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.

The nematode (Ascaris suum) intestine is a location of synergistic anthelmintic effects of Cry5B and levamisole.

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinomimetic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells from the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that there is an irreversible cytoplasmic Ca2+ overload that leads to necrotic cell-death in the enterocyte that is induced by levamisole opening Ca2+ permeable L-subtype nAChRs and the development of Ca2+ permeable Cry5B toxin pores in enterocyte plasma membranes. The effects of levamisole potentiate and speed the actions of Cry5B.

Diethylcarbamazine elicits Ca2+ signals through TRP-2 channels that are potentiated by emodepside in Brugia malayi muscles.

Filarial nematode infections are a major health concern in several countries. Lymphatic filariasis is caused by Wuchereria bancrofti and Brugia spp. affecting over 120 million people. Heavy infections can lead to elephantiasis, which has serious effects on individuals' lives. Although current anthelmintics are effective at killing microfilariae in the bloodstream, they have little to no effect against adult parasites found in the lymphatic system. The anthelmintic diethylcarbamazine is one of the central pillars of lymphatic filariasis control. Recent studies have reported that diethylcarbamazine can open transient receptor potential (TRP) channels in the muscles of adult female Brugia malayi, leading to contraction and paralysis. Diethylcarbamazine has synergistic effects in combination with emodepside on Brugia, inhibiting motility: emodepside is an anthelmintic that has effects on filarial nematodes and is under trial for the treatment of river blindness. Here, we have studied the effects of diethylcarbamazine on single Brugia muscle cells by measuring the change in Ca2+ fluorescence in the muscle using Ca2+-imaging techniques. Diethylcarbamazine interacts with the transient receptor potential channel, C classification (TRPC) ortholog receptor TRP-2 to promote Ca2+ entry into the Brugia muscle cells, which can activate Slopoke (SLO-1) Ca2+-activated K+ channels, the putative target of emodepside. A combination of diethylcarbamazine and emodepside leads to a bigger Ca2+ signal than when either compound is applied alone. Our study shows that diethylcarbamazine targets TRP channels to promote Ca2+ entry that is increased by emodepside activation of SLO-1 K+ channels.

Diethylcarbamazine elicits Ca 2+ signals through TRP-2 channels that are potentiated by emodepside in Brugia malayi muscles.

Filarial nematode infections are a major health concern in several countries. Lymphatic filariasis is caused by Wucheria bancrofti and Brugia spp. affecting over 120 million people. Heavy infections can lead to elephantiasis having serious effects on individuals’ lives. Although current anthelmintics are effective at killing the microfilariae in the bloodstream, they have little to no effect against adult parasites found in the lymphatic system. The anthelmintic diethylcarbamazine is one of the central pillars of lymphatic filariasis control. Recent studies have reported that diethylcarbamazine can open Transient Receptor Potential (TRP) channels on the muscles of adult female Brugia malayi leading to contraction and paralysis. Diethylcarbamazine has synergistic effects in combination with emodepside on Brugia inhibiting motility: emodepside is an anthelmintic that has effects on filarial nematodes and is under trials for treatment of river blindness. Here we have studied the effects of diethylcarbamazine on single Brugia muscle cells by measuring the change in Ca 2+ fluorescence in the muscle using Ca 2+ -imaging techniques. Diethylcarbamazine interacts with the TRPC orthologue receptor TRP-2 to promote Ca 2+ entry into the Brugia muscle cells which can activate SLO-1 Ca 2+ activated K + channels, the putative target of emodepside. A combination of diethylcarbamazine and emodepside leads to a bigger Ca 2+ signal than when either compound is applied alone. Our study shows that diethylcarbamazine targets TRP channels to promote Ca 2+ entry that is increased by emodepside activation of SLO-1 channels.

Diethylcarbamazine, TRP channels and Ca2+ signaling in cells of the Ascaris intestine.

The nematode parasite intestine absorbs nutrients, is involved in innate immunity, can metabolize xenobiotics and as we show here, is also a site of action of the anthelmintic, diethylcarbamazine. Diethylcarbamazine (DEC) is used to treat lymphatic filariasis and activates TRP-2, GON-2 & CED-11 TRP channels in Brugia malayi muscle cells producing spastic paralysis. DEC also has stimulatory effects on ascarid nematode parasites. Using PCR techniques, we detected, in Ascaris suum intestine, message for: Asu-trp-2, Asu-gon-2, Asu-ced-11, Asu-ocr-1, Asu-osm-9 and Asu-trpa-1. Comparison of amino-acid sequences of the TRP channels of B. malayi, and A. suum revealed noteworthy similarity, suggesting that the intestine of Ascaris will also be sensitive to DEC. We used Fluo-3AM as a Ca2+ indicator and observed characteristic unsteady time-dependent increases in the Ca2+ signal in the intestine in response to DEC. Application of La3+ and the TRP channel inhibitors, 2-APB or SKF 96365, inhibited DEC mediated increases in intracellular Ca2+. These observations are important because they emphasize that the nematode intestine, in addition to muscle, is a site of action of DEC as well as other anthelmintics. DEC may also enhance the Ca2+ toxicity effects of other anthelmintics acting on the intestine or, increase the effects of other anthelmintics that are metabolized and excreted by the nematode intestine.

Reduced Ca2+ transient amplitudes may signify increased or decreased depolarization depending on the neuromodulatory signaling pathway.

Neuromodulators regulate neuronal excitability and bias neural circuit outputs. Optical recording of neuronal Ca2+ transients is a powerful approach to study the impact of neuromodulators on neural circuit dynamics. We are investigating the polymodal nociceptor ASH in Caenorhabditis elegans to better understand the relationship between neuronal excitability and optically recorded Ca2+ transients. ASHs depolarize in response to the aversive olfactory stimulus 1-octanol (1-oct) with a concomitant rise in somal Ca2+, stimulating an aversive locomotory response. Serotonin (5-HT) potentiates 1-oct avoidance through Gαq signaling, which inhibits L-type voltage-gated Ca2+ channels in ASH. Although Ca2+ signals in the ASH soma decrease, depolarization amplitudes increase because Ca2+ mediates inhibitory feedback control of membrane potential in this context. Here, we investigate octopamine (OA) signaling in ASH to assess whether this negative correlation between somal Ca2+ and depolarization amplitudes is a general phenomenon, or characteristic of certain neuromodulatory pathways. Like 5-HT, OA reduces somal Ca2+ transient amplitudes in ASH neurons. However, OA antagonizes 5-HT modulation of 1-oct avoidance behavior, suggesting that OA may signal through a different pathway. We further show that the pathway for OA diminution of ASH somal Ca2+ consists of the OCTR-1 receptor, the Go heterotrimeric G-protein, and the G-protein activated inwardly rectifying channels IRK-2 and IRK-3, and this pathway reduces depolarization amplitudes in parallel with somal Ca2+ transient amplitudes. Therefore, even within a single neuron, somal Ca2+ signal reduction may indicate either increased or decreased depolarization amplitude, depending on which neuromodulatory signaling pathways are activated, underscoring the need for careful interpretation of Ca2+ imaging data in neuromodulatory studies.

Adapting techniques for calcium imaging in muscles of adult Brugia malayi.

Brugia malayi is a human filarial nematode parasite that causes lymphatic filariasis or 'elephantiasis' a disfiguring neglected tropical disease. This parasite is a more tractable nematode parasite for the experimental study of anthelmintic drugs and has been studied with patch-clamp and RNAi techniques. Unlike in C. elegans however, calcium signaling in B. malayi or other nematode parasites has not been achieved, limiting the studies of the mode of action of anthelmintic drugs. We describe here the development of calcium imaging methods that allow us to characterize changes in cellular calcium in the muscles of B. malayi. This is a powerful technique that can help in elucidating the mode of action of selected anthelmintics. We developed two approaches that allow the recording of calcium signals in the muscles of adult B. malayi: (a) soaking the muscles with Fluo-3AM, promoting large-scale imaging of multiple cells simultaneously and, (b) direct insertion of Fluo-3 using microinjection, providing the possibility of performing dual calcium and electrophysiological recordings. Here, we describe the techniques used to optimize dye entry into the muscle cells and demonstrate that detectable increases in Fluo-3 fluorescence to elevated calcium concentrations can be achieved in B. malayi using both techniques.

Serotonin Disinhibits a Caenorhabditis elegans Sensory Neuron by Suppressing Ca2+-Dependent Negative Feedback.

Neuromodulators, such as serotonin (5-HT), alter neuronal excitability and synaptic strengths, and define different behavioral states. Neuromodulator-dependent changes in neuronal activity patterns are frequently measured using calcium reporters because calcium imaging can easily be performed on intact functioning nervous systems. With only 302 neurons, the nematode Caenorhabditis elegans provides a relatively simple, yet powerful, system to understand neuromodulation at the level of individual neurons. C. elegans hermaphrodites are repelled by 1-octanol, and the initiation of these aversive responses is potentiated by 5-HT. 5-HT acts on the ASH polymodal nociceptors that sense the 1-octanol stimulus. Surprisingly, 5-HT suppresses ASH Ca2+ transients while simultaneously potentiating 1-octanol-dependent ASH depolarization. Here we further explore this seemingly inverse relationship. Our results show the following (1) 5-HT acts downstream of depolarization, through Gαq-mediated signaling and calcineurin, to inhibit L-type voltage-gated Ca2+ channels; (2) the 1-octanol-evoked Ca2+ transients in ASHs inhibit depolarization; and (3) the Ca2+-activated K+ channel, SLO-1, acts downstream of 5-HT and is a critical regulator of ASH response dynamics. These findings define a Ca2+-dependent inhibitory feedback loop that can be modulated by 5-HT to increase neuronal excitability and regulate behavior, and highlight the possibility that neuromodulator-induced changes in the amplitudes of Ca2+ transients do not necessarily predict corresponding changes in depolarization.SIGNIFICANCE STATEMENT Neuromodulators, such as 5-HT, modify behavior by regulating excitability and synaptic efficiency in neurons. Neuromodulation is often studied using Ca2+ imaging, whereby neuromodulator-dependent changes in neuronal activity levels can be detected in intact, functioning circuits. Here we show that 5-HT reduces the amplitude of depolarization-dependent Ca2+ transients in a C. elegans nociceptive neuron, through Gαq signaling and calcineurin but that Ca2+ itself inhibits depolarization, likely through Ca2+-activated K+ channels. The net effect of 5-HT, therefore, is to increase neuronal excitability through disinhibition. These results establish a novel 5-HT signal transduction pathway, and demonstrate that neuromodulators can change Ca2+ signals and depolarization amplitudes in opposite directions, simultaneously, within a single neuron.

Serotonin differentially modulates Ca2+ transients and depolarization in a C. elegans nociceptor.

Monoamines and neuropeptides modulate neuronal excitability and synaptic strengths, shaping circuit activity to optimize behavioral output. In C. elegans, a pair of bipolar polymodal nociceptors, the ASHs, sense 1-octanol to initiate escape responses. In the present study, 1-octanol stimulated large increases in ASH Ca(2+), mediated by L-type voltage-gated Ca(2+) channels (VGCCs) in the cell soma and L-plus P/Q-type VGCCs in the axon, which were further amplified by Ca(2+) released from intracellular stores. Importantly, 1-octanol-dependent aversive responses were not inhibited by reducing ASH L-VGCC activity genetically or pharmacologically. Serotonin, an enhancer of 1-octanol avoidance, potentiated 1-octanol-dependent ASH depolarization measured electrophysiologically, but surprisingly, decreased the ASH somal Ca(2+) transients. These results suggest that ASH somal Ca(2+) transient amplitudes may not always be predictive of neuronal depolarization and synaptic output. Therefore, although increases in steady-state Ca(2+) can reliably indicate when neurons become active, quantitative relationships between Ca(2+) transient amplitudes and neuronal activity may not be as straightforward as previously anticipated.