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
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
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.
Publications:
· 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.
·
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.
·
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
·
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
·
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.
·
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.).
·
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