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The gene content of a mitochondrial (mt) genome, i.e., 37 genes and a large noncoding region (LNR), is usually conserved in Metazoa. The arrangement of these genes and the LNR is generally conserved at low taxonomic levels but varies substantially at high levels. We report here a variation in mt gene content and gene arrangement among chigger mites of the genus Leptotrombidium. We found previously that the mt genome of Leptotrombidium pallidum has an extra gene for large-subunit rRNA (rrnL), a pseudo-gene for small-subunit rRNA (PrrnS), and three extra LNRs, additional to the 37 genes and an LNR typical of Metazoa. Further, the arrangement of mt genes of L. pallidum differs drastically from that of the hypothetical ancestor of the arthropods. To find to what extent the novel gene content and gene arrangement occurred in Leptotrombidium, we sequenced the entire or partial mt genomes of three other species, L. akamushi, L. deliense, and L. fletcheri. These three species share the arrangement of all genes with L. pallidum, except trnQ (for tRNA-glutamine). Unlike L. pallidum, however, these three species do not have extra rrnL or PrrnS and have only one extra LNR. By comparison between Leptotrombidium species and the ancestor of the arthropods, we propose that (1) the type of mt genome present in L. pallidum evolved from the type present in the other three Leptotrombidium species, and (2) three molecular mechanisms were involved in the evolution of mt gene content and gene arrangement in Leptotrombidium species.

To investigate the evolution pattern and phylogenetic utility of duplicate control regions (CRs) in mitochondrial (mt) genomes, we sequenced the entire mt genomes of three Ixodes species and part of the mt genomes of another 11 species. All the species from the Australasian lineage have duplicate CRs, whereas the other species have one CR. Sequence analyses indicate that the two CRs of the Australasian Ixodes ticks have evolved in concert in each species. In addition to the Australasian Ixodes ticks, species from seven other lineages of metazoa also have mt genomes with duplicate CRs. Accumulated mtDNA sequence data from these metazoans and two recent experiments on replication of mt genomes in human cell lines with duplicate CRs allowed us to re-examine four intriguing questions about the presence of duplicate CRs in the mt genomes of metazoa: (1) Why do some mt genomes, but not others, have duplicate CRs? (2) How did mt genomes with duplicate CRs evolve? (3) How could the nucleotide sequences of duplicate CRs remain identical or very similar over evolutionary time? (4) Are duplicate CRs phylogenetic markers? It appears that mt genomes with duplicate CRs have a selective advantage in replication over mt genomes with one CR. Tandem duplication followed by deletion of genes is the most plausible mechanism for the generation of mt genomes with duplicate CRs. Once duplicate CRs occur in an mt genome, they tend to evolve in concert, probably by gene conversion. However, there are lineages where gene conversion may not always occur, and, thus, the two CRs may evolve independently in these lineages. Duplicate CRs have much potential as phylogenetic markers at low taxonomic levels, such as within genera, within families, or among families, but not at high taxonomic levels, such as among orders.

Ticks were collected from 8 houses in Mvumi Mission village, near Dodoma, Tanzania. All ticks were examined for Borrelia infestation by flagellin gene-based nested polymerase chain reaction. All houses were highly infested with ticks, and all ticks collected were of the Ornithodoros porcinus species. Fifty-one out of 120 ticks were infected with spirochetes, and a flagellin gene sequence comparison showed that most of the spirochetes belonged to Borrelia duttonii, which is the causative agent of tick-borne relapsing fever in East Africa. The rest of the spirochetes were quite different from B. duttonii and instead resembled the New World tick-borne relapsing fever borreliae. Phylogenetic analysis using 16S ribosomal RNA gene sequences also supported the interpretation that the spirochete was a Borrelia species distinct from previously described members of the genus.

There are two major groups of ticks: soft ticks and hard ticks. The hard ticks comprise the prostriate ticks and the metastriate ticks. The mitochondrial (mt) genomes of one species of prostriate tick and two species of metastriate ticks had been sequenced prior to our study. The prostriate tick has the ancestral arrangement of mt genes of arthropods, whereas the two metastriate ticks have rearrangements of eight genes and duplicate control regions. However, the arrangement of genes in the mt genomes of soft ticks had not been studied. We sequenced the mt genomes of two species of soft ticks, Carios capensis and Ornithodoros moubata, and a metastriate tick, Haemaphysalis flava. We found that the soft ticks have the ancestral arrangement of mt genes of arthropods, whereas the metastriate tick, H. flava, shares the rearrangements of mt genes and duplicate control regions with the other two metastriate ticks that have previously been studied. Our study indicates that gene rearrangements and duplicate control regions in mt genomes occurred once in the most recent common ancestor of metastriate ticks, whereas the ancestral arrangement of arthropods has remained unchanged for over 400 million years in the lineages leading to the soft ticks and the prostriate ticks.

We examined the vector competence of the tick, Ornithodoros moubata, using laboratory-reared gerbils as hosts. Transmission of the relapsing fever agent Borrelia duttonii occurred efficiently from infected ticks to uninfected gerbils and from infected gerbils to uninfected ticks. Spirochetes were maintained stably in the ticks for at least 3 months, but they disappeared from the bloodstream of infected gerbils after three episodes of spirochetemia. We also examined transovarial transmission of B. duttonii during the gonotrophic cycle and filial generation. No spirochetes could be detected from the offspring generation of the ticks by culture and polymerase chain reaction (PCR) methods, although spirochetes were still found in the female ticks. The results indicate that, because of the rarity of transovarial infection, the role of transovarial passage of B. duttonii to eggs and larval O. moubata ticks is limited in maintaining B. duttonii. Our findings strongly suggest that B. duttonii is maintained through the O. moubata tick-human transmission cycle in tick-borne relapsing fever endemic areas.

To better understand the evolution of mitochondrial (mt) genomes in the Acari (mites and ticks), we sequenced the mt genome of the chigger mite, Leptotrombidium pallidum (Arthropoda: Acari: Acariformes). This genome is highly rearranged relative to that of the hypothetical ancestor of the arthropods and the other species of Acari studied. The mt genome of L. pallidum has two genes for large subunit rRNA, a pseudogene for small subunit rRNA, and four nearly identical large noncoding regions. Nineteen of the 22 tRNAs encoded by this genome apparently lack either a T-arm or a D-arm. Further, the mt genome of L. pallidum has two distantly separated sections with identical sequences but opposite orientations of transcription. This arrangement cannot be accounted for by homologous recombination or by previously known mechanisms of mt gene rearrangement. The most plausible explanation for the origin of this arrangement is illegitimate inter-mtDNA recombination, which has not been reported previously in animals. In light of the evidence from previous experiments on recombination in nuclear and mt genomes of animals, we propose a model of illegitimate inter-mtDNA recombination to account for the novel gene content and gene arrangement in the mt genome of L. pallidum.

Tick-borne relapsing fever caused by the spirochaete Borrelia duttonii is a common cause of serious illness in central Tanzania. Screening of Ornithodoros sp ticks from infested houses for the presence of B duttonii had detected a previously unidentified species of Borrelia. We investigated whether this species infected the human population in a central Tanzanian village, by use of blood slide examination and PCR. PCR was twice as sensitive in detection of infections, showing Borrelia sp in six (11%) of 54 children with fever, and in 13 (4%) of 307 otherwise healthy children. Genotyping Borrelia from 17 infections identified Borrelia duttonii and an unnamed species. Our findings show that the newly discovered species is a causal agent of tick-borne relapsing fever.

Borrelia duttonii strain Ly, a causative agent of relapsing fever, contains a linear one megabase chromosome and 12 linear plasmid molecules. Here we report that the sequence of the 44-kb linear plasmid of strain Ly is found to contain variable major protein (vmp) genes for antigenic variation of relapsing fever borreliae. The determined sequence is of 44,010 bp except for both ends of the molecule. Of 39 open reading frames (ORFs) found in the sequence, 21 ORFs (named vmpA to U) showed moderate similarities with vmp genes for Borrelia hermsii. However, most of the vmp homologues are apparently nonfunctional because of their frameshifts within the sequence and/or absence of promoter and ribosome-binding signals upstream of their genes. RT-PCR experiments using the specific primer for each vmp gene revealed that vmpE, one of the vmp genes, was expressed at the location of the 44-kb plasmid molecule. The result suggests that the plasmid molecule may play a role in the preservation of the serotype switching of vmp genes in a mammalian host.

In May 1999, field surveys of Lyme disease spirochetes were conducted around the Tianshan Mountains in Xinjiang Uygur Autonomous Region in northwestern People's Republic of China. Ixodes persulcatus ticks were obtained in a Tianchi Lake valley with primary forest, while the tick fauna was poor in the semidesert or at higher altitudes in this region. Species identities were confirmed by molecular analysis in which an internal transcribed spacer sequence was used. Of 55 adult ticks, 22 (40%) were positive for spirochetes as determined by Barbour-Stoenner-Kelly culture passages. In addition, some rodents, including Apodemus uralensis (5 of 14 animals) and Cricetulus longicaudatus (the only animal examined), and some immature stages of I. persulcatus (4 of 11 ticks) that had fed on A. uralensis were positive for spirochetes. Based on 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis and reactivity with monoclonal antibodies, 35 cultures (including double isolation cultures) were identified as Borrelia garinii (20 isolates, including 9 Eurasian pattern B isolates and 11 Asian pattern C isolates), Borrelia afzelii (10 pattern D isolates), and mixed cultures (5 cultures, including isolates that produced B. garinii patterns B and C plus B. afzelii pattern D). These findings revealed that Lyme disease pathogens are distributed in the mountainous areas in northwestern China even though it is an arid region, and they also confirmed the specific relationship between I. persulcatus and genetic patterns of Borrelia spp. on the Asian continent.