A practical miracidial motility assay for assessing Fasciola hepatica sensitivity to compounds in vitro

TL;DR

This paper introduces a new high-throughput assay to test the sensitivity of Fasciola hepatica larvae to drugs, revealing geographic and drug-specific variations in response.

Contribution

The study introduces a novel automated motility assay for assessing drug sensitivity in Fasciola hepatica miracidia.

Findings

CLOS, TCBZ, and TCBZ-SO caused concentration-dependent motility inhibition in miracidia.

Geographic isolates showed varying sensitivity to TCBZ and TCBZ-SO.

MMA is a reproducible, high-throughput platform for drug testing.

Abstract

Fasciola hepatica causes fasciolosis in livestock and humans worldwide, yet reliable tools to assess drug efficacy against the early developmental stages of this parasite are lacking. Here, we developed an automated miracidial motility assay (MMA) using the WMicroTracker ONE infrared detection system to quantify the sensitivity of F. hepatica miracidia to anthelmintic compounds including clorsulon (CLORS), closantel (CLOS), triclabendazole (TCBZ) and triclabendazole-sulphoxide (TCBZ-SO). Systematic optimisation of assay conditions, including inoculum size, observation window and solvent concentration yielded a reliable platform for evaluating the sensitivity of F. hepatica miracidia from diverse geographic isolates to these compounds. Our results demonstrated that three compounds (CLOS, TCBZ and TCBZ-SO) produced concentration-dependent motility inhibition, whereas CLORS had no effect. CLOS displayed the highest potency among isolates from New South Wales (NSW), Tasmania (TAS) and Victoria (VIC), whereas TCBZ and TCBZ-SO exhibited isolate-specific sensitivity patterns. Miracidial responses of the NSW and TAS isolates to TCBZ, TCBZ-SO and CLOS were also compared in vitro with those of newly excysted juveniles (NEJs) produced from the same isolates. Overall, the findings show that MMA provides a reproducible, host-independent and high-throughput phenotypic platform for assessing miracidial sensitivity to compounds.

Image 1

•High-throughput, phenotypic motility assay established for Fasciola hepatica larvae (miracidia).•Significant variation in miracidium responses to different drugs.•Marked geographical variability in miracidial responses to the same drug.•This assay should be adaptable to other motile developmental stages and trematode species.

High-throughput, phenotypic motility assay established for Fasciola hepatica larvae (miracidia).

Significant variation in miracidium responses to different drugs.

Marked geographical variability in miracidial responses to the same drug.

This assay should be adaptable to other motile developmental stages and trematode species.

Figures

Experimental workflow of the miracidial motility assay (MMA) and comparative assessment of compound sensitivity in *Fasciola hepatica*. Miracidia hatched from embryonated *Fasciola hepatica* eggs were exposed to test compounds in 384-well flat-bottom plates. The motility was quantified using the WMicroTracker ONE system to generate motility data and derive half maximal inhibitory concentration (IC_50_) values. The IC_50_ values determined in miracidia were subsequently applied to newly excysted juveniles (NEJs) generated via snail infection and metacercarial excystation. The NEJs were subjecte

Egg hatching dynamics of *Fasciola hepatica* isolates from three geographic regions. Hatching rates for egg isolates from New South Wales (NSW), Tasmania (TAS) and Victoria (VIC) at five time points between 14 and 18 days.Fig. 2

Optimisation of experimental conditions for the miracidial motility assay (MMA). Miracidia hatched from a *Fasciola hepatica* egg isolate from New South Wales (NSW) were used in this assay. (A) Effect of miracidia density per well (per well: 384-well flat-bottom plate with 40 μL miracidia solution per well); (B) Time course of miracidial motility; (C) Effect of DMSO tolerance level on miracidia motility. DMSO, dimethyl sulphoxide; RO, reverse osmosis; TCBZ-SO, triclabendazole-sulphoxide.Fig. 3Fig. 4Statistical comparison of IC_50_ values for three compounds in the miracidial motility assay. Mi

Motility responses of newly excysted juveniles (NEJs) from the New South Wales (NSW) and Tasmania (TAS) isolates at the miracidial IC_50_ concentrations determined at 0 h. (A) Movement profiles of NEJs from the NSW isolate following exposure to DMSO (0.6%), CLOS (1.25 μg/mL), TCBZ (7.50 μg/mL) and TCBZ-SO (15.42 μg/mL); (B) Movement profiles of NEJs from the TAS isolate following exposure to DMSO (0.6 %), CLOS (0.04 μg/mL), TCBZ (4.50 μg/mL) and TCBZ-SO (1.45 μg/mL). Error bars represent standard error of the log^10^-transformed (average movement per NEJ +1) for NSW (n = 4), the log^10^-transf

Comparative sensitivity of excysted juveniles (NEJs) from the New South Wales (NSW) and Tasmania (TAS) exposed to (A) dimethyl sulphoxide (DMSO), (B) closantel (CLOS), (C) triclabendazole (TCBZ) and (D) triclabendazole-sulphoxide (TCBZ-SO). NEJs from the NSW isolate were exposed to DMSO (0.6%), CLOS (1.25 μg/mL), TCBZ (7.50 μg/mL) and TCBZ-SO (15.42 μg/mL). NEJs from the TAS isolate were exposed to DMSO (0.6 %), CLOS (0.04 μg/mL), TCBZ (4.50 μg/mL) and TCBZ-SO (1.45 μg/mL). Error bars represent standard error of the log10-transformed (average movement per NEJ) for DMSO control and of the squar

Full text

Introduction

1

Fasciola hepatica is a globally distributed parasitic trematode that causes fasciolosis in mammals including livestock and humans, leading to major economic and public health burdens (Caravedo and Cabada, 2020). The parasite's complex life cycle alternates between a definitive mammalian host and an intermediate snail host, involving egg, larval (miracidium, sporocyst, redia, cercaria and metacercaria), newly excysted juvenile (NEJ), juvenile and adult stages (Sabourin et al., 2018). Miracidium is the free-living stage that infects the snail intermediate host, and NEJ marks the beginning of parasitic phase in the definitive host (Lalor et al., 2021). Each developmental stage displays distinct morphological and physiological traits and interacts differently with the environment (as a free-living stage), or the host (as a parasitic stage) (Lalor et al., 2021).

In the absence of effective vaccines, the control of fasciolosis relies heavily on anthelmintic treatment (Toet et al., 2014). Most available flukicidal compounds are active only against some developmental stages of F. hepatica (see Mooney et al., 2009). For instance, clorsulon (CLORS) and closantel (CLOS) are primarily active against late immature and adult flukes, with CLORS showing ∼95–100% efficacy against 8-week-old and adult fluke (Malone et al., 1984; Zimmerman et al., 1986) and CLOS achieving ∼70–80% efficacy at 6 weeks and >90% at 8 weeks post-infection (Maes et al., 1988). In contrast, triclabendazole (TCBZ) is effective against both juvenile and adult stages of F. hepatica, with reported efficacies usually ≥90% from ∼2 to 4 weeks post-infection through to late immature and adult flukes (Martínez-Moreno et al., 1997; Richards et al., 1990). Excessive and prolonged use of TCBZ has led to the emergence of resistant F. hepatica populations, first reported in Australia in 1995 and now widespread internationally (Overend and Bowen, 1995; Kelley et al., 2016). Reports of reduced efficacy of CLORS and CLOS in resistant populations (Martínez-Valladares et al., 2014; Novobilský and Höglund, 2015) further indicate the fragility of current treatment options.

These challenges emphasise the need for reliable methods to diagnose anthelmintic resistance and to distinguish resistant from susceptible F. hepatica populations. Given the parasite's complex life cycle and stage-dependent drug sensitivities, diagnostic approaches need to account for both parasite biology and treatment context. Consequently, a range of in vivo, in vitro and molecular tools have been developed to assess resistance and susceptibility, each with distinct applicability and limitations. Field-based tests, such as the faecal egg-count reduction test (FECRT) and the coproantigen reduction test (CRT), assess drug efficacy in animals with patent F. hepatica infection. Their diagnostic performance may be affected by variability in egg shedding and delayed egg clearance, with CRT in particular showing reduced sensitivity during pre-patent infection (Rokni et al., 2002; Fairweather et al., 2012). The egg-hatch assay (EHA) provides a direct *in vitro-*assessment of the inhibition of embryonic (miracidial) development and hatching (Alvarez et al., 2009; Fairweather et al., 2012), but their broader applicability is constrained by variability in eggshell permeability, prolonged incubation time and the need for manual scoring. Although EHA can be applied to benzimidazoles such as albendazole, F. hepatica eggs exhibit little or no responsiveness to TCBZ under standard conditions, making the assay unsuitable for evaluating TCBZ activity (Fairweather et al., 2020). Molecular methods, including PCR and DNA sequencing, potentially offer high specificity and sensitivity for testing but require prior knowledge of resistance-associated mutations and are presently impractical for field evaluations (Brockwell et al., 2014; Carnevale et al., 2015). There is, therefore, a clear imperative to develop an improved, more rapid and practical method that enables reproducible and quantitative phenotypic assessment of drug effects and/or resistance in F. hepatica in the laboratory.

Over the past decade, major advances in phenotypic screening have produced automated, quantitative motility and development assays for nematodes (e.g., Caenorhabditis elegans and Haemonchus contortus) and trematodes (e.g., Schistosoma mansoni) using platforms such as the WMicroTracker system — an automated system that quantifies the movement of small animals over time and space utilising infrared technology (Gunderson et al., 2020). This assay has enabled medium-to high-throughput and reproducible evaluation of compound activity and potency using larval parasite stages. In contrast, equivalent platforms have not been developed for F. hepatica or other liver flukes. The free-living miracidium stage of F. hepatica provides an attractive opportunity to address this gap. Eggs can be isolated from faeces or bile and stored at relatively low temperature for extended periods (Reddington et al., 1982), and embryonated eggs can be maintained for longer durations, providing a source of larvae without the need for live animals (Alvarez et al., 2009). Miracidia are short-lived, highly motile and amenable to quantitative behavioural analysis under defined in vitro conditions (Villa-Mancera et al., 2015), making them a practical and ethically favourable stage for the phenotypic assessment of drug effects or resistance.

In this study, we aimed to establish a practical, repeatable and quantitative in vitro-assay for assessing the effects of selected anthelmintic properties in F. hepatica miracidia. Building on advances in nematode phenotypic screening, we sought to evaluate whether infrared-based motility detection could be adapted to miracidia as a platform for measuring compound effects and supporting future studies of anthelmintic susceptibility and/or resistance in trematodes.

Materials and methods

2

The experimental workflow is depicted in Fig. 1. Miracidia were hatched from embryonated F. hepatica eggs, which were collected from the gallbladders of naturally infected sheep. Newly hatched miracidia were utilised directly for the optimisation and application of a miracidial motility assay (MMA) to quantitatively assess compound sensitivity, or used to infect the snail intermediate host to produce metacercariae and then NEJs. A panel of anthelmintic compounds was prepared and tested in the optimised MMA to assess their in vitro activity or potency based on motility measurements, with selected comparative experiments conducted using NEJs. Statistical analyses were conducted to compare the sensitivity of compounds between developmental stages or geographic isolates.Fig. 1. Experimental workflow of the miracidial motility assay (MMA) and comparative assessment of compound sensitivity in Fasciola hepatica. Miracidia hatched from embryonated Fasciola hepatica eggs were exposed to test compounds in 384-well flat-bottom plates. The motility was quantified using the WMicroTracker ONE system to generate motility data and derive half maximal inhibitory concentration (IC_50_) values. The IC_50_ values determined in miracidia were subsequently applied to newly excysted juveniles (NEJs) generated via snail infection and metacercarial excystation. The NEJs were subjected to comparative compound assessment, and movement responses were quantified using image-based analysis with the EVOS M7000 system. Statistical analyses were conducted to compare the sensitivity of compounds between developmental stages or geographic isolates. IC_50_, half-maximal inhibitory concentration; NEJs, newly excysted juveniles.Fig. 1

Procurement of eggs and miracidia

2.1

Fasciola hepatica eggs were obtained from the gallbladders of naturally infected sheep from abattoirs in Yass, New South Wales (NSW 2582); Powranna, Tasmania (TAS 7300); and Tyrendarra, Victoria (VIC 3285). Eggs were isolated from bile as described in a previous study (Reddington et al., 1982), with modifications to improve egg purity and yield. Briefly, eggs were sedimented and washed extensively in tap water and then stored in ∼200 mL of water in tissue culture flasks (T225, Corning, USA) at 4 °C in complete darkness until further processing.

For embryonation, eggs were incubated in darkness at 25 °C and monitored microscopically until developed miracidia could be observed (cf. Alvarez et al., 2009; Villa-Mancera et al., 2015). On the day of hatching, eggs were exposed to light (full spectrum aquarium light, SATCO NUVO, USA) at room temperature for 1 h to stimulate the release of miracidia (Fairweather et al., 2012), typically within 40–60 min. The phototactic miracidia were immediately used in the MMA or used to infect snails.

Production of metacercariae and newly excysted juvenile worms (NEJs)

2.2

A laboratory line of Austropeplea cf*. brazieri*, maintained in the Department of Veterinary Biosciences, The University of Melbourne, was used for infection. Individual snails (shell length: ∼5 mm) were exposed to F. hepatica miracidia (n = 3 per snail) in 24-well plates containing 1 mL of artificial pond water (Sukee et al., 2024). Following a 1 h incubation at room temperature (22–24 °C), wells were examined under a stereomicroscope to verify penetration. Following miracidial exposure, snails were maintained in groups of 24 within 9 × 13-inch Pyrex trays at ambient room temperature (18–25 °C) under an 8 h light cycle and fed ad libitum with algal wafers (Hikari™, Japan). Cercarial development within snails was monitored from day 40 by microscopic examination (through the shells). Cercariae were harvested using the “terminal release method” (McCusker et al., 2023) by gently crushing snails on dialysis tubing to induce cercarial release and encystment. Metacercariae were stored in reverse osmosis (RO) water containing 100 units/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin B (antibiotic-antimycotic, Gibco, Thermo Fisher Scientific, USA) at 4 °C. Excystment was performed to obtain NEJs as previously described (McCusker et al., 2023).

Preparation of anthelmintic compounds

2.3

Four compounds were tested— three compounds (CLOS, CLORS and TCBZ) and the main active metabolite of TCBZ (triclabendazole sulphoxide, TCBZ-SO); for convenience, we refer to all four as “compounds”. These compounds were purchased in solid form from a commercial supplier (Supelco, USA) and dissolved in 100% dimethyl sulphoxide (DMSO, ≥99.7% HPLC-grade, Sigma-Aldrich, USA). For all assays, compounds were dissolved in two-fold serial dilutions immediately prior to testing using sterile RO water to yield final concentrations ranging from 0 to 120 μg/mL; using the same dilution series, the final DMSO concentrations in the assay wells ranged from 0 to 1.2% (cf. Robinson et al., 2002; Taki et al., 2021).

Optimisation of the miracidial motility assay (MMA)

2.4

To establish a robust phenotypic assay suitable for medium-to high-throughput screening, experimental conditions were first optimised using the F. hepatica isolate from NSW. The resultant density of miracidia per well, incubation time and DMSO tolerance are presented in Sub-section 3.2.

First, to establish the suitable miracidial density range in 384-well flat-bottom microplates (Corning, USA), four densities (5, 10, 15 and 30 miracidia per well) were assessed, each in quadruplicate (n = 4), using the WMicroTracker ONE system (PhylumTech, Sunchales, Argentina; cf. Taki et al., 2021), which measures organismal motility infrared beam scattering (Herath et al., 2022). Second, the time of the motility measurement was optimised in a 384-well flat-bottom plate, with 15 miracidia per well and four technical replicates (n = 4). Blank control-wells contained miracidia and sterile RO water, whereas test-wells contained miracidia plus 15 or 30 μg/mL TCBZ-SO prepared in 0.15% or 0.3% DMSO, respectively. Third, the optimal measurement window that showed the greatest difference in motility between the control and test groups was established. Fourth, to determine the maximum DMSO concentration that did not significantly impair motility, six DMSO concentrations (0%, 0.15%, 0.3%, 0.6%, 1.2% and 2.4%) were each tested in quadruplicate using 15 miracidia per well in a 384-well plate.

The established miracidial motility assay (MMA)

2.5

The effects of compounds on miracidial motility were assessed in wells of 384-well flat-bottom microplates using the WMicroTracker ONE system. All compounds were tested in quadruplicate and repeated on three separate occasions on independent microplates to ensure repeatability. For each replicate, 10 μL of each concentrated compound solution, 10 μL of 2.4% DMSO solution (solvent control) or 10 μL of RO water (blank control) were first added to each well. Then, 30 μL of a freshly prepared suspension of 10–15 newly hatched miracidia were added, resulting in a final volume of 40 μL per well and a miracidial density of 0.25–0.38 per μL. Compound solutions were prepared as two-fold serial dilutions from 100% DMSO stocks, yielding test concentrations from 0 to 120 μg/mL and corresponding DMSO concentrations of 0–1.2%. The final concentration of 0.6% DMSO used routinely in the MMA did not affect miracidia motility (see Sub-section 3.2 and Fig. 4C).

In the WMicroTracker ONE system, motility readings were normalised to the mean motility observed in the DMSO control (0.6%) to minimise plate-to-plate variability. Motility (%) in each well was calculated relative to this baseline, with the blank control (RO water) serving as an internal control to detect any effect of the DMSO solvent alone on background motility in wells. Dose-response curves were produced by fitting a four-parameter nonlinear regression model to estimate the half-maximal inhibitory concentration (IC_50_) values (see Sub-section 2.7).

Comparative assessment of anthelmintic responses in NEJs

2.6

Miracidial responses to TCBZ, TCBZ-SO and CLOS were compared in vitro with those of newly excysted juveniles (NEJs) produced for selected F. hepatica egg isolates. NEJs were exposed for 24 h in a 384-well flat-bottom plate to the respective IC_50_ concentrations of CLOS, TCBZ and TCBZ-SO (as determined for miracidia at 0 h) and to the solvent (DMSO) control (0.6%). Both the motility and viability of NEJs were assessed at 0, 2, 4, 6 and 24 h using an EVOS M7000 imaging system (Thermo Fisher Scientific, USA). This imaging system was used for the NEJ assay because it provides the controlled environmental conditions (37 °C and 5% CO_2_) required for the survival of NEJs and allows reliable detection of motility that cannot be captured by the WMicroTracker ONE due to the slow movement of NEJs. At 24 h, viability was confirmed by Sytox Green staining (Invitrogen, Thermo Fisher Scientific, USA) at a final concentration of 250 nM.

A custom python script (https://github.com/MengweiZHENG/Fasciola-hepatica-NEJs-movement-assessment) was used to analyse sequential TIFF image stacks recorded using the EVOS M7000. Pixel-level changes over 60 frames were recorded by converting the images to an 8-bit grayscale and comparing each frame sequentially against a running average background image. Pixel differences between the current frame and the evolving background were calculated using absolute difference operations, with an intensity threshold of 90 applied to classify whether a pixel had changed. The number of pixels exceeding this threshold was recorded for each frame and expressed as both raw counts and rates per second. Final output of the analysis was a tab-delimited file that summarised pixel changes per well. Data from the compound-treated groups (CLOS, TCBZ and TCBZ-SO) were normalised against the solvent control group (DMSO), consistent with the approach used for the analyses of miracidia.

Statistical analysis

2.7

For miracidia, IC_50_ values were calculated by fitting dose-response curves to a nonlinear four-parameter logistic model in GraphPad Prism v.10.4.0 (GraphPad Software, San Diego, CA, USA). A one-way analysis of variance (ANOVA) was performed to compare drug responses among different isolates, at different miracidia densities per well and various DMSO tolerance levels. A p-value < 0.05 was considered statistically significant. For NEJs, the normality of the raw average pixel change (movement) data was assessed using the Shapiro-Wilk test. Where appropriate, the data were subjected to logarithmic 10 (log^10^) and/or square root transformation to achieve normality, after which the transformed data were analysed using two-way ANOVA. Statistical significance is indicated as follows: ∗ p < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 and ∗∗∗∗ P < 0.0001.

Results

3

Egg hatching and miracidial yields

3.1

The hatch rates of F. hepatica egg batches from three geographically distinct isolates (NSW, TAS and VIC) were evaluated. The NSW eggs exhibited a progressive increase in hatching rates with prolonged incubation (Fig. 2). By day 14, most eggs were at the cell division stage rather than the eye-spot stage (Fairweather et al., 2012) prior to miracidia emergence; the hatching rate reached a maximum of 50.0% ± 0.2 on day 18 (Fig. 2). In contrast, eggs from VIC and TAS hatched earlier, with respective peak rates of 75.0% ± 0.2 and 70.0% ± 0.2 (Fig. 2) on day 14. These rates subsequently declined by day 18, 53.1% ± 0.4 for VIC and 51.2% ± 0.2 for TAS (Fig. 2). The peak hatching time for each isolate was used in all subsequent experiments.Fig. 2. Egg hatching dynamics of Fasciola hepatica isolates from three geographic regions. Hatching rates for egg isolates from New South Wales (NSW), Tasmania (TAS) and Victoria (VIC) at five time points between 14 and 18 days.Fig. 2

Optimisation of experimental conditions for MMA

3.2

The optimal miracidial density in individual wells of 384-well flat-bottom plate was initially assessed for the NSW isolate. Motility measured in wells containing 5 miracidia was significantly lower compared to those with 10, 15 or 30 miracidia (One-way ANOVA; p < 0.05, Fig. 3A); no significant differences were observed among the wells containing 10, 15 and 30 miracidia. Based on these findings, a density range of 10–15 miracidia per well was selected for the MMA.Fig. 3. Optimisation of experimental conditions for the miracidial motility assay (MMA). Miracidia hatched from a Fasciola hepatica egg isolate from New South Wales (NSW) were used in this assay. (A) Effect of miracidia density per well (per well: 384-well flat-bottom plate with 40 μL miracidia solution per well); (B) Time course of miracidial motility; (C) Effect of DMSO tolerance level on miracidia motility. DMSO, dimethyl sulphoxide; RO, reverse osmosis; TCBZ-SO, triclabendazole-sulphoxide.Fig. 3. Fig. 4Statistical comparison of IC_50_ values for three compounds in the miracidial motility assay. Miracidia hatched from three distinct Fasciola hepatica egg isolates from New South Wales (NSW), Tasmania (TAS) and Victoria (VIC) were used. Isolates were exposed to (A) CLOS, (B) TCBZ and (C) TCBZ-SO and results were compared using one-way ANOVA (p < 0.05). Error bars represent standard error of the mean IC_50_ concentration among three independent assays. IC_50_, half-maximal inhibitory concentration; CLOS, closantel; TCBZ, triclabendazole; TCBZ-SO, triclabendazole-sulphoxide; ANOVA, analysis of variance.Fig. 4

Then, the incubation duration in the presence or absence of TCBZ and TCBZ-SO was assessed for NSW miracidia (Fig. 3B). In the control group (DMSO-only), miracidia remained very motile for > 12 h. In contrast, exposure to 15 μg/mL TCBZ-SO resulted in markedly reduced miracidial motility within 1 h, and miracidia became non-motile after 3 h (Fig. 3B). Miracidia showed no activity within a 1 h exposure to 30 μg/ml TCBZ-SO (Fig. 3B). Therefore, 3 h post-exposure was selected as the time for subsequent IC_50_ motility measurement in MMA.

To assess solvent tolerance, the motility of miracidia (NSW isolate) was evaluated at six distinct DMSO concentrations (Fig. 3C). Significant differences in motility compared with the control were observed only at the concentrations of ≥ 0.6% DMSO (One-way ANOVA; p < 0.05). Thus, a maximum DMSO concentration of 0.6% was used as a solvent control in all subsequent experiments.

Differential compound-sensitivity patterns of Fasciola hepatica miracidia from NSW, TAS and VIC

3.3

The sensitivity of F. hepatica miracidia from NSW, TAS and VIC isolates to each of four compounds (CLORS, CLOS, TCBZ and TCBZ-SO) was assessed in MMA. CLOS, TCBZ and TCBZ-SO each reduced miracidial motility in a concentration-dependent manner for each of the isolates, whereas CLORS did not induce a measurable inhibition, so no IC_50_ curves could be produced (Table 1; Figs. S1A, E and I). Subsequent analyses focused on the three active compounds: CLOS, TCBZ and TCBZ-SO.Table 1. The absolute IC_50_ values for four compounds following exposure of three isolates of Fasciola hepatica miracidia at four different time points.Table 1. Geographic isolateTime (h)CLORS (μg/mL)CLOS (μg/mL)TCBZ (μg/mL)TCBZ-SO (μg/mL)New South Wales (NSW)0–1.257.5015.421–0.993.7111.252–0.963.547.683–0.953.727.31Victoria (VIC)0–0.203.923.651–0.341.831.852–0.021.691.803–0.181.841.80Tasmania (TAS)0–0.044.501.451–0.101.181.842–0.021.361.763–0.181.631.72The IC_50_ value is determined from n = 12 (three independent assays). IC_50_, half-maximal inhibitory concentration; data are presented as mean ± standard error of the mean (SEM; n = 12); compounds: clorsulon (CLORS), closantel (CLOS), triclabendazole (TCBZ) and triclabendazole-sulphoxide (TCBZ-SO); time of exposure to each drug: 0, 1, 2 and 3 h.

At 0 h, CLOS consistently exhibited the greatest in vitro-potency across isolates, with respective IC_50_ values of 1.25 μg/mL (NSW), 0.04 μg/mL (TAS) and 0.20 μg/mL (VIC) (Table 1; Fig. S1B, F and J). TCBZ displayed moderate activity, with an IC_50_ of 7.50 μg/mL for the NSW isolate and lower values for TAS (4.50 μg/mL) and VIC (3.92 μg/mL) (Table 1; Fig. S1C, G and K). TCBZ-SO showed most variation in potency among isolates: potency was lower for NSW isolate (IC_50_ = 15.42 μg/mL), but higher than the TAS isolate (IC_50_ = 1.45 μg/mL) and VIC isolate (IC_50_ = 3.65 μg/mL) (Table 1; Figs. S1D, H and L).

Following exposure over 1–3 h, the IC_50_ values for TCBZ remained relatively constant for miracidia from the NSW and VIC isolates, ranging from 3.54 to 3.72 μg/mL (Δ = 0.18 μg/mL, NSW) and 1.69–1.84 μg/mL (Δ = 0.15 μg/mL, VIC), respectively. In contrast, greater variability was observed for the TAS isolate (1.18–1.63 μg/mL, Δ = 0.45 μg/mL). Overall, the IC_50_ values for NSW isolate were 2.17–2.74-fold higher than those for the TAS isolate and 1.92–2.20-fold higher than those for the VIC isolate (Table 1; Fig. S1C, G and K). TCBZ-SO showed time-dependent increases in in vitro-potency for all isolates, with the largest fluctuation observed for the NSW isolate (7.31–11.25 μg/mL, Δ = 3.94 μg/mL), whereas VIC (1.80–1.85 μg/mL, Δ = 0.05 μg/mL) and TAS (1.72–1.84 μg/mL, Δ = 0.12 μg/mL) isolates showed relatively stable IC_50_ ranges. Overall, the IC_50_ values for the NSW isolate were 3.97–6.54-fold higher than the TAS isolate and 3.95–6.25-fold higher than the VIC isolate (Table 1; Fig. S1D, H and L).

For CLOS, miracidia from the NSW isolate exhibited a consistent decline in IC_50_ values over time (0.95–0.99 μg/mL; Table 1; Fig. S1B). Those from the TAS and VIC isolates exhibited a biphasic response: an initial IC_50_ value decrease at 2 h, followed by an increase at 3 h. The IC_50_ values for TAS were 0.10 μg/mL (1 h), 0.02 μg/mL (2 h) and 0.18 μg/mL (3 h), whereas VIC showed 0.34 μg/mL (1 h), 0.02 μg/mL (2 h) and 0.18 μg/mL (3 h). Overall, the IC_50_ values for the NSW isolate were 2.57–49.5-fold higher than those established for the TAS isolate and 5.28–49.5-fold higher than those for the VIC isolate (Table 1; Fig. S1B, F and J).

Statistically, there was no significant difference in TCBZ IC_50_ value for miracidia among the three distinct isolates of F. hepatica (One-way ANOVA, p > 0.05; Fig. 4B). However, miracidia representing the NSW isolate showed significantly lower sensitivity to TCBZ-SO and CLOS compared to the TAS and VIC isolates (One-way ANOVA, p < 0.05; Fig. 4A and C).

Differences in compound-sensitivities between F. hepatica miracidia and NEJs

3.4

To determine whether compound sensitivity patterns identified in MMA were reflected at a subsequent developmental stage, NEJs were exposed to the miracidium-derived IC_50_ concentrations of CLOS, TCBZ and TCBZ-SO (as determined at 0 h). NEJ motility responses were quantified using the EVOS-based imaging system, with average pixel change per NEJ used as a proxy for movement. NEJ assays were not performed for the VIC isolate because the miracidia-based IC_50_ comparison (Fig. 4) revealed the greatest divergence between the NSW and TAS isolates, compared with NSW and VIC isolates; accordingly, the TAS isolate was selected as the primary comparator for NEJ-stage validation.

For NSW-derived NEJs, motility gradually decreased over the first 4–6 h across all treatment groups. From 6 to 24 h, movement stabilised in the DMSO, CLOS and TCBZ-SO groups, whereas TCBZ-treated NEJs showed a marked loss of movement after 4–6 h (Fig. 5A). A viability assay using Sytox Green nucleic acid revealed that 47.2% (17/36) of TCBZ-treated NEJs were non-viable at 24 h (Fig. 6). Two-way ANOVA analysis showed a significant reduction in NEJ movement in the TCBZ group compared with the DMSO control after 4 h (p < 0.05), consistent with the higher sensitivity of NSW-derived miracidia to TCBZ than to TCBZ-SO (Table 1; Fig. 5A).Fig. 5. Motility responses of newly excysted juveniles (NEJs) from the New South Wales (NSW) and Tasmania (TAS) isolates at the miracidial IC_50_ concentrations determined at 0 h. (A) Movement profiles of NEJs from the NSW isolate following exposure to DMSO (0.6%), CLOS (1.25 μg/mL), TCBZ (7.50 μg/mL) and TCBZ-SO (15.42 μg/mL); (B) Movement profiles of NEJs from the TAS isolate following exposure to DMSO (0.6 %), CLOS (0.04 μg/mL), TCBZ (4.50 μg/mL) and TCBZ-SO (1.45 μg/mL). Error bars represent standard error of the log^10^-transformed (average movement per NEJ +1) for NSW (n = 4), the log^10^-transformed (average movement per NEJ) among TAS (n = 4). NEJs, newly excysted juveniles; IC_50_, half-maximal inhibitory concentration; DMSO, dimethyl sulphoxide; CLOS, closantel; TCBZ, triclabendazole; TCBZ-SO, triclabendazole-sulphoxide.Fig. 5. Fig. 6Evaluation of the viability of newly excysted juveniles (NEJs) Fasciola hepatica from New South Wales (NSW) by Sytox Green nucleic acid staining. (A–D): Representative wells from replicates one to four of triclabendazole (TCBZ)-treated NSW NEJs at 24 h. Dead NEJs were identified microscopically by fluorescent staining (GFP filter) at 4 × magnification; insets in panels B and C show individual NEJs from the same well that were outside of the field of view of the main images; scale bar = 500 μm. NEJs, newly excysted juveniles; TCBZ, triclabendazole.Fig. 6

For TAS-derived NEJs, motility in the TCBZ-treated group steadily declined over 6 h. CLOS- and DMSO-treated NEJs exhibited an initial decrease from 0 to 4 h, followed by slight recovery and stabilisation, whereas TCBZ-SO-treated NEJs decreased from 0 to 6 h, and then increased markedly from 6 to 24 h (Fig. 5B). Two-way ANOVA revealed a significant difference between TCBZ-SO and DMSO at 0 and 6 h (p < 0.05), consistent with the higher sensitivity of TAS isolate to TCBZ-SO than to TCBZ (Table 1; Fig. 5B).

Comparing between isolates, untreated NEJs derived from both NSW and TAS exhibited a continuous decline in movement, with significant differences during the first 2 h (Two-way ANOVA, p < 0.05; Fig. 7A). After normalisation, CLOS treatment resulted in a transient decline, followed by a recovery in NEJs from the NSW isolate, whereas NEJs from the TAS isolate showed a gradual increase in movement, but no significant isolate-difference in movement was detected (Two-way ANOVA, p > 0.05; Fig. 7B). In the TCBZ-treated group, the movement of NEJs from the NSW isolate decreased steadily to complete inhibition by 6 h, whereas the movement of NEJs from the TAS isolate increased from 0 to 4 h and then declined, with significant differences observed from 4 to 6 h (Two-way ANOVA, p < 0.05; Fig. 7C). For TCBZ-SO, the motility of NEJs from the NSW and TAS isolates exhibited an initial, slight increase (0–2 h), a decrease (2–4 h) and subsequent recovery (4–24 h), with a significant isolate-difference at 0 h (Two-way ANOVA, p < 0.05; Fig. 7D).Fig. 7. Comparative sensitivity of excysted juveniles (NEJs) from the New South Wales (NSW) and Tasmania (TAS) exposed to (A) dimethyl sulphoxide (DMSO), (B) closantel (CLOS), (C) triclabendazole (TCBZ) and (D) triclabendazole-sulphoxide (TCBZ-SO). NEJs from the NSW isolate were exposed to DMSO (0.6%), CLOS (1.25 μg/mL), TCBZ (7.50 μg/mL) and TCBZ-SO (15.42 μg/mL). NEJs from the TAS isolate were exposed to DMSO (0.6 %), CLOS (0.04 μg/mL), TCBZ (4.50 μg/mL) and TCBZ-SO (1.45 μg/mL). Error bars represent standard error of the log10-transformed (average movement per NEJ) for DMSO control and of the square root-transformed (normalised average movement) for TCBZ, TCBZ-SO and CLOS treated newly excysted juveniles (n = 4 each). NEJs, newly excysted juveniles; CLOS, closantel; DMSO, dimethyl sulphoxide; TCBZ, triclabendazole; TCBZ-SO, triclabendazole-sulphoxide.Fig. 7

Discussion

4

Here, we established a phenotypic assay (MMA) in a 384-well plate format for the quantitative assessment of the sensitivity of Fasciola hepatica miracidia to anthelmintic drugs. Through systematic optimisation of this assay, we identified an optimum inoculum of 10–15 miracidia per well, a 3-h observation window and a solvent (DMSO) threshold of 0.6% as critical parameters for reliable assay performance. Under these conditions, CLORS produced no inhibitory effect, whereas CLOS, TCBZ and TCBZ-SO each caused a concentration-dependent motility reduction with reproducible IC_50_ values. Surprisingly, CLOS induced the strongest inhibition of motility on miracidia, surpassing the activity of TCBZ and TCBZ-SO. Taken together, the findings provide the first empirical evidence that both TCBZ/TCBZ-SO and CLOS act on the free-living miracidium stage of F. hepatica under in vitro conditions, rejecting the hypothesis that CLOS is only effective against immature and adult stages (Hutchinson et al., 2009). These observations extend the known in vitro-activity range of these compounds to the miracidium stage.

Results for miracidia were compared with the activity on the motility of the NEJ stage using the NSW and TAS isolates only. When NEJs were exposed to the miracidium-derived IC_50_ concentrations (at 0 h), the relative activity of TCBZ and TCBZ-SO across isolates reflected the pattern observed at the miracidial stage: TCBZ produced greater inhibition than TCBZ-SO in NEJs from the NSW isolate, whereas TCBZ-SO showed stronger early inhibitory effects than TCBZ for the TAS isolate. In contrast, CLOS was only effective against the miracidial stage. These findings suggest that IC_50_ values might be tightly linked to distinct developmental stages, consistent with previous observations and indicating stage- and isolate-associated variation in the anthelmintic sensitivity of F. hepatica (see Duthaler et al., 2010). These observations suggest that the present miracidial assay could become a practical platform for assessing the susceptibility or resistance of F. hepatica to existing or novel anthelmintics, provided that there is a relationship between their in vitro-activity and potency against miracidia and their efficacy against immature and adult flukes.

To establish the present assay, we first optimised key baseline conditions for miracidium exposure, including per-well inoculum size, observation window and maximum solvent concentration. These parameters vary widely among published studies and are fundamental to achieving reliable motility measurements (Taki et al., 2021; Sun et al., 2025). Optimising conditions enabled a reliable, scalable assay for the free-living stage that uses a small number of miracidia raised from gallbladder-derived eggs from livers at slaughter or from faecal samples. The present MMA in a complementary manner to field-based tools for the diagnosis of anthelmintic resistance/susceptibility (e.g., FECRT and CRT; cf. Alvarez et al., 2009; Elliott et al., 2015; Fissiha and Kinde, 2021) whose performances can be affected by variability in egg shedding (Rokni et al., 2002), delayed egg clearance after treatment (Fairweather et al., 2020) and reduced sensitivity during early or pre-patent infections (Fairweather et al., 2012), the present miracidial assay enables a direct evaluation of the free-living stage with rapid, high-throughput and reproducible results (Alberich et al., 2025).

Applying the optimised assay has revealed that CLOS, TCBZ and TCBZ-SO can act on the miracidial stage of F. hepatica, extending their recognised efficacy to a free-living stage. The strong and rapid activity of CLOS in this stage is particularly noteworthy, as experimental data indicate that its earliest confirmed efficacy in rats and sheep occurs at ∼2–4 weeks post-infection (Mohammed-Ali and Bogan, 1987; Maes et al., 1988). While our data are consistent with the possibility that CLOS interferes with energy homeostasis — since the miracidium is non-feeding, short-lived and relies solely on aerobic utilisation of endogenous glycogen (Boyunaga et al., 2001) — we did not observe structural damage or metabolic disruption in this stage. Further work using ultrastructural or biochemical approaches will be required to determine the precise targets of CLOS in this stage. By contrast, the comparatively slower and weaker responses to TCBZ and TCBZ-SO are consistent with their known requirement for metabolic activation (Fairweather et al., 2020). Miracidia possess limited metabolic capacity (Boyunaga et al., 2001), which may constrain the conversion of TCBZ to its sulphoxide form for full activity (Robinson et al., 2004), potentially explaining the delayed or reduced potency of TCBZ in miracidia. Although early studies proposed that TCBZ may interfere with microtubules, recent genomic mapping has showed that beta-tubulin genes do not fall within the major locus associated with TCBZ resistance, suggesting that amino acid changes in β-tubulin are unlikely to be the principal driver of the resistance phenotype, even though microtubule-associated processes highly likely contribute to the drug's mode of action (Fairweather et al., 2020; Beesley et al., 2023). Taken together, this information highlights clear differences in drug susceptibility among developmental stages of F. hepatica and emphasise the importance of considering stage-specific metabolic capacity and drug transformation when interpreting phenotypic responses or designing interventions targeting early stages. The marked isolate-specific differences in compound-sensitivity, particularly the contrasting patterns observed in miracidia derived from the NSW (Postcode 2582), TAS (7300) and VIC (3285) isolates, point to possible underlying variation in trans-tegumental drug permeability (Stitt and Fairweather, 1993, 1994; Choi et al., 2025), oxidative activation capacity (Fairweather et al., 2020), microtubule isoforms (Stitt et al., 1992; Ryan et al., 2008), drug efflux capacity (Scarcella et al., 2012; Fairweather et al., 2020) and/or genetic background. Such divergence in drug sensitivity between isolates may indicate early signs of reduced susceptibility, which requires further exploration using different developmental stages of F. hepatica from a range of distinct geographical localities.

In this study, based on motility, NEJs appeared less susceptible to the concentration of drug required for 50 % reduction of the motility of miracidia. Using the miracidial IC_50_ concentrations, CLOS did not significantly inhibit motility or viability of NEJs originating from NSW and TAS isolates over 24 h, which contrasts sharply with this compound's rapid and pronounced effect in miracidia. Miracidia rely almost entirely on endogenous glycogen for energy (Boyunaga et al., 2001), whereas the NEJs begin to acquire nutrients from the host environment and engage in more flexible energy metabolism (Dalton et al., 2004; Wilson et al., 2011). These developmental differences in energy utilisation may contribute to the distinct phenotypic responses observed across stages, although this aspect remains to be confirmed experimentally. Notably, when NEJs were exposed to IC_50_ concentrations established in miracidia, only TCBZ killed miracidia representing the NSW isolate, with a loss of viability at 4–6 h. Thus, NEJ responses cannot be directly extrapolated from miracidial IC_50_ values, underscoring the need for stage-specific IC_50_ determinations.

Building on the findings from the present study, it will be important to define the phenotypic and molecular determinants underlying these stage-specific responses. Future work should build upon preliminary observations for NEJs, to establish a more comprehensive framework for F. hepatica phenotypic susceptibility testing across multiple developmental stages and assess which phenotypic characters of specific stages of F. hepatica will be most informative. According to current evidence for the NEJ stage (Ferraro et al., 2016; McCusker et al., 2024, 2025), body elongation (length-width ratio), surface expansion and migration distance could be the most informative indicators for quantifying drug responses, consistent with observations from transcriptomic analysis describing intense neoblast-like cell proliferation and cellular differentiation during early development (Robb et al., 2022). Additionally, in vitro culture studies have demonstrated that the NEJ's and juvenile's growth is accompanied by marked metabolic reprogramming and tegumental restructuring (McCusker et al., 2016; Vitkauskaite et al., 2025). Such developmental and physiological changes are likely to affect drug uptake and susceptibility, supporting the proposition that these dynamic growth-associated indicators may affect the evaluation of anthelmintic action in NEJs. In contrast, the adult stage of F. hepatica exhibits pronounced structural specialisation and damage-associated host interactions under chemical stress (Lalor et al., 2021). Therefore, phenotypic characters related to death or cellular degeneration (e.g., reduction in tegument integrity, cytoskeletal collapse or cell death-linked signals) might serve as indicators of drug efficacy in adult F. hepatica stages (McConville et al., 2009; Ezeta-Miranda et al., 2024).

In future, research should focus on integrating phenotypic profiling of miracidial, NEJ and adult stages with genetic data to establish the molecular basis of drug responsiveness and resistance in F. hepatica. Linking observable phenotypic signatures with genomic markers would likely provide a powerful framework for drug resistance surveillance and monitoring in F. hepatica populations (Beesley et al., 2023; Choi et al., 2025), which would contribute to improved resistance management.

Although more work is needed, current evidence indicates that the WMicroTracker platform has significant potential for assessing drug susceptibility and resistance in F. hepatica and potentially other trematodes. Although originally developed to characterise locomotion in C. elegans (see Simonetta and Golombek, 2007), the WMicroTracker platform has since been adapted for high-throughput screening of compounds on parasitic nematodes (Taki et al., 2021, 2022), including assays of medicinal plants and essential oils against H. contortus (Garbin et al., 2021), as well as investigations into macrocyclic lactone (ML) efficacy and drug-interaction dynamics (Alberich et al., 2025). The latter study, for example, used WMicroTracker ONE to assess dose-responses in C. elegans and H. contortus, validated its ability to quantify motility inhibition by ivermectin, moxidectin and eprinomectin and derived IC_50_ and resistance factors that discriminated field isolates with different resistance status. Compared with other phenotypic methods, such as the automated larval migration assay (ALMA), motility tracking showed high sensitivity and avoided underestimation of effects caused by sheath or cuticular barriers that limit exposure during migration assays. Together, these advances indicate that the WMicroTracker platform has the potential to be a versatile and scalable tool for investigations of drug susceptibility and resistance across a broader range of helminth parasites.

Conclusion

5

This study establishes MMA as the first standardised assay for the rapid and quantitative assessment of compound sensitivity in miracidia of F. hepatica. Following systematic optimisation, we used this assay to identify stage- and isolate-specific responses to CLOS, TCBZ and TCBZ-SO and provided the first evidence that these compounds act on miracidia. Overall, MMA offers an adaptable, sensitive and scalable platform with the potential for early, standardised evaluation of drug efficacy and resistance surveillance in F. hepatica and potentially other trematodes.

CRediT authorship contribution statement

Mengwei Zheng: Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Aya C. Taki: Writing – review & editing, Supervision, Methodology, Investigation, Formal analysis, Conceptualization. Tanapan Sukee: Writing – review & editing, Resources, Methodology, Investigation, Formal analysis. Jane Hodgkinson: Writing – review & editing, Supervision, Investigation. Terry W. Spithill: Writing – review & editing, Supervision, Methodology, Investigation. Robin B. Gasser: Writing – review & editing, Supervision, Resources, Investigation, Conceptualization. Neil D. Young: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Ethics approval

This is not required as the liver specimens were donated to us and no ethics was required for the snail culture.

Funding

This project was supported through a Future Fellowship (FT230100559 to N.D.Y.) and grants to N.D.Y. (DP230100270) and R.B.G. (LP180101085 and LP220200614) from the 10.13039/501100000923Australian Research Council (ARC).

Declarations of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.