Volume 72, Issue 6
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645


An urgent need exists for new agents to control mosquito vectors of disease. Mosquito larvicides based on the bacteria subsp. (Bti) or (Bs) are effective in many habitats, but use is limited by their high cost. Moreover, mosquito resistance evolves rapidly to Bs where it is used intensively. The efficacy of these bacteria is due to a binary protein (BsB) in Bs and four proteins (Cry4A, Cry4B, Cry11A, and Cyt1A) in Bti. Here we report the use of promoters and a 5′ mRNA stabilizing sequence to synthesize high levels of Bs2362 binary toxin in Bti strains. The recombinant BtiIPS-82/BsB showed high potency against fourth instars of , a vector of West Nile virus, being 21-fold as potent as BtiIPS-82, and 32-fold as potent as Bs2362. Similar improved efficacy was obtained against larvae of Moreover, BtiIPS-82/BsB suppressed resistance to Bs2362 in .


Article metrics loading...

Loading full text...

Full text loading...



  1. WHO, 1999. The World Health Report. Geneva: World Health Organization.
  2. WHO, 1999. Guideline Specifications for Bacterial Larvicides for Public Health Use. Geneva: World Health Organization. WHO Document WHO/CDS/CPC/WHOPES/99.2.
  3. Rao DR, Mani TR, Rajendran R, Joseph AS, Gajanana A, Reuben R, 1995. Development of a high level of resistance to Bacillus sphaericus in a field population of Culex quinquefasciatus from Kochi, India. J Am Mosq Control Assoc 11 : 1–5.
  4. Silva-Filha M-H, Regis L, Nielsen-LeRoux C, Charles J-F, 1995. Low-level resistance to Bacillus sphaericus in a field-treated population of Culex quinquefasciatus (Diptera: Culicidae). J Econ Entomol 88 : 525–530.
  5. Yuan Z, Zhang Y, Cia Q, Liu E-Y, 2000. High-level field resistance to Bacillus sphaericus C3-41 in Culex quinquefasciatus from southern China. Biocontr Sci Technol 10 : 41–49.
  6. Federici BA, Lüthy P, Ibarra JE, 1990. Parasporal body of Bacillus thuringiensis israelensis: structure, protein composition, and toxicity. de Barjac H, Sutherland DJ, eds. Bacterial Control of Mosquitoes and Blackflies. New Brunswick, NJ: Rutgers University Press, 11–44.
  7. Baumann P, Clark MA, Baumann L, Broadwell AH, 1991. Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxins. Microbiol Rev 55 : 425–436.
  8. Wu D, Chang F, 1985. Synergism in mosquitocidal activity of 26 and 65 kDa proteins from Bacillus thuringiensis subsp. israelensis crystal. FEBS Lett 190 : 232–236.
  9. Ibarra JE, Federici BA, 1986. Isolation of a relatively nontoxic 65-kilodalton proteins inclusion from the parasporal body of Bacillus thuringiensis subsp. israelensis. J Bacteriol 165 : 527–533.
  10. Crickmore N, Bone EJ, Williams JA, Ellar DJ, 1995. Contribution of the individual components of the δ-endotoxin crystal to the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis. FEMS Microbiol Lett 131 : 249–254.
  11. Georghiou GP, Wirth MC, 1997. Influence of exposure to single versus multiple toxins of Bacillus thuringiensis subsp. israelensis on development of resistance in the mosquito Culex quinquefasciatus (Diptera: Culicidae). Appl Environ Microbiol 63 : 1095–1101.
  12. Wirth MC, Walton WE, Federici BA, 2000. Cyt1A from Bacillus thuringiensis restores toxicity of Bacillus sphaericus against Culex quinquefasciatus (Diptera: Culicidae). J Med Entomol 37 : 401–407.
  13. Wirth MC, Walton WE, Federici BA, 2000. Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti. Appl Environ Microbiol 66 : 1093–1097.
  14. Trisrisook M, Pantuwatana S, Bhumiratana A, Panbangred W, 1990. Molecular cloning of the 130-kilodalton mosquitocidal δ-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus. Appl Environ Microbiol 56 : 1710–1716.
  15. Bar E, Lieman-Hurwitz J, Rahamin E, Keynan A, Sandler N, 1991. Cloning and expression of Bacillus thuringiensis israelensis δ-endotoxin DNA in B. sphaericus. J Invertebr Pathol 57 : 149–158.
  16. Poncet S, Delécluse A, Anello G, Klier A, Rapoport G, 1994. Transfer and expression of the CryIVB and CryIVD genes of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus 2297. FEMS Microbiol Lett 117 : 91–96.
  17. Bourgouin C, Delécluse A, de La Torre F, Szulmajster J, 1990. Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression. Appl Environ Microbiol 56 : 340–344.
  18. Servant P, Rosso M-L, Hamon S, Poncet S, Delécluse A, Rapoport G, 1999. Production of Cry11A and Cry11Ba toxins in Bacillus sphaericus confers toxicity towards Aedes aegypti and resistant Culex populations. Appl Environ Microbiol 65 : 3021–3026.
  19. Thiéry I, Hamon S, Delécluse A, Orduz S, 1998. The introduction into Bacillus sphaericus of the Bacillus thuringiensis subsp. medellin Cyt1Ab1 gene results in higher susceptibility of resistant mosquito larva populations to B. sphaericus. Appl Environ Microbiol 64 : 3910–3916.
  20. Li T, Sun F, Yuan Z, Zhang Y, Yu J, Pang Y, 2000. Coexpression of cyt1Aa of Bacillus thuringiensis subsp. israelensis with Bacillus sphaericus binary toxin gene in acrystalliferous strain of B. thuringiensis. Curr Microbiol 40 : 322–326.
  21. Park H-W, Ge B, Bauer LS, Federici BA, 1998. Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence. Appl Environ Microbiol 64 : 3932–3938.
  22. Park H-W, Bideshi DK, Johnson JJ, Federici BA, 1999. Differential enhancement of Cry2A versus Cry11A yields in Bacillus thuringiensis by use of the cry3A STAB mRNA sequence. FEMS Microbiol Lett 181 : 319–327.
  23. Lereclus D, Arantes O, Chaufaux J, Lecadet MM, 1989. Transformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis. FEMS Microbiol Lett 60 : 211–218.
  24. Agaisse H, Lereclus D, 1996. STAB-SD: a Shine-Dalgarno sequence in the 5′ untranslated region is a determinant of mRNA stability. Mol Microbiol 20 : 633–643.
  25. Kalfon A, Larget-Thiéry I, Charles J-F, de Barjac H, 1983. Growth, sporulation and larvicidal activity of Bacillus sphaericus. Eur J Appl Microbiol Biotechnol 18 : 168–173.
  26. Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 277 : 680–685.
  27. Georghiou GP, Metcalf RL, Gidden FE, 1966. Carbamate-resistance in mosquitoes. Selection of Culex pipiens fatigans Wiedemann (= C. quinquefasciatus Say) for resistance to Baygon. Bull World Health Organ 35 : 691–708.
  28. Finney D, 1971. Probit Analysis. Cambridge, United Kingdom: Cambridge University Press.
  29. Raymond M, Prato G, Ratsira D, 1993. Probability Analysis of Mortality Assays Displaying Quantal Response. Version 3.3. Saint Georges D’Orques, France: Praxeme.
  30. Becker N, 2000. Bacterial Control of vector-mosquitoes and black flies. Charles J-F, Delécluse A, Nielsen-LeRoux, C, eds. Entomopathogenic Bacteria: From Laboratory to Field Application. Dordrecht, The Netherlands: Kluwer Academic Publishers, 383–398.
  31. Charles J-F, Silva-Filha MH, Nielsen-LeRoux C, 2000. Mode of action of Bacillus sphaericus on mosquito larvae: incidence on resistance. Charles J-F, Delécluse A, Nielsen-LeRoux, C, eds. Entomopathogenic Bacteria: From Laboratory to Field Application. Dordrecht, The Netherlands: Kluwer Academic Publishers, 237–252.
  32. González-Pastor JE, Hobbs EC, Losick R, 2003. Cannibalism by sporulating bacteria. Science 301 : 510–513.
  33. Engelberg-Kulka H, Hazan R, 2003. Cannibals defy starvation and avoid sporulation. Science 301 : 467–468.
  34. Wirth MC, Jiannino JA, Federici BA, Walton WE, 2004. Synergy between toxins of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus. J Med Entomol 41 : 935–941.
  35. Wirth MC, Georghiou GP, Federici BA, 1997. CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus. Proc Natl Acad Sci USA 94 : 10536–10540.
  36. Mani GS, 1985. Evolution of resistance in the presence of two insecticides. Genetics 109 : 761–783.
  37. Tabashnik BE, 1994. Evolution of resistance to Bacillus thuringiensis. Annu Rev Entomol 39 : 47–79.
  38. Becker N, Ludwig M, 1993. Investigations on possible resistance in Aedes vexans after a 10-year application of Bacillus thuringiensis israelensis. J Am Mosq Control Assoc 9 : 221–224.

Data & Media loading...

  • Received : 03 Sep 2004
  • Accepted : 14 Dec 2004

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error