Comparative sialomics between hard and soft ticks: implications for the evolution of blood-feeding behavior

Ticks evolved various mechanisms to modulate their host's hemostatic and immune defenses. Differences in the anti-hemostatic repertoires suggest that hard and soft ticks evolved anti-hemostatic mechanisms independently, but raise questions on the conservation of salivary gland proteins in the ancestral tick lineage. To address this issue, the sialome (salivary gland secretory proteome) from the soft tick, Argas monolakensis, was determined by proteomic analysis and cDNA library construction of salivary glands from fed and unfed adult female ticks. The sialome is composed of approximately 130 secretory proteins of which the most abundant protein folds are the lipocalin, BTSP, BPTI and metalloprotease families which also comprise the most abundant proteins found in the salivary glands. Comparative analysis indicates that the major protein families are conserved in hard and soft ticks. Phylogenetic analysis shows, however, that most gene duplications are lineage specific, indicating that the protein families analyzed possibly evolved most of their functions after divergence of the two major tick families. In conclusion, the ancestral tick may have possessed a simple (few members for each family), but diverse (many different protein families) salivary gland protein domain repertoire.     

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

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.     

A novel clade of cysteinyl leukotriene scavengers in soft ticks

Inflammation is an important vertebrate defense mechanism against ecto-parasites for which ticks have evolved numerous mechanisms of modulation. AM-33 and TSGP4, related lipocalins from the soft ticks Argas monolakensis and Ornithodoros savignyi bind cysteinyl leukotrienes with high affinity as measured by isothermal titration calorimetry. This was confirmed in a smooth muscle bioassay that measured contraction of guinea pig ileum induced by leukotriene C(4) where both proteins inhibited contraction effectively. Conservation of this function in two diverse soft tick genera suggests that scavenging of cysteinyl leukotrienes evolved in the ancestral soft tick lineage. In addition soft ticks also evolved mechanisms that target other mediators of inflammation that include scavenging of histamine, serotonin, leukotriene B(4), thromboxane A(2), ATP and inhibition of the complement cascade. Inhibitors of blood-coagulation and platelet aggregation were also present in the ancestral soft tick lineage. Because histamine and cysteinyl leukotrienes are mainly produced by mast cells and basophils, and these cells are important in the mediation of tick rejection reactions, these findings indicate an ancient antagonistic relationship between ticks and the immune system. As such, modulation of the hemostatic system as well as inflammation was important adaptive responses in the evolution of a blood-feeding lifestyle in soft ticks.     

Nuttalliella namaqua: a living fossil and closest relative to the ancestral tick lineage: implications for the evolution of blood-feeding in ticks

Ticks are monophyletic and composed of the hard (Ixodidae) and soft (Argasidae) tick families, as well as the Nuttalliellidae, a family with a single species, Nuttalliella namaqua. Significant biological differences in lifestyle strategies for hard and soft ticks suggest that various blood-feeding adaptations occurred after their divergence. The phylogenetic relationships between the tick families have not yet been resolved due to the lack of molecular data for N. namaqua. This tick possesses a pseudo-scutum and apical gnathostoma as observed for ixodids, has a leathery cuticle similar to argasids and has been considered the evolutionary missing link between the two families. Little knowledge exists with regard to its feeding biology or host preferences. Data on its biology and systematic relationship to the other tick families could therefore be crucial in understanding the evolution of blood-feeding behaviour in ticks. Live specimens were collected and blood meal analysis showed the presence of DNA for girdled lizards from the Cordylid family. Feeding of ticks on lizards showed that engorgement occurred rapidly, similar to argasids, but that blood meal concentration occurs via malpighian excretion of water. Phylogenetic analysis of the 18S nuclear and 16S mitochondrial genes indicate that N. namaqua grouped basal to the main tick families. The data supports the monophyly of all tick families and suggests the evolution of argasid-like blood-feeding behaviour in the ancestral tick lineage. Based on the data and considerations from literature we propose an origin for ticks in the Karoo basin of Gondwanaland during the late Permian. The nuttalliellid family almost became extinct during the End Permian event, leaving N. namaqua as the closest living relative to the ancestral tick lineage and the evolutionary missing link between the tick families.     

蜱类系统学研究进展

本文介绍了近年来蜱的编目和分类及系统发育和演化等方面的研究进展。对软蜱科(Argasidae)说明了属的变动;硬蜱科(Ixodidae)介绍了璃眼蜱亚科(Hyalomminae)和凹沟蜱亚科(Bothriocrotoninae)以及相应变动的属(新建的凹沟蜱属Bothriocroton和须角蜱属Cornupalpatum,合并的牛蜱属Boophilus、暗眼蜱属Anocentor和盲花蜱属Aponomma)。根据新的分类变更对已知种类进行了分析。至2006年,世界已知蜱类有3科18属897种,中国有2科10属119种。阐明了有关蜱类系统发育研究的主要观点,并讨论了其不足和有待深入研究的问题。目前,把形态学和分子生物学数据结合在一起的全证据方法,并结合蜱类和不同宿主之间的关系、动物地理学、古生物学以及比较寄生虫学的资料,成为解决蜱类系统发育问题的有效途径。     

Characterization of anti-hemostatic factors in the argasid, Argas monolakensis: implications for the evolution of blood-feeding in the soft tick family

To date, the only anti-hemostatic factors characterized for softs ticks are for Ornithodoros moubata and Ornithodoros savignyi, ticks that feeds mainly on mammals. This includes thrombin (ornithodorin and savignin), fXa (TAP and fXaI) and platelet aggregation (disagegin and savignygrin) inhibitors that belong to the BPTI-Kunitz protein family. This raises the question on how well anti-hemostatic factors will be conserved in other soft tick genera that feeds on other vertebrates such as birds. We characterized the anti-hemostatic factors from Argas monolakensis, a soft tick that feeds mainly on Californian gulls. The main anti-clotting factor (monobin) is an ortholog of ornithodorin and savignin and shows similar slow tight-binding kinetics. The main anti-platelet activities are apyrase and fibrinogen receptor antagonists (monogrins). The monogrins are orthologs of disagregin and savignygrin and like savignygrin presents the RGD integrin-recognition motif on the BPTI substrate-binding presenting loop. This implies that the anti-hemostatic factors evolved in the ancestral soft tick lineage and has been maintained in soft tick species from two distinct genera with different host preferences. The Argas derived anti-hemostatic factors bind to mammalian targets with affinities similar to that observed for their orthologs in the Ornithodoros genus. This cross-reactivity could have facilitated the switching of soft ticks from avian to mammalian hosts and can explain in part the ability of Argas ticks, to feed on humans, thereby remaining a possible health risk.     

蜱源抗凝血因子研究进展

蜱是一种人畜共患体表寄生虫,通过叮咬宿主和吸血,将病原体传播给宿主,引发多种疾病。凝血反应是人和动物的重要生理过程,是生理性止血的重要环节。蜱叮咬和吸食宿主血液周期长,在吸血过程中分泌多种抗凝物质,抑制凝血反应,可帮助蜱长时间保持吸血状态。目前,已知的蜱源抗凝物质依据其功能主要包括蛋白酶抑制剂、纤维蛋白(原)溶解剂、血小板聚集抑制剂和血管活性蛋白4大类。这些抗凝血物质可分别作用于凝血级联反应中内源性通路、外源性通路、共同通路中的关键步骤,以及促进纤蛋白溶解和抑制血小板激活,从而抑制宿主血管中的凝血反应。蛋白酶抑制剂主要通过抑制凝血级联反应共同通路中凝血酶和Xa因子活性;纤维蛋白(原)溶解剂引起纤维蛋白原的水解并延迟纤维蛋白凝块的形成;血小板聚集抑制剂通过降解血小板聚集激动剂,并结合血栓素A2(thromboxane A2, TXA2)和血小板上的αIIbβ3整合素抑制血小板聚集;血管活性蛋白抑制宿主血管收缩以及伤口愈合和血管生成。此外,还有一些蜱分泌的其他蛋白分子可通过不同的通路来实现抗凝血作用。本文对迄今为止各类蜱中发现的具有抗凝血活性的蛋白和小分子及其抗凝血作用机制进行总结阐述,将...     

Evolutionary changes in symbiont community structure in ticks

Ecological specialization to restricted diet niches is driven by obligate, and often maternally inherited, symbionts in many arthropod lineages. These heritable symbionts typically form evolutionarily stable associations with arthropods that can last for millions of years. Ticks were recently found to harbour such an obligate symbiont, Coxiella‐LE, that synthesizes B vitamins and cofactors not obtained in sufficient quantities from blood diet. In this study, the examination of 81 tick species shows that some Coxiella‐LE symbioses are evolutionarily stable with an ancient acquisition followed by codiversification as observed in ticks belonging to the Rhipicephalus genus. However, many other Coxiella‐LE symbioses are characterized by low evolutionary stability with frequent host shifts and extinction events. Further examination revealed the presence of nine other genera of maternally inherited bacteria in ticks. Although these nine symbionts were primarily thought to be facultative, their distribution among tick species rather suggests that at least four may have independently replaced Coxiella‐LE and likely represent alternative obligate symbionts. Phylogenetic evidence otherwise indicates that cocladogenesis is globally rare in these symbioses as most originate via horizontal transfer of an existing symbiont between unrelated tick species. As a result, the structure of these symbiont communities is not fixed and stable across the tick phylogeny. Most importantly, the symbiont communities commonly reach high levels of diversity with up to six unrelated maternally inherited bacteria coexisting within host species. We further conjecture that interactions among coexisting symbionts are pivotal drivers of community structure both among and within tick species.     

Variegin, a novel fast and tight binding thrombin inhibitor from the tropical bont tick

Tick saliva contains potent antihemostatic molecules that help ticks obtain their enormous blood meal during prolonged feeding. We isolated thrombin inhibitors present in the salivary gland extract from partially fed female Amblyomma variegatum, the tropical bont tick, and characterized the most potent, variegin, one of the smallest (32 residues) thrombin inhibitors found in nature. Full-length variegin and two truncated variants were chemically synthesized. Despite its small size and flexible structure, variegin binds thrombin with strong affinity (K(i) approximately 10.4 pM) and high specificity. Results using the truncated variants indicated that the seven residues at the N terminus affected the binding kinetics; when removed, the binding characteristics changed from fast to slow. Further, the thrombin active site binding moiety of variegin is in the region of residues 8-14, and the exosite-I binding moiety is within residues 15-32. Our results show that variegin is structurally and functionally similar to the rationally designed thrombin inhibitor, hirulog. However, compared with hirulog, variegin is a more potent inhibitor, and its inhibitory activity is largely retained after cleavage by thrombin.     

Co-phylogenetic analysis of Anaplasma phagocytophilum and its vectors, Ixodes spp. ticks

The coevolutionary history of Ixodes spp. ticks, the obligately tick-transmitted bacterial pathogen Anaplasma phagocytophilum, and its various rodent reservoir hosts world-wide is not known. According to coevolution theory, the most recently evolved of tick-bacterial complexes could have difficulty maintaining A. phagocytophilum in nature, because transmissibility has not been efficiently maximized. This study was intended to examine the phylogeographic history of I. ricinus-subgroup ticks and A. phagocytophilum, provide an estimate for the date of the divergence of A. marginale and A. phagocytophilum, and evaluate whether there is correspondence between tick and Anaplasma spp. trees. Analysis of Ixodes spp. ticks showed a New World clade consisting of I. scapularis and I. pacificus, European I. ricinus as a sister group to this clade, and Asian I. persulcatus as basal. Of the three A. phagocytophilum genes evaluated, the most resolution was provided by the ankA gene. ankA sequences formed an Old World clade with eastern North America strains as a sister clade. California strains were highly diverse and did not form a clade. Base substitution rates were very comparable along both A. marginale and A. phagocytophilum lineages. Based on 16S rDNA analysis, maximum and minimum divergence times of A. phagocytophilum and A. marginale were calculated to be 78,296,703 and 43,415,708 years, respectively. If A. phagocytophilum did closely coevolve with specific I. ricinus-subgroup tick species, then A. phagocytophilum strains could have specialized on local tick species and optimized local infectivity in the Old World and eastern US. However, lack of absolute resolution of tick trees and conflicting prevalence data (with low prevalence in Asia and western North America) preclude us from inferring a tight coevolutionary relationship of tick species from this phylogeographic analysis.     

Microbial community structure reveals instability of nutritional symbiosis during the evolutionary radiation of Amblyomma ticks

Mutualistic interactions with microbes have facilitated the adaptation of major eukaryotic lineages to restricted diet niches. Hence, ticks with their strictly blood‐feeding lifestyle are associated with intracellular bacterial symbionts through an essential B vitamin supplementation. In this study, examination of bacterial diversity in 25 tick species of the genus Amblyomma showed that three intracellular bacteria, Coxiella‐like endosymbionts (LE), Francisella‐LE and Rickettsia, are remarkably common. No other bacterium is as uniformly present in Amblyomma ticks. Almost all Amblyomma species were found to harbour a nutritive obligate symbiont, Coxiella‐LE or Francisella‐LE, that is able to synthesize B vitamins. However, despite the co‐evolved and obligate nature of these mutualistic interactions, the structure of microbiomes does not mirror the Amblyomma phylogeny, with a clear exclusion pattern between Coxiella‐LE and Francisella‐LE across tick species. Coxiella‐LE, but not Francisella‐LE, form evolutionarily stable associations with ticks, commonly leading to co‐cladogenesis. We further found evidence for symbiont replacements during the radiation of Amblyomma, with recent, and probably ongoing, invasions by Francisella‐LE and subsequent replacements of ancestral Coxiella‐LE through transient co‐infections. Nutritional symbiosis in Amblyomma ticks is thus not a stable evolutionary state, but instead arises from conflicting origins between unrelated but competing symbionts with similar metabolic capabilities.     

Coevolutionary analyses of the relationships between piroplasmids and their hard tick hosts

Host–parasite coevolution is a key driver of biological diversity. To examine the evolutionary relationships between piroplasmids and their hard tick hosts, we calculated the molecular clock and conducted phylogenetic analyses of both groups. Based on our results, we conclude that the divergence time of piroplasmids (~56 Mya) is later than divergence time of their hard tick hosts (~86 Mya). From analyses of the evolution of both piroplasmid and vector lineages and their association, we know that hard ticks transmit piroplasmids with high genus specificity and low species specificity.     

Tick-derived Kunitz-type inhibitors as antihemostatic factors

Endogenous Kunitz-type inhibitors target a large number of serine proteinases, including coagulation factors VIIa and Xa, but not thrombin. By contrast, several two-domain Kunitz inhibitors of this major procoagulant proteinase have been isolated from both soft ticks (e.g., ornithodorin from Ornithodoros moubata) and hard ticks (e.g., boophilin from Rhipicephalus (Boophilus) microplus). Surprisingly, these anticoagulants do not follow the canonical mechanism of proteinase inhibition. Instead, their N-terminal residues bind across the thrombin active-site cleft, while C-terminal modules interact with the basic exosite I. The reactive-site loop of boophilin remains fully accessible in its complex with thrombin, and might interact with FXa according to the standard mechanism. A conceptually similar inhibition mechanism is employed by a related inhibitor of the TF–FVIIa complex isolated from Ixodes scapularis, ixolaris. Significant variations to the Kunitz fold are encountered in several of these factors, and are particularly evident in the single-domain FXa inhibitor, O. moubata TAP, and in soft tick-derived platelet antiaggregants (e.g., O. moubata disagregin). Altogether, these antihemostatic factors illustrate the divergence between hard and soft ticks. The unsurpassed versatility of tick-derived Kunitz inhibitors establishes them as valuable tools for biochemical investigations, but also as lead compounds for the development of novel antithrombotics.     

The influence of tick behavior,biotope and host specificity on concerted evolution of the platelet aggregation inhibitor savignygrin,from the soft tick Ornithodoros savignyi

Ticks are obligate blood-feeding parasites that secrete anti-hemostatic components during feeding to enable control of the hemostatic system of the host. Complex interactions at the tick-host interface are an indication of the important role that the host played during tick evolution. The question is to what extent interaction with the host and the environment influences tick evolution. Previously, two isoforms (97% sequence identity) of savignygrin, an alphaIIbbeta3 antagonist, have been described. The presence of both isoforms within 20 random individuals confirmed that these isoforms must be recent gene duplicates. Analysis of the sequence differences between the isoforms shows a Kn/Ks ratio of 1, which indicates neutral selection for the isoforms. However, the biased localization of differences within the 3' end of the genes suggests that concerted evolution acts on the isoforms. Calculation of the divergence date between the isoforms (1.6-5.2 MYA) also indicates purifying selection, as ample time had passed after duplication, for inactivation of one gene copy. We conclude that concerted evolution has functioned to maintain a high copy number of the savignygrins in order for Ornithodoros savignyi to parasitize a wide host range. This contrasts with O. moubata that expresses the savignygrin homolog, disagregin, as a single copy at lower concentration levels and correlates with the confined habitat and consequently narrow host range of O. moubata. Recent "domestication" of O. savignyi due to animal husbandry practices could however, have reduced the selection constraints acting to maintain the gene copies as evidenced by the structural instability of one of the isoforms. Our results suggest that environmental factors and host associations do play an important role in the evolution of anti-hemostatic components in ticks.     

Inhibition mechanisms of hematophagous invertebrate compounds acting on the host blood coagulation and platelet aggregation pathways

To facilitate blood feeding, hematophagous invertebrates have evolved a sophisticated array of physiological compounds that counteract homeostatic systems and inflammatory reactions of the vertebrate host. For this reason, hematophagous invertebrates possess a variety of anticoagulation components that are inhibitors of coagulant factors or antagonists of the platelet receptor. The examination of kinetic data and the crystal structure analysis have exposed the inhibition mechanisms for many of these anticoagulant reagents. Here, we attempt to classify the antihemostatic molecules and to focus on the kinetic approaches that have been instrumental in defining these mechanisms.     

Evolution of Thrombin and Other Hemostatic Proteases by Survey of Protochordate, Hemichordate, and Echinoderm Genomes

Protochordate genomes enable a prevalence of hemostasis evolution. Broad searches were performed for homologs of human serine proteases of hemostasis on the genomes of Branchiostoma floridae, Saccoglossus kowalevskii, and Strongylocentrotus purpuratus. Sequences were analyzed by multiple bioinformatic tools. The survey revealed numerous homologous components. Amphioxus was rich in some serine proteases not accompanied by gamma-carboxyglutamic or kringle domains similar more to thrombin than to other coagulation factors. The serine proteases found in amphioxus exhibited the attributes similar to those of thrombin by phylogeny relationships, sequence conservation, gene synteny, spatial structure, and ligand docking. A few plasminogen- and plasminogen activators-like proteases with kringles were also present. Those serine proteases demonstrated the greatest proximity rather to plasminogen or plasminogen activators than to thrombin. Searching for homologs of serine protease hemostatic factors in acorn worm and sea urchin revealed several components similar to those found in amphioxus. Hypothetically, the common ancestor of chordates had three separate serine proteases that evolved independently into immunoglobulin-like and kringle proteases in lancelets, and prothrombin, plasminogen activators, and plasminogen in vertebrates. Ancestral proteases evolved in vertebrates into hemostasis factors after merging the proper N-terminal domains and duplications.     

Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species

The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.     

Autocidal control of ticks by silencing of a single gene by RNA interference

Ticks impact human and animal health worldwide and new control methods are needed to circumvent drawbacks of tick control by acaricide application including selection of drug resistant ticks and environmental pollution. Using RNA interference we silenced the expression of a single gene, subolesin, and produced ticks with diminished reproductive performance and prevented successful mating and production of viable offspring. We propose a sterile acarine technique (SAT) for reduction of tick populations by release of subolesin-silenced ticks. Conservation of subolesin among tick species suggests that SAT may be useful for control of many medically and economically important tick species.     

Phylogeny of the hard ticks (Ixodidae) inferred from 18S rRNA indicates that the genus Aponomma is paraphyletic

We examined the phylogeny of ticks (Acari:Parasitiformes:Ixodida) and their closest known mite relatives (Acari:Parasitiformes:Mesostigmata and Holothyrida) using 18S rRNA sequences. In our analyses, we included sequences from 36 taxa. Sequences for 13 hard ticks (Family Ixodidae), 5 soft ticks (Family Argasidae), and 2 mesostigmatid mites were obtained from the GenBank database and we generated sequences for 15 hard ticks and 1 holothyrid mite. Ten of these tick species were endemic to Australia. Our analyses indicated that the suborder Holothyrida is more closely related to Ixodida than to Mesostigmata, the group used as outgroup in earlier molecular studies. This finding is consistent with Lehtinen's (1991) hypothesis that the Holothyrida rather than the Mesostigmata is the sister-group to the Ixodida. Within the hard ticks the genus Aponomma and thus the family Amblyomminae were paraphyletic. Taxonomic revision of these taxa is needed. The genus Amblyomma was paraphyletic without the inclusion of "typical" Aponomma species (Ap. latum and Ap. fimbriatum). There was a basal divergence between endemic Australian and other species in both the Metastriata and the Prostriata divisions of the hard ticks.