Expression of Bacillus thuringiensis toxin Cyt2Ba in the entomopathogenic fungus Beauveria bassiana increases its virulence towards Aedes mosquitoes

Background The entomopathogenic fungus Beauveria bassiana has been widely used to kill mosquito larvae and adults in the laboratory and field. However, its slow action of killing has hampered its widespread application. In our study, the B. bassiana fungus was genetically modified to express the Bacillus thuringiensis (Bt) toxin Cyt2Ba to improve its efficacy in killing mosquitoes. Methodology/Principal findings The efficacy of the wild type (WT) of B. bassiana and a transgenic strain expressing Cyt2Ba toxin (Bb-Cyt2Ba) was evaluated against larval and adult Aedes mosquitoes (Aedes aegypti and Aedes albopictus) using insect bioassays. The Bb-Cyt2Ba displayed increased virulence against larval and adult Aedes mosquitoes compared with the WT: for Ae. aegypti adults, the median lethal time (LT50) was decreased by 33% at the concentration of 1x 10(8) conidia/ml, 19% at 1x 10(7) conidia/ml and 47% at 1x 10(6) conidia/ml. The LT50 for Ae. albopictus adults was reduced by 20%, 23% and 29% at the same concentrations, respectively. The LT50 for Ae. aegypti larvae was decreased by 42% at 1x 10(7) conidia/ml and 25% at 1x 10(6) conidia/ml, and that for Ae. albopictus larvae was reduced by 33% and 31% at the same concentrations, respectively. In addition, infection with Bb-Cyt2Ba resulted in a dramatic reduction in the fecundity of Aedes mosquitoes. Conclusions/Significance In conclusion, our study demonstrated that the virulence of B. bassiana against mosquitoes can be significantly improved by introducing the Bt toxin gene Cyt2Ba into the genome to express the exogenous toxin in the fungus. The transgenic strain Bb-Cyt2Ba significantly reduced the survival and fecundity of Ae. aegypti and Ae. albopictus compared with the WT strain, which suggested that this recombinant B. bassiana has great potential for use in mosquito control. Author summary Mosquito vectors transmit many diseases to humans and animals, causing illness and death and resulting in substantial socio-economic burdens in endemic countries. The control of mosquitoes has almost exclusively relied on the use of chemical insecticides, which has recently led to the broad resistance of important mosquito vectors worldwide. Entomopathogenic fungi, such as Beauveria bassiana, are an important alternative or complement to chemical insecticides. However, the relatively slow action of fungal pathogens in killing mosquitoes, compared with chemical insecticides, has hampered their widespread application. To improve the insecticidal efficacy of the entomopathogen B. bassiana, the fungus was genetically modified to express the Bacillus thuringiensis toxin Cyt2Ba. The mitotically stable transformant (Bb-Cyt2Ba) successfully expressed Cyt2Ba toxin, and the virulence of this strain against adults and larvae of Aedes aegypti and Aedes albopictus mosquitoes was significantly improved. In addition, egg laying was significantly affected by Bb-Cyt2Ba infection. Infection with this fungus resulted in a dramatic reduction in fecundity of the target mosquitoes. Therefore, this recombinant B. bassiana has great potential for use in mosquito control.