Insect Biochemistry and Molecular Biology 34 (2004) 1–17 www.elsevier.com/locate/ibmb Minireview Adaptation of ticks to a blood-feeding environment: evolution from a functional perspective Ben J. Mans , Albert W.H. Neitz Department of Biochemistry, University of Pretoria, Pretoria 0002, South Africa Received 5 June 2003; accepted 8 September 2003 Abstract Ticks had to adapt to an existing and complex vertebrate hemostatic system from being free-living scavengers. A large array of anti-hemostatic mechanisms evolved during this process and includes blood coagulation as well as platelet aggregation inhibitors. Several questions regarding tick evolution exist. What was the nature of the ancestral tick? When did ticks evolve blood-feeding capabilities? How did these capabilities evolve? Did host specificity influence the adaptation of ticks to a blood-feeding environment? What are the implications of tick evolution for future research into tick biology and vaccine development? We investigate these questions in the light of recent research into protein superfamilies from tick saliva. Our conclusions are that the main tick families adapted independently to a blood-feeding environment. This is supported by major differences observed in all processes involved with blood-feeding for hard and soft ticks. Gene duplication events played a major role in the evolution of novel protein functions involved in tick–host interactions. This occurred during the late Cretaceous and was stimulated by the radiation of birds and placental mammals, which provided numerous new niches for ticks to adapt to a new lifestyle. Independent adaptation of the main tick families to a blood-feeding environment has several implications for future tick research in terms of tick genome projects and vaccine development. 2003 Published by Elsevier Ltd. Keywords: Tick; Evolution; Feeding; Hemostasis 1. Evolution of hematophagy Hemostasis is an efficient defense mechanism that prevents blood loss through an open wound and is found in all organisms that have a hemostatic system. The ver- tebrate hemostatic system originated approximately 400 million years ago (MYA) and is a formidable barrier for hematophagous parasites (Doolittle and Feng, 1987). In contrast, hematophagy (blood-feeding behavior) evolved independently at least six times in the approximately 15000 species of 400 genera of the hematophagous arthropods during the Cretaceous and Jurassic eras (145– 65 MYA) (Balashov, 1984; Ribeiro, 1995). Elucidation of the evolutionary mechanisms of blood-feeding arthro- pods could further our understanding of the evolution Corresponding author. Tel.: +27-12-420-2011; fax: +27-12-362- 5302. E-mail address: [email protected] (B.J. Mans). 0965-1748/$ - see front matter 2003 Published by Elsevier Ltd. doi:10.1016/j.ibmb.2003.09.002 of complex systems as is exhibited at the blood-feeding interface. Ticks have been shown to be excellent models for the study of parasite–host relationships. Tick–host interaction, the biology of tick salivary glands and sali- vary gland components secreted by ticks during feeding have been described in several excellent reviews (Binnington and Kemp, 1980; Ribeiro, 1987; Law et al., 1992; Ribeiro, 1995; Sauer et al., 1995; Bowman et al., 1997; Sauer et al., 2000). These reviews have indicated that ticks are organisms that have adapted a complex system of efficient counter-measures by which the host’s defense mechanisms are regulated. No review, however, has integrated this knowledge with what is known about general tick evolution. The current review considers the adaptation of ticks to a blood-feeding environment from both an evolutionary as well as functional perspective. Understanding tick evolution could allow for a more integrated view of tick biology and thereby assist in the search for vaccine targets.

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Page 1: Adaptation of ticks to a blood- feeding enviroment: evolution from a functional perspective

Insect Biochemistry and Molecular Biology 34 (2004) 1–17www.elsevier.com/locate/ibmb


Adaptation of ticks to a blood-feeding environment: evolution froma functional perspective

Ben J. Mans∗, Albert W.H. NeitzDepartment of Biochemistry, University of Pretoria, Pretoria 0002, South Africa

Received 5 June 2003; accepted 8 September 2003


Ticks had to adapt to an existing and complex vertebrate hemostatic system from being free-living scavengers. A large array ofanti-hemostatic mechanisms evolved during this process and includes blood coagulation as well as platelet aggregation inhibitors.Several questions regarding tick evolution exist. What was the nature of the ancestral tick? When did ticks evolve blood-feedingcapabilities? How did these capabilities evolve? Did host specificity influence the adaptation of ticks to a blood-feeding environment?What are the implications of tick evolution for future research into tick biology and vaccine development? We investigate thesequestions in the light of recent research into protein superfamilies from tick saliva. Our conclusions are that the main tick familiesadapted independently to a blood-feeding environment. This is supported by major differences observed in all processes involvedwith blood-feeding for hard and soft ticks. Gene duplication events played a major role in the evolution of novel protein functionsinvolved in tick–host interactions. This occurred during the late Cretaceous and was stimulated by the radiation of birds and placentalmammals, which provided numerous new niches for ticks to adapt to a new lifestyle. Independent adaptation of the main tickfamilies to a blood-feeding environment has several implications for future tick research in terms of tick genome projects andvaccine development. 2003 Published by Elsevier Ltd.

Keywords: Tick; Evolution; Feeding; Hemostasis

1. Evolution of hematophagy

Hemostasis is an efficient defense mechanism thatprevents blood loss through an open wound and is foundin all organisms that have a hemostatic system. The ver-tebrate hemostatic system originated approximately 400million years ago (MYA) and is a formidable barrier forhematophagous parasites (Doolittle and Feng, 1987). Incontrast, hematophagy (blood-feeding behavior) evolvedindependently at least six times in the approximately15000 species of 400 genera of the hematophagousarthropods during the Cretaceous and Jurassic eras (145–65 MYA) (Balashov, 1984; Ribeiro, 1995). Elucidationof the evolutionary mechanisms of blood-feeding arthro-pods could further our understanding of the evolution

∗ Corresponding author. Tel.:+27-12-420-2011; fax:+27-12-362-5302.

E-mail address: [email protected] (B.J. Mans).

0965-1748/$ - see front matter 2003 Published by Elsevier Ltd.doi:10.1016/j.ibmb.2003.09.002

of complex systems as is exhibited at the blood-feedinginterface. Ticks have been shown to be excellent modelsfor the study of parasite–host relationships. Tick–hostinteraction, the biology of tick salivary glands and sali-vary gland components secreted by ticks during feedinghave been described in several excellent reviews(Binnington and Kemp, 1980; Ribeiro, 1987; Law et al.,1992; Ribeiro, 1995; Sauer et al., 1995; Bowman et al.,1997; Sauer et al., 2000). These reviews have indicatedthat ticks are organisms that have adapted a complexsystem of efficient counter-measures by which the host’sdefense mechanisms are regulated. No review, however,has integrated this knowledge with what is known aboutgeneral tick evolution. The current review considers theadaptation of ticks to a blood-feeding environment fromboth an evolutionary as well as functional perspective.Understanding tick evolution could allow for a moreintegrated view of tick biology and thereby assist in thesearch for vaccine targets.

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2. The Ixodida

Ticks are obligate hematophagous organisms and themost important ectoparasites of domestic animals. TheIxodida comprises three families, the Ixodidae or hardticks, Argasidae or soft ticks and Nuttalliellidae that ismonotypic (Hoogstraal, 1956). The Ixodida are part ofthe phylum Arthropoda and group with the spiders, scor-pions, whip-scorpions, sun spiders, harvestmen, hoodedtick spiders and false scorpions in the class Arachnida.Ticks are a small group within the much larger group ofmites that group in the subclass Acari, and form with theHolothyrida and Mesostigmata, the order parasitiformes(Evans, 1992).

3. The origin of ticks

Ticks were considered to be of the earlier lineages ofterrestrial arachnids, with proposed origins in the lateSilurian (443–417 MYA) (Lindquist, 1984), Devonian(417–362 MYA) (Oliver, 1989), late Permian (290–248MYA) (Hoogstraal and Aeschlimann, 1982) and Triassic(248–206 MYA) (Hoogstraal, 1985; Balashov, 1989;Balashov, 1994). It has also been proposed that ticksoriginated more recently in the Cretaceous (144–65MYA) (Fillipova, 1977) or late Cretaceous (120 MYA)(Klompen et al., 1996). The latter is based on compari-son of the distribution of ixodids worldwide, where someof the presumably basal lineages are exclusively Aus-tralian and suggests a major role of Australia in the evol-ution of ixodids. The late Cretaceous was the last timethat Australia was part of Gondwanaland and indicatesthat this period played an important role in the origin ofthe Australian lineages, and by extension the entire tickfamily (Klompen et al., 2000). This period also saw thedivergence of early birds and mammals and was closelyfollowed by dinosaur extinction (65 MYA). This pro-vokes tantalizing suggestions that ticks evolved at thedawn of the emergence of mammals. An alternative cur-rent viewpoint is that ticks have originated on the Aus-tralian mainland in the Devonian with the first hostsbeing tetrapods (amphibians) (Dobson and Barker,1999). The resolution of this debate will be veryimportant, as the adaptation to a blood-feeding environ-ment is intimately linked with the original tick host orhosts.

4. A Devonian or Cretaceous origin for ticks?

Origins in the Devonian would have been stimulatedby the appearance of the first terrestrial vertebrates inthe form of amphibians. Support for this hypothesis isprovided by the fact that one tick species (Amblyommarotandatum) feed on the neotropical giant toad, Bufo

marinus (Oliver, 1989). Tetrapods and amphibians dohave the rudimentary components of the hemostatic sys-tem that include thrombin and fXa, as well as platelets(Lewis, 1996). Such an early appearance of hemato-phagy with a limited set of hosts would have suggestedthat ticks have adapted to a blood-feeding environmentbefore divergence. In contrast, it was recently suggestedthat ticks evolved anti-hemostatic mechanisms indepen-dently and that this was precipitated due to a radiationof both mammals and birds during the late Cretaceous(Mans et al., 2002a). Recent studies based on the mol-ecular clock also defined the evolution of hematophagyin reduvidd bugs to the early Cretaceous (Gaunt andMiles, 2002). This timeframe also fits in with other esti-mates for the evolution of hematophagy in other organ-isms (Balashov, 1984). The radiation of early mammalsand birds during this period provides a strong leitmotiffor the general evolution of hematophagy. Evolution ofhematophagy in the Devonian would however maketicks the earliest organisms to evolve blood-feedingcapabilities.

5. Tick fossil records

Acariform fossils have been found as far back as theDevonian, while parasitiform fossils have so far onlybeen found up to the early Eocene (35–40 MYA), untilthe discovery of the argasid tick (Carios jerseyi) foundin New Jersey amber, dated 90–94 MYA (Klompen andGrimaldi, 2001). This implies that if ticks originated 120MYA, they had already speciated by ~92 MYA into themain tick families, as well as into argasid genus level.Ixodid fossils were found in Baltic amber (Ixodes succi-neus, 30–40 MYA), Oligocene deposits (Ixodes tertiar-ius, 30 MYA), Dominican amber (Amblyomma testud-inis, Ornithodoros antiquus, 30–40 MYA) and in the earof a woolly rhinoceros (Dermacentor reticulates, 1–3MYA (Scudder, 1885; Weidner, 1964; Lane and Poinar,1986; Poinar, 1995). While, the absence of tick fossilsand parasitiform fossils in general in earlier periods can-not refute an early origin of ticks, it can be consideredcircumstantial evidence for a recent origin.

6. Tick systematics

Analysis of ixodid and argasid relationships, using18S RNA indicated strong support for the monophyly ofArgasidae and Ixodidae (Black et al., 1997). It alsoplaced Argasinae and Ornithodorinae as single mono-phyletic groups, respectively, inside the Argasidae,which is concurrent with original morphologically basedphylogenies (Hoogstraal and Aeschlimann, 1982). Cur-rent research on tick systematics is mainly involved withthe determination of the genus relationships of the ixod-

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ids (Klompen et al., 2000; Murrel et al., 2001). One ofthe most interesting relationships discovered is that thegenus Boophilus emerged from within the genus Rhip-icephalus (Beati and Keirans, 2001). Others are therecent origins of the Dermacentor–Anocentor lineage(50 MYA) in Africa and the Nosomma–Hyalomma lin-eage (19 MYA) in the Orient (Murrel et al., 2001).

7. Ticks as free-living scavengers or predators

Phylogenetic analysis indicates that the Holothyridarather than Mesostigmata are a sister group to ticks(Dobson and Barker, 1999; Klompen et al., 2000). It isof interest that the Holothyrida are distributed only inareas that were part of the Gondwanaland, which corre-sponds with the current ideas on tick origins (Klompenet al., 2000). Holothyrida is a group of free-living mites,which mainly live on body fluids of dead organisms. Ithas been suggested that ticks shared this same traitbefore adaptation to a blood-feeding environment(Walter and Proctor, 1998). Of interest is the phenom-enon of cannibalism observed in some soft ticks. It wassuggested that this behavior is a vestigial remnant of anentomophagous ancestor (Balashov, 1972). Suchbehavior correlates with the idea that the ancestral tickmight have lived on lymph fluids from perhaps not onlyarthropods, but vertebrates as well. The question thatarises is what properties would a lymph feeding ancestorshare with the hematophagous tick.

8. The nature of the ancestral tick

A scavenger ancestor that feeds on dead arthropodsneeds matrix-degrading proteins, such as hyaluronidaseand proteases to make the food source more accessibleas well as aid in digestion. Hyaluronidase activity hasbeen indicated in tick saliva, where it probably functionsin the development of the hematoma during feeding(Neitz and Vermeulen, 1987). A range of protease activi-ties has been described in the SGE of O. savignyi(Mahlaku et al., manuscript in preparation). Serine pro-teases are also part of the clotting mechanisms of arthro-pod hemolymph (Jiang and Kanost, 2000). Clotting ofhemolymph during feeding could be problematic andserine protease inhibitors could have played a role ininhibiting any protease activity present in the body fluidsof the dead organisms it fed on. These serine proteaseinhibitors could have served as precursors for the devel-opment of the anti-hemostatic factors found in tick sal-iva. Depending on the complexities of the food sources,it could be speculated that the salivary glands at the timeof divergence between the Holothyrida and Ixodida hadalready been differentiated organs. As such, the state ofthe primitive salivary gland could have influenced the

protein repertoire of the ancestral tick. Proteins must notonly be expressed in the glands, but also need the signalsnecessary for secretion. This would have meant that acertain subset of proteins were expressed in the salivaryglands and that the number targeted to the secretorypathway would have been limited to those used duringtheir feeding process. As such, ticks could have startedwith a limited set of proteins, from which new diversityhad to be generated. This could account for the predomi-nance of certain protein folds found in tick saliva suchas the BPTI-like proteins (basic pancreatic trypsininhibitor) and the lipocalins. As yet, no comparativestudy has considered differences between holothyrid andixodid salivary glands. Such a study could, however,start to investigate the nature of the ancestral tick sali-vary gland. In order to understand the transformation ofa scavenger to a hematophagous organism, it isimportant to identify those components evolved in thisprocess. This entails an assessment of tick–host interac-tions.

9. Tick–host interactions

Ticks are pool feeders and to acquire a blood meal,ticks need to penetrate the host’s skin and damageenough blood vessels for the release of blood. Slow andfast feeding ticks have different mouthparts that causedifferent degrees of damage to the host (Binnington andKemp, 1980). In ixodid ticks (slow feeders), the lesiondevelops gradually with the formation of a hematoma.It has been proposed that the main damage at the feedinglesion is caused by neutrophils attracted to the feeding-site where they degranulate and cause inflammation(Ribeiro, 1987). Argasids feed rapidly with deep pen-etration of the host’s skin and cause considerable dam-age so that blood loss can still occur long after a tick hasstopped feeding (Binnington and Kemp, 1980). Duringprobing for blood, capillary and small blood vessels arelacerated, host cells are ruptured and hemorrhage occurs.This increases the blood volume at the site of feedingand leads to activation of the host’s defense mechanismsthat include the hemostatic (blood coagulation and plate-let aggregation) and the immune systems (Ribeiro,1987). Platelet aggregation represents the most immedi-ate defense and is sufficient to arrest blood flow fromsmall vessels (Law et al., 1992). It is possible that plate-let aggregation directed the evolution of the salivary pro-teins of all blood sucking arthropods to a greater extentthan did blood coagulation (Ribeiro, 1987). Coagulationmay thus be of lesser significance in the host’s defenseagainst blood sucking arthropods. It may play a moreimportant role during the ingestion of the blood mealand it has been suggested that anti-coagulant inhibitorsin ticks have their main function in the gut where theyserve to keep the blood in a fluid form (Bowman et al.,

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1997). It should however be considered that bloodcoagulation can occur within 3–4 min after vasculardamage. In contrast, soft ticks feed within 30–60 minand hard ticks feed up to a week or longer. This leavesample time for blood coagulation to play a significantrole in tick feeding. It was recently indicated that in softticks, inhibitors of blood coagulation preceded those forplatelet aggregation, suggesting that blood coagulationplayed an important role during the early adaptation ofticks to a blood-feeding environment (Mans et al.,2002a).

10. Platelet aggregation and its role in hemostasis

Several reviews on specific mechanisms of plateletactivation and aggregation are available (Siess, 1989;Gachet and Cazenave, 1991; Jamaluddin, 1991; Clemet-son, 1995; Gachet et al., 1997). Platelets in an unacti-vated form have a smooth surface and a discoid shape.Upon vascular damage, the platelets are activated by avariety of compounds (ADP, collagen, thrombin, throm-boxane A2, epinephrine, platelet activating factor,thrombospondin) that bind to specific membrane recep-tors present on the platelet surface. Activation ismediated by signal transduction of the different receptorsthat activate either the cyclo-oxygenase or phospholipaseC pathway, or inhibit adenylate cyclase. This leads tocalcium mobilization from the open canaliculary systemand cytoskeletal rearrangement that leads to a shapechange to a spherical form. Shape change isaccompanied by the extension of numerous pseudopodson the platelet surface. Focal adhesion points form on thepseudopods, with a concomitant activation of the plateletintegrin αIIbβ3 that acts as fibrinogen receptor. αIIbβ3

(GPIIbIIIa) is the most thoroughly characterized integrinand is the major integrin of platelets and the onlyadhesion receptor capable of mediating platelet aggre-gation by binding of fibrinogen or von Willebrand’s fac-tor (Calvete, 1994; Calvete, 1995; Shattil et al., 1997;Plow et al., 2000; Plow et al., 2001). On resting platelets,αIIbβ3 exists in an inactive conformation. Upon plateletactivation by various agonists, αIIbβ3 undergoes a con-formational change that allows binding of fibrinogen, aplasma protein involved in the blood-coagulation cas-cade. Binding of fibrinogen allows cross-linking of plate-lets and subsequent aggregation. Activation and initialaggregation of platelets lead to secretion of platelet gran-ule contents, which in turn, activate other platelets andinduce inflammation. Compounds of special interest fortick biology, include ADP that activates platelets andATP that causes mast cell degranulation and neutrophilaggregation. This leads to local inflammation and vaso-constriction (Ribeiro, 1989). Serotonin and tromboxaneA2 stimulate vasoconstriction, while serotonin also pro-motes vascular permeability for the infiltration of mast

cells and macrophages. The platelet aggregation cascadecan be targeted by ticks at several stages of the plateletaggregation processes and can be divided into pre-acti-vation, post-activation and post-aggregation inhibition(Fig. 1). The various platelet aggregation inhibitors aresummarized in Table 1.

11. Pre-activation inhibition (PrAI) of plateletaggregation

Agonists of platelet aggregation can be removedbefore activation of platelets takes place. ADP isremoved through hydrolysis by apyrase, an ATP-diphos-phohydrolase enzyme that has been identified in mosthematophagous organisms studied to date (Law et al.,1992). Apyrase activity has been found in the salivarygland extracts or saliva of the hard tick, I. scapularis,and the soft ticks, O. moubata and O. savignyi (Ribeiroet al., 1985; Ribeiro et al., 1991; Mans et al., 1998a, b).Apyrase activity appears, however, to be absent from thesaliva of A. americanum (Bowman et al., 1997). PGI2

and PGD2 inhibit platelet aggregation by preventingADP secretion during platelet activation (Ribeiro et al.,1988; Bowman et al., 1995). The thrombin inhibitorsinvolved in the inhibition of the coagulation cascade alsoserve to inhibit thrombin-induced platelet aggregation(Nienaber et al., 1999). Instead of removing the agonist,

Fig. 1. Various stages of platelet aggregation targeted by ticks. (a)Pre-activation inhibition of platelet aggregation involves either theremoval of the platelet agonist or occupation of the agonist receptorthereby preventing agonist binding and subsequent platelet activation.(b) Platelets may still be activated (as indicated by platelet shapechange), although aggregation is inhibited by targeting the fibrinogenreceptor. (c) Aggregated platelets can be disaggregated by removal offibrinogen from the fibrinogen receptor by competitive binding of anantagonist to the fibrinogen receptor. Proteolysis of fibrinogen can alsolead to platelet disaggregation. An outside–inside signal transductionevent can induce a conformation change in the fibrinogen receptor thatleads to release of fibrinogen. This can be accompanied by a reversibleplatelet shape change back to the original discoid form.

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Table 1Platelet aggregation inhibitors from ticks. An updated list adapted from Bowman et al. (1997). PrAI, PtAI and PAgI indicate pre-activation, post-activation and post-aggregation inhibition, respectively. ND is not determined

Species Compound Target Stage Mr (kDa) Reference

O. moubata Apyrase ADP PrAI NA Ribeiro et al. (1991)O. savignyi Apyrase ADP PrAI, PAgI 67 Mans et al. (1998a, b)O. moubata Moubatin Collagen receptor PrAI 17 Waxman and Connolly (1993)O. moubata TAI Collagen receptor PrAI 15 Karczewski et al. (1995)O. moubata Disagregin αIIbβ3 PtAI 6 Karczewski et al. (1994)O. savignyi Savignygrin αIIbβ3 PtAI, PAgI 7 Mans et al. (2002b)D. variabilis Variabilin αIIbβ3 PtAI 5 Wang et al. (1996)I. scapularis PGI2 PGI2-receptor PrAI ND Ribeiro et al. (1988)A. americanum PGD2 PGI2/D2-receptor PrAI ND Bowman et al. (1995)H. longicornis Longicornin Collagen receptor PrAI 16 Yuanguo et al. (1999)

the inhibitors can also occupy the agonist receptor, ther-eby preventing binding of agonist and subsequent acti-vation. Activation of platelets by collagen is preventedby moubatin, a collagen specific inhibitor identified inthe tick O. moubata, while tick adhesion inhibitor (TAI)inhibits the adhesion of platelets to matrix collagen(Waxman and Connolly, 1993; Karczewski et al., 1995).

12. Post-activation inhibition (Pt-AI) of plateletaggregation

If platelets are activated, aggregation can still beinhibited by targeting the platelet fibrinogen receptor.Binding of fibrinogen to integrin αIIbβ3 is inhibited bythe RGD-containing inhibitors variabilin from the hardtick, D. variabilis, and savignygrin from the soft tick,O. savignyi, as well as the RGD lacking inhibitor, disag-regin from the soft tick O. moubata (Karczewski et al.,1994; Karczewski and Connolly, 1997; Wang et al.,1996; Mans et al., 2002b).

13. Post-aggregation inhibition (PAgI) of plateletaggregation

If inhibition of aggregation at pre- or post-activationlevel fails, aggregated platelets may still be disaggre-gated. The αIIbβ3 antagonists can displace fibrinogenfrom its receptor thereby allowing disaggregation (Manset al., 2002c). Apyrase disaggregates aggregated plate-lets, after platelet activation and degranulation, possiblyvia removal of ADP and a subsequent signal transduc-tion event that leads to a reversal of platelet shape, froma spherical to a discoid shape (Mans et al., 2000). Aggre-gated platelets can also be disaggregated by proteolysisof fibrinogen (Mahlaku et al., manuscript in preparation).Disaggregation of platelet aggregates is an importantback-up mechanism that ticks can use if first-line defense

mechanisms fail to inhibit platelet aggregation com-pletely.

14. Blood coagulation and its role in hemostasis

The blood-clotting cascade consists of a series of ser-ine proteases that sequentially activate each other (Fig.2). The intrinsic pathway of blood coagulation startswith collagen-induced activation of fXII which activatesfXI as well as kallikrein. Kallikrein cleaves a precursorto form bradikinin, a peptide causing inflammation, thesensation of pain and irritation (Ribeiro, 1989). Theextrinsic pathway starts with the release of thromboplas-tin (tissue factor) from damaged endothelial cells, whichactivates factor VII (Fig. 2) (Bevers et al., 1993). Bothpathways eventually coalesce in the formation of factorXa that in turn produces thrombin. Fibrinogen is thencleaved by thrombin to fibrin, which forms a network,the main constituent of the blood clot together withplatelets and erythrocytes (Jackson and Nemerson,1980). The whole blood coagulation cascade depends ona negatively charged membrane surface where the differ-ent serine proteases involved in the cascade can dock tobe sequentially activated (Esmon, 1995). Normally, theouter membranes of cells are depleted of negativelycharged phospholipids. The platelets provide this mem-brane surface by the translocation of phosphatidyl serinefrom its inner to outer membrane during platelet acti-vation and aggregation. Clotting proteins (fVII, fX andprothrombin) subsequently bind to the membranethrough a Ca2+ bridge by means of the modified aminoacid γ-carboxyglutamate (Gla) and is targeted andlocalized to the site of damage (Tans and Rosing, 1986).These events ultimately lead to edema, one of the signsmany tick resistant hosts display. Edema and associatedirritation lead to host grooming, an important factor inthe reduction of the tick burden (Wikel, 1996).

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Fig. 2. The blood coagulation cascade. The intrinsic pathway is initiated by the binding of fXII to a negatively charged surface like collagen orthe activated platelet (PL) surface and is then activated by kallikrein. fXIIa then activates fXI as well as prekallikrein. This leads to eventualactivation of fX. The extrinsic pathway is activated by trauma that releases tissue factor. This binds to fVII, which is activated by thrombin andtogether activates fX. Prothrombin is activated to thrombin by fXa that cleaves fibrinogen to fibrin that forms a network. Thrombin also activatesfXIII that stabilizes the fibrin clot by cross-linkage. Coagulation factors for which inhibitors have been identified in ticks are circled.

15. Blood-clotting inhibitors from ticks

Numerous inhibitors of the different serine proteasesinvolved in the clotting cascade have been described forhard and soft ticks (Table 2). Inhibitors display a varietyof molecular masses, targets and inhibitory mechanisms.In some cases, the absence of inhibitors to specific anti-coagulants was also noted. Inhibitors of fV and fVIIhave been described for the hard tick, D. andersoni(Gordon and Allen, 1991). The same paper states thatno significant effect was noted for the inhibition ofthrombin or fXa by salivary gland extracts. Significantinhibition of fXa activity was also absent in saliva fromBoophilus microplus (Horn et al., 2000). Similarly, noeffect was noted for inhibition of the intrinsic pathwayby saliva from I. scapularis, although the extrinsic path-way was inhibited. It was concluded that an activity mustexist that targets the extrinsic pathway before activationof fXa (Ribeiro et al., 1985). Recently, this observationwas confirmed by the characterization of ixolaris, atissue factor pathway inhibitor that targets thefVIIa/tissue factor complex (Francischetti et al., 2002).An inhibitor of fXa that also inhibited the intrinsic path-way was, however, recently discovered for I. scapularis(Narasimhan et al., 2002). This inhibitor showedsequence similarity to Salp9 and Salp14, proteins ident-ified as immunodominant proteins (Das et al., 2001).These proteins have not yet been assigned to a proteinfamily and differ from other tick anti-coagulants.

The extrinsic pathway was also the only pathway

inhibited by salivary gland extracts from Haemaphysalisinermis, while it was also more significantly targetedthan the extrinsic pathway by SGE from Ripicephalusappendiculatus and D. reticulates (Kazimirova et al.,2000). This suggests that there are more inhibitors forthe extrinsic pathway than those of the intrinsic pathwayin salivary gland extracts of hard ticks. fXa and thrombininhibitors have been described in a variety of hard ticks(Table 2). Inhibitors differ from one another acrossgenus level in terms of molecular mass and kineticmechanism. It is tempting to suggest that these differ-ences confirm the independent adaptation of tick familiesto a blood-feeding environment. Unfortunately, it is notknown whether these studies represent an exhaustivedescription of the anti-coagulant capacities of these tickspecies. An interesting phenomenon is observed for thefXa inhibitor from H. dromedarii, that inhibits fXa non-competitively, but inhibits thrombin competitively(Ibrahim et al., 2001a, b). This could however, be anartifact, as it was not determined whether this inhibitorexhibits tight-binding kinetics or not. Tick anti-coagulantpeptide (TAP) and fXaI (factor Xa inhibitors), as wellas ornithodorin and savignin (thrombin inhibitors), havebeen described for the soft ticks, O. moubata and Orni-thodoros savignyi, respectively (Waxman et al., 1990;Gaspar et al., 1995; Gaspar et al., 1996; Joubert et al.,1998; Van de Locht et al., 1996; Nienaber et al., 1999;Mans et al., 2002d). The soft tick inhibitors inhibitthrombin by targeting both the thrombin active site aswell as the fibrinogen-binding exosite. In contrast, two

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Table 2Anti-coagulants from ticks. Adapted from Bowman et al. (1997). The coagulation cascade target can be a specific serine protease, complex or anindication of intrinsic pathway (PT) or extrinsic pathway (APTT) inhibition. The kinetic mechanism of inhibition (ST, slow-tight binding; C,competitive; UC, uncompetitive; NC, non-competitive), determined Ki values and molecular masses are also indicated. ND is not determined.

Species Target Kinetic mechanism (Ki) Mr (kDa) Reference

Soft ticksO. moubata fXa ST,C (Ki ~0.58 nM) 7 Waxman et al. (1990)O. moubata Thrombin ST,C (Ki ~low pM) 12 Van de Locht et al. (1996)O. savignyi fXa ST,C (Ki ~0.83 nM) 7 Gaspar et al. (1996)O. savignyi Thrombin ST,C (Ki ~5 pM) 12 Nienaber et al. (1999)O. savignyi TF (possibly) ND 9.1, 9.3 Ehebauer et al. (2002)

Hard ticksR. appendiculatus fXa ND 65 Limo et al. (1991)H. truncatum fXa UC (Ki ~0.7 nM) 17 Joubert et al. (1995)H. dromedarii FXa UC (Ki ~134 nM) 15 Ibrahim et al. (2001a)H. dromedarii Thrombin NC (Ki ~11.7 µM) 3.2 Ibrahim et al. (2001b)H. dromedarii Thrombin C (Ki ~211 nM) 15 Ibrahim et al. (2001b)B. microplus Thrombin ND ND Anastopoulos et al. (1991)B microplus Kallikrein Not ST(Ki ~120 nM) 18 Tanaka et al. (1999)B. microplus Thrombin Not ST (ND) 60 Horn et al. (2000)I. holocyclus Thrombin ND ND Anastopoulos et al. (1991)H. longicornis Thrombin ND ND Anastopoulos et al. (1991)H. longicornis Thrombin NC (Kd ~3–4µM) 7 Iwanaga et al. (2003)A. americanum fXa, thrombin ND 16 Zhu et al. (1997)A. americanum Thrombin ST, C (Ki ~73 pM) 12 Zhu et al. (1997)A. variegatum Thrombin NC ~3770 Kazimirova et al., 2002I. ricinus Thrombin ND 7 Hoffmann et al. (1991)D. andersoni fV, fVII ND ND Gordon and Allen (1991)I. scapularis FVIIa-TF C 15.7 Francischetti et al. (2002)I. scapularis fXa Not ST 9.8 Narasimhan et al. (2002)

thrombin inhibitors (madanins) from the hard tick, Hyal-omma longicornis, inhibit thrombin by targeting thefibrinogen-binding exosite alone (Iwanaga et al., 2003).

16. Immunomodulatory components in tick salivathat affect blood-feeding

Saliva contains prostaglandin E2 (PGE2) which pro-motes vasodilation, inhibits platelet aggregation, mastcell degranulation and T-lymphocyte activation. PGE2

has been identified in the saliva of Ixodes scapularis,Amblyomma americanum and B. microplus (Dickinsonet al., 1976; Ribeiro et al., 1985; Ribeiro et al., 1988;Ribeiro et al., 1992; Bowman et al., 1995). This alonecounteracts a whole range of the host’s defenses. Anti-histamine is also present which prevents inflammation.Histamine-binding proteins that sequestrate histaminefrom the feeding site have been identified in R. append-iculatus Paesen et al., 1999; Paesen et al., 2000). Thesaliva of I. scapularis contains a carboxypeptidaseactivity that cleaves bradykinin and thus prevents irri-tation and pain (Ribeiro et al., 1985; Ribeiro and Mather,1998). An anti-complement protein from the tick I.scapularis that inhibits rabbit erythrocyte lysis by humanserum has also been described (Valenzuela et al., 2000).

17. Molecular evolution as mechanism ofadaptation to a blood-feeding environment

The obvious question is how did the anti-hemostaticand immunomodulatory mechanisms evolve. The answerto this lies at molecular level. Intrinsic to the word adap-tation is the notion of change and applied to novelenvironments, the idea of acquisition of novel propertiesor functions. In biochemical terms, the study of the adap-tation of organisms lies in the realm of molecular andprotein evolution. As such adaptation entails the acqui-sition of novel protein functions. It is clear that therewas a definite selective pressure on ticks to adapt to ablood-feeding environment and that an impressive arrayof anti-hemostatic components were evolved by ticks.The question that can be asked is what evolutionarymechanism allowed ticks to generate such a diversityof functions.

Several different modes for the acquisition of newprotein function exist. An organism can utilize an exist-ing function in a new way, for example, if apyrase hadan intracellular function in the endoplasmic reticulum orGolgi apparatus (Gao et al., 1999; Zhong and Guidotti,1999) and it acquired the signals for extracellular export,it might now also be involved in the regulation of plate-let aggregation. Alternately, a protein can through

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mutation acquire a novel function while still retainingits old function. This is referred to as gene sharing andsuch “moonlighting” proteins with multiple functions arediscovered frequently (Jeffery, 1999). It has been esti-mated that there exist approximately a 1000–7000 differ-ent protein folds or topologies (Chothia, 1992; Orengoet al., 1994). It has been predicted that there are at least1709, 6241, 13601, 18424 and 40580 protein codingsequences in Haemophilus, yeast, fly, worm and humangenomes, respectively (Rubin et al., 2000; Li et al.,2001). It should thus be clear that protein folds are util-ized for more than one function. The problem that arisesthough, is how are new functions evolved from old folds.Protein function tends to be conserved, so that exceptfor the case of gene sharing, proteins would not losetheir original function to gain a new function. Thisphenomenon is referred to as purifying or negative selec-tion in that any mutation that would be deleterious forprotein function would be removed from the population.The answer to this lies in gene duplication, proposed byOhno (1970) to be the single most important event inthe generation of new protein functions. Genome-widecomparisons of the occurrence of gene duplication andthe presence of multi-gene protein families with diversefunctions have validated this view in recent years (Lynchand Conery, 2000; Wagner, 2001; Liberles et al., 2001;Kondrashov et al., 2002).

18. Gene duplication and the acquisition of novelprotein function

Genes can duplicate via unequal crossing-over duringrecombination, polyploidy (duplication of wholegenomes), non-homologous chromosomal breakage andreunion or transposition (Maeda and Smithies, 1986). Ithas been estimated that gene duplication is a very com-mon event during adaptive radiation (Ohno, 1970). Inthis regard, it has been estimated that there are at least284, 1858, 8971, 5536 and 4519-15121 duplicated genesin the genomes of Haemophilus, yeast, fly, worm andhumans, respectively (Rubin et al., 2000; Li et al., 2001).Gene duplication rates of 0.02–0.2 genes per millionyears have been estimated for different species, so thatat least 50% of all genes in a genome are expected toduplicate and increase in frequency at least once every35–350 million years. With a genome size of 15 000genes, it can be expected that at least 60–600 duplicategenes arise in a pair of sister taxa per million years(Lynch and Conery, 2000). The genome size for A.americanum has been estimated at 1.04 × 109 bp (Palmeret al., 1994). This would place it with other genomesthat have ~30 000–60 000 genes (Grauer and Li, 2000).Assuming a divergence time between 120 and 92 millionyears for hard and soft tick species, a variety of novelprotein functions restricted to hard and soft ticks,

respectively, could thus be expected. This emphasizesthe importance of the question, whether the ticks evolvedanti-hemostatic strategies before or after divergence intothe main tick families.

19. Case studies of gene duplication in ticks

Evidence of major duplication events within the ticklineage comes from comparisons of chromosome num-bers of ticks and other parasitiformes mites. Mesostig-mata generally possess 6–10 diploid chromosomes.Chromosome numbers for ticks in contrast are muchhigher and vary more with the numbers ranging from16–28 diploid chromosomes (Oliver, 1989). This impliesthat at least one genome duplication event occurred forticks, which could have provided the necessary rawmaterial for the evolution of blood-feeding capabilities.Gene duplication and subsequent gain/loss of proteinfunction has been indicated for two main tick proteinfamilies, a family of bovine pancreatic trypsin inhibitor(BPTI)-like anti-hemostatic factors and a family of pro-teins that are part of the lipocalin protein family. Proteinfamily is defined here as proteins that share a commonstructural protein fold, although their functions mightdiffer, i.e. paralogous proteins.

20. The tick BPTI-like family

The BPTI-like fold (basic trypsin inhibitor fold) ischaracteristic of a family of serine protease inhibitors.These inhibitors are small (50–65 residues), with sixcysteines arranged in a characteristic disulfide bond pat-tern (Laskowski and Kato, 1980). The basic structureconsists of an N-terminal 310-helix around the first cyst-eine, a central double stranded anti-parallel β-sheetlinked by a hairpin loop and a C-terminal three turn α-helix. In ticks, several proteins have been identified thatpossess the BPTI-like fold. Kunitz inhibitors (BmTI)have been identified from larvae of the hard tick B.microplus that inhibit trypsin, elastase and kallikrein(Tanaka et al., 1999). There is also a BPTI-like sequencefrom B. microplus deposited in the Genbank named car-rapatin (P81162), for which a BLAST search indicatesvery high similarity with the second domain of tissuefactor plasminogen inhibitor (TFPI, unpublishedobservation). Ixolaris, the fVIIa/tissue factor complexinhibitor, is also a double-domain BPTI-like protein(Francischetti et al., 2002).Of interest is the fact that thesecond BPTI-domain of ixolaris lacks the Cys14–Cys38disulfide bond that constricts the substrate-bindingpresenting loop of the canonical BPTI inhibitors. In softticks, both fXa (TAP and fXaI) and thrombin(ornithodorin and savignin) inhibitors belong to the

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BPTI-family as well as platelet fibrinogen receptorantagonists (Table 1 and 2).

21. Inhibition mechanisms of the tick BPTI-likeinhibitors

Canonical Kunitz inhibitors inhibit their respectiveserine proteases by presentation of a substrate-bindingloop (located around the second cysteine) to the activesite of their enzymes (Fig. 3), where they bind in an anti-parallel β-sheet fashion similar to natural serine proteasesubstrates (Laskowski and Kato, 1980; Bode and Huber,1992). BmTI is a double-domain BPTI inhibitor, and itseems as if its mechanism of action is similar to thecanonical inhibitors (Tanaka et al., 1999). Ixolarisinhibits the fVIIa/TF complex by first binding to theexosite of fXa by its second BPTI-like domain, thendocks with the fVIIa/TF complex and binds in the activesite of fVIIa via its first domain (Fig. 3). It was proposedthat binding in the active site occurs via the canonicalmechanism (Francischetti et al., 2002). In contrast to thecanonical BPTI inhibitors, the soft tick inhibitors, inserttheir N-terminal sequences into the enzymes active sitein a manner reminiscent of hirudin (Fig. 3). The fXa

Fig. 3. Inhibition mechanisms of BPTI-like proteins. (a) The seconddomain of TFPI in complex with fXa (Burgering et al., 1997). Notethe presentation of the BPTI-substrate-binding loop to the active site.(b) The proposed inhibition mechanism for ixolaris (Francischetti etal., 2002). (c) Binding of a soft tick fXa inhibitor (TAP) into the activesite of fXa (Wei et al., 1998). Note the insertion of the N-terminalresidues into the active site and interaction of the C-terminal α-helixwith fXa exosite. (d) Binding of a soft tick thrombin inhibitor(ornithodorin) with thrombin (Van de Locht et al., 1996). Note theinsertion of the N-terminal residues of the first BPTI-like domain intothe active site of thrombin and the interaction of the C-terminal α-helix of the second BPTI domain with thrombin’s exosite.

inhibitors (fXaI) consist of a single BPTI-like domain(60 amino acids) that interacts with fXa via the N-ter-minal as well as secondary interactive sites in its C-ter-minal α-helix (Wei et al., 1998). The thrombin inhibitorshave two BPTI-like domains (60 amino acids each witha 8 residue linker), of which the N-terminal domain(NTI) interacts with the active site via the N-terminalresidues and secondary interaction sites, while the C-terminal domain (CTI) interacts via its C-terminal α-helix with the fibrinogen-binding exosite of thrombin(Van de Locht et al., 1996; Mans et al., 2002d). Theintegrin αIIbβ3 antagonists, disagregin and savignygrin,have recently been shown to belong to the BPTI-family(Mans et al., 2002b). In the case of savignygrin, anRGD-motif is presented on the classical BPTI substrate-binding presenting loop.

22. An evolutionary pathway for the soft tick BPTIinhibitors

Phylogenetic analysis showed that the hard tick BPTIinhibitors grouped with insect hemolymph derivedinhibitors (proposed to play a role in the regulation ofinsect hemolymph coagulation), instead of soft tick BPTIinhibitors. In contrast, phylogenetic analysis showed thatthe soft tick BPTI inhibitors descended from a commonancestor (Mans et al., 2002a). Common ancestry in thissense can only mean that there were multiple gene dupli-cation events and subsequent mutation that led to a gainor loss of protein function. An evolutionary model thatindicates a sequential evolution of new protein functionwas proposed (Fig. 4). This model accounts for the non-canonical mechanism of inhibition displayed by the ser-ine protease inhibitors and indicates that platelet aggre-gation inhibitory function of disagregin and savignygrinevolved from the serine protease inhibitors. A higher rateof non-synonymous to synonymous substitution of thefXa and platelet aggregation inhibitors also indicates theoccurrence of positive Darwinian selection that suggestsevolutionary pressure to evolve these functions (Mans etal., 2002a). In the case of the savignygrins, four isoformshave been described of which two have been shown tobe recent gene duplication events that occurred afterdivergence of O. savignyi and O. moubata (Mans et al.,2003a). These isoforms are maintained by gene conver-sion in order to maintain high expression levels. This isprobably due to the wide host range of O. savignyi,where high expression levels allow this tick species totarget a wide range of αIIbβ3 levels found in differenthosts. This is one of the few examples where host speci-ficity and habitat could be related to events occurring atthe molecular level of the tick–host interface.

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Fig. 4. The acquisition of new anti-hemostatic functions in soft ticks.(a) Due to conformational restriction loops around thrombin’s activesite, canonical BPTI inhibitors cannot inhibit thrombin. The C-terminaldomain of the soft tick thrombin inhibitors evolved to target thefibrinogen-binding exosite of thrombin. (b) A tandem gene duplicationevent leads to a homo-dimeric inhibitor. A new mechanism for com-petitive serine protease inhibition evolves, whereby the N-terminalresidues of the soft tick thrombin inhibitors are inserted into thrombin’sactive site thereby avoiding the restrictive loops. (c) A gene duplicationof the N-terminal domain leads to utilization of the novel serine pro-tease inhibitory activity to inhibit fXa. (d) Gene duplication of fXaleads to utilization of the existing but redundant BPTI-substratepresenting loop to target the platelet fibrinogen receptor. An alternativeevolutionary pathway is direct gene duplication from the C-terminaldomain of the thrombin inhibitors (dotted line).

23. Independent evolution of anti-hemostaticinhibitors in hard and soft ticks

It is interesting that BPTI inhibitors from the hard tick,B. microplus, are not grouped with the anti-hemostaticfactors of soft ticks, but rather with insect hemolymphderived protease inhibitors, that inhibit their respectiveenzymes via the classical BPTI mechanism. fXa andthrombin inhibitors from hard ticks have also beendescribed with molecular masses (17–65 kDa) that differsignificantly from that of the BPTI fold (Bowman et al.,1997; Table 2). The recently described madanins fromH. longicornis that inhibit thrombin are small 7 kDa pro-teins with no homologs in the sequence databases(Iwanaga et al., 2003). The protein family to which thefXa inhibitor homologue of Salp9 and Salp14 belongsis also unique although it would seem as if several paral-ogs exist for this family (Narasimhan et al., 2002). Fur-thermore, variabilin, a platelet aggregation inhibitorfrom the hard tick, Dermacentor variabilis, does notresemble the platelet aggregation inhibitors from softticks at all (Wang et al., 1996). Its cysteine pattern aswell as the localization of its RGD-motif differs com-pletely from the observed BPTI-fold and the motif foundin savignygrin. The molecular masses and kinetic mech-

anisms of hard tick and soft tick coagulation inhibitorsalso differ to such an extent that a common protein foldcan be ruled out (Table 2). There is even a large variationin coagulation targets within the hard tick family. Thiswould suggest that soft tick derived BPTI inhibitors onlyacquired their specific mechanisms of action after thedivergence of hard and soft ticks, as well as suggestingindependent adaptation to a blood-feeding environment.This is of interest because it would have been expectedthat ticks, being monophyletic, would have adapted to ablood-feeding environment before divergence. It alsoraises the question whether anti-hemostatic functionsobserved in the Ornithodoros genus are represented inother soft tick families. It has been mentioned that blood-feeding behavior evolved at least six times indepen-dently (Ribeiro, 1995). Most of the convergent evolutionoccurred at family level and as has been argued, is notthe exception for ticks.

24. Independent adaptation to a blood-feedingenvironment

If hard and soft ticks adapted independently to ablood-feeding environment and are also monophyletic,the question is raised what specific features did the maintick lineages share before their divergence. It should beobvious that it could not have been blood feeding per se.Feeding on host lymph would, however, have providedcommon ground and a start-off point to adapt indepen-dently to the more lucrative lifestyle of the blood feeder.Remnants of this might be seen in hard tick feeding,where the initial feeding phase that can last up to 4 days,does not entail the engorgement of blood, but rather lym-phatic fluid (Balashov, 1972). This feeding style leads,however, to a prolonged exposure to the host’s immunesystem and soft ticks have aborted this mode of feedingand opted for the faster mode in order to escape thisconfrontation. Hard ticks, on the other hand, had toevolve immunosuppressive strategies as exemplified bythe lipocalins (Paesen et al., 1999). In order to feed fast,soft ticks needed a soft cuticle that allows for rapidengorgement. Hard ticks, in contrast, need a hard cuticleto protect it against the long periods of grooming bythe host.

If the main tick families adapted independently totheir new blood-feeding environments, other features intheir biology that are also dependent on a blood-feedinglifestyle, should corroborate this. To this end, propertiesthat are intimately involved in the blood-feeding processdiffer quite remarkably (Table 3). Differences are in factso distinct that it could only have evolved on inde-pendent trajectories, probably at an early stage of adap-tation to a blood-feeding environment. While inde-pendent acquisition of novel anti-hemostatic componentsby the two main tick families has occurred, the presence

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Table 3Properties of hard and soft ticks intimately associated with blood-feeding. Data obtained from Balashov (1972), Obenchain and Galun (1982) andSonenshine (1991). Specific advantages of evolved property are indicated

Soft ticks (Argasidae) Hard ticks (Ixodidae)

Integument Leathery integument allows for fast feeding Sclerotized scutum protects during longattachment periods but needs a growth phase toallow for rapid engorgement

Feeding Multiple fast feeding events: escapes host’s Single slow feeding event: Locates host onlyimmune response once and imbibes large blood meal

Mating strategy Mate while unfed or when fed: does not limit Only mate after initial attachment. Matingfeeding event to host location essential for rapid engorgement

Oviposition strategy Lay several egg batches, once after every feeding Lay one batch of eggs, after which female diesevent

Salivary gland morphology One simple granular acinus fully developed Multiple granular acini, that develop and changebefore feeding before rapid engorgement

Regulation of water balance Secrete excess water from blood meal via the Secrete excess water from blood meal viacoxal organs salivary glands

Blood meal digestion Feed first, digest later. First stage of digestion Low rate of feeding and digestion duringincludes development of midgut epithelium preparatory feeding phase when tick grows to

allow rapid engorgement

of apyrase in both families has been indicated (Law etal., 1992). Apyrase (ATP-diphosphohydrolase; EC3.6.1.5) inhibits platelet aggregation induced by ADP aswell as is able to disaggregate platelets aggregated byADP (Mans et al., 1998b; Mans et al., 2000). It has beenidentified in all the hematophagous arthropod familiesinvestigated so far (Ribeiro, 1995), which suggests thatthis enzyme might have been present in the ancestralnon-hematophagous tick. Of interest is the absence ofapyrase in the saliva of the tick, A. americanum(Bowman et al., 1997). In the latter case, this might bean example of gene loss. Alternatively, apyrase mighthave been independently co-opted as an anti-hemostaticfactor in hard and soft ticks.

25. Independent adaptation and host specificity

It has been argued that the emergence of hematophagyin ticks was triggered by the divergence of early modernbirds and mammals (Mans et al., 2003a). This providesand interesting counterpoint to suggestions that evol-ution of ticks was not so much influenced by host speci-ficity as by ecological factors (Klompen et al., 1996).While, this might be the case for adaptation to theenvironment, independent evolution of anti-hemostaticstrategies would suggest that host diversity could haveinfluenced the adaptation of ticks to the vertebrate hemo-static system. Positive Darwinian selection would indeedsuggest that the hemostatic system of the host played adecisive role in the evolution of hematophagy in ticks.The filling of various niches by placental mammals andearly avians could have imposed geographical or evenecological isolation necessary for independent adaptationto a blood-feeding environment by ticks. It could be

argued that this could be in favor of an argument forhost specificity. However, vertebrates share a similarhemostatic system, so that even if ticks did adapt toblood-feeding environments independently, they wouldnot be prohibited to feed on different hosts if encoun-tered. It is, however, known that under laboratory set-tings, certain tick shows a host preference. This is mostprobably due to tick rejection by some hosts, normallyvia immune reactions that might be a limiting factor forsome ticks to acquire a wide host range. A hypothesisthat could be advanced is that there was an initial iso-lation of the tick families due to host preference. Assuch, hard ticks might have adapted to a blood-feedingenvironment via placental mammals, while soft ticksmight initially have fed on avians.

26. The tick lipocalin family

Lipocalins are a family of small (150–183 amino acidresidues), secretory proteins, that exhibit a variety offunctions that includes transport of small molecules,arthropod coloration, pheromone transport, prostaglan-din synthesis, smell reception, regulation of cell growth,tissue development, metabolism and regulation of theimmune response (Flower, 1996; Flower et al., 2000).The lipocalin protein fold consists of an eight (A-H)stranded, anti-parallel β-barrel with a (+1)7 topology.One end is closed off by an N-terminal 310 helix. Theother end is open to solvent and a ligand-binding pocketis formed here via the binding cavity and the exposedloops. A C-terminal α-helix packs against one side ofthe barrel and is followed by a short β-sheet (I). Lipocal-ins have been found in the salivary gland secretions fromvarious hematophagous organisms (Montford et al.,

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2000). In the triatomine bug, Rhodnius prolixus, thenitrophorins (NP1–NP4) carry heme that transport nitricoxide, which is released at the feeding site, causingsmooth muscle relaxation and vasodilation (Champagneet al., 1995). All four proteins can also regulate inflam-mation and the host’s immune response by binding hista-mine. NP2 (prolixin-S) can also inhibit the conversionof fX to fXa (Ribeiro et al., 1995). Three other lipocalinsfrom R. prolixus do not contain heme, but inhibit plateletaggregation by sequestration of the agonist ADP(Francischetti et al., 2000). The triatomine bug, Triatomapallidipennis, inhibits specifically collagen-induced plat-elet aggregation via the lipocalin, pallidipin (Noeske-Jungblut et al., 1994). Triabin is another lipocalin thatinhibits thrombin’s activity by targeting the fibrinogen-binding exosite (Noeske-Jungblut et al., 1995). In thecase of triabin, an interesting deviation in its topologyoccurred with an exchange of β-strands B and C of theβ-barrel (Fuentes-Prior et al., 1997).

In hard ticks, the histamine-binding proteins (HBP1-3) function in the regulation of inflammation during thelong feeding periods of hard ticks have been identifiedin R. appendiculatus (Paesen et al., 1999; Paesen et al.,2000). In soft ticks, moubatin and four highly abundantproteins (TSGPs) proposed to function in tick salivarygland granule biogenesis have been identified as beinglipocalins (Fig. 5) (Mans et al., 2001; Mans et al.,2003b). Two of the TSGPs have also been identified asbeing the toxins involved in the pathogenesis caused bythe tick, O. savignyi (Mans et al., 2002e). Identity ofTSGP2 and TSGP3 with moubatin suggested that theTSGPs might be involved in anti-hemostatic mech-

Fig. 5. Alignment of the tick histamine-binding proteins and the soft tick TSGPs. Indicated are the secondary structure motifs and proposeddisulfiide bonds of the TSGPs.

anisms (Mans et al., 2003b). However, no platelet aggre-gation or blood coagulation inhibitory properties couldbe assigned to the TSGPs. Database analysis indicate,that an extensive family of tick lipocalins already existswith 20 current members. This number will probablyincrease in the near future with more lipocalins withdiverse functions being found in different tick species.Phylogenetic analysis indicated that most tick lipocalinsevolved after the divergence of hard and soft ticks andthat might even have occurred up to genus level (Manset al., 2003b).

27. Origins of tick toxicoses

An interesting phenomenon is the ability of some ticksto cause pathogenic changes in their host. This couldtake the form of paralysis, anaphylaxis or cardiac failure(Mans et al., 2002e). From an evolutionary perspective,it could be considered whether tick toxins have a com-mon ancestor shared with toxins from other toxic arthro-pods or even if tick toxins share a common ancestor spe-cific to the tick lineage. Toxins might also have specificfunctions related to their toxicity that was specificallyacquired during adaptation to a blood-feeding environ-ment. Toxicity could also be a by product of proteinsbeing in a novel environment and recognition of hosttargets, a chance event. To investigate these possibilities,it is important to clearly delineate various forms of tox-icoses and find their shared properties or differences(mechanism of pathogenesis, homology), as this willgive valuable information as to their origins. TSGP2 and

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TSGP4 have been shown to be toxins that affect the car-diac system (Mans et al., 2002e). TSGP2, TSGP3 andTSGP4 are absent in the salivary gland extracts of O.moubata (Mans et al., 2003b). Phylogenetic analysis hasalso indicated that TSGP2 and TSGP3 may be recentgene duplication events that occurred after divergenceof O. savignyi and O. moubata (Mans et al., 2003b).These results suggest that the majority of observed ticktoxicoses may be recently acquired traits. This could bedue to the fact that ticks are non-permanent feeders thatcan survive without their host for extended periods oftime. This relieves much of the burden other permanentparasites face, which have to depend on the host’s fitnessand viability for their own survival. This adaptation ofticks to survive away from their host also led to ticksbeing more aggressive than ectoparasites living perma-nently on their host (Lehmann, 1993). Due to this lackof dependence of ticks on host fitness, the tick burdenitself can severely affect the host. As such, the emerg-ence of toxins that affect the health of the host may notconfer an evolutionary disadvantage to the tick.

28. Independent adaptation and transmission oftick-borne diseases

Hard ticks transmit most tick-borne diseases and it canbe argued that this is because of their prolonged feedinghabits that facilitate both delivery and uptake of blood-borne parasites. The multiple feeding sessions of softticks, on the other hand, are also a valid strategy usedby some parasites for transmittance. Short feeding timesdo not really matter that much if mosquitoes are con-sidered effective disease vectors. If ticks did adapt to ablood-feeding environment before divergence, then para-sitic diseases would have been more widely spreadamong the tick population. Independent adaptation to ablood-feeding environment could, however, have influ-enced which ticks the parasites eventually exploited asvectors.

29. The tick genome project

Much interest was recently expressed at the FourthInternational Conference on Ticks and Tick-BornePathogens in Banff, Alberta 2002 to sequence a modeltick genome. The choice of which genome to sequencewas one of the main questions raised and whether sucha chosen genome would be representative of the Ixodidaas a whole. The recent studies on the gene duplicationevents of the BPTI and lipocalin families and their impli-cations for the independent adaptation of ticks to ablood-feeding environment have specific bearing on achoice of a tick genome. The high level of sequencedivergence and the indications that many functional pro-

teins might only have acquired novel functions after thedivergence of hard and soft ticks would suggest that onetick species would not be an adequate model for tickevolution. This implies that even if the genome of a hardtick species are sequenced to serve as a model for tickgenome studies, we might still miss some of the mostimportant molecules involved in tick feeding from otherspecies. The choice of a model tick or ticks for genomesequencing will remain a central problem. Choosing atick closest to the ancestral tick (if we can predict themore ancient lineages correctly) will allow us to studyearly evolutionary events although we will miss thoseproteins recently diverged. The most fruitful endeavorwould be to sequence the genomes of representativesfrom both hard and soft ticks as well as the Nuttallielli-dae. The latter is probably one of the most importanttargets, as it will provide us with an external referencepoint for the other tick families. As the Nuttalliellidaeis also the only monotypic family and occurred only inGondwanaland (the suggested cradle of tick origins), thistick might be the closest living fossil that we have tothe ancestral tick. The scarcity of living specimens ofthis tick makes it a priority in the study of tick biodivers-ity. If this tick species, should be lost or becomes extinct,we might have lost one of the most valuable tick speciesstill living in the study of tick evolution. Sequencing thegenome of a soft as well as hard tick could also highlightthose differences that make each family unique.

30. Implications of tick evolution for vaccinedevelopment

The search for universal vaccine targets that will pro-vide a wide range of cross-protection in ticks has beengoing for quite a long time. As yet, only one commercialvaccine that shows cross-protection between Boophilusand Hyalomma species has been developed. This vaccineis based on the gut-membrane protein Bm86 from B.microplus. While a Bm86 homologue has been identifiedin R. evertsi evertsi, no cross-protection was observed(De Vos et al., 2001). It should be clear that for extensivecross-species protection to be viable, the vaccine targetshould be present in most tick species targeted and suf-ficiently conserved, to be recognized by the host’simmune system. Early radiation of ticks and independentadaptation of ticks to a blood-feeding environment sug-gest that a universal vaccine target that will work againstall tick species is non-viable. The best that we couldhope for is to find conserved targets within subsets ofclosely related tick genera or even species. It is foreseenthat studies into the evolutionary relationships of ticksat functional level will greatly facilitate this approach.

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31. Conclusions

The study of how ticks adapted to a blood-feedingenvironment allows for a better understanding of basictick biology and the general mechanisms by which hem-atophagy evolved. It also gives us a specific referenceframework by which we can approach complex issuessuch as the tick genome project and vaccine develop-ment strategies. Understanding ticks at molecular levelwill allow us to combat them on their own turf.


This paper was completed using grants by the SouthAfrican National Research Foundation and the Univer-sity of Pretoria.


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