This is an open access article distributed under the terms of the Creative Commons Attribution License.-- et al. ; Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor- SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found. ; This research was supported by grants BFU2011-23896 and the European Union FP7 ANTIGONE project number 278976 (JdlF). NA was funded by Ministerio de Educacion y Ciencia, Spain. VN was funded by the European Social Fund and the Junta de Comunidades de Castilla-La Mancha (Program FSE 2007–2013), Spain. RS was supported by the project Postdok_BIOGLOBE (CZ.1.07/2.3.00/30.0032) and by the Grant 13-12816P (GA CR). OH was supported by the Grant Agency of the Czech Republic grant no. 13-27630P and European Union FP7 MODBIOLIN project number 316304. CR and LBS were supported by the United Kingdom Biotechnology and Biological Sciences Research Council's National Capability Grant to the Pirbright Institute. ; Peer Reviewed