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Additional file 9: Figure S3. Maximum-likelihood phylogenetic trees of different sub-groups of cys-loop LGICs. Phylogenies includes sequences from different ticks species (labels and branches in blue) and other arthropods: P. tepidariorum (house spider), Acariformes, Parasitiformes, and D. melanogaster. Labels indicate accession numbers of protein sequences and species name. Accessions of I. ricinus are listed in Table S6. Filled circles on branches indicate bootstrap support (support increases with circle width, ranging from 80 to 100). Trees were rooted based on the complete phylogeny (Fig. 5). A: GABA group, B: «Insect group 1», C: pHCL, D: GluCls, E: Gly, F: His.

Additional file 7: Figure S1. Genomic structure of the gene GABA-1-Rdl, with three predicted isoforms and their relative expression. A: Reannotation of the gene XP_042145571.1 located on a genomic scaffold of Ixodes scapularis, NW_024609839.1. The gene (drawing not to scale) has a span of ~247 kbp, on a scaffold of ~92 Mbp. The whole gene is on the minus frame. Boxes correspond to exons. Grey-filled boxes correspond to exons which we consider incorrect (over-predictions in both cases, based on the conserved sequence of GABA-1-Rdl) and by comparison with the homologous sequence in other tick species. Exons in blue correspond to a predicted triplication of one exon (exon 7a, 7b, 7c), whereas only one exon (7b) was annotated for XP_042145571.1. For each numbered exon, the positions indicate the start and end. Numbers followed by a star correspond to reannotations and differ from the published sequence. The translated sequence of each exon is given. B: Alignment of the translated sequences for the three alternative exons 7, including both the sequences for I. scapularis based on our reannotation and the homologous sequences from the I. ricinus alternative transcripts identified in the synganglion transcriptome (this study). Two trans-membrane domaines are indicated, and an arrow shows the A->S mutations known to confer resistance to dieldrin. C: Relative expression (y-axis) of the three isoforms of GABA-1-Rdl. Expressions was counted as counts per millions with RSEM, and normalized to evaluate relative expression. In x-axis, different synganglion libraries produced in this study (described in Table 4). In blue, red and yellow, estimated relative expression of exon 7a, 7b and 7c respectively.

Additional file 8: Figure S2. Genomic organization of Histamine-gated-like sequences in Ixodes scapularis. We used the Histamine gated-like sequences obtained from our meta-transcriptome of Ixodes ricinus to search homologous genes in I. scapularis (homology inferred from near-identity of protein sequences), and to locate them on the genome. The figure shows the entire scaffold NW_024609883 (109.621.045 bp) from the I. scapularis genome, which has homologs to His1 to His12 in I. ricinus. The upper scale indicates positions in Mbp. The lower scale represents a focus on a smaller region containing clusters of His-like sequences, with a scale in Kbp. Annotated genes and their orientations are indicated by filled triangles, with below, the accession of the protein sequence in I. scapularis, and the name of its homologous sequence in I. ricinus (this study). A star indicates that the gene model is probably incorrect: XP_042142979 matches with His1 only over the first four exons, while its remaining sequence appears to represent a chimeric fusion with a totally different gene, XP_040070355 is missing an N-term, XP_040070356 is incomplete and matches only the beginning of His3, whereas XP_040068404 matches only the end of His3 and is in opposite frame of XP_ 040070356 (we interpret this as a likely error of the genome assembly, the two accession probably representing respectively the start and end of the same gene). Open triangles indicate regions where no gene has been annotated in I. scapularis, but where we detected high similarities with I. ricinus genes (for His4 and His9 respectively). For His13 to His18, homologous regions were detected on different scaffolds.

Additional file 10: Figure S4. Concentration–response relationship of GABA on the I. ricinus RDL receptor expressed in Xenopus oocytes. A. Representative current traces of a single oocyte micro-injected with Iri-rdl cRNA perfused with increasing concentrations of GABA for 10 seconds (short bars). The concentration of GABA (μM) is indicated above each trace. B. Concentration–response curve for GABA on the Iri-RDL channel. All current responses are normalized to 300 μM and shown as the mean ± SEM.

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Ticks are strict blood-feeding arthropods (Acari), which represent a major health issue for wild or domesticated animals and humans, due to their potential to transmit disease agents. Control of ticks is increasingly difficult due to the development of drug-resistant parasites. Ligand-gated ion channels of the tick central nervous system are the primary targets of acaricides. Among those receptors, the γ-aminobutyric acid-gated chloride ion channels (GABACls) are the main synaptic inhibitory receptors. Lotilaner is a recently developed parasiticide from the isoxazoline chemical class that was shown to be a non-competitive antagonist of GABACls from the livestock tick Rhipicephalus microplus. In the present study, we characterized the GABACls from the European tick species Ixodes ricinus. We extracted RNAs from Ixodes ricinus nymphs. Taking advantage of the phylogenetic closeness of I. ricinus and R. microplus in the Arthropoda phylum, we identified the I. ricinus GABACl subunit homologue. The cDNA encoding the Iri- GABACl was cloned and the corresponding in vitro synthesized cRNAs were micro-injected into Xenopus laevis oocytes to investigate its pharmacological properties. Functional expression and two-electrode voltage clamp studies demonstrated that the GABACl subunit formed a homomeric receptor gated by GABA. Importantly, the insecticides like lotilaner, fipronil and picrotoxin efficiently blocked the GABA currents as previously observed for the R. microplus GABACl. Surprisingly, I. ricinus GABACl was not sensitive to the pesticide dieldrin, suggesting a potential naturally existing resistance mechanism involving alternative exons. Here we report the functional characterization of the first GABACl of I. ricinus demonstrating that it is an important molecular target for lotilaner. Transcriptomic analysis of I. ricinus are in progress to identify new acaricidal targets.