Toxins from Bacillus thuringiensis subspecies Israelensis sorbed on clays: Persistence and activity against the mosquito Culex pipiens

Developments in Soil Science, Volume 28B Editors: A. Violante, P.M. Huang, J.-M. Bollag and L. Gianfreda © 2002 Elsevier Science B.V. All rights reserved.

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TOXINS FROM BACILLUS THURINGIENSIS SUBSPECIES ISRAELENSIS SORBED ON CLAYS: PERSISTENCE AND ACTIVITY AGAINST THE MOSQUITO CULEXPIPIENS P. Gonzalez^ O. L. Pantani^, G.G. Ristori^and A. Fereres^ ^ Consejo Superior de Investigaciones Cientificas Centro de Ciencias Medioambientales Serrano, 115 dpdo, 28006, Madrid, Spain ^ Istituto per la Genesi e I'Ecologia del Suolo, CNR Firenze, P.le delle Cascine 28, 50144 Firenze, Italy

The parasporal crystal of the isolate of Bacillus thuringiensis (Bt) subsp. israelensis (Bti) H14 is v^idely used to control larvae of mosquitoes and blackflies. Its outstanding insecticidal property is the result of the synergistic activity of 4 major proteins (Cry4A, Cry4B, Cryl 1A and CytlA). Hov^ever, the persistence of the insecticidal property is quite low due to a \o^ stability under field conditions (the crystals settle rapidly). There are some examples in the literature indicating that Bt toxins degrade at a slow^er rate w^hen they are sorbed or bound on clays. Also, the sorption oiBti toxins on slow^-settling clays could be a v^ay of increasing their presence near the air-water interface, where larvae of several mosquito species are usually feeding. We investigated the activity and persistence of^Bti crystals for a period of up to 29 days against the 4*^ instar larvae of Culexpipiens (LC50 = 0.003 ^g/ml of water) when mixed with a suspension of Na-bentonite. For comparison purposes, a similar experiment was conducted with toxins that were dissolved from the crystals and then sorbed on clay. We developed a method to improve the dissolution of the crystals by using some chemicals that were not toxic to the larvae of C pipiens. On the first day, the mortality was significantly higher using crystals alone than in the clay + crystal mixtures. The efficacy of crystals declined over time in all treatments, but after 29 days, the mortality increased in some of the treatments to which clay had been added. Conversely, the mortality rate of C. pipiens declined over time when no clay was added to the crystals. The overall cumulative mortality was highest for crystals alone, and there were no significant differences when a low concentration of clay (0.05 mg/ml) was present. The cumulative mortality was significantly reduced when the clay concentration was increased. The LC50 and LC90 of the solubiHzed toxins in the absence of clays were 34.0 ng/ml and 131.6 ng/ml, respectively, indicating that their toxicity was about 10 times less than that of the intact crystals (3.3 ng/ml and 54.6 ng/ml, respectively). In all cases, the solubilized clay-sorbed toxins had lower toxicity to C. pipiens than intact crystals mixed with clays (at equal concentrations).

1. INTRODUCTION The parasporal body of the isolate H14 oi Bacillus thuringiensis subsp. israelensis (Bti) is the most toxic per unit weight of all knovm Bt isolates [1] because it combines 4 major proteins

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with synergistic activity: Cry4A, Cry4B, CryllA and CytlA [2, 3]. It is used to control larvae of mosquito and blackfly, although its persistence is quite low due to its low stability under field conditions. This lack of residual activity is the result of photodegradation by UV light [4], heat, desiccation or changes in pH [5], together with a rapid settlement of crystals into the mud or consumption by microorganisms [6]. The efficacy of Bti crystals varies depending on the mosquito species, Anopheles spp. being more tolerant than Culex spp. and Aedes spp., probably because of the low filtration rate of the larvae of Anopheles [7]. It is possible to increase the efficacy oiBti crystals by mixing them with com oil or wheat flour to provide a floating bait to Anopheles larvae [8]. It is also possible to increase the stability of Bti crystals to UV radiation by adding a powder of melanin pigment into a spore suspension and preparations of 6endotoxin of Bti [9]. There are some examples in the literature indicating that toxins of Bt (obtained fi*om subspecies kurstaki and tenebrionis) bound or sorbed to clays with a high cationic exchange capacity can delay toxin degradation and may even increase its insecticidal activity [10]. Conversely, it has also been reported that the efficacy of commercial formulations of Bti decreases in aqueous environments containing soil or clay particles at concentrations between 10 and 0.1 mg/ml [11, 12]. Nevertheless, Ramoska et al. [11] did not use materials that were able to form stable colloidal suspensions. A sieve was used for separating the mineral material, and particles smaller than 100 jiim were considered as clay. To prevent Bti crystalsfi*omsettling into the mud of ponds, it would be very practical to solubilize them and attach the resulting toxins to particles with a greater capacity to remain in suspension. To adsorb Bti toxins to solid particles, the crystals need to be solubilized first, which results in a drastic reduction in toxicity; in fact, solubilized crystals were more than 7000 times less toxic to Aedes aegypti larvae than intact crystals [6], probably because mosquito larvae, as filter feeders, selectively concentrate particles of 0.5 to 10 |xm in diameter while excluding water and soluble molecules fi-om the gut [13]. Schnell et al. [6] were able to demonstrate that solubilized Bti crystals remain intrinsically toxic to mosquito larvae by adsorbing them to 0.8-^m latex beads; the toxicity of sorbed material was only 27 times less than intact crystals. Therefore, attachment or sorption of Bti toxins to particles with a greater capacity to remain in suspension in water, such as clay minerals, should enhance its persistence while retaining most of its toxicity. The objective of our work was to assess the efficacy and persistence of Bti crystals when mixed with different concentrations of clays (stable colloidal suspensions) against C. pipiens. Another objective was to dissolve the crystals and test the bioactivity of solubilized toxins after they are sorbed on clays.

2. MATERIALS AND METHODS 2.1. Persistence oiBti crystals mixed witli clays and bioactivity against C, pipiens Crystals of Bti were separated fi-om the commercial product (CP) Turbac CD (Abbott Laboratories) by isopycnic gradient centrifiigation as described by Ang and Nickerson [14]. The crystals were found to float at a density of 1.33-1.35 g/cc. The procedure was improved to increase the yield of extraction. The CP was diluted with distilled water, allowed to settle in a tube (30 cm high, 1000 ml) for 24 hours and the supernatant was removed. This procedure was repeated several times (4-5) to remove most of the light material. Sodium bromide was added to the resulting suspension until the density of 1.34 g/cc was reached (approx. 37% NaBr). This

71 suspension was centrifliged (Rotor Beckman JCF-Z at 15,000 rpm) and the supernatant discarded. The pellet (crystals) was recovered, washed and centrifliged until no NaBr was detected in the supernatant. Eventually, the dried suspension was photographed using scanning electron microscopy as described by Yamamoto et al. [15] to confirm the final result. To work with stable colloidal suspensions, the reference material (SWy-1, Crook County, NaMontmorillonite, obtainedfi-omthe Source Clay Repository, Univ. of Missouri) was allowed to settle for 1 week in a bucket whose height was 38 cm; only the material in suspension was recovered. Five different concentrations of Bti crystals (0.03, 0.06, 0.12, 0.15 and 0.3 \ig/m\) were combined with 4 different concentrations of clays (0, 0.05, 0.5 and 5 mg/ml). After thoroughly mixing each of the clay and crystal suspensions in a beaker, the suspensions were poured into plastic cups (200 ml/cup). The mosquito larvae used for the experiments were started fi'om an autogenic population of C. pipiens originallyfi-omBaix Llobregat (Barcelona, Spain), which was kindly supplied by C. Aranda and R. Eritja. Ten 4^^-instar larvae of C. pipiens were placed into each experimental plastic cup. The mortality was assessed 24 h after exposure to the insecticidal suspension. Mosquitoes (dead and alive) were removed from the cups every time after counting, and a new set of 10 larvae was introduced at different time intervals (1,5, 15, and 29 days after the suspension was prepared). There were twelve repHcates of each clay-crystal combination. All experiments were carried out inside an environmentally controlled chamber at a constant temperature of 25 ± 1°C and a relative humidity of 75 ± 10%. The LC50 and LC90 for C. pipiens of the intact crystals alone were determined by using five concentrations that were determined in preliminary tests. Before the bioassays, it was determined that the clays alone were not toxic to C pipiens at a concentration of 5 mg/ml under the same experimental conditions. 2.2. Persistence of solubilized clay-sorbed toxins from Bti crystals and bioactivity against C. pipiens The procedure to dissolve the toxins in the crystals has been used in the past for identification of toxins and analytical purposes, and as far as the authors are aware, no attempt has been made to dissolve completely the crystals for evaluation of their toxicity. Insell and Fitz-James [16] studied the solubility of crystals under different conditions (pH, chemicals and so on). Our main purpose was to compare the activity of intact crystals with equivalent amounts of solubilized clay-sorbed proteins. The procedure finally adopted was able to dissolve completely the crystals. The procedure used for solubilization was similar to the one described by Ishii and Ohba [17], although we used ascorbic acid instead of P-mercaptoethanol as a reducing agent, to avoid toxic effects on mosquito larvae [6]. In summary, 3 mg oiBti crystals, 10 ml of 0.01 M EDTA, and 0.176 g of ascorbic acid were added in a beaker with stirring. The pH was adjusted to 10 by dropwise addition of O.IN Na2C03. Finally, the volume was brought to 100 ml. The suspension was kept at 37°C for 1 h to promote the dissolution of crystals. The resultant solution was centrifiiged (15,000 g for 15 minutes) and no pellet was detected, demonstrating the complete dissolution of the solid material. The composition of this solution was similar to intact crystals, as demonstrated by SDS-PAGE analysis (data not shown). Appropriate amounts of the above solution and of clay were mixed, and the pH was adjusted to 7.0 with diluted HCl, to eliminate any effect on mortality due to alkaline conditions and to perform all the experiments under constant conditions. Some preliminary measurements indicated that the Crook County clay used for the sorption experiments is capable of depleting from solution around 75% of the solubilized proteins at pH 7.0 (1 ml of solution containing 0.5 mg of clay and 2 mg of solubilized crystals).

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The bioassays with C. pipiens were carried out in the same way as described above. The number of rephcates was 10, and the concentrations of crystals and clays used were the same except that an extra concentration of clay equivalent to 1 mg/ml and 0.6 ng/ml of crystals was also used. The LC50 and LC90 of the solubilized crystals for C pipiens were also determined using 5 different concentrations. 2.3. Analysis of data The corrected mortality of C. pipiens in all treatments was calculated using Abbott's formula [18] and the LC50 and LC90 were calculated for both intact and solubilized crystals using the POLO software for probit analysis [19]. The mean cumulative mortality (defined as the sum of mosquitoes dead after each count made on day 1, 5, 15, and 29) was calculated for each pair of clay-5/z concentrations. Cumulative mortality (in %) was subjected to a 2-factor analysis of variance (ANOVA) using the concentrations of toxin and clay as two independent factors. The means obtained for each pair of clay-5/z concentrations were compared to each other by orthogonal contrasts (plaimed comparisons of pair of means) at the P = 0.05 level. All data were transformed before running the ANOVA according to the transformation: X-arcsin X/100. The variation over time of the insecticidal activity (calculated as corrected mortality) was plotted, and the persistence of Bti mixed or sorbed to clays was analyzed by a 1-factor ANOVA for each pair of clay-Bti concentrations (in this case, the variable was time). Mean comparisons were then made according to Fisher's unprotected LSD test (P=0.05), after transforming the mortality values by X'=arcsin Vx/100.

3. RESULTS AND DISCUSSION 3.1. Persistence of Bti crystals mixed with clays and bioactivity against C pipiens The LC50 and LC90 for the intact crystals against C pipiens were 3.3 ng/ml and 54.6 ng/ml, respectively. The LC50 value is not very different fi-om the one reported in the literature for intact crystals SLgdinst Aedes aegypti (7.5 ng/ml) [6]. Table 1A shows the cumulative mortality (%) of C. pipiens over the entire experiment when exposed to different concentrations of clay-crystal. In this case, the mean comparisons were made across concentrations of clays. The data show that when the concentration of clay was increased, the toxicity of Bti crystals decreased. However, there were no significant differences (P 0.05) in mortality between the treatments with crystals alone and those containing 0.05 mg/ml of clay (for all of the concentrations of crystals used). Moreover, when crystal concentration was low (0.03 or 0.06 |il/ml), there were no significant differences in cumulative mortahty between 0.05 and 0.5 mg/ml of clay; clay significantly reduced the insecticidal activity of Bti crystals at concentrations > 0.5 mg/ml. These results are consistent with the ones reported by Ramoska et al. [11], who showed a significant reduction in toxicity with clay concentrations higher than 0.5 mg/ml. Table IB shows the cumulative mortality of C. pipiens over the entire experiment when the mean comparison was made across the concentration of Bti crystals. In this case, there was a clear increase in toxicity with increasing concentrations of crystals. This increase was larger when low concentrations of clays were used.

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Table 1 Cumulative mortality (%) (Mean ± SEM) of C pipiens when using mixtures of 5// intact crystals and clay at different concentrations over the entire experiment A) comparisons across [clay] Clay mg

mr^ 0.00 0.05 0.50 5.00

0.03 34.37 ± 2.8 a 27.50 ± 1.9 ab 23.54 ± 2.9 b 10.83 c± 2.2 c

0.06 46.87 ± 2.7 a 43.54 ± 2.5 ab 35.21 ±3.7b 21.04 ± 4.2 c

B) comparisons across [Bti crystals] Bti \ig ml"'

Bti \ig ml'^ 0.12 62.71 ±2.3 a 64.58 ± 2.6 a 43.12 ± 2.4 b 24.79 ± 2.6 c

0.15 74.79 ±1.7 a 71.04 ±2.8 a 49.79 ± 2.3 b 25.83 ± 2.6 c

0.30 85.21 ±2.9 a 89.79 ±1.8 a 72.71 ± 2.6 b 32.29 ± 3.7 c

Clay mg ml"^

5.00 0.00 0.50 0.05 0.03 10.83 ±2.2 a 23.54 ±2.9 a 34.37 ±2.8 a 27.50 ±2.0 a 0.06 21.04 ± 4.2 b 46.87 ± 2.7 b 43.54 ± 2.5 b 35.21 ± 3.7 b 0.12 24.79 ± 2.6 be 62.71 ± 2.3 c 43.12 ±2.4 be 64.58 ± 2.6 c 0.15 25.83 ±2.6 be 74.79 ± 1.7 d 49.79 ± 2.3 c 71.04 ± 2.8 c 0.30 32.30 ± 3.7 c 85.21 ± 2.8 e 72.71 ± 2.6 d 89.79 ± 1.8 d Different letters within columns indicate significant differences according to a 2-factor ANOVA and orthogonal contrasts (planned comparisons of pair of means) (P<0.05). Data were transformed before running a 2-factor ANOVA according to X'=arcsin X/100. The activity of Bti crystals over time was quantified by calculating the mortality of C. pipiens at different periods of time (1, 5, 15 and 29 days after the clay-crystal suspensions were prepared). When the period of time after the preparation of the suspensions increased, the bioactivity of the mixtures was maintained in the presence of clays, whereas the activity of the preparations without clays declined significantly over time (Figure 1). On the first day, the insecticidal activity of the crystals alone was higher than when adding clays to the suspension. However, on day 29, the Bti activity was in general greater in the presence of clays. In some cases, the activity of the toxin was significantly higher on day 29 than on the first day after preparation of the suspension ([Bti]= 0.06 and 0.12 ^g/ml data not reported combined with [clay]= 5 mg/ml). These results indicate that clays can increase the persistence of Bti crystals over time. Altematively, the clays may hamper the ingestion of crystals, which results in lower activity but higher persistence. To this point, it should be emphasized that depletion of crystals ingested by dead mosquitoes may reduce the activity of a Bti suspension over time [4]. Therefore, the exposure of 5 different sets of mosquito larvae to the crystal suspensions, such as the ones used in the experiments, could result in a reduction in the persistence of their insecticidal properties, hi our experiments, the high mortality obtained on the first date in the non-clay treatments possibly resulted in a partial depletion of the crystals by the first set of mosquitoes that was

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Crystals at 0.03 |Lig/ml

^0

5

10 15 20 25 30 0 DAYS

5

10 15 20 25 30 DAYS

Figure 1. Variation of mortality of C pipiens over time when exposed to different concentrations oiBti solid crystals mixed with clays (error bars = SEM). tested. This depletion could explain the sharp reduction observed in the insecticidal activity of the non-clay suspensions over time, since their toxicity was very low when the fifth set of mosquitoes was tested on day 29. 3.2. Bioactivity and persistence of solubilized Bti crystals sorbed to clays against C pipiens The LC50 and LC90 of the solubilized crystals in the absence of clays were 34.0 ng/ml and 131.6 ng/ml, respectively, indicating that the toxicity was about 10 times less than that of the intact crystals. However, our solubihzation protocol seems to be much more efficient than the ones previously pubhshed; the dissolution procedure decreased the activity of solid crystals only 10 times instead of 7000 times, as previously reported [6]. This difference could have been the result of the incomplete dissolution obtained by previous authors, which could result in a lack of some toxins and, consequently, in a reduction of activity because of the lack of synergistic activity of all toxins together. Tables 2A and 2B show the cumulative mortality (%) of C. pipiens over the entire experiment when exposed to different concentrations of solubilized clay-sorbed toxin, across all

Table 2 Cumulative mortality (%) (Mean ± SEM) of C. pipiens when using solubilized (from Bti crystals) clay-sorbed toxins at different concentrations over the entire experiment. A) comparisons across [clay] Claymg/ml 0.00 0.05 0.50 1.00 5.00

0.03 15.50 ± 0.3a 14.00 ±0.8ab 17.00 ± 1.1a 13.50 ±0.7ac 10.00 ± 0.5bc

0.06 24.25± 1.5a 19.25 ±2.1ab 14.50 ±1.7bc 14.50 ±1.2bc 13.50± 1.3c

Bti jxg/ml 0.12 0.15 29.00 ± 0.9a 33.25 ± 0.8a 27.25 ± 1.7a 33.50 ± 2.5a 21.25 ± 1.5b 30.00 ± 3.1a 19.75 ±3.4cb 17.50 ± 1.5b 14.50 ± 1.6c 14.25± 1.2b

0.30 47.25 ± 2.99a 46.00 ± 4.41a 37.75 ± 4.52b 30.75 ± 2.47c 26.25 ± 2.69c

0.60 61.50 ± 0.7a 59.50 ± 1.0a 58.50 ± 0.7a 49.00 ± 0.8b 41.00 ± 1.1c

B) comparisons across [solubilized clay-sorbed Bti\ Clay mg/ml 0.50 Bti |Lig/ml 1.00 5.00 0.00 0.05 0.03 13.50 ± 0.7a 14.00 ± 0.8a 17.00 ±1.lab 10.00 ± 0.5a 15.50 ± 0.3a 0.06 14.50 ± 1.7a 19.25 ± 2.1b 14.50 ±1.8ab 24.25 +1.5b 13.50 ±1.3ab 0.12 29.00 ± 0.9cb 27.25 ± 1.7c 21.25 ± 1.5b 19.75 ±3.4bc 14.50 ±1.6bc 0.15 30.00 ± 3.1c 33.25 ± 0.8c 33.50 ±2.5d 17.50 ±1.5ac 14.25 ±1.2ac 0.30 37.75 ±4.5d 47.25 ± 3.0d 46.00 ± 4.4e 30.75 ±2.5d 26.25 ± 2.7d 0.60 59.50 ±1.0f 58.50 ±0.7e 49.00 ± 0.8e 61.50 ± 0.7e 41.00±l.le Different letters within columns indicate significant differences according to a 2-factor ANOVA and orthogonal contrasts (planned comparisons of pair of means) (P<0.05). Data were transformed before running a 2 factor ANOVA according to X'=arcsin X/100.

76 the concentrations of clays and toxins used, respectively. The data show that when the concentration of clay was increased, the toxicity of the toxins sorbed on clay decreased. However, as reported for the case of the intact crystals and clay mixtures, there were no significant differences (P 0.05) in mortality when using a concentration of 0.05 mg/ml of clay and solubilized clay-sorbed toxins alone. In all cases, the toxins sorbed on clays had lower toxicity than the crystals mixed with clays (at equal concentrations), indicating that the solubilized clay-sorbed toxins were not as efficient as intact crystals mixed with clays in controlling C pipiens. A similar result was reported for solubilized toxins sorbed on latex beads that were much less active against A. aegypti than the intact crystals mixed with latex beads [6]. The mortality of C pipiens at 1, 5, 15, and 29 days after the suspensions were prepared was used to monitor over time the activity of solubilized clay-sorbed toxins. The results (Figure 2) indicate that with time, the bioactivity is maintained in a similar way as for clay-solid crystal mixtures, whereas the activity of the preparations without clays declined earlier. In some cases, the activity of the toxins on day 29 was higher for the clay-sorbed toxin than for the toxin solution alone.

100

.

P 80 -

o

Bf/at0.03)ig/ml A~A 0.00 • - • 0.05 A—A 0.50 mg/ml of clay

60 -

• - • 1.00 -. • - • 5.00

CC

P 40 20 0 100

*^^^--===t^E==:=t er/at0.15|ig/m

ef/at0.30^ig/ml

5

5

.o 80

10 15 20 25 30 0 DAYS

10 15 20 25 30 DAYS

Figure 2. Variation of mortality of C. pipiens over time when exposed to different concentrations of solubilized clay-sorbed toxins from Bti (error bars = SEM).

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ACKNOWLEDGMENTS This work was funded by the European Union INCO grant n° ERBIC18CT970135. The authors wish to thank C. Aranda and R. Eritja for providing the mosquito colonies and for their expertise in mosquito-rearing techniques and bioassays.

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15. Yamamoto, T., lizuka, T., Aronson, J.N., 1983. Mosquitocidal protein of Bacillus thuringiensis subsp. israelensis: identification and partial isolation of the protein. Curr. Microbiol. 9,279-284. 16. Insell, J.P., Fitz-James, P.C, 1985. Composition and toxicity of the inclusion o^ Bacillus thuringiensis subsp. israelensis. Appl. Environ. Microbiol. 50, 56-62. 17. Ishii, T., Ohba, M., 1993. Diversity of Bacillus thuringiensis environmental isolates showing larvicidal activity specific mosquitoes. J. Gen. Microbiol. 139, 2849-2854. 18. Abbott, W.S., 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18,265-267. 19. Russell, R.M., Robertson, J.L., Savin, N.E., 1977. POLO: A new computer program for Probit analysis. ESA Bulletin 23, 209-213.