In vitro sexual dimorphism establishment in schistosomes

Reviewer #1 (Public review):

Pichon, Rémi et al. describe an in vitro method for transforming Schistosoma cercariae into mature adult worms. The authors show that human serum (HS) supports parasite growth and differentiation more effectively than fetal bovine serum (FBS). They also observed differences in parasite growth and activity, with worms cultured in HS efficiently digesting human red blood cells (hRBC). Cultured worms were able to pair with ex vivo adult worms and produce eggs, indicating functional maturation suitable for downstream applications such as drug screening. While the experimental approach is comprehensive and supports the advantages of HS culture conditions, the pairing efficiency was low (≈7%) and required long culture periods (70-80 days), highlighting limitations that may affect reproducibility.

A major strength of the study, in particular, is that the authors clearly differentiate the effects of FBS versus HS on developmental progression. The conversion rate observed in HS cultures is significant and consistent with previously published data.

While the study has several strengths, some aspects of the work are not fully explored. In particular, the role of hRBC supplementation requires further clarification. Although HS-cultured worms were shown to digest hRBC more readily, the implications of this observation remain unclear. Specifically, it would be useful to understand whether hRBC supplementation influences (1) long-term culture stability, (2) molecular pathways associated with development and differentiation, or (3) the pairing capacity of the worms. While addressing these questions may not be the main objective of the study, further discussion of these points would strengthen the manuscript.

The manuscript is clearly written and represents a valuable contribution to the field. Overall, the experimental approach is sound, and the results support a useful methodological framework for the in vitro culture of Schistosoma worms and the attainment of sexual maturity, particularly for adult male worms.

Reviewer #2 (Public review):

Summary:

The authors perform confirmation studies of Paul Basch's seminal schistosome work from 1981, demonstrating the development of transformed schistosomules into sexually dimorphic adult parasites, albeit without successful egg production. In addition to the findings from Basch's earlier work, the authors add some new molecular data in the form of an analysis of proliferative cells in in-vitro-derived animals.

Strengths:

The authors successfully confirm experimental results from earlier schistosome researchers, providing a potential new tool for studying schistosome biology without the need for vertebrate hosts.

Weaknesses:

The display of data from the authors is sometimes difficult to follow/understand where it comes from. For example:

(1) Line 136: The authors claim that parasites in HS and FBS conditions have substantially different mortality rates (11.3 +/- 2.7 vs 5 +/- 2.3) but a quite high p-value (0.8). Analyzing the raw data myself, I obtained a mean of 8.2 +/- 1.7% vs 4.8% +/- 4.3% with a p-value of 0.15. Either the data are not clearly presented, and I did not follow them, or the data presented in the text do not match the raw data in the supplemental files.

(2) Line 187/Figure 4: Though it is not clearly stated, it appears that the authors treat their EdU counts as an ordinal data set of 61 steps (from 0 to >60) rather than a continuous measure of EdU+ cells per animal. In this author's opinion, the graph strongly suggests a continuous data set, and the fact that this reviewer had to dig through poorly-labeled raw data to discover the nature of the data is problematic. The authors should either switch to a continuous data set or make it explicit that the data shown are ordinal. If counting EdU+ cells is too arduous, the authors could consider comparing the amount of EdU+ area to the amount of DAPI+ area in maximum intensity projections of their confocal images, as this would roughly approximate the amount of proliferative cells in the animals.

There are some minor issues as well:

(1) Line 122: It is perhaps incorrect to refer to humans as "the" definitive host of schistosomes, as S. japonicum is primarily considered a zoonotic infection with water buffalo/cows being the primary definitive host.

(2) Line 185/298: The authors refer to EdU pulse-chase experiments, but the experiments described here are EdU pulse experiments.

Reviewer #3 (Public review):

Summary:

This study is significant as it established a protocol for the long-term culture of Schistosoma mansoni newly transformed cercariae, which developed in vitro into sexually dimorphic forms. The impact of two different sera, Fetal Bovine Serum (FBS) and Human Serum (HS), added to the culture medium supplemented with human red blood cells was evaluated. The authors demonstrated that HS-cultured parasites were able to digest red blood cells, a critical step for long-term parasite development. Furthermore, while most FBS-cultured parasites did not progress beyond an early liver stage, sexual dimorphism was clearly evident in the HS-cultured worms, albeit delayed compared to in vivo development.

Strengths:

This study could contribute to further in vitro studies for a better understanding of the unique sexual biology of Schistosoma mansoni and for screening novel schistosomicidal compounds. By increasing parasite development in in vitro studies, this protocol could have a positive impact on the principles of the 3Rs (Replacement, Reduction and Refinement) for animal research.

Weaknesses:

As the authors mentioned, "pairing between male and female parasites was rare. Pairing was observed in approximately ~7% of the experiments, usually after day ~ 80 in culture. Egg production was also not achieved with this protocol.

Author response:

eLife Assessment

This useful study presents an improved protocol for long-term in vitro culture of Schistosoma mansoni that enables progression toward sexually dimorphic stages, representing a meaningful advance for studying parasite development and reducing reliance on animal models. The findings show that host-specific culture conditions support essential developmental and metabolic functions required for parasite maturation, although development remains delayed compared to in vivo conditions. The evidence is solid overall, but limited pairing efficiency and the absence of egg production indicate that the system does not yet fully recapitulate complete reproductive development.

On behalf of the co-authors, we thank the three reviewers and the editors for their complimentary remarks as well as the major and minor comments/ concerns. Addressing these concerns have led to revisions that improved the manuscript. In particular, further analyses have generated an updated Figures 3 and 4, and Supplementary Tables S1, and S4-S6.

Public Reviews:

Reviewer #1 (Public review):

Pichon, Rémi et al. describe an in vitro method for transforming Schistosoma cercariae into mature adult worms. The authors show that human serum (HS) supports parasite growth and differentiation more effectively than fetal bovine serum (FBS). They also observed differences in parasite growth and activity, with worms cultured in HS efficiently digesting human red blood cells (hRBC). Cultured worms were able to pair with ex vivo adult worms and produce eggs, indicating functional maturation suitable for downstream applications such as drug screening. While the experimental approach is comprehensive and supports the advantage of HS culture conditions, the pairing efficiency was low (≈7%) and required long culture periods (70-80 days), highlighting limitations that may affect reproducibility.

We acknowledge the reviewer for the positive highlights. Regarding the low in vitro pairing efficiency, we have now edited the manuscript to clarify a misleading statement related to 7%. We decided to remove the value of 7% — which corresponds to the percentage of experiments in which couples were observed, as it does not accurately represent the actual number of observed worm pairs and it is probably misleading. We have updated the text as follows:

Results, lines 230 ff.:

“While the establishment of sexual dimorphism was robust and reproducible across more than 15 independent experiments, pairing between male and female parasites was rare. Pairing was observed only in experiments lasting more than 80 days in which we were only able to observe a few couples. In addition, these pairings were temporary (Figures 6A, B; Supplementary Video S4).”

We also agree with the reviewer that the extended culture periods required to obtain fully sexually dimorphic parasites remain a limitation. As elaborated in Discussion (see below), key factors, probably derived from the host, are missing in the in vitro system explaining both the slow in vitro development and low rate of spontaneous pairing between in vitro developed, sexually dimorphic male and female worms. This was discussed as follows (lines 340-343): “That said, while our system was highly efficient in producing sexually dimorphic worms, spontaneous pairing between male and female parasites was extremely rare, mainly in aged in vitro cultures (from 80 to 100 days in culture) indicating that other factors, e.g., cholesterol, may be missing[35].”

A major strength of the study, in particular, is that the authors clearly differentiate the effects of FBS versus HS on developmental progression. The conversion rate observed in HS cultures is significant and consistent with previously published data.

While the study has several strengths, some aspects of the work are not fully explored. In particular, the role of hRBC supplementation requires further clarification. Although HS-cultured worms were shown to digest hRBC more readily, the implications of this observation remain unclear. Specifically, it would be useful to understand whether hRBC supplementation influences (1) long-term culture stability, (2) molecular pathways associated with development and differentiation, or (3) the pairing capacity of the worms. While addressing these questions may not be the main objective of the study, further discussion of these points would strengthen the manuscript.

We agree that deciphering the role of the human Red Blood Cells (hRBCs) supplementation is critical. Regarding the influence of hRBCs on the long-term culture stability in parasite development it has been well established for more than four decades that schistosomes do need red blood cells to grow in culture [Basch, P. F. Cultivation of Schistosoma mansoni in vitro. II. production of infertile eggs by worm pairs cultured from cercariae. J Parasitol 67, 186-190 (1981); Basch, P. F. Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing. J. Parasitol. 67, 179-185 (1981)]. The molecular pathways underlying development, sexual differentiation and pairing and modulated by hRBCs in culture is currently being investigated by our team. We decided not to include these data and analyses in the current manuscript, as they fall outside its scope.

The manuscript is clearly written and represents a valuable contribution to the field. Overall, the experimental approach is sound, and the results support a useful methodological framework for the in vitro culture of Schistosoma worms and the attainment of sexual maturity, particularly for adult male worms.

We thank the reviewer for highlighting the manuscript’s strengths.

Reviewer #2 (Public review):

Summary:

The authors perform confirmation studies of Paul Basch's seminal schistosome work from 1981, demonstrating the development of transformed schistosomules into sexually dimorphic adult parasites, albeit without successful egg production. In addition to the findings from Basch's earlier work, the authors add some new molecular data in the form of an analysis of proliferative cells in in-vitro-derived animals.

Strengths:

The authors successfully confirm experimental results from earlier schistosome researchers, providing a potential new tool for studying schistosome biology without the need for vertebrate hosts.

We thank the reviewer for highlighting the manuscript’s strengths.

Weaknesses:

The display of data from the authors is sometimes difficult to follow/understand where it comes from. For example:

(1) Line 136: The authors claim that parasites in HS and FBS conditions have substantially different mortality rates (11.3 +/- 2.7 vs 5 +/- 2.3) but a quite high p-value (0.8). Analyzing the raw data myself, I obtained a mean of 8.2 +/- 1.7% vs 4.8% +/- 4.3% with a p-value of 0.15. Either the data are not clearly presented, and I did not follow them, or the data presented in the text do not match the raw data in the supplemental files.

We thank the reviewer for pointing this out; we have now edited Supplementary Tables S1 and S6 by turning them into a long format for the sake of clarity. Accordingly, Results, Methods sections, and indicated supplementary tables were edited as follows:

Results, lines 142 ff.:

“No morphological differences were observed between parasites cultured either in FBS or HS within the first week in culture; in both conditions most parasites were classified as early schistosomula [category 1: 76% ± 30 (average ± SD) in FBS and 73% ± 29 (average ± SD) in HS] with few lung (category 2) and early liver schistosomula (category 3) (Figure 1B, week 1; Supplementary Figure S1). The mean mortality (category 0) at week 1 was slightly higher, but not statistically significant (P= 0.42), in worms cultured in HS [9.75% ± 2.76 (average ± SD)] compared to the mortality registered in FBS-cultured parasites [5.52% ± 5.18 (average ± SD), Supplementary Table S6], consistent with previous findings[39].”

Methods, lines 463-465:

“To evaluate differences in mortality between HS- and FBS-cultured parasites, data from 5 experiments were combined and analysed using a Shapiro-Wilk normality test to test normality of the data and a non-parametric Wilcoxon rank sum exact test (Supplementary Tables S1 and S6).”

Supplementary Tables:

Supplementary Table S1. “Raw counts of parasites within each developmental stage category. Each row corresponds to a picture of parasites in culture medium containing FBS or HS. Each column corresponds to the raw parasite counts at indicated stage development (categories 0 to 5), time in culture (Time in days - D), and experimental condition.”

Supplementary Table S6. “Summary of all statistical tests employed in this study. 1. Statistical tests of parasite mortality and the raw data table used for this test. 2. Statistical tests for worm size comparisons (correspond to Figure 2). 3. Statistical tests for worm black gut comparisons (correspond to Figure 3). BG: Black gut. 4. Statistical tests for EdU positive cells comparisons (correspond to Figure 4). Replicate code: E, M and L correspond to day 2, 8 and 15 respectively; R and W correspond to the presence (R) or absence (W) of RBCs added 13 days after transformation.”

For clarity, in Author response image 1 we provide the R script used to perform the statistical tests on the data shown in Supplementary Table S6 (column Raw count of parasite developmental category per image and experiment)

Author response image 1.

(2) Line 187/Figure 4: Though it is not clearly stated, it appears that the authors treat their EdU counts as an ordinal data set of 61 steps (from 0 to >60) rather than a continuous measure of EdU+ cells per animal. In this author's opinion, the graph strongly suggests a continuous data set, and the fact that this reviewer had to dig through poorly-labeled raw data to discover the nature of the data is problematic. The authors should either switch to a continuous data set or make it explicit that the data shown are ordinal. If counting EdU+ cells is too arduous, the authors could consider comparing the amount of EdU+ area to the amount of DAPI+ area in maximum intensity projections of their confocal images, as this would roughly approximate the amount of proliferative cells in the animals.

As the reviewer correctly pointed out, the data were treated as ordinal because counting worms with more than 60 Edu+ cells became extremely difficult and highly inaccurate. Therefore, we decided to group in a single category, “60 EdU+ cells”, all worms showing more than 60 EdU+ cells. We have now updated Figure 4 where medians are shown instead of media values, Supplementary Table S5 to provide more comprehensive access to the raw counts, and Supplementary Table S6 to indicate the data for EdU+ cells per worm were considered ordinal. Accordingly, we have revised the corresponding sections as follows:

Results, lines 211 ff.:

“HS-cultured schistosomula showed higher numbers of proliferating stem cells, with a median of >48 and >60 EdU+ cells per worm at days 8 and 15, respectively (Figure 4). On the other hand, most FBS-cultured parasites displayed no more than an average of 20 EdU+ cells per worm (Figure 4).”

Methods, lines 520 ff.:

“EdU+ cells per parasite were counted for an average of 100 parasites across three independent experiments (Supplementary Table S5). Worms were grouped based on the number of cells per individual, but all those showing ⪰ 60 EdU+ cells were counted in the same group named ‘60 EdU+ cells'. Therefore, the data were considered ordinal data. Statistical analysis was performed by Kruskal-Wallis test with Dunn multiple comparison post-hoc test, with P≤0.05 considered significant (Supplementary Table S6).”

Figure 4 legend, lines 830 ff.:

“A. Violin plots showing the number of Edu+ cells per worm at indicated time points (2, 8, and 15 days post cercarial transformation) in parasites cultured either in Foetal Bovine Serum (FBS, blue) or Human Serum (HS, light brown). Human Red Blood Cells (hRBCs) were added in the culture at day 13 post cercarial transformation. The small black dots indicate individual worms, and the big black point indicates the median of EdU+ cells per worm. All worms showing ⪰ 60 EdU+ cells were counted and clustered together in the group named ‘60 EdU+ cells’. Hence, the data were treated as ordinal and statistical analysis performed by Kruskal-Wallis test with Dunn multiple comparison post-hoc test, with P≤0.05 (*) considered significant (Supplementary Tables S5 and S6).”

We thank the reviewer for the very interesting suggestion to quantify cell proliferation by calculating the ratio between EdU+ area to DAPI+ area in maximum intensity projections images. Measuring the fluorescence area for each worm in maximum projection is an excellent idea; however, due to the number of EdU+ cells present in some samples, we think this technique would not provide additional information or produce more detailed data compared with our analysis when the number of Edu+ cells exceeds 60 per worm. We will certainly consider this approximation for future studies.

There are some minor issues as well:

(1) Line 122: It is perhaps incorrect to refer to humans as "the" definitive host of schistosomes, as S. japonicum is primarily considered a zoonotic infection with water buffalo/cows being the primary definitive host.

We thank the reviewer for pointing this out; we have now replaced “schistosomes” with “Schistosoma mansoni” (current line 131)

(2) Line 185/298: The authors refer to EdU pulse-chase experiments, but the experiments described here are EdU pulse experiments.

This is a very good point, we thank the reviewer for bringing this up and have accordingly edited by replacing “EdU pulse-chase” with “EdU pulse” experiments in lines 37, 204, and 321.

Reviewer #3 (Public review):

Summary:

This study is significant as it established a protocol for the long-term culture of Schistosoma mansoni newly transformed cercariae, which developed in vitro into sexually dimorphic forms. The impact of two different sera, Fetal Bovine Serum (FBS) and Human Serum (HS), added to the culture medium supplemented with human red blood cells was evaluated. The authors demonstrated that HS-cultured parasites were able to digest red blood cells, a critical step for long-term parasite development. Furthermore, while most FBS-cultured parasites did not progress beyond an early liver stage, sexual dimorphism was clearly evident in the HS-cultured worms, albeit delayed compared to in vivo development.

Strengths:

This study could contribute to further in vitro studies for a better understanding of the unique sexual biology of Schistosoma mansoni and for screening novel schistosomicidal compounds. By increasing parasite development in in vitro studies, this protocol could have a positive impact on the principles of the 3Rs (Replacement, Reduction and Refinement) for animal research.

We thank the reviewer for highlighting the manuscript’s strengths.

Weaknesses:

As the authors mentioned, "pairing between male and female parasites was rare. Pairing was observed in approximately ~7% of the experiments, usually after day ~ 80 in culture. Egg production was also not achieved with this protocol.

Following the reviewer’s point and to clarify a misleading point, we have now decided to remove the value of 7% — which corresponds to the percentage of experiments in which couples were observed. However, this value does not accurately reflect the actual number of observed worm pairs, and it is probably misleading. We have updated the text as follows:

Results, lines 230 ff.:

“While the establishment of sexual dimorphism was robust and reproducible across more than 15 independent experiments, pairing between male and female parasites was rare. Pairing was observed only in experiments lasting more than 80 days in which we were only able to observe a few couples. In addition, these pairings were temporary (Figures 6A, B; Supplementary Video S4).”