Aedes aegypti strain selected with Bacillus thuringiensis svar. israelensis larvicide for 50 generations remains susceptible and exhibited increased fitness.

Abstract

Background: Bacillus thuringiensis serovar israelensis (Bti) was the first bacterial larvicide developed for dipteran control. Its insecticidal crystal, composed of protoxins such as Cry11Aa, Cry4Ba, Cry4Aa, and Cyt1Aa, exhibits a selective and complex mode of action and a low potential for resistance development. Most resistance studies have focused on populations from regions where larvicide applications are seasonal, while Aedes aegypti populations in tropical endemic areas may face continuous selection pressure, raising concerns about potential selection of resistance. This study evaluated an Ae. aegypti strain (RecBti), Subjected to strong Bti selection for 50 generations under laboratory conditions, as part of a longitudinal study for assessing in vivo susceptibility to Bti and also biological traits that could be potentially influenced by this chronic larvicide exposure.

Methods: The RecBti strain, continuously selected using a commercial Bti larvicide for 50 generations, was compared with reference strains. Bioassays with Bti, Cry11Aa, and Cry4Ba were performed to assess the susceptibility of larvae (F₄₀ and F₅₀) to these compounds. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were carried out to investigate the expression of midgut-bound proteins reported as Cry toxin receptors in F₅₀ larvae. Energy reserves (lipids and reducing sugars) were quantified in larval pools and adult females from F₅₀. Additionally, biological parameters of egg viability after varying quiescence periods and adult longevity, were also evaluated in F₅₀.

Results: RecBti larvae remained susceptible to the Bti crystal and its individual Cry11Aa and Cry4Ba toxins after 50 generations of chronic exposure, compared with the reference susceptible strain. The transcriptional analysis revealed that three genes encoding Cry midgut receptors-aminopeptidase, alkaline phosphatase, and cadherin-were expressed at significantly higher levels in RecBti larvae compared with the reference strain. RecBti larvae and adults exhibited significantly higher lipid reserves, although no significant difference in reducing sugar levels was observed compared with the reference individuals. Egg viability of RecBti females was higher, and adults showed increased longevity.

Conclusions: This study provides evidence for the low risk of resistance development of Ae. aegypti to Bti, on the basis of a prolonged and intensive selection process. The levels of expression of Cry toxin receptors found in RecBti larvae, compared with reference larvae, are consistent with the status of in vivo susceptibility. However, long-term Bti exposure was associated with physiological and biological changes that may have implications for mosquito fitness and vectorial competence. These findings show that Bti is the larvicide that has a low potential to select resistance, compared with other compounds available, and highlight the importance of monitoring other effects of larvicide on the biology of mosquitoes.