Molecular & Biochemical Parasitology 147 (2006) 118–125

Caenorhabditis elegans ivermectin receptors regulate locomotor behaviourand are functional orthologues of Haemonchus contortus receptors

Alan Cook a,1, Nathalie Aptel b,1, Virginia Portillo b,2, Elodie Siney a, Rajinder Sihota a,Lindy Holden-Dye a, Adrian Wolstenholme b,∗

a Neurosciences Research Group, School of Biological Sciences, Bassett Crescent East, University of Southampton, Southampton SO16 7PX, UKb Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK

Received 23 December 2005; received in revised form 30 January 2006; accepted 1 February 2006Available online 24 February 2006

Abstract

The target site for the anthelmintic action of ivermectin is a family of nematode glutamate-gated chloride channel alpha subunits (GluCl�) thatbind the drug with high affinity and mediate its potent paralytic action. Whilst the action of ivermectin on the pharyngeal muscle of nematodes isrelatively well understood, its effect on locomotor activity is less clear. Here we use RNAi and gene knockouts to show that four GluCl� subunitsaHoetf©

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re involved in regulating the pattern of locomotor activity in Caenorhabditis elegans. A Haemonchus contortus orthologue of these subunits,cGluCl�3, has been shown to be expressed in the motor nervous system and here we have shown that it is a functional, as well as a structural,rthologue by virtue of the observation that it can restore normal motor movement in the C. elegans GluCl� mutant, avr-14(ad1032), whenxpressed under the control of the avr-14 promoter. This supports the contention that ivermectin exerts its paralytic action on parasitic nematodeshrough activation of GluCl channels in the motor nervous system. Furthermore, functional complementation in C. elegans provides a method tourther the understanding of this important class of anthelmintic targets.

2006 Elsevier B.V. All rights reserved.

eywords: Glutamate-gated chloride channels; Nematode; Anthelmintic; Ivermectin

. Introduction

Glutamate-gated chloride channels (GluCl) are receptors forhe macrocyclic lactone anthelmintics such as ivermectin [1].hey are a class of inhibitory receptor that appear to be unique to

he invertebrate phyla. Despite the importance of these channelss targets for ivermectin, little is known of their physiologicalole. Furthermore, establishing the properties of these channelsn parasites of economic importance is hampered by the technicalifficulty of such studies on the target species.

The GluCl gene family has been most extensively studied inhe model genetic animal Caenorhabditis elegans (for reviewee [2]). There are five confirmed genes that encode receptorubunits, avr-14, avr-15, glc-1, glc-2 and glc-3. The nomencla-

∗ Corresponding author. Tel.: +44 1225 386553; fax: +44 1225 386779.E-mail address: bssajw@bath.ac.uk (A. Wolstenholme).

1 These authors made an equal contribution to the work reported in this paper.2 Present address: Faculty of Life Sciences, University of Manchester,ackville St., Manchester M60 1QD, UK.

ture for the respective subunits is GluCl�3, GluCl�2, GluCl�1,GluCl� and GluCl�4. In addition a sixth gene, glc-4, is relatedto these sequences but has yet to be confirmed to encode a func-tional GluCl. An initial insight into the physiological roles of thedifferent GluCl receptor subunits has been provided by an anal-ysis of the gene expression patterns for avr-14 and avr-15. Thesedata indicate that these receptors are expressed in the neuronalcircuits that regulate feeding and locomotion [3,4], consistentwith their involvement in mediating the inhibitory effects ofivermectin on these behaviours. avr-15 is also expressed in thepharyngeal muscle [3] with glc-2 [5] and it is likely that GluCl�2forms a hetero-oligomeric receptor with GluCl� in the pharynx.The expression patterns of glc-1, glc-3 and glc-4 have not beendetermined and their physiological role is unknown.

The role of C. elegans GluCl in mediating the actions ofivermectin has been indicated by the observation of ivermectinresistance in strains with mutations in the different genes encod-ing GluCl. Mutations in avr-14 and avr-15 reduce the sensitivityof the animals to ivermectin. However, whilst mutations inavr-15 render the pharyngeal muscle completely insensitive to

166-6851/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.molbiopara.2006.02.003

A. Cook et al. / Molecular & Biochemical Parasitology 147 (2006) 118–125 119

ivermectin [6], the intact animal only shows high level resis-tance to ivermectin if all three genes, avr-14, avr-15 and glc-1,are mutated [4].

GluCl genes have also been identified in a wide range ofspecies of parasitic nematodes, but the information on theirproperties and physiological roles is less extensive than for C.elegans [7]. Some efforts have focused on identifying the GluClreceptors in Haemonchus contortus because of the economicimportance of this parasite, and the emergence of resistanceto ivermectin [8]. Three genes have been identified. Two ofthese are clear orthologues of C. elegans genes and have beendesignated Hcglc-2 and Hcavr-14 [9,10]. Immunostaining forHcGluCl� (encoded by Hcglc-2) and for HcGluCl�3 (the geneproduct of Hcavr-14) revealed expression in the motor nervoussystem, consistent with a role for these channels in locomotion.Intriguingly avr-14 is alternately spliced, and this processing isconserved in all species of nematode where this has been stud-ied, including H. contortus [10]. Specific antibodies raised to thesplice products have shown that HcGluCl�3A and HcGluCl�3Bare differentially expressed in the anterior region of the wormwith the former present in chemosensory amphidial neuronesand the latter in pharyngeal neurones [11]. Overall, this suggeststhat the role for GluCl receptors is similar in H. contortus to C.elegans, i.e. in both nematodes the inhibitory channels regulatelocomotion and feeding.

It is clear that a number of important questions remain to beacwm1His

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assays were carried out ‘blind’ at room temperature (18–22 ◦C).The strains to be tested were scored on the same day andapproximate time as wild-type controls, to allow for variationcaused by slight changes in environmental conditions. In thestudies described here, measurements of the duration of forwardmovement in wild-type controls varied between mean values ofapproximately 15–30 s (Table 1). Individual young adult wormswere transferred to NGM plates (prepared according to stan-dardised protocols and equilibrated at room temperature), withno food, and allowed to acclimatise for 5 min. Following this,the duration of forward movement, between reversals or direc-tion changes, was timed for 10 consecutive periods. This wasperformed for at least 10 animals per strain. For the experimentsin Figs. 1b and 3, the number of reversals for each animal wascounted in a 5-min period. Data are presented as mean ± S.E.M.Statistical analysis was performed using Student’s unpairedt-test.

2.2. RNAi constructs

E. coli (HT115) was engineered to express double-strandedRNA (dsRNA) for the gene of interest as described in [14]. Theexperiments described here used exon rich fragments (∼1 kb) ofthe genes of interest, glc-1, glc-2, avr-15, avr-14A and avr-14B,which were amplified by PCR from C. elegans N2 genomicDt1t

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ddressed. Not the least of these is defining the role of the GluClhannels in regulating locomotor behaviour in nematodes. Heree have addressed this by analyzing the motor behaviour ofutant and/or RNAi-treated C. elegans for avr-14, avr-15, glc-

, glc-2 and glc-3. Furthermore, we provide evidence that thecavr-14 can rescue the locomotor deficit in C. elegans avr-14,

ndicating that these two subunits are functional, as well as atructural, orthologues.

. Methods

Wild-type and mutant strains of C. elegans were cul-ured according to standard techniques at 20 ◦C on nematoderowth medium (NGM) agar plates seeded with OP50 bacteria12].

.1. Behavioural analysis

A preliminary phenotypic analysis of the GluCl mutant ani-als included in this study did not reveal any gross abnormalities

n behaviour. However, the animals did appear to have an alteredattern of locomotion. In the absence of food, wild-type wormsxhibit a stereotypical pattern of behaviour characterised by aelatively long duration of forward movement interspersed withhorter periods of reverse movement or turns. The frequencyf the spontaneous reversals in the absence of food is relativelyonstant, approximately 10 times in 3 min, i.e. the duration oforward movement of approximately 18 s [13]. In the GluClutants, the frequency of reversals appeared to be increasedith a concomitant decrease in the duration of forward move-ent. To quantify this, behavioural assays were performed. The

NA. For the latter two, which are splice variants of avr-14,he splice variant specific exons were amplified. The amplifiedkb products were cloned into the L4440 feeding vector and

ransformed into HT115.

.3. RNAi treatment

Wild-type (N2), avr-14(ad1032) or avr-15(ad1051) mutantsere fed on bacteria expressing double-stranded RNA (dsRNA)

or either glc-1 or glc-2 after the method of ref. [15]. Briefly,GM plates containing 50 �g ml−1 ampicillin and 1mM IPTGere seeded with HT115 bacteria expressing dsRNA for theene of interest. Individual L4 larvae were placed onto theselates and allowed to grow for 48 h. L4 larvae were replica platednto individual plates and young adults subjected to behaviouralnalysis. The control was the empty vector, also induced withPTG.

.4. Transformation constructs

C. elegans and H. contortus GluCl subunit cDNAs wereloned downstream of a 1.5 kbp putative ‘promoter’ fragment ofvr-14. The promoter fragment of C. elegans avr-14 was clonednto the SacI/XbaI sites of pBluescript and the cDNAs into thebaI/KpnI sites. The promoter fragment was obtained by PCRf genomic DNA incorporating the relevant restriction sites.ermline transformation was achieved using standard microin-

ection techniques [16]. avr-14(ad1032) worms were injectedith DNA at a concentration of 75–100 ng �l−1 for marker myo-:gfp (pPD118.33 Fire Vector Kit 1997) and 30 ng �l−1 for testNA.

120 A. Cook et al. / Molecular & Biochemical Parasitology 147 (2006) 118–125

Table 1Summary of the locomotion data obtained for wild-type and GluCl mutant worms

Strain Treatment Duration of forward movement (s) Reversals in 5 min Control (%)

Wild type None 23.3 ± 2.0 100avr-14 None 12.6 ± 3.3* 53.2Wild type RNAi avr-14A 12.7 ± 0.8** 54.5Wild type RNAi avr-14B 11.4 ± 1.0*** 48.9avr-15 None 12.3 ± 1.1* 51.2Wild type RNAi avr-15 10.6 ± 1.0*** 45.4avr-14::avr-15 None 8.3 ± 1.1*** 35.0

Wild type None 27.8 ± 1.6 100Wild type RNAi glc-1 18.8 ± 1.4*** 67.6

Wild type None 19.9 ± 3.6 100Wild type RNAi glc-2 19.5 ± 3.0 97.6

Wild type None 8.4 ± 1.1 100glc-3 None 3.1 ± 0.4*** 37

Wild type None 7.2 ± 0.6 100avr-15 None 10.4 ± 1.8** 144Wild type RNAi glc-1 9.6 ± 0.8* 133avr-15 RNAi glc-1 7.1 ± 0.7 98.6

Wild type None 5.4 ± 0.6 100avr-14 None 8.5 ± 0.6*** 157Wild type RNAi glc-1 10.1 ± 0.8 187avr-14 RNAi glc-1 5.0 ± 0.8*** 92.6

AVA, AVD and AVE are ventral cord interneurones that synapse onto the VA and DA motor neurones responsible for backwards movement. AVB and PVC are alsoventral cord interneurones, but these synapse onto the VB and DB motor neurones responsible for forward movement.

* p ≤ 0.05.** p ≤ 0.01.

*** p ≤ 0.001.

2.5. Strains

Behavioural analyses were performed on wild-type (Bris-tol N2), avr-14(ad1032) DA1371, avr-15(ad1051) DA1051 andavr-14(ad1032); avr-15(ad1051) DA1302 which were providedby Joseph Dent and Leon Avery. avr-14(ad1032) and avr-15(ad1051) are putative functional null mutations [3,4]. Thedeletion strain glc-3(ok321) RB594 was obtained from the C.elegans Genetics Center (CGC). The mutation in glc-3 is apredicted functional null since it has a deletion of several hun-

dred base pairs. RB594 was out-crossed four times prior tobehavioural analysis.

3. Results

3.1. Mutations in GluClα subunits affect locomotorbehaviour

Mutations in two of the genes encoding GluCl channels,avr-14 and avr-15, resulted in a significant reduction in the

F e wos is acci

ig. 1. GluCl subtype mutants move forward for a shorter duration than wild-typignificantly shorter duration of forward movement than wild-type worms. Thisn glc-3 shows significantly fewer reversals than wild type (p ≤ 0.001).

rms. Null or functional null mutants for avr-14 or avr-15 (p < 0.05) (a), exhibit aentuated in the avr-14; avr-15 double mutant (p < 0.001). (b) A deletion mutant

A. Cook et al. / Molecular & Biochemical Parasitology 147 (2006) 118–125 121

Fig. 2. The effects of gene mutation can be phenocopied by RNAi. (a) The deficit in the duration of forward movement in avr-14(ad1302) mutants is phenocopiedby feeding wild-type worms RNAi for either avr-14A (p < 0.01) or avr-14B (p < 0.001). (b) Feeding wild-type worms RNAi for glc-1 (p < 0.001) but not glc-2 (c)results in a significant reduction in the duration of forward movement.

duration of forward movement compared to wild-type controlanimals (Fig. 1a). This reduction was accentuated in the avr-14; avr-15 double mutant (Fig. 1a). There are two predictedisoforms of AVR-14, AVR-14A and AVR-14B, obtained byalternative gene splicing. They differ from each other in theregion predicted to encode the chloride channel, but have thesame glutamate-binding domain. To test whether either of theisoforms of AVR-14 is specifically involved in the control of theduration of forward movement, we used RNAi directed againstthe two splice variants avr-14A and avr-14B. RNAi for either ofthese splice variants resulted in a similar reduction in forwardmovement duration as observed for avr-14(ad1302) (Fig. 2a).RNAi was also used to investigate the role of the GluCl� sub-unit, glc-1 (Fig. 2b), and showed a reduction in the duration offorward movement compared to wild type. In contrast, RNAi forthe GluCl� subunit, glc-2 (Fig. 2c), did not affect the durationof forward movement. This could either be because the dsRNAiwas not effective in the neurones that control this behaviour,or that glc-2 is not involved in this behaviour. Latterly a geneknockout for glc-2 (VC722; ok1047) became available. This

strain does not have an aberrant pattern of locomotion, arguingagainst a role for GluCl� in the regulation of this behaviour (datanot shown).

The results with the glc-3 mutant were rather different, asthese worms showed a marked reduction in reversal frequency(Fig. 1b) and a concomitant increase in the duration of theirforward movements.

3.2. glc-1 RNAi reveals an interaction with avr-14 andavr-15

In a further series of experiments, the frequency of reversalswas compared between wild type, GluCl mutants and RNAi-treated animals. The frequency of reversals was significantlyincreased in avr-14 (Fig. 3a) and avr-15 mutant worms (Fig. 3b)compared to wild-type animals, consistent with their decreasedduration of forward movement. A similar observation was madefor wild-type animals treated with RNAi for glc-1 (comparedto animals treated with empty vector). However, and somewhatanomalously, RNAi for glc-1 in avr-14 animals restored the fre-

F 0.01)f r glc-i

ig. 3. Functional interaction between multiple GluCl subunits. (a) avr-14 (p <oraging than wild type. This is also the case for wild-type worms fed RNAi fon an apparent reversion to a wild-type number of reversals.

or (b) avr-15 (p < 0.05) mutants, exhibit a greater number of reversals during1 (a and b). Feeding RNAi for glc-1 to avr-14 (a) or avr-15 (b) mutants results

122 A. Cook et al. / Molecular & Biochemical Parasitology 147 (2006) 118–125

Fig. 4. Rescue of avr-14 deficit in forward movement duration by C. elegans or H. contortus subunits. Transformation of avr-14 mutants with native GluCl�3A orH. contortus HcGluCl�3A (a) or GluCl�3B or H. contortus HcGluCl�3B (b) under the avr-14 promoter rescues the deficit in forward movement duration.

quency of reversals back to wild type (Fig. 3a). This was alsoobserved following RNAi for glc-1 in avr-15 mutants (Fig. 3b).Further investigations revealed that this change in reversal fre-quency did not result from a reduction in the duration of theforward movements but, in part, from an increase in the durationof reversals from 0.6 ± 0.05 s in control avr-14 to 1.34 ± 0.2 sin avr-14 treated with glc-1 dsRNA (p ≤ 0.001). Visual obser-vation of avr-14 worms treated with RNAi for glc-1 suggestedthat they frequently had difficulty resuming forward movementafter a reversal and were rendered temporarily immobile. Takentogether, these extended reversals and short-term immobilisa-tions explained the apparent reversion to a wild-type phenotypeseen in these animals.

These data provide evidence that genes encoding GluCl�subunits are involved in regulating the pattern of locomotorbehaviour in C. elegans.

3.3. avr-14(ad1032) deficit can be rescued by cDNAsencoding C. elegans and H. contortus GluClα3 subunits

Germ line transformation of avr-14(ad1032) mutants withC. elegans or H. contortus genes encoding GluCl�3A (Fig. 4a)or GluCl�3B (Fig. 4b) expressed under the avr-14 promoterresulted in a rescue of the duration of forward movement backto wild-type values. This demonstrates the ability of H. contortusGluCl�3 subunits to functionally substitute for their C. elegansc1apnlgtamt

4. Discussion

Previous studies have shown that GluCl receptors are widelyexpressed in the neural circuits in C. elegans that regulate feedingand locomotion [4]. Furthermore, it is well-established that theactivity of the pharyngeal muscle is controlled in part by glu-tamatergic signalling through a receptor containing the GluClsubunit, AVR-15 [3,6]. The latter also confers sensitivity to theinhibitory action of ivermectin. However, less is known of therole of GluCl in the regulation of locomotion. Here we haveprovided evidence that these inhibitory channels are involvedin determining the duration of forward movement. This patternof movement underpins foraging behaviour in C. elegans and isinfluenced by a number of environmental variables [17] througha well-defined neuronal circuit (Fig. 5) in which sensory inputswill modulate the activity of a small set of command interneu-rones. During food search, in the early phase of food deprivation,the animal intersperses periods of forward movement with rever-sals and a change of direction. Excitatory ionotropic glutamatereceptors have been shown to play an important role in the sig-nalling pathways of this circuit [18,19]. For example, null muta-tions in the NMDA receptor nmr-1(allele) lead to an increase inthe duration of forward movement [18]. The behaviour of thesemutants is consistent with the proposal that forward movementis maintained until excitatory glutamate signalling depolarizesa command interneurone to a threshold value that triggers theriattmtroti

ounterparts. Two stably transformed lines were tested for avr-4::GluCI�3A, avr-14::GluCI�3B and avr-14::HcGluCl�3And one line for avr-14::HcGluCl�3B. The avr-14(ad1032)henotype was rescued by both lines transformed using theative receptor under the avr-14 promoter, one of the twoines transformed using avr-14::HcGluCl�3A and in the sin-le line transformed with avr-14::HcGluCl�3B. In the lineransformed with avr-14::HcGluCl�3B, the worms exhibited

‘super-rescue’ phenotype, with the duration of the forwardovements becoming approximately double that of the wild

ype.

eversal. In other words, there is a glutamate-dependent ‘switch’n the circuit that terminates forward movement and allows thenimal to reverse. Therefore, mutations which reduce the excita-ory glutamatergic drive, increase the time taken for the switcho be activated and therefore increase the duration of forward

ovement [20]. Interestingly, we have shown here that muta-ions in three out of four GluCl� subunit genes resulted in aeduction in the duration of forward movement, a phenotypepposite to that observed for the excitatory glutamate recep-ors. In the model as described, this would be consistent withnhibitory GluCl signalling prolonging the time taken for the

A. Cook et al. / Molecular & Biochemical Parasitology 147 (2006) 118–125 123

Fig. 5. A simplified model for the control of reversal behaviour based on thecircuitry described in [27,20]. Both excitatory and inhibitory ionotropic gluta-mate receptors are expressed in the circuit, for example, in AVA. Excitation ofAVA leads to initiation of backward movement whereas inhibition, e.g. by GluClchannels, leads to an increase in the time spent moving forward. Mutations inthe GluCl genes will therefore lead to increased reversal frequencies due to thereduced expression of these inhibitory channels. AVA, AVB, AVD, AVE andPVC are ventral cord ‘command’ interneurones that synapse onto the motorneurones that drive forward and reverse movement.

switch to be activated, and therefore mutations would be pre-dicted to decrease the duration of forward movements. Morerecently, a GluCl current has been recorded from a neurone inthe circuit that regulates reversal frequency interneurone [21],lending support to the hypothesis that the effects of GluCl muta-tion observed here occur as a result of a direct effect on theexcitability of this group of command interneurones.

There is only one C. elegans gene predicted to encode aGluCl� subunit, glc-2, and this study did not reveal any rolefor this subunit in the regulation of reversal behaviour. In con-trast gene mutations or RNAi for three of the GluCl� subunitsresulted in a decrease in the duration of forward movement;GluCl�1 (glc-1), GluCl�2 (avr-15) and GluCl�3B (avr-14B).These three subunits are also predicted to be components ofivermectin receptors as they have previously been shown to beactivated by ivermectin [1,3,4]. Gene knockouts, or RNAi, forGluCl�3A also decreased the duration of forward movement,but unlike GluCl�3B this is not predicted to be an ivermectinreceptor [4]. Mutations in the fourth GluCl� subunit gene, glc-3,resulted in an opposite phenotype. GluCl�4 channels are alsosensitive to ivermectin [22] and the gene encoding this channelcontains a 16-base-pair motif predicted to drive expression in theAIY interneurone [23]. This interneurone has been shown to sup-press turns and reversals [24]. The loss of a GluCl�4-containingGluCl from AIY may lead to its constitutive activation, resultingin markedly fewer reversals.

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(Table 1), in each case decreasing the duration by about 50%,is consistent with the idea that they may all be an essentialfunctional element of the same hetero-oligomeric receptor. Thedouble mutant for GluCl�2 and GluCl�3, avr-14::avr-15, had aslightly shorter duration of forward movement than for either ofthe single mutants. However, the effect of the double mutant wasmuch less than the sum of the single mutants, again consistentwith the idea that both subunits, when regulating locomotion,function as part of the same receptor complex.

The role of GluCl�1 in the control of reversal behaviour israther more complex than that of the other subunits. AlthoughRNAi for glc-1 resulted in an increase in the number of reversals,i.e. similar to the effect observed for the other GluCl� subunits,in an avr-14 mutant background the effect was to restore thenumber of reversals to the wild-type frequency. Some explana-tion of this somewhat anomalous observation was provided bya closer inspection of the phenotype of the glc-1 RNAi-treatedavr-14 animals. Although they score at wild-type levels for thenumber of reversals, their locomotor behaviour is aberrant. Theymoved backwards for longer and appeared to hesitate prior tothe commencement of a forward run. A similar observation wasmade for glc-1 RNAi-treated avr-15 animals. This suggests thatglc-1 may have a slightly different role in the neuronal circuitscontrolling locomotion compared to the other GluCl� subunits,i.e. in addition to regulating reversals it may also be involved ininitiating forward movement.

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There may be multiple subtypes of GluCl receptor that con-ain different combinations of � subunits. However, the obser-ation that gene deletions, or RNAi, for several of the GluClsxerts a similar effect on the duration of forward movement

In the studies described here we have used both RNAi andene deletion mutants to investigate the role of the GluCl chan-els in behaviour. The results obtained using either approachere similar, and both support a role for GluCl� subunits in the

ocomotor circuits. The similarity of the results obtained usingredicted null mutations and RNAi treatments gives us confi-ence that the RNAi treatments are both effective and specificn this case, though it is impossible to absolutely discount theossibility that the RNAi may also affect other genes. This is anxample of the efficacy of RNAi in the nervous system of C. ele-ans and may possibly result from a relatively low abundance,nd/or high turnover, of the receptors compared to other neu-onally expressed genes that have been unsuccessfully targetedith RNAi [25].C. elegans has previously been exploited as a model for the

xpression and characterisation of genes from parasitic nema-odes [26]. Here we also used this approach to test whether a generedicted to encode a GluCl�3 subunit in H. contortus could acts a functional subunit in C. elegans. First, we showed that theltered pattern of locomotion in the avr-14 mutant was rescued inransgenic animals expressing either avr-14A or avr-14B behindhe native C. elegans promoter. Thus either, or both, of theseplice variants of the C. elegans GluCl�3 subunit are impli-ated in this behaviour. The C. elegans avr-14 promoter washen used to drive expression of either H. contortus GluCl�3Ar GluCl�3B in the C. elegans avr-14 mutant. The observationhat expression of either of these H. contortus genes in the avr-4 genetic background could rescue the aberrant behaviour of C.legans indicates that they can restore function in the neuronalircuits regulating locomotion. The simplest explanation of thiss that they restore GluCl signalling that is defective in avr-14

124 A. Cook et al. / Molecular & Biochemical Parasitology 147 (2006) 118–125

and are thus functional orthologues of the C. elegans GluCl�3subunits. Intriguingly, expression of the H. contortus GluCl�3Bsubunit generated a transgenic animal which had twice the dura-tion of forward movement compared to the wild-type controls.This could be due to the presence of multiple copies of the trans-gene and simply result from the high levels of over-expressionof GluCl�3. As we have not integrated the transgene we can-not resolve whether or not this is the case. However, we did notobserve this effect for any of the other lines of transgenic animalsand it is interesting to note the high affinity of the H. contortusGluCl�3B subunit for glutamate when expressed in Xenopusoocytes (A. Rogers, D. Yates, A.J. Wolstenholme, unpublishedobservations). This may provide a pharmacological explanationfor the “super-rescue” of avr-14 observed following expressionof the avr-14::HcGluCl�3B construct. It should be noted thatwe did not attempt to use the H. contortus promoter to driveHcGluCl�3B expression in C. elegans: it is possible that theexpression patterns of the gene may differ in the two speciesand the use of the parasite promoter may yield a different phe-notype due to expression of HcGluCl�3B in different cell typesor at different levels.

In summary, we have provided evidence that the GluCl� sub-units, GluCl�1, GluCl�2, GluCl�3 and GluCl�4 are involvedin the reversal behaviour of C. elegans. One question that arisesfrom this study is whether any of the subunits are part of thesame hetero-oligomeric receptor complex, or whether there aredodbHwtoGanaaCtb

A

R

R

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[5] Laughton DL, Lunt GG, Wolstenholme AJ. Reporter gene constructssuggest that the Caenorhabditis elegans avermectin receptor beta-subunitis expressed solely in the pharynx. J Exp Biol 1997;200:1509–14.

[6] Pemberton D, Franks C, Walker R, Holden-Dye L. Characterisationof glutamate-gated chloride channels in the pharynx of wild-typeand mutant Caenorhabditis elegans delineates the role of the sub-unit GluCl-�2 in the function of the native receptor. Mol Pharmacol2001;59:1037–43.

[7] Cully D, Wilkinson HA, Vassilatis DK, Etter A, Arena JP. Molecularbiology and electrophysiology of glutamate-gated chloride channels ofinvertebrates. Parasitology 1997;113:S191–200.

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ifferent receptor subtypes comprising different combinationsf these subunits. A definitive answer to this will require a moreetailed analysis of the expression pattern of the subunits com-ined with electrophysiological analysis of the native receptors.owever, it is notable that the decrease in the duration of for-ard movement was very similar for all of the mutants, with

he exception of glc-3, which might indicate that they are partf the same receptor complex. It is also notable that four of theluCl� subunits involved in regulating reversal behaviour are

lso known to be activated by ivermectin, indicating that thiseuronal circuit is likely to be an important site of anthelminticction. Using this behavioural assay in C. elegans we have beenble to demonstrate the functional equivalence of orthologous. elegans and H. contortus GluCl subunits. This provides fur-

her insight into the role of these channels in the regulation ofehaviour in the parasitic nematodes.

cknowledgement

This work was funded by the Biotechnology and Biologicalesearch Council GAIN Initiative, UK (award GAN13134).

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