Composition and Seasonal Variation of Rhipicephalus turanicus and Rhipicephalus sanguineus Bacterial Communities Itai Lalzar, Shimon Harrus, Kosta Y. Mumcuoglu and Yuval Gottlieb Appl. Environ. Microbiol. 2012, 78(12):4110. DOI: 10.1128/AEM.00323-12. Published Ahead of Print 30 March 2012. Downloaded from Updated information and services can be found at: http://aem.asm.org/content/78/12/4110 These include: SUPPLEMENTAL MATERIAL Supplemental material http://aem.asm.org/ REFERENCES This article cites 44 articles, 17 of which can be accessed free at: http://aem.asm.org/content/78/12/4110#ref-list-1 CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» on June 10, 2013 by guest Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ Composition and Seasonal Variation of Rhipicephalus turanicus and Rhipicephalus sanguineus Bacterial Communities Itai Lalzar,a Shimon Harrus,a Kosta Y. Mumcuoglu,b and Yuval Gottlieba Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel,a and Department of Microbiology and Molecular Genetics, The Kuvin Center for the Study of Infectious and Tropical Diseases, Hadassah Medical School, The Institute for Medical Research Israel-Canada, The Hebrew University of Jerusalem, Jerusalem, Israelb A 16S rRNA gene approach, including 454 pyrosequencing and quantitative PCR (qPCR), was used to describe the bacterial com- munity in Rhipicephalus turanicus and to evaluate the dynamics of key bacterial tenants of adult ticks during the active questing season. The bacterial community structure of Rh. turanicus was characterized by high dominance of Coxiella and Rickettsia and extremely low taxonomic diversity. Parallel diagnostic PCR further revealed a novel Coxiella species which was present and nu- Downloaded from Coxiella sp. densities were significantly higher in female versus male .(187 ؍ merically dominant in all individual ticks tested (n ticks and were overall stable throughout the questing season. In addition, we revealed the presence of the novel Coxiella sp. in Rh. sanguineus adult ticks, eggs, and hatched larvae, indicating its vertical transmission. The presence of both spotted fever group Rickettsia spp. (SFGR) and non-SFGR was verified in the various individual ticks. The prevalence and density of Rickettsia spp. were very low compared to those of Coxiella sp. Furthermore, Rickettsia sp. densities were similar in males and females and significantly declined toward the end of the questing season. No correlation was found between Coxiella sp. and Rickettsia sp. densities. These results suggest different control mechanisms in the tick over its different bacterial populations and point to an obligatory and facultative association between the two tick species and Coxiella sp. and Rickettsia spp., respectively. http://aem.asm.org/ any pestiferous arthropods carry obligatory and facultative to develop future strategies for the prevention and control of tick- Mbacterial symbionts, which fundamentally shape their biol- borne diseases. ogy and may determine their efficiency as disease vectors and their In Israel, the brown dog tick, Rhipicephalus sanguineus (Acari: performance as pests (2, 35). Ticks are considered among the most Ixodidae), is most common and is often found with a second important vectors of pathogens of medical and veterinary impor- sympatric species, Rhipicephalus turanicus (16, 30). While Rh. san- tance (10). guineus is the known vector of Rickettsia conorii (9, 22), several Incidences of arthropod-borne epidemics, such as the Crime- other bacterial pathogens, such as Coxiella burnetii, Ehrlichia ca- on June 10, 2013 by guest an-Congo hemorrhagic fever transmitted by ticks (48), are ex- nis, and Anaplasma platys, have also been found in these two tick pected to increase due to global climate changes and other drivers species, making them potential vectors (10, 20). In addition, Rh. associated with modern life (reviewed in references 9, 21, and 28). sanguineus also harbors a Coxiella-like endosymbiont (34). Indeed, the prevalence of tick-borne pathogens such as Borrelia In this study, we adopted a 16S rRNA gene-based approach to burgdorferi and Anaplasma phagocytophilum has been correlated describe the composition and dynamics of the bacterial commu- with seasonal climatic changes (14). Therefore, it is expected that nities in Rh. turanicus and Rh. sanguineus. PCR-DGGE and 454 other microbial tenants within tick hosts are affected in the same pyrosequencing were used to describe the tick bacterial commu- manner. nity, and quantitative PCR (qPCR) assays were used to follow the In studies based on low-throughput techniques such as clone seasonal changes in bacterial populations. libraries and denaturing gradient gel electrophoresis (DGGE), the MATERIALS AND METHODS bacterial communities of several ixodid tick species, among them Amblyomma americanum, Ixodes scapularis, and Ixodes ricinus, Tick collection and rearing. Questing ticks were collected by dragging a 1-m2 white cotton cloth over the vegetation in two consecutive years. In were found to be dominated by a single bacterial genus along with 2009, ticks were collected in March and April from the outskirts of Kib- other less dominant but diverse bacterial species, such as Arseno- butz Hulda, Israel (31° 49= 56.28Љ N, 34° 53= 0.24Љ E) and in May from the phonus sp., Serratia grimesii, and Klebsiella oxytoca. While each sand dunes in Caesarea (32° 30= 0Љ N, 34° 54= 0Љ E). In 2010, ticks were individual tick usually showed relatively few bacteria, a higher collected weekly between March and July from the outskirts of Kibbutz diversity of bacterial taxa was found at the tick population level (4, Hulda. Collected ticks were immediately preserved in 100% ethanol and 8, 23, 31, 39, 44). More recent studies based on high-throughput kept at Ϫ20°C until further use. techniques have revealed higher bacterial diversity of individual ticks. However, to the best of our knowledge, a combined quanti- tative and qualitative assessment of tick bacterial communities has Received 2 February 2012 Accepted 26 March 2012 not been performed to date. Such a dual analysis might be impor- Published ahead of print 30 March 2012 tant for the evaluation of dynamic interactions within bacterial Address correspondence to Yuval Gottlieb, [email protected]. populations (27, 50). Determining seasonal effects on an entire Supplemental material for this article may be found at http://aem.asm.org/. bacterial community within a tick vector might result in better Copyright © 2012, American Society for Microbiology. All Rights Reserved. resolution of host-bacterium interactions and of bacterium-bac- doi:10.1128/AEM.00323-12 terium interactions within the host. This approach could be used 4110 aem.asm.org Applied and Environmental Microbiology p. 4110–4116 June 2012 Volume 78 Number 12 Tick-Associated Bacterial Communities In addition, several fully engorged female ticks were collected in 2010 Mass sequencing of 16S rRNA gene fragments. To characterize the from dogs located in a dog kennel (Rehovot, Israel [31° 53= 38.03Љ N, 34° Rh. turanicus bacterial community, 36 representative samples, 17 male 48= 59.04Љ E]). They were kept in a moistened plastic tube at 25°C until and 19 female ticks collected from Hulda in 2010, were sent to the Re- eggs were laid. A batch from each tick clutch was then surface sterilized in search and Testing Laboratory (Lubbock, TX) for mass-sequencing anal- soapy water and 70% ethanol and preserved in 100% ethanol for DNA ysis. 16S rRNA bacterial tag-encoded FLX amplicon pyrosequencing was extraction. Most of the eggs were incubated until the larvae hatched. The performed using the Roche 454 sequencing system as previously described newly hatched larvae were preserved in 100% ethanol for DNA extraction (12). Amplicons originating from the 530F–1100R (V4–V6) region, num- as described further on. bered in relation to the E. coli 16S rRNA gene, were sequenced. Retrieved Tick species classification. Ticks were first classified as Rh. sanguineus sequences were analyzed using MOTHUR (40). Sequences shorter than or Rh. turanicus according to their morphological characteristics based on 200 bp, as well as those of low quality, were omitted. Sequences were then the work of Walker et al. (45). aligned using the Silva-compatible alignment database, and a distance To ascertain the identity of the collected ticks used in this study, a matrix was calculated (36). Sequences were grouped into operational taxo- specific gene-based assay was developed. Partial fragments of the 12S mi- nomic units (OTUs) at a 97% sequence similarity threshold (i.e., se- tochondrial rRNA gene were sequenced from 10 randomly selected ticks quences that differ by Յ3% are clustered in the same OTU). Representa- as previously described (3). Phylogenetic analysis (detailed below) tives of each OTU were classified with the MOTHUR classify.seqs module, showed that some of the ticks were closely related to Rh. turanicus and and affiliation, down to the genus level, was verified by the ARB program others to Rh. sanguineus (see Fig. S1 in the supplemental material). Based package (25), the Ribosomal Database Project (http://rdp.cme.msu.edu/) Downloaded from on the aligned sequences, a new primer specific to Rh. sanguineus ticks (46),