All experiments were approved by the Animal Ethics Committee (ethics number P242) of the QIMR Berghofer Medical Research Institute. The study was conducted according to the guidelines of the National Health and Medical Research Council of Australia, as published in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 7th edition, 2004 (www.nhmrc.gov.au). All work involving live S. mansoni life cycle stages was performed in quarantine-accredited laboratories as required by Australian Biosecurity law.

Swiss mice (female, 6 weeks old) were infected subcutaneously with 100 S. mansoni cercariae. Seven weeks post-infection the mice were euthanised and adult worms were obtained by portal perfusion with 37°C pre-warmed RPMI Medium 1640 (Gibco, Sydney, Australia). Mouse livers were removed at necropsy and liver eggs were isolated (41, 42), with immature and mature liver eggs separated as described (43). Miracidia were harvested by hatching mature eggs in deionized water under light (41). S. mansoni cercariae were obtained by shedding infected Biomphalaria glabrata snails under bright light. Schistosomula were obtained by mechanical transformation of cercariae in vitro as described (2), and cultured in Basch’s medium (2) for five days to produce 5-day old schistosomula.

Searches for SmFGFRs sequences were conducted using genome version Smansoni_v7, GCA_000237925.3 (44, 45) on the WormBase ParaSite website (https://parasite.wormbase.org/Schistosoma_mansoni_prjea36577/Info/Index). Searches for homologous FGFR protein sequences were performed using the Basic Local Alignment Search Tool (BLAST) on the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the WormBase ParaSite website (http://parasite.wormbase.org/Multi/Tools/Blast). Protein sequence identity was analyzed using EMBOSS Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) (46, 47). Signal peptides were predicted by the SignalP-5.0 Server (http://www.cbs.dtu.dk/services/SignalP/). Protein molecular weight and isoelectric point were calculated using the ExPASy-Compute pI/Mw tool (http://web.expasy.org/compute_pi/). Domain predictions were conducted using the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/). Prediction of SmFGFRA 3D structures was performed using I-TASSER (https://zhanglab.dcmb.med.umich.edu/I-TASSER/) (48–50). Searching the S. mansoni single-cell atlas was carried out using the SchistoCyte resource (http://www.collinslab.org/schistocyte/) (39, 51).

Total RNAs were extracted from S. mansoni eggs, miracidia, cercariae, schistosomula and adult male and female worms using RNeasy Mini Kits (Qiagen, Melbourne, Australia), followed by cDNA synthesis using QuantiTect Reverse Transcription Kits (Qiagen). Quantitative real-time PCR (qPCR) was performed with QuantiNova SYBR^® Green PCR Kits (Qiagen) using a Corbett Rotor Gene 6000 Real-Time PCR system. Forward primer (5’-ATGGGACTCAATTACGCATT-3’) and reverse primer (5’-CACCACTGTCTTCCGACCTG-3’) for SmfgfrA were designed using the Primer 3 software (http://frodo.wi.mit.edu/), and the specificity of the primer sequences was confirmed by BLAST. The S. mansoni Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) housekeeping gene was used as reference gene (52). The qPCR reactions comprised 10 µl 2xSYBR Green PCR Master Mix, 100 ng cDNA, and 0.7 µM of each primer. The cycling parameters were as follows: 95°C for 2 min, 40 cycles of 95°C for 5 s and 58°C for 10 s. Relative gene transcriptional levels were normalized to the GAPDH gene and determined using the 2^-ΔΔCt calculation.

The SmFGFRA extracellular ligand binding domain (from T³⁹ to L³⁸⁶, SmFGFRA-L) coding sequence (excluding signal peptide) was amplified from S. mansoni cDNA using a forward primer (5’-TACTTCCAATCCAATGCAACTTTACACTGTGCGTGTGACGC-3’) and a reverse primer (5’-TTATCCACTTCCAATGTTATTATCACAATCCACTATCCCTATAGGACAAATTTTC-3’). The full length SmFGFRA (from L²⁷ to H⁹¹⁸) coding gene, without signal peptide, was amplified from S. mansoni cDNA using a forward primer (5’-TACTTCCAATCCAATGCACTTGAGTGTAAATCACAATCAATGTACGAA-3’) and a reverse primer (5’-TTATCCACTTCCAATGTTATTATCACTAGTGTAAATACTGTCGCGGTTCCAAGT-3’). Both fragments were expressed in Escherichia coli after cloning into the pET His6 TEV LIC cloning vector (1B) (a gift from Scott Gradia, Addgene, Watertown, Massachusetts, USA); a Ligation Independent Cloning (LIC) fusion tag was added at the 5’ end of each primer (underlined in each primer sequence). The vector was linearized using the SspI-HF restriction enzyme (New England Biolabs, Melbourne, Australia) and then purified using a QIAquick Gel Extraction Kit (Qiagen). The linearized vector and insert were treated with T4 polymerase and then annealed at a molecular ratio of 1:3 at room temperature for 5 min followed by the addition of 1 μl 25 mM EDTA to stop the reaction. After sequence confirmation, the reconstructed vectors were transformed into E. coli (Rosseta strain) for expression induced with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) (Sigma-Aldrich, Sydney, Australia) at 37°C for 3 h. Recombinant protein was purified using a Ni-NTA His-tag affinity kit (Qiagen). HALT protease inhibitor cocktail (Thermo Fisher Scientific) was added during protein purification. The His-tag on the purified protein was then removed by ProTEV Plus (Promega, Sydney, Australia) according to the manufacturer’s instructions.

A polyclonal antibody against the recombinant SmFGFRA-L (rSmFGFRA-L) was generated in SWISS mice (8 weeks old, female). Briefly, five mice were injected intraperitoneally with rSmFGFRA-L protein (25 μg/each mouse) adjuvanted with Montanide ISA 720 VG (SEPPIC, Courbevoie, France) three times at 2-weekly intervals (3). Blood was collected 2 weeks after the third injection. The titre of the antibody was determined using an enzyme-linked immunosorbent assay (ELISA) as described (53–55). In brief, an ELISA plate (Thermo Fisher Scientific, Brisbane, Australia) was coated with rSmFGFRA-L protein (1 µg/ml, 100 µl/well) in 0.05 M Carbonate-Bicarbonate coating buffer (pH 9.6) overnight at 4°C, followed by blocking at 37°C for 1 h with blocking buffer [1% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich) in PBS containing 0.05% (v/v) Tween-20 (PBST)]. Serially diluted serum in blocking buffer (100 µl/well) was added and the plate incubated at 37°C for 1 h. As negative controls, naïve mouse serum was used. Following 3 x washes with 0.05% PBST, a secondary antibody, Novex™ Goat anti-Mouse IgG (H+L) - cross-adsorbed, horseradish peroxidase (HRP) conjugate (Thermo Fisher Scientific), was added (1:2,000, 100 µl/well) and the plate was incubated at 37°C for 1 h. After 3 x washes with 0.05% PBST, 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added (50 μl/well) followed by 10 min incubation at room temperature prior to stopping the reaction with 2 M sulphuric acid (50 μl/well). The absorbance of each well was measured at 450 nm using a POLARstar OPTIMA multi-detection microplate reader (BMG LABTECH, Victoria, Australia). A positive antibody response was defined as an OD450 higher than 2.1 times the mean of the OD450 of serum samples from control mice.

S. mansoni, S. japonicum, and S. haematobium soluble worm antigen preparation (SWAP) (53) and S. mansoni soluble egg antigen (SEA) (56) were prepared as described. The mouse anti-rSmFGFRA-L serum was utilized in western blots to probe electrophoresed schistosome SWAP and S. mansoni SEA. Protein samples were separated on 12% SDS-PAGE gels and transferred to an Immun-Blot low fluorescence-PVDF membrane (Bio-rad, Sydney, Australia). The membrane was first blocked with Odyssey Blocking Buffer (PBS) (LI-COR Biosciences, Lincoln, Nebraska, USA) for 1 h at room temperature. Then, the membrane was incubated with the mouse anti-rSmFGFRA-L serum (1:100 diluted in Odyssey buffer with 0.1% Tween-20) for 1 h followed by four washes in phosphate-buffered saline (PBS) plus 0.1% Tween-20 (0.1% PBST). Subsequently, the membrane was incubated with IRDye-labeled 680LT goat anti-mouse IgG antibody (Li-COR Biosciences) (1:15,000 diluted in Odyssey buffer with 0.1% Tween-20 and 0.01% SDS) for 1 h with shaking in a dark chamber. After washing (4X) with 0.1% PBST, the membrane was dried in the dark and visualized using the Odyssey CLx Infrared Imaging System (53).

S. mansoni adult worms, immature eggs, mature eggs, miracidia, cercariae, and 5-day old schistosomula were fixed in 10% (w/v) formalin in PBS prior to paraffin embedding. Sections (3-4 µm) prepared from the paraffin blocks were affixed to positively charged adhesive slides, air-dried overnight at 37°C and then dewaxed and rehydrated through xylol and descending graded alcohols to water. Subsequently, the sections were transferred to Dako Target Retrieval Buffer (pH 9.0) (Dako, Carpinteria, California, USA) and subjected to 30 min heat antigen retrieval at 95°C. This was followed by washing three times in 0.1% PBST. Sections were blocked in Biocare Medical Background Sniper with 2% (w/v) BSA (Sigma-Aldrich) for 15 min to stop non-specific binding and then incubated at room temperature overnight with mouse anti-rSmFGFRA-L antibody (1:50 dilution). After washing three times with 0.1% PBST, the sections were incubated with Alexa Fluor^® 555 donkey anti-mouse IgG (Invitrogen, Melbourne, Australia) (1:300 dilution) for 2 h at room temperature.

To determine the co-localization of SmFGFRA and stem cells in adult S. mansoni, freshly perfused worms were cultured overnight in RPMI complete medium [RPMI Medium 1640 (Gibco, Sydney, Australia) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco) and 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco)] at 37°C in 5% CO_2. Then the worms were incubated for 24 h with 10 µM EdU (thymidine analog 5-ethynyl-2’-deoxyuridine) (Thermo Fisher Scientific), which only stains stem cells in schistosomes (7). The stained worms were fixed in 10% formalin, paraffin embedded and sectioned. Sections (4 µm) of EdU-labeled adult worms were subjected to EdU detection using a Click-iT™ EdU Cell Proliferation Kit (Alexa Fluor™ 488 dye) (Thermo Fisher Scientific) according to the manufacturers’ instructions. Then the same sections were subjected to SmFGFRA immunolocalization as described above. Nuclei in all tissue sections were also stained with diamidino-2-phenylindole (DAPI) gold (Invitrogen) and visualized using a Zeiss 780 NLO confocal microscope (Zeiss, Oberkochen, Germany).

The binding affinity between rSmFGFRA-L/human FGFR1 and human acidic FGF (aFGF)/basic FGF (bFGF) was determined using the Octet RED 96 System (FortéBio, Menlo Park, California, USA) in standard Greiner black 96-well microplates (Sigma-Aldrich) as described (53). Briefly, to investigate the binding between rSmFGFRA-L and human aFGF/bFGF, the rSmFGFRA-L protein was biotinylated using a NHS-PEO4-biotin kit (Thermo Fisher Scientific), desalted with a Zeba Spin Desalting Column (Thermo Fisher Scientific), and then the protein was immobilized to a Streptavidin Biosensor (FortéBio). Prior to the assay, the Biosensors were hydrated in kinetic buffer (PBS with 15 mM NaCl, 0.1 mg/ml BSA, 0.002% Tween-20) for 60 min. Subsequently, a duplicate set of Biosensors were first incubated in kinetic buffer for 300 s as baseline and followed by immobilization for 600 s in 200 μl of 150 ng/μl biotinylated rSmFGFRA-L protein. Next, the Biosensors were washed in kinetic buffer for another 300 s. Finally, the sensors were exposed to a series (200 μl volume) of diluted concentrations of human aFGF/bFGF (Thermo Fisher Scientific). To further explore whether the binding affinity between human bFGF and rSmFGFRA-L is comparable to that between human bFGF and human FGFR1, human bFGF (8 ng/μl) was biotinylated and immobilized to Biosensors and then exposed to different concentrations (30 ng/μl and 23 ng/μl) of rSmFGFR-L and recombinant extracellular ligand binding domain of human FGFR1 (R²²-I³⁷⁶) (FGFR1-L) (Sigma-Aldrich). PBS was used as a negative control. The association of the two proteins was detected for 1000 s followed by dissociation in the kinetic buffer for 1000 s. Experiments were run at 30°C with the orbital agitation of the microplate set to 1000 rpm. Data were analyzed using the FortéBio Data Analysis 7.1 program and included a double reference subtraction. The sample subtraction was conducted using PBS as a reference control, and sensor subtraction was performed on all samples automatically (53).

An anti-phospho-P44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Cell Signaling Technology, New England Biolabs, Ipswich, USA) was used to detect activated (phosphorylated) extracellular signal regulated kinases 1 and 2 (Erk1/2) in S. mansoni adult worms (53) following stimulation with human aFGF or bFGF. Briefly, freshly perfused S. mansoni adult worms were cultured overnight in RPMI complete medium at 37°C in 5% CO_2. The parasites were then cultivated for 30 min in RPMI complete medium containing 10 nM human aFGF, or 10 nM bFGF, or 10 nM aFGF and bFGF (the same concentration used in a previous study (14)). For the inhibitor treatment group, adult S. mansoni were cultured in RPMI complete medium with 10 μM BIBF 1120 (dissolved in dimethyl sulfoxide (DMSO) and then diluted to different concentrations) (Sigma-Aldrich), or 0.1% DMSO (negative control) for 30 min as described (14). After each treatment, worms were harvested for SWAP extraction as described (53) in the presence of HALT protease inhibitor cocktail. Isolated SWAP was then separated on a 10% SDS-PAGE gel and analyzed by western blotting as described above.

A Universal Tyrosine Kinase Assay Kit (Takara, Melbourne, Australia) was used to determine the enzymatic activity of rSmFGFRA at different concentrations (100 ng/μl - 0.78 ng/μl) in the presence or absence of 5 μM and 10 μM BIBF 1120 as described (14, 36), following the manufacturer’s instructions. DMSO (0.1%) was served as negative control. The activity of rSmFGFRA was determined by comparing its absorbance with that of control protein tyrosine kinase (PTK), supplied with the kit, according to the manufacturer’s instructions. Technical duplicates were performed and the experiment was repeated twice.

S. mansoni eggs were cultured overnight at 37°C in RPMI complete medium under an atmosphere of 5% CO_2. The eggs were then treated with different concentrations of BIBF 1120 (2.5 μM, 5 μM, 10 μM, 20 μM) or 0.1% DMSO (negative control). After seven days, the eggs were collected and miracidia were hatched in deionized water under light as described above. The egg hatching efficiency (%) in each group was calculated by dividing the number of hatched eggs with the total number of examined eggs X 100. The behavior of miracidia hatched from treated and control eggs was also monitored using a published bioassay (57, 58). Briefly, around 30 S. mansoni miracidia in 100 μl deionized water were, using a pipette, evenly distributed to the centre of a microscope slide. Miracidial movement (swimming) was monitored using an Olympus-CKX41 microscope equipped with an Olympus DPI Digital Microscope Camera DP22 (25 frames per second at 2.8-megapixel image quality). Miracidial movement in the field of view (FOV) was recorded for 1 minute by video. Then the videoed miracidial tracks were analyzed using FIJI software to calculate three individual behavioral measurements including velocity of miracidial movement, duration (time) of miracidia staying within the FOV, and tortuosity (the ratio of track length to maximum displacement) of miracidial movement (57). The miracidial movement velocity was calculated in pixel/s using the rolling mean subtraction method (57, 58). Miracidial location was tracked in each frame along an x-y axis and the trajectories were interpolated using the plugin for FIJI software, known as TrackMate (58, 59). Employing the MTrackJ plugin, the average velocity, duration, and tortuosity of miracidia in the FOV were determined. Heatmaps, representing the movement pattern of individual miracidia, were generated as described (58, 60).

All data are displayed as the mean ± SE. Differences between groups were analyzed for statistical significance by One-way ANOVA. GraphPad Prism software (Version 8.2.1, La Jolla, CA, USA) was used for all statistical analyses. A statistically significant difference for a particular comparison was defined as a p value ≤ 0.05. * p value≤ 0.05, ** p value≤ 0.01, *** p value ≤ 0.001, **** p value ≤ 0.0001, not significant (ns).

SmfgfrA (Smp_175590) encodes SmFGFRA and SmfgfrB (Smp_157300) has two transcripts encoding SmFGFRB1 and SmFGFRB2, respectively. SmFGFRA shares 78.4% amino acid sequence identity with S. haematobium FGFR2 (ShFGFR2, MS3_0015372) and 59.9% identity with S. japonicum FGFR2 isoform 2 (SjFGFR2-2, EWB00_002899). In contrast, SmFGFRA shares only 17.4% and 17.9% amino acid sequence identity with SmFGFRB1 and SmFGFRB2, respectively, and their common amino acids are mainly distributed in the conserved TK domain. SmFGFRA shares 21.7% sequence identity with human FGFR1.

The tertiary structures of the SmFGFRs were predicted by the I-TASSER server ( Figure 1 ). The ligand binding residues (L⁵⁸⁸, G⁵⁸⁹, G⁵⁹¹, V⁵⁹⁶, A⁶¹⁷, K⁶¹⁹, I⁶⁵⁰, R⁶⁵¹, F⁶⁵², I⁶⁶⁴, L⁶⁶⁶ - A⁶⁶⁹, G⁶⁷², V⁷⁵⁴, C⁷⁶⁴, D⁷⁶⁵, F⁷⁶⁶) and the enzyme active site residue (R⁷⁵¹) of SmFGFRA were predicted as shown in Figure 1 . The ligand binding residues (l²⁹² - A²⁹⁵, G²⁹⁷, I²⁹⁸, Y³⁰⁰, A³²¹, K³²³, I³⁵³, M³⁶⁹, E³⁷⁰ -A³⁷², N³⁷⁶, R⁴²⁵, L⁴²⁸, A⁴³⁸, D⁴³⁹) and enzyme active site residue (R⁴²⁵) of SmFGFRB1, and the ligand binding residues (L³¹², V³²⁰, A³⁴¹, K³⁴³, E³⁶⁰, V³⁶¹, M³⁶⁴, I³⁷³, F³⁷⁵, L³⁸⁷, M³⁸⁹, E³⁹⁰-A³⁹², R⁴⁴⁵, N⁴⁴⁶, L⁴⁴⁸, A⁴⁵⁸, F⁴⁶⁰) and the enzyme active site residue (R⁴⁴⁵) of SmFGFRB2 are shown in Figure 1 (14). The predicted domain structures of SmFGFRA and SmFGFRB1/2 are shown in Supplementary Figure 1 . Neither signal peptides nor immunoglobulin (IG)-like domains that are responsible for protein-protein and protein-ligand interactions (61, 62) were found in either SmFGFRB1 or SmFGFRB2 ( Supplementary Figure 1 ).

The predicted binding ability of SmFGFRA with its ligand indicated its potential in inducing dimerization of the extracellular domains of FGFRs [as demonstrated in mammalian cells (11, 12, 63)], and subsequent transautophosphorylation of the cytoplasmic TK domain, thereby activating downstream signal transduction in S. mansoni. However, no endogenous genes encoding FGF ligands have been identified in the genomes of S. mansoni (45, 64), suggesting that the parasite might explore host FGF ligands to stimulate its own signaling pathway.

Using real-time PCR assays, transcriptional levels of the SmfgfrA gene were quantified in eggs, miracidia, cercariae, schistosomula, and adult female and male worms of S. mansoni. We found SmfgfrA was transcribed in all the life cycle stages examined with eggs and miracidia having the highest expression levels ( Figure 2 ).

Purified rSmFGFRA-L and rSmFGFRA, expressed in E. coli, were shown by SDS-PAGE to migrate as bands at the predicted sizes of approximately 40 KDa ( Figure 3A , Lane 1) and 105 KDa ( Figure 3A , Lane 2), respectively. The smeared bands observed in the SDS-PAGE with purified full-length protein rSmFGFRA ( Figure 3A , Lane 2) may indicate protein degradation during protein expression and/or purification, although cocktail protease inhibitors were added during the purification procedure.

Specific antibody against rSmFGFRA-L was generated in mice and the titer of the antibody (1:25,600) determined by ELISA. The anti-rSmFGFRA-L serum was used for western blot analysis and immunolocalization.

To determine whether the anti-serum raised against SmFGFRA cross reacted with homologous components in SWAP extracts prepared from adult S. japonicum and S. haematobium, western blot analysis was performed using the generated mouse anti-rSmFGFRA-L serum. Western blots demonstrated that the anti-rSmFGFRA-L antibody (1:80) recognized a band at the expected molecular size of approximately 130 kDa in the SWAPs (50 μg/well) of S. mansoni, S. japonicum and S. haematobium, whereas no bands were evident when the SWAPs were probed with naïve mouse serum ( Figure 3B ). Similarly, native SmFGFRA (approximately 130 kDa) was also recognized by the anti-rSmFGFRA-L antibody in S. mansoni SEA while no band was recognized by the naïve mouse serum ( Figure 3B ).

Immunolocalization was performed on sections of immature and mature eggs collected from S. mansoni infected mouse livers, miracidia, cercariae, 5-day old schistosomula, and adult male and female worms to determine the distribution of SmFGFRA in the different developmental stages. Immunofluorescence showed that native SmFGFRA was localized in the embryonic cells ( Figure 4B ) of immature eggs (egg embryogenesis stages 2-3) (65, 66). In positive cells, the SmFGFRA signal was detected in a perinuclear distribution. In mature eggs, SmFGFRA was detected in the inner envelope (von Lichtenburg’s Layer), the peripheral cellular neural mass, the lateral and apical glands, and germinal cells ( Figure 4D ). SmFGFRA localization was not detected on the egg shell or in the central neuropile of the neural mass where no neuronal cells are present (67). The distribution of SmFGFRA was similar in miracidia to that observed in mature eggs being localized within the ciliated epithelium, neural mass, lateral glands, apical gland, and germinal cells ( Figure 4F ). SmFGFRA was also detectable in the tegument, oral sucker, preacetabular glands, germinal cells, and flame cells of cercariae (6, 68) ( Figure 4H ), and in the tegument and the internal cell masses, including germinal cells (4), of 5-day old schistosomula ( Figure 4J ). Significantly, SmFGFRA fluorescence labelling was detected in almost all tissues of the adult male worms including the tubercles ( Figure 5F ), tegument, parenchyma ( Figure 4L ), oral sucker, ventral sucker, and testes (69) ( Figure 5J ). Similarly, SmFGFRA was amply distributed in the tegument and inner cell masses of female worms including the vitellaria, but not the gastrodermis ( Figure 4N ). Naïve mouse serum served as negative control to probe fixed immature eggs ( Figure 4A ), mature eggs ( Figure 4C ), miracidia ( Figure 4E ), cercariae ( Figure 4G ), schistosomula ( Figure 4I ), adult male worms ( Figure 4K ) and female worms ( Figure 4M ).

EdU is known for its ability to incorporate into newly synthesized cellular DNA, and stains only proliferating stem cells in S. mansoni adults (7). To determine the relationship between EdU⁺ cells (stem cells) and cells that express SmFGFRA (SmFGFRA⁺), we undertook immunolocalization of SmFGFRA using an anti-rSmFGFRA-L polyclonal antibody to probe sections of adult worms stained with EdU. All EdU⁺ cells present in both adult female worms and male worms were SmFGFRA⁺ ( Figure 5 ), emphasizing the critical roles of SmFGFRA in maintaining stem cells at the translational level. However, not all SmFGFRA⁺ cells were EdU⁺, suggesting SmFGFRA is multi-functional in S. mansoni, being not only important in stem cell maintenance but also being involved in other critical biological processes.

We performed real-time binding assays using the Octet RED system with ‘biolayer interferometry’ technology to investigate the binding affinity between rSmFGFRA-L and human bFGF/aFGF ( Figure 6 ). A strong in vitro interaction between rSmFGFRA-L and human bFGF (protein concentration ranging from 5 ng/μl to 29.6 ng/μl) was detected ( Figure 6A ) (KD = 3.6E-10, coefficient of determination (r²) = 0.98). Binding affinity was also evident, albeit less strongly, between rSmFGFRA-L and human aFGF (protein concentration ranging from 24 ng/μl to 68.3 ng/μl) (KD = 5.81E-09, r² = 0.92) ( Figure 6B ). Increasing the concentration of bFGF/aFGF resulted in an increased binding response with the dissociation phase slowly decreasing, demonstrating the specific binding ability presented between rSmFGFRA-L and bFGF/aFGF. To compare the binding affinity of rSmFGFRA-L to human bFGF and aFGF in vitro, rSmFGFRA-L immobilized sensors were exposed to bFGF or aFGF at 14.2 ng/μl and 11 ng/μl, respectively. At the same protein concentration, bFGF exhibited 19-33.7 times stronger binding affinity than aFGF to rSmFGFRA-L ( Figure 6C ). Notably, at the same concentration, human FGFR1-L demonstrated higher binding affinity than rSmFGFRA-L to human bFGF ( Figure 6D ).

To explore whether human FGFs could activate the S. mansoni MAPK pathway by phosphorylating Erk1/2, we incubated adult worms for 30 minutes with either 10 nM aFGF or bFGF individually or in combination. Then, SWAP extracted from the treated parasites was subjected to western blot analysis using an anti-phospho-P44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody. Phosphorylation of Erk1/2 was readily detected at the expected band size (approximately 44 kDa) (53) in SWAP of adult S. mansoni stimulated with either aFGF or bFGF, or co-stimulated with both aFGF and bFGF; stronger band intensity was observed in worms treated with bFGF ( Figures 7A , S2 ). The Erk band was detectable in an extract of control parasites (wild type, i.e. untreated worms; and worms incubated with 0.1% DMSO). By using an anti-actin (control) antibody, no significant differences were evident in band intensity detected in any of the tested or control groups ( Figure 7A , S2 ). Furthermore, 30 minute treatment of adult worms with the TK inhibitor BIBF 1120 (10 μM), an agent considered to selectively inhibit S. mansoni FGF receptors (36), resulted in markedly depleted phosphorylation of Erk ( Figure 7A , s2 ).

SmFGFRA is a receptor tyrosine kinase (RTK) and the RTKs are important enzymes involved in the signal transduction pathway (12). A universal Tyrosine Assay Kit, used to measure the TK activity of rSmFGFRA, indicated, as might be expected, increased activity with increasing concentrations (0-100 ng/μl) of rSmFGFRA ( Figure 7B ). However, the TK activity of rSmFGFRA was considerably inhibited following incubation with the TK inhibitor, BIBF 1120; indeed, when the rSmFGFRA concentration was less than 6.25 ng/μl, TK activity was completely inhibited in the presence of 5 μM or 10 μM BIBF 1120 ( Figure 7B ).

To further understand the effects of inhibiting the FGFRs by BIBF 1120 in S. mansoni, eggs isolated from the livers of infected mice were treated in vitro with different concentrations (2.5 - 20 μM) of the inhibitor, and then the egg hatching efficiency was examined. BIBF 1120 had clear concentration-dependent effects on egg hatching ability. In the presence of 2.5 μM BIBF 1120, egg hatching efficiency was moderately but not significantly reduced. However, egg hatching was markedly decreased by 21.3% (p=0.0077), 54.4% (p ≤ 0.0001), and 65% (p ≤ 0.0001) in the presence of 5 μM, 10 μM, and 20 μM BIBF 1120, respectively, compared with control eggs treated with 0.1% DMSO ( Figure 8 ).

Behavioral changes of miracidia hatched from eggs treated with BIBF 1120 were investigated by analyzing recordings of miracidial movement tracks. Heatmaps of the miracidia movement patterns were created to illustrate the behavior of individual miracidia within the 1 min recording. The heatmaps of the control miracidia [wild type miracidia (WT) and miracidia hatched from eggs treated with 0.1% DMSO] depicted linear soft blue lines suggesting these miracidia had less circular but faster movement ( Figures 9A, B ). In contrast, there were more circular lines and more abundant red and yellow regions in heatmaps of miracidia hatched from the BIBF 1120-treated eggs ( Figures 9C–F ), indicating more turning and circling and relatively slower movement of these miracidia in the FOV. As shown in Figure 9G , the swimming velocity of miracidia hatched from eggs treated with 10 μM and 20 μM BIBF 1120 was significantly decreased by 30.7% (p=0.0023) and 51.3% (p ≤ 0.0001), respectively, compared with that measured in the 0.1% DMSO-treated group. Markedly, the average time duration of miracidia hatched from eggs treated with 10 μM and 20 μM BIBF 1120 in the FOV was elevated by 72.1% (p=0.0006) and 72.7% (p=0.0088), respectively ( Figure 9H ). Similarly, the movement tortuosity of miracidia hatched from 10 μM and 20 μM BIBF 1120 treated eggs was dramatically increased by 61.7% (p=0.0003) and 68.8% (p=0.0024), respectively, compared with that observed in the 0.1% DMSO-treated group ( Figure 9I ). No significant behavioral modifications were evident in miracidia hatched from eggs treated with BIBF 1120 at concentrations of 2.5 μM and 5 μM.