Key strongylid nematodes of animals — Impact of next-generation transcriptomics on systems biology and biotechnology

Biotechnology Advances 30 (2012) 469–488

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Research review paper

Key strongylid nematodes of animals — Impact of next-generation transcriptomics on systems biology and biotechnology Cinzia Cantacessi, Bronwyn E. Campbell, Robin B. Gasser ⁎ Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia

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Article history: Received 19 July 2011 Received in revised form 9 August 2011 Accepted 19 August 2011 Available online 26 August 2011 Keywords: Transcriptomics Strongylid nematodes Bioinformatics Vaccine targets Drug candidates Ancylostoma-secreted proteins Genomics Next-generation sequencing

a b s t r a c t The advent and integration of high-throughput 'omic technologies (e.g., genomics, transcriptomics, proteomics and metabolomics) are becoming instrumental to assist fundamental explorations of the systems biology of organisms. In particular, these technologies now provide unique opportunities for global, molecular investigations of parasites. For example, studies of the transcriptomes (all transcripts in an organism, tissue or cell) of different species and/or developmental stages of parasitic nematodes provide insights into aspects of gene expression, regulation and function, which is a major step to understanding their biology. The purpose of this article was to review salient aspects of the systematics and biology of selected species of parasitic nematodes (particularly key species of the order Strongylida) of socio-economic importance, to describe conventional and advanced sequencing technologies and bioinformatic tools for large-scale investigations of the transcriptomes of these parasites and to highlight the prospects and implications of these explorations for developing novel methods of parasite intervention. © 2011 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The phylum Nematoda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. A background on key strongylid nematodes of major socio-economic importance in animals — biology 2.1.1. Members of the superfamily Ancylostomatoidea . . . . . . . . . . . . . . . . . . . . . 2.1.2. Members of the superfamily Trichostrongyloidea . . . . . . . . . . . . . . . . . . . . . 2.1.3. Members of the superfamily Strongyloidea . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Aspects of immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Treatment strategies and vaccine research . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Genomic and bioinformatic approaches for investigating the transcriptomes of parasitic nematodes . . . . 3.1. Conventional techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Next-generation sequencing (NGS) technologies . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Current bioinformatic tools and pipelines for the analysis of EST datasets . . . . . . . . . . . . . . 3.3.1. Sequence assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Conceptual translation and annotation . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. Similarity searches, current online tools and databases . . . . . . . . . . . . . . . . . . 3.3.4. The free-living nematode, Caenorhabditis elegans, as a reference for parasitic nematodes . . 4. Recent advances in the characterization of the transcriptomes of gastrointestinal strongylid nematodes . . . 5. Conclusions and major prospects in biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author. Tel.: + 61 3 97312283; fax: + 61 3 97312366. E-mail address: [email protected] (R.B. Gasser). 0734-9750/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2011.08.016

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1. Introduction Infectious diseases caused by bacteria, viruses, fungi and parasites have significant social and economic impacts on hundreds of millions of animals and people (e.g., World Health Organization, 2008a,b; www.fao.org). Thanks to the access to an array of effective drugs and vaccines, infectious diseases account for one of ten deaths in the richest countries of the world. However, in developing countries, six of every ten people still die due to complications associated with these diseases (World Health Organization, 2008a,b). Parasitic roundworms (nematodes) of the gastrointestinal tracts of humans and livestock are of particular socio-economic importance worldwide (Artis, 2006; Bethony et al., 2006a; Brooker et al., 2006; de Silva et al., 2003; Nikolaou and Gasser, 2006). Of these nematodes, the soil-transmitted helminths (STHs) Ancylostoma duodenale, Necator americanus, Ascaris spp. and Trichuris trichiura are estimated to infect almost one sixth of all humans (Hotez et al., 2009; O'Harhay et al., 2010), and gastrointestinal parasites of livestock (including Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus spp.) cause substantial economic losses estimated at billions of dollars per annum, due to poor productivity, failure to thrive, the costs of anthelmintic treatment and deaths (Newton and Meeusen, 2003; Newton and Munn, 1999). In addition to their socio-economic impact, genetic resistance in nematodes of livestock against the main classes of anthelmintics (Wolstenholme et al., 2004) has stimulated research towards developing alternative intervention and control strategies against these parasites. Despite the wealth of information on aspects of parasite systematics, biology, epidemiology, immunology and anthelmintics (reviewed by Anderson, 2000; Kennedy and Harnett, 2001), there is a paucity of knowledge of the molecular mechanisms that govern essential biological processes in parasitic nematodes. Gaining an improved understanding of the molecular biology of these organisms offers a possible avenue for the discovery and development of novel methods of treatment and control. Advances in genomic and bioinformatic technologies are now providing the opportunity to explore fundamental aspects of the molecular biology of parasitic nematodes, such as developmental and reproductive processes. In particular, studies of the transcriptomes of parasites (= the study of expressed sequence tags [ESTs]; i.e., short sequences of complementary DNAs [cDNAs] synthesised from the messenger RNA [mRNA]; Adams et al., 1991) have become central to various areas of molecular parasitology, including gene discovery and characterization, and for gaining insights into aspects of gene expression, regulation and function (Nagaraj et al., 2007a; Parkinson and Blaxter, 2009). The purpose of this article is to provide a background on socio-economically important gastrointestinal nematodes of animals and to highlight gaps in our knowledge of their molecular biology, and to review recent progress in transcriptomics of these worms and its implications for exploring their biology and the diseases that they cause, as a foundation for developing new intervention strategies. 2. The phylum Nematoda This is one of the most diverse phyla in the animal kingdom and includes N28,000 species, of which N16,000 are parasites of animals or plants (Anderson, 2000; Hugot et al., 2001). According to the classification proposed by Chitwood (1950), the phylum Nematoda consists of two classes, the Adenophorea and the Secernentea (Chitwood, 1950; Skrjabin et al., 1967). Within the latter class, the order Rhabditida includes both free-living and entomopathogenic species of nematodes, whereas the order Tylenchida includes species of parasitic nematodes of invertebrates, particularly insects, mites and leeches, and plants (Anderson, 2000; Skrjabin et al., 1967). Conversely, species within the orders Ascaridida, Oxyurida, Spirurida and Strongylida are parasitic in humans and other animals (Anderson, 2000). Within the order Strongylida, the superfamily Ancylostomatoidea includes species of blood-feeding nematodes (called hookworms) that inhabit

the small intestines of mammalian hosts and are characterised by large, globular buccal capsules, which enable them to attach to the intestinal wall (Anderson, 2000; Skrjabin et al., 1967). The superfamily Strongyloidea includes, amongst others, intestinal parasites of equines (family Strongylidae), the nodule worms of pigs (Chabertiidae) and the gapeworm of birds (Syngamidae) (Anderson, 2000; Skrjabin et al., 1967). Members of this superfamily are characterised by complex buccal capsules, often with a series of leaf-like structures on the border of the labial region (= corona radiata) (Lichtenfels, 1980). Conversely, the buccal capsule of species of parasitic nematodes of the superfamily Trichostrongyloidea is absent or greatly reduced, and lips and corona radiata are vestigial or absent (Durette-Desset, 1983; Durette-Desset and Chabaud, 1977, 1981). Members of the superfamily Trichostrongyloidea are parasitic in a broad range of mammalian species, particularly wild and domestic ruminants (Anderson, 2000; Skrjabin et al., 1967). According to the classification proposed by Blaxter et al. (1998), parasitic nematodes of the order Strongylida, together with free-living nematodes of the sub-order Rhabditina (e.g., Caenorhabditis elegans) and order Diplogasterida (e.g., Pristionchus pacificus), belong to ‘clade V’ of the phylum Nematoda (Fig. 1). 2.1. A background on key strongylid nematodes of major socio-economic importance in animals — biology and pathogenesis 2.1.1. Members of the superfamily Ancylostomatoidea The hookworms A. duodenale and N. americanus of humans are estimated to infect ~740 million people in rural areas of the tropics and sub-tropics (de Silva et al., 2003), with the highest prevalence (~17%) recorded in areas of China and sub-Saharan Africa (de Silva et al., 2003; Xu et al., 1995), and causing an estimated disease burden of 22 million disability-adjusted life years (DALYs) (Hotez et al., 2006). Whilst N. americanus is the most widely distributed hookworm of humans globally (cf. de Silva et al., 2003), a related species, A. caninum, is a cosmopolitan hookworm of the small intestine of dogs and other canids (Anderson, 2000; Skrjabin et al., 1967). The life cycle of these parasitic nematodes is direct, with female hookworms releasing thin-shelled eggs (50–80 × 36–42 μm in size), which are passed in the faeces of the host (Schad and Warren, 1990; Skrjabin et al., 1967). Under suitable environmental conditions (i.e., 23–33 °C), the first-stage larvae (L1s), characterised by a pointed, tapered tail, an elongated buccal cavity and an oesophagus with a valved bulb, hatch from the eggs (Schad and Warren, 1990; Skrjabin et al., 1967). The L1s feed on bacteria and, within 2 days, moult to second (L2)- and, subsequently, to the third-stage larvae (L3s) within 4–5 days. This latter stage retains the cuticle of the L2 (i.e., sheath) and is referred to as a ‘filariform’ larva (Schad and Warren, 1990). The infection occurs when the L3s penetrate the skin of the vertebrate host after cuticular shedding (Looss, 1898); subsequently, larvae enter the subcutaneous tissues and migrate via the circulatory system to the heart and lungs, where they moult to fourth-stage larvae (L4s). From the lungs, the larvae migrate (via the trachea and pharynx) to the small intestine, where they develop to adult males and females within 2–7 weeks, depending on the species (Anderson, 2000; Bruni and Passalaqua, 1954; Lewert and Lee, 1954; Skrjabin et al., 1967). The adult worms (dioecious) attach by their buccal capsule to the intestinal mucosa, rupture capillaries and feed on blood (Gasser et al., 2008a; Gilman, 1982). Although skin penetration is considered the main route, ingestion of some hookworms L3s may also lead to infection (Foster and Cross, 1934). L3s of some hookworms can undergo developmental arrest (= hypobiosis) in the somatic tissues of the mammalian host and, following activation during pregnancy, undergo transmammary transmission to the offspring (Arasu and Kwak, 1999; Gibbs, 1986; Schad et al., 1973). The pathogenesis of hookworm disease is mainly a consequence of the blood loss, which is caused by tissue damage and direct ingestion of blood by the adult worms (Gilman, 1982). These focal lesions are characterised by local haemorrhage, tissue cytolysis and neutrophilic

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Strongylida Rhabditida Diplogasterida Rhabditida Rhabditida Rhabditida Rhabditida Apelenchida Tylenchida Oxyurida Spirurida Rhigonematida Ascaridida Plectidae Chromadorida Monhysterida Enoplida Triplonchida Trichocephalidae Dorylaimida (Longidoridae) Mermithida

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L3s from contaminated pastures. Once ingested, the L3s pass through the forestomachs and undergo an exsheathment process to then establish, via the parasitic L4s, as adult males and females in the abomasum (H. contortus) or small intestine (T. colubriformis) within ~3 weeks (Anderson, 2000; Olsen, 1986; Skrjabin et al., 1967; Veglia, 1915). The exsheathment process is triggered by stimuli from the host and may include (depending on the species of nematode) dissolved, gaseous CO2 and undissociated carbonic acid (H. contortus) or hydrochloric acid and pepsin (T. colubriformis) in the abomasum; the L3s respond to these stimuli by producing an exsheathment fluid which determines the detachment of the sheath from the bodies of the larvae (Nikolaou and Gasser, 2006; Rogers and Sommerville 1963, 1968; Sommerville, 1957). H. contortus infection is typically accompanied by clinical signs linked to the haematophagous activity of this parasite. The main symptoms of acute haemonchosis are anaemia, variable degrees of oedema, lethargy, decreased live-weight gain, impaired wool and milk production as well as decreased reproductive performance, often leading to death in severely affected animals (Noble and Noble, 1982; Waller et al., 1996). The pathogenesis of T. colubriformis infection is triggered by the presence of adult parasites in mucus-covered tunnels in the epithelial surface of the small intestine (Holmes, 1985) and usually consists of extensive villous atrophy combined with hyperplasia of the sub-mucosal glands, mucosal thickening and erosion as well as infiltration of lymphocytes and neutrophils into affected areas (Barker, 1973, 1975; Beveridge et al., 1989; Garside et al., 2000; Holmes, 1985). Clinical signs of trichostrongylosis include malabsorption, weight loss, progressive emaciation and diarrhoea (= scouring or ‘black scour’).

Mononchida Nematomorpha, Priapulida Fig. 1. Evolutionary relationships within the phylum Nematoda based on the analysis of sequences representing the small subunit (SSU) of the ribosomal DNA from 53 nematode taxa, using the neighbour joining and maximum parsimony algorithms. Roman numerals (I–V) represent nematode clades; adapted from Blaxter et al. (1998).

immune response (Gilman, 1982). The clinical manifestations of the disease relate mainly to iron-deficiency anaemia, which can cause physical and mental retardation and sometimes death in children as well as maternal mortality, abortions and impaired lactation (Bethony et al., 2006a; Hotez et al., 2004; Loukas et al., 2006).

2.1.2. Members of the superfamily Trichostrongyloidea The trichostrongyloid nematodes of small ruminants H. contortus and Trichostrongylus spp. (including T. colubriformis) are responsible for substantial production losses in the livestock industries worldwide (O'Connor et al., 2006). H. contortus is the most important parasitic nematode of small ruminants in tropical and sub-tropical areas or summer rainfall areas, whereas Trichostrongylus spp. are often dominant parasites in winter rainfall areas due to their ability to develop and survive at lower temperatures than H. contortus does (Anderson et al., 1978). The life cycles of H. contortus and T. colubriformis are similar and direct, with morulated eggs (66–79× 43–46 and 79–101 × 39– 47 μm in size, respectively) (Monnig, 1926; Skrjabin et al., 1967; Veglia, 1915), being laid by females in the abomasum (H. contortus) or small intestine (T. colubriformis) of the mammalian host (Monnig, 1926; Veglia, 1915). Under suitable environmental conditions (i.e., 100% humidity, 26 °C and 18 to 21 °C for H. contortus and T. colubriformis, respectively) (Olsen, 1986; Veglia, 1915), L1s hatch from eggs to then develop (via the L2s) to infective L3s. The cuticle of the L2 is retained as a sheath around the L3 and protects it from desiccation (Anderson, 2000; Olsen, 1986; Veglia, 1915). Small ruminants acquire the infection by ingesting

2.1.3. Members of the superfamily Strongyloidea The superfamily Strongyloidea includes, amongst others, important parasitic nematodes of equids (e.g., ‘large strongyles’= sub-family Strongylinae and ‘small strongyles’ or cyathostomins =sub-family Cyathostostominae), ruminants (e.g., gastrointestinal nematodes of the subfamily Chabertiinae) and pigs (e.g., the ‘nodule worms’= sub-family Oesophagostominae) (Anderson, 2000; Skrjabin et al., 1967). Within the latter subfamily, Oe. dentatum is a common parasite of the large intestine of domestic and wild pigs, responsible for severe production losses to the livestock industry. The life cycle of nematodes of the genus Oesophagostomum is direct, with gravid female worms releasing thin-shelled eggs (70–75 ×40–45 μm in size) (Anderson, 2000; Skrjabin et al., 1967; Veglia, 1924, 1928), which are passed in the faeces of the host into the environment, where they develop rapidly to L1s and L2s, characterised by a rhabditiform oesophagus and a small number of intestinal cells (Anderson, 2000; Veglia, 1924, 1928). Under optimal environmental conditions (15–20 °C; Fossing et al., 1995), the L2s develop to infective L3s within 3–5 days (Anderson, 2000). These larvae retain the cuticle of the L2s and are characterised by a strongyliform oesophagus and a conical tail (Anderson, 2000; Veglia, 1924, 1928). Pigs acquire infection by ingesting infective L3s from the environment. Larvae exsheath in the small intestine, migrate to the caecum/colon, invade the mucosa/ submucosa to undergo histotropic development (McCracken and Ross, 1970) and then develop (via the L4 stage) to dioecious adults (Anderson, 2000). Eggs appear in host faeces ~20 days following the ingestion of L3s (Talvik et al., 1997). The pathogenesis of oesophagostomiasis relates to the formation of nodules in the mucosa of the intestine, which is often accompanied by mild inflammatory reactions due to the accumulation of eosinophils and neutrophils in the sites of lesions (Stockdale, 1970). Infections are often characterised by a reduction in appetite, growth rate and feed conversion efficiency during the period of nodule formation (Stewart and Gasbarre, 1989). In intense infections (e.g., following the ingestion of N200,000 larvae), necrotic enteritis associated with diarrhoea has been observed (Stockdale, 1970).

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2.2. Aspects of immunology Numerous studies have attempted to define the cells and molecules implicated in host immune responses against gastrointestinal parasitic nematodes (Allen and Maizels, 2011; Anthony et al., 2007; Artis, 2006; Balic et al., 2000; Gasbarre et al., 1993; Gause et al., 2003; Klei, 1997; Liu et al., 2004, 2010; Loukas et al., 2005a; Loukas and Prociv, 2001; Maizels et al., 2004; Maizels and Yazdanbakhsh, 2003; Patel et al., 2009). The primary immunological reactions stimulated by nematodes of the order Strongylida are largely dependent on the processes and mechanisms of invasion and establishment in the host (Artis, 2006). For instance, the migrating L3s of hookworms are known to stimulate a marked peripheral blood eosinophilia in the mammalian host, both systemic and in the lungs (Maxwell et al., 1987; White et al., 1986). Conversely, gastrointestinal nematodes that do not undergo extensive tissue migration stimulate a mucosal immune response at the site of infection (Miller and Horohov, 2006). For instance, the primary invasion of the abomasa of small ruminants by larvae of H. contortus and T. axei leads to a localised IgE-mediated immune response (Miller and Horohov, 2006). However, it has been observed that the infection of pigs with larvae of Oe. dentatum is associated with a systemic production of IgG antibodies (Joachim et al., 1998, 1999; Larsen et al., 1997), followed by the formation of nodules containing the larvae, within the intestinal mucosa (Stockdale, 1970). Despite differences in immune responses stimulated by larvae of strongylid nematodes, adult stages appear to stimulate similar immunological reactions in their mammalian host(s), which include (i) increased production of mucus by the gastrointestinal epithelium of the host, (ii) eosinophilia and increased presence of mast cells and leucocytes in the site of infection and (iii) production of specific antibodies (Artis, 2006; Balic et al., 2000). A consensus seems to have emerged that the immunological reactions against primary infections by gastrointestinal parasitic nematodes are regulated by a T helper (Th) 2-type immune response which, in turn, determines the secretion of several types of cytokines, including IL-4, IL-5, IL-9 and IL-13 (Artis, 2006; Haussler, 1996; Loukas et al., 2005a; McSorley and Loukas, 2010; Patel et al., 2009). In contrast, immunological responses in hosts with chronic infections have been shown to be regulated predominantly by a Th1-type immune response (characterised by the production of IL-2, IL-18 and interferon-γ) (Allen and Maizels, 2011; Maizels et al., 2004; Maizels and Yazdanbakhsh, 2003). In particular, individuals infected chronically by hookworms show a significant alteration of the immune response to helminth infections, characterised by a dysfunction of the antigen-presenting ability of dentritic cells, which results in a ‘hypo-responsiveness’ of the antigen-induced proliferation of Tlymphocytes (McSorley and Loukas, 2010). 2.3. Diagnosis The diagnosis of gastrointestinal nematode infections is usually made based on the detection and identification of the parasite eggs in the faeces from the infected host using, for instance, the formalinethyl acetate sedimentation, the Kato-Katz and/or the McMaster techniques. Such techniques can allow an estimation of the burden of the infection (reviewed by Gasser et al., 2008a). With the exception of the large and thick-shelled eggs of Nematodirus spp. (~152–260 × 67–120 μm) (Skrjabin et al., 1967), eggs of most strongylids are very similar morphologically, often making specific diagnosis of strongylid infections impossible (e.g., Blotkamp et al., 1993; Gasser et al., 2008a; Polderman et al., 1991). To overcome this limitation, strongylid eggs in the faeces can be cultured to allow L3s to develop, so that they can be identified to the genus level (Blotkamp et al., 1993; Gasser et al., 2008a; Polderman et al., 1991; van Wyk et al., 2004). Various serological and immunological methods, such as the complement fixation test, fluorescentantibody test and enzyme-linked immunosorbent assay, have been evaluated for the diagnosis of infections with strongylids, including

hookworms (Bungiro and Cappello, 2005; Ganguly et al., 1988; Gasser et al., 2008a; Shetty et al., 1988; Xue et al., 1999). However, these tests do not permit the accurate diagnosis of infections with multiple species of worms and/or reliably distinguish between current and past infections (reviewed by Gasser et al., 2008a). In addition, cross-reactivity can be a common limitation of serological and immunological assays (Gasser et al., 2008a). Polymerase chain reaction (PCR)-based diagnostic techniques have also been established for the diagnosis of infection by parasitic nematodes (e.g., Bott et al., 2009; Hodgkinson, 2006; Gasser et al., 2008a,b; von Samson-Himmelstjerna et al., 2002). Different molecular markers have been employed that allow specific and sensitive diagnosis; these markers include, amongst others, regions of the ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) (Chilton, 2004). Importantly, numerous studies (e.g., Bott et al., 2009; Chilton and Gasser, 1999; de Gruijter et al., 2002, 2005; Gasser et al., 1996, 1998a,b, 1999; Hoste et al., 1995, 1998; Monti et al., 1998; Palmer et al., 2007; Romstad et al., 1997a,b, 1998; Schindler et al., 2005; Traub et al., 2004; van Lieshout et al., 2005; Verweij et al., 2001; Yong et al., 2007) have demonstrated the utility of the first and second internal transcribed spacers (ITS-1 and ITS-2) of the nuclear rDNA as genetic markers for the specific identification of a range of strongylid nematodes, including hookworms (i.e., N. americanus, A. duodenale, A. caninum, A. ceylanicum and A. braziliense) and other strongylids (including trichostrongyloids and strongyloids) (reviewed by Gasser, 2006; Gasser et al., 2008a). 2.4. Treatment strategies and vaccine research Presently, the control of gastrointestinal nematodes relies heavily on the use of anthelmintic drugs (reviewed by Holden-Dye and Walker, 2007). Such drugs, which are grouped into classes on the basis of their chemical structure and mode of action, include imidazothiazoles/ tetrahydropyrimines (e.g., levamisole and pyrantel), benzimidazoles (e.g., albendazole and mebendazole), macrocyclic lactones (e.g., ivermectin and moxidectin) and amino-acetonitrile derivatives (e.g., monepantel). The imidazothiazoles/tetrahydropyrimidines act by binding to the nicotinic acetylcholine receptors, resulting in an over-stimulation, blockage of the neuromuscular junctions and paralysis of the worms; the parasites are unable to move in the intestinal tract and are eliminated by the peristaltic action of the intestines. Benzimidazole compounds are active against a range of species of parasitic nematodes (Keiser and Utzinger, 2008). They act by blocking the formation of the microtubular matrix by binding to the cytoskeletal protein tubulin, which is essential for various biological processes in the cell, including chromosome movement and cell division (Holden-Dye and Walker, 2007; Stepek et al., 2006; Wolstenholme et al., 2004). The macrocyclic lactones act by opening glutamate-gated chloride channels, increasing the flow of chloride ions, leading to defects in neurotransmission and flaccid paralysis (Stepek et al., 2006). Although the mode of action of the amino-acetonitrile derivatives remains to be fully elucidated, recent studies (Rufener et al., 2009, 2010) have suggested that these compounds cause paralysis of the worms by binding to nematode-specific acetylcholine receptors of the DEG-3 subfamily (Rufener et al., 2009). The relatively low cost, ease of administration and efficacy of anthelmintic drugs against a wide spectrum of gastrointestinal parasitic nematodes of animals has led to their extensive use and, consequently, to the emergence of resistance (Wolstenholme et al., 2004). Indeed, resistance in gastrointestinal parasites of livestock to the main groups of broadspectrum drugs has been reported, particularly in Africa, Australia, New Zealand, Asia and South America (Gilleard, 2006; Gilleard and Beech, 2007; Stepek et al., 2006; Waller, 1997; Wolstenholme et al., 2004). In relation to benzimidazoles, at least three mutations in the gene encoding the beta-tubulin isotype 1 in H. contortus were proposed to be involved in the mechanism of resistance (Kwa et al., 1993). Although it was hypothesised that the less frequent use of anthelmintics in humans (compared with their extensive use in livestock) should negatively

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affect the selection of resistance in parasitic nematodes (Geerts et al., 1997, Geerts and Gryssels, 2001; Horton, 2003; Roos, 1997), some studies (Albonico et al., 1994, 2003; de Clercq et al., 1997; Kotze and Kopp, 2008; Reynoldson et al., 1997; Sacko et al., 1999) have reported a reduction in efficacy of mebendazole and pyrantel in N. americanus and A. duodenale in areas of Mali, Australia and Zanzibar, which had been attributed to emerging anthelmintic resistance. Given the lack of knowledge of the molecular mechanisms linked to resistance in parasitic nematodes (Wolstenholme et al., 2004), much attention is now directed towards the identification of new drug targets and the development of novel and effective nematocides (Besier, 2007; Campbell et al., 2010) as well as new, effective strategies to prevent its emergence (Albonico, 2003, Morgan and Coles, 2010; Smits, 2009). Much research has focused on developing vaccines against a range of parasitic nematodes (Bethony et al., 2006b; Claerebout et al., 2003; Dalton and Mulcahy, 2001; Diemert et al., 2008; Hotez et al., 2008; Knox, 2000; Loukas et al., 2006; Newton and Meeusen, 2003; Newton and Munn, 1999). For instance, irradiated larvae were used as the basis of a vaccine against H. contortus infection in sheep (Jarrett et al., 1959, 1961; Smith and Angus, 1980). In experimental trials, this vaccine was demonstrated to be highly effective, with up to 98% protection being achieved in lambs (7–8 months of age) and adult sheep against challenge infection with L3s of H. contortus (see Jarrett et al., 1959, 1961; Smith and Angus, 1980). However, in field trials, this vaccine did not achieve protection against naturally acquired infection (Silvanathan et al., 1984). These results, together with the discontinuation (for commercial rather than scientific reasons) of the manufacture of an effective irradiated larval vaccine against A. caninum in dogs (Miller, 1971), indicated the need to search for effective immunogenic molecules as anti-nematode vaccines. Various molecular components of the epithelial cell surface membrane of the digestive tract of different species of gastrointestinal nematodes have been evaluated as vaccine candidates both in experimental murine models and in livestock (Bethony et al., 2006b; Diemert et al., 2008; Hotez et al., 2003; Newton and Meeusen, 2003). For instance, a 110 kDa integral membrane aminopeptidase of H. contortus, which is heavily glycosylated and localised in the brush border of the epithelial cells of the gut of the adult worm, was shown to be effective in reducing the intensity of H. contortus infection in different breeds and ages of sheep (Munn et al., 1993; Newton, 1995; Newton et al., 1995; Smith et al., 2001). However, protection is limited to native proteins administered multiple times, usually in Freund's adjuvant (Knox and Smith, 2001). Another peptidase complex (P1), separated from H11 by ion-exchange chromatography, was identified (Smith et al., 1993) and shown to represent an ubiquitous component of the microvillar membrane of the intestinal cells of H. contortus (see Smith et al., 1993). Although vaccination with this protein complex was shown to result in a significant reduction (69%) in the number of H. contortus eggs in the faeces from vaccinated sheep following H. contortus challenge infection, P1 led only to a ~22–38% reduction in the intensity of infection (Newton, 1995). Conversely, vaccination with the glucose-binding glycoprotein complex (H-gal GP complex), separated by lectin affinity chromatography from other integral membrane proteins from the gut of H. contortus, was demonstrated to result in ~53–72% protection and a N 90% reduction in the number of eggs in the faeces from vaccinated sheep (Smith et al., 1994). However, the vaccination of lambs (9 months of age) with a combination of prokaryotically expressed recombinant proteins representing H-gal GP failed to stimulate protective immunity against challenge infection with H. contortus L3s (Cachat et al., 2010). Other vaccine candidates have represented molecules in the excretory/secretory products (ES) from worms (Bethony et al., 2006b; Loukas et al., 2006; Pearson et al., 2010a). For instance, proteases in ES from parasitic nematodes are a major focus of vaccine development because of their inferred roles in the digestion of nutrients acquired from the host and in the penetration and migration through host

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tissues (Pearson et al., 2010a). Of such proteases, metalloproteases and aspartic and cysteine proteases have been the focus of attention for blood-feeding nematodes, such as H. contortus, A. caninum and N. americanus (see Bethony et al., 2006b; Loukas et al., 2004, 2006; Pearson et al., 2009; Redmond and Knox, 2004; Skuce et al., 1999; Smith et al., 2003; Williamson et al., 2003a). For instance, the vaccination of dogs with recombinant forms of an aspartyl- and cyteine-protease from A. caninum (designated Ac-APR-1 and Ac-CP-2, respectively) was shown to result in a partial protection against this hookworm, characterised by an absence of clinical signs and a reduced fecundity of the adult worms in dogs (Loukas et al., 2004, 2005b). In addition, vaccination of hamsters with the N. americanus homologue of Ac-CP-2 (i.e., Na-CP-2) has been shown to induce a partial protection, associated with a ~30– 46% reduction of the intensity of infection following challenge infection with L3s (Xiao et al., 2008). Similarly, vaccination with a cysteine protease-enriched fraction (thiol sepharose-binding fraction), prepared from membrane extracts from the microvillar surface of the intestinal cells of adult H. contortus (see Knox et al., 2005), was shown to reduce the intensity of infection by 47% as well as the number of eggs in faeces by 77% in sheep following a single challenge infection (Knox et al., 1999). Proteases from larval stages have also been the focus of vaccine research, because of their proposed role(s) in the invasion of the host (Pearson et al., 2010b). In hookworms, for example, one of the best characterised proteases in larval ES is an astacin-like zinc metalloprotease from A. caninum, called Ac-MTP-1 (Hotez et al., 1990; Williamson et al., 2006), which has been demonstrated to degrade fibronectin, laminin and collagen (Williamson et al., 2006). Based on the results of a vaccine trial conducted in hamsters, this protein has been proposed as a potential candidate for the development of a multi-epitope vaccine (Xiao et al., 2008). Also, two cysteine-rich secretory proteins, known as ‘Ancylostoma-secreted proteins’ (ASPs), which belong to the pathogenesis related protein (PRP) superfamily (Hawdon et al., 1996, 1999; Hawdon and Hotez, 1996) and are a major component of ES of hookworm L3s, represent vaccine candidates (Brooker et al., 2004). Although the function of these molecules has not yet been assessed in detail (Brooker et al., 2004), they have been suggested to play active roles in tissue invasion and the modulation of host immune responses (Datu et al., 2008; Hawdon et al., 1996). A study showed that the vaccination of hamsters with a recombinant ASP (i.e., Ac-ASP-2) from the infective L3 of A. ceylanicum, expressed in Pichia pastoris, was effective in reducing significantly the intensity of hookworm infection in hamsters challenged orally with L3s of A. caninum (see Goud et al., 2004). Ac-ASP-2 exhibits a high degree of amino acid sequence similarity with a low-molecular weight antigen from adult H. contortus, designated Hc24. In sheep, vaccination with Hc24 was shown to result in a significant reduction in the number of eggs in faeces and intensity of infection (i.e., ~70%) following challenge infection with L3s of H. contortus (see Schallig et al., 1997). More generally, ASP homologues have been proposed to play key regulatory roles of fundamental biological processes in a range of organisms, including plants (van Loon et al., 2006), arthropods (Kovalick and Griffin, 2005) and trematodes (Chalmers et al., 2008). However, the evolutionary relationships amongst members of this group of molecules are presently unclear. Given the fundamental roles that ASP homologues are proposed to play in a range of eukaryotes, a clear classification, based on molecular analyses of sequence data derived from a range of eukaryote species, should provide a framework to explore aspects of the structure, function and interactions of these molecules (cf. Cantacessi et al., 2009a), which will ultimately provide a foundation for the development of strategies to disrupt key biological pathways linked to ASPs. Taken together, the results of studies focusing on the identification of suitable immunogens and the development of effective vaccines against gastrointestinal parasitic nematodes show that significant progress has been made over the years. However, there is still a paucity of information on parasite–host interactions at the molecular level. A better understanding of these interactions should assist future research aimed

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at identifying novel vaccine and drug targets. Advances in genomic technologies are now providing unique opportunities to explore the molecular biology of parasitic nematodes, parasite–host interactions and diseases on a global scale. Indeed, the advent and integration of highthroughput 'omic technologies (e.g., genomics, transcriptomics, proteomics, metabolomics and lipidomics) are revolutionising the way biology is done, allowing the systems biology of organisms to be elucidated. In particular, sequencing provides a powerful approach for detailed explorations of transcription (i.e., ‘transcriptomics’) and associated molecular processes in an organism, its tissues and its cells (see Table 1). 3. Genomic and bioinformatic approaches for investigating the transcriptomes of parasitic nematodes 3.1. Conventional techniques The genome of any living organism includes coding regions that are transcribed into mRNAs, which are subsequently translated into proteins. Various techniques, such as Northern blot (Alwine et al., 1977), quantitative real-time reverse-transcription PCR (qRT-PCR; Higuchi et al., 1993) and differential display (DD; Liang and Pardee, 1992), have been used to define patterns of transcription of single genes or small numbers of molecules in parasitic nematodes, including T. colubriformis, H. contortus, Oe. dentatum and the ‘brown stomach worm’ of cattle Os. ostertagi (see Boag et al., 2000; Cottee et al., 2006; Hartman et al., 2003; Moore et al., 2000; Nikolaou et al., 2002; Pratt et al., 1990; Sangster et al., 1999; Savin et al., 1990). Recently, techniques that allow ‘global’ analyses of gene transcription have become increasingly popular. For instance, serial analysis of gene expression (SAGE) was one of the first techniques allowing the identification of transcripts and comparison of levels of transcription (Velculescu et al., 1995). The SAGE technique is based on the generation of a short, specific tag (14 bp) from each mRNA present in the sample; these tags are used for the construction of a SAGE library. The sequencing of these tags allows a relatively high-throughput determination of their frequencies in the library, which are correlated with relative amounts of the corresponding mRNAs. Despite its demonstrated utility in investigations of yeast (Velculescu et al., 1997) and humans (Boon et al., 2002; Liang, 2002), the application of the SAGE method for studies of gene transcription in parasitic nematodes of socio-economic importance has remained limited (see Datson et al., 1999). One study (Skuce et al., 2005) used SAGE to sequence and analyse ~3000 transcripts from adult H. contortus, of which ~60% had homologues in public databases. In the last years, the analysis of EST datasets has proven to be the most widely used approach for investigations of the transcriptomes of parasitic nematodes. In vitro, mRNAs are reverse transcribed, resulting in stable complementary DNAs (cDNAs); ESTs usually represent

Table 1 Some milestones in sequencing and analyses of transcriptomes; modified from Morozova et al. (2009). Year

Milestone

1965 Sequencing of the first RNA molecule 1977 Development of the Northern blot technique and the Sanger sequencing method 1989 First report of reverse transcription PCR experiments for transcriptome analysis 1991 First high-throughput EST sequencing study 1992 Development of the differential display (DD) technique for the analysis of differentially transcribed mRNAs 1996 Reports of the use of the microrray and Serial Analysis of Gene Expression (SAGE) methods for transcriptome studies 2005 First next-generation sequencing platform introduced to the market (454/Roche)

References Holley et al. (1965) Alwine et al. (1977); Sanger et al. (1977a,b) Sarkar and Sommer (1989) Adams et al. (1991) Liang and Pardee (1992) DeRisi et al. (1996)

Margulies et al. (2005)

single-pass DNA sequence reads derived from cloned cDNAs (Adams et al., 1991; McCombie et al., 1992). Traditional sequencing (Sanger et al., 1977a,b) involves the use of a DNA polymerase, a primer and four types of deoxynucleotide triphoshatases (dNTPs) to synthesise the complementary strand from the template (Clifton and Mitreva, 2009; Sanger et al., 1977a,b). The advent of EST sequencing marked a revolution in the field of parasitology and has been used in a range of studies aimed at investigating fundamental molecular processes in parasitic nematodes as well as drug and vaccine target discovery (e.g., Blaxter et al., 1996; Daub et al., 2000; Doyle et al., 2010; Hoekstra et al., 2000; McCarter et al., 2000; Parkinson et al., 2001, 2004; Parkinson and Blaxter, 2009; Williams et al., 2000). For nematodes of animals, applications range from analyses of stage- and gender-enriched molecules (e.g., Boag et al., 2000; Campbell et al., 2008; Cottee et al., 2006; Hartman et al., 2001; Hoekstra et al., 2000) to global analyses of gene transcription (e.g., Daub et al., 2000; Parkinson et al., 2001, 2004; Rabelo et al., 2009; Ranjit et al., 2006; Yin et al., 2008). Also cDNA microarray technology (DeRisi et al., 1996) represented a significant advance for large-scale studies of the transcriptomes of parasitic nematodes (reviewed by Grant and Viney, 2001). In cDNA microarrays, several thousands of oligonucleotides, usually cDNAs, EST clones or PCR products (which correspond to previously characterised genes/transcripts) are ‘spotted’ (= ‘arrayed’) on to glass slides or chips in precise positions. The mRNAs from different stages or tissues are labelled with different fluorescent or radioactive markers and hybridised to the spots on the array. The relative abundance of hybridization for each mRNA population is then determined by comparing the relative signal intensity of each fluorescent marker (DeRisi et al., 1996). Supported by the increasing amount of sequence data available in public databases, microarray technology has allowed comparisons of levels of transcription of large numbers of mRNAs in, for instance, different tissues, developmental stages and sexes of these nematodes to be performed, ultimately providing researchers with the opportunity of identifying molecules considered to play essential roles in fundamental biological pathways of survival, development and reproduction (reviewed by Grant and Viney, 2001; Newton and Meeusen, 2003; Gasser et al., 2007). Clearly, the availability of microarray technology has resulted in an expanded knowledge of the transcriptomes of socio-economically important strongylids, including H. contortus, T. vitrinus, Teladorsagia circumcincta, Oe. dentatum and A. caninum (see Campbell et al., 2008; Cottee et al., 2006; Datu et al., 2008; Moser et al., 2005; Nisbet et al., 2008; Nisbet and Gasser, 2004). In addition, the combined use of suppressive-subtractive hybridization (SSH) and microarray analysis has been useful in enabling rapid comparisons of transcriptional profiles between/among life cycle stages, genders and/or species of parasitic nematodes (Cottee et al., 2006; Datu et al., 2008; Huang et al., 2008; Liu et al., 2007; Nisbet et al., 2008; Yang et al., 1999). For instance, SSH-based microarray analyses of both sexes of T. vitrinus and Oe. dentatum (see Cottee et al., 2006; Nisbet and Gasser, 2004) provided initial insights into gender-enriched transcription. These studies showed that transcripts encoding protein kinases and phosphatases, major sperm proteins (MSPs) and enzymes involved in carbohydrate metabolism were consistently male-enriched, whereas vitellogenins, heat-shock proteins and chaperonins were enriched in females (Cottee et al., 2006; Nisbet and Gasser, 2004). Given the economic impact of these nematodes in the livestock industry (cf. Nikolaou and Gasser, 2006; Sackett and Holmes, 2006) and the problems associated with resistance to various classes of nematocides (Cezar et al., 2010; LeJambre et al., 2005; Wolstenholme et al., 2004), an enhanced knowledge of the molecules transcribed in other developmental stages of these parasites could provide insights into molecular mechanisms linked to drug resistance and allow the prediction or identification of novel drug targets. Knowledge of the complement of molecules transcribed in the larval stages of strongylid nematodes should also aid the elucidation of pathways associated with infectivity and interactions with the

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vertebrate host. The molecular mechanisms that regulate the transition from the free-living to the parasitic stage of nematodes might allow the development of novel strategies to disrupt this transition. Previous studies have analysed differences in transcription between the ensheathed, free-living L3 and the exsheathed L3 of H. contortus (see Geldhof et al., 2005; Hartman et al., 2003; Hoekstra et al., 2000) and the related strongylid, A. caninum (see Datu et al., 2008; Moser et al., 2005). The results of a cDNA microarray analysis, complemented by qRT-PCR of differentially transcribed molecules, showed that, amongst others, the majority of transcripts encoding ASPs were up-regulated in free-living L3s compared with parasitic, serum-stimulated larvae of A. caninum (see Moser et al., 2005). However, a recent study using SSH-based microarray analysis (Datu et al., 2008) showed a substantial increase in the numbers and levels of transcripts encoding ASPs in serum-activated L3s (Datu et al., 2008). Considering that A. caninum and H. contortus are both blood-feeding nematodes, deeper investigations of the molecular mechanisms associated with the transition from the free-living to the parasitic stage of H. contortus should provide insights into conserved pathways of development for these strongylid nematodes. In addition, with the exception of A. caninum (see Abubucker et al., 2008; Moser et al., 2005; Wang et al., 2010a), investigations of the transcriptomes of other hookworms are presently limited (Daub et al., 2000; Rabelo et al., 2009; Ranjit et al., 2006). To date, molecular studies of this group of blood-feeding nematodes have mainly involved A. caninum, because of its use as a model for human hookworms (Abubucker et al., 2008; Datu et al., 2008; Mitreva et al., 2005; Moser et al., 2005; Wang et al., 2010a). Clearly, detailed knowledge and understanding of the molecules transcribed in all stages of different species of hookworms, including N. americanus and A. duodenale of humans, should facilitate the identification of conserved pathways linked to development, survival, reproduction, parasite interactions and disease, and could assist in the discovery of new intervention strategies. Recent advances in sequencing technologies (Bentley et al., 2008; Harris et al., 2008; Margulies et al., 2005; Pandey et al., 2008) now provide the unique opportunity to perform de novo analyses of the whole transcriptomes of different species, sexes and/or developmental stages of nematodes of socio-economic importance. Indeed, although ‘next-generation’ (also called ‘massively parallel’) sequencing methods were introduced only recently (Roche 454 GS FLX; Margulies et al., 2005), their capacity to generate millions or hundreds of millions of sequences, in parallel, has placed them at the forefront of the genomic and transcriptomic research (Mardis, 2008a; Marguerat and Bahler, 2010; Wang et al., 2009a). They are, thus, powerful tools for investigating the transcriptomes of parasitic nematodes on a scale like never before. 3.2. Next-generation sequencing (NGS) technologies Next-generation sequencing (NGS) has substantially reduced the costs of generating large sequence datasets. Currently available NGS sequencing platforms include the 454/Roche (Margulies et al., 2005;

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www.454.com), the Illumina/Solexa (Bentley et al., 2008; www. illumina.org) and the SOLiD (= Supported Oligonucleotide Ligation and Detection) (Harris et al., 2008; www.appliedbiosystems.com) (Table 2). The 454/Roche platform (Margulies et al., 2005; www.454.com) uses a sequencing-by-synthesis approach. For transcriptomic studies, cDNA is randomly fragmented (by ‘nebulization’) into 500–1000 base pair (bp) fragments; adaptors are ligated to each end of these fragments, which are then mixed with a population of agarose beads whose surfaces anchor oligonucleotides complementary to the 454specific adapter sequence, such that each bead is associated with a single fragment. Each of these complexes is transferred into individual oil– water micelles containing amplification reagents and is then subjected to an emulsion PCR (emPCR) step, during which ~10 million copies of each cDNA are produced and bound to individual beads. Subsequently, in the sequencing phase, the beads anchoring the cDNAs are deposited on a pico-titre plate, together with other enzymes required for the pyrophosphate sequencing reaction (i.e., ATP sulfurylase and luciferase) and the sequencing is carried out by flowing sequencing reagents (nucleotide and buffers) over a plate (cf. Mardis, 2008b). After the introduction of the 454 technology, the first Illumina (formerly Solexa) sequencer became available (Bentley et al., 2008; www. illumina.com). This technology involves fragmentation of the cDNA sample, followed by the in vitro ligation of Illumina-specific adaptors to each cDNA template; the termini of the template are covalently attached to the surface of a glass slide (or flow cell). Attached to the flow cell are primers complementary to the other end of the template, which bend the cDNAs to form bridge-like structures. During the amplification step (bridge-PCR), clonal clusters, each consisting of ~ 1000 amplicons, are generated; subsequently, the cDNAs are linearised, and the sequencing reagents are directly added to the flow cell, with four fluorescently labelled nucleotides. After the incorporation of a fluorescent base, the flow cell is interrogated with a laser in several locations, which results in several image acquisitions at the end of a single synthesis cycle (cf. Mardis, 2008b). This technology is considered ideal for re-sequencing projects, targeted sequencing, single nucleotide polymorphism (SNP) analyses and gene transcription studies. The sequencing process of the SOLiD platform (Harris et al., 2008; www.appliedbiosystems.com) employs the enzyme DNA ligase, instead of a polymerase (reviewed by Mardis, 2008b). Briefly, after an emPCR step, the adaptor sequences of the cDNA templates bind to complementary primers that are covalently anchored to a glass slide. Subsequently, a set of four fluorescently labelled di-probes (octamers of random sequence, except known dinucleotides at the 3′-terminus) is added to the sequencing reaction. If an octamer is complementary to the template, it will be ligated, and the two specific nucleotides can be called; subsequently, an image is acquired and the fluorescent dye is removed, so that other octamers can be ligated. After multiple ligations (e.g., 7 ligations for a 35 bp read), the newly synthetised cDNA is removed and the primer is inactivated. This process is repeated multiple times from different starting points of the cDNA templates, so that each position

Table 2 Technical features of next-generation sequencing platforms (i.e., 454/Roche, Illumina/Solexa and SOLiD)a.

Platform Sequencing method Sequencing chemistry Base pairs sequenced (per run) Read length Run time Advantages Disadvantages a

454/Roche

Illumina/Solexa

SOLiD

Genome Sequencer FLX Emulsion PCR of bead-bound oligos Pyrosequencing using polymerase 0.5 Gb

Genome Analyzer IIx Isothermal bridge amplification on flowcell Ligation (‘dual-base encoding’ octamers) ~ 30–50 Gb

SOLiD 3 Plus System Emulsion PCR of bead-bound oligos Reversible terminator using polymerase ~ 60 Gb

400–800 bp ~ 12 h Long read length Unreliable determination of homopolymer regions and large repeats

100 bp ~ 2–9 days No emulsion PCR required Long run time, short read length

100 bp ~ 3 days High accuracy due to dual base calls Short read length

Based on information available on July 2011.

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is sequenced at least twice. This technique, known as ‘two-base-calling’, allows the correction of sequencing errors, thus providing accurate base calling (cf. Mardis, 2008b). Because of the short read-length, the range of applications of the SOLiD system is considered similar to that of the Illumina technology and includes (targeted) re-sequencing projects, SNP detection and gene transcription studies. In the last years, numerous studies have demonstrated the utility of NGS technologies for investigating, for instance, aspects of the systematics, population genetics and molecular biology of pathogens, including viruses (Kuroda et al., 2010), bacteria (Tettelin, 2009), arthropods (Wang et al., 2010b), protozoa (Chen et al., 2009; Franzens et al., 2009; Otto et al., 2010) and helminths (Cantacessi et al., 2010a-d, 2011; Jex et al., 2008; Wang et al., 2010a; Young et al., 2010a,b, 2011). In particular, the 454 platform was used recently for the de novo sequencing of the transcriptomes of important trematodes of humans and animals (i.e., Clonorchis sinensis, Opisthorchis viverrini, Fasciola hepatica and F. gigantica; Young et al., 2010a,b, 2011). More than 50,000 unique and novel sequences were characterised for each of these parasites, demonstrating the capacity of this technology to generate huge datasets. The development of bioinformatic tools has become crucial for the detailed analyses of such datasets. 3.3. Current bioinformatic tools and pipelines for the analysis of EST datasets The increasing number of EST datasets in public databases has been accompanied by an expansion of bioinformatic tools for the analysis of sequence datasets, both at the cDNA and protein levels. This expansion has resulted in the development of a number of web-based programmes and/or integrated pipelines (Conesa et al., 2005; Huang and Madan, 1999; Hunter et al., 2009; Iseli et al., 1999; Nagaraj et al., 2007a,b; Soderlund et al., 2009). The principles, methods and protocols for the analysis of EST data, together with currently available bioinformatic tools and pipelines, have been reviewed (Nagaraj et al., 2007a,b). In brief, following the acquisition of sequence data, ESTs are firstly screened for sequence repeats, potential contaminants and/or adaptor sequences (Falgueras et al., 2010; Nagaraj et al., 2007b). Vector sequences (used at the cloning step) are eliminated prior to the assembly using information available in web-based non-redundant vector databases, such as UniVec (http://www.ncbi. nlm.nih.gov/VecScreen/UniVec.html) and EMVEC (http://www.ebi.ac. uk/Tools/blastall/vectors.html). Following the pre-processing of ESTs, sequences are ‘clustered’ (= assembled) into contiguous sequences (of maximum length) based on sequence similarity. 3.3.1. Sequence assembly The main goal of sequence assembly is to determine, with confidence, the sequence of a target transcript/gene. This process involves the alignment and merging of DNA fragments to form long contiguous sequences (i.e., contigs) (Nagaraj et al., 2007b; Ranganathan et al., 2009). Long- (e.g., generated by Sanger sequencing or 454 technology) and short-reads (e.g., Illumina or SOLiD platform) are assembled using the algorithms ‘overlap-layout-consensus’ (Myers, 1995) and ‘de Bruijn graph’ (Idury and Waterman, 1995; Zerbino and Birney, 2008), respectively. For the former algorithm (Myers, 1995), all pairwise overlaps among reads are computed and stored in a graph; all graphs are used to compute a layout of reads and then a consensus sequence of contigs (Miller et al., 2010; Scheibye-Alsing et al., 2009). Some of the assemblers designed to support long-read assembly include PHRAP (Green, 1996), the contig assembly programme v.3 (CAP3; Huang and Madan, 1999), the TIGR assembler (Sutton et al., 1995), the parallel contig assembly programme (PCAP; Huang et al., 2003) and the mimicking intelligent read assembly programme (MIRA; Chevreux, 2005).

For the ‘de Bruijn graph’ (Idury and Waterman, 1995; Zerbino and Birney, 2008), reads are fragmented into short segments, called ‘k-mers’, where ‘k’ represents the number of nucleotides in each segment; overlaps between or among k-mers are captured and stored in graphs, which are subsequently used to generate the consensus sequences (Miller et al., 2010; Scheibye-Alsing et al., 2009). Examples of programmes specifically designed for the assembly of short-reads include the short sequence assembly by k-mer search and 3′-read extension (SSAKE; Warren et al., 2007), Velvet (Zerbino and Birney, 2008), the exact de novo assembler (EDENA; Hernandez et al., 2008), EulerSR (Pevzner et al., 2001), the assembly by short sequencing (ABYSS; Simpson et al., 2009) and the short oligonucleotide analysis package (SOAP; Li et al., 2008). 3.3.2. Conceptual translation and annotation Following assembly, the contigs and single reads (or singletons) are compared with known sequence data available in public databases, in order to assign a predicted identity to each query sequence if significant matches are found (Nagaraj et al., 2007b). In addition, assembled nucleotide sequences are usually conceptually translated into predicted proteins using algorithms that identify protein-coding regions (ORFs) from individual contigs. Examples of such algorithms are OrfPredictor (Min et al., 2005), ESTScan (Iseli et al., 1999) and DECODER (Fukunishi and Hayashizaki, 2001). Once peptides are predicted, protein analyses, including amino acid sequence comparisons with data available in public databases, and known protein domains are then inferred (Iseli et al., 1999). For instance, the software InterProScan (Hunter et al., 2009) provides an integrated tool for the characterization of a protein family, or an individual protein sequence, domain and/or functional site by comparing sequences with information available in the databases PROSITE (Hofmann et al., 1999), PRINTS (Attwood et al., 2000), Pfam (Bateman et al., 2000), ProDom (Corpet et al., 1999), SMART (Schultz et al., 2000) and/or Gene Ontology (GO; Ashburner et al., 2000). In addition, other programmes are available for the prediction of transmembrane domains (e.g., TMHMM; Krogh et al., 2001) and/or signal peptide motifs (e.g., SignalP; Nielsen et al., 1997). 3.3.3. Similarity searches, current online tools and databases Different types of the Basic Local Alignment Software Tool (BLAST; Altschul et al., 1990) are used for comparing the nucleotide sequence data with DNA or cDNA (BLASTn), or amino acid (BLASTx) sequences or conceptually translated peptides with protein sequences (BLASTp), available in databases (Nagaraj et al., 2007b). Public databases represent comprehensive collections of nucleotide and amino acid sequences. Due to the rapid progress in the discovery and characterization of novel genes and proteins, online public databases have become one of the primary resources for sequence data storage, analysis and annotation. For example, the International Nucleotide Sequence Database Collaboration includes three ‘sister’ databases, namely GenBank (Benson et al., 2002), the Enterprise Management Technology Transfer nucleotide database curated by the European Molecular Biology Laboratories (EMBL; Stoesser et al., 2002) and the DNA Databank of Japan (DDBJ; Tateno et al., 2002). In these databases, all publicly available nucleotide sequences are stored and curated; in addition, each sequence is stored as a separate record and linked to information, such as primary source references and predicted and/or experimentally verified biological features. For ESTs, raw sequence data are often stored in sub-divisions of these nucleotide databases, such as UniGene (Wheeler et al., 2001) and the Sequence Read Archive (Shumway et al., 2010). Various databases, which exclusively store known amino acid sequence data, are also available. For instance, the Protein Data Bank (PDB; Berman et al., 2000), maintained by the Research Collaboratory for Structural Bioinformatics, represents the primary source for protein structures, whereas the SWISS-PROT database (Bairoch and Apweiler, 2000) is a protein sequence database for a number of prokaryotes and eukaryotes. The TrEMBL (Bairoch and Apweiler, 1996) division of SWISS-PROT contains a non-redundant

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set of translations for all coding sequences in the EMBL nucleotide sequence database that do not correspond to existing SWISS-PROT entries. In addition to these comprehensive general databases, there is a number of specialised collections of gene and protein information on particular organisms. Examples include the databases for Saccharomyces cerevisiae (yeast) (http://www.yeastgenome.org/; Cherry et al., 1997), Drosophila melanogaster (vinegar fly) (http:// flybase.org/; Tweedie et al., 2009), Mus musculus (mouse) (http:// www.informatics.jax.org/; Bult et al., 2008) and C. elegans (free-living nematode) (WormBase at http://www.wormbase.org; Harris et al., 2010). WormBase is a comprehensive repository of information on C. elegans and related nematodes, such as C. briggsae (see Harris et al., 2010; Harris and Stein, 2006). Here, essentially all information and data on classical genetics, cellular biology, structural and functional genomics of these free-living nematodes are stored and continually curated (Bieri et al., 2006; Harris et al., 2010; Harris and Stein, 2006; Schwarz et al., 2006). A web-based bioinformatic pipeline (called ESTExplorer) was established for the automated analysis and annotation of EST datasets (both at the nucleotide and amino acid levels) (Nagaraj et al., 2007a), and shown to substantially accelerate and facilitate the analyses of sequences (generated using conventional Sanger sequencing) compared with traditional database searches (Nagaraj et al., 2008). However, thus far, this pipeline has not been adapted to the analyses of large transcriptomic (e.g., RNA-seq) datasets generated using NGS technologies. 3.3.4. The free-living nematode, Caenorhabditis elegans, as a reference for parasitic nematodes Presently, the analysis and annotation of sequence data derived from parasitic nematodes relies heavily on information available for C. elegans (in WormBase). The latter nematode is simple in its anatomy (959 somatic cells in the hermaphrodite and 1031 in the male), has a short life cycle (~3 days) and is easy to culture in vitro (Brenner, 1974). The genome of C. elegans is ~97 Mb in size (The C. elegans Sequencing Consortium, 1998); it contains five autosomal and a single sex chromosome. The karyotype is n =12 (10A:2X) for the hermaphrodite and 2n =11 (10A:X) for the male. Since the first publication on the genetics of C. elegans (see Brenner, 1974), the amount of biological and molecular data has expanded massively (O'Connell, 2005), so that the establishment of an up-to-date database to rapidly access information on this free-living nematode had become necessary. Currently, WormBase (www.wormbase.org) contains detailed and curated information on ~19,000 C. elegans genes and associated data on, for instance, transcription/expression profiles in different developmental stages, tissues and cells, mutants and their phenotypes, genetic and physical maps, SNPs, information on gene–gene and protein–protein interactions as well as all peer-reviewed literature pertaining to C. elegans. The introduction of the technique of RNA interference (RNAi; Fire et al., 1998) has represented a revolution in the study of gene function in metazoan organisms and has led to detailed information on the functions of ~96% genes in C. elegans (see Barstead, 2001; Kamath and Ahringer, 2003; Simmer et al., 2003; Sonnichsen et al., 2005; Sugimoto, 2004). The principle of RNAi relies on the introduction of double-stranded RNA (dsRNA) into the cells of a living organism, which induces the degradation of the homologous (target) mRNA (Fire et al., 1998). The dsRNA can be introduced directly into C. elegans by injection (Fire et al., 1998), by soaking worms in solution (Tabara et al., 1998) or by feeding worms Escherichia coli expressing a dsRNA fragment of a target gene (Timmons and Fire, 1998); it can also be introduced using a transgene expressing dsRNA (Tabara et al., 1999; Tavernarakis et al., 2000). This gene silencing approach opened up avenues for large-scale studies of molecular function in C. elegans (see Ashrafi et al., 2003; Barstead, 2001; Kamath and Ahringer, 2003; Maeda et al., 2001; Simmer et al., 2003; Sugimoto, 2004; Tabara et al., 1999) as well as for comparative studies (e.g., comparison with

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parasitic nematodes or humans) (Britton and Murray, 2006; Geldhof et al., 2007; Morris, 2008; Rosso et al., 2009). In addition to the RNAi technique, the transgenesis of C. elegans has been widely used for assessing gene function (Fire, 1986; Stinchcomb et al., 1985). This technique can involve the microinjection of expression constructs, which usually comprise plasmid or cosmid DNA, often incorporating green fluorescent protein (GFP; Chalfie et al., 1994) into the syncytium (mitotically active) region of the adult hermaphrodite gonad (= ‘gonadal microinjection’); alternatively, the DNA constructs can be transferred directly into target cells via high density microparticles of gold or tungsten (= ‘biolistics’ or ‘particle bombardment’) (reviewed by Lok and Artis, 2008). Introduced DNA does not usually integrate into the chromosome, rather it forms a multicopy extrachromosomal array, which can be inherited. GFP allows the study of a number of (temporal and spatial) biological processes, including gene expression, protein localization and dynamics, protein– protein interactions, cell division, chromosome replication and organisation, intracellular transport pathways, organelle inheritance and biogenesis (Hobert and Loria, 2006). In C. elegans, GFP was first used as a marker for gene expression (Chalfie et al., 1994), where the GFP coding sequence was placed under the control of a promoter for the mec-7 tubulin gene; in C. elegans expressing this regulated GFP, the pattern of fluorescence in vivo was similar to that characterised previously using an antibody probe (Chalfie et al., 1994). Since this first study, GFP has been used in gene expression studies of C. elegans and led to a Nobel Prize (to Dr. Martin Chalfie, 2008; http://nobelprize.org/nobel_prizes/ chemistry/laureates/2008/#). Today, GFP is applied widely to analyses of gene expression and localisation in a broad range of biological systems (Chudakov et al., 2010). In addition to studies of gene expression and localization, patterns of gene transcription during key developmental and reproductive processes have also been investigated in C. elegans using microarray technology (Jiang et al., 2001; Kim et al., 2001; Reinke et al., 2000). In the first study (Reinke et al., 2000), various groups of molecules were demonstrated to have high expression levels in the germline tissues of C. elegans, i.e. the ‘germline intrinsic’ molecules (expressed in the germline of hermaphrodites producing either sperm or oocytes, and proposed to play key roles in biological processes linked to meiosis, stem cell recombination and germline development) and molecules highly expressed exclusively in oocytes-producing and sperm-producing hermaphrodites, respectively (Reinke et al., 2000). The latter group included a large number of molecules, such as protein kinases and phosphatases, associated with spermatogenesis, in accordance with other studies investigating gender-enriched transcriptional patterns in parasitic nematodes (e.g., Campbell et al., 2008; Cottee et al., 2006; Nisbet and Gasser, 2004). Previously, genetic studies had indicated that ~50–70% of genes in parasitic nematodes have orthologues in C. elegans (see Blaxter et al., 1998; Parkinson et al., 2004), which led to the grouping of this free-living nematodes into ‘clade V’ of the phylum Nematoda, together with parasitic nematodes of the order Strongylida (Blaxter et al., 1998; Chilton, 2004). These results, together with similarities in various characteristics (such as body plan and moulting) between C. elegans and some parasitic nematodes (e.g., Bürglin et al., 1998; Nikolaou and Gasser, 2006), suggested that this free-living nematode provides a useful system for comparative investigations of many conserved biochemical and molecular pathways linked to development in related nematodes. 4. Recent advances in the characterization of the transcriptomes of gastrointestinal strongylid nematodes The advent of NGS (Table 2) and recent progress in the development of improved bioinformatic tools are providing unparalleled opportunities for global investigations of the transcriptomes of parasitic nematodes. Recent studies have utilised these new technologies to explore the transcriptomes of different developmental stages and both sexes

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of key strongylid nematodes, including T. colubriformis, H. contortus, N. americanus and Oe. dentatum (see Cantacessi et al., 2010a-d) (Table 3). For instance, the transcriptome of adult T. colubriformis was defined using 454 sequencing technology, and bioinformatic analyses and the main biological pathways that key groups of molecules are linked to in this nematode were inferred (Cantacessi et al., 2010a). Molecules encoding peptides predicted to be associated with the nervous system (i.e., ‘transthyretin-like’ and ‘neuropeptide-like'proteins; TTLs and NLPs, respectively), digestion of host proteins or inhibition of host proteases (i.e., proteases and protease inhibitors, respectively) were highly represented in the T. colubriformis transcriptome (Cantacessi et al., 2010a), with serine- and metallo-proteases and ‘Kunitz-type’ protease inhibitors being the vast majority of molecules characterised (Cantacessi et al., 2010a). In strongylid nematodes, these molecules play fundamental roles in the invasion of the vertebrate host by mediating, for example, tissue penetration, feeding and/or immuno-evasion by (i) digesting antibodies; (ii) cleaving cell-surface receptors for cytokines and/or (iii) causing the direct lysis of immune cells (Björnberg et al., 1995; Hotez and Prichard, 1995; Robinson et al., 1990; Shaw et al., 2003; Williamson et al., 2003b). In H. contortus, 454 sequencing and bioinformatic analyses were used to explore differences in gene transcription between the free-living (L3) and the parasitic (xL3) third larval stage and to predict the roles that key transcripts play in the metabolic pathways linked to larval development (Cantacessi et al., 2010b). These analyses revealed that TTLs and calciumbinding proteins were highly represented in the transcriptome of both H. contortus L3 and xL3, whereas selected transcripts encoding collagens and neuropeptides were present exclusively in L3 and proteases in xL3 (Cantacessi et al., 2010b). In nematodes, the synthesis of collagens has been observed to increase significantly prior to a moult (Fetterer, 1996), whereas proteins involved in the development of the nervous system are essential in the cascade of events leading to the growth and development of the larval stages (Quinn et al., 2008). Therefore, increased transcription of neuropeptides in L3s of H. contortus might relate to

axon guidance and synapse formation during the L3's transition to parasitism (Cantacessi et al., 2010b). This statement is supported by the fact that, in H. contortus, the transition from the free-living to the parasitic L3 is triggered by gaseous CO2, detected by chemosensory neurons of amphids, located in the anterior end of the L3 stage, ultimately leading to the secretion of the neurotransmitter noradrenaline (reviewed by Nikolaou and Gasser, 2006). Conversely, the largest number of C. elegans orthologues of H. contortus xL3-specific transcripts encoded peptidases and other enzymes of the amino acid catabolism, supporting previous evidence that cysteine proteases play a crucial role in the catabolism of globin, as is the case for A. caninum and N. americanus (see Pratt et al., 1990; Ranjit et al., 2008, 2009; Williamson et al., 2004). A similar spectrum of proteases and other molecules linked to catalytic activity had been shown also to be highly represented in the transcriptomes of activated xL3 stages of both H. contortus and A. caninum by comparison with their L3s (Cantacessi et al., 2010b; Datu et al., 2008). This finding, for two haematophagous bursate nematodes with differing life histories, is likely to reflect the key roles that these molecules play in host tissue invasion, degradation and/or digestion. Despite biological, ecological and evolutionary differences between H. contortus and hookworms, both of them (at the adult stage) feed on blood in the gastrointestinal tract of the host, causing pathogenic effects. Although human hookworms are of major socio-economic importance (see Bethony et al., 2006a; de Silva et al., 2003; Hotez et al., 2009; O'Harhay et al., 2010), genomic and molecular studies have mainly involved A. caninum (e.g., Abubucker et al., 2008; Datu et al., 2008; Mitreva et al., 2005; Moser et al., 2005; Wang et al., 2010a). Recently, 454 sequencing and bioinformatic analyses were conducted to explore, for the first time on a large scale, the transcriptome of the adult stage of N. americanus (see Cantacessi et al., 2010c). The results showed that transcripts encoding proteases and Kunitz-type protease inhibitors were most abundantly represented in the transcriptome of this nematode, supporting the fundamental roles that these molecules play in multi-enzyme cascades to digest haemoglobin and other

Table 3 Summary of experimental approaches used for investigations of the transcriptomes of key gastrointestinal strongylid parasitic nematodes. Species investigated (alphabetical)

Experimental approach utilised

Ancylostoma caninum

cDNA microarray and Sanger sequencing of expressed sequence tags (ESTs) Suppressive–subtractive hybridization, cRNA microarray and Sanger sequencing of ESTs Next-generation sequencing of ESTs Sanger sequencing of cDNA and Northern blot

Haemonchus contortus

Sanger sequencing of ESTs Sanger sequencing and Southern blot RNA-arbitrarily primed polymerase chain reaction Serial analysis of gene expression cDNA microarray and Sanger sequencing of ESTs

Necator americanus

Oesophagostomum dentatum

Ostertagia ostertagi Teladorsagia circumcincta Trichostrongylus colubriformis Trichostrongylus vitrinus

Number of transcripts characterised 145 602 48,326 13 2145 1 15 2825 333

Next-generation sequencing of ESTs Sanger sequencing of ESTs

23,495 1075

Next-generation sequencing of ESTs RNA-arbitrarily primed polymerase chain reaction and Sanger sequencing Reverse-transcription PCR and Sanger sequencing of ESTs Suppressive–subtractive hybridization, cDNA microarray and Sanger sequencing of ESTs Next-generation sequencing of ESTs Reverse-transcription PCR and Sanger sequencing Suppressive–subtractive hybridization and Sanger sequencing of ESTs Sanger sequencing Next-generation sequencing of ESTs Suppressive–subtractive hybridization, cDNA microarray and Sanger sequencing of ESTs Reverse-transcription PCR and Sanger sequencing of ESTs

19,997 31 5 391

Key references Moser et al. (2005); Cantacessi et al. (2009b) Datu et al. (2008) Wang et al. (2010a) Pratt et al. (1990); Sangster et al. (1999); Nikolaou et al. (2002) Hoekstra et al. (2000); Yin et al. (2008) Hartman et al. (2003) Hartman et al. (2001) Skuce et al. (2005) Campbell et al. (2008); Cantacessi et al. (2009b) Cantacessi et al. (2010b) Daub et al. (2000); Ranjit et al. (2006); Rabelo et al. (2009) Cantacessi et al. (2010c) Boag et al. (2000) Cottee et al. (2004) Cottee et al. (2006)

448,661 4 361

Cantacessi et al. (2010d) Moore et al. (2000) Nisbet et al. (2008)

1 21,259 561

Savin et al. (1990) Cantacessi et al. (2010b) Nisbet and Gasser (2004)

1

Hu et al. (2007)

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serum proteins (Ranjit et al., 2009; Williamson et al., 2004) and in preventing homeostasis and inhibiting host proteases (Furmidge et al., 1996; Milstone et al., 2000), respectively. Using a combination of orthology-mapping and functional data available for C. elegans, Cantacessi et al. (2010c) predicted 18 potential drug targets in the transcriptome of the adult stage of N. americanus and included, for instance, mitochondrial-associated proteins known to be essential in C. elegans (see Grad et al., 2007). In an effort to predict and prioritise molecules that might represent novel drug targets and are expressed across different stages of development, Cantacessi et al. (2010d) employed 454 sequencing and predictive algorithms to explore similarities and differences in the transcriptomes of the L3, L4 and adult male and female of Oe. dentatum (see Cantacessi et al., 2010d). Most of the molecules unique to adult males and females of Oe. dentatum could be linked to pathways associated with reproductive processes. For instance, a large number of Oe. dentatum male-specific molecules encoded majorsperm proteins (MSPs), in accordance with previous studies of male-enriched datasets of other species of trichostrongylid nematodes, including T. vitrinus and H. contortus (see Campbell et al., 2008; Nisbet and Gasser, 2004). Based on the observation that MSPs from various nematodes, including C. elegans, are characterised by a significant amino acid sequence conservation (~67%; cf. Cottee et al., 2004), a similar role has been proposed for these proteins in processes linked to the maturation of oocytes in the uterus of female nematodes (Miller et al., 2001, 2003). In addition, a large proportion (17%) of molecules unique to the larval stages of Oe. dentatum represented proteases that have been reported to evoke immunological and/or inflammatory reactions (including infiltrations of neutrophils and eosinophils) surrounding the encapsulated larvae (cf. Stockdale, 1970; reviewed by Gasser et al., 2007). In addition, somatic extracts of and supernatants from in vitro maintenance cultures of Oe. dentatum L4s have been shown to induce the proliferation of porcine mononuclear cells in vitro (Freigofas et al., 2001), which supports the hypothesis that L4-specific proteases may play an active role in the modulation of the host's immune response (Björnberg et al., 1995; Hotez and Prichard, 1995; Robinson et al., 1990). The results from this study (Cantacessi et al., 2010d) also showed that a high proportion (27–32%) of transcripts encoding protein kinases and phosphatases were common among all developmental stages of Oe. dentatum investigated (see Cantacessi et al., 2010d). Supported by investigations of the free-living nematode, C. elegans, recent studies have predicted, for instance, that some phosphatases and kinases could represent targets for novel nematocidal drugs (Campbell et al., 2011a,b). For instance, some cantharidin/norcatharidin analogues (see Hill et al., 2007; McCluskey et al., 2002; Stewart et al., 2007) are known to display exquisite and specific inhibitory activity against PP1 and PP2A phosphatases, which indicated that some of them could be designed to selectively inhibit essential serine/threonine phosphatase (STPs) of nematodes (Campbell et al., 2011b). Indeed, the nematocidal effect of selected analogues of the phosphatase inhibitor norcantharidin on larvae of H. contortus has been demonstrated recently (Campbell et al., 2011c). In addition to phosphatases, other molecules, such as chitin-binding proteins or proteases, might be interesting drug targets, given that they are proposed to have crucial roles in pathways linked to developmental and reproductive processes in some nematodes (see Cantacessi et al., 2010d; Gasser et al., 2007; Olson et al., 2006). Highly represented in the transcriptomes of T. colubriformis, H. contortus, N. americanus and Oe. dentatum (see Cantacessi et al., 2010a-d) are molecules encoding proteins containing a ‘sperm-coating protein (SCP)-like extracellular domain’ (InterPro: IPR014044), also called SCP/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS; Pfam accession number no. PF00188), or ASPs (reviewed by Cantacessi et al., 2009a). Due to their abundance in the excretory/secretory (ES) products from serumactivated L3s (= aL3s) of A. caninum and high transcriptional levels of mRNAs encoding ASPs in activated L3s compared with non-activated,

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ensheathed L3s, these molecules have been hypothesised to play a major role in the transition from the free-living to the parasitic stages of this hookworm (Datu et al., 2008; Hawdon et al., 1996). Other ASP homologues have been characterised for the adult stage of hookworms, and are proposed to play a role in the initiation, establishment and/or maintenance of the host–parasite relationship (e.g., Datu et al., 2008; Mulvenna et al., 2009; Zhan et al., 2003). Due to their immunogenic properties, one ASP (i.e., Na-ASP-2) has been under investigation as a vaccine candidate against necatoriasis in humans (Bethony et al., 2005, 2008; Loukas et al., 2006; Mendez et al., 2008; Xiao et al., 2008). Whether SCP/TAPS proteins or their genes represent drug target candidates still remains to be determined. For ASPs, a major focus of future research should be on studying their structure and function in parasitic helminths, to pave the way for applied outcomes, such the development of drugs and/or vaccines. 5. Conclusions and major prospects in biotechnology Accurate bioinformatic analyses of sequence data are crucial in providing biological meaningful molecular biological information on organisms, including parasitic nematodes. Until recently, detailed bioinformatic analyses of large nucleotide and protein sequence datasets have been restricted mainly to specialised laboratories with substantial computer and software capacities. The introduction of new integrated bioinformatic systems, such as Bio-cloud (http://cloud. genomics.cn) and Artemis (http://www.sanger.ac.uk/resources/ software/artemis/) for the de novo assembly and annotation of large-scale sequence datasets marks a change in molecular parasitology, because the community can rapidly derive substantial amounts of biological information from data produced by NGS. Recent transcriptomic studies have employed 454 sequencing of normalised cDNA libraries (Cantacessi et al., 2010a-d). The normalisation process allows transcripts to be studied qualitatively, but this approach does not allow differential gene expression to be investigated quantitatively. Exploring differential transcription among stages, sexes and tissues of parasitic nematodes and other helminths provides unique insights into molecular changes occurring, for example, during development and reproduction. Future studies involving the sequencing of non-normalised cDNA libraries by, for instance, Illumina technology (Bentley et al., 2008; www.Illumina.com) will provide an avenue to explore essential biological pathways in parasitic nematodes, such as those linked to the development of neuronal tissue, the formation of cuticle and the digestion of host haemoglobin in H. contortus (cf. Cantacessi et al., 2010b) and in mitochondrial and amino acid metabolism in N. americanus (cf. Cantacessi et al., 2010c). However, the incorporation of gene expression data will inevitably pose new computational challenges for the correct assembly and analysis of sequence datasets and, for instance, for the accurate prediction of alternatively spliced transcripts. Currently, due to the lack of complete genomic sequences for parasitic nematodes, newly generated transcriptomic and genomic sequence datasets need to be assembled de novo, which means that pooled reads are assembled without a bias towards known sequences (e.g., Zerbino and Birney, 2008). Due to the amount of RNA required for NGS (~5–10 μg; Meyer et al., 2007; Jarvie and Harkins, 2008), transcriptomes from small nematodes usually originate from multiple individuals, potentially leading to an increased complexity of the sequence data acquired (linked, for instance, to single nucleotide polymorphisms [SNPs] and other types of sequence variation) and posing some challenges for the assembly. In terms of complexity, computational and time requirements, de novo assemblies are orders of magnitude slower and much more computer-memory intensive than knowledge-based (mapping) assemblies, in which reads are aligned and assembled against an existing “backbone” sequence (Flicek and Birney, 2009). In addition, reliable de novo assemblies are heavily dependent upon the availability of long reads (N100 bases) and of high-coverage, paired-end sequence data

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(Flicek and Birney, 2009; Pepke et al., 2009). In previous studies, the complementary nature of the 454 and Illumina sequencing platforms has allowed the assembly of raw reads into large scaffolds without need for a reference sequence (e.g., Nagarajan et al., 2010; Reinhardt et al., 2009; Tsai et al., 2010). Thus, clearly, the 454 sequence data assembled in previous studies (e.g., Cantacessi et al., 2010a-d) should assist future de novo assemblies of Illumina data (both transcriptomic and genomic) for the species investigated to date. In the absence of a reference genome for a parasitic nematode, a correct assembly of ESTs is a crucial step for examining coding genes and, ultimately, addressing biological questions regarding gene and protein function. Knowledge of the function of genes and gene products from organisms is predicted using a process known as ‘sequence annotation’, which has been defined as “the process of gathering all available information and relating it to the sequence assembly both by experimental and computational means” (Gregory et al., 2006). Currently, the annotation of sequence data from parasitic nematodes is primarily based on comparisons with data available in public databases accessible via multiple portals (see Cantacessi et al., 2010a-d) and updated at different rates. For instance, the SWISSPROT database (http://au.expasy.org/sprot/) accepts corrections from its user community, whereas GenBank (http://www.ncbi.nlm.nih.gov/ genbank/) only accepts corrections from the author of an entry (cf. Karp, 1998), thus significantly affecting the accuracy and speed with which new sequences are annotated. In addition, some informationmanagement systems evolve to efficiently incorporate data from large-scale projects, but often, the annotation of single records from the literature is slow and cumbersome (cf. Benitez-Paez, 2009). Given that, presently, the annotation of sequence data for parasitic nematodes relies heavily on the use of bioinformatic approaches and already annotated/curated sequence data for a wide range of organisms (e.g., Cantacessi et al., 2010a-d), these observations are particularly crucial and deserve further consideration. For instance, the analyses and annotation of large-scale transcriptomic sequence datasets for parasitic nematodes could be considerably facilitated through the establishment of a ‘reference’ website for molecular parasitologists, which could provide regular releases of newly developed and validated bioinformatic pipelines for the analyses of sequence datasets as well as links to regularly updated databases. In the future, the establishment of a ‘centralized’ consortium to facilitate the sharing and optimization of bioinformatic pipelines for sequence processing and annotation and, more broadly, to allow access to new sequence data, as well as experimental protocols and relevant literature would be very useful to the scientific community. Typically, the annotation of peptides inferred from the transcriptomes of parasitic nematodes is performed by assigning predicted biological function/s based on comparison with existing information available for C. elegans and for other organisms in public databases (e.g., WormBase, www.wormbase.org; InterPro, http://www.ebi.ac.uk/ interpro/; Gene Ontology, http://www.geneontology.org/; OrthoMCL, http://www.orthomcl.org/; BRENDA, http://www.brenda-enzymes. org/) (cf. Cantacessi et al., 2010a-d). Using this approach, predictions of key groups of molecules were made in relation to their function and essential role(s) in biological processes (Cantacessi et al., 2010ad). Such groups included the SCP/TAPS proteins and molecules linked to the physiology of the nervous system, to the formation of the cuticle, proteases and protease inhibitors, and protein kinases and phosphatases (Cantacessi et al., 2010a-d). However, in order to support data inferred from bioinformatic analyses of sequence data, experimental validation is now required. In particular, extensive laboratory experiments need to be conducted to evaluate the functions of molecules in the parasites studied and/or in a suitable surrogate organism. RNAi has been applied to a number of strongylid nematodes of animals, but success has been relatively limited (e.g., Boag et al., 2003; Geldhof et al., 2006, 2007; Issa et al., 2005; Knox et al., 2007; Kotze and Bagnall, 2006; Samarasinghe et al., 2011; Visser et al., 2006; Zawadzki et al.,

2006). Current evidence (Geldhof et al., 2007; Zawadzki et al., 2006) suggests that a number of nematodes of animals, including H. contortus, lack critical components of the RNAi machinery (see Geldhof et al., 2007; Knox et al., 2007; Viney and Thompson, 2008). Transgenesis and gene complementation studies have shown considerable promise for evaluating the function of genes from some parasitic nematodes (e.g., Lok, 2009; Hu et al., 2010; Stepek et al., 2010). In particular, a study demonstrating successful transgenesis in the parasitic nematode Parastrongyloides trichosuri (Rhabditida) (see Grant et al., 2006) as well as the use of C. elegans as a surrogate system for the analysis of the function of some genes from selected members of the Strongylida and Rhabditida (see Lok, 2009; Hu et al., 2010; Stepek et al., 2010) provide substantial promise and scope for the application of this methodology to functional genetic studies of selected groups of parasitic nematodes. C. elegans represents a useful model for studying aspects of, for example, the neurobiology of parasitic nematodes, including A. caninum (see Bhopale et al., 2001), H. contortus (see Jagannathan et al., 1999; Li, 2005; Li et al., 2000, 2001; Shompole and Jasmer, 2003) and T. colubriformis (see McVeigh et al., 2006). For instance, due to the similarities in amphid structures between the dauer (arrested or hypobiotic) form of C. elegans (see Riddle and Albert, 1997) and the L3s of various parasitic nematodes, such as H. contortus (see Li et al., 2001), A. caninum (see Bhopale et al., 2001) and Strongyloides stercoralis (see Castelletto et al., 2009), it was proposed that similar molecular mechanisms control the development of these stages (Bürglin et al., 1998; Castelletto et al., 2009; Tissenbaum et al., 2000). The molecular and biochemical pathways that control dauer formation in C. elegans have been the subject of detailed studies (e.g., Riddle and Albert, 1997; reviewed by Li, 2005; Hu, 2007; Fielenbach and Antebi, 2008; Wang et al., 2009b). The formation of dauer in this nematode is mediated by a specific pheromone, whose secretion is determined by parallel insulin-signalling pathways in four types of sensory neurons, namely ADF, ASG, ASI and ASJ (Bargmann and Horvitz, 1991; Bürglin et al., 1998; Riddle and Albert, 1997). One pathway is mediated by the ‘dauer formation’ DAF-2 (insulin-like) receptor (Kimura et al., 1997; Riddle and Albert, 1997), whereas the second is regulated by the combination of DAF-7 (transforming growth factor-ß [TGF-ß]) and DAF-11 (guanylate cyclase) (Riddle and Albert, 1997; reviewed by Li, 2005); loss of function in either pathway results in dauer, indicating that these pathways function independently (reviewed by Li, 2005). The daf-22 gene has been proposed to play a crucial role the synthesis of the dauer-pheromone, based on the observation that daf-22(m130), a dauer-defective mutant C. elegans, accumulates a hydrophilic precursor of precursors of this molecule with similar activity (Golden and Riddle, 1985; Hu, 2007). Although the precise molecular function of daf-22 is still unclear (cf. Hu, 2007), the nuclear receptor daf-12 is hypothesised to represent the final, common target of regulatory pathways for dauer (Hu, 2007). In addition, the fork head boxO (FoxO) transcription factor, encoded by daf-16, is known to stimulate a dauer-specific pattern of expression that promotes the formation of dauer (e.g., Kenyon et al., 1993; Lin et al., 1997; Ogg et al., 1997). For S. stercoralis and H. contortus, homologues of C. elegans daf-16 have been isolated and characterised (designated Ss-daf-16 and Hc-daf-16.1 and Hc-daf16.2, respectively; Massey et al., 2003; Hu et al., 2010). Using transgenesis, both Ss-daf-16 and Hc-daf-16.2 were shown to restore daf-16 function to a C. elegans strain carrying a null mutation at this locus (Hu et al., 2010; Massey et al., 2003). Another study (Datu et al., 2009) demonstrated the involvement of the insulin-like signalling pathway in a cascade of events that lead to the activation of the A. caninum L3s (following serum-stimulation), and the authors postulated the involvement of a daf-16 homologue in the molecular events linked to the resumption of feeding. The results from these studies indicate significant scope for further (functional) explorations of other molecules linked to key biological pathways in parasitic nematodes. For example, the H. contortus datasets analysed to date (Cantacessi

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et al., 2010b) could be investigated for the presence of other members of the insulin-signalling pathway, which could then be subjected to functional studies employing transgenesis, in a similar way as described by Hu et al. (2010), in the first instance. Elucidating, in detail, the function(s) of molecules involved in the transition from free-living to parasitic stages as well as among the different parasitic stages of socio-economically parasitic nematodes should have enormous implications for a fundamental understanding of their biology, ecology and adaptation, and will provide unique insights which could lead to new intervention or control strategies. Currently, bioinformatic methods are increasingly being used to predict proteins as novel anthelmintic targets (Caffrey et al., 2009; Doyle et al., 2010; Krasky et al., 2007; Woods and Knauer, 2010). This approach involves ‘filtering’ (Geary et al., 1999; McCarter, 2004) and usually includes inferring targets based on key principles and requirements (cf. Seib et al., 2009). First, target proteins should play a key (i.e., essential) role in fundamental biological processes of the parasite, such that the distruption of the molecule or its gene will damage and/or kill the parasite. In the absence of functional genomic information for most parasitic nematodes of animals, the essentiality of genes and/or gene products is inferred based on effective knockdown or knockout of homologues/orthologues in C. elegans (see Geary et al., 1999; Gilleard et al., 2005; Rufener et al., 2010). In addition, since drugs against parasite molecules could also theoretically bind to and affect host molecules, candidate proteins should be unique to the parasite or, at least, show significant differences in sequence and structure from host homologues (Linares et al., 2006; Seib et al., 2009). In parasitic nematodes, the prediction of drug target candidates is assisted by using extensive information on function and essentiality in C. elegans, D. melanogaster, M. musculus and/or S. cerevisiae, accessible via public databases (e.g., http://www.wormbase.org, http://flybase. org/, http://www.informatics.jax.org/ and http://www.yeastgenome. org/). In addition, since most effective drugs achieve their activity by competing with endogenous small molecules for a binding site on a target protein (Hopkins and Groom, 2002), the amino acid sequences predicted from essential genes can be screened for the presence of conserved ligand-binding domains (Chang et al., 2009; Hopkins and Groom, 2002) and lists of inhibitors known to specifically bind to such domains can be compiled (Chang et al., 2009). Interestingly, ~50% of the targets predicted to date for N. americanus (see Cantacessi et al., 2010c) and Oe. dentatum (see Cantacessi et al., 2010d) represented proteins belonging to the same categories, i.e. zinc metalloproteases, amino peptidases, guanosine triphosphatases (GTPases), protein tyrosine kinases (PTKs) as well as serine/threonine protein phosphatases (STPs) and kinases (STKs) (Cantacessi et al., 2010c,d). Multiple cellular signalling pathways function through the activity of small GTP-binding proteins (GTPases) to regulate multiple biological processes, such as transmembrane signal transduction, cytoskeletal reorganisation, gene expression, intracellular vesicle trafficking, microtubule organisation and nucleocytoplasmic transport (reviewed by Konstantinopoulos et al., 2007). GTPases are small (~20–28 kDa), monomeric proteins belonging to six families (i.e., Ras, Rho, Rab, Arf, Ran and Rad; Lundquist, 2007) which act as bi-molecular switches between two conformational states (i.e., GDP-bound [“inactive” state] and GTP-bound [“active” state]) and hydrolyze GTP (van Golen, 2009). In humans, the aberrant regulation of GTPases is linked to a number of dysfunctions, including neurological and developmental disorders and cancer (van Golen, 2009). In addition, intracellular pathogenic bacteria, such as Mycobacterium tuberculosis, are known to target host GTPases to evade host immune responses to facilitate the infection process (Meena, 2010). Such information has stimulated efforts to develop novel therapeutic strategies to inhibit the function of GTPases (Konstantinopoulos et al., 2007). For instance, treatments with farnesyltransferase inhibitors, to block the oncogenic properties of Ras GTPases, have been shown to be effective in significantly reducing the progression of

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various forms of cancer, including carcinomas of the colon, pancreas and lung, neurofibrosarcoma and chronic myelogenous leukaemia, in experimental animals (Gibbs et al., 1994; Gibbs and Oliff, 1997) and the migration and organisation of the cytoskeleton of human prostate cancer cells (Virtanen et al., 2010). Although the structure of individual small GTPases is conserved across eukaryotes, analyses of datasets for N. americanus and Oe. dentatum (see Cantacessi et al., 2010c,d) have allowed the identification of significant differences in sequence within specific regions of nematode and host transcripts encoding GTPases. These differences might be considered in future studies, aimed at assessing the possibility of designing and synthesising selective and specific inhibitors against parasite GTPases. Homology modelling (de Beer et al., 2009; Wieman et al., 2004), X-ray crystallography and docking studies (e.g., Asojo et al., 2005a,b; Lorber and Shoichet, 2005; McInnes and Fischer, 2005; Villoutreix et al., 2007; Yarnitzky et al., 2010) should assist in this process. Protein kinases (PTKs) were also consistently predicted as targets for nematocides in N. americanus and Oe. dentatum (see Cantacessi et al., 2010c,d). PTKs belong to a large family of proteins regulating development, cell division, differentiation and metabolism in many organisms; these molecules are considered the second most important group of drug targets after GPCRs (see Cohen, 2002; Dissous et al., 2007). The family of PTKs comprises cell surface receptors (RTK) and non-receptor or cytosolic (CTK) kinases. The unregulated activation or over-expression of PTKs is considered to play a central role in the induction of various forms of cancer (Capdeville et al., 2002; Kim et al., 2009; Lemmon and Schlessinger, 2010; Xu and Huang, 2010; Zwick et al., 2001). Integrated genomic–bioinformatic–chemoinformatic approaches have been employed for the identification and screening effective PTK inhibitors as therapeutic agents for the treatment of human leukaemia (e.g., Peng et al., 2003; Vangrevelinghe et al., 2003). For example, in studies aimed at identifying novel inhibitors of a human tyrosine kinase involved in the development and progression of chronic myelogenous leukaemia, 15 compounds were selected following in silico screening of a database of 200,000 known inhibitors (Peng et al., 2003). Of these compounds, eight were shown to selectively inhibit the growth of leukaemia in vitro (Peng et al., 2003). In another study, novel and selective inhibitors of caseine kinase II (CK2) were identified via in silico screening of a database containing ~400,000 compounds, followed by in silico docking (Vangrevelinghe et al., 2003). These examples indicate the advantages of using computer-aided tools for the rational prediction and design of drugs for subsequent in vitro and in vivo efficacy testing (cf. Hammami and Fliss, 2010). Nonetheless, it is clear that any compound shown to be efficacious must also be rigorously tested for its safety according to international guidelines (reviewed by Dorato and Buckley, 2007; http://www.ich.org/cache/compo/276-254-1.html). Because of the regulatory role that PTKs play in a number of signalling pathways in the cell, interference with their activity can result in the disruption of fundamental homeostatic processes in parasites (reviewed by Liotta and Siekierka, 2010). In the last years, PTKs have received particular attention as drug targets in protozoan parasites, such as species of Plasmodium, Leishmania and Trypanosoma and helminths, including Echinococcus multilocularis and Schistosoma mansoni (reviewed by Liotta and Siekierka, 2010). In the latter two species, PTK inhibitors (i.e., tyrphostins AG1024 and AG538) have been shown to significantly affect the development and survival of the adult parasite through the blockage of glucose uptake (reviewed by Liotta and Siekierka, 2010). In another study, the inactivation of S. mansoni PTKs with herbimicin A (an Src kinase inhibitor) was demonstrated to interfere with mitosis, thus significantly affecting the expression of proteins essential for egg production, including the formation of the eggshell (Knobloch et al., 2006). Although the crystal structures of PTKs from parasitic nematodes have not yet been defined, progress has been made in the identification and design of effective inhibitors based on homology models for protein kinases from humans (Liotta and Siekierka, 2010). There is evidence that the active sites of

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parasite PTKs display subtle differences compared with their human counterparts (Liotta and Siekierka, 2010), which is considered promising for the development of parasite-specific kinase inhibitors. However, much more study is required to establish the potential of PTK inhibitors as nematocides. This is obviously a research area worthy of pursuit. In the future, improved bioinformatic prediction and prioritisation of potential drug targets in parasitic nematodes will depend on the availability of complete genome sequences. Global repertoires of drug targets could be inferred. For instance, the parasite kinome (= the complete set of kinase genes in the genome) could represent a unique opportunity for the design of parasite-selective inhibitors (Liotta and Siekierka, 2010). In addition, the integration of genomic, transcriptomic and proteomic data will be crucial to identify other groups of molecules essential to parasite survival and development, which could represent drug target candidates. Clearly, next-generation sequencing will provide the efficiency and depth-of-coverage required to rapidly define the complete genomes of eukaryotic pathogens of socio-economic importance. In conclusion, the present article has reviewed recent progress on the transcriptomes of some strongylid nematodes of socio-economic importance. These transcriptomic datasets, which are now available via public databases (i.e., http://www.ncbi.nlm.nih.gov and http:// research.vet.unimelb.edu.au/gasserlab/index.html), represent an invaluable resource for the future assembly and annotation of respective genomes. This progress in genomic sequencing and annotation as well as the integrated use of 'omic technologies opens the door to understanding the molecular biology of these parasitic nematodes on an unprecedented scale and major biotechnological outcomes relating to the development of new drugs, vaccines and diagnostic tools. Acknowledgements Funding from the Australian Research Council, the Australian Academy of Science, the Australian–American Fulbright Commission, Melbourne Water Corporation and the National Health and Medical Research Council is gratefully acknowledged (RBG). Support from the Victorian Life Sciences Computation Initiative (VLSCI) and IBM Collaboratory is also acknowledged (RBG). CC wishes to thank the many colleagues who contributed to or supported her research project. References Abubucker S, Martin J, Yin Y, Fulton L, Yang SP, Hallsworth-Pepin K, et al. The canine hookworm genome: analysis and classification of Ancylostoma caninum survey sequences. Mol Biochem Parasitol 2008;157:187–92. Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, et al. Complementary DNA sequencing: expressed sequence tags and human genome project. Science 1991;252:1651–6. Albonico M. Methods to sustain drug efficacy in helminth control programmes. Acta Trop 2003;86:233–42. Albonico M, Smith PG, Hall A, Chwaya HM, Alawi KS, Savioli L, et al. A randomized controlled trial comparing mebendazole and albendazole against Ascaris, Trichuris and hookworm infections. Trans R Soc Trop Med Hyg 1994;88:585–9. Albonico M, Bickle Q, Ramsan M, Montresor A, Savioli L, Taylor M, et al. Efficacy of mebendazole and levamisole alone or in combination against intestinal nematode infections after repeated targeted mebendazole treatment in Zanzibar. Bull World Health Org 2003;81:343–52. Allen JE, Maizels RM. Diversity and dialogue in immunity to helminths. Nat Rev Immunol 2011;11:375–88. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403–10. Alwine JC, Kemp DJ, Stark GR. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci USA 1977;74:5350–4. Anderson RC. Nematode parasites of vertebrate. Their development and transmission 2nd Edition. 2000. Wallingford, UK: CABI Publishing; 2000. Anderson N, Dash KM, Donald AD, Southcott WH, Waller PJ. Epidemiology and control of nematode infections. In: Donald AD, Southcott WH, Dineen JK, editors. The epidemiology and control of gastrointestinal parasites of sheep in Australia. Australia: CSIRO; 1978. p. 23–51.

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