PMC

RNAi was performed in cultured schistosomulae as described in except that motility was measured both in the absence and presence of added serotonin (+5HT) at 10⁻⁴ M. Data are the means and SEM of 40 animals and 2–3 transfections per treatment. **, significantly different from basal activity (measured in the absence of serotonin) at p<0.001. ns, not significant at p<0.05.

Schistosomulae were treated with varying amounts of serotonin (5HT) (A) or o-methyl-serotonin (B). Larval motility was quantified as the frequency of body movements (shortening and elongation) per min of observation, using an imaging assay, as described . (C) Larval motility was measured in the presence of 10⁻⁴ M serotonin (5HT) added alone or together with a serotonin antagonist also at 10⁻⁴ M. Motility data are the means and SEM of 3 experiments each with 10–12 animals per treatment. ^a Significantly different from the untreated controls (p<0.05); ^bSignificantly different from 5HT-induced motility (p<0.05). MIAN, mianserin; CPH, cyproheptadine.

Summary of mechanisms by which serotonin can stimulate worm movement. The results suggest that serotonin works through its Sm5HTR receptor to stimulate interneuronal and/or neuromuscular signaling, indirectly increasing contractility of the musculature. Serotonin may also have direct effects on the muscles but it is unclear if those effects are mediated by Sm5HTR or a different receptor. An additional mechanism by which serotonin may control movement is through modulation of sensory neuronal circuits, where Sm5HTR is abundantly expressed. These could play an important role in mediating locomotory responses to host-derived signals, for example chemotactic responses or other types of host-parasite interaction.

Larvae were treated with 50(mock control) and cultured for 8 days. (A) Motility assays were performed using the same imaging assay described above. The data are the means and SEM of 3 experiments, each performed with at least 20 animals in duplicates. (B) For measurements of RNA expression, total RNA was isolated 8- days post-transfection and oligo-dT reverse-transcribed. Quantitative qPCR was performed with primers targeting Sm5HTR and α-tubulin as a housekeeping gene. Expression was calculated relative to the scrambled control sample, using the comparative Pfaffl method. *** Significantly different from control at p<0.0001; ns, not significant at p<0.05. (C) Representative images of schistosomulae transfected with Sm5HTR siRNA compared to scrambled siRNA or untransfected larvae. Images were obtained at 8 days post-transfection. The controls show the characteristic movement of repeated shortening and elongation, whereas the RNAi-suppressed animals are round in shape and appear unable to elongate the body.

In vitro transformed schistosomulae (3–8 days old) were probed with affinity-purified anti-Sm5HTR antibody and visualized by confocal microscopy. (A) Typical schistosomulum showing Sm5HTR (green) immunoreactivity in varicose nerve fibers along the length of the body (left panel). Animals were co-labelled with TRITC-phalloidin to visualize the musculature (middle panel) and the overlay (right panel) shows punctate bright yellow fluorescence, indicating sites of apparent co-localization between Sm5HTR and the outer circular muscles of the body wall (arrows). (B) Schistosomulae were co-labelled with anti-serotonin antibody (green) and anti-Sm5HTR antibody (red). The overlay shows close proximity between the two signals (solid arrows) in the cerebral ganglia (cg) and the main longitudinal nerve cords (nc). Sites of apparent co-localization are marked by open arrows. Sm5HTR immunoreactivity is also prominent in the developing caecum (ce). (C) Schistosomulum co-labelled with anti-serotonin antibody (green) and anti-Sm5HTR antibody (red). Lateral serotonin-containing nerve fibers (solid arrows) can be seen in close proximity to Sm5HTR immunoreactivity in the body wall region of the larva. cg, cerebral ganglia; ce, caecum; nc, nerve cord. (D) Transmission light and corresponding fluorescence image of a typical negative control. No significant fluorescence could be seen in any of the negative controls probed with secondary antibody only or antigen-preadsorbed antibody. Scale bars, 25 µm.

(A) Adult males and females were electroporated with Sm5HTR siRNA or irrelevant control siRNA and motility was recorded approximately 24 hr post-transfection, using an imaging assay. Worm movement was recorded for periods of 2 min in the presence of added serotonin (10⁻⁴ M) and motility data were normalized relative to the irrelevant siRNA control also treated with serotonin. Each data point is the mean and SEM of 15–19 worms obtained from three separate RNAi experiments. Motility in the RNAi – suppressed animals was significantly different from the scrambled siRNA control at p<0.001 (***) or p<0.01 (**). (B) RNAi knockdown was verified at 24 hr post-transfection by reverse transcription (RT) -qPCR analysis. The % remaining expression was determined by the Pfaffl method and was calculated relative to the corresponding male or female control siRNA group. (C) RNAi knockdown was further verified at the protein level by western blot analysis. Worms were harvested 24 hr post-transfection and membrane proteins were extracted. Equal amounts of membrane proteins from test and control worms (mixed males and females) were probed with anti-Sm5HTR antibody or an antibody against a different membrane protein (SmSERT; ) as a loading control. Worms electroporated with Sm5HTR siRNAs had significantly less western positive Sm5HTR protein than the control worms electroporated with irrelevant siRNA.

The amino acid sequences of known and predicted serotonin receptors were aligned using the ClustalW method and a Neighbor-Joining Best Tree was constructed from the alignment. The alignment included representative examples of all major classes of serotonergic GPCRs but only three clades are shown for simplicity. These include type 2 serotonin receptors (5HT2), type 7 (5HT7) and structurally related type 1 and type 5 receptors (5HT1/5). The outgroup in the alignment (not shown) was a distantly related rat metabotropic glutamate receptor (NP_058707). The tree was rooted to the outgroup and was tested by bootstrap analysis with 1000 iterations. The length of the branches is proportional to the genetic distance between sequences. Sequences were all obtained from the National Center for Biotechnology Information (NCBI). S. mansoni sequences are designated by their S. mansoni GeneDB “smp”designation number and are marked by arrows. Accession numbers for the remaining sequences are as follows: 5HT2_Drosophila (NP_730859); 5HT2_Lymnae (AAC16969.1), 5HT2(ser-1)_C. elegans (AAC15778); 5HT2B_Xenopus (NP_001082744); 5HT2A_human (NP_000612); 5HT2A_mouse (NP_766400); 5HT2B_human (NP_000858); 5HT2C_human (NP_000859); 5HT5B_mouse (NP_034613.2); 5HT5A_human (NP_076917.1); 5HT1_Helisoma (AAQ95277.1); 5HT1A_Xenopus (NP_001079299.1); 5HT1A_human (NP_000515.2); 5HT1A_chicken (NP_001163999.1); 5HT1D_dog (P11614); 5HT1A_pufferfish (O42385); 5HT1_Aplysia (NP_001191550); 5HT1(ser-4)_C. elegans (NP_497452); 5HT_Haemonchus (AAO45883); 5HT7_Xenopus (NP_001079253.1); 5HT7_human (NP_062873.1); 5HT7_chicken (NP_001167605.1) 5HT7_Helisoma (AAQ84306.1); 5HT7_Meleagris (XP_003206102); 5HT7_zebrafish (XP_690599); 5HT7_Clonorchis (GAA29051); 5HT7_Aedes (AF296125); 5HT7 (ser-7)_C. elegans (NP_741730); 5HT_Dugesia.1 (BAA22404); 5HT_Dugesia.4 (BAA22403); 5HT7_Dugesia (BAI44327.1); 5HT4_chicken (XP_414481.2); 5HT4_human (NP_000861.1); 5HT6_chicken (NP_001166911.1); 5HT6_human (NP_000862.1); 5HT6_mouse (NP_067333.1); ratmGlu.8393487; mGluR1_rat (NP_058707).

Adult worms were probed with anti-Sm5HTR antibody followed by a FITC-labeled secondary antibody (green) and then examined by confocal microscopy. In some experiments, animals were co-labeled with TRITC-phalloidin (red) to visualize the musculature or DAPI (blue) to label nucleated cell bodies. (A) A typical adult male showing strong Sm5HTR green fluorescence in the cerebral ganglia (cg) and main longitudinal ventral nerve cords (vnc) of the CNS. Also visible are smaller nerve fibers innervating the oesophagus and caecum (arrows). (B) Sm5HTR immunofluorescence is apparent in anastomosing fine nerve fibers of the oral sucker (os) in a male schistosome. (C) Head region of a male schistosome showing numerous Sm5HTR-containing nerve fibers, including a pair of main nerve cords that extend anteriorly to the oral sucker (arrows). (D) Sm5HTR expression is visible both in the ventral nerve cords (vnc) and lateral longitudinal cords (lnc) in a male worm. (E) Dorsal view of the mid-body of a male worm showing high expression of Sm5HTR in the paired dorsal nerve cords (dnc) and connecting transverse commissures (tc). (F) Large numbers of Sm5HTR immunoreactive fibers in the tail region of a male worm. (G) Sm5HTR is present in the innervation of the ventral sucker (vs) (solid arrows) and the peripheral nerve net (nn) of the worm's body wall. (H) Mid-body view of a male worm showing strong Sm5HTR immuoreactivity in numerous peripheral nerve fibers extending to the worm's body wall. A higher magnification of this peripheral innervation (I) shows that Sm5HTR is present in neuronal elements of the submuscular nerve plexus (solid arrows) and also the subtegumental nerve plexus closer to the surface of the animal (open arrows). Specific immunoreactivity can be seen in apparent sensory nerve fibers (solid arrowhead) connecting the surface of the parasite to the submuscular nerve net below. (J) Sm5HTR was also detected in female schistosomes. A typical adult female shows strong immunoreactivity in the cerebral ganglia (cg) and ventral nerve cords (vnc) of the CNS, as well as smaller nerve fibers innervating the caecum and ventral sucker. No significant immunoreactivity was seen in any of the negative controls, either male worms (K) or females. (L) Diagram of key elements of the schistosome nervous system: cg, cerebral ganglia; vnc, ventral nerve cord; lnc, longitudinal lateral nerve cord; dnc, dorsal nerve cord; tc, transverse commissures; nn, nerve net; os, oral sucker; vs, ventral sucker. Scale bars, 25 µm.

(A) HEK 293 cells were transfected with a plasmid expressing Sm5HTR-FLAG or empty plasmid (mock control) and probed with anti-FLAG antibody, followed by a FITC-conjugated secondary antibody. Typical in situ immunofluorescence results show expression of FLAG-tagged receptor in the test cells. (B) Schematic of the key signaling mechanisms of serotonin-activated GPCRs. Receptors can signal through changes in intracellular cAMP or Ca²⁺, depending on which G protein (Gs, Gi/o, Gq) is activated. Preliminary experiments showed that serotonin activation of Sm5HTR in HEK 293 cells elevated cAMP but had no effect on cytosolic Ca²⁺ (data not shown). AC, adenylate cyclase; PLCβ, phospholipase C-β; IP₃, inositol trisphosphate; ER, endoplasmic reticulum. (C) Sm5HTR activation causes an increase in intracellular cAMP. HEK 293 cells expressing Sm5HTR were treated with biogenic amines, known serotonergic agonists and antagonists, each at 10⁻⁴ M. Antagonists were tested in the presence of 10⁻⁴ M serotonin. After incubation, the cells were lysed and the lysates were assayed for cAMP. The data were normalized relative to control cells transfected with empty plasmid (mock control). Substances tested included: serotonin (5HT), histamine (HA), acetylcholine (ACh), dopamine (DA), octopamine (OA), tyramine (TA), adrenaline (A), metanephrine (MTN), tryptamine (Trp), o-methyl-serotonin (methyl-5HT), buspirone (BUS), 8-Hydroxy-DPAT (DPAT), mianserin (MIAN), chlorpromazine (CHP), cyproheptadine (CPH). ^a significantly different from untreated at p<0.01; ^b significantly different from serotonin –induced cAMP level at p<0.01. Experiments were repeated with variable concentrations of serotonin (D) and o-methyl-serotonin (E) to obtain dose-response curves. Data were baseline-subtracted and normalized relative to the maximum agonist-induced response. Each data point is the mean and SEM of at least three independent experiments performed in duplicates or triplicates.