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Journal of Invertebrate Pathology 99 (2008) 28–34

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Journal of Invertebrate Pathology 99 (2008) 28–34 Journal of Invertebrate Pathology 99 (2008) 28–34 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa New cell lines derived from the black cutworm, Agrotis ipsilon, that support replication of the A. ipsilon multiple nucleopolyhedrovirus and several group I nucleopolyhedroviruses Robert L. Harrison *, Dwight E. Lynn Invasive Insect Biocontrol and Behavior Laboratory, Plant Sciences Institute, USDA, Agricultural Research Service, Building 011A, Room 214, BARC-W, 10300 Baltimore Avenue, Beltsville, MD 20705, USA article info abstract Article history: New cell lines were recently developed from the embryos of the black cutworm, Agrotis ipsilon (Lepidoptera: Received 14 December 2007 Noctuidae). A primary culture was initiated from 4-day-old A. ipsilon eggs in ExCell420 medium supple- Accepted 27 February 2008 mented with 5% fetal bovine serum. This initial culture produced sufficient cell growth to allow subcul- Available online 5 March 2008 tivation and eventually led to the establishment of eight distinct strains. Two of these strains (AiE1611T and AiEd6T) were selected for further characterization. Extracts of these strains were compared to an extract from A. ipsilon eggs by isozyme analysis and shown to be from the same species. Both strains were Keywords: susceptible to infection by the A. ipsilon multiple nucleopolyhedrovirus (AgipMNPV), as well as to lepi- Agrotis ipsilon (Hufnagel) dopteran group I NPVs from A. californica, Anagrapha falcifera, Anticarsia gemmatalis, Galleria mellonella, Black cutworm Agrotis ipsilon multiple nucleopolyhedrovirus Helicoverpa armigera, Plutella xylostella, and Rachiplusia ou, with large numbers of occlusion bodies pro- AgipMNPV duced in most of the inoculated cells. The cell lines did not support the replication of group II NPVs from Baculovirus Helicoverpa zea, Lymantria dispar, and Spodoptera exigua. Both cell lines produced confluent monolayers in Nucleopolyhedrovirus plaque assays and supported the formation of plaques upon infection with AgipMNPV and Autographa Cell lines californica (Ac)MNPV. Twenty AgipMNPV plaques were picked from either AiE1611T or AiEd6T monolay- Biological control ers, and the plaque isolates were serially passaged three times through A. ipsilon cells. Only one isolate from AiE1611T cells exhibited genotypic variation in the form of an altered restriction fragment profile. Our results suggest these new lines can be useful in the study of AgipMNPV and A. ipsilon cellular and molecular biology. Published by Elsevier Inc. 1. Introduction and form the basis for environmentally benign, species-specific microbial insecticides (Hunter-Fujita et al., 1998; Moscardi, The black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: 1999). A nucleopolyhedrovirus has been identified and Noctuidae), is found in many regions worldwide and feeds on a described from the black cutworm (Boughton et al., 1999). This wide range of plants. In the United States, it is a sporadic but sig- virus, the A. ipsilon multiple nucleopolyhedrovirus (AgipMNPV), nificant pest on corn (Clement and McCartney, 1982; Engelken has been shown in greenhouse and field trials on corn (Bough- et al., 1990; Showers et al., 1983), and is considered to be the most ton et al., 2001) and in turfgrass (Prater et al., 2006) to exhibit damaging pest of turfgrass (Potter, 1998). Management of black high insecticidal activity against A. ipsilon larvae and to reduce cutworm infestations typically involves rescue applications of larval feeding. chemical insecticides. Continuously growing insect cell lines derived from Lepidoptera Baculoviruses are a group of arthropod-specific viruses that are widely used in the study of baculoviruses and potentially can have been isolated mostly from larvae of Lepidoptera (Bonning, be developed for the generation of recombinant baculoviruses with 2005). All baculoviruses belong to a single family, the Baculovi- enhanced insecticidal activity and the large-scale production of ridae, which currently is composed of the genera Nucleopolyhe- baculovirus insecticides (Black et al., 1997; Rhodes, 1996). In this drovirus and Granulovirus (Theilmann et al., 2005). The study, two cell lines that support the replication of AgipMNPV virion-containing occlusion bodies (OBs) formed by baculovi- were developed from A. ipsilon embryos. Production and growth ruses during infection are highly virulent against host insects of plaque-purified AgipMNPV clones in these cells was character- ized, and the effects of passage through the cell lines was exam- * Corresponding author. Fax: +1 301 504 5104. ined. These cell lines will allow for the further study of E-mail address: [email protected] (R.L. Harrison). AgipMNPV and its development as an insecticide, and can serve 0022-2011/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.jip.2008.02.015 R.L. Harrison, D.E. Lynn / Journal of Invertebrate Pathology 99 (2008) 28–34 29 as a source of material for further studies into the physiology and 2.3. Isozyme analysis cellular and molecular biology of A. ipsilon. The AuthentikitÒ system (Innovative Chemistry, Marshfield, MA) was used for characterizing the cell lines following the manu- 2. Materials and methods facturer’s directions. Cell extracts were applied to 1% agarose gels, electrophoresed at 160 V for 25 min and then stained for malic en- 2.1. Cell line development zyme (ME, E.C. 1.1.1.40), isocitrate dehydrogenase (ICD, E.C. 1.1.1.42), phosphoglucose isomerase (PGI, E.C. 5.3.1.9), or phospho- One-day-old A. ipsilon eggs on gauze cloth were obtained from glucomutase (PGM, E.C. 2.7.5.1). The stains, buffers, and gels were the rearing colony at the Corn Insects and Crop Genetics Research obtained from Innovative Chemistry. An extract from IPLB-Sf21 Unit, Ames, IA. The eggs were incubated for 3 days at 26 °C prior cells (Vaughn et al., 1977) and another prepared from A. ipsilon to use. Primary cultures were established from 100 eggs as previ- eggs were also included on the gels for comparison. The isozyme ously described (Lynn, 1996, 2007) in Ex-Cell 420 (SAFC Biosciences, migration patterns on these gels were also compared with gels of Lenexa, KY) supplemented with 5% fetal bovine serum (FBS) and other cell lines maintained in our laboratory. 50 lg/ml gentamicin sulfate. The initial subculture was performed by replacing the culture medium with Ca/Mg-free phosphate buf- 2.4. Budded virus (BV) production fered saline that was then replaced with 50 lg/ml VMF-trypsin. After 20 min at room temperature, 2 ml fresh medium was added Black cutworm larvae were reared individually in 1-oz. cups on to the culture and the cells suspended by gentle flushing of medium A. ipsilon diet from Southland Products (Lake Village, AR) at 28 °C. from the pipet. The cell suspension was then transferred to a 12.5- When 4th-instar larvae were preparing to molt (as evident from cm2 tissue culture flask (FalconÒ) and incubated at 26 °C. After the head capsule slippage), diet was removed from the cups and the initial subcultivation, the 2.0 ml of the medium was replaced with larvae were starved overnight. The following morning, larvae that fresh medium after 1 week. Cells in the spent medium were col- had molted to 5th instar were fed an 8 mm3 cube of diet on which lected by low speed (50g) centrifugation and resuspended in fresh 8.75 Â 105 AgipMNPV polyhedra had been pipetted. Larvae that medium to create a suspended strain (designated IPLB-AiE1611S). consumed the diet cube were allowed to feed on diet ad libitum. The attached cells in the first subculture were suspended after Infected larvae at 4 days post-infection were surface-sterilized another week at 26 °C using the trypsinization procedure described with 70% ethanol and bled. Hemolymph from individual larvae in Lynn (2002) for very strongly attached cells, leading to the devel- either had a milky appearance (consistent with NPV infection) or opment of a trypsinized cell strain (IPLB-AiE1611T). contained hemocytes that carried polyhedra. Hemolymph was col- Selection favors the development of a cell line consisting of the lected from 15 larvae, pooled, and diluted 10-fold with Hink’s fast-growing cells from primary culture after relatively few pas- TNM-FH medium (SAFC Biosciences) supplemented with 10% fetal sages (Lynn, 2007). These fast-growing cells may or may not be bovine serum (Invitrogen, Carlsbad, CA) and 0.8 mg/ml glutathi- susceptible to baculovirus infection. Single-cell cloning to preserve one. Hemocytes and polyhedra were pelleted by centrifugation at cell type variety is very difficult at a low passage number. To obtain 3000g and 4 °C for 3 min, and the BV-containing supernatant was strains derived from a variety of cell types, AiE1611T cells at the stored at 0 °C. sixth passage were seeded at a low density in 3.0 ml medium in a 60-mm tissue culture dish (CorningÒ). These dishes were placed 2.5. Infections of cell lines in a plastic box with wet towels and incubated undisturbed for 4 weeks. The resulting colonies of cells were observed with an in- Two dozen cell lines being maintained in the Beltsville labora- verted phase contrast microscope and morphologically distinct tory (Table 1) and the newly established A. ipsilon cell lines were colonies marked. These colonies were scraped loose from the dish exposed to hemolymph from infected A. ipsilon larvae. Cells were with a Rainin pipet tip and transferred to 0.2 ml medium in a 96- distributed into 24-well tissue culture plates (GreinerÒ) using the well tissue culture plate (FalconÒ). Of 24 colonies initially isolated normal subculture procedure and 1.0 ml of medium supplemented in this manner, six continued growing and were maintained as un- with 50 lg/ml gentamicin for each line and 0.2 ml of a 1:10 dilu- ique strains. tion of the hemolymph was added to each cell line. The medium was replaced with 1.0 ml fresh medium after 2 h in some wells.
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