Control of fluid secretion by isolated salivary glands of the lone star tick

r. Insect Physiol., 1975, Vol. 21, pp. 1893 to 1898.

CONTROL

OF FLUID SECRETION BY ISOLATED SALIVARY GLANDS OF THE LONE STAR TICK G.

Department

Pergamon Press. Printed in Great Britain.

of Entomology,

R.

NEEDHAM and J.

Oklahoma

State

R.

University,

SAUER Stillwater,

Oklahoma 74074,

U.S.A.

(Received 6 May 1975) Abstnact-Adrenaline, noradrenaline, cyclic-AMP, and theophylline stimulated salivary fluid secreti,>n in glands isolated from rapidly engorging females of the lone star tick, Amblyomma americanum. Cyclic-AMP and theophylline effectively initiated secretion only after the glands were pre-incubated and pre-stimulated to secrete with adrenaline. Pilocarpine nitrate, 5-hydroxytryptamine r:5-HT), and glutamate did not stimulate glandular secretion. 2,4-Dinitrophenol (DNP) slowed adrenaline’s ability to stimulate secretion. Results obtained after additions of harmaline and ouabain point to the possibility of a (Na++K+)-ATPase system for helping to move fluid across The significance of these findings to the mechanisms and control of tick the glandular epithelium. salivary glandular function is discussed. INTRODUCTION TIC= concentrate ingested blood by eliminating excess salt and water. Ixodid (hard) ticks utilize their paired salivary glands to excrete excess ions and water w:nile still attached and feeding on the host (GREGSON, 1967; TATCHELL, 1969; SAUER and HAIR, 1972; KAUFMAN and PHILLIPS, 1973a, b, c; Hsu and SAUER, 1975). KAUFMAN and PHILLIPS (1973b, c) suggested that in Dermacentor andersoni salivation occurs by means of a secreto.ry rather than a filtration-resorption mechanism and that control of fluid secretion may be neural rather than hormonal, citing the effectiveness of catecholamines in stimulating secretion. They also suggested the possible presence of a chloride pump for driving secretion. Studies by SAUER et al. (1974) on control of 38Cl uptake by isolated salivary glands of Amblyomma americanurn supported the findings of Kaufman and Phillips concerning the importance of catecholamines to glandular function, but cyclic-AMP was found to increase 36C1 uptake in A. americanurn, in contrast to the inability of this same drug to stimulate fluid secretion by glands of D. andersoni (KAUFMAN and PHILLIPS, 19’7313). Differences in the effectiveness of cyclic-AMP to glandular fu:nction in these two species prompted us to additionally investigate the effects of drugs and inhibitors on tick salivary fluid secretion in the lone star tick, A. americanum.

MAl-ERlAlS Salivary

glands

were

AND

METHODS

removed

from

female

A.

*Journal article No. 2997 of the Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma. This research was supported in part by NSF Grant No. BMS-74-24140 from the National Science Foundation.

americanum ticks undergoing the rapid last phase of engorgement while feeding on sheep. Rearing techniques have been described by PATRICK and HAIR (1975). Dissection and bathing media prepalations contained the following constituents : NaCI, 10.00 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; diluted to 1 1. volumetrically with deionized water. Salines had a pH of 6.9 and a freezing point depression of -0.777”C. All solutions were pre-oxygenated with 100% Oa for 30 min prior to use in experiments. To isolate the salivary glands the dorsal cuticle was carefully removed by making a circular lateral incision around the tick with a single edge razor blade. The gut, reproductive, nervous, and tracheal systems, along with any adhering tissue, were carefully removed to fully expose the glands and ducts. The main salivary duct was next detached from the sclerotized mouth parts close to the salivarium. A drop of the oxygenated saline was added to a disposable Petri dish filled with liquid paraffin. The gland was removed from the tick, rinsed, and placed in the drop of saline. The main duct was located and manipulated by a fine pin and drawn from the drop of Ringer saline to the surrounding liquid paraffin. During glandular secretion the fluid accumulated on the insect pin, where it was collected with a finely tapered capillary. After collection and transfer to a distant part of the liquid paraffin, the spherically shaped droplet’s diameter was measured with a dissecting microscope equipped with an ocular micrometer and its volume determined by making the appropriate calculation. Experimental protocol

1893

In preliminary

experiments

adrenaline

stimulated

1894

G. R. NEEDHAM

salivary fluid secretion at concentrations ranging from low3 to 1O-6 M. Consistent success in stimulating salivation was achieved by incorporating 10e5 M adrenaline in the gland’s bathing medium. All experiments were divided into three phases, pre-experimental, experimental, and post-experimental. The pre-experimental phase included incubation of the glands with 10e5 M adrenaline, which served the purpose of determining secretory competence of the glands. Salivary glands were exposed to a fresh or new drug-Ringer solution and rinsed at precisely the same times (every 8 min) in all phases of the experiment for statistical comparisons. Glands were incubated in the drugRinger saline for 5 of the 8 min, while the remaining 3 min were used for measuring the secreted droplet and rinsing the gland. The experimental phase tested the effects of various potential glandular stimulants and/or inhibitors. Two controls were therefore necessary. The stimulating drug types were added with the Ringer saline in the same manner as adrenaline-saline solutions in the preand post-phases ; however, potential fluid inhibitors were added in the presence of adrenaline because the glands will not secrete in the absence of a The drugs harmaline, Sstimulating factor. hydroxytryptamine (serotonin), theophylline, Lglutamate, and adenosine 3’-5’-monophosphate were purchased from Nutritional Biochemicals Corp. K & K Laboratories was the source of L-adrenaline, L-noradrenaline, ouabain, and piloN6-2’-0-dibutyryl adenosine carpine nitrate. 3’-5’-cyclic monophosphate was obtained from from Sigma Chemical Co., and 2,4_dinitrophenol Fisher

Scientific

Co.

RESULTS

Effects of potential

stimulants

Fig. 1 depicts salivary secretion when glands were incubated with a Ringer saline including lo+’ M adrenaline in the pre- and post-experimental phases of the experiment. The rate reached a maximum average of 97 nl/min in the pre-experimental phase, with considerable variation in glandular maximum secretory ability evident. In the experimental phase, which served as the control for the following experiments, secretion rapidly dropped to zero when adrenaline was deleted from the bathing medium. Results show that glandular function can be restored when adrenaline is re-applied in the post-experimental phases of the experiments. Rates averaged 71 f 28, 0, and 49 + 32, respectively (Table 1). In contrast to the above, cyclic-AMP had a stimulatory effect when added during the experimental portion of the experiment, causing glands to secrete at an average rate of 124+42 nl/min (Table 1). This contrasts with results obtained by

J. R. SAL-ER

AND

PM-

EXPERIMENTAL ( PhoreI)

IDO-

POSTEXPERIMENTAL

(PhoseI[) SOlllYZ

Soline Adrenalme

EXPERIMENTAL

Id%

go-

80-

$70z

.z

60-

x ;50z g403020-

Rates of secretion after pm- and postFig. 1. experimental phase exposure of glands to adrenalinesaline solutions. Only a Ringer-saline was added to the bathing medium during the experimental phase. (4 ) Additions of fresh bathing medium; (t ) removal. Five min of glandular incubation was followed by 3 min of glandular rinsing and measurement of the secreted droplet. Results during the experimental phase served as the control for experiments in which the stimulatory effects of other drugs were tested (Table 1). Each point represents the average of four separate experiments. KAUFMAN and PHILLIPS (1973b), who were unsuccessful in getting glands of D. andersoni to secrete after administering cyclic-AMP to the bathing medium. However, these authors did not pre-incubate the glands with adrenaline, as was done in our experiments. Because of this difference, we tested the ability of cyclic-AMP to initiate glandular secretion in the absence of prior adrenaline stimulation (Fig. 2). We, too, were unsuccessful in obtaining secretion under these conditions. To verify that the glands were competent, adrenaline was added to the bathing medium following cyclicAMP administration. Without exception, the glands secreted after administration of the drug (Fig. 2). To further test the possible r61e of cyclic-AMP in glandular secretion, theophylline, an inhibitor of the enzyme phosphodiesterase, was added during the experimental phase of experiments. It, too, stimulated glandular secretion (Table 1). However, like cyclic-AMP, no appreciable rate of secretion was achieved when theophylline was added without prior adrenaline stimulation (Fig. 2). Additions of the dibutyryl derivative of cyclic-AMP in separate experiments were also ineffective, even

Control Table

of fluid secretion

1. Effect of potential stimulants secretion

by salivary glands of the lone star tick

1895

on salivary fluid

race of secretion (nanoliterslmin) D*“p,UsedIn -ma” sxperimental sre-~xprrimentalExperimental AI+II P* P’=t-Exp=i==n=l Phase (II)

Pilast

n

Phase (III)

,Phase (II)

(I)

(-71)

--

49 + 32

2 B 1

124 + 42

(t44)


72 2 53

.$

57 + 11

(+I0


115 + 48

z 3

020

Control

4

71 :t 28

Cyclic-AMP (lO-’ M)

4

80 :t 43

Theo~hylline (loN)

4

48 ,+_16

Piloearpine (10-s ?o

3

36 +_ 14

222

(-34)

'43+ 22

1+1

(-42) NS

?i

Piloearpine (lo-3 HI 3

Noradrenaline (lo-5M) 4 58 + 34 Noradrenaline

(lo-3N)

Glutamate (lo-5

M)

4

a2 + 27

3

104+ 21

NS

91 + 47

55 + 39

191+ 60 (+133)
72 _ + 32

194+ 31 (+lIz)
76 i 28

(-100)

:4+4

*is

128 k 70

Gl”tar@ate

351

C-381,NS

25 + 3

34 + 9

1y1

(-33) NS

28 2 17

63 + 23

121

C-62) NS

18 + II

(lo-3 11)

3

41+

5-HT (10-S M)

4

5-HT (10‘3 11)

4

14

*As co~~pared t,, control compnring

of

rC the

the

secretion

to

daring

bathing

A t-test WO.05

i;

“a.;

secretory

during medium

used

to

rates

treatments.

each phgne.

Ringer-saline the

Statistical

AIJII.

changes

and experimenral

bathing

added

Fhasss. at

average

the control

raee~ of “as

the

to

analyses bctveen

determine

glands

statistical

phase. in

rhe

I and

II

as mean

Dnly the drug indicated

stimulate

Time

were made by phases

Data is expressed

the experimental

2 z

was added

Fig. 2. Rates of secretion after incubating glands with changes (every 8 min) of theophylline and cyclic-AMP Ringer-saline. After 32 min (solid vertical arrow) a Ringer-adrenaline solution was added to the glands in place of the above. Bathing media changes, rinsings, and measurement of secreted droplet were as dsecribed in Fig. 1. Glands were exposed to four changes of dibutyryl cyclic-AMP at 10 min intervals followed by Ringer-adrenaline after 40 min (open arrow). Results indicate the average of three trials with cyclic-AMP and dibutyryl cyclic-AMP and four with theophylline.

Adrenaline pre-

si&ficance

and

post-

of

data

PRE - EXPERIMENTAL (PhoseI)

level.

EXPERIMENTAL

POSTEXPERIMENTAL

(PhoselIJ

(Pharem)

Saline 160-

when the glar.ds were incubated for longer periods of time in the absence of pre-adrenaline stimulation. The protocol for exposure to dibutyryl cyclic-AMP was lengthened because evidence points to the requirement for intracellular esterases to remove the butyryl substituent from the 2’- position before this molecule is active (BERRIDGE and PRINCE, 1972). Interestingly, it appears that this cyclic-AMP analogue retarded subsequent glandular recovery with additions of adrenaline (Fig. 2). When glan.ds were incubated with the parasympathomimetic drug pilocarpine, no secretion during the experimental phase was noted (Table 1). Noradrenaline differs from adrenaline by only one methyl group and appears to be an even more potent stimulant of salivary secretion (Table 1). Glutamate, even at high concentrations, showed no potential ts:, cause glandular secretion (Table 1). Salivary glands of the adult blowfly, Cdiphora erythrocephak, are receptive to both cyclic-AMP and S-hydroxytryptamine (BERRIDGE and PATEL, 1968); however, glands from the lone star tick did not secrete wInen 5-HT was present in the bathing medium (Table 1).

(minl

Adrenaline

Saline

Saline lt5M

Adrenaline

Id%

i Adrenaline

16%

140r C '?120C .ZlOOz B 80s 9y C? 6040ZO-

Time(min.1

Fig. 3. Rate of glandular secretion when an adrenalinesaline solution bathed salivary glands throughout for 70 min. Bathing media changes, rinsings, and measurement of secreted droplet were as described in Fig. 1. Results during the experimental phase served as the control for experiments in which the possible inhibitory effects of drugs were tested (Table 2). Each point represents the average of four separate experiments.

G. R. NEEDHAM AND

1896 Effects of potential

J. R. SAUER

this drug completely blocked fluid secretion (Table 2). At a 10-s M concentration of the drug, inhibition was apparent, but we were unable to demonstrate a significant reduction in the rate of secretion. Recovery of glandular function was substantial at both concentrations during the post-experimental phase.

inhibitors

Insight into possible energy needs for fluid transport and possible mechanisms by which ions move across cells can be obtained by the use of potential inhibitors. In Fig. 3 adrenaline was present during all three experimental phases and served as a control for the remaining results. The rate of fluid secretion increased to 150 nl/min during the first 30 min, then maintained a constant rate for the next 15 min, with considerable variance in maximum rates of secretion between individual glands again evident. After 48 min the rate declined to approximately 100 nl/min in the post-experimental phase. 2,4-Dinitrophenol significantly (P < 0.05) slowed secretion in the experimental phase (Table 2). Additions of ouabain during the experimental phase, even at 10e3 M concentrations, did not significantly inhibit secretion. However, after administration of low3 M ouabain we were unable to restore the gland’s ability to secrete when an adrenaline-Ringer solution was re-added during the post-experimental phase. This result was also observed following administration of lOwaM DNP to the bathing medium. To help clarify these findings the effects of the psychotomimetic drug harmaline were investigated. Harmaline is an inhibitor of (Na++ K+)-ATPase systems by competing with Na ions in the Na+dependent phosphorylation phase of the reaction (CANESSAet al., 1973). At a concentration of 10e3 M

DISCUSSION The results agree with those of KAUFMAN and PHILLIPS (1973b) in that catecholamines stimulate tick salivary fluid secretion. The results are also consistent with data where catecholamines stimulated chloride uptake into lone star tick salivary gland cells (SAUER et al., 1974). The latter findings were cited as evidence in support of the standing gradient hypothesis (DIAMONDand BOSSERT, 1967, 1968) to help explain the mechanisms of salivary fluid secretion. The major difference and finding of present results in contrast to those published by KAUFMAN and PHILLIPS (1973b) concerns the stimulatory effect of cyclic-AMP and the phosphodiesterase inhibitor theophylline. The latter drug’s effect was not investigated by Kaufman and Phillips. The glands of A. americanum secreted in the presence of cyclic-AMP and theophylline, but only after prestimulation with adrenaline. We, like KAUFMAN and PHILLIPS (1973b), were unable to achieve glandular secretion if cyclic-AMP was administered to the glands before adrenaline administration.

Table 2. Effects of potential inhibitors of salivary fluid secretion m-usUsedIn mean rate OfsecreC*on (nanolitersfmin) Experime”talPre-Experimental Experimental AI_ pf Post-Experimental Phase (II)

Adrenaline

n

Phase (I)

Phase (II)

Phase (III)

(control)

4

76+ 40

DNP (10-S a)

4

48 i: IO

50 + 12

(+2)

CO.01

M)

4

45 + lb

41 + 16

C-4)

CO.01

131+

49

(+55)

105 + 39 61 + 10

DNP

(lo-3

2+1

O”tLbd”

M)

4

58 + 6

113 _+.37

(+5s)

NS

Ouabain (lo-3 M)

3

222

11

‘45 + 23

(+23)

NS

l+_l

Harmaline (lo-5 W)

4

62 + 30

76 + 44

(+14)

NS

113 + 70

lfarmaline (lo-3 N)

4

49 i: 17

(lo-5

(control)

the average

phases I and II of the adrenaline Data is expressed line

bathed

cubation determine

as mean rates

the glands

during

with the potential statistical

C-46)
321

* As compared to adrenaline were made by comparing

control

(phase II).

analyses

rates

and experimental during

34 + 13

Statistical

in secretory

A t-test

between

treatments.

each phase.

phases of the experiment,

inhibitor

significance

dI+II.

changes

of secretion

all

60 + 34

Adrena-

includiog

in-

was used to

of data at the PcO.05 level.

Control of fluid secretion by salivary glands of the lone star tick However, both cyclic-AMP and theophylline alone are potent stimulants of chloride uptake into A. americanurn salivary gland cells ( SAUERet al., 1974). The question of importance is by what means does pre-incubation and stimulation with adrenaline render the glands susceptible to stimulation by cyclic-AMP (and theophylline) ? One possible explanation is that adrenaline may inhibit a phosphodiesterase enzyme; this would allow intracellular levels of cyclic-AMP to build up to a sufficient concentration to initiate processes involved in fluid transport. Evidence for catecholaminergic noncompetitive in’hibition of cyclic nucleotide phosphodiesterase has been demonstrated in calf heart (GOREN and ROSEN, 1972). Also, the ability of catecholomines to activate adenyl cyclase is well known (BERRIDGE and PRINCE, 1972) Alternatively, adrenaline might increase the cell’s permeability to molecules such as cyclic-AMP, which is thought to be relatively impermeable (BERRIDGEand PRINCE, 1972). How ever, the cyclic-AMP derivative dibutyryl cyclic-AMP, which reportedly mimics cyclic-AMP and enters cells more readily (BERRIDGE and PRINCE, 1.972), was also ineffective as a lone stimulant of salivary secretion (Fig. 2). Why dibutyryl cyclic-AMP slowed glandular recovery after additions of adrenaline is unclear. It is also well to keep in mind the effects of 5-HT and cyclicAMP on salivary secretion in Callipizwa (BERRIDGE and PRINCE, 1972). These authors found differences in the mode of action of these two drugs, even though both lstimulate fluid secretion. They also implicated calcium as an important intracellular regulator. Another interesting aspect of the present results was the effect noted after additions of ouabain and harmaline during adrenaline-stimulated secretion. Both chemicals have been shown to inhibit (Na++ K+)-ATPase Isystems, but by different mechanisms (CANESSA et al., 1973). Considerable evidence indicates that ouabain inhibits ATPase by binding to the K+-sensitive phosphorylated form of the enzyme (CHAIXNOCK and POTTER, 1969; SEN et al., 1969). Harmaline, however, blocks phosphorylation of the enzyme by competing with Na ions, which are essential for this reaction to occur (CANESSAet al., 1973). Harmaline was an effective inhibitor of fluid secretion at concentrations of low3 M during the experimental phase; and even though an inhibition was apparent, we were unable to demonstrate a signifcant inhibition with 10m3 M ouabain during the experimental phase. However, glandular secretion could not be restored by adrenalineRinger in the post-experimental phase, but was restored in the harmaline-treated glands (Table 2). One might speculate that lack of recovery by ouabain (10M3 M) treated glands is due to a more involved inhibitor-enzyme interaction than that glands. which occun in the harmaline-treated However, mo:re research is required to fully clarify

1897

this possibility. It is also worth noting that KAUFMAN and PHILLIPS (197313) completely inhibited salivary secretion in D. andersoni with 10e6 M ouabain. It may be that ouabain, and to a lesser extent harmaline, are drugs that enter the cells of the gland slowly and the (Naf + K+)ATPase system, if present, is located on the apical side of the fluid secreting cells in A. americanurn. This might explain why only high concentrations of the inhibitors demonstrated an effect. In addition, harmaline’s effect on this system is via competitive inhibition with Na ions (CANESSA et al., 1973), which could also explain why higher concentrations are effective in blocking secretion. The presence of an energy requirement for fluid transport is supported by the reduction in secretion after addition of DNP to the bathing medium. We, like KAUFMANand PHILLIPS (1973b), were unable to stimulate glands to secrete with pilocarpine in the bathing medium. Pilocarpine has been used by a number of workers to stimulate salivary secretion in viva (TATCHELL, 1967; PURNELL et al., 1969; BARKERet al., 1973). The questions of importance regarding this drug is its site of action. We have preliminary information indicating that pilocarpine affects the electrical potential across the tick gut epithelium (EIKENBARY and SAUER, unpublished observation). Whether the gut is the initial site of action of pilocarpine after in viva administration to the tick is still speculative, however. Our results on ineffective stimulation by 5-HT and glutamate are consistent with the findings of KAUFMAN and PHILLIPS (1973b) and the ineffectiveness of these drugs in stimulating chloride uptake into salivary gland cells of the lone star tick (SAUER et al., 1974). In conclusion, adrenaline and noradrenaline stimulate salivary fluid secretion in the lone star tick. Cyclic-AMP and theophylline initiate secretion after glands have been pre-stimulated to secrete fluid with adrenaline. Results obtained after additions of harmaline and ouabain point to the possibility of a (Naf + K+)-ATPase system in the glandular epithelium; but because only high concentrations of both inhibitors were effective, this possibihty awaits further elucidation. Inhibition of salivary secretion by DNP indicates that oxidative metabolism is involved in moving fluid across glandular cells. Acknowledgements-The authors would like to thank

Mrs. P. MINCOLLAand Mrs. D. MIL~TEADfor technical

assistance; also, Drs. CALVINBEAMILS and J. A. HAIRfor critically reviewing the manuscript. REFERENCES

BARKERR. W., BURRISE., SAUERJ. R., and HAIR _I. A. (1973) Composition of tick oral secretion obtained by three different collection methods, J. med. Ent. 10, 198-201.

G. R. NEEDHAM AND J. R. SAUER

1898

BERRIDGE M. J. and PATEL N. G. (1968) Insect salivary glands: stimulation of fluid secretion by 5-hydroxytryptamine and adenosine-3’-5’-monophosphate. Science, Wash. 162, 462463. BERRIDCE M. J. and PRINCE W. T. (1972) The role of cyclic-AMP and calcium in hormone action. A&. Insect Physiol. 9, l-49. CANESA M., JAIMOVICHE., and FUENTE M. DE LA (1973) Harmaline: a competitive inhibitor of Na ion in the (Na++K+)-ATPase system. J. memb. Biol. 13, 263-282. CHARNOCKJ. S. and POTTER H. A. (1969) The effect of Mg++ and ouabain on the incorporation of 32P-ATP from Y-ATP~~ into Na+- and K+-activated adenosine triphosphatase. Biochem. Biophys. Arrk. 134, 42-47. DIAMOND J. M. and BOSSERT W. H. (1967) Standinggradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia. J. gen. Physiol. 50, 2061-2083. DIAMOND J. M. and BOSSERT W. H. (1968) Functional consequences of ultra-structural geometry in “backwards” fluid-transvortine evithelia. 7. Cell Biol. 37, 694-702. GOREN E. N. and ROSEN 0. M. (1972) Inhibition of a cyclic nucleotide from beef heart by catecholamines and related compounds. MO&X. Pfzarnacol. 8, 38C384. GREGSONJ. D. (1967) Observations on the movement of fluids in the vicinity of the mouth parts of naturally feeding Dermacentor andersoni Stiles. Parasitology 57, I-S. Hsu M. H. and SAUER I. R. (1975) Ion and water balance in the feeding lone star tick. damp. Biochem. Physiol. I.

In press.

KAUFMAN W. R. and PHILLIPS J. E. (1973a) Ion and water balance in the ixodid tick Dermacentor andersoni -1. Routes of ion and water excretion. r. exp. Biol. 58, 523-536.

KAUFMAN W. R. and PHILLIPS J. E. (1973b) Ion and water balance in the ixodid tick Dermacentor andersoni -11. Mechanism and control of salivary secretion. J. exp. Biol. 58, 537-547. KAUFMAN W. R. and PHILLIPS J. E. (1973~) Ion and water balance in the ixodid tick Dermacentor andersoni -111. Influence of monovalent ions and osmotic pressure on salivary secretion. r. e.rp. Biol. 58, 549564. PATRICK C. D. and HAIR J. A. (1975) Laboratory rearing procedures and equipment for multi-host ticks (Acarina: Ixodidae). r. med. Ent. In press, PURNELL R. E., BRANAGAND., and RADLEY D. E. (1969) The use of parasympathomimetic drugs to stimulate salivation in the tick Rhipirephalus appendiculatus and the transmission of Theileria parva using saliva obtained by this method from infected ticks. Parasitology 59, 709-718. SAUER J. R., FRICK J. H., and HAIR J. A. (1974) Control of Yl uptake by isolated salivary glands of the lone star tick. r. Insect PJzysiol. 20, 1771-1778. SAUER J. 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. SEN A. K., TOBIN T., and POST R. L. (1969) A cycle for ouabain inhibition of sodiumand potassiumdependent adenosine triphosphatase. J. biol. Chem. 244, 6596-6604. TATCHELL R. J. (1967) A modified method for obtaining tick oral secretion. .r. Parasit. 53, 1106-l 107. TATCHELL R. J. (1965) The ionic- regulatory r6le of salivarv secretions of the cattle tick. Booahilus microplus. j. Insect Physiol. 15, 1421-143’0. 1