Fig 1.
Survival and growth of juvenile Fasciola hepatica in vitro over 29 weeks following excystment.
Juvenile fluke were maintained in: RPMI; 10% Foetal Bovine Serum (FBS) in RPMI; 5%, 10%, 20% or 50% Chicken Serum (CS) in RPMI; and, 100% CS. A—Percentage survival of juvenile F. hepatica over 29 weeks (mean±SEM). Statistical analyses were performed using One Way ANOVA with Dunnett’s post hoc test. ****, P<0.0001. B—Surface area of juvenile F. hepatica in mm2 (mean±SEM). Statistical analyses were performed using Kruskal-Wallis with Dunn’s post hoc test and One Way ANOVA with Dunnett’s post hoc test. **, P<0.01.
Fig 2.
Development of digestive and reproductive tissues in Fasciola hepatica juveniles grown in vitro.
Fluke were maintained in 50% Chicken Serum in RPMI. A—Percentage of juveniles exhibiting different degrees of gut branching at various times post-excystment; B—Percentage of juveniles with evidence of uterine tubing at various times post-excystment; C—Confocal microscope image of newly excysted juvenile exhibiting suckers (oral (OS) and ventral (VS)) and early digestive caeca (DC) where red is indicative of muscle actin staining) (scale bar 50 μm); D—Confocal microscope image of juvenile 3 weeks post-excystment exhibiting primary branching of the digestive caeca (PB) and early uterine tubing (*) (scale bar 50 μm); E—Confocal microscope image of uterine tubing (*) seen in a juvenile 3 weeks post-excystment (scale bar 25 μm); F—Confocal microscope image of juvenile 4 weeks post-excystment showing more pronounced uterine tubing (*) and secondary branching of the digestive caeca (SB) (scale bar 50 μm); G—Confocal microscope image of juvenile 29 weeks post-excystment revealing considerable growth of the juvenile and much extended uterine tubing (*) that proceeds underneath the ventral sucker (VS) (scale bar 50 μm).
Fig 3.
Growth and development of a rapidly growing juvenile Fasciola hepatica maintained in vitro (20% Chicken Serum [CS] in RPMI over 29 weeks).
A—Growth of juvenile liver fluke maintained in 20% CS in RPMI or in 50% CS in RPMI; B—Confocal microscope images of the rapidly growing juvenile exhibiting oral sucker (OS), pharynx (P), gonopore tubing (G), ventral sucker (VS), uterine tubing (U), ootype (O), testes tubing (T) and tertiary branching of digestive caeca (TB) (scale bars 500 μm on main image, 50 μm on smaller, higher magnification images).
Fig 4.
Development of tegument surface in Fasciola hepatica juveniles grown in vitro (in 50% Chicken Serum in RPMI).
A—Change over time post-excystment in mean length of spines surrounding oral sucker of juveniles. Statistical analyses were performed using Kruskal-Wallis with Dunn’s post-hoc test, ** P<0.01; B—SEM image showing underdeveloped spines located between oral and ventral sucker in NEJ (scale bar 5 μm); C—Scanning Electron Microscope (SEM) image showing spines developing between oral and ventral sucker in juvenile 2 weeks post-excystment (scale bar 5 μm); D—SEM image showing spines developing two-tipped points between oral and ventral sucker in juvenile 4 weeks post-excystment (scale bar 5 μm); E—SEM image showing spines exhibiting multi-tipped points on anterior dorsal surface of juvenile 29 weeks post-excystment (scale bar 5 μm); F—SEM image showing absence of spines at posterior ventral surface of NEJ (scale bar 10 μM); G—SEM image showing appearance of developed spines at posterior ventral surface in juvenile 2 weeks post-excystment (scale bar 10 μm).
Fig 5.
Ultrastructure of the developing tegument in juvenile Fasciola hepatica maintained in vitro (in 50% Chicken Serum in RPMI).
A—Change over time post-excystment in mean tegument syncytium thickness of juveniles. Statistical analyses were performed using Kruskal-Wallis with Dunn’s post-hoc test, **** P<0.0001; B—Change over time post-excystment in mean tegument invagination length in juveniles. Statistical analyses were performed using Kruskal-Wallis with Dunn’s post-hoc test, * P<0.05; C—TEM image showing tegument of NEJ with few discernible features and no obvious syncytium; D—TEM image showing the tegument of a juvenile 1 week post-excystment with some T0 bodies and T2 bodies; E—TEM image showing the tegument of a juvenile 2 weeks post-excystment with the presence of both T0 bodies, early T1 bodies and T2 bodies; F—TEM image showing the tegument of a juvenile 3 weeks post-excystment with T0, T1 and T2 bodies present; G—TEM image showing the tegument of a juvenile 4 weeks post-excystment with mature T1 bodies visible and T2 bodies; H—TEM image showing tegument of juvenile 29 weeks post-excystment packed with mature T1 bodies and T2 bodies. All scale bars, 500 nm.
Fig 6.
Proliferating cells in growing juvenile Fasciola hepatica.
Incorporation of 5-ethynyl-2-deoxyuridine (EdU) identifies DNA synthesis occurring during proliferation of cells with neoblast-like morphology. Green fluorescence denotes EdU and blue fluorescence denotes Hoechst 3342 labelling of nuclear DNA. A, B—Distribution of EdU labelled nuclei (EdU+) in fluke grown for 7 days in RPMI+50% chicken serum (A), or unsupplemented RPMI (B); C—Quantification of EdU+ nuclei in non-growing (-CS) vs growing (+CS) specimens; D, E—Morphology of dispersed EdU+ cells; D shows two example cells, E shows single cell and individual fluorescence signals (Hoechst 3342, EdU, brightfield, overlaid); F, G, H—Examples of non-proliferating (EdU-) cells showing distinct morphologies associated with differentiated cells; I, Pulse chase protocols; J, Heatmaps illustrating the change in EdU+ localisation associated with pulse-chase exposure, suggesting that EdU+ nuclei migrate towards differentiated tissue; K-O, example staining patterns of juvenile F. hepatica from each pulse-chase exposure protocol.
Fig 7.
Inhibition of growth with hydroxyurea indicates a role for neoblast-like cells in growth and development.
A—Hydroxyurea (HU) produces a concentration-dependent inhibition of worm growth over a 7 day period in RPMI+50% CS; B—7 days HU (25 mM) exposure slows the rate of growth in juveniles maintained in RPMI+50% CS, following HU removal growth rate increases during a subsequent 3 day recovery period. Numbers beside points refer to figures below; C—Increase in worm size following removal of HU (1 vs 3) suggests recovery of growth; D—EdU accumulation does not recover significantly following HU removal. E—EdU labelled nuclei in juveniles maintained in RPMI+50% CS and 25 mM HU over 7 days as seen in ‘Treatment 1’ in B; F—EdU labelled nuclei in juveniles maintained in RPMI+50% CS over 7 days as seen in ‘Treatment 2’ in B; G—EdU labelled nuclei in juveniles maintained in RPMI+50% CS and 25 mM HU for 7 days before HU is removed as seen in ‘Treatment 3’ in B; H—EdU labelled nuclei in juveniles maintained in RPMI+50% CS over 10 days as seen in ‘Treatment 4’ in B; in all graphs mean +/-SEM is presented (shown in red on scatter graphs); in scatter graphs each data-point represents a measurement from an individual worm. Statistical analyses were performed using One Way ANOVA with Dunnett’s post hoc test against untreated “0” sample (A), t-test on days 3, 7 and 10 (B) or Kruskal-Wallis test with Dunn’s post hoc test to compare medians with all other medians (C, D). **, p<0.01; ***, p<0.001; ****, p<0.0001.
Table 1.
Excretory/secretory proteins of F. hepatica from fluke grown in 20% or 50% CS in RPMI or 100% CS for 29 weeks in vitro.