Identifying the most important research, policy and practice questions for substance use, problematic alcohol use and behavioural addictions in autism (SABA-A): A priority setting partnership.

BACKGROUND: Autistic people are more likely to report problematic alcohol and other substance use when compared to the general population. Evidence suggests that up to one in three autistic adults may have an alcohol or other substance use disorder (AUD/SUD), although the evidence base for behavioural addictions is less clear. Autistic people may use substances or engage in potentially addictive behaviours as a means of coping with social anxiety, challenging life problems, or camouflaging in social contexts. Despite the prevalence and detrimental effects of AUD, SUD and behavioural addictions in community samples, literature focusing on the intersection between autism and these conditions is scarce, hindering health policy, research, and clinical practice. METHODS: We aimed to identify the top 10 priorities to build the evidence for research, policy, and clinical practice at this intersection. A priority-setting partnership was used to address this aim, comprising an international steering committee and stakeholders from various backgrounds, including people with declared lived experience of autism and/or addiction. First, an online survey was used to identify what people considered key questions about Substance use, alcohol use, or behavioural addictions in autistic people (SABA-A). These initial questions were reviewed and amended by stakeholders, and then classified and refined to form the final list of top priorities via an online consensus process. OUTCOMES: The top ten priorities were identified: three research, three policy, and four practice questions. Future research suggestions are discussed.

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.

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.

Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes.

Multiple advances have been made to increase the efficiency of CRISPR/Cas9 editing using the model genetic organism Caenorhabditis elegans (C. elegans). Here we report on the use of co-CRISPR 'marker' genes: worms in which co-CRISPR events have occurred have overt, visible phenotypes which facilitates the selection of worms that harbour CRISPR events in the target gene. Mutation in the co-CRISPR gene is then removed by outcrossing to wild type but this can be challenging if the CRISPR and co-CRISPR gene are hard to segregate. However, segregating away the co-CRISPR modified gene can be less challenging if the worms selected appear wild type and are selected from a jackpot brood. These are broods in which a high proportion of the progeny of a single injected worm display the co-CRISPR phenotype suggesting high CRISPR efficiency. This can deliver worms that harbour the desired mutation in the target gene locus without the co-CRISPR mutation. We have successfully generated a discrete mutation in the C. elegans nlg-1 gene using this method. However, in the process of sequencing to authenticate editing in the nlg-1 gene we discovered genomic rearrangements that arise at the co-CRISPR gene unc-58 that by visual observation were phenotypically silent but nonetheless resulted in a significant reduction in motility scored by thrashing behaviour. This highlights that careful consideration of the hidden consequences of co-CRISPR mediated genetic changes should be taken before downstream analysis of gene function. Given this, we suggest sequencing of co-CRISPR genes following CRISPR procedures that utilise phenotypic selection as part of the pipeline.

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.

Impact of drug solvents on C. elegans pharyngeal pumping.

Caenorhabditis elegans provides a multi-cellular model organism for toxicology and drug discovery. These studies usually require solvents such as dimethyl sulfoxide (DMSO), ethanol or acetone as a vehicle. This raises the need to carefully consider whether the chemical vehicles used in these screens are anodyne towards C. elegans. Here, we use pharyngeal pumping as a bioassay to assess this. Pharyngeal pumping is a visually scoreable behaviour that is controlled by environmental cues activating sensory and integrative neural signalling to coordinate pharyngeal activity. As such it serves as a rich bioassay to screen for chemical modulation. We found that while pumping was insensitive to high concentrations of the widely used drug solvents ethanol and acetone, it was perturbed by concentrations of DMSO above 0.5 % v/v encompassing concentrations used as drug vehicle. This was manifested as an inhibition of pharyngeal pump rate followed by a slow recovery in the continued presence of the solvent. The inhibition was not observed in a neuroligin mutant, nlg-1, consistent with DMSO acting at the level of sensory processing that modulates pumping. We found that bus-17 mutants, which have enhanced cuticle penetration to drugs are more sensitive to DMSO. The effect of DMSO is accompanied by a progressive morphological disruption in which internal membrane-like structures of varying size accumulate. These internal structures are seen in all three genotypes investigated in this study and likely arise independent of the effects on pharyngeal pumping. Overall, these results highlight sensory signalling and strain dependent vehicle sensitivity. Although we define concentrations at which this can be mitigated, it highlights the need to consider time-dependent vehicle effects when evaluating control responses in C. elegans chemical biology.

Investigating autism associated genes in C. elegans reveals candidates with a role in social behaviour.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by a triad of behavioural impairments and includes disruption in social behaviour. ASD has a clear genetic underpinning and hundreds of genes are implicated in its aetiology. However, how single penetrant genes disrupt activity of neural circuits which lead to affected behaviours is only beginning to be understood and less is known about how low penetrant genes interact to disrupt emergent behaviours. Investigations are well served by experimental approaches that allow tractable investigation of the underpinning genetic basis of circuits that control behaviours that operate in the biological domains that are neuro-atypical in autism. The model organism C. elegans provides an experimental platform to investigate the effect of genetic mutations on behavioural outputs including those that impact social biology. Here we use progeny-derived social cues that modulate C. elegans food leaving to assay genetic determinants of social behaviour. We used the SAFRI Gene database to identify C. elegans orthologues of human ASD associated genes. We identified a number of mutants that displayed selective deficits in response to progeny. The genetic determinants of this complex social behaviour highlight the important contribution of synaptopathy and implicates genes within cell signalling, epigenetics and phospholipid metabolism functional domains. The approach overlaps with a growing number of studies that investigate potential molecular determinants of autism in C. elegans. However, our use of a complex, sensory integrative, emergent behaviour provides routes to enrich new or underexplored biology with the identification of novel candidate genes with a definable role in social behaviour.

Molecular Investigation of the Unfolded Protein Response in Select Human Tauopathies.

BACKGROUND: Tauopathies are a group of neurodegenerative diseases associated with the accumulation of misfolded tau protein. The mechanisms underpinning tau-dependent proteinopathy remain to be elucidated. A protein quality control pathway within the endoplasmic reticulum, the unfolded protein response (UPR), has been suggested as a possible pathway modulating cellular responses in a range of neurodegenerative diseases, including those associated with misfolded cytosolic tau. OBJECTIVE: In this study we investigated three different clinically defined tauopathies to establish whether these diseases are accompanied by the activation of UPR. METHODS: We used PCR and western blotting to probe for the modulation of several reliable UPR markers in mRNA and proteins extracted from three distinct tauopathies: 20 brain samples from Alzheimer's disease patients, 11 from Pick's disease, and 10 from progressive supranuclear palsy. In each disease samples from these patients were compared with equal numbers of age-matched non-demented controls. RESULTS: Our investigation showed that different markers of UPR are not changed at the late stage of any of the human tauopathies investigated. Interestingly, UPR signatures were often observed in non-demented controls. CONCLUSION: These data from late-stage human cortical tissue report an activation of UPR markers within the aged brain across all cohorts investigated and further support the emerging evidence that the accumulation of misfolded cytosolic tau does not drive a disease-associated activation of UPR.

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.

C. elegans pharyngeal pumping provides a whole organism bio-assay to investigate anti-cholinesterase intoxication and antidotes.

Inhibition of acetylcholinesterase by either organophosphates or carbamates causes anti-cholinesterase poisoning. This arises through a wide range of neurotoxic effects triggered by the overstimulation of the cholinergic receptors at synapses and neuromuscular junctions. Without intervention, this poisoning can lead to profound toxic effects, including death, and the incomplete efficacy of the current treatments, particularly for oxime-insensitive agents, provokes the need to find better antidotes. Here we show how the non-parasitic nematode Caenorhabditis elegans offers an excellent tool for investigating the acetylcholinesterase intoxication. The C. elegans neuromuscular junctions show a high degree of molecular and functional conservation with the cholinergic transmission that operates in the autonomic, central and neuromuscular synapses in mammals. In fact, the anti-cholinesterase intoxication of the worm's body wall neuromuscular junction has been unprecedented in understanding molecular determinants of cholinergic function in nematodes and other organisms. We extend the use of the model organism's feeding behaviour as a tool to investigate carbamate and organophosphate mode of action. We show that inhibition of the cholinergic-dependent rhythmic pumping of the pharyngeal muscle correlates with the inhibition of the acetylcholinesterase activity caused by aldicarb, paraoxons and DFP exposure. Further, this bio-assay allows one to address oxime dependent reversal of cholinesterase inhibition in the context of whole organism recovery. Interestingly, the recovery of the pharyngeal function after such anti-cholinesterase poisoning represents a sensitive and easily quantifiable phenotype that is indicative of the spontaneous recovery or irreversible modification of the worm acetylcholinesterase after inhibition. These observations highlight the pharynx of C. elegans as a new tractable approach to explore anti-cholinesterase intoxication and recovery with the potential to resolve critical genetic determinants of these neurotoxins' mode of action.

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.

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.

Pathogenic tau does not drive activation of the unfolded protein response.

The unfolded protein response (UPR) is commonly associated with a range of neurodegenerative diseases, and targeting UPR components has been suggested as a therapeutic strategy. The UPR surveys protein folding within the endoplasmic reticulum. However, many of the misfolded proteins that accumulate in neurodegeneration are localized so that they do not directly cause endoplasmic reticulum triggers that activate this pathway. Here, using a transgenic mouse model and primary cell cultures along with quantitative PCR, immunoblotting, and immunohistochemistry, we tested whether the UPR is induced in in vivo and in vitro murine models of tauopathy that are based on expression of mutant tauP301L We found no evidence for the UPR in the rTg4510 mouse model, in which mutant tau is transgenically expressed under the control of tetracycline-controlled transactivator protein. This observation was supported by results from acute experiments in which neuronal cultures expressed mutant tau and accumulated misfolded cytoplasmic tau aggregates but exhibited no UPR activation. These results suggest that the UPR is not induced as a response to tau misfolding and aggregation despite clear evidence for progressive cellular dysfunction and degeneration. We propose that caution is needed when evaluating the implied significance of the UPR as a critical determinant across major neurodegenerative diseases.

Intra Strain Variation of the Effects of Gram-Negative ESKAPE Pathogens on Intestinal Colonization, Host Viability, and Host Response in the Model Organism Caenorhabditis elegans.

In its native environment of rotting vegetation, the soil nematode Caenorhabditis elegans encounters a range of bacteria. This includes species from the ESKAPE group of pathogens that pose a clinical problem in acquired hospital infections. Here, we investigated three Gram-negative members of the ESKAPE group, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. Pathogenicity profiles as measured by time to kill adult C. elegans showed that P. aeruginosa was the most pathogenic, followed by K. pneumoniae, while C. elegans cultured on A. baumannii exhibited the same survival as those on the standard laboratory food source for C. elegans, Escherichia coli OP50. The pathogenicity was paralleled by a reduction in time that C. elegans resided on the bacterial lawn with the most pathogenic strains triggering an increase in the frequency of food-leaving. Previous reports indicate that gut colonization is a feature of pathogenicity, but we found that the most pathogenic strains were not associated with the highest level of colonization. Indeed, clearance of P. aeruginosa strains from the C. elegans gut was independent of bacterial pathogenicity. We show that this clearance is regulated by neuromodulation as C. elegans mutants in unc-31 and egl-3 have enhanced clearance of P. aeruginosa. Intriguingly this is also not linked to their pathogenicity. It is likely that there is a dynamic balance occurring in the C. elegans intestinal environment between maintaining a healthy, beneficial microbiota and removal of pathogenic bacteria.

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.

Yeast two-hybrid screening identifies MPZ-1 and PTP-1 as candidate scaffolding proteins of metabotropic glutamate receptors in Caenorhabditis elegans.

The metabotropic glutamate receptors (mGluRs) are a class of G-protein-coupled receptor that undergo extensive interactions with scaffolding proteins, and this is intrinsic to their function as an important group of neuromodulators at glutamatergic synapses. The Caenorhabditis elegans nervous system expresses three metabotropic glutamate receptors, MGL-1, MGL-2 and MGL-3. Relatively little is known about how the function and signalling of these receptors is organised in C. elegans. To identify proteins that scaffold the MGL-1 receptor, we have conducted a yeast two-hybrid screen. Three of the interacting proteins, MPZ-1, NRFL-1 and PTP-1, displayed motifs characteristic of mammalian mGluR scaffolding proteins. Using cellular co-expression criterion, we show mpz-1 and ptp-1 exhibited overlapping expression patterns with subsets of mgl-1 neurons. This included neurones in the pharyngeal nervous system that control the feeding organ of the worm. The mGluR agonist L-CCG-I inhibits the activity of this network in wild-type worms, in an MGL-1 and dose-dependent manner. We utilised L-CCG-I to identify if MGL-1 function was disrupted in mutants with deletions in the mpz-1 gene. The mpz-1 mutants displayed a largely wild-type response to L-CCG-I, suggesting MGL-1 signalling is not overtly disrupted consistent with a non-obligatory modulatory function in receptor scaffolding. The selectivity of the protein interactions and overlapping expression identified here warrant further investigation of the functional significance of scaffolding of metabotropic glutamate receptor function.

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.

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.

An oxytocin-dependent social interaction between larvae and adult C. elegans.

Oxytocin has a conserved role in regulating animal social behaviour including parental-offspring interactions. Recently an oxytocin-like neuropeptide, nematocin, and its cognate receptors have been identified in the nematode Caenorhabditis elegans. We provide evidence for a pheromone signal produced by C. elegans larvae that modifies the behaviour of adult animals in an oxytocin-dependent manner increasing their probability of leaving a food patch which the larvae are populating. This increase is positively correlated to the size of the larval population but cannot be explained by food depletion nor is it modulated by biogenic amines, which suggest it is not an aversive behaviour. Moreover, the food-leaving behaviour is conspecific and pheromone dependent: C. elegans adults respond more strongly to C. elegans larvae compared to other nematode species and this effect is absent in C. elegans daf-22 larvae which are pheromone deficient. Neurotransmitter receptors previously implicated in C. elegans foraging decisions NPR-1 and TYRA-3, for NPY-like neuropeptides and tyramine respectively, do not appear to be involved in oxytocin-dependent adult food-leaving. We conclude oxytocin signals within a novel neural circuit that regulates parental-offspring social behaviour in C. elegans and that this provides evidence for evolutionary conservation of molecular components of a parental decision making behaviour.