The Tao survivorship of schistosomes: implications for schistosomiasis control.

International Journal for Parasitology xxx (2016) xxx–xxx

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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara

Review Article

The Tao survivorship of schistosomes: implications for schistosomiasis control Pengfei Cai ⇑, Geoffrey N. Gobert, Hong You, Donald P. McManus ⇑ Molecular Parasitology Laboratory, QIMR Berghofer Medical Research Institute, Queensland, Australia

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Article history: Received 27 November 2015 Received in revised form 6 January 2016 Accepted 6 January 2016 Available online xxxx Keywords: Schistosomiasis Host-schistosome interface Immunomodulation microRNAs Epigenetic control Transmission blocking vaccine Drug target identification CRISPR-Cas system

a b s t r a c t Schistosomiasis, caused by blood flukes of the genus Schistosoma, is a major public health problem which contributes substantially to the economic and financial burdens of many nations in the developing world. An array of survival strategies, such as the unique structure of the tegument which acts as a major hostparasite interface, immune modulation mechanisms, gene regulation, and apoptosis and self-renewal have been adopted by schistosome parasites over the course of long-term evolution with their mammalian definitive hosts. Recent generation of complete schistosome genomes together with numerous biological, immunological, high-throughput ‘‘-omics” and gene function studies have revealed the Tao or strategies that schistosomes employ not only to promote long-term survival, but also to ensure effective life cycle transmission. New scenarios for the future control of this important neglected tropical disease will present themselves as our understanding of these Tao increases. Ó 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Schistosomiasis remains one of the most serious public health issues in the developing world, afflicting more than 240 million people, with close to 800 million at risk (Steinmann et al., 2006; Weerakoon et al., 2015). This neglected tropical disease is caused by infection with blood fluke (trematode) worms of the genus Schistosoma. The three main species of clinical relevance are Schistosoma mansoni, Schistosoma japonicum and Schistosoma haematobium. The annual number of disability-adjusted life years (DALYs) lost due to schistosomiasis was estimated to be 3.3 million in 2010, ranking it as third in the list of global neglected diseases (Hotez et al., 2014). Infection with S. mansoni and S. japonicum results in hepatic and intestinal schistosomiasis, associated with the formation of granulomas and fibrosis around trapped eggs lodged in the liver or intestinal wall. Schistosoma haematobium infections result in urogenital schistosomiasis and the associated pathologies include fibrosis of the bladder and bladder cancer, and there is an increased risk of HIV infection and infertility in women with urogenital schistosomiasis (Brindley and Hotez, 2013). The chronic and debilitating symptoms associated with ⇑ Corresponding authors. Tel.: +61 7 3362 0406; fax: +61 7 3362 0104 (P. Cai). Tel.: +61 7 3362 0401; fax: +61 7 3362 0104 (D.P. McManus). E-mail addresses: [email protected] (P. Cai), Don.McManus@ qimrberghofer.edu.au (D.P. McManus).

schistosomiasis contribute significantly to the current cycle of poverty existing in many developing countries in the tropics and subtropics. Schistosomes are dioecious and have a complex lifecycle involving an aquatic snail as an intermediate host and a mammalian definitive host (Weerakoon et al., 2015). Eggs produced by the female adult worm are released from the definitive host and hatch in freshwater. The released miracidia then penetrate the snail host and develop asexually into mother and then daughter sporocysts, within which cercariae are produced that are in turn released into water. The schistosome lifecycle continues when the freeswimming cercariae infect a mammalian host. After skin penetration, the larvae transform into schistosomula which migrate via a route involving the epidermis, the epidermal-dermal basement membrane, and dermis (Mountford and Trottein, 2004). Once in the dermis, the schistosomula rapidly exit after locating capillaries or lymphatic vessels in which they are carried to the heart and lungs, and finally arrive at the hepatic portal system, where the juveniles pair up and mature sexually. Schistosomes in copula then migrate to the mesenteric veins (S. mansoni and S. japonicum) or the pelvic venous plexus (S. haematobium), where the female worms lay eggs intravascularly, with patency periods varying between species. In contrast to S. haematobium and S. mansoni, which both generally only infect humans, S. japonicum is parasitic in humans and more than 40 other species of mammals which act as reservoir hosts. The adult parasites can survive in the harsh

http://dx.doi.org/10.1016/j.ijpara.2016.01.002 0020-7519/Ó 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Cai, P., et al. The Tao survivorship of schistosomes: implications for schistosomiasis control. Int. J. Parasitol. (2016), http://dx.doi.org/10.1016/j.ijpara.2016.01.002

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P. Cai et al. / International Journal for Parasitology xxx (2016) xxx–xxx

microenvironment of the blood vessels of the definitive hosts for several decades. The schistosome lifecycle is shown in Fig. 1. Currently, praziquantel (PZQ)-based chemotherapy, combined with morbidity management, are the predominant strategies adopted for the treatment and control of schistosomiasis (Mutapi et al., 2011b; Chen, 2014). This strategy reflects the fact that no

effective vaccine is available to prevent schistosomiasis, despite a large panel of antigen candidates having been tested over the past decades (McManus and Loukas, 2008). Theoretically, schistosomiasis should be manageable since multiple stages of its causative agents can be targeted for control and even for elimination, but this contrasts with the long-lasting prevalence of the disease in many

Fig. 1. Diagram illustrating the key survival strategies adopted by schistosomes throughout the life cycle. Cercariae sense environmental stimuli and invasion is further mediated by protein kinase signalling pathways at the initial step of penetration. Different histolytic enzymes are employed by Schistosoma mansoni and Schistosoma japonicum (SmCE and SjB2, respectively) as the major invasive peptidase to facilitate penetration after the cercariae adhere to mammalian skin. Schistosomula use a wide variety of neurotransmitters to control their motility via interacting with neurotransmitter receptors or transporters, important factors for their migration. The tegument and digestive tract are two important host-parasite interfaces crucial for the survival of schistosomes. In the tegument (Teg), antigenic variation (Sj-tetraspanin (TSP)-2, -5, -18 and -22) and mimicry (a covered with host non-immune antibodies and complement components) commonly take place. Also, the worms utilise receptors (e.g., insulin receptor (IR) I and transforming growth factor-beta (TGF-b) II receptor) to facilitate their development and fecundity through interaction with host-derived ligands. The schistosome digestive tract is responsible for nutrient acquisition and waste disposal. The oesophageal gland (OG) initiates the digestion of host blood, and several members of the schistosome micro-exon gene (MEG) and venom allergen-like (VAL) families may be involved in this process. A number of proteases (e.g., cathepsin B1, L1, L2, D and C) continue the process in the gut lumen. The excreted/secreted protein (ESP) of mature schistosome eggs is recognised as the main mediator in the triggering and manipulation of the host immune response by orchestrating a range of immune cells, particularly dendritic cells (DCs), regulatory T (Treg) cells and neutrophils. The asexual reproduction of a sporocyst produces a large number of cercariae, a process which greatly increases the chance for infection of definitive hosts. Gene regulatory mechanisms are crucial for schistosomes with their complex life cycle stage transitions For example, bivalent histone H3 methylation (at position K4 and K27 at the N-terminus) only occurs in the cercarial stage, and this process has been suggested to trigger the transcription of approximately 120 genes in S. mansoni. The microRNA (miRNA) content in total small RNAs varies in different developmental stages and a set of miRNAs has been confirmed to be associated with the development and sexual maturation of these parasites, highlighting their pivotal roles in gene regulation during transit of the developmental stages. In schistosome germline cells (posterior ovary (PO) and testes (T)), Argonaute 2 (AGO2) – associated endo-siRNAs suppress transposable element (TE) activity, acting as a guardian for genome stability. An intrinsic apoptosis pathway and a population of neoblast-like cells have been identified in schistosomes, suggesting that a self-renewal mechanism contributes to the vitality of the parasite within the blood vessels of the definitive host. Crucial genes expressed in different stages and tissues/cells are denoted in brackets, while the miRNA content (%) in the total small RNA population is shown in the different developmental stages. FGFR, fibroblast growth factor receptor orthologue; G, gynaecophoric canal; GCP, Gynaecophoral canal protein; GL, gut lumen; HSC, hepatic stellate cells; HSP, heat shock protein; SEA, soluble egg antigen; SSC, stem somatic cells. (Modified from Fig. 1, Cai et al., 2016.)



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endemic areas. This is, in part, the result of the failure to implement well-organised and effective social, economic and educational control policies, including improved sanitation measures and, to a lesser degree, climatic and human-made ecological changes (Molyneux, 2006; Adenowo et al., 2015). But, in addition, the Tao (a Chinese word meaning ‘‘the Way”, or ‘‘Path”) of schistosome survival, evolved over the long-term association of the parasite with its hosts, has contributed significantly to the parasite’s persistence. Completed genome sequencing projects and an increasing array of biological, immunological, high-throughput ‘‘-omics” (Table 1) and gene function studies (Collins et al., 2013; Hagen et al., 2014) have been carried out over the past several years on schistosomes. These studies have provided invaluable, publically available genomic sequences for the three main schistosome species, but also gene (including mRNA and small RNA transcripts) expression profiles for different developmental stages and between sexes (Gobert et al., 2009; Cai et al., 2011). As well, key information on the molecular and genetic characterisation of many features of the tegument, excretory-secretory proteomes (PerezSanchez et al., 2006), the degradome, epigenome, glycome, kinome, immunome and exosome of schistosomes has been obtained (Table 1). These critical data provide novel insights into the biology, pathogenesis and host-parasite interplay of schistosome parasites at the molecular level, thus revealing the survival strategies or Tao of these pathogens. Here, we review recent advances in the Tao of survival adopted by schistosomes, discuss the possibility of exploiting their Achilles’ heel, whereby novel approaches can be developed for the control of the ancient and persistent parasitic disease of schistosomiasis.

step in the mammalian host invasion process remains elusive, and questions remain as to how cercariae sense and are attracted to the skin of the definitive host and what signals trigger them to release the gland contents from the acetabular gland complex to aid in penetration (Haas et al., 2008). It has been proposed that ciliated sensory papillae on the cercarial surface can sense chemicals and temperature changes, which help in the detection of mammalian hosts, although precise details regarding the molecular mechanism involved are lacking (Haeberlein and Haas, 2008; Lee et al., 2013). However, it has been shown recently that the protein kinase signalling pathway is involved in sensory perception by cercariae (Ressurreicao et al., 2015). Cercariae exhibited distinct protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK) activities when exposed to different temperatures (e.g., 37 °C vs 24 °C), or following stimulation by medium-chain free fatty acids such as linoleic acid (LA) (Ressurreicao et al., 2015). After skin adhesion, the larvae secrete invasive peptidases from acetabular glands of cercariae appear to aid in the penetration process (Ingram et al., 2011). It is noteworthy that distinct proteolytic enzymes are employed by S. mansoni and S. japonicum during the penetration process, a feature that may determine the speed of invasion (Ingram et al., 2011). A serine peptidase, cercarial elastase (SmCE), plays a key role in S. mansoni skin invasion, although as in S. japonicum, the cysteine peptidase cathepsin B2 (SjCB2) has also been recently implicated as the major histolytic enzyme employed during invasion (Ingram et al., 2011; Liu et al., 2015a).

2. Cercaria – the invader

During penetration of the definitive host skin, schistosome cercariae shed their external glycocalyx layer and tails, and transform into other larval forms, termed schistosomula (Fig. 1B). These larvae then migrate from the skin, via the heart and lungs, to their final site of development, the hepatic portal vessel; proper motor control and the neuromuscular system are thought to be crucial to this migration (Ribeiro and Geary, 2009). Schistosoma japonicum is distinctive by its higher speed and success rate of migration compared with the other schistosomes (Ruppel et al., 2004). Several studies on S. mansoni have shed light on the molecular

Schistosoma spp. shed cercariae (Fig. 1A) from intermediate snail hosts under the induction of light, implying they are equipped with photoreceptors, such as the guanine protein coupled receptor (GPCR), rhodopsin, which has been implicated in the photoreception process (Hoffmann et al., 2001). These infectious cercariae are covered with a carbohydrate-rich ‘glycocalyx layer’, which protects them from the high osmotic pressure of fresh water (Samuelson and Caulfield, 1985; Nanduri et al., 1991). The initial

3. Schistosomule – the traveller

Table 1 Selected ‘-omics’ studies on Schistosoma mansoni, Schistosoma japonicum and Schistosoma haematobium. -omics

Species

References

Genomics

S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.

The Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium (2009) Berriman et al. (2009) and Protasio et al. (2012) Young et al. (2012) Hu et al. (2003), Liu et al. (2006) and Gobert et al. (2009) Protasio et al. (2012) Young et al. (2012) Liu et al. (2009, 2015a) and Liao et al. (2011) Castro-Borges et al. (2011), Ingram et al. (2011) and Sotillo et al. (2015) Higon et al. (2011) McWilliam et al. (2013) Jang-Lee et al. (2007) and Smit et al. (2015) Geyer et al. (2013) Carneiro et al. (2014) and Roquis et al. (2015) Geyer et al. (2013) Liu et al. (2014) Hao et al. (2010) and Cai et al. (2011, 2013) de Souza Gomes et al. (2011), Simoes et al. (2011) and Marco et al. (2013) Driguez et al. (2016) Gaze et al. (2014) Mutapi et al. (2008, 2011a) Wang et al. (2006) Andrade et al. (2011) Stroehlein et al. (2015) Wang et al. (2015a) Nowacki et al. (2015) and Sotillo et al. (2016)

Transcriptomics

Proteomics

Glycomics Epigenomics

Degradomics Small RNAomics Immunomics

Kinomics

Exosomics

japonicum mansoni haematobium japonicum mansoni haematobium japonicum mansoni haematobium japonicum mansoni japonicum mansoni haematobium japonicum japonicum mansoni japonicum mansoni haematobium japonicum mansoni haematobium japonicum mansoni


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mechanisms involved in the neuronal signalling pathway associated with motor effects based on investigations of neurotransmitters (McVeigh et al., 2009), transporters (Patocka and Ribeiro, 2013) and GPCRs in schistosomula (Hamdan et al., 2002; ElShehabi and Ribeiro, 2010; El-Shehabi et al., 2012; Patocka et al., 2014). Neurotransmission in schistosomes involves a wide range of neurotransmitters, including some ‘classical’ ones: acetylcholine (ACh), glutamate, and biogenic amines (e.g., serotonin (5hydroxytryptamine; 5HT), histamine (HA), dopamine (DA) and noradrenaline (NA)), as well as several families of neuropeptides (Ribeiro and Geary, 2009). These substances mediate neural signalling via their interaction with neurotransmitter receptors and transporters (Ribeiro and Patocka, 2013). RNA interference (RNAi)-immediate knockdown of the serotonin receptor (Sm5HTR) (Patocka et al., 2014) and G protein-coupled acetylcholine receptor (SmGAR) (MacDonald et al., 2015) in S. mansoni schistosomula results in a hypoactive phenotype, indicating the pathways induced by these receptors contribute to larval motility. In contrast, a serotonin transporter (SmSERT) (Patocka and Ribeiro, 2013) and acetylcholine gated chloride channels (SmACCs) in S. mansoni (MacDonald et al., 2014) have been shown as inhibitory modulators of neuromuscular function. These observations indicate that the motility of the juvenile schistosome is coordinated by the nervous system in response to environmental cues. In addition, it should be kept in mind that the neuromuscular system also controls other biological processes in schistosomes such as penetration, feeding, digestion, waste disposal, reproductive activities and egg excretion (Ribeiro and Geary, 2009), highlighting the importance of neuronal signalling pathways in the survival of these parasites. 4. Intra-host stages – the dwellers 4.1. Functional implications of the mammalian host-schistosome interface On entry into the mammalian host, transformation of the underlying tegument, the dynamic host-interactive layer involved in multiple functions crucial for the survival of the parasite, takes place (Jones et al., 2004). The tegumental surface is a unique layer that allows the schistosome to adapt to the host blood circulatory microenvironment. Multiple evasion mechanisms including membrane turnover, antigenic mimicry and secretion of immunomodulatory molecules have been identified at the parasite-host interface, all of which contribute to schistosome survival (Skelly and Alan Wilson, 2006; Ludin et al., 2011; Hagen et al., 2015b). The schistosome digestive tract is another key host-parasite interface which is involved in the uptake of a variety of key nutrients including amino acids, amino sugars, peptides, triglycerides, fatty acids and iron, and in waste disposal (Skelly et al., 2014). A discussion of the complex ‘molecular crosstalk’ which takes place at the host-parasite interface follows. 4.1.1. Antigenic polymorphism The tegumental protein tetraspanin 2 (Sm-TSP-2) has been identified as a promising vaccine candidate against S. mansoni (Tran et al., 2006), and is now being produced under good manufacture practices (GMP) for subsequent evaluation in a Phase I clinical trial (Fonseca et al., 2015). One merit of Sm-TSP-2 as a vaccine candidate is that field isolates exhibit limited variation within the large extracellular loop (LEL) region (Cupit et al., 2011), although individual worms from a wider geographical area should be examined to corroborate these findings. In contrast, the TSP-2 homologue in S. japonicum shows extensive variation in the LEL region. In total, nine subclasses of protein (i.e., Sj-TPS-2a to Sj-TSP-2g)

were identified with no more than three subclasses being expressed in individual adult worms (Cai et al., 2008; Zhang et al., 2011). This sequence variability may represent a molecular mechanism adopted by S. japonicum to enable it to parasitise more than 40 species of mammals as reservoir hosts. Immunisation with several subclasses of Sj-TPS-2 has provided limited and inconsistent protective efficacy in murine vaccine-challenge trials, indicating that the high level of polymorphism in this molecule limits its potential as a vaccine candidate for schistosomiasis japonica (Cai et al., 2008; Zhang et al., 2011). Other tetraspanins showing variation in the LEL region include Sj-TPS-5, Sj-TSP-18 and Sj-TSP-22 (Fig. 1C) (Wu et al., 2011b), implying these proteins likely play important roles in the host-parasite interplay, although their localisation and biological function need to be determined. Analysis of the S. mansoni genome identified at least 45 microexon genes (MEGs) coding for small secreted proteins; these MEGs have an unusual structure that frequently undergoes alternative splicing (Berriman et al., 2009; DeMarco et al., 2010). Microexons make up the majority of the coding sequence of these genes but they are alternatively spliced, thus creating a variable pool of related proteins by a ‘pick and mix’ strategy. Some of these MEGs are expressed exclusively in the oesophageal gland of S. mansoni and may be secreted into the oesophageal lumen (Li et al., 2013). One of these genes, MEG-14, was denoted as a disordered chameleon, and has been implicated in the parasite’s capability of avoiding the host immune system (Dunker, 2013). Further, it has been recently shown that genes encoding polypeptides located at the host-parasite interface (e.g., MEG protein products and venom allergen-like (VAL) proteins) are under biological pressure from the host immune system. As such, these genes have undergone accelerated evolution, which is reflected in the accumulation of transposable elements and higher values of synonymous and non-synonymous substitution rates (Philippsen et al., 2015), which in turn provides a mechanism whereby schistosomes evade immune attack by the definitive host. Extensive alternative splicing events have also been revealed in adult S. japonicum worms (Piao et al., 2014; Wang et al., 2015b). Alternatively spliced genes associated with the processing of environmental information are more actively transcribed in male schistosomes, implying their encoded products are likely more involved in the host-parasite interplay (Piao et al., 2014). 4.1.2. Antigenic mimicry Schistosomes can disguise themselves through non-specific interactions with host Ig (McIntosh et al., 2006), shown by the binding of the Fc region of murine IgG2b and IgG3, by schistosome paramyosin and complement components (Castro-Borges et al., 2011). In addition, it has been shown that human non-immune IgG can bind with the large extracellular domain (LED) of Sjc23, another member of the tetraspanin family of S. japonicum, via the Fc domain (Wu et al., 2011a). More recently, using different human Ig subtypes (IgG, IgM and IgE) as bait, a comprehensive proteomic study has been carried out which revealed that a panel of proteins (Sj31, Sj22.6, tropomyosin and annexin A13) (Fig. 1C), located in the tegument of S. japonicum, were able to selectively bind to the Fc domain of host non-immune Igs, further suggesting that this mechanism is adopted by schistosomes to avoid host immune attack (Wu et al., 2015). 4.1.3. Receptors Transcriptional and genomic analysis has revealed that schistosomes are equipped with mammalian host-like receptors for insulin, progesterone, cytokines and neuropeptides, suggesting that they can monitor and respond to host-derived hormones and growth factors, presumably benefiting their growth and development within the definitive host blood system (Hu et al., 2003;



The Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium, 2009). In this respect, well-studied molecules are insulin receptors (IRs) and transforming growth factor-beta (TGF-b) receptors. The IR plays a crucial role in the regulation of glucose metabolism. In schistosomes, two IRs (IR-1 and IR-2) have been identified, with IR-1 mainly expressed in the tegument of adult worms (Fig. 1C) (Khayath et al., 2007; You et al., 2010). Transcriptional analysis of in vitro maintained male and female adult S. japonicum worms has revealed that, in response to human insulin, genes involved in growth and development are up-regulated (You et al., 2009). Bioinformatics analysis predicted that both receptors possess ligand domains for host insulin and this was confirmed by yeast two hybrid analysis and protein binding assays (You et al., 2010, 2015), implying the activation of the insulin pathway in schistosomes is triggered by the host ligand. In addition, functional studies have revealed that worm glucose levels were significantly decreased after knockdown of the expression of parasite IR genes (You et al., 2015). Furthermore, the fecundity of female worms in mice was reduced following vaccination by S. japonicum IRs (You et al., 2015). Another important type of receptor expressed on the surface of schistosome worms and within the reproductive tissues of the female parasite are TGF-b receptors I and II (Fig. 1C). It has been shown that TGF-b receptor II in S. mansoni can utilise host-derived TGF-b as a ligand and plays a role in the pairing of male and female worms (Osman et al., 2006). It has been further shown that the TGF-b signalling pathway also affects schistosome embryogenesis via a parasite-derived TGF-b homolog, SmInAct, as a ligand (Freitas et al., 2007). 4.1.4. Other tegument-associated and secretory products A selection of tegument-associated proteins, including protease inhibitors and DNase, have been recently characterised in schistosomes, that may play unique physiological functions in promoting parasite survival in the extremely hostile host microenvironment. It has been shown that a Kunitz type protease inhibitor (SmKI-1), a protein localised to the tegument and secreted from adult worms, can inhibit mammalian-derived trypsin, chymotrypsin, neutrophil elastase, FXa and plasma kallikrein, implying an involvement in the protection of the parasite from immune attack by the mammalian host (Ranasinghe et al., 2015). In S. japonicum, the serine protease inhibitor (serpin) SjB6, is a secretory protein highly expressed in the egg stage, and is proposed to help schistosome eggs deposited in host liver and intestinal tissues resist attack from host proteases (Molehin et al., 2014). Further, a novel DNase II homologue (Sjda), which is also predominantly distributed in the tegument of adult female S. japonicum, displays a typical divalent iron-independent DNA catalytic activity and may also play an important role in the host-parasite interaction (Hou et al., 2015). Recently, an analysis of extracellular vesicles released by S. mansoni schistosomula identified a panel of proteins, including heat shock protein (HSP) 70, membrane associated protein 29, and a set of small RNAs, including microRNAs (miRNAs, e.g., miR-61, miR-36a, miR-277 and miR-3479) and tRNA-derived small RNAs, tsRNAs, although the potential roles of these new participants in the host-parasite interaction remain obscure (Nowacki et al., 2015). However, the study at least partially confirmed the potential origin of circulating parasite-derived miRNAs which may serve as novel diagnostic biomarkers for schistosomiasis (Hoy et al., 2014; Cai et al., 2015). 4.1.5. Secretory proteins from the digestive tract The digestive tract of adult schistosomes, comprising the mouth, oesophageal gland, gut lumen and gastrodermis, is involved in nutrient acquisition from the host and in waste disposal. The gut is particularly important in female worms due to their significant nutrient requirements for egg production. Indeed, it has been estimated that 39,000 erythrocytes are ingested by an

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adult male worm of S. mansoni per h, compared with 330,000 erythrocytes acquired by a female worm (10 times more) (Figueiredo et al., 2015). Several protein families have been localised to the oesophageal gland, gut lumen and gastrodermis, and all are suggested to play key biological roles in digestion (Figueiredo et al., 2015). It is noteworthy that the schistosome oesophagus is not simply a conduit but plays a central role in initiating the digestion of host blood (Li et al., 2013). The lysis of host erythrocytes occurs rapidly once they enter into the posterior oesophageal compartment, which contrasts with leucocytes which are tethered in the posterior lumen, and it has been implied that MEGs and VAL may function in these processes (Li et al., 2013; Skelly et al., 2014). After processing in the oesophageal gland, a set of proteases (e.g., cathepsin B1, L1, L2, D and C), which have been shown to have significantly increased expression in the gastrodermis, are responsible for further digestion of host proteins (Caffrey et al., 2004; Liu et al., 2014). In addition, several highly immunogenic saposin homologs are localised to the gastrodermis and these are likely involved in haemolysis and lipid acquisition (Don et al., 2008) but their precise function and biological significance requires further study. 4.2. Reproductive system Male schistosomes have evolved a unique body structure, the gynaecophoric canal, for pairing. Only after constant pairing with the male can the female worm develop its reproductive apparatus and become sexually mature, a process which is a prerequisite for egg production. The precise molecular cross-talk between male and female worms after the onset of pairing remains unclear but evidence regarding the involvement of certain key molecules in this critical physiological activity of schistosomes has been accumulating. Gynaecophoral canal protein (GCP), the expression of which in male worms is limited to the gynaecophoric canal (Fig. 1C) (Osman et al., 2006) and in female worms is dramatically up-regulated post pairing (Cheng et al., 2009), has been implicated in promoting the body contact of worms in copula, thus facilitating sexual development. As mentioned earlier, following induction by host TGF-b, the TGF-b signalling pathway in S. mansoni mediates the development of the female gonads via regulating the expression of GCP (Osman et al., 2006). Also, there is evidence that key components of the TGF-b signalling pathway (TGF-bRI and II, R-Smad, Co-Smad and FKBP12) exhibit their expression in the vitelline tissue (Morel et al., 2014). Further, signal transduction processes, such as those mediated by the gonad-specific Src kinase, SmTK3 (Buro et al., 2013) and Abl tyrosine kinases (SmAbl1 and SmAbl2) (Knobloch et al., 2007; Buro et al., 2014), also play an essential role in schistosome gonad development and eggshell formation, probably in tandem with the TGF-b signalling pathway. 5. Egg – the immune modulator The host immune response during a schistosome infection is characterised primarily by a Th1 response prior to the worms becoming patent that switches to a highly Th2-polarised response upon the onset of oviposition (egg laying) (Pearce and MacDonald, 2002). It is widely accepted that schistosomes modulate the host immune response during infection using intricate molecular mechanisms. Although extensive investigations have been carried out on this important biological process, it is far from being well understood. Excreted/secreted proteins (ESPs) from the mature egg are thought to be the main modulators in triggering and manipulating the host immune response (i.e., the switch from a Th1 to a Th2-polarised response) (Hagen et al., 2015b). Proteomic analysis of ESPs from S. mansoni identified Omega-1 and the


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interleukin-4-inducing principle from S. mansoni eggs (IPSE/alpha1) as the major components of a total of 190 proteins (Mathieson and Wilson, 2010). Further functional studies have also revealed that these heavily glycosylated proteins play key roles in immunoregulation by the mammalian host but via different mechanisms. Omega-1 binds to the mannose receptor via glycans and is then internalised by dendritic cells (DCs) wherein it suppresses DC function by impairing protein (IL-12p70, IL-10 and TNF-alpha) synthesis, thus redirecting the immune response to a Th2 phenotype (Everts et al., 2012). IPSE/alpha-1 mediates IL-4 production by basophils in a novel IgE-dependent but antigen-independent pattern (Schramm et al., 2007; Meyer et al., 2015), implying it has a role in driving the development of a Th2 response (Fig. 1D). However, extreme polarisation of the Th2 response results in severe immunopathology (i.e., granuloma formation and fibrosis) in host tissues, particularly in the liver (Chuah et al., 2014). Both the host and the parasite thus have evolved strategies for modulating the immune response. For the host, the up-regulation of IL-13Ra2, a potent decoy receptor for IL-13, and the recruitment of natural and inducible regulatory T (Treg) cells to the liver, are examples of how the host may limit immunopathology during schistosomiasis (Wilson et al., 2007). Conversely, the effect triggered by the parasite eggs is also extensive via their orchestration of a variety of immune cells (Fig. 1D). For example, IL-10 secreting Treg cells can be induced in vitro by DCs stimulated with S. mansoni-derived lysophosphatidylserine via Toll-like receptor 2 (TLR2) activation (van der Kleij et al., 2002). Similarly, in S. japonicum, CD4+CD25+ Treg cells can be induced by an HSP60 (SjHSP60)-derived peptide, SJMHE1 and by SjHSP60 itself, in a TLR2- and TLR4-dependent way, respectively (Wang et al., 2009; Zhou et al., 2015). Furthermore, soluble egg antigens (SEA) of S. japonicum induce a pro-inflammatory, anti-fibrogenic phenotype in hepatic stellate cells (HSCs), a cell population key for the induction of fibrosis during schistosomiasis (Anthony et al., 2013), observations also observed in an ex vivo model of the disease (Gobert et al., 2015). In addition, the eggspecific secretory antigen of S. japonicum, SjE16.7, is a potent recruiter for neutrophils, and has been proposed as an important pathogenic factor facilitating egg excretion through host gut tissues by mediating local inflammatory responses (Wu et al., 2014). More recently, it has been shown that a distinct subset of B cells (B10), which can negatively regulate T cell immune responses, can be induced by SEA both in vitro and in vivo, and further promote the proliferation of Treg cells (Tian et al., 2015). Collectively, these observations imply that, as part of the parasite’s own sophisticated strategies, the regulatory immune response induced by worm-derived components represents a key component of the host-pathogen interplay which can be beneficial for both the host (pathology limitation) and the parasite (survival and proliferation). However, it is important to emphasise that the deposition of large numbers of S. haematobium eggs in the bladder wall has been found to be strongly associated with the development of squamous cell carcinoma (Rambau et al., 2013).

6. Miracidium – the invader; and sporocyst – the asexual proliferator Once released into freshwater, the mature miracidium (Fig. 1E), enclosed in the egg, can escape and attempts to find and establish an infection in a suitable snail intermediate host. The specificity of the snail infected by miracidia of the three schistosome species of clinical relevance (i.e., S. mansoni infects Biomphalaria pfeifferi (Africa) or Biomphalaria glabrata (the Americas), S. japonicum parasitises Oncomelania spp. (East Asia), S. haematobium infects of Bulinus spp. (Africa)) determines the geographical distribution of

schistosomiasis (Ross et al., 2002). The miracidium is bounded by a ciliated epithelium on which chemoreceptors can sense chemoattractants – miraxone has been proposed as one such chemical (Stibbs et al., 1976) – released from the snail. Once again, the protein kinase signalling pathways are involved in the downstream of the process, such components include the Fes-like tyrosine kinase, SmFes, a protein expressed in the terebratorium of the miracidium (Bahia et al., 2007). The larvae then penetrate the snail with the help of contents secreted from this gland, although the precise process has not been well investigated. After successful penetration, the larva sheds its ciliated plates, forms a new syncytial tegument, and differentiates into a mother sporocyst. Within 2–3 weeks, the mother sporocyst asexually generates daughter sporocysts, which further produce a large number of cercariae from germinative cells for infection of the definitive host (Fig. 1F), another important strategy to promote transmission. 7. Gene regulation Gene regulation plays a crucial role in many aspects of the complex life cycle and biology of schistosomes. In this post-genomics era, a number of transcriptomic studies have been undertaken in this area (Gobert et al., 2009; Piao et al., 2011; Protasio et al., 2012; Liu et al., 2014, 2015b). In addition, several epigenome, including small RNome, studies have provided novel insights on gene regulatory mechanisms in schistosomes (Cai et al., 2011; Roquis et al., 2015). 7.1. Regulatory functions of epigenetic control Epigenetic mechanisms including DNA methylation, histone modifications and those involving small non-coding RNAs can be utilised by an organism in response to external environmental factors, to enable the silencing or over expression of genes without direct impact on the genomic sequence. It remains controversial whether a functional DNA methylation mechanism is present in schistosomes, and the role of histone modifications in gene regulation during blood fluke development is not entirely clear (CabezasCruz et al., 2014). Nonetheless, by employing highly sensitive methods, Geyer et al. (2011) provided strong evidence in support of the phenomenon of DNA methylation in S. mansoni. The components within a functional DNA methylation pathway, including two DNA methyltransferase isoforms (SmDnmt2-1/2) and a methyl-CpG-binding domain (MBD) containing protein (SmMBD), were co-regulated throughout the schistosome lifecycle (Geyer et al., 2011). SmDnmt2 knockdown dramatically reduced the global level of DNA methylation (Geyer et al., 2011). Importantly, cytosine methylation was inhibited in adult worms treated by a ribonucleoside, 5-azacytidine showing an abnormal phenotype in schistosome egg production, morphology, maturation and ovarian architecture, highlighting a pivotal role for 5-methylcytosine in oviposition of the parasite, although the exact molecular mechanism underpinning this phenotype alternation needs to be uncovered. Also, the process of schistosome egg production can be affected by histone modifications. It has been shown that the nuclear receptor heterodimer SmRXR1/SmNR1 regulates the transcription of Smp14, an eggshell protein coding gene, via recruitment of two histone acetyltransferases (HATs), SmGCN5 and SmCBP1 to a specific DNA response element present in the Smp14 promoter (Carneiro et al., 2014). Once chromatin in this region is remodelled by HATs, an RNA Pol II is then recruited to the promoter, triggering the transcription of Smp14 (Carneiro et al., 2014). Recently, by using chromatin immunoprecipitation followed by massive parallel sequencing (ChIPSeq), Roquis et al. (2015) analysed the genome-wide chromatin structure of



S. mansoni at the level of histone modification in three different developmental stages (i.e., cercariae, schistosomula and adult worms). Bivalent histone H3 methylation was over-represented in the cercarial stage (Fig. 1A, B), while the methylation of H3K27 was removed during the transformation into schistosomula and continued to be absent in adult worms. Based on contrasting transcriptional levels before and after cercarial transformation, and the suggested function of bivalent methylation of histones H3K4 and H3K27 in vertebrate embryonic stem cells (Vastenhouw and Schier, 2012), specific H3 modifications in schistosomes are similar to those in stem cells, to poise transcription (Roquis et al., 2015). miRNAs are a group of small RNA molecules, ranging in length between 20 and 24 nucleotides, that are involved in gene expression regulation at the post-transcriptional level. It has been estimated that miRNAs contribute to the regulation of 60% of the human protein coding mRNA population (Friedman et al., 2009). The identification of miRNAs has been performed using deepsequencing technology in schistosomes, with comprehensive miRNA expression profiles revealed in various developmental stages or different sexes with high accuracy and coverage (Hao et al., 2010; Cai et al., 2011, 2013; de Souza Gomes et al., 2011; Marco et al., 2013). These results have denoted the regulatory functions of several miRNAs in parasite development and sexual maturation. For example, miR-7-5p, miR-61, miR-219-5p, miR-125a/b, miR-124-3p, miR-1 and bantam are all associated with sexual dimorphism in schistosomes (Cai et al., 2011; Marco et al., 2013), and a key role for miR-36-3p in embryo development has been suggested (Cai et al., 2013). Consistent with the observations by Roquis et al. (2015), it has been shown that the expression of the miRNA population is gradually decreased during the course of schistosome development following host penetration, but is increased in the egg stage (i.e., miRNAs account for 50%, 35%, 30%, 20, 10% and 25% of total small RNA population in cercariae, lung-stage schistosomula, hepatic schistosomula, adult males, adult females and eggs, respectively (Cai et al., 2013) (Fig. 1)). Another study has shown that a number of miRNAs, including sja-bantam, sja-miR-1, sja-miR-124-3p, sja-miR-2a-3p and sja-miR-36-3p, are all expressed significantly higher in the cercarial stage, compared with lung-stage schistosomula (Cai et al., 2011), implying that the silencing of potential target genes in cercariae is also fulfilled via boosted miRNA expression. 7.2. Genomic stability Piwi-interacting RNA (piRNA) is the largest class of small noncoding RNA molecules expressed in animal cells. The piRNA pathway, which is normally involved in the suppression of transposable elements (TEs) via a ‘ping-pong’ mechanism, has been characterised in Caenorhabditis elegans, fruit fly, zebrafish, and mammals, but is absent in schistosomes (Skinner et al., 2014). However, it has been shown that a key factor in the small non-coding RNA pathway is the Argonaute protein Ago2, and this is expressed in adult female schistosome reproductive tissues, particularly the posterior ovary (Fig. 1C); it predominantly binds endo-short interfering RNAs (siRNAs) derived from particular classes of TEs (Cai et al., 2012), indicating that siRNAs associated with Ago2 may exert this function in germline cells and/or fast developing embryos in schistosomes, thereby representing an important mechanism for the maintenance of genome integrity in the offspring (Cai et al., 2012; Skinner et al., 2014). 8. Apoptosis and self-renewal Apoptosis and self-renewal are fundamental cellular processes which contribute crucially to the development and survival of all

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multicellular organisms. In contrast to necrosis, apoptosis is a highly regulated and controlled process of programmed cell death (PCD), beneficial in maintaining cell populations within tissues during an organism’s lifecycle (Elmore, 2007). There are two major pathways of apoptosis signalling, namely the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. It seems only the latter exists in schistosomes, and the key components (Bcl-2 family proteins, inhibitor of apoptosis proteins and caspases) within this pathway have been identified and characterised (Lee et al., 2011). Also, gene profiling and functional studies have revealed that apoptosis is ubiquitous throughout the schistosome life cycle (Lee et al., 2014). More notably, originally identified in the related free-living flatworms Schmidtea mediterranea (Wagner et al., 2011), a cohort of planarian neoblast-like cells was subsequently identified in S. mansoni following the application of thymidine analogue labelling (Collins et al., 2013). This study demonstrated that these schistosome somatic stem cells can proliferate and differentiate into derivatives of multiple germ layers, and by RNAi it was shown that a fibroblast growth factor receptor orthologue gene (SmfgfrA) is crucial for the maintenance of this cell population (Collins et al., 2013). Future studies characterising the role of neoblast-like cells in schistosome biology could help solve gaps in our knowledge as to how these flukes are able to survive long-term within their definitive mammalian hosts. 9. Implications for schistosomiasis control As a result of the extensive progress that has been made in our understanding of Tao of survival evolved by schistosomes, can an Achilles’ heel of these parasites be found and exploited? Can new strategies be pursued that will result in the development of new interventions and improved control strategies, leading to elimination of schistosomiasis, one of the most persistent of the parasitic diseases? Several scenarios can be proposed. 9.1. Vaccination Faecal production of infected bovines, especially water buffaloes, contributes approximately 90% of the environmental S. japonicum egg contamination in endemic regions of China (McManus and Loukas, 2008), and the Philippines (Gordon et al., 2012). As such for schistosomiasis japonica, a veterinary transmission blocking vaccine may be an ideal option for controlling the disease. This may be achieved by targeting several promising antigens such as IR, since the fecundity of female worms was impaired considerably after vaccinating with Sj-IRs in several trials using the mouse model (You et al., 2015). Based on the observation that Rhesus macaques can self-cure from S. japonicum infection by blocking the function of the parasite oesophagus using intrinsic antibody, the proteins SjMEGs 4.1, 8.2, 9, 11 and VAL-7, all of which are involved in this region of the parasite, may provide additional novel potential vaccine targets (Li et al., 2015). As discussed in Section 4.1.1., S. mansoni-TSP-2 is notable by its evolutionary conservation compared with its S. japonicum homologue, and has a key function in maintaining the turnover of the tegument of the parasite, thereby representing an Achilles’ heel in this species of schistosome (Tran et al., 2010). To date, Sm-TSP-2 is still the most promising vaccine antigen against S. mansoni infection, and will shortly undergo testing in a Phase I clinical trial (Fonseca et al., 2015). For S. haematobium, an encouraging vaccine candidate is Sh-GST28, having resulted in a daily egg output reduction of >50% in vaccinated/challenged monkeys (Boulanger et al., 1995), and an acceptable safety profile in human clinical trials (Riveau et al., 2012). Furthermore, it has been observed that anti-fecundity immunity against S. haematobium is dependent on


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transmission intensity and is associated with the IgG1 isotype to worm-derived antigens (Wilson et al., 2014). Although the precise targets of this response are unknown, these findings encourage further research aimed at the discovery of potential anti-fecundity/ morbidity vaccine candidates against S. haematobium. 9.2. Identification of new drug targets Sustained long-term field use of the single drug PZQ has raised considerable concern about the development of PZQ drug resistant schistosomes. Identification of new drug targets continues to be an important area for researchers in the schistosomiasis field. Neuronal signalling and epigenetic pathways, protein kinases in the reproductive system, proteases and components in pivotal protein-protein interactions present potential targets for identifying anti-schistosomal drugs. As indicated earlier, schistosomes employ a wide panel of neurotransmitters, particularly biogenic amines, to control their motor activities via corresponding neurotransmitter transporters and receptors, which provide novel candidates as drug targets. Indeed, dopamine D2-type antagonists, serotonin reuptake inhibitors, voltage-gated calcium channel antagonists have been shown to inhibit the transformation of miracidia to primary sporocysts following the medium-highthroughput screening of 1280 well-characterised chemicals (Taft et al., 2010). Further, by targeting epigenetic control, it has been shown that histone deacetylases (HDAC) inhibitors can induce an apoptotic phenotype of schistosomula and adult worms of S. mansoni both in vitro and in vivo (Pierce et al., 2011). In relation to proteases, a remarkable reduction in both worm burden and liver pathology has been achieved in the murine model of schistosomiasis by administration of the cysteine protease inhibitor K11777 (Abdulla et al., 2007). Protein interactions represent another novel area for the rational design of potential anti-schistosome drugs targets, a prime example being the non-canonical mediated PSD-95/ Dlg/ZO-1 (PDZ) domain, an important module found in many scaffolding proteins (Mu et al., 2012; Cai et al., 2014). 9.3. Genomic editing based on gene manipulation Schistosomes are equipped with small RNAi machinery, and gene manipulation (based on transgenes and RNAi) has been applied for targeting many of different stages (cercariae, schistosomula, adults, eggs, miracidia and sporocysts) of the major species (Brindley and Pearce, 2007; Rinaldi et al., 2011; Beckmann and Grevelding, 2012; Mann et al., 2014; Hagen et al., 2015a). This approach may guide the future development of novel intervention tools for the elimination of schistosomiasis. Recently, as a novel genome editing tool, the CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas (CRISPR associated proteins) system has attracted considerable attention (Sander and Joung, 2014; Zetsche et al., 2015). In this system, the nuclease Cas9/ Cpf1 is directed by a short guide RNA to cleave target sequences. It is an affordable, yet powerful gene editing technology, which has been applied in many cell lines and organisms including protozoan parasites, e.g., Plasmodium (Lee and Fidock, 2014), Leishmania (Sollelis et al., 2015), and Cryptosporidium (Vinayak et al., 2015). The CRISPR-Cas9/Cpf1-RNA delivered by pseudotyped retroviruses may likewise represent an alternative tool for generating permanent transgenic schistosomes (Hoffmann et al., 2014), via targeting egg and miracidium stages (Rinaldi et al., 2012; Attar, 2015). This new technology may be beneficial not only for drug and vaccine candidate identification, but also for targeting specific proteinencoding schistosome genes implicated in disease pathology (Hagen et al., 2014) and/or morbidity control of the disease. And furthermore, a CRISPR-altered schistosome population with a compromised phenotype may be created and released into the wild,

based on integrated gene drive technology; the approach generates autocatalytic mutations to produce homozygous loss-of-function mutations (Gantz and Bier, 2015). 10. Concluding remarks In this post-genomics era, extensively focused biological, immunological and functional, as well as various ‘‘-omics”, studies have been carried out, which have resulted in the accumulation of considerable new information regarding survival mechanisms in schistosomes. These studies further highlight key biological features and indications as to how schistosomes manipulate the host immune system, all of which represent the Tao of survival adopted by this important group of parasitic worms. With this progress it is important to reconsider future control strategies for this neglected disease. Functional and immunological studies provide a solid basis for the development of anti-schistosome vaccines. Furthermore, the publicly available genomic sequences for the three main human schistosome species provide an important avenue for the identification of new anti-schistosomal drug targets, while the outcomes of gene editing present a potentially novel approach in producing permanent transgenic schistosomes fostering the development of new interventions for the control schistosomiasis. Acknowledgements D.P.M. is a National Health and Medical Research Council of Australia (NHMRC) Senior Principal Research Fellow and Senior Scientist at QIMR Berghofer, Australia. We acknowledge the NHMRC for financial support of our research on schistosomiasis. The authors have declared no conflict of interest. References Abdulla, M.H., Lim, K.C., Sajid, M., McKerrow, J.H., Caffrey, C.R., 2007. Schistosomiasis mansoni: novel chemotherapy using a cysteine protease inhibitor. PLoS Med. 4 e14. Adenowo, A.F., Oyinloye, B.E., Ogunyinka, B.I., Kappo, A.P., 2015. Impact of human schistosomiasis in sub-Saharan Africa. Braz. J. Infect. Dis. 19, 196–205. Andrade, L.F., Nahum, L.A., Avelar, L.G., Silva, L.L., Zerlotini, A., Ruiz, J.C., Oliveira, G., 2011. Eukaryotic protein kinases (ePKs) of the helminth parasite Schistosoma mansoni. BMC Genomics 12, 215. Anthony, B.J., James, K.R., Gobert, G.N., Ramm, G.A., McManus, D.P., 2013. Schistosoma japonicum eggs induce a proinflammatory, anti-fibrogenic phenotype in hepatic stellate cells. PLoS One 8 e68479. Attar, N., 2015. Techniques & applications: Cpf1 makes for a CRISPR cut. Nat. Rev. Microbiol. 13, 660. Bahia, D., Mortara, R.A., Kusel, J.R., Andrade, L.F., Ludolf, F., Kuser, P.R., Avelar, L., Trolet, J., Dissous, C., Pierce, R.J., Oliveira, G., 2007. Schistosoma mansoni: expression of Fes-like tyrosine kinase SmFes in the tegument and terebratorium suggests its involvement in host penetration. Exp. Parasitol. 116, 225–232. Beckmann, S., Grevelding, C.G., 2012. Paving the way for transgenic schistosomes. Parasitology 139, 651–668. Berriman, M., Haas, B.J., LoVerde, P.T., Wilson, R.A., Dillon, G.P., Cerqueira, G.C., Mashiyama, S.T., Al-Lazikani, B., Andrade, L.F., Ashton, P.D., Aslett, M.A., Bartholomeu, D.C., Blandin, G., Caffrey, C.R., Coghlan, A., Coulson, R., Day, T.A., Delcher, A., DeMarco, R., Djikeng, A., Eyre, T., Gamble, J.A., Ghedin, E., Gu, Y., Hertz-Fowler, C., Hirai, H., Hirai, Y., Houston, R., Ivens, A., Johnston, D.A., Lacerda, D., Macedo, C.D., McVeigh, P., Ning, Z., Oliveira, G., Overington, J.P., Parkhill, J., Pertea, M., Pierce, R.J., Protasio, A.V., Quail, M.A., Rajandream, M.A., Rogers, J., Sajid, M., Salzberg, S.L., Stanke, M., Tivey, A.R., White, O., Williams, D. L., Wortman, J., Wu, W., Zamanian, M., Zerlotini, A., Fraser-Liggett, C.M., Barrell, B.G., El-Sayed, N.M., 2009. The genome of the blood fluke Schistosoma mansoni. Nature 460, 352–358. Boulanger, D., Warter, A., Trottein, F., Mauny, F., Bremond, P., Audibert, F., Couret, D., Kadri, S., Godin, C., Sellin, E., et al., 1995. Vaccination of patas monkeys experimentally infected with Schistosoma haematobium using a recombinant glutathione S-transferase cloned from S. mansoni. Parasite Immunol. 17, 361– 369. Brindley, P.J., Hotez, P.J., 2013. Break Out: urogenital schistosomiasis and Schistosoma haematobium infection in the post-genomic era. PLoS Negl. Trop. Dis. 7 e1961. Brindley, P.J., Pearce, E.J., 2007. Genetic manipulation of schistosomes. Int. J. Parasitol. 37, 465–473.


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Role of microRNAs in schistosomes and schistosomiasis.

Apoptosis in schistosomes: toward novel targets for the treatment of schistosomiasis.

Revisiting glucose uptake and metabolism in schistosomes: new molecular insights for improved schistosomiasis therapies.

Predation of Biomphalaria and non-target molluscs by the crayfish Procambarus clarkii: implications for the biological control of schistosomiasis.

The history of schistosomiasis research and policy for its control.

The Tao of myeloma.

Cancer survivorship care: Implications for primary care advanced practice nurses.

Alternative approaches in schistosomiasis control.

Deciphering the glycogenome of schistosomes.

Something old, something new: is praziquantel enough for schistosomiasis control?

Enhancing collaboration between China and African countries for schistosomiasis control.

Prevalence of Schistosomes and Soil-Transmitted Helminths and Morbidity Associated with Schistosomiasis among Adult Population in Lake Victoria Basin, Tanzania.

Changing policy and practice in the control of pediatric schistosomiasis.

Metrifonate in the control of urinary schistosomiasis in Zanzibar.

Historical perspective: snail control to prevent schistosomiasis.

Some personal views on the control of schistosomiasis mansoni.

TRP channels in schistosomes.

Local and International Implications of Schistosomiasis Acquired in Corsica, France.

Longitudinal survey on the distribution of Biomphalaria sudanica and B. choanomophala in Mwanza region, on the shores of Lake Victoria, Tanzania: implications for schistosomiasis transmission and control.

Epidemiology and control of schistosomiasis: present situation and priorities for further research. Scientific Working Group on Schistosomiasis.

Immunity to schistosomes.

The epidemiology of schistosomiasis in Burundi and its consequences for control.

Aligning Task Control with Desire for Control: Implications for Performance.

[Measures for control of schistosomiasis adopted by the National Health Foundation].