An Overview on the Crystal Toxins from Bacillus thuringiensis

Veloorvalappil N. Jisha, Robinson B. Smitha, Sailas Benjamin

.
Enzyme Technology Laboratory, Biotechnology Division, Department of Botany, University of Calicut, Kerala, India.

DOI: 10.4236/aim.2013.35062                

Abstract

Strains of Bacillus thuringiensis (Bt) are known to produce crystalline proteins (δ-endotoxins) concomitantly with sporulation during their stationary phase of growth, which are demonstrated as lethal to lepidopeterous, coleopeterous and dipterous insects in addition to mites, nematodes, protozoa and flukes. Upon ingestion, the δ-nascent endotoxin is an inactive protoxin complex of (Cry alone or Cry and Cyt toxins together) high molecular mass, which is cleaved upon ingestion into the active component proteins at the high alkaline environments in the digestive tract of these agricultural pests. Conventionally, Bt-crystals are being produced employing submerged or liquid fermentation techniques in commercial media, but recently many workers have used solid-state fermentation strategy for the enhanced production of Bt-toxin at low cost. Apart from δ-endotoxin, some isolates of Bt produce another class of insecticidal small molecules called β-exotoxin (thuringiensin), which may be harmful to humans. Moreover, resistance to Bt developed in various target pest is yet another concern for Bt-industry. Following a brief introduction, this review addresses various toxins produced by various strains of Bt, Bt production media and media formulations with emphasis to solid-state fermentation, general structure of Cry toxin, its mode of action, target pests, bioassay, resistance to Bt toxins and resistance management. Briefly, this review would provide the readers an overview on the general aspects of Bt toxin, its general structure and mechanism of action.

Keywords

Bacillus thuringiensis; δ-Endotoxin; Resistanc

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	R. J. Milner, “History of Bacillus thuringiensis,” Agriculture Ecosystems and Environment, Vol. 49, No. 1, 1994, pp. 9-13. doi:10.1016/0167-8809(94)90014-0
[2]	J. C. Lord, “From Metchnikoff to Monsanto and beyond: The Path of Microbial Control,” Journal of Invertebrate Pathology, Vol. 89, No. 1, 2005, pp. 19-29. doi:10.1016/j.jip.2005.04.006
[3]	E. W. Nester, L. S. Thomashow, M. Metz and M. Gordon, “100 Years of Bt, a Critical Scientific Assessment,” American Academy of Microbiology, 2002.
[4]	L. A. Lacey, R. Frutos, H. K. Kaya and P. Vail, “Insect Pathogens as Biological Control Agents: Do They Have a Future?” Biological Control, Vol. 21, No. 3, 2001, pp. 230-248. doi:10.1006/bcon.2001.0938
[5]	R. A. de Maagd, A. Bravo and N. Crickmore, “How Bacillus thuringiensis Has Evolved Specific Toxins to Colonize the Insect World,” TRENDS in Genetics, Vol. 17, No. 4, 2001, pp. 193-199. doi:10.1016/S0168-9525(01)02237-5
[6]	M. Marvier, C. Mc Creedy, J. Regetz and P. Karieva, “A Meta-Analysis of Effects of Bt Cotton and Maize on Nontarget Invertebrates,” Science, Vol. 316, No. 5830, 2007, pp. 1475-1477. doi:10.1126/science.1139208
[7]	R. B. Smitha, V. N. Jisha, S. Pradeep, M. K. Sarath Josh and Sailas Benjamin, “Potato Flour Mediated Solid State Fermentation for Enhanced Production of Bt-Toxin,” Journal of Bioscience and Bioengineering.
[8]	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
[9]	H. Agaisse and D. Lereclus, “How does Bacillus thuringiensis Produce so Much Insecticidal Crystal Protein?” Journal of Bacteriology, Vol. 177, No. 21, 1995, pp. 6027-6032.
[10]	D. G. Mc Dowell and N. H. Mann, “Characterization and Sequence Analysis of a Small Plasmid from Bacillus thuringiensis var. kurstaki Strain HD1-DIPEL,” Plasmid, Vol. 25, No. 2, 1991, pp. 113-120. doi:10.1016/0147-619X(91)90022-O
[11]	C. C. Beegle and T. Yamamoto, “History of Bacillus thuringiensis Berliner Research and Development,” Canadian Entomologist, Vol. 124, No. 4, 1992, pp. 587-616. doi:10.4039/Ent124587-4
[12]	A. Bravo, S. Likitvivatanavong, S. Gill and M. Soberón, “Bacillus thuringiensis: A Story of a Successful Bioinsecticide,” Insect Biochemistry and Molecular Biology, Vol. 41, No. 7, 2011, pp. 423-443. doi:10.1016/j.ibmb.2011.02.006
[13]	A. Sierpinska, “Toxicity of Bacillus thuringiensis to Some Defoliating Forest Lepidoptera,” Folia Forestalia Polonica. Seria A—Lesnictwo, Vol. 39, 1997, pp. 79-92.
[14]	H. Bti, “Production of Bacillus thuringiensis subsp. Israelensis from Agro-Based Product (corncob),” Current Science, Vol. 101, No. 8, 2011, p. 1009.
[15]	S. Kalman, K. L. Kiehne, N. Cooper, M. S. Reynoso and T. Yamamoto, “Enhanced Production of Insecticidal Proteins in Bacillus thuringiensis Strains Carrying an Additional Crystal Protein Gene in Their Chromosomes,” Applied and Environmental Microbiology, Vol. 61, No. 8, 1995, pp. 3063-3068.
[16]	F. Fargette and N. Grelet, “Bacillus thuringiensis Berliner Mutants Resistant to Various Antibiotics and Showing Alterations of Sporulation,” Comptes Rendus Hebdomadaires des Séances de l’Académie des Sciences. Série D: Sciences Naturelles, Vol. 280, No. 4, 1975, p. 499.
[17]	I. Mahler and H. O. Halvorson, “Two ErythromycinResistance Plasmids of Diverse Origin and Their Effect on Sporulation in Bacillus subtilis,” Journal of General Microbiology, Vol. 120, No. 1, 1980, pp. 259-263.
[18]	J. S. Feitelson, J. Payne and J. Kim, “Bacillus thuringiensis: Insects and beyond,” Nature Biotechnology, Vol. 10, No. 3, 1992, pp. 271-275. doi:10.1038/nbt0392-271
[19]	H. E. Schnepf, N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D. R. Zeigler and D. H. Dean, “Bacillus thuringiensis and Its Pesticidal Crystal Proteins,” Microbiolopgy and Molecular Biology Review, Vol. 62, No. 3, 1998, pp. 775-806.
[20]	R. A. de Maagd, A. Bravo, C. Berry, N. Crickmore and H. E. Schnepf, “Structure, Diversity, and Evolution of Protein Toxins from Spore-Forming Entomopathogenic Bacteria,” Annual Review of Genetics, Vol. 37, No. 1, 2003, pp. 409-433. doi:10.1146/annurev.genet.37.110801.143042
[21]	R. B. Smitha, “Dual Production of Endotoxin and Amylase from Bacillus thuringiensis Subsp kurstaki by Fermentation and Efficacy Studies of Endotoxin against Eriophyid Mite,” Ph.D. Thesis, University of Calicut, India, 2010.
[22]	S. Guo, M. Liu, D. Peng, S. Ji, P. Wang, Z. Yu and M. Sun, “New Strategy for Isolating Novel Nematicidal Crystal Protein Genes from Bacillus thuringiensis Strain YBT-1518,” Applied Environmental Microbiology, Vol. 74, No. 22, 2008, pp. 6997-7001. doi:10.1128/AEM.01346-08
[23]	D. L. Berlitz, A. O. D. Azambuja, A. Sebben, J. V. D. Oliveira and L. M. Fiuza, “Mortality of Oryzophagus Oryzae (Costa Lima, 1936) (Coleoptera: Curculionidae) and Spodoptera Frugiperda (JE Smith, 1797) (Lepidoptera: Noctuidae) Larvae Exposed to Bacillus thuringiensis and Extracts of Melia azedarach,” Brazilian Archives of Biology and Technology, Vol. 55, No. 5, 2012, pp. 725-731. doi:10.1590/S1516-89132012000500012
[24]	P. Toledo, T. Liedo, T. Williams and J. Ibarra, “Toxicity of Bacillus thuringiensis-Exotoxin to Three Species of Fruit Flies (Diptera: Tephritidae),” Journal of Economic Entomology, Vol. 92. No. 5, 1999, pp. 1052-1056.
[25]	L. Ruiu, I. Floris, A. Satta and D. J. Ellar, “Toxicity of a Brevibacillus laterosporus Strain Lacking Parasporal Crystals against Musca domestica and Aedes aegypti,” Biological Control, Vol. 43. No. 1, 2007, pp. 136-143. doi:10.1016/j.biocontrol.2007.07.002
[26]	M. Boisvert and J. Boisvert, “Effects of Bacillus thuringiensis var. israelensis on Target and Nontarget Organisms: A Review of Laboratory and Field Experiments,” Biocontrol Science and Technology, Vol. 10, No. 5, 2000, pp. 517-561. doi:10.1080/095831500750016361
[27]	H. Park, D. K. Bideshi, M. C. Wirth, J. J. Johnson, W. E. Walton and B. A. Federici, “Recombinant Larvicidal Bacteria with Markedly Improved Efficacy against Culex Vectors of West Nile Virus,” American Journal of Tropical Medicine and Hygiene, Vol. 72, No. 6, 2005, pp. 732-738.
[28]	S. Cohen, O. Dym, S. Albeck, E. Ben-Dov, R. Cahan, M. Firer and A. Zaritsky, “High-Resolution Crystal Structure of Activated Cyt2Ba Monomer from Bacillus thuringiensis Subsp. Israelensis,” Journal of Molecular Biology, Vol. 380, No. 5, 2008, pp. 820-827. doi:10.1016/j.jmb.2008.05.010
[29]	N. Jiménez-Juárez, C. Munoz-Garay, I. Gómez, G. Saab-Rincon, J. Y. Damian-Almazo, S. S. Gill, M. Sobero and A. Bravo, “Bacillus thuringiensis Cry1Ab Mutants Affecting Oligomer Formation Are Non-Toxic to Manduca sexta Larvae,” Journal of Biological Chemistry, Vol. 282, No. 29, 2007, pp. 21222-21229. doi:10.1074/jbc.M701314200
[30]	R. Peng, A. Xiong, X. Li, H. Fuan and Q. Yao, “A δ-Endotoxin Encoded in Pseudomonas fluorescens Displays a High Degree of Insecticidal Activity,” Applied Microbiology and Biotechnology Vol. 63, No. 3, 2003, pp. 300306. doi:10.1007/s00253-003-1343-2
[31]	B. Lambert, W. Theunis, R. Agouda, K. Van Audenhove, C. Decock, S. Jansens, J. Seurinck and M. Peferoen, “Nucleotide Sequence of Gene Cry IIID Encoding a Novel Coleopteran-Active Crystal Protein from Strain BTI109P of Bacillus thuringiensis Subsp kurstaki,” Gene, Vol. 110, No. 1, 1992, pp. 131-132. doi:10.1016/0378-1119(92)90457-Z
[32]	M. A. F. Abdullah and D. H. Dean, “Enhancement of Cry19Aa Mosquitocidal Activity against Aedes aegypti by Mutations in the Putative Loop Regions of Domain II,” Applied and Environmental Microbiology, Vol. 70, No. 6, 2004, pp. 3769-3771. doi:10.1128/AEM.70.6.3769-3771.2004
[33]	N. Zouari, O. Achour and S. Jaoua, “Productionof Delta-Endotoxin by Bacillus thuringiensis Subsp kurstaki and Overcoming of Catabolite Repression by Using Highly Concentrated Gruel and Fish Meal Media in 2-and 20 dm3 Fermenters,” Journal of Chemical Technology and Biotechnology, Vol. 77, No. 8, 2002, pp. 877-882. doi:10.1002/jctb.650
[34]	F. Rajamohan, O. Alzate, J. A. Cotrill, A. Curtiss and D. H. Dean, “Protein Engineering of Bacillus thuringiensis Delta-Endotoxin: Mutations at Domain II of CryIAb Enhance Receptor Affinity and Toxicity toward Gypsy Moth Larvae,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 93, No. 25, 1996, pp. 14338-14343. doi:10.1073/pnas.93.25.14338
[35]	D. H. Sauka, J. Sánchez, A. Bravo and G. B. Benintende, “Toxicity of Bacillus thuringiensis Delta-Endotoxins against Bean Shoot Borer (Epinotia aporema Wals.) Larvae, a Major Soybean Pest in Argentina,” Journal of Invertebrate Pathology, Vol. 94, No. 2, 2007, pp. 125-129. doi:10.1016/j.jip.2006.09.002
[36]	S. Herrero, J. González-Cabrera, B. E. Tabashnik and J. Ferré, “Shared Binding Sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A Toxins,” Applied and Environmental Microbiology, Vol. 67, No. 12, 2001, pp. 5729-5734. doi:10.1128/AEM.67.12.5729-5734.2001
[37]	G. M. S. Lima, R. W. S. Aguiar, R. F. T. Corrêa, E. S. Martins, A. C. M. Gomes, T. Nagata, M. T. De-Souza, R. G. Monnerat and B. M. Ribeiro, “Cry2A Toxins from Bacillus thuringiensis Expressed in Insect Cells Are Toxic to Two Lepidopteran Insects,” World Journal of Microbiology and Biotechnology, Vol. 24, No. 12, 2008, pp. 2941-2948. doi:10.1007/s11274-008-9836-x
[38]	M. Porcar, A. M. Grenier, B. Federici and Y. Rahbe, “Effects of Bacillus thuringiensis δ-Endotoxins on the Pea Aphid (Acyrthosiphon pisum),” Applied and Environmental Microbiology, Vol. 75, No. 14, 2009, pp. 48974900. doi:10.1128/AEM.00686-09
[39]	J. D. Tang, A. M. Shelton, J. V. Rie, S. D. Roeck, W. J. Moar, R. T. Roush and M. Peferoen, “Toxicity of Bacillus thuringiensis Spore and Crystal Protein to Resistant Diamondback Moth (Plutella xylostella),” Applied and Environmental Microbiology, Vol. 62, No. 2, 1996, pp. 564-569.
[40]	J. Payne, K. E. Narva and J. Fu, “Bacillus thuringiensis Genes Encoding Nematode Active Toxins,” US Patent No. 5831011, 1998.
[41]	M. E. Carter, M. G. Villani, L. L. Allee and J. E. Losey Berlin, “Absence of Non-Target Effects of Two Bacillus thuringiensis Coleopteran Active δ-Endotoxins on the Bulb Mite, Rhizoglypus robini (Claparède) (Acari, Acaridae),” Journal of Applied Entomology, Vol. 128, No. 1, 2004, pp. 56-63. doi:10.1046/j.1439-0418.2003.00788.x
[42]	S. Ranjbari, M. Safaralizadeh and H. S. Aramideh, “Insecticidal Effect of Bacillus thuringiensis Var kurstaki on the Various Instars Larvae of Plutella xylostella L. (Lep.: plutellidae) under Laboratory Condition,” Egyptian Academic Journal of Biological Sciences, Vol. 3, No. 1, 2011, pp. 27-32.
[43]	S. Naimov, M. Weemen-Hendriks, S. Dukiandjiev and R. A. Maagd, “Bacillus thuringiensis Delta-Endotoxin Cry1 Hybrid Proteins with Increased Activity against the Colorado Potato Beetle,” Applied and Environmental Microbiology, Vol. 67, No. 11, 2001, pp. 5328-5330. doi:10.1128/AEM.67.11.5328-5330.2001
[44]	E. Wellman-Desbiens and J. C Cote, “Development of a Bacillus thuringiensis-Based Assay on Lygus Hesperus,” Journal of Economic Entomology, Vol. 98, No. 5, 2005, pp. 1469-1479. doi:10.1603/0022-0493-98.5.1469
[45]	C. Chandi, G. S. Mandal, A. Basu, K. S. Ghosh, S. D. Gupta, M. K. Maiti and S. K. Sen, “Prediction-Based Protein Engineering of Domain I of Cry2A Entomocidal Toxin of Bacillus thuringiensis for the Enhancement of Toxicity against Lepidopteran Insects,” Protein Engineering, Design & Selection, Vol. 20, No. 12, 2007, pp. 599-606. doi:10.1093/protein/gzm058
[46]	J. Wojciechowska, “Two Novel Delta-Endotoxin Gene Families cry26 and cry28 from Bacillus thuringiensis ssp. Finitimus,” FEBS Letters, Vol. 453, No. 1-2, 2009, pp. 46-48. doi:10.1016/S0014-5793(99)00650-X
[47]	I. M. Dubovskii, O. A. Olifirenko and V. V. Glupov, “Level and Activities of Antioxidants in Intestine of Larvae Galleria mellonella L. (Lepidoptera, Pyralidae) at Peroral Infestation by Bacteria Bacillus thuringiensis ssp. Galleriae,” Journal of Evolutionary Biochemistry and Physiology, Vol. 41, No. 1, 2005, pp. 20-25. doi:10.1007/s10893-005-0030-6
[48]	A. Hanan, H. A. A. El-Sadawy, J. M. Georgy, S. Shaaban, E. I. Hossary and H. A. Kassem, “Fusion of Bacillus stearothermophilus Leucine Aminopeptidase II with the Raw-Starch-Binding Domain of Bacillus sp. Strain TS-23 α-Amylase Generates a Chimeric Enzyme with Enhanced Thermostability and Catalytic Activity,” Journal of Indian Microbiology Technology, Vol. 3, 2008, pp. 23-29.
[49]	N. Tigue, J. Jacoby and D. J. Ellar, “The Triple Helix 4 Residue, Asn135, Is Involved in the Oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis Toxins,” Applied and Environmental Microbiology, Vol. 67, No. 12, 2001, pp. 5715-5720. doi:10.1128/AEM.67.12.5715-5720.2001
[50]	C. N. Chilcott, P. J. Wigley, A. H. Broadwell, D. J. Park and D. J. Ellar, “Activities of Bacillus thuringiensis Insecticidal Crystal Proteins Cyt1Aa and Cyt2Aa against Three Species of Sheep Blowfly,” Applied Environmental Microbiology, Vol. 64, No. 10, 1998, pp. 4060-4061.
[51]	T. Iizuka, S. Ishikawa, S. Asano, H. Bando, Z. Zheng and N. Murai, “Insecticidal Activity of the CryIA(a) and Cry IB Delta-Endotoxin of Bacillus thuringiensis Was Retained after the Coding Region of the Gene Was Truncated and Expressed in Escherichia coli,” Journal of Sericulture Science, Vol. 63, No. 4, 1994, pp. 303-309.
[52]	M. Vasquez, C. Parra, E. Hubert, P. Espinoza, C. Theoduloz and L. Meza-Basso, “Specificity and Insecticidal Activity of Chilean Strains of Bacillus thuringiensis,” Journal of Invertebrate Pathology, Vol. 66, No. 2, 1995, pp. 143-148. doi:10.1006/jipa.1995.1077
[53]	H. Kati, S. Kazim, N. Remziye and Z. Demirbag, “A Highly Pathogenic Strain of Bacillus thuringiensis Serovar Kurstaki in Lepidopteran Pests,” The Journal of Microbiology, Vol. 45, No. 6, 2007, pp. 553-557.
[54]	S. Rouis, M. Chakroun, I. Saadaoui and S. Jaoua, “Proteolysis, Histopathological Effects, and Immunohistopathological Localization of Delta-Endotoxins of Bacillus thuringiensis subsp. kurstaki in the Midgut of Lepidopteran Olive Tree Pathogenic Insect Prays oleae,” Molecular Biotechnology, Vol. 35, No. 2, 2007, pp. 141-148. doi:10.1007/BF02686109
[55]	A. Aronson, C. Geng and L. Wu, “Aggregation of Bacillus thuringiensis Cry1A Toxins upon Binding to Target Insect Larval Midgut Vesicles,” Applied and Environmental Microbiology, Vol. 65, No. 6, 1999, pp. 25032507.
[56]	D. M. F. Capalbo, F. H. Valicente and I. M. Oliveira, “Solid-State Fermentation of Bacillus thuringiensis tolworthi to Control Fall Armyworm in Maize,” Electronic Journal of Biotechnology, Vol. 4, No. 2, 2001, pp. 9-10.
[57]	B. Lambert, L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey and M. Peferoen, “A Bacillus thuringiensis Insecticidal Crystal Protein with a High Activity against Members of the Family Noctuidae,” Applied and Environmental Microbiology, Vol. 62, No. 1, 1996, pp. 80-86.
[58]	T. Erban, M. Nesvorna, M. Erbanova and J. Hubert, “Bacillus thuringiensis Var. tenebrionis Control of Synanthropic Mites (Acari: Acaridida) under Laboratory Conditions,” Experimental and Applied Acarology, Vol. 49, No. 4, 2009, pp. 339-346. doi:10.1007/s10493-009-9265-z
[59]	N. Crickmore, D. R. Zeigler, J. Feitelson, E. Schnepf, J. van Rie, D. Lerecus, 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.
[60]	M. H. Hasan, A. Akter, M. Ilias, S. N. Khan and M. M. Hoq, “Growth, Sporulation and Toxin Production by Bacillus thuringiensis Isolates in Media Based on Mustard-Seed Meal,” Bangladesh Journal of Microbiology, Vol. 27, No. 2, 2010, pp. 51-55.
[61]	F. R. Clairmont, R. E. Milne, V. T. Pham, M. B. Carrière, and H. Kapla, “Role of DNA in the Activation of the Cry1A Insecticidal Crystal Protein from Bacillus thuringiensis,” The Journal of Biological Chemistry, Vol. 273, No. 15, 1998, pp. 9292-9296. doi:10.1074/jbc.273.15.9292
[62]	J. Li, J. Carroll and D. J. Ellar, “Crystal Structure of Insecticidal δ-Endotoxin from Bacillus thuringiensis at 2.5A Resolution,” Nature, Vol. 353, 1991, pp. 815-821. doi:10.1038/353815a0
[63]	L. Q. Xia, X. M. Zhao, X. Z. Ding, F. X. Wang and Y. J. Sun, “The Theoretical 3D Structure of Bacillus thuringiensis Cry5Ba,” Journal of Molecular Modeling, Vol. 14, No. 9, 2008, pp. 843-848. doi:10.1007/s00894-008-0318-8
[64]	D. M. Ojcius and I. D. E. Young, “Cytolytic Pore-Forming Proteins and Peptides: Is There a Common Structural Motif?” Trends in Biochemical Sciences, Vol. 16, 1991, pp. 225-229. doi:10.1016/0968-0004(91)90090-I
[65]	H. Hofte and H. R. Whiteley, “Insecticidal Crystal Proteins of Bacillus thuringiensis,” Microbiological Reviews, Vol. 53, No. 2, 1989, pp. 242-255.
[66]	N. M. Rosas-García, “Biopesticide Production from Bacillus thuringiensis: An Environmentally Friendly Alternative,” Recent Patents in Biotechnology, Vol. 3, No. 1, 2009, pp. 28-36. doi:10.2174/187220809787172632
[67]	C. Du, P. A. Martin and K. W. Nickerson, “Comparison of Disulfide Contents and Solubility at Alkaline pH of Insecticidal and Non-Insecticidal Bacillus thuringiensis Protein Crystals,” Applied and Environmental Microbiology, Vol. 60, No. 10, 1994, pp. 3847-3853.
[68]	W. H. McGaughey and M. E. Whalon, “Managing Insect Resistance to Bacillus thuringiensis Toxins,” Science, Vol. 258, No. 5087, 1992, pp. 1451-1455. doi:10.1126/science.258.5087.1451
[69]	J. Carroll, D. Convents, J. Van Damme, A. Boets, J. Van Rie and D. J. Ellar, “Intramolecular Proteolytic Cleavage of Bacillus thuringiensis Cry3A δ-Endotoxin May Facilitate Its Coleopteran Toxicity,” Journal of Invertebrate Pathology, Vol.70, No. 1, 1997, pp. 41-49. doi:10.1006/jipa.1997.4656
[70]	V. Vachon, R. Laprade and J. L. Schwartz, “Current Models of the Mode of Action of Bacillus thuringiensis Insecticidal Crystal Proteins: A Critical Review,” Journal of Invertebrate Pathology, Vol. 111, No. 1, 2012, pp. 1-12. doi:10.1016/j.jip.2012.05.001
[71]	J. Iriarte and P. Caballero, “Biología y Ecología de Bacillus thuringiensis,” Phytoma. Espana, 2001, pp. 15-44.
[72]	J. A. Baum, T. Bogaert, W. Clinton, G. R. Heck, P. Feldmann, O. Ilagan and J. Roberts, “Control of Coleopteran Insect Pests through RNA Interference,” Nature Biotechnology, Vol. 25, No. 11, 2007, pp. 1322-1326. doi:10.1038/nbt1359
[73]	N. Zouari, A. Dhouib, R. Ellouz and S. Jaoua, “Nutritional Requirements of a Bacillus thuringiensis Subsp Kurstaki Strain and Use of Gruel Hydrolysate for the Formulation of a New Medium for Delta-Endotoxin Production,” Applied Biochemistry and Biotechnology, Vol. 69, No. 1, 1998, pp. 41-52. doi:10.1007/BF02786020
[74]	A. Yezza, R. D. Tyagi, J. R. Valéro and R. Y. Surampalli, “Bioconversion of Industrial Wastewater and Wastewater Sludge into Bacillus thuringiensis Based Biopesticides in Pilot Fermentor,” Bioresource Technology, Vol. 97, No. 15, 2006, pp. 1850-1857. doi:10.1016/j.biortech.2005.08.023
[75]	S. Benjamin, R. B. Smitha, V. N. Jisha, S. Pradeep, S. Sajith, S. Sreedevi, P. Priji, K. N. Unni and M. K. Sarath Josh, “A Monograph on Amylases from Bacillus Spp.,” 2013.
[76]	U. Holker and J. Lenz, “Solid-State Fermentation—Are There Any Biotechnological Advantages?” Current Opinion in Microbiology, Vol. 8, No. 3, 2005, pp. 301-306. doi:10.1016/j.mib.2005.04.006
[77]	Z. Tokcaer, W. Bayraktar, U. Mehmetoglu, G. Ozcengiz and N. J. Alaeddinogly, “Response Surface Optimization of Antidipteran Delta-Endotoxin Production by Bacillus thuringiensis Subsp. israelensis HD 500,” Process Biochemistry, Vol. 41, No. 2, 2006, pp. 350-355. doi:10.1016/j.procbio.2005.02.030
[78]	R. R. Farrera, F. Perez-Guevara and M. Torre, “Carbon: Nitrogen Ratio Interacts with Initial Concentration of Total Solids on Insecticidal Crystal Protein and Spore Production in Bacillus thuringiensis HD-73,” Applied Microbiology and Biotechnology, Vol. 49, No. 6, 1998, pp. 758-765. doi:10.1007/s002530051243
[79]	G. Amin, S. Alotaibi, Y. A. Narmen and W. D. Saleh, “Bioinsecticide Production by the Bacterium Bacillus thuringiensis. 1. Pattern of Cell Growth, Toxin Production and By-Product Synthesis,” Archives of Agronomy and Soil Science, Vol. 54, No. 4, 2008, pp. 387-394. doi:10.1080/03650340802158201
[80]	I. Goldberg, B. Sneh, E. Battat and D. Klein, “Optimization of a Medium for a High Yield Production of Spore-Crystal Preparation of Bacillus thuringiensis Effective against the Egyptian Cotton Leaf Worm Spodoptora littoralis Boisd,” Biotechnology Letters, Vol. 2, No. 29, 1980, pp. 419-426. doi:10.1007/BF00162348
[81]	D. P. Sikdar, M. K. Majundar and S. K. Majundar, “Effect of Minerals on the Production of the Delta-Endotoxin by Bacillus thuringiensis,” Biotechnology Letters, Vol. 13, No. 7, 1991, pp. 511-514. doi:10.1007/BF01049209
[82]	O. N. Morries, V. Converse, P. Kanagaratnam and J. S. Davies, “Effect of Cultural Conditions on Spore-Crystal Yield and Toxicity of Bacillus thuringiensis Subsp aizawai HD133,” Journal of Invertebrate Pathology, Vol. 67, No. 2, 1996, pp. 129-136. doi:10.1006/jipa.1996.0020
[83]	H. Bti, “Production of Bacillus thuringiensis Subsp israelensis from Agro-Based Product,” Current Science, Vol. 101, No. 8, 2011, pp. 1009-1010.
[84]	F. H. Valicente, T. E. de Souza, M. I. S. Leite, F. L. Freire and C. M. Vieira, “Production of Bacillus thuringiensis Biopesticide Using Commercial Lab Medium and Agricultural By-Products as Nutrient Sources,” Revista Brasileira de Milho e Sorgo, Vol. 9, No. 1, 2010, pp. 1-11.
[85]	L. Zhuang, S. Zhou, Y. Wang, Z. Liu and R. Xu, “Cost-Effective Production of Bt Biopesticides by Solid-State Fermentation Using Wastewater Sludge: Effects of Heavy Metals,” Bioresource Technology, Vol. 102, No. 7, 2011, pp. 4820-4826. doi:10.1016/j.biortech.2010.12.098
[86]	M. Grove, W. Kimble and W. McCarthy, “Effects of Individual Bacillus thuringiensis Insecticidal Crystal Proteins on Adult Heliothis virescens (F) and Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae),” Biocontrol, Vol. 46, No. 3, 2001, pp. 321-335. doi:10.1023/A:1011424400297
[87]	E. Mizuki, M. Ohba, T. Akao, S. Yamashita, H. Saitoh and Y. S. Park, “Unique Activity Associated with Non-Insecticidal Bacillus thuringiensis Parasporal Inclusions: In Vitro Cell-Killing Action on Human Cancer Cells,” Journal of Applied Microbiology, Vol. 86, No. 3, 1999, pp. 477-486. doi:10.1046/j.1365-2672.1999.00692.x
[88]	Z. Xu, B. Yao, M. Sun and Z. Yu, “Protection of Mice Infected with Plasmodium berghei by Bacillus thuringiensis Crystal Proteins,” Parasitology Research, Vol. 92, No. 1, 2004, pp. 53-57. doi:10.1007/s00436-003-0990-7
[89]	L. D. Marroquin, D. Elyassnia, J. S. Griffitts, J. S. Feitelson and R. V. Aroian, “Bacillus thuringiensis Toxin Susceptibility and Isolation of Resistance Mutants in the Nematode Caenorhabditis elegans,” Genetics, Vol. 155, No. 4, 2000, pp. 1693-1699.
[90]	G.-M. Li, X.-Y. Zhang and L.-Q. Wang, “The Use of Bacillus thuringiensis on Forest Integrated Pest Management,” Journal of Forestry Research, Vol. 12, No. 1, 2001, pp. 51-54. doi:10.1007/BF02856801
[91]	M. Fadel and M. Sabour, “Utilization of Dairy by Product in the Production of Bioinsecticide,” Journal of Biological Sciences, Vol. 2, No. 2, 2002, pp. 116-120.
[92]	S. Buchholz, P. Neumann, K. Merkel and R. R. Hepburn, “Evaluation of Bacillus thuringiensis berliner as an Alternative Control of Small Hive Beetles, Aethina tumida Murray (Coleoptera; Nitidulidae),” Journal of Pest Science, Vol. 79, No. 4, 2006, pp. 251-254. doi:10.1007/s10340-006-0141-x
[93]	N. Helassa, H. Quiquampoix, S. Noinville, W. Szponarski and S. Staunton, “Adsorption and Desorption of Monomeric Bt (Bacillus thuringiensis) Cry1Aa Toxin on Montmorillonite and Kaolinite,” Soil Biology and Biochemistry, Vol. 41, No. 3, 2009, pp. 498-504. doi:10.1016/j.soilbio.2008.12.008
[94]	M. K. Er, S. Karada and C. Mart, “Effectiveness of Bacillus thuringiensis Var. kurstaki on Thaumetopoea solitaria Frey. (Lepidoptera: Thaumetopoeidae) Larvae in Laboratory Conditions,” Turkish Journal of Agriculture and Forestry, Vol. 31, No. 4, 2007, pp. 255-261.
[95]	R. L. Hellmich, B. D. Siegfried, M. K. Sears, D. E. Stanley-Horn, M. J. Daniels, H. R. Mattila, T. Spencer, K. G. Bidne and L. C. Lewis, “Monarch Larvae Sensitivity to Bacillus thuringiensis Purified Proteins and Pollen,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 98, No. 21, 2001, pp. 11925-11930. doi:10.1073/pnas.211297698
[96]	S. Khewa and A. Mukhopadhyay, “Biocontrol Potential of a Newly Isolated Bacterial Agent against Arctornis submarginata (Walker) (Lepidoptera: Lymantriidae) Occurring in Darjeeling Terai Region,” Journal of Biopesticides, Vol. 3, 2010, pp. 114-116.
[97]	P. Yashodha and C. Kuppusamy, “A Bioefficacy of Bacillus thuringiensis Var kurstaki against Eggplant Borer, Leucinodes orbonalis (Pyraustidae: Lepidoptera),” International Journal of Integrative Biology, Vol. 3, No. 3, 2008, pp. 209-213.
[98]	V. Gobatto, S. G. Giani, M. Camassola, A. J. P. Dillon, A. Specht and N. M. Barros, “Bacillus thuringiensis Isolates Entomopathogenic for Culex quinquefasciatus (Diptera: Culicidae) and Anticarsia gemmatalis (Lepidoptera: Noctuidae),” Brazilian Journal of Biology, Vol. 70, No. 4, 2010, pp. 1039-1046. doi:10.1590/S1519-69842010000500018
[99]	J. Ferre, B. Escriche, Y. Bel and J. Van Rie, “Biochemistry and Genetics of Insect Resistance to Bacillus thuringiensis Insecticidal Crystal Proteins,” FEMS Microbiology Letters, Vol. 132, 1995, pp. 1-7. doi:10.1016/0378-1097(95)00271-6
[100]	B. E. Tabashnik, “Evolution of Resistance to Bacillus thuringiensis,” Annual Review of Entomology, Vol. 39, No. 1, 1994, pp. 47-79. doi:10.1146/annurev.en.39.010194.000403
[101]	A. Trisyono and M. E. Whalon, “Fitness Costs of Resistance to Bacillus thuringiensis in Colorado Potato Beetle (Coleoptera: Chrysomelidae),” Journal of Economic Entomology, Vol. 90, No. 2, 1997, pp. 267-271.
[102]	B. E. Tabashnik, Y. B. Liu, N. Finson, L. Masson and D. G. Heckel, “One Gene in Diamondback Moth Confers Resistance to Four Bacillus thuringiensis Toxins,” Proceedings of the National Academy of Sciences, Vol. 94, No. 5, 1997, pp. 1640-1644. doi:10.1073/pnas.94.5.1640
[103]	D. J. Wright, M. Iqbal, F. Granero and J. Ferre, “A Change in a Single Midgut Receptor in the Diamondback Moth (Plutella xylostella) Is Only in Part Responsible for Field Resistance to Bacillus thuringiensis Subsp. kurstaki and B. thuringiensis Subsp. aizawai,” Applied and Environmental Microbiology, Vol. 63, No. 5, 1997, pp. 1814-1819.
[104]	J. L. Fox, “Resistance to Bt Toxin Surprisingly Absent from Pests,” Nature Biotechnology, Vol. 21, No. 9, 2003, pp. 958-959. doi:10.1038/nbt0903-958b
[105]	B. E. Tabashnik, T, Brévault and Y. Carrière, “Insect Resistance to Bt Crops: Lessons from the First Billion Acres,” Nature Biotechnology, Vol. 31, 2013, pp. 510-521.
[106]	D. N. Alstad and D. A. Andow, “Managing the Evolution of Insect Resistance to Transgenic Plants,” Science, Vol. 268, No. 5219, 1995, pp. 1894-1896. doi:10.1126/science.268.5219.1894
[107]	F. Gould, “Potential and Problems with High-Dose Strategies for Pesticidal Engineered Crops,” Biocontrol Science and Technology, Vol. 4, No. 4, 1994, pp. 451-461. doi:10.1080/09583159409355357
[108]	F. Gould, “Genetic Engineering, Integrated Pest Management and the Evolution of Pests,” Trends in Biotechnology, Vol. 6, No. 4, 1988, pp. 15-18. doi:10.1016/0167-7799(88)90007-8