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.
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).
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.
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.
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.
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.
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.