Establishment and Characterization of Insect Cell Lines from 10 Lepidopteran Species

In Vitro Cell. Dev. Biol.—Animal 37:367–373, June 2001 ᭧ 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 amplification fingerprinting 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 flasks. The potential use for these cell lines in baculovirus production is discussed. Key words: DNA fingerprinting; 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 speciﬁc 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 efﬁcacious 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 [Hu¨bner] [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] [Hu¨bner], S. frugiperda), vegetables (P. xylostella, S. exigua, Tri- 2 All programs and services of the U.S. Department of Agriculture are choplusia ni [Hu¨bner] [Noctuidae: Lepidoptera]), and forage crops offered on a nondiscriminatory basis without regard to race, color, national (Anticarsia gemmatalis [Hu¨bner] [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 GOODMAN ET AL. see b c c y), corporate collaborator , in order to ensure that these cells were not BCIRL/AMCY-AgE-CLG BCIRL/AMCY-SeE-CLG4 BCIRL/AMCY-SeE-CLG5 BCIRL/AMCY-HzE-CLG1 BCIRL/AMCY-HzE-CLG2 BCIRL/AMCY-HzE-CLG3 BCIRL/AMCY-HzE-CLG5 BCIRL/AMCY-HzE-CLG6 BCIRL/AMCY-HzE-CLG7 BCIRL/AMCY-AfOV-CLG BCIRL/AMCY-AfTS-CLG BCIRL/AMCY-AgOV-CLG1 BCIRL/AMCY-AgOV-CLG2 BCIRL/AMCY-AgOV-CLG3 BCIRL/AMCY-AiOV-CLG BCIRL/AMCY-AiTS-CLG BCIRL/AMCY-HvE-CLG1 BCIRL/AMCY-HvE-CLG2 BCIRL/AMCY-HvE-CLG3 BCIRL/AMCY-Hv-TS-GES BCIRL/AMCY-HvOV-CLG BCIRL/AMCY-HzE-CLG8 BCIRL/AMCY-HzE-CLG9 BCIRL/AMCY-OnFB-GES1 BCIRL/AMCY-OnFB-GES3 BCIRL/AMCY-PxE-CLG BCIRL/AMCY-PxLP-CLG BCIRL/AMCY-SeE-CLG1 BCIRL/AMCY-SfTS-GES BCIRL/AMCY-TnE-CLG1 BCIRL/AMCY-TnE-CLG1MK BCIRL/AMCY-TnE-CLG2 BCIRL/AMCY-TnE-CLG2MK BCIRL/AMCY-TnE-CLG3 BCIRL/AMCY-TnTS-GES1 BCIRL/AMCY-TnTS-GES3 r Tissue column), cell line initiator (CLG or GES, 7.5 7.5 4.5 8.9 7.0 7.0 3.6 2.1 9.3 6.9 5.4 5.6 4.9 4.9 5.0 5.0 5.1 6.1 18.1 NR 29.3 29.1 29.1 29.0 23.7 24.3 27.1 NR NR 23.1 NR 11.3 22.1 22.3 NR 16.7 ϳ ϳ ϳ passage Complete designation Weeks to first a 20% FBS 20% FBS 20% FBS 20% FBS 20% FBS 20% FBS 20% FBS 20% FBS 20% FBS 20% FBS 20% FBS 20% FBS 10% FBS 10% FBS 10% FBS 10% FBS 10% FBS 10% FBS 10% FBS 10% FBS 20% FBS 10% FBS 10% FBS 10% FBS 10% FBS 10% FBS 10% FBS 20% FBS 20% FBS 20% FBS 20% FBS 20% FBS 10% FBS 10% FBS 10% FBS 10% FBS ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ϩ ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 MGM-448 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 TC199-MK ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 ExCell 401 TNM-FH TC199-MK ExCell 401 ExCell 401 ExCell 401 ExCell 401 TABLE 1 NEWLY ESTABLISHED LEPIDOPTERAN CELL LINES Ovarian/fat body (OV) Testes/fat body (TS) Embryonic (E) Embryonic (E) Testes (TS) Embryonic (E) Embryonic (E) Embryonic (E) Ovarian/fat body (OV) Testes/fat body (TS) Embryonic (E) Embryonic (E) Embryonic (E) Testes (TS) Ovaries (OV) Fat body (FB) Fat body (FB) Embryonic (E) Embryonic (E) Testes (TS) Testes (TS) Ovarian/fat body (OV) Ovarian/fat body (OV) Ovarian/fat body (OV) Embryonic (E) Embryonic (E) Embryonic (E) Embryonic (E) Embryonic (E) Embryonic (E) Embryonic (E) Embryonic (E) Whole insect Embryonic (E) Embryonic (E) Embryonic (E) Adult Egg Egg Larva Egg Egg Egg Adult Larva Egg Egg Larva Adult Egg Larva Larva Adult Adult Egg Egg Egg Egg Egg Egg Larva Pupa (LP) Egg Egg , approximate. Designations include (in order of appearance): initials of the laboratory (BCIRL, Biological Control of Insects Research Laborator ϳ (Ag) Adult (Sf) (Af) Adult (Se) Egg (Px) Egg (Hv) Egg (On) Larva (Hz) Egg Species Stage Tissue Establishment media (Tn) (Tn) Egg (Ai) Adult AgOV3 (with the original designation of AgOV1/2) was generated by combining cells in suspension of both AgOV1 and AgOV2 prior to their first subculture Abbreviations: NR, not recorded; Originally described by Grasela et al. (2000). a b c Trichoplusia ni Anagrapha falcifera Spodoptera frugiperda lost. author list), and cell line number (if more than one exists of the specified type). (AMCY, American Cyanamid Co.), insect species (as indicated in italics in the Species column), insect stage or tissue type (as indicated in the Stage o Agrotis ipsilon Anticarsia gemmatalis Heliothis virescens Helicoverpa zea Ostrinia nubilalis Plutella xylostella Spodoptera exigua Trichoplusia ni 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 ϩ 10% FBS, (C) TnE1 (P 27) in ExCell 401 ϩ 2.5% FBS, and (D) SfTS (P 20) in ExCell 401 ϩ 10% FBS. Magniﬁcation: A, C, ϫ400; B, D, ϫ600. 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). In some cases, different tissue types were co- cultured to determine if this procedure would encourage the proliferation of baculoviruses (Weiss et al., 1992; Goodman and McIntosh, 1994; different cell types (i.e., fat body with ovaries or testes). In general, emphasis Black et al., 1997). Thus, our studies involved numerous aspects was placed on tissues from the egg, larval, and adult stages. For species of of cell culture initiation and development. origin, 10 lepidopteran species were used in primary culture initiations, with the majority of insects obtained from laboratory-reared colonies: A. gemma- MATERIALS AND METHODS talis, H. virescens, H. zea, P. xylostella, T. ni (provided by Paula Peters, BCIRL, USDA, ARS, Columbia, MO); A. ipsilon (provided by Mary Cell line establishment and maintenance. Cultures were initiated from sev- Jackson/Armon Keaster, Department of Entomology, University of Missouri, eral insect tissues, including heterogeneous cultures derived from homoge- Columbia, MO); and O. nubilalis (provided by Jeanette Dyer, USDA, ARS, 370 GOODMAN ET AL.

TABLE 2

DAF-PCR ANALYSIS OF SELECTED NEWLY ESTABLISHED CELL LINES (NUMBER OF BASE PAIRS)a AgOV HvE2,3 HzE1, AfOV AgE 1,2,3 AiOV AiTS HvE1 HvOV 3,5,6 OnFB1 PxE PxLP SeE1 SeE5 SfTS TnE1,2 TnE3 TnE1-MK TnE2-MK

1470 1470 1470 1243 1243 916 916 756 552 552 552 498 498 481 481 481 481 463 463 463 396 396 396 396 348 348 348 348 348 342 308 308 308 308 308 308 295 295 295 261 261 261 221 221 221 221 221 221 203 203 203 174 174 174 174 174 174 174 174 163 163 163 163 163 163 163 130 130 130 130 130 130 130 130 130 130 130 123 123 123 123 123 123 123 123 100 100 100 100 100 100 100 100 83 83 83 83

a A primer for the mammalian aldolase gene was utilized for ampliﬁcation (PCR). Mean number of base pairs of DNA bands from different lanes from 1 to 3 separate gels are presented. Complete cell line designations are given in Table 1.

Ames, IA). Additionally, A. falcifera larvae were obtained from the field by glass Erlenmeyer flasks containing 30 ml ExCell 401. Flasks were rotated Benjamin Puttler (Department of Entomology, University of Missouri). at 130 rpm using an orbital shaker, and maintained at 28Њ C. Cells were The following insect cell culture media were used for primary culture subcultured every 7–10 d at a density of 5 ϫ 105 cells/ml for the first 12 initiations: ExCell 401௢ (a serum-free medium developed for culturing noc- passages, after which they were subcultured at appropriate dilution ratios tuid cells; JRH Biosciences,3 Lenexa, KS); MGM-448 (developed for cultur- which were dependent on the culture density and estimated growth rate ing fat body cells; Mitsuhashi, 1984); and two serum-containing media de- (1:3–1:10). veloped for culturing lepidopteran cells, TC199-MK (McIntosh et al., 1973) Cell line characterization. Two procedures were used for the biochemical and TNM-FH (Sigma Chemical Co., St. Louis, MO). All media used for suc- characterization of the newly established cell lines: deoxyribonucleic acid cessful primary culture initiations contained 10–20% fetal bovine serum (DNA) amplification fingerprinting using polymerase chain reaction (DAF- (FBS) inactivated at 56Њ C for 30 min (Intergen, Purchase, NY), 200 U/ml PCR) and isoenzyme analysis using isoelectric focusing (IEF). DAF-PCR was penicillin, and 0.2 mg/ml streptomycin (Sigma). performed using a primer for mammalian aldolase, with the resulting frag- Insects were sterilized by washing in 70% ethanol (containing 1% Triton ments separated on 2.5% agarose gels, as previously described (McIntosh X-100) followed by 0.53–2.2% sodium hypochlorite (Clorox Co., Oakland, et al., 1996). The DNA was extracted from cells using the protocol outlined CA) (1–2 washes/reagent, 5 min/wash), and then rinsing 1–2 times in sterile in the Puregene௢ extraction kit (Gentra Systems, Inc., Minneapolis, MN), water or Hanks’ balanced salt solution (HBSS) containing 200 U/ml peni- and stored at 4Њ C. Isoenzyme analysis was performed using appropriate cillin, 0.2 mg/ml streptomycin, 0.5 ␮g/ml amphotericin B, and 13 mM 3- enzyme substrates from the AuthentiKit௢ System (Innovative Chemistry, [N-moropholino]propanesulfonic acid (MOPS), pH 6.8–7.0. For dissections, Marchfield, MA) for the isoenzymes listed later. Protein extracts were gen- after the initial incision, the body cavity was thoroughly rinsed with HBSS erated by incubating cells in cell culture lysis reagent (Promega, Madison, to remove hemocytes and other contaminants. Tissues were removed and WI) for 15 min at room temperature, after which the cellular debris was again washed in HBSS. It was found early in our studies that mincing removed by centrifugation (12,000 ϫ g, 1 min). Extracts were stored at Ϫ70Њ C tissues in the absence of enzymes (0.5% trypsin, 0.5–2.5% collagenase, and then subjected to IEF using agarose gels in sodium barbital buffer (as and/or 0.1–0.2% hyaluronidase) generally produced the best results; there- described in the AuthentiKit௢ System manual). The isoenzymes examined fore, for all cultures except whole P. xylostella larvae/pupae (0.5% trypsin were: aspartate aminotransferase (AST), glucose-6-phosphate dehydrogenase with 0.2% ethylenediamine-tetraacetic acid, 5 min; McIntosh et al., 1981), (G6PD), hexokinase (HK), isocitrate dehydrogenase (IDH), lactate dehydro- enzymes were not used. After mincing, tissues were placed directly into T12.5 genase (LDH), malate dehydrogenase (MDH), malic enzyme (ME), phos- or T25 culture flasks (Costar, Cambridge, MA) with either 2.5 or 5 ml medium, phoglucoisomerase (PGI), and phosphoglucomutase (PGM) (Table 3). respectively. For whole-insect (P. xylostella) and egg preparations, specimens were homogenized using a plastic micropestle in a 1.5-ml Eppendorf tube, and centrifuged to pellet the tissues and cells (500 ϫ g, 10 min). The re- RESULTS sulting pellet was placed into disposable T25 flasks (Stiles et al., 1992). Pri- mary cultures were initially fed every 7 d (using ½ medium replacement), Cell line establishment and maintenance. Table 1 lists the cell then once or twice a wk as cell replication increased (using total medium lines generated, and their species and tissue of origin. Cell lines replacement). Additionally, for egg homogenates, 0.7 mg/ml glutathione was were established from 10 lepidopteran species, including eight noc- added to inhibit melanization (Goodwin et al., 1982), followed by frequent tuids, one plutellid, and one pyralid. The success of culture estab- media changes within the first wk. For all cultures, antibiotic concentrations were reduced to 50 U/ml penicillin and 0.05 mg/ml streptomycin upon their lishment was not dependent on the species but on the tissues se- first subculture. lected. Of the tissue types used, whole embryonic homogenates and For suspension cultures, selected cell lines were placed into 125-ml minced adult reproductive tissues produced the highest number of LEPIDOPTERAN CELL LINES 371

TABLE 3

ISOENZYME ANALYSIS OF LEPIDOPTERAN CELL LINESa Cell lineb PGM G6PD MDH PGI HK AST LDH ME IDH

AfOV Ϫ8.5 6.5 4.0 Ϫ0.5 0.5 Ϫ10 1.0 Ϫ3 Ϫ2.5 AgE Ϫ7.0 7.5 Ϫ9.0 3.2 4.3 Ϫ24.3, Ϫ41.3 Ϫ1.0 Ϫ5.5 Ϫ1.5 AgOV1 Ϫ7.0 7.5 Ϫ6.2 Ϫ2 4.0, 9.0 Ϫ4.7 0.5 Ϫ5.0 Ϫ1.0, Ϫ0.5 AgOV2 Ϫ7.5 8.0 Ϫ6.0, 3.5 Ϫ2.0 4.0, 10.0 Ϫ5.0 1.0 Ϫ5.0 Ϫ1.0, Ϫ0.5 AgOV3 Ϫ7.0 9.0 3.0 Ϫ2.5 4.0, 7.5, 0.5 Ϫ4.5 0.0 Ϫ6.0 Ϫ1.5, 2.0 AiOV Ϫ4.0, Ϫ0.5 5.0 3.0 0.0 0.5 Ϫ1.2 Ϫ2.0 Ϫ2.0 Ϫ5.0 AiTS Ϫ2.0 3.5 Ϫ0.5 Ϫ1.0 2.0, 7.0 Ϫ27.5, Ϫ1.0 Ϫ1.5 Ϫ8.0, Ϫ6.0, Ϫ2.0 Ϫ4.0 HvE1 Ϫ4.5 1.0 0.0 3.0 1.0 7.0 Ϫ4.5 3.5 0.0 HvE2 Ϫ6.0 0.5 Ϫ6.5 2.0 Ϫ0.5 Ϫ7.0 Ϫ4.5 2.0 Ϫ8.5, Ϫ2.0 HvE3 Ϫ7.5 2.0 Ϫ3.0 2.0 0.0 Ϫ9.0 Ϫ5.5 2.0 Ϫ7.5, Ϫ3.5 HvOV Ϫ8.0 2.0 Ϫ4.0 2.0 0.5 Ϫ9.2 ND 2.0 Ϫ7.5, Ϫ3.0 HvTS Ϫ7.0 2.0 Ϫ6.0 2.5 0.0 Ϫ9.0 4.5 2.0 Ϫ6.8, Ϫ3.0 HzE1 Ϫ4.0 0.5 Ϫ6.0 Ϫ2.5 0.0 Ϫ4.0 4.0 1.0 Ϫ7.5 HzE2 Ϫ5.0 1.0 Ϫ6.0 Ϫ2.0 0.0 Ϫ5.0 3.5 0.0 Ϫ7.0, Ϫ3.0 HzE3 Ϫ4.0 Ϫ3.0 Ϫ1.3 Ϫ2.0 0.0 Ϫ5.2 5.0 Ϫ0.3 Ϫ8.0, Ϫ4.0 HzE5 Ϫ3.5 0.0 Ϫ6.0 Ϫ2.5 1.5 Ϫ3.5 4.5 1.0 Ϫ7.5 HzE6 Ϫ4.0 0.5 Ϫ7.0 Ϫ1.5 Ϫ1.0 Ϫ5.5 3.5 Ϫ1.0 Ϫ7.0 HzE7 Ϫ4.0 0, 10.0 Ϫ6.0 Ϫ2.0 Ϫ0.5 Ϫ4.5 4.0 0.0 0.5 OnFB1 Ϫ5.5 6.5 Ϫ8.5 0.0 2.0 Ϫ7.3 7.0 Ϫ2.5 Ϫ8.5 PxE Ϫ0.5 8.0 Ϫ6.6 Ϫ1.0 Ϫ1.5 Ϫ3.0 ND Ϫ2.0 Ϫ5.0, Ϫ4.0 PxLP Ϫ1.0 7.5 Ϫ7.5 Ϫ1.0 Ϫ8.0, Ϫ2.0 Ϫ30.3, Ϫ3.2 6.0 Ϫ7.5 Ϫ6.0 SeE1 Ϫ2.0 0.0 Ϫ4.5 0.5 0.0, 2.0 Ϫ1.0 1.0 Ϫ1.5 1.5, 2.5 SeE4 Ϫ2.0 0.0 Ϫ4.5 0.0 0.0, 2.0 Ϫ1.0 1.0 Ϫ1.5 1.5, 2.5 SeE5 Ϫ3.0 0.0 Ϫ4.5 0.5 0.0, 2.0 Ϫ1.0 1.0 Ϫ1.5 1.5, 2.5 SfTS Ϫ2.0 0.0 0.0 0.0 0.5 Ϫ30.5, 0.0 Ϫ3.5 Ϫ0.5 Ϫ0.5 TnE1 Ϫ6.5 6.5 4.0 Ϫ3.5 Ϫ8.5 Ϫ28.0, Ϫ9.0 Ϫ5.0 Ϫ1.0 Ϫ7.0, Ϫ10.0 TnE1MK Ϫ4.0 6.0 Ϫ3.0, 3.0 Ϫ3.0 1.5 Ϫ6.5 Ϫ3.5 0.5 Ϫ7.0 TnE2 Ϫ4.0 6.5 6.0 Ϫ4.5 0.0 Ϫ8.0 Ϫ5.5 Ϫ0.5 ND TnE2MK Ϫ4.0 6.5 Ϫ0.5, 6.0 Ϫ4.5 0.0 Ϫ8.0 Ϫ6.0 Ϫ0.5 ND TnE3 Ϫ4.0, 6.0 6.5 3.0 Ϫ4.0 Ϫ0.5 Ϫ6.5 Ϫ4.5 0.0 Ϫ7.0 TnTS1 Ϫ4.0 7.0 2.0 Ϫ3.0 0.0 Ϫ28.0, Ϫ8.3 Ϫ6.0 Ϫ0.5 Ϫ6.5 TnTS3 Ϫ6.0 5.5 2.0 Ϫ3.0 Ϫ3.0, Ϫ1.0 Ϫ7.0 Ϫ6.0 Ϫ0.5 Ϫ6.5

a Values were determined using Authentikit and indicate distance (mm) from the isoenzyme band of the standard cell line (Sf9) to that of the test cell line. When two Sf9 isoenzymes were noted for a substrate, the distance to the band closest to the origin was used to generate the results of the test cell lines. If the test cell line and Sf9 each had two isoenzymes for a given substrate, then the differences between the distances closest to the origin and those from the farthest origin were used for the calculations. Enzymes tested were: AST, G6PD, HK, IDH, LDH, MDH, ME, PG1, PGM. ND ϭ not determined. b Complete cell line designations are given in Table 1.

continuous cell lines. Additionally, a cell line was generated from erage number of wk to the first subculture was 13.3 Ϯ 1.7 (standard whole P. xylostella homogenates with only two attempts. In contrast, error). numerous cultures were initiated from specific larval tissues (pri- Numerous cell lines were adapted to either serum-free or re- marily from fat body, hemocytes, and testes), but only five were duced-serum conditions using the ExCell 401 medium. Those subcultured (HvTS, OnFB1, OnFB3, TnTS1, TnTS3). adapted to serum-free conditions were: AfOV, AgOV1, AgOV2, In most cases, successful initiations were generated from tissues AgOV3, AgE, AiOV, HvE1, HvE3, HvOV, PxE, PxLP, TnE3, and that were not exposed to exogenous enzymes for tissue disruption. TnE1-MK. Those adapted to reduced-serum conditions were (with Tissues were either minced (for excised tissues) or gently homog- % FBS in medium indicated): HvE2 (1%), TnE1 (1%), and TnTS1 enized (for whole insects/embryos). Additionally, the majority of (5%). the cell lines were established successfully using ExCell 401 with Furthermore, an attempt was made to adapt three cell lines 10–20% FBS (Table 1). Initial culture-establishment attempts (AiOV, HvE1, HvE2) to suspension culture. Under these conditions, were performed using serum-free media for larval tissues, but be- the AiOV and HvE2 lines grew well and were maintained for at cause these initiations yielded negative results, we supplemented least 20 passages, whereas the HvE1 line did not perform well and the media subsequently with serum (which we continued to do for was discontinued in suspension culture after four passages. the tissues from all other stages as well). After establishment, the Cell line characterization. The morphologies of the new cell lines amount of FBS in the medium was slowly decreased, resulting in differed from each other (Fig. 1), with those of most lines consisting many of the cell lines being adapted to grow in either reduced- primarily of spherical cells (Fig. 1A). Some lines consisted of cells serum or serum-free medium. that were elongated (Fig. 1B), with these types of cells tending to The length of time needed for each cell culture to be initially be intolerant to reductions in serum concentrations within the me- subcultured varied greatly, from 3.6 to 29.3 wk (Table 1). The av- dium. Additionally, unique features were also exhibited by some 372 GOODMAN ET AL. cell lines, such as cells that appeared less refractile than other lines al., 2001). Taken together, these data indicate that some of the (TnE1, Fig. 1C) and cells that interacted with each other to form newly established cell lines have the potential to serve in baculo- large membranous vesicles (SfTS, Fig. 1D). virus mass-production schemes. DAF-PCR was performed on the cell lines indicated in Table 2, using a primer to mammalian aldolase. DNA fragment patterns of ACKNOWLEDGMENTS cell lines from the same species exhibited numerous similarities, We express our appreciation to Drs. Charles Kariuki, Dwight E. Lynn, and whereas those from different insect species were distinctly different. Reneé M. Wagner for helpful suggestions on the writing of the manuscript, The cell lines that exhibited the highest intraspecies diversity for and to Mr. Steve Long for technical assistance. We also wish to acknowledge DAF-PCR patterns were those from T. ni, with the cell lines having gratefully the support of American Cyanamid Co. (now BASF Agro Research) the lowest intraspecies diversity being those from H. zea. through Cooperative Research Agreement 58-3K95-4-276. Table 3 lists the cell lines subjected to isoenzyme analysis. Con- trary to the results from the DAF-PCR analysis, the results from REFERENCES isoenzyme analysis indicated that most of the cell lines tested pro- Black, B. C.; Brennan, L. A.; Dierks, P. M.; Gard, I. E. Commercialization duced a unique array of isoenzyme band patterns when all the iso- of baculoviral insecticides. In: Miller, L. K., ed. The baculoviruses. enzymes were examined. New York: Plenum Press; 1997. Fitt, G. P. The ecology of Heliothis species in relation to agroecosystems. Annu. Rev. Entomol. 34:17–52; 1989. DISCUSSION Gelertner, W. D.; Federici, B. A. Continuous cell line from Spodoptera exigua (Lepidoptera: Noctuidae) that supports replication of nuclear poly- Cell line establishment. 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