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.     

Promoter analysis of macrophage- and tick cell-specific differentially expressed Ehrlichia chaffeensis p28-Omp genes

Background  

Ehrlichia chaffeensis is a rickettsial agent responsible for an emerging tick-borne illness, human monocytic ehrlichiosis. Recently, we reported that E. chaffeensis protein expression is influenced by macrophage and tick cell environments. We also demonstrated that host response differs considerably for macrophage and tick cell-derived bacteria with delayed clearance of the pathogen originating from tick cells.     

The major tick salivary gland proteins and toxins from the soft tick,Ornithodoros savignyi,are part of the tick Lipocalin family: implications for the origins of tick toxicoses

The origins of tick toxicoses remain a subject of controversy because no molecular data are yet available to study the evolution of tick-derived toxins. In this study we describe the molecular structure of toxins from the soft tick, Ornithodoros savignyi. The tick salivary gland proteins (TSGPs) are four highly abundant proteins proposed to play a role in salivary gland granule biogenesis of the soft tick O. savignyi, of which the toxins TSGP2 and TSGP4 are a part. They were assigned to the lipocalin family based on sequence similarity to known tick lipocalins. Several other tick lipocalins were also identified using Smith-Waterman database searches, bringing the tick lipocalin family up to 20. Phylogenetic analysis showed that most tick lipocalins group within genus-specific clades, suggesting that gene duplication and divergence of tick lipocalin function occurred after tick speciation, most probably during the evolution of a hematophagous lifestyle. TSGP2 and TSGP3 show high sequence identity and group terminal to moubatin, an inhibitor of collagen-induced platelet aggregation from the tick, O. moubata. However, no platelet aggregation inhibitory activity is associated with the TSGPs using ADP or collagen as agonists, suggesting that TSGP2 and TSGP3 duplicated after divergence of O. savignyi and O. moubata. This timing is supported by the absence of TSGP2-4 in the salivary gland extracts of O. moubata. The absence of TSGP2 and TSGP4 in salivary gland extracts from O. moubata correlates with the nontoxicity of this tick species. The implications of this study are that the various forms of tick toxicoses do not have a common origin, but must have evolved independently in those tick species that cause pathogenesis.     

Two seasons of tick paralysis in Victoria yet one season in Queensland and New South Wales,Australia

We studied 22,840 cases of tick paralysis in dogs and cats that were attributable to infestation with the eastern paralysis tick, Ixodes holocyclus. We report that the mortality rates from the holocyclotoxins of the tick or from euthanasia due to complications arising from tick paralysis in dogs and cats were 10% and 8%, respectively. The distribution of cases of tick paralysis among the 52 weeks of 22 years (1999 to 2020, inclusive) in four regions along the eastern coast of Australia revealed much about how the life-cycle of this tick varied among regions. The four regions in our study were: (i) Cairns, Innisfail, and surrounding postcodes in Far North Queensland; (ii) South East Queensland; (iii) Northern Beaches of Sydney in New South Wales; and (iv) the Shire of East Gippsland in Victoria. We found that the season of tick paralysis started earlier in more northerly latitudes than in more southerly latitudes. We also found that Victoria has two seasons of tick paralysis, one from approximately the third week of February to the first week of May, and another from approximately the third week of September to the third week of December, whereas all of the other regions we studied in eastern Australia only had one season of tick paralysis. When we studied the two seasons of tick paralysis in Victoria, we found a statistically significant negative correlation between the number of cases of tick paralysis between the two seasons: the more cases in one season, the fewer the cases in the next season. One possible explanation for the negative correlation may be immunity to I. holocyclus acquired by dogs and cats in the first season.     

Blocking the secretion of saliva by silencing the HlYkt6 gene in the tick Haemaphysalis longicornis

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) have been identified as the key components of the protein complexes that facilitate vesicle traffic, of which Ykt6 (from Saccharomyces cerevisiae, v-SNARE) is proved to be a multifunctional protein in the membrane fusion. In the present study, a tick homologue of Ykt6 (HlYkt6, predicted 22.6 kDa), was isolated from the ixodid tick Haemaphysalis longicornis. RT-PCR and Western blot analysis indicated that the gene and the encoded protein were expressed ubiquitously in different tissues of the partially fed adult tick. Silencing of the HlYkt6 gene resulted in a significant decrease of the engorged body weight (82.9 ± 26.8 mg vs. 232.17 ± 59.1 mg in the PBS-injected control group and 178.7 ± 57.0 mg in the GFP dsRNA-injected control group) and high mortality of replete ticks (100% in tested group vs. 4.8% in the PBS and 20.4% in GFP dsRNA-injected control groups). Disruption of HlYkt6 mRNA led to the suppression of saliva secretion, and a lower anticoagulant activity of the released liquid from the glands (APTT time: 25.25 ± 1.50 s) than that of the control groups (39.25 ± 0.50 s in the PBS-treated group and 40.0 ± 1.41 s in the GFP dsRNA-treated group). These results suggest the vital role of the HlYkt6 protein in the exocytosis of saliva proteins, the feeding and survival of ticks.     

Concordance of bacterial communities of two tick species and blood of their shared rodent host

High‐throughput sequencing is revealing that most macro‐organisms house diverse microbial communities. Of particular interest are disease vectors whose microbiome could potentially affect pathogen transmission and vector competence. We investigated bacterial community composition and diversity of the ticks Dermacentor variabilis (n = 68) and Ixodes scapularis (n = 15) and blood of their shared rodent host, Peromyscus leucopus (n = 45) to quantify bacterial diversity and concordance. The 16S rRNA gene was amplified from genomic DNA from field‐collected tick and rodent blood samples, and 454 pyrosequencing was used to elucidate their bacterial communities. After quality control, over 300 000 sequences were obtained and classified into 118 operational taxonomic units (OTUs, clustered at 97% similarity). Analysis of rarefied communities revealed that the most abundant OTUs were tick species‐specific endosymbionts, Francisella and Rickettsia, and the commonly flea‐associated bacterium Bartonella in rodent blood. An Arsenophonus and additional Francisella endosymbiont were also present in D. variabilis samples. Rickettsia was found in both tick species but not in rodent blood, suggesting that it is not transmitted during feeding. Bartonella was present in larvae and nymphs of both tick species, even those scored as unengorged. Relatively, few OTUs (e.g. Bartonella, Lactobacillus) were found in all sample types. Overall, bacterial communities from each sample type were significantly different and highly structured, independent of their dominant OTUs. Our results point to complex microbial assemblages inhabiting ticks and host blood including infectious agents, tick‐specific endosymbionts and environmental bacteria that could potentially affect arthropod‐vectored disease dynamics.     

Tick saliva microbiomes isolated from engorged and partially fed adults of Haemaphysalis flava tick females

The tick Haemaphysalis flava is one of the most significant blood‐feeding arthropod parasites and is a vector for numerous human and animal pathogens. However, a comprehensive investigation of the microbial communities found in the saliva of this tick species is lacking. This study used 16S rRNA Illumina sequencing to characterize the compositions of microbiomes present in saliva and whole tick samples isolated from engorged and partially fed adult H. flava females. This revealed that the bacterial diversity present in tick saliva increased after a prolonged blood meal, and that the species diversity found in saliva was significantly higher than that of whole ticks. Three bacteria phyla, in particular, made up more than 80% of the microbial community across all samples—Proteobacteria, Firmicutes and Actinobacteria. Furthermore, some of the genera identified in this study had not previously been reported in ticks before, such as Facklamia, Vagococcus, Ruminococcus, Lachnospira, Bradyrhizobium, Peptostreptococcus, Jeotgalicoccus, Roseburia, Brachybacterium, Sporosarcina, u114, Megamonas and Dechloromonas. Finally, we found that many of the isolated bacteria were opportunistic pathogens, indicating a potential risk to humans and livestock exposed to H. flava. These results will contribute to fully understanding the transmission of tick‐borne pathogens.     

Microbiome changes through ontogeny of a tick pathogen vector

Blacklegged ticks (Ixodes scapularis) are one of the most important pathogen vectors in the United States, responsible for transmitting Lyme disease and other tick‐borne diseases. The structure of a host's microbial community has the potential to affect the ecology and evolution of the host. We employed high‐throughput sequencing of the 16S rRNA gene V3‐V4 hypervariable regions in the first study to investigate the tick microbiome across all developmental stages (larvae, nymphs, adults). In addition to field‐collected life stages, newly hatched laboratory‐reared larvae were studied to determine the baseline microbial community structure and to assess transovarial transmission. We also targeted midguts and salivary glands due to their importance in pathogen maintenance and transmission. Over 100 000 sequences were produced per life stage replicate. Rickettsia was the most abundant bacterial genus across all sample types matching mostly the Ixodes rickettsial endosymbionts, and its proportion decreased as developmental stage progressed, with the exception of adult females that harboured a mean relative abundance of 97.9%. Laboratory‐reared larvae displayed the lowest bacterial diversity, containing almost exclusively Rickettsia. Many of the remaining bacteria included genera associated with soil, water and plants, suggesting environmental acquisition while off‐host. Female organs exhibited significantly different β‐diversity than the whole tick from which they were derived. Our results demonstrate clear differences in both α‐ and β‐diversity among tick developmental stages and between tick organs and the tick as a whole. Furthermore, field‐acquired bacteria appear to be very important to the overall internal bacterial community of this tick species, with influence from the host bloodmeal appearing limited.     

Microbial communities and interactions in the lone star tick,Amblyomma americanum

To quantify microbial composition and interactions, we identified prokaryotic communities in the lone star tick (Amblyomma americanum) based on 16S rRNA gene sequences and direct probing. The lone star tick is the vector of emerging diseases and host to additional symbionts of unknown activity, and is representative of other blood‐sucking arthropods. We evaluated the potential for vertical (transovarial) transmission by molecular analysis of microbial symbionts from egg and larval clutches. Direct probing of adults (N = 8 populations from the southeastern and midwestern USA, 900 ticks total) revealed three vertically transmitted symbionts: a Coxiella symbiont occurred at 100% frequency, Rickettsia species occurred in 45–61% of all ticks in every population and an Arsenophonus symbiont occurred in 0–90% of ticks per population. Arsenophonus and Rickettsia exhibited significant heterogeneity in frequency among populations. The human pathogens Ehrlichia chafeensis and Borrelia lonestari were rare in most populations. Additional microbes were detected sporadically. Most ticks (78%) were co‐infected by two or three microbes but statistical analysis indicated no significant deviation from random co‐occurrence. Our findings indicate that microbial communities within lone star ticks are diverse, and suggest that direct probing for a wider range of prokaryotes and application of quantitative polymerase chain reaction (PCR) may provide further insights into microbial interactions within disease vectors. Our results also emphasize the close phylogenetic relationship between tick symbionts and human pathogens, and consistent differences in their prevalence.     

Immunocytochemical mapping of FMRFamide-like peptides in the argasid tick Ornithodoros parkeri and the ixodid tick Dermacentor variabilis

FMRFamide-like immunoreactivity was studied in the argasid tick Ornithodoros parkeri and the ixodid tick Dermacentor variabilis using immunocytochemistry based on the peroxidase-antigeroxidase method. FMRFamide-like immunoreactive cells are widely distributed in various regions of the tick synganglion including protocerebral, cheliceral, stomodeal, palpal, pedal I–IV, and opisthosomal regions in both species. However, there is one layer of immunoreactive cells located on the dorsal surface of the postoesophageal part of the synganglion that is found only in D. variabilis. Besides the immunoreactivity within the cell body and its axons, the neuropile and the neural lamella (the extracellular sheath of the synganglion) are rich in immunoreactive materials. Some coxal muscles are innervated by the FMRFamide-like immunoreactive processes of the nerve from the pedal ganglion.     

Towards the selection of cattle for tick resistance in Africa

Half-body tick collections and visual assessment of tick burdens were performed monthly over six months on 100 bulls at the Kenya National Boran Stud, Mutara Ranch, Kenya.Boophilus decoloratus predominated among several tick species infesting the animals. Burdens ofB. decoloratus and total tick burdens were highly correlated. Rankings of relative tick resistance among bulls were consistent from month to month. Rankings based on visual assessment were very close to those based on actual tick burdens. Animals with thin skins appeared to carry fewer ticks, but tick burden bore no relationship to coat colour. The results suggest that simple visual inspection of total tick burdens may be a suitable basis for the selection of tick resistance in cattle.     

Potential tick vectors for Theileria equi in Israel

Theileria equi Mehlhorn and Schein, 1998 (Piroplasmida: Babesiidae) is an important tick-borne pathogen of horses that is highly endemic in many parts of the world, including Israel. The present study evaluated the potential roles of five hard tick species [Hyalomma excavatum Koch, 1844; Hyalomma marginatum Koch, 1844; Rhipicephalus turanicus Pomerantsev 1936; Rhipicephalus annulatus Say, 1821; Haemaphysalis parva (Neumann, 1897) (all: Ixodida: Ixodidae)], previously found to infest horses in Israel, in acting as vectors for piroplasmosis. For this, DNA was extracted from whole ticks and, when possible, from the salivary glands in each species (n = 10–59). Polymerase chain reaction amplification and sequencing of the 18S rRNA gene were used to detect T. equi in 48 of the 127 ticks (37.8%) and in 21 of the 90 extracted salivary glands (23.3%) in all five species. All but two sequences were classified as T. equi genotype A; the remaining two were classified as genotype D. The findings of this study point to Ha. parva and R. annulatus as potential novel vectors of T. equi, and suggest that parasite genotype selection occurs within the tick vector.     

Superoxide dismutases from larvae of the camel tick Hyalomma dromedarii

Three superoxide dismutases (EC 1.15.1.1) (TLSOD1, TLSOD2 and TLSOD3) were purified from larvae of the camel tick Hyalomma dromedarii by ammonium sulfate precipitation, ion exchange and gel filtration columns. SDS-PAGE revealed that the subunit molecular masses of the SODs are 40 ± 2 kDa, 67 ± 1.5 kDa and 45 ± 2.6 kDa for TLSOD1, TLSOD2 and TLSOD3, respectively. TLSOD1 and TLSOD2 are monomeric proteins, while TLSOD3 isoenzyme exhibits dimeric structure with native molecular mass of 90 kDa. The pI values are estimated at pH 8.0, pH 7.2 and pH 6.6 for the three SODs which displayed pH optima at 7.6, 8.0 and 7.8, respectively. CuCl₂ and ZnCl₂ increase the activity of TLSOD2 and TLSOD3, while MnCl₂ increases the activity of TLSOD1. KCN inhibits the activity of TLSOD2 and TLSOD3, while a remarkable resistance of TLSOD1 isoenzyme was detected. TLSOD1 is suggested to be a manganese containing isoenzyme while TLSOD2 and TLSOD3 are suggested to be copper/zinc-containing isoenzymes. These results indicate the presence of three different forms of SODs in the larval stage of camel tick. This finding will contribute to our understanding of the physiology of these ectoparasites and the development of non-traditional methods to control them.     

Paradigms in tick evolution

The study of tick evolution may be classified into disciplines such as taxonomy and systematics, biogeography, evolution and development (evo-devo), ecology, and hematophagy. These disciplines overlap and impact each other to various extents. Advances in one field may lead to paradigm shifts in our understanding of tick evolution not apparent to other fields. The current study considers paradigm shifts that occurred, are in the process, or may occur in future for the disciplines that study tick evolution. Some disciplines have undergone significant changes, while others may still be developing their own paradigms. Integration of these various disciplines is essential to come to a holistic view of tick evolution; however, maturation of paradigms may be necessary before this vision can be attained.     

The efficiency of patch sampling for determination of relative tick burdens in comparison with total tick counts

Quanttative data on host tick burdens ar fundamental for the initiation of control strategies and effective management of wildlife populations, but the methods of live sampling employed for domestic animals are unsuitable for sampling wild animals. Despite advances in the use of destructive methods (the scrub and digestion techniques) to obtain measures of the total tick burden on wildlife, these methods are too involved for many field workers, who often need only measures of relative tick burden. Recently, patch sampling methods have been introduced whereby only certain predilection sites are sampled, the presumption being that the number of ticks collected gives an indiccation of the relative degree of infestation. We examined the validity of patch sampling as a measure of relative tick burden by comparing adult ticks collected from the ears, head, neck, foreleg and perianal region of impala (aepyceros melampus) with total tick burdens of the same animals derived from the digestion technique. Adult ticks from patch sampling were positively and significantly correlated with total adults and total ticks (larvae, nymphs, and adults) on impala, with ticks patch sampled from the neck showing the highest correlation with the total tick burden. Comparison of relative tick loads from patch sampling with absolute tick loads from digestion for three classes of impala (females, bachelor males and territorial males) gave qualitatively similar results. We conclude that, when measures of relative tick load are sufficient and destructive sampling is not feasible, patch sampling can provide reliable information on relative tick burdens that are positively correlated with the total tick burden.     

蜱类分类系统的变更

随着分子生物学技术在蜱类研究中的应用,及对蜱类系统发生的深入研究,蜱的分类及命名发生了很大变更。而中国在这一领域的研究还停留在20世纪90年代,分类系统过时。文章对中国曾经采用的蜱类分类系统与现今世界上普遍认可的分类系统进行了详细比较,以期引起注意,从而促进蜱类系统学及其他研究领域的发展。     

Metarhizium anisopliae conidial responses to lipids from tick cuticle and tick mammalian host surface

Conidial germination and the formation of appressoria are important events in the interactions between entomopathogenic fungi and their arthropod hosts. In this study, we demonstrate the effects of lipids extracted from tick epicuticle and the surface of a mammalian host (calf) on conidial germination and the development of appressoria in two subspecies of Metarhizium anisopliae, M. anisopliae var. anisopliae (M.an.an.-7) and M. anisopliae var. acridum (M.an.ac.-5), which have different levels of virulence toward ticks. Pentane extracts of epicuticles of ticks susceptible and resistant to fungal infection always stimulated the germination of M.an.an.-7 conidia and the development of their appressoria; whereas the effects of dichloromethane (DCM) extracts of tick epicuticle varied depending on the tick. The DCM extracts from most of the tick species and developmental stages stimulated conidial germination and/or the formation of appressoria in M.an.an.-7. However, a DCM extract of lipids from the most resistant tick, engorged Hyalomma excavatum female, inhibited the germination of M.an.an.-7 conidia. Conidia of the non-virulent M.an.ac.-5 did not germinate on agarose amended with any of the examined tick extracts. However, when the tick extracts were placed on bactoagar, conidial germination increased 7- to 8-fold. Extracts from the skin, hair and ear secretions of a calf stimulated conidial germination and the formation of appressoria in M.an.an.-7, but not M.an.ac.-5. This study demonstrates that lipids from tick epicuticles and mammalian skin selectively affect the germination of conidia of entomopathogenic fungi. The effects of these lipids may explain the variability in tick control these fungi provide for different hosts.     

Tritrophic interactions between a fungal pathogen,a spider predator,and the blacklegged tick

The blacklegged tick Ixodes scapularis is the primary vector for the bacterium causing Lyme disease in eastern North America and for other medically important pathogens. This species is vulnerable to attack by fungal pathogens and arthropod predators, but the impacts of interactions between biocontrol agents have not been examined. The biocontrol agent Met52^®, containing the entomopathogenic fungus Metarhizium brunneum (=M. anisopliae), controls blacklegged ticks with efficacy comparable to chemical acaricides. The brush‐legged wolf spider Schizocosa ocreata is a predator of I. scapularis that reduces their survival under field conditions. We conducted a field microcosm experiment to assess the compatibility of Met52 and S. ocreata as tick biocontrol agents. We compared the fits of alternative models in predicting survival of unfed (flat) and blood‐fed (engorged) nymphs. We found the strongest support for a model that included negative effects of Met52 and S. ocreata on flat nymph survival. We found evidence for interference between biocontrol agents, with Met52 reducing spider survival, but we did not find a significant interaction effect between the two agents on nymph survival. For engorged nymphs, low recovery rates resulted in low statistical power to detect possible effects of biocontrol agents. We found that nymph questing activity was lower when the spider was active above the leaf litter than when the spider was unobserved. This provides the first evidence that predation cues might affect behavior important for tick fitness and pathogen transmission. This study presents field microcosm evidence that the biopesticide Met52 and spider Schizocosa ocreata each reduced survival of blacklegged ticks Ixodes scapularis. Met52 reduced spider survival. Potential interference between Met52 and the spider should be examined at larger scales, where overlap patterns may differ. Ticks were more likely to quest when the spider was inactive, suggesting the ticks changed their behavior to reduce danger.