Ticks and Tick-borne Diseases 5 (2014) 864–870 Contents lists available at ScienceDirect Ticks and Tick-borne Diseases j ournal homepage: www.elsevier.com/locate/ttbdis Original article The composition and transmission of microbiome in hard tick, Ixodes persulcatus, during blood meal a,1 b,1 a a Xue-Chao Zhang , Zhang-Nv Yang , Bo Lu , Xiao-Fang Ma , a a,∗ Chuan-Xi Zhang , Hai-Jun Xu a Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China b Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310053, China a r t i c l e i n f o a b s t r a c t Article history: The tick Ixodes persulcatus is the predominant tick species in Northeastern China, and it is a major vector Received 19 March 2014 in transmission of tick-borne diseases. By 16S rRNA Illumina sequencing, we investigated the micro- Received in revised form 3 June 2014 biome of I. persulcatus and assessed the variation of the microbiome before and after blood feeding. The Accepted 4 July 2014 prolonged blood meal dramatically altered the composition of the microbiome but did not influence the Available online 20 August 2014 bacterial diversity. Overall, 373 and 289 bacterial genera were assigned to unfed and fed ticks, respec- tively. To investigate microbes that were potentially transmitted to vertebrate hosts during a blood meal, Keywords: we examined the microbiome in rat blood after tick bites. Our data showed that 237 bacterial genera Ixodes persulcatus Microbiome were suspected to be pathogens of vertebrates because they were commonly detected in unfed ticks, fed ticks, and rat blood samples after tick bites. Additionally, the prevalence survey on Borrelia burgdorferi Tick-borne pathogen Blood meal s.l., Ehrlichia chaffeensis, Anaplasma phagocytophilum and Yersinia pestis was performed. We found that 16S ribosomal RNA B. garinii and B. afzelii are the predominant genospecies of the Lyme disease spirochete in I. persulcatus ticks. This is the first time that the microbial composition in this tick species and in rat blood transmitted via tick bites has been reported. These data may ultimately assist in identification of novel pathogens transmitted by I. persulcatus ticks. © 2014 Elsevier GmbH. All rights reserved. Introduction tick-borne diseases in humans in this region (Cao et al., 2003; Wan et al., 1998). Ticks are obligate, blood-sucking ectoparasites that are com- Ticks host a variety of disease-causing bacteria, including monly found all around the world. They transmit a broad range Borrelia, Rickettsia, Anaplasma, Francisella, Coxiella and Ehrlichia of infectious agents to humans and animals, including viruses, genera; and new pathogenic agents continue to be reported. For bacteria and protozoa. They are considered to be second only to instance, a new member of the family Anaplasmataceae, Candida- mosquitoes as worldwide vectors of human disease (de la Fuente tus Neoehrlichia mikuresis (Kawahara et al., 2004) has been found et al., 2008; Jongejan and Uilenberg, 2004). The hard tick Ixodes per- in Ixodes ovatus ticks. This pathogen can cause illness that pre- sulcatus is the most epidemiologically important vector from Russia dominantly develops in immunocompromised patients (Fehr et al., to eastern Asia, which is home to around one-fifth of the world’s 2010; Welinder-Olsson et al., 2010). These findings provide unam- population (Cao et al., 2000). Notably, this species is widely dis- biguous evidence that there are as-yet unidentified pathogens tributed in Northern China and is most commonly responsible for associated with ticks. Additionally, co-infection of pathogens has been recently reported in I. persulcatus ticks. This increases the risk of multiple infections in humans, which could lead to more severe clinical manifestations (Cao et al., 2003; Huang et al., 2006; ∗ Swanson et al., 2006). In contrast to the significance of their medical Corresponding author at: 866 Yu-Hang-Tang Rd., C1039 Nong-Sheng-Huan Building, Zi-Jin-Gang Campus, Zhejiang University, Hangzhou 310058, China. and veterinary importance, the competency of ticks for pathogens Tel.: +86 571 88982996; fax: +86 571 88982991. is largely unexplored (Parola and Raoult, 2001). E-mail addresses: [email protected] (X.-C. Zhang), Besides pathogens, ticks also harbor a variety of endosym- [email protected] (Z.-N. Yang), [email protected] (B. Lu), biotic bacteria that exist in commensal, mutualistic or parasitic [email protected] (X.-F. Ma), [email protected] (C.-X. Zhang), relationship with their arthropod hosts (Noda et al., 1997; Sacchi [email protected] (H.-J. Xu). 1 These authors contributed equally to this work. et al., 2004; Sassera et al., 2006; Scoles, 2004; Zhong et al., 2007). http://dx.doi.org/10.1016/j.ttbdis.2014.07.009 1877-959X/© 2014 Elsevier GmbH. All rights reserved. X.-C. Zhang et al. / Ticks and Tick-borne Diseases 5 (2014) 864–870 865 Table 1 Multiple lines of evidence have demonstrated that interactions Summary of sequencing results of unfed ticks, fed ticks and rat blood. among microbes can affect pathogen fate and transmission (Clay et al., 2008; de la Fuente et al., 2003; Macaluso et al., 2002; Unfed tick Fed tick Rat blood Narasimhan et al., 2014). Hence, the biological importance of Clean reads 119,200 55,833 197,043 microbial interactions within the tick vector together with the OTUs 14,098 8181 30,612 surveillance of well-known and emerging infectious agents associ- Shannon index 6.472 6.325 10.699 Good’s coverage 0.884 0.882 0.837 ated with I. persulcatus warrants the studies of bacterial diversity present in this tick. Given that most microorganisms that exist in nature are V4 16S rRNA amplicon sequencing and data analysis not amenable to culturing, classical culture-dependent methods have only detected a fraction of the microbiome (Amann et al., The genomic DNA of individual unfed female adult ticks (n = 50), 1995). With the advance of molecular techniques, high-throughput fed female adults (n = 10), or rat blood (n = 6) was combined at sequencing technologies such as 16S rRNA amplicon sequencing equimolar concentrations in three pools before 16S rRNA Illumina or the shotgun metagenomic approach have been used to investi- sequencing. The V4 region of the 16S rRNA gene was amplified gate the entire array of bacteria associated with ticks under natural with 515F-806R primers to construct the amplicon libraries accord- conditions (Andreotti et al., 2011; Carpi et al., 2011; Nakao et al., ing to a previously described method (Peiffer et al., 2013). The 2013). The blood meal has been shown to increase bacterial diver- paired-end sequencing was performed on an Illumina Miseq plat- sity and induce bacteria multiplication in tick gut during the feeding form (Novogene, Beijing, China) based on a standard protocol process, suggesting that specific bacteria are more likely to be trans- from the manufacturer. Raw data were screened and assembled mitted to vertebrates (Azad and Beard, 1998; De Silva and Fikrig, by QIIME (Caporaso et al., 2010) and FLASH (Magoc and Salzberg, 1995; Heise et al., 2010). 2011) software packages. The UCLUST method (Caporaso et al., In the present study, we conducted a comprehensive survey of 2010) was used to cluster the sequences into Operational Taxo- the pathogen load and the microbiome associated with I. persulca- nomic Units (OTUs) at an identity threshold of 97%. Meanwhile, tus ticks before and after feeding via 16S rRNA amplicon Illumina the RDP Classifier (Wang et al., 2007) was used to assign each sequencing. Also, we examined the microbiome in rat blood after OTU to a taxonomic level. Additional analyses, such as rarefaction tick bites to detect potential pathogens that are transmitted to curves, Shannon index, and Good’s coverage, were carried out with vertebrate hosts. We found that B. burgdorferi s.l. is the dominant QIIME. In addition, the OTU table produced by the QIIME pipeline bacterial pathogen in I. persulcatus, followed by A. phagocytophilum. was imported into MEGAN 4 and mapped on the NCBI taxonomy These results highlight the need for a thorough understand- database (Huson et al., 2011). The genera shared among the three ing of the medical and veterinary significance of I. persulcatus samples were represented by a Scale-Venn diagram using eulerAPE ticks. (http://www.eulerdiagrams.org/eulerAPE/). Metastats web inter- face (White et al., 2009) was used to detect a statistically significant Materials and methods difference between unfed and fed tick microbiomes at phylum and genus levels. Ethics statement PCR amplification and sequence analysis Experiments and animal care were conducted according to protocols approved by the Institutional Animal Care and Use Com- The prevalence survey on B. burgdorferi s.l., E. chaffeensis, A. mittee of Zhejiang University. phagocytophilum and Y. pestis was carried out via PCR, based on the genomic DNA extracted from individual unfed male ticks (n = 50) and unfed female ticks (n = 50). All primer sequences and gene tar- Tick collection and storage gets are listed in Table S1. The PCR products were separated using 1.5% agarose gel electrophoresis and visualized under UV light. Host-seeking ticks were collected by dragging a white cloth over Amplicons of the 5S-23S rRNA intergenic spacer of B. burgdorferi s.l. vegetation in the woodland near Mudanjiang in May 2012 and ◦ ◦ were cloned with the pMD19-T Vector (TaKaRa, Dalian, China) and 2013 (N 44.916 , E 129.498 ), where B. burgdorferi and A. phagocy- then subjected to conventional Sanger sequencing. The obtained tophilum are known to be endemic (Cao et al., 2000). Field-collected sequences were queried to the NCBI database to find the closest ticks were kept in conditional incubators. A subset of unfed I. per- counterparts. sulcatus adults (50 males and 50 females) were washed with 70% ethanol via gently shaking for 30s.