Anopheles stephensi and Toxorhynchites amboinensis: aseptic rearing of mosquito larvae on cultured cells

Abstract

Aseptic larvae of Anopheles stephensi and Toxorhynchites 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. Toxorhynchites amboinensis eggs of 36 hr or older were placed singly to assure hatching and avoid cannibalism. Hatching rates were over 80%. All larval instars were maintained in L-15 medium at 28 C with a 12-hr photoperiod. Anopheles stephensi larvae were reared in 25-cm2 tissue culture flasks containing 10 ml of L-15 medium with 30 to 50 first and second instar larvae or 10 third and fourth instar larvae per flask. Toxorhynchites amboinensis larvae remained in the 24-well plate in 1.5 ml of medium through the second instar; third 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 fourth instar. First and second instar A. stephensi larvae were fed cultured cells once, and third or fourth instar larvae twice a day. Toxorhynchites 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 X 10(6) cells, and T. amboinensis larvae 10 times more cells before pupating. Anopheles stephensi pupated after 7 to 8 days and adults emerged during days 9 to 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.