Indian Journal of Experimental Biology Vol. 38, December 2000, pp. 1201-1206

A new continuous cell line from larval hemocytes of Spodoptera litura (F.)

U Pant , S S Athavale & V C Vipat

National Inst itute of Virology, 20 A, Dr. Ambedkar Road, Pune 411 00 I, Indi a

Received 3 November /999; revised 20 September 2000

A new cell line has been established fro''' 1 larval hemocytes of the moth, S. filum (tobacco cut worm). It took 147 days to form a mono layer and one year for the first 17 passages. At present, the culture is at 861h passage level and is designated NIV -SU-I 095. Three cell types could be distinguished , viz. plasmatocytes (53%), prohemocytes (36%) and granul ar hemo­cytes ( II %). The chromosome number was very high, 74% metaphase cells showed more than 100 chromosomes. The cells could be cryopreserved. The cells were susceptible to the baculoviruses, AUlogmpha ca fifornica nuclear polyhedrosis virus and S. filUm nuclear polyhedrosis viru s (S LNPV). Plaques could be observed on 71h post infection day with SLNPV. Si x cloned cell lines have been developed of which clone 11-1 F was more sensitive to both the baculoviruses compared to the original cell line.

Lepidopteran cell cultures have become invaluable for biotechnological research with the recent devel­opment of baculovirus expression vector system'. Several foreign genes have been expressed in lepi­dopteran cell lines during the last 15 years2-7 . Lately, recombinant baculoviruses are also being constructed as bioinsecticidesR and efforts are being made to mass cultivate these in lepidopteran cell lines9

. Hence there is a need for continuous establishment of several new, and well characterized lepidopteran cell lines suscep­tible to baculoviruses.

Spodoptera lilura (tobacco cut worm; Lepidoptera : Noctuidae) is an important polyphagous agricultural pest in India and efforts are being made to control this insect using baculoviruses as bioinsecticides.

Establishment and characterization of a new cell line from the larval hemocytes of S. litura moth, in­cluding its susceptibility to baculoviruses are reported in this communication.

Materials and Methods Initiation of cel! line-S. litura larvae were co l­

lected from Bhor, a vill age near Pune in Maharashtra

state. The field collected larvae UIld the moths that

emerged from them were identified as S. lilura by

scienti sts of the Entomology Div ision, The College of

Agriculture, Pune. These larvae were reared in the laboratory and fed on castor leaves. The procedure

follo wed for initiating the primary culture was as de­scri bed by Sohi 10 with slight modifications. In short,

the larvae were washed thoroughly under tap water

followed by two quick washes with 2.5% sodium hy­

pochlorite solution and immersion for 5 min in thi s

solution. The larvae were then washed twice with

sterile glass distilled water and kept in a sterile petri

dish lined with blotting paper. The proleg of the larva

was cut with a sharp scissor and the hemolymph was

allowed to drop direct ly into the culture vessel (Nunc,

50 ml) containing 3.5 ml of the culture medium. The culture medium was Grace's insect tissue culture me­

dium" (GM)-modified by the addition of lactalbumin

hydrolysate (0.3%; Difco) and yeastolate (0.3 %; Difco). Penicillin (50 fl/ml ), streptomycin ( 100 Ilg/ml) and gentamyein (40 Ilg/ml) were from HA,

Pune, were routinely added to the medium. The me­

dium was supplemented with fetal bovine serum

(FBS) at 20% concentration which was reduced to

10% after 10lh passage. While initiating the cultures, GM contained glutathione (I Ilg/ml)' 2 and Cysteine

(340 Ilg/ml)", both from Nutritional Biochemicals, to prevent melanization . Late r, (passage five onwards)

both these ingredients were deleted from the culture

medium because melanization does not occur in the

culture once it gets adapted to in vitro conditions.

Each culture bottle contained approximately 0.5 ml of

hemolymph from five larvae. The culture was left

undisturbed for 10-15 min when most of the hemo­cytes got attached to the culture vessel. The medium ,

including few unattached ce ll s, was then removed ; the culture was rinsed thrice wi th GM lacking FBS and was fed with fresh GM supplemented with 20%

1202 INDI AN J EXP BIOL. DECEMBER 2000

FBS . T he culture was incubated stati onary at 28°C.

At 7- 10 day interva l, the culture was fed by removing

50% of the spent medium and repleni shing with fres h

culture medium. Subcltlti vation-Cell monolayer could be eas il y

d ispersed w ith a Pasteur pipette; a split-rati o of I: 10 was fo ll owed at 7-1 0 day interval and the inoculum per culture bottl e was 0.4 milli on ce ll s/ ml.

Hemocyte preparation from f resh hemolymph­The proleg of a 41h instar larva of S. tituret was cut and the hemolymph co ll ected in 2-3 ml of GM containing 10% FBS and a few crystals of cys te ine. T he mixture was properl y shaken and 1-2 drops were taken on several c lean g lass s lides and a ll owed to stand fo r 30 mi n . When the hemocytes sett led on the glass sur­face, the slides were was hed with Rina ld ini 's sa lt so­lution (RSS) and fi xed in methanol for 15 min . The ce ll s were then stained with Gi emsa ( I : 10 di luti on in dis ti lled water) for 10 min, d ifferentiated briefly in g lass-d ist illed water, dehydrated in two changes of acetone, c leared in xy lene and mounted in DePex.

Morphological studies-The cul ture was grown in Le ighton tubes w ith covers lips. 3-4 days later the covers lips were removed, washed wi th RSS , and sta ined with Giemsa as mentioned above.

Karyological studies-Th ese stud ies were con­ducted as described earlier '4 . In short , 3 day o ld cul­

ture was g iven co lc hec ine ( I 00 ~g/ml ) treatment fo r 16 hr ; then hypotone treatment wi th d isti li ed water fo r 4-5 min and then stained with Giemsa ( I : 10) for 8-10 min . Hundred metaphase spreads were studied fo r frequency estimati on. Karyo logical study was conducted at passage 14 le ve l o nl y and not at higher passage leve ls or of cloned cell lines.

Cloning- (a) Plating efficiency: Before c loning the cell line, the pl ating effici ency of the culture was de­termined in fres h and conditioned medium using 50 mm Petri di shes. Conditioned medium was prepared by growing the culture in fresh medium for 4 days. The medium was removed, spun for 10 min at 1,000 rpm, filtered and kept at 4°C. The pl ating effi c ie ncy of NIV -SU-I 095 was found to be 12.6% in condi ­tioned medium; in fresh medium it was less than 1% .

(b) Cloning experiments: Using dilution plating method and conditi oned medium (gave better pl ating effi c iency) the ce ll s were cloned. The ex periment was conducted in 96 we ll pl ate (Nunc) by inocul ating about 10 cell s per we ll. Around 2 1 days later, onl y those c lones were picked up from well s showing a sing le co lony of ce ll s. In thi s way I I c lones were ob-

tained from two different ex periments. T hese were maintained for 8 subcultures. Later, on the bas is of ce ll morphol ogy, healthy appearance and steady growth of ce ll s, six c loned cell lines were selected . These were:-

I . r - I H 4. n - 8G 2. r - 7G 5. II - IF 3. I- li E 6. II - 6D

These c loned cell li nes were ma inta ined and the ir susceptibi lity to two bacul oviruses was determi ned.

Virus studies-Ex trace llul ar virus (ECY) of Auro­grapha californica nuc lear poly hedros is virus (AcNPY) and S. fitu ra NPY (S LNPY) was used to infect the ce ll cultures as described earlier 's and the NPY were counted in a hemocytomete r after son i­cating the ce ll s. Stock of AcNPY (ECY) was pre­pared in commercia ll y ava il able S. f m giperda (Sf-9) cell culture. T he ce ll s were grown in Nu nc plas ti c bott les (600 ml) containi ng 20 ml GM supp lemented with 10% FBS. On the 3rd day the culture med iu m was removed and the cu ltures were in fected with 2 ml of AcNPY (ECY) (obta ined initi ally from Biochem­istry Di vis ion, NIV). Afte r I hr of adsorption the cultures were fed with the growth medium and incu­bated stat ionary at 2,:"C. On the 5th post-infection day (PID) the ti ssue culture flui d (TCF) was har­

ves ted , Mill ipore fil te red (0.22 ~lm) and stored in small a liquots at -70°C.

The po lyhedra of SLNPY were fed to S. fitura lar­vae and infected hemo lympi. 01 s ick larvae contai ning SLNPY (ECY) was co llected and used to infec t a susceptible S. litura (larva l ovary) cell line (NIV-SU-992)16. The infected TCF was used to prepare stock of SLNPY (ECY) in the same cell line as described above .

Pl aque studies were conducted as desc ribed by Hink' 7.

Results and Discussion A new indi genous ce ll line has been established

from larval hemocytes of S. litura . The culture at present is at 861h passage level and can now be called an establi shed cell line . It is des ignated NIY -SU-1095 . Several primary cultures from larva l hemocytes were ini tiated us ing diffe rent nu mber of larvae per culture vesse l but the cul tures were lost e ither due to darkening of the medium (me lani zati on) or due to contaminati on. F inall y, 15 healthy 4111 instar larvae were used to prepare three primary cultures of hemo­cytes. O f these, one of the cultu res gave ri se to the

PANT el al. : SPODOPTERA LARVAL HEMOCYTE CELL LINE 1203

established cell line. The sequence of events was as follows;- 147 days after initiation, a monolayer of cells had formed in the culture vessel and the I st pas­sage was made. From then on the culture was either fed or subcultured as and when good cell growth was observed. It took more than one year for the first 17 passages. Cell s grew loosely attached to culture bot­tle. Growth of cells in plastic bottles was much better than glass bottles. Initially , at the time of subcultur­ing, the split-ratio followed was 1:2 but became 1:5 by 171h passage level and I : 10 by 77lh passage level .

Majority of the lepidopteran cell lines reported in the world have been developed from embryos, ovaries (adult, pupae, larvae), hemocytes and neonate larvae,

\

•

Fig. 1-5. filum culture from larval hemocytes, NIV-SU-1095

of these the largest number is from neonate larvae '. Although cell lines have been established from hemo­cytes, melanization and its toxic effects cause serious problem in culturing hemocytes. We minimized mel­anization by adding glutathione and cysteine in the medium at early passages of the culture and by usi ng only 5 larvae per culture vessel.

Three cell types were observed in the culture, (Fig. I a), majority of them were plasmatocytes (53 %), followed by prohemocytes (36%) and granular hemo­cytes (II %). The plasmatocytes were of varied, ir­regular shapes, the cytoplasm and nucleus took up light stain. Some of the plasmatocytes were large, multinucleated and laden with phagocytic inclusions.

a) Cell sheet at passage 3, showi ng plasmatocytcs ( I), prohemocytes (2) (shown by arrows & numbers). Giemsa stained (400 x) b) Metaphase chromosomes. Giemsa stained (2,000x) c) Culture infected wi th the baculovirus, SLNPV , on 20th post infecti on day, showing one cell fi lled with polyhedra (shown by arrow). Living (400 x ) d) Pl aques observed with SLNPV infection at 10-4 d ilution of the virus on 7th post infection day

1204 INDIAN J EX P BIOL. DECEMBER 2000

Plasmatocytes could be further differentiated into 3 major types; elongated, large irregular and large phagocytic. (Table I). Prohemocytes were small round cells, which took up deep stain, the nucleus filled almost entire space of the cell. The granular hemocytes were round in shape, the nucleus being half the size of the cytoplasm, the ce ll s took up light stain , showing granules in the cytoplasm.

To determine the cell types that had got established in in vitro condition, these were compared with he­mocytes present in fresh larval hemolymph prepara­tions (Table I). Most of the cel ls in fresh hemolymph preparations (Fig. 2) had a single nucleus, phagocyti c plasmatocytes were not seen, majority of the cell population was granu lar hemocytes (55%) and oeno­cytoids were I %. In NIV -SU-I 095 ce ll culture there was predominance of plasmatocytes (53%), several of them were multinucleated showing phagocytic inclu­sions; oenocytoids were absent and majority of the cells were very much enlarged in size compared to fresh hemocytes. Such observations have also been made in Mal({cosoll7a disstria hemocyte cultures's.

The growth curve of the cell line was determined at passages 15, 56 and 86 by counting only viable cell s (Fig. 3) . The procedure followed was as described earlier'6. At passage 15 the logarithmic phase of growth started fro m day 3 and continued up to day 7. The maximum population of 4.8 million cells/ ml was observed on day 10 at passage 15 , however, growth rate increased with passages and 3.5 to 4 milli on cell s/ ml were observed on i h day itself at higher pas­sages. It was also observed that at h igher passages the number of viable cells started falling from day 7, thi s was however not observed at earl y passages of the culture. Similar observations have also been made by us with the commercially available S. Jrugiperda cloned culture, Sf-9 (unpublished data) .

Cells at passage 14, showed numerous chromo­somes, very often too large in number to be counted (Fig. I b). The chromosomes were small , round with

diffuse centromere i.e. typical lepidopteran chromo­somes. 74% cell s showed more than 100 chromo­somes; the smallest chromosome number cou nted was 24 (Fig. 4) . The chromosome number of S. fitum male larval gonads was also determined; 100 meta­phase spreads were observed. 6 1 % of the metaphase cell s showed 30 chromosomes, whereas the remaining ceil s had 28-3 1 chromosomes.

The culture cou ld be stored and rev i ved success­fully from liquid nitrogen. The culture coul d remain viable for 15 days at 28"C but the via ble cell coun t went down drastically. At 13"C the cul ture cou ld re­main viable for about a month.

The culture was susceptib le to both the baculovi­ruses, AcNPV and SLNPV (Fig. Ic) . At passage 59, the culture was infected with SLNPV. On 6th pm 3 1 % cell s were found infected. Cel ls were well at­tached till 71h PID without apparent cytopath ic effect and the NPV count was 2 million/ml. The culture,

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'. t 1-

Fig. 2-Fresh hemol ymph preparati on of 5. filum larva showing plasmatocyte ( I), prohemocytes (2), granul ar hemocytes (3) and oenocytoids (4) (shown by arrows and numbers). Giemsa stained (400x)

Table 1-5. filllm cell types, their size and number as observed inNIV -SU- I 095 cell cu lture and fresh hemol ymph preparations

Cell types /11 vilro /11 vivo

% cell size (l-lIn) nucleus size (J..llll ) % cell size (~ Ill ) nucleus size (J..lm) Plaslllatocytes

Elongated 2S 66- 105 12-24 5 45-75 9 Large irregular IS 50-55 27 20 45-60 12-15 Large phagocytic 7 ISO- ISO 24-30 NIL

Prohelllocytes 36 21 IS- IS 19 10-12 9 Granular helllocytes \I 35 15 55 10 6 Oenoc~loids NIL 40-45 9-12

PANT el al. : SPODOPTERA LARVAL HEMOCYTE CELL LI NE 1205

6

- . - P 15 -+-P56 -P86

5

4

E 3 --if) ill II 2 c 0

~

3 4 5 6 7

No. of days

Fig. 3-Growth curve of S. lililra cell line (N IV -SU-I 095 ) at passages 15, 56 and 86 (viable cells only)

also at passage 59, was infected with AcNPV, on 6th 80..----------------,-

PID, 15 % cell s were found infected, no CPE was ob­served and the NPV count was 3 million/ml on 7th PID. Thus, although the number of cell s infected with AcNPV was less than SLNPV, the number of pol yhe­dra produced per cell was much hi gher compared to the homologous virus SLNPV.

The culture was not susceptible to B. mor; NPV, H. armigera NPV and potato tuber moth granulosis vi-rus.

It was observed that when the original culture was infected with AcNPV/SLNPV certain cell types showed earl y appearance and hi gher number of poly­hedra (Fig. Ie). Hence cloning was undertaken to ob­tain a cell strain that would give high yield of NPV in a shorter time. Six cloned cell lines were obtained having different percentages of the cell types. Clone II-IF (P-7) had 97% plasmatocytes majority being elongated cell s; 3% prohemocytes; and granular he­mocytes were absent. Thus, clone II-IF had higher percentage of plasmatocytes and low percentage of prohemocytes compared to the original culture. The susceptibility of these 6 cloned cell lines, the original cell line (NIV-SU-I095, P-62) and Sf-9 cell line (commercially available) to SLNPV was determined (Tab le 2 ). The cloned cell line IT-IF (P-21 ) was found to be more susceptible to SLNPV than the original ce ll line and almost equal in susceptibility to Sf-9 culture.

The above experiment was repeated using AcNPV . (Table 2) . It was observed that the same clone II- I F (P-27) was much more susceptible than NIV -SU-I 095 (P-67) and also Sf-9 culture. The NPV count was 4 .1

70

~ 60 QJ U

'0 50 QJ

~ 40 C QJ 30 ~

~ 20

10

Below 50 Between 50-100 Above 100

Range of chromosome nu mber

Fig. 4-Hi stogram of S. lilllret cell line (N IV-SU- 1095) showing distriblilion of chromosomes at passage 14.

Table 2-Susceptibility of NIV -SU-I 095 cell line, it 's six cloned cell lines and Sf-9 culture to the bacu lovirus SLNPV and Ac PV

Cell lines Million NPV/ml (PID -7) SLNPV AcNPV

NIV-SU-1095 1.0 0.2 Sf-9 3.0 2.2 I- IH 0.2 0.6 1-7G O.S 1.6 I-liE 2. 1 2.3 11 -60 1.1 I.S II -I F 2.5 4. 1 11-8G 1.4 1.3

million/ml on 7th PID in Il- I F culture compared to 0.2 and 2.2 million NPV/ml in the other two cell lines respecti vel y.

It should be noted that the results in one experi­ment are comparable but the results of different ex­periments performed at different periods with differ-

1206 INDIA N J EXP BIOL, DECEMBER 2000

ent passage leve ls of cells and of viruses are not nec­essari Iy comparable.

To determine whether the culture could be used for the titration of SLNPV, plaque assay was performed. After several tria ls, when the culture (P-74) was in­fected with SLNPV, on i h PID di stinct plaques were observed in 10.3 and 10-4 dilutions of the virus. (Fig. I d). In thi s experiment agar overl ay (3%) was used. However, in the next experiment, 1% agarose was tried and similar result was obtained, however, on the 13th PID. The number of plaques in both the experiments was too large to be counted. The experi­ment will be repeated again with higher dilutions of the virus, however, it appears that thi s ce ll line can be used for titration of SLNPV by plaque assay.

Thus, a new indigenous cell line from larva l hemo­cytes (NIV -SU-I 095) has been estab li shed . It is at 86th passage level. It's clone II-I F is much more sus­ceptible to AcNPV than the parent ce ll line and the commerc iall y available Sf-9 cell line. The culture may prove valuabl e in baculovirus ex press ion vector stud ies.

Acknowledgement The study was conducted by grants from the Di vi­

sion of Biotechnology, New Delhi . Authors thank Dr. C. Dayaraj for baculovirus AcNPV and Dr. V .M . Pawar (Rahuri ) for SLNPV .

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