Fig 1.
ABC169 supports the maintenance of schistosome vitellaria.
(A) Morphological changes of eggs laid by worm pairs maintained in BM169. After D6, parasites began laying abnormally formed eggs (arrow). These eggs were small, usually did not contain a lateral spine, and did not possess a smooth surface. (B) Fast Blue BB staining (brownish-red labeling) showing loss of mature vitellocytes during culture in BM169. Representative images from 3 experiments with n > 10 parasites. (C) Confocal slice showing Fast Blue BB and BODIPY labeling of vitelline and lipid droplets in the vitellaria, respectively. Shown are freshly recovered parasites (“Fresh”) and schistosomes at D20 of cultivation in BM169 supplemented with RBCs, LDL supplement (Cholesterol), or ascorbic acid. ABC169 represents the combination of RBCs, LDL supplement, and ascorbic acid. Representative images from 3 experiments with n > 10 parasites. (D) Whole-mount in situ hybridization showing expression of vitellaria-enriched genes in freshly perfused female worms and parasites cultured in ABC169 or BM169 for 20 days. Representative images from 3 experiments with n ≥ 9 parasites. (E) Heatmap showing relative expression of vitellaria-enriched transcripts in freshly perfused female worms (“Fresh”) and parasites cultured in ABC169 or BM169 for 20 days. Each column represents an independent biological replicate; samples are normalized to the expression of an arbitrarily chosen biological replicate from the BM169 group. Changes in expression between each of the 11 genes in BM169 and ABC169 were statistically significant (p < 0.05, t test). Underlying primary data can be found in S1 Data. (F) Morphology of eggs laid by paired adult females in ABC169 supplemented with L-ascorbic acid or D-isoascorbic acid on D20. Representative of 3 experiments. Scale bars: A, B, D, F, 100 μm; C, 25 μm. ABC169, Ascorbic Acid, Blood Cells, Cholesterol, and BM169; BM169, Basch’s medium 169; BODIPY, boron dipyrromethene; D, day; LDL, low-density lipoprotein; myb/sant like, Myb/SANT-like DNA-binding domain-containing protein; RBC, red blood cell; sod, extracellular superoxide dismutase [Cu-Zn]; tes, trematode eggshell synthesis protein; y+lat2, Y+L amino acid transporter 2.
Fig 2.
Eggs laid in ABC169 develop and hatch.
(A) Rate of egg production per worm pair in BM169 or medium ABC169 during in vitro culture. Dotted line indicates the point at which parasites begin laying morphologically abnormal eggs. n = 24 worm pairs in BM169 and n = 19 in ABC169, examined in 4 experiments. Error bars represent 95% confidence intervals. (B) Morphology of eggs (left, bright field; right, DIC) laid by paired adult females in BM169 or ABC169 on D20. Red arrows show early embryos in eggs laid in ABC169. (C–E) Quantification of the (C) size, (D) number of DAPI-labeled nuclei, and (E) autofluorescence intensity from eggs laid by freshly perfused female worms (Fresh, n = 41) and parasites cultured in BM169 (n = 61) or ABC169 (n = 59) on D20. In each group, eggs were harvested from 9 worm pairs across 3 separate experiments. ****p < 0.0001, t test. Error bars represent 95% confidence intervals. (F) EdU-labeled embryonic cells of an egg laid by a paired adult female in ABC169 on D20. (G) Percentage of eggs with clusters of cycling EdU+ embryonic cells from eggs laid between D16 and D22 by paired adult females in BM169 or ABC169. >2,000 eggs from 6 experiments. Error bars represent 95% confidence interval. (H) Miracidia from eggs laid in ABC169. Left, miracidium (pseudocolored red) inside an egg laid on D20 of culture in ABC169. Right, hatched miracidium from D20 egg labeled with DAPI and phalloidin. These miracidia appear grossly normal in morphology possessing 2 pairs of flame cells (arrows). Underlying primary data for panels A, C–E, and G can be found in S1 Data. Scale bars: B, F, H, 20 μm; C–E, 50 μm. ABC169, Ascorbic Acid, Blood Cells, Cholesterol, and BM169; BM169, Basch’s medium 169; D, day; DIC, differential interference contrast; EdU, 5-ethynyl-2′-deoxyuridine.
Fig 3.
ABC169 supports the maturation of immature female schistosomes following pairing with a male worm.
(A) Vitellaria development visualized by Fast Blue BB labeling in immature female schistosomes in the presence or absence of male worms in BM169 or ABC169 at D20 of culture. Black pigment in ABC169 parasites represents digested RBCs. (B) Eggs laid by immature female in BM169 or ABC169 on day 20 after pairing with a male. (C) Proportion of females laying eggs (left axis, magenta) and number of eggs laid per worm pair (right axis, green) from D1 to D20 following pairing of immature females with male worms in ABC169. n = 31 worm pairs examined in 4 separate experiments. Error bars represent 95% confidence intervals. Underlying primary data can be found in S1 Data. (D) Eggs laid by previously immature females commence embryonic development. Left, representative developing EdU-labeled embryo from egg laid in ABC169 on D20. Scale bars: A, B, 100 μm; D, 20 μm. AB169, Ascorbic Acid, Blood Cells, Cholesterol, and BM169; BM169, Basch’s medium 169; D, day; EdU, 5-ethynyl-2′-deoxyuridine; RBC, red blood cell.
Fig 4.
Using ABC169 to study schistosome male-induced female sexual maturation.
(A) Pairing of immature females with amputated male segments. Left, cartoon showing approximate positions of male amputations. Most often, we observed posterior segments pairing with posterior region of immature females, shown to right. (B) Fast Blue BB labeling showing female vitellaria development after pairing with posterior male segments. Left panels, female after separation from male segment showing vitellaria development in posterior region. Right panel, female before separation from male segment showing vitellaria development is restricted to paired region. Representative of >40 female parasites examined in 4 separate experiments. (C) Eggs laid by immature female worms paired with male segments are morphologically normal (left) but do not contain developing embryos, as measured by EdU labeling (right). n > 500 eggs examined in 4 separate experiments. (D) DAPI staining to examine ovary development in immature females paired with intact male worms or an amputated posterior male segment. Females paired with intact males possess DOs (n = 23), whereas females paired with male segments produce no DOs (n = 41). (E) Pairing of immature females with decapitated and castrated male segments. Left, cartoon showing approximate positions of amputation, removing both the head and testes. These decapitated and castrated segments paired with females along most of the female body, shown to right. (F) DAPI staining to examine ovary development in immature females paired with decapitated and castrated male segments. These ovaries contain DOs. Representative images from n = 22 parasites examined in 3 separate experiments. (G) Fast Blue BB showing vitellaria development in females after pairing with decapitated and castrated male segments. Representative images n = 22 parasites from 3 separate experiments. (H) EdU labeling showing embryonic development in eggs from females paired with decapitated and castrated male segments. 273/602 eggs laid between D16–D22 contained clusters of EdU+ cells. (I) DAPI labeling of metaphase spreads from eggs laid by (left) fresh ex vivo female parasites paired with intact males and (right) immature female parasites paired with decapitated and castrated male segments. Embryonic cells from fresh ex vivo parasites were diploid (2n = 16), whereas those from unfertilized females are haploid (n = 8). Scale bars: A, B, 500 μm; C, D, F, H, 50 μm; G, 100 μm; I, 10 μm. ABC169, Ascorbic Acid, Blood Cells, Cholesterol, and BM169; BM169, Basch’s medium 169; D, day; DO, differentiated oocyte; EdU, 5-ethynyl-2′-deoxyuridine.
Fig 5.
Smp_248100 is expressed in the primordial vitellaria.
(A) A schematic diagram of protein product encoded by Smp_248100. This protein contains a ZnF-C4 domain similar to other NR proteins. (B) Whole-mount in situ hybridization showing expression of Smp_248100 in a mature female (left), immature female (middle), or male worm (right). Smp_248100 highly expressed in female worms exclusively in the region of vitellaria. Representative images from 3 experiments with n > 35 parasites. Scale bar, 100 μm. (C–D) Two-color FISH for H2B (green) and Smp_248100 (magenta) mRNAs on (C) immature females or (D) sexually mature females. In immature virgin females, Smp_248100 was only expressed in H2B+ cells. In adult females, Smp_248100 was expressed in H2B+ cells as well as some H2B– cells. We did not detect Smp_248100 expression in mature vitellocytes in the vitelline duct (bounded by dashed yellow lines). Representative images from n > 30 parasites examined in 3 separate experiments. Scale bars: B, 100 μm; C, 50 μm. DIC, differential interference contrast; FISH, fluorescent in situ hybridization; H2B, Histone H2B; NR, nuclear receptor; ZnF-C4, Zinc finger, C4 type.
Fig 6.
Smp_248100 is specifically required for vitellaria maturation following pairing with a male worm.
(A) qPCR showing decreased Smp_248100 transcript levels in Smp_248100(RNAi) versus control(RNAi). *p < 0.05. Error bars indicate 95% confidence intervals calculated based on 4 separate experiments. (B) Loss of mature vitellaria in Smp_248100(RNAi) worms. Fast Blue BB labeling following control or Smp_248100 RNAi treatment (top). Whole-mount in situ hybridization showing expression of the sod gene (a marker for vitellocytes) in control RNAi or Smp_248100(RNAi) worms (bottom). Smp_248100(RNAi) parasites produce few vitellocytes. Representative images from 4 separate experiments. (C) Plot showing egg production per worm pair between D12–D14 of RNAi treatment. Representative images from n > 146 parasites for each treatment examined in 6 separate experiments. ***p < 0.001, t test. Error bars represent 95% confidence intervals. (D) EdU labeling of proliferative cells following control or Smp_248100 RNAi treatment. We observed no significant difference in the number of EdU-labeled nuclei between control(RNAi) and Smp_248100(RNAi) animals within the vitellaria. Representative images from n > 41 parasites for each treatment examined in 4 separate experiments. ns, p > 0.05, t test. Error bars represent 95% confidence intervals. (E) DAPI labeling showing ovaries from control and Smp_248100 RNAi-treated female parasites at D14 after pairing with a male worm. Anterior towards the top; arrow indicates differentiated oocytes. Representative images from 4 separate experiments. Underlying primary data for panels A and C–D can be found in S1 Data. Scale bars: B, 100 μm; D, E, 50 μm. D, day; EdU, 5-ethynyl-2′-deoxyuridine; ns, not significant; qPCR, quantitative PCR; RNAi, RNA interference; sod, extracellular superoxide dismutase [Cu-Zn].