ANOPHELES-STEPHENSI AND TOXORHYNCHITES-AMBOINENSIS - ASEPTIC REARING OF MOSQUITO LARVAE ON CULTURED-CELLS
ANOPHELES-STEPHENSI AND TOXORHYNCHITES-AMBOINENSIS - ASEPTIC REARING OF MOSQUITO LARVAE ON CULTURED-CELLS
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DOI：
10.2307/3281096
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发表时间：
1982-01-01
影响因子：
1.3
通讯作者：
MARAMOROSCH, K
中科院分区：
文献类型：
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作者：
MUNDERLOH, UG;KURTTI, TJ;MARAMOROSCH, K
斯氏按蚊和竹按蚊的无菌幼虫在连续细胞系(RU Tae 12V)上饲养，该细胞系悬浮生长为多细胞囊泡。表面灭菌的鸡蛋在24孔培养皿中孵化，每孔装有0.2mlLeibovitz‘’S L-15培养液，在潮湿的气氛中孵化。36小时或以上的竹节虫卵被单独放置，以确保孵化和避免同类相食。孵化率为80%。所有幼虫在L-15培养液中均维持在28度。C，光周期为12小时。斯氏飞虱幼虫培养在25cm2的组织培养瓶中，每瓶含10毫升L-15培养液，1、2龄幼虫30~50条，3、4龄幼虫10条。竹毛虫幼虫停留在24孔板中1.5ml的培养液中直到2龄，3龄幼虫保存在12孔板中(每孔3毫升)，到达4龄时转移到25 cm~2的培养瓶中(每瓶10毫升)。1龄和2龄斯氏飞虱幼虫每天饲喂1次培养细胞，3龄和4龄幼虫每天饲喂2次培养细胞。日本血吸虫幼虫在孵化后的前4天饲喂1次囊泡，此后每隔1~2天饲喂1次。每头斯氏吸浆虫幼虫摄食约2倍。106个细胞和羽化前竹节幼虫10倍以上的细胞。斯氏飞虱化蛹时间为7-8天，成虫羽化时间为9-11天。羽化始于孵化后第21天，成虫羽化时间为5天。从斯氏狸殖吸虫幼虫或血色右头绦虫和硬蜱Anocentor(Dermacentor)nitens胚胎分离的细胞系仅支持斯氏吸虫幼虫有限的生长。去纤维仓鼠(Mesocricus Auratus)的血液虽然容易摄取，但并不支持斯氏疟原虫的生长，而以血细胞和竹节虫细胞饲养的幼虫生长有限。
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.