Academic literature on the topic 'Ticks salivary glands'

SUMMARYTo compare the proteins in salivary glands, saliva and haemolymph of Rhipicephalus appendiculatus female ticks, antisera were prepared from guinea-pigs immunized with soluble denatured salivary gland extracts (SGE). The extracts were derived from R. appendiculatus female ticks that were either unfed (day 0) or partly fed (day 6). The sera were used in immunoblotting, following SDS–polyacrylamide gel electrophoresis, to examine the antigen profiles during the course of tick feeding on guinea-pigs. Day 0 and day 6 SGE antisera appeared to detect common proteins in the different tick samples. For example, haemolymph apparently shared some of the small protein bands (31·5–34 kDa) detected in SGEs. These small proteins appeared in both samples at the same stage of feeding, suggesting that haemolymph and salivary glands not only have common antigens but may also share some functions. Furthermore, a number of protein bands were detected in haemolymph before they were apparent in the salivary glands or saliva. Thus some antigens detected in the salivary glands and saliva may be derived from the haemolymph. The results indicate that the host may be exposed to tick saliva antigens that are also present in the haemolymph. We discuss the significance of these observations with regard to the induction of host immunity to ticks and the development of tick vaccines.

ABSTRACT Infectivity of Anaplasma spp. develops when infected ticks feed on a mammalian host (transmission feed). Specific Anaplasma marginale major surface protein 2 (MSP2) variants are selected for within the tick and are expressed within the salivary glands. The aims of this study were to determine when and where MSP2 variant selection occurs in the tick, how MSP2 expression is regulated in salivary glands of transmission-feeding ticks, and whether the number of A. marginale organisms per salivary gland is significantly increased during transmission feeding. The South Idaho strain of A. marginale was used, as MSP2 expression is restricted to two variants, SGV1 and SGV2, in Dermacentor andersoni. Using Western blot, real-time PCR, and DNA sequencing analyses it was shown that restriction and expression of MSP2 occurs early in the midgut within the first 48 h of the blood meal, when ticks acquire infection. A. marginale is present in the tick salivary glands before transmission feeding is initiated, but the msp2 mRNA and MSP2 protein levels per A. marginale organism increase only minimally and transiently in salivary glands of transmission-feeding ticks compared to that of unfed ticks. A. marginale numbers per tick increase gradually in salivary glands of both transmission-fed and unfed ticks. It is concluded that MSP2 variant selection is an early event in the tick and that MSP2 variants SGV1 and SGV2 are expressed both in the midgut and salivary glands. While MSP2 may be required for infectivity, there is no strict temporal correlation between MSP2 expression and the development of infectivity.

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.

SUMMARYHost immunoglobulin G (IgG) crossed the gut wall into the haemocoel of adult Rhipicephalus appendiculatus female ticks when they fed on guinea-pigs. Guinea-pig IgG was also found in saliva of the feeding ticks. The concentration and antibody activity of IgG in haemolymph, salivary gland extract (SGE) and saliva at different stages of tick feeding were detected by enzyme-linked immunoassay. Specific activity of the IgG in tick samples was determined by feeding ticks on guinea-pigs which were immunized with killed Escherichia coli: 35–42% of the antibody activity in guinea-pig immune serum remained in the tick samples. The high relative concentration of IgG in tick saliva at later stages of feeding suggests that the tick may have a mechanism for getting rid of foreign proteins via the salivary gland. Such a mechanism could involve lgG binding proteins (1GBPs) which were found in both haemolymph and SGE of female ticks at day 6 of feeding using a guinea-pig IgG–agarose affinity column. In female ticks, the Mr of 1GBPs in SGE (23 and 57 kDa) were less than those in haemolymph (78 and > 100 kDa). The existence of 1GBPs in both the tick salivary gland and haemolymph indicate that haemolymph and salivary gland cooperate to remove foreign proteins, e.g. host immunoglobulin, from the body during feeding. This mechanism may be a part of the tick self-defence system.

ABSTRACT Borrelia burgdorferi, the spirochetal agent of Lyme disease, is transmitted by Ixodes ticks. When an infected nymphal tick feeds on a host, the bacteria increase in number within the tick, after which they invade the tick’s salivary glands and infect the host. Antibodies directed against outer surface protein A (OspA) of B. burgdorferi kill spirochetes within feeding ticks and block transmission to the host. In the studies presented here, passive antibody transfer experiments were carried out to determine the OspA antibody titer required to block transmission to the rodent host. OspA antibody levels were determined by using a competitive enzyme-linked immunosorbent assay that measured antibody binding to a protective epitope defined by monoclonal antibody C3.78. The C3.78 OspA antibody titer (>213 μg/ml) required to eradicate spirochetes from feeding ticks was considerably higher than the titer (>6 μg/ml) required to block transmission to the host. Although spirochetes were not eradicated from ticks at lower antibody levels, the antibodies reduced the number of spirochetes within the feeding ticks and interfered with the ability of spirochetes to induceospC and invade the salivary glands of the vector. OspA antibodies may directly interfere with the ability of B. burgdorferi to invade the salivary glands of the vector; alternately, OspA antibodies may lower the density of spirochetes within feeding ticks below a critical threshold required for initiating events linked to transmission.

Abstract The relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae are each maintained and transmitted in nature by their specific tick vectors, Ornithodoros hermsi Wheeler (Acari: Argasidae) and Ornithodoros turicata (Duges), respectively. The basis for this spirochete and vector specificity is not known, but persistent colonization of spirochetes in the tick’s salivary glands is presumed to be essential for transmission by these long-lived ticks that feed in only minutes on their warm-blooded hosts. To examine this hypothesis further, cohorts of O. hermsi and O. turicata were infected with B. hermsii and examined 7–260 d later for infection in their midgut, salivary glands, and synganglion. While the midgut from all ticks of both species at all time points examined were infected with spirochetes, the salivary glands of only O. hermsi remained persistently infected. The salivary glands of O. turicata were susceptible to an early transient infection. However, no spirochetes were observed in these tissues beyond the first 32 d after acquisition. Ticks of both species were fed on mice 112 d after they acquired spirochetes and only those mice fed upon by O. hermsi became infected. Thus, the vector competency for B. hermsii displayed by O. hermsi but not O. turicata lies, in part, in the persistent infection of the salivary glands of the former but not the latter species of tick. The genetic and biochemical mechanisms supporting this spirochete and vector specificity remain to be identified.

ABSTRACT Borrelia burgdorferi-infected ticks were fed on either OspC-immunized mice or normal, nonimmunized mice. After 72 h, the ticks were detached, followed by dissection and subsequent culturing in Barbour-Stoenner-Kelley II medium of the salivary glands from each tick to determine the presence of borreliae. Forty percent (10 of 25) of salivary glands from ticks that had fed on nonimmunized mice were culture positive, while only 7.4% (2 of 27) of salivary glands from ticks that had fed on OspC-immunized mice were culture positive, thus indicating a much reduced borrelial migration from the midgut when the bloodmeal contained anti-OspC antibodies. Fluorescent antibody staining of the corresponding midguts from ticks that had fed on the OspC-immunized mice showed that borreliae were present but did not produce OspC. In contrast, borreliae in midguts from ticks that had fed on normal mice demonstrated substantial ospC expression. This study provides evidence that, during tick feeding on an OspC-immunized host, transmission of borreliae from the tick is prevented; it also suggests that OspC functions in a tick-to-host transmission mechanism.

Female tick salivary glands undergo rapid degeneration several days post engorgement. This degeneration may be caused by the increased concentration of ecdysone in the hemolymph during the fast feeding period and both autophagy and apoptosis occur. In this work, we first proved autophagy-related gene (ATG) and caspase gene expression peaks during degeneration of the tick salivary glands. We explored the regulatory role of Rhipicephalus haemaphysaloides autophagy-related 5 (RhATG5) in the degeneration of tick salivary glands. During the fast feeding phase, RhATG5 was cleaved and both calcium concentration and the transcription of Rhcalpains increased in the salivary glands. Recombinant RhATG5 was cleaved by μ-calpain only in the presence of calcium; the mutant RhATG5191-199Δ was not cleaved. Treatment with 20-hydroxyecdysone (20E) led to programmed cell death in the salivary glands of unfed ticks in vitro, RhATG8-phosphatidylethanolamine (PE) was upregulated in ticks treated with low concentration of 20E. Conversely, RhATG8-PE decreased and Rhcaspase-7 increased in ticks treated with a high concentration of 20E and transformed autophagy to apoptosis. High concentrations of 20E led to the cleavage of RhATG5. Calcium concentration and expression of Rhcalpains were also upregulated in the tick salivary glands. RNA interference (RNAi) of RhATG5 in vitro inhibited both autophagy and apoptosis of the tick salivary glands. RNAi of RhATG5 in vivo significantly inhibited the normal feeding process. These results demonstrated that high concentrations of 20E led to the cleavage of RhATG5 by increasing the concentration of calcium and stimulated the transition from autophagy to apoptosis.

Tick salivary glands play critical roles in maintaining water balance for survival, as they eliminate excess water and ions during blood feeding on hosts. In the long duration of fasting in the off-host period, ticks secrete hygroscopic saliva into the mouth cavity to uptake atmospheric water vapor. Type I acini of tick salivary glands are speculated to be involved in secretion of hygroscopic saliva based on ultrastructure studies. However, we recently proposed that type I acini play a role in resorption of water/ions from the primary saliva produced by other salivary acini (i.e., types II and III) during the tick blood feeding phase. In this study, we tested the function of type I acini in unfed female Ixodes scapularis. The route of ingested water was tracked after forced feeding of water with fluorescent dye rhodamine123. We found that type-I acini of the salivary glands, but not type II and III, are responsible for water uptake. In addition, the ingestion of water through the midgut was also observed. Injection or feeding of ouabain, a Na/K-ATPase inhibitor, suppressed water absorption in type I acini. When I. scapularis was offered a droplet of water, ticks rarely imbibed water directly (5%), while some approached the water droplet to use the high humidity formed in the vicinity of the droplet (23%). We conclude that during both on- and off-host stages, type I acini in salivary glands of female Ixodes scapularis absorb water and ions.

The salivary glands of ixodid ticks are the primary organs of osmoregulation and the source of pathogen transfer from tick to host. Excess fluid is extracted from the blood meal, moved across the gut wall and into the salivary glands, where it is returned to the host. Previously it has been shown in vivo and in vitro that the type III acinus alternately swells as it fills with fluid and then contracts as the acinus empties, and that cytochalasin D prevents contraction of the type III acini.In this investigation, the rhodamine-phalloidin technique was used to localize actin filaments in the salivary glands of fed mated female ticks. Two microfilament inhibitors were used as controls. Samples were also taken for electron microscopy and dot blot analysis.

Aujourd'hui, l'émergence ou la réémergence de maladies transmises par les tiques (TBDs) devient un problème majeur. En raison des problèmes générés par l'utilisation des acaricides (pollution, résistance), il est donc urgent d'identifier de nouvelles approches pour contrôler les populations de tiques. Parmi ces stratégies, la vaccination visant des molécules conservées chez les tiques et impliquées dans leur capacité vectorielle, sont devenues particulièrement attractives. En conséquence, l'identification de cibles antigéniques appropriées est un défi majeur pour la mise en œuvre de ces stratégies de contrôle des tiques et des TBDs. Dans le présent travail, l'objectif principal est d'élucider les interactions moléculaires entre I. ricinus et B. henselae, afin d'identifier des molécules qui pourraient représenter des cibles vaccinales contre les tiques et les agents pathogènes qu'elles transmettent. Dans ce but, nous avons identifié, par séquençage à haut débit, des transcrits d'Ixodes ricinus différentiellement exprimés au niveau des glandes salivaires de la tique en réponse à une infection par B. henselae. Dans un second temps, l'implication d'un de ces transcrits surexprimés lors de l'infection dans la transmission de B. henselae, a été évaluée. Enfin, et en premier lieu, nous avons validé l'utilisation de la technique de gorgement artificiel sur membrane pour infecter I. ricinus par B. henselae et évalué l'impact de différents paramètres sur le gorgement des tiques. Les résultats ont montré que la technique de gorgement sur membrane est bien adaptée à l'infection d'I. ricinus par B. henselae en laboratoire, et que la proportion et le poids des tiques gorgées sont diminués lors de l'infection du sang par la bactérie Le séquençage en 454 des glandes salivaires de tiques a généré une banque de référence contenant 24, 539 transcrits, et la comparaison des glandes salivaires d'I. ricinus infectés et non-infectés par B. henselae a montré que 839 et 517 transcrits étaient respectivement significativement surexprimés et sous-exprimés en réponse à l'infection par des bactéries. Parmi les gènes de fonction connue, 161 transcrits correspondent à 9 familles déjà identifiées, quand les autres correspondent à des gènes de fonction inconnue. L'extinction par RNA interférence du gène le plus surexprimé, IrSPI qui appartient à la famille des inhibiteurs de sérine protéase BPTI/Kunitz, a entraîné une réduction de la taille du repas sanguin prit par les tiques (et donc sa descendance) ainsi que du niveau d'infection au niveau des glandes salivaires. En conclusion, cette étude a démontré que la technique de gorgement artificiel des tiques sur membrane est un outil puissant pour étudier les interactions entre les tiques et les agents pathogènes qu'elles transmettent comme B. henselae. Ce travail apporte aussi une nette avancée en termes de données génétiques sur I. ricinus (dont le génome n'est pas séquencé) et sur les interactions moléculaires entre une bactérie et son vecteur. Enfin, ce travail a permis la mise en évidence d'une molécule représentant un candidat vaccinal très prometteur à la fois pour diminuer la population de tiques et lutter contre les agents pathogènes qu'elles transmettent. Dans le futur, et en fonction de la confirmation du rôle des gènes identifiés ici dans la transmission bactérienne, de nombreux candidats vaccins pourront ainsi être évalués, ouvrant alors de nouvelles perspectives dans la lutte contre les tiques et les maladies dues aux agents qu'elles transmettent
Ticks are obligate blood-feeding ectoparasites of many hosts including mammals, birds and reptiles. After mosquitoes, they are the most important vectors worldwide, and are able to transmit the highest variety of pathogens including virus, bacteria and parasites. Ixodes ricinus (Acari: Ixodidae), the most common tick species in Europe, is a three-life stage hard tick. It is frequently associated with bites in humans, and transmits several pathogens, including Tick-Borne Encephalitis, Babesia spp., Borrellia spp., Anaplasma spp., and to a lesser extent Bartonella spp. Bartonella spp. are facultative intracellular bacteria associated with a number of emerging diseases in humans and animals. It has been demonstrated that I. ricinus is a competent vector for B. henselae that causes cat scratch disease as well as being increasingly associated with a number of other syndromes, particularly ocular infections and endocarditis. Recently, emergence or re-emergence of tick-borne diseases (TBDs) is increasingly becoming a problem. Indeed, and because of the limited success and disadvantages of controlling TBDs via acaricides, new approaches are urgently needed. Therefore, vaccine strategies that target conserved components of ticks that play roles in vector infestation and vector capacity have become particularly attractive. Accordingly, the identification of suitable antigenic targets is a major challenge for the implementation of tick and TBDs control strategies. In the present work, the main objective is to elucidate molecular interactions between I. ricinus and B. henselae in order to identify some targets that may be used as vaccines against ticks and tick-borne pathogens. Two principal points are focused on: primarily, to identify I. ricinus salivary gland differentially expressed transcripts in response to B. henselae infection with next generation sequencing techniques (454 pyrosequencing and HiSeq 2000); secondly, to validate the implication of one of these transcripts in the transmission of B. henselae. For that purpose, and at first, we validated artificial membrane feeding technique for ticks infection by B. henselae and evaluated the impact of several parameters on tick feeding. Results showed that membrane feeding technique is a suitable method to infect I. ricinus with B. henselae and that the proportion and weight of engorged ticks are decreased by B. henselae infection of the blood meal. Transcriptional analysis of the tick salivary glands generated a reference databank containing 24,539 transcripts, and the comparison of B. henselae-infected and non-infected I. ricinus female salivary glands showed that 839 and 517 transcripts were significantly up- and down-regulated in response to bacteria infection, respectively. Among them, 161 transcripts corresponded to 9 groups of ticks salivary gland gene families already described, when the other ones corresponded to genes of unknown function. Silencing the most up-regulated gene IrSPI, which belongs to BPTI/Kunitz family of serine protease inhibitor, resulted in reduction of tick feeding and bacteria load in tick salivary gland. In conclusion, this work demonstrated that artificial-membrane feeding technique is a powerful tool for investigating the interactions between tick and tick-borne pathogens as B. henselae. It also increases the available genomic information for I. ricinus and the knowledge to improve our understanding of the molecular interaction between tick and tick-borne pathogens. At last, it provides a potential vaccine candidate to control tick-borne diseases. In the future, and depending of differentially expressed genes' role confirmation, more and more vaccine candidate will be provided by this work, and the strategy of controlling tick and tick-borne disease will come to a new stage

Doctor of Philosophy
Entomology
Yoonseong Park
Tick salivary secretion is crucial for survival and for successful feeding. Tick saliva includes excretory water/ions and bioactive components for compromising the hosts' immune responses, and provides a direct route for pathogen transmission. Control of the tick salivation involves autocrine/paracrine dopamine, the most potent stimulator of tick salivation. Our research group reported the presence of two dopamine receptors in the salivary glands of the blacklegged tick (Ixodes scapularis): dopamine receptor (D1) and invertebrate specific D1-like dopamine receptor (InvD1L). Dopamine-induced salivary secretion was orchestrated by two distinct physiological roles via activation of the two dopamine receptors (Chapter 2). Low concentration of dopamine activated D1 receptor on epithelial cells of salivary gland acini leading inward fluid transport. High concentration of dopamine activated InvD1L receptors on axonal projections innervating myoepithelial cells modulating pumping/gating actions for emptying luminal saliva into the main duct. Thus, ticks coordinated salivary secretion with duo dopamine receptors. Dopamine-mediated saliva production involves an important downstream component, Na/K-ATPase (Chapter 3). Na/K-ATPase was found in the epithelial cells of all types of acini. However, Na/K-ATPase had two different functions in salivary secretion in different acini: 1) dopamine-mediated production of primary saliva in distally located salivary gland acini type-2/- 3, and 2) dopamine-independent resorption in proximally located salivary gland acini type-1. Type-1 acini were also found to function in direct water absorption of off-host ticks, which could be a potential route for delivery of acaricides. Chapter 4 investigated the comparative transcriptomics of the lone star tick underlying the processes of pathogen acquisition. Differential expression analyses in pathogen-exposed ticks revealed a number of transcripts that are important in the tick-pathogen interaction. These included genes for tick immunity against pathogen and for modulation of tick physiology facilitating a pathogen’s invasion and proliferation. My study expanded the understanding of physiological mechanisms controlling tick salivation. In addition, transcriptomics of ticks in interaction with pathogen identified several genes that are relevant in vector/pathogen interactions. The knowledge obtained in my study will facilitate to the development of novel methods for the disruption of tick feeding and pathogen transmission.

The purpose of this study is to characterise molecules that are secreted in tick salivary glands. A cDNA library of salivary glands was constructed in a lambda phage. Immunoscreening was carried out with a) a hyperimmune antiserum obtained from rabbit immunised with salivary gland extract of unfed A variegatum, b) an antiserum from a rabbit exposed to feeding A variegatum; c) an antiserum from calf repeatedly exposed to feeding A variegatum. All antisera recognised proteins of salivary glands extracts on Western blot analysis, but none detected recombinant salivary gland proteins from the cDNA library. A random selection of cDNA clones were sequenced and compared with those from the Amblyomma variegatum Gene Index (AvGI) and the GenBank of non-redundant sequences database (NCBI). Several clones displayed sequence identity with already known sequences from other tick species. One clone displayed 31% identity with a consensus sequence of the AvGI EST, which shared 28% identity with a 36kDa immunosuppressant molecule isolated from Dermacentor andersoni. Screening of the cDNA library, using the Signal Sequence Trap technique, identified a clone with 99% identity with a glycine rich protein from the AvGI database. This gene was designated FLAv41E and had a molecular weight of 30.9kDa. Reverse transcription PCR (polymerase chain reaction) amplification of FLAv41E gene revealed that it is expressed in the salivary glands of A variegatum female from day 3 to 10 of feeding. Its secondary structure revealed domain similarities with a 29kDa cement protein characterised in Haemaphysalis longicornis ticks. The recombinant FLAv41E protein was expressed in insect cells using a baculovirus system. FLAv41E appears to be a cement protein.

The dopamine receptor is critical in fluid secretion from tick salivary glands and, as such, was perceived as a potential drug target.  Homology cloning was employed to isolate a dopamine receptor from R. sanguineus salivary glands.  A dopamine receptor was successfully cloned from locust salivary glands although not from R. sanguineus salivary glands. A cDNA library was constructed from feeding female R. sanguineus salivary glands and random mass sequencing of 1440 clones was performed. Sequences were identified from database searches and classified according to gene ontology.  Several enzymes were identified as potential drug targets and a glutathione S-transferase (GST) was chosen for further study.  The dopamine receptor was not identified, as were no other G protein-coupled receptors. A mu class GST from R. sanguineus salivary glands was expressed as a recombinant protein in E. coli and was found to have differences when compared to other tick GSTs, namely low catalytic activity with a model substrate.  Preliminary studies suggested that the GST is not involved in prostaglandin synthesis and the GST bound haematin with high affinity.  Of several tissues investigated within female ticks, the GST mRNA was only expressed in the salivary glands and only in glands from feeding female ticks indicating a potential role in blood feeding. Two approaches were employed to identify drug targets in R. sanguineus salivary glands.  It is concluded that whilst random mass sequencing of a cDNA library yields many potential targets, because of the nature of the salivary gland tissue with high repetition of mitochondrial and secretory protein RNA, homology cloning would be the recommended approach for scientific, low abundance targets.

Bacteria of the genus Rickettsia belong to the family Rickettsiaceae in the order Rickettsiales and have for long been described simply as short, Gram-negative, strict intracellular rods that retain basic fuchsin when stained by the method of Gimenez (Raoult and Roux 1997). These bacteria are associated with ticks, lice, fleas or mites. To date the Rickettsia genus contains 24 recognized species classified into three groups based on their antigenic, morphological, and ecologic patterns: 1) the typhus group, 2) the spotted fever group and 3) Rickettsia bellii (Fournier and Raoult 2007). Most spotted fever group (SFG) rickettsiae are closely associated with ticks belonging to the family Ixodidae (also called “hard” ticks) (Parola et al. 2005). Ticks can act as vectors, reservoirs, and/or amplifiers of SFG rickettsiae and require optimal environmental conditions which determine the geographic distribution of the vectors and consequently the risk areas for rickettsioses. Many Rickettsia species are strictly associated with one genus of ticks and the transmittion to people is made through the tick bite, which generally implies that the Rickettsia can localize to their salivary glands. Therefore, since larvae, nymphs, and adults may all be infective for susceptible vertebrate hosts, the ticks must be regarded as the main reservoir host of rickettsiae. Humans are not considered as good reservoirs for Rickettsiae, as they are seldom infested with ticks for long periods and rickettsiaemia has normally short duration, especially with antibiotic intervention.