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Establishment and Characterization of Insect Cell Lines from 10 Lepidopteran Species

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Establishment and Characterization of Insect Cell Lines from 10 Lepidopteran Species In Vitro Cell. Dev. Biol.ÐAnimal 37:367±373, June 2001 q 2001 Society for In Vitro Biology 1071-2690/01 $10.00+0.00 ESTABLISHMENT AND CHARACTERIZATION OF INSECT CELL LINES FROM 10 LEPIDOPTERAN SPECIES CYNTHIA L. GOODMAN,1 GALAL N. EL SAYED, ARTHUR H. MCINTOSH, JAMES J. GRASELA, AND BRAD STILES Biological Control of Insects Research Laboratory (BCIRL), U.S. Department of Agriculture (USDA),2 Agricultural Research Service (ARS), Columbia, Missouri 65203-3535 (C. L. G., A. H. M., J. J. G.), Department of Entomology, University of Missouri, Columbia, Missouri 65211 (G. N. E.), and BASF Agro Research (formerly American Cyanamid Co., Agricultural Research Div.), P.O. Box 400, Princeton, New Jersey 08540 (B. S.) (Received 27 December 2000; accepted 16 March 2001) SUMMARY Cell lines from selected lepidopteran species were established for the overall purpose of use in baculovirus production. A total of 36 new cell lines from 10 lepidopteran species were generated, including cell lines from a pyralid, the European corn borer, Ostrinia nubilalis, a plutellid, the diamondback moth, Plutella xylostella, as well as eight noctuids: the black cutworm, Agrotis ipsilon, the celery looper, Anagrapha falcifera, the velvetbean caterpillar, Anticarsia gemmatalis, the corn earworm, Helicoverpa zea, the tobacco budworm, Heliothis virescens, the beet armyworm, Spodoptera exigua, the fall armyworm, Spodoptera frugiperda, and the cabbage looper, Trichoplusia ni. Tissues used for cell line establishment included fat bodies, ovaries, testes, or whole embryos/larvae/pupae. All the cell lines were subcultured numerous times, characterized by isoenzyme analysis and/or deoxyribonucleic acid ampli®cation ®ngerprinting using polymerase chain reaction, and stored in liquid nitrogen. Many of the cell lines were adapted to grow in serum-free medium, with cell lines from A. ipsilon and H. virescens being adapted to suspension culture using shaker ¯asks. The potential use for these cell lines in baculovirus production is discussed. Key words: DNA ®ngerprinting; isoenzymes; baculovirus; noctuidae; plutellidae; pyralidae. INTRODUCTION The purpose of the present study was to establish cell lines from lepidopteran species and tissues that are not well represented by Insect cell lines can be utilized in many areas of biological re- the existing cell lines, with the expectation that some of these new search, including physiology, toxicology, and pathology. In the area lines would have properties important for the mass production of of pathology, an important use of insect cell lines is in the inves- selected baculoviruses. The under-represented areas include cell tigation of virus±cell interactions. These studies have led to the lines from speci®c differentiated tissues known to be susceptible to application of cell lines for the mass production of viral pesticides, viral infection (e.g., larval fat body; Vail and Jay, 1973) and selected notably baculoviruses (Goodman and McIntosh, 1994; Murhammer, agricultural pest insects which may be susceptible to viral pesti- 1996; Black et al., 1997). Within this framework, the development cides (e.g., black cutworm, Agsrotis ipsilon [Hufnagel] [Noctuidae: of new insect cell lines is known to lead to improvements in virus- Lepidoptera]). The latter list also includes insect species, or their production levels and/or virus stability (Hink and Strauss, 1976; relatives, known to produce ef®cacious baculoviruses in vivo (e.g., Miltenburger et al., 1984; Goodman and McIntosh, 1994). In fact, Plutella xylostella (L.) [Plutellidae: Lepidoptera], Kariuki and Mc- numerous cell lines have been previously established from pest in- Intosh, 1999 and Anagrapha falcifera [Kirby] [Noctuidae: Lepidop- sects of agricultural importance for the primary purpose of studying tera], a close relative of the original host of AcMNPV, Autographa and/or optimizing baculovirus production (e.g., McIntosh and Ig- californica). Overall, the insects selected for cell culture initiation noffo, 1981; Gelertner and Federici, 1986; Lynn and Shapiro, 1998; represented pests of numerous crop plants, including corn (A. ips- McKenna et al., 1998; Kariuki et al., 2000). ilon, Helicoverpa zea [Boddie] [Noctuidae: Lepidoptera], Ostrinia nubilalis [HuÈbner] [Pyralidae: Lepidoptera], Spodoptera frugiperda 1 To whom correspondence should be addressed at Biological Control of [J. E. Smith] [Noctuidae: Lepidoptera]), cotton (Heliothis virescens Insects Research Laboratory, U.S. Department of Agriculture, Agricultural [Fabricius] [Noctuidae: Lepidoptera], H. zea, Spodoptera exigua Research Service, 1503 S. Providence Road, Columbia, Missouri 65203- 3535. E-mail: [email protected] [HuÈbner], S. frugiperda), vegetables (P. xylostella, S. exigua, Tri- 2 All programs and services of the U.S. Department of Agriculture are choplusia ni [HuÈbner] [Noctuidae: Lepidoptera]), and forage crops offered on a nondiscriminatory basis without regard to race, color, national (Anticarsia gemmatalis [HuÈbner] [Noctuidae: Lepidoptera], P. xy- origin, religion, sex, age, marital status, or handicap. lostella) (Turnipseed, 1973; Trumble and Baker, 1984; Fitt, 1989; 3 Names are necessary to report factually on the available data; however, Talekar, 1990). the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the A second goal of our studies was to determine the adaptability exclusion of others that may also be suitable. of each cell line to serum-free medium and, in selected cases, to 367 368 TABLE 1 NEWLY ESTABLISHED LEPIDOPTERAN CELL LINESa Weeks to ®rst Species Stage Tissue Establishment media passage Complete designation Anagrapha falcifera (Af) Adult Ovarian/fat body (OV) ExCell 401 1 20% FBS 18.1 BCIRL/AMCY-AfOV-CLG Adult Testes/fat body (TS) ExCell 401 1 20% FBS NR BCIRL/AMCY-AfTS-CLG Anticarsia gemmatalis (Ag) Adult Ovarian/fat body (OV) ExCell 401 1 20% FBS 29.3 BCIRL/AMCY-AgOV-CLG1 Adult Ovarian/fat body (OV) ExCell 401 1 20% FBS 29.1 BCIRL/AMCY-AgOV-CLG2 Adult Ovarian/fat body (OV) ExCell 401 1 20% FBS 29.1 BCIRL/AMCY-AgOV-CLG3b Egg Embryonic (E) ExCell 401 1 20% FBS 7.0 BCIRL/AMCY-AgE-CLG Agrotis ipsilon (Ai) Adult Ovarian/fat body (OV) ExCell 401 1 20% FBS 3.6 BCIRL/AMCY-AiOV-CLG Adult Testes/fat body (TS) ExCell 401 1 20% FBS 29.0 BCIRL/AMCY-AiTS-CLG Heliothis virescens (Hv) Egg Embryonic (E) ExCell 401 1 20% FBS 23.7 BCIRL/AMCY-HvE-CLG1 Egg Embryonic (E) ExCell 401 1 20% FBS 2.1 BCIRL/AMCY-HvE-CLG2 Egg Embryonic (E) ExCell 401 1 20% FBS 24.3 BCIRL/AMCY-HvE-CLG3 Larva Testes (TS) MGM-448 1 10% FBS 27.1 BCIRL/AMCY-Hv-TS-GES Adult Ovaries (OV) ExCell 401 1 20% FBS 9.3 BCIRL/AMCY-HvOV-CLG Helicoverpa zea (Hz) Egg Embryonic (E) ExCell 401 1 10% FBS 6.9 BCIRL/AMCY-HzE-CLG1 Egg Embryonic (E) ExCell 401 1 10% FBS ;7.5 BCIRL/AMCY-HzE-CLG2 Egg Embryonic (E) ExCell 401 1 10% FBS ;7.5 BCIRL/AMCY-HzE-CLG3 GOODMAN ET AL. Egg Embryonic (E) ExCell 401 1 10% FBS ;4.5 BCIRL/AMCY-HzE-CLG5 Egg Embryonic (E) ExCell 401 1 10% FBS 8.9 BCIRL/AMCY-HzE-CLG6 Egg Embryonic (E) ExCell 401 1 10% FBS 7.0 BCIRL/AMCY-HzE-CLG7 Egg Embryonic (E) ExCell 401 1 10% FBS NR BCIRL/AMCY-HzE-CLG8 Egg Embryonic (E) ExCell 401 1 10% FBS NR BCIRL/AMCY-HzE-CLG9 Ostrinia nubilalis (On) Larva Fat body (FB) TNM-FH 1 10% FBS 23.1 BCIRL/AMCY-OnFB-GES1 Larva Fat body (FB) ExCell 401 1 20% FBS NR BCIRL/AMCY-OnFB-GES3 Plutella xylostella (Px) Egg Embryonic (E) ExCell 401 1 10% FBS 5.4 BCIRL/AMCY-PxE-CLG Larva Pupa (LP) Whole insect ExCell 401 1 10% FBS 11.3 BCIRL/AMCY-PxLP-CLG Spodoptera exigua (Se) Egg Embryonic (E) ExCell 401 1 10% FBS 5.6 BCIRL/AMCY-SeE-CLG1c Egg Embryonic (E) ExCell 401 1 10% FBS 4.9 BCIRL/AMCY-SeE-CLG4 Egg Embryonic (E) ExCell 401 1 10% FBS 4.9 BCIRL/AMCY-SeE-CLG5c Spodoptera frugiperda (Sf) Larva Testes (TS) ExCell 401 1 10% FBS 5.0 BCIRL/AMCY-SfTS-GES Trichoplusia ni (Tn) Egg Embryonic (E) ExCell 401 1 20% FBS 5.0 BCIRL/AMCY-TnE-CLG1 Egg Embryonic (E) TC199-MK 1 10% FBS 22.1 BCIRL/AMCY-TnE-CLG1MK Egg Embryonic (E) ExCell 401 1 20% FBS 5.1 BCIRL/AMCY-TnE-CLG2 Trichoplusia ni (Tn) Egg Embryonic (E) TC199-MK 1 10% FBS 22.3 BCIRL/AMCY-TnE-CLG2MK Egg Embryonic (E) ExCell 401 1 20% FBS 6.1 BCIRL/AMCY-TnE-CLG3 Larva Testes (TS) ExCell 401 1 20% FBS NR BCIRL/AMCY-TnTS-GES1 Larva Testes (TS) ExCell 401 1 20% FBS 16.7 BCIRL/AMCY-TnTS-GES3 a Abbreviations: NR, not recorded; ;, approximate. Designations include (in order of appearance): initials of the laboratory (BCIRL, Biological Control of Insects Research Laboratory), corporate collaborator (AMCY, American Cyanamid Co.), insect species (as indicated in italics in the Species column), insect stage or tissue type (as indicated in the Stage or Tissue column), cell line initiator (CLG or GES, see author list), and cell line number (if more than one exists of the speci®ed type). b AgOV3 (with the original designation of AgOV1/2) was generated by combining cells in suspension of both AgOV1 and AgOV2 prior to their ®rst subculture, in order to ensure that these cells were not lost. c Originally described by Grasela et al. (2000). LEPIDOPTERAN CELL LINES 369 FIG. 1. Cell lines were photographed using a Zeiss MC 63 camera attached to a Zeiss ICM 405 inverted microscope: (A) AiOV (P 38) in ExCell 401, (B) AiTS (P 20) in ExCell 401 1 10% FBS, (C) TnE1 (P 27) in ExCell 401 1 2.5% FBS, and (D) SfTS (P 20) in ExCell 401 1 10% FBS. Magni®cation: A, C, 3400; B, D, 3600. suspension-culture conditions; i.e., characteristics that would be nized embryos, larvae, or pupae as well as cultures from isolated tissues (e.g., important for the use of these cell lines in the mass production of fat body, ovaries, and testes).
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