The bacterial endosymbiont Wolbachia pipientis protects its hosts from a range of pathogens by limiting their ability to form infections inside the insect. This “pathogen blocking” could be explained by innate immune priming by the symbiont, competition for host-derived resources between pathogens and Wolbachia, or the direct modification of the cell or cellular environment by Wolbachia. Recent comparative work in Drosophila and the mosquito Aedes aegypti has shown that an immune response is not required for pathogen blocking, implying that there must be an additional component to the mechanism. Here we have examined the involvement of cholesterol in pathogen blocking using a system of dietary manipulation in Drosophila melanogaster in combination with challenge by Drosophila C virus (DCV), a common fly pathogen. We observed that flies reared on cholesterol-enriched diets infected with the Wolbachia strains wMelPop and wMelCS exhibited reduced pathogen blocking, with viral-induced mortality occurring 2–5 days earlier than flies reared on Standard diet. This shift toward greater virulence in the presence of cholesterol also corresponded to higher viral copy numbers in the host. Interestingly, an increase in dietary cholesterol did not have an effect on Wolbachia density except in one case, but this did not directly affect the strength of pathogen blocking. Our results indicate that host cholesterol levels are involved with the ability of Wolbachia-infected flies to resist DCV infections, suggesting that cholesterol contributes to the underlying mechanism of pathogen blocking. Wolbachia pipientis is an intracellular bacterium that naturally infects many insect species. These bacteria can block the replication and dissemination of a variety of pathogens that coinfect the insect. In mosquitoes this effect applies to viruses including dengue and the parasite that causes malaria. This makes Wolbachia a promising method of controlling a number of insect-transmitted diseases of humans. Some studies suggest that pathogen blocking is due to Wolbachia stimulating the host innate immune system, however in the fly Drosophila melanogaster, blocking occurs in the absence of such an immune effect. This suggests that there is an additional component to the mechanism. Host nutrients such as cholesterol are a limiting factor in virus replication and may serve as a source of competition that underlies pathogen blocking. Here we show that competition over cholesterol contributes to the pathogen blocking effect, with higher available cholesterol levels causing less effective blocking, and increased viral titres in Wolbachia-infected D. melanogaster challenged with the pathogenic Drosophila C virus. These results suggest that competition over cholesterol is an important part of the pathogen blocking mechanism.

The tick microbiome comprises communities of microorganisms, including viruses, bacteria and eukaryotes, and is being elucidated through modern molecular techniques. The advent of next-generation sequencing (NGS) technologies has enabled the genes and genomes within these microbial communities to be explored in a rapid and cost-effective manner. The advantages of using NGS to investigate microbiomes surpass the traditional non-molecular methods that are limited in their sensitivity, and conventional molecular approaches that are limited in their scalability. In recent years the number of studies using NGS to investigate the microbial diversity and composition of ticks has expanded. Here, we provide a review of NGS strategies for tick microbiome studies and discuss the recent findings from tick NGS investigations, including the bacterial diversity and composition, influential factors, and implications of the tick microbiome.

The Australian paralysis tick (Ixodes holocyclus) is of significant medical and veterinary importance as a cause of dermatological and neurological disease, yet there is currently limited information about the bacterial communities harboured by these ticks and the risk of infectious disease transmission to humans and domestic animals. Ongoing controversy about the presence of Borrelia burgdorferi sensu lato (the aetiological agent of Lyme disease) in Australia increases the need to accurately identify and characterise bacteria harboured by I. holocyclus ticks. Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes present in DNA samples from I. holocyclus and I. ricinus ticks, collected in Australia and Germany respectively. The 16S amplicons were purified, sequenced on the Ion Torrent platform, and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomy. Initial analysis of I. holocyclus and I. ricinus identified that > 95 % of the 16S sequences recovered belonged to the tick intracellular endosymbiont “Candidatus Midichloria mitochondrii” (CMM). A CMM-specific blocking primer was designed that decreased CMM sequences by approximately 96 % in both tick species and significantly increased the total detectable bacterial diversity, allowing identification of medically important bacterial pathogens that were previously masked by CMM. Borrelia burgdorferi sensu lato was identified in German I. ricinus, but not in Australian I. holocyclus ticks. However, bacteria of medical significance were detected in I. holocyclus ticks, including a Borrelia relapsing fever group sp., Bartonella henselae, novel “Candidatus Neoehrlichia” spp., Clostridium histolyticum, Rickettsia spp., and Leptospira inadai. Abundant bacterial endosymbionts, such as CMM, limit the effectiveness of next-generation 16S bacterial community profiling in arthropods by masking less abundant bacteria, including pathogens. Specific blocking primers that inhibit endosymbiont 16S amplification during PCR are an effective way of reducing this limitation. Here, this strategy provided the first evidence of a relapsing fever Borrelia sp. and of novel “Candidatus Neoehrlichia” spp. in Australia. Our results raise new questions about tick-borne pathogens in I. holocyclus ticks.

The genus Borrelia, originally described by Swellengrebel in 1907, contains tick- or louse-transmitted spirochetes belonging to the relapsing fever (RF) group of spirochetes, the Lyme borreliosis (LB) group of spirochetes and spirochetes that form intermittent clades. In 2014 it was proposed that the genus Borrelia should be separated into two genera; Borrelia Swellengrebel 1907 emend. Adeolu and Gupta 2014 containing RF spirochetes and Borreliella Adeolu and Gupta 2014 containing LB group of spirochetes. In this study we conducted an analysis based on a method that is suitable for bacterial genus demarcation, the percentage of conserved proteins (POCP). We included RF group species, LB group species and two species belonging to intermittent clades, Borrelia turcica Güner et al. 2004 and Candidatus Borrelia tachyglossi Loh et al. 2017. These analyses convincingly showed that all groups of spirochetes belong into one genus and we propose to emend, and re-unite all groups in, the genus Borrelia.

To date, little has been documented about microorganisms harboured within Australian native ticks or their pathogenic potential. Recently, a Borrelia sp. related to the Relapsing Fever (RF) group was identified in a single tick removed from a wild echidna (Tachyglossus aculeatus). The present study investigated the presence of Borrelia in 97 Bothriocroton concolor ticks parasitizing echidnas in Queensland, New South Wales, and Victoria, Australia, using nested PCR with Borrelia-specific primers targeting the 16S rRNA (16S) and flaB genes. Borrelia-specific PCR assays confirmed the presence of a novel Borrelia sp. related to the RF and reptile-associated (REP) spirochaetes in 38 (39 %) B. concolor ticks. This novel Borrelia sp. was identified in 41 % of the B. concolor ticks in Queensland and New South Wales, but not in any ticks from Victoria. The resulting flaB sequences (407 bp) were 88 and 86 % similar to the flaB sequences from Borrelia turcica and Borrelia hermsii, respectively. Of the ticks confirmed as Borrelia-positive following the flaB assay, 28 were positive with the 16S assay. Phylogenetic analysis of the 16S sequences (1097 bp) suggests that these sequences belong to a novel Borrelia sp., which forms a unique monophyletic clade that is similar to, but distinct from, RF Borrelia spp. and REP-associated Borrelia spp. We conclude that the novel Borrelia sp. identified in this study does not belong to the Borrelia burgdorferi (sensu lato) complex, and that the phylogenetic analysis of the partial 16S gene sequences suggests it forms a unique monophyletic cluster in the genus Borrelia, potentially forming a fourth major group in this genus associated with monotremes in Australia. However, a thorough molecular characterisation will be required to confirm the phylogenetic position of this unique Borrelia sp. The zoonotic potential and pathogenic consequences of this novel Borrelia sp. are unknown at the current time.