S. mansoni miracidia respond to snail-derived biomolecules [46], although the precise identity of the active biomolecule(s) has not been clearly defined. One study implicated “miracidia-attracting glycoproteins” present within the snail mucus [12], while in silico analysis from B. glabrata snail conditioned-water proteins predicted interactions of uncharacterized S. mansoni proteins with B. glabrata proteins [46]. Peptides have also been implicated, whereby a snail-derived novel peptide (named P12) stimulated changes in the behaviour of the S. mansoni miracidia [47].
In this study, to narrow down biomolecules potentially involved in the parasite and host interplay, we utilised gene resources from both the B. glabrata and S. mansoni to identify ortholog GPCRs that are likely used by each organism to detect similar ligands. We reported that 8 S. mansoni miracidia GPCRs share significant identity with 8 B. glabrata GPCRs, not only in GO mapping, but also within regions corresponding to putative TM domains. These include GPCRs with similarity to neuropeptide GPCRs that bind FMRFa, AST-A/buccalin and sulfakinin peptides. We propose that the miracidial ortholog GPCRs may be used for neural signaling, requiring a common ligand, and/or are used to detect semiochemical biomolecules present within the water. The latter expectation was validated by miracidial behaviour changes in the presence of snail FMRFamide and AST-A/buccalin peptides.
The AST-A and its receptor have been characterised in various insects [48] where they are involved in multiple functions such as inhibition of juvenile hormone biosynthesis and reduction of food intake [48], AST-A-like neuropeptides have been identified in gastropods and bivalve molluscs, including Lottia gigantea, Theba pisana, Aplysia californica and Crassostrea gigas [49–52]. Buccalin, named following its first identification in the accessory radula closer muscle of A. californica [53], has been implicated in various activities in molluscs such as the inhibition of muscle contraction, regulation of feeding and spawning [53–55]. Also in gastropods and bivalves, the AST-A/buccalin receptor was identified through in silico analysis of publicly available genomic datasets including that of B. glabrata [56]. In our study, we identified an AST-A/buccalin receptor ortholog in S. mansoni, although there are no reports that S. mansoni has a buccalin-like peptide. In fact, a comprehensive neuropeptide investigation of 10 platyhelminth species showed that only the free-living turbellarian Macrostomum lignano has a buccalin-like peptide (npp-9 gene; GAYSGFLG) [57]. We identified a buccalin-like peptide in the B. glabrata conditioned water. Despite the presence of neuropeptides in mucus secretions having not been well investigated, we previously identified neuropeptides (including buccalin) within the salivary gland mucus of T. pisana [58, 59].
The FMRFa was first discovered in the hard clam Mercenaria mercenaria and it is thought to have a pleitropic role in molluscan physiology [60–63]. Extensive studies performed on the freshwater snail Helisoma showed that FMRFa and related peptides are densely concentrated not only in the nervous system but also within the salivary glands [64]. An FMRFa receptor has been identified in the heart and nervous tissue of the land snail Helix [62, 65] and the optic lobe membrane of the squid Loligo pelei [66]. The S. mansoni genome contains a gene encoding a FLP precursor (npp-13 gene) that may be processed to release two RFamide peptides (HFMPQRFa and YTRFVPQRFa) [57]. A synthetic FLP (GNFFRFa) derived from non-schistosome platyhelminth precursors stimulates contraction of S. mansoni muscle fibres in vitro [67]. An FLP receptor has also been reported in the turbellarian flatworm Girardia tigrina based on sequence similarity and a receptor calcium mobilization assay [68]. The homolog receptor in S. mansoni miracidia was investigated in the current study due to its similarity with the B. glabrata FMRFa receptor. Our behaviour assays also indicated that snail-derived FMRFa can be detected by S. mansoni miracidia due to their staying significantly longer in FOV and the increased acceleration of miracidia, supported by the observed presence of an FLP in B. glabrata conditioned water. However, as S. mansoni has the potential to generate endogenous FLPs, we cannot preclude the possibility that the applied FMRFa may stimulate endogenous effects, leading to the observed miracidial behaviour changes.
The monoamine 5-HT plays a critical role in neural transmission and has been very well documented throughout eumetazoans, as has the conservation of 5-HT GPCRs. In adult S. mansoni, 5-HT stimulates motor activity [69], while in the miracidia, an immunofluorescent approach localized 5-HT to within sensory nerves [70]. The 5-HT GPCR was identified within our interspecies GPCR ortholog analysis yet we found that 5-HT at 5 mM did not modify miracidial behaviour, while the significant change in acceleration could be attributed to its high concentration.
Sulfakinin is a sulfated neuropeptide best known for its function as a satiety (food intake) factor [71]. In silico data-mining showed that molluscan SK has the C-terminal RF(W)amide sequence common to insect sulfakinins, as well as the DY motif shared by both insect SKs and vertebrate cholecystokinin (CCK) [72]. Since vertebrate CCKs and insect SKs reveal similar biological function relating to digestive enzyme secretion, satiety and smooth muscle contraction [73], it is possible that their molluscan counterparts have retained similar basic biological activities. In contrast, there is no obvious SK in S. mansoni, suggesting that the parasite may only recognize the B. glabrata SK, either as a secreted semiochemical, or once it penetrates the snail as a guidance peptide to navigate to the hepatopancreas where it proliferates [74]. Our behavior assays demonstrated that SK did not alter miracidial behavior (neither the velocity nor duration present under FOV were affected), and therefore it is more likely to act as an internal stimulus in S. mansoni.
FMRFa and buccalin peptides may contribute to a cocktail of biomolecules that could be used as an effective, species-specific attractant. Our serial dilution assays suggested sustained bioactivity for both buccalin and FMRFa peptides at a concentration of at least 1 µM. We also report 1 orphan peptide GPCR ortholog within B. glabrata and S. mansoni miracida, which is consistent with the possibility that uncharacterized species-specific peptides could help attract miracidia to the appropriate snail host due to its presence in many tissues of B. glabrata and its high expression level in S. mansoni miracidia.