Hyperosmotic Oral Fluid Secretion During Active Water Vapour Absorption and During Desiccation-Induced Storage-Excretion by the Unfed Female Tick Amblyomma Americanum

The exact mechanism of active water absorption in ticks remains undetermined (Needham and Teel, 1986). O’Donnell and Machin (1988) review several mechanisms that are involved in active water uptake, the most common of which is based on the colligative properties of a secreted, hyperosmotic material that can drive water uptake. Previous research on severely desiccated ticks has shown that Na⁺, K⁺ and Cl^- salts are found on and in the mouthparts (Rudolph and Knülle, 1978; Devine, 1982). These deposited salts have been observed to deliquesce at a_N values near the CEA for the tropical bont tick A. variegatum (Rudolph and Knülle, 1978). However, it remains to be demonstrated whether more water is gained from the salt residues than is required to deliver them onto the gnathosoma. Needham and Teel (1986) suggest that desiccation-induced oral secretions in these long-lived ectoparasites may represent a storage-excretion mechanism to reduce elevated haemolymph osmotic pressures (OP) incurred by desiccation, with the advantage that these salts are reclaimed later during rehydration activity. Rudolph and Knülle (1978) used the protozoan parasite Theileria to destroy only types II and III acini of the salivary glands without preventing active uptake, and thereby indirectly implicated the type I agranular acini. Changes in the conformation of mitochondria demonstrated in these salivary acini during rehydration further suggest that the salivary glands are involved in vapour uptake (Needham et al. 1990).

This study employed nano-osmometry and melting-point analysis of frozen sections to measure the osmotic pressure of oral fluid of frozen sections and haemolymph in individual ticks in differing physiological states. Fluid osmolalities exceeding 5.49osmolkg^_J, which would be equal to or greater than the equilibrium vapour activity of 0.91 a_v for unfed female A. americanum (Sigal, 1990), could function as the basis of a solute-driven, water vapour absorption mechanism. Unfed adult females of the ixodid tick Amblyomma americanum (CEA ≥0.88 a_v; Sigal, 1990) were used in this study. Ticks were obtained from a laboratory colony reared at the Acarology Laboratory, The Ohio State University, Columbus, Ohio, maintained at 0.93 a_v and room temperature (26°C). Several 1-month-old female A. americanum were randomly selected from a cohort of ticks that had previously been fed on an adult chicken and used to induce oral fluid secretion as described below. Another group of ticks was also selected and placed in dry air for at least 24h prior to rehydration and analysis of vapour uptake.

In the first fluid collection method we used light and heat stress to induce fluid production after the method of Barker et al. (1973). Ticks secured on a glass slide ventral side up with double-sided sticky tape were observed to exude fluid droplets laterally from the hypostome-cheliceral margin during high-intensity illumination with an incandescent microscope illuminator. Oral fluid samples (0.33-24.00 nl) were collected as soon as they were released by a finely drawn, oil-filled glass capillary inserted down the oral channel. Slight negative pressure was applied to the capillary through a 5 ml oil-filled syringe, connected by flexible polyethylene tubing. After the oral fluid had been collected and measured, a sample of haemolymph was similarly collected from the cut distal segment of leg I or II. Oral fluid samples and haemolymph droplets were injected under oil into the sample wells of a nanolitre osmometer (Clifton Technical Physics, Wanamassa, New Jersey). Melting points of up to four replicate unknowns were compared with those of two standard NaCl solutions either side of the expected range.

In the second method, oral fluid and haemolymph OP values were measured in situ in serially sectioned mouthparts. Prior to analysis, we established that ticks were actively absorbing water vapour by monitoring changes in water mass during rehydration using a Sartorius 4410 recording electrobalance (±1 μg) operating in an airflow-regulated atmosphere at a_v values between 0.80 to 0.96 (Machin and Lampert, 1987). When mass gain was confirmed, the tick was quickly removed from the balance, quick-frozen in hexane cooled to near-freezing by liquid nitrogen and embedded in Tissue-tek embedding medium (Miles Inc., Elkhart, Indiana) on a microtome stub in a cryostat at —20.0°C. Transverse gnathosomal sections (12 qm) of the frozen mouthparts were placed on a slide in a droplet of chilled kerosene under a cover slip and inserted into a Mettler temperature-controlled stage (temperature resolution of 0.1°C=0.054osmolkg^-1) on a compound microscope positioned within the cryostat (Machin, 1979). Melting temperatures of the cheliceral haemolymph and the fluid of salivarium compartments were recorded by direct observation and from photographs taken as the sections were warmed from — 20.0°C to approximately 0.0°C in 0.5-2.0°C increments. The distinct lateral and central compartments of the salivarium, the two separate cheliceral haemolymph compartments and the surrounding haemolymph-filled basis capitulum were identified (Fig. 1B), and the crystalline ice structures of frozen fluids carefully noted. Observations were then made of changes in light refractive qualities and the appearance of liquified curvature of crystal edges during the graded series of intermittent temperature increases. Because of the extremely small size of the lateral compartments, the detection of crystalline particle movement was a useful criterion of melting, since it was difficult to say otherwise whether a single ice crystal in such a small compartment had melted.

Of the 11 ticks that yielded reliable data, seven comparisons between oral fluid and haemolymph osmotic pressures in the same individual were possible. Five of these showed considerably higher oral fluid OP values compared with corresponding haemolymph values. The remaining unpaired measurements were consistent with this pattern. In the light/heat-induced group (Table 1), oral fluid OP was quite variable, either considerably exceeding the more stable haemolymph values or resembling them. The sample sizes were too small to establish differences between hydrated and desiccated animals.

The OP values for some of the thermally induced oral fluid collected during this study were similar to the values simultaneously determined for haemolymph. It should be noted that the lowest OP values from oral fluid were obtained from the largest fluid volumes collected. This inverse correlation may be attributable to mixing with larger volumes of more dilute haemolymph resulting from the inadvertent rupturing of the delicate subcheliceral membrane sheath during insertion of the capillary. In some cases, when the capillary was inserted it was seen to be broken, with the tip lodged in the internal mouthparts of the tick. In these cases, the values were discarded. Nevertheless, the fluid collected may have been mixed with haemolymph following undetected injury, and could account for those OP values that were similar to those of haemolymph. Haemolymph OP values of hydrated ticks in our study were higher than in past determinations (Hsu and Sauer, 1975).

Melting-point analysis of oral fluid from the gnathosoma of rehydrating ticks shows that three compartments associated with the buccal region contained fluid with elevated OP values (Table 2). These were the two lateral compartments of the salivarium which communicate with the third central lumen compartment of the salivarium lying dorsal and anterior to the pharyngeal opening. The sections observed in this series were posterior to the divergence of the hypostome and chelicerae (Fig. 1A). The lateral compartments are seen in the photomicrograph of Fig. 1B. This interpretation was based upon the gnathosomal morphology of Dermacentor andersoni (Gregson, 1960) and Hyalomma asiaticum (Balashov, 1968) and confirmed by our serial sectioning of A. americanum mouthparts. The greatest fluid OP observed was 6.45 osmol kg^-1 (0.896 a_v) in the right lateral compartment of the salivarium (Table 2), while another high OP of 5.38osmol kg^-1 (0.912 a_v) was observed in the left lateral compartment of a separate frozen section. Fluid OP in the central compartment below the chelicerae was 4.84osmol kg^-1 (0.920 a_v). It should be noted that the fluids from tick number 1 were analyzed from an animal that had been exposed to a rehydrating period of over 16 h, which may account for the lower OP values observed in the central compartment. We suggest that the lower OP values (higher a_v values) of the central salivarium fluid represent fluid that had already been diluted by ambient water vapour and is rehydrated oral fluid which could be swallowed for net water gain. Since the size of the compartments observed were extremely small, the differences in the OPs of similar compartments may be due to variations in the in situ visual detection of the first ice crystal melting.

These results represent the first analysis of oral fluid from the gnathosoma of a rehydrating tick. The oral fluid is sufficiently hyperosmotic to absorb water vapour from a_v values reported within the CEA range for unfed, adult females of this species (Sigal, 1990). The OP values are among the highest for any fluid involved in active water vapour absorption in arthropods (O’Donnell and Machin, 1988). The OP of desiccation (heat)-induced oral fluid was also determined, and it indicates a highly concentrated fluid that may serve as a mechanism of storage-excretion for severely desiccated ticks. We conclude that active water uptake by adult A. americanum is a solute-driven process that is dependent upon the production of a hyperosmotic fluid probably by type I agranular acini of the salivary glands.

This work was supported in part by research grants to Professor John Machin from the Natural Sciences and Engineering Research Council of Canada. We acknowledge Bernie Sigal, C. Cihon, C. Harless and R. Kazee of Chroma Studios, Columbus, Ohio, for their phototechnical support. We thank Dr Olaf Kahl for suggestions on a draft of the manuscript and, in particular, Dr Jonathan Wright for his valuable assistance and comments on the manuscript.