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Control of 36Cl uptake by isolated salivary glands of the lone star tick
J. Insect
Physiol.,1974, Vol. 20, pp. 1771 to 1778. Perganwm Press. Printed in Great Britain
CONTROL OF =Cl UPTAKE BY ISOLATED SALIVARY GLANDS OF THE LONE STAR TICK* J. R. SAUER, J. H. FRICK,
and J. A. HAIR
Department of Entomology, Oklahoma State University, Stillwater, Oklahoma 74074, U.S.A. (Received 12 February 1974) Abstract-Salivary glands obtained from non-feeding, adult females of the lone star tick, Amblyomma amticamtm, did not take up significant amounts of Wl after 30 min incubation in vitro. Adenosine-3’,5’-monophosphate (cyclicAMP) and theophylhne slightly enhanced uptake in glands obtained from non-feeding ticks. Cyclic-AMP, theophylline, adrenaline, noradrenaline, and dopamine were potent stimulatants of *sCl uptake; but pilocarpine and glutamate were not stimulanta of uptake in glands obtained from adult females undergoing the rapid last phase of engorgment. Small amounts of haemolymph obtained from engorging females failed to stimulate uptake as compared to control experimental glands that were incubated in the absence of haemolymph. INTRODUCTION
THE SALIVARYglands of hard ticks play a vital rBle in ion and water elimination while the parasite is feeding on the host (GREGSON,1967; TATCHELL, 1969; SAUER and HAIR, 1972; KAUFMANand PHILLIPS, 1973a, b, c). KAUFMANand PHILLIPS (1973b) postulated that salivation occurs by means of a secretory rather than a filtration-resorption mechanism in Dermucentor andersoni, and that control of fluid secretion is probably neural rather than hormonal. The basis for the latter tentative hypothesis was the following: natural haemolymph from salivating ticks did not trigger secretion; 5-hydroxytryptamine (S-HT) and adenosine-3’,5’-monophosphate (cyclic-AMP), known stimulants of hormone mediated secretion in arthropod salivary glands (BERRIDGE, 1970), were weak or non-stimulatory ; but adrenaline, noradrenaline, and dopamine were highly stimulatory. These authors (KAUFMANand PHILLIPS, 1973~) have suggested that a chloride pump may be the principal driving force for fluid secretion. BARKERet al. (1973) have demonstrated that oral secretions obtained from artificially stimulated lone star ticks, Amblyomma awicanum, also contain large amounts of chloride. Recent morphological evidence (KIRKLAND, 1971) suggests that the Type I alveoli in the salivary glands of the rabbit tick, Haemophysalis leporispalustris, or Type III in D. anakrsuni (MEREDITH and KAUFMAN, 1973) possess the usual
* Journal article No. 2804 of the Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74074. This research was supported in part by Grant No. 36420 from the National Science Foundation. 1771
1772
J. R. SAUER,J. H. FRICK, ANDJ. A. HAIR
features of fluid-transporting epithelia, extensive infoldings of the plasma membrane (DIAMOND and BOSSERT, 1967, 1968; BERRIDGE and OSCHMAN, 1969). Similar features have been observed in glands obtained from the lone star tick (BARKER, in preparation). This model proposes the formation of local osmotic gradients within basal infoldings of the tissue brought about, in part, by active uptake of solutes. In view of the possible significance of movements of chloride into cells of the salivary gland epithelium, we decided to investigate the effects of S-HT, cyclicAMP, and theophylline on the uptake of WZl by isolated glands obtained from both engorged and unengorged A. americanum, and the effects of catecholamine compounds, haemolymph, pilocarpine, and glutamate on uptake in glands obtained from engorging A. americanum. MATERIALS
AND METHODS
Salivary glands were carefully removed from 3 to &week-old unfed females and from partially engorged female lone star ticks, A. americanum, after excising the anterior dorsal cuticle and exposing the contents of the haemocoele. In individual experiments pairs of glands were removed from either 4 unfed ticks or one of the pair from a partially engorged female and placed into a pre-aerated medium containing the following components: NaCl, 7.65 g; NaH,PO,H,O, 2.35 g; Na,HP0,.7H,O, 2.35 g; KHCO,, 1.92 g; CaCl,, 0.39 g; MgSO,, 0.34 g; glucose, 5.88 g; inositol, 0.47 g; and bovine albumin, 0.10 g/kg Ha0 with a pH of 6.8 and a freezing-point depression of - 0.675”C. The latter closely approximates that of lone star tick haemolymph as reported by SHIH et al. (1973). Relative amounts of salts and sugars were based on the suggestions of REHACEKand BRZOSTOWSKI(1969). The glands were preincubated for 15 min and transferred to 100 ~1 of saline containing l-58 &i of 36Cl (Amersham-Searle). Radioactive chloride was added in the form of Na3’%l. In addition, 0.044 g/l. of penicillin G sodium and 0.15 g/l. of streptomycin sulphate (Mann Research, Inc.) were added to the incubation medium to inhibit possible microbial contamination. 5-HT, cyclic-AMP, and anhydrous theophylline used in experiments were obtained from Nutritional Biochemicals. L-Adrenaline, dopamine (HCI), Lnoradrenaline, and pilocarpine nitrate used in experiments were obtained from K & K Laboratories. The incubation vessel with medium and glands were placed in an Eberbach Shaker and held in a B.O.D. chamber at 37°C. The medium with glands was agitated on the shaker for the duration of the experiment. At the end of the incubation period the glands were removed, placed in a cloth basket (FRICK et al., in preparation), washed twice in cold saline solution (minus albumin), and homogenized in O-2 ml of O-1 M sodium phosphate buffer, pH 6% Total protein and the was determined (LOWRY et al., 1951) on aliquots of the homogenate remainder was treated with 2.2 vol. of absolute ethanol and allowed to stand overnight at 4°C. Aliquots of the ethanol solution were removed and analysed for radioactivity with a Nuclear-Chicago Gas-flow Planchette Counter.
1773
36c1 UPTAKE BY SALIVARY GLANDS OF LONE STAR TICK
RESULTS
Uptake rate of 3eCl by salivary glands
The rate of s6Cl uptake by the glands of the unengorged ticks is presented in Fig. 1. Chloride rapidly accumulates in the tissue during the first 15 to 30 min. Very little increase in the uptake of 36C1 is observed between 30 and 60 min of incubation. 600.;
14) A
500-
Tl,me.
min
FIG. 1. Uptake rate of 3‘JC1by salivary glands obtained from non-feeding females of A. americanurn.
The inulin space of salivary glands from unfed ticks is approximately 22.4 per cent of the total tissue water of glands taken from unfed females after incubation for 30 min with 14C-inulin (FRICK et al., in preparation). The average weight of one tick salivary gland is approximately 45 pg. The average total protein is equivalent to about 6 pg. Taking the average medium radioactivity (15,000 counts/ min per ~1) into account, the 36Cl in glands (Fig. 1, Table 1) at the end of 30 min incubation is approximately that which can be accounted for (442 counts/min) by the estimated inulin space. This suggests that there is little cellular uptake of chloride by glandular tissue from unengorged ticks when it is incubated for 30 min in Ringer’s saline. Effects of 5-HT
5-HT is a potent stimulant of fluid secretion in arthropod fluid-secreting epithelia (BERRIDGEand PATEL, 1968; MADDRELL,1972; PRINCE and BERRIDGE, 1972). Part of the mode of action of 5-HT across insect salivary glands is to stimulate a serosal to mucosal movement of anions (BERRIDGEand PRINCE, 1972). On the other hand, 5-HT is a weak stimulant of fluid secretion in isolated salivary glands of the tick D. andersoni (KAUFMAN and PHILLIPS, 1973b). When 8 x 1O-4 M 5-HT is added to the bathing medium of isolated glands of the unengorged lone star ticks, there is an apparently higher uptake of Yl after 30 min of incubation,
J. R. SAUER, J. H. FRICK, ANDJ. A. HAIR
1774
TABLE ~-EFFECTS OF DRUGSON UPTAKEOF “C1/20 pg PROTEINPER 30 min BY ISOLATED SALIVARY GLANDSFROMLONESTARTICKS Countsfmin per 20 f”g protein Counts/min per 20 fig protein less (+ S.E.)* estimated inulin space uptake
Drug
pt
Unfed females Control S-HT 8 x 1O-4 M lo-” M Cyclic-AMP (5 x 1O-3 M) Theophylline (7 x 1O-s M)
425 + 65 (12) 561+ 63 (15) 484f 115 (5) 724 + 50 (7)
119 42 282
NSf NSf < 0.01
950f
508
< 0.001
111 (12) Engorging females
Control S-HT (8 x lo-’ M) Cyclic-AMP (5 x 1O-3 M) Theophylline (7 x 1O-s M) Adrenaline (10-s M) Noradrenaline (1O-6 M) Dopamine (1O-6 M) Pilocarpine (10-s M) Haemolymph (0.5-2.2 /.d/50 ~1 bathing medium Glutamate (1O-3 M)
2712 f 565 (13) 4765 + 630 (7)
1536 3589
< 0.05
12,992 + 1929 (15)
11,816
< 0.001
20,524 f 4322 (7)
19,348
< 0.001
14,105 f 7710 (6)
12,929
< 0.05
13,535 * 5000 (7)
12,359
< 0.01
9854 f 3850 (6)
8340
4007 f 947 (6)
2831
NSf
1634 + 211 (7)
458
NSf
1223 + 79 (4)
47
NSf
* Numerals in parentheses indicate numbers of experiments. t t-test. 1 At P< 0.05 level.
but the mean is not significantly different from the control mean (Table 1). Also, when lesser amounts (lOmg M) of 5HT were added to the bathing medium the effect of the addition failed to stimulate uptake as compared to the controls.
Eflects of cyclic-AMP and theophylline The effect of cyclic-AMP and theophylline salivary glands is presented in Table 1. When
upon the uptake of chloride by the 5 x 1O-3 M cyclic-AMP was added
“cl
UPTAKE
to the bathing medium, per pg protein, or about addition of 7 x 1O-3 M of chloride by the tissue
BY
SALIVARY
GLANDS
OF LONE
STAR
TICK
1775
the average amount of radioactivity was 724 counts/min la7 times that taken up by control glands. Likewise, the theophylline to the system stimulates the accumulation of unengorged ticks.
Uptake of 36CI by glands of engorged ticks
The salivary glands of Ixodidae are also known to undergo morphological changes at the onset of feeding, which coincides with the time when they are beginning to function in fluid elimination, in vivo (KIRKLAND, 1971). We have observed an approximate eightfold increase in glandular protein of glands obtained from females undergoing the rapid phase of engorgment, and an approximate twenty-eightfold increase in weight (FRICK et al., in preparation). The inulin space of these salivary glands is approximately 17 per cent of the total tissue water after incubation for 30 min in a medium containing lPC-inulin (FRICK et al., in preparation). Using the same reasoning as that employed earlier for glands obtained from unfed ticks, the estimated inulin space uptake of 3sC1under present conditions by glands obtained from engorging ticks is approximately 1176 counts/ min. The difference between inulin space and actual uptake (I= 2484) (Table 1) indicates that an apparent amount of chloride is taken up by unstimulated glands. This is in contrast to the minimal uptake observed in glands obtained from nonfeeding ticks. Additions of 8 x lo-’ M 5-HT slightly enhanced the uptake of 36C1as compared to the control glands (Table 1). After the addition of 5 x 10m3M cyclic-AMP and 7 x 10~~ M theophylline the amounts of 36C1 taken up were significantly greater than the controls. Additions of haemolymph from females in their rapid phase of engorgement failed to stimulate uptake as compared to the controls (Table 1). These results, however, must be viewed with caution because it was impossible to obtain substantial amounts of haemolymph, even when pooled from numbers of feeding ticks (0.5 to 2.2 ~1 from 5 to 6 ticks). The haemolymph was necessarily greatly diluted when added to the bathing medium. Also, time (15-20 min) was required to collect the haemolymph; and inactivation of possible ‘factors’ cannot be completely ruled out. Injections of the parasympathetic stimulant pilocarpine into engorged hard ticks have been used to stimulate oral secretions (BARKERet al., 1973), but the addition of 10~~ M of this drug to the system failed to enhance the uptake of chloride significantly (Table 1). Additions of the potentially neuroactive substance glutamate (Table 1) failed to enhance uptake; apparently, this drug reduced uptake as compared to the controls, but the variations were such that a significant difference between the two means could not be shown. Because adrenaline, noradrenaline, and dopamine are potent stimulants of salivary secretion in glands obtained from feeding D. andersoni we tested their effect on 36Cl uptake by glands obtained from engorging A. awicanum. All three are potent stimulants of 36Cl uptake as compared to the controls (Table 1).
1776
J. FL SAWER, J. H. FRICK,ANDJ. A. HAIR
DISCUSSION KAUFMANand PHILLIPS (1973~) suggested that the glandular epithelium in D. andevsoni contains a specific chloride pump which may be the principal driving force for fluid secretion. The oral secretions obtained from engorged or partially engorged artificially stimulated lone star ticks contain a large amount of chloride (BARKERet al., 1973), the concentration of which is higher than that measured in the haemolymph of feeding ticks (Hsu and SAUER,in preparation). If the suggested r81e of chloride movements across the cells is responsible for generating fluid movement, then it is probable that control of chloride uptake is essential to the control of fluid secretion. The fact that glands of non-feeding (and non-secreting) ticks fail to exhibit chloride uptake is consistent with this hypothesis. The movement of solutes has been discussed relative to the standing gradient hypothesis (DIAMONDand BOSSERT,1967, 1968), which has been used to explain iso-osmotic flow across fluid transporting epithelia. This hypothesis requires the removal of solutes from the cell’s basal infoldings, creating an area of low solute concentration in the channel and high concentration in the cell. Because of spatial apicalbasal separation, water can then enter the cell osmotically before the solute has a chance to diffuse away. The present results are consistent with this hypothesis. The lack of stimulatory effect by pilocarpine on chloride uptake is interesting because injections of this cholinergic drug in viva stimulate lone star ticks (BARKER et al., 1973) as well as other ticks (HOWELL, 1966 ; TATCHELL,1967 ; PURNELLet al., 1969) to produce oral secretions. As KAUFMANand PHILLIPS (1973b) suggest, the concentrations used to cause this effect in viva are very high and the drug may exert its effect either on the gut epithelium or activate neurons in the CNS, which then stimulate efferent axons innervating the salivary glands. A constituent of the medium used in all experiments was bovine albumin. BERRIDGEand PATEL (1968) have demonstrated that minute quantities of 5-HT are sometimes associated with this protein. It may be that in the case of glands obtained from engorging ticks, a sufficiently high concentration of S-HT adhering to the protein was sufficient to stimulate a small amount of uptake above that of the estimated inulin space. We did not attempt to detect the presence of 5-HT in our bovine albumin nor try experiments in the absence of the protein. The amounts’ needed to stimulate insect salivary secretion by BERRIDGEand PATEL were quite high, and only high concentrations of the biogenic amine apparently stimulate chloride uptake. Also, the albumin was a regular constituent of the medium used in all experiments, and any possible effect would likely be the same for all. The results of these experiments tend to corroborate the results of KAUFMAN and PHILLIPS, who suggest that control of fluid secretion in ticks may be neural rather than hormonal, and that the transmitter is a catecholaminergic substance. The evidence to support the latter conclusion is the observed stimulatory effect of adrenaline, noradrenaline, and dopamine on chloride uptake but absence of stimulatory effect after additions of high concentrations of pilocarpine and glutamate. The recent observations by COONS and ROSHDY (1973) indicating the
“Cl UPTAKEBY SALIVARY GLANDSOF LONESTARTICK
1777
presence of axons with neurosecretory material in close association with the a&i in unfed males of Dermacentor vu&b& may suggest other possibilities and may help explain why stimulatory ‘factors’ cannot be found in the haemolymph. Catecholaminergic molecules could stimulate these axons to release neurosecretory material locally in the sense described by MADDRELL (1968), which could affect functioning of the salivary glands. It should prove interesting to see if similar features are normally present in females and what changes may occur during feeding. The effectiveness as reported here of cyclic-AMP to stimulate chloride uptake in A. americanurn is interesting. Cyclic-AMP has been implicated in many cells as a link between the primary (hormonal) message and the effector system itself (RASMUSSEN, 1970). In insects, for example, cyclic-AMP and theophylline stimulate fluid secretion into isolated salivary glands, mimicking the effect produced by S-HT (BERRIDGE,1970). On the other hand, cyclic-AMP and theophylline are incapable of stimulating fluid secretion in isolated salivary glands of D. a&s& (KAUFMAN and PHILLIPS, 1973b). With the negative results with L). andersoni in mind, the effect of these compounds upon the uptake of 86C1by glands from A. awicanum was unexpected. It may be that the basal membrane of the secretory cell in Dermacentor is only very slightly permeable to exogenous cyclic-AMP, or that the events leading to the final elaboration of the fluid in ixodid ticks is more complex than that described for the insect salivary glands by BERRIDGE (1970). Also, of course, differences in the mechanisms of fluid secretion between the two species of ticks cannot be ruled out. Acknowledgement-Appreciation is extended to Ms. FRANCZNEMARKET for expert technical assistance. Sincere thanks are given to Drs. C. G. Bw, T. L. ARCHER,and R. BERBERETfor critically reviewing the manuscript.
REFERENCES BARKERR. W., BIJRRISE., SAUERJ. R., and HAIR J. A. (1973) Composition of tick oral secretions obtained by three different coIIection methods. r. med. Ent. 10, 198-201. BERRIDGEM. J. (1970) The role of S-hydroxytryptamine and cyclic AMP in the control of fluid secretion by isolated salivary glands. J. eJcp. Biol. 53, 171-186. BERRIDGEM. J. and OSCHMAN J. L. (1969) A structural basis for fluid secretion by MaIpighian tubules. Tissue &f Cell 1, 247-272. BERRIDCEM. J. and PATELN. G. (1968) Insect salivary glands: stimulation of fluid secretion by 5hydroxytryptamine and adenosine 3’,5’-monophosphate. Science, Wash. 162,
462-463. BERRIDGEM. J. and PRINCEW. T. (1972) Transepithelial potential changes during stimulation of isolated salivary glands with 5-hydroxytryptamine and cyclic AMP. J. exg. Biol.
56,139-153. COONSL. B. and ROSHDYM. A. (1973) Fine structure of the salivary glands of unfed male Dnmacenlor variabilis (Say) (Ixodoidea: Ixodidae). J. Parasit. 59, 900-912. DIAMONDJ. M. and BOSSERTW. H. (1967) Standing-gradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia. J. gen. Physiol. 50, 2061-2083. DIAMONDJ. M. and BO~~ERTW. H. (1968) Functional consequences of ultrastructural geometry in ‘backwards’ fluid transporting epithelia. g. Cell Biol. 37, 694-702. GREGSONJ. D. (1967) Observations on the movement of fluids in the vicinity of the mouthparts of naturally feeding Dermacentor amiersoni Stiles. Parasitology 57, l-8. 58
1778
J. R. SA~JER,J. H. FRICK, ANDJ. A. HAIR
HOWELL C. J. (1966) Collection of salivary gland secretion from the argasid Ornithodorus savignyi by use of a pharmacological stimulant. J. S. Afr. vet. med. Assoc. 37, 236-239. KAUFMANW. R. and PHILLIPS J. E. (1973a) Ion and water balance in the ixodid tick Dermacentor andersoni-I. Routes of ion and water excretion. r. exp. Biol. 58, 523-536. KAUFMANW. R. and PHILLIPS J. E. (197313) Ion and water balance in the ixodid tick Dermacentor andersoni-II. Mechanism and control of salivary secretion. J. exp. Biol. 58, 537-547. KAUFMANW. R. and PHILLIPS J. E. (1973~) Ion and water balance in the ixodid tick Dermacentor andersoni-III. Influence of monovalent ions and osmotic pressure on salivary secretion. J. exp. Biol. 58, 549-564. KIRKLANDW. L. (1971) Ultrastructural changes in the nymphal salivary glands of the rabbit tick, Haemaphysalis leporispalustris, during feeding. J. Insect Physiol. 17, 1933-1946. LOWRY 0. H., ROSEBROUGH N. J., FARR A. L., and RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. r. biol. Chem. 193, 265-275. MADDRELLS. H. P. (1968) Neurosecretion in insects. In Insects and Physiology (Ed. by J. W. L. BEAMENTand J. E. TFSHEFNE), pp. 103-l 18. Elsevier, New York. MADDRJXLLS. H. P. (1972) The mechanisms of insect excretory systems. Adv. Insect Physiol. ,8, 199-331. MEREDITHJ. and KAUFMANW. R. (1973) A proposed site of fluid secretion in the salivary gland of the ixodid tick Dermacentor andersoni. Parasitology 67, 205-217. PRINCE W. T. and BERRIDCEM. J. (1972) The effects of S-hydroxytryptamine and cyclic AMP on the potential profile across isolated salivary glands. J. exp. Biol. 56, 323-333. PURNELLR. E., BRANAGAN D., and RADLEYD. E. (1969) The use of parasympathomimetic drugs to stimulate salivation in the tick Rhipicephulus appendiculatus, and the transmission of Theileria parva using saliva obtained by this method from infected ticks. Parasitology 59, 709-718. RASMUSSENH. (1970) Cell communication, calcium ion, and cyclic adenosine monophosphate. Science, Wash. 170, 404-412. REHACEKJ. and BRZOSTOWSKI H. W. (1969) A tick tissue culture medium based on analyses of tick haemolymph. r. Insect Physiol. 15, 1431-1436. SAUERJ. R. and HAIR J. A. (1972) The quantity of blood ingested by the lone star tick (Acarina: Ixodidae). Ann. ent. Sot. Am. 65, 1065-1068. SHIH C., SAIJERJ. R., EIKENBARYP., HAIR J. A., and FRICK J. H. (1973) The effects of desiccation and rehydration on the lone star tick. J. Insect Physiol. 19, 505-514. TATCHELL R. J. (1967) A modified method for obtaining tick oral secretion. J. Parasit. 53, 1106-1107. TATCHELLR. J. (1969) The ionic regulatory r8le of the salivary secretions of the cattle tick, Boophilus microplus. J. Insect Physiol. 15, 1421-1430.
Physiol.,1974, Vol. 20, pp. 1771 to 1778. Perganwm Press. Printed in Great Britain
CONTROL OF =Cl UPTAKE BY ISOLATED SALIVARY GLANDS OF THE LONE STAR TICK* J. R. SAUER, J. H. FRICK,
and J. A. HAIR
Department of Entomology, Oklahoma State University, Stillwater, Oklahoma 74074, U.S.A. (Received 12 February 1974) Abstract-Salivary glands obtained from non-feeding, adult females of the lone star tick, Amblyomma amticamtm, did not take up significant amounts of Wl after 30 min incubation in vitro. Adenosine-3’,5’-monophosphate (cyclicAMP) and theophylhne slightly enhanced uptake in glands obtained from non-feeding ticks. Cyclic-AMP, theophylline, adrenaline, noradrenaline, and dopamine were potent stimulatants of *sCl uptake; but pilocarpine and glutamate were not stimulanta of uptake in glands obtained from adult females undergoing the rapid last phase of engorgment. Small amounts of haemolymph obtained from engorging females failed to stimulate uptake as compared to control experimental glands that were incubated in the absence of haemolymph. INTRODUCTION
THE SALIVARYglands of hard ticks play a vital rBle in ion and water elimination while the parasite is feeding on the host (GREGSON,1967; TATCHELL, 1969; SAUER and HAIR, 1972; KAUFMANand PHILLIPS, 1973a, b, c). KAUFMANand PHILLIPS (1973b) postulated that salivation occurs by means of a secretory rather than a filtration-resorption mechanism in Dermucentor andersoni, and that control of fluid secretion is probably neural rather than hormonal. The basis for the latter tentative hypothesis was the following: natural haemolymph from salivating ticks did not trigger secretion; 5-hydroxytryptamine (S-HT) and adenosine-3’,5’-monophosphate (cyclic-AMP), known stimulants of hormone mediated secretion in arthropod salivary glands (BERRIDGE, 1970), were weak or non-stimulatory ; but adrenaline, noradrenaline, and dopamine were highly stimulatory. These authors (KAUFMANand PHILLIPS, 1973~) have suggested that a chloride pump may be the principal driving force for fluid secretion. BARKERet al. (1973) have demonstrated that oral secretions obtained from artificially stimulated lone star ticks, Amblyomma awicanum, also contain large amounts of chloride. Recent morphological evidence (KIRKLAND, 1971) suggests that the Type I alveoli in the salivary glands of the rabbit tick, Haemophysalis leporispalustris, or Type III in D. anakrsuni (MEREDITH and KAUFMAN, 1973) possess the usual
* Journal article No. 2804 of the Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74074. This research was supported in part by Grant No. 36420 from the National Science Foundation. 1771
1772
J. R. SAUER,J. H. FRICK, ANDJ. A. HAIR
features of fluid-transporting epithelia, extensive infoldings of the plasma membrane (DIAMOND and BOSSERT, 1967, 1968; BERRIDGE and OSCHMAN, 1969). Similar features have been observed in glands obtained from the lone star tick (BARKER, in preparation). This model proposes the formation of local osmotic gradients within basal infoldings of the tissue brought about, in part, by active uptake of solutes. In view of the possible significance of movements of chloride into cells of the salivary gland epithelium, we decided to investigate the effects of S-HT, cyclicAMP, and theophylline on the uptake of WZl by isolated glands obtained from both engorged and unengorged A. americanum, and the effects of catecholamine compounds, haemolymph, pilocarpine, and glutamate on uptake in glands obtained from engorging A. americanum. MATERIALS
AND METHODS
Salivary glands were carefully removed from 3 to &week-old unfed females and from partially engorged female lone star ticks, A. americanum, after excising the anterior dorsal cuticle and exposing the contents of the haemocoele. In individual experiments pairs of glands were removed from either 4 unfed ticks or one of the pair from a partially engorged female and placed into a pre-aerated medium containing the following components: NaCl, 7.65 g; NaH,PO,H,O, 2.35 g; Na,HP0,.7H,O, 2.35 g; KHCO,, 1.92 g; CaCl,, 0.39 g; MgSO,, 0.34 g; glucose, 5.88 g; inositol, 0.47 g; and bovine albumin, 0.10 g/kg Ha0 with a pH of 6.8 and a freezing-point depression of - 0.675”C. The latter closely approximates that of lone star tick haemolymph as reported by SHIH et al. (1973). Relative amounts of salts and sugars were based on the suggestions of REHACEKand BRZOSTOWSKI(1969). The glands were preincubated for 15 min and transferred to 100 ~1 of saline containing l-58 &i of 36Cl (Amersham-Searle). Radioactive chloride was added in the form of Na3’%l. In addition, 0.044 g/l. of penicillin G sodium and 0.15 g/l. of streptomycin sulphate (Mann Research, Inc.) were added to the incubation medium to inhibit possible microbial contamination. 5-HT, cyclic-AMP, and anhydrous theophylline used in experiments were obtained from Nutritional Biochemicals. L-Adrenaline, dopamine (HCI), Lnoradrenaline, and pilocarpine nitrate used in experiments were obtained from K & K Laboratories. The incubation vessel with medium and glands were placed in an Eberbach Shaker and held in a B.O.D. chamber at 37°C. The medium with glands was agitated on the shaker for the duration of the experiment. At the end of the incubation period the glands were removed, placed in a cloth basket (FRICK et al., in preparation), washed twice in cold saline solution (minus albumin), and homogenized in O-2 ml of O-1 M sodium phosphate buffer, pH 6% Total protein and the was determined (LOWRY et al., 1951) on aliquots of the homogenate remainder was treated with 2.2 vol. of absolute ethanol and allowed to stand overnight at 4°C. Aliquots of the ethanol solution were removed and analysed for radioactivity with a Nuclear-Chicago Gas-flow Planchette Counter.
1773
36c1 UPTAKE BY SALIVARY GLANDS OF LONE STAR TICK
RESULTS
Uptake rate of 3eCl by salivary glands
The rate of s6Cl uptake by the glands of the unengorged ticks is presented in Fig. 1. Chloride rapidly accumulates in the tissue during the first 15 to 30 min. Very little increase in the uptake of 36C1 is observed between 30 and 60 min of incubation. 600.;
14) A
500-
Tl,me.
min
FIG. 1. Uptake rate of 3‘JC1by salivary glands obtained from non-feeding females of A. americanurn.
The inulin space of salivary glands from unfed ticks is approximately 22.4 per cent of the total tissue water of glands taken from unfed females after incubation for 30 min with 14C-inulin (FRICK et al., in preparation). The average weight of one tick salivary gland is approximately 45 pg. The average total protein is equivalent to about 6 pg. Taking the average medium radioactivity (15,000 counts/ min per ~1) into account, the 36Cl in glands (Fig. 1, Table 1) at the end of 30 min incubation is approximately that which can be accounted for (442 counts/min) by the estimated inulin space. This suggests that there is little cellular uptake of chloride by glandular tissue from unengorged ticks when it is incubated for 30 min in Ringer’s saline. Effects of 5-HT
5-HT is a potent stimulant of fluid secretion in arthropod fluid-secreting epithelia (BERRIDGEand PATEL, 1968; MADDRELL,1972; PRINCE and BERRIDGE, 1972). Part of the mode of action of 5-HT across insect salivary glands is to stimulate a serosal to mucosal movement of anions (BERRIDGEand PRINCE, 1972). On the other hand, 5-HT is a weak stimulant of fluid secretion in isolated salivary glands of the tick D. andersoni (KAUFMAN and PHILLIPS, 1973b). When 8 x 1O-4 M 5-HT is added to the bathing medium of isolated glands of the unengorged lone star ticks, there is an apparently higher uptake of Yl after 30 min of incubation,
J. R. SAUER, J. H. FRICK, ANDJ. A. HAIR
1774
TABLE ~-EFFECTS OF DRUGSON UPTAKEOF “C1/20 pg PROTEINPER 30 min BY ISOLATED SALIVARY GLANDSFROMLONESTARTICKS Countsfmin per 20 f”g protein Counts/min per 20 fig protein less (+ S.E.)* estimated inulin space uptake
Drug
pt
Unfed females Control S-HT 8 x 1O-4 M lo-” M Cyclic-AMP (5 x 1O-3 M) Theophylline (7 x 1O-s M)
425 + 65 (12) 561+ 63 (15) 484f 115 (5) 724 + 50 (7)
119 42 282
NSf NSf < 0.01
950f
508
< 0.001
111 (12) Engorging females
Control S-HT (8 x lo-’ M) Cyclic-AMP (5 x 1O-3 M) Theophylline (7 x 1O-s M) Adrenaline (10-s M) Noradrenaline (1O-6 M) Dopamine (1O-6 M) Pilocarpine (10-s M) Haemolymph (0.5-2.2 /.d/50 ~1 bathing medium Glutamate (1O-3 M)
2712 f 565 (13) 4765 + 630 (7)
1536 3589
< 0.05
12,992 + 1929 (15)
11,816
< 0.001
20,524 f 4322 (7)
19,348
< 0.001
14,105 f 7710 (6)
12,929
< 0.05
13,535 * 5000 (7)
12,359
< 0.01
9854 f 3850 (6)
8340
4007 f 947 (6)
2831
NSf
1634 + 211 (7)
458
NSf
1223 + 79 (4)
47
NSf
* Numerals in parentheses indicate numbers of experiments. t t-test. 1 At P< 0.05 level.
but the mean is not significantly different from the control mean (Table 1). Also, when lesser amounts (lOmg M) of 5HT were added to the bathing medium the effect of the addition failed to stimulate uptake as compared to the controls.
Eflects of cyclic-AMP and theophylline The effect of cyclic-AMP and theophylline salivary glands is presented in Table 1. When
upon the uptake of chloride by the 5 x 1O-3 M cyclic-AMP was added
“cl
UPTAKE
to the bathing medium, per pg protein, or about addition of 7 x 1O-3 M of chloride by the tissue
BY
SALIVARY
GLANDS
OF LONE
STAR
TICK
1775
the average amount of radioactivity was 724 counts/min la7 times that taken up by control glands. Likewise, the theophylline to the system stimulates the accumulation of unengorged ticks.
Uptake of 36CI by glands of engorged ticks
The salivary glands of Ixodidae are also known to undergo morphological changes at the onset of feeding, which coincides with the time when they are beginning to function in fluid elimination, in vivo (KIRKLAND, 1971). We have observed an approximate eightfold increase in glandular protein of glands obtained from females undergoing the rapid phase of engorgment, and an approximate twenty-eightfold increase in weight (FRICK et al., in preparation). The inulin space of these salivary glands is approximately 17 per cent of the total tissue water after incubation for 30 min in a medium containing lPC-inulin (FRICK et al., in preparation). Using the same reasoning as that employed earlier for glands obtained from unfed ticks, the estimated inulin space uptake of 3sC1under present conditions by glands obtained from engorging ticks is approximately 1176 counts/ min. The difference between inulin space and actual uptake (I= 2484) (Table 1) indicates that an apparent amount of chloride is taken up by unstimulated glands. This is in contrast to the minimal uptake observed in glands obtained from nonfeeding ticks. Additions of 8 x lo-’ M 5-HT slightly enhanced the uptake of 36C1as compared to the control glands (Table 1). After the addition of 5 x 10m3M cyclic-AMP and 7 x 10~~ M theophylline the amounts of 36C1 taken up were significantly greater than the controls. Additions of haemolymph from females in their rapid phase of engorgement failed to stimulate uptake as compared to the controls (Table 1). These results, however, must be viewed with caution because it was impossible to obtain substantial amounts of haemolymph, even when pooled from numbers of feeding ticks (0.5 to 2.2 ~1 from 5 to 6 ticks). The haemolymph was necessarily greatly diluted when added to the bathing medium. Also, time (15-20 min) was required to collect the haemolymph; and inactivation of possible ‘factors’ cannot be completely ruled out. Injections of the parasympathetic stimulant pilocarpine into engorged hard ticks have been used to stimulate oral secretions (BARKERet al., 1973), but the addition of 10~~ M of this drug to the system failed to enhance the uptake of chloride significantly (Table 1). Additions of the potentially neuroactive substance glutamate (Table 1) failed to enhance uptake; apparently, this drug reduced uptake as compared to the controls, but the variations were such that a significant difference between the two means could not be shown. Because adrenaline, noradrenaline, and dopamine are potent stimulants of salivary secretion in glands obtained from feeding D. andersoni we tested their effect on 36Cl uptake by glands obtained from engorging A. awicanum. All three are potent stimulants of 36Cl uptake as compared to the controls (Table 1).
1776
J. FL SAWER, J. H. FRICK,ANDJ. A. HAIR
DISCUSSION KAUFMANand PHILLIPS (1973~) suggested that the glandular epithelium in D. andevsoni contains a specific chloride pump which may be the principal driving force for fluid secretion. The oral secretions obtained from engorged or partially engorged artificially stimulated lone star ticks contain a large amount of chloride (BARKERet al., 1973), the concentration of which is higher than that measured in the haemolymph of feeding ticks (Hsu and SAUER,in preparation). If the suggested r81e of chloride movements across the cells is responsible for generating fluid movement, then it is probable that control of chloride uptake is essential to the control of fluid secretion. The fact that glands of non-feeding (and non-secreting) ticks fail to exhibit chloride uptake is consistent with this hypothesis. The movement of solutes has been discussed relative to the standing gradient hypothesis (DIAMONDand BOSSERT,1967, 1968), which has been used to explain iso-osmotic flow across fluid transporting epithelia. This hypothesis requires the removal of solutes from the cell’s basal infoldings, creating an area of low solute concentration in the channel and high concentration in the cell. Because of spatial apicalbasal separation, water can then enter the cell osmotically before the solute has a chance to diffuse away. The present results are consistent with this hypothesis. The lack of stimulatory effect by pilocarpine on chloride uptake is interesting because injections of this cholinergic drug in viva stimulate lone star ticks (BARKER et al., 1973) as well as other ticks (HOWELL, 1966 ; TATCHELL,1967 ; PURNELLet al., 1969) to produce oral secretions. As KAUFMANand PHILLIPS (1973b) suggest, the concentrations used to cause this effect in viva are very high and the drug may exert its effect either on the gut epithelium or activate neurons in the CNS, which then stimulate efferent axons innervating the salivary glands. A constituent of the medium used in all experiments was bovine albumin. BERRIDGEand PATEL (1968) have demonstrated that minute quantities of 5-HT are sometimes associated with this protein. It may be that in the case of glands obtained from engorging ticks, a sufficiently high concentration of S-HT adhering to the protein was sufficient to stimulate a small amount of uptake above that of the estimated inulin space. We did not attempt to detect the presence of 5-HT in our bovine albumin nor try experiments in the absence of the protein. The amounts’ needed to stimulate insect salivary secretion by BERRIDGEand PATEL were quite high, and only high concentrations of the biogenic amine apparently stimulate chloride uptake. Also, the albumin was a regular constituent of the medium used in all experiments, and any possible effect would likely be the same for all. The results of these experiments tend to corroborate the results of KAUFMAN and PHILLIPS, who suggest that control of fluid secretion in ticks may be neural rather than hormonal, and that the transmitter is a catecholaminergic substance. The evidence to support the latter conclusion is the observed stimulatory effect of adrenaline, noradrenaline, and dopamine on chloride uptake but absence of stimulatory effect after additions of high concentrations of pilocarpine and glutamate. The recent observations by COONS and ROSHDY (1973) indicating the
“Cl UPTAKEBY SALIVARY GLANDSOF LONESTARTICK
1777
presence of axons with neurosecretory material in close association with the a&i in unfed males of Dermacentor vu&b& may suggest other possibilities and may help explain why stimulatory ‘factors’ cannot be found in the haemolymph. Catecholaminergic molecules could stimulate these axons to release neurosecretory material locally in the sense described by MADDRELL (1968), which could affect functioning of the salivary glands. It should prove interesting to see if similar features are normally present in females and what changes may occur during feeding. The effectiveness as reported here of cyclic-AMP to stimulate chloride uptake in A. americanurn is interesting. Cyclic-AMP has been implicated in many cells as a link between the primary (hormonal) message and the effector system itself (RASMUSSEN, 1970). In insects, for example, cyclic-AMP and theophylline stimulate fluid secretion into isolated salivary glands, mimicking the effect produced by S-HT (BERRIDGE,1970). On the other hand, cyclic-AMP and theophylline are incapable of stimulating fluid secretion in isolated salivary glands of D. a&s& (KAUFMAN and PHILLIPS, 1973b). With the negative results with L). andersoni in mind, the effect of these compounds upon the uptake of 86C1by glands from A. awicanum was unexpected. It may be that the basal membrane of the secretory cell in Dermacentor is only very slightly permeable to exogenous cyclic-AMP, or that the events leading to the final elaboration of the fluid in ixodid ticks is more complex than that described for the insect salivary glands by BERRIDGE (1970). Also, of course, differences in the mechanisms of fluid secretion between the two species of ticks cannot be ruled out. Acknowledgement-Appreciation is extended to Ms. FRANCZNEMARKET for expert technical assistance. Sincere thanks are given to Drs. C. G. Bw, T. L. ARCHER,and R. BERBERETfor critically reviewing the manuscript.
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