The complete mitochondrial genome of Dermacentor (Indocentor) auratus (Acari, Ixodidae)

Dermacentor (Indocentor) auratus Supino, 1897 is a prominent ixodid vector of numerous pathogens of public health and veterinary importance. Using long-range PCR of two overlapping regions sequenced on an Illumina MiSeq machine, the complete mitochondrial genome of D. auratus is reported here. The resulting contigs were able to be assembled into a complete and circularised genome which had the general organisation of the mitochondrial genomes of the Metastriates. It had a total length of 14,766 bp and contained 37 genes, including 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes, as well as 2 non-coding control regions and 3 tick-boxes. The phylogenetic analysis on the whole mitogenome confirmed the position of D. auratus within the Dermacentor clade.


Introduction
Ticks are the second largest pathogen transmitters globally, after mosquitoes [7], and tick-borne diseases (TBDs) have a colossal impact on livestock industries worldwide, and also constitute an important public health threat for humans [20]. The recent increase in TBD infections in the United States and globally, added to the increase travel fluxes and mobility, accentuates the risk of import of TBDs into new areas, as recently highlighted with the first imported case of human babesiosis in Singapore [15]. Following import, the risk of introduction and establishment of a TBD locally depends on the vector competency of the local tick populations. It is therefore important to inventory the tick fauna, particularly in areas where this has been overlooked or neglected [13] to perform risk assessments. Renewed interest in this field has lead to interesting reports such as the recent discovery of Dermacentor (Indocentor) auratus Supino, 1897 [24] in Singapore, from multiple hosts, including humans [14]. Dermacentor auratus is a competent vector of the highly pathogenic zoonotic Kyasanur Forest disease (KFD) virus [21] and the murine Lanjan Virus [25]. Several other pathogens have also been isolated from D. auratus such as Anaplasma spp., Rickettsia spp., Francisella spp., Borrelia spp. and Hepatozoon spp. [22,23,27], highlighting the veterinary and public health risks related to this species [14,22,23,27].
Along with the report of the discovery of D. auratus in Singapore, preliminary molecular data on its mitogenome were provided with sequences of the cytochrome c oxidase I (cox1) and the large subunit ribosomal RNA (lsu rRNA) genes [14]. The sequences were deposited in GenBank under accession numbers MT371767 and MT371768 (cox1) and MT371591 and MT371592 (lsu rRNA), respectively [14] and were used as the starting point for the present work that reports the entire mitochondrial genome of D. auratus.

Materials and methods
DNA material from a D. auratus TKL1 isolate obtained in [14] was used. Assuming that the mitogenome of D. auratus is circular, similar to other Metastriate ticks, its amplification was attempted using two long-range PCRs targeting fragments of %9700 bp and %6200 bp spanning the whole, and overlapping at their extremities by 457 bp and 709 bp. The primers proposed by [4] and [9] and successfully used in [14] were cross-paired. The PCRs were run in a total volume of 30 lL containing 1X High Fidelity PCR Buffer, 2 mM of MgSO 4 , 250 lM of each dNTP, 250 gM of each oligonucleotide primer, 0.06 U/lL of Platinum Ò Taq High Fidelity DNA Polymerase (Invitrogen™) and 3 lL of DNA template. The PCR assays were performed on a Veriti thermal cycler (Applied Biosystems), with the following conditions: initial denaturation at 95°C for 2 min, followed by 35 cycles of annealing at 55°C for 1 min, extension at 68°C for 6 or 10 min depending on the fragment size (increased by 20 s at each cycle from the 11th cycle), and denaturation at 94°C for 30 s, then terminated with a final cycle of annealing at 55°C for 2 min and extension at 72°C for 15 min. PCR products were visualised by capillary electrophoresis on a QIAxcel Ò Advanced System (QIAgen Ò ) equipped with a QIAxcel Ò DNA High Resolution Kit (QIAgen Ò ), then purified using a QIAquick Ò PCR Purification Kit (QIAgen Ò ), eluted in EB and stored at À30°C until sequencing. Both fragments were prepared for next-generation sequencing (NGS) by a Nextera XT DNA Library Preparation Kit (Illumina™) and sequenced on a MiSeq System (Illumina™) to generate 300 bp paired-end reads. Fastq raw reads were trimmed using Trimmomatic v.0.36 [5] to remove low-quality bases and assembled with Shovill v.1.0.4 with SPAdes as the  [1]. Contigs were aligned and scaffolded using the overlapping fragments to reconstruct the full mitogenome of D. auratus using CLC Main Workbench v8.0.1 (QIAgen Ò ). Annotation was performed using MITOS [3] and manual search of homology and comparison. Transfer RNA secondary structure prediction was obtained using the Vienna RNA Websuite [10]. Multiple alignments of the D. auratus mitochondrial genomes obtained in this study with several mitogenomes (39 Ixodidae, 1 Argasidae and 1 Nuttalliellidae) retrieved from GenBank were performed with MUSCLE [8]. Phylogenetic analysis, performed with MEGAX [12], was inferred by the maximum likelihood (ML) method based on the GTR + C + I model of evolution [26]. The most appropriate model of nucleotide substitution was selected based on BIC score [19]. Reliability of tree topology, branch support and nodal robustness were assessed by non-parametric bootstrap using 1000 replicates. The mitochondrial genome sequence obtained in this study was deposited in GenBank under accession number MW034677.

Results and discussion
Long-range PCRs, sequencing, and de novo assemblies generated 2 contigs of 9700 bp and 6194 bp, respectively with an average sequencing depth of 25-and 44-fold coverage, respectively. After reconstruction, the complete mitogenome of D. auratus was confirmed to be circular and showed a total length of 14,766 bp, with AT/GC contents of 77.2% and 22.8%, respectively. Genome annotation revealed homology to the other hard-tick mitochondrial genomes, where it contained 37 genes, including 13 protein-coding genes (cox1 to cox3, nad1 to nad6, nad4L, atp6, atp8 and cytb), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), as well as 2 non-coding control regions (CR) and 3 tick-boxes. Details about the organisation, orientation and size of the genes and non-coding elements are listed in Table 1 and mapped in Figure 1.
The complete and annotated mitochondrial genome of D. auratus has been deposited in GenBank under the accession number MW034677.

Ribosomal RNA genes
Both ssu rRNA and lsu rRNA genes were found with a length of 700 bp and 1166 bp, respectively similar to other ticks [6,11,16,18,28]. Their AT/GC compositions were 78.7%/21.3% and 81.6%/18.4% for ssu rRNA and lsu rRNA, respectively. They were both located on the negative strand between the tRNA-L2 gene and the CR1 with the tRNA-V gene intercalated in between, this organisation follows the other Metastriates [18] (Fig. 1).

Transfer RNA genes
Twenty two tRNA genes were identified including 2 tRNA-L and 2 tRNA-S (Figs. 1, 2). Their size ranged from 56 bp (tRNA-S1) to 68 bp (tRNA-M) ( Table 1). The majority of the tRNA genes (tRNA-M, tRNA-W, tRNA-K, tRNA-D, tRNA-G, tRNA-A, tRNA-R, tRNA-N, tRNA-S1, tRNA-E, tRNA-I, tRNA-T, tRNA-S2 and tRNA-C) were encoded on the positive strand, while the remaining eight were encoded on the negative strand (Fig. 1). The AT/GC content of the tRNAs ranged from 70.1%/29.9% (tRNA-K) to 90.2%/9.8% (tRNA-G). Prediction of the tRNA secondary structure showed that 20 of the tRNAs have the standard cloverleaf structure, while tRNA-S1 (anticodon TCT) and tRNA-C were missing the D-arm (Fig. 2). Mitochondrial tRNA-S secondary structure lacking the D-arms is a common feature in most animal species, including ticks [28]. Mitochondrial tRNA-C secondary structures are variable among tick species with some missing D-arm and/or T-arm and some having standard cloverleaf structure; D. auratus was only lacking the D-arm, a feature previously observed in other tick species [27,28] (Fig. 2). The sizes, coding strand and arrangement of the tRNA genes were similar to what is observed in the mitogenome of other Dermacentor species [11] and other ticks [6,16,18,28].

Non-coding sequences
Duplicate CRs were found; they contained the regulatory elements of mitochondrion transcription and replication. CR1 and CR2 spanned 311 bp and 304 bp in length, respectively and shared a common identical core sequence of 249 bp (position 9080-8832 for CR1 and 14692-14444 for CR2). Their AT/GC content was 65.9%/34.1% and 66.8%/33.2%, respectively on their full length and 64.3%/35.7% for the core sequence. CR1 was located between ssu rRNA and tRNA-I and CR2 between tRNA-L1 and tRNA-C, both on the negative strand. These locations have been observed in many other Metastriates [6,16,18,28]. Three non-coding motifs called "tick-boxes" were identified, a number identical to what was reported in Metastriates [18]. They are short post-transcriptional regulatory elements of 18 bp [18]. Tick-Boxes 1 and 2 shared an identical sequence, while Tick-Box 3 showed only 1 nucleotide difference. Tick-Box 1 was located on the negative strand at the Figure 3. Maximum likelihood phylogeny inferred on the mitochondrial genomes of Dermacentor (Indocentor) auratus obtained in this study (MW034677) (highlighted) and 41 other tick mitochondrial genomes retrieved from GenBank. Accession numbers of the sequences are provided between vertical bars. The mitogenomes of the Argasidae and Nuttalliellidae were used as outgroups to root the tree. The ML analysis used the GTR + C + I model of evolution [26]. Reliability of tree topology, branch support and nodal robustness were assessed by non-parametric bootstrap using 1000 replicates (only >70% shown). Analysis performed with MEGA X [12].
3 0 end of the nad1 gene, a position widely overlooked in many published tick mitochondrial genomes where the termination of the nad1 gene is extended to a full TAA stop codon and abuts or overlaps with the tRNA-E gene incorporating Tick-Box 1, as pointed out by [18]. Tick-Box 2 was located between the lsu RNA and the tRNA-L2 genes on the negative strand, while Tick-Box 3 was located between tRNA-Q and tRNA-F on the positive strand, following the general organisation of the other Metastriates [18].

Phylogenetic analysis
The ML analysis was performed on the mitochondrial genomes of D. auratus obtained in this study (MW034677) and 39 other Ixodidae, as well as 1 Argasidae and 1 Nuttalliellidae used as outgroups to root the tree (Fig. 3). The phylogenetic tree of the Ixodidae was strongly supported with the major genera clearly separated (bootstrap values 100%) (Fig. 3) and was monophyletic, following the recent establishment of the genera Robersticus and Archaeocroton to accommodate Amblyomma elaphense and Amblyomma sphenodonti [2], which previously had controversial phylogenetic positions (Fig. 3) [2,6,16,28]. Expectedly, D. auratus is clustered among the other Dermacentor species (Fig. 3).

Dermacentor subgenera
Up to seven subgenera were recognised within the genus Dermacentor: Dermacentor, Indocentor, Asiacentor, Americentor, Serdjukovia, Kohlsiella and Olenvenia, with the validity of the last three remaining controversial [29]. The type species of the subgenus Indocentor is D. auratus. The mitochondrial genome of D. auratus obtained here constitutes a useful reference for future studies on Dermacentor subgeneric classification. This will become particularly true when more sequences from different species become available, and reaching this point could be accelerated with the use of the simple protocol adopted here.

Conclusion
This study reports the complete mitochondrial genome of D. (Indocentor) auratus obtained using a simple method of amplification and sequencing based on two long-range PCRs and next generation sequencing. The mitogenome of D. auratus is circular and has the typical Metastriates tick mitochondrial genome organisation. All the genes were annotated and the secondary structure of the tRNAs was determined. Phylogenetic analyses indicated that D. auratus clustered with other Dermacentor species. The availability of the complete mitochondrial genome sequence of D. auratus will provide a useful reference to help in the rapid and accurate identification of this important vector of TBDs, as well as for further studies on the phylogeny and evolution history of hard ticks.