Size Quantification of Blood and Sugar Meals in Aedes aegypti Mosquitoes

Abstract

Both male and female mosquitoes consume sugar-rich nectar meals required for metabolic energy, but only females consume protein-rich blood meals, which are required for egg development. The size of each meal consumed has subsequent effects on behavior and reproduction; therefore, precise quantification is an important aspect of mosquito feeding behavior studies. This protocol describes a high-throughput, end-point assay to quantify meal volumes ingested by individual mosquitoes. The addition of a fluorescent dye to the meal allows for meal size quantification. Individual mosquitoes that have been fed this meal are homogenized in 96-well plates, and the fluorescence levels are measured with a plate reader. This protocol can also be adapted to determine if alteration of meal composition affects the ingested meal volume, if mosquito strain or genotype dictates consumption, or if meals are derived from multiple sources.

Automated analysis of feeding behaviors of females of the mosquito Aedes aegypti using a modified flyPAD system

“…For the fluorescent tracer added to the meals, either sugar solution or blood, a 0.2% (w/v) stock solution of aqueous fluorescein sodium salt (VWR Radnor, PA, USA, 0681-100G) was prepared 77,78 , and the tube was then wrapped in aluminum foil to avoid light exposure. All sugar meals were prepared as 10% sucrose and 0.002% (w/v) fluorescein as final concentrations.…”

“…The total volume of sucrose solution or blood ingested by each female was estimated using the fluorescein quantification method as described 77,78 . Briefly, after the 30 min assays, females were anesthetized using humid CO 2 and individually transferred to 2 mL polypropylene tubes with screw caps and silicon O-rings, containing five 1.4 mm ceramic beads (Omni International, 19-645-3) and 100 µL of phosphate-buffered saline (PBS) (655090, Greiner Bio-One, Kremsmünster, Austria).…”