Ingestion, dissolution, and proteolysis of the Bacillus sphaericus toxin by mosquito larvae

JOURNAL

OF INVERTEBRATE

Ingestion,

PATHOLOGY

53, 12-20 (1989)

Dissolution, and Proteolysis Toxin by Mosquito

of the Bacillus sphericus Larvae

CHRISTOPHALY, MIR S. MULLA, AND BRIAN A. FEDERIC? Department of Entomology, University of California, Riverside, California 92521 Received December 2, 1986; accepted May 2, 1988 Larvae of Culex quinquefasciatus are much more susceptible to the toxin of Bacillus sphaericus than are larvae of Aedes aegypti. In the present study, the rate of ingestion, dissolution, and the cleavage by midgut proteases of the B. sphaericus toxin were compared in larvae of these species to determine whether these factors account for the differences in susceptibility. During filter feeding, larvae of both species removed significant quantities of B. sphaericus toxin from suspensions. Filtration rates for 1 hr, the time at which C. quinquefasciatus exhibited marked intoxication, were higher for A. aegypti (576-713 @arva/hr) than for C. quinquefasciatus (44fj-544 @trva/hr). Within 24 hr of exposure, A. aegypti larvae ingested 97-99% of the toxin particulates and suffered not more than 10% mortality in suspensions which induced complete mortality in C. quinquefasciatus within 2 hr of exposure. Quantification of the particulate toxin present in larvae after exposure to B. sphaericus suspensions revealed that larvae of both species contained only minor amounts of the toxin, suggesting the larvae had been able to solubilize the toxin after ingestion. Proteases recovered from the feces of larvae cleaved a 43-kDa protein isolated from B. sphaericus toxin extract to 40 kDa in both species. Thus, differences in susceptibility to the B. sphaericus toxin between A. aegypti and C. quinquefasciatus are not due to differences in rates of ingestion, dissolution, or the specificity of proteases. B 1989 Academic press, IIIC. KEY WORDS: Aedes aegypti; Bacillus sphaericus; Culex quinquefasciatus; ingestion rate; insecticidal protein; insecticidal toxin; midgut enzymes; proteolysis.

INTRODUCTION

velopment of this pathogen as a control agent. Certain important mosquitoes such as the yellow fever mosquito, Aedes aegypti L., are almost completely insensitive to the toxin of all known strains of B. sphaericus, as are the larvae of blackflies, which vector major filarial diseases (Yousten, 1984). Lack of sensitivity to the toxin of B. sphaericus could be due to one or a combination of several factors including slower rates of toxin ingestion or dissolution, proteolytic degradation of the toxin by midgut proteases, or the lack of appropriate midgut cell receptor sites. Ramoska and Hopkins (1981) demonstrated that larvae of A. aegypti and C. quinquefasciatus, the latter a species highly susceptible to B. sphaericus, ingest spores of strain 1593 at similar rates. Thus, rate of ingestion would not appear to be responsible for differences in sensitivity. However, the fate of the toxin in the larval gut was not followed in this study. In larvae

The bacterium Bacillus sphaericus, strain 2362 (Weiser, 1984), is a promising candidate for microbial control of mosquito larvae. Preparations from sporulated cultures used as larvicides yield excellent control of many species belonging to the genera Anopheles, Culex, and Psorophora, as well as of some species of the genus Aedes (reviewed by Mulla, 1985; Lacey and Undeen, 1986). Toxicity to these mosquitoes is due to a remarkably selective protein toxin located primarily in a parasporal inclusion. When ingested by larval mosquitoes, this inclusion dissolves in the midgut and the toxin destroys the midgut epithelium. Although the high selectivity of B. sphaericus is an advantage with respect to human health and environmental safety, it also constitutes a major impediment to the de’ To whom reprint requests should be addressed. 12 0022-2011/89 $1.50 Copyright 6 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

B. sphaericus

TOXIN

IN

of C. quinquefasciatus, Yousten and Davidson (1982) have shown that inclusions containing the toxin dissolve rapidly in the midgut. In another study using C. pipiens, Baumann et al. (1985) provided strong evidence that the native B. sphaericus toxin was a high-molecular-weight precursor which was cleaved to a 43-kDa active toxin by midgut proteases, and could be further degraded to 40 kDa by these proteases, but was quite resistant to further degradation in the mosquito midgut. In a study relevant to the fate of the B. sphaericus toxin after ingestion, Mian and Mulla (1984) found that strain 1593 lost toxicity during incubation in the gut juices obtained from larvae of A. aegypti, but not during incubation in gut juices obtained from larvae of C. quinquefusciatus, thus suggesting that the lack of sensitivity in the former species may be due to proteolytic degradation of the toxin. Proteases in larvae of Aedes and Culex do exhibit differences in molecular mass and activity (Spiro-Kern, 1974; Kunz, 1978). It is, therefore, possible that differences in dissolution or proteolytic cleavage of the B. sphaericus toxin between larvae of A. aegypti and C. quinquefusciatus may account for their marked differences in sensitivity. In the present study, we compared ingestion rates, dissolution, and proteolytic cleavage products of the toxin of B. sphaericus strain 2362 by larvae of A. aegypti and C. quinquefasciatus. Feeding studies demonstrated that larvae of both species ingest and dissolve the toxin particulates in their gut. By digestion of semipurified toxin with midgut proteases we determined that in both species the products of proteolytic cleavage are similar in size. These results indicate that the differences in susceptibility between these two species are not due to differences in ingestion rates, rates of toxin dissolution or the specificity of midgut proteases, thereby suggesting differences in receptors and/or site-of-action may account for differences in the species specificity of B. sphaericus.

MOSQUITO

13

LARVAE

MATERIALS Ingestion

AND METHODS

and dissolution

of toxin by lar-

vae. The ingestion and dissolution of the B. sphaericus toxin in vivo was studied using an experimental formulation of strain 2362 (Solvay BSP-I, Solvay Group, Brussels, Belgium). This aqueous suspension had a particle content of 15% (w/w), and, according to information supplied by the manufacturer, a spore titer of 2 X lo7 spores/ml. Stock suspensions containing 1 mg/ml of formulation/ml distilled water were prepared, agitated, and examined under a phase contrast microscope to ensure that the distribution of spores and toxin particles was homogeneous. For experimental exposure of larvae, 5 ml of the stock suspension was introduced into cups containing 100 or 50 ml of deionized water at 25°C. Early fourth instars of C. quinquefusciatus or A. aegypti, reared under conditions described previously (A. aegypti: Aly et al., 1985; C. quinquefusciarus: Aly and Mulla. 1987) were placed in the suspensions in groups of 50 larvae/container. For each experiment, three suspensions were prepared, two for a test series, and one for a control. The tests were (A) toxin suspension in 100 ml of water plus 50 larvae, (B) toxin suspension in 50 ml of water plus 50 larvae, and (C) toxin suspension in 100 ml of water with no larvae. A total of six replicates was carried out for each test with each mosquito species. After exposure times of 1 or 4 hr (C. quinquefusciutus), or 4 or 24 hr (A. uegypti), larval mortality was recorded. Subsequently, larvae were separated from the remaining suspension using a plastic strainer, rinsed under running water (tap), and homogenized in 50 ml of an ice-cold detergent solution (0.01% Tween 80 in 2.5 mM phosphate buffer, pH 7.2) in a cell mortar. Test suspensions were refilled to the original volume and stored on ice. Toxin in the larval homogenates, and remaining in the suspensions, was quantified through bioassays against larvae of C. quin-

14

ALY,

MULLA,

quefasciatus. For these assays, groups of 30 early fourth instars were placed in 100 ml of deionized water containing larval food (60 mg/liter), a 1:2 mixture of powdered dog food and yeast (Aly et al., 1985). Each suspension and larval homogenate was tested in four logarithmically increasing concentrations; each concentration was tested in duplicate. Concentrations were established which induced between 5 and 95% mortality in the larval groups by 48 hr posttreatment. At this time, the surviving larvae were counted, and LCs, values were determined using a computerized program for the dosage-mortality regression analysis (Ray, 1982). Day-to-day variation in the larval response to bacterial toxins in bioassays is the primary source of variability. We eliminated this source of variation by calibrating the sensitivity of each larval group against a toxin control (cup C) as described above. Since the toxin control was maintained under the same conditions as the suspensions containing the larvae, other factors which might have contributed to a loss of toxicity over time (e.g., bacterial degradation, adhesion to container walls) were considered not to have significantly influenced our results. Proteolysis Enzymes

of Toxin by Larval in Vitro

Gut

Toxin preparation. An aqueous preparation of toxin extracted from I?. sphaericus was kindly provided by Dr. E. Davidson, Arizona State University, Tempe. This extract had been prepared by alkaline extraction of strain 2362 spores and paraspores, followed by neutralization and sterile filtration (Davidson, 1983). To separate toxin from most of the other proteins, the proteins in the extract were precipitated by the stepwise addition of ammonium sulfate. Concentrations of 20, 40, 60, 80, and 100% saturation were added to the extract. After each stepwise addition, the precipitates were centrifuged from the extract (10 min, 18,000 r-pm), washed with an ammonium

AND

FEDERICI

sulfate solution of equal concentration, and then dissolved and dialyzed in 0.2 M TrisHCI buffer (PH 9). The molecular masses of proteins present in individual fractions were determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) as described below. To determine the toxin content of each fraction, a microbioassay using second instars of C. quinquefasciatus was developed. Groups of five larvae were incubated at 27°C in 5 ml of artificial well water (Humason, 1972), containing 100 mglliter of yeast as food. Fractions were diluted and tested at the following concentrations with respect to the original fraction: 10w2, 10p3, 10p4, 10e5, and lo-?ml. Mortality was recorded 2 days post-treatment. Enzyme preparation. Since mosquito larvae egest their gut contents at the onset of pupation, gut enzymes can be recovered from water in which large numbers of larvae have pupated (Yang and Davies, 1971). In our experiments, approximately 2000 late fourth instars were kept in 500 ml of water (25°C) containing l-2 g of larval food. As a control preparation, equal amounts of the larval food were incubated in 500 ml of water without larvae. Every 24 hr the number of pupae was recorded, and the water was filtered through cheese cloth and cooled on ice. Particulate matter was removed by centrifugation, and the solution was buffered at pH 9 with 0.2 M Tris and stored at - 67°C. Prior to use in experiments, three collections of enzyme solutions from each mosquito species were combined and made up with Tris buffer to a concentration of enzyme calculated to be the amount contained by one larvae in each 1.7 ml of the solution. Protein content. Protein content of toxin extracts and enzyme preparations was quantified using the Bio-Rad test (Bio-Rad Laboratories, Richmond, California). In this test, the shift of absorbance of Coomassie blue from 465 to 595 nm due to binding to proteins is used to quantify pro-

B. sphaericus

TOXIN

IN

teins photometrically. Calibration graphs with bovine albumin as substrate were prepared at pH 7.2 and 9. Adsorption in the presence of toxin extract, enzyme, and control preparations was determined after reaction with the dye. Protein concentration was determined using the calibration graph obtained under the corresponding pH conditions. Proteolytic activity. Proteolytic activity of enzyme preparations was quantified using Hideazure as a substrate. Upon digestion, this water insoluble substrate releases a dye which can be quantified photometrically at 595 nm. The proteolytic activity of gut enzyme preparations was compared to the activity of Trypsin 1:250 as a standard (Difco Laboratories, Detroit, Michigan). Solutions of Trypsin 1:250 were prepared in ice-cold 0.2 M Tris buffer, pH 7.2, at concentrations of 0.001, 0.01, 0.1, and 1 mg/ml. Hideazure was added to a series of test tubes, 20 mg/tube, along with 4 ml of Tris buffer. The buffer pH was 7.2 for the trypsin digestions and 9 for the digestions with the gut enzyme preparations. Digestion was started by the addition of 1 ml of Trypsin solution or enzyme preparation, followed by vortexing and immediate incubation of the tubes at 40°C in a water bath. After 10 min of incubation, the digestion was terminated by the addition of 100 ~1 of 10 N HCVtube. Hideazure particles were removed by filtration through a filter membrane with pores of 3 mm diameter, and adsorption of solutions was measured at 595 nm in a Zeiss spectrophotometer. Temperature and pH of optimal proteolytic activity of gut enzymes was determined in similar experiments. Temperature conditions tested were 30”, 35”, and 40°C at pH 9; the pH conditions tested were pH 8, 8.5, 9, 9.5, or 10 at 40°C. The pH was measured using a glass electrode before and immediately after incubation. Proteolysis in vitro. Gut enzyme preparations were diluted 1:4, 1:16, and 1:64 with 0.2 M Tris buffer, pH 9. A 100~~1 sample of undiluted enzyme preparation, or of each

MOSQUITO

15

LARVAE

dilution thereof, was cooled on ice in a I.5 ml Eppendorf tube. As substrate, 100 ~1 of the toxin fraction obtained from the 20% ammonium sulfate precipitation step was added to the tubes. Samples were mixed by vortexing and incubated in a water bath at 40°C. After 1 hr of incubation, the reaction was stopped by the addition of 2000 ~1 of SDS-mercaptoethanol disruption buffer (Hames and Rickwood, 1981) and then boiled for 2 min. Determination of cleavage products. The molecular masses of proteins present in digests were determined by SDS-PAGE. Running gels of 10% polyacrylamide were prepared according to the method of Hames and Rickwood (1981) with electrophoresis carried out at pH 9. Samples of 10 ~1 of the digested proteins were applied per well. Electrophoresis was carried out at 60 V for 10 min, followed by 120 V for 90 min. The gels were stained with Coomassie blue. RESULTS

Ingestion and Dissolution by Larvae

of the Toxin

The results of the feeding studies showed that larvae of both A. aegypti and C. quinquefasciatus removed toxin from the B. sphaericus suspensions by feeding on the suspended particulates. This was demonstrated by a significant reduction in the toxicity of the suspensions in which larvae were allowed to feed in comparison to the control suspensions held for identical time periods without larvae. Whereas the control suspensions had LC&‘s in the range 3 l109 tJ/lOO ml when assayed against C. quinquefusciatus, the LC,,‘s of suspensions previously exposed to larvae of A. aegypti for 4 hr (24 hr) were in the range 179361 t&O0 ml. Calculation of toxin content of test suspensions on the basis of control suspensions revealed that during the 4-hr period, 71-81% had been removed, whereas, during the 24-hr period of exposure, 97-99% of the toxin had been removed (Table 1). Tests with higher concen-

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INGESTION

MULLA,

AND

FEDERICI

TABLE 1 Bacillus sphaericus TOXIN ASSESSED THROUGH THE RELATIVE TOXICITY OF B. sphaericus SUSPENSIONS AND HOMOGENATES OF TREATED LARVAE”

AND DISSOLUTION

Species Aedes aegypti

OF

Concentration of B. sphaericus suspensions 5 mg/lOO ml 5 mg/50 ml

Culex quinquefasciatus

5 mg/lOO ml

% B. sphaericusb in Exposure time (hr) 4 24 4 24

24 f 5’ 2+-l

1

8 8 6 6

4 5

mg/SO ml

Suspension

1 4

10 2 3 3k2 80 + 72 2 58 f 46 +

Treated larvae 522

121 521 1 * 0.5 9-tl 921 11 +6 12 f 5

Total 29 3

15 4 89 81 69 58

a Toxicity of test suspensions, larval homogenates, and suspensions held without larvae (controls) tested against fourth instars of C. quinquefasciatus. Removal of toxin from the suspensions was determined by comparison of LC,,‘s obtained from test and control suspensions within the same replicates. b Relative to the toxicity of control suspensions within replicates. c Means and standard deviations for six replicates.

trations of toxin confirmed that toxin ingestion by larvae was responsible for toxin removal: when the same amount of toxin was suspended in 50 ml instead of 100 ml of water, larvae were able to remove a higher proportion of toxin (87-93% within 4 hr, Table 1). The same effects were noted in feeding experiments with larvae of C. quinquefasciatus. When placed in 100 or 50 ml of the B. sphaericus suspension for 1 hr, larvae of this species removed, respectively, 12-28 or 3U8% of the toxicity. Due to the onset of intoxication and a concomitant cessation of feeding at around 1 hr, only slightly higher rates of clearance were observed by 4 hr (Table 1). To determine the rate of ingestion by larvae in the presence of B. sphaericus toxin, filtration rates were calculated from the clearance of suspensions (Coughlan, 1969). Based on the data presented in Table 1, larvae of C. quinquefasciatus filtered 446 + 190 pl/larva/hr (100~ml test) or 545 + 99 pl/larva/hr (50-ml test) during the first hour of exposure. Larvae of A. aegypti exhibited filtration rates of 714 + 95 kl/larva/hr (looml test) or 576 f 66 tJ/larva/hr @O-ml test) during the first 4 hr of exposure. Data obtained with other exposure times appeared

unsuitable for calculation of filtration rates since larvae had either ceased feeding due to intoxication (C. quinquefasciatus), or the toxin was removed to an extent that made calculations less accurate (A. aegypti) .

Only relatively small amounts of toxin were detected in larvae homogenized after being allowed to feed on B. sphaericus suspensions. In larvae of A. aegypti allowed to feed for 4 hr, from 0.3-1.2 larvae contained an amount of toxin sufficient to induce 50% mortality in tests with C. quinquefasciatus. As calculated from toxicity of control suspensions, larvae contained 3-7% of the amount of toxin originally present in test suspensions (Table 1). Since these larvae had removed 71-81% of the toxin, it was evident that a large proportion of the ingested toxin had been dissolved in the larval guts. This conclusion was further substantiated by the results obtained with exposure times of 24 hr. Larvae exposed for this length of time contained a maximum of 2% of the toxin present in control suspensions, although they had removed at least 95% of toxin from suspensions (Table 1). Larvae of C. quinquefasciatus generally contained higher amounts of undissolved toxin, with the LC,, equivalent present in

B.

sphaericus

TOXIN

0.1-0.3 larva. Nevertheless, comparison of toxin content in larvae and test suspensions with the toxin content of control suspensions showed that an average of 11% (looml test) or 42% (50-ml test) of the toxin had been dissolved during the gut passage (Table 1).

IN

MOSQUITO

LARVAE

17

this precipitate were toxic at concentrations of 10p4/ml. In contrast, concentrations of 10p3/ml and fractions obtained with higher ammonium sulfate saturation were not toxic at the highest concentration tested ( 10K2/ml). Analysis by SDS-PAGE demonstrated that the 20% fraction consisted of two major proteins with molecular masses of 40 and 43 kDa (Fig. 1, Lane 2). Proteolysis of the B. sphaericus Toxin Gut enzyme preparations. The total in Vitro amount of protein in the gut enzyme preparations was low. In preparations from larToxin preparation. The toxicity of the al- vae of A. aegypti, the concentration was 30 kaline extract in tests against second instars pglml, whereas in the preparations from C. of C. quinquefusciatus was detectable at quinquefusciatus the concentration was 60 concentrations of 10e4/ml. The extract conkg/ml. The enzymatic activity with Hideatained numerous proteins (Fig. 1, Lane 1). zure as a substrate was equivalent to 4.0However, most of the toxicity could be pre- 4.7 pg of trypsin/ml (C. quinquefusciatus) cipitated from the extract at a concentraand 5.0-5.5 &ml (A. aegypti). In control tion of 20% ammonium sulfate. Solutions of samples, no detectable amount of soluble protein, and no proteolytic activity, was apparent. Maximal proteolytic activity in the enzyme preparations was found under pH conditions of 9.1-10.0 (C. quinquefusciatus) and 8.3-9-O (A. aegypti). When different temperature conditions were compared, enzymatic activity was 1.3-1.4 times or 2.6-2.9 times higher at 4O”C, than at, respectively, 30” and 35°C without marked differences being apparent between the two species of mosquitoes. Therefore, pH 9 and 40°C were selected as reaction conditions for the in vitro digestion of the B. sphaericus toxin. Digestion in vitro. Proteases from both test species were able to cleave the 43-kDa protein, as well as the other proteins in the samples, except for the 40-kDa protein. In Fig. 2, proteins present in the toxin samples incubated without larval proteases (Lane 2) -, : are compared to proteins present in samples after incubation with the gut enzymes (Lanes 3-6). Increasing the protease conFIG. 1. Polyacrylamide gel electrophoresis of fraccentration resulted in a concomitant reductions obtained by precipitation of proteins from a toxin tion in the number of undegraded proteins extract of Bacillus sphaericus. Lane 1, whole extract; Lane 2, 20% fraction; Lane 3, 40% fraction; Lane 4, apparent in the gels. At the higher concen60% fraction; Lane 5, 80% fraction, and Lane 6, fractrations of proteases, only the 40-kDa protion obtained with ammonium sulfate at 100% saturatein was apparent in the gels for both spetion. Molecular masses of the standards are located in cies (Lane 5). A “side-by-side” analysis of the furthest lane to the right, with masses indicated in the products of digestion obtained using the kilodaltons.

18

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1

2

6

4

n:

.,

6

MULLA,

.6’

AND

1

FEDERICI

2

3

-4

5

6

7

8

cx

Ae

FIG. 2. Polyacrylamide gel electrophoresis of protein obtained by proteolytic cleavage of the Bacillus sphaericus toxin with proteases isolated from mosquito feces. Lane 1, gut enzymes without toxin; Lane 2, toxin preparation without enzymes; Lanes 3-6, toxin preparation plus an increasing amount of enzyme with dilutions as follows: Lane 3, 164; Lane 4, 1:16; Lane 5, 1:4; and Lane 6, 1:l. The last two lanes, labeled 7 and 8, contain, respectively, 1:l dilutions of enzyme from Culex plus toxin (Lane 7) and 1:l dilutions of enzymes from Aedes plus toxin (Lane 8). The conditions for incubation were 4o”C, pH 9, for 1 hr. Molecular masses of the standards are located toward the middle of the gel and are indicated in kilodaltons.

the B. sphaericus toxin. Ramoska and Hopkins (1981) drew a similar conclusion based on a study of spore uptake by C. quinquefasciatus and A. aegypti using B. sphaericus strain 1593. Extending the scope of this earlier study, we demonstrated that during feeding larvae of both species not only ingest toxinDISCUSSION containing particles of B. sphaericus strain 2362, but are capable of dissolving the toxin In the present study, we have demonunder the alkaline strated that the larvae of both sensitive (C. from the particulates quinquefusciatus) and nonsensitive (A. ae- conditions present in their guts (Dadd, ingest partigypti) species of mosquitoes 1975). Only minor amounts of the ingested cles of the B. sphaericus strain 2362 toxin. toxin could be detected in the homogenates This was shown by quantifying the amount of whole larvae. However, one general limof toxin/toxic activity in suspension prior to itation of the data obtained from bioassays and after larvae were allowed to feed in sus- must be noted. In bioassays employing pensions of a commercial formulation of B. mosquitoes as the test insect, toxin partisphaericus. The rate of toxin removal, cles appear up to 1000 times more active though different for each species, indicated than dissolved toxin of the same amount. consumption of the toxin and that A. ae- This is mainly an effect of the feeding begypti did so at a rate higher than that which havior of mosquito larvae. Whereas in the resulted in the death of larvae of C. quin- present study larvae of A. aegypti and C. of feeding, quinquefascititus were able to clear toxin quefusciatus. Thus, inhibition for example, induced by Bacillus thurin- particulates at a rate of several hundred mispecies (Yen- croliters per larva per hour, drinking rates giensis in some lepidopteran do1 et al., 1975), does not account for the of these species were 140-210 (C. quinquenl/larva/hr (A. aelow susceptibility of A. aegypti larvae to fusciatus) and l-20

enzymes from each species demonstrated that the proteases of both species acted similarly on the toxin substrate. In both cases, after digestion, only the 40-kDa protein remained (Fig. 2, Lanes “Cx” and “Ae”).

B. sphaericus TOXIN

IN MOSQUITO

gypli) (Aly and Dadd, unpubl.). Therefore, results obtained with particulate toxins cannot be compared directly with results derived from assays of solubilized substances. Thus, the observed loss in activity of B. sphaericus toxin after ingestion could be a result of simple solubilization or the result of solubilization followed by proteolytic degradation. To distinguish between these two possibilities, we characterized the products of proteolytic cleavage using enzymes obtained from each species of mosquito. Gut enzymes were collected from feces of pupating mosquitoes, a method first developed by Yang and Davies (1971). Compared to enzyme preparations obtained by dissection of larvae (Dadd, 1975; Mian and Mulla, 1984), the major advantage of this method is the absence of intracellular proteases in the samples. According to Yang and Davies (1971), feces of pupating mosquito larvae contain almost the same level of proteolytic activity as preparations of dissected midguts, an indication that gut enzymes are almost completely recovered from feces. Since larval proteases are almost inactive at the pH conditions under which we collected the feces, loss of certain groups of proteases during collection was also highly unlikely. The solubilized toxin was digested in vitro and molecular masses of the digestion products were determined by electrophoresis. Analysis by SDS-PAGE showed that proteases of both test species acted similarly on the toxin, cleaving the major 43kDa protein to one of 40 kDa. Baumann et al. (1985) identified a 43-kDa protein as the toxin in B. sphaericus strain 2362, and also showed that this was cleaved to one of 40 kDa by the gut proteases of C. pipiens. Several laboratories have already reported different molecular masses for the B. sphaericus toxin (Tinelli et al., 1982: 55 kDa; Davidson, 1983: 54 kDa; Baumann et al., 1985: 40 kDa). Based on the observed resistance to further proteolytic cleavage by enzymes from mosquitoes, we conclude that the 40kDa protein observed in our gels is the

LARVAE

19

same as the 40-kDa protein reported by Baumann et al. (1985). The 43-kDa protein in the 20% ammonium sulfate precipitation step is very likely the precursor of this protein, as indicated by the digestion experiments and gel analyses. The primary purpose of the present study was to determine whether gut enzymes from different species of mosquitoes cleaved the toxin differently. Since the proteases obtained from A. aegypti and c’. quinquefasciatus produced products of identical molecular mass, it is concluded that differential proteolytic degradation leading to different peptide cleavage products is not responsible for the differences in sensitivity to the B. sphaericus toxin exhibited by these two mosquito species. In summary, we concluded that neither differential rates of ingestion and dissolution nor differences in the specificity of midgut proteases account for the difference in sensitivity to the B. sphaericus toxin exhibited by larvae of A. aegypti and C. quirzquefasciatus. We hypothesize, therefore, that the difference is probably due to still unknown processes of toxin binding to a gut cell receptor or differences in toxin metabolism within the cell. ACKNOWLEDGMENTS We thank Kathleen Stritzke for laboratory assistance and Dr. Elizabeth W. Davidson for the supply of B. sphaericus toxin extract. This study received partial financial support from the WHO/World Bank/UNDP Special Program for Research and Training in Tropical Diseases and the University of California Mosquito Control Research Program.

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molecular weight precursors. .Z. Bacterial., 163, 738-747. COUGHLAN, J. 1969. The estimation of filtering rate from the clearance of suspensions. Mar. Biol., 2, 356-358. DADD, R. H. 1975. Alkalinity within the midgut of mosquito larvae with alkaline-active digestive enzymes. J. Insect Physiol., 21, 1847-1853. DAVIDSON, E. W. 1983. Alkaline extraction of toxin from spores of the mosquito pathogen Bacillus sphaericus strain 1593. Canad. J. Microbial., 29, 271-275. HAMES, B. D., AND RICKWOOD, D. 1981. “Gel Electrophoresis of Proteins.” IRL Press, Washington. HUMASON, G. L. 1972. “Animal Tissue Techniques.” Freeman, San Francisco. KUNZ, P. A. 1978. Resolution and properties of the proteinases in the larva of the mosquito, Aedes aegypti. Insect Biochem., 8, 43-51. MIAN, L. S., AND MULLA, M. S. 1984. Effect of proteolytic enzymes on the activity of the microbial agent Bacillus sphaericus against Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae). Bull. Sot. Vector Ecol., 8, 122-127. MULLA, M. S. 1985. Field evaluation and efficacy of bacterial agents and their formulations against mosquito larvae. In “Integrated Mosquito Control Methodologies” (M. Laird and J. W. Miles, Eds.), Vol. 2. Academic Press, New York/London.

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RAMOSKA, W. A., AND HOPKINS, T. L. 1981. Effects of mosquito larval feeding behavior on Bacillus sphaericus efficacy. J. Znvertebr. Pathoi, 37, 26% 272. RAY, A. A. 1982. “SAS User Guide: Statistics.” SAS Institute, Cary, North Carolina. SPIRO-KERN, A. 1974. Untersuchungen iiber die Proteasen bei Culex pipiens. J. Comp. Physiol., 90, 5370. TINELLI, R., AND BOURGOUIN, C. 1982. Larvicidal toxin from Bacillus sphaericus spores. FEBS Lett., 142, 155-158. WEISER, J. 1984. A mosquito-virulent Bacillus sphaericus in adult Simulium damnosum from northern Nigeria. Zentralbl. Microbial., 13, 57-60. YANG, Y. J., AND DAVIES, D. M. 1971. Digestive enzymes in the excreta of Aedes aegypti larvae. J. Znsect Physiol., 17, 2119-2123. YENDOL, W. G., HAMLEN, R. A., AND ROSARIO, S. B. 1975. Feeding behavior of gypsy moth larvae on Bacillus thuringiensis-treated foliage. J. Econ. Entomol.,

68, 25-27.

YOUSTEN, A. A. 1984. Bacillus sphaericus: Microbiological factors related to its potential as a microbial larvicide. Adv. Biotechnol. Processes, 3, 315-343. YOUSTEN, A. A., AND DAVIDSON, E. W. 1982. Ultrastructural analysis of spores and parasporal crystals formed by Bacillus sphaericus 2297. Appl. Environ. Microbial., 44, 144!%1455.