Proteins in the saliva of the Ixodida (ticks): Pharmacological features and biological significance

Abstract

The saliva of ticks (Suborder Ixodida) is critical to their survival as parasites. A tick bite should result in strong responses from the host defence systems (haemostatic, immune and inflammatory) but tick saliva appears to have evolved to counter these responses. We review current knowledge of tick saliva components, with emphasis on those molecules confirmed to be present in the secreted saliva but including some that have only been confirmed to be present in salivary glands. About 50 tick saliva proteins that are well described in the literature are discussed. These saliva components include enzymes, enzyme inhibitors, amine-binding proteins and cytokine homologues that act as anti-haemostatic, anti-inflammatory or immuno-modulatory agents. Sequence comparisons are illustrated. The importance of tick saliva and the significance of the findings to date are also discussed.

Introduction

Ticks live on all continents of the world. Many of the 899 or so species of ticks are associated with disease in humans, livestock and wild life (Barker and Murrell, 2004), but research into the role of ticks in disease, primarily or as vectors, has only become a priority in some countries relatively recently. In part, this is due to the fact that compared to the bites and stings of other arachnids, the ‘bite’ of a tick is rarely dramatic and sometimes unnoticed resulting in uncertainty regarding the possible pathogenicity of ticks. The ‘bite’ of a tick involves the insertion of cutting, tube-like mouthparts through the hosts skin and anchoring of the mouthparts in place with cement-like material and/or backwards pointing barbs allowing the tick to feed on the hosts blood for hours, days or even weeks. Surprisingly this process is usually not noticed by the host. The past twenty years has seen a surge of interest in ticks in medical, veterinary, entomological and biochemical fields such that there is now a large and growing body of literature on ticks that we will now review.

We begin by discussing current ideas on the evolution of ticks, followed by a discussion of the functional importance of the saliva and salivary glands to these animals. This is the logical place to begin as, until further data is available on tick genomes¹ and phylogenetics, the function or activity of tick saliva is the greatest clue to predicting the composition of the saliva of any given species. This is followed by a detailed review of the components of tick saliva that have been isolated to date. We focus on the proteinaceous components, which are often referred to as tick ‘toxins’. The significance of these components to the tick, to the host and to scientific research is discussed. To avoid ambiguous nomenclature, preference is given to those proteins for which a protein sequence has been determined and can be retrieved with NCBI Entrez² (thereby providing a unique accession number). Preference is also given to those proteins whose activity and presence in saliva has been confirmed. Components other than proteins are discussed only briefly.

The Arthropoda is a diverse phylum of animals, representing over 80% of the known animal species (Savory, 1977). Almost all of the arthropods lacking mandibles (subphylum Chelicerata) are classified as Arachnids, the spiders, mites, ticks and their kin. The spiders are the best-known arachnids but one of the largest subclasses of the Arachnida is the Acari, the ticks and mites.

The evolution of ticks is still unclear because there are few fossils. The earliest estimates have the origins of ticks in the Devonian period (around 400 million years ago), parasitising amphibians. Fossil evidence from this period (Oliver, 1989) and phylogenetic and zoogeographical evidence (Murrell et al., 2001, Barker and Murrell, 2002) seem to support this hypothesis. However, Hoogstraal (1985) suggests a late Palaeozoic or early Mesozoic era evolution (around 250 million years ago) parasitising reptiles and Klompen et al. (2000) argue for an even more recent evolution in the Cretaceous period (around 100 million years ago). Our working hypothesis for the evolution of ticks, based on the cumulated evidence of the authors listed above plus other reviews (Ribeiro, 1987, Sonenshine, 1993a, Klompen et al., 2000, Barker and Murrell, 2002, Dusbabek, 2002, Mans and Neitz, 2004) is as follows.

The wide range of studies of extant ticks and mites, including phylogenetics and examination of salivary gland secretions, suggest to us that ticks evolved from a saprophytic to a haematophagous lifestyle by adaptation of behaviour, anatomy and salivary gland activity. Intermediate ‘tick-like’ parasitic mites may have lived ca. 390 million years ago (Devonian period), were probably not fully haematophagous and may have parasitised tetrapods on Gondwanaland (Dobson and Barker, 1999). Salivary secretions that facilitated feeding on oozing wounds may have been the same enzymes and enzyme inhibitors that had previously enabled the ancestors of these acarines to feed on dead invertebrates and vertebrates. The ancestors of ticks may have developed host and wound-seeking abilities and additional saliva components that enabled them to specialise in serum and blood ingestion and digestion. In turn, it seems likely to us that the need to counter haematophagous ‘ticks’, and other arthropods, influenced the evolution of the vertebrate haemostatic systems. Obligate tick haematophagy, however, probably did not evolve until around 140 MYA (Cretaceous period) since the phylogenetic evidence suggests it evolved independently into what eventually became the hard (Ixodidae ticks: ticks with a sclerotised scutum on the dorsal cuticle; ca. 713 species) and soft (Argasidae ticks; ca. 185 species) ticks. Fossil evidence of hard and soft ticks from before that time has not been found³.

We speculate that the evolution of the two main tick families⁴, the soft ticks (Argasidae) and hard ticks (Ixodidae), may have been a result of the need for mechanisms to cope with host inflammatory and immune defences. Interestingly, this might have coincided with the radiation of early mammals and birds that would have resulted in a greater variety of hosts and host defences to drive the divergent evolution of ticks. It seems that the soft ticks fed rapidly to help overcome the host defences whereas the hard ticks took longer to feed but developed saliva that could modulate host (particularly mammalian) immune and inflammatory responses (Ribeiro, 1987, Sonenshine, 1993a).

Tick phylogeny and evolution, saliva composition and host-specificity are probably inter-related in a complex parasite-host interface. Accurate phylogenies (evolutionary histories) assist the study of the evolution of phenotypes like immune systems, the components of the saliva and host-specificity. When phenotypes like these are mapped onto a phylogeny we may discover, for example, how many times an enzyme activity evolved. Fig. 1 shows the current working hypotheses for the phylogeny of the ticks.

The action of the tick chelicerae and the insertion of the hypostome (see Fig. 2) through the host's skin causes epidermal and dermal damage including the rupture of local blood vessels. This mechanical damage would be expected to lead to host responses that are activated by damage from other similar sized foreign bodies, including other arthropod bites⁵. These would include haemostatic (pathways in red in Fig. 3) and inflammatory pathways (blue pathways in Fig. 3). Secretion of saliva by the tick could also introduce antigens that would activate the host immune responses (pathways in green in Fig. 4). An effective haemostatic response from the host would be expected to result in poor tick feeding and reproduction, due to insufficient protein ingestion by the female tick for egg production. Effective inflammatory and immune responses would be expected to result in tick rejection (death of the tick while feeding) and/or a grooming response by the host that would result in the removal of the offending tick. These outcomes are not seen in all cases of tick feeding, as will be discussed below.

The schematics (Fig. 3, Fig. 4) have been included in this review as a hypothetical centre-point around which to discuss tick saliva function. As will be shown in this review, tick saliva contains components that counter these host haemostatic, inflammatory and immune responses, enabling ticks to feed for days to weeks at one site (Ribeiro and Francischetti, 2003), compared with the minutes to hours for other haematophagous parasites such as lice and leeches. These components have been found to exist in numerous forms including enzymes, enzyme inhibitors, immunoglobulin-binding proteins, amine-binding lipocalins and integrin inhibitors as will be discussed in the following sections.

There are three possible outcomes for the tick and the host after a tick begins to feed. In the case of natural hosts⁶, the anti-inflammatory and immuno-modulatory components of the tick saliva may prevent the rejection of the tick, thus the tick is able to continue to imbibe blood. For example, when Rhipicephalus sanguineus feed on dogs (their natural host) the host reaction is restricted to an immediate cutaneous immune response with the mononuclear cell and delayed hypersensitivity reactions apparently reduced (Ferreira, Szabó et al., 2003). Today, after apparently millions of years of co-evolution between this tick and canids, the saliva components do not have serious or long-term effects on the natural host/s except possibly the facilitation of transmission of pathogens.

Ticks are thought to have evolved saliva that inhibits the cutaneous immune response of their most common host/s (Ribeiro, 1987). As the cutaneous immune response differs among species⁷, there is the possibility that when a tick feeds on unnatural hosts, that host will begin to generate a cutaneous immune response that the tick cannot counter and that this response may generate sufficient antibody, histamine, serotonin, complement and/or lymphokines that the tick will be rejected or reproduce poorly. In some cases the first exposure of such a host to a tick may not lead to rejection of the tick. Rather this exposure may sensitise the host so that subsequent exposure leads to tick rejection. That is the host acquires tick resistance (as first studied by Trager (1939)). Studies on Rhipicephalus sanguineus feeding on guinea-pigs (not a natural host) show that guinea pigs can acquire resistance to the ticks after only a few bites, with a delayed, cell-mediated hypersensitivity response involving basophils and T_H1 cells (in contrast to the response in dogs) which results in the ticks having reduced weight at the time of detachment, reduced egg laying capacity or even death in situ (Ferreira et al., 2003). This immunity can last for several months (Askenase et al., 1982).

In an apparent adaptation to avoid rejection, some species of ticks have become host-specific. These ticks refuse to attach to a host of the ‘wrong’ species, relying on odour and other chemical information from the host sensed by receptor organs on the mouthparts and first pair of legs of the tick, while other ticks have remained more or less opportunistic with catholic feeding habits (Sonenshine, 1993c). A third situation can therefore be hypothesised. This is where an opportunistic tick begins to feed on a host whose cutaneous immune response is being suppressed by the tick but the host did not co-evolve with that species of tick. In these cases, components of the tick saliva may also have severe effects on the host, a situation unlikely to arise through co-evolution. A possible example of this is the effect of Ixodes holocyclus (Australian paralysis tick) on the domestic dog. Whereas native marsupials, especially bandicoots, can carry large burdens of this tick with no apparent ill-effect, a single female tick can induce a rapidly ascending, flaccid, paralysis syndrome in a host dog (Dodd, 1921, Clunies-Ross, 1935), which can be fatal even with treatment (Atwell et al., 2001). On repeated challenge by I. holocyclus the majority of dogs develop some degree of paralysis, only relatively mild cutaneous reactions (sometimes referred to as ‘craters’) and the ticks can feed to repletion if the dog does not die first (Clunies-Ross, 1935). A small percentage of individual dogs have been observed to not develop paralysis, develop a greater reaction at the bite site and ‘kill’ the ticks in situ. The factors underlying this difference between individuals is not understood and is the focus of current research (Atwell, R.B.—personal communication). In general, however, it can be said that dogs do not acquire immunity to this tick without constant, repeated exposure for several weeks (Stone et al., 1983a), suggesting suppression of the immune and inflammatory responses of dogs by the saliva of this tick species

The types and extent of immune and inflammatory response to a feeding tick seem to determine the degree of trauma observed in the skin of the host. For example Dermacentor andersoni feeding on guinea-pigs that have acquired immunity to this tick can cause severely inflammed, weeping wounds while the same species of tick feeding on a natural host will result in minimal skin reaction even with repeated bites (Trager, 1939) (see also the review by Sonenshine (1993c)). Bos indicus cattle are said to suffer less damage to the hide as a result of tick bites, especially Rhipicephalus (Boophilus) microplus ticks, compared with Bos taurus cattle. Although not fully understood, it is possible that this could be partially as a result of inflammatory tolerance by these cattle relative to Bos taurus cattle⁸, that is the tropical breeds of cattle do not react as greatly to the tick bite as do temperate breeds and the hide is therefore left with less damage. In general, however, it could be said that a tick bite leaves a more significant wound than other haematophagous parasites, which could be due to the length of feeding and the anchoring mechanisms (including barbed hypostome and cement proteins) employed by ticks.

Despite the varied life-cycles of ticks (see Fig. 5) and the likely variety of functions to be carried out by the salivary glands as a result, the general morphological appearance of the salivary glands is conserved amongst ticks. Detailed reviews of the anatomy and physiology of the salivary glands of ticks can be found elsewhere (Binnington and Stone, 1981, Kemp et al., 1982, Sonenshine, 1993a, Sauer et al., 1995); a brief summary only will be given here.

The paired salivary glands have lateral positions in the tick. Each gland has a main salivary duct that passes cranially where the two ducts fuse into a structure termed the salivarium. The salivarium, a passive saliva storage area, in turn fuses with the pharynx of the tick to form the oral cavity, through which both saliva (outwards) and the blood meal (inwards) flow. The intake of blood from the feeding lesion and the outflow of saliva into the feeding lesion occur via the mouthparts (hypostomal gutter ventrally and chelicerae dorsally). The feeding apparatus of a hypothetical hard tick is diagrammatically illustrated in Fig. 6 (Sonenshine, 1993b).

The salivary glands consist of an anterior region of acini (roughly spherical arrangements of secretory cells around a central secretory pore) attached directly to the main duct; more caudally the acini are arranged in lobules connected by intralobular and interlobular ducts to the main salivary duct. Although the number and variety of salivary gland acini varies among species and even life-stages of a species, the distribution of acinar types is conserved amongst the ticks studied so far. The acini attached directly to the main duct at the anterior end of the gland are generally agranular and involved primarily with osmo-regulation. The more caudal acini are involved in secretion of the bioactive components of the saliva (Binnington and Stone, 1981, Kemp et al., 1982).

The salivary glands are under neuro-hormonal control that includes at least two different catecholamine based pathways (Kaufman and Harris, 1983, Sonenshine, 1993b, Sauer et al., 1995), and nitric oxide controlled duct dilations (Lamoreaux et al., 2000). This enables ticks to increase salivary gland activity and saliva secretion in response to stimuli such as feeding. At their peak the glands of a feeding tick can be twenty-five times the size of the unfed state, mainly through an increase in the number and size of secretory granules within certain cells as seen on histological examination (Binnington and Stone, 1981). Once the tick is engorged the glands degenerate through a process of cell apoptosis (Bowman and Sauer, 2004).

The salivary glands of ticks produce what has been called a ‘cocktail’ of molecules with different functions (Ribeiro and Francischetti, 2003, Valenzuela, 2004). Proteins discussed below are listed with their common names followed by the NCBI Entrez accession number in brackets where available. Although preference is given to components that have been confirmed to be present in saliva, some have only been confirmed to the level of salivary gland or salivary gland extract (SGE).