EXPERIMENTAL
PARASITOLOGY
4,
Permacentor
106-114
(1977)
andersoni: R.
WILLIAM
School
KAUFMAN
of Veterinary
Madingley
Culture l AND Medicine,
STEPHEN University
Road, Cambridge
(Accepted
of Whole
for publication
Salivary F.
BARNETT
of Cambridge,
CB3 OES, England, 13 October
Glands
U.K.
1976)
KAUFMAN, W. R., AND BARNETT, S. F. 1977. Dermucentor andersoni: Culture of whole salivary glands. Experimental Pamsitology 42, 106-114. Whole salivary glands were dissected from Dermacentor andersoni (Stiles) female ticks in the slow phase of engorgement and were exposed to various culture conditions. The rate of fluid secretion in vitro was used as an assay for survival time in culture. Our best results were obtained when glands were submerged in TC 199 supplemented with 10% newborn calf serum, the atmosphere being 4% CO% in oxygen. Under these conditions glands secreted at a rate of 73% after 3 days and 25% after 7 days in culture compared to freshly excised glands. Supplements of insulin and cortisol were beneficial. INDEX DESCRIPTORS: Dermacentorandersoni Stiles; Atari; Ixodidae; Ticks; Organ culture; Salivary glands; Salivary secretion in vitro; Salivary gland degeneration; Oxygen; Insulin; Cortisol; Babesidae; Theileridae; Piroplasms; Culture media; Manduca sexta hemolymph.
is not known, however, to what extent the observed co-development of parasite and salivary gland are interdependent. For example, are developmental processes within the parasite simply triggered by some early event following attachment of the tick, thereafter proceeding independently, or is there continual flow of information between the vector and parasite? Investigation of this problem would be greatly facilitated were one able to culture intact salivary glands for extended periods. The purpose of this communication is to provide a basis for such an organ culture method.
The Babesidae and Theileridae are protozoan parasites of mammals which cause considerable economic loss in domestic stock. These parasites are transmitted chiefly (if not solely) by ixodid ticks and gain access to the definitive host via the tick salivary secretions (Barnett 1968; Riek 1968). During the final stages of development in the unfed tick the parasites reside in the salivary glands where they remain in a noninfective state until such time as the tick begins feeding on a new host (Martin, Barnett, and Vidler 1964). The immature parasites then enter a phase of development which culminates in the stage which is infective to the blood cells of the host. During the same period the salivary glands themselves enter a phase of development (Till 1961; Balashov 1965). It still
MATERIALS
AND
METHODS
Adult Dermacentor andersoni Stiles (Acari:Ixodidae) were taken from a laboratory colony maintained according to the method of Wilkinson ( 1971). The ticks were allowed to commence feeding on a
1 Present address: Institut de Zoologie, Universite de Neuchgtel, 2000 Neuchgtel, Switzerland. .06 Copyright All rights
0 1977 by Academic of reproduction
in any
Press, Inc. form reserved.
ISSN
0014-4894
Dermacentor
andersoni:
CULTURE
rabbit, and the females were taken off at a stage (about 80 to 200 mg) in which the salivary glands are known to be fully functional (Kaufman 1976). The ticks were rinsed in 70% ethanol for 30 set, dabbed dry on absorbent tissue paper, and immobilized in sterile dissecting dishes by means of uv-treated Plasticine modeling clay. They were then covered with sterile dissecting medium (Hanks’ balanced salt solution-composition in mg/liter: NaCI, 8000; Na2HP04, 48; KCl, 400 KH2P04, 60; CaC12, 140; MgSO+ 98; in-glucose, 1600; phenol red, 10; with 200 units/ml of penicillin and 100 @g/ml of streptomycin sulfate added), and the salivary glands were dissected out within a sterile-air flow cabinet (Bass Aire, England). The extirpated gland pairs were then rinsed in three washes of sterile Hanks’ solution and one wash of the sterile culture medium. The gland pairs were then transferred to fresh culture medium (generally 1 ml) held in wells of Linbro plastic culture trays (Biocult Ltd., Scotland). These covered trays were then placed over water in air-tight chambers. For experiments in which glands were exposed to an atmosphere of 4% COZ in oxygen, the gas mixture (BOC medical grade) was introduced by flushing the air from the chamber for 3 min. The chamber containing the culture trays was manually agitated for 2 min twice daily and the culture medium was compIetely renewed every other day. Success of culture was assayed quantitatively by monitoring the subsequent ability of the glands to secrete fluid in &TO. Briefly, glands were transferred in a drop of bathing medium (a slightly modified version of TC 199) to a petri dish filled with liquid paraffin (mineral oi1). By means of a fine silk thread tied to a fragment of cuticle, part of the main duct of the gland could be pulled out of the bathing medium into the surrounding liquid paraffin, Thus, when salivation was stimul’ated by the introduction of dopamine into
OF
SALIVARY
GLANDS
107
the bathing medium (final concentration was 1O-6 M), the rate of secretion could be calculated from the diameter of the resultant spherical droplet of saliva which appeared at the orifice of the duct. Kaufman and Phillips (1973b) and Kaufman ( 1976) may be consulted for further details. Experimental
Me,dia
All media contained penicillin G (200 units/ml) and streptomycin sulfate (100 pg/ml) and were sterilized by membrane filtration. TC 199 consisted of unmodified TC medium 199 as supplied by Burroughs-Wellcome, supplemented with newborn calf serum ( 10 % ) . TC 199 x 2: double-strength TC 199 supplemented with 10% calf serum. Medium A: TC 199, to which an equimolar amount of NaCl replaced the recommended NaHC03, supplemented with 10% calf serum and 5 ml/l of a 2% Difco bactoyeast extract. Medium Asucrose: Medium A supplemented with 27.5 g/l of sucrose to render it isosmotic with tick hemoIymph (375 mOsm/l). Medium D: tissue culture medium of Rehacek and Brzostowski (1969) supplemented with 1% Manduca serta hemolymph (heat-treated) taken from the last larval stage. Antherea pernyi hemolymph (the species they used) was not available to us. Medium Dpupa: as Medium D except that pupal instead of Iarval hemolymph was used. Medium D,, : as Medium D but 10% calf serum replaced both the hemolymph extract and purified bovine serum albumin contained in Medium D. A Aocculent precipitate was unavoidable when the pHs of the three latter media were brought to neutrality. This precipitate was filtered off before membrane filtration, but we did not determine its composition.
108
KAUFMAN
AND
BABNETT
TABLE
I
Culture of Dermucentor andersmi Salivary Glands in Various Media, the Ambient Atmosphere Being Air Medium
Number of days in culture 3
A D DD D*, F FW
5
7
14
ROSa
SE
N
ROS
SE
N
ROS
SE
N
ROS
SE
N
33.9
10.8
10
30.4
10.2
11
47.3 s.9* 23.9 33.2* 1.9 3.3
11.9 3.3 7.1 7.1 0.7 1.0
15 8 14 16 12 12
16.3 0 1.0 1.5
4
17 4 4 4
a ROS. rate of fluid secretion in TC 199 and 10-G M dopamine following * Signkcantly different (P < 0.05).
Medium F: Berridge’s (1966) complex medium with phenol red (10 mg/liter) added as a pH indicator. Medium Fcs: Medium F supplemented with 10% calf serum. Medium H: as Medium A with the calf serum supplement augmented to 35%. Medium J: Medium H supplemented with 50 pg/ml of crystalline bovine insulin ( BDH ) and 0.1 pg/ml of cortisol (hydrocortisone hemisuccinate; Glaxo). Medium L: as Medium J but with 10% calf serum supplement. RESULTS
Salivary glands from Dermacentor andersoni ticks weighing 80 to 200 mg secrete fluid in vitro at a rate in excess of 200 nl/min when tested immediately following dissection, The rates to be presented for cultured glands may thus be compared to the latter figure as a rough index of viability. In our preliminary experiments we surveyed a number of nonrelated culture media using air as the ambient atmosphere (Table I). Medium A seemed to support the glands in culture somewhat better than the D series (based on a tick tissue culture medium) and the F series media [based on Berridge’s (1966) medium for
0.6 1.5
culture.
physiological studies on in vitro Malpighian tubules of the dipteran, CaZZiphoTa]. Since the above-mentioned glands were completely submerged in culture medium we considered that they may, under such conditions, suffer from a relative lack of oxygen, Our first approach to remedy this was simply to bring the tissue closer to the air-medium interface (Wilde&al 1971). Small “tables” made from Minimesh (fine stainless steel mesh; Expanded Metal Co., Ltd., West Hartlepool Co., Durham, England) were sterilized and placed in the culture wells and Medium A was added so as to just reach the level ‘of the table. Under these conditions, the fluid secretory rates were certainly no higher and, if anything, were lower than those for submerged glands (Fig. 1). Our second approach was to increase the partial pressure of oxygen in the ambient atmosphere. For this experiment we used an atmosphere of 4% CO’2 in oxygen and cultured the glands in normal TC 199 with the usual supplement of 10% calf serum. Some glands were submerged and some rested on the wire mesh tables. The results in Fig. 1 indicate that increased oxygen did improve performance markedly, at least during the initial few days of culture
Dermacentor
andersoni:
CULTURE
OF
SALIVARY
GLANDS
I
I
I
I
0
5
10
15
Days
in
109
culture
FIG. 1. The rate of fluid secretion as a function of duration in culture for a variety of treatments. ( 0) D. ande~soni control glands: Rate of secretion determined immediately following dissection. ( l ) Glands submerged in Medium A; atmosphere is air. ( 0) Glands on wire-mesh tables in Medium A; atmosphere is air. ( n ) Glands on wire-mesh tables in TC 199 ~2; atmosphere is 96% 02, 4% CO*. (A) Glands submerged in TC 199; atmosphere is 96% 02, 4% CO,. (A) Glands on wire-mesh tables in TC 199; atmosphere is 96% 02, 4% CO,. The SE and N for each mean are indicated.
(cf. Table I), but in our experiment the rate of secretion was not significantly higher at 7 days. In unfed ticks the osmotic pressure of hemolymph is considerably higher (about 525 mOsm/liter) than that of the hemolymph from feeding ticks (375 mOsm/ liter), as are the concentrations of at least some of the major ions (Kaufman and Phillips 1973a). Since salivary glands remain for long periods of time in this con-
centrated milieu, albeit in an inactive state, we asked whether mimicking these conditions in vitro would improve subsequent performance. The results were disappointing since the rate of secretion after 7 days in double-strength TC 199 was only one tenth the value for control glands in single-strength medium (Fig. 1). Wildenthal ( 1971)) in evolving a culture technique suitable for whole embryonic mouse hearts, found that the hearts re-
110
KAUFMAN
Percent
calf
Am
Serum
FIG. 2. The effects of newb’om calf serum, bovine insulin, and cortisol on D. andersoni glands cultured for ‘7 days submerged in medium; atmosphere is 96% O,, 4% CO,. ( l ) TC 199 with calf serum as indicated; (A) Medium H; (A) Medium J; ( n ) Medium L. The SE and iV for each mean are indicated.
mained alive and functioning far longer when the calf serum concentration was increased to 35%, higher concentrations affording no advantage. With the serum supplement at 35%, longevity was further increased with the addition of insulin and cortisol. We thus compared the effects of Medium H (35% serum) and Medium J (35% serum plus 50 pg/ml of insulin and 0.1 pg/ml of cortisol). The rate of secretion after 7 days in Medium H was 19.0 -t- 6.0 (meNan+ SE; N = 10) and in Medium J was 60.7 + 15.1; iV = 15 (0.025 > p > 0.01). Thus, although cortisol and insulin appeared to improve performance over calf serum alone, the relatively low rate in Medium H relative to that in normal TC 199 (Fig. 1) suggested also that the high concentration of calf serum itself may have been detrimental to the glands. Consequently, we tested the effect of varying the calf serum supplement. The results
BARNEY
(Fig. 2) suggest that up to 10% calf serum results in little if any improvement over unsupplemented TC 199, and that higher concentrations, especially whole serum, are deleterious. Reverting to 10% calf serum and once ‘again testing the effect of insulin and cortisol (Medium L) we found that the glands tended to do better in Medium L than in TC 199 (Fig. 2) but in this experiment the difference proved to be statistically insignificant. We next asked whether the glands would survive longer if the medium were in continual motion, thus allowing a more efficient exchange between nutrients and metabolites in the medium and in the tissue. Glands were, accordingly, cultured in test tubes which were continually rotated on a roller drum at 1 revolution every 4 min. In most cases the glands adhered to the glass and thus spent approximately half the time within the medium. Medium A and #air were used in some experiments and TC 199 and 4% CO2 in oxygen in other experiments. Figure 3 shows that culture of the glands in roller tubes was, if anything, worse than culture in stationary trays. Finally, we considered that a consequence of the organ culture could possibly be reduced sensitivity to exogenously applied dopamine and that we had misinterpreted this for tissue degeneration. Thus with a number of assays we exposed the glands to 10~~M dopamine once the rates in lO-‘j M dopamine had equilibrated. The rate of secretion in 1O-5 M (37 * nI/min; iV = 26) was insignificantly different (pairs t test) than thlat in 1O-6M (41 -+ 8 nl/min; N = 26). DISCUSSION
Under our best culture conditions Dermacentor andersoni glands secreted on average at a rate of 73% after 3 days and 25% after 7 days of the rate of noncultured glands. The saliva to medium ratios for sodium and potassium ions were
Dermacentor
mUh?TsOni:
Stationary
Roller
Medium
FIG. 3. A comparison in roller
tubes
A
and
CULTURE
tubes
SALIVARY
TC
199
Roller and
cultures of D. andersoni SE and N for each mean
similar to those for noncultured glands (unpublished observations). In our experiments many glands, cultured under air, were still viable after 14 days of culture although the rates of secretion by this time were only 8% of maximum (Fig. 1). As they stand these results are, nonetheless, encouraging since viability is well-maintained throughout the period that piroplasms develop to maturity in parasitized salivary glands (generally 2 to 5 days after attachment of the tick; Martin, Bamett, and Vidler 1964). Experiments using the above culture method (Fig. 1, upper curve) have already been performed in our laboratory with Hyalomma excavatum Koch nymphal salivary glands infected with Nuttalliu danii (Tsur, Hadani, and Pipano 1960; Hadani and Kaufman, in preparation). The latter work suggests that not only do the parasites remain alive in cultured glands, but that there is also some development toward the infective particle stage. An interesting observation of the present work was that the tissue culture medium devised by Rehacek and Brzostowski (1969) with tick cells particularly in mind, proved not to be an ideal medium, al-
111
GLANDS
Stationary
air
between stationary ( 1 revolution/4 min). The
OF
4% CO,,
96%
salivary glands are indicated.
tubes 0,
and cultures
though we tested it only with air as the ambient atmosphere (Table I). Although the addition of insect hemolymph to the above medium was beneficial, it seemed that calf serum was at least as good in this regard. In harmony with the latter observation Vaughn (1971) points out that for many insect tissues mammalian serum substitutes adequately for insect serum. AIthough the in vitro culture of tick cells has received some attention and has enjoyed some degree of success (Martin and Vidler 1962; Varma, Pudney, and Leake 1975), there seemsto be less information available on organ culture. Hoffmann, Schein, and Jagow (1970) cultured explanted organs from a variety of ticks and examined their progress by histological methods. Salivary glands taken from fully fed females degenerated in culture before (8-12 days) those from earlier stages (2650 days). In a subsequent work Hoffmann (1972) noted that explants which were infected with Bahia bigemina fared less well in culture than pathogen free tissue. Of particular interest was that B. bigemina did not develop in cultured salivary glands although in cultures of intestine, ovary, and hemacytes, schizonts and merozoites could be
112
KAUFMAN
identified. One possible explanation is that, at least for fed females up to 9 days postrepletion, the salivary glands would have been in Ia degenerating condition even before culture began. We have attempted to keep a fully differentiated organ alive in culture with as little loss in function as possible, so we chose an assay to monitor this function. In this regard our work parallels &at of Wildenthal (1971) who succeeded in keeping fetal mouse hearts alive in culture for at least 21 days. He found that a number of conditions were indispensable for successful culture, notably ensuring that the liquid-air interface was not too high, for even partial immersion of the organ caused early death presumably due to oxygen starvation. Contrary to Wildenthal’s findings, submersion of the glands within the nutrient fluid was superior to leaving them at the liquid-air interface. One possibility for this difference is that the delicate salivary tissue suffers more from the lack of mechanical support normally afforded by the liquid than does the muscular heart. Consideration should also be given to the fact that oxygen can be toxic in excessive amounts (Dickens 1962; Haugaard 1968). What constitutes an excessive amount depends on a variety of factors, such as tissue thickness, metabolic activity, and so forth. For example, recent work on culturing rat embryos in vitro shows that development depends largely on the availability of oxygen and that the optimum amount varies considerably from stage to stage (New 1970; New and Coppola 1970a, 1970b; New, Coppola, and Terry 1973). In the tick salivary ,gland, no cell is more than a few microns depth from the bathing medium. In the absence of a catecholamine stimulant, fluid secretion does not occur (Kaufman 1976), and the salivary glands may not be very active. Indeed we have noticed in this study that there was very little acidification of the medium between changes. Although the glands ‘did fare
AND
BARNETT
better submerged in medium with an oxygen-rich atmosphere than either supported on wire-mesh tables (Fig. 1) or revolving in and out of the culture medium (Fig. 3), Ia more extensive survey is indicated to show whether an optimum O2 concentration exists between 20 and 95%. The primary action of insulin is the promotion of sugar entry into the cell, many of its other effects being an indirect consequence of this effect on carbohydrate metabolism (Goth 1974). Cortisol also has far-reaching effects on the metabolic disposition of proteins, fats, land carbohydrates, and also influences water and electrolyte balance. Schryver (1965) and Reynolds (1966) demonstrated the beneficial action of cortisol on embryonic tibiotarsi of chicks which they attributed to the hormone’s ability to prevent overhydration of the tissue in culture, possibly by reducing the leakage of hydrolases from lysosomes (Weissmann and Dingle 1961; Schryver 1965). Insulin, cortisol, or both together may also be beneficial in our culture system (Fig. 2), but these experiments should be extended. In enumerating the possible causes of degeneration in culture we point out that in vitro salivary glands have been released from nervous influence. The gradual decline in secretory response could not be attributed to a progressive hyposensitivity to exogenous dopamine. This was not surprising, since the denervation of mammalian salivary glands results in supersensitivity of secretory cells to cholinomimetics (Emmelin 1964; Emmelin and Thulin 1973) and not hyposensitivity. This supersensitivity phenomenon results from an increased synthesis of receptor sites on the postjunctional membrane (Lavoie, Collier, and Tenenhouse 1976; Pestronk, Drachman, ‘and Grifhn 1976). On the other hand, selective denervation in other systems does often result in tissue degeneraJuorio, tion (see, for example, Barlow, and Martin, 1974; Lockshin 1971). Follow-
Demacentor
andersoni:
ing engorgement of the female ixodid ticks, autolysis of the salivary glands occurs conprogresses. comitantly Ias vitellogenesis Perhaps a tropic influence of nerves to the salivary glands is withheld in engorged females. Alternatively, or in concert with the latter, degeneration may occur by the direct mediation of a hormone, perhaps even the same one which triggers vitellogenesis. We have already alluded to the experiments of Hoffmann, Schein, and Jagow (1970) in which glands taken from the phase of Hochsekretion (rapid phase of engorgement) degenerate in culture after 8-12 days. In vivo, salivary glands show clear signs of degeneration even 1 day postrepletion (Till 1961)) thus the 8-12-day survival time in culture suggests that the in vitro tissue might have been spared prolonged exposure to an “autolytic factor” which normally appears in the replete tick. The present culture method for D. andersoni salvary glands may be powerful enough to investigate this intriguing problem. ACKNOWLEDGMENTS We thank Glaxo Laboratories Ltd., Greenford, Middlesex, U.K. for their gift of hydrocortisone hemisuccinate, and Mr. B. Gardiner of the Zoology Department, Cambridge, U.K. for providing us with specimens of Manduca sexta. We are pleased to acknowIedge The Wellcome Trust for their generous financial support in the form of an operating grant and a postdoctoral veterinary fellowship awarded to W.R.K. during the course of this work.
REFERENCES BALASHOV, Yu. S. 1965. Mechanism of salivation and the morphologic-histochemical peculiarities of the salivary glands in ixodid ticks (Acarina, Ixodoidea) . Entomological Reviews 44, 462472. BARLOW, J., Juomo, A., AND MARTIN, R. 1974. Monoamine transport in the octopus posterior salivary gland nerves. Journal of Comparative Physiology 89, 105-122. BARNETT, S. F. 1968. Theileriasis. In “Infectious Blood Diseases of Man and Animals” (D. Weinman and M. Ristic, eds.), Vol. 2, Chap. 20. Academic Press, New York.
113
CULTURE OF SALIVARY GLANDS BEFWDGE, M.
J. 1966. Metabolic pathways of isolated Malpighian tubules of the blody functioning in an artificial medium. IoumoZ of Insect Physiology 12, 1523-1538. DICKENS, F. 1962. The toxic effects of oxygen on nervous tissue. In “Neurochemistry” (K. Elliott, I. Page and J. Quastel, eds.), 2nd ed., Chap. 35. Charles Thomas, Illinois. EMMELIN, N. 1964. Secretory nerves of the salivary glands. In “International Series of Monographs on Oral Biology. Vol. III, Salivary Glands and Their Secretions” (L. Sreebny and J. Meyer, eds.), Pergamon Press, New York. EMMELIN, N., AND THULIN, A. 1973. Action of drugs on denervated myoepithelial cells of salivary glands. British Journal of Pharmacology 48, 73-79. Goss, R. 1969. “Principles of Regeneration.” Academic Press, New York. GOTH, A. 1974. “Medical Pharmacology.” 7th ed. C. V. Mosby, St. Louis, MO. HAUGAARD, N. 1968. Cellular mechanisms of oxygen toxicity. Physiological Reviews 48, 311373. HOFFMANN, G. 1972. Haltung babesieninfizierter Zeckengewebe in kiinstlichem Nahrmedium. ‘Zeitschrift fiir angewandte Entomologie 71, 26-34. HOFFMANN, G., SCHEIN, E., AND JAGOW, M. 1970. Untersuchungen an exstirpierten und in der Kultur gehaltenen Zeckengeweben. Zeitsch-ift fiir Tropenmedizin und Parasitologic 21, 46-61. KAUFMAN, W. 1976. The influence of various factors on fluid secretion by in vitro salivary glands of ixodid ticks. Journal of Experimental Biology 64, 727-742. KAUFMAN, W., AND PHILLIPS, J. 1973a. Ion and water balance in the ixodid tick, Dermacentor andersoni. I. Routes of ion and water excretion. Journal of Experimental Biology 58, 523-536. KAUFMAN, W., AND PHILLIPS, J. 197313. Ion and water balance in the ixodid tick Dermacentor andersoni. II. Mechanism and control of salivary secretion. Iournal of Experimental Biology 58, 537-547. LAVOIE, P.-A., COLLIER, B., AND TENENHOUSE, A. 1976. Comparison of ru-bungarotoxin binding to skeletal muscles after inactivity or denervation. Nature 260, 349-350. LOCKSHIN, R. A. 1971. Programmed cell death: Nature of the nervous signal controlling breakdown of intersegmental muscles. Journal of Insect Physiology 17, 149-158. MARTIN, Cyclic
H., BARNETT, S., AND VIDLER, B. 1964. development and longevity of Theileria parva in the tick Rhipicephalus appendiculatus. Experimental Parasitology 15, 527-555.
MARTIN,
H., AND VIDLER,
B. 1962.
In vitro
growth
114
KAUFMAN
tick tissues (Rhipicephalus appendiculatus Neumann, 1901) . Experimental Parasitology 12, 192-203. NEW, D. 1970. Culture of fetuses in vitro. In “Advances in the Biosciences. Vol. 6. Schering Symposium on Intrinsic and Extrinsic Factors in Early Mammalian Development, Venice, 1970” (G. Raspe, ed.), pp. 367-380. Pergamon Press, Vieweg. NEW, D., AND COPPOLA, P. 1970a. Development of explanted rat fetuses in hyperbaric oxygen. Teratology 3, 153-162. NEW, D., AND COPPOLA, P. 1970b. Effects of different oxygen concentrations on the development of rat embryos in culture. Journal of Reproductive Fertility 21, 109-118. NEW, D., COPPOLA, P., AND TERRY, S. 1973. Culture of explanted rat embryos in rotating tubes. Journal of Reproductive Fertility 35, 135-138. PESTRONK, A., DRACHMAN, D., AND GRIFFIN, J. 1976. Effect of muscle disuse on acetylcholine receptors. Nature 260, 352-353. REHACEK, J., AND BFZOSTOWSKI, H. 1969. A tick tissue culture medium based on analyses of tick hemolymph. Journal of Insect Physiology 15, 431436. REYNOLDS, J. 1966. The effect of hydrocortisone on the growth of chick bone rudiments in chemically defined medium. Experimental Cell Research 41, 174-189. RIEK, R. 1968. Babesiosis, In “Infectious Blood Diseases of Man and Animals” (D. Weinmann and M. Ristic, eds.), Chap. 19. Academic Press, New York. of
AND
BARNETT
SCHRYVER, cortisone
H.
1965.
on
the
The
water
influence uptake
of
of hydroembryonic
chick tibio-tarsi in organ culture. Experimental Cell Research 37, 327-337. TILL, W. 1961. A contribution to the anatomy and histology of the brown ear tick, Rhipicephalus appendiculatus ( Neumann). Memoirs of the Entomological Society of South Africa, N’o. 6. TSUR, I., HADANI, A., AND PIPANO, E. 1960. Nuttallia danii n.sp .--a haemoprotozoon from the gerbil (Meriones tristrami shawii). Refuah Veterinarith, Quarterly of the Israel Veterinary Medical Association 17, 236-244. VARMA, N., PUDNEY, M., AND LEAKE, C. 1975. The establishment of three cell lines from the tick Rhipicephalus appendiculatus and their infection with some arboviruses. Journal of Medical Entomology 11, 698-706. VAUGHN, J. 1971. Cell cuture media and methods. In “Invertebrate Tissue Culture” (C. Vago, ed.), Chap. 1. Academic Press, New York. WEISSMANN, G., AND DINGLE, J. 1961. Release of lysosomal protease by ultraviolet irradiation and inhibition by hydrocortisone. Experimental Cell Research 25, 207-210. WILDENTHAL, K. 1971. Long-term maintenance of SpontaneousIy beating mouse hearts in organ culture. Journal of Applied Physiology 30, 153157. WILKINSON, P. 1971. Termination of diapause in laboratory-reared Dermacentor andersoni adults. In ‘Proceedings of the 3rd International Congress of Acarology, Prague.” pp. 803-805. International Publications Service.
PARASITOLOGY
4,
Permacentor
106-114
(1977)
andersoni: R.
WILLIAM
School
KAUFMAN
of Veterinary
Madingley
Culture l AND Medicine,
STEPHEN University
Road, Cambridge
(Accepted
of Whole
for publication
Salivary F.
BARNETT
of Cambridge,
CB3 OES, England, 13 October
Glands
U.K.
1976)
KAUFMAN, W. R., AND BARNETT, S. F. 1977. Dermucentor andersoni: Culture of whole salivary glands. Experimental Pamsitology 42, 106-114. Whole salivary glands were dissected from Dermacentor andersoni (Stiles) female ticks in the slow phase of engorgement and were exposed to various culture conditions. The rate of fluid secretion in vitro was used as an assay for survival time in culture. Our best results were obtained when glands were submerged in TC 199 supplemented with 10% newborn calf serum, the atmosphere being 4% CO% in oxygen. Under these conditions glands secreted at a rate of 73% after 3 days and 25% after 7 days in culture compared to freshly excised glands. Supplements of insulin and cortisol were beneficial. INDEX DESCRIPTORS: Dermacentorandersoni Stiles; Atari; Ixodidae; Ticks; Organ culture; Salivary glands; Salivary secretion in vitro; Salivary gland degeneration; Oxygen; Insulin; Cortisol; Babesidae; Theileridae; Piroplasms; Culture media; Manduca sexta hemolymph.
is not known, however, to what extent the observed co-development of parasite and salivary gland are interdependent. For example, are developmental processes within the parasite simply triggered by some early event following attachment of the tick, thereafter proceeding independently, or is there continual flow of information between the vector and parasite? Investigation of this problem would be greatly facilitated were one able to culture intact salivary glands for extended periods. The purpose of this communication is to provide a basis for such an organ culture method.
The Babesidae and Theileridae are protozoan parasites of mammals which cause considerable economic loss in domestic stock. These parasites are transmitted chiefly (if not solely) by ixodid ticks and gain access to the definitive host via the tick salivary secretions (Barnett 1968; Riek 1968). During the final stages of development in the unfed tick the parasites reside in the salivary glands where they remain in a noninfective state until such time as the tick begins feeding on a new host (Martin, Barnett, and Vidler 1964). The immature parasites then enter a phase of development which culminates in the stage which is infective to the blood cells of the host. During the same period the salivary glands themselves enter a phase of development (Till 1961; Balashov 1965). It still
MATERIALS
AND
METHODS
Adult Dermacentor andersoni Stiles (Acari:Ixodidae) were taken from a laboratory colony maintained according to the method of Wilkinson ( 1971). The ticks were allowed to commence feeding on a
1 Present address: Institut de Zoologie, Universite de Neuchgtel, 2000 Neuchgtel, Switzerland. .06 Copyright All rights
0 1977 by Academic of reproduction
in any
Press, Inc. form reserved.
ISSN
0014-4894
Dermacentor
andersoni:
CULTURE
rabbit, and the females were taken off at a stage (about 80 to 200 mg) in which the salivary glands are known to be fully functional (Kaufman 1976). The ticks were rinsed in 70% ethanol for 30 set, dabbed dry on absorbent tissue paper, and immobilized in sterile dissecting dishes by means of uv-treated Plasticine modeling clay. They were then covered with sterile dissecting medium (Hanks’ balanced salt solution-composition in mg/liter: NaCI, 8000; Na2HP04, 48; KCl, 400 KH2P04, 60; CaC12, 140; MgSO+ 98; in-glucose, 1600; phenol red, 10; with 200 units/ml of penicillin and 100 @g/ml of streptomycin sulfate added), and the salivary glands were dissected out within a sterile-air flow cabinet (Bass Aire, England). The extirpated gland pairs were then rinsed in three washes of sterile Hanks’ solution and one wash of the sterile culture medium. The gland pairs were then transferred to fresh culture medium (generally 1 ml) held in wells of Linbro plastic culture trays (Biocult Ltd., Scotland). These covered trays were then placed over water in air-tight chambers. For experiments in which glands were exposed to an atmosphere of 4% COZ in oxygen, the gas mixture (BOC medical grade) was introduced by flushing the air from the chamber for 3 min. The chamber containing the culture trays was manually agitated for 2 min twice daily and the culture medium was compIetely renewed every other day. Success of culture was assayed quantitatively by monitoring the subsequent ability of the glands to secrete fluid in &TO. Briefly, glands were transferred in a drop of bathing medium (a slightly modified version of TC 199) to a petri dish filled with liquid paraffin (mineral oi1). By means of a fine silk thread tied to a fragment of cuticle, part of the main duct of the gland could be pulled out of the bathing medium into the surrounding liquid paraffin, Thus, when salivation was stimul’ated by the introduction of dopamine into
OF
SALIVARY
GLANDS
107
the bathing medium (final concentration was 1O-6 M), the rate of secretion could be calculated from the diameter of the resultant spherical droplet of saliva which appeared at the orifice of the duct. Kaufman and Phillips (1973b) and Kaufman ( 1976) may be consulted for further details. Experimental
Me,dia
All media contained penicillin G (200 units/ml) and streptomycin sulfate (100 pg/ml) and were sterilized by membrane filtration. TC 199 consisted of unmodified TC medium 199 as supplied by Burroughs-Wellcome, supplemented with newborn calf serum ( 10 % ) . TC 199 x 2: double-strength TC 199 supplemented with 10% calf serum. Medium A: TC 199, to which an equimolar amount of NaCl replaced the recommended NaHC03, supplemented with 10% calf serum and 5 ml/l of a 2% Difco bactoyeast extract. Medium Asucrose: Medium A supplemented with 27.5 g/l of sucrose to render it isosmotic with tick hemoIymph (375 mOsm/l). Medium D: tissue culture medium of Rehacek and Brzostowski (1969) supplemented with 1% Manduca serta hemolymph (heat-treated) taken from the last larval stage. Antherea pernyi hemolymph (the species they used) was not available to us. Medium Dpupa: as Medium D except that pupal instead of Iarval hemolymph was used. Medium D,, : as Medium D but 10% calf serum replaced both the hemolymph extract and purified bovine serum albumin contained in Medium D. A Aocculent precipitate was unavoidable when the pHs of the three latter media were brought to neutrality. This precipitate was filtered off before membrane filtration, but we did not determine its composition.
108
KAUFMAN
AND
BABNETT
TABLE
I
Culture of Dermucentor andersmi Salivary Glands in Various Media, the Ambient Atmosphere Being Air Medium
Number of days in culture 3
A D DD D*, F FW
5
7
14
ROSa
SE
N
ROS
SE
N
ROS
SE
N
ROS
SE
N
33.9
10.8
10
30.4
10.2
11
47.3 s.9* 23.9 33.2* 1.9 3.3
11.9 3.3 7.1 7.1 0.7 1.0
15 8 14 16 12 12
16.3 0 1.0 1.5
4
17 4 4 4
a ROS. rate of fluid secretion in TC 199 and 10-G M dopamine following * Signkcantly different (P < 0.05).
Medium F: Berridge’s (1966) complex medium with phenol red (10 mg/liter) added as a pH indicator. Medium Fcs: Medium F supplemented with 10% calf serum. Medium H: as Medium A with the calf serum supplement augmented to 35%. Medium J: Medium H supplemented with 50 pg/ml of crystalline bovine insulin ( BDH ) and 0.1 pg/ml of cortisol (hydrocortisone hemisuccinate; Glaxo). Medium L: as Medium J but with 10% calf serum supplement. RESULTS
Salivary glands from Dermacentor andersoni ticks weighing 80 to 200 mg secrete fluid in vitro at a rate in excess of 200 nl/min when tested immediately following dissection, The rates to be presented for cultured glands may thus be compared to the latter figure as a rough index of viability. In our preliminary experiments we surveyed a number of nonrelated culture media using air as the ambient atmosphere (Table I). Medium A seemed to support the glands in culture somewhat better than the D series (based on a tick tissue culture medium) and the F series media [based on Berridge’s (1966) medium for
0.6 1.5
culture.
physiological studies on in vitro Malpighian tubules of the dipteran, CaZZiphoTa]. Since the above-mentioned glands were completely submerged in culture medium we considered that they may, under such conditions, suffer from a relative lack of oxygen, Our first approach to remedy this was simply to bring the tissue closer to the air-medium interface (Wilde&al 1971). Small “tables” made from Minimesh (fine stainless steel mesh; Expanded Metal Co., Ltd., West Hartlepool Co., Durham, England) were sterilized and placed in the culture wells and Medium A was added so as to just reach the level ‘of the table. Under these conditions, the fluid secretory rates were certainly no higher and, if anything, were lower than those for submerged glands (Fig. 1). Our second approach was to increase the partial pressure of oxygen in the ambient atmosphere. For this experiment we used an atmosphere of 4% CO’2 in oxygen and cultured the glands in normal TC 199 with the usual supplement of 10% calf serum. Some glands were submerged and some rested on the wire mesh tables. The results in Fig. 1 indicate that increased oxygen did improve performance markedly, at least during the initial few days of culture
Dermacentor
andersoni:
CULTURE
OF
SALIVARY
GLANDS
I
I
I
I
0
5
10
15
Days
in
109
culture
FIG. 1. The rate of fluid secretion as a function of duration in culture for a variety of treatments. ( 0) D. ande~soni control glands: Rate of secretion determined immediately following dissection. ( l ) Glands submerged in Medium A; atmosphere is air. ( 0) Glands on wire-mesh tables in Medium A; atmosphere is air. ( n ) Glands on wire-mesh tables in TC 199 ~2; atmosphere is 96% 02, 4% CO*. (A) Glands submerged in TC 199; atmosphere is 96% 02, 4% CO,. (A) Glands on wire-mesh tables in TC 199; atmosphere is 96% 02, 4% CO,. The SE and N for each mean are indicated.
(cf. Table I), but in our experiment the rate of secretion was not significantly higher at 7 days. In unfed ticks the osmotic pressure of hemolymph is considerably higher (about 525 mOsm/liter) than that of the hemolymph from feeding ticks (375 mOsm/ liter), as are the concentrations of at least some of the major ions (Kaufman and Phillips 1973a). Since salivary glands remain for long periods of time in this con-
centrated milieu, albeit in an inactive state, we asked whether mimicking these conditions in vitro would improve subsequent performance. The results were disappointing since the rate of secretion after 7 days in double-strength TC 199 was only one tenth the value for control glands in single-strength medium (Fig. 1). Wildenthal ( 1971)) in evolving a culture technique suitable for whole embryonic mouse hearts, found that the hearts re-
110
KAUFMAN
Percent
calf
Am
Serum
FIG. 2. The effects of newb’om calf serum, bovine insulin, and cortisol on D. andersoni glands cultured for ‘7 days submerged in medium; atmosphere is 96% O,, 4% CO,. ( l ) TC 199 with calf serum as indicated; (A) Medium H; (A) Medium J; ( n ) Medium L. The SE and iV for each mean are indicated.
mained alive and functioning far longer when the calf serum concentration was increased to 35%, higher concentrations affording no advantage. With the serum supplement at 35%, longevity was further increased with the addition of insulin and cortisol. We thus compared the effects of Medium H (35% serum) and Medium J (35% serum plus 50 pg/ml of insulin and 0.1 pg/ml of cortisol). The rate of secretion after 7 days in Medium H was 19.0 -t- 6.0 (meNan+ SE; N = 10) and in Medium J was 60.7 + 15.1; iV = 15 (0.025 > p > 0.01). Thus, although cortisol and insulin appeared to improve performance over calf serum alone, the relatively low rate in Medium H relative to that in normal TC 199 (Fig. 1) suggested also that the high concentration of calf serum itself may have been detrimental to the glands. Consequently, we tested the effect of varying the calf serum supplement. The results
BARNEY
(Fig. 2) suggest that up to 10% calf serum results in little if any improvement over unsupplemented TC 199, and that higher concentrations, especially whole serum, are deleterious. Reverting to 10% calf serum and once ‘again testing the effect of insulin and cortisol (Medium L) we found that the glands tended to do better in Medium L than in TC 199 (Fig. 2) but in this experiment the difference proved to be statistically insignificant. We next asked whether the glands would survive longer if the medium were in continual motion, thus allowing a more efficient exchange between nutrients and metabolites in the medium and in the tissue. Glands were, accordingly, cultured in test tubes which were continually rotated on a roller drum at 1 revolution every 4 min. In most cases the glands adhered to the glass and thus spent approximately half the time within the medium. Medium A and #air were used in some experiments and TC 199 and 4% CO2 in oxygen in other experiments. Figure 3 shows that culture of the glands in roller tubes was, if anything, worse than culture in stationary trays. Finally, we considered that a consequence of the organ culture could possibly be reduced sensitivity to exogenously applied dopamine and that we had misinterpreted this for tissue degeneration. Thus with a number of assays we exposed the glands to 10~~M dopamine once the rates in lO-‘j M dopamine had equilibrated. The rate of secretion in 1O-5 M (37 * nI/min; iV = 26) was insignificantly different (pairs t test) than thlat in 1O-6M (41 -+ 8 nl/min; N = 26). DISCUSSION
Under our best culture conditions Dermacentor andersoni glands secreted on average at a rate of 73% after 3 days and 25% after 7 days of the rate of noncultured glands. The saliva to medium ratios for sodium and potassium ions were
Dermacentor
mUh?TsOni:
Stationary
Roller
Medium
FIG. 3. A comparison in roller
tubes
A
and
CULTURE
tubes
SALIVARY
TC
199
Roller and
cultures of D. andersoni SE and N for each mean
similar to those for noncultured glands (unpublished observations). In our experiments many glands, cultured under air, were still viable after 14 days of culture although the rates of secretion by this time were only 8% of maximum (Fig. 1). As they stand these results are, nonetheless, encouraging since viability is well-maintained throughout the period that piroplasms develop to maturity in parasitized salivary glands (generally 2 to 5 days after attachment of the tick; Martin, Bamett, and Vidler 1964). Experiments using the above culture method (Fig. 1, upper curve) have already been performed in our laboratory with Hyalomma excavatum Koch nymphal salivary glands infected with Nuttalliu danii (Tsur, Hadani, and Pipano 1960; Hadani and Kaufman, in preparation). The latter work suggests that not only do the parasites remain alive in cultured glands, but that there is also some development toward the infective particle stage. An interesting observation of the present work was that the tissue culture medium devised by Rehacek and Brzostowski (1969) with tick cells particularly in mind, proved not to be an ideal medium, al-
111
GLANDS
Stationary
air
between stationary ( 1 revolution/4 min). The
OF
4% CO,,
96%
salivary glands are indicated.
tubes 0,
and cultures
though we tested it only with air as the ambient atmosphere (Table I). Although the addition of insect hemolymph to the above medium was beneficial, it seemed that calf serum was at least as good in this regard. In harmony with the latter observation Vaughn (1971) points out that for many insect tissues mammalian serum substitutes adequately for insect serum. AIthough the in vitro culture of tick cells has received some attention and has enjoyed some degree of success (Martin and Vidler 1962; Varma, Pudney, and Leake 1975), there seemsto be less information available on organ culture. Hoffmann, Schein, and Jagow (1970) cultured explanted organs from a variety of ticks and examined their progress by histological methods. Salivary glands taken from fully fed females degenerated in culture before (8-12 days) those from earlier stages (2650 days). In a subsequent work Hoffmann (1972) noted that explants which were infected with Bahia bigemina fared less well in culture than pathogen free tissue. Of particular interest was that B. bigemina did not develop in cultured salivary glands although in cultures of intestine, ovary, and hemacytes, schizonts and merozoites could be
112
KAUFMAN
identified. One possible explanation is that, at least for fed females up to 9 days postrepletion, the salivary glands would have been in Ia degenerating condition even before culture began. We have attempted to keep a fully differentiated organ alive in culture with as little loss in function as possible, so we chose an assay to monitor this function. In this regard our work parallels &at of Wildenthal (1971) who succeeded in keeping fetal mouse hearts alive in culture for at least 21 days. He found that a number of conditions were indispensable for successful culture, notably ensuring that the liquid-air interface was not too high, for even partial immersion of the organ caused early death presumably due to oxygen starvation. Contrary to Wildenthal’s findings, submersion of the glands within the nutrient fluid was superior to leaving them at the liquid-air interface. One possibility for this difference is that the delicate salivary tissue suffers more from the lack of mechanical support normally afforded by the liquid than does the muscular heart. Consideration should also be given to the fact that oxygen can be toxic in excessive amounts (Dickens 1962; Haugaard 1968). What constitutes an excessive amount depends on a variety of factors, such as tissue thickness, metabolic activity, and so forth. For example, recent work on culturing rat embryos in vitro shows that development depends largely on the availability of oxygen and that the optimum amount varies considerably from stage to stage (New 1970; New and Coppola 1970a, 1970b; New, Coppola, and Terry 1973). In the tick salivary ,gland, no cell is more than a few microns depth from the bathing medium. In the absence of a catecholamine stimulant, fluid secretion does not occur (Kaufman 1976), and the salivary glands may not be very active. Indeed we have noticed in this study that there was very little acidification of the medium between changes. Although the glands ‘did fare
AND
BARNETT
better submerged in medium with an oxygen-rich atmosphere than either supported on wire-mesh tables (Fig. 1) or revolving in and out of the culture medium (Fig. 3), Ia more extensive survey is indicated to show whether an optimum O2 concentration exists between 20 and 95%. The primary action of insulin is the promotion of sugar entry into the cell, many of its other effects being an indirect consequence of this effect on carbohydrate metabolism (Goth 1974). Cortisol also has far-reaching effects on the metabolic disposition of proteins, fats, land carbohydrates, and also influences water and electrolyte balance. Schryver (1965) and Reynolds (1966) demonstrated the beneficial action of cortisol on embryonic tibiotarsi of chicks which they attributed to the hormone’s ability to prevent overhydration of the tissue in culture, possibly by reducing the leakage of hydrolases from lysosomes (Weissmann and Dingle 1961; Schryver 1965). Insulin, cortisol, or both together may also be beneficial in our culture system (Fig. 2), but these experiments should be extended. In enumerating the possible causes of degeneration in culture we point out that in vitro salivary glands have been released from nervous influence. The gradual decline in secretory response could not be attributed to a progressive hyposensitivity to exogenous dopamine. This was not surprising, since the denervation of mammalian salivary glands results in supersensitivity of secretory cells to cholinomimetics (Emmelin 1964; Emmelin and Thulin 1973) and not hyposensitivity. This supersensitivity phenomenon results from an increased synthesis of receptor sites on the postjunctional membrane (Lavoie, Collier, and Tenenhouse 1976; Pestronk, Drachman, ‘and Grifhn 1976). On the other hand, selective denervation in other systems does often result in tissue degeneraJuorio, tion (see, for example, Barlow, and Martin, 1974; Lockshin 1971). Follow-
Demacentor
andersoni:
ing engorgement of the female ixodid ticks, autolysis of the salivary glands occurs conprogresses. comitantly Ias vitellogenesis Perhaps a tropic influence of nerves to the salivary glands is withheld in engorged females. Alternatively, or in concert with the latter, degeneration may occur by the direct mediation of a hormone, perhaps even the same one which triggers vitellogenesis. We have already alluded to the experiments of Hoffmann, Schein, and Jagow (1970) in which glands taken from the phase of Hochsekretion (rapid phase of engorgement) degenerate in culture after 8-12 days. In vivo, salivary glands show clear signs of degeneration even 1 day postrepletion (Till 1961)) thus the 8-12-day survival time in culture suggests that the in vitro tissue might have been spared prolonged exposure to an “autolytic factor” which normally appears in the replete tick. The present culture method for D. andersoni salvary glands may be powerful enough to investigate this intriguing problem. ACKNOWLEDGMENTS We thank Glaxo Laboratories Ltd., Greenford, Middlesex, U.K. for their gift of hydrocortisone hemisuccinate, and Mr. B. Gardiner of the Zoology Department, Cambridge, U.K. for providing us with specimens of Manduca sexta. We are pleased to acknowIedge The Wellcome Trust for their generous financial support in the form of an operating grant and a postdoctoral veterinary fellowship awarded to W.R.K. during the course of this work.
REFERENCES BALASHOV, Yu. S. 1965. Mechanism of salivation and the morphologic-histochemical peculiarities of the salivary glands in ixodid ticks (Acarina, Ixodoidea) . Entomological Reviews 44, 462472. BARLOW, J., Juomo, A., AND MARTIN, R. 1974. Monoamine transport in the octopus posterior salivary gland nerves. Journal of Comparative Physiology 89, 105-122. BARNETT, S. F. 1968. Theileriasis. In “Infectious Blood Diseases of Man and Animals” (D. Weinman and M. Ristic, eds.), Vol. 2, Chap. 20. Academic Press, New York.
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CULTURE OF SALIVARY GLANDS BEFWDGE, M.
J. 1966. Metabolic pathways of isolated Malpighian tubules of the blody functioning in an artificial medium. IoumoZ of Insect Physiology 12, 1523-1538. DICKENS, F. 1962. The toxic effects of oxygen on nervous tissue. In “Neurochemistry” (K. Elliott, I. Page and J. Quastel, eds.), 2nd ed., Chap. 35. Charles Thomas, Illinois. EMMELIN, N. 1964. Secretory nerves of the salivary glands. In “International Series of Monographs on Oral Biology. Vol. III, Salivary Glands and Their Secretions” (L. Sreebny and J. Meyer, eds.), Pergamon Press, New York. EMMELIN, N., AND THULIN, A. 1973. Action of drugs on denervated myoepithelial cells of salivary glands. British Journal of Pharmacology 48, 73-79. Goss, R. 1969. “Principles of Regeneration.” Academic Press, New York. GOTH, A. 1974. “Medical Pharmacology.” 7th ed. C. V. Mosby, St. Louis, MO. HAUGAARD, N. 1968. Cellular mechanisms of oxygen toxicity. Physiological Reviews 48, 311373. HOFFMANN, G. 1972. Haltung babesieninfizierter Zeckengewebe in kiinstlichem Nahrmedium. ‘Zeitschrift fiir angewandte Entomologie 71, 26-34. HOFFMANN, G., SCHEIN, E., AND JAGOW, M. 1970. Untersuchungen an exstirpierten und in der Kultur gehaltenen Zeckengeweben. Zeitsch-ift fiir Tropenmedizin und Parasitologic 21, 46-61. KAUFMAN, W. 1976. The influence of various factors on fluid secretion by in vitro salivary glands of ixodid ticks. Journal of Experimental Biology 64, 727-742. KAUFMAN, W., AND PHILLIPS, J. 1973a. Ion and water balance in the ixodid tick, Dermacentor andersoni. I. Routes of ion and water excretion. Journal of Experimental Biology 58, 523-536. KAUFMAN, W., AND PHILLIPS, J. 197313. Ion and water balance in the ixodid tick Dermacentor andersoni. II. Mechanism and control of salivary secretion. Iournal of Experimental Biology 58, 537-547. LAVOIE, P.-A., COLLIER, B., AND TENENHOUSE, A. 1976. Comparison of ru-bungarotoxin binding to skeletal muscles after inactivity or denervation. Nature 260, 349-350. LOCKSHIN, R. A. 1971. Programmed cell death: Nature of the nervous signal controlling breakdown of intersegmental muscles. Journal of Insect Physiology 17, 149-158. MARTIN, Cyclic
H., BARNETT, S., AND VIDLER, B. 1964. development and longevity of Theileria parva in the tick Rhipicephalus appendiculatus. Experimental Parasitology 15, 527-555.
MARTIN,
H., AND VIDLER,
B. 1962.
In vitro
growth
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tick tissues (Rhipicephalus appendiculatus Neumann, 1901) . Experimental Parasitology 12, 192-203. NEW, D. 1970. Culture of fetuses in vitro. In “Advances in the Biosciences. Vol. 6. Schering Symposium on Intrinsic and Extrinsic Factors in Early Mammalian Development, Venice, 1970” (G. Raspe, ed.), pp. 367-380. Pergamon Press, Vieweg. NEW, D., AND COPPOLA, P. 1970a. Development of explanted rat fetuses in hyperbaric oxygen. Teratology 3, 153-162. NEW, D., AND COPPOLA, P. 1970b. Effects of different oxygen concentrations on the development of rat embryos in culture. Journal of Reproductive Fertility 21, 109-118. NEW, D., COPPOLA, P., AND TERRY, S. 1973. Culture of explanted rat embryos in rotating tubes. Journal of Reproductive Fertility 35, 135-138. PESTRONK, A., DRACHMAN, D., AND GRIFFIN, J. 1976. Effect of muscle disuse on acetylcholine receptors. Nature 260, 352-353. REHACEK, J., AND BFZOSTOWSKI, H. 1969. A tick tissue culture medium based on analyses of tick hemolymph. Journal of Insect Physiology 15, 431436. REYNOLDS, J. 1966. The effect of hydrocortisone on the growth of chick bone rudiments in chemically defined medium. Experimental Cell Research 41, 174-189. RIEK, R. 1968. Babesiosis, In “Infectious Blood Diseases of Man and Animals” (D. Weinmann and M. Ristic, eds.), Chap. 19. Academic Press, New York. of
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BARNETT
SCHRYVER, cortisone
H.
1965.
on
the
The
water
influence uptake
of
of hydroembryonic
chick tibio-tarsi in organ culture. Experimental Cell Research 37, 327-337. TILL, W. 1961. A contribution to the anatomy and histology of the brown ear tick, Rhipicephalus appendiculatus ( Neumann). Memoirs of the Entomological Society of South Africa, N’o. 6. TSUR, I., HADANI, A., AND PIPANO, E. 1960. Nuttallia danii n.sp .--a haemoprotozoon from the gerbil (Meriones tristrami shawii). Refuah Veterinarith, Quarterly of the Israel Veterinary Medical Association 17, 236-244. VARMA, N., PUDNEY, M., AND LEAKE, C. 1975. The establishment of three cell lines from the tick Rhipicephalus appendiculatus and their infection with some arboviruses. Journal of Medical Entomology 11, 698-706. VAUGHN, J. 1971. Cell cuture media and methods. In “Invertebrate Tissue Culture” (C. Vago, ed.), Chap. 1. Academic Press, New York. WEISSMANN, G., AND DINGLE, J. 1961. Release of lysosomal protease by ultraviolet irradiation and inhibition by hydrocortisone. Experimental Cell Research 25, 207-210. WILDENTHAL, K. 1971. Long-term maintenance of SpontaneousIy beating mouse hearts in organ culture. Journal of Applied Physiology 30, 153157. WILKINSON, P. 1971. Termination of diapause in laboratory-reared Dermacentor andersoni adults. In ‘Proceedings of the 3rd International Congress of Acarology, Prague.” pp. 803-805. International Publications Service.
