Investigation of vertical and horizontal transmission of Spiroplasma in ticks under laboratory conditions.

Many arthropods harbour bacterial symbionts, which are maintained by vertical and/or horizontal transmission. Spiroplasma is one of the most well-known symbionts of ticks and other arthropods. It is still unclear how Spiroplasma infections have spread in tick populations despite its high prevalence in some tick species. In this study, Ixodes ovatus, which has been reported to harbour Spiroplasma ixodetis at high frequencies, was examined for its vertical transmission potential under experimental conditions. Next, two isolates of tick-derived Spiroplasma, S. ixodetis and Spiroplasma mirum, were experimentally inoculated into Spiroplasma-free Haemaphysalis longicornis colonies and the presence of Spiroplasma in their eggs and larvae was tested. Our experimental data confirmed that S. ixodetis was transmitted to eggs and larvae in a vertical manner in the original host I. ovatus. In the second experiment, there was no significant difference in engorged weight, egg weight, and hatching rate between Spiroplasma-inoculated and control H. longicornis groups. This suggested that Spiroplasma infection does not affect tick reproduction. Spiroplasma DNA was only detected in the eggs and larvae derived from some individuals of S. ixodetis-inoculated groups. This has demonstrated the potential of horizontal transmission between different tick species. These findings may help understand the transmission dynamics of Spiroplasma in nature and its adaptation mechanism to host arthropod species.

Comparative mitogenomics elucidates the population genetic structure of Amblyomma testudinarium in Japan and a closely related Amblyomma species in Myanmar.

Ticks are the second most important vector capable of transmitting diseases affecting the health of both humans and animals. Amblyomma testudinarium Koch 1844 (Acari: Ixodidae), is a hard tick species having a wide geographic distribution in Asia. In this study, we analyzed the composition of A. testudinarium whole mitogenomes from various geographical regions in Japan and investigated the population structure, demographic patterns, and phylogeographic relationship with other ixodid species. In addition, we characterized a potentially novel tick species closely related to A. testudinarium from Myanmar. Phylogeographic inference and evolutionary dynamics based on the 15 mitochondrial coding genes supported that A. testudinarium population in Japan is resolved into a star-like haplogroup and suggested a distinct population structure of A. testudinarium from Amami island in Kyushu region. Correlation analysis using Mantel test statistics showed that no significant correlation was observed between the genetic and geographic distances calculated between the A. testudinarium population from different localities in Japan. Finally, demographic analyses, including mismatch analysis and Tajima's D test, suggested a possibility of recent population expansion occurred within Japanese haplogroup after a bottleneck event. Although A. testudinarium has been considered widespread and common in East and Southeast Asia, the current study suggested that potentially several cryptic Amblyomma spp. closely related to A. testudinarium are present in Asia.

Applications of Blocker Nucleic Acids and Non-Metazoan PCR Improves the Discovery of the Eukaryotic Microbiome in Ticks.

Ticks serve as important vectors of a variety of pathogens. Recently, the viral and prokaryotic microbiomes in ticks have been explored using next-generation sequencing to understand the physiology of ticks and their interactions with pathogens. However, analyses of eukaryotic communities in ticks are limited, owing to the lack of suitable methods. In this study, we developed new methods to selectively amplify microeukaryote genes in tick-derived DNA by blocking the amplification of the 18S rRNA gene of ticks using artificial nucleic acids: peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). In addition, another PCR using non-metazoan primers, referred to as UNonMet-PCR, was performed for comparison. We performed each PCR using tick-derived DNA and sequenced the amplicons using the Illumina MiSeq platform. Almost all sequences obtained by conventional PCR were derived from ticks, whereas the proportion of microeukaryotic reads and alpha diversity increased upon using the newly developed method. Additionally, the PNA- or LNA-based methods were suitable for paneukaryotic analyses, whereas the UNonMet-PCR method was particularly sensitive to fungi. The newly described methods enable analyses of the eukaryotic microbiome in ticks. We expect the application of these methods to improve our understanding of the tick microbiome.