John R. Sauer

Salivary glands in ixodid ticks: control and mechanism of secretion.

The salivary glands are vital to the biological success of ixodid ticks and the major route for pathogen transmission. Important functions include the absorption of water vapor from unsaturated air by free-living ticks, excretion of excess fluid for blood meal concentration, and the secretion of bioactive protein and lipid compounds during tick feeding. Fluid secretion is controlled by nerves. Dopamine is the neurotransmitter at the neuroeffector junction regulating secretion via adenylate cyclase and an increase in cellular cAMP. Dopamine also affects the release of arachidonic acid which is subsequently converted to prostaglandins. Prostaglandin E(2) (PGE(2)) is secreted at extremely high levels into tick saliva for export to the host where it impacts the host physiology. Additionally, PGE(2) has an autocrine or paracrine role within the salivary gland itself where it interacts with a PGE(2) receptor to induce secretion (exocytosis) of bioactive saliva proteins via a phosphoinositide signalling pathway and an increase in cellular Ca(2+). Regulation of fluid secretion has been extensively studied, but little is known about the mechanism of fluid secretion. Continuing advances in tick salivary gland physiology will be made as key regulatory and secretory gland proteins are purified and/or their genes cloned and sequenced.

Tick saliva: recent advances and implications for vector competence

Abstract . Secretions of the tick salivary glands are essential to the successful completion of the prolonged feeding of these ectoparasites as well as the conduit by which most tick‐borne pathogens are transmitted to the host. In ixodid ticks the salivary glands are the organs of osmoregulation, and excess water from the bloodmeal is returned via saliva into the host. Host blood must continue to flow into the feeding lesion as well as remain fluid in the tick mouthparts and gut. The hosts haemostatic mechanisms are thwarted by various anti‐platelet aggregatory, anticoagulatory and anti‐vasoconstrictory factors in tick saliva. Saliva components suppress the immune and inflammatory response of the host permitting the ticks to remain on the host for an extended period of time and, adventitiously, enhancing the transmission and establishment of tick‐borne pathogens. Over the years much work has been done on the numerous enzyme and pharmacological activities found in the tick saliva. The present article reviews the most recent work on salivary gland secretionith special emphasis on how they favour pathogen transmission.

Tick salivary glands: function, physiology and future

The salivary glands are the organs of osmoregulation in ticks and, as such, are critical to the biological success of ticks both during the extended period off the host and also during the feeding period on the host. Absorption of water vapour from unsaturated air into hygroscopic fluid produced by the salivary glands permit the tick to remain hydrated and viable during the many months between blood-meals. When feeding, the tick is able to return about 70% of the fluid and ion content of the blood-meal into the host by salivation into the feeding site. This saliva also contains many bioactive protein and lipid components that aid acquisition of the blood-meal. The salivary glands are the site of pathogen development and the saliva the route of transmission. The importance of the multifunctional salivary glands to tick survival and vector competency makes the glands a potential target for intervention. Here we review the cell biology of tick salivary glands and discuss the application of new approaches such as expressed sequence tag projects and RNA interference to this important area in the field of tick and tick-borne pathogen research.

RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum.

RNA interference (RNAi), a gene silencing process, has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned putative Amblyomma americanum histamine binding protein (HBP) to test the significance of using this methodology in the assessment of the function and importance of gene products in ectoparasitic ticks. The female salivary glands incubated in vitro with HBP dsRNA had a significantly lower histamine binding ability. In addition, the injection of HBP dsRNA into the unfed females led both to a reduced histamine binding ability in the isolated salivary glands and to an aberrant tick feeding pattern or host response. Molecular data demonstrated less expression of the HBP mRNA in the RNAi group. Taken together, these results suggest that RNAi might be an important tool for assessing the significance of tick salivary gland secreted proteins modulating responses at the tick–host interface.

Amblyomma americanum: Characterization of salivary prostaglandins E2 and F2α by RP-HPLC/bioassay and gas chromatography-mass spectrometry

Secretagogue-induced saliva of the tick, Amblyomma americanum (L.) was fractionated by reversed-phase-high-performance liquid chromatography (RP-HPLC) and bioassayed in smooth muscle preparations. Material with retention times of authentic prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) were found to cause contraction of preparations of rat colon and rat stomach strips. Gas chromatography-mass spectra of selected ions of both HPLC-purified fractions confirmed the existence of PGE2 and PGF2 alpha. Bioassay of individual samples obtained from ticks stimulated to salivate with pilocarpine, dopamine + theophiline, or dopamine + theophiline + GABA indicated that all these secretagogues induced similar amounts of prostaglandin secretion, averaging 469 ng PGE2/ml. These pharmacological doses of prostaglandin are hypothesized to assist in tick feeding by inducing vasodilation and/or other pharmacological events in their hosts.

Protein changes in the salivary glands of the female lone star tick, Amblyomma americanum, during feeding.

Several new proteins, as determined by SDS-polyacrylamide gel electrophoresis, appeared in the salivary glands of female Amblyomma americanum soon after attachment of the tick to a host. Other proteins, present in unfed ticks, increased in quantity during tick feeding. The newly synthesized proteins remained undiminished through the remainder of tick feeding, suggesting incorporation, as structural components, into enlarging alveoli Types II and III. All but one of these kinds of protein were found in the salivary glands of unmated, partially fed females attached to a host for equal periods of time; however, total gland protein was much less than that observed in glands of mated females. Another group of proteins, present in the salivary glands of unfed females, were secreted or converted to other substances by the salivary glands of engorging females during various phases of feeding. All but two of these remained in the salivary glands of females if mating did not occur. One protein, present in unfed ticks, increased in quantity during early phases of feeding, but during later phases of feeding it was secreted or converted to other substances. This protein remained in the glands of females if mating did not take place. One other protein found in the glands of unfed ticks decreased during early phases of feeding but increased during later phases of feeding. This protein was also seen in the glands of partially fed and unmated females. Results suggested that following attachment, ixodid females were stimulated to feed and synthesize new kinds of salivary gland protein. Mating stimulated additional female feeding and an increase in the amounts but not the number of salivary gland proteins. Following mating, salivary glands were also stimulated to secrete considerable protein.

Biosynthesis of salivary prostaglandins in the lone star tick, Amblyomma americanum

Dopamine-induced saliva from ticks fed [3H]arachidonic acid contained the radiolabelled prostaglandins E2, F2 alpha, D2, and B2, the latter probably derived from PGE2 owing to the alkalinity of tick saliva. Prostaglandin synthetase (PGS) activity in the salivary gland homogenate from the lone star tick, Amblyomma americanum, could not be detected by standard radiometric methodologies successfully employed for tissues from many animal species, including numerous arthropods. Modifications to the assay conditions had no effect. The presence of a PGS-inhibitor in the salivary glands was ruled out. It is postulated that the PGS in A. americanum salivary glands may be considerably different from that found in other animals, including vertebrate hosts.

A Proteomics Approach to Characterizing Tick Salivary Secretions

The saliva of ticks contains a complex mixture of bioactive molecules including proteins that modulate host responses ensuring successful feeding. The limited amount of saliva that can be obtained from ticks has hampered characterization of salivary proteins using traditional protein chemistry. Recent improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize small amounts of protein. These methods were employed to characterize salivary proteins from Amblyomma americanum and Amblyomma maculatum. Salivation was induced by injection of dopamine and theophylline. It was necessary to desalt and concentrate saliva before analysis by 2-D electrophoresis. Comparison of 1-D and 2-D gel patterns revealed that the major protein component of saliva did not appear on 2-D gels. Characterization of this protein showed that it was identical to the major protein present in the hemolymph of both tick species. Protein profiles obtained by 1-D and 2-D gel electrophoresis were similar for both tick species, however, higher concentrations of lower molecular weight proteins were present in A. maculatum. Protein analysis by MALDI-TOF mass spectrometry and western blot analysis showed that except for the most abundant protein with a molecular weight of 95 kDa, all of the proteins detected were of host origin. It is not known if this is an artifact of the collection method or has physiological significance. In either case, in these species of ticks, host proteins will have to be removed from saliva samples prior to 2-D analysis in order to characterize lower abundance proteins of tick origin.

Possible role in uptake of water vapour by ixodid tick salivary glands.

Abstract The mouth is confirmed as the site of water vapor uptake in the lone star tick, Amblyomma americanum . It was shown that the level of chloride ( 36 Cl) increased in the mouthparts of desiccated ticks. The highest levels of 36 Cl 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 then swallowed and absorbed from the lumen of the gut.

Changes in lipids of the salivary glands of the lone star tick, Amblyomma americanum, during feeding

The lipid composition of salivary glands from male and female lone star ticks, Amblyomma americanum, was investigated at progressive stages of tick feeding. The amounts of fatty acids from both phospholipid and neutral lipid fractions increased dramatically during the initial stage of feeding and peaked in partially fed females weighing 100-250 mg. Percentage compositions of myristic (14:0) and palmitic acid (16:0) decreased, but stearic (18:0), oleic (18:1), linoleic (18:2), and arachidonic acid (20:4) increased during tick feeding. Arachidonic acid, the precursor to eicosanoids including the 2-series of prostaglandins, increased from 1.3% of all fatty acids in salivary glands from unfed female ticks to 8.2% in salivary glands from fully engorged female ticks. Arachidonic acid was found in the triglyceride fraction of unfed and fed virgin females but only in phosphatidylcholine and phosphatidylethanolamine from salivary glands of other fed female ticks. Comparisons between fed and unfed male ticks and fed/virgin, fed/mated, and unfed females demonstrate that feeding is necessary for accumulation of arachidonic acid in salivary gland phospholipids.