Description of a new tick species, closely related to Amblyomma javanense (Supino, 1897), associated with Varanus bengalensis (Squamata: Varanidae) in Pakistan.

The genus Amblyomma contains the highest percentage of reptile-associated ticks, and comprises approximately nine subgenera. One of these subgenera is Adenopleura, which also encompasses Amblyomma javanense, and its type species Amblyomma compressum. This study describes a new Amblyomma species associated with Bengal monitor lizards (Varanus bengalensis) based on morphology and its mitogenome in Khyber Pakhtunkhwa, Pakistan. Reptiles belonging to different genera were examined for Amblyomma ticks and only the monitor lizard was infested with ticks in the District Bajaur. Collected Amblyomma cf. javanense ticks were analyzed and formally described as a new species. Overall, 57 A. cf. javanense ticks were collected on monitor lizards (4/27) with a 15% prevalence of infestation, 2.1 mean abundance, and 14.3 mean intensity. Ticks comprised males (n = 23, 40%), females (n = 14, 25%) and nymphs (n = 20, 35%), while no larvae were found. BLAST analysis of A. cf. javanense sequences showed the following maximum identities; 98.25% with undetermined Amblyomma species based on 12S rRNA, 96.07% with A. javanense based on 16S rRNA, 99.56% and 90.95% with an Amblyomma sp. and A. javanense, respectively, based on ITS2. Moreover, the mitochondrial genome of A. cf. javanense showed maximum identities of 80.75%, 80.48% and 79.42% with Amblyomma testudinarium, A. javanense, and Amblyomma sp., respectively. The phylogenetic analysis of A. cf. javanense revealed that its 12S rRNA and 16S rRNA are closely related to an Amblyomma sp. and A. javanense, respectively, from Sri Lanka, its ITS2 is closely related to A. javanense from China and an Amblyomma sp. from Sri Lanka, and its mitogenome is closely related to A. javanense and Amblyomma sp. from China. The pairwise distance analysis resulted in divergence of 0-1.71% (12S rRNA), 0-17.5% (16S rRNA), 0-9.1% (ITS2) and 0-20.5% (mitochondrial genome). We also contributed the full-length mitochondrial genome sequence of A. compressum and showed that this species does not share a most recent common ancestor with A. javanense. As the subgenus Adenopleura is paraphyletic, this study could help to understand the systematics and phylogeny of this taxon.

Transcriptomic Analysis of Salivary Glands of Ornithodoros brasiliensis Aragão, 1923, the Agent of a Neotropical Tick-Toxicosis Syndrome in Humans.

Tick salivary glands produce and secrete a variety of compounds that modulate host responses and ensure a successful blood meal. Despite great progress made in the identification of ticks salivary compounds in recent years, there is still a paucity of information concerning salivary molecules of Neotropical argasid ticks. Among this group of ticks, considering the number of human cases of parasitism, including severe syndromes and hospitalization, Ornithodoros brasiliensis can be considered one of the major Neotropical argasid species with impact in public health. Here, we describe the transcriptome analysis of O. brasiliensis salivary glands (ObSG). The transcriptome yielded ~14,957 putative contigs. A total of 368 contigs were attributed to secreted proteins (SP), which represent approximately 2.5% of transcripts but ~53% expression coverage transcripts per million. Lipocalins are the major protein family among the most expressed SP, accounting for ~16% of the secretory transcripts and 51% of secretory protein abundance. The most expressed transcript is an ortholog of TSGP4 (tick salivary gland protein 4), a lipocalin first identified in Ornithodoros kalahariensis that functions as a leukotriene C4 scavenger. A total of 55 lipocalin transcripts were identified in ObSG. Other transcripts potentially involved in tick-host interaction included as: basic/acid tail secretory proteins (second most abundant expressed group), serine protease inhibitors (including Kunitz inhibitors), 5' nucleotidases (tick apyrases), phospholipase A2, 7 disulfide bond domain, cystatins, and tick antimicrobial peptides. Another abundant group of proteins in ObSG is metalloproteases. Analysis of these major protein groups suggests that several duplication events after speciation were responsible for the abundance of redundant compounds in tick salivary glands. A full mitochondrial genome could be assembled from the transcriptome data and confirmed the close genetic identity of the tick strain sampled in the current study, to a tick strain previously implicated in tick toxicoses. This study provides novel information on the molecular composition of ObSG, a Brazilian endemic tick associated with several human cases of parasitism. These results could be helpful in the understanding of clinical findings observed in bitten patients, and also, could provide more information on the evolution of Neotropical argasids.

Nuclear (18S-28S rRNA) and mitochondrial genome markers of Carios (Carios) vespertilionis (Argasidae) support Carios Latreille, 1796 as a lineage embedded in the Ornithodorinae: re-classification of the Carios sensu Klompen and Oliver (1993) clade into its respective subgenera.

Argasid systematics remains controversial with widespread adherence to the Hoogstraal (1985) classification scheme, even though it does not reflect evolutionary relationships and results in paraphyly for the main genera of soft ticks (Argasidae), namely Argas and Ornithodoros. The alternative classification scheme, proposed by Klompen and Oliver (1993), has problems of its own: most notably paraphyly of the subgenus Pavlovskyella and the controversial grouping together of the subgenera Alectorobius, Antricola, Carios, Chiropterargas, Nothoaspis, Parantricola, Reticulinasus and Subparmatus into the genus Carios. Recent phylogenetic analyses of 18S/28S rRNA sequences and mitochondrial genomes agree with the scheme of Klompen and Oliver (1993), with regard to the paraphyly of Pavlovskyella, placement of Alveonasus, Ogadenus, Proknekalia and Secretargas in the Argasinae and placement of Carios and Chiropterargas in the Ornithodorinae (Mans et al., 2019). The Carios clade and its constituent subgenera remain controversial, since the phylogenetic position of its type species Carios (Carios) vespertilionis Latreille, 1796 (formerly Argas vespertilionis) has not been determined with confidence. The current study aimed to resolve Carios sensu lato Klompen and Oliver, 1993, and Carios sensu stricto Hoogstraal, 1985, by determining and analysing phylogenetic nuclear and mitochondrial markers for C. (C.) vespertilionis. Both the nuclear and mitochondrial markers support placement of Carios s.s. within the subfamily Ornithodorinae, but to the exclusion of the clade that includes the 6 other subgenera that are part of Carios s.l. Klompen and Oliver (1993), namely Alectorobius, Antricola, Nothoaspis, Parantricola, Reticulinasus and Subparmatus. These 6 subgenera form a monophyletic clade that might be placed as new subgenera within the genus Alectorobius, or elevated to genera. Given the substantial differences in biology among these subgenera, we propose that these 6 subgenera be elevated to genera. Thus, we propose to modify the classification scheme of Mans et al. (2019) so that the subfamily Argasinae now has six genera, Alveonasus, Argas (subgenera Argas and Persicargas), Navis, Ogadenus, Proknekalia and Secretargas, and the subfamily Ornithodorinae has nine genera, Alectorobius, Antricola (subgenera Antricola and Parantricola), Carios, Chiropterargas, Nothoaspis, Ornithodoros (subgenera Microargas, Ornamentum, Ornithodoros, Pavlovskyella and Theriodoros), Otobius, Reticulinasus and Subparmatus (genera indicated in bold).

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.

Argasid and ixodid systematics: Implications for soft tick evolution and systematics, with a new argasid species list.

The systematics of the genera and subgenera within the soft tick family Argasidae is not adequately resolved. Different classification schemes, reflecting diverse schools of scientific thought that elevated or downgraded groups to genera or subgenera, have been proposed. In the most recent classification scheme, Argas and Ornithodoros are paraphyletic and the placement of various subgenera remains uncertain because molecular data are lacking. Thus, reclassification of the Argasidae is required. This will enable an understanding of soft tick systematics within an evolutionary context. This study addressed that knowledge gap using mitochondrial genome and nuclear (18S and 28S ribosomal RNA) sequence data for representatives of the subgenera Alectorobius, Argas, Chiropterargas, Ogadenus, Ornamentum, Ornithodoros, Navis (subgen. nov.), Pavlovskyella, Persicargas, Proknekalia, Reticulinasus and Secretargas, from the Afrotropical, Nearctic and Palearctic regions. Hard tick species (Ixodidae) and a new representative of Nuttalliella namaqua (Nuttalliellidae), were also sequenced with a total of 83 whole mitochondrial genomes, 18S rRNA and 28S rRNA genes generated. The study confirmed the utility of next-generation sequencing to retrieve systematic markers. Paraphyly of Argas and Ornithodoros was resolved by systematic analysis and a new species list is proposed. This corresponds broadly with the morphological cladistic analysis of Klompen and Oliver (1993). Estimation of divergence times using molecular dating allowed dissection of phylogeographic patterns for argasid evolution. The discovery of cryptic species in the subgenera Chiropterargas, Ogadenus and Ornithodoros, suggests that cryptic speciation is common within the Argasidae. Cryptic speciation has implications for past biological studies of soft ticks. These are discussed in particular for the Ornithodoros (Ornithodoros) moubata and Ornithodoros (Ornithodoros) savignyi groups.

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.

De novo assembly and annotation of the salivary gland transcriptome of Rhipicephalus appendiculatus male and female ticks during blood feeding.

Tick secretory proteins modulate haemostasis, inflammation and immune responses of the host and are attractive recombinant anti-tick vaccine candidates. Yet, many of the proteins have not been characterised due to the limited sequence availability for ticks and other arthropods for homology-based annotation. To address this limitation, we sequenced the salivary glands of the economically important adult male and female Rhipicephalus appendiculatus ticks during feeding. The quality-filtered Illumina sequencing reads were de novo assembled to generate a R. appendiculatus sialotranscriptome of 21,410 transcripts. A non-redundant set of 12,761 R. appendiculatus proteins was predicted from the transcripts, including 2134 putative secretory and 8237 putative housekeeping proteins. Secretory proteins accounted for most of the expression in the salivary gland transcriptome (63%). Of the secretory protein class, the Glycine-rich superfamily contributed 66% and the Lipocalin family 12% of the transcriptome expression. Differential expression analysis identified 1758 female and 2346 male up-regulated transcripts, suggesting varying blood-feeding mechanisms employed between female and male ticks. The sialotranscriptome assembled in this work, greatly improves on the sequence information available for R. appendiculatus and is a valuable resource for potential future vaccine candidate selection.

Ancestral reconstruction of tick lineages.

Ancestral reconstruction in its fullest sense aims to describe the complete evolutionary history of a lineage. This depends on accurate phylogenies and an understanding of the key characters of each parental lineage. An attempt is made to delineate our current knowledge with regard to the ancestral reconstruction of the tick (Ixodida) lineage. Tick characters may be assigned to Core of Life, Lineages of Life or Edges of Life phenomena depending on how far back these characters may be assigned in the evolutionary Tree of Life. These include housekeeping genes, sub-cellular systems, heme processing (Core of Life), development, moulting, appendages, nervous and organ systems, homeostasis, respiration (Lineages of Life), specific adaptations to a blood-feeding lifestyle, including the complexities of salivary gland secretions and tick-host interactions (Edges of Life). The phylogenetic relationships of lineages, their origins and importance in ancestral reconstruction are discussed. Uncertainties with respect to systematic relationships, ancestral reconstruction and the challenges faced in comparative transcriptomics (next-generation sequencing approaches) are highlighted. While almost 150 years of information regarding tick biology have been assembled, progress in recent years indicates that we are in the infancy of understanding tick evolution. Even so, broad reconstructions can be made with relation to biological features associated with various lineages. Conservation of characters shared with sister and parent lineages are evident, but appreciable differences are present in the tick lineage indicating modification with descent, as expected for Darwinian evolutionary theory. Many of these differences can be related to the hematophagous lifestyle of ticks.

Next-generation sequencing as means to retrieve tick systematic markers, with the focus on Nuttalliella namaqua (Ixodoidea: Nuttalliellidae).

Nuclear ribosomal RNA (18S and 28S rRNA) and mitochondrial genomes are commonly used in tick systematics. The ability to retrieve these markers using next-generation sequencing was investigated using the tick Nuttalliella namaqua. Issues related to nuclear markers may be resolved using this approach, notably, the monotypic status of N. namaqua and its basal relationship to other tick families. Four different Illumina datasets (∼55 million, 100 bp reads each) were generated from a single tick specimen and assembled to give 350k-390k contigs. A genome size of ∼1 Gbp was estimated with low levels of repetitive elements. Contigs (>1000 bp, >50-fold coverage) present in most assemblies (n=69), included host-derived 18S and 28S rRNA, tick and host-derived transposable elements, full-length tick 18S and 28 rRNA, the mitochondrial genome in single contig assemblies and the histone cassette. Coverage for the nuclear rRNA genes was above 1000-fold confirming previous sequencing errors in the 18S rRNA gene, thereby maintaining the monotypic status of this tick. Nuclear markers for the soft tick Argas africolumbae were also retrieved from next-generation data. Phylogenetic analysis of a concatenated 18S-28S rRNA dataset supported the grouping of N. namaqua at the base of the tick tree and the two main tick families in separate clades. This study confirmed the monotypic status of N. namaqua and its basal relationship to other tick families. Next-generation sequencing of genomic material to retrieve high quality nuclear and mitochondrial systematic markers for ticks is viable and may resolve issues around conventional sequencing errors when comparing closely related tick species.

The mitochondrial genomes of Nuttalliella namaqua (Ixodoidea: Nuttalliellidae) and Argas africolumbae (Ixodoidae: Argasidae): estimation of divergence dates for the major tick lineages and reconstruction of ancestral blood-feeding characters.

Ixodida are composed of hard (Ixodidae), soft (Argasidae) and the monotypic Nuttalliellidae (Nuttalliella namaqua) tick families. Nuclear 18S rRNA analysis suggested that N. namaqua was the closest extant relative to the last common ancestral tick lineage. The mitochondrial genomes of N. namaqua and Argas africolumbae were determined using next generation sequencing and de novo assembly to investigate this further. The latter was included since previous estimates on the divergence times of argasids lacked data for this major genus. Mitochondrial gene order for both was identical to that of the Argasidae and Prostriata. Bayesian analysis of the COI, Cytb, ND1, ND2 and ND4 genes confirmed the monophyly of ticks, the basal position of N. namaqua to the other tick families and the accepted systematic relationships of the other tick genera. Molecular clock estimates were derived for the divergence of the major tick lineages and supported previous estimates on the origins of ticks in the Carboniferous. N. namaqua larvae fed successfully on lizards and mice in a prolonged manner similar to many argasids and all ixodids. Excess blood meal-derived water was secreted via the salivary glands, similar to ixodids. We propose that this prolonged larval feeding style eventually gave rise to the long feeding periods that typify the single larval, nymphal and adult stages of ixodid ticks and the associated secretion of water via the salivary glands. Ancestral reconstruction of characters involved in blood-feeding indicates that most of the characteristics unique to either hard or soft tick families were present in the ancestral tick lineage.