Distinctive Genome Reduction Rates Revealed by Genomic Analyses of Two Coxiella-Like Endosymbionts in Ticks.

Genome reduction is a hallmark of symbiotic genomes, and the rate and patterns of gene loss associated with this process have been investigated in several different symbiotic systems. However, in long-term host-associated coevolving symbiont clades, the genome size differences between strains are normally quite small and hence patterns of large-scale genome reduction can only be inferred from distant relatives. Here we present the complete genome of a Coxiella-like symbiont from Rhipicephalus turanicus ticks (CRt), and compare it with other genomes from the genus Coxiella in order to investigate the process of genome reduction in a genus consisting of intracellular host-associated bacteria with variable genome sizes. The 1.7-Mb CRt genome is larger than the genomes of most obligate mutualists but has a very low protein-coding content (48.5%) and an extremely high number of identifiable pseudogenes, indicating that it is currently undergoing genome reduction. Analysis of encoded functions suggests that CRt is an obligate tick mutualist, as indicated by the possible provisioning of the tick with biotin (B7), riboflavin (B2) and other cofactors, and by the loss of most genes involved in host cell interactions, such as secretion systems. Comparative analyses between CRt and the 2.5 times smaller genome of Coxiella from the lone star tick Amblyomma americanum (CLEAA) show that many of the same gene functions are lost and suggest that the large size difference might be due to a higher rate of genome evolution in CLEAA generated by the loss of the mismatch repair genes mutSL. Finally, sequence polymorphisms in the CRt population sampled from field collected ticks reveal up to one distinct strain variant per tick, and analyses of mutational patterns within the population suggest that selection might be acting on synonymous sites. The CRt genome is an extreme example of a symbiont genome caught in the act of genome reduction, and the comparison between CLEAA and CRt indicates that losses of particular genes early on in this process can potentially greatly influence the speed of this process.

Tissue tropism and vertical transmission of Coxiella in Rhipicephalus sanguineus and Rhipicephalus turanicus ticks.

Arthropod symbionts present tissue tropism that corresponds to the nature of the association and the mode of transmission between host generations. In ticks, however, our knowledge of symbiont tissue tropism and function is limited. Here, we quantified and localized previously described Coxiella-like symbionts in several organs of the tick Rhipicephalus turanicus. Quantitative polymerase chain reaction revealed high densities of Coxiella in the female gonads, and both male and female Malpighian tubules. Using fluorescence in situ hybridization and transmission electron microscopy, we further showed that in the gonads of both Rh. turanicus and Rh. sanguineus, Coxiella does not colonize the primary oocytes but is found later in young and mature oocytes in a specific distribution, suggesting controlled vertical transmission. This method revealed the presence Coxiella in the distal part of the Malpighian tubules, suggesting a possible role in nitrogen metabolism. While testing Rickettsia symbionts, no specific tissue tropism was found, but a slightly higher densities in the tick gut. The low density of Rickettsia in the female ovaries suggests competition between Rickettsia and Coxiella for vertical transmission. The described tissue distribution supports an obligatory role for Coxiella in ticks.

Composition and seasonal variation of Rhipicephalus turanicus and Rhipicephalus sanguineus bacterial communities.

A 16S rRNA gene approach, including 454 pyrosequencing and quantitative PCR (qPCR), was used to describe the bacterial community in Rhipicephalus turanicus and to evaluate the dynamics of key bacterial tenants of adult ticks during the active questing season. The bacterial community structure of Rh. turanicus was characterized by high dominance of Coxiella and Rickettsia and extremely low taxonomic diversity. Parallel diagnostic PCR further revealed a novel Coxiella species which was present and numerically dominant in all individual ticks tested (n = 187). Coxiella sp. densities were significantly higher in female versus male ticks and were overall stable throughout the questing season. In addition, we revealed the presence of the novel Coxiella sp. in Rh. sanguineus adult ticks, eggs, and hatched larvae, indicating its vertical transmission. The presence of both spotted fever group Rickettsia spp. (SFGR) and non-SFGR was verified in the various individual ticks. The prevalence and density of Rickettsia spp. were very low compared to those of Coxiella sp. Furthermore, Rickettsia sp. densities were similar in males and females and significantly declined toward the end of the questing season. No correlation was found between Coxiella sp. and Rickettsia sp. densities. These results suggest different control mechanisms in the tick over its different bacterial populations and point to an obligatory and facultative association between the two tick species and Coxiella sp. and Rickettsia spp., respectively.