Establishment and characterization of a cell line from embryos of the Indianmeal moth, Plodia interpunctella

JOCIKNAL

OF INVEKTEBKA~II

Establishment

PATHOLOG\I

46. Iti& 188 (1985,

and Characterization of a Cell Line from the Indianmeal Moth, Plodia interpunctellal

K. ROGER TSANG,?

GORDON B. WARD.~ ALI H. MAKDAN,~ PHILLIP MARION A. BROOKS,> AND LAWRENCE JACOBSON’:’

Embryos

of

K. HAKEIN,

Received February 13. 1985: accepted March 25. 1985 A cell line. UMN-PIE-1181. initiated in November. 1981. from embryos of a malathion-resistant strain of Indianmeal moth, P/o& ittre~prrttc~tello. was in the 83rd passage on January 28. 1985. The line consists of single, small. fibroblastlike cells that are polyploid with chromosome numbers ranging from 56 to 180. Growth rate is dependent on seeding density. there being no growth at or below seeding densities of 2 x 10% ml; optimum growth requires a fetal bovine serum concentration of at least 5%. Twenty-nine isozymes were examined. Five enzymes from the cell lines resolved well and subsequently were compared to enzymes extracted from 4-day-old embryos and other life stages of the insects. Phosphomannose isomerase, malic enzyme. malate dehydrogenase. phosphoglucose isomerase, and glucose-6-phosphate dehydrogenase in extracts from the cultured cells and from the insects had identical patterns. Two bands for glutamate-oxalacetate transaminase, present in the cell line. were not observed in the tissue extracts. Furthermore. lactate dehydrogenase from the cultured cells appeared as four bands but was not detectable in any of the samples run from the various life stages of the insects. I 19X? Academic Pre\\. Inc. KEY WORDS: P/o&r itttrrpunc~tc~llu embryonic cell line: insect cell seeding density: serum dependency of cultured insect cells: isozymes in insect cell line.

establishment and characterization of a cell line from P. interpunctella with the intent of making the cells available from studying replication of granulosis viruses and microsporida. In this paper we report the successful establishment of a line from embryos of this insect, and give its karyotype analysis, growth kinetics at various seeding densities and serum concentrations, and isozyme characteristics.

INTRODUCTION

Plodia interpunctella, the Indianmeal moth (IMM), causes a significant amount of damage to stored grain and food products. Since this insect has developed resistance to malathion and synergized pyrethin (Zettler et al., 1973; Armstrong and Soderstrong, 1975), control of it by conventional methods is difficult. A granulosis virus may be used as an alternative control agent (Hunter et al., 1977). The main objective of our work was the

MATERIALS

Insect rearing. Pupae were obtained from the Stored Product Insects Research and Development Laboratory, Savannah, Georgia, in 1980. Larvae were reared on a medium consisting of turkey mash (1200 g), honey (300 ml), glycerin (300 ml), sorbic acid (2.4 g), and water (150 ml), each autoclaved separately and then combined and mixed to a uniform texture. Eggs were surface-sterilized with 10% formalin for 20 min, rinsed three times in sterile water, and

’ Mention of a proprietary product does not constitute an endorsement by the Minnesota Agricultural Experiment Station. ? Present address: Protatek International, 1491 Energy Park Drive West. St. Paul, Minn. 55108. 3 Present address: Metabolism and Radiation Research Laboratory. U.S. Department of Agriculture. Fargo, N.D. 5.5103. 4 Present address: 4339 Columbus Ave.. Minneapolis, Minn. 5.5407. 5 To whom correspondence should be addressed. 180 0022-2011185 $1.50 Copyright All rights

(r; 1985 hy Academx Prea\. Inc. of reproduction in any form resrved.

AND METHODS

f’lodicr CELL

placed on the food in sterile quart jars. Adults that emerged were briefly anesthetized with carbon dioxide and transferred to empty quart jars closed with wire screen caps and inverted over empty Petri dishes. After 2 days of incubation at 27”C, 70% RH, and 12:12 photoperiod, the eggs that had fallen through the screens were collected from the Petri dishes. Preparing eggs and initiating cell cultures. Approximately 1000 eggs were allowed to develop for 4 days (embryogenesis is completed in 5 days). To initiate cultures, half of the eggs were placed in each of two sterile, screw-cap, graduated conical cen-

trifuge tubes of 12 ml capacity and rinsed with distilled water to remove moth scales. Then the eggs were gently shaken several times over a period of 7 min in 10 ml 2% sodium hypochlorite. The liquid was decanted off and the eggs were rinsed twice in 10 ml of sterile, double-distilled water. Ten milliliters aqueous 0.2% Hyamine (Rohm and Haas, Philadelphia, Pa) was added and left on the eggs for I5 min with occasional agitation. This was followed by two rinses with sterile, double-distilled water which was finally replaced with 10 ml of fresh sterile culture medium, UMN-M4 (Table 1).

TABLE CELL

Component

CULTURE

mg/liter

181

LINE

1

MEDIUM

UMN-M4

Component

Amino acids sugars B-Alanine 164 Fructose L-Alanine 184 Glucose L-Arginine-HCI 573 Sucrose L-Asparagine 286 Organic acids L-Aspartic acid 286 o-Ketoglutaric L-Cystine 21 Fumaric L-Glutamic acid 491 Malic L-Glutamine 491 Succinic Glycine 531 Vitamins L-Histidine 2046 &Biotin L-Isoleucine 41 Calcium pantothenate L-Leucine 61 Choline chloride L-Lycine-HCI 511 Folic acid L-Methionine 41 myo-lnositol L-Phenylalanine 123 Niacin L-Proline 286 p-Aminobenzoic acid oL-Serine 900 Pyridoxine-HCI L-Threonine 143 Riboflavin L-Tryptophan 82 Thiamin-HCI L-Tyrosine 41 Cyanocobalamin L-Valine 82 Inorganic salts CaClz 818 KCI 1833 MgClz .6 Hz0 933 MgSO, . 7 Hz0 1138 NaHCO, 286 NaH?PO, . H,O 826 FBS (heat-inactivated), 100 ml/liter pH adjusted to 6.40 with KOH Osmotic pressure ca. 310 mOsm/kg Hz0 Final medium filter-sterilized with O.?+m membrane filter (AcroSOA) and stored at 4°C.

mgiliter 327 513 21829 302 49 548 49 0.01 0.02 0.20 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.5

182

TSANG

The eggs in the culture medium were crushed and homogenized with 15 up-anddown strokes with a sterile Teflon plunger (3431-G04: Arthur H. Thomas. Philadelphia, Pa). The supernatant suspension of cells was collected and centrifuged once at 275a for 5 min to remove excess yolk material. Each cell pellet was then resuspended in 10 ml of culture medium and divided into two culture flasks (25 cm2 T-30 Falcon; (Falcon, Div. Becton. Dickenson and Co., Oxnard, Calif.). After a IO-min rest, the cells had settled on the bottom of the flask and were well attached. The seeding densities in the flasks, assessed with a calibrated ocular grid in a phase-contrast inverted microscope (Unitron), were estimated to be l-2 x lOh cells/flask. The cultures were incubated at 26°C and examined daily using phase-contrast microscopy. After 7 days, half of the medium was pipetted off of the cell layer in each flask and replaced with an equal amount of fresh medium. This partial change was continued for 3 weeks when the cultures were divided for the first time. They were subcultured at 3week intervals until the 15th passage, after which they were subcultured at IO-day intervals. Karyotypr analysis. At passages 18 and 20, aliquots of cells in logarithmic growth phase were treated with colcemid, 0.3 kg/ ml culture medium, for 8 hr, then osmotically shocked in 15 ml 75 ITIM KC1 per flask. After 20 min, the cells were loosened, removed from the flask, and centrifuged at 275g for 5 min. The cell pellet was suspended in 5 ml of fixative consisting of 1 part glacial acetic acid and 3 parts methanol for 10 min, and then centrifuged at 275a for 5 min. The cell pellet was resuspended three more times in fixative and centrifuged. Finally the pellet was suspended in 5 ml of fixative and 2-3 drops of the suspension was placed on each of several clean glass slides. The fixative was ignited by passing an alcohol lamp beneath the slides, and the smears were allowed to air-dry

ET AL.

thoroughly. Metaphase spreads were located with phase-contrast optics at 200x and 400 x , and Giemsa’s stain was used for viewing under brightfield optics. The chromosomes, which are very small, were counted under oil immersion. Seeding density irlflrrence. Three cultures in 17th passage (UMN-PIE-118lSl7) in stationary growth were pooled to obtain l-2 x 10’ cells. These cells were used to make seven dilutions with seeding densities ranging from 4.87 x IO4 to 14.5 x 10’ cells/ 5 ml/flask. Each seeding density subculture was replicated three times and incubated at 26°C. Growth in the flasks was recorded by counting the cells at 200 x with a calibrated ocular grid in an inverted phase-contrast microscope. Ten regularly spaced fields, determined by precise positioning of the flask on the microscope stage, were counted in every flask at 2- to 3-day intervals for a period of 13 days. Serum dependency. Three cultures of UMN-PIE-118lSl7 in logarithmic growth were pooled and yielded approximately 1 x 10’ cells, which were apportioned into 12 flasks. Thus, each flask was seeded with approximately 8 x 10” cells/5 ml. The FBS content in medium UMN-M4 was varied in four ways: 10. 5, 1, or O%, and each serum concentration was tested in triplicate flasks. Since the FBS had an osmotic pressure of 305 mOsm/kg, decreasing it made no significant change in the osmotic pressure of the medium, which ordinarily is 3 10. Starch gel electrophoresis oj’ isogmes. Isozymes were run on starch gels composed of 37.5 g of Electrostarch (Electrostarch Co., Madison, Wis.) and 21.3 g of Sigmastarch (Sigma Chemical Co., St. Louis, MO.) dissolved in 420 ml of gel buffer. Four buffers were tested for each enzyme. These buffers were: (1) ClaytonTretiak buffer system (CT): electrode buffer, 0.04 M citrate-N-(3-aminopropyl)morpholine, pH 6.1; gel buffer, 0.002 M citrate-N-(3-aminopropyl)-morpholine, pH 6.0 (Clayton and Tretiak, 1972). (2) Ridgway buffer system (R): electrode

Ploditr CELL

to electrophoresis cells from two flasks were pooled (yielding about 6 x 1Ohcells), washed in serum-free medium, and pelleted. Tissue extracts, used for comparison to cell lines, consisted of about 500 4-dayold embryonated eggs, five large 4th-instar larvae, five pupae, and five adults of each sex. The cell pellet, the eggs, and the insects were each homogenized in groundglass tissue homogenizers with smail amounts of 0.1 M Tris-HCI buffer, pH 7.0 (Selander et al., 1971). Twice the specimens were frozen at - 70°C and thawed. Samples were then sonicated for 5 min at setting 7 on a Branson Sonifier cell disruptor Model

buffer, 0.3 M lithium borate, pH 8.1; gel buffer, 0.003 M Tris, 0.005 M citric acid, pH 8.5 (Ridgway et al., 1970). (3) Tris-citrate buffer system (TC): electrode buffer, 0.13 M Tris, 0.043 M citrate, pH 7.0; gel buffer, 0.009 M Tris, 0.003 M citrate, pH 7.0 (Siciliano and Shaw, 1976). (4) Tris-verseneborate buffer system (TVB): electrode buffer, 0.5 M Tris, 0.016 M versene, 0.65 M borate, pH 8.0; gel buffer, 0.05 M Tris, 0.0016 M Versene, 0.065 M borate, pH 8.0 (Sicilian0 and Shaw, 1976). Two initial screening runs were done on cultured cells to identify useful enzymes and cell types. For the third run, just prior

TABLE ENZYMES

SCREENED

IN Plodiu

CELL

183

LINE

2

LINE

WITH

ELECTROPHORESIS

GELS

Enzyme tested

Ref.”

Results

Adenylate kinase (EC 2.7.4.3) Alcohol dehydrogenase (EC 1.1.1. I) Catalase (EC 1.11.1.6) Creatine phosphokinase I EC 2.7.3.2) Diaphorase (EC 1.6.99) Fructose-1,6-diphosphatase (EC 3.1.3.11) Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) Glutamate dehydrogenase (EC 1.4.1.3) Glutamate oxalacetate transaminase (EC 2.6.1.1) Glutamate pyruvate transaminase (EC 2.6. I .2) Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) Glycerate-2-dehydrogenase (EC 1.1.1.29) a-Glycerophosphate dehydrogenase (EC 1.1. I .8) Hexokinase (EC 2.7.1.1) Isocitrate dehydrogenase (EC 1.1.1.42) Lactate dehydrogenase (EC 1. I. 1.27) Malate dehydrogenase (EC 1.1.1.37) Malic enzyme (EC 1.1.1.40) Nucleoside phosphorylase (EC 2.4.2.1) Peptidase (EC 3.4.3.1) Peroxidase (EC 1. II. 1.7) Phosphoglucomutase (EC 2.7.5.1) h-Phosphogluconate dehydrogenase (EC 1.1.1.44) Phosphoglucose isomerase (EC 5.3.1.9) Phosphoglycerate kinase (EC 2.7.2.3) Phosphomannose isomerase (EC 5.3.1.8) Sorbitol dehydrogenase (EC 1.1.1.15) Succinate dehydrogenase (EC 1.3.99.1) Xanthine dehydrogenase (EC 1.2. I .37)

1 1 2 1 1 1 1 1 2 2 2 2 1 1 2 1 1 I 2 2 2 1 1 1 I 1 1 1 1

+ + + ++ ++ + ++ ++ ++ + ++ ++ -

I’ Staining for enzyme identification Shaw (1976).

followed protocols of (1) Allendorf et al. (1977) and (2) Sicilian0 and

184

I-SANG

ET AI,.

FIGS. l-4. Morphological characteristics of cells of IMM during establishment of cell line I-‘MNPIE-l 181. Heterogeneous types of cells grew in early passages. I-IO. Epithelial-like cells (a~ -rows) were present (Fig. I) and contracting myoblasts were abundant (Fig. 21. As the culture\ ht xame established fusiform fibroblast-like cells predominated (Pig. 3). By the 25th passage only the fibtx rblastlike cell type was in evidence (Fig. 4). The bar on all figures = 50 km.

P/o&

CELL

185 (Heat Systems, Plainview, N.Y.) in an ice bath. Samples analyzed in TC and TVB gels were run for 3 hr at 250 V (60 mAlge1). Samples in R and CT gels were run for 5 hr at 250 V (60 mA/gel). Stains for enzymes are referenced in Table 2. All staining chemicals were obtained from Sigma Chemical Company. RESULTS Cell line establishment. In the four original cultures derived from a batch of 1000 eggs, several heterogeneous types of cells were present during the first 10 passages, evidenced by distinct phenotypic differences. These were epithelial-like cells (Fig. l), contractile myoblasts (Fig. 2), and fusiform fibroblasts (Figs. 3, 4). The first two types gradually died out and were no longer apparent after the 10th passage. The fusiform fibroblast-like cells gave rise to the present cell line (Fig. 4), designated as UMN-PIE118 1. Currently (January 28, 1985) this line is in the 83rd passage. Karyotype analysis. At the time of anal-

185

LINE

ysis the cells were polyploid, with chromosome numbers ranging from 56 to 180. In 129 metaphases examined, the distribution of chromosomes was approximately bimodal: in 56% of the metaphases there were 56-92 chromosomes while in 22% there were 134- 152 chromosomes (Fig. 5). The first group is thought to be near diploid. Seeding density optimum. The growth rate (Fig. 6) varied greatly depending on the seeding density. There was little, if any, growth during the 13-day incubation period if the seeding density was 2 x lo5 cells/ flask or less. Population doublings increased progressively with increased seeding densities above 2 x lo6 cells/flask. In the first 8 days there were 1.30 doublings in flasks inoculated with 14.5 x IO’ cells while there was no increase in flasks inocmated with 1.93 x lo5 cells. Serum dependency. During the first 7 days at 5% FBS, the population doubling was 1.69 and at 10% FBS, it was 1.89. There was little or no growth in medium

% 10 9 PASSAGE % 18.20 6 7 z Z6, ul $5 L4 3 2 1

2

0 Lll 50

FIG. 5. mosomes contained mosomes.

60

Primary karyology of per cell is shown for chromosome numbers The remainder of the

C Ii ROMOSOM ES /SPREAD UMN-PIE-1181. The frequency distribution of the number of chrothe 18th and 20th passages. One subpopulation, 56% of the cells, ranging from 56 to 92, while another 22% contained 134-152 chrocells were highly heteroploid.

186

TSANG

ET AL

106 ‘9 6 7 6 5 4 2 Ly

3

“r 22

DAYS

IN

CULTURE

FIG. 6. Relationship of growth rate to seeding density. Growth rate of the cells is shown increasing as the seeding density was increased above a threshold inoculum of 2 x IO? cells/flask. When the seeding density was too low (less than 105) the cultures did not survive.

containing 1% or less of FBS. The minimal effective FBS concentration was not determined (Fig. 7). Zsozyme analysis. The best staining reactions of the cells were for phosphomannose isomerase (PMI), malic enzyme (ME), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), glucose-6-phosphate dehydrogenase (G6PDH), glutamateoxalacetate transaminase (GOT), and lactate dehydrogenase (LDH) (Fig. 8). The adult, pupal, larval, and 4-day-old egg isozyme patterns for PMI, ME, MDH, PGI, and G6PDH were comparable to those of

the cultured cells. Of these enzymes, only MDH was resolved in two staining bands on all buffer systems. These five enzymes may be useful in characterizing a cell line and verifying its identity. Cells from the line showed two GOT bands that were not seen in the tissue samples. LDH appeared as four bands in the cell line, while there was no evidence of it in any of the tissue samples. Five other enzymes were visualized on gels but were poorly resolved and the staining intensity was slight. These poorly resolved enzymes were creatine phosphokinase (1 band), peptidase (1 band), and di-

P/Aitr CELL

LINE

PGI

(TVB) --II-

t 0

DAYS

IN

CULTURE

I GIPDH

FIG. 7. Serum-dependent growth rates of UMNPIE. Growth rate of the cells is shown increasing as the serum concentration was raised.

(TVB) +

m-a--

aphorase (2 bands) on the R buffer system, and adenylate kinase (1 band) and isocitrate dehydrogenase (I band) on the TC buffer system. DISCUSSION

The long period of adaptation generally required in the establishment of insect cell lines was not experienced in this case. Presumably the high seeding density (1-2 x lo6 cells/5 ml) used in initiation of the primary cultures was responsible for immediate establishment. The moth medium UMN-M4 that we have adapted from Grace’s medium seems to have been very favorable for growth. Regular and careful monitoring of cell growth and thus optimum medium changes and subculturing probably contributed significantly to the early and successful initiation of this cell line. A cell line (IAL-PID2) was originated from imaginal wing discs of IMM by Lynn and Oberlander (1983). Although the cells could not be regularly subcultured for the first 14 months, this was nevertheless a breakthrough since it is rare that cells growing out from organ culture ever reach high enough numbers to constitute cell lines. The reported chromosome numbers

LDH

(TC)

i t

1 ICM

FIG. 8. The isozyme patterns identified in cultured cells of IMM are diagrammatically represented. The upper five enzymes gave homologous patterns for all stages of the insect while the lower two enzymes were not observed in tissue extracts. Enzymes: PMI. phosphomannose isomerase; ME, malic enzyme; MDH, malate dehydrogenase; PGI. phosphoglucose isomerase; G6PDH, glucose-6-phosphate dehydrogenase: GOT, glutamate-oxalacetate transaminase; LDH, lactate dehydrogenase. Buffer systems: CT, Clayton-Tretiak; R, Ridgway; TC, Tris-citrate; TVB. Tris-versene-borate.

of the line are more variable and of higher ploidy than the chromosomes in UMNPIE- I 18 I. Isozyme differences also exist. On a pH 7.0 system, IAL-PID2 shows

188

TSANG ET AL.

two malic enzyme bands while UMN-PIE1 I81 shows a single band. Similarly, IALPID2 has two isocitrate dehydrogenase isozymes while UMN-PIEI18 I has but a single, poorly staining band. These discrepancies may be due to culture conditions, adaptation period, or differences in tissue origins. Lactate dehydrogenase activity is strong in our cells but not in tissue extracts. The significance of enzyme activity in cultured cells and comparison with activity in tissue extracts is difficult to assess. Selection of cells with particular functions in the early stages and enzyme induction during prolonged culture may both be operative. The IAL-PID2 line is expected to be useful in physiological investigations such as hormonal control of cell proliferation. Both IAL-PID2 and UMN-PIE-I I81 may be exploited for studies of viruses and other pathogens ofP. interpunctella, an important pest insect. ACKNOWLEDGMENTS These investigations were supported in part by the Minnesota Agricultural Experiment Station. This is Paper No. 13,778 of the Scientific Journal Series.

REFERENCES F. W.. MITCHELL, N.. RYMAN, N.. AND STAHL, G. 1977. Isozyme loci in brown trout (S&IO

ALLENDORF.

/r/ift(~ L.): Detection and interpretation from population data. Here&tr~.s. 86, 179- 190. ARMSTKONG. J. W.. AND SOIIFKSI.KON(;. E. L. 1975. Malathion resistance in some populations of the Indian meal moth infesting dried fruits and tree nuts in California. J. Ecott. Enrottzol., 68, 505-507. CLA’~.TON. J. W.. AND TKETIAK. D. N. 1972. Aminecitrate buffers for pH control in starch gel electrophoresis. +l. Fish. RPS. Bmwd Crttrctd.. 29, l1691172. HUNTER. D. K.. COLLIEK. S. S.. ANI) HOFFMAN. D. F. 1977. Granulosis virus of the Indian meal moth as a protectant for stored inshell almonds. J. Ec,on. Entotnol.. 70, 493-494. LYNN. D. E.. AND OBEKLANDER.

H. 1983. The establishment of cell lines from imaginal wing discs of Spodopteru~tcgiperdrl and Plodia it~~erptmctellrr. J. Insect

Physiol..

29, 591-596.

RIDGWAY. G. J., SHERBLIRNE. S. W., AND LEWIS. R. D. 1970. Polymorphisms in the esterases of Atlantic herring. Tram. Amer. Fish. Sm.. 99, 147-151. SELANDER. R. K.. SMITH. M. H.. YANG. S. Y., JOHNSON, W. E.. ANDGENTRY. J. B. 1971. Biochemical polymorphism and systematics in the genus Perotnyscrrs. 1. Variation in the old field mouse (Perotnysctts polionofus). Itt “Studies in Genetics VI.” pp. 49-90. Univ. Texas Publ. 7103, Austin. Texas. SICILIANO. M. J., AND SHAW. C. R. 1976. Separation and visualization of enzymes on gels. In “Chromatogrdphic & Electrophoretic Techniques. Vol. 2, Zone Electrophoresis” (I. Smith, ed.). pp. 185-209. William Heinemann Medical Books. London. ZETTLER.

J. L.,

MCDONALD,

L. L..

REDLINGEK,

L. M.. AND JONES. R. D. 1973. Plodia iwierpwzctella and Cadra c,crlctellaresistance in strains to malathion and synergized pyrethrins. J. Eron. Ent~tn~~l., 66, 1049- 10.50.