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The culture of embryonic cells from the bug Triatoma maculata (Erichson) (Hemiptera: Reduviidae)
8 1967 by Academic Experimental
Press Inc.
Cell Research
THE CULTURE
45, 671-675
OF EMBRYONIC CELLS FROM THE BUG MACULATA (ERICHSON) (HEMIPTERA: REDUVIIDAE)
TRIATOMA
M. G. R. VARMA London
671
(1967)
School
of Hygiene
and MARY
and Tropical
Medicine,
PUDNEY London,
W.C. I., England
Received August 9, 1966l
THE
growing importance of arthropod-borne virus (arbovirus) infections of man and his domestic animals has, in recent years, renewed interest in the cell culture of arthropods and in the possibility of growing the viruses in them. Most of the attempts at in vitro cultivation of tissues from bloodsucking arthropods have been confined to mosquitoes and ticks because of their importance in the transmission of human and animal diseases. We have cultured embryonic cells from the blood-sucking bug Triatoma maculata (Erichson) (Family Reduviidae: sub-family Triatominae). These triatomine bugs, which are capable of transmitting the pathogenic trypanosome, Trypanosoma cruzi, are easy to maintain in the laboratory and because of their relatively large size yield more tissue suitable for cultivation than most other blood sucking insects. Earlier attempts at cell culture of triatomine bugs were not successful [a], although Liischer [S] showed that cells from the cut leg of Rhodnius prolixus Stdl grew out into a fine glass tube fixed to the cut end. More recently, Vago and Flandre [lo] cultivated cells from the ovary and Malpighian tubules of Triatoma infestans (Klug) and Rhodnius prolixus in plasma clots. In view of the relatively large size of T. maculata eggs (1.5 to 1.75 mm long) and the ease with which the embryos could be removed, we decided to use the embryos as a source of cells to be cultivated. While embryonic cells from non-blood-sucking insects have been grown in vitro [l, 3, 4, 7, 91, there has been no comparable work with embryos of blood-sucking insects. MATERIALS Preparation
of cell
suspensions.-Eggs
from Colombia and maintained School of Hygiene and Tropical 1 Revised
version
received
October
AND
METHODS
of T. maculata were from a strain originating in the Entomology Department of the London Medicine since 1954. Only embryos at the post4, 1966. Experimental
Cell Research
45
672
M. G. R. Varma
and Mary Pudney
blastokinetic stage were used. The earlier pre-blastokinetic stages did not yield sufficient cells and later stages when the eyes had turned black proved unsuitable for cultivation. The eggs were surface sterilized by shaking them in a tube in a 1 in 40 solution of Roccal (a product of Bayer Products Company of Surbiton-upon-Thames, England, containing 1 per cent benzalkonium chloride) and washed in running tap-water. They were then placed in a sterile petri dish containing a small quantity of sterile phosphate buffered saline, pH 7.2 (PBS) containing 1000 units of penicillin and 100 [mcg of streptomycin per ml]. The embryos were removed from the egg shell by using two sterile cataract knives and placed in about IO ml of the culture medium. They were washed by 4 changes of PBS containing antibiotics and cut into small pieces with a pair of spring scissors. The cells were dissociated by placing the tissues in 5 ml of a 0.1 per cent solution of the protease, Pronase (B grade, obtainable from Calbiochem Ltd., Basingstoke, England) in a calcium and magnesium free diluent. Five ml was sufficient for dissociating tissues from 100 to 200 embryos. The pronase with the embryonic tissues was left at 28°C for 15 min and inactivated at the end of this period by adding 0.5 ml of calf serum. None of the adverse effects of pronase on mammalian cells observed by Kahn et al. [5] was seen: on the contrary, for dissociating embryonic cells of the bugs, pronase was found to be more satisfactory than 0.25 per cent trypsin. The cell suspension, after filtering through sterile gauze, was centrifuged at 800 rpm for 8 min at room temperature. The cells were washed once in 5 ml of PBS and suspended in the culture medium (see below) at the rate of 1.0 ml of medium to cells from 42 to 90 embryos. This gave an average cell count of 2.29 x IO6 cells/ml (range 1.74 x IO6 to 2.93 x IO6 cells/ml). Cells from 50 to 60 embryos in 1.0 ml was usually adequate for setting up cultures. The suspension was seeded into Leighton tubes with cover glasses, 1.0 ml to each tube, bunged and incubated stationary at 28°C. Culture medium--The culture medium was a modification of Kitamura’s medium [6] for the cultivation of tissues from the mosquito, CuZex pipiens var. molestus. To 100 ml of Kitamura’s basal medium was added 0.1 ml of a penicillin and streptomycin mixture to give a concentration of 1000 units of penicillin and 100 mcg of streptomycin per ml of medium. After adding 25 ml of synthetic medium 199 (Glaxo Laboratories Ltd., Greenford, England) to 100 ml of the basal medium, the pH was adjusted to 7.0-7.2 with 7.5 per cent NaHCO,. The medium was sterilized by filtering through sintered glass and finally completed by adding, just before use, foetal calf serum (from Microbiological Associates Inc., Bethesda, U.S.A.) to produce a concentration of 20 per cent of the calf serum. About the sixth day, the medium with any cell debris present, was completely removed and replaced with fresh culture medium containing IO per cent foetal calf serum. Thereafter, the culture was maintained by removing half the medium and replacing it with fresh medium containing IO per cent foetal calf serum, usually twice a week. RESULTS
The cells became adherent to the glass within 24 hr. By the first day after seeding, the attached cell clumps with fibroblast-like outgrowths at the periExperimental
Cell Research
45
Embryonic
Figs.
cells from the bug Triatoma
1-6.-Cell
culture
of bug
embryos.
673
maculata
Stained
H & E.
Fig.
l.-Epithelial-type
cells in a 7-day-old
culture.
x 800.
Fig.
2.-Fibroblast-type
cells in a 7-day-old
culture.
x 800.
Experimental
Ce
Research
45
M. G. R. Varma
and Mary Pudney
Figs. 3%6.-Progressive mitotic phases in bug cells in culture. Fig. 3, Prophase. 7-day-old culture. x 480; Fig. 4, Metaphase. 40-day-old culture. x 1000; Fig. 5, Anaphase. 7-day-old culture. x 750; Fig. 6, Telophase. 40.day-old culture. x 800.
phery, could be made out as red patches due to the naturally occurring pigment in the cells. At this stage, the clumps are more than one cell thick. By about the fourth day, the fibroblast-like outgrowths from contiguous cell clumps had joined together to form a loose network. The pigment gradually disappeared and 7-10 days after seeding, the cells had become clear. In a good culture, the cover glass at this stage is covered with numerous close-set patches of epithelial-type cells (Fig. 1) and libroblast-type cells (Fig. 2) which are interconnected to form a network. Most of the cell clumps have by this time, flattened out to patches one cell thick. Although a confluent sheet was not obtained, the compact network formed by the interconnected patches of cells completely covered the surface of the cover glass. The cultures were maintained in a healthy state for up to about 40 days. After this period, the cells became vacuolated and a slow process of degeneration started, although peristaltic movements of some cell clumps which were seen as early as the seventh day, continued up to 43 days and 49 days in two cultures. In one culture, a few healthy cells were seen as late as 74 days after seeding. Experimental
Cell Research
45
Embryonic
cells from the bug Triatoma
maculata
655
In cultures which were well established, the number of tibroblast-type cells appeared to be slightly in excess of the epithelial-type cells, although the area covered by the tlbroblasts was greater, thus giving the impression of larger numbers. Some of the tibroblast-type cells were multinucleate with, in some cases, several nuclei arranged inside the cells like peas in a pod. Others were myoblasts since their cytoplasm eventually became striated and the cells themselves showed contractile movements. A few giant cells with nuclei three to four times larger than those of the ordinary cells were also seen. Mitoses were seen as early as the fourth day. They became more frequent by about the seventh day and in one culture numerous mitoses were seen as late as 40 days after seeding (Figs. 336). Attempts at subculturing the cells were unsuccessful.
SUMMARY
The culture of embryonic cells of a blood-sucking bug, Triatoma macdata, is described. The culture medium was a modification of one used for cultivating tissues from the culicine mosquito, Culex pipiens var. molestus. Good dissociation of the embryonic cells was obtained by using a 0.1 per cent solution of the protease, Pronase. Frequent mitoses were observed at least up to 40 days. Cultures were maintained in a healthy condition up to this period after which they started degenerating. Attempts at subculturing the cells were unsuccessful. The authors wish to thank Professor D. S. Bertram, Director of the Department of Entomology at the School for his interest and constant encouragement, Miss Judy Merryweather for technical assistance, Mr C. J. Webb for advice in preparing the photographs and Calbiochem Ltd., for a free sample of Pronase. The investigation was supported by a grant from the Medical Research Council of Great Britain. REFERENCES 1.
ECHALIER,
G.,
OHANESSIAN,
A.
and
BRUN,
G.,
Compt.
Rend.
Acad.
Sci.
Paris
261,
3211
(1965). 2.
A. J. P., Nature 173, 504 (1954). and MARAMOROSCH, K., Exptl Cell Res. 36, 625 (1964). HORIKAWA, M., LING, L.-N. and Fox, A. S., Nature 210, 183 (1966). KAHN, J., ASHWOOD-SMITH, M. J. and ROBINSON, D. M., Exptl Cell KITAMURA, S., Kobe J. Med. Sci. 11, 23 (1965). LANDUREAU, J. C., Exptl Cell Res. 41, 545 (1966). L~SCHER, M., Nature 160, 873 (1947). MITSUHASHI, J., Jap. J. Appl. Ent. Zool. 9, 107 (1965). VAGO, C. and FLANDRE, O., Ann. Epiphyt. 14, 127 (1963). GOODCHILD,
3. HIRUMI, 4.
5. 6. 7. 8. 9. 10.
H.
Experimental
Res. 40, 445 (1965).
Cell Research
45
Press Inc.
Cell Research
THE CULTURE
45, 671-675
OF EMBRYONIC CELLS FROM THE BUG MACULATA (ERICHSON) (HEMIPTERA: REDUVIIDAE)
TRIATOMA
M. G. R. VARMA London
671
(1967)
School
of Hygiene
and MARY
and Tropical
Medicine,
PUDNEY London,
W.C. I., England
Received August 9, 1966l
THE
growing importance of arthropod-borne virus (arbovirus) infections of man and his domestic animals has, in recent years, renewed interest in the cell culture of arthropods and in the possibility of growing the viruses in them. Most of the attempts at in vitro cultivation of tissues from bloodsucking arthropods have been confined to mosquitoes and ticks because of their importance in the transmission of human and animal diseases. We have cultured embryonic cells from the blood-sucking bug Triatoma maculata (Erichson) (Family Reduviidae: sub-family Triatominae). These triatomine bugs, which are capable of transmitting the pathogenic trypanosome, Trypanosoma cruzi, are easy to maintain in the laboratory and because of their relatively large size yield more tissue suitable for cultivation than most other blood sucking insects. Earlier attempts at cell culture of triatomine bugs were not successful [a], although Liischer [S] showed that cells from the cut leg of Rhodnius prolixus Stdl grew out into a fine glass tube fixed to the cut end. More recently, Vago and Flandre [lo] cultivated cells from the ovary and Malpighian tubules of Triatoma infestans (Klug) and Rhodnius prolixus in plasma clots. In view of the relatively large size of T. maculata eggs (1.5 to 1.75 mm long) and the ease with which the embryos could be removed, we decided to use the embryos as a source of cells to be cultivated. While embryonic cells from non-blood-sucking insects have been grown in vitro [l, 3, 4, 7, 91, there has been no comparable work with embryos of blood-sucking insects. MATERIALS Preparation
of cell
suspensions.-Eggs
from Colombia and maintained School of Hygiene and Tropical 1 Revised
version
received
October
AND
METHODS
of T. maculata were from a strain originating in the Entomology Department of the London Medicine since 1954. Only embryos at the post4, 1966. Experimental
Cell Research
45
672
M. G. R. Varma
and Mary Pudney
blastokinetic stage were used. The earlier pre-blastokinetic stages did not yield sufficient cells and later stages when the eyes had turned black proved unsuitable for cultivation. The eggs were surface sterilized by shaking them in a tube in a 1 in 40 solution of Roccal (a product of Bayer Products Company of Surbiton-upon-Thames, England, containing 1 per cent benzalkonium chloride) and washed in running tap-water. They were then placed in a sterile petri dish containing a small quantity of sterile phosphate buffered saline, pH 7.2 (PBS) containing 1000 units of penicillin and 100 [mcg of streptomycin per ml]. The embryos were removed from the egg shell by using two sterile cataract knives and placed in about IO ml of the culture medium. They were washed by 4 changes of PBS containing antibiotics and cut into small pieces with a pair of spring scissors. The cells were dissociated by placing the tissues in 5 ml of a 0.1 per cent solution of the protease, Pronase (B grade, obtainable from Calbiochem Ltd., Basingstoke, England) in a calcium and magnesium free diluent. Five ml was sufficient for dissociating tissues from 100 to 200 embryos. The pronase with the embryonic tissues was left at 28°C for 15 min and inactivated at the end of this period by adding 0.5 ml of calf serum. None of the adverse effects of pronase on mammalian cells observed by Kahn et al. [5] was seen: on the contrary, for dissociating embryonic cells of the bugs, pronase was found to be more satisfactory than 0.25 per cent trypsin. The cell suspension, after filtering through sterile gauze, was centrifuged at 800 rpm for 8 min at room temperature. The cells were washed once in 5 ml of PBS and suspended in the culture medium (see below) at the rate of 1.0 ml of medium to cells from 42 to 90 embryos. This gave an average cell count of 2.29 x IO6 cells/ml (range 1.74 x IO6 to 2.93 x IO6 cells/ml). Cells from 50 to 60 embryos in 1.0 ml was usually adequate for setting up cultures. The suspension was seeded into Leighton tubes with cover glasses, 1.0 ml to each tube, bunged and incubated stationary at 28°C. Culture medium--The culture medium was a modification of Kitamura’s medium [6] for the cultivation of tissues from the mosquito, CuZex pipiens var. molestus. To 100 ml of Kitamura’s basal medium was added 0.1 ml of a penicillin and streptomycin mixture to give a concentration of 1000 units of penicillin and 100 mcg of streptomycin per ml of medium. After adding 25 ml of synthetic medium 199 (Glaxo Laboratories Ltd., Greenford, England) to 100 ml of the basal medium, the pH was adjusted to 7.0-7.2 with 7.5 per cent NaHCO,. The medium was sterilized by filtering through sintered glass and finally completed by adding, just before use, foetal calf serum (from Microbiological Associates Inc., Bethesda, U.S.A.) to produce a concentration of 20 per cent of the calf serum. About the sixth day, the medium with any cell debris present, was completely removed and replaced with fresh culture medium containing IO per cent foetal calf serum. Thereafter, the culture was maintained by removing half the medium and replacing it with fresh medium containing IO per cent foetal calf serum, usually twice a week. RESULTS
The cells became adherent to the glass within 24 hr. By the first day after seeding, the attached cell clumps with fibroblast-like outgrowths at the periExperimental
Cell Research
45
Embryonic
Figs.
cells from the bug Triatoma
1-6.-Cell
culture
of bug
embryos.
673
maculata
Stained
H & E.
Fig.
l.-Epithelial-type
cells in a 7-day-old
culture.
x 800.
Fig.
2.-Fibroblast-type
cells in a 7-day-old
culture.
x 800.
Experimental
Ce
Research
45
M. G. R. Varma
and Mary Pudney
Figs. 3%6.-Progressive mitotic phases in bug cells in culture. Fig. 3, Prophase. 7-day-old culture. x 480; Fig. 4, Metaphase. 40-day-old culture. x 1000; Fig. 5, Anaphase. 7-day-old culture. x 750; Fig. 6, Telophase. 40.day-old culture. x 800.
phery, could be made out as red patches due to the naturally occurring pigment in the cells. At this stage, the clumps are more than one cell thick. By about the fourth day, the fibroblast-like outgrowths from contiguous cell clumps had joined together to form a loose network. The pigment gradually disappeared and 7-10 days after seeding, the cells had become clear. In a good culture, the cover glass at this stage is covered with numerous close-set patches of epithelial-type cells (Fig. 1) and libroblast-type cells (Fig. 2) which are interconnected to form a network. Most of the cell clumps have by this time, flattened out to patches one cell thick. Although a confluent sheet was not obtained, the compact network formed by the interconnected patches of cells completely covered the surface of the cover glass. The cultures were maintained in a healthy state for up to about 40 days. After this period, the cells became vacuolated and a slow process of degeneration started, although peristaltic movements of some cell clumps which were seen as early as the seventh day, continued up to 43 days and 49 days in two cultures. In one culture, a few healthy cells were seen as late as 74 days after seeding. Experimental
Cell Research
45
Embryonic
cells from the bug Triatoma
maculata
655
In cultures which were well established, the number of tibroblast-type cells appeared to be slightly in excess of the epithelial-type cells, although the area covered by the tlbroblasts was greater, thus giving the impression of larger numbers. Some of the tibroblast-type cells were multinucleate with, in some cases, several nuclei arranged inside the cells like peas in a pod. Others were myoblasts since their cytoplasm eventually became striated and the cells themselves showed contractile movements. A few giant cells with nuclei three to four times larger than those of the ordinary cells were also seen. Mitoses were seen as early as the fourth day. They became more frequent by about the seventh day and in one culture numerous mitoses were seen as late as 40 days after seeding (Figs. 336). Attempts at subculturing the cells were unsuccessful.
SUMMARY
The culture of embryonic cells of a blood-sucking bug, Triatoma macdata, is described. The culture medium was a modification of one used for cultivating tissues from the culicine mosquito, Culex pipiens var. molestus. Good dissociation of the embryonic cells was obtained by using a 0.1 per cent solution of the protease, Pronase. Frequent mitoses were observed at least up to 40 days. Cultures were maintained in a healthy condition up to this period after which they started degenerating. Attempts at subculturing the cells were unsuccessful. The authors wish to thank Professor D. S. Bertram, Director of the Department of Entomology at the School for his interest and constant encouragement, Miss Judy Merryweather for technical assistance, Mr C. J. Webb for advice in preparing the photographs and Calbiochem Ltd., for a free sample of Pronase. The investigation was supported by a grant from the Medical Research Council of Great Britain. REFERENCES 1.
ECHALIER,
G.,
OHANESSIAN,
A.
and
BRUN,
G.,
Compt.
Rend.
Acad.
Sci.
Paris
261,
3211
(1965). 2.
A. J. P., Nature 173, 504 (1954). and MARAMOROSCH, K., Exptl Cell Res. 36, 625 (1964). HORIKAWA, M., LING, L.-N. and Fox, A. S., Nature 210, 183 (1966). KAHN, J., ASHWOOD-SMITH, M. J. and ROBINSON, D. M., Exptl Cell KITAMURA, S., Kobe J. Med. Sci. 11, 23 (1965). LANDUREAU, J. C., Exptl Cell Res. 41, 545 (1966). L~SCHER, M., Nature 160, 873 (1947). MITSUHASHI, J., Jap. J. Appl. Ent. Zool. 9, 107 (1965). VAGO, C. and FLANDRE, O., Ann. Epiphyt. 14, 127 (1963). GOODCHILD,
3. HIRUMI, 4.
5. 6. 7. 8. 9. 10.
H.
Experimental
Res. 40, 445 (1965).
Cell Research
45