Anopheles stephensi and Toxorhynchites amboinensis: Aseptic Rearing of Mosquito Larvae on Cultured Cells

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Abstract
Aseptic larvae of A. stephensi and T. amboinensis were reared on a continuous cell line (RU TAE 12 V) from the mosquito, T. amboinensis, that grew in suspension as multicellular vesicles. Surface-sterilized eggs were hatched in a 24-well plate containing 0.2 ml of Leibovitz''s L-15 medium per well and incubated in a humidified atmosphere. T. amboinensis eggs 36 h or older were placed singly to assure hatching and avoid cannibalism. Hatching rates were > 80%. All larval instars were maintained in L-15 medium at 28.degree. C with a 12-h photoperiod. A. stephensi larvae were reared in 25-cm2 tissue culture flasks containing 10 ml of L-15 medium with 30 to 50 1st and 2nd instar larvae or 10 third and fourth instar larvae per flask. T. amboinensis larvae remained in the 24-well plate in 1.5 ml of medium through the 2nd instar, 3rd instar larvae were kept in 12-well plates (3 ml of medium per well) and transferred to 25-cm2 flasks (10 ml per flask) when they reached the 4th instar. First and 2nd instar A. stephensi larvae were fed cultured cells once, and 3rd or 4th instar larvae twice a day. T. amboinensis larvae were fed vesicles once during the first 4 days after hatching, and every 1 or 2 days thereafter. Each A. stephensi larva consumed approximately 2 .times. 106 cells and T. amboinensis larvae 10 times more cells before pupating. A. stephensi pupated after 7-8 days and adults emerged during days 9-11. Pupation in T. amboinensis began on day 21 after hatching and adults emerged 5 days later. Cell lines isolated from A. stephensi larvae or embryos of the ticks Rhipicephalus sanguineus and Anocentor (Dermacentor) nitens supported only limited growth of A. stephensi larvae. Defibrinated hamster (Mesocricetus auratus) blood, though readily ingested, did not support the growth of A. stephensi whereas larvae reared on blood cells plus T. amboinensis cells showed limited growth.