Haemonchus contortus Acetylcholine Receptors of the DEG-3 Subfamily and Their Role in Sensitivity to Monepantel

Gastro-intestinal nematodes in ruminants, especially Haemonchus contortus, are a global threat to sheep and cattle farming. The emergence of drug resistance, and even multi-drug resistance to the currently available classes of broad spectrum anthelmintics, further stresses the need for new drugs active against gastro-intestinal nematodes. A novel chemical class of synthetic anthelmintics, the Amino-Acetonitrile Derivatives (AADs), was recently discovered and the drug candidate AAD-1566 (monepantel) was chosen for further development. Studies with Caenorhabditis elegans suggested that the AADs act via nicotinic acetylcholine receptors (nAChR) of the nematode-specific DEG-3 subfamily. Here we identify nAChR genes of the DEG-3 subfamily from H. contortus and investigate their role in AAD sensitivity. Using a novel in vitro selection procedure, mutant H. contortus populations of reduced sensitivity to AAD-1566 were obtained. Sequencing of full-length nAChR coding sequences from AAD-susceptible H. contortus and their AAD-1566-mutant progeny revealed 2 genes to be affected. In the gene monepantel-1 (Hco-mptl-1, formerly named Hc-acr-23H), a panel of mutations was observed exclusively in the AAD-mutant nematodes, including deletions at intron-exon boundaries that result in mis-spliced transcripts and premature stop codons. In the gene Hco-des-2H, the same 135 bp insertion in the 5′ UTR created additional, out of frame start codons in 2 independent H. contortus AAD-mutants. Furthermore, the AAD mutants exhibited altered expression levels of the DEG-3 subfamily nAChR genes Hco-mptl-1, Hco-des-2H and Hco-deg-3H as quantified by real-time PCR. These results indicate that Hco-MPTL-1 and other nAChR subunits of the DEG-3 subfamily constitute a target for AAD action against H. contortus and that loss-of-function mutations in the corresponding genes may reduce the sensitivity to AADs.


Introduction
Throughout the world, successful livestock production of ruminants is hampered by gastro-intestinal nematodes. Haemonchus contortus in particular is responsible for substantial losses to the global sheep industry [1]. Haemonchus contortus is a blood-feeding nematode that inhabits the abomasum of sheep, producing in acute infections, severe anemia that can lead to the death of infected animals.
Broad spectrum chemotherapy against gastro-intestinal nematodes is restricted to 3 anthelmintic classes: the benzimidazoles, such as albendazole and oxfendazole, the imidazothiazoles, including levamisole and tetramisole and the macrocyclic lactones (e.g. ivermectin, moxidectin, abamectin and doramectin). The increased usage of anthelmintics has contributed to the spread of resistant nematodes with increasing reports of nematodes insensitive to most if not all of the available classes of anthelmintics [2][3][4][5][6][7][8][9][10]. In some countries in the southern hemisphere, sheep farming is severely endangered by such populations [4], further increasing the need for a new class of anthelmintic [11].
Recently, a new class of compounds, the Amino-Acetonitrile Derivatives (AADs) was discovered [12] with good tolerability in mammals and promising activity against drug-resistant nematodes. The AADs are low molecular mass compounds bearing different aryloxy and aroyl moieties on an amino-acetonitrile core [13]. Further studies [14] have allowed the selection of a drug candidate, AAD-1566 (monepantel). In order to investigate the mode of action of this new class of compounds, AAD-resistant Caenorhabditis elegans mutants were generated by EMS mutagenesis. Classical forward genetics revealed that the majority of recuperated AAD-resistant mutants carried mutations in the gene acr-23, a member of the nematode-specific DEG-3 subfamily of nicotinic acetylcholine receptor (nAChR) alpha subunits [12]. Preliminary data had already indicated an involvement of similar acetylcholine receptors in AAD action against H. contortus [12]. Here we report the identification of the gene monepantel-1 (Hco-mptl-1, formerly named Hc-acr-23H) and other members of the DEG-3 subfamily of ACR genes from H. contortus. A panel of different mutations, mis-splicing in particular, in Hco-mptl-1 transcripts from AAD-resistant worms indicates that Hco-MPTL-1 is a target for monepantel action against H. contortus.

Haemonchus contortus isolates
The drug-susceptible H. contortus CRA (Hc-CRA) was received in 1984 from the Veterinary Institute of Onderstepoort, Republic of South Africa and has since been passaged in sheep 75 times. The H. contortus Howick isolate (Hc-Howick) was received from the same institute in 2001. This is a multidrug-resistant isolate that is completely resistant to albendazole, rafoxanide, morantel, ivermectin and trichlorfon [6,15]. The isolate has been passaged in sheep 9 times since being received. The mutant lines Hc-CRA AAD M and Hc-Howick AAD M were selected from Hc-CRA and Hc-Howick, respectively, by in vitro exposure to increasing doses of AAD-1566 alternatively with propagation in sheep [12].

Collection of nematode eggs
Haemonchus contortus isolates were propagated in 3-6 month old sheep ('Blanc des Alpes'), which had been experimentally infected with the nematode. The sheep were kept in groups of 4 and housed indoors off pasture to prevent natural infection. After 14 days, they were transferred to individual cages. Starting on day 21 after infection, eggs were collected from homogenized feces and filtered several times through a 32 mm sieve. Eggs were further purified by floating on 50% sucrose solution, rinsed with water and counted microscopically.

In vivo determination of drug sensitivity
Sheep studies were performed with approval of a Cantonal animal welfare committee (permit number FR 25A/05). Anthelmintic efficacy tests in sheep were performed according to the guidelines of the World Association for the Advancement of Veterinary Parasitology [16]. Each animal was infected intraruminally on study day 221 with 3000 L 3 -larvae of H. contortus (cultivated in coprocultures). On study day 0, the sheep were treated with single anthelmintics or combinations thereof as an oral drench at the recommended dose. A sheep was classified as 'cured' when no more eggs were counted in the feces and no adults were found in the abomasum at necropsy.

Recovery of adult Haemonchus contortus and isolation of nucleic acids
Adult worms were recovered from the abomasum of freshly euthanized sheep, washed in Hank's Buffered Salt Solution (HBSS; Invitrogen) and immediately shock-frozen in liquid nitrogen. While frozen, the worms were crushed with a Kontes pellet pestle (Fisher Scientific). The powder was resuspended in 600 ml of lysis buffer (10 mM Tris pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.5% SDS, 100 mg/ml RNase A) and incubated at 37uC for 1 hour. Pronase (100 mg/ml) was added to the mixture and the tubes were incubated at 37uC until the solution became clear. The samples were extracted with equal volumes of phenol:chloroform (1:1) and chloroform. The DNA was ethanol precipitated, washed and resuspended in 50 ml of Tris-Cl (pH 7.5). For RNA extraction, worms were homogenized in TRIzol and processed according to the instructions of the supplier (Invitrogen). To remove DNA contamination, the RNA samples were treated with a TURBO DNA-free kit (Ambion). To generate cDNA, 1 mg of total RNA was reverse transcribed to cDNA using a d(T) 30 primer and a Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT; SMART cDNA library construction kit from Clontech).

Construction and screening of a Haemonchus contortus cDNA library
A total of 4 mg of mRNA was isolated from a mixture of male and female Hc-CRA using a Oligotex kit from Qiagen. A cDNA library was constructed with the ZAP-cDNA Cloning kit and Gigapack III Gold packaging kit. The library was screened at high stringency (hybridization at 65uC in 56SSC, 56 Denhardt's solution, 0.1% SDS, 0.1% sodium pyrophosphate, 100 mg/ml salmon sperm DNA; final wash at 60uC in 0.26SSC, 0.1% SDS) with a 32 P-labeled 456 bp fragment of Hco-mptl-1. This fragment had been amplified from cDNA with the primers Hco-mptl-1_frw3 and Hco-mptl-1_rev1 and cloned into pCRH2.1-TOPOH (Invitrogen). Positive phages were taken through 3 rounds of plaque purification with this probe and the phagemid (pBluescript SK+) was excised using the ExAssist helper phage in the E. coli SOLR strain. Inserts were sequenced in both directions with standard M13 forward and reverse primers and the internal primers Hcomptl-1_frw4 and Hco-mptl-1_rev3. The sequences were read and assembled using 4Peaks (by A. Griekspoor and T. Groothuis; http://mekentosj.com).

PCR
The primers used for PCR-amplification, real-time PCR or for cDNA first strand synthesis of H. contortus nAChR genes are summarized in Table S1. For nested PCR on cDNA with spliced leader (SL) primers, the primary products were diluted 50-fold and 2 ml were used for the second PCR with nested primers. The annealing temperature was fixed at 55uC for cDNA and 58uC for genomic DNA template. PCR products were gel purified using the NucleoSpinH ExtactII kit (Macherey-Nagel) and cloned into either pGEM-T easy (Promega) or pCRH2.1-TOPOH (Invitrogen). Plasmid DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced using the standard primers M13 forward and reverse and, if necessary, an additional internal primer to cover long products. For rapid amplification of cDNA ends by PCR (RACE-PCR), an internal reverse primer (Table S1)

Author Summary
Worldwide, sheep and cattle farming are threatened by anthelmintic-resistant gastro-intestinal nematodes. A novel chemical class of synthetic anthelmintics was recently discovered, the Amino-Acetonitrile Derivatives (AADs), which exhibit excellent efficacy against various species of livestock-pathogenic nematodes and, more importantly, overcome existing resistances to the currently available anthelmintics. Haemonchus contortus, the largest nematode found in the abomasum of sheep and cattle, is a blood-feeding parasite that causes severe anemia that can lead to the sudden death of the infected animal; H. contortus is highly susceptible to AADs. In order to elucidate the mode of action of the AADs, we have developed 2 independent H. contortus mutants with reduced sensitivity to monepantel (AAD-1566). Both mutants were affected in their acetylcholine receptor (ACR) genes of the DEG-3 subfamily. In particular, we discovered a panel of mutations in the gene monepantel-1 (Hco-mptl-1) including deletions leading to mis-splicing, insertions and point mutations leading to premature termination of translation of the protein. These findings support the notion that Hco-MPTL-1 and other nAChR subunits of the DEG-3 subfamily are targets of the AADs. The fact that the DEG-3 subfamily of acetylcholine receptors is nematode-specific may explain the good therapeutic index of AADs in mammals.
was combined with splice leader sequence (1 or 2) to obtain the 59 UTR, or an internal forward primer combined with a poly-dT primer for the 39 UTR of the transcript.
For real-time PCR, 1 mg of total RNA from adult H. contortus was used to synthesize first-strand cDNA by random priming using Superscript II reverse transcriptase (Invitrogen) in a final volume of 20 ml following the manufacturer's instructions. Reversetranscribed material corresponding to 40 ng RNA was amplified in 25 ml MESA GREEN qPCR MasterMix Plus for SYBR Assay (Eurogentec) by using the ABI SDS7000 Sequence Detection System under the following conditions: 1 cycle of 95uC for 15 minutes followed by 40 cycles of 95uC for 15 seconds and 60uC for 1 minute. The primer pairs used for the amplification are listed in Table S1 and target the following genes: b-tubulin, Hco-mptl-1, Hco-des-2H and Hco-deg-3H. Three independent total RNA extractions were performed and each was tested in duplicate.
Relative expression values were calculated according to Livak and Schmittgen [17]; a 136 bp region within the phosphoglucose isomerase gene was used for normalization, a 122 bp region within the b-tubulin gene was used as a (presumably) non-affected control, and no reverse transcriptase and no template reactions as negative controls. The specificity and identity of individual amplicons were verified by melt curve analysis and visualized on a 2% agarose gel.

In vivo sensitivity of Haemonchus contortus AAD mutants
In order to study the mode of action of the AADs, we used 2 mutant isolates, Hc-CRA AAD M and Hc-Howick AAD M selected from parent Hc-CRA and Hc-Howick isolates, respectively. Both mutant isolates showed reduced sensitivity to AAD-1566 (monepantel) in vitro [12]. To test whether the observed loss of susceptibility to AAD-1566 in vitro was relevant for the situation in vivo, Hc-CRA, Hc-Howick and their AAD M derivatives were challenged in vivo with single compounds or combinations thereof; AAD-1566 and the commercial compounds were applied at their recommended doses to sheep. Sheep were infected intraruminally with Hc-CRA AAD M . Following treatment with AAD-1566 at the proposed minimum dose rate of 2.5 mg/kg body weight [18] eggs were found in the feces and adults seen at necropsy (Table 1). Likewise, nematode eggs and adults were also found in sheep infected with Hc-Howick AAD M larvae when treated either with AAD-1566 or albendazole or a combination of AAD-1566 and ivermectin ( Table 1). The offspring from the Hc-Howick AAD M isolate that survived the AAD-1566 and ivermectin treatment were cultured and challenged with albendazole and levamisole over the following generations (data not shown). Finally, Hc-Howick AAD M was able to survive a full simultaneous in vivo treatment with albendazole, levamisole, ivermectin and AAD-1566, administered at their recommended doses (Table 1). Thus the reduction of sensitivity to AAD-1566 induced in vitro was also relevant in vivo for the mutant lines. The AAD-mutant H. contortus apparently did not show any alterations in motility, infectivity to sheep (determined by the numbers of adult H. contortus recovered at necropsy) or egg production, and did not exhibit any phenotype with respect to the ultrastructure (by electron microscopy) of the cuticle, head or tail.

Cloning of Haemonchus contortus Hco-mptl-1
To obtain the full length coding sequence of the Hco-mptl-1 gene, a lambda phage cDNA library from mRNA of adult H. contortus was constructed and screened at high stringency with a radioactive probe from a partial Hco-mptl-1 sequence. After 3 rounds of selection, a clone with the full-length coding sequence, Hco-mptl-1, was isolated and sequenced. The Hco-mptl-1 mRNA is composed of at least 17 exons and 16 introns (1992 bp) with a short 59 UTR and 39 UTR (21 bases and 42 bases, respectively). The transcript is trans-spliced as the splice leader 1 (SL1) is present at its 59 end. Interestingly, a start codon (AUG) is present in exon 1 but is followed after 8 amino acids by a stop codon in frame (UGA). This is a feature found in many other organisms [20][21][22] and it is assumed to play a role in the regulation of translation efficiency. In most cases, upstream AUGs decrease mRNA translation efficiency and have a strong, negative regulatory effect [23]. The longest open reading frame (ORF) in the Hco-mptl-1 gene is obtained when the translation is initiated at the second AUG codon in exon 3 and extends over 1695 bases. Overlapping long range PCR was performed in order to estimate the total size of Hco-mptl-1. The gene was found to be approximately 18.5 kb long with a large intron (about 7 kb) between exons 3 and 4 (see below). The predicted Hco-MPTL-1 protein consists of 564 amino acids and possesses motifs typical for Cys-loop ligand-gated ion channels, including an N-terminal signal peptide of 18 amino acids [24], 4 transmembrane domains and the Cys-loop (2 cysteines separated by 13 amino acids). Loops A to F, which are involved in ligand binding [25] are also present in the protein ( Figure S1). In loop C, there are 2 adjacent cysteines, defining Hco-MPTL-1 as a nAChR alpha subunit.
The predicted Hco-MPTL-1 protein shares 48.5% identity and 66.8% similarity with C. elegans ACR-23 and 60.2% identity and 70.7% similarity with C. elegans ACR-20. The novel H. contortus nAChR was originally named Hc-ACR-23H based on a partial sequence that was most closely related to C. elegans ACR-23 [12].
In the light of the full-length sequence, this nomenclature seems to have been premature since the Haemonchus nAChR turned out to be more closely related to C. elegans ACR-20 ( Figure 1). In the absence of a complete record of ACR paralogues from H. contortus, and in analogy to levamisole-insensitive (lev-) mutants in C. elegans [26], we propose to name the gene monepantel-1 (Hco-mptl-1) due to its apparent involvement in monepantel sensitivity.

Hco-mptl-1 mutations associated with the AAD-mutant phenotype
In order to compare the Hco-mptl-1 sequences from the AADsusceptible isolates and their AAD-mutant progeny, primers were designed at each extremity of the ORF (Hco-mptl-1_59_frw3 and Hco-mptl-1_39end_rev1) and the full length Hco-mptl-1 coding sequences amplified from cDNA from adults. A product of about 1800 bp was obtained for all isolates apart from the Hc-CRA AAD M , which produced a shorter product of 1650 bp ( Figure 3B). Sequencing clones of the latter revealed that they lacked either exon 4 or exon 15 (Figure 4, Hco-MPTL-1-m2 and m3). This was confirmed with primers flanking either exon 4 (Hco-mptl-1_59_frw2 and Hco-mptl-1_rev8; Figure 3C) or exon 15 (Hco-mptl-1_frw6 and Hco-mptl-1_rev6; Figure 3D). PCR with a SL1 forward primer and a reverse primer in the Hco-mptl-1 coding sequence (Hco-mptl-1_rev1, product of about 1200 bp; Figure 3A) also produced shorter products (1000 bp and 850 bp; Figure 3A Mutation cause mis-splicing of the Hco-mptl-1 transcript in Hc-CRA AAD M mutants To understand the molecular basis of exon loss in the Hc-CRA AAD M isolate, PCR primers Hco-mptl-1_frw8 and Hco-mptl-1_rev6 (Table S1) were designed to flank the mis-spliced exon 15. PCR was performed using genomic DNA as a template. Sequencing of cloned PCR products revealed a 10 bp deletion upstream of exon 15 in the Hc-CRA AAD M mutant that encompasses the predicted splice acceptor site (UUUCAG; Figure 5). Presumably, the splicing machinery is not able to identify the end of intron 14 and uses the next splice acceptor site (intron 15). This would explain why exon 15 is skipped (Figure 4, Hco-MPTL-1-m3). Joining of exon 14 to exon 16 causes a frame-shift leading to a premature stop codon. With primers flanking exon 4 (Hco-mptl-1_frw10/gDNA and Hcomptl-1_rev8; Table S1), a 323 bp deletion was detected consisting of the end of intron 3 (206 bp) and most of exon 4 (117 bp). Again, loss of the predicted splice acceptor site at the end of intron 3 may explain the observed loss of exon 4 (Figure 4, Hco-MPTL-1-m2), since the splicing machinery will use the next available splice acceptor site (intron 4), joining exon 3 and exon 5. The resulting frame-shift causes a premature stop at codon 19 (TGA), terminating translation after the signal peptide (Figure 4, Hco-MPTL-1-m2). Sheep were treated orally with commercial compounds at the recommended doses. An animal was considered to have been effectively treated when no more eggs were counted in the feces and no adults were found in the abomasum at necropsy. doi:10.1371/journal.ppat.1000380.t001 Detection of the Hco-mptl-1 E93* point mutation in the Hc-Howick AAD M nematodes No obvious mutations such as mis-spliced exons were detected in the Hc-Howick AAD M isolates. When sequencing the Hco-mptl-1 coding regions (SL1 and Hco-mptl-1_rev6) from both susceptible and AAD-1566-mutant Howick isolates, a transversion from G 277 to T in exon 6 of the Hco-mptl-1 gene was observed that led to a premature stop codon (E93*; Figure 6). Direct sequencing of RT-PCR products (using Hco-mptl-1_frw4 and Hco-mptl-1_rev1 primers) revealed that about 80% of the Hc-Howick AAD M cDNAs, as estimated from the electropherogram [27], carried a T at position 277 ( Figure 6A). The point mutation underlying E93* creates a restriction site for the endonuclease BfrI (recognition site: CTTAAG) that lent itself for RFLP analysis. Only the PCR product amplified from cDNA of Hc-Howick AAD M was digested by BfrI ( Figure 6B). As expected from the sequencing, a small proportion (about 20%) of the product was not cut, indicating that not all of the Hco-mptl-1 genes from Hc-Howick AAD M population carried the G277T mutation. When this BfrI-unrestricted product from Hc-Howick AAD M was excised from an agarose gel, cloned and sequenced, a further polymorphism was detected that led to skipping of exon 8 (Figure 4, Hco-MPTL-1-m6). As this exon is very short (22 bases), it was impossible to discriminate between mutant and parental wild type PCR products (Figure 3). Loss of exon 8 causes a frame-shift leading to a premature stop codon and a predicted Hco-MPTL-1 protein truncated at amino acid 166 ( Figure 4). A minority of the Hco-mptl-1 PCR products obtained from Hc-Howick AAD M did not contain any major mutations. These sequences could come from AAD-susceptible individuals within the H. contortus Howick AAD M populations or from AADmutant individuals that carry other, yet to be identified mutations.

An insertion in the 59 UTR of the des-2 homologue of Haemonchus contortus AAD mutants
As the DEG-3 subfamily gene Hco-des-2H has also been implicated in AAD action in H. contortus [12], we cloned and sequenced the full-length Hco-des-2H coding sequence from H. contortus cDNA by RACE-PCR. Using primers NheI_des2_frw1 and XhoI_des2_rev1 (Table S1), 2 products were obtained from the four H. contortus isolates. Cloning and sequencing revealed the smaller transcript to lack 168 bases coding for part of the internal loop between TM3 and TM4, possibly indicating alternative splicing of the Hco-des-2H gene. The predicted protein (full version) consists of 534 amino acids and shows 69% identity and 80% similarity with C. elegans DES-2. Hco-DES-2H possesses motifs typical for Cys-loop ligand-gated ion channels (4 transmembrane domains, a Cys-loop and loops A to F) and the 2 adjacent cysteines in the C-loop, defining Hco-DES-2H as a nAChR alpha subunit ( Figure S2).     When comparing Hco-des-2H coding sequences (Table 2) obtained from Hc-CRA and Hc-CRA-AAD M , respectively Hc-Howick and Hc-Howick-AAD M , no mutation was found to correlate perfectly with AAD-susceptibility. Nevertheless, using the SL1 primer and 2 internal reverse primers (Hco-AcRa_rev3 and Hco-AcRa_rev2) in a nested PCR experiment, an insertion of 135 bp was detected in the 59 UTR of the Hco-des-2H gene from the Hc-CRA AAD M and Hc-Howick AAD M isolates, creating 2 additional start codons. Both start codons are followed by an early stop codon in frame.
In the C. elegans genome, DES-2 and DEG-3 are encoded on the same operon and both subunits are co-expressed to form a functional channel [28,29]. Performing RACE-PCR on H. contortus (adults) cDNA we identified Hco-deg-3H encoding a protein of 569 amino acids that shows 68.4% identity and 78% similarity to C. elegans DEG-3. Again, Hco-DEG-3H carried all the hallmarks of nAChR alpha subunits ( Figure S3). No mutations were detected for Hco-deg-3H in the AAD-mutant H. contortus isolates compared to the parental isolates. The Hco-deg-3H mRNA carries a spliced leader type 2 (SL2) sequence at its 59 end. To test whether Hco-des-2H and Hco-deg-3H are also on an operon in H. contortus, a long range PCR was performed using a forward primer designed at the end of Hco-des-2H (Hco-des2_frw11) and a reverse primer at the beginning of Hco-deg-3H (Hco-deg3_2r). A band of approximately 6 kb was obtained for the 4 isolates confirming that Hco-des-2H and Hco-deg-3H are encoded on a single operon. However, the distance between the 2 genes is 10 times larger in H. contortus than in C. elegans.

Discussion
A new chemical class of synthetic anthelmintics, the AADs, was recently discovered [12]. The AADs exhibit excellent efficacy against various species of livestock-pathogenic nematodes and more importantly, can control nematodes resistant to the currently available anthelmintics [30,31]. To get insights into the mode of action of the new AADs, a classical 'forward genetic' screen for AAD-resistant C. elegans mutants was performed previously [12]. As a result, AADs were proposed to act through the nAChR ACR-23, a member of the nematode-specific DEG-3 subfamily [32]. By screening the currently available (but incomplete) H. contortus genome sequence for DEG-3 nAChR homologues, it was found that this subfamily is conserved between C. elegans and H. contortus. Six paralogous proteins out of 8 in C. elegans or C. briggsae were identified (Figure 1), in contrast to only 2 in the genome of B. malayi [33]. The AADs possess a unique mode of action: the nAChR subunits involved in AAD action are different from those targeted by imidazothiazoles [34,35] and there is no crossresistance between the 2 chemical classes [12].
Two independent AAD-mutant H. contortus lines were used to screen for mutations in ACR genes of the DEG-3 subfamily. Two genes were found to be affected: The H. contortus des-2 homologue Hco-des-2H, where all AAD-mutant H. contortus carried an insertion in the 59 UTR introducing 2 additional, out-of-frame start codons, and the gene monepantel-1 (Hco-mptl-1), for which a panel of different mutations were detected in AAD-mutant (AAD M ) H. contortus. Apart from 1 nonsense mutation discovered in Hc-Howick AAD M nematodes (Hco-MPTL-1-m5; Figure 4), the detected mutations all involved mis-splicing resulting in loss of exon(s) from the mRNA as indicated by shortened reverse transcriptase-PCR products (Figure 3). This unusual mechanism has not been described before in H. contortus. In the genetic screen performed on AAD-resistant C. elegans [12], 2 mutants bearing a G-to-A transition of the conserved G nucleotide in the 39 splice acceptor sites of either the second or third introns were described; these mutations are predicted to cause an increase in the size of the mRNA due to the lack of splicing of the affected intron. In another study [36], a single base pair change in the first intron of the lev-8 subunit gene was identified in a partially levamisole-resistant C. elegans mutant. This mutation leads to alternative splicing and introduction of a premature stop codon. In the case of mutations Hco-MPTL-1-m2 (loss of exon 4), Hco-MPTL-1-m3 (loss of exon 15) or Hco-MPTL-1-m6 (loss of exon 8), exon skipping creates a frame-shift that leads to a premature stop codon ( Figure 4). These mutations, including the Hco-MPTL-1-m5 (stop codon) are predicted to result in a truncated, non-functional Hco-MPTL-1 protein and/or, if the mutant mRNA is recognized by the nonsense-mediated mRNA decay (NMD) machinery [37], degradation of the mRNA (some known genes of the NMD machinery in C. elegans have orthologues in the H. contortus genome; Rufener and Mä ser, unpublished). Measuring the expression levels of the 3 DEG-3 subfamily genes Hco-mptl-1, Hco-des-2H and Hco-deg-3H in adult H. contortus, we found statistically significant differences in the steady state level of mRNA in AAD mutant worms. In the Hc-CRA AAD M isolate, a significant increase of the Hco-deg-3H transcript was observed. A possible explanation may be that this compensates for the loss of the Hco-MPTL-1 subunit since no more full-length Hco-mptl-1 transcript was detectable in Hc-CRA AAD M . In the case of Hc-Howick AAD M , all 3 nAchR genes were down-regulated compared to Hc-Howick. Although we cannot give a result-based explanation, we interpret the finding that the expression of DEG-3 subfamily nAChR genes is affected in H. contortus as further evidence for the involvement of these genes in AAD susceptibility.
The mutations Hco-MPTL-1-m1 (loss of exon 2 and 3) and Hco-MPTL-1-m4 (partial loss of exon 4 and 15) did not cause a frameshift, but the loss of the signal peptide and the first 39 amino acids of the extracellular loop for the first mutation, and a truncated protein for the second mutation. Interestingly, 1 of the previously identified AAD-resistant C. elegans mutants also carried a mutation in the signal peptide of the Cel-ACR-23 protein [12]. Receptors are assembled in the endoplasmic reticulum (ER) [38] and interference with the signal peptide could result in mis-localization of the protein or in improper interactions with ER-resident, ACRspecific chaperones [25,[39][40][41]. Furthermore, it is known that the expression, assembly and transport to the surface of ACR subunits is subject to stringent quality controls that guarantee the functional competence of the final product [42][43][44]. Truncated nAChR proteins are likely to be targeted to the lysozyme and degraded.
In summary, we have detected a large number of different mutations affecting the Hco-mptl-1 gene and transcript, respectively, from AAD mutant H. contortus (Table 2). For the benzimidazoles, a variety of different mutations in the target protein ßtubulin are associated with drug resistance, 3 so far from H. contortus [15,45,46] and many more from phytopathogenic fungi [47]. These are point mutations, that are thought to interfere with benzimidazole binding while preserving microtubular function. The mutations have less drastic effects on the predicted protein than those described here for Hco-mptl-1 of H. contortus. At present, we do not know whether Hco-mptl-1 is an essential gene in H. contortus, but our findings indicate that it may not be. There were no mutations in common between H. contortus CRA-AAD M and n.d.
Howick-AAD M , indicating that the genetic screen was not saturated. However, for Hco-des-2H, an insertion of 135 bp creating 2 additional start codons was present in the 59 UTR from both AAD M isolates. While Hco-des-2H mRNA levels were significantly lower in Hc-Howick AAD M (compared to Hc-Howick), no effect was observed on Hco-des-2H expression in Hc-CRA AAD M . It is interesting to note that in C. elegans, mutant worms lacking a functional DES-2 did not exhibit any AAD resistance [12]. The in vitro protocol used to breed AAD-mutant H. contortus is very focused using a large number of individuals and a border line subcurative exposure concentrations over extended time period. This protocol is different from the situation in the field, e.g. after multiple generations exposed to subcurative treatment in sheep, we have so far not been able to obtain AAD-resistant H. contortus (Pradervand and Kaminsky, unpublished data).
In conclusion, several independent mutations were found in the Hco-mptl-1 gene from H. contortus mutants with reduced sensitivity for monepantel, implicating Hco-MPTL-1 as a likely target for AAD action against H. contortus. However, this hypothesis is difficult to test since H. contortus is not readily amenable to genetic manipulation [48]. The AADs are very well tolerated by sheep or cattle [14]. The absence of DEG-3 subfamily acetylcholine receptors in mammals might explain the selective toxicity of AADs to nematodes.  Text S1 All the sequences as submitted to GenBank.