were obtained from Avanti Polar Lipids, Alabaster, Alabama, USA.

Effects of B. thuringiensis toxins

The spruce budworm is susceptible to several Cry toxins (Van Frankenhuyzen et al. 1991, 1993), some of which also show in vitro activity against Cf1 cells (Schwartz et al. 1993; McCarthy, 1994a), as summarised in Table 1. Cell Ca2+ measurements were conducted with 0.35 μmol l−1 Cry1Aa, Cry1Ab, Cry1Ac and Cry1C, four lepidopteran-specific toxins, and 0.35 μmol l−1 Cry3A, a coleopteran-specific toxin. In NBS, i.e. in the presence of 6.8 mmol l−1 extracellular Ca2+, neither Cry1Aa (N=9) nor Cry3A (N=6) elicited a Ca2+ surge in Cf1 cells (results not shown). However, the cells responded to Cry1Ac, Cry1Ab and Cry1C exposure (Fig. 1). Cry1Ac triggered a large, sustained Ca2+ surge. A 260 % increase in fluorescence ratio was observed. The signal reached 90 % of its maximum amplitude after 132±95 s (mean ± S.E.M., N=35). Cry1C also induced a sustained Ca2+ surge, but the fluorescence ratio was only 20–60 % greater than the basal level and it took 405±110 s (mean ± S.E.M., N=13) to reach 90 % of the final level. The response to Cry1Ab was transient. Its peak amplitude was between those of Cry1Ac and Cry1C (a 130 % increase) and the time to 90 % peak was 221±123 s (mean ± S.E.M., N=18).

Table 1.

Ca2+ response to, and toxicity of, Cry toxins

Ca2+ response to, and toxicity of, Cry toxins
Ca2+ response to, and toxicity of, Cry toxins
Fig. 1.

Ca2+ surges in single, Fura-2 loaded Cf1 cells in response to exposure to Cry toxin. The cells were bathed in a Ca2+-rich physiological saline solution (NBS). Cry1Ab, Cry1Ac or Cry1C (0.35 μmol l−1) was added to the bath at the time indicated by the arrows. Traces are representative of 35 experiments with Cry1Ac, 18 with Cry1Ab and 13 with Cry1C.

Fig. 1.

Ca2+ surges in single, Fura-2 loaded Cf1 cells in response to exposure to Cry toxin. The cells were bathed in a Ca2+-rich physiological saline solution (NBS). Cry1Ab, Cry1Ac or Cry1C (0.35 μmol l−1) was added to the bath at the time indicated by the arrows. Traces are representative of 35 experiments with Cry1Ac, 18 with Cry1Ab and 13 with Cry1C.

In the absence of extracellular Ca2+, Cry1Ac triggered a small, but significant, Ca2+ surge (Fig. 2). The response was sustained over the measurement period. Its amplitude was 20 % greater than the basal fluorescence ratio (N=7). When tested in 0NBS, Cry1Aa, Cry1Ab, Cry1C and Cry3A did not affect the intracellular Ca2+ concentration of the cells.

Fig. 2.

Ca2+ transient in response to exposure to Cry1Ac toxin in the absence of extracellular Ca2+. The cell was bathed in 0NBS (see Materials and methods). Toxin addition is indicated by the arrow. The Ca2+ surge is much smaller than that observed with Ca2+ in the bath. The trace is representative of seven experiments.

Fig. 2.

Ca2+ transient in response to exposure to Cry1Ac toxin in the absence of extracellular Ca2+. The cell was bathed in 0NBS (see Materials and methods). Toxin addition is indicated by the arrow. The Ca2+ surge is much smaller than that observed with Ca2+ in the bath. The trace is representative of seven experiments.

Effects of Ca2+ transport modulators on Cry1Ac-induced Ca2+ surges

Ca2+ transport through the cell membrane

Ca2+ entry through the plasma membrane was investigated using D600, Co2+, Ni2+ and La3+. D600 is an efficient blocker of voltage-dependent Ca2+ channels (Triggle, 1990). Co2+, Ni2+ and La3+ are general inhibitors of Ca2+ transport across cell membranes (Tsien, 1990). The addition of 50 μmol l−1 D600, either before or after Cry1Ac application, had no effect on the sustained [Ca2+] elevation observed in the presence of extracellular Ca2+ (N=11, results not shown).

Intracellular Ca2+ concentration was unaffected by 5 mmol l−1 Co2+ or 5 mmol l−1 Ni2+ when added to NBS in the absence of Cry1Ac toxin. However, following Cry1Ac application in Co2+-or Ni2+-containing NBS, both 340 nm and 380 nm fluorescence intensities decreased (results not shown). It was verified in control experiments that the ions alone did not induce cell swelling or dye leakage. Thus, it appeared that, in the presence of Cry1Ac, Ni2+ and Co2+ entered the cells and quenched the Ca2+ fluorophore. In fact, it has been reported that Ni2+ and Co2+ can enter melanotrophs through Ca2+ channels and that Fura-2 is indeed quenched by these ions (Shibuya and Douglas, 1992).

When 5 mmol l−1 LaCl3 was added to NBS before the toxin, Cf1 cell Ca2+ concentration was unaffected by exposure to Cry1Ac (Fig. 3). When LaCl3 was added during the plateau phase of the Ca2+ response to Cry1Ac, the surge was immediately interrupted, but the intracellular Ca2+ concentration did not return completely to the baseline.

Fig. 3.

Effect of La3+ on the Cry1Ac-induced Ca2+ response in single Cf1 cells. The cells were bathed in Ca2+-rich physiological saline solution (NBS). Toxin addition is shown by the leftmost, upward-pointing arrow. Trace 1: LaCl3 (5 mmol l−1) was added to the bath during the plateau phase of the Ca2+ surge (rightmost, downward-pointing arrow). It immediately inhibited the Ca2+ response. Trace 2: the same effect was observed when La3+ was added during the fast rising phase of the surge (middle, downward-pointing arrow). Trace 3: in this experiment, the cell was bathed in La3+-containing NBS and then exposed to Cry1Ac. The trivalent cation prevented the development of the Ca2+ surge. Traces are representative of five experiments.

Fig. 3.

Effect of La3+ on the Cry1Ac-induced Ca2+ response in single Cf1 cells. The cells were bathed in Ca2+-rich physiological saline solution (NBS). Toxin addition is shown by the leftmost, upward-pointing arrow. Trace 1: LaCl3 (5 mmol l−1) was added to the bath during the plateau phase of the Ca2+ surge (rightmost, downward-pointing arrow). It immediately inhibited the Ca2+ response. Trace 2: the same effect was observed when La3+ was added during the fast rising phase of the surge (middle, downward-pointing arrow). Trace 3: in this experiment, the cell was bathed in La3+-containing NBS and then exposed to Cry1Ac. The trivalent cation prevented the development of the Ca2+ surge. Traces are representative of five experiments.

Cry1Ac channels are permeable to Ca2+

Cry1Ac forms K+-selective channels in planar lipid bilayers (Slatin et al. 1990; Schwartz et al. 1997) and permeabilises Cf1 cells (Knowles and Ellar, 1987; B. Escriche, N. De Decker and E. Van Kerkhove, personal communication). The hypothesis that Cry1Ac channels are also permeable to divalent ions was tested in experiments using planar lipid bilayers. Under both symmetrical and non-symmetrical conditions (50 mmol l−1 CaCl2 in the cis chamber and either 50 mmol l−1 or 450 mmol l−1 CaCl2 in the trans chamber), and with 0.3 μmol l−1 Cry1Ac added to the cis chamber, discrete current jumps were observed (Fig. 4A, upper trace, non-symmetrical conditions). Under non-symmetrical conditions, the zero-current voltage was shifted to the right by 17 mV (Fig. 4B), consistent with Ca2+ selectivity. The conductance of the channel was 141±27 pS (N=6) under symmetrical conditions and 185±32 pS (means ± S.E.M., N=5) under non-symmetrical conditions (Fig. 4A). The addition of 5 mmol l−1 CoCl2 to the cis chamber affected the kinetics of the channel dramatically without blocking it (Fig. 4A, middle trace). However, subsequent addition of 5 mmol l−1 of LaCl3 abolished channel activity (Fig. 4A, lower trace). These data demonstrate that Ca2+ passes through Cry1Ac channels and that the trivalent ion La3+ is an efficient blocker of the channel. They suggest that Co2+ enters the channel and introduces at least one additional short-lived state to the kinetic behaviour of the channel: current jumps of similar amplitude but significantly shorter duration.

Fig. 4.

(A) Single-channel current flowing through Cry1Ac toxin channels formed in planar lipid bilayers under non-symmetrical conditions (50 mmol l−1 CaCl2 in the cis chamber, 450 mmol l−1 CaCl2 in the trans chamber). The membrane potential was held at −20 mV (with respect to the trans chamber, which was held at ground potential). The three traces are from the same experiment in which 5 mmol l−1 CoCl2 and 5 mmol l−1 LaCl3 were sequentially added to the cis side of the membrane. The upper trace shows distinct current jumps, indicative of channel switching between the open and the closed states (indicated by the letter c and the dashed line to the left of the traces). There were at least three equally conducting channels in the phospholipid membrane. Subconducting levels were also observed (asterisks). The middle trace shows the effects of Co2+ on Cry1Ac channel activity. Less than 60 s after addition of the divalent ion, channel current kinetics was significantly affected and assumed fast transitions between the open and closed states. Subsequent addition of La3+ resulted in complete current inhibition, as shown in the lower trace. The data are representative of three experiments. (B) Current–voltage relationships for the Cry1Ac channel. The amplitude of the current (I) was plotted against applied voltage (V) for an experiment conducted under symmetrical (50 mmol l−1 CaCl2 in the cis chamber, 50 mmol l−1 CaCl2 in the trans chamber, triangular symbols) and under non-symmetrical (50 mmol l−1 CaCl2 in the cis chamber, 450 mmol l−1 CaCl2 in the trans chamber, square symbols) conditions. The IV relationships were rectilinear (r2 > 0.98) over the range of voltage tested and were shifted to the right when an ionic gradient was established across the channel, indicative of channel selectivity to Ca2+. The channel conductance was 141±27 pS under symmetrical conditions (N=6) and 185±32 pS under non-symmetrical conditions (N=5) (means ± S.E.M.).

Fig. 4.

(A) Single-channel current flowing through Cry1Ac toxin channels formed in planar lipid bilayers under non-symmetrical conditions (50 mmol l−1 CaCl2 in the cis chamber, 450 mmol l−1 CaCl2 in the trans chamber). The membrane potential was held at −20 mV (with respect to the trans chamber, which was held at ground potential). The three traces are from the same experiment in which 5 mmol l−1 CoCl2 and 5 mmol l−1 LaCl3 were sequentially added to the cis side of the membrane. The upper trace shows distinct current jumps, indicative of channel switching between the open and the closed states (indicated by the letter c and the dashed line to the left of the traces). There were at least three equally conducting channels in the phospholipid membrane. Subconducting levels were also observed (asterisks). The middle trace shows the effects of Co2+ on Cry1Ac channel activity. Less than 60 s after addition of the divalent ion, channel current kinetics was significantly affected and assumed fast transitions between the open and closed states. Subsequent addition of La3+ resulted in complete current inhibition, as shown in the lower trace. The data are representative of three experiments. (B) Current–voltage relationships for the Cry1Ac channel. The amplitude of the current (I) was plotted against applied voltage (V) for an experiment conducted under symmetrical (50 mmol l−1 CaCl2 in the cis chamber, 50 mmol l−1 CaCl2 in the trans chamber, triangular symbols) and under non-symmetrical (50 mmol l−1 CaCl2 in the cis chamber, 450 mmol l−1 CaCl2 in the trans chamber, square symbols) conditions. The IV relationships were rectilinear (r2 > 0.98) over the range of voltage tested and were shifted to the right when an ionic gradient was established across the channel, indicative of channel selectivity to Ca2+. The channel conductance was 141±27 pS under symmetrical conditions (N=6) and 185±32 pS under non-symmetrical conditions (N=5) (means ± S.E.M.).

Cell Ca2+ mobilisation

The results described above obtained using 6.8 mmol l−1 Ca2+ in the bath indicated that the Ca2+ surge observed in response to Cry1Ac exposure was probably due to the influx of the divalent ion through toxin-made pores. However, a Ca2+ signal was also recorded when the toxin was applied to the cells in the absence of extracellular Ca2+, suggesting that Ca2+ was released from intracellular pools into the cytosol. In an attempt to identify the origin of the Cry1Ac-induced signal recorded in 0NBS, experiments were conducted using thapsigargin and TMB-8. These compounds modulate Ca2+transport across the membranes of Ca2+ stores. Thapsigargin inhibits the Ca2+-ATPase of Ca2+ pools, thus increasing the level of cytosolic Ca2+ by preventing Ca2+ uptake into cellular stores (Thastrup et al. 1990). TMB-8 is an antagonist of Ca2+ release from the endoplasmic reticulum (Chiou and Malagodi, 1975). Fig. 5 shows the effect of thapsigargin. In NBS, 100 nmol l−1 thapsigargin elicited a large Ca2+ transient. Subsequent addition of Cry1Ac resulted in a sustained elevation of cell [Ca2+] similar to that observed with the toxin alone. In 0NBS, thapsigargin triggered a transient small rise in [Ca2+]. Subsequent addition of Cry1Ac had no effect on the intracellular Ca2+ concentration, which remained at the basal level. The same experimental protocol was used with TMB-8 (50 μmol l−1), and the results are illustrated in Fig. 6. In NBS, there was no response to TMB-8 alone, and Cry1Ac induced its typical Ca2+ surge in the presence of the drug. In 0NBS, TMB-8 had no effect on cell [Ca2+]. Further addition of Cry1Ac failed to trigger a Ca2+ surge.

Fig. 5.

Effect of thapsigargin on Cf1 intracellular [Ca2+] and the Cry1Ac-induced Ca2+ surge. Thapsigargin (100 nmol l−1) was applied to the cells at the time indicated by the leftmost, upward-pointing arrow. Trace 1: in the presence of extracellular Ca2+, the agent triggered a large Ca2+ transient. Subsequent application of 0.35 μmol l−1 Cry1Ac (downward-pointing arrow) elicited a second Ca2+ surge. Trace 2: in the absence of extracellular Ca2+, a small Ca2+ transient was induced by thapsigargin. There was no further Ca2+ response to the addition of Cry1Ac toxin at the time indicated by the downward-pointing arrow. Traces are representative of six experiments.

Fig. 5.

Effect of thapsigargin on Cf1 intracellular [Ca2+] and the Cry1Ac-induced Ca2+ surge. Thapsigargin (100 nmol l−1) was applied to the cells at the time indicated by the leftmost, upward-pointing arrow. Trace 1: in the presence of extracellular Ca2+, the agent triggered a large Ca2+ transient. Subsequent application of 0.35 μmol l−1 Cry1Ac (downward-pointing arrow) elicited a second Ca2+ surge. Trace 2: in the absence of extracellular Ca2+, a small Ca2+ transient was induced by thapsigargin. There was no further Ca2+ response to the addition of Cry1Ac toxin at the time indicated by the downward-pointing arrow. Traces are representative of six experiments.

Fig. 6.

Response to Cry1Ac in TMB-8-treated cells. Top trace: in Ca2+-rich physiological solution, 50 μmol l−1 TMB-8 was applied to the cell (downward-pointing arrow). The addition of 0.35 μmol l−1 Cry1Ac (upward-pointing arrow) evoked a large Ca2+ surge. The trace is representative of three experiments. Bottom trace: when the cell was bathed in Ca2+-free medium and 50 μmol l−1 TMB-8 was added to the bath (downward-pointing arrow), 0.35 μmol l−1 Cry1Ac (upward-pointing arrow) failed to trigger a Ca2+ signal. The trace is representative of three experiments

Fig. 6.

Response to Cry1Ac in TMB-8-treated cells. Top trace: in Ca2+-rich physiological solution, 50 μmol l−1 TMB-8 was applied to the cell (downward-pointing arrow). The addition of 0.35 μmol l−1 Cry1Ac (upward-pointing arrow) evoked a large Ca2+ surge. The trace is representative of three experiments. Bottom trace: when the cell was bathed in Ca2+-free medium and 50 μmol l−1 TMB-8 was added to the bath (downward-pointing arrow), 0.35 μmol l−1 Cry1Ac (upward-pointing arrow) failed to trigger a Ca2+ signal. The trace is representative of three experiments

This study demonstrates that cytotoxic B. thuringiensis proteins triggered Ca2+ surges in Cf1 cells, and the results are similar to previous studies in our laboratory in which we reported comparable Ca2+ responses in Sf9 cells (Schwartz et al. 1991) and UCR-SE-1a cells (Monette et al. 1994) exposed to Cry1C toxin, to which both cell lines are susceptible (McCarthy, 1994a,b). Table 1, which summarises Cf1 toxicity data (Van Frankenhuyzen et al. 1991; Schwartz et al. 1993), and the results of the present study show that non-cytotoxic proteins (Cry1Aa and Cry3A) failed to elicit a change in Cf1 intracellular Ca2+ concentration, whereas Cry1Ab, Cry1Ac and Cry1C, which are active against the cell line, induced Ca2+ surges in the cells. The correlation between toxicity and the extent of intracellular Ca2+ activity was not perfect: Cry1C, which is more toxic than Cry1Ab, induced smaller Ca2+ surges in the cells. However, of the three proteins, Cry1Ac, the most toxic to Cf1 cells, triggered the largest Ca2+ response, suggesting that the increase in cell [Ca2+] participates in the mode of action of the toxin. This is consistent with our previous work on Sf9 cells, which demonstrated that Cry1C toxicity was related to extracellular Ca2+ concentration in a dose-dependent manner and that this effect was influenced by several Ca2+ transport modulators, implying that changes in intracellular [Ca2+] may be related to cytotoxicity (Monette et al. 1997).

The results of this study clearly show that the Cry1Ac-induced Ca2+ surge had two components: a large Ca2+ surge was recorded in the presence of Ca2+ in the bath, and a small transient Ca2+ signal took place in a Ca2+-free environment. Our data indicate that the large surge was due to the influx of Ca2+ into the cell. La3+, which is known to inhibit the cell Ca2+ extrusion mechanisms (Triggle, 1990), had no effect on cell [Ca2+] in the absence of Cry1Ac. However, La3+ prevented the Ca2+ surge when added to the bath before the toxin and terminated the toxin-induced response when applied after the toxin. This suggests that the trivalent ion blocked the pathway used by Ca2+ to enter the cells. Experiments with D600, a general inhibitor of voltage-dependent Ca2+ channels (Tsien, 1990), showed that such channels were not involved in toxin-induced Ca2+ influx, either because Cf1 cells do not possess voltage-dependent Ca2+ channels or because, under our experimental conditions, the cells were fully depolarised and the channels were inactivated. Indeed, preliminary whole-cell patch-clamp experiments in our laboratory and elsewhere (B. Escriche, N. De Decker and E. Van Kerkhove, personal communication) indicated that the resting potential of Cf1 cells was close to 0 mV. While other Ca2+-permeable channels that could be activated by Cry1Ac and inhibited by La3+ may exist in Cf1 cells, the more likely explanation for the large rise in Ca2+ response to toxin exposure is that Ca2+ entered the cells through the pores formed by the insertion of toxin into the cell membrane. Several studies have shown that Cry toxins formed ion channels in planar lipid bilayers (Slatin et al. 1990; Schwartz et al. 1993, 1997; English et al. 1991; Von Tersch et al. 1994; Grochulski et al. 1995; Lorence et al. 1995) and single insect cells (Schwartz et al. 1991; Monette et al. 1994). The toxins also permeabilised liposomes (Yunovitz and Yawetz, 1988; Haider and Ellar, 1989; English et al. 1991; Butko et al. 1994), midgut brush-border membranes vesicles (Sacchi et al. 1986; Hendrickx et al. 1989; Wolfersberger, 1989; Uemura et al. 1992; Carroll and Ellar, 1993; Lorence et al. 1995; Martin and Wolfersberger, 1995) and isolated midguts (Harvey and Wolfersberger, 1979; Liebig et al. 1995; Peyronnet et al. 1997). In the present study, we have provided evidence that divalent ions entered toxin-exposed cells, most probably through the toxin pores, as demonstrated by Fura-2 quenching by Ni2+ and Co2+. Furthermore, using planar lipid bilayers, we have demonstrated that Cry1Ac channels are indeed permeable to Ca2+.

Interestingly, in the absence of extracellular Ca2+, Cry1Ac induced a small Ca2+ transient in Cf1 cells, a clear indication that the effect of the toxin on intracellular [Ca2+] could not be solely attributed to the influx of Ca2+ through toxin-made pores. Such a response when Ca2+ was omitted from the bath was also observed in a previous study on the effects of Cry1C on UCR-SE-1a cells (Monette et al. 1994). It has been reported that, in both the Cf1 cell line and in a Mamestra brassica (cabbage moth) cell line, adenylate cyclase was activated by B. thuringiensis proteins that are toxic to these cells (Knowles and Farndale, 1988). However, in the M. brassica cells, this effect could not be related to the cytolytic mechanism, and it was suggested that the toxins interacted with cell membrane components, possibly lipids, thus affecting adenylate cyclase activity. In Cf1 cells, hormone-mediated receptor responses include the activation of adenylate cyclase and phospholipase C (Orr et al. 1988). Adenylate cyclase, which produces cyclic AMP, is sensitive to intracellular Ca2+ concentration and protein kinase C, a Ca2+-activated, phospholipid-dependent enzyme. This protein is stimulated by diacylglycerol, one of the two second messengers produced by phosphoinositide metabolism (Rasmussen and Barrett, 1984). Protein kinase C has recently been characterised in Cf1 cells (Gupta and Downer, 1993). Our data on B. thuringiensis-induced Ca2+ mobilisation from thapsigargin- and TMB-8-sensitive stores indicate that the inositol trisphosphate second messenger may also be produced in Cf1cells. The mechanism by which Ca2+ is released from organelles in response to toxin exposure has yet to be investigated. It is tempting to speculate that, upon binding to a specific, as yet poorly identified, surface receptor (Knowles and Ellar, 1986), which may be coupled to both adenylate cyclase and phospholipase C, B. thuringiensis toxins induce intracellular Ca2+ signalling and the production of protein kinase C, which in turn modulates cyclic AMP levels, as observed by Knowles and Farndale (1988). Further studies are needed to examine the role that such signals may play in the mode of action of the B. thuringiensis protein.

We are indebted to S. Sohi, from the Canadian Forest Service, Natural Resources Canada, Sault Sainte Marie, Ontario, Canada, for providing the Cf1 cells. The authors wish to thank M. Beauchemin and A. Mazza, from the Biotechnology Research Institute, National Research Council, Montreal, for expert technical assistance, G. Guihard, R. Brousseau and L. Masson, from the Biotechnology Research Institute, National Research Council, Montreal, V. Vachon, from the Groupe de recherche en transport membranaire, Université de Montréal, and D. Baines, from the Canadian Forest Service, Natural Resources Canada, Sault Sainte Marie, Ontario, Canada, for stimulating suggestions and critical reading of the manuscript. This work was supported in part by Strategic Research Grant STR0167557 from the Natural Sciences and Engineering Research Council of Canada to J.L.S. and R.L. NRCC publication no. 4048

Butko
,
P.
,
Cournoyer
,
M.
,
Pusztai-Carey
,
M.
and
Surewicz
,
W. K.
(
1994
).
Membrane interactions and surface hydrophobicity of Bacillus thuringiensis δ-endotoxin CryIC
.
FEBS Lett
.
340
,
89
92
.
Cannon
,
R. J. C.
(
1996
).
Bacillus thuringiensis use in agriculture: a molecular perspective
.
Biol. Rev
.
71
,
561
636
.
Carroll
,
J.
and
Ellar
,
D. J.
(
1993
).
An analysis of Bacillus thuringiensis δ-endotoxin action on insect-midgut-membrane permeability using a light-scattering assay
.
Eur. J. Biochem
.
214
,
771
778
.
Chiou
,
C. Y.
and
Malagodi
,
M. H.
(
1975
).
Studies of the mechanism of action of a new Ca2+ antagonist, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride in smooth and skeletal muscles
.
Br. J. Pharmac
.
53
,
279
285
.
Dent
,
D. R.
(
1993
).
The use of Bacillus thuringiensis as an insecticide
.
In Exploitation of Microorganisms
(ed.
D. G.
Jones
), pp.
19
44
.
London
:
Chapman & Hall
.
English
,
L. H.
,
Readdy
,
T. L.
and
Bastian
,
A. E.
(
1991
).
Delta-endotoxin-induced leakage of 86Rb+-K+ and H2O from phospholipid vesicles is catalyzed by reconstituted midgut membrane
.
Insect Biochem
.
21
,
177
184
.
Federici
,
B. A.
(
1995
).
The future of microbial insecticides as vector control agents
.
J. am. Mosquito Contr. Ass
.
11
,
260
268
.
Gill
,
S. S.
,
Cowles
,
E. A.
and
Pietrantonio
,
P. V.
(
1992
).
The mode of action of Bacillus thuringiensis endotoxins
.
A. Rev. Ent
.
37
,
615
636
.
Gole
,
J. W. D.
,
Orr
,
G. L.
and
Downer
,
R. G. H.
(
1987
).
Forskolin-insensitive adenylate cyclase in cultured cells of Choristoneura fumiferana (Insecta)
.
Biochem. biophys. Res. Commun
.
145
,
1192
1197
.
Grochulski
,
P.
,
Masson
,
L.
,
Borisova
,
S.
,
Pusztai-Carey
,
M.
,
Schwartz
,
J. L.
,
Brousseau
,
R.
and
Cygler
,
M.
(
1995
).
Bacillus thuringiensis CrylA(a) insecticidal toxin: crystal structure and channel formation
.
J. molec. Biol
.
254
,
447
464
.
Grynkiewicz
,
G.
,
Poenie
,
M.
and
Tsien
,
R. Y.
(
1985
).
A new generation of Ca2+ indicators with greatly improved fluorescence properties
.
J. biol. Chem
.
260
,
3440
3450
.
Gupta
,
J.
and
Downer
,
R. G. H.
(
1993
).
Partial characterization of protein kinase C from an insect cell line
.
Biochim. biophys. Acta
1203
,
210
214
.
Haider
,
M. Z.
and
Ellar
,
D. J.
(
1989
).
Mechanism of action of Bacillus thuringiensis insecticidal δ-endotoxin: interaction with phospholipid vesicles
.
Biochim. biophys. Acta
978
,
216
222
.
Harvey
,
W. R.
and
Wolfersberger
,
M. G.
(
1979
).
Mechanism of inhibition of active potassium transport in isolated midgut of Manduca sexta by Bacillus thuringiensis endotoxin
.
J. exp. Biol
.
83
,
293
304
.
Höfte
,
H.
and
Whiteley
,
H. R.
(
1989
).
Insecticidal crystal proteins of Bacillus thuringiensis
.
Microbiol. Rev
.
53
,
242
255
.
Hendrickx
,
K.
,
De Loof
,
A.
and
Van Mellaert
,
H.
(
1990
).
Effects of Bacillus thuringiensis δ-endotoxin on the permeability of brush border membrane vesicles from tobacco hornworm (Manduca sexta) midgut
.
Comp. Biochem. Physiol
.
95C
,
241
245
.
Hu
,
Y.
,
Rajan
,
L.
and
Schilling
,
W. P.
(
1994a
).
Ca2+ signaling in Sf9 insect cells and the functional expression of a rat brain M5 muscarinic receptor
.
Am. J. Physiol
.
266
,
C1736
C1743
.
Hu
,
Y.
,
Vaca
,
L.
,
Zhu
,
X.
,
Birnbaumer
,
L.
,
Kunze
,
D. L.
and
Schilling
,
W. P.
(
1994b
).
Appearance of a novel Ca2+ influx pathway in Sf9 insect cells following expression of the transient receptor potential-like (trpl) protein of Drosophila
.
Biochem. biophys. Res. Commun
.
201
,
1050
1056
.
Johnson
,
D. E.
(
1994
).
Cellular toxicities and membrane binding characteristics of insecticidal crystal proteins from Bacillus thuringiensis toward cultured insect cells
.
J. Invert. Pathol
.
63
,
123
129
.
Knowles
,
B. H.
(
1994
).
Mechanism of action of Bacillus thuringiensis insecticidal δ-endotoxins
.
Adv. Insect Physiol
.
24
,
275
308
.
Knowles
,
B. H.
and
Ellar
,
D. J.
(
1986
).
Characterization and partial purification of a plasma membrane receptor for Bacillus thuringiensis var. kurstaki lepidopteran-specific δ-endotoxin
.
J. Cell Sci
.
83
,
89
101
.
Knowles
,
B. H.
and
Farndale
,
R. W.
(
1988
).
Activation of insect cell adenylate cyclase by Bacillus thuringiensis δ-endotoxins and melittin
.
Biochem. J
.
253
,
235
241
.
Li
,
J.
,
Carroll
,
J.
and
Ellar
,
D. J.
(
1991
).
Crystal structure of insecticidal δ-endotoxin from Bacillus thuringiensis at 2 å resolution
.
Nature
353
,
815
821
.
Liebig
,
B.
,
Stetson
,
D. L.
and
Dean
,
D. H.
(
1995
).
Quantification of the effect of Bacillus thuringiensis toxins on short-circuit current in the midgut of Bombyx mori
.
J. Insect Physiol
.
41
,
17
22
.
Lorence
,
A.
,
Darszon
,
A.
,
Diaz
,
C.
,
Lievano
,
A.
,
Quintero
,
R.
and
Bravo
,
A.
(
1995
).
δ-Endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers
.
FEBS Lett
.
360
,
217
222
.
Martin
,
F. G.
and
Wolfersberger
,
M. G.
(
1995
).
Bacillus thuringiensis δ-endotoxin and larval Manduca sexta midgut brush-border membrane vesicles act synergistically to cause very large increases in the conductance of planar lipid bilayers
.
J. exp. Biol
.
198
,
91
96
.
Masson
,
L.
,
Préfontaine
,
G.
,
Péloquin
,
L.
,
Lau
,
P. C. K.
and
Brousseau
,
R.
(
1989
).
Comparative analysis of the individual protoxin components in P1 crystals of Bacillus thuringiensis subsp. kurstaki isolates NRD-12 and HD-1
.
Biochem. J
.
262
,
507
512
.
Mccarthy
,
W. J.
(
1994a
).
Application of insect cell culture to the study of Bacillus thuringiensis toxins
.
In Insect Cell Biotechnology
(ed.
K.
Maramorosch
and
A.
Mcintosh
), pp.
71
88
.
Boca Raton, FL
:
CRC Press
.
Mccarthy
,
W. J.
(
1994b
).
Cytolytic differences among lepidopteran cell lines exposed to toxins of Bacillus thuringiensis subsp. kurstaki (HD-263) and aizawai (HD-112): effect of aminosugars and N-glycosylation
.
In Vitro cell. devl. Biol
.
30A
,
690
695
.
Monette
,
R.
,
Potvin
,
L.
,
Baines
,
D.
,
Laprade
,
R.
and
Schwartz
,
J. L.
(
1997
).
Interaction between calcium ions and Bacillus thuringiensis toxin activity against Sf9 cells (Spodoptera frugiperda, Lepidoptera)
.
Appl. env. Microbiol
.
63
,
440
447
.
Monette
,
R.
,
Savaria
,
D.
,
Masson
,
L.
,
Brousseau
,
R.
and
Schwartz
,
J. L.
(
1994
).
Calcium-activated potassium channels in the UCR-SE-1a lepidopteran cell line from the beet armyworm (Spodoptera exigua)
.
J. Insect Physiol
.
40
,
273
282
.
Morris
,
O. N.
,
Trottier
,
M.
,
Mclaughlin
,
N. B.
and
Converse
,
V.
(
1994
).
Interaction of caffeine and related compounds with Bacillus thuringiensis ssp. kurstaki in Bertha armyworm (Lepidoptera: Noctuidae)
.
J. econ. Ent
.
87
,
610
617
.
Orr
,
G. L.
,
Gole
,
J. W. D.
,
Gupta
,
J.
and
Downer
,
R. G. H.
(
1988
).
Modulation of octopamine-mediated production of cyclic AMP by phorbol-ester-sensitive protein kinase C in an insect cell line
.
Biochim. biophys. Acta
970
,
324
332
.
Peyronnet
,
O.
,
Vachon
,
V.
,
Brousseau
,
R.
,
Baines
,
D.
,
Schwartz
,
J. L.
and
Laprade
,
R.
(
1997
).
Effect of Bacillus thuringiensis toxins on the membrane potential of lepidopteran insect midgut cells
.
Appl. env. Microbiol
.
63
,
1679
1684
.
Rasmussen
,
H.
and
Barrett
,
P. Q.
(
1984
).
Calcium messenger system: an integrated view
.
Physiol. Rev
.
64
,
938
984
.
Sacchi
,
V. F.
,
Parenti
,
P.
,
Hanozet
,
G. M.
,
Giordana
,
B.
,
Luthy
,
P.
and
Wolfersberger
,
M. G.
(
1986
).
Bacillus thuringiensis toxin inhibits K+-gradient-dependent amino acid transport across the brush border membrane of Pieris brassicae midgut cells
.
FEBS Lett
.
204
,
213
218
.
Schwartz
,
J. L.
,
Garneau
,
L.
,
Masson
,
L.
and
Brousseau
,
R.
(
1991
).
Early response of cultured lepidopteran cells to exposure to δ-endotoxin from Bacillus thuringiensis: involvement of calcium and anionic channels
.
Biochim. biophys. Acta
1065
,
250
260
.
Schwartz
,
J. L.
,
Garneau
,
L.
,
Savaria
,
D.
,
Masson
,
L.
and
Brousseau
,
R.
(
1993
).
Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers
.
J. Membr. Biol
.
132
,
53
62
.
Schwartz
,
J. L.
,
Lu
,
Y.-J.
,
Söhnlein
,
P.
,
Brousseau
R.
,
Laprade
R.
,
Masson
,
L.
and
Adang
,
M. J.
(
1997
).
Ion channels formed in planar lipid bilayers by Bacillus thuringiensis toxins in the presence of Manduca sexta midgut receptors
.
FEBS Lett
.
412
,
270
276
.
Shibuya
,
I.
and
Douglas
,
W. W.
(
1992
).
Calcium channels in rat melanotrophs are permeable to manganese, cobalt, cadmium and lanthanum, but not to nickel: evidence provided by fluorescence changes in Fura-2-loaded cells
.
Endocrinology
131
,
1936
1941
.
Slatin
,
S. L.
,
Abrams
,
C. K.
and
English
,
L.
(
1990
).
Delta-endotoxins form cation-selective channels in planar lipid bilayers
.
Biochem. biophys. Res. Commun
.
169
,
765
772
.
Tabashnik
,
B. E.
(
1994
).
Evolution of resistance to Bacillus thuringiensis
.
A. Rev. Ent
.
39
,
47
79
.
Thastrup
,
O.
,
Cullen
,
P. J.
,
Drøbak
,
B. K.
,
Hanley
,
M. R.
and
Dawson
,
A. P.
(
1990
).
Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase
.
Proc. natn. Acad. Sci. U.S.A
.
87
,
2466
2470
.
Triggle
,
D. J.
(
1990
).
Calcium, calcium channels and calcium channel antagonists
.
Can. J. Physiol. Pharmac
.
68
,
1474
1481
.
Tsien
,
R. W.
(
1990
).
Calcium channels, stores and oscillations
.
A. Rev. Cell Biol
.
6
,
715
760
.
Uemura
,
T.
,
Ihara
,
H.
,
Wadano
,
A.
and
Himeno
,
M.
(
1992
).
Fluorometric assay of potential change of Bombyx mori midgut brush border membrane induced by δ-endotoxin from Bacillus thuringiensis
.
Biosci. Biotech. Biochem
.
56
,
1976
1979
.
Vachon
,
V.
,
Paradis
,
M. J.
,
Marsolais
,
M.
,
Schwartz
,
J. L.
and
Laprade
,
R.
(
1995a
).
Endogenous K+/H+ exchange activity in the Sf9 insect cell line
.
Biochemistry
34
,
15157
15164
.
Vachon
,
V.
,
Paradis
,
M. J.
,
Marsolais
,
M.
,
Schwartz
,
J. L.
and
Laprade
,
R.
(
1995b
).
Ionic permeabilities induced by Bacillus thuringiensis in Sf9 cells
.
J. Membr. Biol
.
148
,
57
63
.
Van Frankenhuyzen
,
K.
,
Gringorten
,
J. L.
,
Gauthier
,
D.
,
Milne
,
R. E.
,
Masson
,
L.
and
Peferoen
,
M.
(
1993
).
Toxicity of activated CryI proteins from Bacillus thuringiensis to six forest Lepidoptera and Bombyx mori
.
J. Invert. Path
.
62
,
295
301
.
Van Frankenhuyzen
,
K.
,
Gringorten
,
J. L.
,
Milne
,
R. E.
,
Gauthier
,
D.
,
Pusztai-Carey
,
M.
,
Brousseau
,
R.
and
Masson
,
L.
(
1991
).
Specificity of activated CryIA proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for defoliating forest Lepidoptera
.
Appl. env. Microbiol
.
57
,
1650
1655
.
Villalon
,
M.
,
Vachon
,
V.
,
Brousseau
,
R.
,
Schwartz
,
J. L.
and
Laprade
,
R.
(
1997
).
Video imaging analysis of the plasma membrane permeabilizing effects of Bacillus thuringiensis insecticidal toxins in Sf9 cells
.
Biochim. biophys. Acta
1368
,
27
34
.
Von Tersche
,
M. A.
,
Slatin
,
S. L.
,
Kulesza
,
C. A.
and
English
,
L. H.
(
1994
).
Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide
.
Appl. env. Microbiol
.
60
,
3711
3717
.
Wolfersberger
,
M. G.
(
1989
).
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
.
Archs Insect Biochem. Physiol
.
12
,
267
277
.
Yunovitz
,
H.
and
Yawetz
,
A.
(
1988
).
Interaction between the δ-endotoxin produced by Bacillus thuringiensis ssp. entomocidus and liposomes
.
Eur. J. Biochem
.
230
,
105
108
.