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Establishment of a cell line derived from embryos of the diamondback moth, Plutella xylostella (L.)
JOURNAL
OF INVERTEBRATE
PATHOLOGY
59,
174-177 (1992)
Establishment of a Cell Line Derived from Embryos of the Diamondback Moth, Plutella xylostella (L.) SHENG-HUEY Department
of Entomology, National
LEE AND ROGER F. Haul
Chung Hsing University, Taichung,
Taiwan 40227, Republic of China
Received February 26, 1991; accepted June 14, 1991 MATERIALS
Cells from embryonic tissues of the diamondback moth, Plutella xylostella CL.), were cultured with a modified Hink’s medium. The primary culture was initiated on October 17, 1987 and the first passagewas made 30 days after culturing. Continuous cultures had undergone 90 passages by May 1989.The cell line designated as “PX-1187” is a new line established from this moth. It formed a monolayer and had a cell population composedof cells of various types with the spherical cells predominating. The cultured cells were polyploid with a chromosome number ranging from 20 to 180. The doubling time was 27 hr. Optimal cell growth was obtained between 25 and 28°C in medium containing 10% FBS. The isozyme profiles of PX-1187 were different from those of silkworm lines; viz., Bm-N and Bm-0. This cell line could be stored in liquid nitrogen (-196OC) for over 10 months. 0 1992 Academic Press, Inc. KEY WORDS: Diamondback moth; Plutella xylostella; cell culture; cell line; PX-1181; embryo.
INTRODUCTION
Over 100 cell lines have been established from various insect tissues of several orders of insects, for example, Lepidoptera, Diptera, Homoptera, Hemiptera, Orthoptera (Hink, 1980; Mitsuhashi, 1981). Cell culture is generally considered a useful technique for some fundamental studies in cell physiology, genetics, and biochemistry and for the study of insect pathogens and parasites. An in vitro culture system can be especially useful for biotechnological studies in relation to invertebrate pathology (see Maramorosch, 1987). In applied aspect, cell culture is widely used in producing viruses for the purpose of biological control (Grace, 1982). Although a cell line was established from pupae of the diamondback moth, Plutella xylostella (L.) (see Chen and Dai, 19871, the present communication reports a new line derived from embryonic tissues of this moth.
’ To whom correspondence
$1.50
The initial explant for primary culture was obtained from a-day-old eggs of P. xylostella which had been reared in the laboratory for several generations. Approximately 0.03-g eggs were surface sterilized by immersing into 70% ethanol for 5 min and then in 0.2% sodium hypochlorite for 5 min. The sterilized eggs were washed in double-distilled water several times and finally rinsed with the culture medium. The eggs were crushed and homogenized in the medium with a Teflon homogenizer, and then centrifuged at 1OOOg for 10 min. The supernatant was discarded and the pellet was resuspended in 5 ml medium. The fragmented embryos were finally transferred into a sterile flask (25 cm21 and incubated at 28°C. The culture medium was changed by replacing a half volume with fresh one every 1 week. The culture medium used was modified from Hink (1970) and had the following composition: 75 ml Grace’s medium (GIBCO), 0.3 g lactalbumin hydrolysate (Difco), 0.3 g TC yeastolate (Difco), 4 ml whole egg ultraflltrate (GIBCO), 20 ml fetal bovine serum (FBS) (GIBCO), and 1 ml antibiotic mixture containing penicillin G, streptomycin S, Kanamycin S, and Nystatin in 100 ml liquid medium. The pH value was adjusted to 6.4 with 2 N KOH.
Subculture was carried out by detaching the cells from the wall surface of the culture flask using a rubber policeman. The detached cells were dispersed with a pipet and transferred to a new flask containing 5 ml fresh medium. Before the 10th passage, subculture was done by a split ratio of 1:2 in an interval of 1 week. However the split ratio was increased gradually up to 1:lO and subculture was made every 5-6 days, and beginning from the 10th passage the FBS was also reduced gradually from 20 to 15% and finally to 10%
should be addressed.
Copyright 8 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Culture
Subculture
174 0022-2011192
Primary
AND METHODS
ESTABLISHMENT
OF CELL LINE FROM P.
after the 31st passage. In addition, the whole egg ultrafiltrate was omitted from the medium thereafter. Karyotype Analysis
To a culture flask (25 cm2), 0.1 ml colchicine at 20 Fg/ml (Sigma) was added after culturing for 24 hr. The cells were detached after reacting for 3 hr and centrifuged at 1OOOg for 10 min. The pellet was suspended in 0.56% KC1 for 30 min, centrifuged again, and finally resuspended in the fresh fixatives (methyl alcohol:acetic acid = 3:l). The cell suspension was kept at 4°C overnight. It was then subjected to several cycles of centrifugation and resuspension in fresh fixative. The fixed cells were stained with 3% Giemsa solution for 15 min on a glass slide. The chromosome number was counted by sampling ca. 200 cells under oil immersion. Isozyme Analysis
Isozyme analysis was performed for the P. xylostella cell line together with two Bomby~ mori cell lines, viz., Bm-N (from Kyushu University, Japan) and Bm-0 (Sun and Hou, 1989). The procedures were modified from Tabachnick and Knudson (1980). The cultured cells at log phase were frozen in liquid nitrogen for 10 set and thawed rapidly at 65°C. This freezing and thawing process was repeated five times. The disrupted cells in grinding buffer were centrifuged at ll,OOOg for 6 min and the supernatants were collected for electrophoresis. Two isozymes, i.e., lactate dehydrogenase (LDH) and malate dehydrogenase (MDH), were analyzed. The gels were stained by soaking for 30 min in a solution containing sodium lactate, 3nicotinamide adenine dinucleotide (NAD+), nitro blue tetrazolium (NBT-R), and Tris-HCl (pH 8.5) for LDH or in a solution containing sodium malate, NAD+ , NBT-R, and Tris-HCl (pH 8.5) for MDH. After staining, 1 ml phenazine methosulfate (4mg/ml) was added to 100 ml of the above staining solution. The gels were fixed in the fixative fluid (50% methanol:acetic acid = 1:lO).
xylo.steZZu
175
days. The explant shrank gradually and cell mass liberated from it. Then more cells migrated from the explant and a number of cells were mitotic. At this moment cell size and morphology were variable (Fig. la). The early migrated cells are mostly spindle in shape with prominent cytoplasmic processes. They formed various networks attaching to the bottom of the culture flask. These networks are either muscle-like and nerve-like structures, or composed of fibrous connective tissues. However, some spindle cells can also grow freely. The spherical cells can either attach to the flask wall or remain in a suspending state in the medium. Spherical, giant, and vacuolated cells, and cells of irregular shape were seen in this primary culture (Fig. la). Subculture
The first subculture was carried out when the bottom of the culturing flask was covered with masses of network cells. The initial explant still existed in the early subcultures, but it became shrunken gradually and
Measurement of Cell Growth
The cultured cells in a dish (10 x 35 mm) at log phase were adjusted to 2 X 106cells/ml. For testing the effect of different FBS concentrations on cell growth, media were prepared by adding 0, 1,5, or 10% FBS. For testing temperature effect, the cells were incubated at 15,20,25,32, or 36°C. The cultured cells were stained with trypan blue and counted daily using a hemocytometer under a microscope. RESULTS Primary
Culture
Most tissue fragments to attach to the culture
of P. xylostella embryos began flask after incubating for 2-3
FIG. 1. Primary culture and cell line of P. xyhtellu embryos. (a) Primary culture; 01) PX-1187 cell line. E, explanted tissues; G, giant cell; I, irregular cell; S, spindle cell; SL, spherical cell; T, tadpoleshaped cell; V, cell with vacuoles. Bar: (a) 1000 pm; (b) 50 Km.
176
LEE AND HOU
then disappeared. The cytoplasmic processes of the spindle cells shortened and no more network structures formed. Later spherical cells became dominant in the culture (Fig. lb). The primary culture was set up on October 17,1987 and the first subculture was done after culturing for 30 days. In the subsequent 19 months, this culture was given more than 90 subculturing passages, resulting in the establishment of the first cell line of P. xylostella in May 1989, and this line was designated “PX-1187.” The cultured cells grew slowly in the beginning, the interval of subculture being 2-3 weeks at a split ratio of 1:2. Later the cells multiplied faster and were subcultured every 5-6 days at a 1:lO split ratio. Until the 13th passage, the culture medium was only partially replaced with a fresh one upon subculturing. For all subsequent subculturings, complete replacement of culture medium was made. The FBS concentration was reduced from 15 to 10% starting from the 31st passage. The line had been cultured for more than 120 passages in the same medium by the end of 1990.
1
20
16
-
6-
Characteristics of the PX-1187 Line
This line has a heterogeneous cell population comprising spherical, spindle, tadpole-shaped, and irregular cells (Fig. lb). Most cells can attach to the bottom of the flask, forming monolayer; however, some may clump together, suspending in the medium while the cell number is increased. The spherical cells are predominant among the various cell types. The cell population doubling time at 28°C was 27 hr for a seeding density of 2 x lo5 cells/ml. The cells did not proliferate at 15 or 36°C and at 36°C all cells were dead by the 7th day after culturing. The optimal incubation temperature was found to be 25 and 28°C (Fig. 2). The optimal FBS concentration for growth was 10%. The cell was polyploid and its chromosome number ranged from 20 to 180 at metaphase. The number was increasing with passages; for example, it was 50-120 at the 16th passage and was 80-140 at the 86th passage. Comparison of LDH and MDH isozyme profiles between PX-1187 and B. mori lines showed differences in band formations (Fig. 3). The cells could be stored by suspending with the culture medium containing 10% dimethyl sulfoxide in liquid nitrogen ( - 196”C), and could remain viable for at least 10 months as evidenced from their ability to multiply normally upon subculturing with growth medium.
1
2
3
4
5
6
7
Days after culturing FIG. 2. Growth curves of PX-1187 cells at 37th paeaage incubated under different temperatures. (A) 15°C; (A) 20°C; (0) 25°C; (0) 28°C; (0) 32°C; (WI 36°C.
which Mitsuhashi (1981) first established from fat bodies, most lepidopterous cell lines were cultured from embryos or ovaries. Therefore, we also selected embryonic tissues for initial explants in an attempt to obtain a continuous cell culture of P. xyZosteZZu. In this study, we found that cell growth was not inferior when the whole egg ultrafiltrate was removed from the medium and also the FBS concentration was reduced from 15 to 10%. These changes make the culture medium cheaper. In culturing many other cell lines, FBS was commonly used at 10% (Sieburth and
MDH PX-1187 Blu-Nam-0
m-0
LDH Bill-N
PX-1187
DISCUSSION
Although more than 20 lepidopterous cell lines have been established (Hink, 1980), only one of them designated as BCIRL-PXB-HNU3 was derived from pupae of P. xylostellu (see Chen and Dai, 1987). The present line should be the second cell line established from this species. Except the cell line of Mumestru brussicue
FIG. 3. Malate dehydrogenase (MDH) and lactate dehydrogenaee (LDH) ieozyme proties of PX-1187, Bm-N, and Bm-0 lines.
ESTABLISHMENT
OF CELL LINE FROM P. xylosteEla
Maruniak, 1988). In addition to economical considerations, virus infection was better in B. mori cell lines cultured with a medium containing 10% FBS than in media having a higher FBS content (Watanabe, 1987). The PX-1187 line has its derivation from the embryo and a population composed of various cell types. Similar observations have been reported for different species (Hink, 1970; Kurtti and Brooks, 1977; Dwyer, 1988). The chromosome number of this line is 20-180 comparable to 56-180 in the Plodia interpunctellu line (Tsang et al., 1985) and 31-230 in the B. mori line (Imanishi and Ohtsuki, 1988). The doubling time of this line is similar to that of IPLB-SF-21AE line (Vaughn et al., 1977) but is much shorter than that of UM-BGE-1 line (Kurt&i and Brooks, 1977). The growth curve went down on the first day after subculturing, probably due to cell damage by crushing but it was going up again after 2 days. Differences in LDH and MDH isozyme profiles between PX-1187 and silkworm lines prove that this line is distinct from those two cell lines. ACKNOWLEDGMENTS This study was financially supported by a research grant from the National Science Council, and in part by the Council of Agriculture, Republic of China. We thank Dr. R. J. Chiu of National Chung Hsing University for critical reading of the manuscript, and Dr. C. H. Wang of Fu-Jen University and Dr. T. C. Wang of Academia Sinica for their technical assistance.
REFERENCES Chen, Q., and Dai, X. 1987. Replication of Pieris m;pae granulosis virus in an insect cell line. In “Invertebrate and Fish Tissue Culture” (Y. Kuroda, E. Kursta, and K. Maramorosch, Eds.), pp. 179172. Japan Scientific Societies Press, Tokyo. Dwyer, K. G. 1988. Establishment of cell lines from Pieris rapae
177
embryos characterization and susceptibility to baculoviruses. J. Invert&r. Path&. 52, 268-274. Grace, T. D. C. 1982. Development of insect cell culture. In “Invertebrate Cell Culture, Applications” (K. Maramorosch, and J. Matsuhashi, Eds.), pp. l-8. Academic Press, New York. Hink, W. F. 1970. Established insect cell line from the cabbage looper, Trichoplusia ni. Nature 226, 466-467. Hink, W. F. 1980. The 1979 compilation of invertebrate cell lines and culture media. In “Invertebrate Systems in Vitro”. (E. Kurstak, K. Maramorosch, and A. Dubendorfer, Eds.), pp. 553-578. Elsevier/ North-Holland Biochemical Press, Amsterdam. Imanishi, S., and Ohtsuki, Y. 1988. Characteristics of cell lines established from embryonic tissues of several races of the silkworm, Bombyx mori, cultured in vitro. J. Seric. Sci. Jpn. 57, 184-188. Kurtti, T. J., and Brooks, M. A. 1977. Isolation of cell lines from embryos of the cockroach, BlatteLla germanica. In Vitro 13, 11-17. Maramorosch, K. (Ed.). 1987. “Biotechnology in Invertebrate Pathology and Cell Culture.” Academic Press, San Diego, 511 pp. Mitsuhashi, J. 1981. Establishment and some characteristics of a continuous cell line derived from fat body of the cabbage armyworm (Lepidoptera, Noctuidae). Dev. Growth Differ. 23, 63-72. Sieburth, P., and Maruniak, J. 1988. Growth characteristics of a continuous cell line for the velvetbean caterpillar, Anticarsiu gemmata& Hubner (Lepidoptera: Noctuidae). In Vitro 24, 195-198. Sun, S. Y., and Hou, R. F. 1989. In vitro culturing of cells derived from larval ovaries of the silkworm, Bombyx mori L. J. Agric. Fores (Taiwan) 38, 163-179. (In Chinese with English summary) Tabachnick, W. J., and Knudson, D. L. 1980. Characterization of invertebrate cell lines II: Isosyme analyses electrophoresis. In Vitro 16, 392-298. Tsang, K. R., Ward, G. B., Mardan, A. H., Harein, P. K., Brooks, M. A., and Jacobson, L. 1985. Establishment and characterization of a cell line from embryos of the Indian meal moth, Plodia interpunctella. J. Znvertebr. Pathol. 46, 186-188. Vaughn, J. L., Goodwin, R. H., Tompkins, G. J., and McCawley, P. 1977. The establishment of two cell lines from the insect Spodoptera frugiperdu. In Vitro 13, 213-217. Watanabe, H. 1987. Effect of the concentration of fetal bovine serum in a culture medium on the susceptibility of Bombyx mori cells to a nuclear polyhedrosis virus. Appl. Ent. Zool. 22, 397-398.
OF INVERTEBRATE
PATHOLOGY
59,
174-177 (1992)
Establishment of a Cell Line Derived from Embryos of the Diamondback Moth, Plutella xylostella (L.) SHENG-HUEY Department
of Entomology, National
LEE AND ROGER F. Haul
Chung Hsing University, Taichung,
Taiwan 40227, Republic of China
Received February 26, 1991; accepted June 14, 1991 MATERIALS
Cells from embryonic tissues of the diamondback moth, Plutella xylostella CL.), were cultured with a modified Hink’s medium. The primary culture was initiated on October 17, 1987 and the first passagewas made 30 days after culturing. Continuous cultures had undergone 90 passages by May 1989.The cell line designated as “PX-1187” is a new line established from this moth. It formed a monolayer and had a cell population composedof cells of various types with the spherical cells predominating. The cultured cells were polyploid with a chromosome number ranging from 20 to 180. The doubling time was 27 hr. Optimal cell growth was obtained between 25 and 28°C in medium containing 10% FBS. The isozyme profiles of PX-1187 were different from those of silkworm lines; viz., Bm-N and Bm-0. This cell line could be stored in liquid nitrogen (-196OC) for over 10 months. 0 1992 Academic Press, Inc. KEY WORDS: Diamondback moth; Plutella xylostella; cell culture; cell line; PX-1181; embryo.
INTRODUCTION
Over 100 cell lines have been established from various insect tissues of several orders of insects, for example, Lepidoptera, Diptera, Homoptera, Hemiptera, Orthoptera (Hink, 1980; Mitsuhashi, 1981). Cell culture is generally considered a useful technique for some fundamental studies in cell physiology, genetics, and biochemistry and for the study of insect pathogens and parasites. An in vitro culture system can be especially useful for biotechnological studies in relation to invertebrate pathology (see Maramorosch, 1987). In applied aspect, cell culture is widely used in producing viruses for the purpose of biological control (Grace, 1982). Although a cell line was established from pupae of the diamondback moth, Plutella xylostella (L.) (see Chen and Dai, 19871, the present communication reports a new line derived from embryonic tissues of this moth.
’ To whom correspondence
$1.50
The initial explant for primary culture was obtained from a-day-old eggs of P. xylostella which had been reared in the laboratory for several generations. Approximately 0.03-g eggs were surface sterilized by immersing into 70% ethanol for 5 min and then in 0.2% sodium hypochlorite for 5 min. The sterilized eggs were washed in double-distilled water several times and finally rinsed with the culture medium. The eggs were crushed and homogenized in the medium with a Teflon homogenizer, and then centrifuged at 1OOOg for 10 min. The supernatant was discarded and the pellet was resuspended in 5 ml medium. The fragmented embryos were finally transferred into a sterile flask (25 cm21 and incubated at 28°C. The culture medium was changed by replacing a half volume with fresh one every 1 week. The culture medium used was modified from Hink (1970) and had the following composition: 75 ml Grace’s medium (GIBCO), 0.3 g lactalbumin hydrolysate (Difco), 0.3 g TC yeastolate (Difco), 4 ml whole egg ultraflltrate (GIBCO), 20 ml fetal bovine serum (FBS) (GIBCO), and 1 ml antibiotic mixture containing penicillin G, streptomycin S, Kanamycin S, and Nystatin in 100 ml liquid medium. The pH value was adjusted to 6.4 with 2 N KOH.
Subculture was carried out by detaching the cells from the wall surface of the culture flask using a rubber policeman. The detached cells were dispersed with a pipet and transferred to a new flask containing 5 ml fresh medium. Before the 10th passage, subculture was done by a split ratio of 1:2 in an interval of 1 week. However the split ratio was increased gradually up to 1:lO and subculture was made every 5-6 days, and beginning from the 10th passage the FBS was also reduced gradually from 20 to 15% and finally to 10%
should be addressed.
Copyright 8 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Culture
Subculture
174 0022-2011192
Primary
AND METHODS
ESTABLISHMENT
OF CELL LINE FROM P.
after the 31st passage. In addition, the whole egg ultrafiltrate was omitted from the medium thereafter. Karyotype Analysis
To a culture flask (25 cm2), 0.1 ml colchicine at 20 Fg/ml (Sigma) was added after culturing for 24 hr. The cells were detached after reacting for 3 hr and centrifuged at 1OOOg for 10 min. The pellet was suspended in 0.56% KC1 for 30 min, centrifuged again, and finally resuspended in the fresh fixatives (methyl alcohol:acetic acid = 3:l). The cell suspension was kept at 4°C overnight. It was then subjected to several cycles of centrifugation and resuspension in fresh fixative. The fixed cells were stained with 3% Giemsa solution for 15 min on a glass slide. The chromosome number was counted by sampling ca. 200 cells under oil immersion. Isozyme Analysis
Isozyme analysis was performed for the P. xylostella cell line together with two Bomby~ mori cell lines, viz., Bm-N (from Kyushu University, Japan) and Bm-0 (Sun and Hou, 1989). The procedures were modified from Tabachnick and Knudson (1980). The cultured cells at log phase were frozen in liquid nitrogen for 10 set and thawed rapidly at 65°C. This freezing and thawing process was repeated five times. The disrupted cells in grinding buffer were centrifuged at ll,OOOg for 6 min and the supernatants were collected for electrophoresis. Two isozymes, i.e., lactate dehydrogenase (LDH) and malate dehydrogenase (MDH), were analyzed. The gels were stained by soaking for 30 min in a solution containing sodium lactate, 3nicotinamide adenine dinucleotide (NAD+), nitro blue tetrazolium (NBT-R), and Tris-HCl (pH 8.5) for LDH or in a solution containing sodium malate, NAD+ , NBT-R, and Tris-HCl (pH 8.5) for MDH. After staining, 1 ml phenazine methosulfate (4mg/ml) was added to 100 ml of the above staining solution. The gels were fixed in the fixative fluid (50% methanol:acetic acid = 1:lO).
xylo.steZZu
175
days. The explant shrank gradually and cell mass liberated from it. Then more cells migrated from the explant and a number of cells were mitotic. At this moment cell size and morphology were variable (Fig. la). The early migrated cells are mostly spindle in shape with prominent cytoplasmic processes. They formed various networks attaching to the bottom of the culture flask. These networks are either muscle-like and nerve-like structures, or composed of fibrous connective tissues. However, some spindle cells can also grow freely. The spherical cells can either attach to the flask wall or remain in a suspending state in the medium. Spherical, giant, and vacuolated cells, and cells of irregular shape were seen in this primary culture (Fig. la). Subculture
The first subculture was carried out when the bottom of the culturing flask was covered with masses of network cells. The initial explant still existed in the early subcultures, but it became shrunken gradually and
Measurement of Cell Growth
The cultured cells in a dish (10 x 35 mm) at log phase were adjusted to 2 X 106cells/ml. For testing the effect of different FBS concentrations on cell growth, media were prepared by adding 0, 1,5, or 10% FBS. For testing temperature effect, the cells were incubated at 15,20,25,32, or 36°C. The cultured cells were stained with trypan blue and counted daily using a hemocytometer under a microscope. RESULTS Primary
Culture
Most tissue fragments to attach to the culture
of P. xylostella embryos began flask after incubating for 2-3
FIG. 1. Primary culture and cell line of P. xyhtellu embryos. (a) Primary culture; 01) PX-1187 cell line. E, explanted tissues; G, giant cell; I, irregular cell; S, spindle cell; SL, spherical cell; T, tadpoleshaped cell; V, cell with vacuoles. Bar: (a) 1000 pm; (b) 50 Km.
176
LEE AND HOU
then disappeared. The cytoplasmic processes of the spindle cells shortened and no more network structures formed. Later spherical cells became dominant in the culture (Fig. lb). The primary culture was set up on October 17,1987 and the first subculture was done after culturing for 30 days. In the subsequent 19 months, this culture was given more than 90 subculturing passages, resulting in the establishment of the first cell line of P. xylostella in May 1989, and this line was designated “PX-1187.” The cultured cells grew slowly in the beginning, the interval of subculture being 2-3 weeks at a split ratio of 1:2. Later the cells multiplied faster and were subcultured every 5-6 days at a 1:lO split ratio. Until the 13th passage, the culture medium was only partially replaced with a fresh one upon subculturing. For all subsequent subculturings, complete replacement of culture medium was made. The FBS concentration was reduced from 15 to 10% starting from the 31st passage. The line had been cultured for more than 120 passages in the same medium by the end of 1990.
1
20
16
-
6-
Characteristics of the PX-1187 Line
This line has a heterogeneous cell population comprising spherical, spindle, tadpole-shaped, and irregular cells (Fig. lb). Most cells can attach to the bottom of the flask, forming monolayer; however, some may clump together, suspending in the medium while the cell number is increased. The spherical cells are predominant among the various cell types. The cell population doubling time at 28°C was 27 hr for a seeding density of 2 x lo5 cells/ml. The cells did not proliferate at 15 or 36°C and at 36°C all cells were dead by the 7th day after culturing. The optimal incubation temperature was found to be 25 and 28°C (Fig. 2). The optimal FBS concentration for growth was 10%. The cell was polyploid and its chromosome number ranged from 20 to 180 at metaphase. The number was increasing with passages; for example, it was 50-120 at the 16th passage and was 80-140 at the 86th passage. Comparison of LDH and MDH isozyme profiles between PX-1187 and B. mori lines showed differences in band formations (Fig. 3). The cells could be stored by suspending with the culture medium containing 10% dimethyl sulfoxide in liquid nitrogen ( - 196”C), and could remain viable for at least 10 months as evidenced from their ability to multiply normally upon subculturing with growth medium.
1
2
3
4
5
6
7
Days after culturing FIG. 2. Growth curves of PX-1187 cells at 37th paeaage incubated under different temperatures. (A) 15°C; (A) 20°C; (0) 25°C; (0) 28°C; (0) 32°C; (WI 36°C.
which Mitsuhashi (1981) first established from fat bodies, most lepidopterous cell lines were cultured from embryos or ovaries. Therefore, we also selected embryonic tissues for initial explants in an attempt to obtain a continuous cell culture of P. xyZosteZZu. In this study, we found that cell growth was not inferior when the whole egg ultrafiltrate was removed from the medium and also the FBS concentration was reduced from 15 to 10%. These changes make the culture medium cheaper. In culturing many other cell lines, FBS was commonly used at 10% (Sieburth and
MDH PX-1187 Blu-Nam-0
m-0
LDH Bill-N
PX-1187
DISCUSSION
Although more than 20 lepidopterous cell lines have been established (Hink, 1980), only one of them designated as BCIRL-PXB-HNU3 was derived from pupae of P. xylostellu (see Chen and Dai, 1987). The present line should be the second cell line established from this species. Except the cell line of Mumestru brussicue
FIG. 3. Malate dehydrogenase (MDH) and lactate dehydrogenaee (LDH) ieozyme proties of PX-1187, Bm-N, and Bm-0 lines.
ESTABLISHMENT
OF CELL LINE FROM P. xylosteEla
Maruniak, 1988). In addition to economical considerations, virus infection was better in B. mori cell lines cultured with a medium containing 10% FBS than in media having a higher FBS content (Watanabe, 1987). The PX-1187 line has its derivation from the embryo and a population composed of various cell types. Similar observations have been reported for different species (Hink, 1970; Kurtti and Brooks, 1977; Dwyer, 1988). The chromosome number of this line is 20-180 comparable to 56-180 in the Plodia interpunctellu line (Tsang et al., 1985) and 31-230 in the B. mori line (Imanishi and Ohtsuki, 1988). The doubling time of this line is similar to that of IPLB-SF-21AE line (Vaughn et al., 1977) but is much shorter than that of UM-BGE-1 line (Kurt&i and Brooks, 1977). The growth curve went down on the first day after subculturing, probably due to cell damage by crushing but it was going up again after 2 days. Differences in LDH and MDH isozyme profiles between PX-1187 and silkworm lines prove that this line is distinct from those two cell lines. ACKNOWLEDGMENTS This study was financially supported by a research grant from the National Science Council, and in part by the Council of Agriculture, Republic of China. We thank Dr. R. J. Chiu of National Chung Hsing University for critical reading of the manuscript, and Dr. C. H. Wang of Fu-Jen University and Dr. T. C. Wang of Academia Sinica for their technical assistance.
REFERENCES Chen, Q., and Dai, X. 1987. Replication of Pieris m;pae granulosis virus in an insect cell line. In “Invertebrate and Fish Tissue Culture” (Y. Kuroda, E. Kursta, and K. Maramorosch, Eds.), pp. 179172. Japan Scientific Societies Press, Tokyo. Dwyer, K. G. 1988. Establishment of cell lines from Pieris rapae
177
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