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

Abstract

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 1× 108 conidia/ml, 19% at 1× 107 conidia/ml and 47% at 1× 106 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 1× 107 conidia/ml and 25% at 1× 106 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.

Fig 1. Evidence for Cyt2Ba expression in the Bb-Cyt2Ba strain.

(A) RT-PCR detection of Cyt2Ba gene transcription in Bb-Cyt2Ba collected from the infected dead female Ae. albopictus mosquitoes and the CDA with positive results and in the WT control with negative results. The 18S rRNA gene was detected for the loading control. (B) Western blot detection of Cyt2Ba expression in Bb-Cyt2Ba and the WT with a polyclonal antibody against Cyt2Ba from different samples. Lane 1, culture supernatant from Bb-Cyt2Ba grown in CDA for 4 days with a positive result; Lane 2, culture supernatant from WT grown in CDA for 4 days with a negative result; and Lane 3, the dead female Ae. albopictus mosquitoes infected by Bb-Cyt2Ba and incubated at 25°C for another 4 days; Lane 4, the dead female mosquitoes infected by the WT and incubated at 25°C for another 4 days; Lane 5, loading control.

Fig 2. Survival curves of the adult mosquitoes for different Bb-Cyt2Ba and WT treatments.

The survival curves of Ae. aegypti (A) and Ae. albopictus (B) females when treated with (C1) 1 × 10⁶, (C2) 1 × 10⁷ and (C3) 1 × 10⁸ conidia/ml suspensions of Bb-Cyt2Ba and WT. Each treatment contained 90 mosquitoes, performed three times.

Fig 3. Survival curves of the mosquito larvae for different Bb-Cyt2Ba and WT treatments.

Fig 4. Effects of Bb-Cyt2Ba infection on mosquito reproduction.

Ae. aegypti (A) and Ae. albopictus (B) females infected with a suspension of 1 × 10⁸ conidia/ml of the WT or Bb-Cyt2Ba strain were fed a blood meal 3 h after fungal infection. Noninfected, blood-fed mosquitoes were used as controls. Each bar represents the mean of eggs per mosquito. Mann-Whitney tests were used. Error bars indicate ± standard error (SE).