Rearing and Bioassay of Ae. aegypti Mosquitoes

Ae. aegypti (Rockefeller) eggs were provided by a reference laboratory for insecticide susceptibility tests at the Oswaldo Cruz Institute (Laboratório de Biologia, Controle e Vigilância de Insetos Vetores; IOC-Fiocruz). Eggs were hatched in filtered tap water with a yeast pallet (500 mg). Larvae were maintained under controlled laboratory conditions (i.e., 28 ± 2°C, 12/12 regulated light with white fluorescent lamps), and were fed approximately 250 mg/day of fish food (TetraMin, Tetra, Spectrum Brands Company, WI, USA) per 2,000 larvae daily. Adults were maintained in cages at controlled laboratory conditions with a 10% sucrose solution. Blood-feeding was offered to mosquitoes after 48 h of emergence. Human blood was provided to females by a cotton path on a flask with warm water for approximately 30–45 min. All engorged females were separated and maintained with 10% sucrose for 3–4 days until the beginning of oviposition bioassays. Mosquito rearing and blood feeding was performed under license L-4/2008 of the Animal Use and Ethics Committee of the Oswaldo Cruz Institute (CEUA-IOC/Fiocruz).
Yeast encapsulated orange oil (YEOO) was synthesized by encapsulation of Citrus sinensis EO (CAS Number: 8008-57-9; Cold pressed from peel fruit; Limonene as main component) inside S. cerevisiae (Red Star fresh baker’s yeast) as described by Workman et al. [9 (link)]. Lyophilized YEOO was rehydrated to 50 mg/L of orange oil in water. For the bioassays, the working concentration was 160 mg/L, equivalent to 10x the LC₉₀ determined for third instar (L3) Ae. aegypti larvae [9 (link)]. Inactivated yeast (IY) was prepared by mixing 1 g S. cerevisiae in 4.5 ml water. The yeast was inactivated by heating (≈70°C) and mixing the solution for 20-30min without boiling [9 (link)].