Abstract
Insect cell lines are used to study cellular interactions and gene functions in vitro in several research areas. However, suitable cell lines for experiments are not always available, especially in non-model species. Here, we established novel cell lines derived from fat bodies of six lepidopteran insects: Cydia kurokoi (named NARO-Cyku), Cephonodes hylas (NARO-Cehy), Haritalodes basipunctalis (NARO-Haba), Theretra oldenlandiae (NARO-Thol), Lymantria dispar (NARO-Lydi), and Hyphantria cunea (NARO-Hycu) collected in the field. The larval fat body was a promising tissue for the starting material when samples were limited due to field collection. It was critical that the medium volume was kept to a minimum for primary culture to maintain adherence of the fat body cells to the flask. The flask was coated with poly-L-lysine for effective induction of adherence and cell division. The identities of cell lines were confirmed using DNA barcoding with the mitochondrial cytochrome c oxidase I gene after cultures were passaged over 50 times. All lines except for NARO-Lydi and NARO-Hycu are adherent 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 revealed by transfection of expression vectors and found the immediate early 2 and the Bombyx actin 3 were effective gene promoters. The results suggest that these cell lines are capable of gene functional analysis. Thus, establishments of cell line using our methods for non-model lepidopterans could make a practical contribution to pest management and insect utilization.
Change history
12 June 2020
This article was originally published with the final word of the title, “field”, omitted.
References
Ahmed I, Huebner H, Mamoori YI, Buchholz R (2017) Identification of newly established Spodoptera littoralis cell lines by two DNA barcoding markers. In Vitro Cell Dev Biol Anim 53:288–292. https://doi.org/10.1007/s11626-016-0118-x
Cooper JK, Sykes G, King S, Cottrill K, Ivanova NV, Hanner R, Ikonomi P (2007) Species identification in cell culture: a two-pronged molecular approach. In Vitro Cell Dev Biol Anim 43:344–351. https://doi.org/10.1007/s11626-007-9060-2
Goodwin RH, Tompkins GJ, McCawley P (1978) Gypsy moth cell lines divergent in viral susceptibility - I. Culture and identification. In Vitro 14:485–494. https://doi.org/10.1007/BF02616088
Grace TD (1962) Establishment of four strains of cells from insect tissues grown in vitro. Nature 195:788–789. https://doi.org/10.1038/195788a0
Iwanaga M, Arai R, Shibano Y, Kawasaki H, Imanishi S (2009) Establishment and characterization of the Bombyx mandarina cell line. J Invertebr Pathol 101:124–129. https://doi.org/10.1016/j.jip.2009.05.004
Kayukawa T, Tateishi K, Shinoda T (2013) Establishment of a versatile cell line for juvenile hormone signaling analysis in Tribolium castaneum. Sci Rep 3:1570. https://doi.org/10.1038/srep01570
Kobayashi I, Kojima K, Uchino K, Sezutsu H, Iizuka T, Tatematsu K, Yonemura N, Tanaka H, Yamakawa M, Ogura E, Kamachi Y, Tamura T (2011) An efficient binary system for gene expression in the silkworm, Bombyx mori, using GAL4 variants. Arch Insect Biochem Physiol 76:195–210. https://doi.org/10.1002/arch.20402
Lynn DE, Dougherty EM, McClintock JT, Loeb M (1988) Development of cell lines from various tissues of Lepidoptera. In: Kuroda Y, Kurstak E, Maramorosch K (eds) Invertebrate and fish tissue culture. Japan Scientific Societies Press, Tokyo, pp 239–242
Lynn DE, Harrison RL (2016) Available lepidopteran insect cell lines. In: Murhammer DW (ed) Baculovirus and insect cell expression protocols. Humana Press, New York, pp 119–142. https://doi.org/10.1007/978-1-4939-3043-2_6
Mitsuhashi J (1981) Establishment and some characteristics of a continuous cell line derived from fat bodys of the cabbage armyworm (Lepidoptera, Noctuidae). Develop Growth Differ 23:63–72. https://doi.org/10.1111/j.1440-169X.1981.00063.x
Mitsuhashi J (1983) A continuous cell line derived from fat bodies of the common armyworm, Leucania separata (Lepidoptera: Noctuidae). Appl Entomol Zool 18:533–539. https://doi.org/10.1303/aez.18.533
Mitsuhashi J (2001) Development of highly nutritive culture media. In Vitro Cell Dev Biol Anim 37:330–337. https://doi.org/10.1007/BF02577566
Mitsuhashi J (2002) Invertebrate tissue culture methods. Springer, Tokyo. https://doi.org/10.1007/978-4-431-67875-5
Mitsuhashi J, Hayasaka S, Imanishi S (2003) Continuous cell lines from the common white, Pieris rapae crucivora Boisduval. In Vitro Cell Dev Biol Anim 39:114–116. https://doi.org/10.1007/s11626-003-0003-2
Mitsuhashi J, Inoue H (1988) Obtainment of a continuous cell line from the larval fat bodies of the mulberry tiger moth, Spilosoma imparilis (Lepidoptera:Arctiidae). Appl Entomol Zool 23:488–490. https://doi.org/10.1303/aez.23.488
Sakamoto H (1994) Establishment of a continuous cell line, NSC-HyCu15B, from the larval fat body of the fall webworm, Hyphantria cunea, (Lepidoptera: Arctiidae). Bull Nagano Sericult Cent 3:11–14 (in Japanese)
Smagghe G, Goodman CL, Stanley D (2009) Insect cell culture and applications to research and pest management. In Vitro Cell Dev Biol Anim 45:93–105. https://doi.org/10.1007/s11626-009-9181-x
Suzuki MG, Imanishi S, Dohmae N, Nishimura T, Shimada T, Matsumoto S (2008) Establishment of a novel in vivo sex-specific splicing assay system to identify a trans-acting factor that negatively regulates splicing of Bombyx mori dsx female exons. Mol Cell Biol 28:333–343. https://doi.org/10.1128/MCB.01528-07
Tamura T, Thibert C, Royer C, Kanda T, Eappen A, Kamba M, Kômoto N, Thomas J-L, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme J-C, Couble P (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18:81–84. https://doi.org/10.1038/71978
Tateishi K, Kasahara Y, Watanabe K, Hosokawa N, Doi H, Nakajiima K, Adachi H, Nomoto A (2015) A new cell line from the fat body of Spodoptera litura (Lepidoptera, Noctuidae) and detection of lysozyme activity release upon immune stimulation. In Vitro Cell Dev Biol Anim 51:15–18. https://doi.org/10.1007/s11626-014-9808-4
Theilmann DA, Stewart S (1992) Molecular analysis of the trans-activating IE-2 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology 187:84–96. https://doi.org/10.1016/0042-6822(92)90297-3
Yavin E, Yavin Z (1974) Attachment and culture of dissociated cells from rat embryo cerebral hemispheres on polylysine-coated surface. J Cell Biol 62:540–546. https://doi.org/10.1083/JCB.62.2.540
Acknowledgments
We thank Dr. S. Imanishi for technical advice. We also thank T. Nakakura and Y. Itoh for technical assistance with the experiments.
Author information
Authors and Affiliations
Corresponding author
Additional information
Editor: Tetsuji Okamoto
The original version of this article was revised: This article was originally published with the final word of the title, “field”, omitted.
Rights and permissions
About this article
Cite this article
Watanabe, K., Kobayashi, I., Hatakeyama, M. et al. Establishment and characterization of novel cell lines derived from six lepidopteran insects collected in the field. In Vitro Cell.Dev.Biol.-Animal 56, 425–429 (2020). https://doi.org/10.1007/s11626-020-00438-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11626-020-00438-5