Scorpion neurotoxin AaIT-expressing Beauveria bassiana enhances the virulence against Aedes albopictus mosquitoes

Abstract

To improve the insecticidal efficacy of this entomopathogen Beauveria bassiana, the fungus was genetically modified to express an insect-specific scorpion neurotoxin AaIT. The virulence of the recombinant B. bassiana strain (Bb-AaIT) against Aedes albopictus adults (which occurs via penetration through the cuticle during spore germination or by conidia ingestion), and the larvae (by conidia ingestion) was measured with bioassays. The median lethal concentration (LC₅₀) of Bb-AaIT against A. albopictus larvae was 313.3-fold lower on day 4 and 11.3-fold lower on day 10 than that of the wild type (WT). Through conidia feeding or body contact, Bb-AaIT killed 50% of adult female mosquitoes at 3.9- or 1.9-fold reduced concentrations on day 4 and at 2.1- or 2.4-fold reduced concentrations on day 10. Compared with the results for the WT, the median lethal time (LT₅₀) of Bb-AaIT was reduced by 28.6% at 1 × 10⁷ conidia ml^-1 and 34.3% at 1 × 10⁶ conidia ml^-1 in the larvae bioassay by conidia ingestion, while it decreased 32.3% at 1 × 10⁷ conidia ml^-1 by conidia ingestion and 24.2% at 1 × 10⁸ conidia ml^-1 by penetrating through the cuticle in the adult bioassay. All the differences were significant. Our findings indicated that Bb-AaIT had higher virulence and faster action than the WT in killing the larval and adult mosquitoes, and therefore, it is valuable for development as a commercial mosquito pesticide.

Keywords: Aedes albopictus; Beauveria bassiana; Scorpion neurotoxin; Virulence.

Fig. 1

Evidence for AaIT expression in the Bb-AaIT strain. a RT-PCR detection of AaIT gene transcription in Bb-AaIT collected from the infected dead mosquitoes and the CDA with positive results, and in the WT control with negative results. The 18S rRNA was detected for the loading control. b Western blot detection of AaIT expression in Bb-AaIT and the WT with a polyclonal antibody against AaIT from different samples. Lane 1, culture supernatant from Bb-AaIT 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 mosquitoes infected by Bb-AaIT 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. c The dead female mosquitoes were infected by Bb-AaIT or WT through conidial ingestion. Fungal outgrowths were not observed in the uncultured dead mosquitoes (1 and 3), and typical fungal outgrowths were observed on the dead mosquitoes that were infected by Bb-AaIT (2) and the WT (4) after 4 days of incubation at 25 °C and saturated humidity. Bars, 50 µm

Fig. 2

Survival curves of larval and adult mosquitoes for different Bb-AaIT and WT treatments. a The survival curve of Aedes albopictus larvae when treated with (C1) 1 × 10⁵, (C2) 1 × 10⁶ and (C3) 1 × 10⁷ conidia ml⁻¹ suspensions of Bb-AaIT and the WT. b The survival curve of Aedes albopictus female adults that were infected by conidia ingestion with (C1) 1 × 10⁵, (C2) 1 × 10⁶ and (C3) 1 × 10⁷ conidia ml⁻¹ suspensions of Bb-AaIT and WT. c The survival curve of Aedes albopictus female adults that were infected through cuticle contact with 3 ml of (C1) 1 × 10⁶, (C2) 1 × 10⁷ and (C3) 1 × 10⁸ conidia ml⁻¹ suspensions containing Bb-AaIT and WT that were sprayed on 50 cm² filter paper

Fig. 3

LC₅₀ of WT and Bb-AaIT strains against larval or adult mosquitoes. Error bars: ± SE. Three bioassays were conducted to compare the virulence of the Bb-AaIT and WT strains as follows: a assay 1, the second-instar larvae of Aedes albopictus infection through conidia ingestion. (b, c) 3-day-old (3 days after emergence) female adult mosquito infection through cuticle contact (b, assay 2) or through conidia ingestion (c, assay 3)