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Different strains of Bacillus thuringiensis produce insecticidal crystal proteins (ICPs) that are highly and specifically toxic for some insect species. The mode of action can be summarized as follows. Susceptible larvae ingest ICPs, which are converted to active toxins by solubilization and proteolytic processing in the insect midgut. The "activated" ICPs bind to specific receptors in the apical membrane of the midgut cells, and they produce pores, which leads to colloid osmotic lysis of the cells and insect death (4). Brush border membrane vesicles (BBMVs) from the midguts of target insects have been used in an assay to monitor osmotic swelling (1, 9) based on changes in scattered-light intensity (SLI). A good relationship has been found between ICP toxicity and the ability to make BBMVs leaky. In a hyperosmotic medium, the vesicles initially shrink. Subsequently, if the substances in the medium can cross the membrane, water follows and the vesicles swell. Swelling decreases the SLI.

The present work was carried out with the Egyptian cotton leafworm (Spodoptera littoralis Boisduval), an important agricultural pest. The relationship between vesicle leakiness and ICP toxicity in vivo was studied with three marginally active ICPs (Cry1Aa, Cry1Ab, and Cry1Ac) and a much more active ICP (Cry1Ca). The 50% lethal concentration of Cry1Ca is at least 10 times lower than that of Cry1A proteins (2a, 8).

The data reported below are means ± standard errors of the means, and n indicates the number of determinations. Values were compared by using a one-way analysis of variance test with Dunnett posttests. Differences were considered statistically significant if P < 0.05. Calculations were performed with the Prism computer program (GraphPad Software, Inc.).

Permeability characteristics of BBMVs from S. littoralis. The assay used was adapted from the technique developed by Carroll and Ellar (1). BBMVs prepared (10) from guts of last-instar S. littoralis larvae were suspended in BBMV buffer (17 mM Tris-HCl, pH 7.5) at a concentration of 0.6 mg of protein/ml and incubated overnight at 4°C. A 4-ml quartz cuvette containing a 3-ml sample of BBMVs was placed in the sample compartment of a luminescence spectrophotometer (model LS-5B; Perkin-Elmer Co.) at room temperature. The intensity of 450-nm light 90° from a 450-nm incident beam was recorded. Stock solutions (2.5 M) of sucrose, KCl, potassium gluconate, and NaCl were prepared in BBMV buffer. Hyperosmotic conditions were generated by adding 0.1 ml of a concentrated stock solution. BBMVs from S. littoralis were incubated with different solutes (final concentration, 80 mM) in order to determine which solute produced the greatest initial shrinking (i.e., the greatest increase in the SLI).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Time course of SLI. S. littoralis midgut BBMVs were shrunken by exposure to a hyperosmotic KCl solution (data not shown). A subsequent decrease (from time zero to 25 min) in SLI was observed due to KCl (and water) that leaked into the vesicles. The values shown are means ± standard errors of the means (n = 3). (A) Swelling of the vesicles monitored in the absence () or in the presence of Ba2+ () or nystatin (). Incubation of BBMVs with nystatin in the absence of a hyperosmotic KCl solution was used as a control (). (B) Swelling accelerated in the presence of Cry1Ca (0.9 nmol/mg of BBMV protein) (), but not in the presence of Cry1Aa (), Cry1Ab (), or Cry1Ac (). The Cry1Ab and Cry1Ac curves coincide with the relative SLI in the presence of KCl alone (see panel A []).

ICP effects. Activated ICPs (Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ca) were prepared as described previously (8), diluted in 20 mM Tris-HCl-150 mM NaCl (pH 8.6) (ICP buffer), and added 2 min after exposure of the BBMVs to a hypertonic KCl solution. Figure 1B shows the relative SLI of samples in the presence of the ICPs. Adding ICP buffer alone (n = 3) had no effect (data not shown). The results revealed a direct relationship with toxicity. Thus, the marginally active ICPs Cry1Aa (n = 4), Cry1Ab (n = 3), and Cry1Ac (n = 3) at a concentration of 35 µg/ml (0.9 nmol of ICP/mg of BBMV protein) did not seem to accelerate swelling. In contrast addition of Cry1Aa resulted in an increase in the SLI. Only Cry1Ca (n = 3) significantly accelerated the decrease in SLI compared with exposure to hypertonic KCl alone, suggesting that there was an increase in K⁺ permeability. The effects were slower than those of nystatin; at least 5 min of incubation with Cry1Ca was necessary before a significant difference could be detected. A 10-fold-lower concentration of Cry1Ca (0.09 nmol of ICP/mg of BBMV protein; n = 3) had no effect compared to the control (data not shown). The results obtained suggest that at the concentrations tested, Cry1Ca, but not Cry1Aa, Cry1Ab, or Cry1Ac, made S. littoralis BBMVs permeable to KCl. We cannot eliminate the possibility, however, that (much) higher doses of Cry1A may result in pore formation. These findings support the hypothesis that toxic ICPs increase membrane permeability (4). The protection from KCl entry by Cry1Aa might be due to some interference of this protein with K⁺ or Cl channels. The slower effects of Cry1Ca compared to the effects of nystatin could indicate that the mode of action of this ICP is more complex. Fast effects of Cry1Ac (1) and Cry1Ca (6) on M. sexta have been reported, but there may be differences between ICP-insect interactions.