Parasitic nematodes infect many species of animals throughout the phyla, including humans. Moreover, nematodes that parasitise plants are a global problem for agriculture. As such, these nematodes place a major burden on human health, on livestock production, on the welfare of companion animals and on crop production. In the 21 st century there are two major challenges posed by the wide-spread prevalence of parasitic nematodes. First, many anthelmintic drugs are losing their effectiveness because nematode strains with resistance are emerging. Second, serious concerns regarding the environmental impact of the nematicides used for crop protection have prompted legislation to remove them from use, leaving agriculture at increased risk from nematode pests. There is clearly a need for a concerted effort to address these challenges. Over the last few decades the free-living nematode Caenorhabditis elegans has provided the opportunity to use molecular genetic techniques for mode of action studies for anthelmintics and nematicides. These approaches continue to be of considerable value. Less fruitful so far, but nonetheless potentially very useful, has been the direct use of C. elegans for anthelmintic and nematicide discovery programmes. Here we provide an introduction to the use of C. elegans as a &#;model&#; parasitic nematode, briefly review the study of nematode control using C. elegans and highlight approaches that have been of particular value with a view to facilitating wider-use of C. elegans as a platform for anthelmintic and nematicide discovery and development.
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Also of importance are species of nematode that infect plants. These plant parasitic nematodes cause major crop losses and are a threat to food security. Chemicals termed nematicides are used to protect crops from infestation. C. elegans has been used to provide insight into mechanisms for controlling these nematodes ( Costa et al., ), albeit in a rather limited fashion to date. This review will also discuss the study of the action of the organophosphate and carbamate nematicides in C. elegans .
Despite the prevalence of parasitic worms, anthelmintic drug discovery is the poor relation of the pharmaceutical industry. The simple reason is that the nations that suffer most from these tropical diseases have little money to invest in drug discovery or therapy. It comes as no surprise therefore that the drugs available for human treatment were first developed as veterinary medicines. There is thus a pitifully small repertoire of chemotherapeutic agents available for treatment (see ). In some respects, this situation has been exacerbated by the remarkable success of ivermectin over the last thirty years ( Geary, ), which has decreased motivation for anthelmintic drug discovery programmes ( Geary et al., ). This prompts concern, as anthelmintic resistance has been widely reported in livestock and it may also only be a matter of time before this phenomenon also occurs widely in parasites of humans ( Osei-Atweneboana et al., ; Churcher et al., ; Osei-Atweneboana et al., ).
The majority of anthelmintics and nematicides are limited in their action between trematodes, cestodes, and nematodes, for example, praziquantel, a drug used in the treatment of most humans infected with trematodes or cestodes and thought to act by disrupting calcium homeostasis ( Greenberg, ), has no activity against nematodes (see ). Only benzimidazoles have cross-phyla activity and even then are more active against nematodes than against cestodes or trematodes.
Anthelmintic is the term used to describe a drug used to treat infections of animals with parasitic worms. This includes both flat worms, e.g., flukes (trematodes) and tapeworms (cestodes) as well as round worms (nematodes). The parasites are of huge importance for human tropical medicine and for veterinary medicine. The World Health Organization estimates that 2 billion people harbour parasitic worm infections ( http://www.who.int/mediacentre/factsheets/fs366/en/ ) causing increased morbidity and mortality, while parasitic worms that infect livestock are an important animal welfare issue and place a major economic burden on food production. Domestic pets are also susceptible to parasitic worm infection and it is of note that the companion animal market is a key economic consideration for animal health companies undertaking drug discovery programmes.
A comparative analysis of the physiology and pharmacology of C. elegans with plant parasitic nematodes is more difficult as there is very little information available for these species in this regard. However, compared with most animal parasitic nematodes, plant parasitic nematodes are more amenable to maintenance in the laboratory. A recent review summarises the current knowledge concerning the plant parasitic nematodes and broadly speaking in terms of neurotransmitters there would appear to be good conservation with C. elegans ( Holden-Dye and Walker, ) although the pharmacology of the respective neurotransmitter receptors has yet to be explored.
Neuropeptides are present throughout the animal phyla and also subserve pivotal roles in the nervous systems of the mammalian hosts of parasitic nematodes. However, neuropeptides, unlike the small molecule &#;classical&#; transmitters, (e.g., acetylcholine), have diverged considerably in structure during evolution and this presents the promise that drugs targeting peptidergic signalling in nematodes may have low mammalian toxicity. There has thus been considerable interest in the prospect of developing peptidomimetics as a novel approach for nematode control. A comparative analysis of the neuropeptide content for a range of nematodes has been comprehensively reviewed ( Marks and Maule, ; McVeigh et al., ) and may become an important consideration if this strategy is to be pursued. Progress has been made towards pairing the neuropeptide ligands with their cognate receptors in C. elegans ( Frooninckx et al., ) and this may point to new molecular targets for nematode control.
Parasitic nematodes are very difficult to work with, requiring passage through their host for maintenance of their parasitic life-cycle. This greatly complicates quantitative experiments in their natural habitat. Methods for conducting forward and reverse genetics are also at best primitive. Thus C. elegans has routinely been exploited as a more &#;user-friendly&#; model system that is also highly tractable to molecular genetic techniques. However, the life-style of the free-living worm C. elegans is very different to that of the parasites, and therefore parasitologists have given careful thought to its relevance ( Holden-Dye and Walker, ; Geary and Thompson, ). In terms of overall &#;body plan&#; there is no doubt that all the species in the phylum Nematoda exhibit similarity despite their very different habitats. More detailed consideration has been given to a comparison of the genetics, for example, the arrangement of the genome, synteny, and homology between specific genes ( Mitreva et al., ). Overall, there would seem to be considerable molecular diversity between the different species in the phylum. It is probably safe to conclude that C. elegans is no more dissimilar to parasitic nematodes than each individual species of parasite is to another. Using C. elegans permits the application of powerful molecular genetic approaches and it has been extensively, and successfully, exploited as a model system to define molecular components of signalling pathways that underpin nematode physiology.
Once the effect of a particular drug on C. elegans has been defined, two different strategies may be adopted to investigate the molecular basis for its biological activity. The first follows a hypothesis-led approach in which strains with mutations in genes of known function are tested for altered sensitivity to the drug. The alternate strategy is to conduct a forward genetic screen. This is a powerful and objective approach that provides novel insight into the signalling pathways that mediate anthelmintic action. Often the impact of these studies extends beyond the interests of parasitologists and into the broader context of cellular and molecular neuroscience. This is because the vast majority of anthelmintics exert their effects in the neuromuscular system and key transduction molecules in the nervous system are highly conserved across the phyla from worm to human. Thus anthelmintic resistance screens can promote the discovery and characterisation of genes that have important roles in neurotransmission. An excellent example of the utility of this approach is provided by genetic screens employing the organophosphate cholinesterase inhibitors, in particular aldicarb which provides an excellent example of &#;genetic pharmacology&#; and the interested reader is directed to Nguyen et al. () , Miller et al. () , and Sieburth et al. () .
There is a large body of literature describing the study of bioactive compounds in C. elegans and the proposal to use it for the study of anthelmintics precedes the publication of the C. elegans genome sequence by nearly 20 years. Rand and Johnson () coined the term &#;genetic pharmacology&#; to describe this approach. These studies generally hinge on the ability of a drug to elicit a significant, ideally quantifiable, change in the worm's growth, development, metabolism, and/or behaviour. Pharmacokinetic considerations include the method and duration of drug exposure. For the vast majority of experiments the anthelmintics are applied to intact C. elegans . There are thus two ways in which the drug can gain access to target tissues, namely by ingestion or by diffusion across the cuticle. In this regard it should be noted that for many drugs the cuticle presents a significant permeability barrier. Thus the lipophilicity of drugs has a strong bearing on the concentration that is achieved in target tissues following external application. It is not uncommon for polar drugs to be applied at a concentration fold higher than their predicted affinity for the target. It may be possible to ameliorate this problem to some extent by employing animals that have a compromised cuticle ( Gravato-Nobre et al., ). A number of possible strategies for overcoming C. elegans xenobiotic resistance and enhancing its value in identifying new anthelmintics have been reviewed recently by Burns and Roy () .
Anthelmintics and nematicides are separated into classes on the basis of similar chemical structure and mode of action. There are only a few main classes and each is briefly discussed in turn below. For the most part, information on the physiological and pharmacological actions of these compounds has been obtained from studies on the large parasitic nematode A. suum. C. elegans, on the other hand, has been valuable in defining molecular targets and downstream signalling cascades.
Piperazine was first used as an anthelmintic in the s and it is still the active constituent of over the counter remedies for thread worm infection in children. Its mode of action has primarily been studied in A. suum where it acts as a weak GABA-mimetic and causes a flaccid, reversible paralysis of body wall muscle. Single channel recordings provide evidence that it is a low efficacy, partial agonist at GABA-gated chloride channels (Martin, ). Recently the action of piperazine has been investigated in C. elegans by comparing its action on wild type and mutants for unc-49, a gene which encodes C. elegans GABAA-like receptor subunits expressed at the body wall neuromuscular junction (Miltsch et al., ). Although very high concentrations were required (50-100 mM), piperazine had a similar inhibitory action on both wild type and unc-49 mutant animals. In a development assay for C. elegans, piperazine also had the same inhibitory effect on both wild type and unc-49 mutants. It is possible that piperazine does not interact with UNC-49 but may possibly interact with other putative GABA-gated chloride channel receptors identified in the C. elegans genome, for example, LGC-37, LGC-38, and GAB-1.
The first of this class, thiabendazole, was discovered in and subsequently a number of further benzamidazoles were introduced as broad spectrum anthelmintics. There is an extensive literature on these compounds reporting a number of different biochemical effects. Nonetheless, it is clear that their anthelmintic efficacy is due to their ability to compromise the cytoskeleton through a selective interaction with β-tubulin (Borgers and de Nollin, ; for review see Lacey, ). The effects of benzimidazoles on C. elegans, which include impaired locomotion and reproduction, and a detrimental effect on oocytes, are consistent with disruption of processes requiring integral microtubules. The sensitivity of C. elegans to benzimidazoles is mediated by a single gene, ben-1, which encodes β-tubulin (Driscoll et al., ). This has provided a platform to investigate the molecular basis of benzimidazole resistance in parasitic nematodes. It has been noted that benzimidazole resistance in Haemonchus contortus seems to be associated with the presence of specific alleles for β-tubulin in the drug resistant isolates (Kwa et al., ). Whether or not a specific β-tubulin isoform could confer resistance to the drug was tested by experiments that showed that the sensitivity of C. elegans ben-1 mutants to benzimidazole can be rescued by expressing a H. contortus allele of β-tubulin from benzimidazole susceptible isolates, but cannot be rescued by the allele present in the resistant isolates (Kwa et al., ). This unequivocally demonstrated that a single amino acid substitution, Y for F, in β-tubulin, can confer anthelmintic resistance. This is the first elegant example of a &#;model hopping&#; approach in which the genetic tractability of C. elegans is directly exploited to define gene function for a parasitic worm. The relationship between β-tubulin genes in H. contortus and C. elegans is explored in greater detail in Saunders et al. ().
Paraherquamide A and marcfortine A are both members of the oxindole alkaloid family, originally isolated from Penicillium paraherquei and P. roqueforti, respectively (Zinser et al., ). Their chemistry, efficacy, and mode of action have been reviewed (Lee et al., ). A specific, high affinity binding site for paraherquamide has been identified in a membrane preparation isolated from C. elegans, with an apparent Kd of 263 nM (Schaeffer et al., ). Paraherquamide and its derivative, 2-deoxy-paraherquamide (derquantel), induce flaccid paralysis in parasitic nematodes in vitro. Pharmacological analysis of the effects of these drugs on acetylcholine-stimulated body wall muscle contractions in A. suum muscle strips in vitro has shown that they act as typical competitive antagonists, shifting the concentration-response curves to the right in a parallel fashion (Robertson et al., ). These drugs have no apparent direct effect on A. suum body wall muscle tension or membrane potential (Lee et al., ). Paraherquamide also blocks the actions of other nicotinic agonists, but not equipotently (Robertson et al., ; Zinser et al., ). Interestingly, this antagonist seems to distinguish nicotinic receptor subtypes on the muscle, and has a greater affinity for the receptors mediating the response to levamisole and pyrantel than the receptors that mediate the response to nicotine. Patch-clamp recording from A. suum body wall muscle has found at least three subtypes of nAChRs based on conductance, namely, N, L, and B, with conductances of 22/24 pS, 33/35 pS, and 45 pS, respectively (Qian et al., ). Confirming previous studies, paraherquamide failed to antagonize the N (nicotine-sensitive) subtype, the L (levamisole-sensitive) subtype was antagonized by paraherquamide, and the B (bephenium-sensitive) subtype was antagonized more by 2-deoxyparaherquamide than by paraherquamide. Using a cut preparation of C. elegans, 2-deoxyparaherquamide has been shown to paralyse C. elegans and to antagonize the actions of bephenium in preference to pyrantel and, to a lesser extent, levamisole on this preparation; that is, bephenium>pyrantel>levamisole, suggesting a preference for B subtype nAChRs (Ruiz-Lancheros et al., ). 2-Deoxyparaherquamide only antagonized 10 µM nicotine and so it was difficult to compare sensitivity of nicotine to 2-deoxyparaherquamide in relation to the other nicotinic agonists. Deoxyparaherquamide appeared to be more potent as an antagonist compared to mecamylamine. These authors raised the possibility that the cuticle of C. elegans may be less permeable than the cuticles of parasitic nematodes, which might influence the value of C. elegans as a primary screen for anthelmintics.
Importantly, the mode of action of this class of anthelmintics differs from the more established drugs that interfere with cholinergic transmission (e.g., levamisole) in that they act as competitive antagonists rather than cholinomimetics. The use of paraherquamide in forward genetic screens has not yet been reported but could potentially generate interesting new mutants. As it is a competitive inhibitor of the body wall nACh receptor, it would be predicted that mutations that increase transmitter release should confer resistance. Thus a forward genetic screen might reveal further negative regulators of neurotransmitter release. For a review that covers the discovery, mode of action, and use of 2-deoxyparaherquamide in combination with abamectin as the anthelmintic, Startect, see Woods et al. ().
Nitazoxanide, a pyruvate:ferredoxin oxidoreductase inhibitor, acts against a broad spectrum of protozoa and helminths that occur in the intestinal tract. The enzyme it targets is required to maintain electron transfer during anaerobic respiration. It is currently used for the treatment of protozoal infections (and is therefore not listed in ). The site of action of this compound has not been established in nematodes although anaerobic electron transport enzymes may be a potential target (Gilles and Hoffman, ). The effect of nitazoxanide has been examined on growth and development of C. elegans (Fonseca-Salamanca et al., ). After seven days culture nitazoxanide (100 μM) only reduced population growth by 33%. In contrast mebendazole (5 μM) and albendazole (1 μM) reduced growth by over 90%. Nitazoxanide (100 μM) had no effect on either embryonation or hatching in Heligmosomoides polygyrus. Therefore the efficacy of this compound is relatively low compared to other anthelmintic agents. Nitazoxanide also failed to show much activity against Trichuris muris or Ancylostoma ceylanicum in vivo (Tritten et al., ). Nitazoxanide has been found to inhibit ATP synthesis in C. elegans (Hemphill et al., ).
C. elegans has been used to screen flavonoids for anthelmintic activity. In a study involving 13 flavones, apigenin was found to inhibit larval growth (Yoon et al., ). When the first generation developed in the presence of apigenin there was a slight reduction in body size but larval development in the F1 generation was severely impaired in the presence of the flavone. The mechanism associated with this inhibition of larval growth in C. elegans was investigated by Kawasaki et al. () and found to be associated with DAF-16 activation. These authors proposed that apigenin acts as a stressor to either stimulate DAF-16 activity directly or inhibit DAF-2/insulin signalling, which reduces the inhibitory effect of DAF-2 on DAF-16. In either case DAF-16 is activated which leads to larval arrest. Flavone (2-phenyl chromone) has been shown to induce embryonic and larval lethality in both C. elegans and a plant parasitic nematode, the pinewood nematode, Bursaphelenchus xylophilus (Lee et al., ).
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Fluensulfone (5-chloro-2-(3,4,4-trifluorobut-3-enylsulfonyl)-1,3-thiazole) is a member of the fluoroalkenyl thioether group and has proven potential as a new nematicide to control plant parasitic nematode infections (Oka et al., ; Oka et al., ). For example, application of fluensulfone through both soil drenching and foliar spray has been shown to significantly reduce root infection and penetration by plant parasitic nematodes, Meloidogyne javanica (Oka et al., ) and M. incognita (Oka et al., ). Fluensulfone has marked effects on the development and behaviour of C. elegans and the profile of its effects is different from the other main classes of anthelmintic and nematicides including levamisole, ivermectin, and the organophosphates, suggesting a distinct mode of action (Kearn et al., ).
Spiroindolines have both insecticidal and anthelmintic activity, with SYN351 being the lead compound for studies using C. elegans (Sluder et al., ). SYN351 induces an uncoordinated &#;loopy&#;, coiling locomotion in C. elegans, and also inhibits pharyngeal pumping frequency. cha-1 mutants, which have a defect in their ability to synthesise acetylcholine, are hypersensitive in the presence of the spiroindoline SYN876 while sublethal doses of SYN876 suppress the effect of the cholinesterase inhibitor, aldicarb. These experiments suggest that spiroindolines exert their effects on nematode behaviour by effectively reducing the levels of available acetylcholine at the synapse. A chemical mutagenesis screen employing C. elegans generated mutants which were resistant to spiroindolines. These resistant strains harboured mutations in the gene encoding the vesicular acetylcholine transporter (VAChT), unc-17 (Sluder et al., ), providing evidence that UNC-17 might be the target for the compound. Further studies, in which the vesicular acetylcholine transporter from Drosophila was expressed in PC12 cells, demonstrated a spiroindoline binding site that could be displaced by the classical acetylcholine vesicular transport inhibitor vesamicol. Taken together these data are consistent with the proposal that mutations in unc-17 confer C. elegans resistance to spiroindolines. Therefore spiroindolines are a group of compounds which appear to target a novel site for anthelmintic action, namely the vesicular acetylcholine transporter (Sluder et al., ).
Parasitic diseases are major constraints in fish mariculture. The anthelmintic praziquantel (PZQ) can effectively treat a range of flatworm parasites in a variety of fish species and has potential for broader application than its current use in the global aquaculture industry. In this review we report on PZQ's current use in the aquaculture industry and discuss its efficacy against various flatworm parasites of fish. Routes of PZQ administration are evaluated, along with issues related to palatability, pharmacokinetics and toxicity in fish, while PZQ's effects on non-target species, environmental impacts, and the development of drug-resistance are discussed.
Keywords:
Praziquantel, Aquaculture, Parasite
Worldwide food fish consumption continues to increase, with aquaculture predicted to account for 54% of total fish production and 60% of fish for human consumption by (Kobayashi et al., ; FAO, ). However, diseases are one of the main constraints on aquaculture, with a range of pathogenic organisms, including parasites, able to detrimentally impact fish health (Lafferty et al., ). Fish are common definitive and intermediate hosts for a variety of platyhelminth parasites (for reviews of fish parasites see Woo and Buchmann, and Ogawa, ) and several factors in aquaculture systems can exacerbate the consequences of parasite infections that would otherwise have minimal impacts on the health of wild fish populations (Lafferty et al., ).
PZQ has been an obvious control measure for platyhelminth parasites in the aquaculture industry. However, PZQ for the treatment of fish for human consumption is only registered for use in a number of jurisdictions worldwide, for only certain parasites under specific conditions. In Japan, PZQ is the active ingredient of Hadaclean (Bayer Ltd.), Benesaru (ASKA Animal Health Co., Ltd.), and Praziguard flavour for fish (Riken Vets Pharma Inc.), and there are clear guidelines on how it can be used. In the first of these products was approved by the Japanese government for the treatment of skin fluke Benedenia seriolae in perciform fish by oral administration and to treat sea-caged Japanese amberjack Seriola quinqueradiata; however, fish farmers have tended to prefer hydrogen peroxide bath treatment over PZQ because of its palatability problem. Since , these products have been approved for use in Japan to treat the blood fluke Cardicola opisthorchis infecting cultured Pacific bluefin tuna Thunnus orientalis and are commonly used by tuna farmers as the only available treatment measure. Several other Asian countries including Vietnam, Thailand, Malaysia, and the Philippines allow PZQ use in fish for food consumption (ASEAN, ).
Another example of PZQ use in aquaculture occurs in Norway, where it is used as an oral treatment for tapeworms in Salmonidae, such as rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar (Lunestad et al., ). However, PZQ is not listed for use in fish for human consumption by all governments and its use in aquaculture is typically &#;off-label&#; under special veterinary justification. For example, in Australia at the time of writing there are currently four valid permits granted by the Australian Pesticides and Veterinary Medicines Authority (APVMA) that allow use of PZQ in aquaculture&#;for the treatment of blood flukes in Southern bluefin tuna Thunnus maccoyii and other Thunninae, and for the treatment of B. seriolae and gill fluke Zeuxapta seriolae in yellowtail kingfish Seriola lalandi (APVMA, , a, b, c).
Within this restricted regulatory environment, PZQ is used in the treatment of various fish parasites and is an essential anthelmintic with a distinct and valuable role to play in aquaculture (Ogawa, ; Bader et al., ). However, the efficacy of PZQ treatment varies depending on a range of factors, including the parasite, host, delivery route (including palatability in oral delivery), and environmental conditions, and evaluation of efficacy should be performed in the host-parasite target system (Bader et al., ). Additionally, maximum residue limits, withdrawal periods, and safety in the target fish species needs to be considered when making evidence-based treatment decisions.
PZQ has efficacy against a broad range of flatworm parasites and is used by the aquaculture industry to treat several parasites in different fish species. PZQ's high efficacy against several parasites of considerable importance, combined with its low toxicity and rapid metabolism and elimination from fish, presents it as a therapeutic that should continue to enjoy wide utility in aquaculture in coming years. While regulated for treatment of fish for human consumption with clear guidelines on use and withdrawal periods in several jurisdictions, PZQ enjoys a somewhat restricted regulatory environment and is used off-label under veterinary justification in certain parts of the world.
Variation exists regarding the efficacy of PZQ treatments. Certain parasites are more innately susceptible to PZQ, while mode of delivery and fish species also influencing treatment efficacy. To ensure PZQ can be optimally utilised by the aquaculture industry, further work is required to refine administration and dosage against a wider range of parasites, with specific emphasis on target host-parasite systems and safety in target fish species. To ensure PZQ remains a sustainable, effective control option, administration should ideally be informed by a knowledge of the target-parasites lifecycle and should be used in conjunction with other parasite management strategies to avoid development of parasite resistance to PZQ.
The current issues with palatability in feed are an obstacle that needs to be overcome before PZQ can be further embraced by the aquaculture industry. High PZQ treatment doses necessitating high dietary inclusion levels currently limit oral PZQ administration for treatment of certain parasites. As in feed delivery can be the most convenient form of administration in commercial aquaculture this represents an obvious problem. Bath treatments can represent a viable alternative; however, logistical complexity, costs and stress on fish can limit their utility in commercial settings. Feeding techniques, different formulations to mask taste, and methods to increase bioavailability have been trialled with only limited success and further work is needed to overcome this issue.
As with any medication used by the aquaculture industry, PZQ needs to be used in a prudent and responsible manner to minimise potential negative impacts. While often considered highly specific to flatworms PZQ can produce adverse impacts in other organisms and non-target species. The high probability of release of PZQ into the aquatic environment following use in aquaculture systems means monitoring for environmental impacts or resistance development is vital. Resistance to PZQ among Eubothrium sp. already impacts salmon farming in Norway, while the discovery that even a single mutation to TRP (the possible site of PZQ action in parasites) ablates responsiveness highlights the ease with resistance development may be possible. Further work needs to be done to better understand and address the environmental concerns and the ecological impacts of PZQ.
While PZQ is undoubtably an important component in some current aquaculture systems, and has potential to continue to be used by the aquaculture industry to treat a range of fish parasites, further work is required to ensure PZQ's potential is fully realised and it can remain a viable treatment option in the future.
The authors declare no conflicts of interest.
This review was made possible by funding to NJB by the Australian Government through the Fisheries Research and Development Corporation (FRDC-170).
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