Novel Genetic Lineages of Rickettsia helvetica Associated with Ixodes apronophorus and Ixodes trianguliceps Ticks

Ixodes apronophorus is an insufficiently studied nidicolous tick species. For the first time, the prevalence and genetic diversity of Rickettsia spp. in Ixodes apronophorus, Ixodes persulcatus, and Ixodes trianguliceps ticks from their sympatric habitats in Western Siberia were investigated. Rickettsia helvetica was first identified in I. apronophorus with a prevalence exceeding 60%. “Candidatus Rickettsia tarasevichiae” dominated in I. persulcatus, whereas I. trianguliceps were infected with “Candidatus Rickettsia uralica”, R. helvetica, and “Ca. R. tarasevichiae”. For larvae collected from small mammals, a strong association was observed between tick species and rickettsiae species/sequence variants, indicating that co-feeding transmission in studied habitats is absent or its impact is insignificant. Phylogenetic analysis of all available R. helvetica sequences demonstrated the presence of four distinct genetic lineages. Most sequences from I. apronophorus belong to the unique lineage III, and single sequences cluster into the lineage I alongside sequences from European I. ricinus and Siberian I. persulcatus. Rickettsia helvetica sequences from I. trianguliceps, along with sequences from I. persulcatus from northwestern Russia, form lineage II. Other known R. helvetica sequences from I. persulcatus from the Far East group into the lineage IV. The obtained results demonstrated the high genetic variability of R. helvetica.

In many locations, several Ixodes species occur simultaneously. Thus, in the south taiga subzone of Western Siberia, I. persulcatus often occurs in sympatry with I. trianguliceps; moreover, in some locations, three Ixodes species, I. persulcatus, I. trianguliceps, and I. apronophorus, can coexist together [7,8]. All these tick species have three-host developmental cycles. Small mammals are the main hosts for the preimaginal stages of I. persulcatus, whereas I. persulcatus adults feed mainly on middle-size and large mammals [5,9]. All stages of I. trianguliceps and I. apronophorus feed predominantly on small mammals. European water vole (Arvicola amphibious) is considered to be one of the main hosts for I. apronophorus [6,7,9].
Ixodes persulcatus and I. trianguliceps have been tested for the presence of different bacterial agents in a number of studies. Both tick species can be infected with Anaplasma  Wild rodents were captured in the site Om-Bo in June 2016 and in the site Om-Zn from June-September 2014-2015. Voles and mice of the genera Myodes, Microtus and Apodemus were caught using live traps, and European water voles were captured by steel traps. The species of trapped animals were determined based on morphological features. The animals were examined for the presence of attached ticks (larvae, nymphs and adults), which were removed with forceps.
The species and stage of ticks collected from animals were preliminarily determined using a stereo microscope MC-800 (Micros, Hunnenbrunn/Gewerbezone, Austria), according to morphological keys [9,20]. The tick species of all Ixodes spp. ticks were additionally determined using the multiplex PCR assay by ITS2 fragments as described previously [8]. For a subset of specimens, the species identities were confirmed by sequencing of ITS2 and/or mitochondrial cox1 gene fragments as previously described [8].
Some engorged and nearly engorged larvae and nymphs were stored at 10-15 • C for 1-2 weeks and then transported to the laboratory and allowed to molt into nymphs or adults, respectively. Other ticks were placed in sealed plastic tubes, which were then stored in liquid nitrogen until species determination and DNA extraction.
Hereinafter, ticks molted in the laboratory are named "molted ticks", whereas ticks examined without preliminary molting are named "non-molted ticks".

Tick Metamorphosis
For successful molting, partially engorged larvae and nymphs were fed to repletion on laboratory white mice. Each engorged tick was placed individually in a glass tube and incubated in the dark at 100% relative humidity at 24-26 • C until completion of molting. Molted ticks were individually frozen four weeks after molting and stored at −70 • C until DNA extraction.

DNA Extraction
Frozen ticks were homogenized with the MagNA Lyser Instrument using the MagNa Lyser Green Beads (Roche Diagnostics, Basel, Switzerland). Total DNA was extracted from crushed individual ticks using the Proba NK kit (DNA-Technology, Moscow, Russia) according to the manufacturer's protocol. To prevent cross-contamination, DNA/RNA extraction, amplification, and PCR product detection were carried out in separate rooms. Aerosol-free pipette tips were used at each stage.

Detection and Genotyping of Rickettsia spp.
To detect Rickettsia spp. DNA, nested PCR was performed for the gltA gene with primers glt1-glt4. For correct species determination in the case of probably mixed infection, additional nested reactions were performed independently using primers RT1 and RT2, specific to "Ca. R. tarasevichiae", and primers RH1 and RH3, specific to spotted fever group rickettsiae (SFGR) ( Table 1). The amplified gltA gene fragments from all specimens positive for SFGR and some specimens positive for "Ca. R. tarasevichiae" were sequenced. For a number of specimens, fragments of 16S rRNA, ompA, ompB, sca4, and htrA genes, as well as groESL operon and 23S-5S IGS, were additionally amplified using primers specified in Table 1 and sequenced.

Sequencing and Phylogenetic Analysis
The PCR products were purified using GFX Columns (Amersham Biosciences, Piscataway, NJ, USA). Sanger reactions were performed using the BigDye Terminator V. 3.1 Cycling Sequencing Kit (Applied Biosystems, Foster City, CA, USA) in standard conditions specified in the BigDye Terminator V. 3.1 Cycling Sequencing Kit User Guide. Sanger reaction products were purified using CentriSep spin columns (Princeton Separations, Freehold, NJ, USA) and visualized with a 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with BlastN (http: //www.ncbi.nlm.nih.gov/BLAST, accessed on 3 April 2023) and BioEdit (http://www. mbio.ncsu.edu/BioEdit/bioedit.html, accessed on 3 April 2023). Phylogenetic trees were constructed using the Maximum likelihood (ML) method based on the Tamura-Nei model in MEGA 7.0 with 1000 bootstrap replicates [21].

Statistical Analysis
Differences in the prevalence of causative agents between tick species were computed with the Pearson χ 2 goodness-of-fit test (http://www.socscistatistics.com/tests/chisquare/, accessed on 20 February 2023). p < 0.05 was regarded as statistically significant.

Sampling
In this study, ticks were collected in two sites (Om-Bo and Om-Zn) of Omsk province, Western Siberia. The investigation included 145 ticks collected from small mammals in the site Om-Bo and examined without preliminary molting. In addition, 20 ticks from the site Om-Bo and 115 ticks from the site Om-Zn collected from mammals and molted under laboratory conditions were tested ( Table 2). As Rickettsia spp. is efficiently transmitted transovarially, the study of non-molted ticks of all stages can be informative.  Table 2). Among infected I. apronophorus, 44 ticks were infected with R. helvetica, and single ticks carried DNA of "Ca. R. tarasevichiae" or both R. helvetica and "Ca. R. tarasevichiae". This was the first finding of rickettsiae in I. apronophorus. Forty-eight I. persulcatus carried DNA of "Ca. R. tarasevichiae", and one tick contained DNA of both "Ca. R. tarasevichiae" and R. raoultii. As for I. trianguliceps ticks, R. helvetica was identified in nine larvae, while "Ca. R. uralica" was determined in all infected nymphs and adults (n = 6) and one larva ( Table 2).
Rickettsia raoultii and a new rickettsial genetic variant were revealed in single ticks; they were genotyped only by gltA gene. Rickettsia raoultii isolate from an I. persulcatus larva (726 bp) differed by one mismatch from R. raoultii RpA4 genotype (DQ365803). A new genetic variant Rickettsia sp. Om-113/4_Iper_m from a molted I. persulcatus (373 bp) differed by two nucleotide substitutions from R. heilongjiangensis and R. slovaca (CP002912 and U59725, respectively).

Genotyping of R. helvetica
All R. helvetica isolates were genetically characterized by sequencing gltA fragments with 840 bp length, and five sequence variants were found. For a subset of specimens with various gltA sequences, the ompB (1255 bp), sca4 (783 bp), and 16S rRNA (684 bp) gene fragments were additionally sequenced. A comparison of the obtained sequences showed the presence of six sequence variants, which varied by 2-8 substitutions. All obtained sequences differed from those of prototype R. helvetica strain C9P9 (AICO01000001) and isolates from the Russian Far East (OQ209952, OQ209953, OQ257004) ( Figure 2A). eron (1481 bp) and 23S-5S IGS (354 bp) were determined for four "Ca. R. uralica" samples from I. trianguliceps, including one molted tick (Table S1). The obtained sequences of each genetic locus showed 100% identity.
Rickettsia raoultii and a new rickettsial genetic variant were revealed in single ticks; they were genotyped only by gltA gene. Rickettsia raoultii isolate from an I. persulcatus larva (726 bp) differed by one mismatch from R. raoultii RpA4 genotype (DQ365803). A new genetic variant Rickettsia sp. Om-113/4_Iper_m from a molted I. persulcatus (373 bp) differed by two nucleotide substitutions from R. heilongjiangensis and R. slovaca (CP002912 and U59725, respectively).

Genotyping of R. helvetica
All R. helvetica isolates were genetically characterized by sequencing gltA fragments with 840 bp length, and five sequence variants were found. For a subset of specimens with various gltA sequences, the ompB (1255 bp), sca4 (783 bp), and 16S rRNA (684 bp) gene fragments were additionally sequenced. A comparison of the obtained sequences showed the presence of six sequence variants, which varied by 2-8 substitutions. All obtained sequences differed from those of prototype R. helvetica strain C9P9 (AI-CO01000001) and isolates from the Russian Far East (OQ209952, OQ209953, OQ257004) ( Figure 2A). Phylogenetic analysis based on gltA-ompB-sca4 concatenated sequences demonstrated that the obtained sequences belong to three genetic lineages I-III (Figure 3). Specimens from lineage I (European lineage) clustered together with R. helvetica str. C9P9, a prototype R. helvetica strain isolated from I. ricinus from Switzerland. Studied specimens differed from the C9P9 strain by single substitutions in the ompB or gltA genes (Figure 2A). Sequences from this lineage were identified in site Om-Zn in three molted I. apronophorus and one molted I. persulcatus (Table 3). Sequences from lineage II (I. trianguliceps lineage) were identical to those previously found in two feeding I. trianguliceps nymphs from Omsk Province (Gen-Bank KR150775, KR150777, KR150781, KR150786) [14]. In this study, nine I. trianguliceps larvae and one molted I. apronophorus from site Om-Bo contained DNA of R. helvetica from lineage II (Tables 3 and S2). Lineage III (I. apronophorus lineage) was the most abundant and contained only novel sequences that were determined in 48 I. apronophorus, mainly from the site Om-Bo (Tables 3 and S1). This lineage was genetically diverse; the sequence of a specimen Om-103_Iapr differed from others by one substitution in the gltA gene, whereas sequences of five specimens differed by one substitution in the highly conserved 16S rRNA gene (Figure 2A). Notably, all sequences with a unique substitution in the 16S rRNA gene were found in larvae collected from vole 79. In addition, some other sequences previously detected in I. persulcatus from the Far East formed lineage IV (the Far Eastern lineage) on the constructed phylogenetic tree (Figure 3).
Specimens from lineage I (European lineage) clustered together with R. helvetica str. C9P9, a prototype R. helvetica strain isolated from I. ricinus from Switzerland. Studied specimens differed from the C9P9 strain by single substitutions in the ompB or gltA genes (Figure 2A). Sequences from this lineage were identified in site Om-Zn in three molted I. apronophorus and one molted I. persulcatus (Table 3). Sequences from lineage II (I. trianguliceps lineage) were identical to those previously found in two feeding I. trianguliceps nymphs from Omsk Province (GenBank KR150775, KR150777, KR150781, KR150786) [14]. In this study, nine I. trianguliceps larvae and one molted I. apronophorus from site Om-Bo contained DNA of R. helvetica from lineage II (Tables 3 and S2). Lineage III (I. apronophorus lineage) was the most abundant and contained only novel sequences that were determined in 48 I. apronophorus, mainly from the site Om-Bo (Tables 3 and S1). This lineage was genetically diverse; the sequence of a specimen Om-103_Iapr differed from others by one substitution in the gltA gene, whereas sequences of five specimens differed by one substitution in the highly conserved 16S rRNA gene (Figure 2A). Notably, all sequences with a unique substitution in the 16S rRNA gene were found in larvae collected from vole 79. In addition, some other sequences previously detected in I. persulcatus from the Far East formed lineage IV (the Far Eastern lineage) on the constructed phylogenetic tree (Figure 3).     Since many R. helvetica isolates from the GenBank database were characterized by ompB gene, we used this genetic locus to analyze available R. helvetica sequences from other regions. The phylogenetic tree, which was reconstructed using the ompB gene fragment with a length of 2684 bp, demonstrated the presence of the same four wellsupported genetic lineages I-IV (Figure 4). As a result of phylogenetic analysis, genetic lineage I was supplemented with R. helvetica specimens from I. ricinus from Germany (MF163037, HQ232244-HQ232251) and I. persulcatus from Western Siberia (Novosibirsk province) (KU310591) (Figure 4) Lineage II additionally included 32 R. helvetica specimens identified in I. persulcatus from Komi Republic [31]. Sequences from the Komi Republic differed from the studied R. helvetica sequences from I. trianguliceps by one substitution in each of the ompB and gltA genes (Figures 2B and 4). As for genetic lineage IV, sequences from I. persulcatus from the continental part of the Far East (KT825966, KT825970) also corresponded to this lineage in addition to those from Sakhalin and Putyatin islands [15].
Since many R. helvetica isolates from the GenBank database were characterized by ompB gene, we used this genetic locus to analyze available R. helvetica sequences from other regions. The phylogenetic tree, which was reconstructed using the ompB gene fragment with a length of 2684 bp, demonstrated the presence of the same four well-supported genetic lineages I-IV (Figure 4). As a result of phylogenetic analysis, genetic lineage I was supplemented with R. helvetica specimens from I. ricinus from Germany (MF163037, HQ232244-HQ232251) and I. persulcatus from Western Siberia (Novosibirsk province) (KU310591) (Figure 4) Lineage II additionally included 32 R. helvetica specimens identified in I. persulcatus from Komi Republic [31]. Sequences from the Komi Republic differed from the studied R. helvetica sequences from I. trianguliceps by one substitution in each of the ompB and gltA genes (Figures 2B and 4). As for genetic lineage IV, sequences from I. persulcatus from the continental part of the Far East (KT825966, KT825970) also corresponded to this lineage in addition to those from Sakhalin and Putyatin islands [15]. For more detailed genotyping, sequences of the 16S rRNA (1070 bp), gltA (1037 bp), ompA (1417 bp), ompB (3100 bp), sca4 (2398 bp) and htrA (499 bp) genes, as well as 23S-5S IGS region (489 bp) and groESL operon (1528 bp), were determined for five R. helvetica isolates, belonging to different lineages. Notably, sequences of the groESL operon were identical to all known R. helvetica samples and these sequences were not used for phylogenetic analysis. All other examined genetic loci have polymorphic sites, with the ompB gene being the most variable. Among coding sequences, nucleotide substitutions in 15/25 polymorphic sites were non-synonymous ( Figure 2B). The obtained concatenated sequence of isolate Om-74_Iapr_m from lineage I differed from the sequence of R. helvetica str. C9P9 by one substitution in the ompB gene ( Figure 2B). Sequences of the specimens For more detailed genotyping, sequences of the 16S rRNA (1070 bp), gltA (1037 bp), ompA (1417 bp), ompB (3100 bp), sca4 (2398 bp) and htrA (499 bp) genes, as well as 23S-5S IGS region (489 bp) and groESL operon (1528 bp), were determined for five R. helvetica isolates, belonging to different lineages. Notably, sequences of the groESL operon were identical to all known R. helvetica samples and these sequences were not used for phylogenetic analysis. All other examined genetic loci have polymorphic sites, with the ompB gene being the most variable. Among coding sequences, nucleotide substitutions in 15/25 polymorphic sites were non-synonymous ( Figure 2B). The obtained concatenated sequence of isolate Om-74_Iapr_m from lineage I differed from the sequence of R. helvetica str. C9P9 by one substitution in the ompB gene ( Figure 2B). Sequences of the specimens from lineages II and III varied between themselves by 20 substitutions and differed from the sequences of R. helvetica str. C9P9 and Far Eastern isolate Skh-7_Iper (OQ209950-OQ209956, OQ257004) by 12-16 substitutions ( Figure 2B). The comparison of polymorphic sites from different genetic loci showed that ompB and sca4 gene fragments could be used to reliably differentiate specimens from various genetic lineages ( Figure 2B). Notably, phylogenetic analysis based on the sca4 gene demonstrated that R. helvetica isolate from I. persulcatus from Japan (FJ358501) [27] can also be referred to as the Far Eastern lineage.
Phylogenetic tree based on 16S-gltA-ompA-ompB-sca4-htrA-IGS concatenated sequences (9779 bp) ( Figure 5) showed the presence of four well-supported genetic lineages, which correspond to those that were identified based on analysis of shorter gltA-ompB-sca4 concatenated sequences and the ompB gene fragment with a length of 2684 bp (Figures 3 and 4). isolate from I. persulcatus from Japan (FJ358501) [27] can also be referred to as the Far Eastern lineage.
Phylogenetic tree based on 16S-gltA-ompA-ompB-sca4-htrA-IGS concatenated sequences (9779 bp) ( Figure 5) showed the presence of four well-supported genetic lineages, which correspond to those that were identified based on analysis of shorter gltA-ompB-sca4 concatenated sequences and the ompB gene fragment with a length of 2684 bp (Figures 3 and 4).
The study of rickettsiae agents in ticks from sympatric areas is of particular interest because it makes it possible to compare pathogen-tick association for different tick species from the same location. This study includes I. apronophorus, I. persulcatus and I. trianguliceps ticks collected in two sites in the Omsk Province. In site Om-Bo, the abundance of all these tick species was high, whereas in site Om-Zn, I. persulcatus dominated and the prevalence of I. apronophorus was low [8].
In this study, Rickettsia spp. were first found in I. apronophorus. Rickettsia helvetica was found in 70-80% of molted and non-molted I. apronophorus from both locations, indicating a close association of R. helvetica with I. apronophorus. In addition to R. helvetica, "Ca. R. tarasevichiae" was identified in 3% of non-molted I. apronophorus (Table 2).
Expectedly, "Ca. R. tarasevichiae" prevailed in I. persulcatus, occurring in more than 80% of molted and non-molted ticks. Other Rickettsia spp. were found in I. persulcatus only in single cases. Previously, a similarly high prevalence of "Ca. R. tarasevichiae" in questing adult I. persulcatus has been observed in various regions of the Asian part of Russia in Omsk Province, but not in I. persulcatus from Sakhalin Island, Komi Republic, and Estonia [11,[14][15][16]31,32]. Surprisingly, one molted I. persulcatus was infected with
The study of rickettsiae agents in ticks from sympatric areas is of particular interest because it makes it possible to compare pathogen-tick association for different tick species from the same location. This study includes I. apronophorus, I. persulcatus and I. trianguliceps ticks collected in two sites in the Omsk Province. In site Om-Bo, the abundance of all these tick species was high, whereas in site Om-Zn, I. persulcatus dominated and the prevalence of I. apronophorus was low [8].
In this study, Rickettsia spp. were first found in I. apronophorus. Rickettsia helvetica was found in 70-80% of molted and non-molted I. apronophorus from both locations, indicating a close association of R. helvetica with I. apronophorus. In addition to R. helvetica, "Ca. R. tarasevichiae" was identified in 3% of non-molted I. apronophorus (Table 2).
Expectedly, "Ca. R. tarasevichiae" prevailed in I. persulcatus, occurring in more than 80% of molted and non-molted ticks. Other Rickettsia spp. were found in I. persulcatus only in single cases. Previously, a similarly high prevalence of "Ca. R. tarasevichiae" in questing adult I. persulcatus has been observed in various regions of the Asian part of Russia in Omsk Province, but not in I. persulcatus from Sakhalin Island, Komi Republic, and Estonia [11,[14][15][16]31,32]. Surprisingly, one molted I. persulcatus was infected with "Ca. R. uralica", despite "Ca. R. uralica" was not found in any of the over 500 previously analyzed questing I. persulcatus from Omsk province [14].
As for I. trianguliceps, three Rickettsia species, "Ca. R. uralica", "Ca. R. tarasevichiae" and R. helvetica, were found in this tick species. Notably, the prevalence of Rickettsia spp. substantially varied depending on the sampling site and developmental stage of I. trianguliceps ( Table 2). Association of "Ca. R. uralica" with I. trianguliceps has been previously recorded in other locations of Omsk province and Estonia [14,17], whereas "Candidatus R. thierseensis" (a genetic variant of "Ca. R. uralica") was found in one I. ricinus in Austria [24,36]. The findings of "Ca. R. uralica" in human-biting I. persulcatus and I. ricinus may indicate the potential threat of this rickettsial species to humans. "Candidatus R. tarasevichiae", recently recognized as a pathogenic species [37][38][39][40], is reliably associated with I. persulcatus; however, in rare cases, it has been found in other tick species, namely I. pavlovskyi, Dermacentor spp., and Haemaphysalis spp. [11,16,22,41,42]. In this study, "Ca. R. tarasevichiae" was found in two molted I. trianguliceps in the Om-Zn site (Table 2), which is consistent with the previous detection of "Ca. R. tarasevichiae" in