Bacterial Larvicides


Bacterial larvicides are perhaps the most promising method of chemical-based mosquito control currently available, particularly in treatment wetlands.  Here the "chemicals" are the toxin precursors produced during sporulation by two naturally occurring bacteria.  Two species are currently used for mosquito control in California; however, because Bacillus thuringiensis subsp. israelensis (Bti) is comparatively less effective against mosquitoes inhabiting the organically enriched waters of treatment wetlands, Lysinibacillus sphaericus currently offers a viable alternative for microbial control of mosquitoes in organically-enriched treatment wetlands (Walton et al. 1998). Unlike Bti which contains multiple toxins that limit the potential for the rapid evolution of resistance in mosquitoes, the two toxin precursors in L. sphaericus act as a single toxin following ingestion and partial digestion by mosquito larvae.  Bti has been used for nearly 40 years in large-scale mosquito and black fly control programs.  Resistance had not been detected in mosquito populations in nature which had been subjected to selection from Bti toxins; however, resistance to Bti was recently detected in Culex pipiens in Syracuse, New York (see: Paul et al. 2005. Journal of the American Mosquito Control Association 21: 305-309).  Subsequent testing has failed to confirm evidence of sustained resistance in nature.  Despite what appears to be an anomalous finding, Bti remains an effective control agent for mosquitoes. Nevertheless, mosquitoes, such as the southern house mosquito Culex quinquefasciatus can evolve resistance to the full complement of Bti toxins when under strong selection pressure in the laboratory.  In contrast to the findings for Bti, resistance to L. sphaericus has been observed in mosquito populations in several places (Brazil, China, France, India and Thailand).  Mosquitoes can evolve high levels of resistance to B. sphaericus very rapidly (up to 50,000-fold resistance in several generations), especially when the mosquitoes commonly found often in polluted urban environments, such as catch basins and wastewater contaminated by human sewage, are routinely exposed to L. sphaericus toxins. 


Our research addresses questions such as, how do we prevent or forestall the evolution of resistance to bacterial toxins in mosquitoes?  If a mosquito population exhibits significant levels of resistance to bacterial toxins, then what measures can we use to increase susceptibility in the mosquitoes?  How do mosquito populations resistant to particular Bacillus toxins respond to toxins from closely related species? How effective are genetically engineered bacteria against susceptible and resistant populations of mosquitoes?  How do we design constructed treatment wetlands to enhance the effectiveness of control measures using current formulations of bacterial larvicides?


Our studies of mosquito dispersal (Walton et al. 1999) demonstrated that the predominant mosquito, Culex erythrothorax, occurring at thickly vegetated wetlands does not disperse very far from developmental sites.  Consequently, one would surmise that there is a greatly reduced potential for a resistant population to exchange genes with a nearby population that is susceptible to L. sphaericus.  Surprisingly, collaborative work done with Dr. Andrew Bohonak and Justin Hoesterey at San Diego State University looking at the molecular ecology of Cx. erythrothorax found that there is very little differentiation among populations throughout San Diego County.  Culex tarsalis, however, is thought to be most important vector of West Nile virus and other flaviviruses in the western U.S., is one of the prevalent mosquitoes collected at wetlands in southern California and disperses widely across the landscape.


Our collaborative studies with Dr. Brian Federici and his laboratory have made several important findings related to the efficacy of B. sphaericus against mosquitoes.  First, we demonstrated that Culex quinquefasciatus larvae selected for a high level of resistance to Lysinibacillus (formerly Bacillus) sphaericus toxins become susceptible again to L. sphaericus after combining B. sphaericus and the cytolytic toxin (Cyt1A) from Bacillus thuringiensis subsp. israelensis (Wirth et al. 2000).  We studied the effects of component toxins in Bti (Wirth et al. 2003, 2004a, 2004b, 2010) and L. sphaericus (Wirth et al. 2007) on toxicity and resistance as well as investigating Cyt1A's role in moving the cytolytic toxins into the cells of the digestive tract.  Second, our studies demonstrated that the number of species susceptible to L. sphaericus increased when B. sphaericus was combined with Cyt1A (Wirth et al. 2000b, 2005).  For example, larvae of the yellow fever mosquito, Aedes aegypti, that are refractory to the toxic effects of B. sphaericus become susceptible when exposed to the combination of L. sphaericus and Cyt1A.  We have evaluated the cross-resistance of individual toxins from subspecies closely-related to Bti (Wirth et al. 1998b, 2001a, 2001b, 2004a).  Whereas, toxins such as Cry 11B from B. t. subsp. jegathesan (Btj) exhibit significant levels of cross-resistance in Culex quinquefasciatus larvae resistant to various combinations of Bti toxins, other Btj toxins such as Cry 19A exhibit little cross-resistance in Bti-resistant mosquitoes.  Identification of active polypeptides against resistant mosquitoes will assist in the development of resistance management strategies for these important bacterial toxins.  This work has been carried out in collaboration with colleagues in the Department of Entomology at UCR and at the Pasteur Institute in Paris. 


Dr. Federici's research group has developed recombinant larvicidal bacteria that we have tested against the strains of resistant Culex quinquefasciatus and other mosquito species maintained in my laboratory.  A recombinant strain that expressed toxins from Bti and L. sphaericus was comparatively more toxic to larvae of the southern house mosquito than were the commercially available strains of both species (24-hour LC50 for Bti and 48-hour LC50 for L. sphaericus: 0.37 ng mL-1 vs. 8.1 ng mL-1 for Bti IPS-82 and 11.9 ng mL-1 for L. sphaericus strain 2362: Park et al. 2005). We also carried out collaborative studies with a team of Israeli scientists who have genetically modified Escherichia coli and cyanobacteria to express genes for Bti toxins (Wirth et al. 2004c). The findings of these studies have important implications for genetic engineering of bacterial larvicides and resistance management in programs using bacterial larvicides as an environmentally-friendly approach to mosquito control. 





·   Wirth, M. C., W. E. Walton and B. A. Federici. 2015. Evolution of resistance in Culex quinquefasciatus Say (Diptera: Culicidae) selected with a recombinant Bacillus thuringiensis strain producing Cyt1Aa and Cry11Ba and the binary toxin, Bin, from Lysinibacillus sphaericus. Journal of Medical Entomology: 52: 1028-1035. [PDF available by request]


·   Duguma, D., M. Hall, P. Rugman-Jones, R. Stouthamer, J. D. Neufeld and W. E. Walton. 2015.  Microbial communities and nutrient dynamics in experimental microcosms are altered after application of a high dose of Bti.  Journal of Applied Ecology. doi:10.1111/1365-2664.12422 [link to Wiley]. [Pre-publication version] [Supplemental material]


·   Wirth, M. C., C. Berry, W. E. Walton and B. A. Federici. 2014. Mtx toxins from Lysinibacillus sphaericus enhance mosquitocidal Cry activity and suppress Cry-resistance in Culex quinquefasciatus (Diptera: Culicidae). Journal of Invertebrate Pathology 115: 62-67. [PDF]


·   Mogren, C. L., W. E. Walton and J. T. Trumble.  2014. Tolerance to individual and joint effects of arsenic and Bacillus thuringiensis var. israelensis or Lysinibacillus sphaericus in Culex mosquitoes. Insect Science 21: 477-485. [PDF]


·   Wirth, M. C., B. A. Federici, and W. E. Walton. 2012. Inheritance, stability, and dominance of Cry-resistance in Culex quinquefasciatus (Diptera: Culicidae) selected with the three Cry toxins of Bacillus thuringiensis subsp. israelensis. Journal of Medical Entomology 48: 886-894. [PDF]


·   Subramaniam, J., K. Kovendan, P. Kumar, K. Murugan, and W. E. Walton. 2012. Mosquito larvicidal activity of Aloe vera (Family: Liliaceae) leaf extract and Bacillus sphaericus, against Chikungunya vector, Aedes aegypti. Saudi Journal of Biological Sciences 19: 503-509. [PDF]


·   Wirth, M.C., W. E. Walton, and B. A. Federici.  2010.  Inheritance patterns, dominance, stability and allelism of insecticide resistance and cross-resistance in two colonies of Culex quinquefasciatus (Diptera: Culicidae) selected with Cry-toxins from Bacillus thuringiensis subsp. israelensis. Journal of Medical Entomology 47: 814-822. [PDF]


·   Wirth, M. C., W. E. Walton and B. A. Federici. 2010. Evolution of resistance to the Bacillus sphaericus Bin toxin is phenotypically masked by combination with the mosquitocidal proteins of Bacillus thuringiensis subspecies israelensis. Environmental Microbiology 12: 1154-1160. [PDF]


·   Wirth, M. C., Y. Yang, W. E. Walton, B. A. Federici, and C. Berry. 2007. Mtx toxins synergize Bacillus sphaericus and Cry11Aa against susceptible and insecticide-resistant Culex quinquefasciatus. Applied and Environmental Microbiology 73 (19): 6066-6071. [PDF]


·    Wirth, M. C., A. Zaritsky, E. Ben-Dov, R. Manasherob, V. Khasdan, S. Boussiba, and W. E. Walton. 2007. Cross-resistance spectra of Culex quinquefasciatus resistant to mosquitocidal toxins of Bacillus thuringiensis toward recombinant Escherichia coli expressing genes from B. thuringiensis subsp. israelensis. Environmental Microbiology 9: 1393-1401. [PDF]


·   Wirth, M. C., J. A. Jiannino, B. A. Federici, and W. E. Walton. 2005.  Evolution of resistance to Bacillus sphaericus or a mixture of B. sphaericus + Cyt1A from Bacillus thuringiensis in the mosquito Culex quinquefasciatus (Diptera: Culicidae). J. Invertebrate Pathology 88: 154-162. [PDF]


·   Park, H.-W., D. K. Bideshi, M. C. Wirth, J. J. Johnson, W. E. Walton, and B. A. Federici. 2005. Recombinant larvicidal bacteria with markedly improved efficacy against Culex Vectors of West Nile virus.  American Journal of Tropical Medicine and Hygiene 72: 732-738. [Abstract][PDF]


·   Wirth, M. C., H.-W. Park, W. E. Walton, and B. A. Federici. 2005. Cyt1A of Bacillus thuringiensis delays the evolution of resistance to Cry11A in the mosquito, Culex quinquefasciatus. Appl. Environ. Microbiol. 71: 185-189. [PDF]


·   Wirth, M.C., W. E. Walton, R. Manasherob, V. Khasdan, E. Ben-Dov, S. Boussiba, and A. Zaritsky. 2004. Larvicidal activities of transgenic Escherichia coli against susceptible and Bacillus thuringiensis israelensis-resistant strains of Culex quinquefasciatus. Symposium on the "Ecological Impact of Genetically Modified Organisms." IOBC/WPRS Bulletin 27: 171-176.


·   Wirth, M. C., J. A. Jiannino, B. A. Federici, and W. E. Walton. 2004. Synergy between toxins from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus. J. Med. Entomol. 41: 935-941. [PDF]


·   Wirth, M. C., A. Delécluse, and W. E. Walton.  2004.  Laboratory selection for resistance to Bacillus thuringiensis subsp. jegathesan or a component toxin, Cry 11B, in Culex quinquefasciatus Say (Diptera: Culicidae). J. Med. Entomol. 41: 435-441. [PDF]


·   Walton, W. E.  2003.  Managing mosquitoes in surface-flow constructed treatment wetlands. University of California, Division of Agriculture and Natural Resources. Davis, CA. Publ. No. 8117. 11 pp.  Available at:


·   Wirth, M. C., W. E. Walton, and A. Delécluse.  2003.  Deletion of the Cry11A or the Cyt1A toxin from Bacillus thuringiensis subsp. israelensis: Effect on toxicity against resistant Culex quinquefasciatus (Diptera: Culicidae). J. Invertebrate Pathol. 82: 133-135. [PDF]


·    Knight, R. L., W. E. Walton, G. F. O’Meara, W. K. Reisen, and R. Wass.  2003.  Strategies for effective mosquito control in constructed treatment wetlands. Ecological Engineering 21: 211-232. [PDF]


·   Wirth, M. C., A. Delécluse, and W. E. Walton.  2001.  Cyt1Ab1 and Cyt2Ba1 from Bacillus thuringiensis subsp. israelensis and subsp. medellin synergize Bacillus sphaericus against Aedes aegypti and resistant Culex quinquefasciatus (Diptera: Culicidae). Appl. Environ. Microbiol. 67: 3280-3284. [PDF]


·   Wirth, M. C., A. Delécluse, and W. E. Walton.  2001. Lack of cross-resistance to Cry19A from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) resistant to Cry toxins from Bacillus thuringiensis subsp. israelensis. Appl. Environ. Microbiol. 67: 1956-1958. [PDF]


·   Wirth, M. C., W. E. Walton, and B. A. Federici.  2000. Cyt1A from Bacillus thuringiensis restores toxicity of Bacillus sphaericus against resistant Culex quinquefasciatus (Diptera: Culicidae).  J. Med. Entomol. 37: 401-407. [PDF]


·   Wirth, M. C., B. A. Federici, and W. E. Walton.  2000. Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae). Applied and Environmental Microbiology 66: 1093-1097. [PDF]


·   Walton, W. E., P. D. Workman, and C. Tempelis.  1999. Dispersal, survivorship, and host selection of Culex erythrothorax (Diptera: Culicidae) associated with a constructed wetland in southern California.  Journal of Medical Entomology 36: 30-40. [PDF]


·   Wirth, M. C., A. Delécluse, B. A. Federici, and W. E. Walton.  1998. Variable cross-resistance to Cry 11B from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) resistant to single or multiple toxins of Bacillus thuringiensis subsp. israelensis.  Applied Environ. Microbiol. 64: 4174-4179. [PDF]


·   Walton, W. E., P. D. Workman, L. A. Randall, J. A. Jiannino, and Y. A. Offill.  1998. Effectiveness of control measures against mosquitoes at a constructed wetland in Southern California.  J. Vector Ecology 23: 149-160.


·   Wirth, M. C., A. Delécluse, B. A. Federici, W. E. Walton, and G. P. Georghiou.  1998.  Resistance to Bacillus thuringiensis israelensis in Culex quinquefasciatus and prospects for management.  In:  Proceedings of VIIth International Colloquium on Invertebrate Pathology and Microbial Control.  IVth International Conference on Bacillus thuringiensis.  Sapporo, Japan.  Aug. 23-28, 1998.  pp. 292-294.


·   Walton, W. E. and M. S. Mulla.  1992.  Impacts and fates of microbial pest control agents in the aquatic environment. In:  “Dispersal of Living Organisms into Aquatic Ecosystems.” (A. Rosenfield and R. Mann, eds.).  Maryland Sea Grant.  University of Maryland, College Park, MD. pp. 205-237. 


·   Walton, W. E. and M. S. Mulla.  1991.  Integrated control of Culex tarsalis larvae using Bacillus sphaericus and Gambusia affinis:  Effects on mosquitoes and nontarget organisms in field mesocosms.  Bull. Soc. Vector Ecol. 16: 203-221.


·   Walton, W. E., M. S. Mulla, M. J. Wargo, and S. L. Durso.  1991.  Efficacy of a microbial insecticide and larvivorous fish against Culex tarsalis in duck club ponds in southern California.  Proc. Papers Calif. Mosq. Vector Control Assoc. 58: 148-156.