Zhang et al. Parasites Vectors (2019) 12:325 https://doi.org/10.1186/s13071-019-3582-9 Parasites & Vectors RESEARCH Open Access The bacterial microbiome of feld-collected Dermacentor marginatus and Dermacentor reticulatus from Slovakia Yan‑Kai Zhang1, Zhi‑Jun Yu1, Duo Wang1, Víchová Bronislava2, Peťko Branislav2,3 and Jing‑Ze Liu1* Abstract Background: The important roles of microbial fora in tick biology and ecology have received much attention. Der- macentor marginatus and Dermacentor reticulatus are known vectors of various pathogens across Europe, including Slovakia. However, their bacterial microbiomes are poorly explored. Methods: In this study, bacterial microbiomes of feld‑collected D. marginatus and D. reticulatus from Slovakia were characterized using 16S rRNA high‑throughput sequencing. Results: Diferent analyses demonstrated that the D. marginatus and D. reticulatus microbiomes difer in their diver‑ sity and taxonomic structures. Furthermore, species‑ and sex‑specifc bacteria were detected in the two species. A possible bacterial pathogen “Candidatus Rhabdochlamydia sp.” was detected from D. marginatus males. Among the observed bacteria, Rickettsia showed high abundance in the two species. Several maternally inherited bacteria such as Coxiella, Arsenophonus, Spiroplasma, Francisella and Rickettsiella, were abundant, and their relative abundance varied depending on tick species and sex, suggesting their biological roles in the two species. Conclusions: The bacterial microbiomes of feld‑collected D. marginatus and D. reticulatus were shaped by tick phylogeny and sex. Maternally inherited bacteria were abundant in the two species. These fndings are valuable for understanding tick‑bacteria interactions, biology and vector competence of ticks. Keywords: Bacterial microbiome, Dermacentor marginatus, Dermacentor reticulatus, Slovakia Background pathogens cause over 100,000 cases of human diseases Ticks are obligate blood-sucking parasitic arthropods, yearly [1]. Every year about 65,000 people are infected feeding on mammals, reptiles, birds and amphibians. in EU countries (https ://ecdc.europ a.eu/en/tick-borne More than 900 tick species have been identifed world- -disea ses). Habitat changes, climate changes, human wide, and many species are of great economic and epi- activities and globalization are responsible for the emer- demiological importance [1, 2]. Ticks carry and transmit gence, spreading and migration of hosts, vectors, para- various pathogens, including bacteria, viruses, proto- sites and pathogens as well as for the rising incidence zoans and helminths [3]. Tick-borne diseases (TBDs) and diversity of vector-borne infections [5–7]. To date, caused by these pathogens, such as human granulocytic at least 33 new tick-borne pathogens (TBPs) have been anaplasmosis (HGA), Lyme disease, tick-borne encepha- found in China [8]. Similarly, in Europe, climate change litis (TBE), babesiosis, etc., are distributed worldwide most predominantly afects seasonal range expansions and resulting in serious harms [4]. Globally, tick-borne and contractions of vector-borne diseases even in small areas [9]. For example, TBE cases moved from lowlands *Correspondence: [email protected] to sub mountainous areas in Slovakia since 1980, most 1 Hebei Key Laboratory of Animal Physiology, Biochemistry likely because of rising temperature [10]. and Molecular Biology, College of Life Sciences, Hebei Normal University, Given the importance of ticks as vectors of patho- Shijiazhuang 050024, Hebei, China Full list of author information is available at the end of the article gens, aspects of tick biology and ecology have received © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. Parasites Vectors (2019) 12:325 Page 2 of 10 much attention [2, 11, 12]. The tick microbiome com- Methods prises of communities of TBPs, viruses, bacteria and Tick collection and sample preparation eukaryotes [13]. The rapid development of DNA and Dermacentor marginatus and D. reticulatus were col- RNA sequencing platforms, especially high-through- lected in the area of Slovak Karst, which is one of the put next-generation sequencing (NGS) technologies, mountain ranges of the Slovenské Rudohorie Moun- have served as key drivers in our ability to realize the tains in the Carpathians in southern Slovakia. It consists complexity of the tick microbiome in great detail [13, of a complex of huge karst plains and plateaus. Te area 14]. A series of studies have suggested that these non- has been a protected landscape area since 1973, and in pathogenic microorganisms are also abundant in ticks 2002, the Slovak Karst National Park was established. Te and have important roles in affecting tick biology and park is also a UNESCO Biosphere Reserve and forms a pathogen transmission [4, 13, 15–19]. A typical exam- UNESCO World Heritage site. Several endemic species ple is Coxiella-like endosymbiont, which has been of animals and plants live in this region, which has warm reported as essential for tick survival and reproduc- and moderately humid climate [38]. Tick collection sites tion in Amblyomma americanum [20], Haemaphysa- were established in a small area situated on the northern lis longicornis [21] and Rhipicephalus microplus [22]. grassy slope covered with scattered islands of xerophilous Recently, empirical evidence of an obligate B vitamin shrubs (212 meters above sea level, 48° 34′53.88″ N, 20° provisioning symbiont in ticks was found [23]. Non- 46′ 44.43″ E), near the Hrhov village in eastern Slovakia. pathogenic microorganisms also influence pathogens A small river and lake to the north and an old oak and in different ways. For example, Ixodes scapularis fed hornbeam forest to the south surround the sampling site. on antibiotic-treated mice exhibited a modified gut Tese areas are usually used as the pastures for livestock microbiome, resulting in increased feeding and low grazing. Ticks were collected by the standard fagging Borrelia burgdorferi colonization rates [24]. Similarly, method in the early spring of 2017 and were identifed Gall et al. [15] found that a disrupted microbiome of into developmental stages, species and sex using the Dermacentor andersoni is correlated with Anaplasma taxonomic key [39]. Before study, ticks were stored at marginale and Francisella novicida susceptibility. − 80 °C. A total of 48 adult ticks were used for this study These findings are paramount to fully exploiting the (D. marginatus, n = 24; D. reticulatus, n = 24). For each microbiome in order to control ticks and TBDs. species, according to tick sex, samples were grouped into Dermacentor marginatus and Dermacentor reticula- three pools of 4 individuals each, and sample names are tus are two key tick vectors of various pathogens [1, 25, shown in Table 1. 26]. They are widespread in Europe, ranging from Por- tugal to Ukraine (and continue to the east of Kazakh- DNA extraction stan) [27]. They are also distributed in China [25] and Prior to DNA extraction, ticks were surface-steri- Russia [28]. Slovakia is located in central Europe; its lized in three washes of 70% ethanol followed by one climate lies between the temperate and continental wash of sterile, nuclease-free, deionized water to avoid climate zones with relatively warm summers and cold, cloudy and humid winters. The distributions and vec- tor competences of D. marginatus and D. reticulatus Table 1 Information of tick samples used for bacterial have been fully investigated in Slovakia [29–37]. A sur- microbiome analysis vey found that D. reticulatus has extended its range in Sample name Tick species Sex n the surroundings of its former habitats [31]. In addi- tion, the influences of global climate changes on the DmarF1 Dermacentor marginatus Female 4 structures and dynamics of TBDs in mountain areas DmarF2 Female 4 were assessed under a research project supported by DmarF3 Female 4 governments of China and Slovakia. DmarM1 Male 4 It is evident that D. marginatus and D. reticulatus DmarM2 Male 4 have great importance in medical and animal hus- DmarM3 Male 4 bandry in Slovakia. Investigation of their microbiomes DretF1 Dermacentor reticulatus Female 4 will aid in the control of ticks and TBDs. In this study, DretF2 Female 4 bacterial microbiomes of field-collected D. marginatus DretF3 Female 4 and D. reticulatus from Slovakia were characterized DretM1 Male 4 using 16S rRNA high-throughput sequencing. DretM2 Male 4 DretM3 Male 4 Abbreviation: n, number of individuals Zhang et al. Parasites Vectors (2019) 12:325 Page 3 of 10 contamination from the environment. DNA extraction diversity was examined using weighted and unweighted was performed using a QIAamp Fast DNA Stool Mini UniFrac analysis to compare the diferent groups and Kit (Qiagen, Hilden, Germany). Te concentration and plotted in a principal coordinate analysis (PCoA). Wil- quality of DNA was measured using a Nanodrop 2000 coxon rank-sum test was used to test for diferences of (Termo Fisher Scientifc, Wilmington,