〔Med. Entomol. Zool. Vol. 71 No. 4 p. 279‒288 2020〕 279 reference DOI: 10.7601/mez.71.279

Original Article

Analysis of sequences from Haemaphysalis ava (Acari: Ixodidae) and Tabanus rudens (Diptera: Tabanidae) collected in Ishikawa, Japan

Daisuke K *, Astri Nur F, Michael A -B  , Mamoru W, Yoshihide M, Toshihiko H, Yukiko H, Kyoko S and Haruhiko I

* Corresponding author: [email protected] Department of Medical Entomology, National Institute of Infectious Diseases, 1‒23‒1 Toyama, Shinjuku-ku, Tokyo 162‒8640, Japan

(Received: 26 June 2020; Accepted: 24 August 2020)

Abstract: Trypanosoma are known to be a diverse group of parasites that infect animals belonging to all classes in the subphylum Vertebrata and are important pathogens that aect human and animal health. Although many trypanosomatids have been found in mammals and birds in Japan, information regarding their invertebrate host is currently lacking. During our virome analyses of and horse ies, several trypanosoma-like sequences were found. Further sequence characterization and PCR-based screening revealed trypanosomatids termed Trypanosoma sp. 17ISK-T2 and 17ISK-T22 in the nymphs of Haemaphysalis ava, and T. theileri-like sequences in Tabanus rudens. ese results indicate that virome analysis by next-generation sequencing (NGS) can also be used as a tool for protozoan detection from . Further investigations will assist in understanding the diversity and transmission dynamics of these parasites in Japan.

Key words: Trypanosoma, Trypanosomatids, Trypanosomiasis, Trypanosoma theileri, , horse y

I   been reported in cattle (Sasaki, 1958; Iwata et al., 1959; Ishida et al., 2002; Matsumoto et al., 2011). Although Trypanosomatids in the genus Trypanosoma T. theileri in general shows non-pathogenicity in are a diverse group of parasites that infect animals cattle, the potential for exacerbating pathogenicity belonging to all classes in the subphylum Vertebrata. by concomitant infection with piroplasma or bovine All Trypanosoma, except T. equiperdum Doein, require leukemia virus has been observed (Iwata et al., invertebrate hosts for transmission between 1959; Matsumoto et al., 2011). e prevalence of T. hosts (Kaufer et al., 2017). Trypanosoma includes theileri in cattle is not well understood due to its non- several important pathogens of humans and animals. pathogenicity in cattle occurring in a single infection. For instance, African sleeping sickness caused by T. Furthermore, the vector species of T. theileri have brucei gambiense Dutton and T. b. rhodesiense Stephens remained obscure in Japan thus far. On the other hand, and Fantham is endemic in several African countries multiple Trypanosoma parasites have been observed in (Büscher et al., 2017), while Chagas disease caused by T. Japanese birds (Table 1). However, these parasites have cruzi Chagas is a public health concern in Latin America not been classied into species, and their sequence (Pérez-Molina and Molina, 2018). Furthermore, atypical information is not available (Table 1). is might be human infections of animal trypanosomes such as T. due to their low or unknown pathogenicity in the host b. brusei Plimmer and Brandford, T. evansi Steel, or T. (Kano, 1950; Hirayama et al., 2014). Moreover, human lewisi (Kent) have been reported and recent molecular pathogenic T. lewisi was reported in Japan more than a diagnosis technique advances have allowed more century ago (reviewed by Irikura, 1906), and the recent frequent detection of these atypical infections (Truc et distribution and endemic situation of the parasite al., 2013). remains unclear. Conversely, several Trypanosoma While several trypanosomatids have been found parasites have been found in ticks; however, their from mammals and birds in Japan, their invertebrate vertebrate host has not been identied (Table 1). us, hosts have not yet been elucidated (Table 1). In Japan, information on domestic Trypanosoma parasites is several cases of T. theileri (Laveran) infection have limited. 280 Med. Entomol. Zool. et al., 1998 2011. etc. Nagata, 2006 Nagata, 2006 Kano and Kimura, 1950 Nagata, 2006 reviewed by Kano and Kimura, 1950 Hayashi et al., 1998 Nagata, 2006 reviewed by Kano and Kimura, 1950 Nagata, 2006 reviewed by Kano and Kimura, 1950 Kano and Kimura, 1950 Nagata, 2006 Kano and Kimura, 1950 Nagata, 2006 reviewed by Kano and Kimura, 1950 Nagata, 2006 reviewed by Kano and Kimura, 1950; Sakamoto et al., 1981; Hayashi Nagata, 2006 Kano and Kimura, 1950 Nagai, 1954; Nagata, 2006 Nagata, 2006 reviewed by Kano and Kimura, 1950 Kano and Kimura, 1950 Nagata, 2006 Nagata, 2006 Nagata, 2006 reviewed by Kano and Kimura, 1950 Nagata, 2006 Nagata, 2006 ekisoe et al., 2007 Fujita and Watanabe, 2007 Fujita and Watanabe, 2007 Mae et al., 2018 reviewed by Irikura, 1906 Sasaki, 1958; Iwata et al., 1959; Ishida 2002; Matsumoto Hatama et al., 2007 Nakamoto et al., 2014 Ito and Itagaki, 2003; Fujita Watanabe, 2007 Reference Haemaphysalis hystricis Haemaphysalis ava Amblyomma testudinarium Scientic name ** ** ** unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown unknown tick tick tick hemipteran insects ea tabanid unknown unknown unknown Invertebrate host Common name Acrocephalus bistrigiceps Emberiza spodocephala Fringilla montifringilla Hypsipetes amaurotis Lanius bucephalus Corvus corone Emberiza fucata Otus lempiji Turdus naumanni Garrulus glandarius Carduelis spinus Emberiza variabilis Coccothraustes coccothraustes Cettia diphone Bombycilla japonica Zosterops japonicus Corvus macrorhynchos Pycnonotus sinensis Uragus sibiricus Emberiza cioides Acrocephalus arundinaceus Otus scops Carpodacus roseus Leiothrix lutea Tarsiger cyanurus Luscinia cyane Strix uralensis Parus varius Emberiza elegans Miniopterus fuliginosus Rattus norvegicus and R. rattus Bos taurus Cervus nippon yesoensis Apodemus speciosus Apodemus speciosus Scientic name Table 1. Records of trypanosoma parasites in mammals, birds, and arthropods Japan. Black-browed reed warbler Black-faced bunting Brambling Brown-eared bulbul Bull-headed shrike Carrion crow Chestnut-eared bunting Collared scops owl Dusky thrush Eurasian jay Eurasian siskin Grey bunting Hawnch Japanese bush warbler Japanese waxwing Japanese white-eye Large-billed crow Light-vented bulbul Long-tailed rosench Meadow bunting Oriental reed warbler Oriental scops owl Pallas’s rosench Red-billed leiothrix Red-anked buletail Siberian blue robin Ural owl Varied tit Yellow-throated bunting unknown unknown unknown Eastern bent-wing bat Brown rat and black Cattle Hokkaido sika deer Large Japanese eld mouse Large Japanese eld mouse Common name Aves Mammalia Class Vertebrate host * * * * * * * * * * * * * * * * * * * * * * Invertebrate host have been identied in overseas but not yet Japan. Only morphological observation. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. Trypanosoma sp. KG1 Trypanosoma sp. Trypanosoma sp. Trypanosoma lewisi Trypanosoma theileri Trypanosoma sp. TSD1 Trypanosoma grosi Trypanosoma sp. Trypanosoma sp. Species Trypanosoma dionisii * ** Vol. 71 No. 4 2020 281

During our virome analyses of the ticks and horse mixed into the tabanid pool in equal amounts (42.2‒ ies, several trypanosoma-like sequences were found. 190 µL/pool). Preparation of the library for NGS erefore, this study characterized the sequences and was performed with NEB Next RNA rst-strand investigated their infection status among ticks and and second-strand synthesis modules (New England horse ies collected in Japan. Biolabs), NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs), and NEBNext Ultra M   M  II Ligation Module (New England Biolabs) according Tick and horse y collection to the manufacturer’s protocol. Following purication Host questing ticks were collected from vegetation of the libraries by Agencourt AMPure XP (Beckman elds in several sites in the Ishikawa and Toyama Coulter), quantication was performed, and the Prefectures, Japan, in October 2017 by dragging libraries were amplied as required by NEBNext Ultra as described previously (Kobayashi et al., 2020). II Q5 Master Mix (New England Biolabs). e puried e information regarding tick collection sites was libraries were analyzed using a MiniSeq system listed in a previous report (Kobayashi et al., 2020). (Illumina) with a MiniSeq Mid Output kit (300 cycles) Furthermore, from April to June 2018, additional ticks (Illumina). e obtained reads were subjected to were collected at the same sites. trimming and de novo assembly on a CLC Genomics Female tabanids were collected by sweeping in Workbench version 12 (Qiagen). e resultant contigs Ishikawa Prefecture, Japan, in August 2018. e were identied by BLASTN search. e sequence collection sites were as follows: Point Saruyama, analyses were carried out by Genetyx soware version Yoshiura, Monzen-machi, Wajima City (37°19′26.1″N, 13 (Genetyx). 136°43′31.4″E); Fukami, Monzen-machi, Wajima City (37°17′58.4″N, 136°44′16.6″E); and Awazu, Misaki- Screening and sanger-sequencing of machi, Suzu City (37°29′05.0″N, 137°19′52.6″E). e trypanosomatids collected ticks and tabanids were divided into species In order to identify a trypanosomatid-positive by morphology and stored at −80°C until analyses. pool, a polymerase chain reaction (PCR)-based Incidentally, no blood-fed ticks and tabanids were screening was performed. 200‒300 µL of phosphate- contained in the specimens. buered saline (Sigma-Aldrich) was added to the homogenate and crushed again. DNA was extracted Next-generation sequencing from 200 µL of the supernatant of the homogenate Trypanosomatid sequences were detected during using a DNeasy Blood & Tissue kit (Qiagen) according the process of RNA virome analyses for questing and to the manufacturer’s protocol. e partial 18S characterization of viruses of the ticks and tabanids. ribosomal RNA (rRNA) gene was amplied by Prime e next-generation sequencing (NGS) method for Star Max DNA polymerase (Takara) with the primer the ticks was described previously (Kobayashi et al., set SSU-1 (5′-ATC TGC GCA TGG CTC ATT AC- 2020). e fundamental technique of NGS for tabanids 3′) and SSU-2 (5′-CAC ACT TTG GTT CTT GAT was the same as described previously (Kobayashi et TGA-3′) designated by Barratt et al. (2017) (Fig. 1). al., 2020). In brief, tabanid samples were crushed in a PCR was conducted under the following condition; medium by tissue lyser II (Qiagen) and ltrated with 35 cycles at 98°C for 10 seconds, 55°C for 5 seconds, a 0.45 µm lter. en, nuclease cocktail [14 units of and 72°C for 7 seconds. e amplied products TURBO DNase (Invitrogen), 11.5 units of Baseline- were conrmed by agarose gel electrophoresis. For zero DNase (epicentre), and 15 µg of RNase A (Nippon additional PCR-based screening, a total of 114 pools gene)] was added to the 380 µL ltrate, which was composed of at least eight tick species [Amblyomma

Fig. 1. A schematic illustration of the genetic structure of ribosomal RNA (rRNA) of trypanosomatid and the distribution of trypanosoma-like contigs. e black triangles on the illustration of the rRNA gene represent the position of the primer set used for the PCR-based screening. e lower two dotted boxes showed the distribution of contigs obtained from ticks (above) and tabanids (below). 282 Med. Entomol. Zool.

Table 2. Detected contigs related to trypanosomatids by NGS analysis.

Result of blastn search Contig Length Total read Average Source Accession name (nt) count coverage Highest score sequence name e-value identity (%) no. H. ava nT4_c22 705 45 9.41 Trypanosoma sp. KG1 gene for 18S ribosomal RNA, AB281091 3e-106 92.09, 99.10* partial sequence T. rudens YA3_c42 678 40 8.48 Trypanosoma theileri isolate Cow 2095 clone 4 18S JX853185 0.0 100.00 ribosomal RNA gene, partial sequence; and internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence YA3_c43 574 45 10.58 Trypanosoma minasense genes for 18S rRNA, ITS1, AB362411 0.0 93.69 5.8S rRNA, ITS2, 28S rRNA, partial and complete sequence YA3_c45 807 57 9.99 Trypanosoma minasense genes for 18S rRNA, ITS1, AB362411 0.0 96.42 5.8S rRNA, ITS2, 28S rRNA, partial and complete sequence YA3_c49 1057 65 8.59 Trypanosoma theileri gene for 18S rRNA, 5.8S rRNA, AB007814 0.0 100.00 28S rRNA, partial and complete sequence YA3_c56 574 51 11.98 Trypanosoma minasense genes for 18S rRNA, ITS1, AB362411 0.0 95.30 5.8S rRNA, ITS2, 28S rRNA, partial and complete sequence

* Two unknown sequence regions are contained inside one contig. e range of number shows the value in each frame. testudinarium Koch (1 nymph), Haemaphysalis ava Within this sample, one contig (nT4_c22) related to Neumann (252 nymphs, 81 males, and 90 females), the sequence of trypanosomatids was identied via a H. formosensis Neumann (1 male), H. hystricis Supino BLASTN search (Table 1). e sequence showed high (3 nymphs and 4 females), H. longicornis Neumann similarity to the 18S rRNA gene of Trypanosoma sp. (72 nymphs, 1 male, and 5 females), H. megaspinosa KG-1, isolated from H. hystricis collected in Kagoshima Saito (3 nymphs), Haemaphysalis spp. (105 larvae), Prefecture, Japan ( ekisoe et al., 2007). Furthermore, Ixodes ovatus Neumann (6 nymphs, 10 males, and trypanosoma-like sequences were contained in the 23 females), I. turdus Nakatsudai (3 nymphs and 1 resultant contigs produced from a total of 759,868 reads female), and Ixodes spp. (2 nymphs)] and 36 tabanid in the tabanids sample, which was made up of eight T. pools (composed of 6 Tabanus iyoensis Shiraki, 5 T. rudens females. Four contigs similar to the sequences sapporoensis Shiraki, 5 T. mandarinus Schiner, 2 T. of trypanosomatids were identied by the BLASTN trigonus Coquillett, 2 T. chrysurus Loew, and 25 T. search, and two contigs (named YA3_c42 and YA3_ rudens Bigot) were used. e amplicon was sanger- c49) were 100% identical to that of T. theileri (Table sequenced by the same method described previously 1). Other contigs (YA3_c43, YA3_c45, and YA3_c56) (Kobayashi et al., 2016). from the tabanids showed a 93‒96% sequence identity to the genes of T. minasense Chagas (Table 1). Multiple Phylogenetic analysis sequence alignment revealed that two contigs (YA3_c42 Multiple sequence alignment was performed by and YA3_c49) and others (YA3_c43, YA3_c45, and MUSCLE (Madeira et al., 2019). Selections of the YA3_c56) corresponded to the 18S and 28S rRNA genes suitable nucleotide substitution models by MEGA 6 of the Trypanosoma, respectively (Fig. 1). (Tamura et al., 2013). e phylogenetic dendrogram was constructed by the maximum likelihood (ML) PCR-based screening of trypanosomatids from the method with a GTR+G+I or K2+G model by MEGA ticks and tabanids 6 (Tamura et al., 2013). e trypanosomatid sequences PCR-based screening was performed to identify used in this analysis are shown in Appendix 1. the trypanosomatid-positive pool, with the primer set (SSU-1 and SSU-2), which targeted a highly R  conserved region on the 18S rRNA gene among the Detection of trypanosoma-like sequences by NGS trypanosomatids (Fig. 1). e pool no. 17ISK-T22 analyses (made up of nine H. ava nymphs) was identied During the RNA virome analyses of ticks and as the positive pool for trypanosomatid (Table 3). tabanids, trypanosoma-like contigs were detected e sanger-sequenced amplicon possessed the same (Table 2) in the tick sample nT4 (combination of 7 pools sequence as that of contig nT4_c22, which indicated composed of larva, nymph, and adult H. ava; adult that the sequence was derived from this pool. Further H. formosensis; and larval Haemaphysalis spp., further PCR-based screening was performed on homogenate details can be found in Kobayashi et al., 2020), a total of leover from the virus isolation (Kobayashi et al., 1,149,100 reads were obtained (Kobayashi et al., 2020). 2020) and additional tick samples as described above. Vol. 71 No. 4 2020 283

Table 3. Summary of trypanosomatids detected in this study.

GenBank Source Name accession no. Species Collection site Collection date Trypanosoma sp. 17ISK-T2 LC556385 H. ava pooled 6 nymphs Igisu, Monzen-machi, Wajima City, 5 October, 2017 Ishikawa Prefecture, Japan Trypanosoma sp. 17ISK-T22 LC556386 H. ava pooled 9 nymphs Kamo, Tsubata Town, Ishikawa Prefecture, 24 October, 2017 Japan Trypanosoma sp. 18HF29 LC556387 T. rudens 1 adult female Awazu, Misaki-machi, Suzu City, Ishikawa 8 August, 2018 Prefecture, Japan

A trypanosomatid sequence was identied in only lupus familiaris Linnaeus) in Brazil (Madeira et al., the pool 17ISK-T2 (made up of 6 nymphs of H. ava) 2009). Furthermore, the 17ISK-T2, 17ISK-T22, and (Table 3). e sequence exhibited 100% identity to that trypanosomatids from ticks formed a clade in the of 17ISK-T22 (data not shown), which suggested that dendrogram (Fig. 2B). trypanosomatids detected in the two dierent H. ava To conrm the phylogenetic relationships between pools were the same species. Trypanosoma sp. 18HF29 and trypanosomatids in On the other hand, the trypanosomatid-positive pool T. theileri clade, ML tree was constructed based on was identied as the pool no. 18HF29, which consisted partial 18S rRNA gene (548 nt) by K2+G model (Fig. of one female T. rudens, from the pooled NGS sample 3). Although several T. theileri patasites have been (Table 3). PCR-based screening was performed in the isolated in Japan, Trypanosoma sp. 18HF29 formed a same manner to detect trypanosomatids in the other clade with T. theileri strain KM (GenBank accession tabanid pools. However, no positive pool was detected no. AB007814) in the dendrogram (Fig. 3). from the other 36 tabanid pools. D   Phylogenetic relationships among trypanosomatids Several trypanosomatid sequences had been Phylogenetic analysis was conducted based on detected in data from the RNA virome of ticks and about 1,000 nt sequences of the partial 18S rRNA tabanids. e system for RNA virome analysis was gene amplied by SSU-1 and SSU-2. e phylogenetic established by a previous study (Kobayashi et al., 2020) dendrogram was constructed by the maximum and the host microorganisms such as protozoans likelihood (ML) method with a GTR+G+I model or bacteria were assumed to be excluded during (Fig 2A). e trypanosomatids from the H. ava the small-pore ltration step, at least in principle. ticks (named Trypanosoma sp. 17ISK-T2 and However, several bacteria or trypanosomatids were T22, respectively, Table 3) formed a cluster with detected from the resultant contigs, which were almost Trypanosoma parasites from H. hystricis (Trypanosoma all derived from their rRNA genes. Furthermore, sp. KG-1), badger (T. pestanai Bettencourt and Franca many sequence reads derived from host rRNA genes LEM 110), and wombat (T. copemani Auster, Jeeries, were also detected in spite of the nuclease treatment Friend, Ryan, Adams and Reid H26 and wombat of samples. Ribosomes were composed of rRNAs AAP). However, the 17ISK-T2 and 17ISK-T22 are forming higher-ordered structures and ribosomal located apart from these trypanosomatids. Moreover, proteins (Alberts et al., 2014). erefore, it seems that the trypanomatid detected from T. rudens was the rRNA genes were easy to retain because RNase located in the T. theileri clade (data not shown). A used in the nuclease treatment step in this study Recent studies revealed that several Trypanosoma digests single-stranded RNA only. In addition, a typical parasites, which were related to the 17ISK-T2 and eukaryotic cell contains millions of ribosomes in the 17ISK-T22, were found in animals and ticks (Madeira cytoplasm (Alberts et al., 2014). Furthermore, previous et al., 2009; Marotta et al., 2018a, b). However, there studies indicated that a rich rRNA was contained in were short overlap sequences among the 17ISK-T2, the extracellular vesicles derived from Trypanosoma 17ISK-T22, and related species. erefore, further parasite (Bayer-Santos et al., 2014; Garcia-Silva et al., phylogenetic analysis was performed using the 2014). ese extracellular vesicles probably play a role available sequences of each related species. e in protection of inside RNAs from the nuclease. us, dendrogram constructed by the ML method with a detection of trypanosomatid sequences as shown in K2+G model using aligned about 300 nt of 18S rRNA this study seems very probable. genes was shown in Fig. 2B. In the dendrogram, the A previous study reported a novel Trypanosoma 17ISK-T2 and 17ISK-T22 were related to T. caninum parasite named Trypanosoma sp. KG-1 isolated from Madeira, Almeida, Barros, Olireira, Sousa, Alres, pooled adult H. hystricis collected in Kagoshima Miranda, Schubach and Marzochi, a recently described Prefecture in Japan ( ekisoe et al., 2007). Another Trypanosoma species found in a domestic dog (Canis report showed that three trypanosomatid isolates were 284 Med. Entomol. Zool.

Fig. 2. Phylogenetic characterization of trypanosomatids detected from ticks in this study. (A) e phylogenetic dendrogram was constructed based on the nucleotide sequences (about 1,000 nt) by the ML method with the use of the GTR+G+I model. e bootstrap values of more than 50 required by bootstrap test (1000 replicates) (Felsenstein, 1985) are shown in the next to the branches. e origin of each trypanosomatids is indicated in parenthesis. Bodo caudatus and Trypanoplasma borreli were used as an outgroup. e accession numbers of trypanosomatids used in this analysis are shown in Appendix 1. (B) e phylogenetic dendrogram was constructed based on the nucleotide sequences (about 300 nt) by the ML method with the use of the K2+G model. e bootstrap values of more than 50 required by bootstrap test (1000 replicates) (Felsenstein, 1985) are shown in the next to the branches. Trypanosomatids isolated from ticks are represented using illustrations. Trypanosomatids which are identied in this study are indicated by a black circle and bold-faced. e origin of each trypanosomatids is indicated in parenthesis. e accession numbers of the trypanosomatids used in this analysis are shown in Appendix 1. obtained from the nymphs of H. ava collected from trypanosomatids from H. ava are not available. In this Kagoshima, Tokushima, and Fukushima Prefectures study, two trypanosomatid sequences were detected in Japan (Fujita and Watanabe, 2007). e latter study from two pools of H. ava nymphs, and their sequences suggested that the trypanosomatids from H. ava were dierent from that of the Trypanosoma sp. KG- and the KG-1 were two dierent species because 1. Trypanosoma sp. 17ISK-T2 and 17ISK-T22 were they were discriminable by morphology (Fujita and detected in the same tick species and developmental Watanabe, 2007). However, the sequences of these stages indicated that they are probably the same species Vol. 71 No. 4 2020 285

Fig. 3. Phylogenetic relationships between Trypanosoma sp. 18HF29 and related trypanosomatids. e phylogenetic dendrogram was constructed based on the nucleotide sequences (about 548 nt) by the ML method with the use of the K2 + G model. e bootstrap values of more than 50 required by bootstrap test (1000 replicates) (Felsenstein, 1985) are shown in the next to the branches. T. melophagium was used as the root of tree. Trypanosoma sp. 18HF29 which is identied in this study is indicated by a black circle and bold-faced. e origin of each trypanosomatids is indicated in parenthesis. e accession numbers of trypanosomatids used in this analysis are shown in Appendix 1. as that reported by Fujita and Watanabe (2007). In this McDonnel, Sheils, Gilchrist, Votypka and Vogelnest, study, the trypanosomatids were not detected from and T. avium Votýpka, Szabová, Rádrová, Zídková other tick species, suggesting that this Trypanosoma and Svobodová. Interestingly, T. amblyommi Marotta, might be an H. ava-specic species. Dos Santos, Cordeiro, Barros, Bell-Sakyi and Fonseca Trypanosoma sp. 17ISK-T2 and 17ISK-T22 formed and T. rhipicephalis Marotta, Dos Santos, Cordeiro, a clade with trypanosomatids from ticks and a dog Matos, Barros, Madeira, Bell-Sakyi and Fonseca were in the phylogenetic dendrogram in this study. All isolated from ticks collected from white-lipped peccary trypanosomatids from ticks are yet to be detected in (Tayassu pecari) and cattle, respectively (Marotta et al., their vertebrate hosts ( ekisoe et al., 2007; Marotta et 2018a, b). us, these infested animals appear to be al., 2018a, b). However, T. caninum, which was isolated vertebrate hosts of these trypanosomatids. erefore, from dogs in Brazil, was located in the sister clade of to identify the vertebrate host of these tick-associated 17ISK-T2 and 17ISK-T22. Trypanosoma pestanai, trypanosomatids, further investigations are required. which is a related species of 17ISK-T2 and 17ISK-T22, In this study, two contigs shared 100% identity was isolated not only from a badger (isolate LEM 110) to a sequence of T. theileri and were detected from a but also from a dog (German isolate; Dyachenko et al., tabanid sample. However, the other three contigs from 2017). erefore, the vertebrate host of Trypanosoma the same tabanid sample showed 93‒96% identity to sp. 17ISK-T2 and 17ISK-T22 might be dogs or a that of T. minasense as the highest score sequence by related species in the Canidae family such as a racoon BLASTN search. It appeared that all of the contigs dog or fox in Japan. Haemaphysalis ava is known as were derived from a single pool (pool no. 18HF29) a tick species parasitizing various animals including because only one pool was positive by PCR-based large and medium-sized mammals and birds (Takada screening using a universal primer set targeting the et al., 2019), and the species has been collected in 36 18S rRNA gene of trypanosomatids. In fact, only species in ve order of Aves so far (Yamauchi, 2001). partial sequences of the 28S rRNA gene of T. theileri A variety of trypanosomatids was found in Japanese were available on the International Nucleotide birds, but their invertebrate hosts have not been Sequence Database (DDBJ/EMBL/NCBI). erefore, identied. erefore, it cannot deny the possibility no corresponding sequence of T. theileri was found for that Trypanosoma sp. 17ISK-T2 and 17ISK-T22 are the three contigs (YA3_c43, YA3_c 45, and YA3_c 56). an avian Trypanosoma species, although they are Even though several contigs shared 100% identities phylogenetically distant from previously known to the sequence of T. theileri, we could not conclude avian trypanosomatids such as T. corvi Stephens and that the detected sequences were from a T. theileri as Christophers, mend, Baker, T. culicavium Votýpka, morphological data was not available. Szabová, Rádrová, Zídková and Svobodová, T. e phylogenetic analysis between Trypanosoma thomasbancroi Slapeta, Morin-Adeline, ompson, sp. 18HF29 and trypanosomatids in T. theileri clade 286 Med. Entomol. Zool. indicated that Trypanosoma sp. 18HF29 formed a contribute to the identication of the potential risk of clade with Japanese T. theileri strain KM (GenBank zoonotic infections in humans. accession no. AB007814). However, the information A    of this strain (e.g., host species and location) was not available. Interestingly, other Japanese strains is work was supported by grants-in-aid for of T. theileri (Esashi 9, Esashi 12, and Obihiro) Regulatory Science Research from Ministry of Health, formed a dierent cluster, and it was located far from Labor and Welfare and the Research Program on Trypanosoma sp. 18HF29 and T. theileri strain KM. Emerging and Re-emerging Infectious Diseases from e strain Esashi 9, Esashi 12, and Obihiro were Japan Agency for Medical Research and development isolated from cattle in Hokkaido, Japan, suggesting (AMED). e authors would like to thank Enago that T. theileri may be maintained in local transmission (www.enago.jp) for the English language review. cycle. However, the dendrogram was constructed R   based on short 18S rRNA sequence. To understand the host specicity and natural history of T. theileri and Alberts, B., Johnson, A., Lewis, J., Morgan, D., Ra, M., Roberts, K. related species, further investigations are needed. and Walter, P. 2014. Chapter 6: How cells reads the genome: From DNA to protein. In: Molecular Biology of the Cell. Six edition, pp. e vector species of T. theileri have remained 299‒368, Garland Science, New York, NY, U.S. obscure in Japan thus far. erefore, this study is the Barratt, J., Kaufer, A., Bryce Peters, D. C., Lawrence, A., Roberts, rst detection report of T. theileri-like parasite from T., Lee, R., McAulie, G., Stark, D. and Ellis, J. 2017. Isolation eld-caught vector insect (T. rudens). In fact, T. of novel trypanosomatid, Zelonia australiensis sp. nov. rudens is known as a tabanid species sucking the (: Trypanosomatidae) provides support for a Gondwanan origin of dixenous in the Leishmaniinae. blood from large mammals (e.g., cattle and horse) PLoS Negl. Trop. Dis., 11: e0005215. and humans (Nagasawa, 1967; Kano and Shinonaga, Bayer-Santos, E., Lima, F. M., Ruiz, J. C., Almeida, I. C. and da 2003). A previous study showed that the tabanids Silveira, J. F. 2014. Characterization of the small RNA content of belonging to the genus Haematopota, Hybomitra, Trypanosoma cruzi extracellular vesicles. Mol. Biochem. Parasitol., and Tabanus were regarded as a vector species of T. 193: 71‒74. theileri in Germany (Böse et al., 1987). Furthermore, Böse, R., Friedho, K. T., Olbrich, S., Büscher, G. and Domeyer, I. 1987. Transmission of Trypanosoma theileri to cattle by T. theileri-like trypanosomatids were detected from Tabanidae. Parasitol. Res., 73: 421‒424. the tabanid genera Chrysops, Haematopota, Hybomitra, Büscher, P., Cecchi, G., Jamonneau, V. and Priotto, G. 2017. Human and Tabanus in Poland and Russia (Ganyukova et al., african trypanosomiasis. Lancet, 390: 2397‒2409. 2018; Werszko et al., 2020). erefore, various types Dyachenko, V., Steinmann, M., Bangoura, B., Selzer, M., of tabanid species appeared to play a role as vectors Munderloh, U., Daugschies, A. and Barutzki, D. 2017. of T. theileri and related species. Trypanosoma sp. Co-infection of Trypanosoma pestanai and Anaplasma phagocytophilum in a Dog from Germany. Vet. Parasitol. Reg. TSD1, which is a related species of T. theileri, was Stud. Rep., 9: 110‒114. isolated from sika deer in Hokkaido (Hatama et al., Felsenstein, J. 1985. Condence limits on phylogenies: An approach 2007). Although the vector species of this parasite using the bootstrap. Evolution, 39: 783‒791. has not been identied, tabanids seem to contribute Fisher, A. C., Schuster, G., Cobb, W. J., James, A. M., Cooper, to their transmission in nature as T. cervi Kingston S. M., de León, A. A. P. and Holman, P. J. 2013. Molecular and Morton, which is a related Trypanosoma species characterization of Trypanosoma (Megatrypanum) spp. infecting cattle (Bos taurus), white-tailed deer (Odocoileus virginianus), in deer, is also transmitted by tabanids (Fisher et and elk (Cervus elaphus canadensis) in the United States. Vet. al., 2013). us, T. theileri and related Trypanosoma Parasitol., 197: 29‒42. might be transmitted between cattle and deer by the Fujita, H. and Watanabe, Y. 2007. Tick-borne trypanosomatids in tabanids. Additional investigations will assist with Japan. In: Acari and Emerging/Reemerging Infectious Diseases understanding the transmission dynamics of these (ed. Organizing committee of SADI), pp. 243‒245, Zenkoku Noson Kyoiku Kyokai Publishing, Tokyo, Japan (In Japanese). parasites in nature. Ganyukova, A. I., Zolotarev, A. V., Malysheva, M. N. and Frolov, A. In conclusion, two dierent species of trypanoso- O. 2018. First record of Trypanosoma theileri-like agellates in matid sequences were detected during the RNA horseies from Northwest Russia. Protistology, 12: 223‒230. virome analyses of ticks and tabanids. Further se- Garcia-Silva, M. R., das Neves, R. F., Cabrera-Cabrera, F., quence characterizations and PCR-based screening Sanguinetti, J., Medeiros, L. C., Robello, C., Naya, H., Fernandez- revealed that trypanosomatids called Trypanosoma Calero, T., Souto-Padron, T., de Souza, W. and Cayota, A. 2014. 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Appendix 1. List of trypanosomatids used in this study.

Source Clade Species name Accession no. Common name Scientic name Origin Aquatic clade Trypanosoma binneyi Platypus Ornithorhynchus anatinus Australia AJ132351 Trypanosoma chattoni Northern leopard frog Rana pipiens USA AF119807 Trypanosoma cobitis Stone loach Noemacheilus barbatulus England AJ009143 Trypanosoma epinepheli Barramundi Lates calcarifer China MG598625 Trypanosoma fallisi American toad Anaxyrus americanus Canada AF119806 Trypanosoma granulosum European eel Anguilla anguilla United Kingdom AJ620551 Trypanosoma mega African common toad Amietophrynus regularis Africa AJ009157 Trypanosoma neveulemairei Edible frog Rana esculenta Yugoslavia AF119809 Trypanosoma ranarum Bull frog Rana catesbeiana Canada AF119810 Trypanosoma rotatorium Bullfrog Rana catesbeiana Canada AJ009161 Trypanosoma Trypanosoma brucei brucei Tsetse ies Glossina pallidipes Kenya XR_002989632 brucei clade Trypanosoma brucei gambience Human Homo sapiens Nigeria AJ009141 Trypanosoma brucei rhodesiense Human Homo sapiens Uganda AJ009142 Trypanosoma congolense Tsetse ies Glossina fuscipes fuscipes Central African KP307026 Republic Trypanosoma equiperdum Horse Equus caballus China AJ009153 Trypanosoma evansi Water bualo Bubalus arnee ailand AY904050 Trypanosoma godfreyi Tsetse y Glossina morsitans e Gambia AJ009155 submorsitans Trypanosoma simiae Tsetse y Glossina pallidipes Kenya AJ404608 Trypanosoma vivax Zebu cattle Bos primigenius indicus Nigeria KM391829 Trypanosoma Trypanosoma conorhini Rat Rattus rattus Brazil AJ012411 cruzi clade Trypanosoma cruzi Assassin bug Triatoma infestans Brazil AF245383 Trypanosoma dionisii Eastern bent-wing bat Miniopterus fuliginosus Japan LC326397 Trypanosoma erneyi Angolan free-tailed bat Mops condylurus Mozambique JN040989 Trypanosoma leeuwenhoeki Homann’s two-toed sloth Choloepus homanni Colombia AJ012412 Trypanosoma livingstonei Lander’s horseshoe bat Rhinolophus landeri Mozambique KF192983 Trypanosoma rangeli Linnaeus’s two-toed sloth Choloepus didactylus Brazil AY491767 Trypanosoma vespertilionis Common pipistrelle bat Pipistrellus pipistrellus England AJ009166 Trypanosoma wauwau Parnell’s mustached bat Pteronotus parnellii Brazil KR653211 Trypanosoma Trypanosoma anourosoricis Taiwanese mole shrew Anourosorex squamipes Taiwan AB242823 lewisi clade yamashinai Trypanosoma blanchardi Garden dormouse Eliomys quercinus France AY491764 Trypanosoma grosi Striped eld mouse Apodemus agrarius China FJ694763 Trypanosoma kuseli Siberian ying squirrel Pteromys volans China AB175626 Trypanosoma lewisi Brown howler monkey Alouatta guariba Brazil GU252209 Trypanosoma microti Short-tailed vole Microtis agrestis England AJ009158 Trypanosoma musculi House mouse Mus musculus unknown AJ223568 Trypanosoma niviventerae Chinese white-bellied rat Niviventer confucianus China AB242274 Trypanosoma otospermophili Columbian ground squirrel Urocitellus columbianus USA AB190228 Trypanosoma rabinowitschae European hamster Cricetus cricetus France AY491765 Trypanosoma sapaensis White-toothed shrew Crocidura dracula Viet Nam AB242822 Reptile clade Trypanosoma cascavelli South American rattlesnake Crotalus durissus Brazil EU095837 Trypanosoma freitasi Northern red-sided opossum Monodelphis brevicaudata Brazil MF401951 Trypanosoma lainsoni Russet rice rat Euryoryzomys russatus Brazil MF403111 Trypanosoma scelopori Desert lizard Sceloporus jarrovi USA U67182 Trypanosoma sp. Gecko White-spotted wall gecko Tarentola annularis Senegal AJ620548 Trypanosoma varani Savannah monitor Varanus exanthematicus Senegal AJ005279 Trypanosoma Trypanosoma cf. cervi isolate WTD A1 clone Cl 1 White-tailed deer Odocoileus virginianus USA JX178192 theileri clade Trypanosoma cf. cervi isolate WTD A1 clone Cl 4 White-tailed deer Odocoileus virginianus USA JX178193 Trypanosoma cf. cervi isolate WTD A5 clone Cl 1 White-tailed deer Odocoileus virginianus USA JX178196 Trypanosoma melophagium Louse y Melophagus ovinus Croatia HQ664912 Trypanosoma sp. KrSI1 Horse y Hybomitra tarandina Russia MK156791 Trypanosoma sp. KrSI4 Horse y Hybomitra muehlfeldi Russia MK156792 Trypanosoma sp. KrSI7 Horse y Chrysops divaricatus Russia MK156793 Trypanosoma sp. NoVSI2 Horse y Hybomitra tarandina Russia MK156794 Trypanosoma sp. TSD1 Hokkaido sika deer Cervus nippon yesoensis Japan AB569248 Trypanosoma theileri strain KM Not specied Japan AB007814 Trypanosoma theileri isolate G24 Tsetse y Glossina fuscipes fuscipes Central African KR024688 Republic Trypanosoma theileri strain Esahi 9 Cattle Bos taurus Japan AB569249 Trypanosoma theileri strain Esahi 12 Cattle Bos taurus Japan AB569250 Trypanosoma theileri strain Obihiro Cattle Bos taurus Japan LC385952 Others Trypanosoma amblyommi Tick Amblyomma brasiliense Brazil KX711902 Trypanosoma avium Regent honeyeater Anthochaera phrygia Australia KT728401 Trypanosoma bennetti Lesser spotted eagle Clanga pomarina Slovakia JF778738 Trypanosoma caninum Dog Canis lupus familiaris Brazil KF805460 Trypanosoma copemani isolate H26 Wombat Vombatus ursinus Australia AJ009169 Trypanosoma copemani isolate AAP Wombat Vombatus ursinus Australia AJ620558 Trypanosoma corvi Rook Corvus frugilegus frugilegus United Kingdom AY461665 Trypanosoma culicavium Culex pipiens Czech Republic HQ107970 Trypanosoma grayi Tsetse y Glossina morsitans morsitans e Gambia AJ005278 Trypanosoma irwini Phascolarctos cinereus Australia FJ649479 Trypanosoma minasense Golden-handed tamarin Saguinus midas unknown AB362411 Trypanosoma pestanai Dog Canis lupus familiaris Germany KY354582 Trypanosoma pestanai isolate LEM 110 Badger Meles meles France AJ009159 Trypanosoma ralphi Spectacled caiman Caiman crocodilus Brazil KF546521 Trypanosoma rhipicephalis Tick Rhipicephalis microplus Brazil KX711901 Trypanosoma sp. Bratislava 1 Tick Ixodes ricinus Slovakia MT482752 Trypanosoma sp. KG-1 Tick Haemaphysalis hystricis Japan AB281091 Trypanosoma thomasbancroi Regent honeyeater Anthochaera phrygia Australia KT728396 Out group Bodo caudatus* Denmark AY998649 Trypanoplasma borreli Leech Piscicola geometra Czech Republic L14840

*Free-living species.