Epidermal growth factor receptors (Smp_165470; Smp_093930; Smp_152680; Smp_344500)

Epidermal growth factor receptors (EGFRs) are transmembrane glycoprotein and belong to the receptor tyrosine kinases (RTKs) (Grapa et al. Reference Grapa, Mocan, Gonciar, Zdrehus, Mosteanu, Pop and Mocan2019). The human EGFR is associated with the pathogenesis and progression of various types of carcinomas. In urothelial carcinoma associated with Schistosoma infection, a higher level of EGFR is found than in urothelial carcinoma of other causes (AlHariry et al. Reference AlHariry, El Saftawy, Aboulhoda, Abozamel, Alghamdi, Hamoud and Khalil Ghanam2024). In S. mansoni, EGFR homologs are predominantly expressed in the muscle of adult male and female worms, indicating that this receptor may play a role in muscle development (Ramachandran et al. Reference Ramachandran, Skelly and Shoemaker1996). Moreover, the EGFR substrate is expressed in the vitellarium and the ovary of the adult female and in the testes of the adult male worms, suggesting that this receptor may have additional functions in the gonads (Buro et al. Reference Buro, Burmeister, Quack and Grevelding2017). Maharjan et al. (Reference Maharjan, Kirk, Lawton and Walker2023) demonstrated the anterior localization of EGFR in schistosomula.

EGFR contains a conserved intracellular tyrosine kinase domain, a unique transmembrane hydrophobic domain, and an extracellular domain for binding EGF ligands. Human EGF induces EGFR autophosphorylation in adult worms and larvae of S. mansoni and increases protein and DNA synthesis in adult worms, suggesting that host hormones are involved in the regulation of schistosome development. Schistosome EGFR can bind human EGF with the same affinity as human EGFR (Ramachandran et al. Reference Ramachandran, Skelly and Shoemaker1996).

Human growth factors can modulate schistosome-signalling processes such as protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) (Ressurreição et al. Reference Ressurreição, Elbeyioglu, Kirk, Rollinson, Emery, Page and Walker2016; Vicogne et al. Reference Vicogne, Cailliau, Tulasne, Browaeys, Yan, Fafeur, Vilain, Legrand, Trolet and Dissous2004). Human EGF, insulin, and insulin-like growth factor 1 were found to activate PKC and ERK at the schistosomula surface. The stimulation of these signalling by human growth factors is crucial during early host invasion, as the parasite encounters human growth factors for the first time and must rapidly adapt to the host. Host-mediated ERK activation can drive tegument remodeling, ensuring parasite survival while promoting cell growth and differentiation. Depleting of cholesterol from tegument lipid rafts, which are crucial for S. mansoni biology, disrupts EGFR/IR binding on the schistosomula surface and alters several protein kinases signalling pathways within the parasite (Ressurreição et al. Reference Ressurreição, Elbeyioglu, Kirk, Rollinson, Emery, Page and Walker2016).

Insulin receptors (IRs)

During growth and reproduction, schistosomes consume substantial amounts of energy derived primarily from the host’s nutrition. Adult S. mansoni worms absorb large amounts of blood glucose, equivalent to their dry weight every five hours, from the portal and mesenteric veins of the host (Bueding Reference Bueding1950). Glucose uptake occurs primarily through facilitated diffusion across the worm tegument. Human insulin has been shown to enhance glucose uptake in schistosomes (Ahier et al. Reference Ahier, Khayath, Vicogne and Dissous2008). Two glucose transporters, GTP1 and GTP4, play a crucial role in this process and are distributed asymmetrically on the tegument. GTP1 is located within the basal membrane and transports glucose into the underlying tissues, whereas GTP4 is located in the apical membrane. GTP4 is expressed on the parasite surface concurrently with the appearance of the apical membrane bilayer during cercaria to schistosomule transformation and remains on the surface throughout all life stages of the parasite in vertebrate hosts (Khayath et al. Reference Khayath, Vicogne, Ahier, BenYounes, Konrad, Trolet, Viscogliosi, Brehm and Dissous2007). Glucose uptake in schistosomes is mediated by PI3K/Akt/mTOR signal. The Akt protein, also known as protein kinase B, is associated not only with the expression of GTP4 but also with the shuttling of this transporter within the tegument (Abou-El-Naga Reference Abou-El-Naga2021; McKenzie et al. Reference McKenzie, Kirk and Walker2018; Morel et al. Reference Morel, Vanderstraete, Cailliau, Lescuyer, Lancelot and Dissous2014; Skelly and Shoemaker Reference Skelly and Shoemaker1996). Maharjan et al. (Reference Maharjan, Kirk, Lawton and Walker2023) demonstrated that lipid rafts could be crucial for glucose import into the parasite, potentially in response to host insulin.

Adult S. mansoni possesses two insulin receptors (IR1 and IR2) (Smp_341160; Smp_009990) (Khayath et al. Reference Khayath, Vicogne, Ahier, BenYounes, Konrad, Trolet, Viscogliosi, Brehm and Dissous2007). Both receptors are also present in schistosomula (Maharjan et al. Reference Maharjan, Kirk, Lawton and Walker2023). A potential insulin-like peptide has been identified in S. mansoni, although it is still unclear whether this peptide interacts with the IRs of S. mansoni (Wang et al. Reference Wang, Luo, Zhang, Yin, Dou and Cai2014). Each IR has a unique extracellular N-terminal domain that stabilizes the conformation around the bound ligand (Vicogne et al. Reference Vicogne, Pin, Lardans, Capron, Noël and Dissous2003). These receptors differ in essential signalling motifs and expression locations. IR1 is expressed at the tegumental basal membrane, in muscle tissues, and in the epithelial cells of the intestine, whereas IR2 is predominantly found in the parenchymal cells of adult worms (Khayath et al. Reference Khayath, Vicogne, Ahier, BenYounes, Konrad, Trolet, Viscogliosi, Brehm and Dissous2007). Invertebrates typically have a single IR that regulates both metabolism and growth, whereas vertebrates have two receptors: the IR and the insulin-like growth factor 1 receptor, which control glucose uptake and growth, respectively (Kim and Accili Reference Kim and Accili2002). Similarly, in Schistosoma, IR1 is likely specialized for glucose uptake, given its colocalization with glucose transporters GTP1 and GTP4 in the tegument. In contrast, the widespread expression of IR2 in the worm parenchyma suggests a role in growth control, similar to the insulin-like growth factor 1 receptor in mammals. Stimulation of IRs by human insulin activates the phosphatidylinositol 3 kinase/Akt/ mechanistic target of rapamycin (PI3K/Akt/mTOR) leading to increase the glucose uptake in S. mansoni (Abou-El-Naga Reference Abou-El-Naga2021; McKenzie et al. Reference McKenzie, Kirk and Walker2018). Several studies have highlighted the significance of schistosome insulin receptors for nutrition, growth, and reproduction (Abou-El-Naga Reference Abou-El-Naga2021; Elhenawy et al. Reference Elhenawy, Ashour, Nabih, Shalaby, El-Karef and Abou-El-Wafa2017; Vanderstraete et al. Reference Vanderstraete, Gouignard, Ahier, Morel, Vicogne and Dissous2013). RNA interference (RNAi) of the IRs decreases uptake and affects schistosome development (You et al. Reference You, Gobert, Cai, Mou, Nawaratna, Fang, Villinger and McManus2015).

Fibroblast growth factor receptors (FGFRs)

Secreted human fibroblast growth factor (FGF) interacts with surface RTKs of the FGF receptors (FGFRs) family, which includes four isoforms (FGFR1–FGFR4). FGF binds FGF ligand to FGFR, thereby phosphorylating mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/AKT pathways. Human FGF1 (acidic FGF) and FGF2 (basic FGF) are the most active members of the FGF family and are universally expressed in human tissues. They are released in a signal peptide-independent manner (Ornitz and Itoh Reference Ornitz and Itoh2015).

In the genome of S. mansoni, two genes encoding FGFRs were identified. The fgfrA gene (Smp_175590) encodes a predicted protein with two extracellular immunoglobulin domains and a split tyrosine kinase domain, while the fgfrB gene (Smp_157300) product contains only one extracellular immunoglobulin domain. FGFRA and FGFRB are enzymatically active, expressed in the gonads of schistosomes, and upregulated following pairing, suggesting a role in parasite fertility (Hahnel et al. Reference Hahnel, Quack, Parker-Manuel, Lu, Vanderstraete, Morel, Dissous, Cailliau and Grevelding2014). The expression of fgfrA and fgfrB has been demonstrated in neoblast-like somatic stem cells, with evidence indicating that both receptors play a crucial role in maintaining schistosome stem cells (Wang et al. Reference Wang, Collins and Newmark2013). FGFRA is abundantly expressed in germinal/stem cells across various S. mansoni developmental stages including eggs, miracidia, cercariae, schistosomula, and adult worms. The distribution of FGFRA in embryonic cells of immature eggs and in the neural mass of mature eggs and miracidia, and its co-localization with stem cells in adult S. mansoni, strongly suggest its crucial roles in the maintenance of schistosome stem cells, in the development of the nervous and reproductive systems, and in the host-parasite interaction (Wang et al. Reference Wang, Collins and Newmark2013; Wendt and Collins Reference Wendt and Collins2016). In vitro, FGFRA of adult worms binds to human FGFs and activates the mitogen activated protein kinase (MAPK) pathway (Du et al. Reference Du, McManus, Fogarty, Jones and You2022). Inhibition of FGF signalling by the TK inhibitor significantly reduced egg hatching ability and altered the behavior of hatched miracidia from treated eggs, highlighting the critical role of FGF signalling in the life cycle of S. mansoni (Du et al. Reference Du, McManus, Fogarty, Jones and You2022). Inhibition of fgfrA in S. mansoni reduces stem cell signalling and increases cell apoptosis. Intravenous injection of mice with fgfrA-repressed eggs resulted in significantly smaller granulomas and a reduction in serum IgE levels, underscoring the crucial role of FGFRA in regulating the host immune response during schistosome infection (Du et al. Reference Du, McManus, French, Collinson, Sivakumaran, MacGregor, Fogarty, Jones and You2023).

Venus kinase receptors (VKRs)

S. mansoni expresses an unconventional family of receptor tyrosine kinases (RTKs) known as the Venus kinase receptor (VKR) family, which is unique to invertebrates and was first identified in S. mansoni. Typically, invertebrate genomes contain a single VKR gene, but platyhelminths possess two distinct copies of VKR (Vanderstraete et al. Reference Vanderstraete, Gouignard, Ahier, Morel, Vicogne and Dissous2013). VKRs have an extracellular Venus flytrap module, similar to the ligand-binding domain of class C G-protein coupled receptors, connected via a transmembrane segment to an intracellular tyrosine kinase (TK) domain. This Venus flytrap module has two lobes that close upon ligand binding (Vicogne et al. Reference Vicogne, Pin, Lardans, Capron, Noël and Dissous2003). In S. mansoni, the two receptors, VKR1 (Smp_019790) and VKR2 (Smp_153500), are activated by L-arginine and calcium ions, respectively (Gouignard et al. Reference Gouignard, Vanderstraete, Cailliau, Lescuyer, Browaeys and Dissous2012).

S. mansoni beta-integrin receptor Smβ-Int1 interacts with the SmVKRI. The three putative bridging molecule – SmILK, SmPINCH, and SmNck2 – mediate the Smβ-Int1/SmVKR1 cooperation. This process indicates that SmVKR1 can be activated in a ligand independent manner mediated by receptor/complex interaction (Gelmedin et al. Reference Gelmedin, Morel, Hahnel, Cailliau, Dissous and Grevelding2017).

VKRs are abundantly present in the germinal cells of miracidia, in the larval stage of the parasite, and in the oocytes within the ovary and oviduct of adult female worms. They play crucial roles in growth, differentiation, and reproduction through the PI3K/Akt/mTOR and mitogen-activated protein kinase (MAPK) pathways. VKR1 can also activate the c-Jun N-terminal kinase signal transduction pathway (Gouignard et al. Reference Gouignard, Vanderstraete, Cailliau, Lescuyer, Browaeys and Dissous2012; Vanderstraete et al. Reference Vanderstraete, Gouignard, Cailliau, Morel, Hahnel, Leutner, Beckmann, Grevelding and Dissous2014; Vicogne et al. Reference Vicogne, Pin, Lardans, Capron, Noël and Dissous2003). The Smβ-Int1/SmVKR1 signalling complex plays a crucial role in oocytes differentiation and survival of paired schistosomes (Gelmedin et al. Reference Gelmedin, Morel, Hahnel, Cailliau, Dissous and Grevelding2017).

Both S. mansoni VKR1 (Smp_019790) and VKR2 (Smp_153500) genes are highly transcribed in the ovaries of females compared to the testes of male worms, and each gene exhibits a distinct expression profile. The distribution of each VKR in S. mansoni correlates with its role in oocyte maturation. VKR1 is expressed in mature oocytes located in the posterior part of the ovary and is involved in oocyte migration. VKR2, on the other hand, is expressed in immature oocytes in the anterior part of the ovary and is responsible for their proliferation and growth (Gouignard et al. Reference Gouignard, Vanderstraete, Cailliau, Lescuyer, Browaeys and Dissous2012; Vanderstraete et al. Reference Vanderstraete, Gouignard, Cailliau, Morel, Hahnel, Leutner, Beckmann, Grevelding and Dissous2014). Mathavan et al. (Reference Mathavan, Liu, Robinson, El-Sakkary, Elatico, Gomez, Nellas, Owens, Zuercher, Navratilova, Caffrey and Beis2022) identified GSK1520489, GSK986310, GW696155, and SB- 710363 as kinase inhibitors of S. mansoni VKR2. They inhibit the enzymatic activity and induce phenotypic changes in the worm.

Transforming growth factor receptors

The TGF-β superfamily consists of a wide variety of structurally related polypeptide growth factors that are known to mediate numerous physiological processes, including growth and differentiation, cell death, and tissue repair (Chen et al. Reference Chen, Qin and Simons2023). Members of the TGF-β superfamily are divided into two major subfamilies based on sequence homology and the distinct downstream pathways they activate. The two subfamilies are the TGF-β/activin/nodal subfamily and the bone morphogenetic protein/growth and differentiation factor/Muellerian inhibiting substance (BMP/GDF/MIS) subfamily (Baba et al. Reference Baba, Rah, Bhat, Mushtaq, Parveen, Hassan, Hameed Zargar and Afroze2022).

When activated by a ligand, TGF-β family members bind to a group of transmembrane receptor serine/threonine kinases and transmit signals through them. The receptors are divided into two subtypes: transforming growth factor–β type I receptor (TβRI) (Smp_049760) and type II (TβRII) (Smp_144390). Type II receptor is crucial for ligand binding, and upon ligand binding, it activates type I receptor through phosphorylation (Osman et al. Reference Osman, Niles, Verjovski-Almeida and LoVerde2006). Activated TβRI subsequently transmits the signal to a member of the cytoplasmic Smad family, which can transport the signal to the nucleus and regulate the transcription of specific genes in response to the ligand (Freitas et al. Reference Freitas, Jung and Pearce2009). Smads are a group of proteins that act as intracellular signalling transducers for the TGF-β family. In mammals, the two subfamilies of the TGF-β superfamily activate different classes of Smad proteins. Members of the TGF-β subfamily activate Smad2 and Smad3 homologues, while members of the BMP subfamily activate Smad1, Smad5, and Smad8 homologues (Hata and Chen Reference Hata and Chen2016).

Several components of TGF-β signalling have been identified in S. mansoni, including the TGF-β transmembrane receptor serine/threonine kinases, also known as SmTβRI (Davies et al. Reference Davies, Shoemaker and Pearce1998) and SmTβRII (Osman et al. Reference Osman, Niles, Verjovski-Almeida and LoVerde2006), and two ligands of the schistosome TGF-β family: inhibin/Activin (SmInAct) (Freitas et al. Reference Freitas, Jung and Pearce2007) and SmBMP. SmBMP is expressed in the egg, cercariae, and the protonephridia of adult S. mansoni worm (Freitas et al. Reference Freitas, Jung and Pearce2009). The TGF-β signal also includes four Smad proteins (SmSmad1, SmSmad2, SmSmad4, SmSmad1B) (Carlo et al. Reference Carlo, Osman, Niles, Wu, Fantappie, Oliveira and LoVerde2007; Osman et al. Reference Osman, Niles and LoVerde2004), in addition to six scaffolding/regulatory proteins that play an important role in signal regulation in the TGF-β pathway. They comprise SmSARA (Verjovski-Almeida et al. Reference Verjovski-Almeida, DeMarco, Martins, Guimarães, Ojopi, Paquola, Piazza, Nishiyama, Kitajima, Adamson, Ashton, Bonaldo, Coulson, Dillon, Farias, Gregorio, Ho, Leite, Malaquias, Marques, Miyasato, Nascimento, Ohlweiler, Reis, Ribeiro, Sá, Stukart, Soares, Gargioni, Kawano, Rodrigues, Madeira, Wilson, Menck, Setubal, Leite and Dias-Neto2003), SmGCN5, SmCBP (Carlo et al. Reference Carlo, Osman, Niles, Wu, Fantappie, Oliveira and LoVerde2007), SmFKBP12 (Knobloch et al. Reference Knobloch, Rossi, Osman, LoVerde, Klinkert and Grevelding2004), SmeIF2α (McGonigle et al. Reference McGonigle, Beall and Pearce2002), and Sm14-3-3ε (McGonigle et al. Reference McGonigle, Beall, Feeney and Pearce2001).

SmTβRII and SmTβRI are expressed on the surface of the parasite, and in the case of SmTβRI, its expression is upregulated following infection of the mammalian host (Davies et al. Reference Davies, Shoemaker and Pearce1998). TβRII is also localized in the vitelline and gut epithelial cells of female worms and the sub-tegumental cells of male worms (Osman et al. Reference Osman, Niles, Verjovski-Almeida and LoVerde2006). Due to this localization, the schistosome TGF-β signalling pathway may play a crucial role in the development of vitelline cells in female worms and egg embryogenesis (LoVerde et al. Reference LoVerde, Osman and Hinck2007). SmInAct expression is closely associated with the reproductive potential of the parasite. RNAi-mediated knockdown of SmInAct in eggs halted their development, indicating that SmInAct plays a crucial role in embryogenesis (Freitas et al. Reference Freitas, Jung and Pearce2007; Osman et al. Reference Osman, Niles, Verjovski-Almeida and LoVerde2006). SmSmad4 is localized within the epithelia surrounding the gut and vitellarium, as well as in the sub-tegument and muscles of males (Osman et al. Reference Osman, Niles and LoVerde2004). SmSmad2 is found within the vitellarium, developing egg and ovary of the female worm, as well as in the testes and tubercles of the male worm (Osman et al. Reference Osman, Niles and LoVerde2001). Therefore, the TGF-β signalling pathway in S. mansoni has been implicated in host-parasite interactions, parasite reproductive development, and embryogenesis.

SmTβRII is able to activate SmTβRI in the presence of human TGF-β1, which subsequently activates SmSmad2 and promotes its interaction with SmSmad4, thereby facilitating the transfer of the signal from the receptor complex to the Smad proteins. The newly formed Smad complex translocates into the nucleus, where it associates with nuclear proteins that guide the complex to specific promoter sequences, regulating the transcription of target genes (Freitas et al. Reference Freitas, Jung and Pearce2009). Oliveira et al. (Reference Oliveira, Carvalho, Verjovski-Almeida and LoVerde2012) demonstrated that in vitro treatment with human TGF-β1 led to changes in expression levels of 381 S. mansoni genes, including 316 downregulated genes and 65 upregulated genes. Among these genes, there are genes related to morphology, development, and cell cycle that could influence effects of cytokine on the worm. Osman et al. (Reference Osman, Niles, Verjovski-Almeida and LoVerde2006) demonstrated that TGF-β signalling regulates the expression of the gynecophoral canal protein (SmGCP). This protein is located on the surface of the gynecophoric canal of the male where the female resides for sexual maturation. It is also found on the entire surface of females in copula, but not on unmated males or immature females (Bostic and Strand Reference Bostic and Strand1996). Therefore, it could be suggested that SmGCP might be a gene product induced by the TGF-β pathway and could serve as a crucial signalling molecule for worm pairing (Osman et al. Reference Osman, Niles, Verjovski-Almeida and LoVerde2006).

A distinctive biological trait of schistosomes is that sexual maturation of the female depends on continuous pairing contact with the male. After pairing, mitosis and differentiation are triggered in the female, leading to the development of reproductive organs, including the ovary and vitellarium, followed by the production of eggs (Kunz Reference Kunz2001). Eggs are important for propagation of the parasite life cycle and provoking pathogenesis. In S. mansoni, the TGF-β pathway is involved in female reproductive development and egg embryogenesis (Freitas el al. 2007; Knobloch et al. Reference Knobloch, Beckmann, Burmeister, Quack and Grevelding2007; Osman et al. Reference Osman, Niles, Verjovski-Almeida and LoVerde2006). As TβRs are exposed on the surface, this creates the potential for communication between the male and female parasites. In addition to utilizing host growth factors, it is suggested that schistosomes might also encode endogenous growth factor peptides that have a high degree of sequence similarity with their mammalian orthologues as developmental signals (LoVerde et al. Reference LoVerde, Osman and Hinck2007).

S. mansoni nuclear receptors in subfamily 1

The well-characterized proteins of S. mansoni NR1 are S. mansoni thyroid receptors (SmTRs). Two homologs of vertebrate TR have been identified in S. mansoni (SmTRα and SmTRβ) (Smp_134490, Smp_174260). Phylogenetic analysis indicates that these two copies resulted from a gene duplication specific to Schistosoma (Wu et al. Reference Wu, Niles, Hirai and LoVerde2007a). Both proteins exhibit the consensus structure of TR, featuring a conserved N-terminal signature in the A/B domain typical of TRs, along with the specific CEGCKGFFRR sequence of the NR1 subfamily. Like vertebrate members of this family, SmTRs can form a dimer with retinoid X receptor (SmRXR1). SmTRs could bind to vertebrate TR core DNA elements as a monomer, a homodimer, or a heterodimer by binding with RXR (Wu et al. Reference Wu, Niles, El-Sayed, Berriman and LoVerde2006; Wu et al. Reference Wu, Niles, Hirai and LoVerde2007a). Thyroid hormone (TH) binds to the LBD of the TR, inducing a conformational change in C-terminus of the receptor. This change causes the dissociation of corepressors from the TR, allowing coactivators to bind to the C-terminus in a hormone-dependent manner. The TR and coactivator complex then activates target gene expression (Lazar Reference Lazar2017).

An ortholog of the Drosophila ecdysone-induced protein 78 has been identified in S. mansoni (Wu et al. Reference Wu, Niles, El-Sayed, Berriman and LoVerde2006; Wu et al. Reference Wu, Tak and LoVerde2008). It is directly involved in ecdysone signaling. SmE78 (Smp_000340) is expressed throughout schistosome development, with the highest expression levels observed in the miracidia and egg stages (Wu et al. Reference Wu, Tak and LoVerde2008). Nirde et al. (Reference Nirde, Torpier, De Reggi and Capron1983) have shown that S. mansoni can synthesize the steroid hormone ecdysone. Ecdysterone effectively stimulates host location activities in miracidia (Shiff and Dossaji Reference Shiff and Dossaji1991). However, it remains to be demonstrated whether ecdysone-induced protein 78 is involved in the transduction of an ecdysone signal in S. mansoni (Wu and LoVerde Reference Wu and LoVerde2011).

Another potential member of the SmNP1 subfamily is Smp_248100, an uncharacterized protein from S. mansoni. Primary sequence analysis has confirmed that Smp_248100 contains a DNA-binding domain (DBD) with high sequence similarity to DBDs of other vertebrate and invertebrate NRs, including HR96 from Drosophila melanogaster and DAF-12 from Caenorhabditis elegans. SmHR96α and SmHR96β are homologues of Drosophila hormone receptor 96 (DHR96) (Hu et al. Reference Hu, Wu, Niles and LoVerde2006a; Wu et al. Reference Wu, Niles, El-Sayed, Berriman and LoVerde2006). SmHR96α interacts with SmRXR1 (Hu et al. Reference Hu, Niles and LoVerde2006b). The mRNA of SmHR96α is expressed throughout every stage of the S. mansoni life cycle, with particularly high expression levels in eggs and cercariae. The SmHR96α protein is located in subtegumental and parenchymal cells in both male and female worms, as well as in the ovaries, eggs, and vitelline cells of mature female worms (Hu et al. Reference Hu, Wu, Niles and LoVerde2006a). SmHR96β is recently named Vitellogenic Factor 1. This factor is important in vitelline cell development, and this NR is important for female sexual development after pairing with a male worm (Wang et al. Reference Wang, Chen and Collins2019).

The divergent member SmNR1 is a member of NR subfamily I with no known orthologue. The gene of this member is located on chromosome 1 of S. mansoni and is highly expressed in eggs, sporocysts, and juvenile worms (Wu et al. Reference Wu, Niles, Hirai and LoVerde2007c). The divergent member SmNR1 is a partner of SmRXR1. It requires RXR to form a heterodimer that confers binding to hormone response element (Kojetin et al. Reference Kojetin, Matta-Camacho, Hughes, Srinivasan, Nwachukwu, Cavett, Nowak, Chalmers, Marciano, Kamenecka, Shulman, Rance, Griffin, Bruning and Nettles2015). Mutagenesis analysis indicates that SmCBP1, a co-regulatory protein known to interact with SmFTZ-F1 (Bertin et al. Reference Bertin, Oger, Cornette, Caby, Noël, Capron, Fantappie, Rumjanek and Pierce2006), can mediate interactions with the LBD of SmRXR1 and SmNR1 (Fantappié et al. Reference Fantappié, Bastos de Oliveira, de Moraes Maciel, Rumjanek, Wu and LoVerde2008a). The upstream region of the p14 gene has a novel NR response element containing DNA core motif, composed of an atypically spaced direct repeat 17. SmRXR1 and SmNR1 divergent members specifically bound to the p14-direct repeat 17 element as a heterodimer. SmRXR1, but not SmNR1, is bound to the motif as a monomer. The expression of the S. mansoni p14 gene, which is an eggshell precursor gene expressed only in the vitelline cells of sexually mature female worms in response to an as yet unidentified male stimulus, is regulated through NR signalling pathway (Fantappié et al. Reference Fantappié, Furtado, Rumjanek and LoVerde2008b).

S. mansoni nuclear receptors in subfamily 2

S. mansoni NRs in subfamily 2 include SmTR2/4, 9-cis-retinoic acid receptors (RXR), and hepatocyte nuclear factor 4 (HNF4). The SmTR2/4 gene is expressed in all developmental stages of S. mansoni with a higher level in cercaria and may play a role in regulating female reproductive development (Hu et al. Reference Hu, Wu, Niles and LoVerde2006c).

SmRXR1 (Smp_097700) and SmRXR2 (Smp_073470) are the vertebrate RXR homologues present in S. mansoni (de Mendonça et al. Reference de Mendonça, Escriva, Bouton, Zelus, Vanacker, Bonnelye, Cornette, Pierce and Laudet2000; Freebern et al. Reference Freebern, Niles and LoVerde1999a; Freebern et al. Reference Freebern, Osman, Niles, Christen and LoVerde1999b). Both SmRXRs originated from a Schistosoma-specific gene duplication, like SmTRs. RXR is involved in multiple signalling pathways within the cell nucleus, and its heterodimers control the activity of other NRs (Bertin et al. Reference Bertin, Caby, Oger, Sasorith, Wurtz and Pierce2005). SmRXR1 can form heterodimers with SmTRα, SmTRβ (Wu et al. Reference Wu, Niles and LoVerde2007b), SmHR96α (Hu et al. Reference Hu, Niles and LoVerde2006b), and SmNR1 divergent member (Wu et al. Reference Wu, Niles, Hirai and LoVerde2007c). It can also bind to the cis-elements of the S. mansoni p14 gene (Fantappié et al. Reference Fantappié, Furtado, Rumjanek and LoVerde2008b; Freebern et al. Reference Freebern, Osman, Niles, Christen and LoVerde1999b), which is regulated by a stimulus from the male schistosome (LoVerde et al. Reference LoVerde, Niles, Osman and Wu2004). SmRXR1 mRNA is consistently expressed throughout the developmental stages (Fantappié et al. Reference Fantappié, Furtado, Rumjanek and LoVerde2008b). The co-regulatory protein SmCBP1 can mediate interactions with both SmRXR1 and SmNR1 (Fantappié et al. Reference Fantappié, Bastos de Oliveira, de Moraes Maciel, Rumjanek, Wu and LoVerde2008a). In contrast, SmRXR2 fails to form a heterodimer with SmTRα or SmTRβ (Wu et al. Reference Wu, Niles and LoVerde2007b), SmHR96α (Hu et al. Reference Hu, Niles and LoVerde2006b) or SmNR1 divergent member (Wu et al. Reference Wu, Niles, Hirai and LoVerde2007c). SmRXR2 mRNA is expressed at all life cycle stages, with higher levels in cercariae and miracidia – the free-living larval stages (de Mendonça et al. Reference de Mendonça, Escriva, Bouton, Zelus, Vanacker, Bonnelye, Cornette, Pierce and Laudet2000; Freebern et al. Reference Freebern, Niles and LoVerde1999a). However, the protein expression differs significantly from mRNA, showing high levels in schistosomula but much lower levels in cercariae and miracidia (de Mendonça et al. Reference de Mendonça, Escriva, Bouton, Zelus, Vanacker, Bonnelye, Cornette, Pierce and Laudet2000).

HNF4 is a class of NRs responsible for regulation of gluconeogenesis, bile acid synthesis, cholesterol, and lipid metabolism in the liver of mammals (Chen et al. Reference Chen, Vasoya, Toke, Parthasarathy, Luo, Chiles, Flores, Gao, Bonder, Su and Verzi2020). Stem cells from the blood-digesting gut of S. mansoni express the hnf4 gene (Smp_174700), identified by single-cell sequencing. RNAi assay revealed the importance of the hnf4 gene for gut maintenance, nutrient digestion, and pathology induction, and indirectly showed its importance for parasite growth (Wendt et al. Reference Wendt, Zhao, Chen, Liu, O’Donoghue, Caffrey, Reese and Collins2020). In S. japonicum, HNF4 expression is higher in female than in male worms, both at transcriptional and protein levels. HNF4 is expressed in the reproductive system and intestinal tissues of worms, as well as in cercariae and eggs (Wu et al. Reference Wu, Huang, Zhao, Umar, Chen, Yu and Huang2024). Furthermore, HNF4 plays an important role in blood feeding and interaction with vital pathways such as glucose, lipid, and nucleotide metabolism. Schistosomes obtain hemoglobin, plasma proteins, and immunoglobulins from the blood to meet their energy needs. The processing of these proteins is carried out by a complex system comprising various proteases, many of which are associated with HNF4 in S. japonicum. Furthermore, HNF4 is connected to several proteins involved in carbohydrate metabolism (Wu et al. Reference Wu, Huang, Zhao, Umar, Chen, Yu and Huang2024). Glucose, an essential nutrient, serves as the main energy source for schistosomes, providing them with the energy necessary for their growth and reproduction (You et al. Reference You, Stephenson, Gobert and McManus2014). Adult worms possess a higher lipid content and rely on their host for lipid acquisition (Skelly et al. Reference Skelly, Da’dara, Li, Castro-Borges and Wilson2014). A strong correlation between SjHNF4 and the phospholipid metabolism pathway suggests that SjHNF4 also contributes to lipid metabolism in S. japonicum (Wu et al. Reference Wu, Huang, Zhao, Umar, Chen, Yu and Huang2024).

S. mansoni nuclear receptors in subfamily 4

NR4A is the only member of subfamily 4 identified in Schistosoma worms. SmNR4A, like the human and Drosophila members of NR subfamily 4, has an atypical LBD suggesting that SmNR4A is an orthologue of Drosophila and human NR4A (Wu and LoVerde Reference Wu and LoVerde2021). SmNR4A is highly expressed in daughter sporocysts and adult worms, but scarcely in cercariae and early schistosomules (Wu and LoVerde Reference Wu, Tak and LoVerde2008).

S. mansoni nuclear receptors in subfamily 5

Fushi tarazu-factor 1 (FTZ-F1) (Smp_328000) NRs are the only receptors of subfamily 5 that have been characterized in S. mansoni. SmFTZ-F1 NRs contain two NRs: the SmFTZ-F1α belonging to NR5A group and the SmFTZ-F1 belonging to NR5B group (Lu et al. Reference Lu, Wu, Niles and LoVerde2006; Wu et al. Reference Wu, Niles, El-Sayed, Berriman and LoVerde2006). Smftz-f1α is continuously expressed throughout the schistosome life cycle, with the highest expression level observed at the egg stage (Lu et al. Reference Lu, Wu, Niles and LoVerde2006). RT-PCR reveals that Smftz-f1 is expressed at all developmental stages, with higher mRNA levels in miracidia, sporocysts, and cercariae. However, protein expression levels differ, being highest in cercariae, schistosomula, and male worms (de Mendonça et al. Reference De Mendonça, Bouton, Bertin, Escriva, Noël, Vanacker, Cornette, Laudet and Pierce2002). Romero et al. (Reference Romero, Cobb, Collins, Kliewer, Mangelsdorf and Collins2021) identified the micro-exon gene meg-8.3 as a target gene of SmFtz-F1, and this gene is expressed exclusively in the esophageal gland of the worm. They also found that Smftz-f1 and meg-8.3 are essential for maintaining the esophageal gland and preserving the integrity of the worm’s head. The esophageal gland plays a crucial role in protecting the worm from host attacks (Lee et al. Reference Lee, Chong and Newmark2020).

S. mansoni nuclear receptors subfamily with two DBDs and a single LBD (2DBD-NRs) (Figure 1b)

One of the most interesting findings is the isolation of a new group of NRs from S. mansoni, in which each receptor contains two DBDs and a single LBD (2DBD-NRs) (Wu et al. Reference Wu, Niles, El-Sayed, Berriman and LoVerde2006). S. mansoni Genome Project verified its presence (Berriman et al. Reference Berriman, Haas, LoVerde, Wilson, Dillon, Cerqueira, Mashiyama, Al-Lazikani, Andrade, Ashton, Aslett, Bartholomeu, Blandin, Caffrey, Coghlan, Coulson, Day, Delcher, DeMarco, Djikeng, Eyre, Gamble, Ghedin, Gu, Hertz-Fowler, Hirai, Hirai, Houston, Ivens, Johnston, Lacerda, Macedo, McVeigh, Ning, Oliveira, Overington, Parkhill, Pertea, Pierce, Protasio, Quail, Rajandream, Rogers, Sajid, Salzberg, Stanke, Tivey, White, Williams, Wortman, Wu, Zamanian, Zerlotini, Fraser-Liggett, Barrell and El-Sayed2009). These NRs have a novel modular structure: A/B-DBD-DBD-hinge-LBD organization in the NR. Sm2DBD-NRα is able to form a homodimer but cannot form a heterodimer with RXRs. S. mansoni expresses three 2DBD-NRs (Sm2DBD-NRα, Sm2DBD-NRβ, and Sm2DBD-NRγ) located on different chromosomes (Wu et al. Reference Wu, Niles, Hirai and LoVerde2007a). 2DBD-NRs have been identified and/or isolated only in Platyhelminths (Wu et al. Reference Wu, Niles, El-Sayed, Berriman and LoVerde2006; Wu et al. Reference Wu, Niles, Hirai and LoVerde2007a; Wu and LoVerde 2021) and Mollusca (Kaur et al. Reference Kaur, Jobling, Jones, Noble, Routledge and Lockyer2015; Vogeler et al. Reference Vogeler, Galloway, Lyons and Bean2014), suggesting they may be species-specific. Recently, 2DBD-NRs were identified in different animals (Wu and LoVerde Reference Wu and LoVerde2023). As shown by qRT-PCR, the three Sm2DBD NRs are developmentally regulated. Sm2DBD-NRα was found in sporocysts, cercariae, schistosomules, and male and female worms; Sm2DBDNRβ was expressed at high levels in eggs, sporocysts, cercariae, and male worms; and Sm2DBD-NRγ was only found in cercariae and juvenile worms (Wu et al. Reference Wu, Niles, Hirai and LoVerde2007a).

G protein-coupled receptors (GPCRs)

GPCRs represent the largest family of trans-membrane receptors involved in cellular communication in living organisms. These receptors can detect extracellular signalling molecules such as ions, light, hormones, neurotransmitters, amino acids, and neuropeptides, subsequently initiating a series of intracellular signal transduction pathways to produce the corresponding physiological effects (Weis and Kobilka Reference Weis and Kobilka2018). Biogenic amines exert their effects by interacting with cell-surface receptors, most of which belong to the superfamily of GPCRs. These receptors play roles in various biological processes, including growth, differentiation, neuronal signaling, olfaction, metabolism, and reproduction. The significance of GPCRs is underscored by their medical importance, as 30% to 50% of all pharmaceutical compounds target GPCRs and the signalling pathways they mediate (Miao and McCammon Reference Miao and McCammon2016).

GPCRs are composed of an extracellular N-terminus, a bundle of seven transmembrane α-helices (7TM), connected by extracellular and intracellular loops, and an intracellular C-terminus. The extracellular region, which includes the N-terminus, is responsible for ligand binding and varies in size from relatively short and often unstructured sequences in rhodopsin-like receptors to larger globular domains in other GPCR classes (Lagerström and Schiöth Reference Lagerström and Schiöth2008). The intracellular region interacts with G proteins, arrestins, and other downstream effectors (Tobin et al. Reference Tobin, Butcher and Kong2008).

GPCRs can activate guanine nucleotide-binding proteins (G proteins), which are responsible for signal transduction within the cell. G proteins transmit signals within the cell by interacting with various effector molecules, typically leading to changes in second messenger concentrations and subsequent cellular responses (Luttrell Reference Luttrell2008). In addition, GPCRs can activate G protein-independent signalling pathways via adaptor proteins such as arrestins (Bologna et al. Reference Bologna, Teoh, Bayoumi, Tang and Kim2017). Moreover, GPCRs can collaborate with other membrane proteins such as integrins and receptor tyrosine kinases (RTKs) (Cattaneo et al. Reference Cattaneo, Guerra, Parisi, De Marinis, Tafuri, Cinelli and Ammendola2014; Pyne and Pyne Reference Pyne and Pyne2011). When a ligand binds to a GPCR, it undergoes conformational changes and releases membrane-associated G-protein subunits (α, β, and γ). In this activated state, the GPCR functions as a guanine nucleotide exchange factor, promoting the exchange of GDP for GTP in the α subunit, leading to its dissociation from the βγ dimer. Both the dissociated α subunit and the βγ dimer can then trigger downstream signalling pathways (Frooninckx et al. Reference Frooninckx, Van Rompay, Temmerman, Van Sinay, Beets, Janssen, Husson and Schoofs2012).

According to the GRAFS classification, the mammalian GPCR are classified into five main families; Rhodopsin (Class A), Glutamate (Class C), Adhesion (Class B2), Frizzled/taste2 (Class F), and Secretin (Class B) (Bjarnadóttir et al. Reference Bjarnadóttir, Gloriam, Hellstrand, Kristiansson, Fredriksson and Schiöth2006). In addition to these major families, some organisms have lineage-specific receptors that establish distinct GPCR families (Hofmann and Palczewski Reference Hofmann and Palczewski2015). Rhodopsin-like receptors (Class A) are the most common of all known GPCRs. They are distinguished by short N-termini and their ability to interact with a wide range of ligands. The Glutamate receptor family is characterized by long N-termini that function as the binding site for ligands. Similarly, Adhesion receptors possess long N-termini containing a variety of domains, whereas Frizzled receptors feature long, cysteine-rich N-termini (Lagerström and Schiöth Reference Lagerström and Schiöth2008).

All major GPCR subfamilies were represented in schistosomes, and most of them respond to classical biogenic amines and neurotransmitters like dopamine, histamine, and serotonin (El-Shehabi et al. Reference El-Shehabi, Taman, Moali, El-Sakkary and Ribeiro2012; Hahnel et al. Reference Hahnel, Quack, Parker-Manuel, Lu, Vanderstraete, Morel, Dissous, Cailliau and Grevelding2014; MacDonald et al. Reference MacDonald, Kimber, Day and Ribeiro2015; Patocka et al. Reference Patocka, Sharma, Rashid and Ribeiro2014; Ribeiro et al. Reference Ribeiro, Gupta and El-Sakkary2012). Schistosoma GPCRs (SmGPCRs) are detected at the cell membrane and have a typical GPCR structure, an extracellular N-terminus, and an intracellular C-terminus. Most of homologues of SmGPR are characterized by the replacement of the highly conserved aspartate D^3.32 of TM domain 3 with asparagine (Hamdan et al. Reference Hamdan, Abramovitz, Mousa, Xie, Durocher and Ribeiro2002). Zamanian et al. (Reference Zamanian, Kimber, McVeigh, Carlson, Maule and Day2011) identified 117 S. mansoni G PCRs genes that include all major families; 105 Rhodopsin, 2 Glutamate, 3 Adhesion, 2 Secretin, and 5 Frizzled. Among these gene receptors, novel receptor groups have been detected, including a highly diverged Platyhelminth-specific Rhodopsin and atypical Glutamate-like receptors. Genome sequencing of S. mansoni has identified 126 GPCRs (Hahnel et al. Reference Hahnel, Wheeler, Lu, Wangwiwatsin, McVeigh, Maule, Berriman, Day, Ribeiro and Grevelding2018; Kamara et al. Reference Kamara, Thao, Kaur, Wheeler and Chan2023). However, only a few of these GPCRs have been characterized in terms of its molecular and functional properties (Hoffmann et al. Reference Hoffmann, Davis, Fischer and Wynn2001; MacDonald et al. Reference MacDonald, Kimber, Day and Ribeiro2015; Patocka et al. Reference Patocka, Sharma, Rashid and Ribeiro2014; Taman and Ribeiro Reference Taman and Ribeiro2009). The diversity of GPCR genes in S. mansoni indicates a wide array of functions, potentially including reproductive development (Hahnel et al. Reference Hahnel, Wheeler, Lu, Wangwiwatsin, McVeigh, Maule, Berriman, Day, Ribeiro and Grevelding2018).

Rhodopsin receptors

Rhodopsin receptors are prototypical GPCRs (Class A). They are further characterized by a relatively short extracellular N-terminus, which is typically glycosylated, and an intracellular C-terminal tail of variable length (Kristiansen Reference Kristiansen2004). Analysis of mammalian genomes revealed that the Rhodopsin family is divided into four main groups (α, β, γ, and δ) (Fredriksson et al. Reference Fredriksson, Lagerström, Lundin and Schiöth2003). The α and β subfamilies are the only subfamilies present in S. mansoni. Alpha receptors contain amines (the largest group), opsin-like receptors, and melatonin receptors. S. mansoni possesses at least 24 putative aminergic receptors and four melanopsin-like receptors, but no melatonin-like receptors. The β subfamily contains the neuropeptide and peptide hormone GPCRs. S. mansoni contains 36 putative peptide receptors. In addition, unclassified Rhodopsin receptors have been found. A new receptor, Platyhelminth Rhodopsin Orphan Family 1, has been identified. These receptors, although displaying remnants of classical Rhodopsin, do not show homology to any previously identified GPCRs (Zamanian et al. Reference Zamanian, Kimber, McVeigh, Carlson, Maule and Day2011).

Ortholog of Rhodopsin GPCRs identified in S. mansoni miracidia share similarity with Rhodopsin GPCRs of the intermediate host B. glabrata. These GPCRs may detect similar ligands, including snail-derived odorants that could facilitate miracidial host finding (Phan et al. Reference Phan, Liang, Zhao, Wyeth, Fogarty, Duke, McManus, Wang and Cummins2022).

Rhodopsin Alpha (α) subfamily receptors

S. mansoni has histamine receptors belonging to Class A (rhodopsin-like) GPCRs. Histamine is strongly myo-excitatory in S. mansoni and is endogenously biosynthesized (Hamdan and Ribeiro Reference Hamdan and Ribeiro1999). A histamine receptor called SmGPR-1 (Smp_043260) (formerly SmGPCR) was cloned in S. mansoni. SmGPR-1 has a structure characteristic of the amine GPCR family but does not obviously resemble any of the histamine receptors in mammals. Histamine activation of SmGPCR triggered mobilization of intracellular calcium, but not cAMP. Furthermore, SmGPCR-1 showed a glycine (Gly¹⁹⁶) substitution instead of (Asn/Thr) or charged residue (Glu) in TM domain 5 and an asparagine (Asn¹¹¹) instead of aspartate of TM domain 3 (Hamdan et al. Reference Hamdan, Abramovitz, Mousa, Xie, Durocher and Ribeiro2002). El-Shehabi et al. (Reference El-Shehabi, Vermeire, Timothy and Yoshino2009) revealed that this receptor is expressed in the tegument and musculature of both cercariae and adult parasites.

SmGPR-2 (Smp_043340) is a second histamine receptor of S. mansoni. It is an orphan receptor expressed in the vicinity of histamine-containing neurons in the sub-tegumental neuronal plexus. It is developmentally regulated showing up-regulation in the parasitic stages compared to cercaria, with the highest level of expression in young schistosomula. The highly conserved aspartate D^3.32 of TM domain 3 is also absent in SmGPR-2. This receptor has a novel pharmacological profile. It is inhibited by drugs not known to interact with histamine receptors, while classical anti-histamines had no effect on the receptor activity (El-Shehabi and Ribeiro Reference El-Shehabi and Ribeiro2010).

Dopamine receptors are GPCRs of the Class A Rhodopsin family. Mammals and invertebrates possess five dopamine receptors (D1-D5), which are categorized into two classes, D1-type and D2-type, based on their amino acid sequence homology and pharmacological profiles. D1-type dopamine receptors (D1 and D5) are associated with G-stimulatory proteins. Its activation leads to stimulation of adenylyl cyclase, resulting in an increase in cyclic adenosine monophosphate production from adenosine triphosphate (Gurevich et al. Reference Gurevich, Gainetdinov and Gurevich2016). In contrast, D2-type dopamine receptors (D2, D3, and D4) are linked to G-inhibitory proteins, which inhibit adenylyl cyclase and decrease cyclic adenosine monophosphate levels. S. mansoni D2 (SmD2) (Smp_127310) dopamine receptor exhibits an unusual pharmacological profile. Apomorphine, a potent antagonist of mammalian D2-type receptors, acts as an agonist for the SmD2 receptor, while other classic mammalian antagonists have no effect. This receptor is found in the membrane protein fractions of S. mansoni cercaria, schistosomula, and adult worms. SmD2 is also present in the sub-tegumental somatic musculature and acetabulum of cercaria and schistosomula. In adult parasites, SmD2 is enriched in the somatic muscles and, to a lesser extent, in the muscular lining of the caecum (Taman and Ribeiro Reference Taman and Ribeiro2009). In miracidium, antagonists of D2-type receptors have been found to delay miracidial transformation (Taft et al. Reference Taft, Norante and Yoshino2010).

Another neurotransmitter dopaminergic receptor belonging to GPCRs has been identified in S. mansoni and is named SmGPR-3 (Smp_043290) (El-Shehabi et al. Reference El-Shehabi, Taman, Moali, El-Sakkary and Ribeiro2012). This receptor is an orphan amine-like receptor found in schistosomes but not in mammals and has an atypical antagonist profile compared to mammalian receptors. Some mammalian D2 antagonists enhanced the activity of SmGPR-3 (El-Shehabi et al. Reference El-Shehabi, Taman, Moali, El-Sakkary and Ribeiro2012). SmGPR-3 is abundantly expressed in the nervous system of schistosomes, particularly in the main nerve cords and in the peripheral innervation of body wall muscles. Therefore, there are at least two routes of dopaminergic motor control in S. mansoni, involving both direct and indirect mechanisms. One pathway is mediated by SmD2, which is predicted to act directly on the musculature, while the other is a more indirect neuronal pathway mediated by SmGPR-3 (El-Shehabi et al. Reference El-Shehabi, Taman, Moali, El-Sakkary and Ribeiro2012).

In addition to histamine and dopamine, serotonin is one of the best characterized amines in flatworms and causes muscle excitation in S. mansoni. The worm contains a serotonergic receptor (Sm5HTR) that belongs to the Class A Rhodopsin family and is distantly related to serotonergic type 7 (5HT7) receptors found in other species. Sm5HTR signals through an increase in intracellular cAMP. The receptor is distributed in the cerebral ganglia and main nerve cords and in peripheral nerves of the body wall muscles and tegument. The serotonin receptor (Smp_126730) is a crucial component of the motor control system in S. mansoni (Patocka et al. Reference Patocka, Sharma, Rashid and Ribeiro2014). It is worth mentioning that PZQ, the sole treatment of schistosomiasis, has been identified as a GPCR ligand that acts by modulating serotoninergic signalling. PZQ modulates serotonergic signalling within a concentration range adequate to regulate the vascular tone of mesenteric blood vessels, where adult parasites reside in their host. The activity of PZQ on both parasite and host GPCRs likely contributes to its clinical efficacy by combining a harmful paralytic effect on the parasite with favorable effects on the host that aid in worm clearance (Chan et al. Reference Chan, Cupit, Gunaratne, McCorvy, Yang, Stoltz, Webb, Dosa, Roth, Abagyan, Cunningham and Marchant2017).

Octopamine and its precursor tyramine (phenolamines) are invertebrate specific biogenic proteins and neurotransmitter derived from tyrosine. They are considered the invertebrate counterpart of the adrenergic system. The schistosome genome annotated two putative GPCRs; octopamine (Smp_150180) and tyramine (Smp_043290) GPCRs (Protasio et al. Reference Protasio, Tsai, Babbage, Nichol, Hunt, Aslett, De Silva, Velarde, Anderson, Clark, Davidson, Dillon, Holroyd, LoVerde, Lloyd, McQuillan, Oliveira, Otto, Parker-Manuel, Quail, Wilson, Zerlotini, Dunne and Berriman2012). Octopamine receptor is a G protein-coupled receptor (GPCR) belonging to class A Rhodopsin-like subfamily (Hill et al. Reference Hill, Sharan and Watts2018). Octopamine labeling leads to the discovery of two pairs of ganglia in the adult schistosome brain. This neurotransmitter is localized in both ganglia and is also distributed throughout central and peripheral nerves and modulates schistosomula motility and length (El-Sakkary et al. Reference El-Sakkary, Chen, Arkin, Caffrey and Ribeiro2018).

Rhodopsin Beta (β) subfamily receptors

The β subfamily contains the great majority of neuropeptide and neuropeptide hormone GPCRs. Their neuropeptide signalling is known to play an essential role in flatworm locomotion, feeding, reproduction, host-finding, and regeneration (Kreshchenko Reference Kreshchenko2008). The genome of S. mansoni identifies at least 14 potential neuropeptide receptors, including several FLP-like and NPY/F-like receptors (Berriman et al. Reference Berriman, Haas, LoVerde, Wilson, Dillon, Cerqueira, Mashiyama, Al-Lazikani, Andrade, Ashton, Aslett, Bartholomeu, Blandin, Caffrey, Coghlan, Coulson, Day, Delcher, DeMarco, Djikeng, Eyre, Gamble, Ghedin, Gu, Hertz-Fowler, Hirai, Hirai, Houston, Ivens, Johnston, Lacerda, Macedo, McVeigh, Ning, Oliveira, Overington, Parkhill, Pertea, Pierce, Protasio, Quail, Rajandream, Rogers, Sajid, Salzberg, Stanke, Tivey, White, Williams, Wortman, Wu, Zamanian, Zerlotini, Fraser-Liggett, Barrell and El-Sayed2009). The invertebrate neuropeptide F family is related to the neuropeptide Y family of vertebrate peptides (with a C-terminal F instead of a Y) (McVeigh et al. Reference McVeigh, Mair, Atkinson, Ladurner, Zamanian, Novozhilova, Marks, Day and Maule2009). Zamanian et al. (Reference Zamanian, Kimber, McVeigh, Carlson, Maule and Day2011) identified several peptide receptors in S. mansoni, denoting that the peptidergic signalling is important for neurotransmission in the worm. They found that the number of potential flatworm peptide receptors significantly exceeds the peptide ligands identified so far. Most of the identified receptors cannot be associated with specific ligands with certainty. Peptides include FMRF amide-like peptides (FLPs), neuropeptide Fs (NPFs), and various other specific amides, some of which have similarities to peptides found in other phyla, such as neuropeptide FF (NPFF)-like and gonadotropin- or thyrotropin-releasing hormone-like peptides.

S. mansoni neuropeptide Y/F and its receptors have been identified in intramolluscan stages of S. mansoni and have been found to be associated with maintenance of schistosome germinal cell during intramolluscan development (Buddenborg et al. Reference Buddenborg, Kamel, Hanelt, Bu, Zhang, Mkoji and Loker2019). This neuropeptide has been also linked to a reduction in egg production of the infected snails (de Jong-Brink et al. Reference de Jong-Brink, ter Maat and Tensen2001).

Rhodopsin orphan receptor

A transcriptomics study revealed a rhodopsin orphan GPCR20 of S. mansoni (SmGPCR20) (Li et al. Reference Li, Weth, Haimann, Möscheid, Huber and Grevelding2024). This receptor belongs to the paired males-unpaired males-unpaired females subgroup of SmGPCRs, which is differentially transcribed between males and females expressing high transcript levels in paired male worms (bM), unpaired male worms (sM), and unpaired females (sF), whereas low or no transcripts of this subgroup were present in paired female worms (bF) (Hahnel et al. Reference Hahnel, Wheeler, Lu, Wangwiwatsin, McVeigh, Maule, Berriman, Day, Ribeiro and Grevelding2018; Lu et al. Reference Lu, Sessler, Holroyd, Hahnel, Quack, Berriman and Grevelding2016). This SmGPCR20 orphan receptor (Smp_084270) interacts with two neuropeptides – SmNPP26 and SmNPP40 – as potential interaction legends. qRT-PCR revealed that Smgpcr20, Smnpp26, and Smnpp40 genes showed sex- and/or pairing-dependent expression. The combination of SmGPCR20 with these neuropeptides affects egg production, oogenesis, and growth of the S. mansoni females (Li et al. Reference Li, Weth, Haimann, Möscheid, Huber and Grevelding2024).

Adhesion and Secretin receptors

Adhesion and Secretin receptors belong to Class B2 and Class B, respectively. In vertebrates, this family represents the second largest group of GPCRs, following the Rhodopsin family. Adhesion and Secretin receptors were identified in S. mansoni (Zamanian et al. Reference Zamanian, Kimber, McVeigh, Carlson, Maule and Day2011). They share sequence similarity in their 7-TM domains, but they showed structural differences in their N-terminal domains. Adhesion GPCRs possess a long N-terminal domain that features a varied arrangement of functional domains. Secretin GPCRs have N-terminal hormone-binding domains (HBD) that enable them to respond to peptide hormones (Nordström et al. Reference Nordström, Lagerström, Wallér, Fredriksson and Schiöth2009).

Cellular adhesion molecules are involved in the pathogenesis of S. mansoni. Integrins are one of the cellular adhesion molecules that also include cadherins, selectins, and the immunoglobulin (Ig) (Figliuolo et al. Reference Figliuolo da Paz, Figueiredo-Vanzan and Dos Santos Pyrrho2019). Integrins are a family of heterodimeric transmembrane receptors comprising at least 18 α and 8 β subunits in mammals (Hynes Reference Hynes2002). They mediate cell adhesion, functioning as connectors between the extracellular matrix and the cytoskeleton, while transmitting biochemical and mechanical signals between cells and their surroundings. They function bidirectionally across the plasma membrane, facilitating both inside-out and outside-in signalling (Fu et al. Reference Fu, Wang and Luo2012). Integrins are also involved in various immune-related signalling pathways in mammals (Zhang et al. Reference Zhang, Zhang, Chen and Xie2023).

Integrins work synergistically with other molecules, such as VKR1 and RTKs, within complex signalling pathways that regulate growth and differentiation processes (Gelmedin et al. Reference Gelmedin, Morel, Hahnel, Cailliau, Dissous and Grevelding2017). A significant portion of integrin signalling functions is dependent on a cytoplasmic TK (Harburger and Calderwood Reference Harburger and Calderwood2009). In S. mansoni, the genes coding for these signalling transduction protein kinases have been shown to have roles in reproductive organs differentiation process (Knobloch et al. Reference Knobloch, Beckmann, Burmeister, Quack and Grevelding2007). These kinases are cellular tyrosine kinases members that act in a multi-kinase complex (Beckmann et al. Reference Beckmann, Hahnel, Cailliau, Vanderstraete, Browaeys, Dissous and Grevelding2011) such as Src (SmTK3) (Kapp et al. Reference Kapp, Knobloch, Schüssler, Sroka, Lammers, Kunz and Grevelding2004), Syk (SmTK4) (Beckmann et al. Reference Beckmann, Buro, Dissous, Hirzmann and Grevelding2010), and Src/Abl (SmTK6) families (Beckmann et al. Reference Beckmann, Hahnel, Cailliau, Vanderstraete, Browaeys, Dissous and Grevelding2011).

Beckmann et al. (Reference Beckmann, Quack, Dissous, Cailliau, Lang and Grevelding2012) characterized four alpha-integrins (Smα-Int1–Smα-Int4) and one beta-integrin (Smβ-Int1) subunit from S. mansoni. The α (Smα-Int1, Smp_126140 and Smα-Int2, Smp_170280) and β (Smβ-Int1, Smp_089700) subunits are also present in S. mansoni exosomes (Samoil et al. Reference Samoil, Dagenais, Ganapathy, Aldridge, Glebov, Jardim and Ribeiro2018). The α-integrins of the free-living planarian Schmidtea mediterranea differ from those of S. mansoni in having only three α-integrin subunits. The β-integrins of S. mansoni did not bind fibronectin (Beckmann et al. Reference Beckmann, Quack, Dissous, Cailliau, Lang and Grevelding2012). These β-integrins are closely related to the human β4 subunit that binds laminin (Hynes Reference Hynes2002). The Smβ-Int1/Smα-Int1 heterodimer are found in the gonads, whereas the Smβ-Int1/Smα-Int2 heterodimer might fulfill more specialized functions in the area surrounding the ootype. Smβ-Int1 interacts and co-localizes with cellular tyrosine kinases in the reproductive organs of schistosomes with SmTK4, a Syk kinase, being its most significant interaction partner (Beckmann et al. Reference Beckmann, Quack, Dissous, Cailliau, Lang and Grevelding2012). The Smβ-Int1/SmVKR1 signalling complex plays a crucial role in the oocyte differentiation and survival in paired schistosomes (Gelmedin et al. Reference Gelmedin, Morel, Hahnel, Cailliau, Dissous and Grevelding2017). If Schistosoma integrins function in the same way as the mammalian homologues, these proteins could also help the parasite modulate the immune response of host cells (Samoil et al. Reference Samoil, Dagenais, Ganapathy, Aldridge, Glebov, Jardim and Ribeiro2018).

Glutamate receptors (GluRs)

L-glutamate is an important amino acid neurotransmitter in vertebrates and many invertebrates. It exerts its effects through interactions with ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). The iGluRs function as voltage-gated ion channels, while the metabotropic glutamate receptors (mGluRs) belong to the Class C GPCR superfamily, characterized by the typical seven transmembrane domain structure (Reiner and Levitz Reference Reiner and Levitz2018). Glutamate-gated chloride channels (GluCls) (Smp_128940) are pentameric ligand-gated inhibitory ion channels found exclusively in invertebrates. Their absence in vertebrates makes them an ideal target for antiparasitic drugs. However, GluCls of S. mansoni worms differ significantly from the GluCls of nematodes. This is exemplified by ivermectin, which leads to flaccid paralysis or kills roundworms by activating GluCls, while schistosomes are not susceptible to the drug (Dufour et al. Reference Dufour, Beech, Wever, Dent and Geary2013). Callau-Vázquez et al. (Reference Callau-Vázquez, Pless and Lynagh2018) demonstrated that the GluCl-2 from S. mansoni is activated by glutamate with a potency similar to that of nematode GluCls, despite substantial divergence in the ligand-binding C loop that differs in length compared to other pentameric ligand-gated ion channels, as well as the difference in hydrophobic channel gate.

Upon binding glutamate, mGluRs trigger signalling cascades or facilitate cation influx. mGluRs are structurally related to metabotropic gamma-aminobutyric acid (GABA) receptors and calcium-sensing, taste, and pheromone receptors (Niswender and Conn Reference Niswender and Conn2010). mGluRs are divided into three main groups. Group I (mGluR1 and mGluR5) signals through changes in intracellular calcium and the inositol phospholipid pathway, whereas Group II (mGluR2 and mGluR3) and Group III (mGluR4, mGluR6, mGluR7 and mGluR8) signal primarily through inhibition of adenylate cyclase, which in turn decreases intracellular cAMP signalling (Kryszkowski and Boczek Reference Kryszkowski and Boczek2021). mGluRs consist of a large N-terminal extracellular ligand binding domain (LBD), the characteristic 7-transmembrane (7-TM) segment, and a variable-length intracellular C-terminal domain (ICD). The LBD of mGluRs contains the glutamate-binding site within a Venus Flytrap module, which is connected to the 7-TM region by a short cysteine-rich domain (CRD) (Niswender and Conn Reference Niswender and Conn2010) (Figure 3a).

Figure 3.

(a) Schematic diagram of the expected S. mansoni metabotropic glutamate receptor (SmGluR) (Smp_128940) comprising a large N-terminal extracellular ligand binding domain (LBD), a seven-transmembrane (7-TM) anchoring segment, and a C-terminal intracellular domain (ICD) of varying lengths. The LBD is connected to the 7-TM region by a short cysteine-rich domain (CRD). (b) S. mansoni glutamate-binding protein (SmGBP) (Smp_052660) receptor has a conserved ligand binding domain (LBD) but is missing the cysteine-rich domain, the characteristic 7-TM region, and intracellular domain.

Glutamate immunoreactivity in S. mansoni was detected in the nervous system, including the cerebral ganglia, longitudinal nerve cords, and commissures (Mendonça-Silva et al. Reference Mendonça-Silva, Pessôa and Noël2002). The genome of S. mansoni encodes at least three sequences that are homologous to mGluRs from other species (Berriman et al. Reference Berriman, Haas, LoVerde, Wilson, Dillon, Cerqueira, Mashiyama, Al-Lazikani, Andrade, Ashton, Aslett, Bartholomeu, Blandin, Caffrey, Coghlan, Coulson, Day, Delcher, DeMarco, Djikeng, Eyre, Gamble, Ghedin, Gu, Hertz-Fowler, Hirai, Hirai, Houston, Ivens, Johnston, Lacerda, Macedo, McVeigh, Ning, Oliveira, Overington, Parkhill, Pertea, Pierce, Protasio, Quail, Rajandream, Rogers, Sajid, Salzberg, Stanke, Tivey, White, Williams, Wortman, Wu, Zamanian, Zerlotini, Fraser-Liggett, Barrell and El-Sayed2009). Taman and Ribeiro (Reference Taman and Ribeiro2011a) described a mGluR in S. mansoni (SmGluR) in the nervous system of adult worms and cercariae, as well as in the female reproductive tract. SmGluR belongs to the GPCR superfamily and shares a distant relationship with mGluRs found in other species (Figure 3a). However, SmGluR differs from mammalian mGluRs with respect to signalling mechanism and pharmacological profile. SmGluR is activated by glutamate, whereas GABA has no significant effect. Phylogenetic analyses indicated that SmGluR shares a similar degree of sequence homology with mGluRs as it does with other family C GPCRs, such as GABA receptors.

The second metabotropic glutamate receptor identified in S. mansoni is glutamate-binding protein (SmGBP) (Smp_052660) (Taman and Ribeiro Reference Taman and Ribeiro2011b). SmGBP represents a new type of glutamate receptor that may be unique to flatworms. Genes encoding similarly truncated receptors have been found in the S. japonicum genome (The Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium 2009) and the partially annotated genome of the planarian Schmidtea mediterranea (Robb et al. Reference Robb, Ross and Sánchez Alvarado2008), but they are not known to occur in other metazoans. SmGBP receptor is an atypical receptor; it is a C-terminally truncated mGluR with a conserved ligand (glutamate)-binding domain (LBD) located within a Venus Flytrap module but lacking the cysteine-rich domain (CRD), the characteristic 7-TM region, and the intracellular domain (ICD) (Figure 3b). SmGBP is suggested to be either an integral membrane protein or a peripheral protein closely associated with the membrane. This receptor is gender- and stage-specific. SmGBP is localized on the surface of male worms, especially on the dorsal tubercles but not in females or larval stages (Taman and Ribeiro Reference Taman and Ribeiro2011b). In S. japonicum, the related schistosome species, two putative mGluRs, are identified: SjGRM7 and SjGRM. SjGRM7 has been found to be crucial for normal physiological functions, growth, development, and egg production (Wang et al. Reference Wang, Cheng, Chen, Zhang, Xie, Liu, You, Yi, Zhu, Gu, Xu, Lu, Wang and Hu2022).

Frizzled receptors

Frizzled protein receptors belong to GPCRs Class F. They consist of seven trans-membrane proteins with a cysteine-rich domain in the N-terminal extracellular region required for Wnt ligand binding. Most Frizzled receptors share a common C-terminal motif that is a binding site for the cytoplasmic protein domain (Hering and Sheng Reference Hering and Sheng2002). Wnt signalling plays a key role in embryonic development, energy metabolism, and balance (Nusse Reference Nusse2015). Zamanian et al. (Reference Zamanian, Kimber, McVeigh, Carlson, Maule and Day2011) identified four Frizzled sequences S. mansoni. SmFz1 genes (Smp_118970, Smp_173940) are regulated by pairing in gonads. In vitro inhibition of the gene affects the survival of adult worms, decreases the egg production, and affects the gonad differentiation, morphology, and embryogenesis (Hahnel et al. Reference Hahnel, Quack, Parker-Manuel, Lu, Vanderstraete, Morel, Dissous, Cailliau and Grevelding2014). Secreted frizzled-related protein can inhibit Wnt signalling by competitive binding to the frizzled protein-specific receptor (García-Tobilla et al. Reference García-Tobilla, Solórzano, Salido-Guadarrama, González-Covarrubias, Morales-Montor, Díaz-Otañez and Rodríguez-Dorantes2016). Knockdown of S. japonicum secreted frizzled-related protein gene impairs worm growth and development, survival and morphological structure, reproductive ability, and viability of the eggs produced (Cheng et al. Reference Cheng, Li, Qin, Xu, Zhang, Liu, Gu and Jin2019).

Acetylcholine receptors

Acetylcholine (ACh) is a crucial neurotransmitter in both vertebrate and invertebrate species. In vertebrates, ACh functions primarily as an excitatory neurotransmitter, regulating processes such as muscle contraction, glandular secretion, and memory formation. ACh similarly plays an excitatory role in invertebrates, and its involvement in nematode motor function is well documented. However, there is a significant exception in schistosomes, where ACh acts as a major inhibitory neurotransmitter or modulator. Activation of ACh receptors (AChR) in S. mansoni leads to muscle relaxation, resulting in flaccid paralysis (Day et al. Reference Day, Chen, Miller, Tian, Bennett and Pax1996). Metrifonate, an Acetylcholinesterase (AChE) inhibitor, elevates synaptic levels of ACh, resulting in prolonged paralysis of the axial muscles of schistosomes and halting its movement. This action is suggested to be due to the secondary effects of muscle paralysis. The drug demonstrates equal potency and efficacy in vitro against both S. mansoni and S. haematobium but is only effective in vivo against the latter species (Bueding et al. Reference Bueding, Liu and Rogers1972).

Most of AChRs in schistosomes are nicotinic AChRs (nAChRs), so named because of their high affinity for nicotine. However, muscarinic cholinergic receptors are also expected to be present. One of these receptors possesses all the structural characteristics of GPCR (MacDonald et al. Reference MacDonald, Kimber, Day and Ribeiro2015). In vertebrates, nAChRs are invariably cation-selective (Na⁺, Ca2⁺, K⁺) and mediate excitatory responses. In contrast, invertebrates have cation and anion-selective (Cl) ACh-gated channels. These acetylcholine-gated chloride channels (ACC) mediate Cl^--driven membrane hyperpolarization and are believed to play a role in inhibitory responses to ACh. These ACC are structurally related to nAChRs but are selective for chloride ions (Beech et al. Reference Beech, Callanan, Rao, Dawe and Forrester2013). Structurally, nAChRs belong to the superfamily of Cys-loop ligand-gated ion channel. They form homo- and hetero-pentameric structures organized in a barrel shape around a central ion-selective pore (Albuquerque et al. Reference Albuquerque, Pereira, Alkondon and Rogers2009). A key characteristic of ACCs is the presence of a Pro-Ala motif in the pore-lining M2 domains of their subunits. This motif has been shown to convey anion selectivity to other ligand-gated ion channels (LGICs), replacing a Glu residue typically found in cation-selective channels (Keramidas et al. Reference Keramidas, Moorhouse, Pierce, Schofield and Barry2002). These ACCs that appear to be specific to invertebrates are found in S. mansoni (SmACCs) and have an inhibitory modulatory effect on the neuromuscular system of schistosome potentially through a chloride influx produced by the activation of SmACCs and their receptors (SmACC-1 and SmACC-2) (Smp_176310 and Smp_142690) (MacDonald et al. Reference MacDonald, Buxton, Kimber, Day, Robertson and Ribeiro2014). Treatment with ACh antagonists and RNA interference (RNAi) leads to suppression of SmACCs and induces a hypermotile effect. Two of the SmACCs were localized to regions of the peripheral nervous system that innervate the body wall muscles; however, none appear to be directly expressed in the muscle tissue (MacDonald et al. Reference MacDonald, Buxton, Kimber, Day, Robertson and Ribeiro2014).

Muscarinic acetylcholine receptors (mAChRs) belong to GPCR superfamily and are related to Rhodopsin (Family A GPCRs) in their structure. The term ‘muscarinic’ originates from the preference of receptors to bind to and be activated by the fungal toxin muscarine (Dale Reference Dale1914). Schistosome muscarinic acetylcholine receptor is also referred to as G protein-coupled acetylcholine receptors (SmGAR) (Smp_145540). Expression of this receptor is predicted to be high during the early larval stages of schistosomes (Protasio et al. Reference Protasio, Tsai, Babbage, Nichol, Hunt, Aslett, De Silva, Velarde, Anderson, Clark, Davidson, Dillon, Holroyd, LoVerde, Lloyd, McQuillan, Oliveira, Otto, Parker-Manuel, Quail, Wilson, Zerlotini, Dunne and Berriman2012). SmGAR is constitutively active but can be further stimulated by ACh and, to a lesser extent, by the cholinergic agonist carbachol. Anti-cholinergic drugs exhibit an inverse agonist activity towards SmGAR, significantly reducing its basal activity. A phenotypic RNAi assay demonstrated that suppression of SmGAR activity in early-stage larval schistosomula results in a marked decrease in larval motility (MacDonald et al. Reference MacDonald, Kimber, Day and Ribeiro2015).

In addition to its neuromuscular effects, ACh has been linked to glucose transport across the tegument and increased glucose uptake in schistosomes. AChE has been shown to play a role in modulating glucose uptake by schistosomes from the blood of mammalian hosts. Two main molecular forms of AChE are found in S. mansoni. One form is located within the muscle and plays a role in cholinergic processes, while the other form is found on the surface, anchored to the membrane by a covalently bound glycophosphatidylinositol anchor. This surface-localized AChE may participate in non-cholinergic processes and signal transduction (Espinoza et al. Reference Espinoza, Silman, Arnon and Tarrab-Hazdai1991). Glycophosphatidylinositol-anchored AChE can be released from the schistosome surface membrane by a PI-specific phospholipase C, which can remove significant amounts of AChE from the tegument of schistosomula in vitro without affecting the parasite viability (Espinoza et al. Reference Espinoza, Tarrab-Hazdai, Silman and Arnon1988). It has been suggested that release of AChE triggers immediate replenishment of the surface enzyme. However, this process occurs with another glycophosphatidylinositol-anchored protein, alkaline phosphatase, which is also present on the surface of schistosome (Arnon et al. Reference Arnon, Silman and Tarrab-Hazdai1999).

Glucose uptake is regulated through the interaction of ACh with tegumental nAChRs and AChE. The effect of ACh on glucose uptake can be inhibited by blocking any of the ACh cholinergic systems. AChE is thought to regulate interaction of ACh with its receptor since inhibition of AChE produces an effect similar to excessive presence of ligand (Jones et al. Reference Jones, Bentley, Oliveros Parra and Agnew2002). Exposure to the same concentration of ACh present in host blood was found to enhance glucose uptake in S. haematobium and S. bovis, but not in S. mansoni. However, at higher concentrations, ACh inhibited glucose uptake from the host blood into the parasites. The glucose uptake rate in adult S. haematobium and S. bovis is roughly double that of S. mansoni (Camacho and Agnew Reference Camacho and Agnew1995), and the first two species have relatively higher AChE activity on their teguments compared to S. mansoni (Camacho et al. Reference Camacho, Tarrab-Hazdai, Espinoza, Arnon and Agnew1994). These elevated levels of AChE activity contribute to its increased susceptibility to metrifonate (Harder Reference Harder2002), which may explain why metrifonate is effective against S. haematobium and S. bovis but not S. mansoni.

Adult stages of schistosomes possess AChE and nAChR on their teguments, and both components are concentrated on the surface of the adult male, a key site for nutrient uptake for the worm pair (Camacho and Agnew Reference Camacho and Agnew1995). AChR expression increases during parasites pairing and sexual maturation as the pairing state increases the uptake of several host compounds (Camacho et al. Reference Camacho, Tarrab-Hazdai, Espinoza, Arnon and Agnew1994). AChE inhibitors impair the parasite glucose uptake ability, which affects the parasite growth and development (Sundaraneedi et al. Reference Sundaraneedi, Tedla, Eichenberger, Becker, Pickering, Smout, Rajan, Wangchuk, Keene, Loukas, Collins and Pearson2017; You et al. Reference You, Liu, Du, Nawaratna, Rivera, Harvie, Jones and McManus2018).