Oesophagostomum dentatum — Potential as a model for genomic studies of strongylid nematodes, with biotechnological prospects

Biotechnology Advances 25 (2007) 281 – 293 www.elsevier.com/locate/biotechadv

Research review paper

Oesophagostomum dentatum — Potential as a model for genomic studies of strongylid nematodes, with biotechnological prospects Robin B. Gasser a,⁎, Pauline Cottee a , Alasdair J. Nisbet b , Bärbel Ruttkowski c , Shoba Ranganathan d , Anja Joachim c a

Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia Parasitology Division, Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, EH26 0PZ, Scotland Institute of Parasitology and Zoology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, A-1210 Vienna, Austria d Department of Chemistry and Biomolecular Sciences and Biotechnology Research Institute, Macquarie University, Sydney, New South Wales 2109, Australia b

c

Received 11 November 2006; received in revised form 17 January 2007; accepted 23 January 2007 Available online 7 February 2007

Abstract There are substantial gaps in the knowledge of the molecular processes of development and reproduction in parasitic nematodes, despite the fact that understanding such processes could lead to novel ways of treating and controlling parasitic diseases, through blocking or disrupting key biological pathways. Biotechnological advances through large-scale sequencing projects, approaches for the analysis of differential gene and protein expression and functional genomics (e.g., double-stranded RNA interference) now provide opportunities to investigate the molecular basis of developmental processes in some parasitic nematodes. The porcine nodule worm, Oesophagostomum dentatum (order Strongylida), may provide a platform for testing the function of genes from this and related nematodes, given that this species can be grown and maintained in culture in vitro for periods longer than other nematodes of the same order. In this article, we review relevant biological, biochemical and molecular biological and genomic information about O. dentatum and propose that the O. dentatum — pig system provides an attractive model for exploring molecular developmental and reproductive processes in strongylid nematodes, leading toward new intervention methods and biotechnological outcomes. © 2007 Elsevier Inc. All rights reserved. Keywords: Oesophagostomum dentatum (Nematoda: Strongylida); Parasitic nematodes; Genomics; Development; Reproduction; Caenorhabditis elegans

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . O. dentatum — the parasite in vivo and in vitro . . . . . . 2.1. Biology and pathogenesis . . . . . . . . . . . . . . 2.2. Maintenance of defined strains and in vitro cultivation

⁎ Corresponding author. Tel.: +61 3 97312000; fax: +61 3 97312366. E-mail address: [email protected] (R.B. Gasser). 0734-9750/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2007.01.008

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Biochemical aspects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recent insights into the molecular biology of O. dentatum via genomic approaches . 4.1. Application of differential display . . . . . . . . . . . . . . . . . . . . . . . 4.2. Application of suppressive–subtractive hybridization (SSH). . . . . . . . . . 5. Scope for assessing gene function . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Parasitic nematodes are of major socio-economic importance in plants and animals. For instance, more than 1.3 billion people are infected with Ancylostoma duodenale and/or Necator americanus, blood-feeding hookworms of the small intestine (see Crompton, 2001), causing serious effects on human health, particularly in children. Similarly, parasitic nematodes of livestock also cause substantial economic losses due to subclinical and clinical diseases, with billions of dollars spent annually on the control of gastro-intestinal worms (Newton and Munn, 1999). In addition to the socio-economic impact caused by parasitic nematodes, there is a significant problem with resistance in them against all three classes of (nematocidal) compounds used to treat the diseases they cause (Sangster, 1999; Sangster and Gill, 1999; Wolstenholme et al., 2004; Bartley et al., 2006). Therefore, there is an urgent need to discover new compounds to control these parasites. A possible avenue is via an improved understanding of the fundamental aspects of parasite development and reproduction. In contrast to the free-living nematode Caenorhabditis elegans, very little is known about the molecular basis of development, sexual differentiation, maturation and behaviour in parasitic nematodes (Boag et al., 2001; Nisbet et al., 2004). Since the genome sequence of C. elegans was published in 1998 (The C. elegans Sequencing Consortium, 1998), many facets of this nematode's biology have been investigated. For example, microarray analyses have been used to examine developmental and gender-enriched gene expression (Jiang et al., 2001; Kim et al., 2001); yeast two-hybrid screens have been performed to examine protein– protein interactions (Li et al., 2004), and the function of ∼ 96% of the C. elegans genes has been assessed by double-stranded RNA interference (dsRNAi; Fire et al., 1998; Barstead, 2001; Kamath and Ahringer, 2003; Kamath et al., 2003; Simmer et al., 2003; Sugimoto, 2004; Sönnichsen et al., 2005). Sequencing of the small subunit of nuclear ribosomal RNA from various nematode taxa has suggested that parasitic nematodes,

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particularly those from the order Strongylida, are relatively closely related to C. elegans (see Blaxter et al., 1998). Also, many features between C. elegans and parasitic nematodes are conserved (e.g., basic body plan and moulting), suggesting that the molecular pathways utilized by parasites during development and reproduction may be similar to or the same as those in C. elegans (see Bürglin et al., 1998). Understanding the pathways governing basic nematode biology could have significant implications for the development of novel intervention strategies. In spite of the advances in genomic technologies (Gasser and Newton, 2000; Hashmi et al., 2001; Brooks and Isaac, 2002; Boyle and Yoshino, 2003; Cowman and Crabb, 2003; Aboobaker and Blaxter, 2004; Knox, 2004; Foster et al., 2005) and the study of C. elegans, there is a paucity of information on developmentally regulated and gender-specific genes in parasitic nematodes of animals, particularly those of the order Strongylida. Reports indicate that the porcine nodule worm, Oesophagostomum dentatum, is considered to provide a unique model system for studying fundamental aspects of molecular biology in bursate nematodes (Christensen et al., 1995, 1997a,b; Christensen, 1997). Several characteristics, including its short life cycle, mild pathological effects and the ability to maintain worms in vitro for weeks through several moults, suggest that O. dentatum is a valuable model system to investigate reproductive and developmental processes. The present article reviews the state of knowledge about O. dentatum and discusses the potential of this model for genomic studies, with a view toward biotechnological outcomes. 2. O. dentatum — the parasite in vivo and in vitro 2.1. Biology and pathogenesis The life cycle of O. dentatum is simple and direct (Fig. 1). Unembryonated eggs pass out in the faeces and develop into free-living, first- and second-stage larvae (L1s and L2s, respectively). Feeding on nutrients and

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Fig. 1. The life cycle of Oesophagostomum dentatum. Unembryonated eggs produced by adult (A) O. dentatum females are excreted in the faeces into the environment and develop into free-living first- and second-stage larvae (L1s and L2s, respectively). They then develop into the third-stage larvae (L3s; protected within a cuticular sheath), which are infective to the porcine host. Following ingestion, the L3s exsheath in the small intestines of the pig en route to the large intestine, where they burrow into the intestinal wall. Within the submucosa, the L3s moult to fourth-stage larvae (L4s) and stimulate an immune response, leading to their encapsulation. Following the transition to the L4, the larvae emerge from the mucosa into the lumen of the gut where they mature (after a cuticular moult) to adult male and female worms, which mate to produce offspring. The prepatent period (time from the ingestion of infective L3s by the host to the appearance of O. dentatum eggs in the faeces) is ∼ 3 weeks. Life cycle modified from original illustration by W. Hamilton CMI.

bacteria in the faecal matter, they develop into the infective third-stage larvae (L3s) protected within a cuticular sheath. These move out of the faeces into the surrounding environment (pasture), where the porcine host ingests them. The optimal temperature for larval development is 15–20 °C, but successful development has been observed at temperatures as low as 10 °C or as high as 25 °C (e.g., Fossing et al., 1995). 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 third-stage larvae moult to fourthstage larvae (L4s) (McCracken and Ross, 1970) and evoke an immune response which results in the encapsulation of the larvae in a raised nodular lesion, made up of aggregations of neutrophils and eosinophils

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(Stockdale, 1970). Following the transition to the L4, the larvae emerge from the mucosa after 6–17 days. The parasite undergoes another cuticular moult, subsequently maturing to an adult. The prepatent period is ∼ 17– 20 days (Talvik et al., 1997), although longer periods have been observed (Spindler, 1933; Kotlán, 1948). Interestingly, population density can influence the development of the parasite. Studies (Christensen et al., 1995, 1996a) have shown that O. dentatum undergoes a slower rate of development and has a longer prepatent period to produce fewer eggs, when raised in pigs orally inoculated with high numbers (200,000) of L3s compared with those inoculated with lower numbers. In addition to these effects, O. dentatum adults from pigs infected with high numbers of L3s have been observed to be morphologically stunted, measuring only 60% of the length of those derived from pigs with a low intensity of infection (Christensen et al., 1997a). This phenomenon has also been reported for Oesophagostomum quadrispinulatum of pigs (see Kendall et al., 1977). To date; it is unclear whether this difference relates to parasite or host factors or both. Within the subfamily Oesophagostominae, members such as Oesophagostomum radiatum (a pathogen of ruminants) can cause severe nodular lesions in the intestine of their hosts (Stewart and Gasbarre, 1989). The pathological effects resulting from an O. dentatum infection are relatively mild and somewhat less severe than those caused by infection with O. quadrispinulatum (Christensen et al., 1997b). The pathogenic stages are the histotropic larvae, particularly during invasion and evasion. Local inflammation, haemorrhage and oedema, followed by granuloma formation, resulting in the characteristic nodules are present during the histotropic development (Spindler, 1933; Kotlán, 1948; McCracken and Ross, 1970; Scheuermann, 1985; Häussler, 1996). During larval development, the nodule increases in size and central necrosis is seen. The parasite is surrounded by an eosinophilic membrane (Häussler, 1996) which is destroyed when the parasite leaves the intestinal wall. Thereafter, the lesions in the intestinal wall resolve, although some histotropic L3s may remain encysted (Shorb, 1959; Shorb and Shalkop, 1959; Häussler, 1996). The effects of an O. dentatum infection may be typified by a reduction in appetite in the pig, and reduced growth rate and feed conversion efficiency during the period of nodule formation (Stewart and Gasbarre, 1989). Necrotic enteritis associated with diarrhoea following infection with high doses (e.g., ≥ 200,000 infective larvae; Stockdale, 1970) may also be linked to secondary bacterial infections (Stewart

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et al., 1983). Although clinical signs are frequently absent during the infection, the large intestines are clearly impaired in their function; chloride flux and transepithelial conductivity are increased during histotropic development, and the sensitivity to secretagogic agents, such as prostaglandins or histamine, is increased during the invasive stage (2 days after infection) and decreases during histotropic development (LeonardMarek and Daugschies, 1997). In response to the infection, the crypts of Lieberkühn become elongated (Jensen and Christensen, 1997). The immune response elicited by the host to an O. dentatum challenge infection is limited. During a secondary infection, Nickel and Haupt (1969) interpreted intestinal inflammation which hampered larval development as a sign of an immune response. However, this may be a non-specific reaction, since ʻtrickle' infection with high doses also leads to a reduced worm burden (Christensen et al., 1997a), whereas pigs appear to be fully susceptible to reinfection following anthelmintic treatment (Roepstorff et al., 1996). Adaptive systemic immune responses are not consistently found; specific antibodies can be detected (Larsen et al., 1997; Joachim et al., 1998, 1999a), but antibody-independent cell-mediated cytotoxicity in response to antigens from the parasite is limited (Schuberth et al., 2000). The cellular composition of the granulomata surrounding the developing larvae was described by Häussler (1996). The cell types involved (mainly macrophages and neutrophils as well as CD4+-T-lymphocytes) appear not to contribute to the protection of the host, since the cell types central to parasite expulsion (mast cells and eosinophils) are absent. There is no agerelated susceptibility to infection (Larsen et al., 1997). Depression of peripheral lymphocyte proliferation, as described for O. radiatum in cattle (Gasbarre et al., 1985), is not observed in pigs with O. dentatum infection (Freigofas et al., 2001). Whatever the nature of the immunity against Oesophagostomum, it seems to develop mainly against the larval stages (Kendall et al., 1977) and is reported to be stronger against O. quadrispinulatum than O. dentatum (see Talvik et al., 1998). 2.2. Maintenance of defined strains and in vitro cultivation The free-living stages of Oesophagostomum can be cultured from the egg to the infectious L3 using a standard “copro-culture” technique for strongylids (Honer, 1967). The L3 stage carries an uvea (sheath), which is the remainder of the L2 cuticle, and thus cannot actively feed. Exsheathment (ecdysis) can be achieved either by applying pH changes, mimicking the transition

through the gut, or by treatment (under basic conditions) with oxygen or sodium hypochlorite (Joachim et al., 2005). The latter has the advantage of simultaneously disinfecting the cuticular surface of the nematode. Ecdysis marks the transition from the free-living to the parasitic stage. Exsheathed L3s can be cultivated in vitro in a complex, axenic, protein-rich medium under microaerobic conditions (Daugschies and Watzel, 1999) for several weeks to L4s, a proportion of which develops through to sexually differentiated pre-adults. The development in vitro usually takes longer than in vivo and the larvae are smaller, but they are fully capable of developing into adults when transplanted rectally into pigs (Joachim et al., 2001a). Intestinal stages (L4 to adults) can be recovered after an experimental infection of pigs via the agar-gel migration technique (Slotved et al., 1996) and maintained in culture for up to 2 weeks (Joachim et al., 2001a). Since O. dentatum resides in the large intestines, non-surgical, rectal transplantation into pigs can be readily achieved for experimental purposes (Christensen et al., 1996b). The short-term maintenance (days) of any parasite stage recovered from the pig intestines in serum-free culture medium is possible (Joachim et al., 2001a). Such cultivation techniques can be used for a whole range of bioassays on viable worms, such as growth and development (moult) or motility (e.g., for population biological or biochemical investigations; Watzel, 1997; Daugschies and Ruttkowski, 1998; Joachim et al., 2005), but they could have many other applications. For example, gene silencing via RNAi (Fire et al., 1998) would be a valuable tool for testing gene function(s) in cultivated larval or adult stages of O. dentatum, provided this species has the molecular machinery required. 3. Biochemical aspects The characterization of stage-specifically regulated molecules of O. dentatum commenced with the work on eicosanoids (Daugschies, 1995, 1996; Daugschies and Ruttkowski, 1998). These bioactive lipids are present in a range of invertebrates, and they can be detected in homogenates and as excreted/secreted products (ESP) from the L3 and L4 in stage-specific compositions. Since eicosanoids (amongst other molecules) regulate gastric function and have immunomodulatory effects, it was proposed that the presence of parasite prostaglandins and leukotrienes in host tissue could aid parasite invasion and establishment (Daugschies and Joachim, 2000). In the absence of the host, however, eicosanoids seem to display intrinsic functions in the nematode itself, since the inhibition of their production leads to

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impaired ecdysis, motility and growth in vitro (Daugschies, 1995, 1996). In Western blot analyses, two enzymes, lipoxygenases (LOX) and prostaglandin Hsynthase (syn. cyclooxygenase, COX), responsible for the conversion of precursors to biologically active forms could be detected in protein preparations from O. dentatum, the latter enzyme having a stage-specific expression profile (Joachim et al., 2001b). Very little is known about lipids in nematodes (Wenk, 2006). Gas chromatographic separation and analysis of esterified fatty acids (FA) from homogenates of L3s, L4s and adult males and females of O. dentatum and O. quadrispinulatum demonstrated a relative increase in long-chain FA during larval development. Cultured larvae remove arachidonic acid, the main substrate for eicosanoid production, from the medium (Joachim et al., 2000). The FA profiles differ among developmental stages of these two parasites, whereas there is no significant difference between adult males and females. The biological significance of the changes in the FA composition among different stages remains to be elucidated. Several studies of proteins and glycans of Oesophagostomum have also demonstrated species- and stagespecific profiles for different fractions of worm proteins analysed by one-dimensional sodium dodecyl-sulphate polyacrylamide electrophoresis (SDS-PAGE) and Western blot. While these studies have revealed differences in protein expression during development (and in parasites cultured in vitro), such proteins have not yet been characterized in detail (Joachim et al., 1998, 1999a,b, 2001c). 4. Recent insights into the molecular biology of O. dentatum via genomic approaches 4.1. Application of differential display Differential display (originally described by Liang and Pardee, 1992) is a direct polymerase chain reaction (PCR)-based technique employed for isolating differentially transcribed genes, such as stage- and/or sex-specific genes. A modified differential display approach (i.e. using ‘random’ oligonucleotide primers) was employed for the isolation and characterization of a small subset of genderenriched genes from O. dentatum (see Boag et al., 2000), from which two male-enriched genes were investigated in detail (designated Od-mcrp and Od-mpp-1). The gene Od-mcrp (O. dentatum male cysteine rich protein) shared a high level of similarity to a number of predicted cysteine rich proteins in C. elegans and to trypsin-like serine protease inhibitors (TILs) from other

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organisms (Boag et al., 2002). The putative homologue described in O. dentatum is unusual in that it contains two active site domains within its molecular structure, which are believed to interact with separate proteases (Boag et al., 2002). While other examples of protease inhibitors with dual functional sites are not known for the Nematoda, they have been described in other organisms. For example, Grutter et al. (1988) showed (using X-ray crystallography) that the two domains within the human mucous proteinase inhibitor were able to interact with different proteases. One domain was shown to possess elastase and chymotrypsin binding properties, whereas the other domain is believed to contain anti-trypsin activity. The precise role of OdMCRP has yet to be elucidated in O. dentatum. Interestingly, the C. elegans homologues are sperm-enriched (Reinke et al., 2000; Jiang et al., 2001), suggesting a role in spermatogenesis and/or sperm function in this nematode. Given that proteases and protease inhibitors play important roles in spermatogenesis and sperm function in other organisms (see Monsees et al., 1997; Uhrin et al., 2000), these findings suggest that the function of homologues, such as Od-MCRP, may also be linked to the sexual biology of the nematode. Boag et al. (2003) also characterized another genderenriched gene of O. dentatum. The gene, Od-mpp-1, is a male-enriched putative homologue of a serine/threonine phosphatase and shares significant conservation to two predicted genes in C. elegans of unknown function. In order to understand the function of Od-mpp-1, the putative C. elegans gene homologues (W09C3.6 and T03F1) were studied (Boag et al., 2003). Silencing of the C. elegans predicted serine/threonine phosphatases resulted in a 30–40% reduction in the number of progeny produced (Boag et al., 2003), suggesting that, in absence of this enzyme, fertilization occurred at a reduced level. Microarray profiling experiments in C. elegans had also demonstrated sperm-enriched expression for these predicted genes (Reinke et al., 2000; Hanazawa et al., 2001; Jiang et al., 2001), further supporting a role for these predicted phosphatases in sperm production and/or spermatogenesis. However, the precise effect of the silencing of these genes on the sperm remains to be determined. The testis-specific expression of protein kinases and phosphatases has been observed in both vertebrates and invertebrates (Smith et al., 1996; Armstrong et al., 1998; Herrmann et al., 1998). Analysis of the 5'-UTR of the Od-mpp-1 gene showed that two putative GATA transcription factor-binding sites were found within the first 100 nucleotides of the gene, prior to the initiation codon for methionine (Boag et al., 2003).

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Interestingly, these transcription factor-binding sites are also found preceding the initiation codon of other gender- and spatially-regulated nematode genes, such as the major sperm proteins (Klass et al., 1988; Scott et al., 1989) and the vitellogenins (see MacMorris et al., 1992,

1994). This information suggests that the presence of this binding motif may be a common feature of genderand spatially-regulated genes. To further understand the role of the Od-mpp1 homologues, Boag et al. (2003) localized the expression

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of the predicted phosphatases in transgenic C. elegans. Expression of these promoter:reporter constructs was not only seen within the germ-line of the transgenic animals but also in the neuronal or neuronal-associated cells. Specifically, the expression of the predicted phosphatase W09C3.9 promoter construct was detected in the male tail, potentially associated with either the HOA or HOB neurons. LOV-1, a protein abundant in serine and threonine residues, is expressed in a range of male neurons, including the HOA and HOB and is required for male-specific behaviours, for example, the determination of vulva location (Barr and Sternberg, 1999). Although the functions of the C. elegans or O. dentatum proteins have not been fully elucidated, it is possible that they are involved in the signal transduction pathways of chemosensation and/or mechanosensation of the male nematode during mating. 4.2. Application of suppressive–subtractive hybridization (SSH) In spite of its usefulness, the method of differential display has limitations. It is relatively laborious and time-consuming methodology to carry out, and that frequently “false positive” display products are frequently isolated and subsequently cloned and sequenced (e.g., Liao and Freedman, 2002; Stein and Liang, 2002; Boag et al., 2000). The technique of suppressive subtractive hybridization (SSH) (Diatchenko et al., 1996) overcomes the limitations of differential display. This PCR-based approach allows the effective removal of common (e.g., ‘house-keeping’) genes from the RNA population of interest prior to library construction and also has the advantage that rare transcripts are amplified efficiently (i.e., are enriched), which is not the case for the conventional EST sequencing approach. In a recent study, Cottee et al. (2006a) constructed by SSH archives of female- and male-enriched ESTs from adult stages of O. dentatum, and conducted bioinformatic and microarray analyses of a subset of ESTs from these archives as a foundation for future work on the functional aspects of

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genes differentially transcribed between the sexes of this nematode (cf. Fig. 2, steps 1–4). Of the 516 unique ESTs analysed, gender-enriched transcription was confirmed for 76% of the ESTs. These findings, together with a similar study undertaken in the ovine parasite Trichostrongylus vitrinus (using a slightly different methodology; Nisbet and Gasser, 2004), have shown that SSH is a very effective tool for the isolation of genes specific to gender and/or developmental stages. From the male-enriched EST archive, Cottee et al. (2004) isolated and subsequently characterized a nematode-specific molecule involved in sperm motility, the major sperm protein (MSP). The findings have shown that the homologue isolated from O. dentatum was conserved in relation to msp genes (and gene products) from other species of nematodes, and shared a similar gender- and stage-specific transcriptional profile to msp genes of C. elegans. The role of MSP has been shown to be in the movement of nematode sperm (see Roberts and Stewart, 1995, 2000; Theriot, 1996; Bottino et al., 2002) and the resumption of oocyte maturation and ovulation (following developmental arrest) in C. elegans hermaphrodites (Miller et al., 2001). However, it is not known how a cytoplasmic protein (without a signal sequence) involved in the sperm cytoskeleton is released from spermatozoa to bind to the developmentally arrested oocytes (Miller et al., 2001; Villeneuve, 2001; Kuwabara, 2003). A recent study (Kosinski et al., 2005) has shown that C. elegans MSP is released from spermatozoa through a budding process. The MSP molecules are encapsulated between two membranes in a special vesicle (called MSP-vesicle), which buds off the motile spermatozoa. The membrane of the vesicle disintegrates (through an unknown mechanism), releasing MSP into the proximal gonad arm of the hermaphrodite. In its “free form”, MSP is able to bind to the receptor(s) on the surface of the egg and proximal sheath cell membranes to initiate the maturation and ovulation of the mature oocytes (Miller et al., 2003). While one receptor (VAB-1, variable abnormal morphology) for MSP has been identified in C. elegans

Fig. 2. Approach used or proposed for investigating molecules involved in reproduction and development in the porcine nodule worm, Oesophagostomum dentatum. (1) Suppressive–subtractive hybridization (SSH) archives enriched for complementary (c) DNAs predicted to be associated with development and/or reproduction are constructed for O. dentatum. (2) Clones from these archives are sequenced and subjected to cluster analysis to identify the representative subset of expressed sequence tags (ESTs) for detailed bioinformatic analyses against data available in current databases and functions predicted (cf. Nagaraj et al., 2007). In particular, WormBase (http://www.wormbase.org/) representing the free–living nematode Caenorhabditis elegans, provides relevant information regarding homologous molecules and phenotypes of this nematode following double-stranded RNA interference (dsRNAi). (3) Microarray-based profiling of transcription in different genders or developmental stages of O. dentatum is conducted, and ESTs with the highest similarity (at the protein level) to molecules of C. elegans are explored further. (4) For each of these EST, full-length genes are isolated, sequenced and expressed; the gene and gene product are then characterized, for example, utilizing a range of genetic, genomic, biochemical and/or proteomic approaches, and localized. (5) The proposal for the future is that RNAi is attempted in the parasite itself (using the soaking method; Timmons, 2006) and the phenotype evaluated after transplantation into the porcine host, in order to gain insights into gene function.

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(see Kuwabara, 2003; Miller et al., 2003), no VAB-1 homologues have yet been identified for parasitic nematodes, despite numerous EST/genomic sequencing projects being in progress. Experimental data suggest that the sperm–ovum signalling via MSP binding is similar between the nematodes Ascaris suum and C. elegans (see Miller et al., 2001). Together with the amino acid conservation of MSP across many nematode species, this information suggests that the receptor for MSP may also be conserved. A partial gene homologue of the VAB-1 receptor, which shares 71% amino acid identity over 91 amino acids to the C. elegans homologue, has been amplified from adult female O. dentatum cDNA (Cottee et al., unpublished data). Despite homologues of VAB-1 and MSP having been identified in O. dentatum, their precise relationship and proposed role(s) in sperm movement and the initiation of cell signalling remain to be characterized. This provides an exciting area of future research. The role of MSP in nematode sperm movement has been determined in A. suum (see Buttery et al., 2003). However, the function of MSP in other parasites remains to be elucidated. Gene silencing of msp by dsRNAi has been conducted in the plant parasitic nematodes Globodera pallida and Heterodera glycines (see Urwin et al., 2002). These experiments have shown a reduction (but not complete silencing) of the msp transcript(s) in treated worms, but there was no phenotypic effect on the development of the parasite or the sexual fate of the male. While the male worms remained viable following treatment, it is not known how a reduction of the msp transcript affects the reproductive potential of the male. Also, it is not known whether lower MSP levels in spermatozoa affect the rate at which sperm move and fertilize. To address the question as to whether the mobility of nematode sperm is affected following dsRNAi, two approaches could be used. Firstly, male worms could be treated with double-stranded RNA (dsRNA) and the worm dissected to collect sperm. The dynamics of the sperm cytoskeleton could be assessed using an in vitro culturing system similar to that described by Buttery et al. (2003), where the rate of extension of the sperm plasma membrane (caused by the construction of fibres of MSP molecules) in dsRNA-treated nematodes could be measured. Secondly, this question could also be addressed by using O. dentatum in an in vivo experiment in pigs. Using appropriate controls, male L4s could be treated with dsRNA to msp and then transplanted rectally (Christensen et al., 1996b) (with untreated females) into naïve pigs. The effect of reduced MSP levels on the reproductive index could be assessed by measuring the number of O. dentatum eggs in the faeces from ‘treated’

pigs compared with those in controls (with untreated worms) and by examining the reproductive tract of the worms after the autopsy of the pigs. Cottee et al. (2006b) also isolated and characterized a molecule (equally transcribed in both males and females) originating from the SSH archive which is predicted to play a role in nematode development. This molecule, a homologue of the ubiquitin-conjugating enzyme (UBC-2), had a high level (77–99%) of sequence identity and a similar expression profile to other UBCs, suggesting a similar function. Future work should be undertaken to understand the in vivo role of O. dentatum ubc-2, particularly as the gene silencing phenotypes of the closest gene homologue in C. elegans include “embryonic lethality”, “adult lethality” and “protruding vulva” (cf. Kamath et al., 2003; Simmer et al., 2003; Jones et al., 2004). Since dsRNAi technologies have not yet been tested in O. dentatum, the function of this conserved protein may need to be elucidated in a different organism. This may be achievable by examining the role of O. dentatum UBC-2 in the ubc-2 (equivalent) loss-of-function yeast (Saccharomyces cerevisiae). The O. dentatum ubc-2 gene product would be sub-cloned into a plasmid DNA construct (cf. Treier et al., 1992; Zhen et al., 1993) downstream of the S. cerevisiae ubc-4 (ubc-2 gene homologue) promoter. If the O. dentatum UBC-2 homologue were able to rescue the growth of mutagenized yeast cells at an elevated temperature, this result would suggest that it shared a similar structural conformation and could substitute the in vivo function of the yeast homologue. Similarly, the function of O. dentatum UBC-2 could also be elucidated in C. elegans. The C. elegans strain BC2020 (available from the Caenorhabditis Genetics Centre: http://www. cbs.umn.edu/CGC/) are mutagenized worms carrying a ubc-2 loss-of-function phenotype. Without this enzyme/ protein, C. elegans fails to develop beyond the early larval stages (Hodgkin, 1997). Using a strategy similar to that used for the determination of UBC-2 function in yeast, the O. dentatum ubc-2 open reading frame (ORF) could be cloned into a DNA plasmid between the C. elegans ubc-2 upstream promoter sequence and the downstream 3' untranslated sequence. This construct would then be co-injected (with an appropriate marker plasmid to select transgenic worms) into hermaphrodites of the BC2020 strain of C. elegans. First generation progeny would be allowed to self-fertilize. If the second-generation offspring developed normally to adulthood, the mutation could be considered as rescued, thus demonstrating that function of O. dentatum UBC-2 is similar to the homologue in C. elegans.

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5. Scope for assessing gene function While these most recent studies (Boag et al., 2000, 2002, 2003; Cottee et al., 2004, 2006a,b) have provided insights into selected molecular aspects of parasite development and/or reproduction in O. dentatum, we are still in the preliminary stages of understanding these processes. The function of the genes identified could be investigated, for example, using dsRNAi and/or transgenesis. To date, dsRNAi has been assessed in or adapted to multiple species of nematode (see Hussein et al., 2002; Urwin et al., 2002; Aboobaker and Blaxter, 2003; Lustigman et al., 2004; Issa et al., 2005; Kotze and Bagnall, 2006; Pfarr et al., 2006). For these species, gene silencing has been achieved by maintaining the parasite in a short-term in vitro culture and soaking the parasite in double-stranded RNA. Effective gene silencing has been assessed using approaches, such as reverse transcriptase PCR, Western blotting and biochemical assays. The success of gene silencing in some parasites indicates prospects for studying gene function in O. dentatum (cf. Fig. 2, step 5). The latter parasite has the major advantage over other bursate nematodes that it can be cultured in vitro for relatively long periods of time (weeks) (see Daugschies and Watzel, 1999; see Subsection 2.2). However, the application of dsRNAi to bursate nematodes is limited in that they cannot be cultured through all stages of their life cycle and that they do not reproduce in vitro. This means that the phenotypic effects of gene silencing on the first and subsequent generations cannot be observed (unless the treated parasites are transplanted back into their natural host). It remains to be determined how applicable dsRNAi is to O. dentatum, using a range of gene targets and encompassing different approaches for the delivery of dsRNA into the organism. Current information for Haemonchus contortus indicates that there is a considerable variation in the silencing effect using different approaches (Kotze and Bagnall, 2006; Geldhof et al., 2006, in press). Transgenesis offers another possible way of studying the in vivo role and expression of parasite genes. For parasitic nematodes, such as O. dentatum, the related nematode C. elegans may be utilized as a model to examine parasite gene function, expression and the consequences of aberrant gene expression. The technique involves the microinjection of a plasmid DNA construct or ballistic bombardment of plasmid DNA-coated gold particles into the gonad arm of the C. elegans hermaphrodite (Mello et al., 1991; Hashmi et al., 1995a,b; Davis et al., 1999). The plasmid DNA is incorporated as an

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extra-chromosomal array into the genome of the developing progeny. Each plasmid construct contains a ‘reporter’ molecule, such as the green fluorescent protein (GFP) that is used to identify transgenic progeny. Functional substitution is considered successful if the second-generation offspring survive a ‘mutant’ phenotype. For instance, Britton and Murray (2002) have used this approach to deduce the role of a putative H. contortus cathepsin L (Hc-cpl-1) homologue, rescuing the C. elegans embryonic lethal phenotype. Other authors have used transgenesis in C. elegans to examine the expression of genes from A. suum (see Davis et al., 1999), Strongyloides stercoralis (see Lok and Massey, 2002) and Brugia malayi (see Gomez-Escobar et al., 2002; Higazi et al., 2002). A disadvantage of using C. elegans as a model nematode for functional analyses is that many ESTs/genes of parasitic nematodes are specific to them (usually ∼30–40%; Nisbet and Gasser, 2004; Parkinson et al., 2004; Cottee et al., 2006a). The function of such molecules could be tested in parasitic nematodes, such as S. stercoralis (see Li et al., 2006) or Parastrongyloides trichosuri (see Grant et al., 2006), which are both capable of undertaking free-living developmental programs. However, for each species, mutagenized worms would have to be generated, screened to verify appropriate deletion mutants and maintained. Transgenesis may be possible in O. dentatum, since the parasite can be maintained in vitro for several weeks (Daugschies and Watzel, 1999). Gene expression in O. dentatum could be examined by injecting adult females with a DNA construct and rectally transferring the worms into parasite-naïve pigs. Eggs or L3 derived from faeces could be collected and examined for reporter gene activation (e.g., GFP fluorescence). Transgenic worms could then be cultured in vitro to the sexually differentiated pre-adult stages to determine where the gene of interest is expressed within the parasite. The determination of temporal and spatial gene regulation may provide some information regarding the putative function of genes represented by the ESTs isolated recently (Cottee et al., 2006a). 6. Concluding remarks Recent studies of the parasitic nematode O. dentatum have provided some new insights into the molecular biology of development and reproduction. Results have shown that more than half of the gender-enriched ESTs have a homologue in C. elegans, suggesting that the molecular basis of pathways utilized during nematode development and/or reproduction may be conserved (at least for these two species). However, in contrast, many

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ESTs had a gene homologue only in another parasite species or had no known gene homologue. The latter two groups of molecules are interesting, as they may represent genes that are specific to parasitism or to the species, thus being potentially important in the development of new intervention strategies. These molecules are much more challenging to work on, as their putative function cannot be predicted currently using traditional bioinformatic approaches. However, there is also considerable scope for exploring (relatively conserved) nematode-specific molecules using a combination of genomic and proteomic approaches, particularly now that whole genome sequences will become available in the near future for a range of strongylid nematodes, including O. dentatum (see http://www.genome.gov/ 11007951). Their study will also depend on in vitro culture techniques, such that dsRNAi can be applied to deduce gene function in the parasite itself. Therefore, O. dentatum may provide an attractive platform for testing the function of such genes from this and related (strongylid) nematodes, given that this species can be grown and maintained in culture in vitro for periods longer than other nematodes of the same order (cf. Eckert, 1997) and because the parasite can be rectally transplanted (e.g., from in vitro) into the host without the need for surgical intervention, allowing wellcontrolled in vivo molecular studies. With the emerging resistance to current anthelmintics, it is of paramount importance to understand the molecular biology of parasite development and reproduction, in order to be able to work toward new and effective approaches for parasite intervention. Acknowledgements Support from the Australian Research Council (LP0346983 and LP0667795), Genetic Technologies Limited, and Meat and Livestock Australia is gratefully acknowledged (RBG). PC was the recipient of a postgraduate scholarship from the University of Melbourne. AJN is funded by the Scottish Executive Environment and Rural Affairs Department. References Aboobaker AA, Blaxter ML. Use of RNA interference to investigate gene function in the human filarial nematode parasite Brugia malayi. Mol Biochem Parasitol 2003;129:41–51. Aboobaker AA, Blaxter ML. Functional genomics for parasitic nematodes and platyhelminths. Trends Parasitol 2004;20: 178–84. Armstrong CG, Dombradi V, Mann DJ, Cohen PT. Cloning of a novel testis specific protein serine/threonine phosphatase, PPN 58A, from

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