Fig 1.
Fasciola hepatica possesses serotonin signaling pathway genes.
(A) Schematic representation of the bioinformatic pipeline used to identify serotonin signaling genes in F. hepatica. (B) Gene identifiers (IDs) of serotonin-related components identified in the F. hepatica genome assembly PRJEB58756. Abbreviations: FhTPH = tryptophan hydroxylase; FhAADC = aromatic L-amino acid decarboxylase; FhVMAT = vesicular monoamine transporter; FhSERT = serotonin reuptake transporter; Fh5HTR = serotonin-gated G protein-coupled receptors (GPCRs). (C) Illustration of serotonin signaling at a synapse. (1) The precursor amino acid tryptophan (TRP) is converted to 5-hydroxytryptophan (5-HTP) by tryptophan hydroxylase (TPH). (2) 5-HTP is further converted to serotonin (5-hydroxytryptamine, 5-HT) by aromatic L-amino acid decarboxylase (AADC). (3) 5-HT is sequestered into synaptic vesicles via vesicular monoamine transporters (VMAT). (4) Vesicles fuse with the synaptic membrane, releasing 5-HT into the synaptic cleft. (5) Released 5-HT binds to GPCRs on the postsynaptic neuron to propagate signaling. Note: although serotonin can also interact with ligand-gated ion channels (LGICs), no 5-HT-sensitive LGICs were identified in the F. hepatica genome. Magenta-bracketed numbers indicate the number of encoding genes identified in genome PRJEB58756. Created in BioRender. McCusker, P. (2026) https://BioRender.com/28zes5h. (D) Sequence alignments of F. hepatica serotonin signaling genes with homologous sequences from Homo sapiens (Hs-) and Schistosoma mansoni (Sm-). Red lines highlight conserved catalytic domains in TPH, and red arrowhead indicates pyridoxal-phosphate binding site in AADC. Blue lines represent transmembrane domain 1 of VMAT and SERT, while a blue dot marks a key aspartic acid residue implicated in ligand binding. (E) Heatmap displaying expression levels of F. hepatica serotonin signaling genes across different life cycle stages (egg to adult). Z-scores were calculated from transcripts per million (TPM) data obtained from WormBase ParaSite (release 54) and generated by Cwiklinski et al. [44].
Fig 2.
Comparative and phylogenetic analysis of serotonin-gated G protein-coupled receptors (GPCRs) in Fasciola hepatica and other parasitic flatworms.
(A) Multiple sequence alignment of F. hepatica serotonin GPCRs with homologous sequences from Homo sapiens (Hs-) and Schistosoma mansoni (Sm-). The green line highlights transmembrane domain three (TM3), while the purple line highlights transmembrane domain six (TM6). Conserved serotonin-binding motifs (DVXXCT and WXXF) essential for ligand recognition, as well as the canonical ‘DRY’ motif critical for GPCR activation, are outlined in red. (B) Summary table displaying the number of serotonin-gated GPCRs identified across clinically relevant trematode and cestode species. Genomic data were obtained from WormBase ParaSite (version 19) and analyzed for the presence of serotonin GPCR homologs. The species investigated include Echinococcus canadensis (PRJEB8992), Echinococcus granulosus (PRJNA182977 - G1), Echinococcus multilocularis (PRJEB122 - Java), Echinococcus oligarthrus (PRJEB31222 - DaMi1), Hymenolepis diminuta (PRJEB507 - Denmark), Hymenolepis microstoma (PRJEB124), Hymenolepis nana (PRJEB508 - Japan), Mesocestoides corti (PRJEB510 - Specht & Voge 1965), Taenia asiatica (PRJEB532 - South Korea), Taenia multiceps (PRJNA307624 - Gns01), Taenia saginata (PRJNA71493 - TSAYD01), Taenia solium (PRJNA170813 - Mexico), Dibothriocephalus latus (PRJEB1206), Spirometra erinaceieuropaei (PRJEB1202), Schistocephalus solidus (PRJEB527 - NST_G2), Hydatigera taeniaeformis (PRJEB534 - Spain/Canary Islands), Clonorchis sinensis (PRJNA386618 - Cs-k2), Schistosoma bovis (PRJNA451066), Schistosoma japonicum (PRJNA520774 - HuSjv2), Fasciolopsis buski (PRJNA284521 - HT), Fasciola gigantica (PRJNA230515 - Uganda_cow_1), Schistosoma haematobium (PRJNA78265), Schistosoma curassoni (PRJEB519 - Senegal/Dakar), Schistosoma mansoni (PRJEA36577), Trichobilharzia regenti (PRJEB44434 - tdTriRege1.1), Opisthorchis viverrini (PRJNA222628), Paragonimus westermani (PRJNA454344), and Echinostoma caproni (PRJEB1207 - Egypt). (C) Maximum likelihood phylogenetic tree depicting the evolutionary relationships between F. hepatica serotonin-gated GPCRs and homologous GPCR sequences from other species. Reference sequences were obtained from Homo sapiens (5-HT1A-F, 5-HT2A-C, 5-HT3A-E, 5-HT5A, and 5-HT7), Caenorhabditis elegans (5-HT1, 5-HT2, and 5-HT7), and Drosophila melanogaster (5-HT1 and 5-HT2A/B) through UniProt [45]. Additionally, published S. mansoni GPCR sequences (Smp_197700, Smp_245850, and Smp_126730) were included based on prior literature [references 14,29,46]. (D) CLANS (Cluster Analysis of Sequences) diagram visualizing the clustering relationships of serotonin-gated GPCRs from all examined parasitic flatworm species. Clustering groups are color-coded based on sequence similarity and evolutionary relationships. Red shows flatworm 5-HT GPCR cluster 1 sequences; blue shows flatworm 5-HT GPCR cluster 2 sequences; orange boxes show clustering of F. hepatica 5-HT GPCR sequences; yellow box shows human 5-HT1 GPCR sequence; cyan box shows D. melanogaster 5-HT2 GPCR sequences.
Fig 3.
Structural models of putative Fasciola hepatica 5-HT-gated G protein coupled receptors (GPCRs) support the presence of residues critical to serotonin binding.
(A) Predicted structural confirmation of 5HT7AFhep GPCR showing key ligand binding residues. (B) Predicted structural confirmation of 5HT7BFhep GPCR showing key ligand binding residues. (C) Predicted structural confirmation of 5HT7CFhep GPCR showing key ligand binding residues. (D) Predicted structural confirmation of 5HT1AFhep GPCR showing key ligand binding residues. (E) Predicted structural confirmation of 5HT1BFhep GPCR showing key ligand binding residues. All structures predicted and visualized using Phyre2 [60] and PyMOL [57] software. The serotonin (SRO) ligand was imported directly from the Protein Data Bank (https://www.rcsb.org) for binding analysis carried out by DockingPie [58]. (F) Sequence alignments of F. hepatica 5HT7 and 5HT1 receptors showing predicted essential amino acids for ligand binding. Ligand binding predicted by DockingPie [58]. Blue line highlights transmembrane regions and red shading highlights conserved amino acids predicted by docking analysis.
Fig 4.
Serotonin and serotonin-signaling components are widely expressed in neurons in juvenile Fasciola hepatica.
(A) Whole-body immunocytochemistry in 21-day-old juvenile F. hepatica showing widespread serotonin immunoreactivity (IR), especially in the main ventral nerve cords and brain. Green fluorescence represents serotonin IR. (B) Anterior region immunolocalization of serotonin in 21-day-old juvenile F. hepatica, highlighting serotonin in the cerebral ganglia and surrounding neural structures. Green fluorescence indicates serotonin IR. (C) Whole-body schematic representation of F. hepatica adult, illustrating key biological structures and major nervous system components, including nerve cords, commissures, and neuronal cell bodies. Created in BioRender. McCusker, P. (2026) https://BioRender.com/s3petk8. (D) Anterior schematic of F. hepatica, showing essential oral and reproductive structures, including the oral sucker, pharynx, gonopore, and cirrus sac. Created in BioRender. McCusker, P. (2026) https://BioRender.com/s3petk8. (E) Whole-body fluorescent in situ hybridization (ISH) showing F. hepatica tryptophan hydroxylase (FhTPH) in the nervous system. Red fluorescence indicates positive ISH signal, while blue fluorescence (DAPI staining) marks cell nuclei. FhTPH expression is observed in a pattern that aligns to neuronal cells of the nervous systems. (F) In situ hybridization (ISH) showing expression of the F. hepatica serotonin-gated G protein-coupled receptor (5HT7CFhep) in anterior neurons and adjacent regions. Red fluorescence shows receptor gene expression, while blue fluorescence (DAPI staining) highlights nuclei. Receptor localization is evident in the cerebral ganglia and other cells consistent with neuronal patterning. Abbreviations: OS = oral sucker, VS = ventral sucker, VNC = ventral nerve cord, NCB = neuronal cell body, PhN = pharyngeal nerves, RN = reproductive nerves, CG = cerebral ganglion, GP = gonopore, DNC = dorsal nerve cord, LNC = lateral nerve cord, TC = transverse commissure, PN = peripheral nerves, P = pharynx, U = uterus, CS = cirrus sac, NCBC = neuronal cell body cluster.
Fig 5.
Chemical inhibition and gene-silencing of serotonin-signaling components in juvenile Fasciola hepatica alter motility.
(A) Increased motility in newly excysted juvenile F. hepatica following 24-hour exposure to 1 mM serotonin (5-HT) and 1 μM fluoxetine hydrochloride (FluHCL). Each data point represents motility (percentage length change per mm relative to the negative control) of an individual worm. Statistical significance determined by ANOVA; **p ≤ 0.01, ****p ≤ 0.0001; minimum n = 45 NEJs per treatment group. (B) Altered motility of 28-day-old juvenile F. hepatica after RNAi knockdown of serotonin-signaling pathway genes. Targeted genes include FhTPH (tryptophan hydroxylase), FhAADC1 and FhAADC2 (aromatic amino acid decarboxylase enzymes), FhVMAT (vesicular monoamine transporter), and FhSERT (serotonin reuptake transporter). Motility is expressed as percentage movement (mm/min) of individual juveniles compared to untreated controls. dsRNA exposure carried out twice per week as shown. Statistical significance determined by ANOVA; **p ≤ 0.01, ****p ≤ 0.0001; minimum n = 36 juveniles per treatment group. (C) Decreased motility of 28-day-old juvenile F. hepatica following combinatorial RNAi knockdown of serotonin-G protein-coupled receptors (GPCRs). Targeted receptors include type 1 (5HTR1AFhep and 5HTR1BFhep) and type 7 (5HTR7AFhep, 5HTR7BFhep, and 5HTR7CFhep) serotonin receptors. Data points represent motility (percentage, mm/min) of individual juveniles compared to untreated controls. dsRNA carried out twice per week as shown. Statistical significance determined by ANOVA; **p ≤ 0.01.; minimum n = 68 juveniles per treatment group. (D) Serotonin rescues the motility impact of FhTPH-knockdown in F. hepatica juveniles. Black bars represent basal motility (mm/min) of juveniles post repeated double stranded (ds)RNA exposure (dsCTRL or dsTPH) but pre-chemical exposure. Gray bars show basal motility (mm/min) of juveniles post-dsRNA treatment followed by 18-hour exposure to 0.001% dimethyl sulfoxide (DMSO) or 1 mM 5-HT as outlined in red. Statistical significance determined by t-test; *p ≤ 0.05; minimum n = 30 juveniles per treatment group. (E) The silencing of Fh5HT type 7 GPCRs abolishes the stimulatory effects of 1 mM 5-HT on juvenile liver fluke. Black bars represent percentage motility (compared to RPMI) of juveniles post repeated dsRNA exposure (dsCTRL, ds5HTR1 [5HTR1AFhep, 5HTR1BFhep] or ds5HTR7 [5HTR7AFhep, 5HTR7BFhep, and 5HTR7Cfhep]) but pre-chemical exposure. Gray bars show motility after dsRNA treatment followed by 18-hour exposure to 1 mM 5-HT. Statistical significance determined by t-test; *p ≤ 0.05; minimum n = 30 juveniles per treatment group.
Fig 6.
Serotonin and the gene silencing of selected serotonin-signaling components alter juvenile Fasciola hepatica growth.
(A) The growth enhancing effect of prolonged exposure (28 days) to 1 mM serotonin (5-HT) on juvenile F. hepatica. Each data point represents the size of an individual juvenile expressed as percentage area (mm²) relative to the unsupplemented media control. Statistical significance determined by t-test analysis; **p ≤ 0.01; minimum n = 80 juveniles per treatment group. (B) Brightfield images showing the growth enhancing impact of 1 mM 5-HT supplementation on 21-day-old F. hepatica juveniles. Top image shows snapshot of 21 randomly selected juveniles from 80 total cultured juveniles without 5-HT supplementation, bottom image shows snapshot of 19 randomly selected juveniles from 91 total cultured juveniles with 5-HT supplementation. (C) Growth enhancing effect of FhSERT silencing in 28-day-old F. hepatica juveniles. Targeted genes include FhTPH (tryptophan hydroxylase), FhAADC1 and FhAADC2 (aromatic amino acid decarboxylases), FhVMAT (vesicular monoamine transporter), and FhSERT (serotonin reuptake transporter). Growth is expressed as percentage area (mm²) of individual juveniles compared to untreated controls. dsRNA exposure carried out twice a week as shown. Statistical significance determined by ANOVA; *p ≤ 0.05; minimum n = 34 juveniles per treatment group. (D) RNAi-mediated knockdown of type 1 serotonin-G protein-coupled receptors (GPCRs) in 28-day-old juvenile F. hepatica enhanced growth. Targeted receptors include type 1 (5HTR1AFhep and 5HTR1BFhep) and type 7 (5HTR7AFhep, 5HTR7BFhep, and 5HTR7CFhep). Each data point represents the relative area (percentage, mm²) of an individual juvenile compared to untreated controls. dsRNA exposure carried out twice a week as shown. Statistical significance determined by ANOVA; ****p ≤ 0.0001; minimum n = 64 juveniles per treatment group.