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Possible role in uptake of water vapour by ixodid tick salivary glands
J. Insect Physiol.. 1976, Vol. 22, pp. 1281 to 1285. Pergamon
Press. Printed in Great Britain.
POSSIBLE ROLE IN UPTAKE OF WATER VAPOUR BY IXODID TICK SALIVARY GLANDS* HAROLDL. MCMULLEN,JOHN R. SAUER,and ROBERTL. BURTON Department of Entomology, Oklahoma State University, Stillwater, OK 74074, U.S.A. (Received
1 April 1976)
Abstract-The mouth is confirmed as the site of water vapor uptake in the lone star tick, Amblyommo It was shown that the level of chloride (‘%I) increased in the mouthparts of desiccated ticks. The highest levels of 36CI were found in the mouthparts, salivary glands, and gut tissue during rehydration. It is suggested that ions are secreted by the salivary glands into the mouth where water is picked up hygroscopically by the secretion. It is further suggested that the water and ions are americanum.
then swallowed and absorbed from the lumen of the gut.
INTRODUCTION
THE ABILI~ of various arthropods to absorb water from unsaturated air has been extensively studied. EBELING(19743, BURSELL(1974) and BEAMENT(1964) reviewed species and the mechanisms involved in vapor uptake. There have been several proposed sites for the absorption of water vapor by the various arthropods. The anus has been shown to be the site in Thermobia domestica and Tenebrio molitor (NOBLENESBITT,1970; DUNBARand WINSTON,1975). MELLANBY (1932) suggested that in ticks, absorption occurred through the tracheal system. LEES (1946,1948) found that desiccated ixodid ticks absorbed water from unsaturated atmospheres and suggested that absorption probably occurred through the cuticle. More recently, however, RUDOLPH and KN~~LLE (1974) showed that the mouth is the site of vapor absorption in ixodid ticks. In addition to demonstrating that the mouth is the site of vapor uptake in ixodid ticks, they found that crystals containing sodium and potassium accumulated at the mouthparts of desiccated ticks. These crystals, when removed, absorbed water from the atmosphere if the relative humidity was above 75%. They suggested that ‘resorption of the absorbed water could possibly take place in the gut, although the remarkable abilities of the salivary glands in handling water should be kept in mind’. In view of these facts we sought to obtain more information about the tissues involved in enabling ixodid ticks to absorb water from unsaturated air by tracing the movement of chloride in desiccated and * Journal article No. 3119 of the Agricultural Experimental Station, Oklahoma State University, Stillwater, Oklahoma. This research was supported in part by NSF Grant No. BMS-74-24140 from the National Science Foundation.
rehydrated ticks. Chloride was used because it is the chief anion in the salivary secretion of feeding ixodid ticks (Hsu and SAUER,1975) and because an active transport of chloride has been suggested as a principal driving force for salivary fluid secretion in ixodid ticks (KAUFMANand PHILIPS, 1973~). We further sought to confirm the results of RUDOLPH and KN~~LLE (1974) as to the site of vapor uptake in ixodid ticks.
MATERIALS
AND METHODS
Site of vapor uptake
Both male and female unengorged Amblyommu americanum adults, between 8 and 20 weeks after ecdysis, were used in the experiments. In determining the site of vapor uptake the basic procedures of RUDOLPHand KN~~LLE(1974) were followed. Experiments to trace movement of %
into tissues
One $ of Ringer-saline containing 36C1 was injected into adult unengorged female ticks through the posterior dorsal surface with a Hamilton 701N-10~1 syringe. The stock saline contained the following components (per liter): NaCl, 4.4g; NaH2P0,.Hz0, 2.Og; Na2HP04.7Hz0, 2.Og; KHCO,, 1.63 g; CaCl,, 0.33 g; MgSO,, 0.29 g; glucose, 5.Og; inositol, 0.4g; bovine albumin, 0.1 g. Twenty-one fi of Na3%Y1 solution (specific activity, 68.4hCi/ml) was added to 79 ~1 of the above. This final saline had an osmolarity which is approximately that of lone star tick haemolymph (HSU and SAUER,1975). Individual ticks were weighed and then injected with 1~1 of the above saline. The injection site was lightly covered with surgical spray dressing following each injection and the syringe was removed after the dressing had started to set. After the dressing dried the ticks were placed in glass vials covered with gauze.
1282
HAROLDL. MCMULLEN,JOHNR. SAUER,AND
Ticks were next placed in a desiccation chamber containing anhydrous CaCl, (< 5%r.h. at 25°C) or in a humidity chamber containing a saturated solution of KH2P04 (96% r.h. at 25°C). Ticks left in the humidity chamber were removed after either 24, 48, 72,96. or 120 hr for analysis and served as the control ticks. Another group of ticks was placed in the desiccation chamber and removed for analysis after 24 or 48 hr. Other ticks were placed in the desiccation chamber for 48 hr and then placed in the humidity chamber and removed for analysis after either 12, 24, 48 or 72 hr. This latter group is referred to as rehydrated ticks. The spray dressing was removed and the ticks were weighed after completion of the experiments. Haemolymph was collected and placed on aluminum planchettes for measurement of radioactivity after severing a leg and collection with a micro-capillary. Before measuring radioactivity in the mouthparts, palps were removed and discarded and the mouthparts were severed at the base of the hypostome and placed on planchettes. The ticks were halved with a scalpel anterioposterially ; each half was placed in a petri dish in Ringer-saline and the salivary glands and gut tissue were removed, rinsed (< 1 set) in deionized water to remove saline and surface adhering radioactivity, and placed in groups of five on separate pre-weighed aluminum planchettes. To extract radioactivity and accurately weigh the small amounts of available gut and salivary gland tissue, the following procedures were followed: 100% ethanol was added to each planchette with tissue and allowed to evaporate for 48 hr at room temperature. The planchettes were then placed in the desiccation chamber for an additional 24 hr, after which time they were weighed. The amount of radioactivity was analyzed with a Nuclear-Chicago Gas-flow Planchette Counter@ and expressed as counts/min/lOOpg of gut or salivary gland tissue or counts/min/set of mouthparts. Non-radioactive
chloride analysis
Non-radioactive chloride was analyzed in mouthparts by the coulombic silver chloride precipitation method (Fiske-Marius Micro Chloro-ocounter@). RESULTS Site of vapor uptake
Using
methods
similar to those described by (1974), A. americanum ticks were pre-desiccated over anhydrous CaCl, at 20°C for 96 hr, weighed and placed in groups of 20 with either their mouthparts, anus, or dorsal surface covered with paraffin. A control group was without any paraffin covering. Then ticks were exposed to 93xr.h. (saturated KNO, solution, WINSTON and RUDOLPH and KNULLE
BATES, 1960) at 20°C for 96 hr and re-weighed. All groups gained weight when the mouthparts were not covered (Table 1). Ticks whose mouthparts were
ROBERT L. BURTON
Table 1. Per cent weight change in predesiccated ticks with mouthparts, anus or dorsal surface covered or controls without any covering at 20°C and 93% r.h. Structure covered mouthparts anus dorsal surface control (no treatment) *Numerals iments.
in parentheses
% wt change + SD. -2.4 7.1 3.5 5.5
+ * + +
1.2(7)* 3.0(7) 3.9 (7) 1.2 (7)
indicate number of exper-
covered lost 2.4”/, f 1.3 of their initial weight, while those with the anus covered, the dorsal surface covered, and the controls gained 7.1% _t 3.0, 3.5% f 3.9, and 5.5% If: 1.2, respectively. If the per cent weight change values for the anus covered, dorsal surface covered, and the control ticks are compared, no significant difference is seen (P < 0.05, t-test). If each of these values are compared individually to the per cent weight change value obtained after covering the mouthparts (-2.4% + 1.3), the differences are significant (< 0.05).
When the mouthparts of 96 hr pre-desiccated ticks (over anhydrous CaClJ were sealed off from the rest of the body and a saturated solution of KN03 was placed close to them, the KN03 solution became crystalline in appearance, indicating loss of water from the solution. This did not happen to the solution placed on the side of the tube holding the posterior side of the tick. These results support similar findings by RUDOLPHand KN~JLLE(1974). Desiccation
and rehydration of ticks at 25°C
At - 0% r.h. and 25°C non-injected ticks lost 15.9% _t 5.1 of their initial weight after 48 hr, whereas injected ticks lost 14.6% f 4.7 of their pre-injection weight. No significant difference was found between injected and non-injected ticks at any of these humidities when per cent weight losses were compared. After 24 hr of rehydration at 96% r.h. (25°C) predesiccated ticks injected with 36C1 gained 56.3% f 15.1 of the pre-injection weight lost during desiccation, while non-injected ticks regained 55.5% f 22.6 of lost body weight. After 48 hr of rehydration, injected ticks regained 92.5% f 17.8 pre-injected weight lost, while non-injected ticks regained 98.6% + 17.0 of the original weight lost. By 72 hr rehydration, injected ticks regained 99.4% + 8.2~s 101.3% + 10.1 gain for non-injected ticks. Radioactive
levels in tissues of ticks injected with 36Ci
Haenwlymph.
No significant difference could be demonstrated in the amounts of radioactivity present in tick haemolymph after exposing the ticks to various conditions of desiccation and rehydration (Table 2).
1283
Ixodid tick salivary glands
Table 2. Radioactivity in haemolymph, mouthparts, salivary glands, and gut tissue of ticks following injection of ‘%I into the haemocoel Control Time (hr) 24 48 60 12 96 120 24 48 60 12 96 120 24 48 60 12 96 120 24 48 60 12 96 120
Radioactivity counts/mm* Desiccated Rehydrated
ticks-t
tick@
ticksj
Counts/min + S.D./p haemolymph 400 + 101 (5) 363 + 81 (7) 373 & 89 (9) 346 + 104 (I) 366 f 91 (7) 391 & 78 (I) 362 & 126 (7) counts/min f S.D./mouthpart 142 f 35 (28) 141 f 43 (28) 336 k ll(42) 148 k 73(36)
396 411 439 409
+ 70 (6) + 89 (7) k 61 (7) k 19 (6)
324 + 466 k 52 (16) 385 + 46(18) 326 + 30(16) + SD./100 pg salivary gland tissue 109 f 20 (7) 51 (7) 54 (7) 277 k 29 (7) 202 + 400 f 261 k 77 (6) 540 k 246 + 40 (5) 255 + 251 + 41 (8) counts/min k S.D./loO~g gut tissue 125 f 46 (7) 166 * 101 (7) 134 + 42 (7) 165 + 33 (I) 160 k 241 + 169 k 28 (6) 194 f 171 + 41 (5) 139 + 164 + 45 (8)
140 * 151 f 143 + :ounts/mm 138 f 244 &
92(11) 153 (15) 85(20) 116(11)
83 (7) 150 (7) 245 (I) 129 (7)
74 80 88 68
(7) (7) (7) (7)
* Numerals in parentheses indicate numbers of experiments. t Ticks held constantly at 96% r.h. and 25°C. $ Ticks held constantly at 0 to 5% r.h. and 25°C. § Ticks rehydrated at 96% r.h. and 25°C. Ticks were previously desiccated 48 hr at 0 to 5% r.h. and 25°C.
Mouthparts. Radioactivity was found in the mouthparts 24 hr after injection of 36C1 into the haemocoel (Table 2). Whether injected ticks were desiccated or held at 96xr.h. for 24hr, there was no significant difference in radioactivity in the mouthparts (Table 2). However, the amount of radioactivity present in 48 hr desiccated ticks was more than twice the amount present in control ticks. When ticks were desiccated for 48 hr and then rehydrated at 96% r.h., the level of radioactivity increased still further, but gradually declined with increasing rehydration time (Table 2). Salivary glands. Radioactivity was observable in salivary glands 24 hr after injection of the isotope (Table 2). However, the level of radioactivity was higher in salivary glands after 48 hr and remained close to this value (244 to 267 counts/min/lOO pg tissue) in ticks held constantly at 96zr.h. There was no sign&ant difference in the amounts of radioactivity present in the salivary glands of control and
non-rehydrated 24 and 48 hr ticks. The highest levels of radioactivity were observed in salivary glands from ticks previously desiccated for 48 hr and then rehydrated for 24 and 4Xhr (hours 72 and 96) (Table 2). Gut tissue. The only value for 36C1 that was found to vary significantly from any of the other gut tissue values was that found after 24 hr rehydration (hr 72)
(Table 2). In addition, most concentrations of 36Cl in the gut were signticantly lower (P < 0.05) than those found in the salivary glands. Non-radioactive
chloride
To verify the assumption that 36Cl, as introduced to the ticks in the present experiments, is an accurate reflection of ‘normal’ movements of chloride to tissues during desiccation and rehydration, naturally occurring chloride was measured in the mouthparts of nonfeeding ticks. Chloride was present in the mouthparts (Table 3). Furthermore, after similar periods of desiccation and rehydration, the levels change in propor-
I284
HAROLDL. MCMULLEN,JOHN R. SAUER.AND
ROBERTL. BURTON
Table 3. Natural chloride in mouthparts of ticks exposed to desiccating and rehydrating conditions
Time (hr) 24 48 72 96 120 24 48 72 96 120
p-equiv. x 10e3 Cl/l0 mouthparts Controlt Desiccated1 26.0 k 4.9 (5) 15.0 f 10.3 (5) 38.7 + 4.4(S) 21.7 + 6.2(S) 16.8 f 10.2(S) 20.9 f 6.3 (5) pc-equiv. x 10e3 Cl/l0 mouthparts (lumenr 17.1 * 8.6(S) 14.5 i 8.3 (5) 37.4 * 10.5 (4) 11.7 + 14.4 (5) 12.2 k 6.3 (5) 15.4 f 9.6(5)
+ S.D.* Rehydrateds
61.5 f 17.0(6) 55.6 k 18.7 (6) 41.7 + 15.4(5)
51.4 + 11.3(4) 44.5 + 18.4 (4) 35.8 k 10.4(4)
* Numerals in parenthesis indicate numbers of experiments. t Ticks held constantly at 96% r.h. and room temperature. $ Ticks held constantly at 0 to So/,r.h. and room temperature. $ Ticks rehydrated at 96% r.h. and room temperature. Ticks were previously desiccated 48 hr at 0 to 5% r.h. and room temperature. 7 Refers to scrapings taken from the insides of mouthparts (lumens) by forceps which were then analyzed for Cl. tions approximately equal to the changes in 36C1 found in the mouthparts after injection of the isotope into the haemocoel.
water by the gland after a primary salivary secretion (Fig. 1). Alternately, the primary secretion could be highly concentrated with little reabsorption required.
DISCUSSION The results confirm the findings of RUDOLPH and KNYJLLE(1974) who showed that the mouth is the site of vapor uptake in ixodid ticks. We have also shown that 36C1 accumulates in higher concentration in desiccated and rehydrated tick mouthparts than in the control tick mouthparts. It appears likely that the isotope could arrive in the mouthparts only by regurgitation from the gut after movement across the gut epithelium or by secretion from the salivary glands. Our results tend to suggest that the salivary glands are responsible for this process because of the and following: (1) 36C1 moves into the mouthparts salivary glands of the tick after its initial deposition into the haemocoel; (2) desiccation and subsequent rehydration cause the levels of 36C1 to increase in both the mouthparts and salivary glands but not appreciably in other tissues and (3) the level of 36C1 was shown to be the highest in the mouthparts and salivary glands of rehydrating ticks at the time of maximum vapor uptake. Based upon the known secretory abilities of salivary glands in ixodid ticks (TATCHELL, 1967 ; KAUFMAN and PHILLIPS, 1973a, b, c; SAUER et al., 1974; NEEDHAM and SAUER, 1975) and their known morphology (BALASHOV, 1972; GREGSON, 1973), the suggestion that water vapor uptake aided by secretion of ions from the salivary glands seems more logical than regurgitation of substances from the gut. A possible method for ‘net’ uptake of atmospheric water that involves the saliva is the reabsorption of
Gland
Fig. 1. Diagrammatic summary of how salts of high concentration may be secreted by salivary glands into the region of the mouthparts with subsequent uptake and absorption of water vapor by rehydrating ticks,
1285
Ixodid tick salivary glands
If reabsorption is required, it could occur in much the same way that water is thought to be reclaimed by the insect rectum from strongly hyperosmotic rectal fluid (PHILLIPS, 1970). In this way the salts (possibly NaCl and KCI) in the salivary secretion could become sufficiently concentrated to pick up atmospheric water hygroscopitally, which would yield a net increase in body water, to be swallowed later and absorbed through the gut. The important site of energy expenditure would then be the salivary gland epithelia where water reabsorption occurred, possibly the proximal ducts or acini of the salivary glands. Of potential relevance to this hypothesis is the demonstration by COONS and ROSHDY(1973) of anteriorly confined aveoli having direct communication with the main salivary duct in unfed Dermucentor uaruurkzbilis males. The potentially significant aspect is their anterior location and containment of extensive infoldings of the basal cell membrane with associated mitochondria, a feature common to fluid transporting epithelia. What is interesting is the conspicuous presence of these structures in the unfed male where little fluid elimination during feeding would be expected because of the minimal amount of feeding done by the male. Relevant to all of this is the report of SHIH et al. (1973). They found that Na was initially removed from the haemolymph by ‘severely desiccated’ ticks in the process of becoming ‘moderately hydrated’ and that the increase in water content during these events was mostly in other tissues than the blood. The authors felt that sodium may play some important role in the ability to absorb water from subsaturated atmospheres. It may be that movements of Na and Cl are closely linked; each is the chief cation and anion, respectively, in the haemolymph (Hsu and SAUER, 1975). Also, RUDOLPH and KNIJLLE (1974) have observed a high concentration of Na in the oral secretion of desiccated non-feeding ticks. In conclusion, the mouth is confirmed as the site of water vapor uptake in ixodid ticks. Secretion of ions into the mouthparts appears to be an integral part of the mechanism enabling ticks to take up water vapor from unsaturated air. The experiments of RUDOLPHand KN~~LLE(1974) have demonstrated that the secreted substance is hygroscopic and the results of our experiments suggest that the source of the hygroscopic material may be the salivary glands. When the surrounding humidity increases sufficiently, this material could absorb water from the atmosphere. The resulting solution could then be swallowed and the water and solutes absorbed by the gut tissue, enabling the tick effectively to regain the water it lost at lower humidities.
Acknowledgement-The
valuable assistance of Ms. GLEECEand Mr. DANA J. BROWN is gratefully acknowledged. KATHY
REFERENCES BALASHOV Y. S. (1972)Blood sucking ticks (Ixodoidea) vectors of diseases of man and animals. Misc. Pub/. ent. Sot. Am. 8, 161-376. BEAMENT J. W. L. (1964) The active transport and passive movement of water in insects. Adv. Insect. Physiol. 2, 67-129. BURSEL~E. (1974) Environmental aspects-humidity. In The Physiology of Insecta (Ed. by ROCKSTEINM.) 2nd ed. 2, 43-84. Academic Press, New York. COONS,L. B. and ROSHDY,M. A. (1973) Fine structure of the salivary glands of unfed male Dermacemor uariubilis (Say) (Ixodiodea: Ixodidae). J. Parasitol. 59. 90&912. DUNBARB. S. and WINSTONP. W. (1975) The site of active uptake of water in the larvae of Tenebrio molitor. J. Insect Physiol. 21, 495-500.
EBEL~NGW. (1974) Permeability of insect cuticle. In The Physiology of Insecta. (Ed. by ROCKSTEINM.) 2nd ed. 6. 271-343. Academic Press, New York. GR&N J. D. (1973)Tick paralysis: an analysis of natural
and experimental data. Monog. Can. Dept. Agric. 9. Hsu M. H. and SAUERJ. R. (1975) Ion and water balance in the feeding lone star tick. Comp. Biochem. Physiol. 52, 269-276.
KAUFMANW. R. and PHILLIPSJ. E. (1973a) Ion and water balance in the ixodid tick Dermacentor andersoni-I. Routes of ion and water excretion. J. exp. Biol. 58, 523-536. KAUFMANW. R. and PHILLIPSJ. E. (1973b) Ion and water balance in the ixodid tick Dermacentor andersoni-II. Mechanism and control of salivary secretion. J. exp. Biof. 58, 531-547. KAUFMAN W. R. and PHILLIPS J. E. (1973~)Ion and water balance in the ixodid tick Dermucentor andersoni-III.
Influence of Monovalent ions and osmotic pressure on salivary secretion. J. exp. Biol. 58, 549-564. LEESA. D. (1946) The water balance in Zxodes ricinus L. and certain other species of ticks. Parasitology 37, l-20. LEES A. D. (1948) Passive and active water exchange through the cuticle of ticks. Discuss. Faraday Sot. 3, 187-192.
MELLANBYK. (1932) The effect of atmospheric humidity on metabolism of fasting mealworm, (Tenebrio molitor L., Coleoptera). Proc. R. Sot. Lond. (B) 111, 376390. NEEDHAMG. R. and SAUERJ. R. (1975) Control of fluid secretion by isolated salivary glands of the lone star tick. J. Insect. Physiol. 21, 1893-1898. NOBLE-NESBI~J. (1970) Water uptake from subsaturated atmospheres: its site in insects. Nature, Land. 225, 753-754. PHILLIPSJ. E. (1970) Apparent transport of water by insect excretory systems. Am. Zool. 10, 413436. RUDOLPHD. and KN~JLLEW. (1974) Site and mechanism of water vapour uptake from the atmosphere in ixodid ticks. Nature, Lond. 249, 84-85. SAUERJ. R., FLICK J. H., and HAIR J. A. (1974) Control of ?I uptake by isolated salivary glands of the lone star tick. J. Insect Physiol. 20, 1771-1778. SHIHC., SAUERJ. R.. EIKENBARY P., HAIR J. A., and FRICK J. H. (1973) The effects of desiccation and rehydration on the lone star tick. J. Insect Physiol. 19. 505-514. TATCHELLR. J. (1967) A modified method for obtaining tick oral secretion. J. Parasitol. 53. 1106-1107. WINSTONP. W. and BATESD. H. (1960) Saturated solutions for the control of humidity in biological research. Ecology 41, 232-37.
Press. Printed in Great Britain.
POSSIBLE ROLE IN UPTAKE OF WATER VAPOUR BY IXODID TICK SALIVARY GLANDS* HAROLDL. MCMULLEN,JOHN R. SAUER,and ROBERTL. BURTON Department of Entomology, Oklahoma State University, Stillwater, OK 74074, U.S.A. (Received
1 April 1976)
Abstract-The mouth is confirmed as the site of water vapor uptake in the lone star tick, Amblyommo It was shown that the level of chloride (‘%I) increased in the mouthparts of desiccated ticks. The highest levels of 36CI were found in the mouthparts, salivary glands, and gut tissue during rehydration. It is suggested that ions are secreted by the salivary glands into the mouth where water is picked up hygroscopically by the secretion. It is further suggested that the water and ions are americanum.
then swallowed and absorbed from the lumen of the gut.
INTRODUCTION
THE ABILI~ of various arthropods to absorb water from unsaturated air has been extensively studied. EBELING(19743, BURSELL(1974) and BEAMENT(1964) reviewed species and the mechanisms involved in vapor uptake. There have been several proposed sites for the absorption of water vapor by the various arthropods. The anus has been shown to be the site in Thermobia domestica and Tenebrio molitor (NOBLENESBITT,1970; DUNBARand WINSTON,1975). MELLANBY (1932) suggested that in ticks, absorption occurred through the tracheal system. LEES (1946,1948) found that desiccated ixodid ticks absorbed water from unsaturated atmospheres and suggested that absorption probably occurred through the cuticle. More recently, however, RUDOLPH and KN~~LLE (1974) showed that the mouth is the site of vapor absorption in ixodid ticks. In addition to demonstrating that the mouth is the site of vapor uptake in ixodid ticks, they found that crystals containing sodium and potassium accumulated at the mouthparts of desiccated ticks. These crystals, when removed, absorbed water from the atmosphere if the relative humidity was above 75%. They suggested that ‘resorption of the absorbed water could possibly take place in the gut, although the remarkable abilities of the salivary glands in handling water should be kept in mind’. In view of these facts we sought to obtain more information about the tissues involved in enabling ixodid ticks to absorb water from unsaturated air by tracing the movement of chloride in desiccated and * Journal article No. 3119 of the Agricultural Experimental Station, Oklahoma State University, Stillwater, Oklahoma. This research was supported in part by NSF Grant No. BMS-74-24140 from the National Science Foundation.
rehydrated ticks. Chloride was used because it is the chief anion in the salivary secretion of feeding ixodid ticks (Hsu and SAUER,1975) and because an active transport of chloride has been suggested as a principal driving force for salivary fluid secretion in ixodid ticks (KAUFMANand PHILIPS, 1973~). We further sought to confirm the results of RUDOLPH and KN~~LLE (1974) as to the site of vapor uptake in ixodid ticks.
MATERIALS
AND METHODS
Site of vapor uptake
Both male and female unengorged Amblyommu americanum adults, between 8 and 20 weeks after ecdysis, were used in the experiments. In determining the site of vapor uptake the basic procedures of RUDOLPHand KN~~LLE(1974) were followed. Experiments to trace movement of %
into tissues
One $ of Ringer-saline containing 36C1 was injected into adult unengorged female ticks through the posterior dorsal surface with a Hamilton 701N-10~1 syringe. The stock saline contained the following components (per liter): NaCl, 4.4g; NaH2P0,.Hz0, 2.Og; Na2HP04.7Hz0, 2.Og; KHCO,, 1.63 g; CaCl,, 0.33 g; MgSO,, 0.29 g; glucose, 5.Og; inositol, 0.4g; bovine albumin, 0.1 g. Twenty-one fi of Na3%Y1 solution (specific activity, 68.4hCi/ml) was added to 79 ~1 of the above. This final saline had an osmolarity which is approximately that of lone star tick haemolymph (HSU and SAUER,1975). Individual ticks were weighed and then injected with 1~1 of the above saline. The injection site was lightly covered with surgical spray dressing following each injection and the syringe was removed after the dressing had started to set. After the dressing dried the ticks were placed in glass vials covered with gauze.
1282
HAROLDL. MCMULLEN,JOHNR. SAUER,AND
Ticks were next placed in a desiccation chamber containing anhydrous CaCl, (< 5%r.h. at 25°C) or in a humidity chamber containing a saturated solution of KH2P04 (96% r.h. at 25°C). Ticks left in the humidity chamber were removed after either 24, 48, 72,96. or 120 hr for analysis and served as the control ticks. Another group of ticks was placed in the desiccation chamber and removed for analysis after 24 or 48 hr. Other ticks were placed in the desiccation chamber for 48 hr and then placed in the humidity chamber and removed for analysis after either 12, 24, 48 or 72 hr. This latter group is referred to as rehydrated ticks. The spray dressing was removed and the ticks were weighed after completion of the experiments. Haemolymph was collected and placed on aluminum planchettes for measurement of radioactivity after severing a leg and collection with a micro-capillary. Before measuring radioactivity in the mouthparts, palps were removed and discarded and the mouthparts were severed at the base of the hypostome and placed on planchettes. The ticks were halved with a scalpel anterioposterially ; each half was placed in a petri dish in Ringer-saline and the salivary glands and gut tissue were removed, rinsed (< 1 set) in deionized water to remove saline and surface adhering radioactivity, and placed in groups of five on separate pre-weighed aluminum planchettes. To extract radioactivity and accurately weigh the small amounts of available gut and salivary gland tissue, the following procedures were followed: 100% ethanol was added to each planchette with tissue and allowed to evaporate for 48 hr at room temperature. The planchettes were then placed in the desiccation chamber for an additional 24 hr, after which time they were weighed. The amount of radioactivity was analyzed with a Nuclear-Chicago Gas-flow Planchette Counter@ and expressed as counts/min/lOOpg of gut or salivary gland tissue or counts/min/set of mouthparts. Non-radioactive
chloride analysis
Non-radioactive chloride was analyzed in mouthparts by the coulombic silver chloride precipitation method (Fiske-Marius Micro Chloro-ocounter@). RESULTS Site of vapor uptake
Using
methods
similar to those described by (1974), A. americanum ticks were pre-desiccated over anhydrous CaCl, at 20°C for 96 hr, weighed and placed in groups of 20 with either their mouthparts, anus, or dorsal surface covered with paraffin. A control group was without any paraffin covering. Then ticks were exposed to 93xr.h. (saturated KNO, solution, WINSTON and RUDOLPH and KNULLE
BATES, 1960) at 20°C for 96 hr and re-weighed. All groups gained weight when the mouthparts were not covered (Table 1). Ticks whose mouthparts were
ROBERT L. BURTON
Table 1. Per cent weight change in predesiccated ticks with mouthparts, anus or dorsal surface covered or controls without any covering at 20°C and 93% r.h. Structure covered mouthparts anus dorsal surface control (no treatment) *Numerals iments.
in parentheses
% wt change + SD. -2.4 7.1 3.5 5.5
+ * + +
1.2(7)* 3.0(7) 3.9 (7) 1.2 (7)
indicate number of exper-
covered lost 2.4”/, f 1.3 of their initial weight, while those with the anus covered, the dorsal surface covered, and the controls gained 7.1% _t 3.0, 3.5% f 3.9, and 5.5% If: 1.2, respectively. If the per cent weight change values for the anus covered, dorsal surface covered, and the control ticks are compared, no significant difference is seen (P < 0.05, t-test). If each of these values are compared individually to the per cent weight change value obtained after covering the mouthparts (-2.4% + 1.3), the differences are significant (< 0.05).
When the mouthparts of 96 hr pre-desiccated ticks (over anhydrous CaClJ were sealed off from the rest of the body and a saturated solution of KN03 was placed close to them, the KN03 solution became crystalline in appearance, indicating loss of water from the solution. This did not happen to the solution placed on the side of the tube holding the posterior side of the tick. These results support similar findings by RUDOLPHand KN~JLLE(1974). Desiccation
and rehydration of ticks at 25°C
At - 0% r.h. and 25°C non-injected ticks lost 15.9% _t 5.1 of their initial weight after 48 hr, whereas injected ticks lost 14.6% f 4.7 of their pre-injection weight. No significant difference was found between injected and non-injected ticks at any of these humidities when per cent weight losses were compared. After 24 hr of rehydration at 96% r.h. (25°C) predesiccated ticks injected with 36C1 gained 56.3% f 15.1 of the pre-injection weight lost during desiccation, while non-injected ticks regained 55.5% f 22.6 of lost body weight. After 48 hr of rehydration, injected ticks regained 92.5% f 17.8 pre-injected weight lost, while non-injected ticks regained 98.6% + 17.0 of the original weight lost. By 72 hr rehydration, injected ticks regained 99.4% + 8.2~s 101.3% + 10.1 gain for non-injected ticks. Radioactive
levels in tissues of ticks injected with 36Ci
Haenwlymph.
No significant difference could be demonstrated in the amounts of radioactivity present in tick haemolymph after exposing the ticks to various conditions of desiccation and rehydration (Table 2).
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Ixodid tick salivary glands
Table 2. Radioactivity in haemolymph, mouthparts, salivary glands, and gut tissue of ticks following injection of ‘%I into the haemocoel Control Time (hr) 24 48 60 12 96 120 24 48 60 12 96 120 24 48 60 12 96 120 24 48 60 12 96 120
Radioactivity counts/mm* Desiccated Rehydrated
ticks-t
tick@
ticksj
Counts/min + S.D./p haemolymph 400 + 101 (5) 363 + 81 (7) 373 & 89 (9) 346 + 104 (I) 366 f 91 (7) 391 & 78 (I) 362 & 126 (7) counts/min f S.D./mouthpart 142 f 35 (28) 141 f 43 (28) 336 k ll(42) 148 k 73(36)
396 411 439 409
+ 70 (6) + 89 (7) k 61 (7) k 19 (6)
324 + 466 k 52 (16) 385 + 46(18) 326 + 30(16) + SD./100 pg salivary gland tissue 109 f 20 (7) 51 (7) 54 (7) 277 k 29 (7) 202 + 400 f 261 k 77 (6) 540 k 246 + 40 (5) 255 + 251 + 41 (8) counts/min k S.D./loO~g gut tissue 125 f 46 (7) 166 * 101 (7) 134 + 42 (7) 165 + 33 (I) 160 k 241 + 169 k 28 (6) 194 f 171 + 41 (5) 139 + 164 + 45 (8)
140 * 151 f 143 + :ounts/mm 138 f 244 &
92(11) 153 (15) 85(20) 116(11)
83 (7) 150 (7) 245 (I) 129 (7)
74 80 88 68
(7) (7) (7) (7)
* Numerals in parentheses indicate numbers of experiments. t Ticks held constantly at 96% r.h. and 25°C. $ Ticks held constantly at 0 to 5% r.h. and 25°C. § Ticks rehydrated at 96% r.h. and 25°C. Ticks were previously desiccated 48 hr at 0 to 5% r.h. and 25°C.
Mouthparts. Radioactivity was found in the mouthparts 24 hr after injection of 36C1 into the haemocoel (Table 2). Whether injected ticks were desiccated or held at 96xr.h. for 24hr, there was no significant difference in radioactivity in the mouthparts (Table 2). However, the amount of radioactivity present in 48 hr desiccated ticks was more than twice the amount present in control ticks. When ticks were desiccated for 48 hr and then rehydrated at 96% r.h., the level of radioactivity increased still further, but gradually declined with increasing rehydration time (Table 2). Salivary glands. Radioactivity was observable in salivary glands 24 hr after injection of the isotope (Table 2). However, the level of radioactivity was higher in salivary glands after 48 hr and remained close to this value (244 to 267 counts/min/lOO pg tissue) in ticks held constantly at 96zr.h. There was no sign&ant difference in the amounts of radioactivity present in the salivary glands of control and
non-rehydrated 24 and 48 hr ticks. The highest levels of radioactivity were observed in salivary glands from ticks previously desiccated for 48 hr and then rehydrated for 24 and 4Xhr (hours 72 and 96) (Table 2). Gut tissue. The only value for 36C1 that was found to vary significantly from any of the other gut tissue values was that found after 24 hr rehydration (hr 72)
(Table 2). In addition, most concentrations of 36Cl in the gut were signticantly lower (P < 0.05) than those found in the salivary glands. Non-radioactive
chloride
To verify the assumption that 36Cl, as introduced to the ticks in the present experiments, is an accurate reflection of ‘normal’ movements of chloride to tissues during desiccation and rehydration, naturally occurring chloride was measured in the mouthparts of nonfeeding ticks. Chloride was present in the mouthparts (Table 3). Furthermore, after similar periods of desiccation and rehydration, the levels change in propor-
I284
HAROLDL. MCMULLEN,JOHN R. SAUER.AND
ROBERTL. BURTON
Table 3. Natural chloride in mouthparts of ticks exposed to desiccating and rehydrating conditions
Time (hr) 24 48 72 96 120 24 48 72 96 120
p-equiv. x 10e3 Cl/l0 mouthparts Controlt Desiccated1 26.0 k 4.9 (5) 15.0 f 10.3 (5) 38.7 + 4.4(S) 21.7 + 6.2(S) 16.8 f 10.2(S) 20.9 f 6.3 (5) pc-equiv. x 10e3 Cl/l0 mouthparts (lumenr 17.1 * 8.6(S) 14.5 i 8.3 (5) 37.4 * 10.5 (4) 11.7 + 14.4 (5) 12.2 k 6.3 (5) 15.4 f 9.6(5)
+ S.D.* Rehydrateds
61.5 f 17.0(6) 55.6 k 18.7 (6) 41.7 + 15.4(5)
51.4 + 11.3(4) 44.5 + 18.4 (4) 35.8 k 10.4(4)
* Numerals in parenthesis indicate numbers of experiments. t Ticks held constantly at 96% r.h. and room temperature. $ Ticks held constantly at 0 to So/,r.h. and room temperature. $ Ticks rehydrated at 96% r.h. and room temperature. Ticks were previously desiccated 48 hr at 0 to 5% r.h. and room temperature. 7 Refers to scrapings taken from the insides of mouthparts (lumens) by forceps which were then analyzed for Cl. tions approximately equal to the changes in 36C1 found in the mouthparts after injection of the isotope into the haemocoel.
water by the gland after a primary salivary secretion (Fig. 1). Alternately, the primary secretion could be highly concentrated with little reabsorption required.
DISCUSSION The results confirm the findings of RUDOLPH and KNYJLLE(1974) who showed that the mouth is the site of vapor uptake in ixodid ticks. We have also shown that 36C1 accumulates in higher concentration in desiccated and rehydrated tick mouthparts than in the control tick mouthparts. It appears likely that the isotope could arrive in the mouthparts only by regurgitation from the gut after movement across the gut epithelium or by secretion from the salivary glands. Our results tend to suggest that the salivary glands are responsible for this process because of the and following: (1) 36C1 moves into the mouthparts salivary glands of the tick after its initial deposition into the haemocoel; (2) desiccation and subsequent rehydration cause the levels of 36C1 to increase in both the mouthparts and salivary glands but not appreciably in other tissues and (3) the level of 36C1 was shown to be the highest in the mouthparts and salivary glands of rehydrating ticks at the time of maximum vapor uptake. Based upon the known secretory abilities of salivary glands in ixodid ticks (TATCHELL, 1967 ; KAUFMAN and PHILLIPS, 1973a, b, c; SAUER et al., 1974; NEEDHAM and SAUER, 1975) and their known morphology (BALASHOV, 1972; GREGSON, 1973), the suggestion that water vapor uptake aided by secretion of ions from the salivary glands seems more logical than regurgitation of substances from the gut. A possible method for ‘net’ uptake of atmospheric water that involves the saliva is the reabsorption of
Gland
Fig. 1. Diagrammatic summary of how salts of high concentration may be secreted by salivary glands into the region of the mouthparts with subsequent uptake and absorption of water vapor by rehydrating ticks,
1285
Ixodid tick salivary glands
If reabsorption is required, it could occur in much the same way that water is thought to be reclaimed by the insect rectum from strongly hyperosmotic rectal fluid (PHILLIPS, 1970). In this way the salts (possibly NaCl and KCI) in the salivary secretion could become sufficiently concentrated to pick up atmospheric water hygroscopitally, which would yield a net increase in body water, to be swallowed later and absorbed through the gut. The important site of energy expenditure would then be the salivary gland epithelia where water reabsorption occurred, possibly the proximal ducts or acini of the salivary glands. Of potential relevance to this hypothesis is the demonstration by COONS and ROSHDY(1973) of anteriorly confined aveoli having direct communication with the main salivary duct in unfed Dermucentor uaruurkzbilis males. The potentially significant aspect is their anterior location and containment of extensive infoldings of the basal cell membrane with associated mitochondria, a feature common to fluid transporting epithelia. What is interesting is the conspicuous presence of these structures in the unfed male where little fluid elimination during feeding would be expected because of the minimal amount of feeding done by the male. Relevant to all of this is the report of SHIH et al. (1973). They found that Na was initially removed from the haemolymph by ‘severely desiccated’ ticks in the process of becoming ‘moderately hydrated’ and that the increase in water content during these events was mostly in other tissues than the blood. The authors felt that sodium may play some important role in the ability to absorb water from subsaturated atmospheres. It may be that movements of Na and Cl are closely linked; each is the chief cation and anion, respectively, in the haemolymph (Hsu and SAUER, 1975). Also, RUDOLPH and KNIJLLE (1974) have observed a high concentration of Na in the oral secretion of desiccated non-feeding ticks. In conclusion, the mouth is confirmed as the site of water vapor uptake in ixodid ticks. Secretion of ions into the mouthparts appears to be an integral part of the mechanism enabling ticks to take up water vapor from unsaturated air. The experiments of RUDOLPHand KN~~LLE(1974) have demonstrated that the secreted substance is hygroscopic and the results of our experiments suggest that the source of the hygroscopic material may be the salivary glands. When the surrounding humidity increases sufficiently, this material could absorb water from the atmosphere. The resulting solution could then be swallowed and the water and solutes absorbed by the gut tissue, enabling the tick effectively to regain the water it lost at lower humidities.
Acknowledgement-The
valuable assistance of Ms. GLEECEand Mr. DANA J. BROWN is gratefully acknowledged. KATHY
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