Cholinergic signaling at the body wall neuromuscular junction distally inhibits feeding behavior in Caenorhabditis elegans.

Complex biological functions within organisms are frequently orchestrated by systemic communication between tissues. In the model organism Caenorhabditis elegans, the pharyngeal and body wall neuromuscular junctions are two discrete structures that control feeding and locomotion, respectively. Separate, the well-defined neuromuscular circuits control these distinct tissues. Nonetheless, the emergent behaviors, feeding and locomotion, are coordinated to guarantee the efficiency of food intake. Here, we show that pharmacological hyperactivation of cholinergic transmission at the body wall muscle reduces the rate of pumping behavior. This was evidenced by a systematic screening of the effect of the cholinesterase inhibitor aldicarb on the rate of pharyngeal pumping on food in mutant worms. The screening revealed that the key determinants of the inhibitory effect of aldicarb on pharyngeal pumping are located at the body wall neuromuscular junction. In fact, the selective stimulation of the body wall muscle receptors with the agonist levamisole inhibited pumping in a lev-1-dependent fashion. Interestingly, this response was independent of unc-38, an alpha subunit of the nicotinic receptor classically expressed with lev-1 at the body wall muscle. This implies an uncharacterized lev-1-containing receptor underpins this effect. Overall, our results reveal that body wall cholinergic transmission not only controls locomotion but simultaneously inhibits feeding behavior.

Neuroligin dependence of social behaviour in Caenorhabditis elegans provides a model to investigate an autism-associated gene.

Autism spectrum disorder (ASD) is characterized by a triad of behavioural impairments including social behaviour. Neuroligin, a trans-synaptic adhesion molecule, has emerged as a penetrant genetic determinant of behavioural traits that signature the neuroatypical behaviours of autism. However, the function of neuroligin in social circuitry and the impact of genetic variation to this gene is not fully understood. Indeed, in animal studies designed to model autism, there remains controversy regarding the role of neuroligin dysfunction in the expression of disrupted social behaviours. The model organism, Caenorhabditis elegans, offers an informative experimental platform to investigate the impact of genetic variants on social behaviour. In a number of paradigms, it has been shown that inter-organismal communication by chemical cues regulates C. elegans social behaviour. We utilize this social behaviour to investigate the effect of autism-associated genetic variants within the social domain of the research domain criteria. We have identified neuroligin as an important regulator of social behaviour and segregate the importance of this gene to the recognition and/or processing of social cues. We also use CRISPR/Cas9 to edit an R-C mutation that mimics a highly penetrant human mutation associated with autism. C. elegans carrying this mutation phenocopy the behavioural dysfunction of a C. elegans neuroligin null mutant, thus confirming its significance in the regulation of animal social biology. This highlights that quantitative behaviour and precision genetic intervention can be used to manipulate discrete social circuits of the worm to provide further insight into complex social behaviour.

Development of emodepside as a possible adulticidal treatment for human onchocerciasis-The fruit of a successful industrial-academic collaboration.

Current mass drug administration (MDA) programs for the treatment of human river blindness (onchocerciasis) caused by the filarial worm Onchocerca volvulus rely on ivermectin, an anthelmintic originally developed for animal health. These treatments are primarily directed against migrating microfilariae and also suppress fecundity for several months, but fail to eliminate adult O. volvulus. Therefore, elimination programs need time frames of decades, well exceeding the life span of adult worms. The situation is worsened by decreased ivermectin efficacy after long-term therapy. To improve treatment options against onchocerciasis, a drug development candidate should ideally kill or irreversibly sterilize adult worms. Emodepside is a broad-spectrum anthelmintic used for the treatment of parasitic nematodes in cats and dogs (Profender and Procox). Our current knowledge of the pharmacology of emodepside is the result of more than 2 decades of intensive collaborative research between academia and the pharmaceutical industry. Emodepside has a novel mode of action with a broad spectrum of activity, including against extraintestinal nematode stages such as migrating larvae or macrofilariae. Therefore, emodepside is considered to be among the most promising candidates for evaluation as an adulticide treatment against onchocerciasis. Consequently, in 2014, Bayer and the Drugs for Neglected Diseases initiative (DNDi) started a collaboration to develop emodepside for the treatment of patients suffering from the disease. Macrofilaricidal activity has been demonstrated in various models, including Onchocerca ochengi in cattle, the parasite most closely related to O. volvulus. Emodepside has now successfully passed Phase I clinical trials, and a Phase II study is planned. This Bayer-DNDi partnership is an outstanding example of "One World Health," in which experience gained in veterinary science and drug development is translated to human health and leads to improved tools to combat neglected tropical diseases (NTDs) and shorten development pathways and timelines in an otherwise neglected area.

Identification and characterisation of serotonin signalling in the potato cyst nematode Globodera pallida reveals new targets for crop protection.

Plant parasitic nematodes are microscopic pathogens that invade plant roots and cause extensive damage to crops. We have used a chemical biology approach to define mechanisms underpinning their parasitic behaviour: We discovered that reserpine, a plant alkaloid that inhibits the vesicular monoamine transporter (VMAT), potently impairs the ability of the potato cyst nematode Globodera pallida to enter the host plant root. We show this is due to an inhibition of serotonergic signalling that is essential for activation of the stylet which is used to access the host root. Prompted by this we identified core molecular components of G. pallida serotonin signalling encompassing the target of reserpine, VMAT; the synthetic enzyme for serotonin, tryptophan hydroxylase; the G protein coupled receptor SER-7 and the serotonin-gated chloride channel MOD-1. We cloned each of these molecular components and confirmed their functional identity by complementation of the corresponding C. elegans mutant thus mapping out serotonergic signalling in G. pallida. Complementary approaches testing the effect of chemical inhibitors of each of these signalling elements on discrete sub-behaviours required for parasitism and root invasion reinforce the critical role of serotonin. Thus, targeting the serotonin signalling pathway presents a promising new route to control plant parasitic nematodes.

PharmacoGenetic targeting of a C. elegans essential neuron provides an in vivo screening for novel modulators of nematode ion channel function.

Chemical or drug treatments are successfully used to treat parasitic nematode infections that impact human, animal and plant health. Many of these exert their effects through modifying neural function underpinning behaviours essential for parasite viability. Selectivity against the parasite may be achieved through distinct pharmacological properties of the parasite nervous system, as exemplified by the success of the ivermectin which target a glutamate-gated chloride channel found only in invertebrates. Despite the success of the ivermectins, emerging resistance and concerns around eco-toxicity are driving the search for new nematocidal chemicals or drugs. Here, we describe the potential of a 5-HT-gated chloride channel MOD-1, which is involved in vital parasite behaviours with constrained distribution in the invertebrate phyla. This ion channel has potential pharmacophores that could be targeted by new nematocidal chemicals and drugs. We have developed a microtiter based bioassay for MOD-1 pharmacology based on its ectopic expression in the Caenorhabditis elegans essential neuron M4. We have termed this technology 'PhaGeM4' for 'Pharmacogenetic targeting of M4 neuron'. Exposure of transgenic worms harbouring ectopically expressed MOD-1 to 5-HT results in developmental arrest. By additional expression of a fluorescence marker in body wall muscle to monitor growth we demonstrate that this assay is suitable for the identification of receptor agonists and antagonists. Indeed, the developmental progression is a robustly quantifiable bioassay that resolves MOD-1 activation by quipazine, 5-carboxyamidotryptamine and fluoxetine and highlight methiothepin as a potent antagonist. This assay has the intrinsic ability to highlight compounds with optimal bioavailability and furthermore to filter out off-target effects. It can be extended to the investigation of other classes of membrane receptors and modulators of neuronal excitation. This approach based on heterologous modulation of the essential M4 neuron function offers a route to discover new effective and selective anthelmintics potentially less confounded by disruptive environmental impact.

Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: When novel functional validation approaches highlight major differences between the model Caenorhabditis elegans and parasitic species.

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.

The distinct profiles of the inhibitory effects of fluensulfone, abamectin, aldicarb and fluopyram on Globodera pallida hatching.

BACKGROUND: Fluensulfone is a nematicide with a novel mode of action against plant parasitic nematodes. Here, we utilize in vitro hatching assays to investigate fluensufone's ability to inhibit Globodera pallida hatching, relative to the efficacy of other distinct classes of nematicides. RESULTS: Fluensulfone, abamectin, aldicarb and fluopyram inhibit G. pallida hatching from cysts more potently than from isolated eggs. At 1 µM for cysts, the order of potency is fluensulfone> fluopyram> abamectin> aldicarb. At low concentrations of fluensulfone, inhibition of hatching is reversible, however, more than 50% of the juveniles that hatch from cysts pre-treated with fluensulfone have reduced motility. This is observed to a lesser extent with abamectin, fluopyram and aldicarb. When cysts are exposed to higher concentrations of fluensulfone (≥500 µM), abamectin (≥100 µM) and fluopyram (≥50 µM) inhibition of hatching is irreversible. This results from the loss of encysted juvenile structure giving rise to a granulated appearance consistent with necrosis, suggesting a nematicidal effect. Intriguingly, hatching initiated by root diffusate is arrested when egg populations are subsequently exposed to fluensulfone. CONCLUSION: Fluensulfone, abamectin, fluopyram and aldicarb inhibit G. pallida hatching. Fluensulfone is a potent inhibitor of hatching and impacts on the viability of the J2 s emerging from the cysts. This activity, and the previously described impaired motility and metabolism of hatched juveniles, show that fluensulfone's distinct mode of action among existing nematicides intersects at two pivotal steps of the parasitic life cycle.

The Caenorhabditis elegans cysteine-string protein homologue DNJ-14 is dispensable for neuromuscular junction maintenance across ageing.

Maintenance of synaptic function across ageing is vital in sustaining cognitive function. Synaptic dysfunction is a key part of the pathophysiology of a number of neurodegenerative diseases. The synaptic co-chaperone, cysteine-string protein (CSP), is important for synaptic maintenance and function in Drosophila, mice and humans, and disruption of CSP results in synaptic degeneration. We sought to characterise synaptic ageing in Caenorhabditis elegans upon genetic disruption of CSP. To do this, we focused on the worms' neuromuscular junctions, which are the best characterised synapse. CSP mutant worms did not display reduced lifespan or any neuromuscular-dependent behavioural deficits across ageing. Pharmacological interrogation of the neuromuscular synapse of CSP mutant animals showed no sign of synaptic dysfunction even at advanced age. Lastly, patch clamp analysis of neuromuscular transmission across ageing in wild-type and CSP mutant animals revealed no obvious CSP-dependent deficits. Electrophysiological spontaneous postsynaptic current analysis reinforced pharmacological observations that the C. elegans neuromuscular synapse increases in strength during early ageing and remains relatively intact in old, immotile worms. Taken together, this study shows that surprisingly, despite disruption of CSP in other animals having severe synaptic phenotypes, CSP does not seem to be important for maintenance of the neuromuscular junction across ageing in C. elegans.

Neuroligin tuning of pharyngeal pumping reveals extrapharyngeal modulation of feeding in Caenorhabditis elegans.

The integration of distinct sensory modalities is essential for behavioural decision making. In Caenorhabditiselegans, this process is coordinated by neural circuits that integrate sensory cues from the environment to generate an appropriate behaviour at the appropriate output muscles. Food is a multimodal cue that impacts the microcircuits to modulate feeding and foraging drivers at the level of the pharyngeal and body wall muscle, respectively. When food triggers an upregulation in pharyngeal pumping, it allows the effective ingestion of food. Here, we show that a Celegans mutant in the single gene orthologous to human neuroligins, nlg-1, is defective in food-induced pumping. This was not due to an inability to sense food, as nlg-1 mutants were not defective in chemotaxis towards bacteria. In addition, we found that neuroligin is widely expressed in the nervous system, including AIY, ADE, ALA, URX and HSN neurons. Interestingly, despite the deficit in pharyngeal pumping, neuroligin was not expressed within the pharyngeal neuromuscular network, which suggests an extrapharyngeal regulation of this circuit. We resolved electrophysiologically the neuroligin contribution to the pharyngeal circuit by mimicking food-dependent pumping and found that the nlg-1 phenotype is similar to mutants impaired in GABAergic and/or glutamatergic signalling. We suggest that neuroligin organizes extrapharyngeal circuits that regulate the pharynx. These observations based on the molecular and cellular determinants of feeding are consistent with the emerging role of neuroligin in discretely impacting functional circuits underpinning complex behaviours.

The long and the short of it - a perspective on peptidergic regulation of circuits and behaviour.

Neuropeptides are the most diverse class of chemical modulators in nervous systems. They contribute to extensive modulation of circuit activity and have profound influences on animal physiology. Studies on invertebrate model organisms, including the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, have enabled the genetic manipulation of peptidergic signalling, contributing to an understanding of how neuropeptides pattern the output of neural circuits to underpin behavioural adaptation. Electrophysiological and pharmacological analyses of well-defined microcircuits, such as the crustacean stomatogastric ganglion, have provided detailed insights into neuropeptide functions at a cellular and circuit level. These approaches can be increasingly applied in the mammalian brain by focusing on circuits with a defined and identifiable sub-population of neurons. Functional analyses of neuropeptide systems have been underpinned by systematic studies to map peptidergic networks. Here, we review the general principles and mechanistic insights that have emerged from these studies. We also highlight some of the challenges that remain for furthering our understanding of the functional relevance of peptidergic modulation.

Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes.

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.

Multiple excitatory and inhibitory neural signals converge to fine-tune Caenorhabditis elegans feeding to food availability.

How an animal matches feeding to food availability is a key question for energy homeostasis. We addressed this in the nematode Caenorhabditis elegans, which couples feeding to the presence of its food (bacteria) by regulating pharyngeal activity (pumping). We scored pumping in the presence of food and over an extended time course of food deprivation in wild-type and mutant worms to determine the neural substrates of adaptive behavior. Removal of food initially suppressed pumping but after 2 h this was accompanied by intermittent periods of high activity. We show pumping is fine-tuned by context-specific neural mechanisms and highlight a key role for inhibitory glutamatergic and excitatory cholinergic/peptidergic drives in the absence of food. Additionally, the synaptic protein UNC-31 [calcium-activated protein for secretion (CAPS)] acts through an inhibitory pathway not explained by previously identified contributions of UNC-31/CAPS to neuropeptide or glutamate transmission. Pumping was unaffected by laser ablation of connectivity between the pharyngeal and central nervous system indicating signals are either humoral or intrinsic to the enteric system. This framework in which control is mediated through finely tuned excitatory and inhibitory drives resonates with mammalian hypothalamic control of feeding and suggests that fundamental regulation of this basic animal behavior may be conserved through evolution from nematode to human.

Progressive metabolic impairment underlies the novel nematicidal action of fluensulfone on the potato cyst nematode Globodera pallida.

BACKGROUND: Fluensulfone is a new nematicide with an excellent profile of selective toxicity against plant parasitic nematodes. Here, its effects on the physiology and biochemistry of the potato cyst nematode Globodera pallida have been investigated and comparisons made with its effect on the life-span of the free-living nematode Caenorhabditis elegans to provide insight into its mode of action and its selective toxicity. RESULTS: Fluensulfone exerts acute effects (≤1h; ≥100µM) on stylet thrusting and motility of hatched second stage G. pallida juveniles (J2s). Chronic exposure to lower concentrations of fluensulfone (≥3days; ≤30µM), reveals a slowly developing metabolic insult in which G. pallida J2s sequentially exhibit a reduction in motility, loss of a metabolic marker for cell viability, high lipid content and tissue degeneration prior to death. These effects are absent in adults and dauers of the model genetic nematode Caenorhabditis elegans. CONCLUSION: The nematicidal action of fluensulfone follows a time-course which progresses from an early impact on motility through to an accumulating metabolic impairment, an inability to access lipid stores and death.

Metabotropic Glutamate Receptors: MODULATORS OF CONTEXT-DEPENDENT FEEDING BEHAVIOUR IN C. ELEGANS.

Glutamatergic neurotransmission is evolutionarily conserved across animal phyla. A major class of glutamate receptors consists of the metabotropic glutamate receptors (mGluRs). In C. elegans, three mGluR genes, mgl-1, mgl-2, and mgl-3, are organized into three subgroups, similar to their mammalian counterparts. Cellular reporters identified expression of the mgls in the nervous system of C. elegans and overlapping expression in the pharyngeal microcircuit that controls pharyngeal muscle activity and feeding behavior. The overlapping expression of mgls within this circuit allowed the investigation of receptor signaling per se and in the context of receptor interactions within a neural network that regulates feeding. We utilized the pharmacological manipulation of neuronally regulated pumping of the pharyngeal muscle in the wild-type and mutants to investigate MGL function. This defined a net mgl-1-dependent inhibition of pharyngeal pumping that is modulated by mgl-3 excitation. Optogenetic activation of the pharyngeal glutamatergic inputs combined with electrophysiological recordings from the isolated pharyngeal preparations provided further evidence for a presynaptic mgl-1-dependent regulation of pharyngeal activity. Analysis of mgl-1, mgl-2, and mgl-3 mutant feeding behavior in the intact organism after acute food removal identified a significant role for mgl-1 in the regulation of an adaptive feeding response. Our data describe the molecular and cellular organization of mgl-1, mgl-2, and mgl-3. Pharmacological analysis identified that, in these paradigms, mgl-1 and mgl-3, but not mgl-2, can modulate the pharyngeal microcircuit. Behavioral analysis identified mgl-1 as a significant determinant of the glutamate-dependent modulation of feeding, further highlighting the significance of mGluRs in complex C. elegans behavior.

Distinct molecular targets including SLO-1 and gap junctions are engaged across a continuum of ethanol concentrations in Caenorhabditis elegans.

Ethanol (alcohol) interacts with diverse molecular effectors across a range of concentrations in the brain, eliciting intoxication through to sedation. Invertebrate models including the nematode worm Caenorhabditis elegans have been deployed for molecular genetic studies to inform on key components of these alcohol signaling pathways. C. elegans studies have typically employed external dosing with high (>250 mM) ethanol concentrations: A careful analysis of responses to low concentrations is lacking. Using the C. elegans pharyngeal system as a paradigm, we report a previously uncharacterized continuum of cellular and behavioral responses to ethanol from low (10 mM) to high (300 mM) concentrations. The complexity of these responses indicates that the pleiotropic action of ethanol observed in mammalian brain is conserved in this invertebrate model. We investigated two candidate ethanol effectors, the calcium-activated K(+) channel SLO-1 and gap junctions, and show that they contribute to, but are not sole determinants of, the low- and high-concentration effects, respectively. Notably, this study shows cellular and whole organismal behavioral responses to ethanol in C. elegans that directly equate to intoxicating through to supralethal blood alcohol concentrations in humans and provides an important benchmark for interpretation of paradigms that seek to inform on human alcohol use disorders.

Fluensulfone is a nematicide with a mode of action distinct from anticholinesterases and macrocyclic lactones.

Plant parasitic nematodes infest crops and present a threat to food security worldwide. Currently available chemical controls e.g. methyl bromide, organophosphates and carbamates have an unacceptable level of toxicity to non-target organisms and are being withdrawn from use. Fluensulfone is a new nematicide of the fluoroalkenyl thioether group that has significantly reduced environmental impact with low toxicity to non-target insects and mammals. Here, we show that the model genetic organism Caenorhabditis elegans is susceptible to the irreversible nematicidal effects of fluensulfone. Whilst the dose required is higher than that which has nematicidal activity against Meloidogyne spp. the profile of effects on motility, egg-hatching and survival is similar to that reported for plant parasitic nematodes. C. elegans thus provides a tractable experimental paradigm to analyse the effects of fluensulfone on nematode behaviour. We find that fluensulfone has pleiotropic actions and inhibits development, egg-laying, egg-hatching, feeding and locomotion. In the case of feeding and locomotion, an early excitation precedes the gross inhibition. The profile of these effects is notably distinct from other classes of anthelmintic and nematicide: the inhibition of motility caused by fluensulfone is not accompanied by the hypercontraction which is characteristic of organophosphates and carbamates and C. elegans mutants that are resistant to the carbamate aldicarb and the macrocyclic lactone ivermectin retain susceptibility to fluensulfone. These data indicate fluensulfone's mode of action is distinct from currently available nematicides and it therefore presents a promising new chemical entity for crop protection.

Getting around the roundworms: Identifying knowledge gaps and research priorities for the ascarids.

The ascarids are a large group of parasitic nematodes that infect a wide range of animal species. In humans, they cause neglected diseases of poverty; many animal parasites also cause zoonotic infections in people. Control measures include hygiene and anthelmintic treatments, but they are not always appropriate or effective and this creates a continuing need to search for better ways to reduce the human, welfare and economic costs of these infections. To this end, Le Studium Institute of Advanced Studies organized a two-day conference to identify major gaps in our understanding of ascarid parasites with a view to setting research priorities that would allow for improved control. The participants identified several key areas for future focus, comprising of advances in genomic analysis and the use of model organisms, especially Caenorhabditis elegans, a more thorough appreciation of the complexity of host-parasite (and parasite-parasite) communications, a search for novel anthelmintic drugs and the development of effective vaccines. The participants agreed to try and maintain informal links in the future that could form the basis for collaborative projects, and to co-operate to organize future meetings and workshops to promote ascarid research.

SLO-1-channels of parasitic nematodes reconstitute locomotor behaviour and emodepside sensitivity in Caenorhabditis elegans slo-1 loss of function mutants.

The calcium-gated potassium channel SLO-1 in Caenorhabditis elegans was recently identified as key component for action of emodepside, a new anthelmintic drug with broad spectrum activity. In this study we identified orthologues of slo-1 in Ancylostoma caninum, Cooperia oncophora, and Haemonchus contortus, all important parasitic nematodes in veterinary medicine. Furthermore, functional analyses of these slo-1 orthologues were performed using heterologous expression in C. elegans. We expressed A. caninum and C. oncophora slo-1 in the emodepside-resistant genetic background of the slo-1 loss-of-function mutant NM1968 slo-1(js379). Transformants expressing A. caninum slo-1 from C. elegans slo-1 promoter were highly susceptible (compared to the fully emodepside-resistant slo-1(js379)) and showed no significant difference in their emodepside susceptibility compared to wild-type C. elegans (p = 0.831). Therefore, the SLO-1 channels of A. caninum and C. elegans appear to be completely functionally interchangeable in terms of emodepside sensitivity. Furthermore, we tested the ability of the 5' flanking regions of A. caninum and C. oncophora slo-1 to drive expression of SLO-1 in C. elegans and confirmed functionality of the putative promoters in this heterologous system. For all transgenic lines tested, expression of either native C. elegans slo-1 or the parasite-derived orthologue rescued emodepside sensitivity in slo-1(js379) and the locomotor phenotype of increased reversal frequency confirming the reconstitution of SLO-1 function in the locomotor circuits. A potent mammalian SLO-1 channel inhibitor, penitrem A, showed emodepside antagonising effects in A. caninum and C. elegans. The study combined the investigation of new anthelmintic targets from parasitic nematodes and experimental use of the respective target genes in C. elegans, therefore closing the gap between research approaches using model nematodes and those using target organisms. Considering the still scarcely advanced techniques for genetic engineering of parasitic nematodes, the presented method provides an excellent opportunity for examining the pharmacofunction of anthelmintic targets derived from parasitic nematodes.

Pharmacological assays reveal age-related changes in synaptic transmission at the Caenorhabditis elegans neuromuscular junction that are modified by reduced insulin signalling.

Frailty is a feature of neuromuscular ageing. Here we provide insight into the relative contribution of pre- and postsynaptic dysfunction to neuromuscular ageing using the nematode Caenorhabditis elegans. Assays of C. elegans motility highlight a precipitous decline during ageing. We describe a novel deployment of pharmacological assays of C. elegans neuromuscular function to resolve pre- and postsynaptic dysfunction that underpin this decreased motility during ageing. The cholinergic agonist levamisole and the cholinesterase inhibitor aldicarb elicited whole worm contraction and allowed a direct comparison of neuromuscular integrity, from 1 to 16 days old: measurements could be made from aged worms that were otherwise almost completely immobile. The rapidity and magnitude of the drug-induced contraction provides a measure of neuromuscular signalling whilst the difference between levamisole and aldicarb highlights presynaptic effects. Presynaptic neuromuscular transmission increased between 1 and 5 days old in wild-type but not in the insulin/IGF1 receptor mutant daf-2 (e1370). Intriguingly, there was no evidence of a role for insulin-dependent effects in older worms. Notably in 16-day-old worms, which were virtually devoid of spontaneous movement, the maximal contraction produced by both drugs was unchanged. Taken together the data support a maturation of presynaptic function and/or upstream elements during early ageing that is lost after genetic reduction of insulin signalling. Furthermore, this experimental approach has demonstrated a counterintuitive phenomenon: in aged worms neuromuscular strength is maintained despite the absence of motility.

Modelling organophosphate intoxication in C. elegans highlights nicotinic acetylcholine receptor determinants that mitigate poisoning.

Organophosphate intoxication via acetylcholinesterase inhibition executes neurotoxicity via hyper stimulation of acetylcholine receptors. Here, we use the organophosphate paraoxon-ethyl to treat C. elegans and use its impact on pharyngeal pumping as a bio-assay to model poisoning through these neurotoxins. This assay provides a tractable measure of acetylcholine receptor mediated contraction of body wall muscle. Investigation of the time dependence of organophosphate treatment and the genetic determinants of the drug-induced inhibition of pumping highlight mitigating modulation of the effects of paraoxon-ethyl. We identified mutants that reduce acetylcholine receptor function protect against the consequence of intoxication by organophosphates. Data suggests that reorganization of cholinergic signalling is associated with organophosphate poisoning. This reinforces the under investigated potential of using therapeutic approaches which target a modulation of nicotinic acetylcholine receptor function to treat the poisoning effects of this important class of neurotoxins.