Behavioral Alterations in Male Zebrafish After Administration of Androgen Receptor Blockers and an Activator

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Androgen Receptor Blockers and Activator Exposure Induces Behavioral Alterations in Adult Male Zebrafish” presents a systematic pharmacological investigation into the behavioral effects of second-generation androgen receptor inhibitors (enzalutamide, apalutamide) and an activator (dihydrotestosterone) in adult male zebrafish. The methodological approach is well-structured, and the data analysis is comprehensive, offering valuable insights into the neurobehavioral pharmacology of these compounds. However, several key aspects require clarification and refinement to strengthen the causal inference and overall impact of the study. The following points should be addressed:

1. Experimental Concentration: The rationale for selecting the 1 ppm exposure concentration is not clearly justified within the pharmacological context of this study.
2. The conclusion that behavioral alterations are mediated through AR signaling is not sufficiently supported. Computational docking data should be complemented with experimental evidence of AR pathway modulation (e.g., changes in AR target gene expression or protein activity in relevant tissues).
3. The use of an identical tank across all behavioral tests, conducted in a fixed sequential order (Fig. 1), introduces significant potential confounds of order effects and environmental habituation. This methodological limitation must be explicitly addressed.
4. The table in Figure 2 is not clearly legible and requires reformatting for effective data communication.

Author Response

Comments and Suggestions for Authors

The manuscript entitled “Androgen Receptor Blockers and Activator Exposure Induces Behavioral Alterations in Adult Male Zebrafish” presents a systematic pharmacological investigation into the behavioral effects of second-generation androgen receptor inhibitors (enzalutamide, apalutamide) and an activator (dihydrotestosterone) in adult male zebrafish. The methodological approach is well-structured, and the data analysis is comprehensive, offering valuable insights into the neurobehavioral pharmacology of these compounds. However, several key aspects require clarification and refinement to strengthen the causal inference and overall impact of the study. The following points should be addressed:

1. Experimental Concentration: The rationale for selecting the 1 ppm exposure concentration is not clearly justified within the pharmacological context of this study.

We thank the reviewer for raising this important point regarding concentration selection. Actually, the 1 ppm exposure level was chosen based on considerations specific to chronic behavioral neurotoxicity studies in adult zebrafish rather than clinical pharmacological equivalence. In aquatic exposure paradigms, nominal water concentrations do not directly reflect internal or brain tissue levels due to uptake limitations via gills and skin, metabolism, and dilution within body tissues. Consequently, higher aqueous concentrations are commonly required to elicit measurable neurobehavioral effects in adult zebrafish, particularly under chronic exposure conditions. The selected concentration (1 ppm) was lower than those used in previous zebrafish larvae and rodent studies, while remaining sublethal and not inducing overt toxicity. This allowed us to assess subtle behavioral modulation following 14-day exposure without the confounding effects of acute stress or toxicity. Importantly, the inclusion of dihydrotestosterone as a positive control demonstrated that this concentration range was sufficient to produce biologically meaningful and directionally opposite behavioral effects via androgen receptor modulation, supporting the appropriateness of the selected exposure level for comparative analysis. Therefore, we understood that there was a mistake regarding the framing of the study; thus, we revised the manuscript to shift the justification from pharmacological dose equivalence to behaviorally effective, sublethal chronic exposure to resolve the reviewer’s concern (lines 163-170).

2. The conclusion that behavioral alterations are mediated through AR signaling is not sufficiently supported. Computational docking data should be complemented with experimental evidence of AR pathway modulation (e.g., changes in AR target gene expression or protein activity in relevant tissues).

We thank the reviewer for this insightful and constructive comment. We agree that direct experimental evidence of androgen receptor (AR) pathway modulation, such as changes in AR target gene expression or protein activity in relevant neural tissues, would provide stronger mechanistic support for the observed behavioral alterations. In the present study, the inference of AR involvement is based on a convergent line of evidence, including (i) the known AR-specific pharmacology of enzalutamide and apalutamide, (ii) the inclusion of dihydrotestosterone as a biologically relevant AR agonist producing directionally opposite behavioral effects, and (iii) molecular docking analyses indicating strong binding affinity of all three compounds to the zebrafish AR. Together, these data support functional interference with AR signaling, although we acknowledge that they do not constitute direct molecular validation of downstream AR pathway activation or suppression. Unfortunately, at present, further experimental validation at the molecular level (e.g., AR target gene expression or protein-level assays in brain tissue) could not be performed due to limitations in available instrumentation and facilities. In addition, such experiments would require the collection of additional neural tissues, necessitating an expansion of animal usage and further approval from the institutional animal ethics committee, which was beyond the scope of the current study. We have therefore revised the manuscript to (i) temper causal statements regarding AR-mediated mechanisms, (ii) clearly acknowledge this limitation, and (iii) explicitly propose molecular validation of AR signaling as an important direction for future studies (lines 545-554). We appreciate the reviewer’s suggestion and believe it has strengthened the clarity and rigor of the manuscript.

3. The use of an identical tank across all behavioral tests, conducted in a fixed sequential order (Fig. 1), introduces significant potential confounds of order effects and environmental habituation. This methodological limitation must be explicitly addressed.

We thank the reviewer for highlighting this important methodological consideration. We agree that the use of the same testing tank across multiple behavioral assays conducted in a fixed sequential order may introduce potential order effects and environmental habituation. To minimize these confounds, several measures were implemented. First, behavioral tests were distributed across different exposure days (days 13–15), rather than being conducted consecutively within a single session, thereby reducing cumulative stress and fatigue, as mentioned in the manuscript. Second, when multiple tests were conducted on the same day, individual fish were allowed a 4–5 hour resting period between tests to mitigate carryover effects. Third, environmental habituation was provided prior to each behavioral assay to standardize baseline conditions across experimental groups, with the exception of the novel tank test, which by design assesses responses to environmental novelty. Importantly, all experimental groups were subjected to the identical testing sequence and conditions, ensuring that any potential order or habituation effects were systematic rather than treatment-specific. Thus, while these factors may influence absolute behavioral measures, they are unlikely to account for the observed group-dependent differences. We have revised the manuscript to explicitly acknowledge this methodological limitation and to clarify the steps taken to minimize its impact (lines 531-540).

4. The table in Figure 2 is not clearly legible and requires reformatting for effective data communication.

We thank the reviewer for pointing out this issue. We agree that the inclusion of detailed statistical analysis tables alongside the chronological behavioral graphs in Figure 2 resulted in a crowded layout and reduced legibility. To improve clarity and data presentation, we have revised the figure by removing the statistical tables from Figure 2 and consolidating all corresponding statistical results (two-way ANOVA with Geisser–Greenhouse correction and Dunnett’s multiple comparisons test) into a single comprehensive table, now provided as Supplementary Table S3. This revision allows Figure 2 to focus on the visualization of behavioral dynamics, while detailed statistical outcomes are clearly presented in the Supplementary Materials in a readable format. We believe this reformatting substantially improves data communication and appreciate the reviewer’s suggestion.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have compiled an interesting manuscript characterizing behavioral alterations in male zebrafish in response to androgen blockers. The study addresses a relevant biological question and presents potentially valuable behavioral observations. The following comments are provided to further strengthen the manuscript.

Major Comments

1. Figure 2 (Locomotor vs. Exploratory Behavior):
The figure legends and axis labels in Figure 2 should be presented in a larger font size, as they are currently difficult to read. In addition, the authors report an overall increase in locomotor activity following DHT treatment; however, exploratory behavior appears unchanged. This apparent discrepancy requires clarification. 

2. Figure 3C (Fear Response Test):
In the fear response assay, APA treatment appears to selectively increase the fear response compared with ENZ and DHT. The authors should clarify why APA uniquely alters this behavioral outcome. This observation raises the possibility that the effect may be independent of classical androgen receptor signaling or that APA may act through additional signaling pathways. A brief mechanistic discussion and/or supporting references would strengthen the interpretation of this result.

3. PCA Analysis (Treatment-Specific Effects):
The principal component analysis indicates that APA exerts a distinct effect compared with ENZ. This finding should be expanded upon in the Discussion. 

Minor Comments

4. Figure 4 (Heatmap Visualization):
The color scale used in the heatmap should be revised. Currently, the time gradient and different behavioral categories appear to share similar color patterns, which makes interpretation challenging. The use of distinct or clearly separated color scales, or an alternative visualization strategy, would improve clarity and reduce potential misinterpretation.

Author Response

Comments and Suggestions for Authors: The authors have compiled an interesting manuscript characterizing behavioral alterations in male zebrafish in response to androgen blockers. The study addresses a relevant biological question and presents potentially valuable behavioral observations. The following comments are provided to further strengthen the manuscript.

Major Comments

1. Figure 2 (Locomotor vs. Exploratory Behavior):
   The figure legends and axis labels in Figure 2 should be presented in a larger font size, as they are currently difficult to read. In addition, the authors report an overall increase in locomotor activity following DHT treatment; however, exploratory behavior appears unchanged. This apparent discrepancy requires clarification. 

We thank the reviewer for these helpful comments and have revised the manuscript accordingly. First, we agree that the figure legends and axis labels in Figure 2 were previously too small and difficult to read. To improve clarity, we have increased the font size of all figure legends and axis labels in Figure 2. In addition, the statistical analysis tables have been removed from the figure and consolidated into Supplementary Table S3, as noted in the revised Figure 2 caption, to reduce visual crowding. To further enhance readability and better accommodate the horizontal layout of the chronological behavioral data, the orientation of the manuscript has also been changed to landscape format. Second, regarding the apparent discrepancy between increased locomotor activity and exploratory behavior following DHT treatment, we would like to clarify that DHT exposure did not exclusively affect locomotion. As stated in the Abstract, Results (Section 3.1), and Discussion sections, DHT treatment also produced subtle but statistically significant alterations in exploratory behavior. Specifically, a significant change was observed in the “Time in Top Duration” endpoint in the novel tank test (see Supplementary Table S3), indicating altered vertical exploration. As discussed in the manuscript, this behavioral profile—characterized by increased locomotion accompanied by reduced top exploration—may reflect anxiogenic rather than anxiolytic effects in zebrafish. In fish open-tank paradigms, bottom-dwelling behavior, together with hyperactivity or erratic movement, has been widely interpreted as an indicator of anxiety. This pattern differs from the anxiolytic effects reported in male rodents and highlights potential species-specific or context-dependent differences in androgen-mediated behavioral regulation. We have revised the text to further clarify this point and to ensure consistency between the behavioral results and their interpretation.

 Figure 3C (Fear Response Test):

2. In the fear response assay, APA treatment appears to selectively increase the fear response compared with ENZ and DHT. The authors should clarify why APA uniquely alters this behavioral outcome. This observation raises the possibility that the effect may be independent of classical androgen receptor signaling or that APA may act through additional signaling pathways. A brief mechanistic discussion and/or supporting references would strengthen the interpretation of this result.

We thank the reviewer for this insightful comment, prompting a deeper mechanistic discussion. We agree that the divergence in fear response profiles between the ENZ and APA groups warrants further explanation, particularly given their structural similarities. First, we would like to clarify that, as shown in Figure 3, APA treatment resulted in a less pronounced fear response compared to controls. While both compounds are potent AR antagonists, they also exhibit off-target activity as weak GABA_A receptor antagonists, a mechanism associated with fear and anxiety-like behaviors. However, pharmacological studies in mammalian models have demonstrated that, despite comparable binding affinities, APA maintains significantly lower steady-state brain concentrations compared to ENZ at therapeutic doses. This previous finding is consistent with the hypothesis that APA achieves lower effective concentrations in the zebrafish brain or exhibits reduced blood-brain barrier penetration compared to ENZ, thereby exerting weaker antagonism on GABA_A receptors and resulting in a significantly attenuated fear response in the APA-treated fish compared to the ENZ group. We have updated the Discussion section to incorporate this comparative pharmacological context and explicitly acknowledge that the observed APA-specific effect cannot be conclusively attributed to classical AR signaling alone based on the current dataset (lines 429-445). Further molecular and neuroendocrine investigations will be required to delineate the precise mechanisms underlying this differential behavioral response.

 

3. PCA Analysis (Treatment-Specific Effects):
   The principal component analysis indicates that APA exerts a distinct effect compared with ENZ. This finding should be expanded upon in the Discussion. 

We thank the reviewer for highlighting the importance of the Principal Component Analysis (PCA) and clustering results. We agree that while ENZ and APA cluster together as AR antagonists (distinct from the Control and DHT groups), their separation within that cluster indicates biologically significant differences in their behavioral toxicity profiles. As the reviewer noted, this separation is primarily driven by three key behavioral divergences: (1) shoaling behavior: ENZ-treated fish exhibited a significantly tighter shoal (anxiety-like defense), whereas APA-treated fish did not differ from controls, (2) fear response: APA treatment resulted in a unique attenuation of the fear response (less pronounced avoidance) compared to the ENZ group, and (3) locomotor severity: while both agents reduced locomotion, ENZ caused a slightly more severe reduction in locomotor activity in the novel tank test compared to APA. We have expanded the Discussion section to explicitly interpret these PCA findings (lines 445-461). We now argue that this "behavioral divergence" likely reflects the differential pharmacokinetics and off-target GABAergic interactions of the two drugs, as discussed in the revised pharmacology section.

 

Minor Comments

4. Figure 4 (Heatmap Visualization):
   The color scale used in the heatmap should be revised. Currently, the time gradient and different behavioral categories appear to share similar color patterns, which makes interpretation challenging. The use of distinct or clearly separated color scales, or an alternative visualization strategy, would improve clarity and reduce potential misinterpretation.

We thank the reviewer for this helpful suggestion and agree that the color scheme used in the original heatmap could lead to ambiguity in data interpretation. In the previous version, the color scale of the heatmap values overlapped visually with the annotation colors used to denote different behavioral endpoints, which may have caused confusion. To address this issue, we have revised the heatmap by adopting a clearly separated diverging color scale (red to blue) to represent behavioral endpoint values, while retaining distinct categorical annotation colors (cyan, orange, purple, pink, green, and yellow) for behavioral classifications. This separation enhances visual contrast between quantitative data and categorical annotations, thereby improving clarity and reducing the likelihood of misinterpretation. We believe that this revised visualization strategy substantially improves the readability and interpretability of the heatmap and appreciate the reviewer’s constructive feedback.

 

Reviewer 3 Report

Comments and Suggestions for Authors

A brief summary:

This MS by Huang et al. evaluate the behavioral alterations in adult male zebrafish (Danio rerio) following exposure to specific AR blockers-ENZ and APA, and an activator DHT, to provide a comprehensive comparison between each tested drug. The results suggest that both AR blockers were found to slightly compromise the fish locomotion, with contrary results were observed in DHT treated fish that displayed an increased locomotor activity together with slight alterations in fish exploratory behaviors. Furthermore, ENZ also caused a tightened shoal formation in zebrafish, while exposure to APA was observed to slightly diminish the fear response of fish. On the other hand, the DHT-treated group displayed a higher level of aggression compared to the vehicle control group.

 

• General concept comments.

The research provides a theoretical base on behavioral alterations for male zebrafish with androgen receptor. The results of research present insights for the relationship between androgens and behaviors in zebrafish. All these data are crucial in supporting this species for studies on the physiological and neurological mechanisms underlying androgen-mediated behaviors. The text follows the IMRAD structure. Subsections are clear. The results match the research goals. The order of results presentation combines with the methods. I have a couple of suggestions.

 

• Specific comments

Line 76-81 Provide references and try to list examples related to fish, rather than mice.

Line 114-116 List examples related to fish, rather than rats, change to fish references.

Line 133 What about the fish body weight, total length, and quantity?

Line 136 What is the dissolved oxygen content?

Line 138 What is the quantity of lab-grown brine shrimp and commercial dry food for each feeding?

Line 139 How many healthy fish were chosen for this study?

Line 146-149 There is no need to list the molecular formula.

Line 154 How to distinguish male and female zebrafish?

Line 171 How to record their behaviors, using a camera? How long does each recording session last? Make it clear.

Line 232-255 In Fig 2, there is a significant difference some data. The author should indicate in the results, significant difference (P < 0.01), and greatly significant difference (P < 0.001 or P < 0.001).

Line 270-280 In Fig 3A and B, there is a significant difference some data. The author should indicate in the results, significant difference (P < 0.01), and greatly significant difference (P < 0.001 or P < 0.001).

Line 294-298 “In marine species” Which marine species? Make it clear and provide related references.

Line 307 “behaviors-related endpoints did not show any statistical differences from the control group (Figure 3E-H).” However, in Fig 3H, it shows significant difference as p=0.0399 (P < 0.01). Why? In addition, what does abbreviation "ns" mean in Figure 3? Make it clear.

Line 472 I suggest using a separate subheading “5. Conclusion” to indicate this paragraph.

Line 496 I suggest using Figure 5 as a graphic abstract, as it will better attract the author's interest.

Line 602 Check the references again to meet the formatting requirements of the journal.

 

Comments on the Quality of English Language

the author should seek help with writing in English, I hope round of editing with English native language would further improve the quality of the writing.  The English grammar should be checked again by experts in this research field.

Author Response

Comments and Suggestions for Authors

A brief summary: This MS by Huang et al. evaluate the behavioral alterations in adult male zebrafish (Danio rerio) following exposure to specific AR blockers-ENZ and APA, and an activator DHT, to provide a comprehensive comparison between each tested drug. The results suggest that both AR blockers were found to slightly compromise the fish locomotion, with contrary results were observed in DHT treated fish that displayed an increased locomotor activity together with slight alterations in fish exploratory behaviors. Furthermore, ENZ also caused a tightened shoal formation in zebrafish, while exposure to APA was observed to slightly diminish the fear response of fish. On the other hand, the DHT-treated group displayed a higher level of aggression compared to the vehicle control group.

• General concept comments.

The research provides a theoretical base on behavioral alterations for male zebrafish with androgen receptor. The results of research present insights for the relationship between androgens and behaviors in zebrafish. All these data are crucial in supporting this species for studies on the physiological and neurological mechanisms underlying androgen-mediated behaviors. The text follows the IMRAD structure. Subsections are clear. The results match the research goals. The order of results presentation combines with the methods. I have a couple of suggestions.

• Specific comments

Line 76-81 Provide references and try to list examples related to fish, rather than mice.

We thank the reviewer for this insightful suggestion. We agree that providing examples from fish studies would be more appropriate for this manuscript. We have conducted a comprehensive literature search for studies regarding the behavioral effects of Enzalutamide (ENZ) and Apalutamide (APA) in zebrafish and other teleost models. However, to the best of our knowledge, there are currently no published studies explicitly evaluating the behavioral alterations induced by ENZ or APA in adult zebrafish or other fish species. Most existing aquatic studies on these compounds focus on acute toxicity (e.g., LC₅₀) or their use in xenograft cancer models, rather than behavioral phenotyping. Because of this significant data gap in aquatic toxicology, we relied on mammalian literature (mice/rats) to hypothesize the potential neurobehavioral effects of these androgen receptor blockers. We believe this lack of prior data highlights the novelty and importance of the current study.

Line 114-116 List examples related to fish, rather than rats, change to fish references.

We appreciate the reviewer’s request for more taxonomically relevant examples. Similar to ENZ and APA, while DHT is a potent androgen, its behavioral effects have been most extensively characterized in mammalian models, where it is known to enhance masculine behaviors via central nervous system pathways, further highlighting the novelty and importance of the current study.

Line 133 What about the fish body weight, total length, and quantity?

We thank the reviewer for pointing out this omission. We have now updated the "Materials and Methods" section (Section 2.1) to include the specific biometric data and the experimental layout. A total of 120 healthy adult male zebrafish were used, with an average body weight of 0.4 ± 0.1 g and a total length of 3.0 ± 0.5 cm. To ensure statistical power and reproducibility, the fish were randomly assigned to four experimental groups (Control, ENZ, APA, and DHT). Each group consisted of 30 fish, divided into three biological replicates (n=10 fish per replicate). This crucial information had been added to the manuscript as the reviewer’s suggestion (lines 145-146).

Line 136 What is the dissolved oxygen content?

We thank the reviewer for this request for clarification regarding the experimental conditions. We understand that maintaining optimal dissolved oxygen (DO) levels is critical for ensuring that behavioral responses are not confounded by physiological stress. During the maintenance phase, the dissolved oxygen content was monitored daily and maintained at approximately 7 ppm, which is within the recommended range for adult zebrafish. We have added this information to the revised "Materials and Methods" section (line 135).

Line 138 What is the quantity of lab-grown brine shrimp and commercial dry food for each feeding?

We thank the reviewer for the opportunity to clarify our feeding regimen. While the exact biomass of live brine shrimp was not quantified for each feeding due to natural variations in hatch rates, we employed a standardized "feeding to satiation" protocol to ensure nutritional consistency across all experimental groups. For the dry food, each fish was provided with approximately 3–4 pellets per feeding. For live food, the quantity was carefully adjusted so that all provided food was entirely consumed within 1–2 minutes. This approach was used to ensure that all fish received adequate nutrition while preventing overfeeding and the subsequent accumulation of organic waste, which could degrade water quality and interfere with behavioral results (lines 140-143).

Line 139 How many healthy fish were chosen for this study?

We thank the reviewer for the question. As mentioned above, a total of 120 healthy adult male zebrafish were selected for this study. These fish were randomly and equally distributed among the four experimental groups: Control, Enzalutamide (ENZ), Apalutamide (APA), and Dihydrotestosterone (DHT). To ensure reproducibility and account for potential "tank effects," each treatment group (n = 30) was further subdivided into three independent biological replicates, with each replicate containing 10 fish. All behavioral assays were subsequently performed on individuals from these replicates to ensure a robust sample size for statistical comparison.

Line 146-149 There is no need to list the molecular formula.

We agree with the reviewer’s assessment that the inclusion of the full IUPAC molecular formulas in the main text was unnecessary and hindered the readability of the manuscript. As suggested, we have removed these formulas from the "Materials and Methods" section. To ensure experimental reproducibility, we have retained the specific manufacturer and purity levels for each compound (Enzalutamide, Apalutamide, and Dihydrotestosterone) (lines 152-153).

Line 154 How to distinguish male and female zebrafish?

We thank the reviewer for this important question regarding the sexing of the zebrafish. To ensure a uniform experimental population, only adult male zebrafish were used. Sexual dimorphism in zebrafish is well-characterized, and we employed several established morphological criteria to distinguish males from females: (i) Body Shape: Males are typically more slender and "torpedo-shaped," whereas females have a deeper, more rounded ventral profile (belly), especially when gravid. (ii) Coloration: Males often exhibit a more pronounced yellowish or golden hue between their blue horizontal stripes, particularly on the anal fin, while females tend to be more silvery or blue-white. (iii) Genital Papilla: Females possess a small, visible protrusion (genital papilla) anterior to the anal fin, which is absent in males. Furthermore, to ensure the highest degree of accuracy, any individuals with ambiguous morphology—such as exceptionally "fat" males or "slim" females—were excluded from the study. This rigorous selection process ensured that the behavioral and physiological responses observed were specific to the male androgen system (lines 158-163).

Line 171 How to record their behaviors, using a camera? How long does each recording session last? Make it clear.

We thank the reviewer for the opportunity to clarify our behavioral recording protocols. We have revised the "Materials and Methods" section to provide a more cohesive description of the recording setup and session durations (lines 203-208). All behavioral assays were captured using a Canon EOS D600 DSLR camera equipped with a 55–250 mm lens, mounted at a fixed height above or in front of the test tanks to ensure a consistent field of view. The recorded videos were then analyzed using UMATracker software to extract coordinate data for behavioral endpoint calculations. Regarding the session durations, as already mentioned in the manuscript, most tests consisted of a 5-minute recording following a specific acclimation period. The Novel Tank Test followed a time-series protocol to observe habituation, consisting of 1-minute recordings every 5 minutes over a 31-minute period.

Line 232-255 In Fig 2, there is a significant difference some data. The author should indicate in the results, significant difference (P < 0.01), and greatly significant difference (P < 0.001 or P < 0.001).

We thank the reviewer for this suggestion regarding statistical reporting. We have revised the "Results" section regarding Figure 2 to clearly distinguish between levels of statistical significance. In the updated manuscript, we have adopted the convention for P values based on the reviewer’s comment (section 3.1).

Line 270-280 In Fig 3A and B, there is a significant difference some data. The author should indicate in the results, significant difference (P < 0.01), and greatly significant difference (P < 0.001 or P < 0.001).

We thank the reviewer for this guidance. Similar to the point above, we have refined the results description for Figure 3 to precisely reflect the levels of statistical significance observed. Per the reviewer's suggestion, we have distinguished between significant (P < 0.01) and greatly significant (P < 0.001) differences in the text to provide a clearer interpretation of the data (section 3.2).

Line 294-298 “In marine species” Which marine species? Make it clear and provide related references.

We thank the reviewer for this comment; however, we would like to clarify a potential misunderstanding. We have performed a comprehensive search of the entire manuscript and were unable to locate the phrase "in marine species" or any reference to marine organisms. It is possible there was a discrepancy in line numbering or a clerical error. As described in lines 294–298 of our manuscript, the study utilizes zebrafish (Danio rerio) and convict cichlids (Amatitlania nigrofasciata), both of which are freshwater species. To ensure absolute clarity regarding the predator used in the fear response assay, we have updated the text to explicitly state the freshwater nature of the stimulus fish.

Line 307 “behaviors-related endpoints did not show any statistical differences from the control group (Figure 3E-H).” However, in Fig 3H, it shows significant difference as p=0.0399 (P < 0.01). Why? In addition, what does abbreviation "ns" mean in Figure 3? Make it clear.

We thank the reviewer for this careful reading of our data and for pointing out the need for clearer phrasing. Regarding the significance in Figure 3H (P = 0.0399), we would like to clarify that this result pertains exclusively to the ENZ-treated group, which, as stated in the text, exhibited a "statistically lower average farthest neighbor distance.", while the subsequent sentence—"APA and DHT seemed to have no robust effect... since all of the shoaling behaviors-related endpoints did not show any statistical differences"—was intended to refer only to the APA and DHT groups, which both showed "ns" (non-significant) results compared to the control (section 3.4). Additionally, we have added a definition for the abbreviation "ns" (not statistically significant) to the Figure 3 caption.

Line 472 I suggest using a separate subheading “5. Conclusion” to indicate this paragraph.

We thank the reviewer for this organizational suggestion. We agree that a separate "Conclusion" section improves the clarity and flow of the manuscript. As suggested, we have added a new subheading (Section 5. Conclusion) as this paragraph includes the summary of our findings, the significance of the results, and the future research directions to this section. Additionally, this paragraph also discusses the current study limitations (regarding the test sequence and lack of molecular validation) to maintain a focused concluding statement.

Line 496 I suggest using Figure 5 as a graphic abstract, as it will better attract the author's interest.

We sincerely thank the reviewer for this excellent suggestion. We agree that Figure 5 provides a comprehensive and visually clear summary of the differential behavioral impacts of AR blockers (ENZ, APA) and the AR activator (DHT). Following your suggestion, we have designated Figure 5 as the Graphical Abstract for this manuscript. We have ensured that the figure meets the specific formatting requirements of the journal Biology (MDPI), including the appropriate resolution and aspect ratio. By highlighting the distinct phenotypes observed—such as ENZ’s effect on shoaling, APA’s effect on fear response, and DHT’s effect on aggression—this abstract will indeed provide readers with a clear overview of our findings at a glance.

Line 602 Check the references again to meet the formatting requirements of the journal.

We thank the reviewer for this reminder. We have conducted a thorough review of the bibliography to ensure all entries strictly adhere to the MDPI citation and reference guidelines.

Comments on the Quality of English Language

the author should seek help with writing in English, I hope round of editing with English native language would further improve the quality of the writing.  The English grammar should be checked again by experts in this research field.

We sincerely appreciate the reviewer's feedback regarding the manuscript's linguistic clarity. We take the quality of our communication seriously and have taken professional review and expert consultation steps to ensure the manuscript meets the high standards of the journal. We hope that in the current state, the manuscript has an acceptable linguistic quality.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor and Authors,

I have reviewed the revised manuscript and the authors' point-by-point response to the previous comments. The authors have adequately addressed all my concerns, and the revisions have substantially improved the clarity and rigor of the study.

I have no further questions and recommend the manuscript for acceptance.

Reviewer 3 Report

Comments and Suggestions for Authors

The author has made point-by-point revisions according to the reviewer's suggestions. I agree to revise and publish the manuscript according to the journal's format requirements

Comments on the Quality of English Language

the author should seek help with writing in English, I hope round of editing with English native language would further improve the quality of the writing.  The English grammar should be checked again by experts in this research field.