A laboratory strain of wuh15-2 (F. hepatica/gigantica hybrid type), which was isolated from a cow in Wuhan, China, in 2007 (Ichikawa-Seki et al., 2017), was maintained in Wistar rats and Orientogalba ollula (syn. Lymnaea ollula) at the Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, Japan. This strain was also used in a recent study (Sekii et al., 2023). To prepare the adult flukes, 10–20 metacercariae of the strain collected from intermediate host snails were orally infected with a male Wistar rat (6-week-old, SLC, Shizuoka, Japan). After more than two and a half months of infection, adult flukes were obtained from the bile ducts of the infected rats. The obtained flukes were washed with phosphate-buffered saline (PBS) and then rapidly frozen in liquid nitrogen and kept in a freezer (−80°C) until use. All animal experiments were conducted in compliance with the protocol approved by the Institutional Animal Care and Use Committee of Iwate University (A201836).

The mitochondrial fractions of adult Fasciola sp. were prepared according to a previously described protocol for adult A. suum mitochondria (Iwata et al., 2008). The frozen adult flukes (wet weight: 2.0g) were cut into small pieces and suspended in five volumes of mitochondrial preparation buffer (210 mM mannitol, 10 mM sucrose, 1 mM disodium EDTA, and 50 mM Tris-HCl pH 7.5) supplemented with 10 mM sodium malonate to stabilize complex II. The mixture was homogenized using a motor-driven glass/glass homogenizer with 15 strokes. The homogenate was diluted with the mitochondrial preparation buffer up to 10 times the volume of the original sediment and then centrifuged at 800 × g at 4°C for 10 min to precipitate any unbroken tissue and cell nuclei. The supernatant was then centrifuged at 8,000 × g at 4°C for 10 min to obtain the mitochondrial pellet. The pellet was resuspended in mitochondrial preparation buffer without malonate and centrifuged at 12,000 × g at 4°C for 10 min. The resulting mitochondrial fraction was resuspended in a mitochondrial preparation buffer without malonate. Protein concentration was determined according to the method of Lowry et al. (1951), using bovine serum albumin as a standard (Lowry et al., 1951).

The enzyme activity assays and inhibitor effects were essentially performed in accordance with previous reports with slight modifications (Omura et al., 2001; Matsumoto et al., 2008; Enkai et al., 2023). Assays utilizing the adult mitochondrial fractions were carried out in a 1 mL reaction mixture at 25°C. The final mitochondrial protein concentration was 42.6 μg/mL of reaction mixture in each assay. The mean of triplicate assays was calculated to determine the activity level. Each reaction was monitored for 5 minutes to confirm that the initial slope of absorbance change was not originated from the noise.

NADH-quinone reductase (complex I) activity assay was conducted using a reaction mixture containing 50 mM KPi buffer pH 7.4, 1 mM KCN, and 50 μM NADH. The reaction was initiated by introducing 90 μM ubiquinone (UQ₂) to the reaction mixture. The activity was determined by the oxidation rate of NADH (ϵ = 6.2 mM^-1cm^-1) monitoring at 340 nm (JASCO Corporation, spectrophotometer V-760).

Succinate-quinone reductase (complex II) activity was determined by monitoring the reduction rate of UQ₂ (60 μM; ϵ = 12.7 mM⁻¹ cm⁻¹) at 278 nm (V-760) in the presence of 50 mM KPi buffer pH 7.4, 0.1% (w/v) sucrose monolaurate and 1 mM KCN. The reaction was initiated by adding 10 mM sodium succinate to the reaction mixture.

NADH-cytochrome c reductase (complex I–III) activity was measured in the presence of 50 mM KPi buffer pH 7.4) containing 2 mM EDTA and 2 mM KCN. 33.3 μM cytochrome c and 10 mM sodium malonate by measuring the reduction rate of cytochrome c at 550 nm (ϵ = 19 mM^-1cm^-1) (V-760). This reaction was initiated by the addition of 50 μM NADH to the reaction mixture.

Succinate-cytochrome c (complex II–III) activity was measured in the presence of 50 mM KPi buffer pH 7.4 containing 2 mM EDTA, 2 mM KCN, 33.3 μM cytochrome c, and 10 mM sodium malonate by monitoring the reduction rate of cytochrome c at 550 nm (ϵ = 19 mM^-1cm^-1) (V-760). This reaction was initiated by adding 10 mM sodium succinate to the reaction mixture.

NADH-oxidase (complex I–III–IV) activity was determined in the presence of 50 mM KPi (pH 7.4) as reaction buffer by monitoring the oxidation rate of NADH at 340 nm (ϵ = 6.2 mM⁻¹ cm⁻¹) (V-760). This reaction was initiated by the addition of 50 μM of NADH to the reaction mixture.

Ubiquinol-oxidase (complex III–IV) activity was determined by monitoring the oxidation rate of ubiquinol-1 (100 μM) at 278 nm (ϵ = 12.7 mM⁻¹ cm⁻¹) (V-760) in the presence of 50 mM KPi buffer pH 7.4 containing 2 mM EDTA. This reaction was initiated by the addition of 50 μM ubiquinol-1 to the reaction mixture.

NADH-fumarate reductase (complex I–II) activity assay was performed under anaerobic conditions, where the reaction contained degassed 50 mM KPi buffer pH 7.4, 50 μM NADH, 100 μg/mL of glucose oxidase, 2 μg/mL of catalase and 10 mM glucose and left for 3 min to achieve anaerobiosis. The reaction was initiated by the addition of 5 mM sodium fumarate as an electron acceptor and the activity was determined by monitoring the oxidation rate of NADH at 340 nm (ϵ = 6.2 mM⁻¹ cm⁻¹) (SHIMADZU spectrophotometer UV-3000).

Mitochondria were solubilized according to previously described methods (Schägger and von Jagow, 1991) with slight modifications. Briefly, equal volumes of solubilization buffer containing 50 mM Tris-HCl pH 8.0, 4% (w/v) sucrose monolaurate (SML), 40% (v/v) glycerol and 2 mM sodium malonate, and 10 mg/mL mitochondria from the adult Fasciola sp. were mixed and incubated on ice for 1 h and centrifuged at 200,000 × g at 4°C for 30 min. The resulting supernatants were subjected to high-resolution clear native electrophoresis (hrCNE) using Invitrogen’s Native PAGE™ 4−16% Bis-Tris gel in duplicate. The cathode buffer for hrCNE, 50 mM Bis-Tris, 50 mM Tricine, pH 6.8 (Invitrogen, Native PAGE™ Running buffer) was supplemented with 0.05% (w/v) sodium deoxycholate (DOC) and 0.02% (w/v) dodecylmaltoside (DDM), and the gel was run in the cold room (4°C). The initial voltage for the gel run was set to 100 V, and after 1 h, the voltage was raised to 250 V. For molecular weight determination, a NativeMark™ Unstained Protein Standard was used as a reference. Adult A. suum and bovine mitochondria were used as controls. After hrCNE, both gels were used for visualization, one for assessing complex I through NADH dehydrogenase (NDH) activity staining and the other for complex II through succinate dehydrogenase (SDH) activity staining. Both gels were soaked in 5 mM Tris-HCl buffer, pH 7.4 containing 1.5 mM nitro blue tetrazolium (NBT) salt. Subsequently, 100 µM NADH was added for NDH activity staining, or 10 mM sodium succinate and 100 µg/mL phenazine methosulfate (PMS) were added for SDH activity staining at room temperature (25°C). After 3 min, the reagents were washed with distilled water and the resulting bands were documented.

The inhibition potencies of three typical respiratory chain enzyme inhibitors against mitochondrial complexes of adult Fasciola flukes were determined, i.e., each activity assay was repeated as described above in the presence of the inhibitors. The inhibitors were incubated for 2 minutes prior to initiation of enzyme reactions. The inhibitors used were rotenone, atpenin A5, and ascochlorin, which target complexes I, II, and III, respectively ( Figure 1 ). The final concentrations of the inhibitors were 0.1 μM, 1 μM, and 10 μM, respectively. Inhibition was calculated as the reduction in the enzyme activity with addition of the compound, average of 3 technical replicates.

The NEJs excysted from the metacercariae were prepared for in vitro assays according to a previously described protocol (Andrews et al., 2022) with slight modifications. Briefly, the metacercariae were washed with double distilled water and soaked in 2.2% (w/v) sodium hypochlorite to remove the outer cyst wall debris. The metacercariae were then washed thrice with PBS. The excystment was carried out by incubating the samples for 3 h at 39°C in a disposable dish sealed with Parafilm (Bemis Flexible Packaging, Wisconsin, USA). The incubation solution consisted of 120 mM NaHCO₃, 140 mM NaCl, 0.4% (w/v) taurocholic acid, 33 mM l-cysteine, and 50 mM HCl. The resulting NEJs were washed in RPMI-1640 (Sigma-Aldrich, St Louis, MO, USA) and maintained in a 5% CO₂ incubator (Mini CO₂ incubator 4020, Asahi Life Science, Saitama, Japan) at 37°C until use. Each in vitro assay was initiated on the day of excystment.

To examine the killing efficacy of the three inhibitors, rotenone, atpenin A5, and ascochlorin, against living Fasciola NEJs, the parasites were maintained in the culture medium supplemented with 10 and 100 μM of each inhibitor. Each well received 10 NEJs, and the volume of the culture medium was 2 mL. This medium consisted of RPMI-1640, 10% (v/v) fetal bovine serum (Funakoshi, Tokyo, Japan), penicillin-streptomycin solution mix (at concentrations of 100 U/mL and 100 ng/mL, respectively, from Sigma-Aldrich), 500 ng/mL of amphotericin B (Thermo Fisher Scientific, Massachusetts, USA), and 10 mM HEPES (Dojindo, Kumamoto, Japan). In addition to the three inhibitors, positive (triclabendazole; Tokyo Chemical Industry, Tokyo, Japan) and negative (dimethyl sulfoxide (DMSO); FUJIFILM Wako Pure Chemical, Osaka, Japan) controls were prepared. The parasite cultures were stored in 12-well plates and incubated at 37°C for 7 days. Under aerobic conditions, the plates were placed in a 5% CO₂ incubator (Mini CO₂ Incubator 4020) with O₂ concentration equal to air (approximately 20%). The effect of each inhibitor was evaluated daily. For anaerobic conditions, the plates were placed in an anaerobic container containing an Anaero Pack (Mitsubishi Gas Chemical, Tokyo, Japan), in which the CO₂ concentration exceeded 15% and O₂ concentration was maintained at less than 0.1%. The effects of each inhibitor were evaluated on alternate days. Unlike NEJs, adults are not stable in the in vitro condition for 7days. Therefore, we could not perform the in vitro assays for adults.

The mortality scores of NEJs were evaluated under an inverted microscope (IMT, Olympus, Tokyo, Japan) according to the criteria described by Lorsuwannarat et al. (2014), with slight modifications. The scores were as follows: active motility = 3; inactive, but with visible organs = 2; inactive, with dark color and invisible organs (almost dead) = 1; and inactive with tegument disruption = 0 (clearly dead). The mean scores for 10 NEJs within each well were determined. The Mann-Whitney test was employed to evaluate the significance between DMSO and the inhibitors.

The NEJ viability was evaluated using propidium iodide (PI) staining (Dojindo Laboratories, Kumamoto, Japan). PI emits red fluorescence (λ_ex = 530 nm, λ_em = 620 nm) by binding to the nucleic acid of dead cells. The mortality of NEJs was objectively determined, with emission scoring 0 (dead cells) and no emission (living cells) scoring 1. The mean score of 10 NEJs per well was calculated using the following procedure. The culture medium in each well was washed thrice with PBS. After filling each well with 2 mL PBS, 2 μL of 1 mg/mL PI was added and incubated for 30 min at 37°C with 5% CO₂. Finally, the plates were examined under an inverted fluorescence microscope (IX73; Olympus). The Mann-Whitney test was employed to evaluate the significance between DMSO and the inhibitors.

Quinone components from adult Fasciola flukes and NEJs were analyzed according to a method described previously (Shiobara et al., 2015). A frozen adult Fasciola sp. was cut into four pieces and put in BioMasher^® II 1.5-mL tubes (Nippi Inc., Tokyo, Japan). Samples were then homogenized in 20 volumes of 2-propanol and placed in a bath sonicator for 2 min. After centrifugation, 4 µL of the supernatant was injected into two analytical columns (Inertsil ODS-HL, 3 µm, 150 × 2.1 i.d., GL Science, Japan), a reduction column (CQ-R, 20 × 2.0 i.d., Shiseido, Japan) at 40°C. The quinones and quinols were detected using an electrochemical detector at 600 mV against Ag/AgCl (NANOAPACE SI-2, Shiseido, Japan). The mobile phase consisted of 50 mM sodium perchlorate in methanol/2-propanol/water (31/65/4, v/v/v) at a flow rate of 0.18 mL/min. The total UQ₁₀ content was calculated as the sum of quinone and quinol, and the quinone form alone was observed in RQ₉ and RQ₁₀. Extraction from the NEJs (200 individuals) was initiated by sonication in 2-propanol. Since the quinone content was limited, the extracts were once dried in vacuo, then dissolved in 1/10 volume of 2-propanol, and 8 µL was applied to HPLC. Quinone content was determined based on the wet weight of the parasites, and calculations were made using data from three analyses.

In this study, 34.8 mg (protein) of the mitochondrial fraction was obtained from 2.0 g (wet weight) of adult Fasciola flukes. Then specific enzyme activities involved in the mitochondrial respiratory chain of adult Fasciola flukes were examined ( Table 1 ). Adult Fasciola mitochondrial NADH-quinone reductase (complex I) exhibited a specific activity of 204 nmol/min/mg. The activity of succinate quinone reductase (complex II) was 635 nmol/min/mg. NADH-cytochrome c reductase (complex I–III) activity was 172 nmol/min/mg. Adult Fasciola succinate cytochrome c reductase (complex II–III) showed a specific activity of 60.4 nmol/min/mg. NADH-oxidase (complex I–III–IV) activity was found to be 9.34 nmol/min/mg. The activity of ubiquinol-oxidase (complex III–IV) was 7.42 nmol/min/mg. The specific activity of NADH–fumarate reductase (complex I–II) was 68.0 nmol/min/mg, which is higher than that of adult A. suum (51.7 nmol/min/mg), whose fumarate respiration mechanism has been well studied (Iwata et al., 2008).

The hrCNE ( Figure 3 ) was used to compare the properties of complexes I and II from Fasciola flukes with those of bovine and A. suum. Fasciola complexes I and II were distinct in size compared to those of bovines and A. suum. NDH activity staining, which targeted complex I, revealed that multiple bands were stained in Fasciola and bovine mitochondria, but they were different in size. In contrast, only a single band was observed for A. suum. Complex II was visualized via SDH activity staining, and the results showed that complex II of Fasciola was smaller than that of A. suum but larger than that of bovine. These findings indicate that the sizes of complexes I and II in Fasciola flukes are unique and differ from those of bovine and A. suum.

Specific inhibitors of each enzyme were employed in inhibition assays to investigate the properties of the respiratory chain of adult Fasciola flukes. The percentage inhibition of each enzyme in the mitochondrial respiratory chain of adult Fasciola flukes are shown in Table 2 . Rotenone inhibits all activities of complex I. The inhibition of complexes I, I–II, and I–III–IV activities were 91.0%, 94.1%, and 100%, respectively. Similarly, atpenin A5 inhibits complex II-dependent activity. The inhibition at 1 μM were 97.2%, 92.0%, and 90.5% for complexes II, I–II, and II–III, respectively. Again, 1 μM of ascochlorin inhibited complex III-related activities. It inhibited complexes II–III and I–III–IV by 83.9% and 82.6%, respectively. Notably, ascochlorin unexpectedly inhibited complex II-related activity, although it is known to be a specific inhibitor of complex III. The inhibition of ascochlorin was 72.3% and 85.0% against complexes II and I–II, respectively.

To understand which enzyme complexes are essential for juvenile survival, we performed in vitro assays to assess the killing efficacy of three typical inhibitors against NEJs ( Figure 4 ). Killing efficacy was evaluated using the relative mortality score and PI staining. The results obtained using both methods were consistent with each other. Under aerobic conditions, rotenone was effective at both 10 and 100 μM. Ascochlorin killed NEJs at 100 μM but it was not effective at 10 μM. In contrast, atpenin A5 was ineffective at both concentrations. Under anaerobic conditions, rotenone, ascochlorin, and atpenin A5 at 100 μM killed NEJs. Interestingly, the cytotoxic efficacy of atpenin A5 was observed only under anaerobic conditions.

The quinone components of adults and NEJs were investigated to reveal changes in electron mediators between the two stages. The total UQ₁₀, RQ₁₀, and RQ₉ concentrations in adult Fasciola fluke were 1.5 ± 0.24, 55 ± 2.9, and 1.4 ± 0.89 pmol/mg of the parasite wet weight, respectively. In contrast, the content of total UQ₁₀ in NEJs was 1.4 ± 0.041 pmol/mg and that of RQ₁₀ was 1.5 ± 0.53 pmol/mg. The RQ₁₀/UQ₁₀ ratios were 36.7 and 1.07 for adults and NEJs, respectively. Consequently, RQ₁₀ required for fumarate respiration was predominant in adults. In contrast, in addition to RQ₁₀, UQ₁₀ used in oxygen respiration was detected almost equally in NEJs ( Table 3 ).