JOURNAL
OF INVERTEBRATE
PATHOLOGY
60,
10-14 (19%)
Characterization and Toxicity to Mosquito Larvae of Four Bacillus sphaericus Strains Isolated from Brazilian Soils ROSE GOMES MONNERAT SCHENKEL EMBRAPAICENARGEN,
C.P. 102372,
Brasilia
DF,
Brazil
AND
Luc NICOLAS,~EMMANUEL FRACHON,AND SYLVLANE HAMON Institut
Pasteur,
Unit6
des Bactbries
Entomopathoghes,
25 rue du Dr Roux,
75724
Paris
cedex 15, France
Received March 26, 1991; accepted September 17, 1991
experiments and present operational applications rely on these three strains isolated respectively from Nigeria (Weiser, 19841, Indonesia (Singer, 19731, and Sri Lanka (Wric,kemesinghe and Mendis, 1980). However, among the 20 more toxic strains, only 4 strains have been isolated from Latin America, i.e., strains 1691 and 1881 from El Salvador (Singer, 1977) and strains 2532 and 2533 from Guyana (Lysenko et al., 1985). None has been reported from Brazil, which encompasses numerous and various kinds of ecological environments. At the CENARGEN/EMBRAPA of Brasilia, a screening program of B. sphaericus strains isolated in Brazil has been undertaken to develop vector control s5. 0 1992 Academic Press, Inc. with indigenous biological control agents. In this study KEY WORDS:Bacillus sphaericus; Culex pipiens; Anophesoils that Zesstephensi; biological control; mosquito; endotoxins; gas we report on 4 strains isolated from Brazilian are highly toxic to mosquito larvae and compare their chromatography; fatty acids. toxin composition with the most widely used strain, 2362. The two main toxins of mosquitocidal-known B. INTRODUCTION sphaericus are proteins of 43 and 56 kDa (Baumann et Since the discovery of the first weakly mosquitocidal al., 19851, respectively, which are very homologous bestrains of Bacillus sphaericus, K and & (Kellen et al., tween the strains (Berry et al., 1989). It is therefore novel strains within the 1965), a high number of B. sphaericus strains have very difficult to distinguish been isolated. More than 380 strains are now held in same serotype. We show here that the analysis of the the collection of the WHO Collaborating Center for En- bacterial fatty acids by gas chromatography is a rapid tomopathogenic Bacillus at the Institut Pasteur and method for overcoming this problem. approximately half of them are moderately to highly MATERIALSANDMETHODS toxic to mosquito larvae (Thiery and de Barjac, 1989). In general, the B. sphaericus strains are most active against mosquitoes of the genus Culex followed by Bacterial Strains Anopheles sp. and Psorophora sp. and are poorly active The four Bacillus strains used in this study were against Aedes sp. B. sphaericus strains 2362 and 1593 previously isolated from Brazilian soils: Sl from a polare also highly toxic to Mansonia uniformis while luted brook located along the airport of Vitoria (State strain 2297 is not (Yap et al., 1988). Most of the field of Espirito Santa), which was not colonized by mosquito Four Bacillus sphaericus strains, Sl, S2, S5, and L2, isolated from Brazilian soils, were found to be toxic to larvae of the mosquitoes Culex pipiens and Anopheles stephensi at a level similar to that of strain 2362which is now used operationally. Like strain 2362, the four strains belonged to the serotype H5 and produced major proteins of apparent molecular weights of 125,110,56, and 43 kDa. These latter two proteins were immunologically related to toxins of the same molecular weight as B. sphaericus 2362. Although the four Brazilian strains were very similar to strain 2362, gas chromatography analysis of the fatty acids revealed that these strains were different from strain 2362and from each other, except for a possiblesimilarity between strains Sl and
1 To whom correspondence
larvae; S2 from pasture soil near Brasilia D.F.; L2 from noncultivated bush soil (“cerrada”) near Brasilia DF;
should be addressed. 10
0022-2011/92
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
MOSQUITOCIDAL
BRAZILIAN
and S5 from noncultivated soil in the Atlantic forest of Registro (State of Sao Paulo). The strains were isolated according to the recommendations of WHO (WHO, 1985). B. sphaericus strains used for comparisons of toxicity (strain 2362) and for fatty acid analysis (strains 1593,2362, and 2297) came from the collection of the WHO Collaborating Center for Entomopathogenie Bacillus (Institut Pasteur, Paris). Identification
and Serotyping
The four brazilian strains were identified by phasecontrast microscopy observation of sporulated cultures. H-serotypes were determined by flagellar agglutination and cross-saturation was used for checking the antigenic identity (de Barjac, 1981). Larvicidal
Activity
The toxicity of strains Sl, S2, S5, and L2 mosquito larvae was compared to the toxicity of strain 2362. All the strains were grown in l-liter Erlenmeyer flasks containing 100 ml MBS medium (Kalfon et al., 1983) on an orbital shaker at 30°C for 48 hr. Flasks were inoculated with 1 ml of sporulated 72-hr-old precultures synchronized by heat treatment (8o”C, 12 min). Numbers of total bacteria and heat-resistant spores of the cultures were evaluated respectively before and after heat treatment (SOOC, 12 min) by plating on solid MBS medium and incubating the plates for 48 hr at 30°C. All the cultures were stored at -20°C until bioassayed. Bioassays were carried out on Culex pipiens and Aedes aegypti fourth instar larvae and on Anopheles stephensi third instar larvae, using dilutions of the final whole cultures (FWCD). Bioassays were performed on duplicate samples of 25 larvae per dose in 150-ml suspensions in plastic cups, using five to six doses per strain. The tests were repeated three times for each strain and mosquito species. Larval mortality was recorded after 24 and 48 hr of bacterial exposure, maintained at 25°C. The LCsc’s and LCao’s were calculated using a log-probit program on a Macintosh computer. Protein Analysis and Immunological with the Toxins of B. sphaericus
Relationships 2362
Forty microliters of the 48-hr-old cultures of strains Sl, S2, S5, and L2 were centrifuged at 5000g for 10 min and the pellets were submitted to SDS-PAGE (Laemmli, 1970) on a 0.75-mm-thick gel containing 12.5% polyacrylamide (acrylamide:NJV’-methylenebisacrylamide, 1OO:l). Twenty-four micrograms of an extract of B. sphaericus 2362, prepared by alkaline extraction with sodium hydroxyde as described in Nicolas et al. (1990), was applied to the same gel. The samples were run in triplicate and either stained with Coomassie brilliant blue or electrotransferred to a nitrocellulose
B. sphericus
STRAINS
11
sheet with a transfer buffer containing 25 mM Tris, 20 mM glycine, and 20% (v/v> methanol. Bands immunologically related to either the 43- or the 56-kDa toxin of B. sphaericus 2362 were detected by affinity-purified immunoglobulins respectively directed against these proteins. For this, IgG was purified by affinity (Burke et al., 1982) from polyclonal anti-43-kDa or anti-56-kDa antibodies, prepared as described in de Barjac et al. (19881, and used at a dilution of l/100 or l/50. Bands immunologically related to the B. sphaericus 2362 toxins were visualized with a Biosys goat anti-rabbit IgG horseradish peroxidase conjugate and 4-chloro-lnaphtol as substrate in the development solution. Identification by Gas Chromatography Fatty Acids
Analysis
of
Cellular fatty acid composition of strains Sl, S2, S5, and L2 was analyzed by gas-liquid chromatography as described by Miller and Berger (1985) and compared to that of the highly toxic strains 1593, 2362, and 2297. The strains were grown at 28°C for 28 hr on TSBA medium, which was composed of tryptic soy broth (Difco, Detroit, MI, 30 g/liter) and Bacto-agar (Difco; 15 g/liter). The cells were harvested and then saponified, methylated, extracted, and washed as described in Miller and Berger (1985). The extracts were analyzed by using the HewlettPackard microbial identification system (MIS) equipment, which is made of a gas-phase chromatograph (HP 5890A) linked with an automatic injector (HP 7673A) and integrator (HP 3392A), and a computer unit (HP 98561A). MIS Library Generation Software (Microbial ID, Newark, DE) was used for the numerical analysis of the fatty acid results. RESULTS
The four Brazilian isolates had terminal round spores in swollen sporangia and were identified as B. sphaericus strains. All belonged to serotype H5. The toxicity of the strains, grown for 48 hr in MBS medium, was evaluated on mosquito larvae in comparison with strain 2362 (Table 1). In MBS medium the five strains produced 7.1 x 10’ to 1.2 x 10’ cells/ml after 48 hr of culture. However, all Brazilian strains sporulated less than strain 2362. Results of bioassays (Table 1) show that strains Sl, S2, and S5 had a larvitidal activity similar to that of strain 2362 against C. pipiens and A. stephensi, expressed either as dilution of final whole culture or as the number of cells per milliliter. The LC&,‘s after 48 hr of these four strains were ca. 1 x 10e6 FWCD, 9 x 10’ cells/ml, or 150 to 500 spores/ml for C. pipiens. These strains were approximately 10 times less toxic against A. stephensi than against C. pipiens. As with strain 2362, the Brazilian strains exhibited a low toxicity against A. aegypti (ca. 1O-3 FWCD). When the pellets of sporulated cultures were sub-
SCHENKEL
12 Larvicidal
Activity of the Brazilian
TABLE 1 Strains of B. sphaericusagainst Mosquitoes, Compared to the Activity of B. sphaericus 2362 after 48 hr of Exposure
Cells/ml of culture ( x 10s) Strains
Total cells
Spores
2362
11.0
7.1
Sl
10.0
1.7
s2
12.0
1.4
s5
7.1
2.6
L2
7.2
1.4
ET AL.
Culex
LGO 0.70 (O.OW 7.70 (0.88) 4.90 (0.56) 0.89 (0.29) 8.90 (2.90) 1.51(0.49) 0.93 (0.49) 6.60 (3.48) 2.41 (1.27) 0.90 (0.23) 10.80 (2.76) 3.95 (1.01) 1.96 (0.31) 14.10 (2.23) 2.39 (0.38)
pipiens
w?o 1.67 (0.28)” 2.88 (1.41) 2.06 (1.18) 2.45 (0.50) 5.07 (1.00)
Anopheles
LGO 7.31 (2.32)” 80.00 (25.30) 51.00 (16.20) 10.65 (2.34) 106.00 (23.00) 18.00 (3.90) 12.74 (0.96) 90.00 (6.40) 12.70 (0.70) 6.93 (2.71) 82.80 (3.24) 30.30 (11.90) 13.53 (3.44) 97.40 (24.70) 18.90 (4.80)
stephensi
Aedes
LGO 12.05 (7.70)” 21.72 (3.60) 30.55 (2.51) 16.73 (3.00) 38.86 (10.22)
LGO 0.25 (0.08)* 2.79 (0.89jd 1.80 (0.57)f 0.40 (ND) 2.91 (ND) 0.56 (ND) 0.29 (0.05) 3.45 (0.69) 0.40 (0.08) 0.15 (0.01) 1.06 (0.06) 0.39 (0.02) 0.37 (0.27) 3.75 (2.73) 0.64 (0.46)
aegypti
LC90 2.18 (0.12)’ 2.43 (ND) 1.66 (0.19) 0.59 (0.06) 1.84 (1.60)
Note. The mean activity is expressed as dilution of the final whole culture (“x 1Om6 for C. pipiens and A. stephensi; *X lo-’ for A. aegypti) and in number of total cells/ml ("x lo2 for C. pipiens and A. stephensi; do lo6 for A. aegypti) or heat resistant spores (‘x 10’ for C. pipiens and A. stephensi; fx106 for A. mgypti). The numbers in parentheses indicate the standard deviation (n = 3). ND, not determined.
jetted to gel electrophoresis, all the Brazilian strains contained major proteins of 125, 110, 56, and 43 kDa, these latter two being apparently less intense in strain S5. Another major band of apparent molecular weight of 32 kDa was present in strains Sl and L2 (Fig. 1A). The proteins of 43 and 56 kDa migrated exactly the same distance as corresponding proteins of strain 2362 and were detected by affinity-purified polyclonal antibodies of these proteins (Figs. 1B and 10. The slight reaction of antibodies B. sphaericus 2362 43- or 56-kDa protein with the proteins of strain S5 could be due to the low amount of these proteins in this strain. No reaction was observed with the 32-kDa protein of strains Sl and S5. However a very slight reaction of both antibodies with the 125- and llO-kDa proteins kDa
0
A
could be detected if the exposure with 4-chloro-lnaphtol was prolonged (data not shown). The numerical taxonomic analysis of the fatty acid compositions of strains Sl, S2, S5, and L2, compared to those of strains 1593, 2297 and 2362, is shown in Fig. 2. All strains were analyzed in duplicate, except strains Sl and S5, which were analyzed in triplicate. Gas chromatography analysis appeared to be highly reproducible, because the maximum euclidian distance
- 0.7
L
- 0.6
r-Lr
C
- 0.5 - 0.4
mwa
bcde
abcde
a
bc
de
FIG. 1. SDS-PAGE of B. sphuericus samples (A) and the detection of immunorelated proteins by antisera to the 43-kDA (B) and the 56-kDA (C) proteins ofB. sphuericus 2362. Lanes: mw, molecular weight standards; a, B. sphuericus 2362 extract; b to e, cultures of B. sphuericus strains Sl (b), S2 cc), L2 cd), and S5 (e).
BACILLUS
!M+AERKXJS
STRAINS
FIG. 2. Results of a numerical taxonomic analysis based on the fatty acid compositions of the Brazilian strains compared to strains 1593,2362, and 2297 of B. sphuericus using the MIS system. Analyses were performed either in duplicate (strains S2, L2, 1593, 2297, and 2362) or in triplicate (strains Sl and S5).
MOSQUITOCIDAL
BRAZILIAN
between two duplicate analyses of a same strain was only 0.12 and was obtained with strain 2362. The euclidian distance between strains S2 and L2 and all other strains was greater than 0.3. However, the chance for strains Sl and S5 to actually be different is very low since the euclidian distance between fatty acid profiles of both strains was less than 0.12. Therefore, this analysis shows that all Brazilian strains differ from each other and from strain 1593, 2297, or 2362, with the possible exception of strains Sl and S5, which could be the same strain. DISCUSSION
This study shows that four B. sphuericus strains isolated from different sites in Brazil had a level of toxicity to C. pipiens, A. stephensi, and A. aegypti similar to that of strain 2362, which is now used in operational mosquito control. As the most active strains against mosquito larvae, these four strains harbor 43- and 56kDa proteins immunologically related to the toxins of B. sphaericus 2362. It is now established that the 43and 56-kDa proteins, of deduced molecular masses of 41.9 and 51.4 kDa, respectively (Hindley and Berry, 1987; Baumann et al., 19881, act as a binary toxin to mosquito larvae (Broadwell et al., 1990a; 1990b). DNA sequence analyses of the genes encoding these two proteins in strains originating from areas as distant as West Africa (strains 2362 and IAB59), Sri Lanka (strain 2297), Indonesia (strain 1593), or North America (strain 2317.31, are highly homologous between strains (Berry et al., 1989). This is probably also true for strain 1691 originating from El Salvador since it has a restriction pattern similar to that of strains 1593 and 2362 (Baumann et al., 1987). Strains Sl, S2, S5, and L2 harbor, as does strain 2362, the binary toxin composed of the 41.9- and 51.4kDa proteins, and high molecular weight proteins of 125 and 110 kDa, whose roles in toxicity are not clearly established. However, although these strains seem to be very close to the known mosquitocidal strains, regarding their host range, level of toxicity, and protein pattern, gas chromatography analysis of their fatty acids demonstrate that there are strains different from 2362, 2297, and 1593. Nevertheless, this technique does not allow one to confirm that strains Sl and S5 are actually the same strain. The isolation of these new strains from Brazil make the development of insecticides with indigenous entomopathogenic strains possible. Production of strain S2 on cheap media has now been undertaken at CENARGEN with optimistic results (Dias et al., 1990). ACKNOWLEDGMENTS We are grateful to Professor H. de Bajac, in whose laboratory this work was conducted. Thanks are due to J.-F. Charles, I. Thiery, V. Cosmao-Dumanoir (Institut Pasteur), and E. W. Davidson (Arizona State University) for helpful discussion and comments on the manu-
B. sphoericus STRAINS
13
script and to J. M. C. S. Dias (CENARGEN) for helpful advice. This work was partly supported by the United Nations Development Program/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases. R. G. M. Schenkel was supported by a grant from CNPq National Council for Science and Technology Development, Brasilia, during her stay at the Institut Pasteur. REFERENCES de Bajac, H. 1981. Identification of H-serotypes of Bacillus thuringiensis. In “Microbial Control of Pests and Plant Diseases 19701980” (H. D. Burges, Ed.), pp. 3543. Academic Press, New York. de Bajac, H., Thiery, I., Cosmao-Dumanoir, V., Frachon, E., Laurent, P., Charles, J. F., Hamon, S., and Ofori, J. 1988. Another Bacillus sphaericus serotype harhouring strains very toxic to mosquito larvae: Serotype H6. Ann. Inst. PasteurlMicrobioZ. 139, 363377. Baumann, P., Untermann, B., Baumann, L., Broadwell, A. H., Abbene, S. J., and Bowditch, R. D. 1985. Purification of the larvicidal toxin of Bacillus sphaericus and evidence for high molecular weight precursors. J. Bacterial. 163, 738-747. Baumann, P., Baumann, L., Bowditch, R. D., and Broadwell, A. H. 1987. Cloning of the gene for the larvicidal toxin of B. sphuericus 2362: Evidence for a family of related sequences. J. Bacterial. 169, 4061-4067. Baumann, L., Broadwell, A. H., and Baumann, P. 1988. Sequence analysis of the mosquitocidal toxin genes encoding 51.4- and 41.9kilodalton proteins from Bacillus sphaericus 2362 and 2297. J. Bacterial. 170, 2045-2050. Berry, C., Jackson-Yap., J., Oei, C., and Hindley, J. 1989. Nucleotide sequence of two toxin genes from Bacillus sphaericus IAB59: Sequence comparisons between five highly toxinogenic strains. Nucleic Acids Res. 17, 7516. Broadwell, A. H., Baumann, L., and Baumann, P. 1990a. The 42- and 52-kilodalton mosquitocidal proteins of Bacillus sphaericus 2362: Construction of recombinants with enhanced expression and in vivo studies of processing and toxicity. J. Bacterial. 172, 22172223. Broadwell, A. H., Baumann, L., and Baumann, P. 1990b. Larvicidal properties of the 42- and Bl-kilodalton Bacillus sphuericus proteins expressed in different bacterial hosts: Evidence for a binary toxin. Curr. Microbial. 21, 361-366. Burke, B., Griffiths, G., Reggio, H., Louvard, D., and Warren, G. 1982. A monoclonal antibody against a 135-K Golgi membrane protein. EMBO J. 1, 1621-1628. Dias, J. M. C. S., Honda, C. S., and Schenkel, R. G. M. 1990. Development of production process of Bacillus sphaericus, bioinsecticide against mosquitoes. In “Abstracts of 2” Simposio de Controle Biologic0 Siconbiol, de Volta a Natureza, Brasilia DF, 14-18 October 1990,” p. 99. Hindley, J., and Berry, C. 1987. Identification, cloning and sequence analysis of the BaciZlus sphaericus 1593 41.9 kD larvicidal toxin gene. Mol. Microbial. 1, 187-194. Kalfon, A., Larget-Thiery, I., Charles, J.-F., and de Barjac, H. 1983. Growth, sporulation and larvicidal activity of Bacillus sphuericus. Eur. J. Appl. Microbial. Biotechnol. 18, 168-173. Kellen, W. R., Clark, T. B., Lindegren, J. E., Ho, B. C., Rogoff, M. H., and Singer, S. 1965. Bacillus sphaericus Neide as a pathogen of mosquitoes. J. Znvertebr. Pathol. 7, 442-448. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
SCHENKEL Lysenko, O., Davidson, E. W., Lacey, L. A., and You&en, A. A. 1985. Five new mosquito larvicidal strains of Bacillus sphocricus from non-mosquito origins. J. Am. Mosq. Control Assoc. 1, 369-371. Miller, L., and Berger, T. 1985. Hewlett-Packard Gas Chromatography Application Note 228-38. Hewlett-Packard Co., Palo Alto, CA. Nicolas, L., Lecroisey, A., and Charles, J.-F. 1990. Role of the gut proteinases from mosquito larvae in the mechanism of action and the specificity of the Bacillus sphaericus toxin. Can. J. Microbial. 36, 804-807. Singer, S. 1973. Insecticidal activity of recent bacterial isolates and their toxins against mosquito larvae. Nature 244, 110-111. Singer, S. 1977. Isolation and development of bacterial pathogens of vectors. In “Biological Regulation of Vectors,” Publication No. (NIH) 77-1180, pp. 3-18. HEW, Washington, DC. Thiery, I., and de Barjac, H. 1989. Selection of the most potent Ba-
ET AL. cillus sphaericus strains based on activity ratios determined on three mosquito species. Appl. Microbid. Biotechnol. 31, 577681.
Weiser, J. 1984. A mosquito-virulent Bacillus sphaericus Simulium dumnosum from Northern Nigeria. Zentralbl. biol. 139, 57-60.
in adult Mikro-
World Health Organization. “Informal Consultation on the Development of Bacillus sphaericus as a Microbial Larvicide.” WHO Mimeograph Document WHOSPHAERICUS85.3. Wickremesinghe, R. S. B., and Mendis, C. 1980. Bacillus spore from Sri-Lanka demonstrating rapid larvicidal Culex
quinquefasciatus.
Mosq.
News.
sphaericus
activity on
40, 387389.
Yap, H. H., Ng, Y. M., Foo, A. E. S., and Tan, H. T. 1988. Bioassays of Bacillus sphaericus (strains 1593,2297 and 2362) against Mansonia and other mosquitoes of public health importance in Malaysia. Malays. Appl. Biol. 17, 9-13.
OF INVERTEBRATE
PATHOLOGY
60,
10-14 (19%)
Characterization and Toxicity to Mosquito Larvae of Four Bacillus sphaericus Strains Isolated from Brazilian Soils ROSE GOMES MONNERAT SCHENKEL EMBRAPAICENARGEN,
C.P. 102372,
Brasilia
DF,
Brazil
AND
Luc NICOLAS,~EMMANUEL FRACHON,AND SYLVLANE HAMON Institut
Pasteur,
Unit6
des Bactbries
Entomopathoghes,
25 rue du Dr Roux,
75724
Paris
cedex 15, France
Received March 26, 1991; accepted September 17, 1991
experiments and present operational applications rely on these three strains isolated respectively from Nigeria (Weiser, 19841, Indonesia (Singer, 19731, and Sri Lanka (Wric,kemesinghe and Mendis, 1980). However, among the 20 more toxic strains, only 4 strains have been isolated from Latin America, i.e., strains 1691 and 1881 from El Salvador (Singer, 1977) and strains 2532 and 2533 from Guyana (Lysenko et al., 1985). None has been reported from Brazil, which encompasses numerous and various kinds of ecological environments. At the CENARGEN/EMBRAPA of Brasilia, a screening program of B. sphaericus strains isolated in Brazil has been undertaken to develop vector control s5. 0 1992 Academic Press, Inc. with indigenous biological control agents. In this study KEY WORDS:Bacillus sphaericus; Culex pipiens; Anophesoils that Zesstephensi; biological control; mosquito; endotoxins; gas we report on 4 strains isolated from Brazilian are highly toxic to mosquito larvae and compare their chromatography; fatty acids. toxin composition with the most widely used strain, 2362. The two main toxins of mosquitocidal-known B. INTRODUCTION sphaericus are proteins of 43 and 56 kDa (Baumann et Since the discovery of the first weakly mosquitocidal al., 19851, respectively, which are very homologous bestrains of Bacillus sphaericus, K and & (Kellen et al., tween the strains (Berry et al., 1989). It is therefore novel strains within the 1965), a high number of B. sphaericus strains have very difficult to distinguish been isolated. More than 380 strains are now held in same serotype. We show here that the analysis of the the collection of the WHO Collaborating Center for En- bacterial fatty acids by gas chromatography is a rapid tomopathogenic Bacillus at the Institut Pasteur and method for overcoming this problem. approximately half of them are moderately to highly MATERIALSANDMETHODS toxic to mosquito larvae (Thiery and de Barjac, 1989). In general, the B. sphaericus strains are most active against mosquitoes of the genus Culex followed by Bacterial Strains Anopheles sp. and Psorophora sp. and are poorly active The four Bacillus strains used in this study were against Aedes sp. B. sphaericus strains 2362 and 1593 previously isolated from Brazilian soils: Sl from a polare also highly toxic to Mansonia uniformis while luted brook located along the airport of Vitoria (State strain 2297 is not (Yap et al., 1988). Most of the field of Espirito Santa), which was not colonized by mosquito Four Bacillus sphaericus strains, Sl, S2, S5, and L2, isolated from Brazilian soils, were found to be toxic to larvae of the mosquitoes Culex pipiens and Anopheles stephensi at a level similar to that of strain 2362which is now used operationally. Like strain 2362, the four strains belonged to the serotype H5 and produced major proteins of apparent molecular weights of 125,110,56, and 43 kDa. These latter two proteins were immunologically related to toxins of the same molecular weight as B. sphaericus 2362. Although the four Brazilian strains were very similar to strain 2362, gas chromatography analysis of the fatty acids revealed that these strains were different from strain 2362and from each other, except for a possiblesimilarity between strains Sl and
1 To whom correspondence
larvae; S2 from pasture soil near Brasilia D.F.; L2 from noncultivated bush soil (“cerrada”) near Brasilia DF;
should be addressed. 10
0022-2011/92
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
MOSQUITOCIDAL
BRAZILIAN
and S5 from noncultivated soil in the Atlantic forest of Registro (State of Sao Paulo). The strains were isolated according to the recommendations of WHO (WHO, 1985). B. sphaericus strains used for comparisons of toxicity (strain 2362) and for fatty acid analysis (strains 1593,2362, and 2297) came from the collection of the WHO Collaborating Center for Entomopathogenie Bacillus (Institut Pasteur, Paris). Identification
and Serotyping
The four brazilian strains were identified by phasecontrast microscopy observation of sporulated cultures. H-serotypes were determined by flagellar agglutination and cross-saturation was used for checking the antigenic identity (de Barjac, 1981). Larvicidal
Activity
The toxicity of strains Sl, S2, S5, and L2 mosquito larvae was compared to the toxicity of strain 2362. All the strains were grown in l-liter Erlenmeyer flasks containing 100 ml MBS medium (Kalfon et al., 1983) on an orbital shaker at 30°C for 48 hr. Flasks were inoculated with 1 ml of sporulated 72-hr-old precultures synchronized by heat treatment (8o”C, 12 min). Numbers of total bacteria and heat-resistant spores of the cultures were evaluated respectively before and after heat treatment (SOOC, 12 min) by plating on solid MBS medium and incubating the plates for 48 hr at 30°C. All the cultures were stored at -20°C until bioassayed. Bioassays were carried out on Culex pipiens and Aedes aegypti fourth instar larvae and on Anopheles stephensi third instar larvae, using dilutions of the final whole cultures (FWCD). Bioassays were performed on duplicate samples of 25 larvae per dose in 150-ml suspensions in plastic cups, using five to six doses per strain. The tests were repeated three times for each strain and mosquito species. Larval mortality was recorded after 24 and 48 hr of bacterial exposure, maintained at 25°C. The LCsc’s and LCao’s were calculated using a log-probit program on a Macintosh computer. Protein Analysis and Immunological with the Toxins of B. sphaericus
Relationships 2362
Forty microliters of the 48-hr-old cultures of strains Sl, S2, S5, and L2 were centrifuged at 5000g for 10 min and the pellets were submitted to SDS-PAGE (Laemmli, 1970) on a 0.75-mm-thick gel containing 12.5% polyacrylamide (acrylamide:NJV’-methylenebisacrylamide, 1OO:l). Twenty-four micrograms of an extract of B. sphaericus 2362, prepared by alkaline extraction with sodium hydroxyde as described in Nicolas et al. (1990), was applied to the same gel. The samples were run in triplicate and either stained with Coomassie brilliant blue or electrotransferred to a nitrocellulose
B. sphericus
STRAINS
11
sheet with a transfer buffer containing 25 mM Tris, 20 mM glycine, and 20% (v/v> methanol. Bands immunologically related to either the 43- or the 56-kDa toxin of B. sphaericus 2362 were detected by affinity-purified immunoglobulins respectively directed against these proteins. For this, IgG was purified by affinity (Burke et al., 1982) from polyclonal anti-43-kDa or anti-56-kDa antibodies, prepared as described in de Barjac et al. (19881, and used at a dilution of l/100 or l/50. Bands immunologically related to the B. sphaericus 2362 toxins were visualized with a Biosys goat anti-rabbit IgG horseradish peroxidase conjugate and 4-chloro-lnaphtol as substrate in the development solution. Identification by Gas Chromatography Fatty Acids
Analysis
of
Cellular fatty acid composition of strains Sl, S2, S5, and L2 was analyzed by gas-liquid chromatography as described by Miller and Berger (1985) and compared to that of the highly toxic strains 1593, 2362, and 2297. The strains were grown at 28°C for 28 hr on TSBA medium, which was composed of tryptic soy broth (Difco, Detroit, MI, 30 g/liter) and Bacto-agar (Difco; 15 g/liter). The cells were harvested and then saponified, methylated, extracted, and washed as described in Miller and Berger (1985). The extracts were analyzed by using the HewlettPackard microbial identification system (MIS) equipment, which is made of a gas-phase chromatograph (HP 5890A) linked with an automatic injector (HP 7673A) and integrator (HP 3392A), and a computer unit (HP 98561A). MIS Library Generation Software (Microbial ID, Newark, DE) was used for the numerical analysis of the fatty acid results. RESULTS
The four Brazilian isolates had terminal round spores in swollen sporangia and were identified as B. sphaericus strains. All belonged to serotype H5. The toxicity of the strains, grown for 48 hr in MBS medium, was evaluated on mosquito larvae in comparison with strain 2362 (Table 1). In MBS medium the five strains produced 7.1 x 10’ to 1.2 x 10’ cells/ml after 48 hr of culture. However, all Brazilian strains sporulated less than strain 2362. Results of bioassays (Table 1) show that strains Sl, S2, and S5 had a larvitidal activity similar to that of strain 2362 against C. pipiens and A. stephensi, expressed either as dilution of final whole culture or as the number of cells per milliliter. The LC&,‘s after 48 hr of these four strains were ca. 1 x 10e6 FWCD, 9 x 10’ cells/ml, or 150 to 500 spores/ml for C. pipiens. These strains were approximately 10 times less toxic against A. stephensi than against C. pipiens. As with strain 2362, the Brazilian strains exhibited a low toxicity against A. aegypti (ca. 1O-3 FWCD). When the pellets of sporulated cultures were sub-
SCHENKEL
12 Larvicidal
Activity of the Brazilian
TABLE 1 Strains of B. sphaericusagainst Mosquitoes, Compared to the Activity of B. sphaericus 2362 after 48 hr of Exposure
Cells/ml of culture ( x 10s) Strains
Total cells
Spores
2362
11.0
7.1
Sl
10.0
1.7
s2
12.0
1.4
s5
7.1
2.6
L2
7.2
1.4
ET AL.
Culex
LGO 0.70 (O.OW 7.70 (0.88) 4.90 (0.56) 0.89 (0.29) 8.90 (2.90) 1.51(0.49) 0.93 (0.49) 6.60 (3.48) 2.41 (1.27) 0.90 (0.23) 10.80 (2.76) 3.95 (1.01) 1.96 (0.31) 14.10 (2.23) 2.39 (0.38)
pipiens
w?o 1.67 (0.28)” 2.88 (1.41) 2.06 (1.18) 2.45 (0.50) 5.07 (1.00)
Anopheles
LGO 7.31 (2.32)” 80.00 (25.30) 51.00 (16.20) 10.65 (2.34) 106.00 (23.00) 18.00 (3.90) 12.74 (0.96) 90.00 (6.40) 12.70 (0.70) 6.93 (2.71) 82.80 (3.24) 30.30 (11.90) 13.53 (3.44) 97.40 (24.70) 18.90 (4.80)
stephensi
Aedes
LGO 12.05 (7.70)” 21.72 (3.60) 30.55 (2.51) 16.73 (3.00) 38.86 (10.22)
LGO 0.25 (0.08)* 2.79 (0.89jd 1.80 (0.57)f 0.40 (ND) 2.91 (ND) 0.56 (ND) 0.29 (0.05) 3.45 (0.69) 0.40 (0.08) 0.15 (0.01) 1.06 (0.06) 0.39 (0.02) 0.37 (0.27) 3.75 (2.73) 0.64 (0.46)
aegypti
LC90 2.18 (0.12)’ 2.43 (ND) 1.66 (0.19) 0.59 (0.06) 1.84 (1.60)
Note. The mean activity is expressed as dilution of the final whole culture (“x 1Om6 for C. pipiens and A. stephensi; *X lo-’ for A. aegypti) and in number of total cells/ml ("x lo2 for C. pipiens and A. stephensi; do lo6 for A. aegypti) or heat resistant spores (‘x 10’ for C. pipiens and A. stephensi; fx106 for A. mgypti). The numbers in parentheses indicate the standard deviation (n = 3). ND, not determined.
jetted to gel electrophoresis, all the Brazilian strains contained major proteins of 125, 110, 56, and 43 kDa, these latter two being apparently less intense in strain S5. Another major band of apparent molecular weight of 32 kDa was present in strains Sl and L2 (Fig. 1A). The proteins of 43 and 56 kDa migrated exactly the same distance as corresponding proteins of strain 2362 and were detected by affinity-purified polyclonal antibodies of these proteins (Figs. 1B and 10. The slight reaction of antibodies B. sphaericus 2362 43- or 56-kDa protein with the proteins of strain S5 could be due to the low amount of these proteins in this strain. No reaction was observed with the 32-kDa protein of strains Sl and S5. However a very slight reaction of both antibodies with the 125- and llO-kDa proteins kDa
0
A
could be detected if the exposure with 4-chloro-lnaphtol was prolonged (data not shown). The numerical taxonomic analysis of the fatty acid compositions of strains Sl, S2, S5, and L2, compared to those of strains 1593, 2297 and 2362, is shown in Fig. 2. All strains were analyzed in duplicate, except strains Sl and S5, which were analyzed in triplicate. Gas chromatography analysis appeared to be highly reproducible, because the maximum euclidian distance
- 0.7
L
- 0.6
r-Lr
C
- 0.5 - 0.4
mwa
bcde
abcde
a
bc
de
FIG. 1. SDS-PAGE of B. sphuericus samples (A) and the detection of immunorelated proteins by antisera to the 43-kDA (B) and the 56-kDA (C) proteins ofB. sphuericus 2362. Lanes: mw, molecular weight standards; a, B. sphuericus 2362 extract; b to e, cultures of B. sphuericus strains Sl (b), S2 cc), L2 cd), and S5 (e).
BACILLUS
!M+AERKXJS
STRAINS
FIG. 2. Results of a numerical taxonomic analysis based on the fatty acid compositions of the Brazilian strains compared to strains 1593,2362, and 2297 of B. sphuericus using the MIS system. Analyses were performed either in duplicate (strains S2, L2, 1593, 2297, and 2362) or in triplicate (strains Sl and S5).
MOSQUITOCIDAL
BRAZILIAN
between two duplicate analyses of a same strain was only 0.12 and was obtained with strain 2362. The euclidian distance between strains S2 and L2 and all other strains was greater than 0.3. However, the chance for strains Sl and S5 to actually be different is very low since the euclidian distance between fatty acid profiles of both strains was less than 0.12. Therefore, this analysis shows that all Brazilian strains differ from each other and from strain 1593, 2297, or 2362, with the possible exception of strains Sl and S5, which could be the same strain. DISCUSSION
This study shows that four B. sphuericus strains isolated from different sites in Brazil had a level of toxicity to C. pipiens, A. stephensi, and A. aegypti similar to that of strain 2362, which is now used in operational mosquito control. As the most active strains against mosquito larvae, these four strains harbor 43- and 56kDa proteins immunologically related to the toxins of B. sphaericus 2362. It is now established that the 43and 56-kDa proteins, of deduced molecular masses of 41.9 and 51.4 kDa, respectively (Hindley and Berry, 1987; Baumann et al., 19881, act as a binary toxin to mosquito larvae (Broadwell et al., 1990a; 1990b). DNA sequence analyses of the genes encoding these two proteins in strains originating from areas as distant as West Africa (strains 2362 and IAB59), Sri Lanka (strain 2297), Indonesia (strain 1593), or North America (strain 2317.31, are highly homologous between strains (Berry et al., 1989). This is probably also true for strain 1691 originating from El Salvador since it has a restriction pattern similar to that of strains 1593 and 2362 (Baumann et al., 1987). Strains Sl, S2, S5, and L2 harbor, as does strain 2362, the binary toxin composed of the 41.9- and 51.4kDa proteins, and high molecular weight proteins of 125 and 110 kDa, whose roles in toxicity are not clearly established. However, although these strains seem to be very close to the known mosquitocidal strains, regarding their host range, level of toxicity, and protein pattern, gas chromatography analysis of their fatty acids demonstrate that there are strains different from 2362, 2297, and 1593. Nevertheless, this technique does not allow one to confirm that strains Sl and S5 are actually the same strain. The isolation of these new strains from Brazil make the development of insecticides with indigenous entomopathogenic strains possible. Production of strain S2 on cheap media has now been undertaken at CENARGEN with optimistic results (Dias et al., 1990). ACKNOWLEDGMENTS We are grateful to Professor H. de Bajac, in whose laboratory this work was conducted. Thanks are due to J.-F. Charles, I. Thiery, V. Cosmao-Dumanoir (Institut Pasteur), and E. W. Davidson (Arizona State University) for helpful discussion and comments on the manu-
B. sphoericus STRAINS
13
script and to J. M. C. S. Dias (CENARGEN) for helpful advice. This work was partly supported by the United Nations Development Program/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases. R. G. M. Schenkel was supported by a grant from CNPq National Council for Science and Technology Development, Brasilia, during her stay at the Institut Pasteur. REFERENCES de Bajac, H. 1981. Identification of H-serotypes of Bacillus thuringiensis. In “Microbial Control of Pests and Plant Diseases 19701980” (H. D. Burges, Ed.), pp. 3543. Academic Press, New York. de Bajac, H., Thiery, I., Cosmao-Dumanoir, V., Frachon, E., Laurent, P., Charles, J. F., Hamon, S., and Ofori, J. 1988. Another Bacillus sphaericus serotype harhouring strains very toxic to mosquito larvae: Serotype H6. Ann. Inst. PasteurlMicrobioZ. 139, 363377. Baumann, P., Untermann, B., Baumann, L., Broadwell, A. H., Abbene, S. J., and Bowditch, R. D. 1985. Purification of the larvicidal toxin of Bacillus sphaericus and evidence for high molecular weight precursors. J. Bacterial. 163, 738-747. Baumann, P., Baumann, L., Bowditch, R. D., and Broadwell, A. H. 1987. Cloning of the gene for the larvicidal toxin of B. sphuericus 2362: Evidence for a family of related sequences. J. Bacterial. 169, 4061-4067. Baumann, L., Broadwell, A. H., and Baumann, P. 1988. Sequence analysis of the mosquitocidal toxin genes encoding 51.4- and 41.9kilodalton proteins from Bacillus sphaericus 2362 and 2297. J. Bacterial. 170, 2045-2050. Berry, C., Jackson-Yap., J., Oei, C., and Hindley, J. 1989. Nucleotide sequence of two toxin genes from Bacillus sphaericus IAB59: Sequence comparisons between five highly toxinogenic strains. Nucleic Acids Res. 17, 7516. Broadwell, A. H., Baumann, L., and Baumann, P. 1990a. The 42- and 52-kilodalton mosquitocidal proteins of Bacillus sphaericus 2362: Construction of recombinants with enhanced expression and in vivo studies of processing and toxicity. J. Bacterial. 172, 22172223. Broadwell, A. H., Baumann, L., and Baumann, P. 1990b. Larvicidal properties of the 42- and Bl-kilodalton Bacillus sphuericus proteins expressed in different bacterial hosts: Evidence for a binary toxin. Curr. Microbial. 21, 361-366. Burke, B., Griffiths, G., Reggio, H., Louvard, D., and Warren, G. 1982. A monoclonal antibody against a 135-K Golgi membrane protein. EMBO J. 1, 1621-1628. Dias, J. M. C. S., Honda, C. S., and Schenkel, R. G. M. 1990. Development of production process of Bacillus sphaericus, bioinsecticide against mosquitoes. In “Abstracts of 2” Simposio de Controle Biologic0 Siconbiol, de Volta a Natureza, Brasilia DF, 14-18 October 1990,” p. 99. Hindley, J., and Berry, C. 1987. Identification, cloning and sequence analysis of the BaciZlus sphaericus 1593 41.9 kD larvicidal toxin gene. Mol. Microbial. 1, 187-194. Kalfon, A., Larget-Thiery, I., Charles, J.-F., and de Barjac, H. 1983. Growth, sporulation and larvicidal activity of Bacillus sphuericus. Eur. J. Appl. Microbial. Biotechnol. 18, 168-173. Kellen, W. R., Clark, T. B., Lindegren, J. E., Ho, B. C., Rogoff, M. H., and Singer, S. 1965. Bacillus sphaericus Neide as a pathogen of mosquitoes. J. Znvertebr. Pathol. 7, 442-448. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
SCHENKEL Lysenko, O., Davidson, E. W., Lacey, L. A., and You&en, A. A. 1985. Five new mosquito larvicidal strains of Bacillus sphocricus from non-mosquito origins. J. Am. Mosq. Control Assoc. 1, 369-371. Miller, L., and Berger, T. 1985. Hewlett-Packard Gas Chromatography Application Note 228-38. Hewlett-Packard Co., Palo Alto, CA. Nicolas, L., Lecroisey, A., and Charles, J.-F. 1990. Role of the gut proteinases from mosquito larvae in the mechanism of action and the specificity of the Bacillus sphaericus toxin. Can. J. Microbial. 36, 804-807. Singer, S. 1973. Insecticidal activity of recent bacterial isolates and their toxins against mosquito larvae. Nature 244, 110-111. Singer, S. 1977. Isolation and development of bacterial pathogens of vectors. In “Biological Regulation of Vectors,” Publication No. (NIH) 77-1180, pp. 3-18. HEW, Washington, DC. Thiery, I., and de Barjac, H. 1989. Selection of the most potent Ba-
ET AL. cillus sphaericus strains based on activity ratios determined on three mosquito species. Appl. Microbid. Biotechnol. 31, 577681.
Weiser, J. 1984. A mosquito-virulent Bacillus sphaericus Simulium dumnosum from Northern Nigeria. Zentralbl. biol. 139, 57-60.
in adult Mikro-
World Health Organization. “Informal Consultation on the Development of Bacillus sphaericus as a Microbial Larvicide.” WHO Mimeograph Document WHOSPHAERICUS85.3. Wickremesinghe, R. S. B., and Mendis, C. 1980. Bacillus spore from Sri-Lanka demonstrating rapid larvicidal Culex
quinquefasciatus.
Mosq.
News.
sphaericus
activity on
40, 387389.
Yap, H. H., Ng, Y. M., Foo, A. E. S., and Tan, H. T. 1988. Bioassays of Bacillus sphaericus (strains 1593,2297 and 2362) against Mansonia and other mosquitoes of public health importance in Malaysia. Malays. Appl. Biol. 17, 9-13.
