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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.

Macrocyclic lactones (MLs) are widely used drugs to treat and prevent parasitic nematode infections. In many nematode species including a major pathogen of foals, Parascaris univalens, resistance against MLs is widespread, but the underlying resistance mechanisms and ML penetration routes into nematodes remain unknown. Here, we examined how the P-glycoprotein efflux pumps, candidate genes for ML resistance, can modulate drug susceptibility and investigated the role of active drug ingestion for ML susceptibility in the model nematode Caenorhabditis elegans. Wildtype or transgenic worms, modified to overexpress P. univalens PGP-9 (Pun-PGP-9) at the intestine or epidermis, were incubated with ivermectin or moxidectin in the presence (bacteria or serotonin) or absence (no specific stimulus) of pharyngeal pumping (PP). Active drug ingestion by PP was identified as an important factor for ivermectin susceptibility, while moxidectin susceptibility was only moderately affected. Intestinal Pun-PGP-9 expression elicited a protective effect against ivermectin and moxidectin only in the presence of PP stimulation. Conversely, epidermal Pun-PGP-9 expression protected against moxidectin regardless of PP and against ivermectin only in the absence of active drug ingestion. Our results demonstrate the role of active drug ingestion by nematodes for susceptibility and provide functional evidence for the contribution of P-glycoproteins to ML resistance in a tissue-specific manner.

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; 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.

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.

The control of parasitic nematodes impacting animal health relies on the use of broad spectrum anthelmintics. However, intensive use of these drugs has led to the selection of resistant parasites in livestock industry. In that respect, there is currently an urgent need for novel compounds able to control resistant parasites. Nicotine has also historically been used as a de-wormer until modern anthelmintics were marketed. The pharmacological target of nicotine has been identified in nematodes as acetylcholine- gated ion channels. Nicotinic-sensitive acetylcholine receptors (N-AChRs) therefore represent validated pharmacological targets than remain largely under-exploited. In the present study, we developed an automated larval migration assay (ALMA) and showed that nicotinic derivatives (anabasine/nornicotine) efficiently paralyzed a multiple (benzimidazoles/levamisole/ pyrantel/ivermectin) resistant field isolate of Haemonchus contortus. Additionally, using Caenorhabditis elegans as a model, we confirmed that the N-AChRs subtype contributes to the anthelmintic effect of nicotinic analogs. Interestingly, the functional expression of the homomeric N-AChR from C. elegans and the distantly related horse parasite Parascaris equorum in Xenopus oocytes highlighted some striking differences in their respective pharmacological properties towards nicotine derivative sensitivity. Noteworthy, nicotine and anabasine were more potent than ACh in activating the P. equorum N-AChR as revealed by their respective EC50 values (2.9 ± 0.5 μM and 1.7 ± 0.1 μM versus 6.4 ± 1.1 μM, respectively), unlike nornicotine (34.9 ± 7.2 μM) whereas the potency series for the C. elegans N-AChR was Nic > ACh = Ana > Nor. Taken together these results validate the exploitation of the N-AChRs of parasitic nematodes as targets for the development of resistance-breaking compounds.

National audience

National audience

International audience

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.