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Correlation between haemolymph ecdysteroid titre, salivary gland degeneration and ovarian development in the Ixodid tick, Amblyomma hebraeum Koch
3. Insect Physiol. Vol. 31, No. 2, pp. 95-99, 1991 Copyright 0
Printed in Great Britain. All rights reserved
0022-1910/91 $3.00 + 0.00 1991 Pergamon Pressplc
CORRELATION BETWEEN HAEMOLYMPH ECDYSTEROID TITRE, SALIVARY GLAND DEGENERATION AND OVARIAN DEVELOPMENT IN THE IXODID TICK, AMBLYOMMA HEBRAEUM KOCH W. REUBEN KAUFMAN Department of Zoology, University of Alberta, Edmonton, Alberta T6G 2E9, Canada (Received 8 June 1990; revised 4 October 1990)
Abstract-In
the female tick, Amblyomma hebraeum Koch (Atari: Ixodidae), the salivary glands degenerate and vitellogenesis begins, provided the tick exceeds a critical weight (approx. 300-350 mg). The following were measured in ticks: (1) at various times following normal engorgement (1000-3000 mg) and (2) at various stages (weights) of the feeding cycle, 7 days after removal from the host: salivary fluid secretory competence and ecdysteroid content (by radioimmunoassay) of haemolymph and ovary extracts. (1) Haemolymph ecdysteroid titre rose from < 10 ng/ml on the day of engorgement, to 300-400 ng/ml by day 6 after engorgement. Ecdysteroid content of the ovary rose in parallel from less than 10 rig/g wet weight to 1000-2000 rig/g by day 6. (2) In ticks weighing up to 300 mg, there was a progressive rise in fluid secretory competence, and haemolymph ecdysteroid titre remained below 50 ng/ml. In ticks weighing 3004OOmg, haemolymph ecdysteroid titre was approx. 90 ng/ml and salivary fluid secretory competence had declined by 50% relative to ticks weighing 200-300 mg. In ticks weighing 400-500mg, haemolymph ecdysteroid titre was approx. 140 ng/ml and fluid secretory competence had fallen by over 90% compared to ticks weighing 200-300 mg. Haemolymph ecdysteroid titre continued to rise in larger ticks, peaking at about SOOng/ml. Ecdysteroids were more concentrated in the ovary than in the haemolymph, peaking at about 3500 rig/g in large engorged ticks, 7 days after removal. The data support the following hypothesis: when the tick exceeds the critical weight, ecdysteroids are released (from an unknown endocrine organ) into the haemolymph, where they trigger salivary gland degeneration. The hormone also accumulates in the ovary where it may play a role in tick development and/or hatching. Key Word Index:
Amblyomma;
ticks; salivary gland degeneration;
vitellogenesis; ecdysteroids
ecdysone into the haemocoel, or by exposing isolated glands in organ culture to physiological concentrations of ecdysteroids (Harris and Kaufman, 1985). The latter suggest that the natural degeneration factor is an ecdysteroid. This contention is also supported by the demonstration of Connat et al. (1985) that haemolymph ecdysteroid titre rises dramatically following engorgement. In this study, I demonstrate a direct correlation between haemolymph ecdysteroid titre and salivary gland function. This supports the hypothesis that the degeneration factor is an ecdysteroid, but further data also suggest that ecdysteroids probably have other physiological functions in the engorged female.
INTRODUCTION
Female ticks (Atari) of the family Ixodidae utilize their salivary glands to secrete back into the host a hyposmotic fluid derived from the huge blood meal; as a result of this process, haemolymph volume and osmotic concentration are regulated (Kaufman, 1983). Following engorgement, the salivary glands are resorbed (Till, 1961) due to the secretion of a hormone, “tick salivary gland degeneration factor” (Harris and Kaufman, 1981). This hormone is released only after the tick exceeds a “critical weight” which is about 10 times the unfed weight in Amblyomma hebraeum (Harris and Kaufman, 1984) and A. americanurn (Lindsay and Kaufman, 1988). The full degree of degeneration is usually achieved within 4-5 days after removal once the tick exceeds its critical weight. If ticks are removed from the host below the critical weight, salivary gland degeneration does not occur for at least 2-3 weeks (Harris and Kaufman, 1984). However, one can trigger salivary gland degeneration in such ticks by infusing 20-hydroxy-
MATERIALS
AND METHODS
Ticks
Experimental ticks were taken from a laboratory colony maintained at 26°C in darkness at >95% relative humidity. For most experiments (mated 95
96
W. REDEN KAUFMAN
females), 3040 males were placed on the rabbit a day or so before introducing an equal number of females. Prior attachment of males encourages females to attach and feed more readily. Mating occurs on the host. To produce fed virgins, however, the gonopore of each male was first plugged with a droplet of cyanoacrylate adhesive (“Locktite”; Newington, Conn., U.S.A.) before putting them on the host. The next day, females were added, and checked for attachment every few hours. As soon as most of the females had attached, (generally within 12 h if the males had been fed before, or within 24 h if the males were previously unfed), all the males were removed from the host. The virginity of each experimental female was later confirmed by examining the seminal receptacle for absence of spermatophores, and by examining a smear of the seminal receptacle for absence of spermatozoa. Because virgin females do not readily feed beyond the critical weight, the usual level of infestation for experiments involving virgins was 60 females. Assay for salivary gland degeneration
I use fluid secretion in vitro as a routine assay for salivary gland function. Salivary glands were removed from the tick, the main duct was ligated with a fine strand of silk, the tissue was gently blotted, weighed to the nearest 0.01 mg and incubated at room temperature in constantly stirred TC medium 199 containing 10 pm dopamine. [Dopamine stimulates salivary fluid secretion and is probably the neutral transmitter substance (Kaufman and Wong, 1983)]. After incubation (usually 10 or 15min), the glands were again gently blotted and weighed. Net weight increase under these conditions represents fluid secretory competence by the gland, and is thus an index of salivary gland degeneration. Further details are provided in Kaufman (1983) and Harris and Kaufman (1984). Methodfor sampling haemolymph and ovary for radio immunoassay (RIA)
Ticks were removed from the host, rinsed in water, secured ventrum down to a disposable Petri dish by means of the cyanoacrylate adhesive. They were chilled under crushed ice for about 15 min in order to maximize haemolymph volume collected (see Discussion). A cut was made with a razor blade scalpel a few millimetres around the lateral margin of the integument. Following gentle pressure on the dorsum, haemolymph exuding from the wound was collected in a volumetric capillary tube. The incision was then extended around the integument, leaving only the posterior region of the lateral margin intact. Muscle attachments were cut as necessary to enable reflection of the dorsum toward the posterior end. Usually additional haemolymph could be collected by careful application of the capillary tube to avoid contamination with any gut or Malpighian tubule contents. Under these conditions, I normally collected a mini-
Table 1. Haemolymph volume (~1) collected from pre-chilled ticks Tick weight range (mg) <150 150-260 260400 400-550
> 550 Pooled
Mean volume (JJl) 7.8 11.2 9.6 6.0 11.5 9.4
SEM n 1.1 11 1.8 10 1.7
8
1.1 9 1.5 13 0.7 51
mum of 5 ~1 and frequently a few tens of microlitres of haemolymph from each female (see Table 1). After as much haemolymph as practical was collected, the tick was flooded with modified Hank’s medium (composition in g/l: NaCl 11.5; D-glucose 1.6; KC1 0.4; CaCl, 0.14; MgS04 0.10; KH2P04 0.06; Na,HPO, 0.05; phenol red 0.01; 360 mosmolar; pH 7.0) and the dorsum removed. The salivary glands were dissected out for measuring fluid secretory competence as described above. The ovary was dissected out, cleared of tracheae and other adhering tissue and transferred to fresh TC 199 prior to weighing and processing for the ecdysteroid RIA. RIA for ecdysteroidr
Complete details of the RIA and for processing tissue are presented by Kaufman (1990). Modifications were: (a) it was never necessary to pool haemolymph from several individuals in this study for a single RIA sample; (b) the antibody (directed against ecdysone-22-succinylthyroglobulin; a gift from Dr L. I. Gilbert, University of North Carolina) was more concentrated (& was adjusted to 20-30%) than in the previous study. This increased the useful ecdysteroid concentration range detectable by the assay with only a small concomitant increase in the reliable limit of detection (20-30 pg 20-hydroxyecdysone). All other details of the assay were the same as those described by Kaufman (1990). Because the antibody cross-reacts with several ecdysteroid analogues (Warren and Gilbert, 1988), the data is expressed as “20-hydroxyecdysone equivalents”. Drugs and chemicals
TC Medium 199 was purchased from Gibco Inc. (Grand Island, New York), 20-hydroxyecdysone from Simes (Milan, Italy), dopamine and bovine serum albumin (RIA grade) from Sigma (St Louis, MO., U.S.A.), radiolabelled ecdysone (ecdysone, CI[23,24-3H(N)]-, 45.0 Ci/mmol) from New England Nuclear (NEN DuPont Canada, Lachine, Quebec). Other chemicals were from various sources and of the purest grade available. Experiment
1
Ecdysteroid projile of haemolymph and ovary after engorgement. Ticks were permitted to feed to re-
pletion (at least 1000 mg). Haemolymph
and ovary
97
Ecdysteroids and tick salivary gland degeneration
9-13 after engorgement. Ovary content of ecdysteroids in engorged ticks within 2 days after engorgement was also at a low level [9.4 + 9.4(10) rig/g]] and rose more or less in parallel with the haemolymph titre, peaking at 1738 + 355(10) rig/g by day 9-13 after engorgement.
ZO-OHE equivalents bglma
qr ~1)
Experiment 2 (Figs 2 and 3) haemolymph ovary
o-2
were taken
n
7-8 5-6 3-4 days post-engorgement
Fig. 1. Time course after ovary ecdysteroid titre. pooled as indicated on wherever it exceeds samples
0
9-13
engorgement for haemolymph and Ticks. from different days were the abscissa. The SEM is shown the dimension of the symbol. as described
above
from
ticks
O-13 days after engorgement. Haemolymph was added to 100 ~1 methanol. The ovary was blotted, weighed and, if less than about 10-15 mg, the whole ovary was homogenized in 100 ~1 methanol; in the case of larger ovaries, only a piece (about 2-15 mg) was homogenized. The haemolymph and ovary homogenates were stored at - 15°C until processed further for RIA. Experiment 2
There was no apparent correlation between tick weight and salivary gland weight on day 7 after removal for ticks weighing up to 500 mg, although beyond this weight, the glands were too degenerated to dissect out and weigh; they were thus assigned a weight of “zero” (Fig. 2). The glands of some virgins, in contrast, were present in large ticks (500-1200 mg), although none of them secreted fluid in the assay. Ovary weight is a crude index of degree of vitellogenesis. Up to a tick weight of 300_1OOmg, there was no significant sign (t, > 0.05) of vitellogenesis 7 days after removal. Beyond that weight, there was progressively more vitellogenesis. Ovary weights of virgins were similar to those of mated females of similar size (Fig. 2). Data for salivary gland function, haemolymph ecdysteroid titre and ovary ecdysteroid content on day 7 after removal/after engorgement are presented in Fig. 3. Salivary gland function improved progressively in ticks weighing up to 300 mg. Thereafter, fluid transport ability plunged to a low value in ticks weighing 400-5bOmg. -Reduction in salivary gland
Critical weight for salivary gland degeneration. Ticks were removed from the host at various stages (weights) of the feeding cycle and held for 7 days in the colony incubator. Haemolymph and ovary samples were collected as described above and kept at - 15°C awaiting processing for RIA. The salivary glands were dissected out and fluid transport measured. Statistics All data are presented as mean + SEM (n). Statistical significance was determined by one-way analysis of variance using the MIDAS statistical package of the university’s mainframe computer. RESULTS
Haemolymph sampling
100
200
500
tick weight
Table 1 shows the haemolymph volume collected from ticks used in this study. There was no apparent correlation between tick size and haemolymph volume collected, the average haemolymph sample being 9-10 /Ll. Experiment
1 (Fig. 1)
In a population of engorged ticks, sampled within 2 days of engorgement, the haemolymph ecdysteroid titre was 9.0 f 3.6(11) ng/ml. The titre rose dramatically thereafter, peaking at 414 _I 45(9) ng/ml by day
1000
2000
(mg)
Fig. 2. Wet weight of ovary and salivary glands on day 7 after removal/after engorgement as a function of tick weight. The tick weights shown here and in Fig. 3 are those recorded on day zero. (Weight by day 7 was 87.2% + 0.7(66) of initial weight; the degree of weight loss was similar for all ticks in this study, irrespective of their initial weight on day zero.) Samples were pooled as follows: tick weight up to -150 mg (Y = 122 + 6;- 10); 150-200 mg (R = 175 + 5; 10); 200-300 mg (x = 256 _+7; 7); 300-400 mg (x= 354 & 12; 6); 400-500mg (x=440 _+8; 15); 5001200 mg (x = 894 + 77; 8); > 1200 mg (x = 1897 k 224; IO). The SEM is shown wherever it exceeds the dimension of the symbol.
W. REUBEN KAUFMAN
98
800
400 i
160 260 tick
200
‘500 1000 2600 weight
(mg)
Fig. 3. Fluid secretory competence by salivary glands, and ecclysteroid content of ovary and haemolymph 7 days after removal/after engorgement as a function of tick weight. The ticks shown here are the same as those presented in Fig. 2. The SEM is shown wherever it exceeds the dimension of the symbol. function was first associated with a haemolymph ecdysteroid titre of 89 + 36(5) ng/ml. Loss of function was almost complete at a haemolymph ecdysteroid titre of I39 + 15(9) ng/ml. However, haemolymph ecdysteroid titre continued its rise in larger ticks, ultimately reaching an average of 823 + 184(S) ng/ml. The titre of virgin haemolymph was similar to that of mated haemolymph in ticks of similar size [528 + 152(6) ng/ml vs 469 + 60(6); p > 0.051. Ovary ecdysteroid content rose beginning in ticks weighing 300-400 mg [57 ) 19(6) rig/g ovary weight] and peaking in large engorged ticks at 3510 + 529( 10) rig/g.. Ecdysteroid content of virgin ovaries was similar to that of ovaries from mated ticks of similar size [3570 + 1076(8) rig/g vs 2568 & 366(S) rig/g;; p > 0.051.
DISCUSSION
Pre-chilling of ticks before collecting haemolymph appears to maximize the volume of haemolymph one can collect. Low temperature inhibits contraction of the gut, and so lessens the likelihood that the delicate gut wall will rupture on the scalpel blade or sharp edge of the wounded cuticle. (Tendency for the gut to rupture increases greatly with degree of engorgement.) I collected up to 30 ~1 per female compared to “only a few microlitres” reported by Connat et al. (1985) for the same species of tick. However, the technique would obviously not be appropriate if the chilling process itself affected ecdysteroid content. If
one compares data from my study with corresponding data from that of Connat et al. (1985), it appears as if our two sampling methods lead to similar results: (1) in this study, haemolymph ecdysteroid titre of engorged ticks rose from about 10 ng/ml on day 0 to about 400-800 ng/ml on day 7. Equivalent data from Connat et al. ranges from 400-600 ng/ml. Since SDS are quite large in both studies, I regard these figures to be in reasonable agreement. (2) Ovary samples in this study contained about 90 ng ecdysteroid/ovary in small engorged ticks (8= 900 mg) and about 340 ng/ovary in large engorged ticks (x = 1900 mg; data calculated from Fig. 2). These values are similar to the range reported by Connat et al. for engorged ticks (weight unspecified), 6-17 days after engorgement (90-180 ng/ovary). How does one account for the increase in fluid secretory competence associated with an increase in tick weight up to 300 mg (Fig. 3)? Salivary glands of unfed ticks have only limited ability to secrete fluid; fluid secretory competence increases markedly during the first week of feeding (Kaufman, 1976), a phenomenon correlated with ultrastructural development of the fluid secretory labyrinth of the type III acinus (Fawcett et al., 1981). The increase in fluid secretion shown here in Fig. 3 is probably a reflection of this glandular development induced early in the feeding period. The concept of a critical weight for the release of the degeneration factor established earlier for day 4 after removal (Harris and Kaufman, 1984; Lindsay and Kaufman, 1988) is confirmed in this study for day 7 as well, and the concept now can be extended to haemolymph ecdysteroid titre and ovary ecdysteroid content (Fig. 3). A ten-fold rise in haemolymph titre (from < 10 ng/ml to no more than 90 ng/ml) is associated with a significant loss of salivary gland function. This level was seen at a tick weight range of 300400 mg (Fig. 3). By 400-500 mg, loss of salivary gland function was almost complete and the haemolymph ecdysteroid titre was about 140 ng/ml. Glands from large virgin females (50&1200 mg), like those from mated females, did not secrete fluid on day 7 after removal (see p. 97). However, unlike those of mated females, the glands from large virgins could be dissected out and weighed (Fig. 2 and p, 97). What accounts for this discrepancy? An earlier study from my laboratory demonstrated that salivary gland degeneration depends not only on the tick attaining a critical weight, but also on the presence of a “male factor” transferred to the female with the spermatophore (Harris and Kaufman, 1984). More recent work from my laboratory indicates that the male factor is not essential for salivary gland degeneration, but it hastens it considerably; whereas glands from large mated females lose virtually all function by day 4 after removal, similar loss of function occurs in virgin glands only by day 8 (Lomas and Kaufman, unpublished). In this study, all glands
Ecdysteroids and tick salivary gland degeneration were tested on day 7; although this time was sufficient to result in complete loss of function in both virgin and mated females, reabsorption of the tissue itself apparently had not yet progressed in the virgin females. If 140 ng ecdysteroid/ml haemolymph is correlated with a virtually complete loss of function, why does the haemolymph titre continue to rise another 5-6fold, to about 800 ng/ml (Fig. 3)? It suggests that ecdysteroids probably have functions in the tick additional to that of salivary gland degeneration. I confirm here the earlier report by Connat et al. (1985) of a high concentration of ecdysteroids in the developed ovary. One proposed function of ecdysteroids in ticks is as the vitellogenetic hormone, similar to what pertains in some groups of insects (Hagedorn, 1983; Zhu et al., 1983; Postlethwaite and Giorgi, 1985). However, there is no direct evidence for this conjecture in ticks; i.e. nobody has demonstrated that ecdysteroids can provoke vitellogenesis in small partially fed ticks. Indeed, my laboratory now has considerable evidence to the contrary (Lunke and Kaufman, unpublished). A more likely alternative, also proposed by Connat et al., is that ecdysteroids accumulate in the ovary to be used in embryogenesis and/or hatching from the egg (Hagedorn, 1983). Another possible explanation for the high ecdysteroid content of the ovary is that this organ produces ecdysteroids, as does the ovary of mosquitos (Hagedorn, 1983). There is no evidence yet for an analogous system in ticks, however. Attempts in my laboratory to ovariectomize ticks have failed so far. Acknowledgements-1
am most grateful to Mr Stephen Cozens for invaluable technical assistance in all aspects of this study, to Dr L. I. Gilbert, Department of Biology, University of North Carolina at Chapel Hill, for his generous gift of the ecdysone antibody used in the RIA and Dr Jim Campbell, Department of Microbiology, University of Alberta, for preparing the protein A required for the RIA. Finally, research in this laboratory is generously funded with an operating grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada.
99 REFERENCES
Connat J.-L., Diehl P. A., Gfeller H. and Morici M. (1985) Ecdysteroids in females and eggs of the ixodid tick Amblyomma hebraeum 103-l 16.
Int. J. Invertebr. Reprod. Dev. 8,
Fawcett D. W., Doxsey S. and Biischer G. (1981) Salivary glands of the tick vector (Ripicephalus appendiculatus) of East Coast fever-II. Cellular basis for fluid secretion in the type III acinus. Tissue Cell 13, 231-253. Hagedorn H. H. (1983) The role of ecdysteroids in the adult insect. In Endocrinology of Insects (Eds Downer R. G. H. and Laufer H.), pp. 271-304. Alan R. Liss, New York. Harris R. A. and Kaufman W. R. (1981) Hormonal control of salivary gland degeneration in the ixodid tick Amblyomma hebraeum. J. Insect Physioi. 27, 241-248.
Harris R. A. and Kaufman W. R. (1984) Neural involvement in the control of salivary gland degeneration in the ixodid tick Amblyomma hebraeum. J. exp. Biol. 109, 281-290.
Harris R. A. and Kaufman W. R. (1985) Ecdysteroids: possible candidates for the hormone which triggers salivary gland degeneration in the ixodid tick, Amblyomma hebraeum. Experientia 41, 740-742.
Kaufman W. R. (1976) The influence of various factors on fluid secretion by in vitro salivary glands of ixodid ticks. J. exp. Biol. 64, 727-742.
Kaufman W. R. (1983) The function of tick salivary glands. In Current Topics in Vector Research (Ed. Harris K. F.), pp. 215-247. Praeger Scientific, New York. Kaufman W. R. (1990) Effect of 20-hydroxyecdysone on the salivary glands of the male tick, Amblyomma hebraeum Koch. Expl appl. Acarol. 9, 87-95. Kaufman W. R. and Wong D. L.-P. (1983) Evidence for multiple receptors mediating fluid secretion in salivary glands of ticks. Eur. J. Pharmacol. 87, 43-52. Lindsay P. J. and Kaufman W. R. (1988) Action of some steroids on salivary gland degeneration in the ixodid tick, Amblyomma americanum L. J. Insect Physiol. 34,351-359. Postlethwaite J. H. and Giorgi F. (1985) Vitelloginesis in insects. In Developmental Biology: a Comprehensive Synthesis (Ed. Browder L. W.), Vol. 1, pp. 85-125. Plenum Press, New York. Till W. M. (1961) A contribution to the anatomy and histology of the brown ear tick, Rhipicephalus appendiculatus (Neumann). Mem. ent. Sot. sth. Afv., Vol. 6. Warren J. T. and Gilbert L. I. (1988) Radioimmunoassay: ecdysteroids. In Immunological Techniques in Insect Biology (Eds Gilbert L. I. and Miller T. A.), pp. 181-214. Springer, Berlin. Zhu X. X., Gfeller H. and Lanzrein B. (1983) Ecdysteroids during oogenesis in the oviparous cockroach Nauphoeta cinerea. J. Insect Physiol. 29, 225-230.
Printed in Great Britain. All rights reserved
0022-1910/91 $3.00 + 0.00 1991 Pergamon Pressplc
CORRELATION BETWEEN HAEMOLYMPH ECDYSTEROID TITRE, SALIVARY GLAND DEGENERATION AND OVARIAN DEVELOPMENT IN THE IXODID TICK, AMBLYOMMA HEBRAEUM KOCH W. REUBEN KAUFMAN Department of Zoology, University of Alberta, Edmonton, Alberta T6G 2E9, Canada (Received 8 June 1990; revised 4 October 1990)
Abstract-In
the female tick, Amblyomma hebraeum Koch (Atari: Ixodidae), the salivary glands degenerate and vitellogenesis begins, provided the tick exceeds a critical weight (approx. 300-350 mg). The following were measured in ticks: (1) at various times following normal engorgement (1000-3000 mg) and (2) at various stages (weights) of the feeding cycle, 7 days after removal from the host: salivary fluid secretory competence and ecdysteroid content (by radioimmunoassay) of haemolymph and ovary extracts. (1) Haemolymph ecdysteroid titre rose from < 10 ng/ml on the day of engorgement, to 300-400 ng/ml by day 6 after engorgement. Ecdysteroid content of the ovary rose in parallel from less than 10 rig/g wet weight to 1000-2000 rig/g by day 6. (2) In ticks weighing up to 300 mg, there was a progressive rise in fluid secretory competence, and haemolymph ecdysteroid titre remained below 50 ng/ml. In ticks weighing 3004OOmg, haemolymph ecdysteroid titre was approx. 90 ng/ml and salivary fluid secretory competence had declined by 50% relative to ticks weighing 200-300 mg. In ticks weighing 400-500mg, haemolymph ecdysteroid titre was approx. 140 ng/ml and fluid secretory competence had fallen by over 90% compared to ticks weighing 200-300 mg. Haemolymph ecdysteroid titre continued to rise in larger ticks, peaking at about SOOng/ml. Ecdysteroids were more concentrated in the ovary than in the haemolymph, peaking at about 3500 rig/g in large engorged ticks, 7 days after removal. The data support the following hypothesis: when the tick exceeds the critical weight, ecdysteroids are released (from an unknown endocrine organ) into the haemolymph, where they trigger salivary gland degeneration. The hormone also accumulates in the ovary where it may play a role in tick development and/or hatching. Key Word Index:
Amblyomma;
ticks; salivary gland degeneration;
vitellogenesis; ecdysteroids
ecdysone into the haemocoel, or by exposing isolated glands in organ culture to physiological concentrations of ecdysteroids (Harris and Kaufman, 1985). The latter suggest that the natural degeneration factor is an ecdysteroid. This contention is also supported by the demonstration of Connat et al. (1985) that haemolymph ecdysteroid titre rises dramatically following engorgement. In this study, I demonstrate a direct correlation between haemolymph ecdysteroid titre and salivary gland function. This supports the hypothesis that the degeneration factor is an ecdysteroid, but further data also suggest that ecdysteroids probably have other physiological functions in the engorged female.
INTRODUCTION
Female ticks (Atari) of the family Ixodidae utilize their salivary glands to secrete back into the host a hyposmotic fluid derived from the huge blood meal; as a result of this process, haemolymph volume and osmotic concentration are regulated (Kaufman, 1983). Following engorgement, the salivary glands are resorbed (Till, 1961) due to the secretion of a hormone, “tick salivary gland degeneration factor” (Harris and Kaufman, 1981). This hormone is released only after the tick exceeds a “critical weight” which is about 10 times the unfed weight in Amblyomma hebraeum (Harris and Kaufman, 1984) and A. americanurn (Lindsay and Kaufman, 1988). The full degree of degeneration is usually achieved within 4-5 days after removal once the tick exceeds its critical weight. If ticks are removed from the host below the critical weight, salivary gland degeneration does not occur for at least 2-3 weeks (Harris and Kaufman, 1984). However, one can trigger salivary gland degeneration in such ticks by infusing 20-hydroxy-
MATERIALS
AND METHODS
Ticks
Experimental ticks were taken from a laboratory colony maintained at 26°C in darkness at >95% relative humidity. For most experiments (mated 95
96
W. REDEN KAUFMAN
females), 3040 males were placed on the rabbit a day or so before introducing an equal number of females. Prior attachment of males encourages females to attach and feed more readily. Mating occurs on the host. To produce fed virgins, however, the gonopore of each male was first plugged with a droplet of cyanoacrylate adhesive (“Locktite”; Newington, Conn., U.S.A.) before putting them on the host. The next day, females were added, and checked for attachment every few hours. As soon as most of the females had attached, (generally within 12 h if the males had been fed before, or within 24 h if the males were previously unfed), all the males were removed from the host. The virginity of each experimental female was later confirmed by examining the seminal receptacle for absence of spermatophores, and by examining a smear of the seminal receptacle for absence of spermatozoa. Because virgin females do not readily feed beyond the critical weight, the usual level of infestation for experiments involving virgins was 60 females. Assay for salivary gland degeneration
I use fluid secretion in vitro as a routine assay for salivary gland function. Salivary glands were removed from the tick, the main duct was ligated with a fine strand of silk, the tissue was gently blotted, weighed to the nearest 0.01 mg and incubated at room temperature in constantly stirred TC medium 199 containing 10 pm dopamine. [Dopamine stimulates salivary fluid secretion and is probably the neutral transmitter substance (Kaufman and Wong, 1983)]. After incubation (usually 10 or 15min), the glands were again gently blotted and weighed. Net weight increase under these conditions represents fluid secretory competence by the gland, and is thus an index of salivary gland degeneration. Further details are provided in Kaufman (1983) and Harris and Kaufman (1984). Methodfor sampling haemolymph and ovary for radio immunoassay (RIA)
Ticks were removed from the host, rinsed in water, secured ventrum down to a disposable Petri dish by means of the cyanoacrylate adhesive. They were chilled under crushed ice for about 15 min in order to maximize haemolymph volume collected (see Discussion). A cut was made with a razor blade scalpel a few millimetres around the lateral margin of the integument. Following gentle pressure on the dorsum, haemolymph exuding from the wound was collected in a volumetric capillary tube. The incision was then extended around the integument, leaving only the posterior region of the lateral margin intact. Muscle attachments were cut as necessary to enable reflection of the dorsum toward the posterior end. Usually additional haemolymph could be collected by careful application of the capillary tube to avoid contamination with any gut or Malpighian tubule contents. Under these conditions, I normally collected a mini-
Table 1. Haemolymph volume (~1) collected from pre-chilled ticks Tick weight range (mg) <150 150-260 260400 400-550
> 550 Pooled
Mean volume (JJl) 7.8 11.2 9.6 6.0 11.5 9.4
SEM n 1.1 11 1.8 10 1.7
8
1.1 9 1.5 13 0.7 51
mum of 5 ~1 and frequently a few tens of microlitres of haemolymph from each female (see Table 1). After as much haemolymph as practical was collected, the tick was flooded with modified Hank’s medium (composition in g/l: NaCl 11.5; D-glucose 1.6; KC1 0.4; CaCl, 0.14; MgS04 0.10; KH2P04 0.06; Na,HPO, 0.05; phenol red 0.01; 360 mosmolar; pH 7.0) and the dorsum removed. The salivary glands were dissected out for measuring fluid secretory competence as described above. The ovary was dissected out, cleared of tracheae and other adhering tissue and transferred to fresh TC 199 prior to weighing and processing for the ecdysteroid RIA. RIA for ecdysteroidr
Complete details of the RIA and for processing tissue are presented by Kaufman (1990). Modifications were: (a) it was never necessary to pool haemolymph from several individuals in this study for a single RIA sample; (b) the antibody (directed against ecdysone-22-succinylthyroglobulin; a gift from Dr L. I. Gilbert, University of North Carolina) was more concentrated (& was adjusted to 20-30%) than in the previous study. This increased the useful ecdysteroid concentration range detectable by the assay with only a small concomitant increase in the reliable limit of detection (20-30 pg 20-hydroxyecdysone). All other details of the assay were the same as those described by Kaufman (1990). Because the antibody cross-reacts with several ecdysteroid analogues (Warren and Gilbert, 1988), the data is expressed as “20-hydroxyecdysone equivalents”. Drugs and chemicals
TC Medium 199 was purchased from Gibco Inc. (Grand Island, New York), 20-hydroxyecdysone from Simes (Milan, Italy), dopamine and bovine serum albumin (RIA grade) from Sigma (St Louis, MO., U.S.A.), radiolabelled ecdysone (ecdysone, CI[23,24-3H(N)]-, 45.0 Ci/mmol) from New England Nuclear (NEN DuPont Canada, Lachine, Quebec). Other chemicals were from various sources and of the purest grade available. Experiment
1
Ecdysteroid projile of haemolymph and ovary after engorgement. Ticks were permitted to feed to re-
pletion (at least 1000 mg). Haemolymph
and ovary
97
Ecdysteroids and tick salivary gland degeneration
9-13 after engorgement. Ovary content of ecdysteroids in engorged ticks within 2 days after engorgement was also at a low level [9.4 + 9.4(10) rig/g]] and rose more or less in parallel with the haemolymph titre, peaking at 1738 + 355(10) rig/g by day 9-13 after engorgement.
ZO-OHE equivalents bglma
qr ~1)
Experiment 2 (Figs 2 and 3) haemolymph ovary
o-2
were taken
n
7-8 5-6 3-4 days post-engorgement
Fig. 1. Time course after ovary ecdysteroid titre. pooled as indicated on wherever it exceeds samples
0
9-13
engorgement for haemolymph and Ticks. from different days were the abscissa. The SEM is shown the dimension of the symbol. as described
above
from
ticks
O-13 days after engorgement. Haemolymph was added to 100 ~1 methanol. The ovary was blotted, weighed and, if less than about 10-15 mg, the whole ovary was homogenized in 100 ~1 methanol; in the case of larger ovaries, only a piece (about 2-15 mg) was homogenized. The haemolymph and ovary homogenates were stored at - 15°C until processed further for RIA. Experiment 2
There was no apparent correlation between tick weight and salivary gland weight on day 7 after removal for ticks weighing up to 500 mg, although beyond this weight, the glands were too degenerated to dissect out and weigh; they were thus assigned a weight of “zero” (Fig. 2). The glands of some virgins, in contrast, were present in large ticks (500-1200 mg), although none of them secreted fluid in the assay. Ovary weight is a crude index of degree of vitellogenesis. Up to a tick weight of 300_1OOmg, there was no significant sign (t, > 0.05) of vitellogenesis 7 days after removal. Beyond that weight, there was progressively more vitellogenesis. Ovary weights of virgins were similar to those of mated females of similar size (Fig. 2). Data for salivary gland function, haemolymph ecdysteroid titre and ovary ecdysteroid content on day 7 after removal/after engorgement are presented in Fig. 3. Salivary gland function improved progressively in ticks weighing up to 300 mg. Thereafter, fluid transport ability plunged to a low value in ticks weighing 400-5bOmg. -Reduction in salivary gland
Critical weight for salivary gland degeneration. Ticks were removed from the host at various stages (weights) of the feeding cycle and held for 7 days in the colony incubator. Haemolymph and ovary samples were collected as described above and kept at - 15°C awaiting processing for RIA. The salivary glands were dissected out and fluid transport measured. Statistics All data are presented as mean + SEM (n). Statistical significance was determined by one-way analysis of variance using the MIDAS statistical package of the university’s mainframe computer. RESULTS
Haemolymph sampling
100
200
500
tick weight
Table 1 shows the haemolymph volume collected from ticks used in this study. There was no apparent correlation between tick size and haemolymph volume collected, the average haemolymph sample being 9-10 /Ll. Experiment
1 (Fig. 1)
In a population of engorged ticks, sampled within 2 days of engorgement, the haemolymph ecdysteroid titre was 9.0 f 3.6(11) ng/ml. The titre rose dramatically thereafter, peaking at 414 _I 45(9) ng/ml by day
1000
2000
(mg)
Fig. 2. Wet weight of ovary and salivary glands on day 7 after removal/after engorgement as a function of tick weight. The tick weights shown here and in Fig. 3 are those recorded on day zero. (Weight by day 7 was 87.2% + 0.7(66) of initial weight; the degree of weight loss was similar for all ticks in this study, irrespective of their initial weight on day zero.) Samples were pooled as follows: tick weight up to -150 mg (Y = 122 + 6;- 10); 150-200 mg (R = 175 + 5; 10); 200-300 mg (x = 256 _+7; 7); 300-400 mg (x= 354 & 12; 6); 400-500mg (x=440 _+8; 15); 5001200 mg (x = 894 + 77; 8); > 1200 mg (x = 1897 k 224; IO). The SEM is shown wherever it exceeds the dimension of the symbol.
W. REUBEN KAUFMAN
98
800
400 i
160 260 tick
200
‘500 1000 2600 weight
(mg)
Fig. 3. Fluid secretory competence by salivary glands, and ecclysteroid content of ovary and haemolymph 7 days after removal/after engorgement as a function of tick weight. The ticks shown here are the same as those presented in Fig. 2. The SEM is shown wherever it exceeds the dimension of the symbol. function was first associated with a haemolymph ecdysteroid titre of 89 + 36(5) ng/ml. Loss of function was almost complete at a haemolymph ecdysteroid titre of I39 + 15(9) ng/ml. However, haemolymph ecdysteroid titre continued its rise in larger ticks, ultimately reaching an average of 823 + 184(S) ng/ml. The titre of virgin haemolymph was similar to that of mated haemolymph in ticks of similar size [528 + 152(6) ng/ml vs 469 + 60(6); p > 0.051. Ovary ecdysteroid content rose beginning in ticks weighing 300-400 mg [57 ) 19(6) rig/g ovary weight] and peaking in large engorged ticks at 3510 + 529( 10) rig/g.. Ecdysteroid content of virgin ovaries was similar to that of ovaries from mated ticks of similar size [3570 + 1076(8) rig/g vs 2568 & 366(S) rig/g;; p > 0.051.
DISCUSSION
Pre-chilling of ticks before collecting haemolymph appears to maximize the volume of haemolymph one can collect. Low temperature inhibits contraction of the gut, and so lessens the likelihood that the delicate gut wall will rupture on the scalpel blade or sharp edge of the wounded cuticle. (Tendency for the gut to rupture increases greatly with degree of engorgement.) I collected up to 30 ~1 per female compared to “only a few microlitres” reported by Connat et al. (1985) for the same species of tick. However, the technique would obviously not be appropriate if the chilling process itself affected ecdysteroid content. If
one compares data from my study with corresponding data from that of Connat et al. (1985), it appears as if our two sampling methods lead to similar results: (1) in this study, haemolymph ecdysteroid titre of engorged ticks rose from about 10 ng/ml on day 0 to about 400-800 ng/ml on day 7. Equivalent data from Connat et al. ranges from 400-600 ng/ml. Since SDS are quite large in both studies, I regard these figures to be in reasonable agreement. (2) Ovary samples in this study contained about 90 ng ecdysteroid/ovary in small engorged ticks (8= 900 mg) and about 340 ng/ovary in large engorged ticks (x = 1900 mg; data calculated from Fig. 2). These values are similar to the range reported by Connat et al. for engorged ticks (weight unspecified), 6-17 days after engorgement (90-180 ng/ovary). How does one account for the increase in fluid secretory competence associated with an increase in tick weight up to 300 mg (Fig. 3)? Salivary glands of unfed ticks have only limited ability to secrete fluid; fluid secretory competence increases markedly during the first week of feeding (Kaufman, 1976), a phenomenon correlated with ultrastructural development of the fluid secretory labyrinth of the type III acinus (Fawcett et al., 1981). The increase in fluid secretion shown here in Fig. 3 is probably a reflection of this glandular development induced early in the feeding period. The concept of a critical weight for the release of the degeneration factor established earlier for day 4 after removal (Harris and Kaufman, 1984; Lindsay and Kaufman, 1988) is confirmed in this study for day 7 as well, and the concept now can be extended to haemolymph ecdysteroid titre and ovary ecdysteroid content (Fig. 3). A ten-fold rise in haemolymph titre (from < 10 ng/ml to no more than 90 ng/ml) is associated with a significant loss of salivary gland function. This level was seen at a tick weight range of 300400 mg (Fig. 3). By 400-500 mg, loss of salivary gland function was almost complete and the haemolymph ecdysteroid titre was about 140 ng/ml. Glands from large virgin females (50&1200 mg), like those from mated females, did not secrete fluid on day 7 after removal (see p. 97). However, unlike those of mated females, the glands from large virgins could be dissected out and weighed (Fig. 2 and p, 97). What accounts for this discrepancy? An earlier study from my laboratory demonstrated that salivary gland degeneration depends not only on the tick attaining a critical weight, but also on the presence of a “male factor” transferred to the female with the spermatophore (Harris and Kaufman, 1984). More recent work from my laboratory indicates that the male factor is not essential for salivary gland degeneration, but it hastens it considerably; whereas glands from large mated females lose virtually all function by day 4 after removal, similar loss of function occurs in virgin glands only by day 8 (Lomas and Kaufman, unpublished). In this study, all glands
Ecdysteroids and tick salivary gland degeneration were tested on day 7; although this time was sufficient to result in complete loss of function in both virgin and mated females, reabsorption of the tissue itself apparently had not yet progressed in the virgin females. If 140 ng ecdysteroid/ml haemolymph is correlated with a virtually complete loss of function, why does the haemolymph titre continue to rise another 5-6fold, to about 800 ng/ml (Fig. 3)? It suggests that ecdysteroids probably have functions in the tick additional to that of salivary gland degeneration. I confirm here the earlier report by Connat et al. (1985) of a high concentration of ecdysteroids in the developed ovary. One proposed function of ecdysteroids in ticks is as the vitellogenetic hormone, similar to what pertains in some groups of insects (Hagedorn, 1983; Zhu et al., 1983; Postlethwaite and Giorgi, 1985). However, there is no direct evidence for this conjecture in ticks; i.e. nobody has demonstrated that ecdysteroids can provoke vitellogenesis in small partially fed ticks. Indeed, my laboratory now has considerable evidence to the contrary (Lunke and Kaufman, unpublished). A more likely alternative, also proposed by Connat et al., is that ecdysteroids accumulate in the ovary to be used in embryogenesis and/or hatching from the egg (Hagedorn, 1983). Another possible explanation for the high ecdysteroid content of the ovary is that this organ produces ecdysteroids, as does the ovary of mosquitos (Hagedorn, 1983). There is no evidence yet for an analogous system in ticks, however. Attempts in my laboratory to ovariectomize ticks have failed so far. Acknowledgements-1
am most grateful to Mr Stephen Cozens for invaluable technical assistance in all aspects of this study, to Dr L. I. Gilbert, Department of Biology, University of North Carolina at Chapel Hill, for his generous gift of the ecdysone antibody used in the RIA and Dr Jim Campbell, Department of Microbiology, University of Alberta, for preparing the protein A required for the RIA. Finally, research in this laboratory is generously funded with an operating grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada.
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Fawcett D. W., Doxsey S. and Biischer G. (1981) Salivary glands of the tick vector (Ripicephalus appendiculatus) of East Coast fever-II. Cellular basis for fluid secretion in the type III acinus. Tissue Cell 13, 231-253. Hagedorn H. H. (1983) The role of ecdysteroids in the adult insect. In Endocrinology of Insects (Eds Downer R. G. H. and Laufer H.), pp. 271-304. Alan R. Liss, New York. Harris R. A. and Kaufman W. R. (1981) Hormonal control of salivary gland degeneration in the ixodid tick Amblyomma hebraeum. J. Insect Physioi. 27, 241-248.
Harris R. A. and Kaufman W. R. (1984) Neural involvement in the control of salivary gland degeneration in the ixodid tick Amblyomma hebraeum. J. exp. Biol. 109, 281-290.
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Kaufman W. R. (1976) The influence of various factors on fluid secretion by in vitro salivary glands of ixodid ticks. J. exp. Biol. 64, 727-742.
Kaufman W. R. (1983) The function of tick salivary glands. In Current Topics in Vector Research (Ed. Harris K. F.), pp. 215-247. Praeger Scientific, New York. Kaufman W. R. (1990) Effect of 20-hydroxyecdysone on the salivary glands of the male tick, Amblyomma hebraeum Koch. Expl appl. Acarol. 9, 87-95. Kaufman W. R. and Wong D. L.-P. (1983) Evidence for multiple receptors mediating fluid secretion in salivary glands of ticks. Eur. J. Pharmacol. 87, 43-52. Lindsay P. J. and Kaufman W. R. (1988) Action of some steroids on salivary gland degeneration in the ixodid tick, Amblyomma americanum L. J. Insect Physiol. 34,351-359. Postlethwaite J. H. and Giorgi F. (1985) Vitelloginesis in insects. In Developmental Biology: a Comprehensive Synthesis (Ed. Browder L. W.), Vol. 1, pp. 85-125. Plenum Press, New York. Till W. M. (1961) A contribution to the anatomy and histology of the brown ear tick, Rhipicephalus appendiculatus (Neumann). Mem. ent. Sot. sth. Afv., Vol. 6. Warren J. T. and Gilbert L. I. (1988) Radioimmunoassay: ecdysteroids. In Immunological Techniques in Insect Biology (Eds Gilbert L. I. and Miller T. A.), pp. 181-214. Springer, Berlin. Zhu X. X., Gfeller H. and Lanzrein B. (1983) Ecdysteroids during oogenesis in the oviparous cockroach Nauphoeta cinerea. J. Insect Physiol. 29, 225-230.