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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.

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.

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.

Drug resistance in the parasites field, including the cosmopolitan lice (Pediculus humanus), and the prevalence increasing despite the marketing of new therapies are an important challenge for our societies. The major pharmacological targets of insecticides like pyrethrins, malathion, spinosad and ivermectin (also used as nematicide and acaricide) are the ligand gated ion channels present in the nervous system of insects. Currently, targets of these molecules remain largely unknown in body lice. Among those channels receptors, γ-aminobutyric acid gated chloride ion channels (GABACl) are the main synaptic inhibitory receptors in insects, making them pertinent pharmacological targets. In the present study, we identified and characterized the targets of insecticides in lice to decipher the mode of action of insecticides in Pediculidae. Research in the genomic databases of Pediculus humanus allowed us to identify a GABACl subunit encoded by the Resistance to dieldrin (Rdl) gene. We cloned the corresponding full-length cDNA into a transcription vector and performed in vitro synthesis of the cRNAs, which were injected in the Xenopus oocysts system to reconstitute functional channels. Two-electrode voltage clamp recordings showed that Phh-RDL assemble into a homomeric receptor sensitive to different insecticides like fipronil, picrotoxin and lotilaner, a novel class of ectoparasiticide agent using to treat ticks and fleas of dogs (CredelioTM, Elanco). These results correlated with the efficacy of these drugs on lice in vivo. In conclusion, we report the functional characterization of the first GABACl of Pediculus humanus humanus. These results contribute to our understanding of the mode of action of insecticide compounds and will allow the development of new therapeutic strategies to control lice infestations.

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.

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.

"Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access "; International audience

Parascaris sp. is the largest parasitic nematode of horse causing digestive and respiratory disorders to the animal. The control of equine ascaridiosis relies on anthelmintic treatments including macrocyclic lactones (MLs) as the gold standard. However, control of infestation is increasingly difficult due to the emergence of resistant parasites throughout the world. In the free-living model nematode Caenorhabditis elegans, glutamate-dependent chloride channel receptors (GluCls) were identified as the main targets of MLs. However, in Parascaris sp, the mode of action of MLs remain poorly understood. Here we identified the Parascaris sp. GluCls and characterized the effect of a wide range of MLs. Using a candidate gene approach, we identified the orthologs of 6 genes encoding GluCls subunits in Parascaris sp. The complete cDNAs encoding 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. Two-electrode voltage-clamp experiments were performed on recombinant GluCls to investigate their pharmacological properties. Thus, the expression of a single subunit and combination of different subunits in Xenopus oocytes allowed us to obtain the functional homeric and heteromeric GluCls of Parascaris sp. The receptors were both sensitive to glutamate and ivermectin but the effect of seven different ML compounds revealed striking differences. The physiological function and the impact of MLs on these receptors in vivo are in progress. This study, provides a better understanding of the pharmacology of GluCls as well as the mode of action of MLs in nematode parasites.

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.

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 but was removed from the market when modern anthelmintics became available. 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 that remain largely under-exploited. In the present study, using an automated larval migration assay (ALMA), we report that nicotinic derivatives efficiently paralyzed a multiple (benzimidazoles/levamisole/pyrantel/ivermectin) resistant field isolate of H. contortus. Using C. elegans as a model we confirmed that N-AChRs are preferential targets for nornicotine and anabasine. 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. This work validates the exploitation of the nicotine receptors of parasitic nematodes as targets for the development of resistance-breaking compounds., Graphical abstract Image 1, Highlights • Monitoring of H. contortus L3 anthelmintic sensitivity with the automated larval migration assay. • Nicotinic derivatives paralyze multiple anthelmintic-resistant H. contortus. • C. elegans and Parascaris spp ACR-16 N-AChRs are targeted by nicotinic derivatives.

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.

Les méthodes de luttes employées contre les nématodes parasites qui impactent la santé humaine ou animale sont remises en cause par l’émergence d’isolats capables de développer des résistances aux vermifuges (anthelminthiques). Dans ce contexte, la compréhension des mécanismes moléculaires à l’origine de ce phénomène ainsi que l’identification de nouvelles cibles pharmacologiques pour la découverte de nouveaux anthelminthiques sont indispensables au développement de stratégies de gestion du parasitisme efficaces et plus durables., 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 on the mode of action of available anthelmintics as well as to identify novel targets for the development of next generation anthelmintic drugs.

International audience; Eosinophils are one of the major mammalian effector cells encountered by helminths during infection. In the present study, we investigated the effects of eosinophil granule exposure on the sheep parasitic nematode Haemonchus contortus as a model. H. contortus eggs exposed to eosinophil granule products showed increased rhodamine 123 efflux and this effect was not due to loss of egg integrity. Rh123 is known to be a specific P-glycoprotein (Pgp) substrate and led to the hypothesis that in addition to their critical role in xenobiotic resistance, helminth ABC transporters such as Pgp may also be involved in the detoxification of host cytotoxic products. We showed by quantitative RT-PCR that, among nine different H. contortus Pgp genes, Hco-pgp-3, Hco-pgp-9.2, Hco-pgp-11 and, Hco-pgp-16 were specifically up-regulated in parasitic life stages suggesting a potential involvement of these Pgps in the detoxification of eosinophil granule products. Using exsheathed L3 larvae that mimic the first life stage in contact with the host, we demonstrated that eosinophil granules induced a dose dependent overexpression of Hco-pgp-3 and the closely related Hco-pgp-16. Taken together, our results provide the first evidence that a subset of helminth Pgps interact with, and could be involved in the detoxification of, host products. This opens the way for further studies aiming to explore the role of helminth Pgps in the host-parasite interaction, including evasion of the host immune response.

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.

BACKGROUND AND PURPOSE: The cholinergic agonist levamisole is widely used to treat parasitic nematode infestations. This anthelmintic drug paralyses worms by activating a class of levamisole-sensitive acetylcholine receptors (L-AChRs) expressed in nematode muscle cells. However, levamisole efficacy has been compromised by the emergence of drug-resistant parasites, especially in gastrointestinal nematodes such as Haemonchus contortus. We report here the first functional reconstitution and pharmacological characterization of H. contortus L-AChRs in a heterologous expression system. EXPERIMENTAL APPROACH: In the free-living nematode Caenorhabditis elegans, five AChR subunit and three ancillary protein genes are necessary in vivo and in vitro to synthesize L-AChRs. We have cloned the H. contortus orthologues of these genes and expressed them in Xenopus oocytes. We reconstituted two types of H. contortus L-AChRs with distinct pharmacologies by combining different receptor subunits. KEY RESULTS: The Hco-ACR-8 subunit plays a pivotal role in selective sensitivity to levamisole. As observed with C. elegans L-AChRs, expression of H. contortus receptors requires the ancillary proteins Hco-RIC-3, Hco-UNC-50 and Hco-UNC-74. Using this experimental system, we demonstrated that a truncated Hco-UNC-63 L-AChR subunit, which was specifically detected in a levamisole-resistant H. contortus isolate, but not in levamisole-sensitive strains, hampers the normal function of L-AChRs, when co-expressed with its full-length counterpart. CONCLUSIONS AND IMPLICATIONS: We provide the first functional evidence for a putative molecular mechanism involved in levamisole resistance in any parasitic nematode. This expression system will provide a means to analyse molecular polymorphisms associated with drug resistance at the electrophysiological level.