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5269–5274.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 69, No. 9
Diversity of Bacillus thuringiensis Strains from Latin America with Insecticidal Activity against Different Mosquito Species
Jorge E. Ibarra,1 M. Cristina del Rinco ´n,1 Sergio Ordu ´z,2 David Noriega,2 3 4 Graciela Benintende, Rose Monnerat, Leda Regis,5 Cla ´udia M. F. de Oliveira,5 Humberto Lanz,6 Mario H. Rodriguez,6 Jorge Sa ´nchez,7 7 Guadalupe Pen ˜a, and Alejandra Bravo7*
CINVESTAV IPN, Irapuato 36500, Guanajuato,1 and Instituto Nacional de Salud Publica, Centro de Investigacio ´n sobre Enfermedades Infecciosas, Cuernavaca 62508,6 and Instituto de Biotecnología UNAM, Cuernavaca 62250,7 Morelos, Me ´xico; Corporacio ´n para Investigaciones Biologicas, 7378 Medellín, Colombia2; Instituto de Microbiología y Zoología Agrícola-INTA, Buenos Aires, Argentina3; and EMBRAPA-Recursos Gene ´ticos e Biotecnologia, Brasília/DF,4 and Fundac ¸a ˜o Oswaldo Cruz FIOCRUZ, Recife PE,5 Brazil
Received 16 April 2003/Accepted 1 July 2003
The characterization of selected Bacillus thuringiensis strains isolated from different Latin America countries is presented. Characterization was based on their insecticidal activity against Aedes aegypti, Culex quinquefasciatus, and Anopheles albimanus larvae, scanning electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and plasmid proﬁles as well as PCR analysis using novel general and speciﬁc primers for cry and cyt genes encoding proteins active against mosquitoes (cyt1, cyt2, cry2, cry4A, cry4B, cry10, cry11, cry17, cry19, cry24, cry25, cry27, cry29, cry30, cry32, cry39, and cry40). Strains LBIT315, LBIT348, and IB604 showed threefold higher mosquitocidal activity against A. aegypti and C. quinquefasciatus larvae than B. thuringiensis subsp. israelensis and displayed high similarities with the B. thuringiensis subsp. israelensis used in this study with regard to protein and plasmid proﬁles and the presence of cry genes. Strain 147-8906 has activity against A. aegypti similar to that of B. thuringiensis subsp. israelensis but has different protein and plasmid proﬁles. This strain, harboring cry11, cry30, cyt1, and cyt2 genes, could be relevant for future resistance management interventions. Finally, the PCR screening strategy presented here led us to identify a putative novel cry11B gene. Vector-borne diseases are major public health problems, and their prevalence has dramatically increased worldwide (1, 12). Dengue and malaria are transmitted to humans through Aedes spp. and Anopheles spp. mosquitoes, respectively. Bancroftian ﬁlariasis and the West Nile virus are transmitted by Culex quinquefasciatus. At present, the best control methods for these diseases are based on vector control (1) that is mainly accomplished by using synthetic insecticides. Additionally, mosquitoes have developed resistance to the major chemical insecticide groups. For instance, global DDT spraying to control mosquito populations succeeded for only 8 years, as mosquito resistance appeared (1). The use of entomopathogenic bacteria Bacillus thuringiensis and Bacillus sphaericus as biolarvicides is a viable alternative for insect control (12). B. thuringiensis produces proteinaceous inclusions during sporulation that are toxic towards insect larvae upon ingestion (15). The parasporal body of B. thuringiensis consists of one or more insecticidal ␦-endotoxins (named Cry and Cyt) (http://www.biols.susx.ac.uk/Home/Neil _Crickmore/Bt/index.html). These toxins are highly speciﬁc, are harmless to humans, vertebrates, and plants, and are completely biodegradable, so no residual toxic products accumulate in the environment (15). The number of known B. thuringiensis strains active on diptera is growing (6, 19). A feature of all mosquitocidal strains is the presence of Cyt toxin, which is not very toxic by itself. The mosquitocidal activity of a B. thuringiensis strain is not only the additive effect of each toxin but a complex synergistic interaction among them. B. thuringiensis subsp. israelensis produces four Cry toxins (Cry4Aa, Cry4Ba, Cry10Aa, and Cry11Aa) and two Cyt toxins (Cyt1Aa and Cyt2Ba) (6), and synergism between these toxins has been reported (4, 18). Interestingly, the presence of the Cyt toxin delays or prevents the development of resistance to Cry toxins (17). B. thuringiensis subsp. israelensis has been used in the ﬁeld for nearly 20 years with no development of insect resistance (7, 11). The search for native strains with activity against dipteran species could have an impact on the control of mosquitoes worldwide. Here, we characterized selected strains from Latin American B. thuringiensis collections. The characterization included the identiﬁcation of mosquitocidal cry and cyt genes by multiplex PCR analysis with novel general and speciﬁc primers. B. thuringiensis strains containing some of the previously described cry genes as well as potentially novel Cry proteins were identiﬁed. Also, B. thuringiensis strain 147-8906 showed toxic activity against A. aegypti similar to that of B. thuringiensis subsp. israelensis but showed different gene content. This strain has potential interest for insect resistance management.
* Corresponding author. Mailing address: Instituto de Biotecnología, Universidad Nacional Autonoma de Mexico, Apartado Postal 510-3, Cuernavaca 62250, Morelos, Me ´xico. Phone: (52-73) 291635. Fax: (52-73) 172388. E-mail: firstname.lastname@example.org.
Bioassays.ncbi. Coleoptera 100 vs. thuringiensis strains was screened on larvae of the three mosquito species as described previously (10). thuringiensis strains were grown for 12 h on a nutrient medium plate. aegypti (CINVESTAV strain) at CINVESTAV-Irapuato in Mexico and (CIB strain) at Corporacio ´n para Investigaciones Biologicas in Colombia.000 ng/ml) of the spore-crystal complex of each strain were added (four repetitions). quinquefasciatus larvae when compared with the B. Samples were coated with gold in a Fullam EMS-76 M evaporator for 9 min and examined and photographed with a Philips XL30-ESEM scanning electron microscope operating at a voltage of 10 to 15 kV at ϫ2. thuringiensis subsp. Coleoptera SDS-PAGE SDS-PAGE. and 147-8906. SDS-PAGE and plasmid proﬁle analysis. 919 vs. israelensis positive control was included in addition to a dechlorinated water control. and A.1% Casamino Acids (DIFCO) and 0. Other assays performed Brazil Mexico Mexico (reference 3) Colombia 1. albimanus. aegypti. thuringiensis subsp. LBIT348. albimanus.0). and Colombia (Table 1).) (5).000 rpm for 20 min in a Sorvall SS34 centrifuge. LBIT320. Protein standards were carbonic anhydrase (29 kDa). Concentrated spore-crystal suspensions on disruption buffer were boiled for 5 min.424 vs. Cells were washed in TE (50 mM Tris. beta-exotoxin SDS-PAGE. 1% sodium dodecyl sulfate [SDS]) was added and the mixture was incubated for 5 min at 4°C. 0. The sequences were analyzed with BLAST and ClustalW (5. cry11 cry1 cry1 (ϳ49%). LBIT348. 1. Oligonucleotide PCR primers. The mean 50% lethal concentration was estimated by Probit analysis using statistical parameters (9). quinquefasciatus (Recife strain) at Centro de Pesquisas Aggeu Magalha ˜es/FIOCRUZ in Brazil.552 vs. The SDS-PAGE of spore-crystal suspensions of selected strains is shown in Fig.nih. Puriﬁed fragments were sequenced in the facilities of Universidad Nacional Autonoma de Mexico by using the same primers as used for ampliﬁcation (GenBank no. ␤-galactosidase (116. Calif. cry11. Oligonucleotides were synthesized in a DNA synthesizer (Microsyn 1450A. israelensis HD567 and B.000 rpm in an Eppendorf 5415C centrifuge). Ohio). Diluted suspensions of spore-crystal complexes were placed on aluminum stubs and air dried. of strains toxic to the indicated genus) APPL. Scanning electron microscopy. A loopful of cells was transferred to 0. aegypti when compared with B. phosphorylase B (97.780 cry1 (ϳ90%). Inc.000 and ϫ18. Valencia. 87% relative humidity. thuringiensis subsp. annealing (Table 2) for 1 min. B. and PCR was carried out in a PerkinElmer 480 thermal cycler as follows: 2 min at 95°C. cry3. (iii) PCR using reported primers to identify some cry genes. Riverside. Cells were brieﬂy spun (10 s at 10. 30 cycles of 95°C for 1 min. thuringiensis strains were isolated (16) from soil samples from Mexico.4% K2HPO4. Lepidoptera. Table 3 shows the insecticidal activities of selected strains against A.) as speciﬁed by the manufacturer. and a 12:12 dark/light photoperiod as follows: C.8]) and incubated for 30 min at 37°C in 10 mg of lysozyme/ml in 0. 1. quinquefasciatus and A. by using multiple alignments of reported DNA sequences (Table 2) using ClustalW and GeneWorks 2.5 M sucrose. Lepidoptera. Microscopic observation of the crystals.2 kDa).gov/BLAST/). thuringiensis strain collections used in this study were previously characterized by different methodologies (2) (Table 1). dissolved in distilled water. cyt cry1 (55%). thuringiensis subsp.02% MgSO4-7H2O) with 0. Strains LBIT315. http://www.2% NH4SO4. 0. cry10.5% glucose.5270 IBARRA ET AL.4 kDa). and 72°C for 1 min. Figure 1A shows the scanning electron microscopy observation of the crystals produced by strains LBIT315. . The diameter of the crystals was 0. was added and stored for 20 min at Ϫ20°C. and the mixture was incubated for 20 min at Ϫ80°C to precipitate DNA. thuringiensis subsp. Lepidoptera. cry8. Diptera. ovalbumin (45 kDa). After 10 min at 4°C. cry3. RESULTS The B. Twenty early fourth-instar larvae were placed in 100 ml of dechlorinated water. ENVIRON. enzyme-linked immunosorbent assay SDS-PAGE MATERIALS AND METHODS Bacterial strains.3 (Intelligenetics. israelensis control. and 15 l of supernatant was used as DNA template in the PCR.nlm. Two volumes of ethanol were added. Particles were centrifuged at 12. Diptera. Characteristics of B. IB604. A solution of 3 M sodium acetate.25 kDa).1 m. and 10 mM EDTA (pH 8. 25 mM Tris. PCR mixtures were prepared as described previously (2. thuringiensis strain collections used in this study Country No. thuringiensis subsp. thuringiensis strains were grown to an optical density at 600 nm of 0. and IB604 showed threefold-higher activity against A. B. Lepidoptera. and B. SDS-polyacrylamide gel electrophoresis (SDSPAGE) was performed as described previously (14). The B. A 15-l sample was electrophoresed on 2% agarose gel. and 5 min at 72°C. TABLE 1. 0. pH 4.01% yeast extract. B. 420 vs. 320 vs. namely.6% KH2PO4. Sequence of PCR products. Calif. MICROBIOL.8 in Spizizen medium (0. Diptera. quinquefasciatus. cry7. thuringiensis strains active against mosquitoes were selected for further characterization. Western and Southern blot. cry11 229 vs. Lyophilized spore-crystal complexes were used in the bioassays. Insecticidal activity of B.378 683 690 3. S. DNA was centrifuged as above. 3).6% agarose gels. Two types of novel primers were designed. Larvae were incubated at 28°C and examined after 24 h. kurstaki HD1 were from Bacillus Genetic Stock Center (Columbus. and boiled for 10 min in water to lyse the cells. israelensis. We focused on strains that had higher activity than B. and visualized in 0. Gill (University of California—Riverside. 10 mM EDTA [pH 7. of B. and A. thuringiensis strains were grown in M-1 medium (13) at 30°C with agitation at 200 rpm until sporulation was complete. plasmid patterns. 34 vs. 412 vs. albimanus (INSP strain) at Instituto Nacional de Salud Pu ´blica in Mexico.9 to 1. Other B. B. thuringiensis subsp. 425 vs. Finally. and myosin (205 kDa).1% sodium citrate. general primers (gen) from conserved regions of related cry genes and speciﬁc primers (spe) from highly variable regions. cry3. cry4. These strains showed small ovoid crystal inclusions very similar to those found in B. 0. cry9.). israelensis but a different array of Cry proteins. (ii) enzymelinked immunosorbent and Western blot assays using different polyclonal antisera to identify the Cry groups. cry2. thuringiensis subsp. PCR products were puriﬁed from 1% agarose gels with a QIAquick gel extraction kit as described by the manufacturer (Qiagen. Coleoptera 1. israelensis or that showed similar activity to B.000 magniﬁcations. Insecticidal activity. bovine serum albumin (66. C. cry4. thuringiensis subsp. frozen for 20 min at Ϫ70°C. Ten concentrations (2 to 2.2 M NaOH. jegathesan was kindly supplied by S.1 ml of H2O. strain 147-8906 showed similar insecticidal activity against A. lysis buffer (0. and (iv) bioassays against different insect species to identify active strains. Protein electrophoresis. Systec Inc. Mosquitoes were reared at 28°C. cry8. serotyping. 410 vs. AY326510 and AY326511). Brazil. The analysis included (i) SDS-PAGE of spore-crystal to determine the number and size of Cry proteins. LBIT320. Sample preparation and PCR. aegypti and twofoldhigher activity against C. Coleoptera 400 vs. Plasmid patterns. A.8. thuringiensis strains cry genes recognized (% of strains if less than 100%) Toxicity assay results (no. B. Diptera. israelensis but showed lower activity against C.).
cry12. 5Ј CTCATAATTTTCCGTCCATAAAT (r) 5Ј TTATCAATGTTAAGGGATGC (d). cry8. 5Ј CGGCTTGATCTATGTCATAATCTGT (r) 51 50 5Ј TCAATGCTCCATCCAATG (d). cry11-gen. and cry25 genes. 65. In contrast. Strains LBIT315. and IB604 had a protein proﬁle similar to that reported for B. The B. 69. israelensis showed the expected PCR products with cry4-spe. thuringiensis subsp. thuringiensis subsp. 2003 MOSQUITOCIDAL STRAINS OF BACILLUS THURINGIENSIS 5271 TABLE 2. cry5. 5Ј GCTGAAACGGCACGAATATAATA (r) 5Ј CATTGTTCTACTTGGTATAA (d). B. israelensis (Fig. and 26 kDa. Some cry and cyt genes were identiﬁed in the native strains . jegathesan also showed the expected PCR products when ampliﬁed with cry11-gen and cyt2gen primers. 5Ј CTTGTATAGGCCTTCCTCCG (r) 5Ј CGCTTACAGGATGGATAGG (d). In contrast. with major proteins of 130. B. 75. d and r. cry21. cry9. 5ЈGGAAAAGAGAATATAAAAATGGCCAG (r) cry4Aspe cry4Bspe cry10spe cry11gral 50 5Ј TCAAAGATCATTTCAAAATTACATG (d) 5ЈCGTTTTCAAGACCTAATAATATAATACC (d). Characteristics of general and speciﬁc primers for cry1. The PCR methodology described here utilized 27 primers to detect 17 different cry and cyt genes described to codify for proteins active against mosquitoes (Table 2). 1C). thuringiensis subsp. israelensis. cry14. cry11. Figure 2A shows the PCR products obtained with control strains. cry13. 5Ј ATGTTTGCGACACCATTTTC (r) cry17 ϩ 27 cry19 ϩ 39 47 51 cry24 ϩ 40 cry25esp cry29esp cry30esp cry32gral 48 53 50 50 54 a b Position at 5Ј end of direct and reverse primers for each PCR primer pair. strain 147-8906 displayed a different plasmid proﬁle. respectively. and 28 kDa (8). HD1 strain yielded a PCR product only when ampliﬁed with the cry2-gen primers. 5Ј ACTGGATCTGTGTATATTTTCCTAG (r) 5Ј GGCTTCTAGATCAGGAGATGG (d). and IB604 strains also had plasmid proﬁles similar to that of B. cyt1gral 52 5Ј CCTCAATCAACAGCAAGGGTTATT (d). LBIT320. and cyt-gen primers. 1B. 5Ј TTTCAACATCCACAGTAATTTCAAATGC (r) 197–674 85–565 97–574 509–865 529–884 649–1004 196–551 531–1057 376–902 2500–3020 1041–1541 1706–2165 1868–2189 978–1326 990–1332 1025–1368 1048–1400 645–1477 1306–2201 1332–1948 1209–1840 597–1216 595–899 593–959 1146–1706 897–1348 2479–2744 2236–2913 2338–3014 2254–2930 2218–2894 cyt1Aa cyt1Ab cyt1Ba cyt2Aa cyt2Ba cyt2Bb cyt2Ca cry2Aa cry2Ab cry2Ac cry2Ad cry4aA cry4Ba cry10 cry11Aa cry11Ba cry11Bb cry17Aa cry27Aa cry19Aa cry19Ba cry39 cry24Aa cry40Aa cry25 cry29 cry30 cry32Aa cry32Ba cry32Ca cry32D 477 480 477 356 355 355 355 526 526 520 500 459 321 348 342 343 352 832 895 616 631 619 304 366 560 451 265 677 676 676 676 X03182 X98793 U37196 Z14147 U52043 U82519 AAK50455 M31738 M23724 X57252 AF200816 Y00423 X07423 M12662 M31737 X86902 AF017416 X99478 AB023293 Y07603 D88381 BAB72016 U88188 BAB72018 U88189 AJ251977 AJ251978 AY008143 BAB78601 BAB78602 BAB78603 cyt2gral 50 cry2gral 50 5Ј GAGTTTAATCGACAAGTAGATAATTT (d). LBIT348. 70. thuringiensis subsp. direct and reverse primers. 5Ј GATACAATTACATCTCCTCCTGTA (r) 5Ј AAGCTGCGAATCTGCATTTACTTTT (d). LBIT320.VOL. LBIT348. 5Ј ATCGGAAGGCAATCATTCG (r) 5Ј TGGTCGGGAGAGAATGGATGGA (d). 5Ј CATCATAATCAGAGCGCAGG (r) 5Ј TCAGCTCCAATAACTGGTG (d). LBIT315. thuringiensis subsp. cry24. jegathesan was used as a control for cry19. thuringiensis isolates. 5Ј GCATGTCATCCCCTTGTCTA (r) 5Ј AACTCACACATCCTCCATCG (d). Strain 147-8906 had four different protein bands of 100. 5Ј TGCAAACAGGACATTGTATGTGTAATT (r) 5Ј ATTACAAATTGCAAATGGTATTCC (d). Identiﬁcation of speciﬁc cry genes in the B. and cyt genes Primer pair Anneal temp (°C) Sequenceb Positionsa Gene(s) recognized Product size (bp) GenBank accession no.
2B). israelensis HD567 strains.5) 20. thuringiensis subsp. israelensis HD567.8) 12. AY326510) indicated that it corresponds to a new variant of cry11B (72% amino acid identity to Cry11Bb in the sequenced region). israelensis.5–17.9–44.6) 21. megadaltons. and LBIT348 were similar to the B. open circles. MICROBIOL.5) cry4A cry4B cry11 cyt1 cyt2 cry4A cry4B cry11 cyt1 cyt2 cry4A cry4B cry10 cry11 cyt1 cyt2 cry4A cry4B cry11 cyt1 cyt2 cry11 cyt1 cyt2 cry30 cry4A cry4B cry10 cry11 cyt1 cyt2 B.1) 13. 147-8906.3) 14. thuringiensis subsp. this strain produced a PCR product with a different size than expected when assayed with the cry4B-spe primers (Fig. Characterization of putative novel cry genes. However. (Table 3). The direct primer hybridizes to the 3Јregion of this gene. cry11. The BLAST analysis of the sequence of this PCR product (GenBank no.8–4. DISCUSSION The great extension of Latin America. Strains LBIT315. LBIT320.4–4.7–130.8) 4. thuringiensis subsp. thuringiensis strains. (A) Scanning electron microscopy observation of the spore and crystal mixtures produced by LBIT315. (B) SDS-PAGE of spore-crystal suspensions of selected B. BLAST analysis indicated that it corresponds to the cry30 gene (100% identity).1) 31. OC.52 (5.51 (10. albimanus cry and cyt gene proﬁle LBIT315 LBIT320 IB604 LBIT348 147-8906 Btia a 4. thuringiensis strains with higher activity against mosquitoes than the B. 2B). aegypti and C. and diversity of insects provide the opportunity of isolating novel entomopathogenic bacteria. israelensis demonstrated that strain 447BrB produced a different size PCR product with the cry11-gen primers (Fig. thuringiensis strains Strain 50% lethal concn (ng/ml) (95% conﬁdence interval) for: A.15 (13. B. 1. LBIT320. . thuringiensis subsp. However.7) 4. as the novel primers could identify 17 different cry and cyt genes that codify for proteins active against mosquitoes. Arrows point to crystal inclusions (Cry) and spores (Spo).23 (3. LBIT348.08 (4. IB604.9–9. israelensis.72 (23.3–16. thuringiensis strains that had higher activity than B. We identiﬁed B. AY326511). Characterization of the selected B. Dose-response insecticidal activities and cry and cyt gene proﬁle present in the selected B.9) 30. thuringiensis strains that showed lower toxicity against mosquitoes than B.52 (14.2–51. (C) Agarose gel electrophoresis of the plasmid proﬁle present in selected B. thuringiensis strain analyzed in this report represents a sample of this diversity. israelensis strain with the exception of the cry10 gene that was absent. thuringiensis strains.17 (21.7) 93. aegypti C.21 (6.5272 IBARRA ET AL.2–8.8) 7.7–16. thuringiensis subsp. quinque- TABLE 3.61 (5.2) 11.0–14. quinquefasciatus A. the PCR strategy described here could be useful for the characterization of other B. The PCR analysis performed with other B.86 (11.91 (18. and the reverse primer hybridizes outside of the coding region.7–23. strain 447BrB presented low toxicity against the mosquito larvae and thus was not further characterized.6–5. and cry30 genes. APPL. The PCR product of strain 147-8906 obtained with cry4B-spe primers was sequenced (GenBank accession no. israelensis strain.85 (10.16 (3. MD. Strain 147-8906 was positive with PCR primers of cyt1. thuringiensis subsp. its different climatic regions.5–21. and B. thuringiensis subsp.12 (3. ENVIRON.3) 19. FIG.4) 6. thuringiensis strain collections. The B. Strain IB604 had the same cry and cyt genes present in B.13 (8.5–7. Additionally. israelensis against A.1–28.6) 5. thuringiensis and B.3) 17. thuringiensis subsp.90 (76.0) 7.8–28.15 (15. cyt2.5–4.
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