Journal of Invertebrate Pathology 111 (2012) 143–151

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Journal of Invertebrate Pathology

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A new cell line (NTU-SE) from pupal tissues of the beet armyworm, Spodoptera exigua (: Noctuidae), is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV) ⇑ Chih-Yu Wu a,b, Yue-Wen Chen c, Chih-Chun Lin b, Chia-Ling Hsu b, Chung-Hsiung Wang b,c,d, , ⇑ Chu-Fang Lo b, a Center for Biotechnology, National Taiwan University, Taipei, Taiwan, ROC b Institute of Zoology, National Taiwan University, Taipei, Taiwan, ROC c Department of Science, National Ilan University, Ilan, Taiwan, ROC d Department of Entomology, National Taiwan University, Taipei, Taiwan, ROC article info abstract

Article history: A new continuous cell line, NTU-SE, was established from the pupal tissues of an economically important Received 8 May 2012 pest, the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). This cell line contains four major Accepted 20 July 2012 morphologic types: round, polymorphic, spindle-shaped, and comma-shaped cells. The population dou- Available online 31 July 2012 bling time of this new line in TNM-FH medium supplemented with 8% fetal bovine serum (FBS) at 28 °Cis 35.5 h. The chromosomal spread from NTU-SE cells is typical to the chromosomal morphology of lepidop- Keywords: teran cell lines. Confidently, NTU-SE cell line is a new cell line that exhibits distinct isozyme patterns of Spodoptera exigua esterase, lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) from those of the other Insect cell line cell lines. In addition, the DNA sequence of the nuclear ribosomal internal transcribed spacer (ITS) region Isozyme Internal transcribed spacer (ITS) of NTU-SE cells is above 96% identical to that sequence of S. exigua larvae, as compared to only 66% iden- Nucleopolyhedrovirus tical to that of S. litura larvae. The NTU-SE cell line is highly susceptible to S. exigua multiple nucleopoly- hedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV). Therefore, a highly virulent SeMNPV strain, SeMNPV-1, had been successfully isolated and propagated in NTU-SE cells. We conclude that the NTU-SE cell line will be a useful tool for the selection and mass production of highly virulent SeMNPV strains for the S. exigua biocontrol and the baculovirus based recombinant protein expression systems. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction production for the pest control (Goodman et al., 2001; Goodwin et al., 1978; Granados and McKenna, 1995; Lynn, 2007). Some of Insect cell lines are valuable in a wide range of biological re- these cell lines, such as IPLB-Sf-21 (Sf21) (Vaughn et al., 1977), search, including developmental biology, physiology, and pathol- Sf9 a clonal isolate of Sf21 cells (Summers and Smith, 1987), and ogy (Granados and McKenna, 1995; Hink et al., 1991; Maeda BTI-Tn-5B1-4 (Hi5) (Granados et al., 1994; McKenna et al., 1998), et al., 1985; Smith et al., 1983; Vaughn, 1981; Wu et al., 1989). have been commercialized and become the most commonly used Since Gaw (1958) first cultured all types of tissues from the silk- cell lines for the baculovirus expression vector system (BEVS), a worm, Bombyx mori, using the monolayer culture and then Grace versatile and powerful tool first introduced by Smith et al. (1983) established cell lines from the gum emperor , Opodiphthera for recombinant protein expression. (Antheraea) eucalypti (Grace, 1962), over 600 continuous cell lines The beet armyworm Spodoptera exigua (Lepidoptera: Noctui- have been developed from more than 100 different insect species dae) is native to Southeast Asia, but now is a serious, worldwide (van Oers and Lynn, 2010). In particular, numerous cell lines from pest insect to economically important crop plants (Trumble and lepidopteran pests of economical importance were established for Baker, 1984). In Taiwan, the beet armyworm has caused severe the primary purpose of studying and optimizing baculovirus damage in green onion production since the 1980s. Two possible reasons for the outbreak of beet armyworm infestations are the expansion of green onion cultivation and the insect has become ⇑ Corresponding authors. Address: Institute of Zoology, National Taiwan more tolerant or resistant to many chemical insecticides com- University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617 Taiwan, ROC. monly used (Cheng et al., 1988; Cheng and Kao, 1993). In addition, Fax: +886 2 27364329 (C.H. Wang), +886 2 23633562 (C.F. Lo). the insect larvae usually hide inside the hollow, tube-like leaves of E-mail addresses: [email protected] (C.-H. Wang), [email protected] (C.-F. Lo). the green onions so they were difficult to kill by spraying chemical

0022-2011/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2012.07.022 144 C.-Y. Wu et al. / Journal of Invertebrate Pathology 111 (2012) 143–151 insecticides (Cheng et al., 1988). Two NPV species, S. exigua MNPV tute, Council of Agriculture, Executive Yuan. The larvae were (SeMNPV) and a strain of Autographa californica MNPV (named as reared on an artificial diet (Tuan et al., 1994) and allowed to grow AcMNPV-TWN4) have been isolated from diseased beet armyworm to the pupal stage. The 2 to 4-day-old pupae were collected with- larvae showing nucleopolyhedrosis in Taiwan (Hong, 1988; Tuan out gender selection and surface-sterilized by soaking with 10% et al., 1994). Experimental trials show that SeMNPV can be an Clorox solution and then with 70% iodine alcohol. The internal tis- effective microbial insecticide to effectively suppress S. exigua pop- sues (without specific selection) of 2–3 pupae were picked with a ulations either in the laboratory (Kao et al., 1991; Tuan et al., 1994) fine forceps and a pipet, and subsequently transferred into a 25- or in the green onion field (Kao et al., 1997). Therefore this virus is cm2 flask with 4 ml of TNM-FH medium (Hink and Strauss, 1976) being investigated as a possible addition to the integrated pest plus supplements. The supplements contained 100 IU/ml penicillin management (IPM) programs against the beet armyworm. (Gibco), 100 lg/ml streptomycin (Gibco), 1.25 lg/ml amphotericin The success of using SeMNPV as a microbial insecticide depends B (Sigma), and 16% heat-inactivated (56 °C for 30 min) fetal bovine mainly upon the technology of its mass production for cutting serum (FBS, Hyclone). The cultures were then incubated at 28 °C down costs. Although a method for large-scale production of SeM- and the half of spent culture medium was replaced with an equal NPV by the use of host has been developed, it is still essen- amount of fresh medium plus supplements every 10–14 days. tial to develop continuous cell lines of this species for screening The first subculture of the primary cultures was performed after and maintaining highly virulent SeMNPV strains and for optimiz- 2 months when the cells approached confluence. When subcultur- ing culture conditions. Several cell lines have been established ing, the growing cells were detached from the 25-cm2 flask by vig- from the beet armyworm (Gelernter and Federici, 1986; Goodman orous agitation and 2 ml of the suspended cells were transferred to et al., 2001; Hara et al., 1993, 1995; Weng et al., 2009; Yasunaga- another 25-cm2 flask containing 4 ml of fresh medium plus supple- Aoki et al., 2004; Zhang et al., 2006, 2009, 2011) while not all sup- ments. From the initial subculture to the 50th passage, the interval port high levels of the SeMNPV replication and the viral occlusion between subculture ranged from 1 to 3 weeks, as allowed by the bodies (OBs) production (Table 1). In the present study, we de- cell growth rates. After the 50th passage, the cells propagated rap- scribe the establishment and characterization of a new continuous idly, and thereafter the cultures were adapted to 8% FBS with rou- cell line, NTU-SE, derived from the pupal tissues of S. exigua. tine passages (split ratio was about 1:3–1:5) at intervals of 3– Although some of the other S. exigua cell lines showed both a lower 5 days. This new established cell line was designated NTU-SE (Na- doubling time and a higher number of OBs production than NTU- tional Taiwan University) and has undergone approximately 200 SE, we demonstrate this new cell line will be a useful tool for successive passages for two years. The passage numbers of the cells studying the economical production of microbial insecticides and used in the present study were all around 100th passage to 150th foreign proteins. passage.

2. Materials and methods 2.2. Cell morphology

2.1. Primary culture and subculture Images of cells from the NTU-SE cell line were captured with an Olympus IX-71 inverted phase-contrast microscope with a digital A laboratory colony of S. exigua originated from the southern camera. Cell sizes were calculated according to a calibrated magni- area of Taiwan (Yuanlin and Guantian) was obtained from the Tai- fication factor. Average cell dimensions were determined from wan Agricultural Chemicals and Toxic Substances Research Insti- measurements of 30 cells.

Table 1 Cell lines established from Spodoptera exigu.

Names Sources Morphologies of cells Population Susceptibilities to Production of References doubling SpeiMNPV occlusion bodies times (OBs) NTU-SE Pupal tissues Round, polymorphic, spindle- 35.5 h 96 ± 1% 90 OBs/cell In this study shaped, and comma-shaped cells IOZCAS-Spex-XI Pupal ovaries Spherical, spindle-shaped, and 81.7 h 80% 8.2 OBs/cell Zhang et al., macrophage-like cells 2011 IOZCAS-Spex-III Larval fat bodies Spherical, spindle-shaped, and – Low Low Zhang et al., macrophage-like cells 2006 IOZCAS-Spex-II Larval fat bodies Spherical, spindle-shaped, and 109 h 50% 211 ± 11 OBs/cell Zhang et al., macrophage-like cells 2006, 2009 IOZCAS-Spex-II-A Larval fat bodies Spindle-shaped and spherical 28 h >90% 176 ± 13 OBs/cell Zhang et al., (Spex-IIsubclone) cells 2009 SeHe920-1a Larval hemocytes Spherical and spindle-shaped 28 h – – Yasunaga-Aoki cells et al., 2004 Se3FH (Se4FH, Neonate larval Spindle-shaped and spherical 30 ± 5 h 73 ± 8% 130 OBs/cell Hara et al., Se5FH, Se6FHA, tissues cells 1993, 1995 Se6FHB) Se301 Neonate larval Spherical cells 28 h Nearly 100% 83 OBs/cell Hara et al., tissues (Se3FH 1995 subclone) P8-Se301-C1 Neonate larval Spherical cells 37–39 h <10% Low Weng et al., tissues (Se301 2009 subclone) UCR-SE-1 Neonate larval Epithelial-like and spindle- 56 h 30–50% (> 90% in the – Gelernter and tissues shaped cells spindle-shaped cells) Federici, 1986

–, No data was mentioned in the literature. C.-Y. Wu et al. / Journal of Invertebrate Pathology 111 (2012) 143–151 145

2.3. Growth rate of NTU-SE cells at 94 °C for 15 s, annealing at 52 °C for 30 s, and elongating at 72 °C for 3 min. There was a final extension step at 72 °C for 5 min. The Cells in log phase were seeded in 25-cm2 flasks (T-25, Nunc) at a PCR products were commercially sequenced (Genomics Biosci. & density of 6 105 cells per flask, and then cultured in TNM-FH Tech. Company), and the software CLUSTAL_X, Version 1.81 medium with 8% FBS supplementation at 28 °C. Cell number was (Thompson et al., 1997) was used to align the multiple sequences determined by counting the cells within a microscope reticule of (Supplement-Fig. 2). The pairwise identity of the ITS regions which the area at a certain objective was known. The cell densities among the NTU-SE cells and the S. exigua and S. litura larvae was in five areas of each flask were determined at 24 h intervals for then calculated by GeneDoc (Nicholas et al., 1997) with a score ta- 6 days incubation. Cell population doubling time was calculated ble of Blosum 35. using the exponential formula described by Kuchler (1977). 2.7. Virus susceptibility 2.4. Chromosome number The following six viruses were used to test the viral susceptibil- Log phase NTU-SE cells (9 106 cells) cultured in 75-cm2 flasks ity of NTU-SE cells: AcMNPV, LdMNPV (L. dispar MNPV)-like virus (T-75, Nunc) were treated with 10 ll (1.5 mg/ml) demecolcine (Nai et al., 2009), LyxyMNPV (L. xylina MNPV) (Wu and Wang, (Sigma, D-6279) for 4–6 h at 28 °C. The cells were then scraped 2006), PenuMNPV ( nuda MNPV) (Wang et al., 1996), Mav- from the surfaces of flasks, centrifuged at 200g (TOMY MX-305 iMNPV (M. vitrata MNPV) (Yeh et al., 2007), and SeMNPV. AcMNPV Centrifuge) for 10 min, resuspended for 30 min in a hypotonic was supplied by Dr. M.J. Fraser of Notre Dame University. LdMNPV- solution (0.075 M KCl), and then fixed with Carnoy’s solution like virus, LyxyMNPV, PenuMNPV, and MaviMNPV were obtained (3:1methanol:glacial acetic acid) for 10 min at 4 °C. The fixing pro- from their permissive cell lines, IPLB-LD-652Y (Goodwin et al., cess was repeated three times, and the fixed cells were dropped 1978), NTU-LY-1 (Wu and Wang, 2006), NTU-PN-HH (Wang vertically onto slides. After drying at 60 °C in an incubator, the cells et al., 1996), and NTU-MV (Yeh et al., 2007), respectively. SeMNPV were stained with Giemsa stain for 5 min and the number of meta- was initially isolated from a diseased larva of S. exigua that exhib- phase chromosomes was counted under a microscope. The results ited nucleopolyhedrosis in this study. Briefly, this diseased larva were displayed following the style reported by Hara et al. (1993). was homogenized in TNM-FH medium plus 0.6 mg/ml glutathione (to prevent melanization) and centrifuged at 1250g for 10 min. The 2.5. Isozyme analysis supernatant was then sterilized by filtration through a 0.45-lm syringe filter (Millipore). The filtrate was the sources of the SeM- The isozyme patterns of NTU-SE cells were compared with NPV inoculum. The five viruses and their five permissive cell lines, those of IPLB-Sf9 (S. frugiperda cell line), NTU-MV (Maruca vitrata AcMNPV/Sf9, LdMNPV-like virus/IPLB-LD-652Y, LyxyMNPV/NTU- cell line; Yeh et al., 2007), NTU-YB (yellow butterfly Eurema hecabe LY, PenuMNPV/NTU-PN-HH, and MaviMNPV/NTU-MV, were used cell line; Chen et al., 2009), and MTU-LY-1 (Lymantria xylina cell as positive controls. These cell lines and NTU-SE cell line were also line; Wu and Wang, 2006). All five cell lines were cultured in tested for SeMNPV susceptibility following the method previously TNM-FH medium supplemented with 8% FBS. At confluence, the described (Wu and Wang, 2006). Briefly, log phase cells were cells were harvested from each individual 75-cm2 flask, counted seeded in 25-cm2 flasks at a density of 3 106 cells per flask and with a hemocytometer, and centrifuged at 200g (TOMY MX-305 inoculated with 0.5 ml of the respective virus inoculums. After Centrifuge) for 10 min at 4 °C. The supernatants were discarded 1 h of adsorption, the viral inoculums were discarded and the cells and the pellets were resuspended in a grinding buffer (0.125 M were incubated in fresh TNM-FH medium with 8% FBS at 28 °C. The Tris-HCl, 46 mM citric acid, 10% sucrose, 1% Triton X-100, and formation of occlusion bodies (OBs) in the hypertrophied cell nu- 0.02 mM bromophenol blue) to a concentration of 1.5 107 cells cleus, an obvious sign of NPV infection in permissive cells, was per milliliter. The suspended cells were lysed by three times of checked at intervals up to 1 month under an inverted phase-con- freezing in liquid nitrogen and thawing at 37 °C, and then centri- trast microscope. fuged at 15,000g (TOMY MX-305 Centrifuge) for 10 min at 4 °C. The resulting supernatants were loaded with 10 lg of total protein per lane and electrophoresed on 10% polyacrylamide gels at a con- 2.8. Selection of highly virulent SeMNPV strains by quantitative studies stant current of 20 mA for 2 h. The gels were then stained for three isozymes, esterase, lactate dehydrogenase (LDH), and malate dehy- Serial dilutions (from 101 to1010) of the SeMNPV stock were drogenase (MDH), following the protocols of Manchenko (2003). prepared and inoculated onto NTU-SE cultures in 96-well plastic plates. After 10 days incubation, three SeMNPV strains (SeMNPV- 2.6. Internal transcribed spacer (ITS) region 1, -10, -23) were selected at the 106 and 107 dilution according to their properties of high OBs-productivity. These three viral The DNAs of the larvae of S. exigua, the larvae of closely related strains were further conducted to quantitative studies. Briefly, species S. litura, and NTU-SE cells were extracted using a commer- log phase cells (3 106) were seeded in 25-cm2 flasks and inocu- cial kit (GenemarkÒ Tissue & Cell Genomic DNA Purification Kit) lated with each of three viral strains (0.3 ml; MOI = 1). After 1 h following the protocol described by the manufacturer. The amplifi- of adsorption, the viral inocula were discarded and the cell cultures cation of DNA sequences from the nuclear ribosomal ITS regions were incubated in 5 ml TNM-FH medium with 8% FBS at 28 °C. The (Supplement-Fig. 1) was then performed by polymerase chain percentage of OBs-containing cells (infected cells) in each flask was reaction (PCR) in a thermal cycler (Applied Biosystems 2720 Ther- counted by an inverted phase-contrast microscopy at 7 days post- mal Cycler). The primer set, ITS1-1: 50-CCC CAT AAA CGA GGA ATT infection (pi). Furthermore at 2 weeks pi, the cells were scraped CC-30 and ITS4: 50-TCC TCC GCT TAT TGA TAT GC-30, and the PCR from the surface of the flask and then centrifuged at 1250g (TOMY conditions used were following Wu et al. (2011) report. Each PCR MX-305 Centrifuge) for 15 min. The resulting pellets were resus- consisted of a 50 ll total reaction volume that contained 50 ng of pended with 5 ml of RIPA lysis buffer (150 mM NaCl, 1% NP-40, template DNA, 1 reaction buffer (2 mM MgSO4), 200 lM dNTP, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH8.0) and 0.5 lM of, and 2.5 U of Taq DNA polymerase (Bioman Scientific the viral OBs were then counted by hemocytometer. Meanwhile, Co., Ltd.). After a preheat step at 94 °C for 2 min, 40 amplification the supernatants from the centrifugation were used for the titra- cycles were carried out with three steps in each cycle: denaturing tion of the extracellular viruses (ECVs) and the titers of ECVs were 146 C.-Y. Wu et al. / Journal of Invertebrate Pathology 111 (2012) 143–151

determined by the end-point dilution method (TCID50 analysis, Summers and Smith, 1987).

2.9. Electron microscopy

At 5 days pi, the SeMNPV-infected SE cells were scraped from the surfaces of 25-cm2 flasks and centrifuged at 200g (TOMY MX-305 Centrifuge) for 10 min. The resulting pellet was fixed by a 2% paraformaldehyde – 2.5% glutaraldehyde fixative in 0.1 M phosphate buffer (pH 7.2) at 4 °C for 2 h and then post-fixed with

1% OsO4 in the same buffer at room temperature for 1 h. After washing in cold distilled water, the fixed specimen was dehydrated in an alcohol gradient series (70–100%) and then rinsed in propyl- ene oxide. The specimen was then embedded in Spurr’s (Spurr, 1969) resin and placed in 80 °C oven for 2 days. Thin sections were made by a diamond knife on a Reichert OMU3 Ultramicrotome and stained with 2% aqueous uranyl acetate followed by lead citrate. Photomicrographs of the infected cells were taken with a JOEL JEM-1010 transmission electron microscope at 80 kV.

2.10. Restriction enzyme analysis of viral DNAs

After 10 days pi, the SeMNPV-infected NTU-SE cells were scraped from the surfaces of 75-cm2 flasks and centrifuged at 3000g (TOMY MX-305 Centrifuge) for 10 min. The supernatant was layered on a 20% (w/v) sucrose solution and then precipitated by ultracentrifugation at 25,000g for 1.5 h in a P28S rotor (Hitachi Himac Ultracentrifuge CP 80a). The pellets were resuspended in 1 TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6), with a final Fig. 1. (A) Primary culture of Spodoptera exigua pupal tissues. (B) NTU-SE cell line concentration of 1% (w/v) SDS and then incubated with Proteinase (at 100th passage) after plating for 2 h. P, polymorphic cells; R, round cells; SP, K (0.25 mg/ml) at 56 °C for 3 h. The extraction of viral DNAs was spindle-shaped cells; C, comma-shaped cells. Bar: 100 lm. followed previously described by the phenol/chloroform extraction method (Wang et al., 1996; Wu and Wang, 2005). The respective viral DNAs (5 lg) were digested with BamHI, EcoRV, and HindIII in vitro. This cell line has now undergone approximately 200 pas- (New England BioLabs INC.) for 16 h at 37 °C, and then separated sages for two years in TNM-FH medium supplemented with 8% by electrophoresis in 0.8% agarose gels. After electrophoresis, the FBS at 28 °C. As shown in Fig. 1B, The NTU-SE cell line is heteroge- DNA fragments were stained with 0.3% ethidium bromide in neous in nature and consists of four major cell types, round, poly- 1 TAE and photographed under UV light. morphic, spindle-shaped and comma-shaped cells. The characteristics of these four cell types are as follows: 2.11. Foreign protein expression The round cells are the smallest cell type, account for 33.54% of NTU-SE cells, and have a diameter of 19.3 ± 3.81 (mean ± SD) lm. To test the abilities for foreign protein expression, the semi- The polymorphic cells are irregular in shape, vary in size confluent tested cells (log-phase cells) of NTU-SE were inoculated (77.62 ± 27.76 lm) and account for 32.7% of NTU-SE cells. The with a bicistronic AcMNPV vector, vAcD-Pn50539-E (Fig. 8A) in spindle-shaped cells are predominantly ellipsoidal with two exten- which an IRES element from the Perina nuda virus (PnV) 50539- sions on opposite sides. They account for 24.37% of NTU-SE cells, nucleotides, containing 50-untranslated region (UTR) and down- and vary in size (91.36 ± 28.54 lm in length and 13.96 ± 2.3 lm stream 22 codons (66-nucleotides) of open reading frame (ORF), in width). The comma-shaped cells account for 9.39% of NTU-SE are inserted between the DsRed and EGFP genes. These two fluo- cells, have only one extension, and are 52.78 ± 17.46 lm in length rescent protein genes were driven by a polyhedrin (polh) promoter and 16.83 ± 3.99 lm in width. at the polh locus (Wu et al., 2007). After 1 h adsorption, the viral solution was discarded and the cells were incubated in TNM-FH 3.2. Growth rate of NTU-SE cells medium with 8% FBS at 28 °C. At 5 days pi, cells were examined using an Olympus IX71 inverted fluorescence microscope and the Growth curve for the NTU-SE cell line cultured in TNM-FH med- appropriate filter-mirror sets (Ex. 460–480 nm/Em. 495–540 nm ium supplemented with 8% FBS at 28 °C was shown in Fig. 2. A lag for EGFP; Ex. 520–550 nm/Em. 580 nm for DsRed). Cell morphol- phase of 2 days was followed by an exponential growth phase of ogy was examined under a phase-contrast microscopy. The Sf9 3 days. The plateau phase was reached on day 5, when the cell pop- cells (highly susceptible to AcMNPV) were treated in parallel as ulation had increased to approximately 6 106 cells. The popula- the positive control. tion doubling time of NTU-SE cells was estimated to be 35.5 h.

3. Results 3.3. Chromosome number

3.1. Cell morphology The chromosomal spread from NTU-SE cells showed the typical round shape of the lepidopteran chromosome (Fig. 3A). Chromo- A primary culture derived from internal tissues of the S. exigua some number distribution varied widely from 24 to 246 and the pupae (Fig. 1A) was used to establish the NTU-SE cell line (Fig. 1B) most common cells were triploid (Fig. 3B). C.-Y. Wu et al. / Journal of Invertebrate Pathology 111 (2012) 143–151 147

Fig. 2. The growth curve of NTU-SE cell line. The cells (at 110th passage) were cultured at 28 °C in TNM-FH medium supplemented with 8% FBS. The error bars indicate the standard deviation of three independent trials.

Fig. 3. (A) Representative metaphase chromosome spread of an NTU-SE cell at 150th passage. Bar: 10 lm and (B) Chromosome number distribution in 50 of NTU- Fig. 4. The isozyme patterns of NTU-SE cells at passage 125. (A) Esterase, (B) MDH, SE cells. Chromosome number of S. exigua (n = 31) was determined previously by malate dehydrogenase, and (C) LDH, lactate dehydrogenase profiles of NTU-SE (SE) Hara et al. (1993) from the testis of 5th instar larvae. cells were compared with those of IPLB-Sf9 (Sf9), NTU-MV (MV), NTU-YB (YB), and MTU-LY-1 (LY) cells. The cell lines are indicated on top of the pictures correspondingly.

3.4. Isozyme analysis 3.5. Internal transcribed spacer (ITS) region The NTU-SE cells produced distinct esterase, MDH, and LDH iso- zyme patterns from those produced by the NTU-MV, NTU-YB, and The ITS regions of NTU-SE cells, S. exigua larvae (N = 2), and S. NTU-LY cells (Fig. 4). In addition, NTU-SE and Sf9 cells produced a litura larvae (N = 2) were successfully cloned and sequenced (Gen- similar LDH pattern, while they were different patterns both in Bank ID: JN863291 to JN863294). Surprisingly, two types of ITS se- esterase and MDH analysis. quences were found in a single S. exigua larva but only one type of 148 C.-Y. Wu et al. / Journal of Invertebrate Pathology 111 (2012) 143–151

ITS sequences was found in both NTU-SE cells and S. litura larvae (Supplement-Fig. 2). The sequence identities of the ITS regions be- tween NTU-SE cells and one of S. exigua larva type 1 and type 2 were 99% and 96%, respectively, and these results indicate that the NTU-SE cell line was authentically established from S. exigua. In contrast, the ITS identity was only 66% between NTU-SE cells and S. litura larvae, and there was 64% between NTU-SE cells and Sf9 cells (GenBank ID: GQ478352; Wu et al., 2011).

3.6. Virus susceptibility

The NTU-SE cells were highly susceptible to SeMNPV and AcMNPV. Typical cytopathic effects (CPEs) were observed, such as numerous OBs formed in the hypertrophic nucleus of each in- fected cell at 7 days postinfection. However, no CPEs were found in the NTU-SE cells inoculated with LdMNPV-like virus, LyxyMNPV, PenuMNPV, and MaviMNPV (data not shown). In addition, SeMNPV has a very narrow host range in vitro. Of six lepidopteran cell lines (i.e. IPLB-Sf9, IPLB-LD-652Y, NTU-LY, NTU-PN-HH, NTU-MV, and NTU-SE) tested, only the homologous cell line, NTU-SE cell line, was susceptible to the SeMNPV (data not shown).

3.7. Selection of highly virulent SeMNPV strains by quantitative studies

Three SeMNPV strains (SeMNPV-1, -10, and -23) were selected for quantitative studies. As shown in Fig. 5, the strain SeMNPV-1 had the highest infection rate (96 ± 1%, mean ± SE) and the most of viral OBs (90 OBs/cell) production, followed by the strain SeM- NPV-23 (61 ± 3%; 46 OBs/cell), and the strain SeMNPV-10 (57 ± 4%; 43 OBs/cell). The strain SeMNPV-1 attracted the most attentions because this viral strain was highly OBs-productive in the NTU-SE cells (Supplement-Fig. 3). Additionally, the SeMNPV- 1 strain was also capable to produce high amounts of ECVs, and 7 the ECV titer was measured as 1.2 10 TCID50/ml and as 20 TCID50/cell at 2 weeks postinfection. Fig. 6. Transmission electron photomicrographs of (A) a SeMNPV-infected NTU-SE cell at 5 days post-inoculation and (B) a SeMNPV occlusion body. ONM, outer nuclear membrane; INM, inner nuclear membrane; P, polyhedron; V, virion; N, nucleocapsid; VE, virion envelope; Ph, polyhedron matrix; C, calyx. Bars: (A) 1 lm 3.8. Electron microscopy and (B) 500 nm.

As the transmission electron photomicrographs shown in Fig. 6, the morphogenesis and infectivity of SeMNPV in vitro was similar 3.9. Restriction enzyme analysis of viral DNAs to that reported in vivo by Tuan et al. (1994). After 5 days postin- fection, nucleocapsids, multiple nucleocapsid virions, and develop- The restriction fragment profiles of the three strains SeMNPV-1, ing OBs were observed in the hypertrophied nuclei and the nuclear -10, and -23 DNAs purified from the infected NTU-SE cells were envelope were disrupted by the viral infection. In the cytoplasm essentially identical with each of the BamHI, EcoRV, and HindIII there are marked degenerative changes, including extensive mem- digestions (Fig. 7). A slight difference may exist though, the pat- brane vacuolation and mitochondrial swelling and disruption. The terns here both agreed with the restriction fragments of SeMNPV ultrastructural morphogenesis of SeMNPV in vitro is typical of a le- DNA analyzed by Heldens et al. (1996) and the restriction fragment pidopteran NPV.

Fig. 5. Production of SeMNPV occlusion bodies (OBs). NTU-SE cells at 150th passage were infected with the three selected SeMNPV strains, (A) SeMNPV-1, (B) SeMNPV-10, and (C) SeMNPV-23, and pictures were taken at 7 days post-inoculation. Bar: 50 lm. C.-Y. Wu et al. / Journal of Invertebrate Pathology 111 (2012) 143–151 149

Fig. 7. Restriction enzyme profiles of viral DNAs. The genomic DNAs of three selected strains, SeMNPV-1, -10, and -23 (lane 1, 2, and 3, respectively) were analyzed with restriction enzymes BamHI, EcoRV, and HindIII. Restriction fragments are lettered in order of size. M, GeneMarkÒ GenKB-LC DNA Ladder. sizes deduced from the SeMNPV genomic sequence (GenBank ID: NTU-SE; 93% of Sf9), which indicated that the new S. exigua cell line AF169823). NTU-SE has a great potential to be used for foreign protein expressions.

3.10. Foreign protein expression 4. Discussion Both DsRed and EGFP proteins were expressed in vAcD- Pn50539-E-infected NTU-SE cells at 5 days pi (Fig. 8). The high We have described here how internal tissues from S. exigua pu- AcMNPV susceptibility of the NTU-SE cells was comparable to that pae were successfully used to establish a new cell line designated of the Sf9 cells based on the observation of infection rates (90% of NTU-SE (National Taiwan University). The isozyme patterns

Fig. 8. Expression of foreign proteins. (A) Schematic representation of the bi-cistronic construct of the recombinant AcMNPV, vAcD-Pn50539-E, which contains the dual fluorescent protein genes flanking the PnV 50UTR plus the first 22 codons of the ORF (539 nucleotides) (B) NTU-SE cells at 150th passage were infected with vAcD-Pn50539-E and pictures were taken at 5 days post-inoculation. DsRed-specific fluorescence indicates cap-dependent translation while EGFP-specific fluorescence indicates 50-end- independent translation directed by the PnV 50 IRES element. Phase-contrast pictures show the morphology and location of infected cells. The Sf9 cells were treated in parallel as the positive control. Bar: 100 lm. 150 C.-Y. Wu et al. / Journal of Invertebrate Pathology 111 (2012) 143–151

(Tabachnick and Knudson, 1980) of NTU-SE cells were distinct susceptibility to AcMNPV. Furthermore, the Perina nuda virus from those of other cell lines (Fig. 4), while alignment of the nucle- (PnV) 50 end 539-nucleotides, which contain 50-untranslated re- ar rDNA ITS regions (Gunderson et al., 1987) showed a high iden- gion (UTR) and downstream 22 codons (66-nucleotides) of open tity (above 96%) between the NTU-SE cells and S. exigua larvae reading frame (ORF), were first reported possessing an IRES activ- sequences (Supplement-Fig. 2), which confirms that the NTU-SE ity in IPLB-SF21AE cells (Wu et al., 2007). The ability of this PnV cell line was derived from S. exigua. We further showed that the 50IRES element to direct cap-independent translation in the NTU- NTU-SE cell lines was highly susceptible to SeMNPV (Fig. 5, and SE cells was also evaluated in these studies. Cap-dependent trans- Supplement-Fig. 3) and were able to replicate the virus (Figs. 6). lation was monitored by DsRed expression, while IRES-dependent Fig. 5 further shows that almost all of the NTU-SE cells produced translational initiation was monitored by the expression of EGFP. viral OBs when infected with the viral strain SeMNPV-1, but only As shown in Fig. 8, both DsRed and EGFP were efficiently ex- about 60% infected cells produced OBs when infected with either pressed in the vAcD-Pn50539-E-infected cells, and thus we sug- SeMNPV-10 or -23 strains. We believe this was caused by many gested that the PnV 50 IRES element can efficiently initiate 50- cells infected with SeMNPV-10 and -23 strains undergoing cell ly- end-independent translation in the NTU-SE cells. In other words, sis (i.e. necrosis or apoptosis) during the infection, however, further we found that the NTU-SE cells could be a powerful tool for the examinations are needed to confirm this explanation. expression of multiple foreign proteins with an AcMNPV vector. Most studies of the nuclear ribosomal ITS show low levels of Cell lines originating from different insect tissues tend to differ intraspecific variation while there is rapid divergence between in their capacity to virus infections (Hink et al., 1991). It seems that species (Gotoh et al., 1998; Marinucci et al., 1999; Miller et al., the pupal tissues would be an excellent source for the establish- 1996). Based on these findings, we developed a simple method ment of virus susceptible cell lines, because several lepidopteran for diagnostic assays of insect species and their homologous cell cell lines and the NTU-SE cell line established from pupal tissues line identities (Wu et al., 2011). In insects, the ribosomal genes in our laboratory, all showed highly susceptible to their homolo- are present in several copies (Beckingham, 1982). Although the ex- gous NPVs (Wang et al., 1996; Wu and Wang, 2006; Yeh et al., actly mechanism is not known, all copies within and among ribo- 2007). Interestingly, we found that the S. exigua cell lines from em- somal loci are homogenized through a rapid concerted evolution bryos, neonate larvae, larval haemocytes and larval fat bodies have (Arnheim, 1983; Brochmann et al., 1996; Ainouche and Bayer, been reported (Gelernter and Federici, 1986; Goodman et al., 2001; 1997). This genomic mechanism maintains a great homogeneity Hara et al., 1993; Yasunaga-Aoki et al., 2004; Zhang et al., 2006), within the nuclear ribosomal ITS paralogues via continual turnover but no published reports are available for a cell line from pupal tis- of repeat copies by unequal recombination (Ganley and Kobayashi, sues until recently two new cell lines, IOZCAS-Spex XI (Zhang et al., 2007). Interestingly, in this study we found that there is a variation 2011) and NTU-SE, were established (Table 1). The NTU-SE and the (i.e. two types) among the ITS sequences present in a eukaryote other S. exigua cell lines may thus be a good research resource to genome from a single S. exigua larva (Supplement-Fig. 2). We fur- investigate the key factor that dominate the viral susceptibility of ther found that these two types of ITS sequences from S. exigua lar- cells and to explore why cell lines originated from different tissues vae can be easily distinguished by endonuclease HindIII cleavage, produce variable amounts of virus. Taken together, we concluded and the sequence type 1 is dominant in the PCR product pool (type that the SeMNPV-1/NTU-SE cell system is an excellent tool to study 1 vs. type 2 about 3:1; data not shown). According to the NTU-SE the economical production of microbial insecticides, and it could cells have only one type of ITS sequences, which is 99% identical also potentially be developed into a new baculovirus expression to the S. exigua larvae ITS sequence type 1, we suggest the NTU- vector system (BEVS) to product recombinant proteins. SE cell line might be derived from parts of cells within a single S. exigua (or parts of individuals within S. exigua population) in which Acknowledgments a single homogenized type of ITS sequences present in the genome. Also, we noted that even paralogous ITS sequences are present We thank Tai-Chuan Wang for valuable technical assistance in within an individual insect, the variations between them are very the electron microscopy. This research was supported by the low (<4%) and may not affect the routine utilization of ITS se- grants from the Council of Agriculture (100AS-1.2.2-S-a2(1)) and quences as the molecular marker for insect cell line identification. National Science Council, Republic of China (NSC 100-2321-B- In Taiwan, both SeMNPV and AcMNPV occur in S. exigua (Hong, 002-070-MY2). 1988; Tuan et al., 1994). The AcMNPV strain (AcMNPV-TWN4) has been isolated and in vitro propagated in IPLB-SF21 cells (Hong, 1988), but the SeMNPV strains have not been replicated in cell cul- Appendix A. Supplementary material tures until this study. Thereafter, three SeMNPV strains (SeMNPV- 1, -10, and -23) were selected and we confirmed the identities of Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jip.2012.07.022. these viruses by restriction enzyme profiles (Smith and Summers, 1978), which showed that these three viral DNAs were essentially identical to the genomic DNA from the SeMNPV (Fig. 7). Addition- References ally, we found that the strain SeMNPV-1 shows the most virulence Ainouche, M.L., Bayer, R.J., 1997. On the origins of the tetraploid Bromus species among these three viral strains (Fig. 5). This viral strain is also via- (section Bromus, Poaceae): insights from the internal transcribed spacer ble in the S. exigua larvae and shows the highest virulence in our sequences of nuclear ribosomal DNA. Genome 40, 730–743. preliminary trials (Wang et al., unpublished data). Thus, we sug- Arnheim, N., 1983. Concerted evolution of multigene families. In: Nei, M., Koehn, gest the SeMNPV-1 represents a great potential to be used as R.K. (Eds.), Evolution of Genes and Proteins. Sinauer Associates, Inc., Sunderland, MA, pp. 38–61. microbial insecticide for the S. exigua control. Beckingham, K., 1982. Insect rDNA. In: Busch, H., Rothblum, L. (Eds.), The Cell In addition to SeMNPV, the NTU-SE cell line was also highly Nucleus. Academic Press, New York, pp. 205–269. susceptible to AcMNPV (Fig. 8). We note that it is impossible to Brochmann, C., Nilsson, T., Gabrielsen, T.M., 1996. A classic example of postglacial allopolyploid speciation re-examined using RAPD markers and nucleotide distinguish between the infections of AcMNPV and SeMNPV based sequences: Saxifraga osloensis (Saxifragaceae). Symb. Bot. Ups. 31, 75–89. on the observation of cytopathic effects. Therefore, a recombinant Chen, Y.R., Solter, L.F., Chien, T.Y., Jiang, M.H., Lin, H.F., Fan, H.S., Lo, C.F., Wang, C.H., AcMNPV, vAcD-Pn50539-E (Wu et al., 2007) was used here, and the 2009. Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: efficient expression of the fluorescent reporter proteins in the Lepidoptera) and its susceptibility to microsporidia. J. Invertebr. Pathol. 102, infected cells confirmed that the NTU-SE cells have a high 256–262. C.-Y. Wu et al. / Journal of Invertebrate Pathology 111 (2012) 143–151 151

Cheng, E.Y., Kao, C.H., 1993. Insecticide resistance study in Spodoptera exigua McKenna, K.A., Hong, H.Z., van Nunen, E., Granados, R.R., 1998. Establishment of (Hübner). I. Detecting the resistance in a general survey. J. Agric. Res. China 42, new Trichoplusia ni cell lines in serum-free medium for baculovirus and 396–402. recombinant protein production. J. Invertebr. Pathol. 71, 82–90. Cheng, E.Y., Lu, W.T., Lin, W.G., Lin, D.F., Tsai, T.C., 1988. Effective control of beet Miller, B.R., Crabtree, M.B., Savage, H.M., 1996. Phylogeny of fourteen Culex armyworm, Spodoptera exigua (Hübner), of green onion by the ovicidal action of mosquito species, including the Culex pipiens complex, inferred from the bifenthrin. J. Agric. Res. China 37, 320–327. internal transcribed spacers of ribosomal DNA. Insect Mol. Biol. 5, 93–107. Ganley, A.R.D., Kobayashi, T., 2007. Highly efficient concerted evolution in the Nai, Y.S., Wang, T.C., Chen, Y.R., Lo, C.F., Wang, C.H., 2009. A new ribosomal DNA repeats: total rDNA repeat variation revealed by whole-genome nucleopolyhedrovirus strain (LdMNPV-like virus) with a defective fp25 gene shotgun sequence data. Genome Res. 17, 184–191. from Lymantria xylina (Lepidoptera: Lymantriidae) in Taiwan. J. Invertebr. Gaw, S.Y., 1958. Culturing all types of silkworm tissues using the monolayer culture. Pathol. 102, 110–119. Chinese Sci. Bull. 7, 219–220. Nicholas, K.B., Nicholas, H.B., Deerfield II, D.W., 1997. GeneDoc: analysis and Gelernter, W.D., Federici, B.A., 1986. Continuous cell line from Spodoptera exigua visualization of genetic variation. EMBNEW News 4, 14. (Lepidoptera: Noctuidae) that supports replication of nuclear polyhedrosis Smith, G.E., Summers, M.D., 1978. Analysis of baculovirus genomes with restriction viruses from S. exigua and Autographa californica. J. Invertebr. Pathol. 48, 199– endonuclease. Virology 89, 517–527. 207. Smith, G.E., Summers, M.D., Fraser, M.J., 1983. Production of human beta interferon Goodman, C.L., EI Sayed, G.N., McIntosh, A.H., Grasela, J.J., Stiles, B., 2001. in insect cells infected with a baculovirus expression vector. Mol. Cell. Biol. 3, Establishment and characterization of insect cell lines from 10 lepidopteran 2156–2165. species. In Vitro Cell. Dev. Biol. Animal 37, 367–373. Spurr, A.R., 1969. A low-viscosity epoxy resin embedding medium for electron Goodwin, R.H., Tompkins, G.J., McCawley, P., 1978. Gypsy moth cell lines divergent microscopy. J. Ulrastruct. Res. 26, 31–43. in viral susceptibility. I. Culture and identification. In Vitro (Rockville) 14, 485– Summers, M.D., Smith, G.E., 1987. A manual of methods for baculovirus vectors and 493. insect cell culture procedures. Texas Agricult. Exp. Sta. Bull. No. 1555. Gotoh, R., Gutierrez, J., Navajas, M., 1998. Molecular comparison of the sibling Tabachnick, W.J., Knudson, D.L., 1980. Characterization of invertebrate cell lines. II. species Tetranychus pueraricola Ehara & Cotoh and T. urticae Koch (Acari: Isozyme analysis electrophoresis. In Vitro 16, 392–398. Tetranychidae). Entomol. Sci. 1, 55–57. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The Grace, T.D.C., 1962. Establishment of four strains of cells from insect tissues grown CLUSTAL_X windows interface. Flexible strategies for multiple sequence in vitro. Nature 195, 788–789. alignment aided by quality analysis tools. Nucleic Acid Res. 25, 4876–4882. Granados, R.R., McKenna, K.A., 1995. Insect cell culture methods and their use in Trumble, J.T., Baker, T.C., 1984. Flight phenology and pheromone trapping of virus research. In: Schuler, M.L., Wood, H.A., Granados, R.R., Hammer, D.A. Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) in southern and coastal (Eds.), Baculovirus Expression Systems and Biopesticides. Wiley-Liss Press, New California. Environ. Entomol. 13, 1278–1282. York, pp. 13–39. Tuan, S.J., Kao, S.S., Cheng, D.J., 1994. Histopathology and pathogenicity of Granados, R.R., Li, G., Derksen, A.C.G., Mckenna, K.A., 1994. A new insect cell line Spodoprera exigua nuclear polyhedrosis virus isolated in Taiwan. Chinese J. from Trichoplusia ni (BTI-Tn-5B1-4) susceptible to Trichoplusia ni single Entomol. 14, 33–45. enveloped nuclear polyhedrosis virus. J. Invertebr. Pathol. 64, 260–266. van Oers, M.M., Lynn, D.E., 2010. Insect cell culture. In: Encyclopedia of Life Sciences Gunderson, J.H., Elwood, H., Ingold, A., Kindle, K., Sogin, M.L., 1987. Phylogenetic (ELS). John Wiley & Sons, Ltd., Chichester. relationships between chlorophytes, chrysophytes, and oomycetes. Proc. Natl. Vaughn, J.L., 1981. Insect cells for insect virus production. In: Maramorosch, K. (Ed.), Acad. Sci. USA 16, 5823–5827. Advances in Cell Culture, vol. 1. Academic Press, New York, pp. 281–295. Hara, K., Funakoshi, M., Tsuda, K., Kawarabata, T., 1993. New Spodoptera exigua cell Vaughn, J.L., Goodwin, R.H., Thompkins, G.J., McCawley, P., 1977. The establishment lines susceptible to Spodoptera exigua nuclear polyhedrosis virus. In Vitro Cell. of two insect cell lines from the insect Spodoptera frugiperda (Lepidoptera: Dev. Biol. Animal 29, 904–907. Noctuidae). In Vitro 13, 213–217. Hara, K., Funakoshi, M., Kawarabata, T., 1995. A cloned cell line of Spodoptera exigua Wang, C.H., Chou, C.M., Liu, H.C., Kau, S.L., Kou, G.H., Lo, C.F., 1996. Continuous cell has a highly increased susceptibility to the Spodoptera exigua nuclear line from pupal ovary of Perina nuda (Lepidoptera: Lymantriidae) that is polyhedrosis virus. Can. J. Microbiol. 41, 1111–1116. permissive to nuclear polyhedrosis virus from P. nuda. J. Invertebr. Pathol. 67, Heldens, J.G.M., van Strien, E.A., Feldmann, A.M., Kulcsár, P., Munoz, D., Leisy, D.J., 199–204. Zuidema, D., Goldbach, R.W., Vlak, J.M., 1996. Spodoptera exigua multicapsid Weng, Q., Yang, K., Xiao, W., Yuan, M., Zhang, W., Pang, Y., 2009. Establishment of an nucleopolyhedrovirus deletion mutants generated in cell culture lack virulence insect cell clone that harbours a partial baculoviral genome and is resistant to in vivo. J. Gen. Virol. 77, 3127–3134. homologous virus infection. J. Gen. Virol. 90, 2871–2876. Hink, W.F., Strauss, E., 1976. Growth of the Trichoplusia ni (TN-368) cell line in Wu, C.Y., Wang, C.H., 2005. Characterization and polyhedrin gene cloning of Lyman- suspension culture. In: Kurstak, E., Maramorosch, K. (Eds.), Invertebrate Tissue tria xylina multiple nucleopolyhedrovirus. J. Invertebr. Pathol. 88, 238–246. Culture, Applications in Medicine, Biology, and Agriculture. Academic Press, Wu, C.Y., Wang, C.H., 2006. New cell lines from Lymantria xylina (Lepidoptera: New York, pp. 297–300. Lymantriidae): characterization and susceptibility to baculovirus. J. Invertebr. Hink, W.F., Thomsen, D.R., Davidson, D.J., Meyer, A.L., Castellino, F.J., 1991. Pathol. 93, 186–191. Expression of three recombinant proteins using baculovirus vectors in 23 Wu, J., King, G., Daugulis, A.J., Faulkner, P., Bone, D.H., Goosen, M.F.A., 1989. insect cell lines. Biotechnol. Prog. 7, 9–14. Engineering aspects of insect cell suspension culture: a review. Microbiol. Hong, X.W., 1988. Characterization of sub-clonal populations of the spodoptera Biotechnol. 32, 249–255. frugiperda cell line SF-21AE. Master Thesis Fu Jen Catholic University, New Wu, T.Y., Wu, C.Y., Chen, Y.J., Chen, C.Y., Wang, C.H., 2007. The 50 untranslated region Taipei City, Taiwan, R.O.C. of Perina nuda virus (PnV) possesses a strong internal translation activity in Kao, S.S., Hsia, W.T., Huang, L.H., 1991. Effectiveness of adjuvants for nuclear baculovirus-infected insect cells. FEBS Lett. 581, 3120–3126. polyhedrosis virus against the beet armyworm, Spodoptera exigua. Chinese J. Wu, C.Y., Lin, H.F., Wang, C.H., Lo, C.F., 2011. Identification of insect cell lines and Entomol. 11, 330–334. cell-line cross-contaminations by nuclear ribosomal ITS sequences. J. Appl. Kao, S.S., Huang, L.H., Tsai, Y. S., Tuan, S.J., 1997. Implementation of control program Entomol. 135, 601–610. against Spodoptera exigua with NPV in green onion field. In: The 3rd Asian– Yasunaga-Aoki, C., Imanishi, S., Iiyama, K., Kawarabata, T., 2004. Establishment of Pacific Conference of Entomology. Taichung, Taiwan, ROC. November 16–22, phagocytic cell lines from larval hemocytes of the beet armyworm, Spodoptera 1997. p. 167. exigua. In Vitro Cell. Dev. Biol. Animal 40, 183–186. Kuchler, R.J., 1977. Development of animal cell populations in vitro. In: Kuchler, R.J. Yeh, S.C., Lee, S.T., Wu, C.Y., Wang, C.H., 2007. A cell line (NTU-MV) established from (Ed.), Biochemical Methods in Cell Culture and Virology. Dowden, Hutchingon, Maruca vitrata (Lepidoptera: Pyralidae): characterization, viral susceptibility, and Ross, Inc. Press, Stroudsburg, pp. 90–113. and polyhedra production. J. Invertebr. Pathol. 96, 138–146. Lynn, D.E., 2007. Available lepidopteran insect cell lines. In: Murhammer, D.W. Zhang, H., Zhang, Y.A., Qin, Q.L., Li, X., Miao, L., Wang, Y.Z., Yang, Z.Q., Ding, C., 2006. (Ed.), Methods in Molecular Biology, Baculovirus and Insect Cell Expression New cell lines from larval fat bodies of Spodoptera exigua, characterization and Protocols, vol. 338. Humana Press Inc., Totowa, NJ, pp. 117–137. susceptibility to baculoviruses (Lepidoptera: Noctuidae). J. Invertebr. Pathol. 91, Maeda, S., Kwai, T., Obinata, M., Fujiwara, H., Horiuchi, T., Saeki, Y., Furusawa, M., 9–12. 1985. Production of human interferon in silkworm using a baculovilrus vector. Zhang, H., Zhang, Y.A., Qin, Q., Li, X., Miao, L., Wang, Y.Z., Qu, L.J., Zhang, A.J., Yang, Q., Nature 315, 592–594. 2009. A cell strain cloned from Spodoptera exigua cell line (IOZCAS-Spex II) Manchenko, F.P., 2003. Handbook of Detection of Enzymes on Electrophoretic Gels. highly susceptible to S. exigua nucleopolyhedrovirus infection. In Vitro Cell. Dev. CRC Press, London, p. 285. Biol. Animal 45, 201–204. Marinucci, M., Romi, R., Mancini, P., Di Lusa, M., Severini, C., 1999. Phylogenetic Zhang, A., Li, X., Zhang, H., Wang, H., Miao, L., Zhang, J., Qin, Q., 2011. A new cell line relationships of seven paleartic members of the maculipennis complex inferred from Spodoptera exigua (Lepidoptera: Noctuidae) and its differentially from ITS2 sequence analysis. Insect Mol. Biol. 8, 469–480. expressed genes. J. Appl. Entomol. (published on-line).