Lipid Extraction from Mosquito Ovaries

Female mosquitoes maintained on the two different diets were collected at different times after adult eclosion (2, 3, 4, 6 and 7 days) and immobilized by exposure to ice. Ovaries were dissected by performing an incision in the thorax, cutting the last abdominal segment and pulling out the ovaries^{28 (link)}. Triplicates samples of 10 ovaries each were placed in 1.5 mL Eppendorf tubes, and 10 µL of a mix of labeled internal standard were added (EquiSplash Lipidomix, Avanti Polar Lipids, Alabaster, AL). After adding 100 µL of butanol/methanol and 3 µL of butylated hydroxytoluene, samples were homogenized for 10 s using polypropylene pestles (Fisher Scientific, Pittsburgh, PA), and a handheld cordless motor. The pestles were then rinsed with 200 µL of butanol/methanol. All tubes were sonicated for 30 min, and then centrifuged for 10 min. The supernatant was transferred into autosampler vials with 300 µL silanized glass inserts (Thermo Fisher Scientific, Waltham, MA).