Serotonin signaling modulates growth and motility in juvenile Fasciola hepatica

Fasciola hepatica causes fasciolosis, a parasitic disease that poses significant animal and human health challenges. Control relies on flukicides, most of which are adulticides, with only triclabendazole effective against the pathogenic migratory juvenile. Classical neurotransmitter pathways are widely targeted by anthelmintics yet remain underexplored for flukicide development. Here we explore the importance of serotonin (5-HT) signaling in juvenile fluke. In silico analyses confirmed all F. hepatica life stages express a complete 5-HT signaling pathway encompassing genes encoding proteins for 5-HT synthesis, transport, and reuptake, as well as five putative 5-HT G protein-coupled receptors (GPCRs). Homology and binding motif analyses supported the presence of two 5-HT1 (Fh5HT1A, Fh5HT1B) and three 5-HT7 (Fh5HT7A, -7B, -7C) GPCRs. Immunocytochemistry and in situ hybridization revealed widespread neuronal expression of 5-HT, its synthetic enzyme tryptophan hydroxylase (FhTPH), and the GPCR Fh5HT7C. 5-HT addition stimulated juvenile fluke motility; consistent with this observation, serotonin reuptake inhibition, which causes 5-HT persistence at synaptic junctions, also enhanced juvenile movement. Silencing of FhTPH, a key enzyme in 5-HT synthesis, blunted juvenile motility, a phenotype reversed by the addition of 5-HT. Silencing the fluke vesicular monoamine transporter (FhVMAT), which packages 5-HT into synaptic vesicles, reduced juvenile motility, whilst silencing the 5-HT reuptake transporter (FhSERT) which recycles synaptic 5-HT increased juvenile motility and growth, consistent with 5-HT accumulation enhancing effects. Whilst combinatorial silencing of Fh5HT1 receptors reduced fluke motility, silencing Fh5HT7 receptors led to a greater reduction in motility. Exogenous addition of 5-HT partially rescued motility deficits of juveniles with silenced Fh5HT1 receptors, but 5-HT excitation was abolished in Fh5HT7-RNAi juveniles, exposing their importance to fluke motility. Notably, sustained 5-HT exposure promoted juvenile growth, but these effects were not blunted by receptor-RNAi. The findings emphasize a central role of serotonin signaling in both juvenile motility and growth, exposing novel aspects of receptor function and encouraging therapeutic exploitation for liver fluke control. Author SummaryThe liver fluke, Fasciola hepatica, causes fasciolosis, a neglected tropical disease that poses a significant burden on human and animal health. There is no vaccine for fasciolosis and treatment relies on a single drug, triclabendazole, to control the early stages of infection which cause liver pathology whilst migrating through the mammalian host. Single drug reliance has increased the incidence of drug resistance in both human and animal populations, such that there is a pressing need for the characterization of novel drug targets and development of new anthelmintics targeting liver fluke. The focus of this research is to examine the role of the serotonin signaling system of liver fluke, bridging a gap in knowledge to enable the exploitation of this signaling pathway for flatworm drug development. Here, bioinformatic analysis has characterized the pathway components and receptors in multiple clinically relevant flatworm parasite species. Chemical and functional genomic methods have been used to prove the integral function of serotonin in liver fluke biology, regulating motility and growth, both essential for parasite infection and survival. This work provides data that help validate the serotonergic system of liver fluke as a potential target for future anthelmintic development.