Tick Lineage Research Articles

Efficacy of hydroethanolic extract of Randia aculeata seed against the southern cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) on naturally infested cattle under field conditions.

The cattle tick Rhipicephalus (Boophilus) microplus is a major problem of concern for cattle industry in tropical and subtropical areas. Control of cattle tick is based mainly on the use of chemical acaricides, which has contributed to the emerging problem of selection of resistant tick lineages. Plants have been used as an alternative to conventional acaricidal drugs. On the other hand, the acaricidal activity of hydroethanolic extract of Randia aculeata seed (EHRA) has been demonstrated against R. microplus under laboratory conditions. However, the utility of EHRA seed as a potential acaricidal needs to be determined under field conditions. For this reason, the aim of this study was to evaluate the efficacy of the EHRA against R. microplus sprayed on naturally infested calves, determine the effect of theEHRA seedon acetylcholinesterase activity in R. microplus larval and identify the chemical composition of EHRA. Forty-five male calves were divided in three groups and treated with: G1 water; G2 EHRA 20% w/v and G3 coumaphos 0.2% v/v. Acetylcholinesterase (AChE) activity in R. microplus larvae was determined by a colorimetric assay. The chemical composition of EHRA was accessed through HPLC/MS. Significantly fewer ticks were observed after 24h on the treated group compared to control group. EHRA significantly inhibitedin vitroAChE activity inR.microplus at all tested concentrations. Chlorogenic acid, vanillinic acid, p-coumaric acid, caffeic acid. rutin, quercetin, (-)-epicatechin, 4-hydroxybenzoic acid, quercetin, vanillin, 2,4-dimethoxy-6-methylbenzoic acid, scopoletin and ferulic acid were identified in the extract. The results provided new data for the elucidation of the mechanisms of EHRA acaricide action and to further evaluate the use as a new alternative control agent againstR. microplus under in vivo conditions.

Geographic Distribution and Seasonality of Brown Dog Tick Lineages in the United States.

Two lineages of brown dog ticks (Rhipicephalus sanguineus sensu lato (s.l.), Latreille [Acari: Ixodidae]) have been described in North America: temperate and tropical. To characterize the distribution of these lineages across this region and evaluate seasonal activity, a 12S rRNA mitochondrial gene fragment was sequenced from R. sanguineus s.l. collected from hundreds of dogs and cats from different locations across 25 of the 50 states from 2018 to 2021. Infestations with temperate lineage predominated (78.5%) and were identified on pets from 20 states, with most (83.5%) from areas with annual mean daily average temperature <20°C. Tropical lineage submissions were less common (19.3%), submitted from 15 states, and most (80.0%) tropical lineage ticks were from areas with an annual mean daily average temperature >20°C. Although travel history was not obtained for all dogs, when tropical lineage infestations were found in colder regions, follow up conversations with veterinarians suggested some of these infestations may have resulted from recent travel of dogs. A limited number (2.2%) of dogs from Arizona and Texas were co-infested with both lineages. Both temperate and tropical lineage ticks were collected from pets in every month of the year. Temperate lineage infestations were primarily collected March through August while tropical lineage infestations were more often collected June through November. These data confirm at least two lineages of R. sanguineus s.l. are present in the United States, each predominating in distinct, overlapping geographies, and suggest that peak activity of each lineage occurs at different times of the year.

Tropical and Temperate Lineages of Rhipicephalus sanguineus s.l. Ticks (Acari: Ixodidae) Host Different Strains of Coxiella-like Endosymbionts.

Nonpathogenic bacteria likely play important roles in the biology and vector competence of ticks and other arthropods. Coxiella, a gram-negative gammaproteobacterium, is one of the most commonly reported maternally inherited endosymbionts in ticks and has been associated with over 40 tick species. Species-specific Coxiella-like endosymbionts (CLEs) have been reported in the brown dog tick, Rhipicephalus sanguineus sensu lato (Acari: Ixodidae), throughout the world, while recent research suggests low Coxiella diversity among tick species. We investigated CLE diversity among R. sanguineus s.l. ticks across Arizona. We detected 37 recurrent sequence variants (SVs) of the symbiont, indicating greater diversity in these symbiotic bacteria than previously reported. However, two SVs accounted for the vast majority of 16S rRNA amplicon reads. These two dominant CLEs were both closely related to Candidatus C. mudrowiae, an identified symbiont of Rhipicephalus turanicus ticks. One strain strongly associated with the tropical lineage of R. sanguineus s.l. while the other was found almost exclusively in the temperate lineage, supporting the conclusion that CLEs are primarily vertically transmitted. However, occasional mismatches between tick lineage and symbiont SV indicate that horizontal symbiont transfer may occur, perhaps via cofeeding of ticks from different lineages on the same dog. This study advances our understanding of CLE diversity in Rh. sanguineus s.l.

Characterization of the complete mitochondrial genome of Ixodes granulatus (Ixodidae) and its phylogenetic implications.

Ticks are deemed to be second only to mosquitoes as the most common vector of human infectious diseases worldwide that give rise to human and animal diseases and economic losses to livestock production. Our understanding of the phylogenetic analysis between tick lineages has been restricted by the phylogenetic markers of individual genes. Genomic data research could help advance our understanding of phylogenetic analysis and molecular evolution. Mitochondrial genomic DNA facilitated the phylogenetic analysis of eukaryotes containing ticks. In this study, we sequenced and assembled the circular complete mitogenome information of Ixodes granulatus. The 14,540-bp mitogenome consists of 37 genes, including 13 protein-coding genes (PCGs), two genes for ribosomal RNA (rRNAs), and 22 genes for transfer RNA (tRNAs), and the origin of the L-strand replication region. The directions of the coding strand and component genes in the non-Australasian Ixodes mitochondrial genome were similar to those found in most other Australasian Ixodes, except for the loss of a lengthy control region. The phylogenetic tree based on maximum likelihood (ML) and Bayesian inference (BI) computational algorithms showed that I. granulatus exhibits a close relationship with I. hexagonus and I. ricinus. To our knowledge, this is the first study exploring the complete mitogenome for the species I. granulatus. Our results provide new insights for further research on the evolution, population genetics, systematics, and molecular ecology of ticks.

De novo assembled salivary gland transcriptome and expression pattern analyses for Rhipicephalus evertsi evertsi Neuman, 1897 male and female ticks

Ticks secrete proteins in their saliva that change over the course of feeding to modulate the host inflammation, immune responses, haemostasis or may cause paralysis. RNA next generation sequencing technologies can reveal the complex dynamics of tick salivary glands as generated from various tick life stages and/or males and females. The current study represents 15,115 Illumina sequenced contigs of the salivary gland transcriptome from male and female Rhipicephalus evertsi evertsi ticks of early, mid and late feeding stages from 1320 separate assemblies using three short read assemblers. The housekeeping functional class contributed to the majority of the composition of the transcriptome (80%) but with lower expression (51%), while the secretory protein functional class represented only 14% of the transcriptome but 46% of the total coverage. Six percent had an unknown status contributing 3% of the overall expression in the salivary glands. Platelet aggregation inhibitors, blood clotting inhibitors and immune-modulators orthologous to the ancestral tick lineages were confirmed in the transcriptome and their differential expression during feeding in both genders observed. This transcriptome contributes data of importance to salivary gland biology and blood feeding physiology of non-model organisms.

Sentinel surveillance of Lyme disease risk in Canada, 2019: Results from the first year of the Canadian Lyme Sentinel Network (CaLSeN).

Lyme disease is an emerging vector-borne zoonotic disease of increasing public health importance in Canada. As part of its mandate, the Canadian Lyme Disease Research Network (CLyDRN) launched a pan-Canadian sentinel surveillance initiative, the Canadian Lyme Sentinel Network (CaLSeN), in 2019. To create a standardized, national sentinel surveillance network providing a real-time portrait of the evolving environmental risk of Lyme disease in each province. A multicriteria decision analysis (MCDA) approach was used in the selection of sentinel regions. Within each sentinel region, a systematic drag sampling protocol was performed in selected sampling sites. Ticks collected during these active surveillance visits were identified to species, and Ixodes spp. ticks were tested for infection with Borrelia burgdorferi, Borrelia miyamotoi, Anaplasma phagocytophilum, Babesia microti and Powassan virus. In 2019, a total of 567 Ixodes spp. ticks (I. scapularis [n=550]; I. pacificus [n=10]; and I. angustus [n=7]) were collected in seven provinces: British Columbia, Manitoba, Ontario, Québec, New Brunswick, Nova Scotia and Prince Edward Island. The highest mean tick densities (nymphs/100 m2) were found in sentinel regions of Lunenburg (0.45), Montréal (0.43) and Granby (0.38). Overall, the Borrelia burgdorferi prevalence in ticks was 25.2% (0%-45.0%). One I. angustus nymph from British Columbia was positive for Babesia microti, a first for the province. The deer tick lineage of Powassan virus was detected in one adult I. scapularis in Nova Scotia. CaLSeN provides the first coordinated national active surveillance initiative for tick-borne disease in Canada. Through multidisciplinary collaborations between experts in each province, the pilot year was successful in establishing a baseline for Lyme disease risk across the country, allowing future trends to be detected and studied.

Mitochondrial sequences of Rhipicephalus and Coxiella endosymbiont reveal evidence of lineages co-cladogenesis.

Rhipicephalus ticks are competent vectors of several pathogens, such as Spotted Fever Group Rickettsiae (SFGR) and many Babesia species. Within this genus, different R. sanguineus s.l. lineages show an unequal vector competence and resistance regarding some pathogenic strains. Current literature supports that tick endosymbionts may play an essential role in the transmission ability of a vector. Indeed, the microbial community of Rhipicephalus seems to be dominated by Coxiella-like endosymbionts (CLE). Still, their co-evolutionary associations with the complicated phylogeny of Rhipicephalus lineages and their transmissible pathogens remain unclear. We performed a phylogenetic congruence analysis to address whether divergent R. sanguineus s.l. lineages had a different symbiont composition. For that, we applied a PCR based approach to screen part of the microbial community present in 279 Rhipicephalus ticks from the Iberian Peninsula and Africa. Our analyses detected several qPCR-positive signals for both SFGR and Babesia species, of which we suggest R. sanguineus-tropical lineage as a natural vector of Babesia vogeli and R. sanguineus-temperate lineage of SFGR. The acquisition of 190 CLE sequences allowed to evaluate co-phylogenetic associations between the tick and the symbiont. With this data, we observed a strong but incomplete co-cladogenesis between CLE strains and their Rhipicephalus tick lineages hosts.

Contribution of Canine‐Based Factors to the Spread of RMSF in Arizona and Mexico

Prior to 2003, the number of annual cases of Rocky Mountain spotted fever (RMSF) reported in Arizona was reliably close to zero. However, since an initial RMSF outbreak in 2003, case rates have rapidly increased in Arizona and continue to rise. This outbreak has been tied to a novel vector, Rhipicephalus sanguineus, the brown dog tick. Since then, the vectoring capacity of the brown dog tick is seemingly spreading as Northern Mexico across the border from Arizona has had over 4000 reported cases since 2008, with mortality rates as high as 38%. This discovery was somewhat unsettling considering the continental‐wide distribution of these ticks. Given this is a zoonotic disease, canines act as an amplifier for the bacterial agent Rickettsii rickettsii and then brown dog ticks pass it on to humans as an incidental host. Therefore, the need to understand the canine‐based factors that drive the spread of RMSF in this region has become more urgent.Previously, we have identified three distinct lineages of R. sanguineus within the region with variation in rickettsial infection rates between clades. This could lead to the distribution of ticks on a canine patient, the tick infection rate, and canine seroprevalence driving the human case rate that we see across Arizona and New Mexico. To test this hypothesis, we have focused on the canine as the unit of interest in our epidemiological approach. To this end, we collected all of the ticks and serum from a series of canine patients from five locations across distributed across Arizona and New Mexico. These ticks were phylogenetically categorized based on sequencing of mitochondrial genes and tested for the presence of rickettsial DNA. The serum was tested for rickettsial antibodies using immuno‐fluorescence assay.The results of this study show that the population of ticks found on an individual dog tend to be monophyletic belonging to a single clade, meaning ticks taken off the same canine are more closely related to one another than to ticks taken off other canines. This implies that the distribution of tick clades across the region plays a fundamental role in the transmission of RMSF. Moreover, while the rickettsial prevalence differs between these different tick lineages, we have found that the likelihood of one tick being infected on a canine is directly correlated to the number of other ticks infected on the canine. Therefore, transmission among feeding groups and co‐localized ticks is a major determinant of canine infection rates. Given these two factors drive the canine infection rate, it is likely they drive the human rate as well. These data were then used to evaluate the feasibility of a canine‐based vaccine by calculating the basic reproduction rate among other variables, which allows us to assess the plausibility of achieving herd immunity through vaccination campaigns. These methods will help to control the spread of RMSF to humans as well as prevent future outbreaks.Support or Funding InformationMWU One Health Award to JVB, MWU REAP Award to JVB, MWU Kenneth A. Suarez Award to SM, MWU CGS Funding to AO

Notes on maternal behaviour in soft ticks: Specifically observed in Argas (Argas) striatus Bedford, 1932 and Argas (Secretargas) transgariepinus White, 1846

Maternal behaviour (carrying of larvae on the opisthosoma) in ticks has thus far only been observed in Antricola (Parantricola) marginatus and was considered a unique derived adaptation of this genus. The authors extend this observation to two additional argasid species, namely Argas (Argas) striatus and Argas (Secretargas) transgariepinus. In addition, brooding behaviour over eggs were observed with A. (S.) transgariepinus. Maternal behaviour may be an evolutionary adaptation to ecological challenges in habitats unsuited for larval survival and may be related to the presence of pulvilli in larvae. This adaptation might have been present in the ancestral tick lineage since pulvilli occur in all tick families, and may have been derived from a more ancient adaptation in chelicerates where maternal behaviour was common. Female A. (S.) transgariepinus also possess a unique area on their ventral abdomen that is absent in males and may be a unique adaptation for maternal behaviour in this species. Phylogenetic analysis of the 16S rRNA genes for both species indicate that they are unique lineages that group basal to other members of the Argas genus, supporting the possibility that they harbour ancestral traits for this group.

Multiple Acquisitions of Pathogen-Derived Francisella Endosymbionts in Soft Ticks.

Bacterial endosymbionts of ticks are of interest due to their close evolutionary relationships with tick-vectored pathogens. For instance, whereas many ticks contain Francisella-like endosymbionts (FLEs), others transmit the mammalian pathogen Francisella tularensis. We recently sequenced the genome of an FLE present in the hard tick Amblyomma maculatum (FLE-Am) and showed that it likely evolved from a pathogenic ancestor. In order to expand our understanding of FLEs, in the current study we sequenced the genome of an FLE in the soft tick Ornithodoros moubata and compared it to the genomes of FLE-Am, Francisella persica—an FLE in the soft tick Argus (Persicargas) arboreus, Francisella sp. MA067296—a clinical isolate responsible for an opportunistic human infection, and F. tularensis, the established human pathogen. We determined that FLEs and MA067296 belonged to a sister taxon of mammalian pathogens, and contained inactivated versions of virulence genes present in F. tularensis, indicating that the most recent common ancestor shared by FLEs and F. tularensis was a potential mammalian pathogen. Our analyses also revealed that the two soft ticks (O. moubata and A. arboreus) probably acquired their FLEs separately, suggesting that the virulence attenuation observed in FLEs are not the consequence of a single acquisition event followed by speciation, but probably due to independent transitions of pathogenic francisellae into nonpathogenic FLEs within separate tick lineages. Additionally, we show that FLEs encode intact pathways for the production of several B vitamins and cofactors, denoting that they could function as nutrient-provisioning endosymbionts in ticks.

Gene Duplication and Protein Evolution in Tick-Host Interactions.

Ticks modulate their hosts' defense responses by secreting a biopharmacopiea of hundreds to thousands of proteins and bioactive chemicals into the feeding site (tick-host interface). These molecules and their functions evolved over millions of years as ticks adapted to blood-feeding, tick lineages diverged, and host-shifts occurred. The evolution of new proteins with new functions is mainly dependent on gene duplication events. Central questions around this are the rates of gene duplication, when they occurred and how new functions evolve after gene duplication. The current review investigates these questions in the light of tick biology and considers the possibilities of ancient genome duplication, lineage specific expansion events, and the role that positive selection played in the evolution of tick protein function. It contrasts current views in tick biology regarding adaptive evolution with the more general view that neutral evolution may account for the majority of biological innovations observed in ticks.

Molecular phylogeny of soft ticks (Ixodida: Argasidae) inferred from mitochondrial genome and nuclear rRNA sequences

The genus-level classification of soft ticks (Argasidae) is controversial. A previous phylogenetic analysis of morphological and developmental characters found that the genus Ornithodoros was paraphyletic and raised a new genus, Carios, for species previously in the genera Antricola, Argas, Ornithodoros, and Nothoaspis (Klompen and Oliver, 1993). Genetic analyses of soft ticks to date have been limited to 16S rRNA, which is not highly phylogenetically informative for this group. We sequenced the entire mitochondrial genomes of 7 species of soft ticks, and the partial mitochondrial genomes of a further 5 species of soft ticks. We used these sequences to test the genus-level classification of soft ticks. Our analyses strongly support a clade of Neotropical species (mostly bat-associated) within the subfamily Ornithodorinae. This clade, which we call Neotropical Ornithodorinae, has species from 2 genera, Antricola and Nothoaspis, and 2 subgenera, Ornithodoros (Alectorobius) and Ornithodoros (Subparmatus). We also addressed the phylogenetic position of Ornithodoros savignyi, the type species of the genus Ornithodoros. Our analysis strongly supports a clade consisting of Ornithodoros savignyi and 4 other Ornithodoros species: Or. brasiliensis, Or. moubata, Or. porcinus, and Or. rostratus. This clade, Ornithodoros sensu stricto, did not contain the Alectorobius and Subparmatus species, Or. (Alectorobius) fonsecai, Or. (Alectorobius) capensis, and Or. (Subparmatus) marinkellei, which in traditional classification schemes have been placed in the genus Ornithodoros. Our comparison of mitochondrial rRNA, nuclear rRNA, and mitochondrial genome analyses show that only mitochondrial genome sequences have the potential to resolve the controversial phylogenetic relationships within the major soft tick lineages, such as the taxonomic status of Carios sensu Klompen and Oliver (1993).

The host preferences of Nuttalliella namaqua (Ixodoidea: Nuttalliellidae): a generalist approach to surviving multiple host-switches

Nuttalliella namaqua has been described as a "living fossil" and the closest extant species to the ancestral tick lineage. It was previously proposed that the Nuttalliella lineage parasitized reptile-like mammals in the Permian and had to switch hosts several times due to mass or host lineage extinctions. Extant hosts include girdled lizards and murid rodents. The current study extends knowledge on the extant host range of N. namaqua using gut meal analysis of field collected specimens. Nymphs and females can parasitize a variety of reptiles that includes skinks, geckos and girdled lizards. Blood-meal from a hyrax was also detected in a specimen suggesting that N. namaqua could parasitize a broader range of mammals than the previously suggested murid rodents. Rather than being host specific, N. namaqua is proposed to be a generalist and the ability to switch and parasitize multiple hosts allowed it to survive multiple mass and host lineage extinctions.

Why so many unknown genes? Partitioning orphans from a representative transcriptome of the lone star tick Amblyomma americanum

BackgroundGenomic resources within the phylum Arthropoda are largely limited to the true insects but are beginning to include unexplored subphyla, such as the Crustacea and Chelicerata. Investigations of these understudied taxa uncover high frequencies of orphan genes, which lack detectable sequence homology to genes in pre-existing databases. The ticks (Acari: Chelicerata) are one such understudied taxon for which genomic resources are urgently needed. Ticks are obligate blood-feeders that vector major diseases of humans, domesticated animals, and wildlife. In analyzing a transcriptome of the lone star tick Amblyomma americanum, one of the most abundant disease vectors in the United States, we find a high representation of unannotated sequences. We apply a general framework for quantifying the origin and true representation of unannotated sequences in a dataset and for evaluating the biological significance of orphan genes.ResultsExpressed sequence tags (ESTs) were derived from different life stages and populations of A. americanum and combined with ESTs available from GenBank to produce 14,310 ESTs, over twice the number previously available. The vast majority (71%) has no sequence homology to proteins archived in UniProtKB. We show that poor sequence or assembly quality is not a major contributor to this high representation by orphan genes. Moreover, most unannotated sequences are functional: a microarray experiment demonstrates that 59% of functional ESTs are unannotated. Lastly, we attempt to further annotate our EST dataset using genomic datasets from other members of the Acari, including Ixodes scapularis, four other tick species and the mite Tetranychus urticae. We find low homology with these species, consistent with significant divergence within this subclass.ConclusionsWe conclude that the abundance of orphan genes in A. americanum likely results from 1) taxonomic isolation stemming from divergence within the tick lineage and limited genomic resources for ticks and 2) lineage-specific genes needing functional genomic studies to evaluate their association with the unique biology of ticks. The EST sequences described here will contribute substantially to the development of tick genomics. Moreover, the framework provided for the evaluation of orphan genes can guide analyses of future transcriptome sequencing projects.

Evolution of the tissue factor pathway inhibitor-like Kunitz domain-containing protein family in Rhipicephalus microplus

One of the principle mechanisms utilised by ticks to obtain a blood meal is the subversion of the host’s haemostatic response. This is achieved through the secretion of saliva containing anti-haemostatic proteins into the feeding lesion. Lineage-specific expansion of predicted secretory protein families have been observed in all previously studied ticks and occurred in response to adaptation to a blood-feeding environment. Of these, the predominant families are common between both hard and soft ticks. One of these families, namely the Kunitz domain-containing protein family, includes proven tissue factor pathway inhibitor-like (TFPI-like) anti-haemostatics such as ixolaris and penthalaris that play a crucial role during tick feeding. Although Kunitz-type proteins have been found in Rhipicephalus microplus, the TFPI-like Kunitz protein family has not yet been studied. We report a comprehensive search for TFPI-like Kunitz domain-containing proteins in R. microplus expressed sequence tag libraries, resulting in the identification of 42 homologues. The homologues were bioinformatically and phylogenetically studied, including the application of an intensive Bayesian Markov Chain Monte Carlo (MCMC) analysis of the individual Kunitz domain nucleotide sequences. We show that the R. microplus TFPI-like Kunitz protein family groups into two main clades that presumably underwent ancient duplication, which indicates that a whole genome duplication event occurred at least 150 million years ago. Evidence for recent and ancient gene and domain duplication events was also found. Furthermore, the divergence times of the various tick lineages estimated in this paper correspond with those presented in previous studies. The elucidation of this large protein family’s evolution within R. microplus adds to current knowledge of this economically important tick.

Tick-box for 3'-end formation of mitochondrial transcripts in Ixodida, basal chelicerates and Drosophila.

According to the tRNA punctuation model, the mitochondrial genome (mtDNA) of mammals and arthropods is transcribed as large polycistronic precursors that are maturated by endonucleolytic cleavage at tRNA borders and RNA polyadenylation. Starting from the newly sequenced mtDNA of Ixodes ricinus and using a combination of mitogenomics and transcriptional analyses, we found that in all currently-sequenced tick lineages (Prostriata, Metastriata and Argasidae) the 3′-end of the polyadenylated nad1 and rrnL transcripts does not follow the tRNA punctuation model and is located upstream of a degenerate 17-bp DNA motif. A slightly different motif is also present downstream the 3′-end of nad1 transcripts in the primitive chelicerate Limulus polyphemus and in Drosophila species, indicating the ancient origin and the evolutionary conservation of this motif in arthropods. The transcriptional analyses suggest that this motif directs the 3′-end formation of the nad1/rrnL mature RNAs, likely working as a transcription termination signal or a processing signal of precursor transcripts. Moreover, as most regulatory elements, this motif is characterized by a taxon-specific evolution. Although this signal is not exclusive of ticks, making a play on words it has been named “Tick-Box”, since it is a check mark that has to be verified for the 3′-end formation of some mt transcripts, and its consensus sequence has been here carefully characterized in ticks. Indeed, in the whole mtDNA of all ticks, the Tick-Box is always present downstream of nad1 and rrnL, mainly in non-coding regions (NCRs) and occasionally within trnL(CUN). However, some metastriates present a third Tick-Box at an intriguing site - inside the small NCR located at one end of a 3.4 kb translocated region, the other end of which exhibits the nad1 Tick-Box - hinting that this motif could have been involved in metastriate gene order rearrangements.

Nuttalliella namaqua (Ixodoidea: Nuttalliellidae): First Description of the Male, Immature Stages and Re-Description of the Female

Nuttalliella namaqua is the only species of the enigmatic third tick family. Females possess features of hard and soft ticks and have been designated as the “missing link” between the main tick families. Its position at the base of the tick tree suggests that some of the features unique to hard and soft ticks were present in the ancestral tick lineage. Larvae, nymphae and males have not been described to date and questions regarding their biological affinities to the main tick families remain unclear. The current study addressed these questions via the description of larvae, nymphae and males and resolved issues pertaining to female morphology. Field collected as well as laboratory-engorged females laid eggs and viable larvae subsequently hatched. The larvae possess morphological structures not present in subsequent stages: namely, a sclerotized scutum, pores on the dorsal surface of legs and a dentate anal plate. The last two characters are not present in ixodids and argasids. N. namaqua larvae and nymphae show a similar morphology to females: a unique hypostomal structure i.e., bluntly rounded apically in nymphae and females and ball-like in the larvae. A re-description of some structures in female N. namaqua has resolved differences in the original descriptions, namely that N. namaqua have 4 palpal segments as found in ixodids and argasids and posthypostomal setae. The male was discovered for the first time and described. Characteristic male features include: a pseudoscutum over most of the dorsum, an outgrowth on the chelicerae forming a unique rod-like structure similar to a spematodactyl in mites and medial extension of palpal segment 2 forming a large ventral crib for segment 4. All life stages possess some features found in hard and soft ticks and its status as the “missing link” between the tick families remains.

Phylogenetic analysis of ticks (Acari: Ixodida) using mitochondrial genomes and nuclear rRNA genes indicates that the genus Amblyomma is polyphyletic

Our understanding of the phylogenetic relationships among tick lineages has been limited by the lack of resolution provided by the most commonly used phylogenetic markers. Mitochondrial genomes are increasingly used to address controversial phylogenetic relationships. To date, the complete mitochondrial genomes of eleven tick species have been sequenced; however, only three of these species are metastriate ticks, the most speciose lineage of ticks. In this study, we present the nucleotide sequences of the complete mitochondrial genomes of five more species of metastriate ticks: Amblyomma elaphense, Amblyomma fimbriatum, Amblyomma sphenodonti, Bothriocroton concolor and Bothriocroton undatum. We use complete mitochondrial genome sequences to address the phylogenetic placement of two morphologically ‘primitive’ species –Am. elaphense and Am. sphenodonti – with respect to the genus Amblyomma. Our analysis of these five mitochondrial genomes with the other eleven tick mitochondrial genomes, as well as analysis of nuclear rRNA genes, provides strong evidence that the genus Amblyomma is polyphyletic with the inclusion of Am. sphenodonti and Am. elaphense. A new genus or two new genera may be required to describe Am. sphenodonti and Am. elaphense. It is also possible that these two species are sisters to two established genera, Bothriocroton in the case of Am. sphenodonti, and Haemaphysalis in the case of Am. elaphense. However, other arrangements of these taxa cannot be excluded with the current data. Thus, while Am. sphenodonti and Am. elaphense do not belong in the genus Amblyomma, the phylogenetic placement of these two species cannot be resolved without more data from metastriate ticks, either greater sampling of mitochondrial genomes, or a large data set of nuclear genes.

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.

Experimental infection of the rabbit tick, Haemaphysalis leporispalustris, with the bacterium Rickettsia rickettsii, and comparative biology of infected and uninfected tick lineages

The present study consisted of two experiments that evaluated experimental infections of Haemaphysalis leporispalustris ticks by a Brazilian strain of Rickettsia rickettsii, and their effect on tick biology. In experiment I, ticks were exposed to R. rickettsii during the larval, nymphal or adult stages by feeding on rabbits (Oryctolagus cuniculus) needle-inoculated with R. rickettsii, and thereafter reared on uninfected rabbits for the entire next tick generation. Regardless of the tick stage that acquired the infection, all subsequent tick stages were shown to be infected by PCR (infection rates varying from 1.3 to 41.7%), and were able to transmit R. rickettsii to uninfected rabbits, as demonstrated by rabbit seroconversion, guinea pig inoculation with rabbit blood, and PCR on rabbit blood. In Experiment II, ticks were exposed to R. rickettsii during the larval stage by feeding on rabbits co-infested with R. rickettsii-infected adult ticks, and thereafter reared on uninfected rabbits until the next generation of larvae. Again, all subsequent tick stages were shown to be infected by PCR (infection rates varying from 3.0 to 40.0%), and were able to transmit R. rickettsii to uninfected rabbits. Thus, it was demonstrated that larvae, nymphs, and adults of H. leporispalustris were able to acquire and maintain the R. rickettsii infection by transstadial and transovarial transmissions within the tick population, with active transmission of the bacterium to susceptible rabbits by all parasitic stages. Analyses of biological parameters of uninfected and R. rickettsii-infected tick lineages were performed in order to evaluate possible deleterious effects of R. rickettsii to the infected tick lineages. Surprisingly, all but one of the four R. rickettsii-experimental groups of the present study showed overall better biological performance than their sibling uninfected control ticks. Results of the present study showed that H. leporispalustris could support infection by a high virulent strain of R. rickettsii for at least two generations, in which infected tick lineages tended to have better performance than uninfected ticks. Our results support a possible role of H. leporispalustris in the enzootic maintenance of R. rickettsii in Latin America, as previously suggested by earlier works.