JOURNAL OF INVERTEBRATE PATHOLOGY 52,268-274 (1988)

Establishment of Cell Lines from Pieris rapae Embryos: Characterization and Susceptibility to Baculoviruses

KATHLEEN G. DWYER

Boyce Thompson Institute for Plant Research at Cornell University, Tower Road, Ithaca,New York 14853

SUSAN E. WEBB AND ANTHONY M. SHELTON

Entomology Department, New York State Agricultural Experiment Station, Geneva, New York 14456

AND

ROBERT R. GRANADOS

Boyce Thompson Institute for Plant Research at Cornell University, Tower Road, Ithaca New York 14853

Received October 15, 1987; accepted February 28, 1988

Many embryonic Pieris rapae (imported cabbageworm) cell lines were established from 24-hr-old P. rapae eggs. Some were characterized by growth curve analysis and by malate and lactate dehydrogenase isozyme comparison with other lepidopteran and mosquito cell lines. Most of the cell lines were screened for susceptibility to P. rapae granulosis virus using the peroxidase antiperoxidase assay and DNA slot blot hybridization with negative results. Inoculation of some cell lines with tissue culture-derived budded virus from Autographa californica nuclear polyhe- drosis virus (AcMNPV) had a toxic effect on most of the cells. AcMNPV-inoculated cell lines lysed within 24 h, but there was no evidence of virus replication. The Trichoplusia nis NPV did not replicate in or cause lysis of inoculated cells. o 1988 Academic press, IX.

KEY WORDS: Pieris rapae; embryonic cell lines; baculovirus in vitro replication.

INTRODUCTION

The imported cabbageworm, Pieris ra- pae, is a serious agricultural pest of crucif- erous crops. The baculovirus, P. rapae granulosis virus (PrGV) which infects this insect could be developed as a biocontrol agent for this pest. As such it would offer obvious advantages over chemical pesti- cides in its narrow host range and safety for nontarget organisms. Such development would require knowledge of its molecular biology and its genetic manipulation. Using larvae as a source of viral DNA, RNA, and protein, we have made some progress in the study of PrGV molecular biology (Dwyer and Granados, 1987a,b) but further work and genetic manipulation necessitates es- tablishment of a cell culture system which would replicate the virus in vitro.

Miltenburger et al. (1984) and Naser et al. (1984) have established several cell lines derived from Cydiu pomonella (Cp) eggs

that were susceptible to CpGV infection. Using the same methods, we have estab- lished many Trichoplusia ni (Tn; cabbage looper) and P. rapae primary cell lines. Our work concerning the T. ni primary cell lines and their susceptibility to TnGV and T. ni nuclear polyhedrosis virus (TnSNPV) has been previously reported (Granados et al., 1986). In this paper, we describe some characteristics of the P. rupae primary cell lines, i.e., process of establishment, growth curves, isozyme analysis, and their re- sponse to inoculation with PrGV and other baculoviruses, Autographa californica multiple nuclear polyhedrosis virus (AcM- NPV), and TnSNPV.

MATERIALS AND METHODS

Primary cell culture. P. rapae primary cell lines were established with the methods of Miltenburger et al. (1984). Briefly, 24- hr-old P. rapae eggs, which had been col-

0022-201 Ii88 $1.50 Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

268

Pieris rupne EMBRYONIC CELL LINES 269

lected on Parafilm, were surface sterilized in 2% Chlorox for 10 min, 70% ethanol for 4 min, and rinsed in GTClOO medium (Gra- nados et al., 1986). These eggs were crushed through a sterile 100~pm sieve with a rubber policeman, and the resulting em- bryonic cells and cell aggregates were col- lected in fresh GTClOO medium with 100 kg/ml of gentamicin sulfate (Sigma). Corn- ing tissue culture flasks (25 cm3) were seeded with the equivalent of 300 to 400 eggs in 5 ml of medium and incubated at 28°C. Subsequent subculturing was accom- plished using a rubber policeman to detach cells from the flask so that all cell types could be passed on to daughter flasks.

Determination of growth curve and pop- ulation doubling time. For every cell line analyzed, 16 flasks were seeded with 1 x

lo6 cells/5 ml and incubated at 28°C. Using two flasks, 10 independent cell counts were taken daily with a Neubauer hemocytome- ter for the next 8 days. Growth was seen as the number of cells per milliliter plotted against time. Population doubling time was calculated using the exponential formula described by Hayflick (1973).

Isozyme analysis. A T-25 flask of log phase cells was prepared for isozyme anal- ysis by pelleting the cells at 900g for 5 min, resuspending in 50 t.~l of cell lysis buffer (0.15 M Tris-HCl, pH 7.1, 46 mM citric acid, 10% sucrose, 1% Triton X-100, and 0.02 mM brophenol blue), and lysing with a sonicator. The mixture was microfuged for 2 min and the supernatant stored in aliquots at - 100°C until needed. For sample protein preparation, 4.75%/0.25% acrylamidel bisacrylamide 0.75-mm vertical gels in buffer 39 mM Tris-HCl, pH 7.1, 8.5 mM sodium citrate were used. Five to 20 ~1 of lysate were loaded onto each well, and the samples were separated by electrophoresis for 1000 Vh. Staining for the malate dehy- drogenase (MDH) and lactate dehydroge- nase (LDH) isozymes was done as de- scribed in Steiner and Joslyn (1979) and Shaw and Prasad (1970), respectively.

Inoculation of P. rapae cell lines with

PrGV, TnSNPV, and AcMNPV. PrGV and TnSNPV were obtained from Robert Jacques (Res. Station Agric., Harrow, On- tario, Canada) and M. Semel (Cornell Uni- versity), respectively. PrGV inoculum was made in the following way. P. rapae larvae, reared on a high wheat germ diet (Bell et al., 1979) in 16-0~ Styrofoam cups, were in- fected with PrGV by surface contamination of the diet with lo9 OBs when larvae were in their late fourth instar. Three days later, the hemolymph of 30 larvae was collected by cutting a proleg and bleeding into 10 ml of GTC 100 containing 100 pg/ml gentamicin sulfate and 1 mM cysteine. The mixture was centrifuged for 15 min at 2000g and the cell- free supernatant reserved. This inoculum was infectious when injected into healthy P. rapae larvae. The inoculum was either used immediately or stored at - 100°C.

AcMNPV and TnSNPV inocula were made from cell cultures. TN368 and BTI- TNSBI cell cultures inoculated with AcM- NPV and TnSNPV infectious hemolymph, respectively, resulted in infected cells. The cells were pelleted and the supernatant used to inoculate fresh cell cultures again. The resulting second passage infectious su- pernatant was used to inoculate P. rapae cell lines. The AcMNPV infectious super- natant was further fractionated by pelleting most of the budded virus (BV) in a mi- crofuge for 30 min at 4°C and filtering the supernatant through a 0.22~p,rn filter twice. The BV pellet was resuspended in the orig- inal volume of inoculum. TN368 spent me- dium was obtained from confluent TN368 cell cultures as the supernatant after the cells were pelleted.

P. rapae cell lines to be tested for virus susceptibility were grown in 24-well plates. When the cells were in log phase of growth, the medium was removed, 0.5 ml of inocu- lum was added, and the cells were kept at 28°C. After rocking for 4 hr, 0.5 ml of GTClOO medium was added to all cell lines inoculated with PrGV. After rocking for 1 hr, the inoculum of TN368 spent medium, AcMNPV, and TnSNPV was removed

270 DWYER ET AL.

from P. rupae cell lines, and 1 ml of fresh GTC 100 medium was added.

Assays for PrGV infection of P. rapae primary cell lines. Two methods were used to determine PrGV replication in P. rupae primary cell lines. First, both healthy and PrGV inoculated cell lines were immuno- logically stained by the peroxidase- antiperoxidase (PAP) procedure (Volkman and Goldsmith, 1982) using a polyclonal an- tiserum raised in rabbits against PrGV oc- cluded virus (OV) in this laboratory at a l/200 dilution. Second, DNA slot blot hy- bridization done as described by Burand and Wood (1986) was used for detection of any PrGV DNA synthesis in the PrGV in- oculated cell lines. The cell lines were as- sayed 2 weeks postinoculation.

RESULTS

Primary cell line. From 1984 and 1985, we seeded 75 flasks with the embryonic cells and cell aggregates derived from 24- hr-old P. rapae eggs. Within 2 weeks, the cell aggregates had attached to the surface of the flask. Often, these aggregates would contract, suggesting the presence of mus- cular or nerve tissue. After attachment, half of the medium was replaced with fresh me- dium every week. Within the first 6 weeks, multicellular fibers, radiating from these growing aggregates, would form a net-like structure over the entire bottom of the flask. Late in this process, smaller, distinc- tive cells of many different morphologies would appear attached to and underneath the fibers. Some various morphologies were epithelial-like cells, fibroblast-like cells, highly refractive smaller cells, large cells containing light or dark vacuoles, and strongly attached cells with indistinct cell borders. The first subculture was made when the fibrous network had begun to de- tach from the flask. The contracting fibers did not subculture well, but the smaller cells thrived. The morphologically different types of smaller cells that would predomi- nate varied between daughter flasks and continued to do so with the next few sub-

cultures. Subsequently, we acquired over 250 sublines with different cell populations ready to be tested for virus susceptibility.

Most of the cell lines which were not pos- itive for PrGV replication in vitro were dis- continued. At present, we are maintaining five morphologically different cell lines, BTI-PR8A1, BTI-PR8A2, BTI-PR9A, BTI- PRlOB, and NYAES-PR4A, which repre- sent the most stable of the primary cell lines. (Table 1). Most of these sublines have been subcultured more than 100 times.

Cell lines BTI-PR8Al and BTI-PRlOB were further characterized by growth curve analysis. The growth curve experiment was done in the 62nd and 56th subculture of these sublines, respectively, when they were both being subcultured at a I: 10 dilu- tion weekly. Growth curves for both cell lines (Fig. 1) showed a lag phase of 3.5 to 4 days followed by exponential growth until day 7, whereupon cell population growth plateaued. The population doubling time for cell lines BTI-PR8Al and BTI-PRlOB were calculated to be 41 and 40 hr, respec- tively.

The MDH and LDH isozymes of five P. rupae primary cell lines, BTI-PR8A, BTI- PR8B, BTI-PR9A, BTI-PRIOA, and BTI- PRlOB were compared with those of cell

TABLE 1 MORPHOLOGICAL CHARACTERISTICS OF SELECTED

ESTABLISHED Pieris rapae EMBRYONIC CELL LINES

Cell line designation Morphological description

BTI-PR8Al Loosely attached, small fibroblast-shaped cells that grow in clumps when concentrated

BTI-PRSA2 Firmly attached, small epithelial-like shaped cells growing in a smooth monolayer

BTI-PR9A Firmly attached, small fibroblast-shaped cells growing in a monolayer

BTI-PRlOB Very loosely attached, small, rounded refractive cells detaching when concentrated

NYAES-PR4A Firmly attached, long, forming net-like monolayer

Pieris rqne EMBRYONIC CELL LINES 271

DAYS IN CULTURE

FIG. 1. Growth curves for Pieric rapae embryonic cell lines BTI-PRIAl (0) and BTI-PRlOB (0). Each point is the average of 10 cell counts made from two flasks daily.

lines from lepidopterans Estigmene acrea (salt marsh caterpillar) BTI-EAA (Grana- dos, unpubl.), Heliothis zea (cotton boll- worm) IPLB-1075 (Goodwin et al., 1982), T. ni TN368 (Hink, 1970) BTI-TN52V2P, BTI-TNSBI, BTI-TNSB14 (Granados et al., 1986), and mosquitoes Aedes aegypti Aa and Aedes albopictus Aap (Singh, 1967). The mobility of the lepidopteran isozyme bands relative to the mobility of the mos- quito bands (Table 2) showed no difference between any of the intraspecies cell lines; i.e., all five P. rupae primary cell lines had indistinguishable comigrating isozymes. However, it can be seen that the mobility of MDH and LDH isozymes, when compared

TABLE 2 MOBILITY OF LEPIDOPTERAN ISOZYME BANDS

RELATIVE TO THE MOBILITY OF MOSQUITOS

ISOZYME BANDS~

Cell line Malate Lactate designation dehydrogenase dehydrogenase

BTI-EAA 0.66 0.69

IPLB-1075 0.60

TN368 0.25 BTI-TN52V2P 0.25 BTI-TNSBl 0.25 BTI-TNSBIS 0.25 BTI-TN5B14 0.25

BTI-PRSA BTI-PRSB BTI-PR9A BTI-PRlOA BTI-PRIOB

0.57 0.57 0.57 0.57 0.57

0.90

0.47 0.47 0.47 0.47 0.47

0.31 0.31 0.31 0.31 0.31

0 Results are the average of two separate experiments.

between species, were clearly different. These MDH and LDH isozymes are char- acteristically distinct for every species an- alyzed. The P. rapae primary cell lines have clearly different origins from any of the other lepidopteran cell lines carried in this laboratory.

Screening of P. rapae primary cell lines for PrGV susceptibility. Over 150 P. rapae primary sublines were inoculated with PrGV infectious hemolymph and analyzed for PrGV replication by light microscopy and electron microscopy (EM), immuno- logical staining via the PAP assay, and DNA slot blot hybridization. Cytopathic ef- fects were not observed in any of the cell lines tested. By DNA slot blot hybridiza- tion, the DNA of the PrGV inoculated cell lines were all found to contain less PrGV DNA than was contained in the original in- oculum (data not shown). The PAP assay revealed some of the PrGV inoculated cell lines to include positively stained cells that were not seen in their healthy counterparts. These cells were always a minor portion of the total population, 5% or less. In the case of some cell lines, for example, BTI- TNlOA, PAP stainings were performed upon several different subcultures. Early in its adaptation to tissue culture, this cell line had a significant amount of PrGV suscepti- ble cells. It is difficult to make an estimate on the percentage because these cells were distributed heterogeneously throughout the flask, reflective of colony growth. Within the next live subcultures, the PrGV suscep- tible cells could no longer be detected. In- deed, we found from these assays that ini- tially the primary cell lines contained PrGV susceptible cells that were subsequently lost after further subculturing.

Response of P. rapae primary cell lines to TnSNPV and AcMNPV inoculation. BTI- PR8Al and BTI-PRlOB were inoculated with second passage TnSNPV and AcM- NPV infectious cell culture supernatant. The two cell lines were not affected by TnSNPV, but their response to AcMNPV inoculum was very interesting. Within 2

272 DWYER ET AL.

days, over 90% of the cells in each flask were lysed (Fig. 2). There were no classical signs of AcMNPV replication seen either by light or electron microscopy before lysis occurred. The remaining cells survived and grew. These cell lines were not affected when incubated with spent media from TN368 cells (medium taken from confluent TN368 cell cultures), indicating lysis to be the result of the viral inoculum. To deter- mine if the lysis factor was associated with AcMNPV BV physically or was due to some other protein in the inoculum, AcM- NPV BV in the inoculum was pelleted in the microfuge and the pellet resuspended in the original volume with fresh medium. The supernatant was filtered through a 0.22~p,rn Millipore filter twice. Both fractions were used as P. rupae cell inoculum. The super- natant fraction had no effect, but the AcM-

NPV BV fraction lysed the P. rapae cells. Also, we found that a AcMNPV infectious cell culture supernatant of higher titer would lyse P. rapae cells more effectively than a lower titer inoculum. Thus, AcM- NPV BV will lyse most P. rupae cells in cultures of BTI-PRSAl and BTI-PRlOB. P. rapae cells in these cell lines which were not lysed following the initial virus inocula- tion evolved into cell lines that were more resistant to lysis by AcMNPV with subse- quent exposure.

DISCUSSION

In this paper, we report the establish- ment of many P. rupae embryonic cell lines derived from crushed 24-hr-old P. rapae eggs and sustained in GTClOO medium. The growth kinetics and population doubling time for two of these cell lines were char-

FIG. 2. Light micrograph of healthy cells from Pieris rapae primary cell lines BTI-PRSAI (A) and BTI-PRlOB (B). BTI-PR8Al (C) and BTI-PRlOB (D) cells 48 hr postinoculation with second passage AcMNPV infectious cell culture supematant. The bar in (A) = 50 pm.

Pieris rapae EMBRYONIC CELL LINES 273

acterized. The MDH and LDH isozymes of five of these P. rupae cell lines were com- pared and found to be different from those of other lepidopteran and mosquito cell cul- tures. At present, five morphologically dif- ferent cell lines, BTI-PR8A1, BTI-PRSA2, BTI-PR9A, BTI-PRlOB, and NYAES- PR4A, are being maintained.

When screening these P. rapae embry- onic cell lines for PrGV susceptible cells with the PAP assay and/or DNA slot blot hybridization, we did detect PrGV permis- sive cell types in some of the early passage sublines. Continued screening revealed that these GV permissive cell types became less abundant as the cell lines were passaged. PrGV replication in larvae is tissue specific. Indeed, Miltenburger et al. (1984) suggest that the cellular substrate is very important to the success of GV in vitro replication. By using embryos as a source of cell cultures, we hoped to acquire cell types or their pro- genitors, which would have the tissue “factors” necessary for PrGV in vitro rep- lication, but which would also be able to grow continuously in cell culture. In our primary cell cultures, these susceptible cell types were lost upon passage. Perhaps these cells continued to differentiate and became unadaptable to cell culture. Alter- natively, they may have been out-competed by other nonsusceptible cell types present which are more adaptable and/or faster growing. Survival of the susceptible cell types might be improved by altering the cell culture medium or by cloning out the de- sired cell types from the nonsusceptible cell types.

We discovered that second passage Ac- MNPV infectious cell culture supernatant lysed most of the P. rupae cells in cell lines BTI-PR8Al and BTI-PRlOB. We further found that this lysing activity was associ- ated with the AcMNPV BV. Two possibil- ities are (1) that there is a viral structural protein associated with the AcMNPV BV which is toxic to most P. rupae cells, or (2) that AcMNPV BV commences an infection

in most P. rupae cells which has toxic ef- fects upon the cells, but does not progress far enough to produce classic cytopathic ef- fect, progeny virus, or occlusion bodies. UV inactivation of inoculating AcMNPV BV might resolve these different possibili- ties.

ACKNOWLEDGMENTS

We thank Guo-xun Li, Ana Arevalo, William T. Wilsey, and Anja Derksen for excellent technical as- sistance. This work was supported by a grant from the Cornell Biotechnology Program which is supported by the New York Science and Technology Foundation and a consortium of industries.

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