Munderloh U G, Kurtti T J, Maramorosch K
J Parasitol. 1982 Dec;68(6):1085-91.
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.
斯蒂芬斯按蚊和amboinensis巨蚊的无菌幼虫在源自amboinensis巨蚊的连续细胞系（RU TAE 12 V）上饲养，该细胞系以多细胞囊泡形式悬浮生长。经表面消毒的卵在每个孔含有0.2 ml Leibovitz's L - 15培养基的24孔板中孵化，并在湿润的环境中培养。将36小时及以上的amboinensis巨蚊卵单独放置以确保孵化并避免同类相食。孵化率超过80%。所有幼虫龄期均在28℃、12小时光周期的L - 15培养基中饲养。斯蒂芬斯按蚊幼虫饲养在含有10 ml L - 15培养基的25 cm²组织培养瓶中，每个培养瓶中有30至50只一龄和二龄幼虫或10只三龄和四龄幼虫。amboinensis巨蚊幼虫在二龄前一直饲养在含有1.5 ml培养基的24孔板中；三龄幼虫饲养在12孔板（每孔3 ml培养基）中，四龄时转移到25 cm²培养瓶（每瓶10 ml）中。斯蒂芬斯按蚊一龄和二龄幼虫每天喂食一次培养细胞，三龄或四龄幼虫每天喂食两次。amboinensis巨蚊幼虫在孵化后的前4天喂食一次囊泡，之后每1或2天喂食一次。每只斯蒂芬斯按蚊幼虫在化蛹前消耗约2×10⁶个细胞，而amboinensis巨蚊幼虫消耗的细胞数量是其10倍。斯蒂芬斯按蚊在7至8天后化蛹，成虫在第9至11天羽化。amboinensis巨蚊在孵化后第21天开始化蛹，5天后成虫羽化。从斯蒂芬斯按蚊幼虫或血红扇头蜱和微小牛蜱（革蜱属）胚胎中分离的细胞系仅支持斯蒂芬斯按蚊幼虫的有限生长。去纤维蛋白的仓鼠（金黄仓鼠）血液虽然容易被摄取，但不支持斯蒂芬斯按蚊的生长，而以血细胞加amboinensis巨蚊细胞饲养的幼虫生长有限。
