Distribution of cry and cyt Genes among Indigenous Bacillus thuringiensis Isolates with Mosquitocidal Activity

Ayyasamy Mahalakshmi, Kabilan Sujatha, Poornima Kani, Rajaiah Shenbagarathai

PG and Research Department of Zoology and Biotechnology, Lady Doak College, Madurai, India.
UGC-NRCBS, School of Biological Sciences, Madurai Kamaraj University, Madurai, India.

DOI: 10.4236/aim.2012.23026                

Abstract

Bacillus thuringiensis strains isolated from Madurai, TamilNadu, India were evaluated for their mosquitocidal activity, as well as cry and cyt genes diversity. It revealed that 99% of the parasporal crystal morphology of these isolates was spherical in nature and a variable percentage (0% - 100%) of toxicity was observed against Culex quinquefasciatus and Aedes aegypti. PCR analysis revealed that 53% of the isolates were positive for the various cry and cyt genes tested, whereas 47% did not produce any PCR product for the cry gene analyzed. Diverse pattern of cry and cyt genes distribution was observed even in the isolates from the same sample. B. thuringienis subsp. LDC-9 showed three-fold higher toxicity against Culex quinquefasciatus than that of B. thuringiensis var israelensis which might be used as a potential strain to control mosquitoes in near future after field evaluation.

Keywords

Bacillus thuringiensis; Crystal Endotoxin; Culex quinquefasciatus; Aedes aegypti

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	L. Regis, M. H. Silva-Filha, C. Nielsen-LeRoux and J. F. Charles, “Bacterial Larvicides of Dipteran Disease Vectors,” Trends in Parasitology, Vol. 17, No. 8, 2001, pp. 377-380. doi:10.1016/S1471-4922(01)01953-5
[2]	B. A. Federici, H. W. Park, D. K. Bideshi, M. C. Wirth, J. J. Johnson, Y. Sakano and M. Tang, “Developing Recombinant Bacteria for Control of Mosquito Larvae,” Journal of the American Mosquito Control Association, Vol. 23, No. 2, 2007, pp.164-175. doi:10.2987/8756-971X(2007)23[164:DRBFCO]2.0.CO;2
[3]	G. I. Giraldo-Calderón, M. Pérez, C. A. Morales and C. B. Ocampo, “Evaluation of the Triflumuron and the Mixture of Bacillus thuringiensis plus Bacillus sphaericus for Control of the Immature Stages of Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae) in Catch Basins,” Biomedica, Vol. 28, No. 2, 2008, pp. 224-233.
[4]	K. V. Frankenhuyzen, “Insecticidal Activity of Bacillus thuringiensis Crystal Proteins,” Journal of Invertebrate Pathology, Vol. 101, No. 1, 2009, pp. 1-16. doi:10.1016/j.jip.2009.02.009
[5]	N. Raddadi, A. Belaouis, I. Tamagnini, B. M. Hansen, N. B. Hendriksen, A. Boudabous, A. Cherif and D. Daffonchio, “Characterization of Polyvalent and Safe Bacillus thuringiensis Strains with Potential Use for Biocontrol,” Journal of Basic Microbiology, Vol. 49, No. 3, 2009, pp. 293-303. doi:10.1002/jobm.200800182
[6]	G. Armengol, M. C. Escobar, M. E. Maldonado and S. Orduz, “Diversity of Colombian Strains of Bacillus thuringiensis with Insecticidal Activity against Dipteran and Lepidopteran Insects,” Journal of Applied Microbiology, Vol. 102, No. 1, 2007, pp. 77-88. doi:10.1111/j.1365-2672.2006.03063.x
[7]	N. Crickmore, D. R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum and D. H. Dean, “Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins,” Microbiology and Molecular Biology Reviews, Vol. 62, No. 3, 1998, pp. 807-813.
[8]	N. Crickmore, D. R. Zeigler, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, A. Bravo and D. H. Dean, “Bacillus thuringiensis Toxin Nomenclature,” 2009. http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt
[9]	M. Soberon, L. E. Fernández, C. Pérez, S. S. Gill and A. Bravo, “Mode of Action of Mosquitocidal Bacillus thuringiensis Toxins,” Toxicon, Vol. 49, No. 5, 2007, pp. 597-600. doi:10.1016/j.toxicon.2006.11.008
[10]	A. Bravo, S. S. Gill and M. Soberón, “Mode of Action of Bacillus thuringiensis Cry and Cyt Toxins and Their Potential for Insect Control,” Toxicon, Vol. 49, No. 4, 2007, pp. 423-435. doi:10.1016/j.toxicon.2006.11.022
[11]	A. Guerchicoff, R. A. Ugalde and C. P. Rubinstein, “Identification and Characterization of a Previously Undescribed cyt Gene in Bacillus thuringiensis subsp. israelensis,” Applied and Environmental Microbiology, Vol. 63, No. 7, 1997, pp. 2716-2721.
[12]	D. Wu, J. J. Johnson and B. A. Federeci, “Synergism of Mosquitocidal Toxicity between CytA and CryIV Proteins Using Inclusions Produced from Cloned Genes of Bacillus thuringiensis subsp. israelensis,” Molecular Microbiology, Vol. 13, No. 6, 1994, pp. 965-972. doi:10.1111/j.1365-2958.1994.tb00488.x
[13]	L. Allwin, J. S. Kennedy and V. Radhakrishnan, “Characterization of Different Geographical Strains of Bacillus thuringiensis from Tamil Nadu,” Research Journal of Agriculture and Biological Sciences, Vol. 3, No. 5, 2007, pp. 362-366.
[14]	J. Das and T. K. Dangar, “Diversity of Bacillus thuringiensis in the Rice Field Soils of Different Ecologies in India,” Indian Journal of Microbiology, Vol. 47, No. 4, 2007, pp. 364-368. doi:10.1007/s12088-007-0065-z
[15]	R. Lezama-Gutiérrez, J. J. Hamm, J. Molina-Ochoa, M. López-Edwards, A. Pescador-Rubio, M. González-Ramirez and L. E. Styer, “Occurrence of Entomopathogens of Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Mexican States of Michoacán, Colima, Jalisco and Tamaulipas,” Florida Entomologist, Vol. 84, No. 1, 2001, pp. 23-30.
[16]	S. B. Alves, “Isolamento de patógenos,” In: S. B. Alves, Ed., Controle Microbiano de Insetos, Manole, S?o Paulo, 1986.
[17]	S. Kalman, K. L. Kiehne, J. L. Libs and T. Yamamoto, “Cloning of a Novel cryIC Type Gene from a Strain of Bacillus thuringiensis subsp. galleriae,” Applied and Environmental Microbiology, Vol. 59, No. 4, 1993, pp. 1131-1137.
[18]	J. E. Ibarra and B. A. Federici , “Parasporal Bodies of Bacillus thuringiensis subsp. morrisoni (PG-14) and Bacillus thuringiensis subsp. israelensis are Similar in Protein Composition and Toxicity,” FEMS Microbiology Letters, Vol. 34, No. 1, 2006, pp. 79-84.
[19]	P. H. Johnson and L. I. Grossman, “Electrophoresis of DNA in Agarose gel. Optimizing Separations of Conformational Isomers of Double and Single-Stranded DNAs,” Biochemistry, Vol. 16, No. 19, 1977, pp. 4217-4225.
[20]	J. Spizizen, “Transformation of Biochemically Deficient Strains of Bacillus subtilis by Deoxyribonucleate,” Proceedings of the National Academy of Sciences USA, Vol. 44, No. 10, 1958, pp. 1072-1078. doi:10.1073/pnas.44.10.1072
[21]	M. Meadows, D. Ellis, J. Butt, P. Jarrett and H. Burges, “Distribution, Frequency and Diversity of Bacillus thuringiensis in an Animal Feed Mill,” Applied and Environmental Microbiology, Vol. 58, No. 4, 1992, pp. 1344-1350.
[22]	H. De Barjac and M. M. Lecadet, “Biochemical Determination of Bacillusthuringiensis Thermostabile, Exotoxin, Using the Inhibition of Bacterial RNA Polymerases,” Comptes Rendus Hebdomadaires des Seances de l Academie des Sciences. D: Sciences Naturelles, Vol. 282, No. 23, 1976, pp. 2119-2122.
[23]	W. A. Smirnoff and D. C. O’Connell, “Detection of Polyhedra from Insect Virus Diseases on Filter Membranes,” Stain Technology, Vol. 37, No. 4, 1962, pp. 207-210.
[24]	C. Johnson, A. H. Bishop and C. L. Turner, “Isolation and Activity of Strains of Bacillus thuringiensis Toxic to Larvae of the Housefly (Diptera: Muscidae) and Tropical Blowflies,” Journal of Invertebrate Pathology, Vol. 71, No. 2, 1998, pp. 138-144. doi:10.1006/jipa.1997.4720
[25]	K. Higuchi, H. Saitoh, E. Mizuki, T. Ichimatsu and M. Ohba, “Larval Susceptibility of the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae), to Bacillus thuringiensis H Serovars Isolated in Japan,” Microbiological Research, Vol. 155, No. 1, 2000, pp. 23-29. doi:10.1016/S0944-5013(00)80018-X
[26]	D. Finney, “Probit Analysis,” 3rd Edition, Cambridge University Press, Cambridge, 1971.
[27]	U. K. Laemmli, “Cleavage of Structural Proteins during Assembly of Head of Bacteriophage-T4,” Nature, Vol. 227, 1970, pp. 680-685. doi:10.1038/227680a0
[28]	F. Al-Momani, M. Obeidat, I. Saadoun and M. Meqdam, “Serotyping of Bacillus thuringiensis Isolates, Their Distribution in Different Jordanian Habitats and Pathogenicity in Drosophila melanogaster,” World Journal of Microbiology and Biotechnology, Vol. 20, No. 7, 2004, pp. 749-753. doi:10.1007/s11274-004-4517-x
[29]	J. H. Wang, A. Boets, J. Van Rie and G. X. Ren, “Characterization of cry1, cry2 and cry9 Genes in Bacillus thuringiensis Isolates from China,” Journal of Invertebrate Pathology, Vol. 82, No. 1, 2003, pp. 63-71. doi:10.1016/S0022-2011(02)00202-1
[30]	M. A. Hossain, S. Ahmed and S. Hoque, “Abundance and Distribution of Bacillus thuringiensis in the Agricultural Soil of Bangladesh,” Journal of Invertebrate Pathology, Vol. 70, No. 3, 1997, pp. 221-225. doi:10.1006/jipa.1997.4694
[31]	S. Hastowo, B. W. Lay and M. Ohba, “Naturally Occurring Bacillus thuringiensis in Indonésia,” Journal of Applied Bacteriology, Vol. 73, No. 2, 1992, pp. 108-113. doi:10.1111/j.1365-2672.1992.tb01695.x
[32]	K. Bernhard, P. Jarrett, M. Meadows, J. Butt, D. J. Ellis, G. M. Roberts, S. Pauli, P. Rodgers and H. G. Burges, “Natural Isolates of Bacillus thuringiensis: World Wide Distribution, Characterization, and Activity against Insect Pests,” Journal of Invertebrate Pathology, Vol. 70, No. 1, 1997, pp. 59-68. doi:10.1006/jipa.1997.4669
[33]	M. Obeidat, “Toxicity of Local Bacillus thuringiensis Isolates against Drosophila melanogaster,” World Journal of Agricultural Sciences, Vol. 4, No. 2, 2008, pp. 161- 167.
[34]	M. Porcar and V. Juarez-Perez, “PCR Based Identification of Bacillus thuringiensis Pesticidal Crystal Genes,” FEMS Microbiology Reviews, Vol. 26, 2003, pp. 419-432. doi:10.1111/j.1574-6976.2003.tb00624.x
[35]	S. Ibanez-Bernal, D. Strickman and C. Martínez-Campos “Culidae (Diptera),” In: J. Llorente Bousquets, A. N. García Aldrete and E. González Soriano, Eds., Biodiversidad, Taxonomía y Biogeografía de Artrópodos en México, Universidad Nacional Autónoma, México City, 1996, pp. 591-602.
[36]	A. Bravo, S. Sarabia, L. Lopez, H. Ontiveros, C. Abarca, A. Ortiz, M. Ortiz, L. Lina, J. V. Francisco, P. Guadalupe, N. V. María-Eugenia, S. Mario and Q. Rodolfo, “Characterization of cry Genes in a Mexican Bacillus thuringiensis Strain Collection,” Applied and Environmental Microbiology, Vol. 64, No. 12, 1998, pp. 4965-4972.
[37]	A. Uemori, M. Maeda, K. Yasutake, A. Ohgushi, K. Kagoshima and M. Ohba, “Ubiquity of Parasporin-1 Producers in Bacillus thuringiensis Natural Population of Japan,” Naturwissenschaften, Vol. 94, No. 1, 2007, pp. 34-38. doi:10.1007/s00114-006-0153-7
[38]	M. Ohba, “Bacillus thuringiensis in Nature: Ubiquity and Diversity” In: M. Ohba, O. Nakamura, E. Mizuki and E. Akao, Eds., Proceedings of a Centennial Symposium Commemorating Ishiwata’s Discovery 416 of Bacillus thuringiensis, Kurume, Japan, 2001, pp. 141-145.
[39]	C. Martinez and P. Caballero, “Contents of cry Genes and Insecticidal Toxicity of Bacillus thuringiensis Strains from Terrestrial and Aquatic Habitats,” Journal of Applied Microbiology, Vol. 92, No. 4, 2002, pp. 745-752. doi:10.1046/j.1365-2672.2002.01579.x