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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 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 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 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.

Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode Caenorhabditis elegans, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: unc-38, unc-63, unc-29, lev-1 and lev-8. These subunits form a functional L-AChR when heterologously expressed in Xenopus laevis oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to C. elegans lev-8. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the C. elegans L-AChR when expressed in Xenopus oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a ‘model hopping’ approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode Haemonchus contortus to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of C. elegans lev-8 null mutants. The critical role of acr-8 to in vivo drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for Hco-acr-8 which reduced the sensitivity of H. contortus larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus C. elegans. More broadly, this highlights the limits of C. elegans as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds., Author summary Parasitic nematodes have global health and economic impacts. They infect animals, including livestock, humans, and plants including all major food crops. Their control in human and veterinary medicine is reliant on anthelmintic drugs but this is now challenged by resistant worms especially in livestock. Importantly, for anthelmintics such as levamisole and other cholinergic agonists, resistance appears to be less frequent stressing the need to investigate their molecular target in parasitic nematodes. The levamisole receptor was first identified in the free-living model nematode C. elegans but it is now becoming apparent that this is not a good predictor for many parasitic species. In particular we have found that the LEV-8 subunit which is involved in levamisole sensitivity in C. elegans, is not present in many levamisole-sensitive parasitic species. Here we used heterologous expression systems and gene silencing to provide the functional in vivo demonstration that the ACR-8 subunit, which is not an essential component of the levamisole receptor in C. elegans, has a critical role in the levamisole sensitivity of parasitic nematodes. This has important significance for understanding the molecular targets of cholinergic anthelmintics and addresses the increasing challenge of drug resistance.

Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR., Author Summary The control of parasitic nematode infections in humans, livestock and companion animals is critically dependent on anthelmintic treatment. However, the indiscriminate use of anthelmintic drugs has inevitably led to the selection of resistant parasites. In this respect there is currently an urgent need to increase our knowledge of the mode of action of available anthelmintics as well as to identify novel targets for the development of next generation anthelmintic compounds. In the present study we report the functional and pharmacological characterization of a novel subtype of nematode acetylcholine-gated ion channel in two distantly related parasitic nematode species: Haemonchus contortus and Parascaris equorum. Using the Xenopus laevis oocyte as an expression system, we showed that these receptors are composed of subunits encoded by two closely related genes, acr-26 and acr-27 that are widely distributed in parasitic nematodes infecting humans and animals. We further demonstrate that these receptors represent a molecular target for the anthelmintics morantel and pyrantel. The H. contortus and P. equorum receptors expressed as transgenes in the nematode model Caenorhabditis elegans both confer morantel and pyrantel sensitivity in-vivo. For parasitic nematodes of veterinary and medical importance, this novel class of cholinergic receptor is of prime interest for target-based drug screening and the development of relevant anthelmintic combinations.