Recombinant Larvicidal Bacteria With Markedly Improved Efficacy Against Culex Vectors of West Nile Virus

Abstract

An urgent need exists for new agents to control mosquito vectors of disease. Mosquito larvicides based on the bacteria Bacillus thuringiensis subsp. israelensis (Bti) or B. sphaericus (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 cyt1A 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 Culex quinquefasciatus, 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 Cx. tarsalis. Moreover, BtiIPS-82/BsB suppressed resistance to Bs2362 in Cx. quinquefasciatus.

A 1.1-Kilobase Region Downstream of the bin Operon in Bacillus sphaericus Strain 2362 Decreases Bin Yield and Crystal Size in Strain 2297

“…In addition, phase-contrast microscopy showed that the 1.1-kb insertion also reduced the size of the Bin crystal (data not shown). When B. sphaericus strains were tested against fourth-instar larvae of Culex quinquefasciatus using established methods (18)(19)(20), the 50% and 95% lethal concentrations of recombinant strain 2297DSIN were significantly higher than those of wild-type strain 2297, supporting the results obtained with SDS-PAGE, spore count data, and phase-contrast microscopy ( Table 1). Despite many attempts, very little has been revealed regarding the molecular mechanisms that regulate Bin synthesis and crystallization in B. sphaericus (1,6,8,24).…”

“…The number of spores produced per unit medium by each B. sphaericus strain was also measured to confirm that the observation was not simply due to the increase of the sporulated cell numbers (20). The results showed clearly that there was no statistical difference in the number of spores per milliliter between strains 2297 (5.8 ϫ 10 8 ) and 2297DSIN (5.4 ϫ 10 8 ), whereas production by strain 2362 was significantly higher than those of the other two (9.1 ϫ 10 8 ).…”

Evolution of Resistance inCulex quinquefasciatus(Say) Selected With a RecombinantBacillus thuringiensisStrain-Producing Cyt1Aa and Cry11Ba, and the Binary Toxin, Bin, FromLysinibacillus sphaericus

“…That recombinant strain consisted of B. thuringiensis subsp. israelensis engineered to express the Bin toxin of L. sphaericus in addition to its native toxin complex (Park et al 2005). Selection over 35 generations led to a maximum resistance level of 5-fold in Generation 21, but resistance declined to around 2-fold thereafter (Wirth et al 2010).…”

“…All materials were in the form of lyophilized, crystal/spore powders. The strains were grown on liquid media as described previously (Park et al 2005). Sporulated cells were washed in distilled water, pelleted, lyophilized, and stored in the refrigerator.…”

Mosquito larvicidal potential of potash alum against malaria vector Anopheles stephensi (Liston)

“…Moreover, concerns have also been put forward about their long term usage due to resistance. Furthermore, efforts have been made to improve their efficacy by employing genetic recombinations (Park et al 2005;Wirth et al 2004Wirth et al , 2007.…”