Solubilized crystals of Bacillus thuringiensis var. israelensis were 7,000 times less toxic to Aedes aegypti larvae than intact crystals, presumably because mosquito larvae are filter feeders and selectively concentrate particles while excluding water and soluble molecules. A procedure is described whereby soluble toxins are adsorbed to 0.8- micrometer latex beads, with retention of toxicity. The latex bead assay should make it possible to analyze the structure and mode of action of the mosquito toxin

Nickerson, Kenneth W.

Pfannenstiel, Mary Ann

Schnell, Danny J.

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Microbiology Papers in the Biological Sciences March 1984 Bioassay of Solubilized Bacillus thuringiensis var. israelensis Crystals by Attachment to Latex Beads Danny J. Schnell University of Nebraska-Lincoln Mary Ann Pfannenstiel University of Nebraska-Lincoln Kenneth W. Nickerson University of Nebraska-Lincoln, knickerson1@unl.edu Follow this and additional works at: https://digitalcommons.unl.edu/bioscimicro  Part of the Microbiology Commons Schnell, Danny J.; Pfannenstiel, Mary Ann; and Nickerson, Kenneth W., "Bioassay of Solubilized Bacillus thuringiensis var. israelensis Crystals by Attachment to Latex Beads" (1984). Papers in Microbiology. 75. https://digitalcommons.unl.edu/bioscimicro/75 This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Microbiology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Control of several human diseas-es is based on controlling their mosquito or black fl y vectors. Mosquitoes transmit such devastating diseases as malaria, en-cephalitis, yellow fever, dengue, and fi la-riasis, while black fl ies transmit onchocer-ciasis. Biological control of these disease vectors has been made possible by the re-cent discovery of Bacillus thuringiensis var. israelensis (Bti). This bacterium pro-duces a protein crystal during sporulation that is toxic to the larval stage of many mosquitoes and black fl ies. Purifi ed par-ticulate crystals are toxic to larvae of the mosquito Aedes aegypti at concentrations as low as 1 ng/ml (1). However, it has not been possible to determine the mode of ac-tion of the Bti toxin and the subunit of the multicomponent crystal (1, 2) containing the toxin. Larval toxicity disappears after the crystal is solubilized, probably because mosquito larvae, as fi lter feeders, selective-ly concentrate particles 0.5 to 10 μm in di-ameter while excluding water and soluble molecules (3). We report here a procedure whereby potentially toxic soluble proteins are ad-sorbed to latex beads with retention of tox-icity. The soluble toxin gains access to the larval gut because the particles to which it is attached are of the appropriate size to be concentrated during fi lter feeding. This procedure should prove effective for use with other soluble, gut-active compounds toxic to fi lter-feeding mosquito and black fl y larvae and to netmaking caddis fl ies, midges, and burrowing mayfl ies. We confi rmed that solubilized Bti pro-teins are still intrinsically toxic to mosquito larvae by adsorbing them to 0.8- μm latexbeads (Table 1) . The procedure was adapt-ed from that described for the attachment of antibodies to latex beads (4). Bioassays were performed with second- to third-in-star larvae of A. aegypti (5). The bead con-centration was kept constant while the tox-in concentration was varied through six or more dilutions covering the range from 100 to 0 percent mortality. Microscopic exami-nation revealed larval guts gorged with in-gested beads. The presence of excess beads minimized the effect of slight variations in larval number and size. Values of median lethal concentration (LC50) were calculated after 4 hours instead of the more standard 24 hours because 4 hours approximates the larval transit time for nonnutritive particles in Culex pipiens (6) and because we want-ed to avoid recycling of the beads. Val-ues reported were calculated from three or more independent bioassays. Uncoatedbeads were not toxic to the larvae for at least 48 hours. We confi rmed that the solubilized Bti toxin was adsorbed by centrifuging the beads and washing them three times with 0.1M NaPO4 buffer (pH 7.4) containing 0.01 percent polyvinylpyrrolidone. Larval toxicity remained constant even after re-peated washings. The amount of toxin at-tached to the beads was determined by la-beling the proteins with 125I and measuring bead-adsorbed radioactivity after the attach-ment and centrifugation procedures (Figure 1). The percentage of bead-attached proteins decreased from 80 to 45 percent as the pro-tein concentration was increased from 2 to 37 μg/ml. However, there was no preferen-tial attachment of individual proteins to the beads. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) pro-fi les of the solubilized proteins before and after attachment were virtually identical. Further bioassays in the absence of beads showed that solubilized Bti crystals were about 7,000 times less toxic than in-tact crystals (Table 1). When the solubilized proteins were adsorbed to latex beads of the appropriate size, the LC50 was reduced from 53,000 to 190 ng/ml, a 280-fold increase in toxicity. However, the LC50 for bead-at-tached toxin was still 25 times higher than that for intact crystals. This difference may refl ect a decrease in toxicity on solubiliza-tion or an accumulation of physical factors due to the presence of the beads. Three pos-sible physical factors are (i) decreased tox-in ingestion due to the gut volume occupied by the beads and the longer transit time (6) caused by their nonnutritive status [merely mixing intact crystals with latex beads dou-bled their LC50 (Table 1)], (ii) decreased toxin ingestion by the bottom-feeding Published in Science, New Series, Vol. 223, No. 4641. (Mar. 16, 1984), pp. 1191–1193. Published by the American Association for the Advancement of Science. Used by permission.Submitted October 14, 1983; accepted December 28, 1983Bioassay of Solubilized Bacillus thuringiensis var. israelensis Crystals by Attachment to Latex BeadsDanny J . Schnell*, Mary Ann Pfannenstiel, and Kenneth W. NickersonSchool of Biological Sciences, University qf Nebraska–Lincoln, Lincoln NE 68588* Present address: Department of Biochemistry and Biophysics, University of California, Davis 95616 Abstract: Solubilized crystals of Bacillus thuringiensis var. israelensis were 7,000 times less toxic to Aedes aegypti larvae than intact crystals, presumably because mosquito larvae are fi lter feeders and selectively concentrate particles while excluding water and soluble molecules. A procedure is described whereby soluble toxins are adsorbed to 0.8-micrometer latex beads, with retention of toxicity. The latex bead assay should make it possible to analyze the structure and mode of action of the mosquito toxin. 1191Table 1. Toxicity of soluble and crystalline Bti toxin to Aedes aegypti larvae. Crystals were pre-pared as described by Ang and Nickerson (9). The crystals (2 mg/ml) were solubilized overnight at 4°C in 0.05M NaOH (pH 11.7) containing 10 mM EDTA and were then centrifuged at 300,000 g for 30 minutes (10).The solubilized supernatant proteins were adjusted to 25 times the desired bioassay concentration in 1 ml of 0.1M tris buffer (pH 7.4) and mixed with 10 μl of latex beads (0.8 μm, 10 percent solids; Sigma). After incubation at room temperature for 1 hour the beads were centrifuged at 15,000 g for 10 minutes and the pellet was resuspended in 1 ml of tris containing 0.01 percent polyvinylpyrrolidone (11). Test solutions were diluted with 24 ml of deionized water for the bio-assays. At least six dilutions of each toxin preparation were used; whenever latex beads were em-ployed their fi nal concentrations were identical. Values of LC50 were determined 4 hours after the addition of 10 to 20 larvae and are presented as means ± 2 standard errors. All the experiments were conducted with crystal toxin from a single purifi cation (12). The LC50 values for crystals remained constant over at least a 10-month period. 1192 SCHNELL, PFANNENSTIEL, & NICKERSON IN SCIENCE 223 (1984)A. aegypti larvae because the latex beads re-main suspended during the 4-hour assay pe-riod while intact crystals settle to the bot-tom, and (iii) failure of the ingested toxin to be released from the beads. These factors in combination could account for most of the 25-fold difference in LC50 values between intact crystals and bead-adsorbed proteins. To determine the infl uence of these factors, we measured the levels of ingest-ed, iodinated crystal proteins necessary to kill individual larvae (Table 2). Lar-vae were fed intact crystals, a mixture of beads and intact crystals, or bead-adsorbed solubilized proteins. After 4 hours the lar-vae were removed and the amount of la-beled proteins in the guts of dead and live larvae was determined. Table 2 lists the amounts of toxin found in dead larvae that had been fed the three toxin preparations at concentrations approximating their re-spective LC50 values. We chose these con-centrations because the amount of crystal proteins present in dead larvae decreased with decreasing external toxin concentra-tion until leveling off near the LC50 values. At high toxin concentrations all the larvae were dead within 4 hours, whereas at con-centrations near the LC50 values both live and dead larvae were observed in the same assay vessel. We draw three conclusions from the data in Table 2: 1) The presence of latex beads in-creased the lethal dosage for intact crystals about 2.4 times. 2) In the presence of an equivalent con-centration of beads, solubilized Bti protein required only about 2.6 times more toxin than did intact crystals. Although the cause of this difference remains unknown, it is much less than the 25-fold difference in external LC50 values seen in Table 1. 3) Larval mortality was a threshold phenomenon. Discrete threshold levels ofcrystal protein were necessary to kill indi-vidual A. aegypti larvae. When exposed to bead-attached solubilized protein, larvae that eventually died ingested 16.8 ng of protein at high Bti concentrations and 5.0 ng at concentrations nearer the LC50 val-ue. In contrast, larvae that survived always contained ≤ 4.6 ng protein. Thus it appears that the solubilized Bti proteins retain their toxicity if they can gain access to the larval gut and that at-tachment to latex beads of an appropriate size permits such access. We now address some of the implications that this bioassay has for research on mosquito abatement. As mentioned earlier, the solubilized proteins were 7,000 times less toxic than the intact crystals. Previous estimates of the relative ability of water and particles to displace visible gut contents, such as acti-vated charcoal, had put this factor at only 50 for C. pipiens larvae (3). Assurance that solubilized crystals are still intrinsically toxic should allow more accurate measure-ment of the larval fi lter-feeding factor in all insect species sensitive to Bti. Analysis of the Bti crystal by SDS-PAGE indicates that it consists of a sin-gle band at 28 kilodaltons (kD); a trip-let at 38, 39, and 40 kD; a single band at 53 kD; a doublet at 68 and 70 kD; and a doublet at 135 and 140 kD, with the per-centage of each component varying among strains and batches. Availability of the la-tex bead bioassay will allow the separat-ed protein chains to be bioassayed individ-ually and in combination. Once the identity of the toxic protein is known, 125I-labeled toxin can be used to quantify the threshold level of toxin needed to kill a larva at each instar stage. We found that our standard solubiliza-tion condition, 50 mM NaOH (pH 11.7) with 10 mM EDTA, provided only variable and incomplete solubilization of the 135- and 140-kD protein bands. These bands could be completely solubilized by includ-ing 0.5M dithioerythritol, but this was not accompanied by enhanced larval toxicity. In agreement with others (2), we conclude that it is unlikely that the high molecular weight components of the crystal contrib-ute to larval toxicity (7). We recently showed (8) that the toxici-ty of intact Bti crystals to A. aegypti larvae could be reversed up to 100-fold by lev-els of K2CO3 as low as 0.15 percent. At the time there was no way to determine wheth-er this reversal was directly related to the crystal’s mode of action or was merely pre-venting solubilization in the larva by low-ering the gut pH below a critical value. However, the fact that K2CO3 achieved a similar toxicity reversal with bead-attached solubilized proteins indicates that the car-bonate does not act by preventing eventual crystal solubilization. Long-term control of mosquitoes with Bti is currently impractical because Bti does not persist in nature more than 1 or 2 days. Apparently the toxin settles into the mud or is consumed by other microorgan-isms. Attachment of the toxin to nonnutri-tive particles with a greater tendency to re-main dispersed in water should enhance its persistence. Figure 1. Attachment of 125I-labeled Bti pro-teins to latex beads. Solubilized Bti protein was iodinated in vitro (13) to a specifi c activity of 4.9 × 107 count/min-μg. Unincorporated 125I was removed by chromatography on a Bio-Gel P6 column. The iodinated proteins were ad-sorbed to latex beads as described in the legend to Table 1 and the amount attached at each pro-tein concentration was determined after centrif-ugation by measuring the radioactivity associ-ated with the beads on a Beckman 5500 gamma counter. Assessment of bead-adsorbed proteins by dye binding (14) would have been inappro-priate because this method  indicates protein concentrations only 20 percent as high as those of three other methods. This difference is probably due to the fact that solubilized Bti proteins have a tendency to precipitate at acidic pH values. Each point is the mean ± standard error for four deter-minations. The effi ciency of attachment remained constant for live toxin purifi cations from three different Bti strains over a 10-month period. Table 2. Amounts of Bti proteins ingested by dead Aedes aegypti larvae. Solubilized proteins were iodinated as described in the legend to Fig. 1. Intact crystals were labeled in vitro with Na125I (13). After iodination the crystals were dialyzed for 24 hours against 0.1M NaPO4 buffer (pH 7.4) to remove unincorporated 125I. A specifi c acticity of 5.0 × 107 count/min-μg was obtained for the intact crystals. The LC50 value for labeled intact crystals was identical with that of the unlabeled crystals. The labeled toxin was diluted with unlabeled toxin to concentrations approximating their respective LC50 values (Table 1) and fed to larvae for 4 hours. The guts were dissected from dead larvae, which had been washed in 0.5 percent NaCl containing 0.1 percent SDS, and radioactivity was measured with a Reckman 5500 gamma counter. Data are means ± standard errors.BIOASSAY OF SOLUBILIZED BTI CRYSTALS BY ATTACHMENT TO LATEX BEADS 1193References and Notes 1. D. J. Tyrell. L. I. Davidson, L. A. Bulla, W. A. Ramoska, Appl. Environ. Micro-biol. 38, 656 (1979): D. J. Tyrell et al., J. Bacteriol. 145, 1052 (1981). 2. W. E. Thomas and D. J. Ellar, J . Cell Sci. 60, 181 (1983). 3. R. H. Dadd, J. Exp. Zool. 191, 395 (1975); Ann. Entomol. Soc. Am. 64, 687 (1971). 4. H. S. Abu Salih, A. F. Murant, M. J. Daft, J. Gen. Virol. 3, 299 (1968). 5. K. W. Nickerson and D. J. Schnell, J. In-vertebr. Pathol. 42, 407 (1983). 6. R. H. Dadd, Entomol. Exp. Appl. 13, 407 (1970). 7. Dithioerythritol would interfere with io-dination (13), and some sulfhydryl re-agents exhibit larval toxicity in their own right [V. C. Kramer, D. J. Schnell, K. W. Nickerson, J. Invertebr. Pathol. 42, 285 (1983)]. Therefore we did not include dithioerythritol in our standard crystal solubilization conditions. 8. D. J. Schnell and K. W. Nickerson, Appl. Environ. Microbiol. 45, 1691 (1983). 9. B. J. Ang and K. W. Nickerson, ibid. 36, 625 (1978). 10. M. A. Pfannenstiel, E. J. Ross, V. C. Kramer, K. W. Nickerson, Fed. Eur. Microbiol. Soc. Microbiol. Lett. 21, 39 (1984) found that this procedure solubilized 80 percent of the crys-tal proteins, minimized proteolyt-ic degradation (SDS-PAGE analysis gave gel profi les identical to those of intact crystals), and maximized the general cytolytic activity (2) ex-hibited by solubilized but not intact Bti crvstals toward human erythro-cytes. Considering the multicompo-nent nature of the Bti crvstal, there is no guarantee that the mosquito larval toxin and the general cytolytic factor are identical. Also, since the crystal is incompletely solubilized and the pellet is still highly toxic to mosqui-to larvae, centrifugation at 100,000 g is necessary to defi ne the solubilized toxin. Insuffi cient centrifugation probably explains the erroneous con-clusion (1) that solubilized Bti crys-tals have LC50 values similar to those of whole crystals. 11. The LC50 values for solubilized pro-teins adsorbed to latex beads were identical in the presence and absence of the polyvinylpyrrolidone coating. The toxin-coated beads do not need to be purifi ed before routine bioassays because the unattached solubilized proteins contribute negligibly to over-all toxicity. 12. We have found that, when individu-al batches of purifi ed Bti crystals are bioassayed for 4 hours against A. ae-gypti larvae, the LC50 ranges from 5 to 10 ng/ml. Spontaneous loss of toxici-ty in Bti is common [H. Kamdar and K. Jayaraman, Biochem. Biophys. Res. Commun. 110, 477 (1983)]. 13. M. A. K. Markwell and C. F. Fox, Bio-chemistry 17, 4807 (1978). 14. M. Bradford, Anal. Biochem. 72, 248 (1976). 15. K.W.N. is an NIH research career de-velopment awardee (A1 00327-TMP). This research was supported in part by University Genetics Company.   

Bioassay of Solubilized \u3ci\u3eBacillus thuringiensis\u3c/i\u3e var. \u3ci\u3eisraelensis\u3c/i\u3e Crystals by Attachment to Latex Beads

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Papers in Microbiology Papers in the Biological Sciences 
March 1984 
Bioassay of Solubilized Bacillus thuringiensis var. israelensis 
Crystals by Attachment to Latex Beads 
Danny J. Schnell 
University of Nebraska-Lincoln 
Mary Ann Pfannenstiel 
University of Nebraska-Lincoln 
Kenneth W. Nickerson 
University of Nebraska-Lincoln, knickerson1@unl.edu 
Follow this and additional works at: https://digitalcommons.unl.edu/bioscimicro 
 Part of the Microbiology Commons 
Schnell, Danny J.; Pfannenstiel, Mary Ann; and Nickerson, Kenneth W., "Bioassay of Solubilized Bacillus 
thuringiensis var. israelensis Crystals by Attachment to Latex Beads" (1984). Papers in Microbiology. 75. 
https://digitalcommons.unl.edu/bioscimicro/75 
This Article is brought to you for free and open access by the Papers in the Biological Sciences at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Microbiology by an 
authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
Control of several human diseas-
es is based on controlling their mosquito 
or black fl y vectors. Mosquitoes transmit 
such devastating diseases as malaria, en-
cephalitis, yellow fever, dengue, and fi la-
riasis, while black fl ies transmit onchocer-
ciasis. Biological control of these disease 
vectors has been made possible by the re-
cent discovery of Bacillus thuringiensis 
var. israelensis (Bti). This bacterium pro-
duces a protein crystal during sporulation 
that is toxic to the larval stage of many 
mosquitoes and black fl ies. Purifi ed par-
ticulate crystals are toxic to larvae of the 
mosquito Aedes aegypti at concentrations 
as low as 1 ng/ml (1). However, it has not 
been possible to determine the mode of ac-
tion of the Bti toxin and the subunit of the 
multicomponent crystal (1, 2) containing 
the toxin. Larval toxicity disappears after 
the crystal is solubilized, probably because 
mosquito larvae, as fi lter feeders, selective-
ly concentrate particles 0.5 to 10 μm in di-
ameter while excluding water and soluble 
molecules (3). 
We report here a procedure whereby 
potentially toxic soluble proteins are ad-
sorbed to latex beads with retention of tox-
icity. The soluble toxin gains access to the 
larval gut because the particles to which 
it is attached are of the appropriate size to 
be concentrated during fi lter feeding. This 
procedure should prove effective for use 
with other soluble, gut-active compounds 
toxic to fi lter-feeding mosquito and black 
fl y larvae and to netmaking caddis fl ies, 
midges, and burrowing mayfl ies. 
We confi rmed that solubilized Bti pro-
teins are still intrinsically toxic to mosquito 
larvae by adsorbing them to 0.8- μm latex
beads (Table 1) . The procedure was adapt-
ed from that described for the attachment 
of antibodies to latex beads (4). Bioassays 
were performed with second- to third-in-
star larvae of A. aegypti (5). The bead con-
centration was kept constant while the tox-
in concentration was varied through six or 
more dilutions covering the range from 100 
to 0 percent mortality. Microscopic exami-
nation revealed larval guts gorged with in-
gested beads. The presence of excess beads 
minimized the effect of slight variations in 
larval number and size. Values of median 
lethal concentration (LC50) were calculated 
after 4 hours instead of the more standard 
24 hours because 4 hours approximates the 
larval transit time for nonnutritive particles 
in Culex pipiens (6) and because we want-
ed to avoid recycling of the beads. Val-
ues reported were calculated from three 
or more independent bioassays. Uncoated
beads were not toxic to the larvae for at 
least 48 hours. 
We confi rmed that the solubilized Bti 
toxin was adsorbed by centrifuging the 
beads and washing them three times with 
0.1M NaPO4 buffer (pH 7.4) containing 
0.01 percent polyvinylpyrrolidone. Larval 
toxicity remained constant even after re-
peated washings. The amount of toxin at-
tached to the beads was determined by la-
beling the proteins with 125I and measuring 
bead-adsorbed radioactivity after the attach-
ment and centrifugation procedures (Figure 
1). The percentage of bead-attached proteins 
decreased from 80 to 45 percent as the pro-
tein concentration was increased from 2 to 
37 μg/ml. However, there was no preferen-
tial attachment of individual proteins to the 
beads. Sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) pro-
fi les of the solubilized proteins before and 
after attachment were virtually identical. 
Further bioassays in the absence of 
beads showed that solubilized Bti crystals 
were about 7,000 times less toxic than in-
tact crystals (Table 1). When the solubilized 
proteins were adsorbed to latex beads of the 
appropriate size, the LC50 was reduced from 
53,000 to 190 ng/ml, a 280-fold increase 
in toxicity. However, the LC50 for bead-at-
tached toxin was still 25 times higher than 
that for intact crystals. This difference may 
refl ect a decrease in toxicity on solubiliza-
tion or an accumulation of physical factors 
due to the presence of the beads. Three pos-
sible physical factors are (i) decreased tox-
in ingestion due to the gut volume occupied 
by the beads and the longer transit time (6) 
caused by their nonnutritive status [merely 
mixing intact crystals with latex beads dou-
bled their LC50 (Table 1)], (ii) decreased 
toxin ingestion by the bottom-feeding 
Published in Science, New Series, Vol. 223, No. 4641. (Mar. 16, 1984), pp. 1191–1193. Published by 
the American Association for the Advancement of Science. Used by permission.
Submitted October 14, 1983; accepted December 28, 1983
Bioassay of Solubilized Bacillus thuringiensis var. 
israelensis Crystals by Attachment to Latex Beads
Danny J . Schnell*, Mary Ann Pfannenstiel, and Kenneth W. Nickerson
School of Biological Sciences, University qf Nebraska–Lincoln, Lincoln NE 68588
* Present address: Department of Biochemistry and Biophysics, University of California, Davis 95616 
Abstract: Solubilized crystals of Bacillus thuringiensis var. israelensis were 7,000 times 
less toxic to Aedes aegypti larvae than intact crystals, presumably because mosquito 
larvae are fi lter feeders and selectively concentrate particles while excluding water and 
soluble molecules. A procedure is described whereby soluble toxins are adsorbed to 0.8-
micrometer latex beads, with retention of toxicity. The latex bead assay should make it 
possible to analyze the structure and mode of action of the mosquito toxin.
 1191
Table 1. Toxicity of soluble and crystalline Bti toxin to Aedes aegypti larvae. Crystals were pre-
pared as described by Ang and Nickerson (9). The crystals (2 mg/ml) were solubilized overnight at 
4°C in 0.05M NaOH (pH 11.7) containing 10 mM EDTA and were then centrifuged at 300,000 g for 
30 minutes (10).The solubilized supernatant proteins were adjusted to 25 times the desired bioassay 
concentration in 1 ml of 0.1M tris buffer (pH 7.4) and mixed with 10 μl of latex beads (0.8 μm, 10 
percent solids; Sigma). After incubation at room temperature for 1 hour the beads were centrifuged 
at 15,000 g for 10 minutes and the pellet was resuspended in 1 ml of tris containing 0.01 percent 
polyvinylpyrrolidone (11). Test solutions were diluted with 24 ml of deionized water for the bio-
assays. At least six dilutions of each toxin preparation were used; whenever latex beads were em-
ployed their fi nal concentrations were identical. Values of LC50 were determined 4 hours after the 
addition of 10 to 20 larvae and are presented as means ± 2 standard errors. All the experiments were 
conducted with crystal toxin from a single purifi cation (12). The LC50 values for crystals remained 
constant over at least a 10-month period. 
1192 SCHNELL, PFANNENSTIEL, & NICKERSON IN SCIENCE 223 (1984)
A. aegypti larvae because the latex beads re-
main suspended during the 4-hour assay pe-
riod while intact crystals settle to the bot-
tom, and (iii) failure of the ingested toxin to 
be released from the beads. These factors in 
combination could account for most of the 
25-fold difference in LC50 values between 
intact crystals and bead-adsorbed proteins. 
To determine the infl uence of these 
factors, we measured the levels of ingest-
ed, iodinated crystal proteins necessary 
to kill individual larvae (Table 2). Lar-
vae were fed intact crystals, a mixture of 
beads and intact crystals, or bead-adsorbed 
solubilized proteins. After 4 hours the lar-
vae were removed and the amount of la-
beled proteins in the guts of dead and live 
larvae was determined. Table 2 lists the 
amounts of toxin found in dead larvae that 
had been fed the three toxin preparations 
at concentrations approximating their re-
spective LC50 values. We chose these con-
centrations because the amount of crystal 
proteins present in dead larvae decreased 
with decreasing external toxin concentra-
tion until leveling off near the LC50 values. 
At high toxin concentrations all the larvae 
were dead within 4 hours, whereas at con-
centrations near the LC50 values both live 
and dead larvae were observed in the same 
assay vessel. We draw three conclusions 
from the data in Table 2: 
1) The presence of latex beads in-
creased the lethal dosage for intact crystals 
about 2.4 times. 
2) In the presence of an equivalent con-
centration of beads, solubilized Bti protein 
required only about 2.6 times more toxin 
than did intact crystals. Although the cause 
of this difference remains unknown, it is 
much less than the 25-fold difference in 
external LC50 values seen in Table 1. 
3) Larval mortality was a threshold 
phenomenon. Discrete threshold levels of
crystal protein were necessary to kill indi-
vidual A. aegypti larvae. When exposed to 
bead-attached solubilized protein, larvae 
that eventually died ingested 16.8 ng of 
protein at high Bti concentrations and 5.0 
ng at concentrations nearer the LC50 val-
ue. In contrast, larvae that survived always 
contained ≤ 4.6 ng protein. 
Thus it appears that the solubilized Bti 
proteins retain their toxicity if they can 
gain access to the larval gut and that at-
tachment to latex beads of an appropriate 
size permits such access. We now address 
some of the implications that this bioassay 
has for research on mosquito abatement. 
As mentioned earlier, the solubilized 
proteins were 7,000 times less toxic than 
the intact crystals. Previous estimates of 
the relative ability of water and particles to 
displace visible gut contents, such as acti-
vated charcoal, had put this factor at only 
50 for C. pipiens larvae (3). Assurance that 
solubilized crystals are still intrinsically 
toxic should allow more accurate measure-
ment of the larval fi lter-feeding factor in all 
insect species sensitive to Bti. 
Analysis of the Bti crystal by SDS-
PAGE indicates that it consists of a sin-
gle band at 28 kilodaltons (kD); a trip-
let at 38, 39, and 40 kD; a single band at 
53 kD; a doublet at 68 and 70 kD; and a 
doublet at 135 and 140 kD, with the per-
centage of each component varying among 
strains and batches. Availability of the la-
tex bead bioassay will allow the separat-
ed protein chains to be bioassayed individ-
ually and in combination. Once the identity 
of the toxic protein is known, 125I-labeled 
toxin can be used to quantify the threshold 
level of toxin needed to kill a larva at each 
instar stage. 
We found that our standard solubiliza-
tion condition, 50 mM NaOH (pH 11.7) 
with 10 mM EDTA, provided only variable 
and incomplete solubilization of the 135- 
and 140-kD protein bands. These bands 
could be completely solubilized by includ-
ing 0.5M dithioerythritol, but this was not 
accompanied by enhanced larval toxicity. 
In agreement with others (2), we conclude 
that it is unlikely that the high molecular 
weight components of the crystal contrib-
ute to larval toxicity (7). 
We recently showed (8) that the toxici-
ty of intact Bti crystals to A. aegypti larvae 
could be reversed up to 100-fold by lev-
els of K2CO3 as low as 0.15 percent. At the 
time there was no way to determine wheth-
er this reversal was directly related to the 
crystal’s mode of action or was merely pre-
venting solubilization in the larva by low-
ering the gut pH below a critical value. 
However, the fact that K2CO3 achieved a 
similar toxicity reversal with bead-attached 
solubilized proteins indicates that the car-
bonate does not act by preventing eventual 
crystal solubilization. 
Long-term control of mosquitoes with 
Bti is currently impractical because Bti 
does not persist in nature more than 1 or 2 
days. Apparently the toxin settles into the 
mud or is consumed by other microorgan-
isms. Attachment of the toxin to nonnutri-
tive particles with a greater tendency to re-
main dispersed in water should enhance its 
persistence. 
Figure 1. Attachment of 125I-labeled Bti pro-
teins to latex beads. Solubilized Bti protein was 
iodinated in vitro (13) to a specifi c activity of 
4.9 × 107 count/min-μg. Unincorporated 125I 
was removed by chromatography on a Bio-Gel 
P6 column. The iodinated proteins were ad-
sorbed to latex beads as described in the legend 
to Table 1 and the amount attached at each pro-
tein concentration was determined after centrif-
ugation by measuring the radioactivity associ-
ated with the beads on a Beckman 5500 gamma 
counter. Assessment of bead-adsorbed proteins 
by dye binding (14) would have been inappro-
priate because this method  indicates protein concentrations only 20 percent as high as those of 
three other methods. This difference is probably due to the fact that solubilized Bti proteins have a 
tendency to precipitate at acidic pH values. Each point is the mean ± standard error for four deter-
minations. The effi ciency of attachment remained constant for live toxin purifi cations from three 
different Bti strains over a 10-month period. 
Table 2. Amounts of Bti proteins ingested by 
dead Aedes aegypti larvae. Solubilized proteins 
were iodinated as described in the legend to 
Fig. 1. Intact crystals were labeled in vitro 
with Na125I (13). After iodination the crystals 
were dialyzed for 24 hours against 0.1M NaPO4 
buffer (pH 7.4) to remove unincorporated 125I. A 
specifi c acticity of 5.0 × 107 count/min-μg was 
obtained for the intact crystals. The LC50 value 
for labeled intact crystals was identical with that 
of the unlabeled crystals. The labeled toxin was 
diluted with unlabeled toxin to concentrations 
approximating their respective LC50 values 
(Table 1) and fed to larvae for 4 hours. The guts 
were dissected from dead larvae, which had 
been washed in 0.5 percent NaCl containing 0.1 
percent SDS, and radioactivity was measured 
with a Reckman 5500 gamma counter. Data are 
means ± standard errors.
BIOASSAY OF SOLUBILIZED BTI CRYSTALS BY ATTACHMENT TO LATEX BEADS 1193
References and Notes 
1. D. J. Tyrell. L. I. Davidson, L. A. Bulla, 
W. A. Ramoska, Appl. Environ. Micro-
biol. 38, 656 (1979): D. J. Tyrell et al., 
J. Bacteriol. 145, 1052 (1981). 
2. W. E. Thomas and D. J. Ellar, J . Cell 
Sci. 60, 181 (1983). 
3. R. H. Dadd, J. Exp. Zool. 191, 395 
(1975); Ann. Entomol. Soc. Am. 64, 
687 (1971). 
4. H. S. Abu Salih, A. F. Murant, M. J. 
Daft, J. Gen. Virol. 3, 299 (1968). 
5. K. W. Nickerson and D. J. Schnell, J. In-
vertebr. Pathol. 42, 407 (1983). 
6. R. H. Dadd, Entomol. Exp. Appl. 13, 407 
(1970). 
7. Dithioerythritol would interfere with io-
dination (13), and some sulfhydryl re-
agents exhibit larval toxicity in their 
own right [V. C. Kramer, D. J. Schnell, 
K. W. Nickerson, J. Invertebr. Pathol. 
42, 285 (1983)]. Therefore we did not 
include dithioerythritol in our standard 
crystal solubilization conditions. 
8. D. J. Schnell and K. W. Nickerson, Appl. 
Environ. Microbiol. 45, 1691 (1983). 
9. B. J. Ang and K. W. Nickerson, ibid. 36, 
625 (1978). 
10. M. A. Pfannenstiel, E. J. Ross, V. C. 
Kramer, K. W. Nickerson, Fed. Eur. 
Microbiol. Soc. Microbiol. Lett. 21, 
39 (1984) found that this procedure 
solubilized 80 percent of the crys-
tal proteins, minimized proteolyt-
ic degradation (SDS-PAGE analysis 
gave gel profi les identical to those 
of intact crystals), and maximized 
the general cytolytic activity (2) ex-
hibited by solubilized but not intact 
Bti crvstals toward human erythro-
cytes. Considering the multicompo-
nent nature of the Bti crvstal, there is 
no guarantee that the mosquito larval 
toxin and the general cytolytic factor 
are identical. Also, since the crystal 
is incompletely solubilized and the 
pellet is still highly toxic to mosqui-
to larvae, centrifugation at 100,000 g 
is necessary to defi ne the solubilized 
toxin. Insuffi cient centrifugation 
probably explains the erroneous con-
clusion (1) that solubilized Bti crys-
tals have LC50 values similar to those 
of whole crystals. 
11. The LC50 values for solubilized pro-
teins adsorbed to latex beads were 
identical in the presence and absence 
of the polyvinylpyrrolidone coating. 
The toxin-coated beads do not need 
to be purifi ed before routine bioassays 
because the unattached solubilized 
proteins contribute negligibly to over-
all toxicity. 
12. We have found that, when individu-
al batches of purifi ed Bti crystals are 
bioassayed for 4 hours against A. ae-
gypti larvae, the LC50 ranges from 5 to 
10 ng/ml. Spontaneous loss of toxici-
ty in Bti is common [H. Kamdar and 
K. Jayaraman, Biochem. Biophys. Res. 
Commun. 110, 477 (1983)]. 
13. M. A. K. Markwell and C. F. Fox, Bio-
chemistry 17, 4807 (1978). 
14. M. Bradford, Anal. Biochem. 72, 248 
(1976). 
15. K.W.N. is an NIH research career de-
velopment awardee (A1 00327-TMP). 
This research was supported in part by 
University Genetics Company.   


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Bioassay of Solubilized \u3ci\u3eBacillus thuringiensis\u3c/i\u3e var. \u3ci\u3eisraelensis\u3c/i\u3e Crystals by Attachment to Latex Beads

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