Role of Cyclic Nucleotides and Calcium in Controlling Tick Salivary Gland Function

The paired salivary glands of female ixodid ticks are essentialorgans of osmoregulation. As the female feeds, the rate of salivaryfluid secretion increases greatly enabling the tick to concentrateits bloodmeal by returning excess water and ions to the hostvia the salivary ducts. The glands are controlled by nervesand the neurotransmitter at the neuroeffector junction is dopamine.Cyclic AMP is a "second messenger" of the fluid secretory process.Specific endogenous salivary gland proteins are phosphorylatedby cyclic AMP-dependent protein kinases which facilitate insome way the fluid secretory process. Fluid secretory capabilityand dopamine sensitive adenylate cyclase activity in glandsof feeding females are dependent on weight of the feeding tickfrom which they are obtained. Conversely, cyclic AMP-dependentphosphodiesterase is inversely related to the magnitude of fluidsecretory capability of the glands. Deletion of calcium or additionof verapamil to the bathing medium during experiments with isolatedglands inhibits dopamine-stimulated fluid secretion. The preciserole(s) of calcium in secretion is(are) unknown but it may helpregulate cyclic AMP by regulating activator and inhibitor proteinsof cyclic AMP phosphodiesterase. The inhibitor modulators areat much higher concentrations in salivary glands of ticks inthe rapid phase of feeding.     

Saliva-activated transmission (SAT) of Thogoto virus: dynamics of SAT factor activity in the salivary glands ofRhipicephalus appendiculatus,Amblyomma variegatum,andBoophilus microplus ticks

Thogoto (THO) virus is transmitted from infected to uninfected ticks when co-feeding on uninfected guinea-pigs, even though the guinea-pigs do not develop a detectable viraemia. This form of non-viraemic transmission is potentiated by a factor (s) secreted by the saliva of ticks and hence has been termed saliva-activated transmission (SAT). The synthesis of the SAT factor by the salivary glands of three ixodid tick species was determined by placing uninfected nymphal ticks on guineapigs that were subsequently inoculated with a mixture of THO virus and salivary gland extract (SGE) derived from one of the tick species. SAT factor activity was measured by determining the number of nymphs that acquired THO virus. For the three-host ixodid species,Rhipicephalus appendiculatus andAmblyomma variegatum, maximum enhancement of THO virus transmission was observed when salivary glands were derived from uninfected, female ticks that had fed for a period of 6 or 8 days, respectively. In contrast, when salivary glands were derived form uninfected femaleBoophilus microplus, a one-host ixodid tick species, enhancement of THO virus transmission was observed throughout the tick feeding period. Thus, the natural feeding behaviour of ticks appears to be an important factor in determining the relative importance of these vectors in mediating SAT.     

Factors influencing the toxicity of salivary gland extracts of Ixodes holocyclus Neumann

A bioassay using mice was developed to compare the toxin content of extracts of salivary glands of I. holocyclus at various stages of feeding. The quantity of toxin increased rapidly from the third day of feeding. Toxin production continued and increased in ticks removed after 3–5 days on mice and held at 30°C at 92% RH for 24 h, whereas no toxin was detected in the salivary glands of ticks fed for 3 days and treated similarly. It is suggested that major physiological changes occur in the salivary glands of I. holocyclus on the third day, which once stimulated continue independently of feeding. Toxin production in ticks was not suppressed by passively immunizing mice with anti-tick toxin but was in ticks fed upon hosts with a previous experience of tick feeding.Thus, to obtain salivary glands containing high concentrations of toxin for chemical analysis, it is necessary for salivary glands to develop 5 days from the initial attachment of the tick to a host with no previous experience of tick feeding. This can be achieved by passively immunizing mice against toxin, thus enabling the tick to feed 5 days without killing the mouse or by keeping the tick for 24 h at 30°C at 92% RH following the death of the mouse on the fourth day.     

Putative new expression of genes in ixodid tick salivary gland development during feeding.

Poly(A+) mRNA-enriched fractions from salivary glands of partially fed Amblyomma americanum female ticks were translated in vitro with a rabbit reticulocyte translation system. Translated proteins were labeled with [35S]methionine, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and identified by autoradiography. Thirty major identifiable polypeptides with molecular weights ranging from 14 to 136 kDa were synthesized from mRNA isolated from salivary glands of ticks weighing less than 100 mg. Polypeptides that comigrated at the same molecular weight were translated by mRNA from ticks at a more advanced stage of feeding (more than 300 mg) as were 8 others with molecular weights of 31, 71, 91, 106, 113, 118, and 128 kDa. Results demonstrated that differential gene expression may be stimulated in the developing salivary glands as the tick feeds.     

Brain factor induced formation of inositol phosphates in tick salivary glands

A factor from tick brain increases inositol phosphates in isolated, whole tick salivary glands. The factor is sensitive to trypsin and heat (5 min, 100°C) suggesting that it may be a neuropeptide or protein. The salivary glands undergo growth and differentiation accompanied by considerable proliferation of plasma membranes during tick feeding. Salivary glands from ticks in later stages of feeding produce higher levels of inositol phosphates than glands from ticks in early stages of feeding. Cyclic AMP modulates the formation of inositol phosphates suggesting interaction of salivary gland function by the transducing system that employs cyclic AMP as a “second messenger” and that which employs inositol phosphates.     

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.     

Rhipicephalus appendiculatus feeding on rabbits and cattle: Salivary-gland responses to varying host resistance

AdultRhipicephalus appendiculatus ticks were fed as three sequential infestations on both rabbits and cattle. The feedings at first infestation on naive hosts were optimum for the ticks, whereas at third infestation the hosts were resistant, as expressed by reduced tick feeding performance. Ticks from naive and resistant hosts were examined for histological differences of salivary glands. In ticks fed on resistant rabbits there was a large increase in the synthesis of glycoprotein secretory granules in thec ₁ cells compared with ticks fed on naive rabbits. In ticks fed on naive and resistant cattle, the activity of thec ₁ cells was less than in ticks fed on naive and resistant rabbits. It was concluded that the salivary glands are able to respond selectively to conditions at the feeding site, and that this may be advantageous to the tick.     

“Male factors” in ticks: their role in feeding and egg development

Summary

Female ticks of the family Ixodidae utilize their salivary glands as the major organs for fluid balance, secreting back into the host a dilute saliva. Feeding is composed of three phases: a preparatory phase (1–2 days) during which the tick establishes the feeding lesion, a slow phase (~7 days) during which body weight increases 10-fold, and a rapid phase (~1 day) in which body weight increases a further 10-fold. Following engorgement, the salivary glands are resorbed by an autolytic process triggered by an ecdysteroid hormone. If a female is removed from the host prior to repletion, her subsequent behaviour depends mostly on two factors: the degree of engorgement achieved and whether or not she has mated. If removed during the preparatory or slow phase of engorgement, the salivary glands are not resorbed, the tick will lay virtually no eggs and she will reattach to a host if given the opportunity, all of this irrespective of whether she is virgin or mated. If removed during the rapid phase of engorgement, however, mated females will not reattach to a host even if given the opportunity. Instead, they will resorb the salivary glands within 4 days post-removal and lay a batch of eggs. Virgin females removed after exceeding 10-fold the unfed weight likewise refuse to resume feeding if given the opportunity, but salivary gland reabsorption is delayed (to 8 days post-removal); if any eggs are laid, they are infertile. A number of chemical “factors” entering the female during copulation influence her feeding behaviour and egg development. Here we discuss the complexities of these interactions and suggest how they might be adaptive to ticks in nature.     

The influence of various factors on fluid secretion by in vitro salivary glands of ixodid Ticks.

1. Salivary glands of the female ixodid tick, Dermacentor andersoni, secrete fluid in vitro when bathed in a slightly modified version of the mammalian tissue culture medium 'TC 199'. 2. Rate of salivation in vitro increases with progression of feeding, but there is no comparable increase in dry weight of the salivary glands during the early phase of engorgement. Engorged ticks secreted at only 25% the rate of 90-250 mg ticks, indicating that salivary gland degeneration has already begun in the very early post-engorgement stage. 3. A salivary gland stimulating factor can be detected in the nervous system but not in other tissues. 4. Male salivary glands secrete at only 1/20th the rate of female glands. Thus males probably do not use their salivary glands as osmoregulatory organs. 5. From the uniform lack of response to ACh and uniform response to DA in 7 ixodid tick species, it is suggested that the control of salivation is similar throughout the ixodid family.     

Adenosine-3',5'-monophosphate in salivary glands of unfed and feeding female lone star ticks,Amblyomma americanum (L.)

1. Basal levels of cAMP in salivary glands of female lone star ticks were found to be about 5 pmoles/mg protein during all stages of feeding.2. Glands stimulated with 10⁻⁵M dopamine and 10⁻⁵M dopamine plus theophylline exhibited significant increases in cAMP/mg protein.3. After stimulation by 10⁻⁵ M dopamine was removed, cAMP decreased faster in glands from slowly feeding ticks (< 200 mg) than in glands of rapidly feeding ticks ( > 200 mg).     

Microarray analysis of gene expression changes in feeding female and male lone star ticks,Amblyomma americanum (L)

A collection of EST clones from female tick Amblyomma americanum salivary glands was hybridized to RNA from different feeding stages of female tick salivary glands and from unfed or feeding adult male ticks. In the female ticks, the expression patterns changed dramatically upon starting feeding, then changed again towards the end of feeding. On beginning feeding, genes possibly involved in survival on the host increased in expression as did many housekeeping genes. As feeding progressed, some of the survival genes were downregulated, while others were upregulated. When the tick went into the rapid feeding phase, many of the survival genes were downregulated, while a number of transport‐associated genes and genes possibly involved in organ degeneration increased. In the males, the presence of females during feeding made a small difference, but feeding made a larger difference. Males showed clear differences from females in expression, as well. Protein synthesis genes were expressed more in all male groups than in the partially fed females, while the putative secreted genes involved in avoiding host defenses were expressed less. © 2009 Wiley Periodicals, Inc.     

Role of an aquaporin in the sheep tick Ixodes ricinus: Assessment as a potential control target

Ticks undergo tremendous osmoregulatory stress as they take on up to 100 times their body weight in blood, returning about 75% of the ingested water and ions via their saliva into the host. We postulated that water channels, or aquaporins, involved in this mass water transport might be good targets for acaricide development. An aquaporin (IrAQP1) identified in the sheep tick, Ixodes ricinus, was present only in tissues involved in mass water flux, namely the gut, rectal sac and especially abundant in the salivary glands. IrAQP1 was localised by in situ hybridisation in specific cell and acini types, possibly Type III acini, but absent from the type I acini that are responsible for rehydration of ticks in the non-feeding phase. Gene knockdown of IrAQP1 in isolated salivary glands completely inhibited dopamine-stimulated secretion. Further, IrAQP1 knockdown adult females had 50% reduced body weight gains over the first 5 days feeding on an artificial feeding apparatus and 21% at the point of engorgement on hosts. Haemolymph osmolarity was increased in the IrAQP1-knockdown ticks. Importantly, the blood volume ingested per body weight was reduced by 30%. Overall, it would appear that water passage from the gut to the saliva was disrupted and tick guts were simply too “full” to ingest more blood. However, double-stranded RNA interference of IrAQP1 did not affect mortality of the ticks which successfully fed to detachment at day 9. Overall, our data indicate that IrAQP1 plays a pivotal role in blood meal water handling through the gut and salivary gland, and although its disruption by double-stranded RNA interference dramatically affects feeding performance, ticks remained feeding on the host with subsequent potential pathogen transmission and, therefore, IrAQP1 is not a suitable candidate target for tick control.     

Amblyomma americanum (L.): protein kinase C-independent fluid secretion by isolated salivary glands.

Protein kinase C activity was partially purified from tick salivary glands by fast protein liquid chromatography anion-exchange chromatography. Enzyme activity was stimulated by Ca2+, phosphatidylserine, and diacylglycerol with the highest activity observed in the presence of all three modulators. Enzyme activity was inhibited by a synthetic pseudosubstrate peptide with an amino acid sequence resembling the protein kinase C substrate phosphorylation site. The protein kinase C activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG), when added to whole in vitro salivary glands previously prelabeled with 32P, stimulated the phosphorylation of salivary gland proteins. Activators of protein kinase C (phorbol ester or OAG) did not stimulate fluid secretion by isolated tick salivary glands. OAG and phorbol ester had only minimal affects on the ability of dopamine to stimulate secretion by isolated salivary glands and dopamine's ability to increase salivary gland cyclic AMP.     

Tick antigens recognized by serum from a guinea pig resistant to infestation with the tick Rhipicephalus appendiculatus

Immune resistance to infestation by an ixodid tick, Rhipicephalus appendiculatus, the vector of the cattle disease East Coast Fever, was induced in a guinea pig by repeated tick infestation. This resistance is expressed as the ability of the host to interfere with tick feeding. Resistance to ixodid tick feeding is an acquired response mediated by host antibody. We report the use of antibodies from a resistant host animal, in immunoblotting, to characterize the tick antigens recognized. The major tick antigens identified had molecular weights of 120,000, 94,000, 88,000, 77,000, 58,000, 46,000, 35,000, 31,000, 28,000, 25,000, 20,000 and 16,000. Most of these antigens were found in tick salivary glands. The presence and concentration of many tick salivary antigens appeared to vary with relation to the tick feeding cycle. Many of the antigens present in salivary glands were also detected in tick cement. Tick gut extract, although a poorer source of antigens, contained more of the 31,000 dalton antigen than salivary glands. Larval and nymphal tick extract lacked many of the antigens present in adult ticks. The data suggest that tick resistance is a complex phenomenon probably elicited by several different tick antigens.     

Biochemical differentiation of lone star tick,Amblyomma americanum (L), salivary glands: Effects of attachment,feeding and mating

Salivary glands of female Amblyomma americanum (L.) are stimulated to differentiate by attachment to a host, subsequent feeding and mating. Incorporation of [³H]uridine into ribosomal and transfer RNAs as well as the synthesis of poly(A⁺)mRNA and protein parallel the pattern of increasing enzymatic activity and secretory ability of the glands. Unfed ticks contained 3.5 ± 0.47 ng poly(A⁺)mRNA/gland pr. By the second day of feeding this had increased more than 5-fold. The greatest amount of poly(A⁺)mRNA found in rapid-feeding phase females (body wt > 100 mg) was 370 ± 80 ng/gland pr. Poly(A⁺)mRNA mass doubles on the final day of feeding, just as the ticks exceeded 100 mg in wt. Ticks attached 1 to 10 days had increasingly greater amounts of salivary monosomes, 60 and 40S ribosomal subunits, and polysomes. Polysomal mass/gland pr also attained its maximum above 100 mg tick wt at the slow/rapid-feeding phase boundary; exceeding by 20 times that of unfed ticks. Degenerating glands from replete ticks continued to synthesize protein. In vitro incorporation of [³H]leucine was greatest within 24 h of attachment. Fluorographs of [³H]leucine labeled protein showed that mating caused a drop in incorporation after the 4th day of feeding. Glands from unmated females attached the same number of days continued to incorporate [³H]leucine at higher levels than those from mated females.     

A dopamine sensitive adenylate cyclase in the salivary glands of Amblyomma americanum (L.)

1. Enzyme activity, basal or dopamine-stimulated (10 microM), was linear with time to 25 min and with protein concentration to 0.8 mg protein/ml of final assay volume. Activity was maximal between pH 7.0 and 7.5. 2. Mg2+ maximally stimulated basal or dopamine-sensitive adenylate cyclase activity at about 4 mM. 3. Adenylate cyclase had a Km of 0.042 mM for ATP and maximum velocities for basal and dopamine-stimulated activity of 107 and 179 pmol cyclic AMP formed/mg protein per min, respectively. 4. Half-maximal stimulation of the enzyme occurred at about 4.2 x 10(-7) M dopamine with the threshold being less than 10(-9) M. Dopamine increased the Vmax but had no effect on the Km of ATP. 5. Eighty-five to 90% of the adenylate cyclase activity was found in the particulate fraction. 6. Calcium ion produced a marked inhibition of adenylate cyclase activity above 0.04 mM and half-maximal inhibition occurred near 0.1-0.2 mM.     

The Mitochondrial Genomes of Nuttalliella namaqua (Ixodoidea: Nuttalliellidae) and Argas africolumbae (Ixodoidae: Argasidae): Estimation of Divergence Dates for the Major Tick Lineages and Reconstruction of Ancestral Blood-Feeding Characters

Ixodida are composed of hard (Ixodidae), soft (Argasidae) and the monotypic Nuttalliellidae (Nuttalliella namaqua) tick families. Nuclear 18S rRNA analysis suggested that N. namaqua was the closest extant relative to the last common ancestral tick lineage. The mitochondrial genomes of N. namaqua and Argas africolumbae were determined using next generation sequencing and de novo assembly to investigate this further. The latter was included since previous estimates on the divergence times of argasids lacked data for this major genus. Mitochondrial gene order for both was identical to that of the Argasidae and Prostriata. Bayesian analysis of the COI, Cytb, ND1, ND2 and ND4 genes confirmed the monophyly of ticks, the basal position of N. namaqua to the other tick families and the accepted systematic relationships of the other tick genera. Molecular clock estimates were derived for the divergence of the major tick lineages and supported previous estimates on the origins of ticks in the Carboniferous. N. namaqua larvae fed successfully on lizards and mice in a prolonged manner similar to many argasids and all ixodids. Excess blood meal-derived water was secreted via the salivary glands, similar to ixodids. We propose that this prolonged larval feeding style eventually gave rise to the long feeding periods that typify the single larval, nymphal and adult stages of ixodid ticks and the associated secretion of water via the salivary glands. Ancestral reconstruction of characters involved in blood-feeding indicates that most of the characteristics unique to either hard or soft tick families were present in the ancestral tick lineage.     

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 host's 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.