<Emphasis Type="Italic">In vitro</Emphasis> splenocyte proliferation responses of BALB/c mice to salivary gland extracts of three ixodid tick species (Acari: Ixodidae)

In vitro proliferation and cytokine production were investigated in BALB/c mice splenic cell cultures that were stimulated with concanavalin A (ConA) and lipopolysaccharide (LPS) and simultaneously exposed to salivary gland extracts (SGE) of unfed and partially fed adult ixodid ticks (Ixodes ricinus, Rhipicephalus appendiculatus, Amblyomma variegatum). Generally, tick SGE enhanced proliferation of unstimulated splenocytes and SGE of unfed ticks suppressed mitogen induced proliferation. Partially fed R. appendiculatus and A. variegatum suppressed ConA responses, while partially fed I. ricinus stimulated both ConA and LPS induced proliferation. A. variegatum and R. appendiculatus females slightly enhanced LPS responses 2 days after attachment but suppressed them at the end of the slow feeding phase. In 72 h ConA induced cell cultures, interferon gamma (IFN-γ) production was suppressed by SGE of all ticks, interleukin (IL)-10 production was enhanced by unfed I. ricinus and partially fed A. variegatum males and IL-5 production was enhanced by feeding R. appendiculatus females and A. variegatum males. The study revealed variability in the responsiveness of murine splenocytes to SGE of different ixodid tick species, whereby patterns of host immunomodulation within one tick species differed between sexes and changed during feeding.     

Application of RNA interference in tick salivary gland research.

Ticks are obligate ectoparasites that feed on a variety of hosts including mammals, birds and reptiles. Prolonged attachment on the host and an ability to transmit a wide variety of pathogens are the special features of tick feeding. Salivary glands are the major route for secretion of excess fluid, several proteins, and factors that counteract the host immune response and hence play a significant role in the success of tick feeding. RNA interference (RNAi) enables scientists to silence genes encoding proteins in an absolutely sequence specific manner at the mRNA level. This technique has already been successfully employed in analyzing roles of proteins of important functions or in assigning roles to several proteins of unknown functions in a variety of animals. In this review, we outline the process of RNAi and the applicability of RNAi in tick salivary gland research.     

Quantification and cellular localization of dopamine in the salivary gland of the ixodid tick Amblyomma hebraeum

Dopamine (DA) content of the salivary glands in partially fed female and fed male ticks, Amblyomma hebraeum Koch (Acari: Ixodidae), was measured by high-performance liquid chromatography with electrochemical detection or by a radioenzymatic assay for catecholamines following experimental treatment. Some glands were held in vitro for up to 3 days. Other preparations (backless explants) allowed one side to be surgically denervated, the contralateral side serving as control. Normal ticks were sampled for up to 4 days post-removal from the host (rabbits). In the backless explants, there was little if any difference in DA content between denervated and control sides, even after 4 days in vitro, indicating that unilateral denervation did not eliminate the major salivary gland pool of DA. High doses of reserpine (333 g per g body weight) and 6-hydroxydopamine (1000 g per g body weight) did not significantly reduce the DA content of the salivary gland, also suggesting that only a minor component of the DA pool is within axons innervating the salivary gland. A dispersed population of cells rich in tyrosine hydroxylase immunoreactivity (an enzyme marker for catecholamine-synthesizing cells) was found in close association with the granular acini. This further suggests that the major DA pool in the salivary gland may be in cells other than the dopaminergic nerves arising from the central nervous system. © Rapid Science Ltd. 1998     

Ixodes scapularis salivary gland protein P11 facilitates migration of Anaplasma phagocytophilum from the tick gut to salivary glands

Ixodes ticks harbour several human pathogens belonging to the order Rickettsiales, including Anaplasma phagocytophilum, the agent of human anaplasmosis. When ticks feed on A. phagocytophilum-infected mice, the pathogen enters the ticks' gut. The bacteria then migrate from the gut to infect the salivary glands of the ticks and are transmitted to the next host via the saliva. The molecular mechanisms that enable the migration of A. phagocytophilum from the gut to the salivary glands are poorly understood. Here we show that a secreted tick protein, P11, is important in this process. We show that P11 enables A. phagocytophilum to infect tick haemocytes, which are required for the migration of A. phagocytophilum from the gut to the salivary glands. Silencing of p11 impaired the A. phagocytophilum infection of tick haemocytes in vivo and consequently decreased pathogen infection of the salivary glands. In vitro experiments showed that P11 could bind to A. phagocytophilum and thus facilitate its infection of tick cells. This report provides new insights into A. phagocytophilum infection of ticks and reveals new avenues to interrupt the life cycle of Anaplasma and related Rickettsial pathogens.     

Enhancement of tick-borne encephalitis virus transmission by tick salivary gland extracts

Abstract. To investigate the role of ticks in TBE virus transmission, salivary gland extract (SGE) was derived from partially fed female Ixodes ricinus, Dermacentor reticulatus and Rhipicephalus appendiculatus ticks. Guinea-pigs were infested with uninfected R.appendiculatus nymphs and inoculated with a mixture of TBE virus and SGE or with virus alone. The number of ticks which on average acquired virus from feeding on animals inoculated with TBE virus and SGE from partially fed ticks was 4-fold greater than the number that became infected by feeding on animals inoculated with virus alone or virus plus SGE from unfed I.ricinus. Viraemia was detected in 67% of guinea-pigs inoculated with virus plus SGE compared to 30% of guinea-pigs inoculated with virus alone. Virus titres in the blood were similar for both groups of animals [range 2.0-2.8 log₁₀ plaque-forming units (PFU)/ml of blood]; however, the number of ticks that became infected was significantly higher on animals inoculated with virus plus SGE from partially fed ticks. No significant difference was observed with respect to the tick species used to derive SGE. The results indicate that TBE virus transmission is enhanced by factor(s) associated with the salivary glands of feeding ticks, and that these factor(s) may facilitate efficient transmission of TBE virus between infected and uninfected ticks even when they feed on hosts that have no detectable viraemia.     

Genetic polymorphism of human salivary proline-rich proteins: Further genetic analysis

Electrophoresis of concentrated parotid saliva on slab polyacrylamide gels negatively stained with 3,3-dimethoxybenzidine and hydrogen peroxide (DMB stain) showed nine phenotypes among the proline-rich proteins. These phenotypes are the expression of four autosomal codominant alleles. Gene frequencies are, for Caucasians, Pr ¹=0.640, Pr ¹=0.005, Pr ²=0.080, Pr ²=0.275; for Negroes, Pr ¹=0.700, Pr ¹=0.050, Pr ²=0.080, Pr ²=0.170; for Chinese, Pr ¹=0.770, Pr ¹=0, Pr ²=0, Pr ²=0.230. The presence or absence of another pair of proteins giving the same negative staining is inherited as an autosomal dominant trait (Db). Homozygous Db + + and heterozygous Db + – individuals could not be distinguished. The genetic determinant (Db) for this pair of proteins is either closely linked to or part of the Pr locus. Gene frequencies are, for Caucasians, Db ⁺=0.12, Db ^–=0.88; for Negroes, Db ⁺=0.56, Db ^–=0.44; for Chinese, Db ⁺=0.07, Db ^–=0.93.This study was supported by a grant from the National Institutes of Dental Research (9-R01-DE-03685-08) and in part by grant GM 15422 from the National Institutes of Health.     

Drosophila salivary gland proteins and pupation

Pulse-labeling experiments of salivary glands from the prepupal stages of development showed selectively high rates of synthesis of a set of low molecular weight proteins (6K–12K). These proteins are stably maintained in the salivary glands during prepupal development and are subsequently transported to the pupation fluid (found between the pupal case and the prepupal cuticle) when pupation occurs. These small polypeptides are very basic with the major components having isoelectric points of 8.6–8.7 and the minor components having isoelectric points of 9.1–9.5. This study shows the continuing function of the salivary glands—specifically, the synthesis and secretion of a set of proteins with a putative role in pupation.     

Ixodid tick salivary gland extracts inhibit production of lipopolysaccharide-induced mRNA of several different human cytokines

Extracts prepared from the salivary glands (SGE) of partially fed adult female Rhipicephalus appendiculatus ticks reduced the expression by human peripheral blood leukocytes (PBLs) of lipopolysaccharide (LPS)-stimulated cytokine mRNA. Treatment with SGE had no obvious effect on cytokine mRNA production when compared with untreated PBLs. LPS treatment induced or increased mRNA production for IFN, IFN, TNF-, IL-1, IL-1, IL-5, IL-6, IL-7 and IL-8. All the LPS-stimulated cytokine mRNAs were reduced when treated with a mixture of LPS and SGE. The results indicate the potential of ticks in modulating the cytokine network of their vertebrate hosts, possibly to facilitate blood feeding.     

Morphofunctional changes of salivary glands of female ixodid ticks of subfamilies Ixodinae and Amblyomminae (Acari: Ixodidae) during feeding and their significance

Using light and electron microscopy, a dynamics of functioning of salivary glands of female ticks of the genus Ixodes was studied. Comparative analysis of morphofunctional changes and role of the salivary gland secretions of females of subfamilies Ixodinae and Amblyomminae during feeding was performed on the basis of literature data and our own studies.     

Salivary gland degeneration and vitellogenesis in the ixodid tick Amblyomma hebraeum: Surpassing a critical weight is the prerequisite and detachment from the host is the trigger

The normal engorged body weight of female ixodid ticks (Acari: Ixodidae) is about 100× the unfed weight. Virgin female Amblyomma hebraeum normally do not feed beyond 10× the unfed weight. However, about 10–20% of a population of virgins will feed to perhaps 20× the unfed weight, but not much beyond that. In A. hebraeum, when females surpass about 10× the unfed weight, the following changes in physiology occur if they are removed from the host: (a) they will not reattach if given the opportunity, (b) their salivary glands (SGs) will undergo autolysis within 4 days if they are mated or 8 days if they are virgin, and (c) egg maturation and oviposition will occur in due course. Mated or virgin female ticks removed from the host below about 10× the unfed weight do not experience the latter changes (Kaufman, W.R., Lomas, L., 1996. ‘Male Factors’ in ticks: their role in feeding and egg development. Invertebrate Reproduction and Development 30, 191–198). In 1984 we named this transitional weight, the ‘critical weight’ (CW). Its absolute value is probably a species-specific characteristic (Kaufman, W.R., 2007. Gluttony and sex in female ixodid ticks: how do they compare to other blood-sucking arthropods? Journal of Insect Physiology 53, 264–273). Although mated females tend to engorge within a day of surpassing the CW, virgin females surpassing the CW can remain attached to the host for at least several weeks more. It is not known whether the physiological changes in the SGs and ovaries listed above occur in those large virgins that remain attached, although we suppose that this would be maladaptive. Instead, we hypothesize in this study that surpassing the CW is only a prerequisite for inducing these changes, and that detachment is the actual trigger. We support our hypothesis by demonstrating that large virgins, remaining attached to a host for 8 days, did not undergo SG degeneration nor complete egg maturation during the attachment period. Those changes occurred only within 8 days following detachment. So some type of sensory information associated with attachment to the host, and still undefined, inhibits expression of the physiological changes hitherto associated merely with surpassing the CW.     

Molecular individuality and adaptation of the tick Rhipicephalus appendiculatus in changed feeding environments

The tick Rhipicephalus appendiculatus Neumann (Acari: Ixodidae) naturally infests many host species. However, the mechanisms that enable it to feed on such a wide range of hosts are unclear. One possibility is that a tick population maintains molecular (genotypic and/or phenotypic) diversity among individuals such that individuals vary in their competency in taking bloodmeals under different feeding conditions. As a first step in testing this hypothesis, we showed that the polymorphism of salivary gland proteins, previously demonstrated in unfed ticks, was maintained during feeding on guinea-pigs. We then compared feeding performance under standard laboratory rearing conditions: one instar (adults or nymphs) feeding on guinea-pigs, with three changed conditions: (1) two instars (adults and nymphs) feeding together on guinea-pigs; (2) one instar (adults or nymphs) feeding on hamsters; and (3) two instars (adults and nymphs) feeding together on hamsters. The mean engorged weight of adult females was significantly reduced under all changed conditions, indicating that most of the adult individuals were significantly challenged by the changed conditions. However, some individuals achieved successful engorgement, indicating competence to the changed condition, and demonstrating variation in adaptive ability among individuals. Engorged females produced egg masses positively correlated to the engorged weights. More interestingly, the correlation coefficient (R) increased when feeding condition was changed. This may lead to more efficient selection for population adaptation under the changed conditions. As the feeding success of ixodid ticks depends on the efficiency of the cocktail of immunomodulatory saliva, the relevance of the polymorphism of salivary gland proteins and host adaptation is discussed.     

Transepithelial transport of nonelectrolytes in the rabbit mandibular salivary gland

Summary The characteristics of nonelectrolyte secretion by the rabbit mandibular salivary gland have been investigated in anin vitro perfused preparation. The concentrations of¹⁴C-labeled nonelectrolytes were measured in saliva samples collected over a range of flow rates during the secretory response of the gland to continuous acetylcholine infusion. Of the nine nonelectrolytes studied, the two particularly lipid-soluble molecules, ethanol and antipyrine, appeared in the saliva at approximately the same concentration as in the perfusate, regardless of the secretory flow rate. The more polar molecules (urea, ethanediol, thiourea, glycerol, erythritol, mannitol and sucrose) appeared at saliva/perfusate concentration ratios () which showed a strong dependence on flow. With the exception of thiourea, this could be attributed to the combined contributions of diffusion and solvent drag.For the polar nonelectrolytes, estimates have been obtained of both the permeability coefficients of the gland (P) and the solvent-drag filtration coefficients (1–). The relation between 1– and molecular radius suggests that small polar nonelectrolytes and the bulk of the secreted water cross the epithelium via aqueous channels that are approximately 0.8 nm in width. The location of the channels remains uncertain because tissue space measurements indicate that the nonelectrolytes most affected by solvent drag have access to both transcellular and paracellular pathways.     

Fine needle aspiration cytology of minor salivary gland tumours of the palate

Fine needle aspiration cytology of minor salivary gland tumours of the palate This retrospective study was carried out to review aspirates from minor salivary gland tumours of the palate and to assess the problems encountered in their diagnosis, especially the cytological diagnosis of newer entities such as polymorphous low grade adenocarcinoma (PLGA). Fifty-five cases of palatal salivary gland tumours aspirated over a period of 16 years were reviewed. Histology was available in 26 cases. Pleomorphic adenoma (27 cases) was the most common benign cytodiagnosis. Eleven aspirates were malignant tumours of which eight cases were adenoid cystic carcinoma and three cases were mucoepidermoid carcinoma. Seven cases were diagnosed on fine needle aspiration as suggestive of PLGA. However histological confirmation was available in only one of these cases. Concordance between the initial and revised typings of the tumours was seen in only 28 cases (54%) in the present study. Initially 18 of the 51 tumours (35.3%) could not be typed; and after review, only three could not be typed. Three cases of oncocytoma could be diagnosed on review only. Palatal salivary gland tumours, although relatively uncommon, are difficult to diagnose cytologically. This is more so in cases of newer entities such as PLGA, as their cytological diagnosis is still not well characterized.     

DNA binding properties of the ecdysteroid receptor in the salivary gland of the female ixodid tick, Amblyomma hebraeum

Salivary gland degeneration in the female tick, Amblyomma hebraeum Koch (Acari: Ixodidae) is controlled by an ecdysteroid hormone. In an earlier study (Mao, H., McBlain, W.A., Kaufman, W.R., 1995. Some properties of the ecdysteroid receptor in the salivary gland of the ixodid tick, Amblyomma hebraeum. Gen. Comp. Endocrinol. 99, 340–348), we demonstrated that a protein component of a salivary gland extract binds to ponasterone A (Pon A) with high affinity (Kd1 nM), suggesting a tick ecdysteroid receptor (EcR). In this study, the Pon A binding protein bound to calf thymus DNA; this binding could be dissociated by Drosophila hsp27 EcRE. The binding protein shifted the [³²P]hsp27 EcRE band on a gel mobility shift assay; formation of the complex with hsp27 EcRE required KCl (optimal concentration was approximately 75 mM). A number of physiologically effective ecdysteroids enhanced the binding with the following order of potency: Pon A>Mur A>Mak A>20E>ecdysone, whereas vertebrate steroids (estradiol, cholesterol, corticosterone, progesterone, testosterone) had no such effect. Using monoclonal antibodies against Drosophila EcR and USP, we found that AG10.2 recognized three bands (90.5, 87.3 and 84 kDa for EcR) and AB11 recognized at least two major bands (50.3 and 47.1 kDa for USP) in the salivary gland extract by western blot analysis. In addition, AB11 supershifted the tick EcR-hsp27 EcRE band on a gel mobility shift assay, indicating that the tick EcR heterodimerized with a USP-like protein for DNA binding. Furthermore, selective mutations to the 15-basepair palindrome of hsp27 EcRE at positions −5, +2, or adding a base to the spacer, resulted in considerably reduced affinity to the tick EcR/USP. We thus propose a sequence similarity of EcREs between A. hebraeum and its insect counterpart.     

Structural and histochemical changes in the salivary glands of Rhipicephalus appendiculatus during feeding

The salivary glands of the brown ear tick of cattle, R. appendiculatus, from both sexes and at all stages of feeding, were examined as whole glands and as sections for ultrastructural and histochemical changes. The type 1 acinus consists of a basal labyrinth formed by the interdigitations of a central cell and four peripheral cells. These cells form a specialized border with a central constrictor cell which surrounds the acinar duct. The plasma membrane of the central cell is exposed to the duct. The type 1 acini do not appear to secrete active saliva components involved in feeding. The type 2 acini undergo a great increase in synthetic and secretory activity during feeding in both sexes and secrete a lipoprotein probably to form part of the attachment cone and also glycoproteins and esterases of unknown functions. The type 3 acini of both sexes also secrete a lipoprotein probably to form part of the attachment cone. The f cells of these acini in the females transiently secrete a glycoprotein of unknown function and then transform to become part of a water excreting unit. In the males the secretory activity of the granular cells of the type 2 and 3 acini is maintained for further attachments. The type 4 acini of the males accumulate masses of proteinaceous granules. The system of interstitial cells and intercellular spaces in types 2, 3 and 4 acini is large and increasingly active during feeding.     

Effects of octopamine on fluid secretion by isolated salivary glands of a feeding ixodid tick

Octopamine elicited a dose-related secretory response by salivary glands isolated from the feeding female tick Amblyomma americanum. Half-maximal stimulation occurred at about 60 μM. Phentolamine (10 μM) failed to inhibit the octopamine-mediated response; however, thioridazine (50 μM) inhibited both octopamine (1,000 μM) and dopamine-stimulated (0.1 μM) secretion. Maximal stimulation by dopamine (1.0 μM) showed no further increase in the rate of secretion after adding octopamine (1,000 or 0.1 μM). Glands responded to octopamine (100 μM) with rates significantly lower than controls following exposure to amphetamine (1,000 μM). Octopamine receptors do not appear to mediate the secretory response, and octopamine may stimulate secretion by releasing catecholamines from presynaptic neurons. These results support the hypothesis that dopamine is the natural transmitter mediating fluid secretion in the feeding tick salivary gland.     

Molecular polymorphism of ciliary proteins from different species of the ciliate Tetrahymena

Ciliary proteins from five different Tetrahymena species were analyzed by means of SDS-polyacrylamide gel electrophoresis. Of at least 32 different polypeptides, only 2 were found to have identical molecular weights in all species. In any comparison of 2 species, a maximum of 60% and at least 20% of the proteins had indistinguishable molecular weights, depending on the combination examined. Extensive codominance of ciliary proteins was found in a F₁ hybrid. No interspecific cross-reactions occurred in double-diffusion tests involving the cilia from different species and antisera produced against them in rabbits.Supported by grants from the U.S. Public Health Service (AG-00010 and GM-07779 to D. L. Nanney and AG-00248 to J.H.W.). H.M.S. was sponsored by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft to work in D. L. Nanney's laboratory.     

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.     

Ultrastructural investigation of a salivary gland in a centipede: structure and origin of the maxilla I-gland of Scutigera coleoptrata (Chilopoda, Notostigmophora)

The maxilla I-gland of Scutigera coleoptrata was investigated using light and electron microscopy methods. This is the first ultrastructural investigation of a salivary gland in Chilopoda. The paired gland opens via the hypopharynx into the foregut and extends up to the third trunk segment. The gland is of irregular shape and consists of numerous acini consisting of several gland units. The secretion is released into an arborescent duct system. Each acinus consists of multiple of glandular units. The units are composed of three cell types: secretory cells, a single intermediary cell, and canal cells. The pear-shaped secretory cell is invaginated distally, forming an extracellular reservoir lined with microvilli, into which the secretion is released. The intermediary cell forms a conducting canal and connects the secretory cell with the canal cell. Proximally, the intermediary cell bears microvilli, whereas the distal part is covered with a distinct cuticle. The cuticle is a continuation of the cuticle of the canal cells. This investigation shows that the structure of the glandular units of the salivary maxilla I-gland is comparable to that of the glandular units of epidermal glands. Thus, it is likely that in Chilopoda salivary glands and epidermal glands share the same ground pattern. It is likely that in compound acinar glands a multiplication of secretory and duct cells has taken place, whereas the number of intermediary cells remains constant. The increase in the number of salivary acini leads to a shifting of the secretory elements away from the epidermis, deep into the head. Comparative investigations of the different head glands provide important characters for the reconstruction of myriapod phylogeny and the relationships of Myriapoda and Hexapoda.