A unified approach to investigating 4 dpf zebrafish larval behaviour through a standardised light/dark assay.

Zebrafish are a dynamic research model in the domains of neuropsychopharmacology, biological psychiatry and behaviour. Working with larvae ≤4 days post-fertilisation (dpf) offers an avenue for high-throughput investigation whilst aligning with the 3Rs principles of animal research. The light/dark assay, which is the most widely used behavioural assay for larval neuropharmacology research, lacks experimental reliability and standardisation. This study aimed to formulate a robust, reproducible and standardised light/dark behavioural assay using 4 dpf zebrafish larvae. Considerable between-batch and inter-individual variability was found, which we rectified with a normalisation approach to ensure a reliable foundation for analysis. We then identified that 5-min light/dark transition periods are optimal for locomotor activity. We also found that a 30-min acclimation in the light was found to produce significantly increased dark phase larval locomotion. Next, we confirmed the pharmacological predictivity of the standardised assay using ethanol which, as predicted, caused hyperlocomotion at low concentrations and hypolocomotion at high concentrations. Finally, the assay was validated by assessing the behavioural phenotype of hyperactive transgenic (adgrl3.1-/-) larvae, which was rescued with psychostimulant medications. Our standardised assay not only provides a clear experimental and analytical framework to work with 4 dpf larvae, but also facilitates between-laboratory collaboration using our normalisation approach.

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.

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.

StyletChip: a microfluidic device for recording host invasion behaviour and feeding of plant parasitic nematodes.

Plant parasitic nematodes (PPNs) infest the roots of crops and cause global losses with a severe economic impact on food production. Current chemical control agents are being removed from use due to environmental and toxicity concerns and there is a need for new approaches to crop protection. A key feature of parasitic behaviour for the majority of PPNs is a hollow stomastyle or odontostyle required for interaction with the host plant and feeding. This lance-like microscopic structure, often called a stylet, protrudes from the mouth of the worm and thrusts in a rhythmic manner to stab the host root. Studying stylet activity presents technical challenges and as a consequence the underlying biology is poorly understood. We have addressed this by designing a microfluidic chip which traps the PPN Globodera pallida and permits the recording of an electrophysiological signal concomitant with stylet thrusting. The PDMS chip incorporates a precisely designed aperture to trap the nematode securely around a mid-point of its body. It is fabricated using a novel combination of conventional photolithography and two photon polymerization. The chip incorporates valves for rapid application of test compounds and integral electrodes to facilitate acquisition of electrical signals. We show that stylet thrusting can be induced by controlled application of 5-HT (serotonin) to the worm. Each thrust and retraction produces an electrical waveform that characterises the physiological activity associated with the worm's behaviour. The ability to reproducibly record the stylet activity of PPNs provides a new platform for nematicide screening that specifically focuses on a behaviour that is integral to the parasite host interaction. This is the first report of a microfluidic chip capable of electrophysiological recording from nematodes other than Caenorhabditis elegans. The unique approach is optimised for trapping and recording from smaller worms or worms with distinct anterior body shapes and may be applied to other species of economic or medical importance.

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.

NeuroChip: a microfluidic electrophysiological device for genetic and chemical biology screening of Caenorhabditis elegans adult and larvae.

Genetic and chemical biology screens of C. elegans have been of enormous benefit in providing fundamental insight into neural function and neuroactive drugs. Recently the exploitation of microfluidic devices has added greater power to this experimental approach providing more discrete and higher throughput phenotypic analysis of neural systems. Here we make a significant addition to this repertoire through the design of a semi-automated microfluidic device, NeuroChip, which has been optimised for selecting worms based on the electrophysiological features of the pharyngeal neural network. We demonstrate this device has the capability to sort mutant from wild-type worms based on high definition extracellular electrophysiological recordings. NeuroChip resolves discrete differences in excitatory, inhibitory and neuromodulatory components of the neural network from individual animals. Worms may be fed into the device consecutively from a reservoir and recovered unharmed. It combines microfluidics with integrated electrode recording for sequential trapping, restraining, recording, releasing and recovering of C. elegans. Thus mutant worms may be selected, recovered and propagated enabling mutagenesis screens based on an electrophysiological phenotype. Drugs may be rapidly applied during the recording thus permitting compound screening. For toxicology, this analysis can provide a precise description of sub-lethal effects on neural function. The chamber has been modified to accommodate L2 larval stages showing applicability for small size nematodes including parasitic species which otherwise are not tractable to this experimental approach. We also combine NeuroChip with optogenetics for targeted interrogation of the function of the neural circuit. NeuroChip thus adds a new tool for exploitation of C. elegans and has applications in neurogenetics, drug discovery and neurotoxicology.

Meeting report: 2012 Caenorhabditis elegans Neurobiology meeting, EMBL Advanced Training Centre, Germany.

Some of the finest minds in the field of Caenorhabditis elegans neurobiology were brought together from 14 June to 17 June 2012 in the small, quaint and picturesque German city of Heidelberg for the biannual C. elegans neurobiology conference. Held at the EMBL Advanced Training Centre and wonderfully organised by Diah Yulianti, Jean-Louis Bessereau, Gert Jansen and William Schafer, the meeting contained 62 verbal presentations and hundreds of posters that were displayed around the double-helical walkways that looped throughout the conference centre. Presentations on recent advances in microfluidics, cell ablation and targeted gene expression exemplified the strengths of C. elegans as a model organism, with these advances allowing detailed high-throughput analysis and study. Interesting behaviours that were previously poorly characterised were widely discussed, as were the advantages of C. elegans as a model for neurodevelopment and neurodegeneration and the investigation of neuropeptide function. The examples discussed in this meeting report seek to illustrate the breadth and depth of presentations given on these recurring topics.