Proteomic Analysis of Oesophagostomum dentatum (Nematoda) during Larval Transition, and the Effects of Hydrolase Inhibitors on Development

In this study, in vitro drug testing was combined with proteomic and bioinformatic analyses to identify and characterize proteins involved in larval development of Oesophagostomum dentatum, an economically important parasitic nematode. Four hydrolase inhibitors ο-phenanthroline, sodium fluoride, iodoacetamide and 1,2-epoxy-3-(pnitrophenoxy)-propane (EPNP) significantly inhibited (≥90%) larval development. Comparison of the proteomic profiles of the development-inhibited larvae with those of uninhibited control larvae using two-dimensional gel electrophoresis, and subsequent MALDI-TOF mass spectrometric analysis identified a down-regulation of 12 proteins inferred to be involved in various larval developmental processes, including post-embryonic development and growth. Furthermore, three proteins (i.e. intermediate filament protein B, tropomyosin and peptidyl-prolyl cis-trans isomerase) inferred to be involved in the moulting process were down-regulated in moulting- and development-inhibited O. dentatum larvae. This first proteomic map of O. dentatum larvae provides insights in the protein profile of larval development in this parasitic nematode, and significantly improves our understanding of the fundamental biology of its development. The results and the approach used might assist in developing new interventions against parasitic nematodes by blocking or disrupting their key biological pathways.


Introduction
Parasitic roundworms (nematodes) of animals and humans are of major socioeconomic importance worldwide [1][2][3][4][5]. Of these nematodes, the soil-transmitted helminths (STHs) Ancylostoma duodenale, Necator americanus, Trichuris trichiura and Ascaris spp. are estimated to infect almost one sixth of the global human population [6,7]. Also parasites of livestock, including species of Haemonchus, Ostertagia, Teladorsagia, Trichostrongylus and Oesophagostomum, collectively cause substantial economic losses estimated at billions of dollars per annum, due to poor productivity, failure to thrive, deaths and the cost of anthelmintic treatment [8][9][10][11]. In addition to their socioeconomic impact, widespread resistance in nematodes of livestock against the main classes of anthelmintics [12][13][14] has stimulated research toward designing alternative intervention and control strategies against these parasites. Central to this effort should be the discovery of new drug targets through an improved understanding of the fundamental biology of parasite development.
Despite the advances in '-omics' and computer-based technologies [15], and extensive studies of the free-living nematode Caenorhabditis elegans [16][17][18], there is a paucity of information on developmental processes in parasitic nematodes of animals, particularly those of the order Strongylida, which are of major socioeconomic importance. Numerous studies (reviewed in [19]) show that the porcine nodule worm, Oesophagostomum dentatum, is a unique model for studying fundamental developmental and reproductive processes in strongylid nematodes because of its short life cycle and, particularly, an ability to maintain worms in vitro for weeks through multiple moults.
The life cycle of O. dentatum is simple and direct [20]. Unembryonated eggs are released in host faeces and develop into free-living, first-and second-stage larvae (L1s and L2s, respectively). Feeding on nutrients and microbes in the faecal matter, they develop into the infective, third-stage larvae (L3s) which are protected within a cuticular sheath. These larvae migrate from the faeces into the surrounding environment (pasture or soil), where the porcine host ingests them. Once ingested, the L3s exsheath in the small intestines of the pig en route to the large intestine. Upon reaching the large intestine, they burrow into the mucosal layer of the intestinal wall and subsequently produce lesions. Within the submucosa, the L3s moult to fourth-stage larvae (L4s) [21] and evoke an immune response that results in the encapsulation of the larvae in raised nodular lesions, made up mainly of aggregates of neutrophils and eosinophils [22]. Following the transition to the L4s, the larvae emerge from the mucosa within 6-17 days. The parasite undergoes another cuticular moult, subsequently maturing to an adult. The pre-patent period of O. dentatum is ,17-20 days [23], although longer periods have been observed [20].
Recent transcriptomic studies [15,24] have provided first insights into the molecular biology of different developmental stages of O. dentatum, leading to the characterization of a range of structural and functional molecules. In some studies of nematodes [25][26][27][28][29][30][31], various hydrolases (including cysteine, metallo-, serine and aspartic proteases, and pyrophosphatases) have been identified as key molecules likely to play essential and specific roles in parasite development, cuticle collagen processing and/or moulting processes and thus represent potential drug targets for nematocides. Having available a practical in vitro culture system for O. dentatum [32,33] provides a unique opportunity to assess the effects of specific and selective inhibitors on protein expression in this parasite. In the present study, we selected inhibitors of the most relevant hydrolase groups involved in the development and moulting of parasitic nematodes [25][26][27]30,31,34,35], based on their ability to inhibit these processes without affecting viability and motility. We investigated the effects of these hydrolase inhibitors on the (phenotypic) proteomic profile of O. dentatum during its transition from the L3 to L4 stage using an integrated two-dimensional gel electrophoretic, mass spectrometric and bioinformatic approach, taking advantage of all of the currently available transcriptomic datasets for this parasitic nematode.

Ethics Statement
Experiments were conducted in accordance with the Austrian Animal Welfare Regulations and approved (permit GZ 68.205/ 103-II/10b/2008) by the Animal Ethics Committee of the University of Veterinary Medicine Vienna and the Ministry of Science.

Parasite Material
A monospecific strain (OD-Hann) of O. dentatum was maintained routinely in experimentally infected pigs at the Institute of Parasitology, University of Veterinary Medicine Vienna. The faeces were collected to harvest L3s from coprocultures [23] and stored in distilled water at 11uC for a maximum of six months.
To test for the reversibility of inhibition of development, the larvae were first cultured for seven days in medium containing inhibitors at concentrations with a known inhibitory effect of $90%. Then, the medium was replaced with inhibitor-free medium following several washes, and the larvae maintained until day 14.

Protein Extraction
L3s of O. dentatum (n = 500,000), cultured in vitro for four days with or without the effective hydrolase inhibitors, were harvested, washed three times in phosphate-buffered saline (PBS; pH 7.4), snap frozen in liquid nitrogen and ground to fine powder with mortar and pestle pre-frozen in liquid nitrogen. Proteins were resuspended in ice-cold 10% (v/v) TCA in acetone at 220uC and precipitated for 90 min. After precipitation, proteins were centrifuged at 4uC at 17,500 g for 15 min. The supernatant was discarded, and the pellet washed twice with chilled (220uC) 100% acetone and centrifuged to remove any traces of TCA. Finally, acetone was removed by evaporation at 22uC. Proteins were resuspended overnight in 250-500 ml solubilisation buffer [7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPS; Carl Roth) and 30 mM Tris-Base (Carl Roth)] at 22uC. Insoluble material was removed by centrifugation at 241,800 g at 20uC for 30 min. The supernatant was collected and the total protein content of each sample determined [38] using bovine serum albumin (BSA) as a standard.

Two-dimensional Electrophoresis
For separation in the first dimension, an aliquot of 120 mg of parasite protein was diluted in a final volume of 300 ml of rehydration solution [8 M urea, 2% (w/v) CHAPS, 12.7 mM dithiothreitol (DTT), 2% immobilized pH gradient (IPG) buffer 3-10 non-linear (GE Healthcare Life Sciences, Freiburg, Germany)] and used to rehydrate 13 cm IPG strips with a non-linear gradient pH 3-10 (Immobiline, GE Healthcare Life Sciences) for 18 h at 22-24uC. Isoelectric focusing (IEF) was carried out (300 V ascending to 3,500 V for 90 min, followed by 3,500 V for 18 h) using a Multiphor II electrophoresis chamber (GE Healthcare Life Sciences). After IEF, the IPG strips were incubated at 22uC for 20 min in an equilibration buffer (6 M urea, 2% (w/v) sodium dodecyl-sulphate (SDS), 30% (v/v) glycerol, 150 mM Tris-HCl, pH 8.8, 64 mM DTT). A second equilibration was performed in the same buffer, except that DTT was replaced by 135 mM iodoacetamide. The IPG strips were then washed with deionized water. In the second dimension, SDS-PAGE was performed in vertical slab gels (1.5 mm; T = 12%, C = 2.6%, 1.5 M Tris-HCl, pH 8.8, 10% SDS) under reducing conditions at 15 mA for 15 min, followed by 25 mA in a Protean II electrophoresis chamber (Bio-Rad Laboratories, Hercules, USA). Gels were stained with silver [39]. Two technical replicates and one biological replicate were run for each treatment. Gels were scanned using the program ImageMaster TM 2D platinum v.7.0 (GE Healthcare Life Sciences). Proteins that were significantly (p#0.05) differentially expressed were selected for further analyses by mass spectrometry combined with bioinformatics.

Sample Preparation for Mass Spectrometric Analysis
Protein spots were excised manually from silver-stained twodimensional electrophoretic (2-DE) gels. 'Negative' control spots were also excised from blank regions of each gel and processed in parallel. Spots were washed, reduced with DTT and alkylated with iodoacetamide. In-gel digestion with trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega, Madison, USA) was carried out [40]. In order to enhance peptide ionisation and the sensitivity of mass spectrometry, dried peptides were de-salted using Zip-Tips mC 18 (Millipore, Billerica, USA) according to the manufacturer's instructions.

Spotting and Mass Spectrometry
Peptides were annotated using a Matrix Assisted Laser Desorption Ionisation Tandem Time-of-Flight (MALDI-TOF/ TOF) mass spectrometer (Ultraflex II; Bruker Daltonics, Bremen, Germany) in MS and MS/MS modes. De-salted peptides (0.5 ml) were spotted on to a disposable AnchorChip MALDI target plate pre-spotted with a-cyano-4-hydroxycinnamic acid (PAC target; Bruker Daltonics). MS spectral data were acquired from the samples and a MS/MS list was generated for further analysis based on the most intense ions present (trypsin and major keratin ions were excluded). Spectra processing and peak annotation were carried out using FlexAnalysis and Biotools (Bruker Daltonics).

Data Analysis
All peptide mass fingerprint (PMF) data were refined using MS/ MS data. Protein sequence matches with a significant MS/MS score (p,0.05) were used for further analysis. Ion mass data and amino acid sequences characterized from processed spectra were compared with data in public eukaryotic databases, including an inferred protein database of O. dentatum (www.nematode.net), the UniProt database (http://www.uniprot.org/) and the National Center for Biotechnology Information (NCBI) non-redundant (nr) database (http://www.ncbi.nlm.nih.gov) using the following search parameters: global modifications carbamido-methylation on cysteine; variable modifications oxidation on methionine; peptide mass tolerance = 100 ppm; MS/MS tolerance = 1 Da; one missed cleavage allowed. The programs Proteinscape (v.2.1; Bruker Daltonics) and MASCOT (www.matrixscience.com) were used for data management and analyses. Proteins identified by MS were annotated based on their closest homologue (BLASTp, Evalue cut-off #1610 25 ) in the UniProt database and Gene Ontology (GO). Subsequent GO analyses were performed using the AmiGO BLAST tool [41]. Protein sequences were also mapped to known biological pathways (KOBAS algorithm) [42] using BLASTp (E-value cut-off #10 25 ) based on sequence homology to molecules in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [43].

Statistical Analysis
Statistical calculations were performed using SPSS (v.20.0) for windows. To test for significant differences in the percentage of L4 development, the development-inhibited groups and the controls were compared using the Kruskal-Wallis test and Mann-Whitney U-test.

Testing of Inhibitors
We assessed the inhibitory effects of each compound on the development from the exsheathed L3 to the L4 stage of O. dentatum ( Figure 1) by culturing larvae for 14 days in medium containing seven different hydrolase inhibitors (Table 1). A significant inhibitory effect (p#0.01) was detected for four of the seven hydrolase inhibitors tested. By adding iodoacetamide (125 mM), the development of L3s to L4s was inhibited by 93.965.1% compared with untreated controls. The addition of 1.4 mM EPNP resulted in 93.065.6% inhibition. Complete inhibition of nematode development was achieved by adding 12.5 mM o-phenanthroline to the in vitro cultures. Sodium fluoride (5 mM) resulted in a 90.863.2% inhibition. No significant inhibitory effect was observed for the aspartic protease inhibitor pepstatin A or either serine protease inhibitors (AEBSF and aprotinin) tested (data not shown). The addition of higher concentrations of each of the four inhibitory compounds did not result in an increased inhibitory effect, but did lead to a higher mortality of larvae. The reversibility of the inhibitory effect of each inhibitor was assessed. The removal of iodoacetamide, EPNP, o-phenanthroline and sodium fluoride resulted in 90.5%, 85.6%, 89.5%, and 106.3%, respectively, of L3s developing through to L4s with respect to the controls.

Two-dimensional Gel Electrophoretic Analysis
We explored the induced changes in the O. dentatum larval proteome, following treatment with inhibitors and associated inhibition of development. For this purpose, protein extracts were prepared from whole worms for 2-DE; the average recovery was 7.4 mg protein per 1,000 larvae. The results of 2-DE showed that the protein expression profile varied between the developmentinhibited and control cultures. Protein spots were distributed throughout the whole pH range (3-10), but more spots were in the neutral and acidic pH ranges. The proteins differentially expressed between the development-inhibited and the control larvae were displayed and then annotated.

Annotation of Proteins
The significantly (p#0.05) differentially expressed spots were excised from the 2-DE gels, digested with trypsin and then analysed by MALDI-TOF-MS/MS. The most intense spot (no. 13) representing an actin isoform was present on all gels, was constitutively expressed and was thus selected as reference spot. This approach allowed us to reproducibly resolve 29 spots representing 22 different proteins ( Figure 2, Tables 2 and 3), of which 21 (except spots 2 and 3) shared sequence homology to individual conceptually translated proteins of O. dentatum. Spots 2 and 3 had significant amino acid sequence homology to propionyl-CoA carboxylase alpha-chain of C. elegans (Table 2). Both spots independently yielded a significant MASCOT MS/MS score of 166.4 (spot 2) and 148.5 (spot 3), inferring that these results are highly reliable.

KEGG Analysis
To further categorize key differentially expressed proteins and identify specific pathways in which these proteins are predicted to be involved, homologues to curated proteins in the KEGG database were identified (see Tables S3 and S4). In depth analysis revealed 'cellular processes', 'genetic information processing' and 'metabolism' as the three main protein classification terms (Table  S3A). The class 'cellular processes', including the subclass 'cytoskeleton proteins' comprised the structural protein actin     The KEGG pathway classification included the biological pathway terms 'cellular processes' (n = 4), 'environmental information processing' (n = 2), 'genetic information processing' (n = 3), 'metabolism' (n = 5) and 'organismal systems' (n = 5) (Table S3B)

Gene Ontology (GO) Analysis
In order to better understand the biological processes and molecular functions in which the identified proteins are inferred to be involved, GO analyses were performed using the AmiGO BLAST tool. Of the 22 proteins, 19 (86.4%) could be assigned to GO classifications associated with biological processes and 18 proteins (81.8%) were assigned to certain molecular functions. Only GO annotations found in related nematode species (particularly C. elegans) were taken into account using a cut-off of p#2610 225 (see Tables S5 and S6). GO classification analysis revealed associations of the proteins identified to a range of biological processes (Table S5A), including 'reproduction' (n = 8), 'metabolic process' (n = 10), 'cellular process' (n = 9), 'multicellular organismal growth' (n = 11), 'developmental process' (n = 11), 'growth' (n = 11), 'locomotion' (n = 8), 'response to stimulus' (n = 8), 'localization' (n = 4) and 'biological regulation' (n = 8). Most proteins assigned to the GO term 'metabolic process' related to 'cellular metabolic process' (representing 70% of the enzymes identified). No GO classifications for biological processes were found for three of the 22 proteins, namely DIM-1 (spots 20 and 21), aspartyl protease inhibitor (spot 25) and phosphatidylethanolamine binding protein (spot 27). 'binding' (n = 11) and 'catalytic activity' (n = 10) were the two main functional GO categories (Table S5B); additional GO categories included 'structural molecule activity' (n = 2), 'transporter activity' (n = 1) and 'antioxidant activity' (n = 1). In depth analysis revealed that the majority (63.6%) of proteins assigned to 'binding' are involved in 'protein binding' processes. No GO annotation for molecular functions was found for troponin T (spot 9), DIM-1 (spots 20 and 21), aspartyl protease inhibitor (spot 25) or phosphatidylethanol-amine binding protein (spot 27).

Discussion
Drug resistance represents a major concern in the control of parasitic nematodes [12][13][14]44]. Therefore, much research is directed towards the development of new agents in the treatment of nematode infections. Proteins involved in fundamental developmental processes in nematodes represent promising targets for the design of new and selective interventions. Hence, this study aimed at identifying and characterizing proteins involved in the larval development of O. dentatum, a model organism representative of parasitic nematodes of major socioeconomic impact. We applied an integrative approach combining in vitro drug testing with proteomic and bioinformatic analyses to provide first insights into larval development in O. dentatum.
To generate a development-inhibited phenotype for O. dentatum, hydrolase inhibitors were selected based on their ability to impede the moulting and development of larvae without affecting their viability and motility. Enzyme inhibitors were tested to cover the most relevant hydrolase classes known to be involved in the highly sophisticated moulting process in various nematode species [25][26][27]30,31,34,35]. Significant inhibition of moulting and development has been described previously in a range of nematodes and could be confirmed in O. dentatum for the three inhibitors, ophenanthroline [27,34,45], sodium fluoride [25,26] and iodoacetamide [28]. The inhibitory effect of EPNP had not been tested before on the development of nematode larvae. In malaria parasites, however, this protease inhibitor disintegrates gametocyte membranes [46], and it has been used frequently as an inhibitor of peptidases of the A1 family [47]. This is particularly interesting, since the other aspartic protease inhibitor tested, pepstatin A, was neither effective in our model nor in related nematodes [27,34], probably due to its limited half-life.
Since the development of nematode larvae and the sophisticated moulting process require a series of structural, biochemical, metabolic and physiological changes, we assumed that the proteins essential for fundamental developmental processes in development-inhibited larvae are less abundantly expressed compared with those of uninhibited controls in which normal development occurred. Thus, protein spots that were significantly differentially expressed between development-inhibited and control larvae were subjected to mass spectrometric and bioinformatic analyses. Using this approach, we identified 29 spots representing 22 different proteins. Several spots located at different positions in the same gel were inferred to be distinct protein isoforms or the same protein with varying post-translational modifications. Furthermore, 27 out of 29 identified spots were encoded in O. dentatum contigs derived from ESTs generated by 454 sequencing (FLX GS20 sequencer) [48] and analyzed using an advanced bioinformatic pipeline [49] for the interference of key biological processes [50], such as development and moulting. The O. dentatum contigs were further subjected to homology-based functional annotation. All O. dentatum amino acid sequences characterised had homologous proteins in other nematodes, including species from the orders Strongylida, Rhabditida, Spirurida, and Ascaridida.
The proteins identified and annotated included structural and cytoskeletal proteins (intermediate filament protein B, troponin T, actin, tropomyosin, DIM-1), enzymes involved in metabolism (propionyl-CoA carboxylase, phosphoenolpyruvate carboxy kinase GTP, 4-hydroxybutyrate coenzyme A transferase, fructose-bisphosphate aldolase, malate dehydrogenase, pyruvate dehydrogenase E1, 'probable voltage-dependent anion-selective channel' and peptidyl-prolyl cis-trans isomerase), stress-associated peptides (HSP-60, HSP-70, calreticulin and peroxiredoxin), regulatory and interacting peptides (LIM domain protein, RACK-1, 14-3-3 protein and phosphatidylethanol-amine binding protein) and one protease-inhibiting protein (aspartyl protease inhibitor). The expression of the vast majority (n = 19) of the 22 proteins identified was down-regulated in the development-inhibited group compared with controls, whereas three proteins (DIM-1, LIM domain protein and phosphatidylethanol-amine binding protein) were shown to be up-regulated. We functionally annotated 19 (86. KEGG analysis identified seven proteins (propionyl-CoA carboxylase, phosphoenolpyruvate carboxy kinase GTP, fructose-bisphosphate aldolase, malate dehydrogenase, pyruvate dehydrogenase E1, peroxiredoxin and peptidyl-prolyl cis-trans isomerase) as enzymes involved in amino acid, carbohydrate and/or energy metabolic pathways. Phosphoenolpyruvate carboxy kinase GTP, fructose-bisphosphate aldolase, malate dehydrogenase and pyruvate dehydrogenase E1 are known to be involved in the glucose associated energy metabolic pathways in nematodes [51][52][53][54], whereas propionyl-CoA carboxylase is involved in fatty acid synthesis [55]. The 'probable voltage-dependent anionselective channel' protein likely plays pivotal roles in the parasite's metabolisms, since it is involved in calcium signalling pathways and ion transport [56]. Peroxiredoxins are a ubiquitous family of antioxidant proteins and contribute to the oxidative-stress response of multicellular organisms. In nematodes, antioxidant enzymes and stress-associated proteins are central to the protection against oxygen radicals [57]. The expression of all enzymes and all stress-associated proteins identified in our study was downregulated in development-inhibited larvae compared with larvae exhibiting physiological moulting and growth patterns. We hypothesize that the moulting process is accompanied by increased energy consumption as well as augmented metabolic and physiological activities. These changes were particularly evident when the protein profiles of larvae with physiological developmental patterns were compared with those whose moulting had been inhibited with one of the abovementioned hydrolase inhibitors, respectively.
Intermediate filament protein B, actin, troponin T, tropomyosin and DIM-1 are structural proteins ubiquitously expressed in all eukaryotic cells. They play essential roles in myofibril assembly and muscle contraction [58][59][60], and provide structure and stability [61][62][63]. In C. elegans (wild type strain of variation Bristol N2), the silencing of the gene encoding for tropomyosin, lev-11, results in embryonic lethality and worms that are paralyzed and showed abnormal muscle filament assembly [64,65]. The overexpression of DIM-1 in the development-inhibited larvae might be a response to the reduced expression of the other structural proteins identified. Thus, DIM-1 could be part of a regulatory system and might be over-expressed to ensure stability and structure in O. dentatum larvae. The multifaceted and crucial functions of the abovementioned proteins indicate their importance in fundamental developmental processes. Interestingly, the two structural proteins, namely intermediate filament protein B and tropomyosin are known to play pivotal roles in the moulting process of C. elegans by remodelling the attachments between the muscle, the hypodermis and the exoskeleton [16,18]. Additionally, the protein peptidyl-prolyl cis-trans isomerase is proposed to be involved in the moulting process in nematodes [66]. Besides their function as molecular chaperones and their involvement in stress responses and cell signalling [67][68][69], peptidyl-prolyl cis-trans isomerases of C. elegans are responsible for proper collagen biosynthesis [66] during the moulting process. These proteins represent a large multi-gene family which has also been identified in B. malayi as well as in other parasitic and free-living nematodes, including Onchocerca volvulus, H. contortus, Caenorhabditis briggsae and C. elegans [67,[70][71][72]. Interestingly, we observed a down-regulation of expression of the proteins intermediate filament protein B, tropomyosin and peptidyl-prolyl cis-trans isomerase, all homologues of which are involved in the moulting process in C. elegans. The moulting-and development-inhibited larvae expressed these three proteins to a lesser extent compared with the control larvae, which exhibited normal development and need the involvement of the moulting-associated proteins to accomplish the initiation and completion of their moulting. These three proteins, that are known to be involved in the moulting process, represent particularly promising candidate drug targets against parasitic nematodes, since they are essential for one of the most determining parts in the parasite's development, appear to be conserved across nematode species and are not present in the mammalian host (pig).
Two proteins, LIM domain protein and phosphatidylethanolamine binding protein (PEBP), are associated with ion and lipid binding, respectively. LIM domain proteins are essential in a variety of fundamental biological processes [73] by mediating protein-protein interactions [74]. PEBPs are highly conserved and function in lipid binding, serine protease inhibition [75] and the regulation of several signalling pathways, such as the MAP kinase [76] and the NF-kappaB [77] pathways. Members of the PEBP family include the Ov-16 antigen of O. volvulus [78] and the excretory-secretory antigen 26 of Toxocara canis [79]. In B. malayi, a homologue of this protein is highly abundant in excretory/ secretory products [72,80]. Since both of these proteins were overexpressed in the development-inhibited O. dentatum, we hypothesize that the addition of the hydrolase inhibitors might lead to an up-regulation of specific interactions and signalling pathways, for reasons that remain to be elucidated. These modified processes might be associated with an increased involvement of LIM domain protein and phosphatidylethanol-amine binding protein, in which they might fulfil various protein interaction and/or pathway regulatory functions.

Conclusion
The findings of this study support the hypothesis that, in development-inhibited larvae of O. dentatum, proteins essential for developmental processes are less abundantly expressed compared with the uninhibited controls. Twelve proteins putatively involved in various larval developmental processes as well as three proteins involved in the moulting process were identified to be downregulated in the moulting-and development-inhibited O. dentatum larvae. A better understanding of such key developmental processes paves the way to new interventions against parasitic nematodes by blocking or disrupting key biological pathways in them. Thus, this study provides a foundation on which future research on fundamental developmental processes in parasitic nematodes could be built. Furthermore, the present findings enable us to focus on specific proteins involved in the moulting cascade in O. dentatum. In future studies, the protein expression of exsheathed, ensheathed and moulting O. dentatum larvae could be compared using DIGE technologies [81] and supported by realtime PCR analysis of transcription. We aim to identify and characterize proteins involved in the moulting process and assess their merit as drug targets. The discovery of new drug targets is of particular importance, considering the increasing prevalence and distribution of drug resistance in parasitic nematodes of major socioeconomic importance.

Supporting Information
Table S1 Detailed list of protein homologues including the five best matches for each protein identification. The table lists the the ID number of the Oesophagostomum dentatum contig (Contig ID) and the individual protein identities (Protein identity), the five highest scoring putative proteins identified in homologybased search using the UniProt database (Protein homologue), the species in which these proteins occur (Species), the UniProt accession number of these proteins (UniProt ID), the score for sequence similarity (Score), the per cent of sequence identity (Identitiy [%]) and the expectation value (E-value). (PDF)   Gene Ontology (GO) assigments of proteins identified. The conceptually translated amino acid sequences of the identified Oesophagostomum dentatum contigs were applied to GO analyses using the AmiGO tool. Only annotations found in related nematode species were taken into account using a cut-off of p#2610 225 . (PDF)