Ethics statement

All animal experiments were conducted in compliance with the protocols approved by the Institutional Animal Care and Use Committee of Iwate University (A202108, A202109, A202425, and A202426).

Laboratory strain

A laboratory strain of wuh15–2 (F. hepatica/gigantica hybrid type, triploid), isolated from a cow in Wuhan, China, in 2007 [28], has been maintained in rats (SLC: Wistar, male, 6 weeks old) and Orientogalba ollula (syn. Lymnaea ollula) at the Laboratory of Veterinary Parasitology, Iwate University, Japan. This strain has been used in other recent studies [25,26,29]. The number of metacercariae and mice used and the number of juveniles collected were summarized in S1 Table.

Juveniles collected from mice for normal in vivo developmental controls of the liver-stage

Normal in vivo developmental controls of liver-stage juveniles in mammalian hosts were obtained to evaluate the growth, sexual differentiation, and reproduction of the liver flukes. These three processes can be monitored by measuring the increase in area, changes in body shape, and the development of reproductive organs.

The laboratory strain metacercariae were infected into mice (SLC: ddY, female, 6 weeks old). Juveniles of the laboratory strain were collected from mice at 1, 3, 7, 11, 14, and 21 dpi. The 21 dpi represents the latest point at which Fasciola flukes can be stably recovered from mice. Additionally, we focused on the period before 14 dpi, examining intervals of 3–4 days to monitor morphological changes of reproductive organs as they grew. Juveniles were collected from the abdominal cavity of mice at 1 dpi by flushing with phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄; pH 7.4). Juveniles at 3 dpi and later were collected from the liver via crushed with forceps. Juveniles were placed in a disposable Petri dish (Becton Dickinson, Franklin Lakes, NJ, USA) containing PBS and temporarily stored in an incubator (SMA-80DRS, ASTEC, Osaka, Japan) at 37°C and 5% CO₂ until use. Juveniles treated with PBS were transferred to 12-well plates (Cat. No. 3737, Corning, Corning, NY, USA) or 96-well plates (Cat. No. 260860, Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the juveniles were photographed using a digital camera system (NY-X8i Super System, Canon, Tokyo, Japan) attached to an inverted microscope (IX73, Olympus, Tokyo, Japan or CKX53, Evident, Tokyo, Japan) or a stereomicroscope (SZ40, Olympus, Tokyo, Japan). The outline of a juvenile’s body was traced on a photograph using a UGEE U1200 Pen Display (Hanvon Ugee Technology Co., Ltd., Shenzhen, Guangdong, China), and the body area was measured with ImageJ software [30]. Calibration in ImageJ was performed using a stage micrometer photographed at the same magnification as the samples. The scale was set using the “Set Scale” function in ImageJ based on this reference image prior to measurement.

The area posterior to the ventral sucker enlarged during the days after infection, and the shoulders became pronounced, resulting in a prominent oral cone structure of the flukes. In this study, the oral cone index was designed to monitor changes in body shape. This index increased according to the time course of days post-infection. The maximum body width and width at the top of the ventral sucker were measured for each photographed juvenile (n = 10) using ImageJ software [30] to calculate the oral cone index.

Stained specimens (n = 73, 20, 133, 49, 46, and 19 for each time point) were prepared to visualize the internal organs. After washing with PBS, fresh juveniles were flattened between glass slides or under a cover glass and fixed with 70% ethanol. After staining with hematoxylin and carmine, the specimens were dehydrated, treated with xylene (Fujifilm Wako Chemicals, Osaka, Japan), and mounted with Canada balsam (Fujifilm Wako Chemicals). The internal structure of the juveniles was observed using a light microscope (CX31LBSF, Olympus) or a stereomicroscope.

In vitro culture of juveniles collected from mice in the basic medium

For the initial experiment, juveniles at 3, 7, and 11 dpi collected from mice were incubated in the basic medium. The numbers of infected metacercariae and juveniles collected are summarized in S1 Table. The juveniles at 7 and 11 dpi were the remaining specimens after preparing the staining samples for the in vivo developmental controls. The juveniles at 3 dpi required two additional mice to obtain sufficient numbers for incubation. The basic medium used for the culture was prepared by RPMI 1640 (Nacalai tesque, Kyoto, Japan) with, at final concentration, respectively, 10 mM HEPES (Dojin Chemical Laboratory, Kumamoto, Japan), 50% French fetal bovine serum (Bovogen Biologicals, Melbourne, Australia) or 50% Brazilian fetal bovine serum (Corning), 200 U/mL penicillin and 200 µg/mL streptomycin (Sigma-Aldrich, Saint Louis, MO, USA), and 500 ng/mL amphotericin B (Thermo Fisher Scientific), and was sterilized by a filter (Millex-GSISilter Unit 0.22 µm, MERCK, Darmstadt, Germany, or Rapid Filter Max Set, BM Equipment, Tokyo, Japan). The medium was stored at 5°C and pre-warmed to 37°C prior to use.

The juveniles were cultured in 96-well plates (Thermo Fisher Scientific). The number of juveniles per well was four, one, and four at 3, 7, and 11 dpi, respectively, at the beginning of the culture. S2 Table provides a summary of the total number of juveniles prepared in multiple wells for each dpi. After 25 days of culture, the 11 dpi juveniles were individually placed into new wells to make one juvenile per well, and the assay was continued. This was not due to contamination or other artificial problems. This assay was the first attempt, and the protocol was not refined yet. Therefore, a complex methodology has no meaning other than a simple preliminary trial for testing different methods. Nevertheless, presenting the results of this first trial is necessary because this assay served as the foundation for this study. Each well contained 250 µL of the basic medium and was maintained in an incubator (37°C, 5% CO₂). The medium was changed three times per week by removing 150 µL of medium from each well and adding 150–180 µL of new medium. At the same time as the medium change, the juveniles were photographed and measured as described above, and dead juveniles were removed. Juveniles were considered dead when they exhibited persistent immobility, accompanied by darkening or structural disintegration of the body.

Supplementation of red blood cells in the in vitro culture of juveniles

As the liver fluke is considered a blood feeder [27], the effects of red blood cells (RBC) supplementation on juveniles were examined in vitro, with the expectation that it would serve as a nutrient source. NEJ, 7 dpi, and 11 dpi juveniles were supplemented with RBC.

NEJ was obtained from the metacercariae of a laboratory strain (wuh15–2). The excystment protocol was performed based on previous study [31]. Briefly, the metacercariae were washed with ultrapure water and soaked in 2.2% (w/v) sodium hypochlorite to remove the outer cyst wall debris. The metacercariae were washed three times with PBS. Excystment was performed by incubating the samples for 3 h at 39°C in a disposable dish sealed with Parafilm (Amcor, Melbourne, Australia) in a reagent consisting of 60 mM NaHCO₃ (Kanto Chemical, Tokyo, Japan), 70 mM NaCl (Fujifilm Wako Chemicals), 0.2% (w/v) taurocholic acid (Sigma-Aldrich), 16.5 mM L-cysteine (Fujifilm Wako Chemicals), and 50 mM HCl. The activity of NEJ was confirmed by the sufficient excystment rate (65%, S1 Table) and active movement and then placed in RPMI 1640 and stored in a 37°C, 5% CO₂ incubator until use for around 1 h. Juveniles were collected from mice at 7 and 11 dpi, as described above. The numbers of infected metacercariae and juveniles collected are summarized in S1 Table. After washing with PBS, they were placed in RPMI 1640 and stored under the same conditions as the NEJ until use.

Four juveniles were incubated in one well of a 96-well plate (Thermo Fisher Scientific) at 37°C and 5% CO₂ under the two conditions (one well per condition without replication, n = 4). RBC: 250 µL of the basic medium supplemented with 0.5 µL of bovine defibrinated blood (bovine RBC) (Japan BioSerum, Hiroshima, Japan). Here, the volume of RBC was adjusted to a level where juveniles were easily visible under the microscope, and the number of RBC/µL was not counted before use. The control group was treated with 250 µL of the basic medium. The medium was changed three times a week by removing 150 µL of medium from each well and adding 180 µL of fresh medium. For the RBC group, 0.5 µL of bovine RBC was newly added to the medium. At the same time as the changing medium, juveniles were photographed and measured as described above, and dead juveniles were removed.

The NEJ incubation was over when all of them died. In contrast, for 7 and 11 dpi juveniles, one juvenile was removed from each group after 91 days of incubation to prepare stained specimens, as described above. At 98 days of incubation, when only one active juvenile was left in a 7 dpi group, all remaining juveniles were removed and stained, which was the endpoint of the assays.

Evaluation of higher concentration of red blood cells

The higher concentration of RBC was examined using 11 dpi juveniles, as they showed superior development compared to those at 7 dpi juveniles in the previous assay. A different batch of bovine RBC (Japan BioSerum) was used, and the number of RBC was counted using a hemocytometer (Erma Inc., Saitama, Japan) and was 5.21 × 10⁶/µL at the beginning of the culture. The numbers of infected metacercariae and juveniles collected are summarized in S1 Table. This assay was performed after the supplementation of sex-inducing substances described below, and the method for collecting 11 dpi juveniles was modified to save time. The metacercariae were infected as described above, and juveniles were collected at 11 dpi from the liver by soaking it in the basic medium for at least 4 h in an incubator at 37°C (CN-25C; Mitsubishi Electric Engineering, Tokyo, Japan). This method was adapted from a previous study [32]. It was an easier method that did not alter the collection rate. Prior to soaking, the liver was artificially injured using forceps. The juveniles were then washed with PBS, placed in the basic medium, and stored in an incubator until further use.

Four juveniles per well and two wells for each condition (n = 8) were incubated in a 96-well plate (Thermo Fisher Scientific) at 37°C and 5% CO₂ under the following five conditions. The control group was treated with 250 µL of basic medium. 0.5 µL-RBC: 250 µL of the basic medium supplemented with 0.5 µL of bovine RBC. 1.5, 3.5, and 5.5 µL-RBC: supplemented with 1.5, 3.5, and 5.5 µL of bovine RBC, respectively. The medium was changed three times per week by removing 150 µL of medium from each well and adding 170 µL of new medium. The respective volumes of bovine RBC were added to the RBC group. While changing medium, juveniles were photographed and measured as described above, and dead juveniles were removed. After 30 days of incubation, the remaining juveniles were removed to prepare the stained specimens, as described above.

Extraction of sex-inducing substances

The SIS used in this study were fractionated from Calicophoron calicophorum, a parasitic flatworm that is more easily obtained in Japan compared to Fasciola flukes. Previous studies [25,26] showed that the SIS fraction prepared from this trematode had an excellent ability to induce sexualization in the free-living flatworm, planarian (D. ryukyuensis).

As batch #1, approximately 8.0 g wet weight of C. calicophorum were used according to a previous study [26]. The samples were then homogenized in 480 mL ultrapure water using a Potter-type homogenizer (ASONE, Osaka, Japan). The suspension was crushed on ice using an ultrasonic crusher (TOMY SEIKO, Tokyo, Japan) for 2 min 30 s and centrifuged (16,000 × g, 30 min, 4°C). The supernatant was filtered through a 0.2 µm PES membrane bottle-top filter (Thermo Fisher Scientific). The filtered supernatant was ultracentrifuged (120,000 × g, 30 min, 4°C). The resulting supernatant was freeze-dried and dissolved in water to yield 100 mL of extract. The extract was further separated via open-column chromatography using 50 g of ODS (COSMOSIL 75 C18–OPN; Nacalai Tesque) packed in an Econo-Column (50 φ × 500 mm; Bio-Rad, Hercules, CA, USA). A peristaltic pump (EYELA, Tokyo, Japan) was used to pump the liquids. The 100 mL extract was filtered through a 0.22 µm syringe filter (Waters, Milford, MA, USA) and applied to the ODS column, and the resulting product was collected as the flow-through fraction. Next, 300 mL of water was passed through; the first 60 mL was collected as the flow through fraction, and 240 mL was collected as Fr. M0. Next, 300 mL of 10, 30, and 50% methanol was sequentially loaded, and the eluate was collected as Fr. M10, Fr. M30, and Fr. M50, respectively, and dried using a rotary evaporator (VC-15S, TAITEC, Saitama, Japan). The Fr. M30 derived from approximately 2.0 g wet weight of C. calicophorum was used for feeding bioassays using asexual D. ryukyuensis to confirm sex-inducing activity (S1 Fig) [25,26]. The remaining Fr. M30 from 6.0 g wet weight of C. calicophorum was dissolved in 6 mL of water and stored at −80°C as the SIS fraction (batch #1) until use.

Supplementation of sex-inducing substances in the in vitro culture of the juveniles

As the SIS fraction can induce sexualization in the free-living platyhelminth, planarian (D. ryukyuensis) [25], the effects of SIS supplementation on Fasciola juveniles were examined in vitro. SIS supplementation was performed in NEJ, 7, and 11 dpi juveniles. Juveniles were prepared as described above. These juveniles were from the same batch as those used in the first RBC supplementation assay (S1 Table). A total of eight juveniles were collected for NEJ or each dpi and separated for culturing, as shown below. Four juveniles were incubated in one well of a 96-well plate (Thermo Fisher Scientific) at 37°C and 5% CO₂ under two conditions (one well per condition without replication, n = 4). Water: 240 µL of the basic medium supplemented with 10 µL of sterile ultrapure water. SIS: 240 µL of the basic medium supplemented with 10 µL of the SIS fraction. The medium was changed three times per week by removing 150 µL or 180 µL of medium from each well and adding 172.8 µL of new medium and 7.2 µL (4%) of water or the SIS fraction to each group. While changing medium, juveniles were photographed and measured as described above, and dead juveniles were removed. One juvenile was removed from each group after 91 days of incubation to prepare stained specimens, as described above. Similarly, all remaining juveniles were removed and stained after 98 days of incubation. These time points were the same as those for the initial RBC supplementation assay.

Examination of higher concentrations of sex-inducing substances

The higher concentration of SIS was examined using 11 dpi juveniles, as they showed superior development compared to those at 7 dpi juveniles in the previous assay. A different batch (batch #2) of the SIS fraction was prepared as described above, starting from 8.0 g wet weight of C. calicophorum. The Fr. M30 derived from 2.0 g was used to assess the activity in planarians (S1 Fig) [25,26]. The remaining Fr. M30 (6.0 g) was divided into 1.5 g and 4.5 g, both of which were dissolved in 1.5 mL of water (1 × SIS and 3 × SIS. respectively).

Juveniles at 11 dpi were collected from the liver via crushed with forceps. The numbers of infected metacercariae and juveniles collected are summarized in S1 Table. Four juveniles per well and two wells per condition (n = 8) were incubated in a 96-well plate (Thermo Fisher Scientific) at 37°C and 5% CO₂ under the following conditions. Water: 250 µL of the basic medium supplemented with 10 µL of sterile ultrapure water. 1 × SIS: 240 µL of the basic medium supplemented with 10 µL of 1 × SIS fraction. 3 × SIS: 240 µL of the basic medium supplemented with 10 µL of 3 × SIS fraction. The medium was changed three times per week by removing 150 µL from each well and adding 170 µL of new medium that was prepared in advance by mixing the basic medium with ultrapure water, 1 × SIS, and 3 × SIS, respectively, at a final concentration of 4% (v/v). When the medium was changed, the juveniles were photographed and measured as described above. After 30 days of incubation, the remaining juveniles were removed to prepare the stained specimens, as described above.

Evaluation of the combined effect of sex-inducing substances and red blood cells

The combined effects of SIS and RBC on 11 dpi juveniles were investigated. Juveniles at 11 dpi were collected from the liver by soaking it in the basic medium. The numbers of infected metacercariae and juveniles collected are summarized in S1 Table. The batches of RBC, 1 × SIS, and 3 × SIS were the same as those used in the previous assays. The number of RBC was 4.09 × 10⁶/µL at the beginning of the culture and decreased from the previous assay due to hemolysis during storage. Therefore, the number of RBC supplemented in this assay (4.4 μL) was approximately the same as 3.5 µL-RBC in the previous assay.

Four juveniles per well and two wells per condition were incubated in a 96-well plate (Thermo Fisher Scientific) at 37°C and 5% CO₂ under the following conditions. The assay was performed in duplicate by preparing two plates of the same design (n = 16 for each condition). Water: 250 µL of the basic medium supplemented with 10 µL of sterile ultrapure water. RBC: 250 µL of the basic medium supplemented with 10 µL of water and 4.4 µL of bovine RBC (Japan BioSerum). 1 × SIS: 250 µL of the basic medium supplemented with 10 µL of 1 × SIS fraction. 3 × SIS: 250 µL of the basic medium supplemented with 10 µL of 3 × SIS fraction. 1 × SIS + RBC: 250 µL of the basic medium supplemented with 10 µL of 1 × SIS and 4.4 µL bovine RBC. 3 × SIS + RBC: 250 µL of the basic medium supplemented with 10 µL of 3 × SIS and 4.4 µL bovine RBC.

The medium was changed three times per week by removing 150 µL from each well and adding 170 µL of new medium that was prepared in advance by mixing the basic medium with ultrapure water, 1 × SIS, and 3 × SIS, respectively, at a final concentration of 4% (v/v). In addition, 4.4 µL bovine RBC were added to the respective RBC groups. When the medium was changed, juveniles were photographed and measured as described above. After 30 days of incubation, the remaining juveniles were removed to prepare the stained specimens, as described above.

Statistical analysis

All graphs of juvenile area and oral cone index are presented as median and range. The Mann–Whitney U test, the Kruskal–Wallis test, and the long-rank test were employed in this study using Graph Pad Prism 10 (Graph Pad Software, San Diego, CA, USA), and P < 0.05 was considered a significant difference. When there were fewer than two juveniles in a group, the test could not be performed because the software determined that the sample size was insufficient. All the P values in this study were summarized in S3 Table.

Juveniles collected from mice for normal in vivo developmental controls of the liver-stage

Juveniles of the laboratory strain (wuh15–2, F. hepatica/gigantica hybrid type, triploid) [25] were collected from mice at 1, 3, 7, 11, 14, and 21 dpi to obtain normal in vivo developmental controls of liver-stage juveniles in mammalian hosts. The growth, sexual differentiation, and reproduction of liver flukes were monitored by measuring the increase in area, changes in body shape, and development of reproductive organs. Juveniles were found in the abdominal cavity at 1 dpi and in the liver from 3 dpi onwards (S1 Table). The median body area was 0.02 mm² at 1 dpi, gradually increased to 0.11 mm² at 7 dpi, and then drastically increased to 2.39 mm² at 21 dpi. Inter-sample variation increased over the course of infection (Fig 1).

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Fig 1. Juvenile area of Fasciola flukes collected from mice for normal developmental controls of the liver-stage.

Body areas of juveniles at 1, 3, 7, 11, 14, and 21 days post-infection (dpi). Notably, 1 dpi juveniles were obtained from the abdominal cavity, and 3 dpi juveniles or later were collected from the liver (median ± range). The numbers of samples were 146, 40, 196, 117, 75, and 26 for each time point. Representative images are shown in the graph at the same scale. Please refer to Fig 2C, which illustrates the morphological characteristics of the internal organs of the juveniles.

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The oral cone index (Fig 2A) was used in this study to evaluate changes in the body shape. Ten juveniles were randomly selected from each day post-infection to calculate the oral cone index. The median value of the oral cone index was stable from 1.01 to 1.04 up to 7 dpi but increased to 2.24 at 21 dpi (Fig 2B), and the oral cone was clearly recognizable in live juveniles at 14 dpi (Fig 2C). Although inter-sample variation was observed among individuals, the typical development of the internal organs was as follows: primary and secondary bifurcations of the intestinal tract were observed at 3 and 11 dpi, respectively, which then developed in a complex manner (Fig 2C and 2D). Reproductive organs were difficult to observe in live juveniles, except for the ootype, which was visible at 21 dpi (Fig 2C), and staining was required to visualize them (Fig 2D). In the stained specimens, the genital rudiment gradually developed over time into the hermaphroditic reproductive organs, including testis, ovary, Mehlis’ gland (identical to the ootype location), and other organs (Fig 2D).

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Fig 2. Oral cones and internal organs of Fasciola juveniles from mice for normal developmental controls.

(A) Calculation of the oral cone index. Light green indicates the area of the oral cone after the shoulders are formulated. (B) Median oral cone index value at each time point (median ± range, n = 10). (C) Representative images of live juveniles at each time point with distinct morphological features. (D) Representative images of stained juveniles. n = 73, 20, 133, 49, 46, and 19 for each time point. vs: ventral sucker; s: the beginning of the shoulder; pb: primary bifurcation of the intestinal tract; sb: secondary bifurcation of the intestinal tract; oc: oral cone; ot: ootype; gr: genital rudiment; csr: cirrus sac rudiment; fror: female reproductive organ rudiment; tr: testis rudiment; ur: uterus rudiment; cs: cirrus sac; u: uterus; o: ovary; mg: Mehlis’ gland; t: testis. Bars: 100 µm (1–11 dpi), 500 µm (14 and 21 dpi).

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These observations were consistent with previous descriptions of F. hepatica [18] and Fasciola sp. [33]. Compared to the body area and oral cone index, monitoring reproductive development using stained specimens tends to be more subjective and less reliable. This is owing to the inter-sample variations that naturally occur between individuals and are artificially caused during sample preparation. Specifically, observation of reproductive organs is difficult because the preparation of specimens depends on the technician’s skill, such as the degree of staining and whether their eyes are sufficiently trained to detect testicular branching. Although all stained specimens in this study were in good condition, reliable molecular markers must be established to objectively evaluate reproductive development. However, in the absence of reliable markers, we had no choice but to perform morphological evaluations of the stained specimens in this study.

In vitro culture of juveniles derived from mice in basic medium

Juveniles collected from mice at 3, 7, and 11 dpi were incubated in basic medium (RPMI 1640 + 50% fetal bovine serum [FBS]) (Fig 3) for the initial experiment. Because the evaluation protocol was not refined at the beginning of the research project, the number of juveniles varied among the groups (S2 Table), and we could not always photograph the same individuals in this experiment, resulting in no survival curve being generated for the assay. Nevertheless, in contrast to 3 dpi juveniles, 7 and 11 dpi juveniles appeared to grow and survive for a long time in vitro.

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Fig 3. In vitro culture of juveniles derived from mice in basic medium.

Juveniles at 3 dpi (red), 7 dpi (blue), and 11 dpi (green) were collected from the liver of mice and incubated at 37°C, 5% CO₂ in 250 µL of the basic medium (RPMI 1640 + 50% FBS) (median ± range). Number of samples used is summarized in S2 Table. Representative images of juveniles before and after culture in each group are shown at the same scale. sb: secondary bifurcation of the intestinal tract.

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The median body area of juveniles at 3 dpi was 0.05 mm² on the first day and increased to a median of 0.11 mm² after 7 days in culture. Juveniles gradually weakened, shrank, and all died after 40 days in culture (Fig 3). In this study, the 3 dpi juveniles were collected from the liver (S1 Table). However, their growth in vitro was smaller compared to that at 7 dpi or 11 dpi, suggesting that merely migrating to the liver did not enhance their growth and survival in vitro.

In 7 dpi juveniles (Fig 3), the maximum survival time was 80 days. The median body area was 0.11 mm² on the first day and increased to 1.36 mm² after 63 days in culture. The increase in area was constantly observed in the early phase of the culture but almost stopped after 24 days. The juveniles maintained their area for 48 days but gradually weakened, shrank, and eventually died. In 11 dpi juveniles (Fig 3), the maximum survival time was 143 days. The median body area was 0.55 mm² on the first day and increased to 1.77 mm² after 92 days of culture. The increase in area slowed after eight days of culture. The juveniles were stably active for 99 days. The last juvenile was active for up to 139 days in culture but gradually weakened, shrank, and eventually died. The intestinal tract beyond the secondary bifurcation was observed at 7 and 11 dpi (Fig 3), but development was arrested when the area increase slowed down. The oral cone was never recognized during the time course in any group, indicating that the change in body shape was not induced by the basic medium alone.

A previous study [2] reported that 10 dpi juveniles collected from mice survived for 78 days in vitro; however, detailed growth data were not documented. This study showed the long-term survival of 7 and 11 dpi juveniles in vitro with a drastic increase in body area. Although 3 dpi juveniles grew slightly, the growth rate was completely different from the growth observed at 7 dpi or 11 dpi. The physiological function of juveniles, such as their metabolic system, may change during the first 4–6 days after infection, and juveniles after 7 dpi can acquire the ability to survive for a long time in the basic medium. Previous studies [3 3,4 5] reported metabolic fluctuations in F. hepatica, including energy utilization capacity during growth. However, no reports have been found on 4–6 dpi juveniles. Therefore, further studies are needed to identify the unknown key factors for in vitro survival. In contrast, the primary advantage of NEJ is that it does not require laboratory animals. Therefore, NEJ was included in the subsequent in vitro culture experiments.

Supplementation of red blood cells in the in vitro culture of juveniles

The effects of RBC supplementation on juveniles were examined in vitro (Fig 4) to evaluate its potential as a nutrient source. NEJ, 7 dpi, and 11 dpi juveniles from mice were incubated in the basic medium supplemented with 0.5 µL of bovine RBC (number of RBC/µL was not counted) and compared with the controls.

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Fig 4. Supplementation of red blood cells (RBC) in the in vitro culture of juveniles.

Juvenile area, oral cone index, and survival rate of (A) newly excysted juveniles (NEJ), (B) 7 dpi juveniles, and (C) 11 dpi juveniles. Juvenile area and oral cone index are presented as median ± range. Number of juveniles at the beginning of the culture was four for all groups, and they were incubated at 37°C and 5% CO₂ in a total medium volume of 250 µL. The convexity of the survival curve indicates the artificial removal of juveniles from the culture. RBC: the basic medium (RPMI 1640 + 50% FBS) supplemented with 0.5 µL of bovine RBC (number of RBC was not counted). control: the basic medium. Statistical analyses were performed using the Mann–Whitney U test. *P < 0.05. No significant differences were observed in the survival rates (log-rank test). Representative images of (D) NEJ, (E) 7 dpi, and (F) 11 dpi juveniles after incubation. Number of days after incubation of each sample is labeled under the image. For live juveniles, images were taken on the day with the highest group median body area. The individual corresponding to the median was selected; if the image was unsuitable for presentation (e.g., because of suboptimal positioning), a comparable median-sized individual was used instead. Notably, RBC were ingested by the juveniles. Bars: 100 µm for NEJ and 500 µm for 7 and 11 dpi. pb: primary bifurcation of the intestinal tract; sb: secondary bifurcation of the intestinal tract.

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In NEJ culture (Fig 4A), survival time was not prolonged by RBC supplementation. The maximum survival time was 33 days in NEJ with RBC, which was shorter than the 35 days in the basic medium alone. The increase in area of the RBC group was not significant compared to that of the control group during incubation. The median body area was 0.02 mm² on the first day, and the maximum was 0.07 mm² after 13 days in the basic medium and 0.06 mm² after 15 days with RBC. Subsequently, the juveniles gradually weakened, shrank, and eventually died. Intestinal development was limited to primary bifurcation in both groups (Fig 4D). RBC were ingested by the juveniles and were found in the intestine (Fig 4D).

In the culture of 7 dpi juveniles (Fig 4B), one of the four juveniles (1/4) with RBC died at 65 days of incubation. Another 1/4 juvenile was fixed at 91 days because the body area of the RBC group began shrinking. The remaining juveniles survived for an additional week, and 1/4 juvenile died at 98 days. We then determined this day as the endpoint, and the last active 1/4 juvenile was fixed. In the basic medium, 1/4 juvenile was fixed at 91 days of incubation for comparison with RBC, and the remaining 3/4 juveniles survived until the endpoint and were fixed for staining. The median body area was 0.16 mm² on the first day and increased to 1.72 mm² after 95 days and 1.58 mm² after 70 days in the basic medium and with RBC, respectively. The areas of the two groups were not statistically different until 63 days of culture. Although statistical analysis could not be performed at 98 days owing to the death of the juvenile, the areas of the two groups were not significantly different during incubation. Juveniles with RBC became weaker and shrank toward the end of the culture period, whereas the body area of the juveniles in the basic medium was maintained until the endpoint. The body shape did not change during culture, which was supported by a stable oral cone index in both groups. The oral cone index did not differ between the groups during the statistically significant period, except on day 44. Secondary or greater bifurcations of the intestinal tract were observed in both groups (Fig 4E). In the stained specimens (Fig 4E), the reproductive organs of the juveniles in both groups appeared less developed and more atrophic than those at the beginning of the culture period (Fig 2D, 7 dpi). However, when enlarged under the optical microscope, primary testis branching was observed in both groups (S2 Fig), but the structure of the testicular duct was weak (S3 Fig) compared with that in the in vivo 14 dpi controls (S4 Fig). A detailed comparison between the two groups was not performed here because a comparison after a long incubation time seemed inappropriate, and the number of samples was insufficient (n = 4).

In the culture of 11 dpi juveniles (Fig 4C), 1/4 juvenile with RBC died after 44 days of incubation, 1/4 juvenile was fixed at 91 days, and the remaining 2/4 juveniles were active until the endpoint of 98 days (S1 Movie, RBC group). In the basic medium, 1/4 juvenile was fixed at 91 days of incubation for comparison with RBC, and the remaining 3/4 juveniles survived until the endpoint. The median body area was 0.62 mm² on the first day and increased to 1.54 mm² and 2.46 mm² after 94 days in the basic medium and with RBC, respectively. Although the areas of the two groups were not statistically different, a prominent increase in area was induced by RBC compared to that induced by the basic medium alone. In contrast, the oral cone index was stable, and the body shape did not change in either group. Statistically significant differences were observed only at 6 and 24 days after incubation, but no difference was observed at any other time point until 98 days. The development of the intestinal tract and reproductive organs after incubation was almost the same as that observed in the 7 dpi juveniles after incubation. The reproductive organs appeared less developed and atrophied (Fig 4F), but testis branching was observed in the enlarged images (S2 Fig). However, the structure of the testicular duct was weak (S3 Fig) compared with that in the in vivo 14 dpi controls (S4 Fig). A detailed comparison was not performed here because of the long incubation period and insufficient number of samples.

The RBC-induced increase in the area was observed only at 11 dpi. This indicates that juveniles at 7 dpi or earlier could not utilize RBC for growth, although they ingested RBC. A previous study by Cwiklinski et al. [36] reported that F. hepatica exhibits high levels of cathepsin L1 at 21 dpi compared to NEJ, which is associated with blood feeding. The observed differences in the effects of RBC supplementation on NEJ, 7 dpi, and 11 dpi in this study suggest that early-stage juveniles may have immature physiological functions until 7 dpi. Probably, the capacity to utilize RBC develops along with their age. In contrast, oral cone development was not observed in 11 dpi juveniles, even after the RBC-induced increase in the area; therefore, we speculated that factors other than RBC are required for changes in body shape; however, this could not be concluded here, because the amount of supplemented RBC was possibly insufficient and because of the low number of replicates in this assay. Subsequently, because the effect of RBC was clearer in 11 dpi juveniles than NEJ or 7 dpi, an examination of the higher concentration of RBC was performed in the 11dpi juveniles (S5 Fig). The trend of the RBC-induced increase in area was reproduced in 11 dpi juveniles; however, there was no effect on the oral cone development. The ratio of juveniles whose testes exhibited secondary branching or onwards seemed to be slightly increased in a concentration-dependent manner, that is, 2/6 in the control and 4/6 in 5.5 µL-RBC (S5 Fig). However, the development was incomplete, unlike in vivo 14 dpi juveniles (S4 Fig), particularly the structure of the testicular duct was weak (S3 Fig), as described in the previous assay (S2 Fig). Nevertheless, structural branching of the testicular duct was clearly observed in the cultured juveniles compared with that in freshly recovered 11 dpi juveniles (S3 and S4 Figs).

Supplementation of sex-inducing substances in the in vitro culture of juveniles

The effects of SIS supplementation on Fasciola juveniles were examined in vitro (Fig 5). Notably, the biologically active substances in the SIS fraction have not yet been identified, and the actual amount of the compounds used in this assay is unknown. NEJ, 7 dpi, and 11 dpi juveniles from mice were incubated in the basic medium supplemented with the SIS fraction (10 µL) (S1 Fig, batch #1) and compared with the controls, in which water was added instead of SIS.

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Fig 5. Supplementation of sex-inducing substances (SIS) in the in vitro culture of juveniles.

Juvenile area, oral cone index, and survival rate for (A) newly excysted juveniles (NEJ), (B) 7 dpi juveniles, and (C) 11 dpi juveniles. Juvenile area and oral cone index are presented as median ± range. The number of juveniles at the beginning of the culture was four for all groups, and incubated at 37°C, 5% CO₂ in a total medium volume of 250 µL. The convexity of the survival curve indicates an artificial removal of a juvenile from the culture. SIS: the basic medium (RPMI 1640 + 50% FBS) supplemented with the SIS fraction (10 µL). Water: the basic medium supplemented with water (10 µL). Statistical analyses were performed using the Mann–Whitney U test. *P < 0.05. A significant difference was observed only in the survival rates of NEJ (P < 0.05, log-rank test). Representative images of (D) NEJ, (E) 7 dpi, and (F) 11 dpi juveniles after incubation. The days after incubation of each sample are labeled under the image. For live juveniles, images were taken on the day with the highest group median body area. The individual corresponding to the median was selected; if the image was unsuitable for presentation (e.g., because of suboptimal positioning), a comparable median-sized individual was used instead. Bars: 100 µm for NEJ, 500 µm for 7 and 11 dpi. pb: primary bifurcation of the intestinal tract; sb: secondary bifurcation of the intestinal tract.

https://doi.org/10.1371/journal.pntd.0014113.g005

In the NEJ culture (Fig 5A), survival time was prolonged by supplementation with the SIS fraction; the maximum survival times were 31 and 41 days in the water and SIS groups, respectively. The increase in the area of the SIS group was not significant compared to that of the water group during incubation. The median body area was 0.02 mm² on the first day and increased to 0.05 mm² after 20 and 33 days in both groups. Subsequently, the juveniles gradually weakened, shrank, and died. Intestinal development was limited to the primary bifurcation (Fig 5D).

In the culture of 7 dpi juveniles (Fig 5B), 1/4 juvenile with SIS died at 81 days of incubation, another 1/4 juvenile died at 84 days, and 1/4 juvenile was fixed at 91 days for the comparative purpose, and the last 1/4 juvenile survived until the endpoint of 98 days. In the water group, 1/4 juvenile died at 91 days of incubation. Another 1/4 juvenile was fixed at 91 days, and the remaining 2/4 juveniles survived until the endpoint. The median body area was 0.14 mm² on the first day and increased to 1.54 mm² after 95 days and 2.06 mm² after 52 days in the water and SIS groups, respectively. Significant differences were observed intermittently between days 32 and 67. Subsequently, the body area decreased because of the existence of weakened (shrunken) juveniles, but after the juveniles died and were removed, the area appeared to increase again. The oral cone index was significantly greater in the SIS group on day 28 of culture, and statistical differences were observed intermittently until day 79. The oral cone was clearly visible in the SIS group, and the body shape changed (Fig 5E). Secondary bifurcation of the intestinal tract was observed in both groups. In the stained specimens (Fig 5E), the reproductive organs of the juveniles in both groups were not clearly recognized compared to the beginning of the culture (Fig 2D, 7 dpi). However, when enlarged under the optical microscope, testis branching was observed in both groups (S2 Fig), but the structure of the testicular duct was weak (S3 Fig) compared with that in the in vivo 14 dpi controls (S4 Fig). A detailed comparison between the groups was not performed here because the long incubation time seemed inappropriate for the comparison, and the number of samples was insufficient (n = 4).

In the culture of 11 dpi juveniles (Fig 5C), 1/4 juvenile was removed from both SIS and control groups and fixed at 91 days for comparative purposes. All remaining 3/4 juveniles survived and actively moved until the endpoint of 98 days (S1 Movie, SIS group). The median body area was 0.68 mm² on the first day and increased to 1.48 mm² after 56 days and 2.97 mm² after 94 days in the water and SIS groups, respectively. Significant differences were observed intermittently from 13 to 91 days in culture until one juvenile was artificially removed for staining. The degree of area increase decreased after 46 days of culture. The oral cone index increased over time in the SIS group, with significant differences observed intermittently from 9 to 91 days of culture. The body shape of the SIS group was markedly altered (Fig 5F). The intestinal tract developed well, but the reproductive organs of both groups were not clear after incubation (Fig 5F), but testis branching was observed in the enlarged images (S2 Fig). However, the structure of the testicular duct was weak (S3 Fig) compared with that in the in vivo controls (S4 Fig). A detailed comparison was not performed here because of the long incubation period and insufficient number of samples (n = 4).

Notably, two out of three sexualization processes of the liver-stage juveniles were successfully achieved in this assay, and the maximum values of body area and oral cone index were comparable to those of the 21 dpi juveniles in vivo (Fig 2). Although the development of reproductive organs appeared to be only structural in this assay, it should be emphasized that this is the first report to show that the body shape of juveniles was altered in vitro by supplementation with the SIS fraction, which indicates that SIS stimulates sexualization in a parasitic flatworm, similar to a free-living flatworm, planarians (D. ryukyuensis). The ability to accept SIS differed depending on the age of the juveniles, as the effect on 11 dpi juveniles was evident earlier than that on 7 dpi juveniles, and NEJ did not show this ability. Similarly, a greater change in the oral cone index was observed in 11 dpi juveniles than in 7 dpi juveniles, with significant differences between the values observed from 2 to 77 days of incubation.

Evaluation of higher concentrations of sex-inducing substances

Eleven dpi juveniles showed a clearer effect of SIS supplementation compared with NEJ or 7 dpi juveniles. Therefore, to save SIS and reduce the number of animals used, the higher concentration of SIS supplementation was examined in vitro at only 11 dpi (Fig 6). The endpoint was shortened to 30 days to evaluate the effects on the reproductive organs. A different batch of the SIS fraction was used for this assay (S1 Fig, batch #2). The SIS fraction (1 × SIS) and triple-concentrated fraction (3 × SIS) were used for comparisons. The 1 × SIS group exhibited a slightly slower growth than the SIS group in the previous assay (Fig 5C and S4 Table), possibly due to batch switching of the SIS fraction (S1 Fig). Nevertheless, significant increases in body area and oral cone index were consistently observed in the SIS groups compared to the negative control groups (Figs 5C and 6A and S5 Table). These findings support the robust SIS effects on the sexualization of juveniles. However, 3 × SIS did not show any enhancement compared to 1 × SIS. Both 1 × SIS and 3 × SIS induced testis development in all juveniles (Fig 6), but the effects were incomplete, as described in the previous assay (S3 Fig).

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Fig 6. Evaluation of higher concentrations of sex-inducing substances.

(A) Juvenile area, oral cone index, and survival rate at 11 dpi of juveniles supplemented with 1 × SIS and 3 × SIS fractions. Juvenile area and oral cone index are presented as median ± range. The number of juveniles at the beginning of the culture was eight for all groups and incubated at 37°C, 5% CO₂ in a total medium volume of 250 µL. Water: the basic medium (RPMI 1640 + 50% FBS) with water (10 µL). 1 × SIS: the basic medium with 1 × SIS fraction (10 µL). 3 × SIS: the basic medium with 3 × SIS fraction (10 µL). Statistical analysis was performed using the Kruskal–Wallis test with Dunn’s multiple comparisons test. *P < 0.05, between 3 × SIS and water; †P < 0.05, between 1 × SIS and water. The convexity on the survival curve before the endpoint indicates an accidental death of a juvenile by escape from a well to the outside. (B) Representative images of live and stained juveniles at the endpoint with the same scale. For live juveniles, the individual corresponding to the median body area was selected; if the image was unsuitable for presentation (e.g., because of suboptimal positioning), a comparable median-sized individual was used instead. Arrowhead: ovary or testicular duct. Bars: 500 µm for whole images, 100 µm for magnified images. Number of juveniles with primary testis branching/the total number of specimens is shown in the images. sb: secondary bifurcation of intestinal tract; cs: cirrus sac; o: ovary; t: testis.

https://doi.org/10.1371/journal.pntd.0014113.g006

The median body area was 0.68 mm² on the first day and increased to 0.93 mm², 1.72 mm², and 1.76 mm² in the water, 1 × SIS, and 3 × SIS groups, respectively, after 30 days in culture (Fig 6A). Significant differences between the water groups were observed intermittently from 14 days in culture for 1 × SIS and from 14 to 30 days (endpoint) for 3 × SIS. The oral cone index increased according to the time course in the 1 × SIS and 3 × SIS groups and was statistically significant intermittently from 9 days and 7 days to the endpoint of the culture, respectively, compared to the water group (Fig 6A). The body shapes of both the 1 × SIS and 3 × SIS groups clearly changed, and the oral cone was clearly visible. However, the concentration dependency of SIS was unclear, as no statistical difference was observed between 1 × SIS and 3 × SIS for either the body area or oral cone index.

No juveniles died in the culture medium over time (Fig 6A). However, two juveniles in 1 × SIS accidentally escaped from the well and moved into the space between the wells after 9 days in culture (Fig 6A), indicating the active movement of juveniles during incubation.

The intestinal tract developed well in all groups (Fig 6B). In the stained specimens (Fig 6B), the cirrus sac and ovary were atrophied in all juveniles at the endpoint compared to the normal growth controls (S4 Fig, 14 dpi). Although primary testis branching was observed in six of the eight (6/8) juveniles from the water group (control), the structure of the testicular duct was weak (S3 Fig), and testis development was absent in the remaining two juveniles. In contrast, all eight juveniles in both the 1 × SIS and 3 × SIS groups showed robust development of testis branching (Fig 6B), including secondary branching; however, the testicular ducts were less stained (S3 Fig) and different from the in vivo controls (S4 Fig, 14 dpi), which had well-stained primary testis branching in 39/46 juveniles. It was difficult to find a morphological difference in testis development between the SIS (Fig 6B) and RBC (S5 Fig) supplemented groups (S3 Fig). However, SIS induced robust branching of the testes in all juveniles, whereas the effects of RBC were limited, indicating that SIS is superior to RBC in inducing testis development. Here, we need to emphasize that the staining of the testicular duct was consistently weak across all in vitro samples in this study (S3 Fig), suggesting incomplete functional maturation rather than absence of structural development. This was not attributable to any staining error, as other internal structures within the same specimens (e.g., the intestine) were clearly visible. Notably, the testicular duct was morphologically identifiable and exhibited branching structures (S3 Fig) that were not observed in freshly recovered 11 dpi juveniles (S4 Fig). The reduced staining intensity may reflect the absence of sperm cells within the duct lumen. Further analysis using histological sections will be necessary to elucidate the detailed status of the testicular duct contents.

In conclusion, SIS enhanced an increase in area, change in body shape, and reproductive organ development, although the last one was incomplete. In this assay, no concentration-dependency of SIS was observed. Consequently, it was speculated that SIS does not serve as a nutrient source but rather functions at low concentrations, inducing unknown biological changes that lead to the development of oral cones and testes. A limitation of this assay is that the active compounds in the SIS fraction remain unidentified, making it difficult to investigate the effects of SIS at higher concentrations. The identification of active compounds will facilitate the discovery of important molecules, such as SIS receptors, in liver flukes. However, the identification of the receptor or related molecules in free-living planarians (D. ryukyuensis) is currently challenging, and research is ongoing. Thus, the analysis of liver flukes is much more challenging. Therefore, additional experiments were not performed to elucidate the underlying molecular mechanisms. Nevertheless, the molecular mechanisms underlying SIS-induced sexualization are a promising research topic, as they are potential drug targets for preventing the sexualization of liver flukes, which may contribute to the development of a transmission blocker.

Evaluation of the combined effect of sex-inducing substances and red blood cells

Compared with NEJ and 7 dpi, the effects of single supplementation of SIS and RBC were obvious at 11 dpi. Therefore, the combined effect of SIS and RBC on 11 dpi juveniles was investigated in vitro for 30 days (Fig 7) in anticipation of further development by supplementation with a nutrient source. Statistical analysis was performed by pooling the set of two plates, as the results were almost identical (Mann–Whitney U test). The batches of RBC and SIS fraction (S1 Fig, batch #2) were the same as those used in the higher concentration assays, but the number of RBC was reduced to 4.09 × 10⁶/µL during storage due to hemolysis. Therefore, the number of RBC supplemented in this assay was 4.4 µL, which was approximately the same as 3.5 µL-RBC in the previous assay (S5 Fig), which tended to increase body area for 11 dpi juveniles. Single supplementation with 4.4 µL-RBC, 1 × SIS, and 3 × SIS almost reproduced the previous results of area increase, body shape alteration, and survival rates (Fig 7A-7C).

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Fig 7. Evaluation of the combined effect of sex-inducing substances and red blood cells.

The number of juveniles at the beginning of the culture was 16 for all groups, and they were incubated at 37°C and 5% CO₂ in a total medium volume of 250 µL. (A) Check for the reproducibility of single RBC supplementation using 11 dpi juveniles (median ± range). Water: the basic medium (RPMI 1640 + 50% FBS) with water (10 µL), RBC: the basic medium with water (10 µL) + 4.4 µL-RBC (4.09 × 10⁶/µL). Statistical analysis was performed using the Mann–Whitney U test. §P < 0.05. (B) Combined effects of SIS and RBC (median ± range). Water: the basic medium with water (10 µL), 1 × SIS: the basic medium with 1 × SIS fraction (10 µL). 3 × SIS: the basic medium with 3 × SIS fraction (10 µL). 1 × SIS + RBC: the basic medium with 1 × SIS fraction (10 µL) and 4.4 µL-RBC. 3 × SIS + RBC: the basic medium with 3 × SIS fraction (10 µL) and 4.4 µL-RBC. Statistical analysis was performed using the Kruskal–Wallis test with Dunn’s multiple comparisons test. *P < 0.05, between 1 × SIS or 3 × SIS and water; †P < 0.05, between 1 × SIS + RBC or 3 × SIS + RBC and water; ‡P < 0.05, between 1 × SIS + RBC or 3 × SIS + RBC and the respective single SIS fractions. (C) Survival rates of all groups. The convexity of the survival curve before the endpoint indicates accidental death of a juvenile by escaping from the well to the outside. A significant difference was observed using the log-rank test (P < 0.05). (D) Representative images of live and stained juveniles at the endpoint, with the same scale. For live juveniles, the individual corresponding to the median body area was selected; if the image was unsuitable for presentation (e.g., because of suboptimal positioning), a comparable median-sized individual was used instead. Arrowhead: ovary or testicular duct. Bars: 500 µm for whole images, 100 µm for magnified images. The number of juveniles with primary testis branching/the total number of specimens is shown in the images. sb: secondary bifurcation of intestinal tract; cs: cirrus sac; o: ovary; t: testis.

https://doi.org/10.1371/journal.pntd.0014113.g007

In the control group, 2/16 juveniles escaped from the well at 7 days of incubation, 2/16 juveniles escaped at 11 and 16 days, respectively, and the remaining 12/16 juveniles survived until the endpoint. In the 1 × SIS group, 2/16 juveniles escaped at 9 days, 1/16 juvenile escaped at 14 days, and the remaining 13/16 juveniles survived until the endpoint. In the 3 × SIS group, 2/16 juveniles escaped at 7 and 9 days, respectively, and the remaining 14/16 juveniles survived until the endpoint. In summary, no juvenile died in either SIS fraction, which was consistent to the previous assays (Figs 5 and 6).

The proportion of juveniles with testis branching was lower than in the previous assay. In the 1 × SIS and 3 × SIS groups, 1/13 and 3/14 juveniles, respectively, which were smaller than the other juveniles, failed to develop testicular branching (Fig 7D). The other juveniles, 12/13 and 11/14 juveniles in the 1 × SIS and 3 × SIS groups, respectively, had robust testis branching, similar to the results of the previous assay (S3 Fig), and the induction rate was superior to that of RBC (9/13). Notably, the combined effect of SIS and RBC was not observed in this assay.

In the combined incubation, the median body area increased to 1.17 mm² and 1.68 mm² in the 1 × SIS + RBC and 3 × SIS + RBC groups, respectively, which did not exceed the results of single supplementation of the SIS fractions (Fig 7B). The body area of 1 × SIS + RBC was smaller than that of 1 × SIS alone, with significant differences between 9 and 21 days of culture (Fig 7B), whereas no significant difference was observed between 3 × SIS and 3 × SIS + RBC (Fig 7B). The oral cone indices of 1 × SIS + RBC and 3 × SIS + RBC were smaller than those of the respective SIS fractions alone (Fig 7B).

In the RBC group, 1/16 juvenile died at 9 days of incubation, 1/16 juvenile died at 11 days, and 1/16 juvenile escaped at 16 days, and the remaining 13/16 juveniles survived until the endpoint. In the 1 × SIS + RBC group, 1/16 juvenile died at 4 days of incubation, and the remaining 15/16 juveniles survived until the endpoint. In the 3 × SIS + RBC group, 2/16 juveniles died at 4 days of incubation, 1/16 juvenile died at 7 days, 1/16 juvenile escaped at 7 days, 1/16 juvenile died at 11 days, 1/16 juvenile escaped at 14 days, and the remaining 10/16 juveniles survived until the endpoint. In summary, two, one, and four juveniles died during the culture period in the RBC, 1 × SIS + RBC, and 3 × SIS + RBC treatments, respectively, whereas no juveniles died in either SIS fraction (Fig 7C). Accidental escape from the well was not considered death during culture. The intestinal tract was well developed in all groups (Fig 7D). Regarding the development of reproductive organs (Fig 7D), the ovary and cirrus sacs were atrophic in all the groups. The number of juveniles with robust testis branching was reduced to 4/15 and 4/10 juveniles in the combination group. These results indicate that combined supplementation is not beneficial.

In this assay, some 11 dpi juveniles died with RBC, regardless of the presence or absence of the SIS combination, whereas no juveniles died with SIS alone, suggesting the toxicity of RBC to the juveniles. This speculation is supported by the observation of dead juveniles in all RBC-related assays in this study. Although the results of this study alone do not permit definitive conclusions, the toxicity of RBC may explain the elimination of this combined effect. One of the expected toxicities of RBC is oxidative stress caused by free iron derived from heme [37]. We speculated that the effects of SIS might be inhibited by oxidative stress; however, the detailed mechanism of this inhibition could not be investigated in this study. Anaerobic cultures without oxygen or antioxidant supplementation should be tested in the future to determine whether they can rescue juveniles from RBC-induced oxidative stress. For example, because ascorbic acid is supplemented in the in vitro culture of schistosomes [3 3,8 9], the medium composition can be improved by including antioxidants to prevent oxidative stress. Nevertheless, the results of this study showed that SIS supplementation alone is currently the best method for inducing the sexualization of liver flukes in vitro.