Peptidomic Identification of Behaviour-Modulating Putative Neuropeptides in Schistosoma mansoni Miracidia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Fogarty et al. present the identification and partial functional characterization of putative neuropeptides in Schistosoma mansoni miracidia. The work integrates in silico analyses, peptidomics, and behavioral assays. The study is timely and effectively revitalizes the field of schistosome neuropeptides, which has perhaps received limited attention in recent years. This is a well-designed and conceptually interesting study. Addressing the points below would strengthen the manuscript and broaden its impact.

 

MAJOR ISSUES

1. The authors suggest that chemical interventions interfering with miracidial infection processes (L51–52, L318–319) could represent potential control strategies. While I fully agree that understanding neuropeptide signaling is biologically important, it would be helpful if the authors could realistically discuss the feasibility of such approaches under field conditions.

How could these biocontrol targets be translated into practical applications in endemic regions? What delivery mechanisms could be envisioned? What ecological or economic constraints would apply? A brief yet realistic discussion of implementation challenges would significantly strengthen the translational perspective of the manuscript.

2. In Figure 2B, the authors should consider including expression data from eggs. Neuropeptides may already be expressed in miracidia within immature or mature eggs, potentially contributing to miracidial development before hatching. If no expression is detected in eggs, this would further support the interpretation that these neuropeptides are primarily involved in host-seeking behavior rather than early developmental processes.

Additionally, recent studies have shown differences in expression profiles and phenotypes between eggs/miracidia derived from intestinal versus hepatic origins (https://doi.org/10.1371/journal.ppat.1012268, https://doi.org/10.1101/2025.08.15.670345). These findings should be acknowledged and discussed in the context of the present expression analysis. If relevant datasets are available, incorporating this comparison would further strengthen the manuscript.

Furthermore, if the source transcriptomic data permit, it would be informative to distinguish expression between male and female worms rather than grouping them simply as “mature worms.”

3. The neuropeptide identity of the proposed molecules would be more strongly supported by evidence of spatial localization within miracidia, particularly in association with neuronal structures. Did the authors attempt localization analyses for the most promising candidates? Even preliminary localization data would substantially reinforce the functional interpretation.

 

MINOR ISSUES

L3: “Schistosoma” should be capitalized.

L45: The association with bladder cancer is primarily linked to Schistosoma haematobium, not S. mansoni.

L60–61: A brief explanation of what “F-type” and “Y-type” neuropeptides are would help readers outside the immediate neuropeptide field.

L74: The abbreviation “GPCRs” should be defined at first mention.

L205: NP14 is described as hydrophobic and therefore excluded from behavioral assays. Could the authors speculate on its potential biological role in miracidia? Does its hydrophobic nature limit its diffusion and signaling capacity, or might it suggest membrane-associated or paracrine activity?

L212–214: The sentence beginning “The absence of any variation…” is difficult to interpret and should be rephrased for clarity.

Figure 3: The color code should be explained directly in the figure and in the legend. The blue baseline (presumably the control) appears unequal across groups within the same experiment. Please clarify.

L243: Define the abbreviation “FOV.”

Figure 4: Include the color code directly in the figure for clarity.

L329–331: The authors should comment on why related neuropeptides were identified only in the neuropathogenic avian schistosome. Is this a biological signal or a sampling/database bias?

L418: Please specify the strain of S. mansoni used.

Author Response

MAJOR ISSUES

1. The authors suggest that chemical interventions interfering with miracidial infection processes (L51–52, L318–319) could represent potential control strategies. While I fully agree that understanding neuropeptide signaling is biologically important, it would be helpful if the authors could realistically discuss the feasibility of such approaches under field conditions.

How could these biocontrol targets be translated into practical applications in endemic regions? What delivery mechanisms could be envisioned? What ecological or economic constraints would apply? A brief yet realistic discussion of implementation challenges would significantly strengthen the translational perspective of the manuscript.

Response: Thank you for this important comment. We agree that translating neuropeptide-based interference strategies into field applications requires careful consideration of feasibility and ecological constraints. We have therefore expanded the Discussion to clarify how these targets could potentially be implemented for biocontrol (Lines 317–328).

 

2. In Figure 2B, the authors should consider including expression data from eggs. Neuropeptides may already be expressed in miracidia within immature or mature eggs, potentially contributing to miracidial development before hatching. If no expression is detected in eggs, this would further support the interpretation that these neuropeptides are primarily involved in host-seeking behavior rather than early developmental processes. Recent studies have shown differences in expression profiles and phenotypes between eggs/miracidia derived from intestinal versus hepatic origins (https://doi.org/10.1371/journal.ppat.1012268, https://doi.org/10.1101/2025.08.15.670345). These findings should be acknowledged and discussed in the context of the present expression analysis. If relevant datasets are available, incorporating this comparison would further strengthen the manuscript. Furthermore, if the source transcriptomic data permit, it would be informative to distinguish expression between male and female worms rather than grouping them simply as “mature worms.”

Response: Thank you for this constructive suggestion. We agree that including expression data from the egg stage could provide valuable insight into whether these neuropeptides are already expressed during miracidial development within immature or mature eggs, and whether their primary function relates to developmental processes prior to hatching or to host-seeking behaviour after emergence.

At present, the transcriptomic dataset used in this study does not include egg-stage samples. While recent studies suggested by the reviewer have reported differences in expression profiles and phenotypes between eggs and miracidia derived from intestinal versus hepatic origins, the raw transcriptomic datasets required for direct comparison were not accessible at the time of analysis. In addition, integrating RNA-seq datasets generated under different experimental conditions may introduce biases arising from differences in parasite collection methods, developmental stage definitions, RNA preparation, sequencing platforms, and data processing pipelines. For these reasons, we chose not to combine heterogeneous datasets in the present analysis. We have now cited and briefly discussed these studies in the revised Discussion to acknowledge the reported differences in transcriptional profiles and phenotypes between eggs and miracidia derived from intestinal versus hepatic origins, and to place our expression analysis in the context of these recent findings (Line 342-345).

Importantly, stage-resolved transcriptomic comparisons between eggs and miracidia are currently being undertaken in our group. These analyses aim to clarify the temporal expression patterns of miracidial neuropeptides and to better distinguish potential roles in early developmental processes versus post-hatching host-seeking behaviour. We have added a brief statement in the Discussion to highlight this as an important direction for future work (Lines 347–354).

Regarding the suggestion to distinguish expression between male and female worms, the dataset used in the present analysis reports transcript levels for the adult stage without sex-specific separation. Because the primary focus of this study is the functional impact of these neuropeptides on miracidia behaviour, adult sex-specific expression was not explored further here; however, we agree that sex-resolved transcriptomic analyses could provide additional insights into the broader regulation of these neuropeptide precursors.

 

3. The neuropeptide identity of the proposed molecules would be more strongly supported by evidence of spatial localization within miracidia, particularly in association with neuronal structures. Did the authors attempt localization analyses for the most promising candidates? Even preliminary localization data would substantially reinforce the functional interpretation.

Response: Thank you for this valuable suggestion. We agree that spatial localisation of these peptides within miracidia, particularly in association with neuronal structures, would provide supporting evidence for their classification as neuropeptides and would further strengthen the functional interpretation of their behavioural effects.

At present, we have not performed immunolocalisation or in situ hybridisation analyses for these candidates and therefore do not have preliminary localisation data to include in this study. The primary aim of the current work was to identify putative neuropeptides using peptidomic analysis and to evaluate their functional effects through behavioural bioassays.

We agree that spatial localisation will be an important next step to confirm the neuronal association of these peptides. Future studies will therefore aim to perform localisation analyses, such as antibody-based immunostaining or transcript localisation approaches, to determine their spatial distribution within miracidia and to further clarify their roles in the parasite nervous system. We have added a brief statement in the Discussion to acknowledge this as an important direction for future work (Line 452-457).

 

MINOR ISSUES

L3: “Schistosoma” should be capitalized.

Response: Corrected.

 

L45: The association with bladder cancer is primarily linked to Schistosoma haematobium, not S. mansoni.

Response: Thank you for picking this up. The statement has now been removed.

 

L60–61: A brief explanation of what “F-type” and “Y-type” neuropeptides are would help readers outside the immediate neuropeptide field.

Response: A brief mention that Y is primarily in vertebrates and F are in Platyhelminthes has been added (L 61)

 

L74: The abbreviation “GPCRs” should be defined at first mention.

Response: Now updates (L 74)

 

L205: NP14 is described as hydrophobic and therefore excluded from behavioral assays. Could the authors speculate on its potential biological role in miracidia? Does its hydrophobic nature limit its diffusion and signaling capacity, or might it suggest membrane-associated or paracrine activity?

Response: In our previous study, we observed that highly hydrophobic peptides cause the water droplet on the glass slide to separate from the peptide solution, preventing effective interaction between the peptide and the miracidia during behavioural assays.

 

L212–214: The sentence beginning “The absence of any variation…” is difficult to interpret and should be rephrased for clarity.

Response: We have now rephrased this sentence.

 

Figure 3: The color code should be explained directly in the figure and in the legend. The blue baseline (presumably the control) appears unequal across groups within the same experiment. Please clarify.

Response: The colour code has now been added to the figure.

 

L243: Define the abbreviation “FOV.”

Response – This has now been updated.

 

Figure 4: Include the color code directly in the figure for clarity.

Response: The colour code has now been added to the figure.

 

L329–331: The authors should comment on why related neuropeptides were identified only in the neuropathogenic avian schistosome. Is this a biological signal or a sampling/database bias?

RESPONSE: Thank you for this insightful comment. The observation that related neuropeptides were identified only in the neuropathogenic avian schistosome may reflect either biological divergence among trematodes or limitations in currently available sequence databases. One possibility is that these neuropeptides represent lineage-specific adaptations within certain schistosome clades, potentially associated with differences in host range, life cycle strategies, or parasite–host interactions. Alternatively, the apparent restriction to the avian schistosome may result from incomplete sampling or annotation of neuropeptide precursor genes in other trematodes, as neuropeptide sequences are often highly divergent and can be difficult to identify through standard homology searches.

To clarify this point, we have added a brief statement in the revised Discussion acknowledging that the current observation may reflect either genuine biological specificity or biases arising from incomplete genomic and transcriptomic resources (Line 340-345).

 

L418: Please specify the strain of S. mansoni used.

RESPONSE: The strain information added, which is the Puerto Rican strain.

Reviewer 2 Report

Comments and Suggestions for Authors

General assessment

In the presented work, the authors conducted a comprehensive study of neuropeptides in Schistosoma mansoni miracidia. Combining peptidomics, in silico analysis, and quantitative behavioral bioassays, they identified ten putative neuropeptides, five of which had not been previously detected in this life stage. The most significant finding is the demonstration that two peptides, NP6 (ASLSYF-OH) and NP13 (FLLGLPPSLRQH-OH), induce pronounced and prolonged changes in miracidial behavior (reduced velocity, increased angular deviation). This suggests a key role for these peptides in larval neuroregulation and positions them as potential targets for interrupting schistosomiasis transmission.

The work is executed at a high methodological standard, includes thorough statistical analysis, and is logically structured. The results obtained are novel and significant for understanding schistosome neurobiology and for developing new approaches to disease control.

Specific comments

Novelty: This is the first targeted functional analysis of endogenous neuropeptides in miracidia. The identification of new peptides and the demonstration of their bioactivity at a stage critical for infecting the intermediate host opens new perspectives for transmission-blocking therapies.

Methodology: The use of modern proteomic methods (LC-MS/MS) combined with predictive algorithms (NeuroPred, SignalP) for peptide identification is robust. The design of behavioral experiments with acute and prolonged exposure, along with the use of ART-ANOVA for statistical processing, is highly commendable.

Systematic approach: Integrating peptidomics data with gene expression analysis, phylogenetic analysis (to assess species specificity), and protein-protein interaction (PPI) analysis allowed the authors to reasonably prioritize candidates for further study.

Clear demonstration of effect: The data convincingly show that the effects of NP6 and NP13 are not a non-specific response to high peptide concentrations but represent a dose- and time-dependent specific action. The comparison with previous data on cercariae adds depth to the work and highlights the stage-specificity of neuropeptide signaling.

Physiological significance of the observed behavior:

The authors interpret the reduced velocity and increased "circular movement" (angular deviation) as signs of "lethargy" (lines 230, 315) or "behavioral dysregulation" (line 284). It is not entirely clear whether this behavior indicates stress, disorientation, or a specific pattern potentially related to host-seeking in a natural environment. It is known from the literature that miracidia slow down and change their trajectory when approaching a snail host. The discussion of this point should be expanded. It should be explicitly stated that the observed behavior in vitro likely disrupts the normal locomotion required for successful infection, making NP6 and NP13 promising targets. Hypotheses about the mechanism could also be discussed: do these peptides affect the locomotor apparatus or the sensory systems involved in orientation?

Mechanism of peptide penetration:

The authors note that relatively small peptides (<2 kDa) were used to ensure their effective penetration into miracidia (lines 192, 548-549). However, miracidia are covered with ciliated plates, which are part of the epidermis. How feasible and efficient is passive diffusion of peptides across this barrier? Perhaps the peptides interact with surface receptors rather than penetrating internally. Alternatively, external application might trigger a cascade of reactions leading to the observed changes. This point is important for understanding how these compounds might work as potential biocides in a natural environment.

Connection with PPI analysis results:

In section 2.2, the authors emphasize Smp_176700 (the precursor of NP10), predicting a "significant role in regulating miracidia behavior" (lines 172-173) due to its connections with infection-related proteins (annexin, leishmanolysin). However, in behavioral tests, NP10 (LLMSVAGLHH-OH) showed low bioactivity (line 290). Conversely, for the highly active NP6 and NP13, no such predicted interactions were found (line 400). It may indicate that 1) the precursor Smp_176700 performs other functions not related to the direct behavioral response measured; 2) NP10 might be inactive in vitro but important in vivo in a different context; or 3) there are limitations in the current PPI data due to the large number of uncharacterized proteins. Discussing this nuance would demonstrate the authors' critical approach to their data and outline avenues for future research.

Technical issues:

Units of Measurement: In section 4.10 (line 610), it is stated that the final concentration after adding 8 µL of peptide to 500 µL of medium was 157 µg/mL. However, the text above (line 595) indicates that the stock solution for prolonged exposure was 0.01 mg/mL. Please verify the calculations: (0.01 mg/mL * 0.008 mL) / (0.5 mL + 0.008 mL) ≈ 0.000157 mg/mL = 0.157 µg/mL, not 157 µg/mL. There may be a missing "nano" prefix or an error in the calculation. This is a critical point for interpreting the dosages.

This manuscript represents a valuable and well-executed study that makes a significant contribution to parasite neurobiology and opens new potential targets for combating schistosomiasis. The experimental design, methods employed, and data analysis are of a high standard. Considering the above, I recommend the manuscript for publication after minor revisions.

Author Response

General assessment

In the presented work, the authors conducted a comprehensive study of neuropeptides in Schistosoma mansoni miracidia. Combining peptidomics, in silico analysis, and quantitative behavioral bioassays, they identified ten putative neuropeptides, five of which had not been previously detected in this life stage. The most significant finding is the demonstration that two peptides, NP6 (ASLSYF-OH) and NP13 (FLLGLPPSLRQH-OH), induce pronounced and prolonged changes in miracidial behavior (reduced velocity, increased angular deviation). This suggests a key role for these peptides in larval neuroregulation and positions them as potential targets for interrupting schistosomiasis transmission.

The work is executed at a high methodological standard, includes thorough statistical analysis, and is logically structured. The results obtained are novel and significant for understanding schistosome neurobiology and for developing new approaches to disease control.

Specific comments

Novelty: This is the first targeted functional analysis of endogenous neuropeptides in miracidia. The identification of new peptides and the demonstration of their bioactivity at a stage critical for infecting the intermediate host opens new perspectives for transmission-blocking therapies.

Methodology: The use of modern proteomic methods (LC-MS/MS) combined with predictive algorithms (NeuroPred, SignalP) for peptide identification is robust. The design of behavioral experiments with acute and prolonged exposure, along with the use of ART-ANOVA for statistical processing, is highly commendable.

Systematic approach: Integrating peptidomics data with gene expression analysis, phylogenetic analysis (to assess species specificity), and protein-protein interaction (PPI) analysis allowed the authors to reasonably prioritize candidates for further study.

Clear demonstration of effect: The data convincingly show that the effects of NP6 and NP13 are not a non-specific response to high peptide concentrations but represent a dose- and time-dependent specific action. The comparison with previous data on cercariae adds depth to the work and highlights the stage-specificity of neuropeptide signaling.

Physiological significance of the observed behavior:

The authors interpret the reduced velocity and increased "circular movement" (angular deviation) as signs of "lethargy" (lines 230, 315) or "behavioral dysregulation" (line 284). It is not entirely clear whether this behavior indicates stress, disorientation, or a specific pattern potentially related to host-seeking in a natural environment. It is known from the literature that miracidia slow down and change their trajectory when approaching a snail host. The discussion of this point should be expanded. It should be explicitly stated that the observed behavior in vitro likely disrupts the normal locomotion required for successful infection, making NP6 and NP13 promising targets. Hypotheses about the mechanism could also be discussed: do these peptides affect the locomotor apparatus or the sensory systems involved in orientation?

Response: Thank you for this thoughtful comment and for highlighting the importance of clarifying the physiological significance of the behavioural changes observed. We agree that reduced swimming velocity and increased angular deviation could reflect multiple biological processes, including stress responses, disorientation, or altered host-seeking behaviour. In natural conditions, miracidia are known to reduce swimming speed and alter their trajectory when approaching a suitable snail host, suggesting that these behavioural parameters are closely linked to host detection and infection success.

To address this point, we have expanded the Discussion to clarify that the behavioural changes induced by NP6 and NP13 likely disrupt the coordinated locomotion required for effective host-seeking and infection. Although the observed reductions in velocity and increases in turning behaviour were initially described as “lethargy” or behavioural dysregulation, these patterns may instead reflect interference with the normal locomotor and sensory processes that guide miracidia towards their intermediate host.

We have also added a brief discussion of potential mechanisms, noting that these neuropeptides may influence components of the locomotor apparatus or sensory pathways involved in environmental orientation (Line 380-387). While the precise mechanism remains to be determined, these findings suggest that NP6 and NP13 may alter behavioural programs that are essential for successful host localisation and infection.

 

Mechanism of peptide penetration:

Comment: The authors note that relatively small peptides (<2 kDa) were used to ensure their effective penetration into miracidia (lines 192, 548-549). However, miracidia are covered with ciliated plates, which are part of the epidermis. How feasible and efficient is passive diffusion of peptides across this barrier? Perhaps the peptides interact with surface receptors rather than penetrating internally. Alternatively, external application might trigger a cascade of reactions leading to the observed changes. This point is important for understanding how these compounds might work as potential biocides in a natural environment.

Response: Thank you for highlighting this important point. We agree that the presence of ciliated plates forming part of the miracidial epidermis may represent a barrier that limits passive diffusion of externally applied peptides. In the present study, peptides smaller than 2 kDa were selected partly to increase the likelihood of interaction with the parasite; however, we acknowledge that the exact mechanism by which these peptides influence miracidia behaviour remains unclear.

As suggested, it is possible that the peptides interact with receptors located on the parasite surface rather than penetrating through the ciliated plates. Alternatively, external exposure may trigger signalling cascades or neuromodulatory responses that lead to the observed behavioural changes without requiring intracellular uptake. To clarify this issue, we have added a statement in the revised Discussion noting that future studies should investigate whether these peptides penetrate the miracidial tegument or instead act through surface receptor-mediated mechanisms (Lines 452–457).

 

Connection with PPI analysis results:

Comments: In section 2.2, the authors emphasize Smp_176700 (the precursor of NP10), predicting a "significant role in regulating miracidia behavior" (lines 172-173) due to its connections with infection-related proteins (annexin, leishmanolysin). However, in behavioral tests, NP10 (LLMSVAGLHH-OH) showed low bioactivity (line 290). Conversely, for the highly active NP6 and NP13, no such predicted interactions were found (line 400). It may indicate that 1) the precursor Smp_176700 performs other functions not related to the direct behavioral response measured; 2) NP10 might be inactive in vitro but important in vivo in a different context; or 3) there are limitations in the current PPI data due to the large number of uncharacterized proteins. Discussing this nuance would demonstrate the authors' critical approach to their data and outline avenues for future research.

Response: We agree that the discrepancy between the predicted importance of the precursor Smp_176700 in the PPI analysis and the relatively low bioactivity of its derived peptide NP10 in behavioural assays highlights an important nuance in interpreting these results. The PPI analysis suggested that Smp_176700 is connected with proteins implicated in infection-related processes, including annexin and leishmanolysin-like peptidases; however, the behavioural assays indicated that NP10 itself had limited effects on miracidial locomotion under the conditions tested.

Several explanations may account for this observation. First, the precursor protein Smp_176700 may perform functions unrelated to the specific behavioural responses measured in this study. Second, NP10 may have biological roles that are not readily captured in our in vitro behavioural assays but may become relevant under in vivo conditions or in different developmental contexts. Finally, the current PPI network includes a large proportion of uncharacterized proteins, which may limit the predictive power of interaction analyses. To clarify this point, we have added a brief discussion acknowledging these possibilities and the limitations of the current PPI data (Line 432-440).

 

Technical issues:

Comments: Units of Measurement: In section 4.10 (line 610), it is stated that the final concentration after adding 8 µL of peptide to 500 µL of medium was 157 µg/mL. However, the text above (line 595) indicates that the stock solution for prolonged exposure was 0.01 mg/mL. Please verify the calculations: (0.01 mg/mL * 0.008 mL) / (0.5 mL + 0.008 mL) ≈ 0.000157 mg/mL = 0.157 µg/mL, not 157 µg/mL. There may be a missing "nano" prefix or an error in the calculation. This is a critical point for interpreting the dosages.

Response: Thank you for noticing this. We have read through all units in the paper to ensure accurate prefixes. The concentrations stated in line 217 now match those in line 613.

 

Comments: This manuscript represents a valuable and well-executed study that makes a significant contribution to parasite neurobiology and opens new potential targets for combating schistosomiasis. The experimental design, methods employed, and data analysis are of a high standard. Considering the above, I recommend the manuscript for publication after minor revisions.

Response: Thank you for your positive evaluation of our work and for your constructive comments. We appreciate your recognition of the study’s contribution to parasite neurobiology. We have carefully addressed all of your comments and have revised the manuscript accordingly. The suggested clarifications and additional discussion points have been incorporated into the revised version of the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I was pleased to read the revised version of the manuscript. The authors addressed the vast majority of my points. Below are a few minor issues that could be adjusted.

L59-60: I appreciate the addition of vertebrates/platyhelminths. However, my original question was mainly about the name. Do "F" and "Y" stand for something? Are there some structural/functional differences? Or are they just random letters? Sorry for not being clear; it was just a minor detail.

L205-208: Thank you for this explanation. Therefore, I conclude that the biological effects of NP14 are likely limited. Also, if the mentioned study has been published, it should be cited.

Figure 3: Sorry, but I cannot see the color code in the current version. It was also not addressed whether the blue baseline represents a control, and why it appears uneven across groups within the same experiment.

L342: Thank you for the clarification. A reference on neuropathogenic avian schistosomes (Trichobilharzia regenti) would be helpful, as neuropathogenicity is uncommon in this group (and in schistosomes in general).

Author Response

Comment1: L59-60: I appreciate the addition of vertebrates/platyhelminths. However, my original question was mainly about the name. Do "F" and "Y" stand for something? Are there some structural/functional differences? Or are they just random letters? Sorry for not being clear; it was just a minor detail.

Response 1: Thank you for the clarification and for raising this point. The letters “F” and “Y” refer to the conserved C-terminal amino acids of these neuropeptides, namely phenylalanine (F) and tyrosine (Y), respectively. These residues are characteristic features used to classify neuropeptide families and are often important for receptor binding and biological activity. While both groups share structural similarities, differences in the terminal residue may contribute to variations in receptor specificity and functional roles. We have revised this statement and added a brief clarification in the revised manuscript to explain this nomenclature (Line 59-62).

 

Comment 2: L205-208: Thank you for this explanation. Therefore, I conclude that the biological effects of NP14 are likely limited. Also, if the mentioned study has been published, it should be cited.

Response 2: The previously mentioned study has now been appropriately cited in the revised version to support this observation.

 

Comment 3: Figure 3: Sorry, but I cannot see the color code in the current version. It was also not addressed whether the blue baseline represents a control, and why it appears uneven across groups within the same experiment.

Response 3: The colour code has been included in the revised figure, positioned below the panels and labelled as “Key: Blue, pre-exposure; Red, post-exposure.” To improve clarity, we have added the colour code description in the updated figure legend.

The blue baseline represents the pre-exposure measurements for each neuropeptide treatment rather than a shared control across all groups. The variation in baseline values between groups reflects biological variability, as different batches of miracidia were used for each treatment to avoid confounding effects associated with repeated use of the same organisms. We have added a clarification in the figure legend to explain this point.

 

Comment 4: L342: Thank you for the clarification. A reference on neuropathogenic avian schistosomes (Trichobilharzia regenti) would be helpful, as neuropathogenicity is uncommon in this group (and in schistosomes in general).

Response 4: Thank you for this helpful suggestion. We agree that neuropathogenicity is relatively uncommon among schistosomes and that appropriate context is important. To address this, we have added two relevant references on the neuropathogenic avian schistosome Trichobilharzia regenti in the revised manuscript to support this statement.