Amplification and sequencing of entire tick mitochondrial genomes for a phylogenomic analysis.

The mitochondrial genome (mitogenome) has proven to be important for the taxonomy, systematics, and population genetics of ticks. However, current methods to generate mitogenomes can be cost-prohibitive at scale. To address this issue, we developed a cost-effective approach to amplify and sequence the whole mitogenome of individual tick specimens. Using two different primer sites, this approach generated two full-length mitogenome amplicons that were sequenced using the Oxford Nanopore Technologies' Mk1B sequencer. We used this approach to generate 85 individual tick mitogenomes from samples comprised of the three tick families, 11 genera, and 57 species. Twenty-six of these species did not have a complete mitogenome available on GenBank prior to this work. We benchmarked the accuracy of this approach using a subset of samples that had been previously sequenced by low-coverage Illumina genome skimming. We found our assemblies were comparable or exceeded the Illumina method, achieving a median sequence concordance of 99.98%. We further analyzed our mitogenome dataset in a mitophylogenomic analysis in the context of all three tick families. We were able to sequence 72 samples in one run and achieved a cost/sample of ~ $10 USD. This cost-effective strategy is applicable for sample identification, taxonomy, systematics, and population genetics for not only ticks but likely other metazoans; thus, making mitogenome sequencing equitable for the wider scientific community.

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.

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.

An update of the tsetse fly (Diptera: Glossinidae) distribution and African animal trypanosomosis prevalence in north-eastern KwaZulu-Natal, South Africa.

An unpredicted outbreak of African animal trypanosomosis or nagana in 1990 in north-eastern KwaZulu-Natal necessitated an emergency control programme, utilising the extensive cattledipping system in the area, as well as a reassessment of the tsetse and trypanosomosis problem in the province. Since 1990, sporadic blood sampling of cattle at the dip tanks in the naganainfested areas were undertaken to identify trypanosome species involved and to determine the infection prevalence in cattle. The distribution and species composition of the tsetse populations in the area were also investigated. From November 2005 to November 2007 selected dip tanks were surveyed for trypanosome infection prevalence. During April 2005 to August 2009 the distribution and abundance of tsetse populations were assessed with odour-baited H traps. The tsetse and trypanosome distribution maps were updated and potential correlations between tsetse apparent densities (ADs) and the prevalence of trypanosomosis were assessed. Glossina brevipalpis Newstead and Glossina austeni Newstead were recorded in locations where they have not previously been collected. No significant correlation between tsetse relative abundance and nagana prevalence was found, which indicated complex interactions between tsetse fly presence and disease prevalence. This was epitomised by data that indicated that despite large differences in the ADs of G. austeni and G. brevipalpis, trypanosome infection prevalence was similar in all three districts in the area. This study clearly indicated that both tsetse species play significant roles in trypanosome transmission and that it will be essential that any control strategy, which aims at sustainable management of the disease, should target both species.

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.

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.