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Hormonal control of salivary gland degeneration in the ixodid tick Amblyomma hebraeum
J. Insect Ph_vsiol., Vol. 27. No. 4. pp. 241-248, Printed in Great Britain.
0022-1910/81/040241-08 SO2.00/0 CC;198 1 Pergamon Press Ltd.
1981.
HORMONAL CONTROL OF SALIVARY GLAND DEGENERATION IN THE IXODID TICK AMBLYOMMA HEBRAEUM ROBERT A. HARRIS and WILLIAM R. KAUFMAN Department of Zoology,
University of Alberta, Edmonton, Alberta T6G 2E9, Canada
(Received 29 September 1980: revised 3 1 October 1980) Abstract--Under normal circumstances. salivary glands of female ixodid ticks begin degenerating within hours of completing the blood meal. We have monitored cytological, functional and biochemical changes in the tissue which are diagnostic of the degenerative process. Although ultimately degeneration also befalls salivary glands of partially fed ticks removed prematurely from the host, the process is considerably delayed. When we transplanted salivary glands from partially fed ticks into the haemocoels of replete specimens, autolysis was induced in the donor tissue. whereas such was not the case when similar glands were transplanted to the haemocoeis of other partially fed ticks. We thus suggest that a humoral factor is involved in postprandial resorption of the salivary glands. Succinate dehydrogenase activity decreases, and acid phosphatase activity increases in the salivary glands as a function of time post-engorgement. However. these enzyme assays are not sensitive enough to detect the earliest stages of autolysis. Key Word Inde.v Ixodid ticks, salivary glands, hormonal control of degeneration
INTRODUCTION
THE FEEDING cycle of ixodid ticks does not run a straightforward pattern of engorgement followed by excretion as appears, superficially at least, to be the case for some haematophagous insects. Unlike the latter. ixodid female ticks feed at a most leisurely pace, usually requiring 7-10 days to complete a meal. This feeding period can be divided into a slow phase of engorgement, occupying about N-950,/, of the feeding duration, and a final rapid phase occurring within the last 12-24 hr prior to detachment (LEES, 1946). During the slow phase, many synthetic processes occur, including the laying down of endocuticle (LEES, 1952), development of the salivary glands (TILL. 1961; BINNINGTON, 1978), Genk’s organ, the ovaries and the male genital accessory glands (TILL, 1961); undoubtedly many other as yet undescribed developmental processes occur at this time as well. During the rapid phase of engorgement, there occurs a most dramatic increase in body weight which at repletion may, in some species, exceed 100 times the unfed weight (reviewed by BALASHOV, 1972). During the feeding period, the nutrient portion of the blood meal is concentrated as a result of selective elimination of the excess fluid-principally ions and insects it is the water. !n most haematophagous Malpighian tubule system which is concerned with fluid excretion, but in ixodid ticks the salivary glands function in this capacity while the parasite is still attached to the host. In this way, the many pathogenic agents transmitted by ticks gain access to the host. Thus, on account of its function in osmoregulation and its role in disease transmission, the salivary gland system has recently attracted some attention as a potential focus for exerting biological control over
this medically and economically important group of arthropods. It has been known for some time that following drop-off from the host, the salivary glands undergo resorption within a few days. The histological changes associated with this involution have been described in some detail by TILL (1961) for Rhipicephalus appendiculatus, but only within the last decade has the electron microscope been utilized in the study of this tissue (KIRKLAND, 1971; MEREDITH and KAUFMAN, 1973; COINS and ROSHDY, 1973; MEGAW and BEADLE, 1979). These modern studies have revealed that there is one cell-type in particular which probably secretes the bulk of the fluid portion of saliva. This fluid-secreting cell is virtually nonfunctional before the tick attaches to the host. Within a few days it rapidly gains secretory competence, a phenomenon accompanied by striking changes in ultrastructure which are documented elsewhere (MEGAW and BEADLE, 1979). Following engorgement there is a rapid loss of secretory competence (KAUFMAN. 1976). Here we describe some of the ultrastructural and functional changes which can be taken as diagnostic of salivary gland degeneration, and we present evidence indicating that the signal which initiates autolysis is hormonal.
MATERIALS Experimental
AND
METHODS
animals
The ticks used in this study (Amblyomma hebraeum Koch) came from a colony established here with specimens originating from the Veterinary Research Institute, Onderstepoort, Republic of South Africa. 241
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ROBERTA.
HARRIS AND
The colony is maintained at 26°C in darkness, relative humidity being approx. 95%. When adults are confined to the backs of rabbits as described by KAUFMAN and PHILLIPS (1973), within 7-10 days they will usually feed to repletion (about 1200-3000 mg) and then detach voluntarily. In the text of this paper, such ticks are referred to as ‘gorged’ or ‘replete’. The ticks referred to as ‘partially fed’ are specimens forcibly removed from the rabbit after achieving a weight of between 120 and 450 mg. As will be shown later, subsequent fate of the salivary glands depends on the degree of engorgement. Method for
organ transplant
To test whether degeneration could be associated with any humoral factors, salivary glands of partially fed ticks (120-280 mg) were transplanted to the haemocoels of replete females which had dropped from the host within the previous 12-24 hr. Salivary glands similarly transplanted into the haemocoels of other partially fed females served as controls. Donor ticks were immobilized with ‘Plasticine’ modelling clay (Harbutt’s, England) and flooded with modified Hank’s balanced saline (composition in g/l: NaCl, 11.5; D-glucose, 1.6; KCI, 0.4; CaCI,, 0.14; MgSO,, 0.098; KH,HPO,, 0.06; Na,HPO,, 0.048; phenol red, 0.01; 360 mosm; pH 7.0). The salivary glands were then dissected out as described by KAUFMAN and BARNETT (1977). All recipient ticks were chilled on ice for 20 min. This period of anaesthesia was mandatory to prevent the delicate gut diverticula from gaping through the incision and bursting. For the control series of recipients, a small semicircular incision was made in the dorsum. A
Fig. 1. Diagram of apparatus used to prevent escape of gut contents from gorged females during organ transplant procedure. The apparatus was assembled from part of a Sartorius plastic filter holder normally used for sterile filtration of organ culture media. Adhesive tape, secured to the tick’s dorsal integument by means of an annulus of ‘Super Glue’, was placed over the funnel portion of the filter holder. The filter holder was then connected to the laboratory vacuum line. The degree of vacuum could be regulated by means of an inlet valve so as to just maintain the gut contents within the abdomen during surgery. Access to the haemocoel was achieved by slicing through the tape and integument in the desired region by means of a fine razor-blade scalpel. Following implantation of the salivary gland, the wound was sealed with ‘Super Glue’; most of the excess tape was then cut away and discarded.
WILLIAMR. KAUFMAN gland from a partially fed donor was delivered through this flap of integument into the haemocoel and pushed some way distant from the wound. The incision was sealed with a cyano-acrylate compound (‘Super Glue’, Bondo) and the ticks returned to the 26°C incubator and kept in vials held over saturated KNO, at a relative humidity of 93:,,. In the case of the gorged female recipients, chilling alone was not usually sufficient to prevent escape of gut contents on cutting the dorsal integument. After chilling, engorged females were suspended in a small vacuum chamber as shown and described in Fig. 1. In this apparatus, the tick could be positioned in such a way that, except for the small area of cuticle to be wounded, the remainder of the tick could be maintained under slightly hypobaric conditions. The degree of vacuum was not measured, but was adjusted so as to just retain the gut contents within the tick. After implanting the salivary gland, the wound was sealed with ‘Super Glue’ and the tick returned to the incubator. Ticks appeared to suffer no obvious adverse effects from these operations. Two to four days following implantation, the salivary glands of the donor and recipient ticks were recovered and fixed for subsequent observation under the electron microscope. Electron microscop) Salivary glands were dissected out under Hank’s balanced saline, the large tracheae dissected away and the glands fixed for 12 hr in 2.5% glutaraldehyde in 0.05 M cacodylate buffer made up in 2.5% sucrose (total osmotic pressure = 360 mosm). After briefly washing in cacodylate buffer containing 4% sucrose, the tissue was post-fixed in 4% OsO,-O.O5 M cacodylate buffer, pH 7.4 for 1 hr and washed 3 times with fresh buffer. Tissue was stained for 20 min in 2% uranyl acetate and then passed through an ascending series of ethanol concentrations followed by two changes of 100yO propylene oxide. Tissues were kept overnight in Araldite 502-propylene oxide (1 : 1, v/v), then washed twice with pure Araldite 502 and transferred to plastic moulds. Polymerization proceeded at 60°C for 48 hr. Thin sections were taken on a Porter-Blum Ultramicrotome. Grids (150 mesh coated with Formvar) were stained in 5% uranyl acetate in methanol for 25 min and counterstained in lead citrate for 2 min (REYNOLDS, 1963). Grids were examined on a Phillips 300 transmission electron microscope. Enzyme assays Assays were carried out on crude homogenates prepared as follows: salivary glands were dissected out under 1.27, NaCl (isosmotic to tick haemolymph) and carefully cleared of large tracheae and extraneous tissue. Wet weights of tissue were recorded on a Sartorius 2474 microbalance 90 set after removing the tissue from the dissection medium. During this 90-set period, glands were blotted for 30 set on filter paper according to a standard procedure. After weighing, glands were homogenized in 500-750 ~1 ice-cold 11.2% sucrose containing 5 mM ethylenediaminetetraacetate (EDTA). After removing an aliquot for determination of protein concentration,
Salivary gland autolysis each homogenate was transferred to a plastic vial (1 ml total capacity), capped and frozen in methanol-dry ice. Homogenates were stored at - 17°C for up to 7 weeks before assay. Succinate dehydrogenase, a mitochondrial marker enzyme, was assayed according to a procedure modified after PENNINGTON (1961) and PORTEUS and CLARK (1965). One hundred microlitres of homogenate was mixed in a glass-stoppered centrifuge tube with 1 ml buffer-substrate (composition: O.loo p-iodonitrotetrazolium violet, 25 mM sucrose. 2 mM EDTA, 50 mM sodium succinate, all in 50 mM potassium phosphate buffer, pH 7.4) and pre-incubated on ice for 30 min. The tubes were again vortexed briefly and incubated at 42OC for 20 min. The reaction was stopped with 1 ml ice-cold 10“; trichloroacetic acid. four millitres water-saturated ethylacetate was then added and the tubes vortexed for 30 sec. Following a brief
in ticks
243
centrifugation to separate the phases, absorbance of the organic phase was read at 490 pm on a Beckman DU-2 spectrophotometer. Parallel samples stopped at zero time served as blanks. Acid phosphatase was assayed according to the method of DINGLE (1977) with slight modifications. One hundred microliters of homogenate was mixed in a glass-stoppered centrifuge tube with 200 ~1 buffered glycerophosphate (composition: 5 mg sodium glycero-2-phosphate in 1 ml 0.1 M acetate buffer, pH 5.0) and incubated at 42°C for 20 min. The reaction was stopped with 2 ml ice-cold lo”,, trichloroacetic acid and the tubes left on ice for 15 min. The tubes were centrifuged at 500 g for 10 min. Two millilitres supernatant was mixed with 2 ml ammonium molybdate reagent (composition: 2.5 g (NH,),Mo,0,;4 H,O in 100 ml 0.5 M H,SO,) and 1 ml Elon reagent (Eastman Kodak; composition: 0.5 g 4-methylaminophenol sulphate in 100 ml lo”,,
_A
Partlal ly fed, n=7
Day of
engorgement, n =7
I day post- engorgement, n= 4
3
I 0
,
Minutes
after
IO
,
I
20 injecting
1
I
I
40
days post- engorgement? n=3
,
60 410 @M/kg b.w
pllocarplne,
(b) Partially
PF
,I
fed,
n= IO
day post - removal, n= 5
Day of engorgement, n= 5
I
20
0 Minutes
after
40
injecting dopamine,
day post- engorgement, n = 6
3 days,
n=5
5 days,
n=3
60
530 PM/kg
bw
Fig. 2. (a) Salivation response (cumulative saliva as ‘,” b.w.) to pilocarpine as a function of phase of the feeding cycle. Means, S.E.‘s and N for each group are indicated. Replete ticks show a weaker response to pilocarpine than partially fed ticks and the response decreases as a function of time postengorgement. (b) As in (a) for dopamine.
244
ROBERTA. HARRISAND WILLIAMR. KAUFMAN
(w/v) sodium metabisulphite). After 10 min (and within 1 hr) absorbance at 750 pm was recorded. Amount of inorganic phosphate (Pi) was calculated from the absorbance by means of a standard curve. Samples stopped at zero time served as blanks. Protein was measured by the dye-binding method of BRADFORD (1976) using bovine serum albumin as standard. The 10-30 ~1 aliquots taken for protein determination were first dissolved in an equal volume of 1 N NaOH to solubilize the protein. Injection of drugs The method for introducing pilocarpine and dopaminedrugs which elicit salivation-into the haemocoel is described in full by KAUFMAN (1978). In brief, a 30-gauge needle, fitted to an ‘Agla’ micrometer syringe unit (Burroughs-Wellcome), was introduced a few millimetres into the haemocoel via the articulation joining the capitulum to the thorax. Drugs were dissolved in 1.2% NaCl and were delivered in 1 pl/lOO mg by weight doses. Vehicle-induced salivation was zero. Saliva was collected from the mouthparts in capillary tubes of constant bore such that volumes could be calculated from the length of the fluid column. See further details in KAUFMAN (1978).
RESULTS Dopamine is known to elicit fluid secretion in tick salivary glands by direct post-junctional stimulation (KAUFMAN, 1976, 1977), whereas pilocarpine does so only indirectly (KAUFMAN, 1978). When partially fed ticks (150-350 mg) were removed prematurely from the host and injected with pilocarpine or dopamine, there was only a slight decline in re?ponse as a function of the time elapsed between removal from the host and performance of the injection (Fig. 2). But this decline in response was far more striking and more rapid in replete ticks (> 1000 mg) subjected to the same treatment.Thuswehaveasimple,physiologicaldemonstration of post-prandial salivary gland degeneration. These physiological events can be correlated with characteristic ultrastructural changes in the secretory epithelium (MEGAW and BEADLE, 1979). For a full description of the morphology and ultrastructure of salivary glands from partially fed Dermacentor andersoni females, see MEREDITH and KAUFMAN (1973). Comparing Figs. 3a and b, we see that the fluid-secretory cell of glands taken from partially fed ticks, shows little, if any, change in cytological appearance up to at least 2 days post-removal from the host. Just following engorgement (Fig. 3c), one notices the early stages of formation of autophagic vacuoles, composed of whorls of cell membrane surrounding cytoplasm. By 2 days post-engorgement (Fig. 3d) these autophagic vacuoles are more numerous and contain various inclusion bodies including degenerating remains of mitochondria. We routinely find such structures very early in the postengorgement phase, and on occasion, although not shown here, even in large ticks which are still attached to the host. In the latter cases, the autophagic vacuoles are smaller, less well-developed and are fewer in number. The remaining 2 micrographs in Fig. 3 show the
results of experiments in which a gland from a partially fed tick was transplanted into the haemocoel (see Materials and Methods) of either another partially fed specimen (control, Fig. 3e) or into an engorged specimen (experimental, Fig. 39. The transplanted glands were recovered 2 days later and prepared for observation under the electron microscope. The host glands were also examined. The control glands (Fig. 3e) appear virtually indistinguishable from those of normal partially fed ticks (compare with Figs. 3a, b), indicating that the trauma of the operation does not adversely influence cytology of the fluid-secretory cell. In the experimental ticks (Fig. 30, however, the presence of autophagic vacuoles is clear evidence that autolysis has been induced. We have observed similar results for 7 replicates of the control and 5 replicates of the experimental. The transplanted glands, however. seem to be at a less-advanced stage of degeneration than the host glands of the experimental series, even though there appears to be little difference in the stage of degeneration between host glands of the experimental series and normal glands in gorged ticks 2 days postrepletion. Inasmuch as an obvious sign of gland degeneration cell is the appearance in the fluid-secretory of autophagic vacuoles and the isolation of mitochondria, we tested whether there was a change in specific activity of certain ‘marker enzymes’ for these structures. Succinate dehydrogenase is believed to reside chiefly, if not exclusively, in the inner mitochondrial membrane (LEHNINGER, 1975) and acid phosphatase has long been associated with the presence of lysosomes (DINGLE, 1977). In Fig. 4 we present the changing titre of these two enzymes in crude homogenates of glands in both partially fed and replete ticks as a function of time. In partially fed ticks, there is no significant change in activity for either enzyme up to 5 days post-removal. For engorged ticks, on the other hand, there is a marked rise in acid phosphatase activity and a precipitous fall in succinate dehydrogenase activity within the same time period. In order to see how early in the feeding cycle one would notice enzyme changes characteristic of the engorged tissue, we removed partially fed ticks at various stages of engorgement and monitored succinate dehydrogenase and acid phosphatase activities in the salivary glands. Glands taken from ticks < 450 mg showed little change in enzyme activity within 5 days. Beyond that approximate weight, however, changes in enzyme titre with time resembled those pertaining to normal replete specimens. Normally. ticks of this weight range (400-600 mg) feed to repletion within 12-24 hr.
DISCUSSION Although there have been a number of good histological studies on tick salivary glands since the turn of the century, TILL (1961) seems to have been the first to follow histological changes in this tissue with both development and feeding cycles, and hence to describe the degeneration phenomenon in some detail. We present here the first demonstration that degeneration is triggered, and perhaps supported, by a
Fig. 3. Ultrastructural appearance of the fluid-secretory cell of the type III acinus as a result of various treatments. (a) Salivary gland of a partially fed tick dissected out and fixed within a few hours of removing tick from the host. (b) Gland of a tick similar to that in (a) fixed 2 days post-removal from host. No important changes take place at least during this 2-day period. Scale as in 3(a). Cc) Fluid-secretory cell of an engorged tick fixed on the same day as the tick engorged. Precise time of engorgement was not determined (see text). Fixation could have been as much as 17 hr after drop-off from the host. Arrow denotes an isolation body. Scale as in 3(a). (d) Gland of a tick similar to that in (c). but 2 days post-engorgement. Note the appearance of various inclusion bodies within the autophagic vacuoles. (e) Fluid-secretory cell of a control tick. Salivary gland of this partially fed tick had resided in the haemocoel of another partially fed specimen for 2 days prior to fixation. This fluid-secretory cell shows no obvious signs of degeneration. (f) Fluid-secretory cell of an experimental tick. Salivary gland of this partially fed specimen had resided in the haemocoel of a replete tick for 2 days prior to fixation. Autophagic vacuoles containing inclusion bodies (arrow) indicate that degeneration is proceeding in this gland. Magnification 19,200 Y
Salivary gland autolysis in ticks 60~
(a)
5 2;$
Acid phosphatase
( b 1 Succlnate
dehydrogenase
Days post removal / post engorgement
Fig. 4. Changes in activity of two enzymes as a function of time post-removal (for partially fed ticks, l) or postengorgement (for replete ticks, A). means, S.E.‘s and N are indicated for each point. (a) Acid phosphatase activity. (b) Succinate dehydrogenase activity. For both enzymes, there is little change in activity up to 5 days post-removal for the partially fed ticks. In the engorged ticks. there is a progressive rise in acid phosphatase activity and a decline in succinate dehydrogenase activity with time post-engorgement. Statistical differences between partially fed and engorged specimens are indicated for each day as follows: *0.05 > p z 0.01; **p < 0.01.
blood-borne factor (here tentatively named ‘salivary gland degeneration factor’): namely, glands can be induced to degenerate in isolation from all nervous influence by placing them in a milieu (haemocoel of a replete tick) in which resident glands are degenerating. The proof that this autolysis is a normal physiological process rather than a pathological necrosis, resulting from isolating the tissue from neural influence or its extensive tracheal supply, is clear from a variety of data. First and foremost, the controls did not degenerate, although lack of neural and tracheal input applied to them equally. Apparently, the transplanted tissue receives sufficient oxygen for its needs from that remaining in the attached stumps of tracheae and/or from whatever pool is circulating in the host haemolymph. Secondly, we regard it as significant that the timecourse for degeneration in the transplanted tissue lagged somewhat behind that pertaining to the host glands. Although as yet we have no experimental evidence to bear on the matter, this lag could be explained in at least two ways. (A) Hormone-receptor interaction may be disrupted or the subsequent metabolic events associated with autolysis may proceed less efficiently in the transplanted tissue. (B) The transplanted tissue was most likely exposed to the
247
hormone for a far briefer time than the host tissue. We mentioned earlier that the initial release of salivary gland degeneration factor probably occurs during the last day or so of engorgement while the tick is still attached to the host. Also, for some tick species. including A. hebraeum. there is a diurnal rhythm to drop-off from the host (RECHAV, 1978) with the peak period of drop-off occurring toward the end of the photophase. Since the engorged specimens which we used for our experiments had been collected from the rabbits in the morning, the host glands of the experimental ticks could well have had some 12-24 hr exposure to salivary gland degeneration factor before the transplantations were performed. This fact may easily have contributed to the more advanced degree of degeneration apparent for the host tissue. Thirdly, we noticed in a separate group of control specimens, that both the host and donor glands showed definite signs of degeneration. These particular ticks had been feeding very slowly. A number of mammalian species, including rabbits, are known to develop a strong immunity to tick infestation under certain circumstances (ALLEN and HUMPHREYS, 1979; BOWE~SIDJAOUet al.. 1977). Ticks attached to such immune hosts tend to feed very slowly, and eventually die in situ if they do not detach voluntarily or are not removed by the experimenter. On occasion. we have dissected,out the salivary glands of such slow-feeding ticks and have prepared them for in vitro fluid secretion as described by KAUFMAN (1977), but in most cases secretory rates were abnormally low (unpublished observations). Perhaps one result of this abnormal feeding behaviour on immune rabbits is an untimely release of salivary gland degeneration factor. In any event, we underline the apparently ‘negative result’ of the above-mentioned group of controls as indicating that transplanted glands assume the behaviour of the host glands, be it degeneration or otherwise. We have considered the possibility that postengorgement degeneration is due to the withholding of a ‘salivary gland maintenace factor’, rather than the secretion of a degeneration factor. However, from some preliminary experiments, we believe this explanation to be unlikely. Salivary glands from recently engorged ticks held in organ culture for 5 days failed to degenerate as judged from the activities of succinate dehydrogenase and acid phosphatase. Unless the putative maintenance factor happens to be one of the normal ingredients of TC 199 (we did not supplement the medium with undefined additives), these experiments speak against the existence of a specific maintenance factor. With a view towards developing a simple, to monitor salivary gland quantitative assay degeneration for future experiments, we tested succinate dehydrogenase and acid phosphatase activities as putative candidates, and found that succinate dehydrogenase activity did indeed fall, and acid phosphatase activity rose in glands from engorged ticks, as anticipated (Fig. 4). Although the fall in succinate dehydrogenase activity in engorged specimens was precipitous over the first day or two, for some as yet unexplained reason succinate dehydrogenase activity in freshly gorged specimens is much higher than that in partially fed ticks
ROBERTA. HARRISAND WILLIAMR. KAUFMAN
248
(200400 mg), despite the fact that isolated glands from partially fed ticks secrete fluid in vitro more rapidly (KAUFMAN, 1976). In the final analysis, differences in succinate dehydrogenase activity between the glands of partially fed and engorged ticks do not become statistically significant until the second or third day post-engorgement/removal (Fig. 4). Similarly for acid phosphatase, significant differences between engorged and partially fed tissues are apparent only after day 2. Hence, although the activities of these two enzymes may well reflect progress of degeneration, physiological (Fig. 2) and ultrastructural (Fig. 3) changes are detected much earlier. We are now investigating other methods as putative assays for the release of salivary gland degeneration factor. Acknowledgemenzs-This research was nenerouslv supported by NSERC Canada and the Canadian National Sportsmen’s fund (Grant No. 3-R72). We are indebted to Dr. E. J. SANDERS, Department of Physiology, University of Alberta for allowing us the use of his electron microscope facility and for reviewing the manuscript.
REFERENCES ALLENJ. R. and HUMPHREYS S. J. (1979) Immunisation of guinea pigs and cattle against ticks. Nature. Lond. 280, 491493.
BALASHOV Yu. S. (1972) Blood-sucking ticks (Ixodoidea)Vectors of diseases of man and animals. Misc. Publs. ent. Sot. Am. 8 (5).
BINNINGTONK. C. (1978) Sequential changes in salivary gland structure during attachment and feeding of cattle tick, Boophilus microplus. Int. J. Parasitol. 8, 97-115. BOWE~SIDJAOU J., BROSSARD M. and AFSCHLIMANN A. (1977) Effects and duration of resistance acquired by rabbits on feeding and egg laying in Ixodes ricinus L. Experientia 33, 528-530.
BRADFORD M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. COONS L.
B. and ROSHDYM. A. (1973) Fine structure of the salivary glands of unfed male Dermacentor variabilis (Say) (Ixodoidea : Ixodidae). J. Parasit. 59, 900-912.
DINGLE J. T. (Ed.) (1977) Lysosomes. A Laboratory Handbook. 2nd edition. North-Holland, Amsterdam. KAUFMANW. (1976) The influence of various factors on fluid secretion by in vitro salivary glands of ixodid ticks. J. exp. Biol. 64, 727-742.
KAUFMANW. R. (1977) The influence of adrenergic agonists and their antagonists on isolated salivary glands of ixodid ticks. Eur. J. Pharmacol. 45, 61-68. KAUFMANW. (1978) Actions of some transmitters and their antagonists on salivary secretion in a tick. Am. J. Physiol. 235, R76-R8
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KAUFMANW. R. and BARNETTS. F. (1977) Dermacentor andersoni: culture of whole salivary glands. Exp. Parasit. 42, 106-I 14.
KAUFMAN W. and PHILLIPSJ. E. (1973) Ion and water balance in the ixodid tick Dermacentor andersoni. I. Routes of ion and water excretion. J. exp. Biol. 58, 523-536. KIRKLANDW. L. (1971) Ultrastructural changes in the nymphal salivary glands of the rabbit tick, Haemaphysalis leparispalustris. J. Insect Physiol. 17, 1933-1946.
LEESA. D. (1946) The water balance in Ixodes ricinus L. and certain other species of ticks. Parasitology 37, l-20. Lens A. D. (1952) The role of cuticle growth in the feeding process of ticks. Proc. zool. Sot. Lond. 121, 759-772. LEHMNGERA. L. (1975) Biochemistry. 2nd edition. Worth, New York. MEGAWM. W. J. and BEADLED. J. (1979) Structure and function of the salivary glands of the tick, Boophilus microplus Canestrini (Acarina : Ixodidae). Int. J. Insect Morphol. Embryol. 0, 67-83.
MEREDITHJ. and KAUFMANW. (1973) A proposed site of fluid secretion in the salivary gland of the ixodid tick Dermacentor andersoni. Parasitology 67,205-217.
PENNINGTONR. J. (1961) Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem. J. 80, 649-654. PORTEOUSJ. W. and CLARK B. (1965) The isolation and characterization of subcellular components of the epithelial cells of rabbit small intestine. Biochem. J. 96, 159-171.
RECHAVY. (1978) Drop-off rhythms of engorged larvae and nymphs of the bont tick, Amblyomma hebraeum (Atari : Ixodidae), and the factors that regulate them. J. med. Em. 14, 677-687.
REYNOLDS E. S. (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-21 I.
TILL W. M. (1961) A contribution to the anatomy and histology of the brown ear tick, Rhipicephalus appendiculatus (Neumann). Mem ent. Sot. sth. Afr. 6, April.
0022-1910/81/040241-08 SO2.00/0 CC;198 1 Pergamon Press Ltd.
1981.
HORMONAL CONTROL OF SALIVARY GLAND DEGENERATION IN THE IXODID TICK AMBLYOMMA HEBRAEUM ROBERT A. HARRIS and WILLIAM R. KAUFMAN Department of Zoology,
University of Alberta, Edmonton, Alberta T6G 2E9, Canada
(Received 29 September 1980: revised 3 1 October 1980) Abstract--Under normal circumstances. salivary glands of female ixodid ticks begin degenerating within hours of completing the blood meal. We have monitored cytological, functional and biochemical changes in the tissue which are diagnostic of the degenerative process. Although ultimately degeneration also befalls salivary glands of partially fed ticks removed prematurely from the host, the process is considerably delayed. When we transplanted salivary glands from partially fed ticks into the haemocoels of replete specimens, autolysis was induced in the donor tissue. whereas such was not the case when similar glands were transplanted to the haemocoeis of other partially fed ticks. We thus suggest that a humoral factor is involved in postprandial resorption of the salivary glands. Succinate dehydrogenase activity decreases, and acid phosphatase activity increases in the salivary glands as a function of time post-engorgement. However. these enzyme assays are not sensitive enough to detect the earliest stages of autolysis. Key Word Inde.v Ixodid ticks, salivary glands, hormonal control of degeneration
INTRODUCTION
THE FEEDING cycle of ixodid ticks does not run a straightforward pattern of engorgement followed by excretion as appears, superficially at least, to be the case for some haematophagous insects. Unlike the latter. ixodid female ticks feed at a most leisurely pace, usually requiring 7-10 days to complete a meal. This feeding period can be divided into a slow phase of engorgement, occupying about N-950,/, of the feeding duration, and a final rapid phase occurring within the last 12-24 hr prior to detachment (LEES, 1946). During the slow phase, many synthetic processes occur, including the laying down of endocuticle (LEES, 1952), development of the salivary glands (TILL. 1961; BINNINGTON, 1978), Genk’s organ, the ovaries and the male genital accessory glands (TILL, 1961); undoubtedly many other as yet undescribed developmental processes occur at this time as well. During the rapid phase of engorgement, there occurs a most dramatic increase in body weight which at repletion may, in some species, exceed 100 times the unfed weight (reviewed by BALASHOV, 1972). During the feeding period, the nutrient portion of the blood meal is concentrated as a result of selective elimination of the excess fluid-principally ions and insects it is the water. !n most haematophagous Malpighian tubule system which is concerned with fluid excretion, but in ixodid ticks the salivary glands function in this capacity while the parasite is still attached to the host. In this way, the many pathogenic agents transmitted by ticks gain access to the host. Thus, on account of its function in osmoregulation and its role in disease transmission, the salivary gland system has recently attracted some attention as a potential focus for exerting biological control over
this medically and economically important group of arthropods. It has been known for some time that following drop-off from the host, the salivary glands undergo resorption within a few days. The histological changes associated with this involution have been described in some detail by TILL (1961) for Rhipicephalus appendiculatus, but only within the last decade has the electron microscope been utilized in the study of this tissue (KIRKLAND, 1971; MEREDITH and KAUFMAN, 1973; COINS and ROSHDY, 1973; MEGAW and BEADLE, 1979). These modern studies have revealed that there is one cell-type in particular which probably secretes the bulk of the fluid portion of saliva. This fluid-secreting cell is virtually nonfunctional before the tick attaches to the host. Within a few days it rapidly gains secretory competence, a phenomenon accompanied by striking changes in ultrastructure which are documented elsewhere (MEGAW and BEADLE, 1979). Following engorgement there is a rapid loss of secretory competence (KAUFMAN. 1976). Here we describe some of the ultrastructural and functional changes which can be taken as diagnostic of salivary gland degeneration, and we present evidence indicating that the signal which initiates autolysis is hormonal.
MATERIALS Experimental
AND
METHODS
animals
The ticks used in this study (Amblyomma hebraeum Koch) came from a colony established here with specimens originating from the Veterinary Research Institute, Onderstepoort, Republic of South Africa. 241
242
ROBERTA.
HARRIS AND
The colony is maintained at 26°C in darkness, relative humidity being approx. 95%. When adults are confined to the backs of rabbits as described by KAUFMAN and PHILLIPS (1973), within 7-10 days they will usually feed to repletion (about 1200-3000 mg) and then detach voluntarily. In the text of this paper, such ticks are referred to as ‘gorged’ or ‘replete’. The ticks referred to as ‘partially fed’ are specimens forcibly removed from the rabbit after achieving a weight of between 120 and 450 mg. As will be shown later, subsequent fate of the salivary glands depends on the degree of engorgement. Method for
organ transplant
To test whether degeneration could be associated with any humoral factors, salivary glands of partially fed ticks (120-280 mg) were transplanted to the haemocoels of replete females which had dropped from the host within the previous 12-24 hr. Salivary glands similarly transplanted into the haemocoels of other partially fed females served as controls. Donor ticks were immobilized with ‘Plasticine’ modelling clay (Harbutt’s, England) and flooded with modified Hank’s balanced saline (composition in g/l: NaCl, 11.5; D-glucose, 1.6; KCI, 0.4; CaCI,, 0.14; MgSO,, 0.098; KH,HPO,, 0.06; Na,HPO,, 0.048; phenol red, 0.01; 360 mosm; pH 7.0). The salivary glands were then dissected out as described by KAUFMAN and BARNETT (1977). All recipient ticks were chilled on ice for 20 min. This period of anaesthesia was mandatory to prevent the delicate gut diverticula from gaping through the incision and bursting. For the control series of recipients, a small semicircular incision was made in the dorsum. A
Fig. 1. Diagram of apparatus used to prevent escape of gut contents from gorged females during organ transplant procedure. The apparatus was assembled from part of a Sartorius plastic filter holder normally used for sterile filtration of organ culture media. Adhesive tape, secured to the tick’s dorsal integument by means of an annulus of ‘Super Glue’, was placed over the funnel portion of the filter holder. The filter holder was then connected to the laboratory vacuum line. The degree of vacuum could be regulated by means of an inlet valve so as to just maintain the gut contents within the abdomen during surgery. Access to the haemocoel was achieved by slicing through the tape and integument in the desired region by means of a fine razor-blade scalpel. Following implantation of the salivary gland, the wound was sealed with ‘Super Glue’; most of the excess tape was then cut away and discarded.
WILLIAMR. KAUFMAN gland from a partially fed donor was delivered through this flap of integument into the haemocoel and pushed some way distant from the wound. The incision was sealed with a cyano-acrylate compound (‘Super Glue’, Bondo) and the ticks returned to the 26°C incubator and kept in vials held over saturated KNO, at a relative humidity of 93:,,. In the case of the gorged female recipients, chilling alone was not usually sufficient to prevent escape of gut contents on cutting the dorsal integument. After chilling, engorged females were suspended in a small vacuum chamber as shown and described in Fig. 1. In this apparatus, the tick could be positioned in such a way that, except for the small area of cuticle to be wounded, the remainder of the tick could be maintained under slightly hypobaric conditions. The degree of vacuum was not measured, but was adjusted so as to just retain the gut contents within the tick. After implanting the salivary gland, the wound was sealed with ‘Super Glue’ and the tick returned to the incubator. Ticks appeared to suffer no obvious adverse effects from these operations. Two to four days following implantation, the salivary glands of the donor and recipient ticks were recovered and fixed for subsequent observation under the electron microscope. Electron microscop) Salivary glands were dissected out under Hank’s balanced saline, the large tracheae dissected away and the glands fixed for 12 hr in 2.5% glutaraldehyde in 0.05 M cacodylate buffer made up in 2.5% sucrose (total osmotic pressure = 360 mosm). After briefly washing in cacodylate buffer containing 4% sucrose, the tissue was post-fixed in 4% OsO,-O.O5 M cacodylate buffer, pH 7.4 for 1 hr and washed 3 times with fresh buffer. Tissue was stained for 20 min in 2% uranyl acetate and then passed through an ascending series of ethanol concentrations followed by two changes of 100yO propylene oxide. Tissues were kept overnight in Araldite 502-propylene oxide (1 : 1, v/v), then washed twice with pure Araldite 502 and transferred to plastic moulds. Polymerization proceeded at 60°C for 48 hr. Thin sections were taken on a Porter-Blum Ultramicrotome. Grids (150 mesh coated with Formvar) were stained in 5% uranyl acetate in methanol for 25 min and counterstained in lead citrate for 2 min (REYNOLDS, 1963). Grids were examined on a Phillips 300 transmission electron microscope. Enzyme assays Assays were carried out on crude homogenates prepared as follows: salivary glands were dissected out under 1.27, NaCl (isosmotic to tick haemolymph) and carefully cleared of large tracheae and extraneous tissue. Wet weights of tissue were recorded on a Sartorius 2474 microbalance 90 set after removing the tissue from the dissection medium. During this 90-set period, glands were blotted for 30 set on filter paper according to a standard procedure. After weighing, glands were homogenized in 500-750 ~1 ice-cold 11.2% sucrose containing 5 mM ethylenediaminetetraacetate (EDTA). After removing an aliquot for determination of protein concentration,
Salivary gland autolysis each homogenate was transferred to a plastic vial (1 ml total capacity), capped and frozen in methanol-dry ice. Homogenates were stored at - 17°C for up to 7 weeks before assay. Succinate dehydrogenase, a mitochondrial marker enzyme, was assayed according to a procedure modified after PENNINGTON (1961) and PORTEUS and CLARK (1965). One hundred microlitres of homogenate was mixed in a glass-stoppered centrifuge tube with 1 ml buffer-substrate (composition: O.loo p-iodonitrotetrazolium violet, 25 mM sucrose. 2 mM EDTA, 50 mM sodium succinate, all in 50 mM potassium phosphate buffer, pH 7.4) and pre-incubated on ice for 30 min. The tubes were again vortexed briefly and incubated at 42OC for 20 min. The reaction was stopped with 1 ml ice-cold 10“; trichloroacetic acid. four millitres water-saturated ethylacetate was then added and the tubes vortexed for 30 sec. Following a brief
in ticks
243
centrifugation to separate the phases, absorbance of the organic phase was read at 490 pm on a Beckman DU-2 spectrophotometer. Parallel samples stopped at zero time served as blanks. Acid phosphatase was assayed according to the method of DINGLE (1977) with slight modifications. One hundred microliters of homogenate was mixed in a glass-stoppered centrifuge tube with 200 ~1 buffered glycerophosphate (composition: 5 mg sodium glycero-2-phosphate in 1 ml 0.1 M acetate buffer, pH 5.0) and incubated at 42°C for 20 min. The reaction was stopped with 2 ml ice-cold lo”,, trichloroacetic acid and the tubes left on ice for 15 min. The tubes were centrifuged at 500 g for 10 min. Two millilitres supernatant was mixed with 2 ml ammonium molybdate reagent (composition: 2.5 g (NH,),Mo,0,;4 H,O in 100 ml 0.5 M H,SO,) and 1 ml Elon reagent (Eastman Kodak; composition: 0.5 g 4-methylaminophenol sulphate in 100 ml lo”,,
_A
Partlal ly fed, n=7
Day of
engorgement, n =7
I day post- engorgement, n= 4
3
I 0
,
Minutes
after
IO
,
I
20 injecting
1
I
I
40
days post- engorgement? n=3
,
60 410 @M/kg b.w
pllocarplne,
(b) Partially
PF
,I
fed,
n= IO
day post - removal, n= 5
Day of engorgement, n= 5
I
20
0 Minutes
after
40
injecting dopamine,
day post- engorgement, n = 6
3 days,
n=5
5 days,
n=3
60
530 PM/kg
bw
Fig. 2. (a) Salivation response (cumulative saliva as ‘,” b.w.) to pilocarpine as a function of phase of the feeding cycle. Means, S.E.‘s and N for each group are indicated. Replete ticks show a weaker response to pilocarpine than partially fed ticks and the response decreases as a function of time postengorgement. (b) As in (a) for dopamine.
244
ROBERTA. HARRISAND WILLIAMR. KAUFMAN
(w/v) sodium metabisulphite). After 10 min (and within 1 hr) absorbance at 750 pm was recorded. Amount of inorganic phosphate (Pi) was calculated from the absorbance by means of a standard curve. Samples stopped at zero time served as blanks. Protein was measured by the dye-binding method of BRADFORD (1976) using bovine serum albumin as standard. The 10-30 ~1 aliquots taken for protein determination were first dissolved in an equal volume of 1 N NaOH to solubilize the protein. Injection of drugs The method for introducing pilocarpine and dopaminedrugs which elicit salivation-into the haemocoel is described in full by KAUFMAN (1978). In brief, a 30-gauge needle, fitted to an ‘Agla’ micrometer syringe unit (Burroughs-Wellcome), was introduced a few millimetres into the haemocoel via the articulation joining the capitulum to the thorax. Drugs were dissolved in 1.2% NaCl and were delivered in 1 pl/lOO mg by weight doses. Vehicle-induced salivation was zero. Saliva was collected from the mouthparts in capillary tubes of constant bore such that volumes could be calculated from the length of the fluid column. See further details in KAUFMAN (1978).
RESULTS Dopamine is known to elicit fluid secretion in tick salivary glands by direct post-junctional stimulation (KAUFMAN, 1976, 1977), whereas pilocarpine does so only indirectly (KAUFMAN, 1978). When partially fed ticks (150-350 mg) were removed prematurely from the host and injected with pilocarpine or dopamine, there was only a slight decline in re?ponse as a function of the time elapsed between removal from the host and performance of the injection (Fig. 2). But this decline in response was far more striking and more rapid in replete ticks (> 1000 mg) subjected to the same treatment.Thuswehaveasimple,physiologicaldemonstration of post-prandial salivary gland degeneration. These physiological events can be correlated with characteristic ultrastructural changes in the secretory epithelium (MEGAW and BEADLE, 1979). For a full description of the morphology and ultrastructure of salivary glands from partially fed Dermacentor andersoni females, see MEREDITH and KAUFMAN (1973). Comparing Figs. 3a and b, we see that the fluid-secretory cell of glands taken from partially fed ticks, shows little, if any, change in cytological appearance up to at least 2 days post-removal from the host. Just following engorgement (Fig. 3c), one notices the early stages of formation of autophagic vacuoles, composed of whorls of cell membrane surrounding cytoplasm. By 2 days post-engorgement (Fig. 3d) these autophagic vacuoles are more numerous and contain various inclusion bodies including degenerating remains of mitochondria. We routinely find such structures very early in the postengorgement phase, and on occasion, although not shown here, even in large ticks which are still attached to the host. In the latter cases, the autophagic vacuoles are smaller, less well-developed and are fewer in number. The remaining 2 micrographs in Fig. 3 show the
results of experiments in which a gland from a partially fed tick was transplanted into the haemocoel (see Materials and Methods) of either another partially fed specimen (control, Fig. 3e) or into an engorged specimen (experimental, Fig. 39. The transplanted glands were recovered 2 days later and prepared for observation under the electron microscope. The host glands were also examined. The control glands (Fig. 3e) appear virtually indistinguishable from those of normal partially fed ticks (compare with Figs. 3a, b), indicating that the trauma of the operation does not adversely influence cytology of the fluid-secretory cell. In the experimental ticks (Fig. 30, however, the presence of autophagic vacuoles is clear evidence that autolysis has been induced. We have observed similar results for 7 replicates of the control and 5 replicates of the experimental. The transplanted glands, however. seem to be at a less-advanced stage of degeneration than the host glands of the experimental series, even though there appears to be little difference in the stage of degeneration between host glands of the experimental series and normal glands in gorged ticks 2 days postrepletion. Inasmuch as an obvious sign of gland degeneration cell is the appearance in the fluid-secretory of autophagic vacuoles and the isolation of mitochondria, we tested whether there was a change in specific activity of certain ‘marker enzymes’ for these structures. Succinate dehydrogenase is believed to reside chiefly, if not exclusively, in the inner mitochondrial membrane (LEHNINGER, 1975) and acid phosphatase has long been associated with the presence of lysosomes (DINGLE, 1977). In Fig. 4 we present the changing titre of these two enzymes in crude homogenates of glands in both partially fed and replete ticks as a function of time. In partially fed ticks, there is no significant change in activity for either enzyme up to 5 days post-removal. For engorged ticks, on the other hand, there is a marked rise in acid phosphatase activity and a precipitous fall in succinate dehydrogenase activity within the same time period. In order to see how early in the feeding cycle one would notice enzyme changes characteristic of the engorged tissue, we removed partially fed ticks at various stages of engorgement and monitored succinate dehydrogenase and acid phosphatase activities in the salivary glands. Glands taken from ticks < 450 mg showed little change in enzyme activity within 5 days. Beyond that approximate weight, however, changes in enzyme titre with time resembled those pertaining to normal replete specimens. Normally. ticks of this weight range (400-600 mg) feed to repletion within 12-24 hr.
DISCUSSION Although there have been a number of good histological studies on tick salivary glands since the turn of the century, TILL (1961) seems to have been the first to follow histological changes in this tissue with both development and feeding cycles, and hence to describe the degeneration phenomenon in some detail. We present here the first demonstration that degeneration is triggered, and perhaps supported, by a
Fig. 3. Ultrastructural appearance of the fluid-secretory cell of the type III acinus as a result of various treatments. (a) Salivary gland of a partially fed tick dissected out and fixed within a few hours of removing tick from the host. (b) Gland of a tick similar to that in (a) fixed 2 days post-removal from host. No important changes take place at least during this 2-day period. Scale as in 3(a). Cc) Fluid-secretory cell of an engorged tick fixed on the same day as the tick engorged. Precise time of engorgement was not determined (see text). Fixation could have been as much as 17 hr after drop-off from the host. Arrow denotes an isolation body. Scale as in 3(a). (d) Gland of a tick similar to that in (c). but 2 days post-engorgement. Note the appearance of various inclusion bodies within the autophagic vacuoles. (e) Fluid-secretory cell of a control tick. Salivary gland of this partially fed tick had resided in the haemocoel of another partially fed specimen for 2 days prior to fixation. This fluid-secretory cell shows no obvious signs of degeneration. (f) Fluid-secretory cell of an experimental tick. Salivary gland of this partially fed specimen had resided in the haemocoel of a replete tick for 2 days prior to fixation. Autophagic vacuoles containing inclusion bodies (arrow) indicate that degeneration is proceeding in this gland. Magnification 19,200 Y
Salivary gland autolysis in ticks 60~
(a)
5 2;$
Acid phosphatase
( b 1 Succlnate
dehydrogenase
Days post removal / post engorgement
Fig. 4. Changes in activity of two enzymes as a function of time post-removal (for partially fed ticks, l) or postengorgement (for replete ticks, A). means, S.E.‘s and N are indicated for each point. (a) Acid phosphatase activity. (b) Succinate dehydrogenase activity. For both enzymes, there is little change in activity up to 5 days post-removal for the partially fed ticks. In the engorged ticks. there is a progressive rise in acid phosphatase activity and a decline in succinate dehydrogenase activity with time post-engorgement. Statistical differences between partially fed and engorged specimens are indicated for each day as follows: *0.05 > p z 0.01; **p < 0.01.
blood-borne factor (here tentatively named ‘salivary gland degeneration factor’): namely, glands can be induced to degenerate in isolation from all nervous influence by placing them in a milieu (haemocoel of a replete tick) in which resident glands are degenerating. The proof that this autolysis is a normal physiological process rather than a pathological necrosis, resulting from isolating the tissue from neural influence or its extensive tracheal supply, is clear from a variety of data. First and foremost, the controls did not degenerate, although lack of neural and tracheal input applied to them equally. Apparently, the transplanted tissue receives sufficient oxygen for its needs from that remaining in the attached stumps of tracheae and/or from whatever pool is circulating in the host haemolymph. Secondly, we regard it as significant that the timecourse for degeneration in the transplanted tissue lagged somewhat behind that pertaining to the host glands. Although as yet we have no experimental evidence to bear on the matter, this lag could be explained in at least two ways. (A) Hormone-receptor interaction may be disrupted or the subsequent metabolic events associated with autolysis may proceed less efficiently in the transplanted tissue. (B) The transplanted tissue was most likely exposed to the
247
hormone for a far briefer time than the host tissue. We mentioned earlier that the initial release of salivary gland degeneration factor probably occurs during the last day or so of engorgement while the tick is still attached to the host. Also, for some tick species. including A. hebraeum. there is a diurnal rhythm to drop-off from the host (RECHAV, 1978) with the peak period of drop-off occurring toward the end of the photophase. Since the engorged specimens which we used for our experiments had been collected from the rabbits in the morning, the host glands of the experimental ticks could well have had some 12-24 hr exposure to salivary gland degeneration factor before the transplantations were performed. This fact may easily have contributed to the more advanced degree of degeneration apparent for the host tissue. Thirdly, we noticed in a separate group of control specimens, that both the host and donor glands showed definite signs of degeneration. These particular ticks had been feeding very slowly. A number of mammalian species, including rabbits, are known to develop a strong immunity to tick infestation under certain circumstances (ALLEN and HUMPHREYS, 1979; BOWE~SIDJAOUet al.. 1977). Ticks attached to such immune hosts tend to feed very slowly, and eventually die in situ if they do not detach voluntarily or are not removed by the experimenter. On occasion. we have dissected,out the salivary glands of such slow-feeding ticks and have prepared them for in vitro fluid secretion as described by KAUFMAN (1977), but in most cases secretory rates were abnormally low (unpublished observations). Perhaps one result of this abnormal feeding behaviour on immune rabbits is an untimely release of salivary gland degeneration factor. In any event, we underline the apparently ‘negative result’ of the above-mentioned group of controls as indicating that transplanted glands assume the behaviour of the host glands, be it degeneration or otherwise. We have considered the possibility that postengorgement degeneration is due to the withholding of a ‘salivary gland maintenace factor’, rather than the secretion of a degeneration factor. However, from some preliminary experiments, we believe this explanation to be unlikely. Salivary glands from recently engorged ticks held in organ culture for 5 days failed to degenerate as judged from the activities of succinate dehydrogenase and acid phosphatase. Unless the putative maintenance factor happens to be one of the normal ingredients of TC 199 (we did not supplement the medium with undefined additives), these experiments speak against the existence of a specific maintenance factor. With a view towards developing a simple, to monitor salivary gland quantitative assay degeneration for future experiments, we tested succinate dehydrogenase and acid phosphatase activities as putative candidates, and found that succinate dehydrogenase activity did indeed fall, and acid phosphatase activity rose in glands from engorged ticks, as anticipated (Fig. 4). Although the fall in succinate dehydrogenase activity in engorged specimens was precipitous over the first day or two, for some as yet unexplained reason succinate dehydrogenase activity in freshly gorged specimens is much higher than that in partially fed ticks
ROBERTA. HARRISAND WILLIAMR. KAUFMAN
248
(200400 mg), despite the fact that isolated glands from partially fed ticks secrete fluid in vitro more rapidly (KAUFMAN, 1976). In the final analysis, differences in succinate dehydrogenase activity between the glands of partially fed and engorged ticks do not become statistically significant until the second or third day post-engorgement/removal (Fig. 4). Similarly for acid phosphatase, significant differences between engorged and partially fed tissues are apparent only after day 2. Hence, although the activities of these two enzymes may well reflect progress of degeneration, physiological (Fig. 2) and ultrastructural (Fig. 3) changes are detected much earlier. We are now investigating other methods as putative assays for the release of salivary gland degeneration factor. Acknowledgemenzs-This research was nenerouslv supported by NSERC Canada and the Canadian National Sportsmen’s fund (Grant No. 3-R72). We are indebted to Dr. E. J. SANDERS, Department of Physiology, University of Alberta for allowing us the use of his electron microscope facility and for reviewing the manuscript.
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BALASHOV Yu. S. (1972) Blood-sucking ticks (Ixodoidea)Vectors of diseases of man and animals. Misc. Publs. ent. Sot. Am. 8 (5).
BINNINGTONK. C. (1978) Sequential changes in salivary gland structure during attachment and feeding of cattle tick, Boophilus microplus. Int. J. Parasitol. 8, 97-115. BOWE~SIDJAOU J., BROSSARD M. and AFSCHLIMANN A. (1977) Effects and duration of resistance acquired by rabbits on feeding and egg laying in Ixodes ricinus L. Experientia 33, 528-530.
BRADFORD M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. COONS L.
B. and ROSHDYM. A. (1973) Fine structure of the salivary glands of unfed male Dermacentor variabilis (Say) (Ixodoidea : Ixodidae). J. Parasit. 59, 900-912.
DINGLE J. T. (Ed.) (1977) Lysosomes. A Laboratory Handbook. 2nd edition. North-Holland, Amsterdam. KAUFMANW. (1976) The influence of various factors on fluid secretion by in vitro salivary glands of ixodid ticks. J. exp. Biol. 64, 727-742.
KAUFMANW. R. (1977) The influence of adrenergic agonists and their antagonists on isolated salivary glands of ixodid ticks. Eur. J. Pharmacol. 45, 61-68. KAUFMANW. (1978) Actions of some transmitters and their antagonists on salivary secretion in a tick. Am. J. Physiol. 235, R76-R8
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KAUFMANW. R. and BARNETTS. F. (1977) Dermacentor andersoni: culture of whole salivary glands. Exp. Parasit. 42, 106-I 14.
KAUFMAN W. and PHILLIPSJ. E. (1973) Ion and water balance in the ixodid tick Dermacentor andersoni. I. Routes of ion and water excretion. J. exp. Biol. 58, 523-536. KIRKLANDW. L. (1971) Ultrastructural changes in the nymphal salivary glands of the rabbit tick, Haemaphysalis leparispalustris. J. Insect Physiol. 17, 1933-1946.
LEESA. D. (1946) The water balance in Ixodes ricinus L. and certain other species of ticks. Parasitology 37, l-20. Lens A. D. (1952) The role of cuticle growth in the feeding process of ticks. Proc. zool. Sot. Lond. 121, 759-772. LEHMNGERA. L. (1975) Biochemistry. 2nd edition. Worth, New York. MEGAWM. W. J. and BEADLED. J. (1979) Structure and function of the salivary glands of the tick, Boophilus microplus Canestrini (Acarina : Ixodidae). Int. J. Insect Morphol. Embryol. 0, 67-83.
MEREDITHJ. and KAUFMANW. (1973) A proposed site of fluid secretion in the salivary gland of the ixodid tick Dermacentor andersoni. Parasitology 67,205-217.
PENNINGTONR. J. (1961) Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem. J. 80, 649-654. PORTEOUSJ. W. and CLARK B. (1965) The isolation and characterization of subcellular components of the epithelial cells of rabbit small intestine. Biochem. J. 96, 159-171.
RECHAVY. (1978) Drop-off rhythms of engorged larvae and nymphs of the bont tick, Amblyomma hebraeum (Atari : Ixodidae), and the factors that regulate them. J. med. Em. 14, 677-687.
REYNOLDS E. S. (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-21 I.
TILL W. M. (1961) A contribution to the anatomy and histology of the brown ear tick, Rhipicephalus appendiculatus (Neumann). Mem ent. Sot. sth. Afr. 6, April.