Insect cell lines are used to study cellular interactions and gene functions in vitroin several research areas. However, suitable cell lines for experiments are not alwaysavailable, especially in non-model species. Here, we established novel cell lines derivedfrom fat bodies of six lepidopteran insects: Cydia kurokoi (named NARO-Cyku), Cephonodes hylas (NARO-Cehy), Haritalodes basipunctalis (NARO-Haba), Theretraoldenlandiae (NARO-Thol), Lymantria dispar (NARO-Lydi), and Hyphantria cunea (NARO-Hycu) collected in the field. The larval fat body was a promising tissue for thestarting material when samples were limited due to field collection. It was critical thatthe medium volume was kept to a minimum for primary culture to maintain adherenceof the fat body cells to the flask. The flask was coated with poly-L-lysine for effectiveinduction of adherence and cell division. The identities of cell lines were confirmedusing DNA barcoding with the mitochondrial cytochrome c oxidase I gene after cultureswere passaged over 50 times. All lines except for NARO-Lydi and NARO-Hycu areadherent cells, and population doubling time of six cell lines ranged from 1.03 to 2.49. Induction of gene expression was practicable in the four adherent cell lines as revealedby transfection of expression vectors and found the immediate early 2 and the Bombyxactin 3 were effective gene promoters. The results suggest that these cell lines arecapable of gene functional analysis. Thus, establishments of cell line using our methodsfor non-model lepidopterans could make a practical contribution to pest managementand insect utilization.

在几个研究领域中，昆虫细胞系用于体外研究细胞相互作用和基因功能。然而，并非总是可获得合适的用于实验的细胞系，尤其是在非模型物种中。在这里，我们建立了来自六种鳞翅目昆虫脂肪体的新型细胞系：Cydia kurokoi（命名为NARO-Cyku），Cephonodes hylas（NARO-Cehy），Haritalodes basipunctalis（NARO-Haba），Theretraoldenlandiae（NARO-Thol），Lymantria dispar（ NARO-Lydi）和豚鼠（NARO-Hycu）在野外收集。当样品由于田间采集而受到限制时，幼虫的脂肪体是起始材料的有希望的组织。至关重要的是，对于初次培养，应将培养基体积保持在最低水平，以保持脂肪细胞粘附于烧瓶。将烧瓶用聚-L-赖氨酸包被以有效诱导粘附和细胞分裂。使用线粒体细胞色素C氧化酶I的DNA条形码确认细胞系的身份培养后的基因传代了50多次。除NARO-Lydi和NARO-Hycu区域的贴壁细胞外，所有细胞系以及六个细胞系的群体倍增时间在1.03至2.49之间。如通过表达载体的转染所揭示的，在四个贴壁细胞系中诱导基因表达是可行的，并且发现立即早期2和Bombyxactin 3是有效的基因启动子。结果表明这些细胞系能够进行基因功能分析。因此，使用我们的非模型鳞翅目动物的方法建立细胞系可以为害虫管理和昆虫利用做出实际贡献。