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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.

The human whipworm, Trichuris trichiura, is estimated to infect 289.6 million people globally. Control of human trichuriasis is a particular challenge, as most anthelmintics have a limited single-dose efficacy, with the striking exception of the narrow-spectrum anthelmintic, oxantel. We recently identified a novel ACR-16-like subunit from the pig whipworm, T. suis which gave rise to a functional acetylcholine receptor (nAChR) preferentially activated by oxantel. However, there is no ion channel described in the mouse model parasite T. muris so far. Here, we have identified the ACR-16-like and ACR-19 subunits from T. muris, and performed the functional characterization of the receptors in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. We found that the ACR-16-like subunit from T. muris formed a homomeric receptor gated by acetylcholine whereas the ACR-19 failed to create a functional channel. The subsequent pharmacological analysis of the Tmu-ACR-16-like receptor revealed that acetylcholine and oxantel were equally potent. The Tmu-ACR-16-like was more responsive to the toxic agonist epibatidine, but insensitive to pyrantel, in contrast to the Tsu-ACR-16-like receptor. These findings confirm that the ACR-16-like nAChR from Trichuris spp. is a preferential drug target for oxantel, and highlights the pharmacological difference between Trichuris species.

Parascaris sp. is the only ascarid parasitic nematode in equids and one of the most threatening infectious organisms in horses. Only a limited number of compounds are available for treatment of horse helminthiasis, and Parascaris sp. worms have developed resistance to the three major anthelmintic families. In order to overcome the appearance of resistance, there is an urgent need for new therapeutic strategies. The active ingredients of herbal essential oils are potentially effective antiparasitic drugs. Carvacrol is one of the principal chemicals of essential oil from Origanum, Thymus, Coridothymus, Thymbra, Satureja and Lippia herbs. However, the antiparasitic mode of action of carvacrol is poorly understood. Here, the objective of the work was to characterize the activity of carvacrol on Parascaris sp. nicotinic acetylcholine receptor (nAChR) function both in vivo with the use of worm neuromuscular flap preparations and in vitro with two-electrode voltage-clamp electrophysiology on nAChRs expressed in Xenopus oocytes. We developed a neuromuscular contraction assay for Parascaris body flaps and obtained acetylcholine concentration-dependent contraction responses. Strikingly, we observed that 300 µM carvacrol fully and irreversibly abolished Parascaris sp. muscle contractions elicited by acetylcholine. Similarly, carvacrol antagonized acetylcholine-induced currents from both the nicotine-sensitive AChR and the morantel-sensitive AChR subtypes. Thus, we show for the first time that body muscle flap preparation is a tractable approach to investigating the pharmacology of Parascaris sp. neuromuscular system. Our results suggest an intriguing mode of action for carvacrol, being a potent antagonist of muscle nAChRs of Parascaris sp. worms, which may account for its antiparasitic potency.

International audience; Background and purpose: Macrocyclic lactones (MLs) are the most widely used broad-spectrum anthelmintic drugs for the treatment of parasitic nematodes impacting both human and animal health. MLs are known to act as agonist of the nematode glutamate-gated chloride channels (GluCls). However, for many important nematode species, the GluCls subunit composition and pharmacological properties remain largely unknown. In order to get new insights about the GluCl diversity and MLs mode of action, we identified and pharmacologically characterized receptors made of highly conserved GluCl subunits from the model nematode Caenorhabditis elegans, the human filarial nematode Brugia malayi and the horse parasite Parascaris univalens.Experimental approach: AVR-14, GLC-2, GLC3 and GLC-4 are the most conserved GluCl subunits throughout the Nematoda phylum. For each nematode species, we investigated the ability of these subunits to form either homomeric or heteromeric GluCls when expressed in Xenopus laevis oocytes and performed the detailed pharmacological characterization of the functional channels.Key results: Here, a total of 14 GluCls have been functionally reconstituted and heteromers formation was inferred from pharmacological criteria. Importantly, we report that the GLC-2 subunit plays a pivotal role in the composition of heteromeric GluCls in nematodes. In addition, we describe a novel GluCl subtype, made of the GLC-2/GLC-3 subunit combination, for which a high concentration of the anthelmintics ivermectin and moxidectin reversibly potentiate glutamate-induced response.Conclusion and implications: This study brings new insights into the diversity of GluCl subtypes in nematodes and promote novel drug targets for the development of next generation anthelmintic compounds.

International audience; Background and purpose: Anthelmintic resistance and/or decreased susceptibility to anthelmintics is a major global problem in parasitic nematodes of livestock and is of great concern, mainly in the Soil Transmitted Helminths (STHs), where decreased susceptibility to albendazole has already been reported for some parasites, amongst these, the human whipworm, Trichuris triciura. Trichuris is an interesting nematode genus due to its broad host spectrum and its low susceptibility to several anthelmintic drug classes. However, a noteworthy exception is the cholinergic agonist oxantel, which has a high efficacy against Trichuris spp. Despite this knowledge, the molecular target(s) of oxantel within Trichuris is still unknown. In Ascaris suum, oxantel has a low, but some effect on the homomeric nicotinic Ach receptor ACR-16 (Asu-ACR-16). Ascaris suum is a Clade III parasitic nematode, whereas Trichuris is included in Clade I. We therefore hypothesized that the sensitivity of Tsu-ACR-16 to oxantel is different from the Asu-ACR-16 and could be a target for oxantel within Trichuris.Methodology: A Tsu-acr-16-like receptor was identified, amplified by PCR, cloned, functionally expressed in Xenopus laevis oocytes and pharmacologically characterized under two-electrode voltage-clamp. Results: In electrophysiological experiments, oxantel produced a high and robust current response of the Tsu-ACR-16-like receptor whereas pyrantel activated the receptor moderately. The cholinergic anthelmintic compounds morantel and levamisole did not activate the receptor significantly and nAChR agonists, including epibathidine, nicotine, cytisine, dimethylpiperazine and 3-bromocytisine resulted in minor responses.Conclusion and perspective: The Tsu-ACR-16-like receptor is more sensitive to oxantel than Asu-ACR-16. This receptor may not be the only target of oxantel, but the functional expression of this Tsu-ACR-16-like receptor can prove a valuable screening tool to evaluate the effect of new synthetic or natural compounds against this genus.

International audience; Levamisole and pyrantel are important drugs of the anthelmintic arsenal used in livestock and humans. Anthelmintic resistance is a growing issue and we do believe that unravelling anthelmintic target sites of action constitutes a pre-requisite step towards the identification of drug-resistance mechanisms. Levamisole-sensitive acetylcholine receptors (L-AChRs), are hetero-pentameric ligandgated ion-channels that mediate fast excitatory neurotransmission at the nematode neuromuscular junctions. The canonical Caenorhabditis elegans L-AChR is made up of five different subunits, the three α-type UNC-63, UNC-38, LEV-8 and the two non-α UNC-29 and LEV-1. In contrast to C. elegans, three main subunit changes occurred in the gastrointestinal nematode of small ruminants Haemonchus contortus: the UNC-29 subunit duplicated in four copies, the LEV-1 subunit had no signal peptide and there was no orthologue of LEV-8. We previously characterized several L-AChR subtypes of H. contortus using the Xenopus oocyte expression system. The L-AChR subtype 1.1 is composed of the UNC-29.1, UNC-38, UNC-63 and ACR-8 subunits and is more sensitive to levamisole than acetylcholine. The replacement of UNC-29.1 by the UNC-29.3 or UNC-29.4 copies gave rise to the respective L-AChR subtypes 1.3 and 1.4, which both exhibited dissimilar pharmacology patterns than the L-AChR subtype 1.1. Interestingly, the omission of ACR-8 led to the L-AChR subtype 2.1 which was more sensitive to pyrantel than levamisole. However the UNC-29.2 copy failed to replace UNC-29.1 and LEV-1 was never proven to be functional. In addition, we identified the expression of a truncated form of UNC-63 (Hco-UNC-63b) in the levamisole- and pyrantel-resistant isolate RHS6 evidencing a strong dominant negative effect on the functioning of the L-AChR. Taking the C. elegans L-AChR as a reference receptor led us to assemble new subunit combinations including both Hco-UNC-29.2 and Hco-LEV-1 subunits. Hence, we reconstituted two novel subtypes of H. contortus L-AChRs showing distinct pharmacologies. These data reinforced the critical role for Hco-ACR-8 in levamisole sensitivity. Furthermore, we identified a truncated spliced form of Hco-LEV-1 in the Kokstadt multidrug-resistant isolate and showed that it hampered the function of L-AChRs when co-expressed with its full-length counterpart. Altogether, these studies highlight the wide repertoire of L-AChR subtypes in parasitic nematodes and bring new insight to our understanding of levamisole resistance.

International audience

Parascaris sp. est le plus grand nématode parasite des équins (chevaux, ânes, poneys…) et est responsable de l’ascaridiose équine. Cette maladie se traduit par des troubles digestifs et respiratoires chez l’animal pouvant conduire à sa mort. Le contrôle de ce parasite repose essentiellement sur l’utilisation d’anthelminthiques tels que les lactones macrocycliques (MLs). Cependant, le contrôle de l'infestation est de plus en plus difficile en raison de l’émergence de parasites résistants à travers le monde. Chez le nématode modèle Caenorhabditis elegans, les récepteurs canaux chlorure glutamate-dépendants (GluCls) ont été identifiés comme étant les cibles principales des MLs. Toutefois, chez Parascaris sp., le mode d'action des MLs reste largement méconnu. Dans cette étude, nous avons identifié les GluCls de Parascaris sp. et caractérisé l’effet d’une large gamme de MLs.En utilisant une approche de type gène-candidat, nous avons identifié les orthologues de 6 gènes codant pour des sous-unités de GluCls chez Parascaris sp. Les ADNc codant pour ces sous-unités ont été amplifiés par PCR et clonés dans un vecteur de transcription. Les ARNc correspondants ont été synthétisés in vitro puis microinjectés dans des ovocytes de Xenopus laevis. Des expériences de « two-electrode voltage-clamp » ont été effectuées sur des GluCls recombinants afin d'étudier leurs propriétés pharmacologiques. Ainsi, l’expression d’une seule sous-unité et la combinaison de différentes sous-unités dans les ovocytes de xénope nous ont permis d’obtenir les GluCls homériques et hétéromèriques fonctionnels de Parascaris sp. Les récepteurs étaient sensibles au glutamate et à l'ivermectine, mais présentaient des sensibilités très différentes face aux autres MLs appliquées. L’étude des fonctions physiologiques et l’impact des MLs sur ces récepteurs in vivo est en cours.Cette étude fournit une meilleure compréhension de la pharmacologie des GluCls ainsi que du mode d'action des lactones macrocycliques chez les nématodes parasites.

National audience; Parascaris sp. is the largest nematode parasite of horses and is responsible of equine ascaridiosis causing digestive and respiratory disorders to the animal. The fight against gastro-intestinal nematodes relies on anthelmintic treatments including macrocyclic lactones which are massively used worlwide to control human and veterinary parasites. However, control of infestation is increasingly difficult due to the emergence of resistant parasites throughout the world. The main targets of macrocyclic lactones in insects and nematodes have been identified as glutamate-gated chloride channel receptors (GluCls). However, in Parascaris sp, the mechanisms of action of macrocyclic lactones and the mechanisms of resistance remain largely unknown. First, using a candidate gene approach, we identified the orthologs of 5 genes encoding 6 GluCls subunits in Parascaris sp. The cDNAs of these subunits were amplified by PCR and cloned into a transcription vector. The corresponding cRNAs were synthesized in vitro and then microinjected into Xenopus laevis oocytes. This heterologous expression system is a powerfull tool to studied the composition and the pharmacology of recombinant receptors by recording currents after application of drugs (macrocyclic lactones) by two-electrode voltage-clamp. Thus, expression of the subunit singly or in combination in the xenopus oocytes allowed us to obtain the first functional caracterization of homeric and new heteromeric GluCl subtypes of Parascaris sp. All receptors identified are sensitive to glutamate but showed different affinity for macrocyclic lactones applied (ivermectin, moxidectin, selamectin, doramectin, emamectin, eprinomectin and abamectin). The physiological function and the impact of macrocyclic lactones on these receptors in vivo are in progress. In this study, we show the first functional characterization of Parascaris sp. GluCls sensitive to macrocyclic lactones. This research provides a better understanding of the pharmacology of GluCls as well as the mode of action of macrocyclic lactones.

International audience; Recent studies have described the involvement of Caenorhabditis elegans p-glycoprotein-2 in lysosome formation and lipid storage. In that respect, we are carefully trying to identify in Heligmosomoides polygyrus bakeri the free-living nematode model Caenorhabtidis elegans p-glycoprotein homologs using degenerate primers. These experiments have allowed the identification of five partial p-glycoprotein sequences. Among those Hba-pgp-2 has particular interest. Since, C. elegans PGP-2 is involved in the transport of cholesterol. Cholesterol is needed for molting and growth of the nematodes. In this study, a full-length cDNA sequence corresponding to Hba-pgp-2 was obtained. It presents high homologies with the Cel-pgp-2 gene sequence. Moreover, we showed that Hba-pgp-2 is expressed among the eggs, fourth-stage larvae and adult and their expression could be induced by cholesterol. In order to characterize the Hba-pgp-2 function, the Hba-pgp-2 promoter was analyzed using the nematode model C. elegans and complementation of C. elegans pgp-2 mutant by Hba-pgp-2 was performed. This opens the way for studying the involvement of Pgps in parasitic biological process that can lead to develop new way to control nematode parasites.

The growing range of drug resistant parasitic nematode populations threatens the sustainability of ruminant farming worldwide. In this context, nutraceuticals, animal feed that provides necessary dietary requirements while ensuring parasite control, could contribute to increase farming sustainability in developed and low resource settings. In this study, we evaluated the anthelmintic potential of lupin seed extracts against the major ruminant trichostrongylids, Haemonchus contortus and Teladorsagia circumcincta. In vitro observations showed that seed extracts from commercially available lupin varieties could significantly but moderately inhibit larval migration. This anthelmintic effect was mediated by the seed alkaloid content and was potent against both fully susceptible and multidrug resistant H. contortus isolates as well as a susceptible T. circumcincta isolate. Analytical chemistry revealed a set of four lupanine and sparteine-derivatives with anthelmintic activity, and electrophysiology assays on recombinant nematode acetylcholine receptors suggested an antagonistic mode of action for lupin alkaloids. An in vivo trial in H. contortus infected lupin-fed ewes and goats failed to demonstrate any direct anthelmintic effect of crude lupin seeds but infected lupin-fed goats suffered significantly less parasite-mediated blood losses. Altogether, our findings suggest that the anthelmintic potential of lupin remains limited. However, the potent alkaloids identified could lead to the development of novel drugs or may be used in combination with current anthelmintics to improve their efficacy.

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.