Arginine-Vasotocin Neuronal System in Steindachneridion parahybae (Siluriformes: Pimelodidae) and Its Influence on Artificially Induced Spawning in Captivity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This research paper presents detailed findings on the reproductive behavior of Steindachneridion parahybae under artificial induction conditions. The study reveals that AVT not only potentially associates with aggressive behavior at the central nervous system level, functioning as neurotransmitter, but also possibly acts as a neuropeptide hormone and a potential pituitary hormone-releasing factor, influencing the sex steroid biosynthesis pathway in S. parahybae. Overall, this article provides novel and valuable scientific data regarding the artificially-induced reproduction of S. parahybae, contributing significantly to our understanding of the reproductive physiology of this endangered species. I have only a few concerns on this manuscript.

1、The research employed a setup with a control group composed of a solitary female and an experimental group housing two females together, with the intention to mimic differing social competitive scenarios in order to discern behavioral discrepancies in female S. parahybae during induced spawning. It would be beneficial if the authors could elaborate in the methods section on the reasons behind selecting these specific group arrangements and the measures taken to ensure that the intensity of competition among females within the experimental group approximated that found in their natural environment.

2、Please reconfirm the data for 'Total weight' presented in both Table 1 and Table 2 to verify whether the discrepancies observed are due to typographical errors in punctuation, or if indeed the differences between females and males, as well as between the D (Dominant) and ND (Non-Dominant) groups within the experimental setup, are inherently substantial.

3、In Figure 3 and Figure 7, it is suggested that the authors label the P-values directly within the figures themselves, as this approach would present the statistical significance more intuitively than describing them in the text.

Author Response

Thank you very much for the suggestions. All answers are attached.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

COMMENTS TO THE EDITOR

COMMENTS TO THE AUTHORS

In the present manuscript “Arginine-vasotocin neuronal system in Steindachneridion parahybae (Siluriformes: Pimelodidae) and its influence during artificial induced spawning in captivity” by Honji and colleagues characterize the distribution of vasotocin positive cells and hormone levels in captive female Steindachneridion parahybae catfish. Little is known about the reproductive neuroendocrinology or behavior of this endangered species.

Overall, the results of potential applied and basic scientific interest and the data collection seems sound. My primary concerns are with the presentation of ideas and some methods, as well as some methodological and analysis issues that need clarification.

As written, the introduction does not make the main aim of the study clear. The title suggests characterization of the AVT system, but many different ideas are presented in the introduction without being well connected to one another or the big picture. The introduction should be streamlined to highlight the central question, rationale for the study, and some specific predictions.

It would also be helpful to provide some more overview of the specific biology of the study species in the introduction.

Further, there is a disconnect between the methods and analyses, the results, and the interpretation in the discussion. All aspects addressed in the methods should be clearly presented in the results (or a reason for their omission provided), and all results should be addressed in the discussion. Adjusting these issues will improve the overall flow and clarity, as well as making the importance and impact of the findings clearer. A conceptual diagram summarizing the authors’ current thinking about the relationship between behavior, hormones, and neuropeptides could be especially valuable here.  

I have several questions and concerns about the aggressive behavior:

-      It is stated that fish harm their partners. Please clarify if this is only for same sex or also opposite sex partners.

-      Related to the above, please clarify the justification for housing animals in this way if it is known they harm one another. Was the assessment of dominant versus non-dominant females a goal, and if so with what predictions?

-      Many of the behavioral findings appear to be casual observations rather than systematic. Please clarify how dominant/non-dominant status was assessed (behavioral observations or only physical damage) and whether behavioral observations were systematic (e.g., controlled for duration, time of day, etc.). As presented now, the behavioral component of the paper seems inadequate to draw conclusions about the aggressive behavior of females in this species.

For the analyses:

-      Please clarify how optical density was measured – on individual cells or on the whole brain region?

-      Please clarify the decision to use optical density rather than cell counts. This measure is not quantitative and can be biased. Counts of stained cells would be more rigorous and should be added if possible.

-      Description of statistical models is insufficient. Only very general statements are made when many models were run. Further details are needed to evaluate whether all models were appropriate and to connect results back to analyses.

-      Given the differences in body size between groups, was this controlled for in the analyses? This may not be necessary depending on how the analyses were done, but additional information is needed to evaluate this, in particular given the reported differences in body size between groups of interest. Please justify the decision in either case.

-      Please clarify what is meant by “semi-quantitative” in reference to some analyses.

Given the wealth of data presented, there are a few additional questions that could be addressed that would strengthen the paper.

First, it would be interesting to consider not only individual hormones but also the relationships among them. Specifically, there are well-known trade-offs between glucocorticoids and testosterone and reporting these relationships would be particularly interesting in the context of the arguments about (social) stress and aggressive behavior.

Second, although the sample sizes are small, it could be worth while to look at correlations between hormones and AVT within individuals. Indeed, the potential for a relationship between – for example – cortisol and AVT is suggested by the authors but no analyses to test this idea are presented.

Two additional important points to consider in the discussion:

First, what is the relationship between RNA and protein and how might this influence the patterns observed in the present study versus those using an RNA expression approach.

Second, what is the role of activity in AVT neurons vs the number of AVT neurons. Specifically, if larger fish tent to be more aggressive this might not be a fixed trait, but rather dependent on the current partner. In this scenario, it may be the activity of AVT neurons that is most important as that can change more rapidly depending on environmental conditions. (This line of reasoning is nicely addressed by studied that pair nonapeptides with immediate early genes).

For figures, consider a visualization that better displays the data. For example, boxplots with individual dots overlaid on the plots. Given the size of the error bars, there is no need to start the plots at zero and this makes the spread of the data harder to see.

MINOR COMMENTS

Line 102: Please clarify what is meant by ‘reproductive dysfunction’.

Figure 1: The scale bar appears to be incorrect given the size of the fish listed in Table 1.

Comments on the Quality of English Language

The English is fine but the flow of ideas and narrative of the 'story' needs to be strengthened. 

Author Response

Thank you very much for the suggestions. All answers are attached.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Editor,

The study by Honji and colleague explores induced spawning in an endangered Neotropical catfish, focusing on female aggressive behavior and the vasotocinergic system's role in reproductive physiology. Results indicate that dominant females exhibit more aggression and larger body size, with distinct AVT immunoreactive neurons in the preoptic area. Steroid plasma profiles differ between dominant and non-dominant females. Dominant females showed a higher level of 17α-20β- 611 DHP and a lower level of Cortisol than None dominant females suggesting AVT's involvement in behavioral and physiological changes during induced spawning. Overall, the study is sound and can be publish in Fishes after revision

Line 96-97: it states “Nevertheless, little is known about the potential physiological roles of Neotropical fish “. Do you intend to say physiological roles of AVT neurons?

Author Response

We would like to acknowledge for the critical appraisal and suggestions made by the reviewers and the Editors to our manuscript, fishes-2990344. We are now submitting the modified version of the manuscript according to all these suggestions. You will find the changes made in the revised version listed below (according to the lines described) (yellow marks). Additionally, the manuscript had already been revised according to the attached certificate. If there is still a need, we will send it again for English review, but in this case, we would need more time.

Yours sincerely,

Renato Massaaki Honji

(corresponding author)

Reviewer 3.

Line 96-97: it states “Nevertheless, little is known about the potential physiological roles of Neotropical fish “. Do you intend to say physiological roles of AVT neurons?

Answer: Thanks for the comment. As requested, we have included “AVT neurons” (line: 123).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

COMMENTS TO THE AUTHORS

In the present manuscript “Arginine-vasotocin neuronal system in Steindachneridion parahybae (Siluriformes: Pimelodidae) and its influence during artificial induced spawning in captivity” by Honji and colleagues characterize the distribution of vasotocin positive cells and hormone levels in captive female Steindachneridion parahybae catfish.

I reviewed a previous version of the manuscript and find the current version improved. I thank the authors for their efforts in addressing my comments.

Overall, I think the introduction could still be improved by making the information presented more focused to the study. Some of the information seems tangential to the work presented here, and the primary goal of the authors with this study can be further clarified.

For the presentation of results, upon re-reading it seems that sample size in some groups are N=3? To make this information easier to find, I suggest adding it to Table 2. I also suggest showing the data in the figures as individual data points with sample sizes this small. If I am not understanding properly and N>6, please retain the box plots.

In the methods section 2.2.4, please clarify whether the number of AVT, GnRH and LH cells analyzed for nuclear and cellular area are per animal or total. If these are the total sample sizes, they are too low to draw reliable conclusions.

The discussion remains poorly aligned with the rest of the paper. There are results that are not addressed and no interpretation provided in the discussion. Specifically, the findings presented in Figure 7 which seems central to the original question. These differences in OD should also be interpreted in light of the minimal differences in cell size and number. There does not seem to be any mention of the GnRH or LH cell analysis results.

More generally, the discussion jumps around between AVT, steroid hormone, and behavioral results. I suggest adding some sub sections to help with organization and considering how to rearrange the presentation of ideas for improved clarity.

Comments on the Quality of English Language

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Author Response

Thank you very much for your considerations. The rebuttal letter is attached.

Author Response File: Author Response.pdf