Digging deep into intramitochondrial symbiosis: dual transcriptomics of the hard tick Ixodes ricinus and its bacterial symbiont Midichloria mitochondrii

Abstract

INTRODUCTION. Ixodes ricinus is a hard tick, widespread in Europe and in the Mediterranean basin, that can act as vector of multiple diseases of human and veterinary importance. Midichloria mitochondrii is a bacterial symbiont of this tick, capable of living in the intermembrane space of mitochondria (Beninati et al., 2004, Appl. Environ. Microbiol., 70:2596\u20132602). The bacterium was found to be present in most tick individuals (Lo et al., 2006, Environ. Microbiol., 8:1280-1287) and to be vertically transmitted to the progeny (Sassera et al., 2008, Appl. Environ. Microbiol., 74:6138-6140). To investigate the relationship between host and symbiont we designed an ad-hoc protocol of dual RNA-Seq to sequence the transcriptomes of both organisms in different phases of tick engorgement. MATERIALS AND METHODS. Ticks were collected on roe deer, cut in half and stored in RNALater on site, in order to freeze the transcription patterns in each stage. Ticks were dissected in RNALater and total RNA was extracted from ovaries and salivary glands. A custom library construction kit was designed and used in order to preserve transcripts from both organisms and to limit the prevalence of rRNA sequences in the sample (normally over 85%). The kit protocol included the use of custom designed probes for rRNA depletion. Libraries were sequenced on Illumina machines and the resulting reads were assembled to obtain a reference transcriptome to be used for downstream analyses. RESULTS AND CONCLUSIONS. Ticks were subjected to a custom protocol for RNA sequencing which allowed to obtain an average of 40 million reads per sample and over 50% of the reads were transcripts of host or symbiont. After assembly, over 18,000 tick transcripts and over 1,200 bacterial transcripts were obtained. Differential expression analysis will be performed in the upcoming months; so far, the main result of this work is the development of a sound protocol for dual RNA-seq in this system