Environmentally Realistic Waterborne Atrazine Exposure Affects Behavior in Poecilia latipinna
Round 1
Reviewer 1 Report
Dear author it is my pleasure to review the manuscript titled “Environmentally realistic waterborne atrazine exposure affects behavior in Poecilia latipinna”
I have the following comments on the paper which needs your attention.
1) Data presentation is very poor, and graphs and figures not formatted or labeled clearly, it is not easy to follow the data.
2) It is same with the data provided in supplementary files there is no proper formatting and very difficult to follow.
3) Regarding the title of the manuscript, it is mentioned “behavior” but does not specify what kind of behavior as the behavior is very broad term and needs to be specified.
4) In line 11 “Molly behavior is well documented” this sentence incomplete.
5) The manuscript does not contain any figure to show the track path or heatmaps of the fish said behavior to understand better.
6) In behavioral analysis of the videos, it is not clear how the videos were analysed like using any software tool or manual?
7) Even though behavioral results show promising results the claims of the authors need to be supported some of the more details analysis like blood hormone levels or gene expression analysis for stereognosis pathway.
8) It is also noted that ATZ levels in water or accumulated levels in fish were not analysed which is very important to establish the true impact.
Author Response
Dear Ms. Isla Xu,
Thank you once again for all your time and efforts. Based on your feedback, received on December 22nd 2022, I have made the recommended revisions to manuscript " Environmentally realistic waterborne atrazine exposure affects behavior in Poecilia latipinna" (Manuscript ID: water-2083366). I have made efforts to address all concerns articulated in reviewer #1’a comments outlined below. Should the reviewers feel the paper is in need of further changes, I will be more than happy to make any further modifications they see fit.
Again, thank you very much for your time and consideration, and for granting me the extension on resubmission during the holiday season. It is much appreciated. Please let me know if you have any further questions or concerns.
Sincerely,
David MacLaren Ph.D.
Professor of Biology
Merrimack College
315 Turnpike St. North Andover, MA 01845
maclarenr@merrimack.edu
Reviewer #1
1) Data presentation is very poor, and graphs and figures not formatted or labeled clearly, it is not easy to follow the data.
I have rewritten the descriptive caption accompanying Fig. 3 for better clarity (lines 306-311, pg 8).
Figure 4 has been deleted and replaced with a pair of graphs representing the same data as those from the original figure, only with the data for females (from expt. I) and males (from expt. II) split into separate graphs. The accompanying figure caption has also been revised for better clarity (lines 326-329, pg 9).
Section 3.3 from the Results section of the MS has been significantly revised and expanded as follows (lines 345-357, pg 9-10):
3.3. Experiment III: Comparison of preferences for male body size in AZT-exposed vs unexposed females
Paired samples t-tests comparing the total time control females (n = 26) spent in association with the larger- vs. smaller-bodied male dummy revealed that females spent significantly more time with the larger of the paired males in each of the three trials (L vs S; L vs I; and I vs S; Table 1). However, the only trial in which HD females (n = 20) preferred the larger male was ‘L vs S’ – the trial in which the difference in body size was the largest of the three pairwise combinations. Females from the LD group (n = 18) showed no preferences for either male in any of the three trials (Table 1).
Mean SOP for larger size in the ‘L vs S’, ‘L vs I’, and ‘I vs S’ trials all differed among the HD, LD, and control groups (Figure 5). Specifically, control females SOP for larger male body size was significantly greater than that of LD females in the ‘L vs S’, ‘L vs I’, and ‘I vs S’ trials (Figure 5). The SOP of control females in the ‘L vs I’, and ‘I vs S’ trials was significantly greater than that of HD females as well. However, no difference in SOP was observed between the control and HD females in their ‘L vs S’ trials.
These changes were made in an effort to improve descriptive clarity and properly introduce the reader to Table 1 as well as a remade version of figure 5 that better organizes/summarizes the data from expt. III. The descriptive caption accompanying figure 5 has also been significantly revised (lines 369-374, pg 11).
Minor changes to the descriptive captions accompanying figures 6 and 7 have also been made in the revised MS.
2) It is same with the data provided in supplementary files there is no proper formatting and very difficult to follow.
My sincere apologies for not confirming the raw data submitted in the supplementary files was formatted correctly once uploaded. I have pasted them into a different file format that should be far easier to view and interpret as part of the revised submission.
3) Regarding the title of the manuscript, it is mentioned “behavior” but does not specify what kind of behavior as the behavior is very broad term and needs to be specified.
Use of the broad term “behavior” was chosen based on the variety of behaviors analyzed in the study (reproductive behavior, aggression, anxiety and boldness).
4) In line 11 “Molly behavior is well documented” this sentence incomplete.
The sentence in question has been revised as follows: “The behavior of sailfin mollies is well documented in the scientific literature.”
5) The manuscript does not contain any figure to show the track path or heatmaps of the fish said behavior to understand better.
Track paths and heatmaps were not employed in the present study. Although such methods might offer additional value to the results and may be considered in future studies, the data gathered on total time spent within specified zones documented herein is well established protocol that provides compelling evidence of ATZ’s impact on the various aspects of behavior analyzed in this project.
6) In behavioral analysis of the videos, it is not clear how the videos were analyzed like using any software tool or manual?
Section 2.6 from the Methods section of the MS has been significantly revised and expanded (lines 242-293, pg 4-5) in efforts to: 1. describe more clearly the methods of video analysis used in assessment of fish behavior; 2. provide more information regarding the statistical tests, p-level and statistical software used; and 3. Provide a rationale for the use of T-tests including a statement about the normality of your data and use of non-parametric equivalents.
2.6. Behavioral measures and statistical analyses
For expts. I-III, test subject association preferences were based on total time spent with one dummy relative to the total time spent with the other dummy of the pair. The video data collected on iPads in expt. I-III were analyzed as follows: The total amount of time the subject spent in the preference zones of each of the two dummy stimuli during the course of the 20 min. testing period (irrespective of the number of separate visits made to a given dummy preference zone) were summed for each subject. These measures served as the raw data for all statistical analyses that followed.
For expts. I and II, paired samples t-tests were used to compare the total time subjects spent in association with the male versus the female dummy stimulus. Mean strength of preference (SOP), defined as time spent with the male stimulus—time with the female stimulus per 20 min test, were then calculated and compared among the LD, HD, and control groups of expts. I-III using One-way Repeated Measures ANOVAs, where ‘SOP’ and ‘treatment’ served as the dependant and independent variables, respectively [59, 62, 66]. Where appropriate, the Holm-Sidak method for multiple comparisons was used to test for differences in SOP among treatments.
For expt. III, paired samples t-tests were used to compare the total time female subjects spent in association with the larger- versus smaller male dummy stimulus for all three trials (L vs S; L vs I; and I vs S) for all three treatment groups (LD, HD, and controls). Mean strength of preference (SOP), defined as time spent with the larger male—time with the smaller male per 20 min test, were then calculated and compared among the three paired combinations of dummy males (L vs S; L vs I; and I vs S) for each of the three treatment groups (LD, HD, and controls) again using One-way Repeated Measures ANOVAs, where ‘SOP’ and ‘treatment’ served as the dependant and independent variables, respectively [59, 66, 75]. As described for expts. I and II above, the Holm-Sidak method for multiple comparisons was used to test for differences in SOP among treatments where appropriate. Because each female received three trials (L vs S; L vs I; and I vs S), all significance levels were adjusted to p<.0167 (Bonferroni procedure; Sokal and Rohlf 1981).
The ‘overall responsiveness’ of subjects to the conspecific stimuli (defined as total time spent in association with both dummy stimuli during the course of the 20 min test period) was calculated for each subject and compared among the LD, HD, and control groups for each of the first three experiments (I-III) using One-way Repeated Measures ANOVAs, where ‘overall responsiveness’ and ‘treatment’ served as the dependant and independent variables, respectively. Once again, where appropriate, the Holm-Sidak method for multiple comparisons was used to test for differences in overall responsiveness among treatments.
For expt. IV, iPad video data were used to quantify the total time (s) male subjects spent interacting with their mirror image (within the 10 cm mirror zone). These data were then used to compare total miror zone time for males among the LD, HD, and control groups using One-way Repeated Measures ANOVAs, where ‘time spent in the mirror zone’ and ‘treatment’ served as the dependant and independent variables, respectively. The Holm-Sidak method for multiple comparisons was then used to test for differences in mirror zone time (a proxy for agression) among treatments where appropriate.
Lastly, video data collected during each subject’s 10 min. acclimation period were analyzed for BZT and LTZ. These data were complied across all four experiments and compared among the LD, HD, and control groups of each experiment using One-way Repeated Measures ANOVAs, with ‘BZT’ and ‘LTZ’ the dependant variables and ‘treatment’ as the independent variable. The probabilities reported for all statisical analyses are two-tailed. Standard parametric tests were applied to the data as no violations of the assumptions of normality and/or equal variance were identified. All statistical tests were conducted using SigmaStat (version 11.2).
7) Even though behavioral results show promising results the claims of the authors need to be supported some of the more details analysis like blood hormone levels or gene expression analysis for stereognosis pathway.
I strongly agree that the inclusion of “blood hormone levels” and/or “gene expression analysis for stereognosis pathways” among other molecular and/or physiological measures would offer a more comprehensive analysis of the impact of ATZ exposure beyond the behavioral effects documented in the present study. However, my research students and I were not in a position where such data could be easily gathered and quantified. We feel the behavioral results alone serve as a strong and compelling example of the impacts of AZT exposure on P. latipinna. I follow-up study investigating the potential underlying mechanistic (molecular/physiological) basis for the behavioral effects observed is well worth pursuing.
8) It is also noted that ATZ levels in water or accumulated levels in fish were not analyzed which is very important to establish the true impact.
Regarding analytical confirmation of dosing concentrations used in the present study, I agree the protocol would have been greatly improved by including intermittent/periodic quantification of ATZ levels in the water for all subjects throughout the 12-week exposure period in order to more regularly verify the accuracy of the waterborne levels prepared for each experimental tank. However, my colleagues and I did in fact measure and confirm ATZ levels in the water prepared for a subset of three LD, HD, and control tanks at the end of the 1st week of exposure. Those data should have been incorporated into the initial submission. They have been added to the methods section of the revised MS as follows (lines 144-150; 154-155, pg 4):
“The concentration of atrazine in the water column was ascertained from three randomly selected LD, HD, and control tanks at the end of the first of the 12-week exposure period using methodology employed in a similar study of ATZ exposure in guppies [2]. The average concentration was determined to be 0.35 ppb and 13.22 ppb for the low- and high-dose, respectively, with negligible loss over the 7 days. No atrazine was detected in the control samples.”
Regarding assessment of accumulated ATZ levels in the fish themselves, again this would have made a valuable contribution to the project. However, we did not have the expertise nor the resources available in our laboratory facilities to conduct such analyses on our fish. That said, as previously stated, we feel the behavioral results alone serve as a strong and compelling example of the impacts of AZT exposure on P. latipinna.
Reviewer 2 Report
I have nothing to add to the author, except congratulations for writing an excellent piece of scientific research.
Author Response
Dear Ms. Isla Xu,
Thank you once again for all your time and efforts. Based on your feedback, received on December 22nd 2022, I have made the recommended revisions to manuscript " Environmentally realistic waterborne atrazine exposure affects behavior in Poecilia latipinna" (Manuscript ID: water-2083366). I have made efforts to address all concerns articulated in the reviewer comments. Should the reviewers feel the paper is in need of further changes, I will be more than happy to make any further modifications they see fit.
Again, thank you very much for your time and consideration, and for granting me the extension on resubmission during the holiday season. It is much appreciated. Please let me know if you have any further questions or concerns.
Sincerely,
David MacLaren Ph.D.
Professor of Biology
Merrimack College
315 Turnpike St. North Andover, MA 01845
maclarenr@merrimack.edu
Reviewer #2
I have nothing to add to the author, except congratulations for writing an excellent piece of scientific research.
Thank you for your time and efforts in reviewing the MS and for your kind words of praise and support.
Reviewer 3 Report
General comment
In the manuscript water-2083366 entitled “Environmentally realistic waterborne atrazine exposure affects behavior in Poecilia latipinna” MacLaren examined the effects of environmentally realistic exposure to atrazine on reproductive behavior, aggression, anxiety and boldness of sailfin mollies, Poecilia latipinna. Generally, the manuscript is well written with many details. However, the text needs to be better structured. Moreover, most major publications these days require analytical confirmation of dosing concentrations used in toxicity studies. I strongly affirm the need for analytical confirmation data in such studies. Use of nominal concentrations is insufficient. How can you be sure you treated your subjects with the appropriate dosages/treatment concentrations in the first place?
Specific comments
· Abstract is too long and needs to be better structured. One or two sentences providing a basic introduction to the field, comprehensive to a scientist in any discipline. Two to three sentences of more detailed background, comprehensible to scientists in related disciplines. One sentence clearly stating the general problem being addressed by this particular study. One sentence summarizing the main result (with the words here we show or their equivalent). Two or three sentences explaining what the main result reveals in direct comparison to what was thought to be the case previously, or how the main result adds to previous knowledge. One or two sentences to put the results into a more general context.
· Keywords: the keywords of the paper should not contain any words already in the title but can include abbreviated terms or location information not suitable for the title. Please, rewrite.
· Line 51: Please, remove “also see review”
· Line 54. Please, remove “but see”
· Line 240. Please, provide the statistical tests, p-level and statistical software.
· Line 270. Why did you use T-test? Did you check the normality of your data? Otherwise, you should use non-parametric tests.
Author Response
Dear Ms. Isla Xu,
Thank you once again for all your time and efforts. Based on your feedback, received on December 22nd 2022, I have made the recommended revisions to manuscript " Environmentally realistic waterborne atrazine exposure affects behavior in Poecilia latipinna" (Manuscript ID: water-2083366). I have made efforts to address all concerns articulated in reviewer #3’s comments outlined below. Should the reviewers feel the paper is in need of further changes, I will be more than happy to make any further modifications they see fit.
Again, thank you very much for your time and consideration, and for granting me the extension on resubmission during the holiday season. It is much appreciated. Please let me know if you have any further questions or concerns.
Sincerely,
David MacLaren Ph.D.
Professor of Biology
Merrimack College
315 Turnpike St. North Andover, MA 01845
maclarenr@merrimack.edu
Reviewer #3
General comment
In the manuscript water-2083366 entitled “Environmentally realistic waterborne atrazine exposure affects behavior in Poecilia latipinna” MacLaren examined the effects of environmentally realistic exposure to atrazine on reproductive behavior, aggression, anxiety and boldness of sailfin mollies, Poecilia latipinna. Generally, the manuscript is well written with many details. However, the text needs to be better structured. Moreover, most major publications these days require analytical confirmation of dosing concentrations used in toxicity studies. I strongly affirm the need for analytical confirmation data in such studies. Use of nominal concentrations is insufficient. How can you be sure you treated your subjects with the appropriate dosages/treatment concentrations in the first place?
Regarding analytical confirmation of dosing concentrations used in the present study, I agree the protocol would have been greatly improved by including intermittent/periodic quantification of ATZ levels in the water for all subjects throughout the 12-week exposure period in order to more regularly verify the accuracy of the waterborne levels prepared for each experimental tank. However, my colleagues and I did in fact measure and confirm ATZ levels in the water prepared for a subset of three LD, HD, and control tanks at the end of the 1st week of exposure. Those data should have been incorporated into the initial submission. They have been added to the methods section of the revised MS as follows (lines 147-152; 157-158, pg 4):
“The concentration of atrazine in the water column was ascertained from three randomly selected LD, HD, and control tanks at the end of the first of the 12-week exposure period using methodology employed in a similar study of ATZ exposure in guppies [2]. The average concentration was determined to be 0.35 ppb and 13.22 ppb for the low- and high-dose, respectively, with negligible loss over the 7 days. No atrazine was detected in the control samples.”
Specific comments
- Abstract is too long and needs to be better structured. One or two sentences providing a basic introduction to the field, comprehensive to a scientist in any discipline. Two to three sentences of more detailed background, comprehensible to scientists in related disciplines. One sentence clearly stating the general problem being addressed by this particular study. One sentence summarizing the main result (with the words here we show or their equivalent). Two or three sentences explaining what the main result reveals in direct comparison to what was thought to be the case previously, or how the main result adds to previous knowledge. One or two sentences to put the results into a more general context.
The abstract has been shortened from 405 to 300 words and further revised with reviewer feedback in mind to read as follows:
Abstract: The present study examined the effects of environmentally realistic exposure to atrazine (ATZ) on the behavior of sailfin mollies, Poecilia latipinna. ATZ is one of the most commonly used pesticides in the US and a known endocrine disrupting chemical (EDC). The behavior of sailfin mollies is well documented in the scientific literature. Moreover, they are ecologically important indicators of environmental health, widely distributed among the mangroves along the Gulf Coast of the Southeastern US where significant amounts of ATZ are introduced via runoff and storm water drainage from coastal households, businesses, and farms. Four sets of experiments designed to assess ATZ’s impact on various aspects of male and female reproductive behavior, aggression, anxiety and boldness were conducted following 12 weeks of exposure to either 1 or 15 ppb water-borne ATZ, along with a no-exposure control group. Results indicated the behavior of ATZ-exposed individuals differed from those of controls: ATZ exposure affected which stimulus fish (a male vs a female) subject females preferred to associate with while also affecting female strength of preference for males of larger body size and their sexual receptivity to conspecific males in general. ATZ-exposed males also showed reduced overall responsive to conspecific stimuli and directed significantly less aggression towards their mirror image compared with controls. Lastly, ATZ exposure affected multiple aspects of male and female behavior that are often used as proxies for boldness and anxiety. Overall, ATZ exposure resulted in alterations across a variety of behaviors attributed to sexual receptivity, mate choice and motivation to mate, aggression, as well as boldness and anxiety. Such ATZ-induced behavioral changes may adversely affect the long-term health of natural populations exposed to similar, environmentally realistic concentrations and add to a growing body of empirical data demonstrating substantial fitness consequences of exposure to sub-lethal concentrations of this know EDC.
- Keywords: the keywords of the paper should not contain any words already in the title but can include abbreviated terms or location information not suitable for the title. Please, rewrite.
Eliminated “P. latipinna” and “atrazine” because they are already in the title.
Added “Florida Gulf Coast” as it was the location where the test subjects were initially sampled.
- Line 51: Please, remove “also see review”
Done
- Line 54. Please, remove “but see”
Done
- Line 240. Please, provide the statistical tests, p-level and statistical software.
- Line 270. Why did you use T-test? Did you check the normality of your data? Otherwise, you should use non-parametric tests.
Section 2.6 from the Methods section of the MS has been significantly revised in efforts to: 1. describe more clearly the methods of video analysis used in assessment of fish behavior; 2. provide more information regarding the statistical tests, p-level and statistical software used; and 3. Provide a rationale for the use of T-tests including a statement about the normality of your data and use of non-parametric equivalents.
2.6. Behavioral measures and statistical analyses
For expts. I-III, test subject association preferences were based on total time spent with one dummy relative to the total time spent with the other dummy of the pair. The video data collected on iPads in expt. I-III were analyzed as follows: The total amount of time the subject spent in the preference zones of each of the two dummy stimuli during the course of the 20 min. testing period (irrespective of the number of separate visits made to a given dummy preference zone) were summed for each subject. These measures served as the raw data for all statistical analyses that followed.
For expts. I and II, paired samples t-tests were used to compare the total time subjects spent in association with the male versus the female dummy stimulus. Mean strength of preference (SOP), defined as time spent with the male stimulus—time with the female stimulus per 20 min test, were then calculated and compared among the LD, HD, and control groups of expts. I-III using One-way Repeated Measures ANOVAs, where ‘SOP’ and ‘treatment’ served as the dependant and independent variables, respectively [59, 62, 66]. Where appropriate, the Holm-Sidak method for multiple comparisons was used to test for differences in SOP among treatments.
For expt. III, paired samples t-tests were used to compare the total time female subjects spent in association with the larger- versus smaller male dummy stimulus for all three trials (L vs S; L vs I; and I vs S) for all three treatment groups (LD, HD, and controls). Mean strength of preference (SOP), defined as time spent with the larger male—time with the smaller male per 20 min test, were then calculated and compared among the three paired combinations of dummy males (L vs S; L vs I; and I vs S) for each of the three treatment groups (LD, HD, and controls) again using One-way Repeated Measures ANOVAs, where ‘SOP’ and ‘treatment’ served as the dependant and independent variables, respectively [59, 66, 75]. As described for expts. I and II above, the Holm-Sidak method for multiple comparisons was used to test for differences in SOP among treatments where appropriate. Because each female received three trials (L vs S; L vs I; and I vs S), all significance levels were adjusted to p<.0167 (Bonferroni procedure; Sokal and Rohlf 1981).
The ‘overall responsiveness’ of subjects to the conspecific stimuli (defined as total time spent in association with both dummy stimuli during the course of the 20 min test period) was calculated for each subject and compared among the LD, HD, and control groups for each of the first three experiments (I-III) using One-way Repeated Measures ANOVAs, where ‘overall responsiveness’ and ‘treatment’ served as the dependant and independent variables, respectively. Once again, where appropriate, the Holm-Sidak method for multiple comparisons was used to test for differences in overall responsiveness among treatments.
For expt. IV, iPad video data were used to quantify the total time (s) male subjects spent interacting with their mirror image (within the 10 cm mirror zone). These data were then used to compare total miror zone time for males among the LD, HD, and control groups using One-way Repeated Measures ANOVAs, where ‘time spent in the mirror zone’ and ‘treatment’ served as the dependant and independent variables, respectively. The Holm-Sidak method for multiple comparisons was then used to test for differences in mirror zone time (a proxy for agression) among treatments where appropriate.
Lastly, video data collected during each subject’s 10 min. acclimation period were analyzed for BZT and LTZ. These data were complied across all four experiments and compared among the LD, HD, and control groups of each experiment using One-way Repeated Measures ANOVAs, with ‘BZT’ and ‘LTZ’ the dependant variables and ‘treatment’ as the independent variable. The probabilities reported for all statisical analyses are two-tailed. Standard parametric tests were applied to the data as no violations of the assumptions of normality and/or equal variance were identified. All statistical tests were conducted using SigmaStat (version 11.2).
Reviewer 4 Report
In the manuscript ID water-2083366, the author reported an interesting study about the effects of atrazine (a well-known endocrine disruptor) on some behavioural patterns of sailfin molly, and found sub-lethal effects mainly at the lowest concentration tested (1 ppb). Although the approach used in this laboratory study has some limitations, the author clearly addressed and discussed each issue. Moreover, the manuscript is well written, and the experimental design correct and adequately described.
I only suggest adding a description of the statistical tests used to analyse the data in the subchapter 2.6. Did you use both ANOVA and paired-samples T-tests as reported in the results and figure captions?
Minor comments
Line 52 – environmental effects
Line 58 – fish [18],
Line 123 – environmental contaminants
Line 296 – Figure 4
Table 1 – Use S, I and L abbreviations and specified the meaning in the caption
Line 320 – response
Author Response
Dear Ms. Isla Xu,
Thank you once again for all your time and efforts. Based on your feedback, received on December 22nd 2022, I have made the recommended revisions to manuscript " Environmentally realistic waterborne atrazine exposure affects behavior in Poecilia latipinna" (Manuscript ID: water-2083366). I have made efforts to address all concerns articulated in the reviewer comments. Should the reviewers feel the paper is in need of further changes, I will be more than happy to make any further modifications they see fit.
Again, thank you very much for your time and consideration, and for granting me the extension on resubmission during the holiday season. It is much appreciated. Please let me know if you have any further questions or concerns.
Sincerely,
David MacLaren Ph.D.
Professor of Biology
Merrimack College
315 Turnpike St. North Andover, MA 01845
maclarenr@merrimack.edu
Reviewer #4
In the manuscript ID water-2083366, the author reported an interesting study about the effects of atrazine (a well-known endocrine disruptor) on some behavioural patterns of sailfin molly, and found sub-lethal effects mainly at the lowest concentration tested (1 ppb). Although the approach used in this laboratory study has some limitations, the author clearly addressed and discussed each issue. Moreover, the manuscript is well written, and the experimental design correct and adequately described.
I only suggest adding a description of the statistical tests used to analyse the data in the subchapter 2.6. Did you use both ANOVA and paired-samples T-tests as reported in the results and figure captions?
Section 2.6 from the Results section of the MS has been significantly revised in efforts to: 1. describe more clearly the methods of video analysis used in assessment of fish behavior; 2. provide more information regarding the statistical tests, p-level and statistical software used; and 3. Provide a rationale for the use of T-tests including a statement about the normality of your data and use of non-parametric equivalents.
2.6. Behavioral measures and statistical analyses
For expts. I-III, test subject association preferences were based on total time spent with one dummy relative to the total time spent with the other dummy of the pair. The video data collected on iPads in expt. I-III were analyzed as follows: The total amount of time the subject spent in the preference zones of each of the two dummy stimuli during the course of the 20 min. testing period (irrespective of the number of separate visits made to a given dummy preference zone) were summed for each subject. These measures served as the raw data for all statistical analyses that followed.
For expts. I and II, paired samples t-tests were used to compare the total time subjects spent in association with the male versus the female dummy stimulus. Mean strength of preference (SOP), defined as time spent with the male stimulus—time with the female stimulus per 20 min test, were then calculated and compared among the LD, HD, and control groups of expts. I-III using One-way Repeated Measures ANOVAs, where ‘SOP’ and ‘treatment’ served as the dependant and independent variables, respectively [59, 62, 66]. Where appropriate, the Holm-Sidak method for multiple comparisons was used to test for differences in SOP among treatments.
For expt. III, paired samples t-tests were used to compare the total time female subjects spent in association with the larger- versus smaller male dummy stimulus for all three trials (L vs S; L vs I; and I vs S) for all three treatment groups (LD, HD, and controls). Mean strength of preference (SOP), defined as time spent with the larger male—time with the smaller male per 20 min test, were then calculated and compared among the three paired combinations of dummy males (L vs S; L vs I; and I vs S) for each of the three treatment groups (LD, HD, and controls) again using One-way Repeated Measures ANOVAs, where ‘SOP’ and ‘treatment’ served as the dependant and independent variables, respectively [59, 66, 75]. As described for expts. I and II above, the Holm-Sidak method for multiple comparisons was used to test for differences in SOP among treatments where appropriate. Because each female received three trials (L vs S; L vs I; and I vs S), all significance levels were adjusted to p<.0167 (Bonferroni procedure; Sokal and Rohlf 1981).
The ‘overall responsiveness’ of subjects to the conspecific stimuli (defined as total time spent in association with both dummy stimuli during the course of the 20 min test period) was calculated for each subject and compared among the LD, HD, and control groups for each of the first three experiments (I-III) using One-way Repeated Measures ANOVAs, where ‘overall responsiveness’ and ‘treatment’ served as the dependant and independent variables, respectively. Once again, where appropriate, the Holm-Sidak method for multiple comparisons was used to test for differences in overall responsiveness among treatments.
For expt. IV, iPad video data were used to quantify the total time (s) male subjects spent interacting with their mirror image (within the 10 cm mirror zone). These data were then used to compare total miror zone time for males among the LD, HD, and control groups using One-way Repeated Measures ANOVAs, where ‘time spent in the mirror zone’ and ‘treatment’ served as the dependant and independent variables, respectively. The Holm-Sidak method for multiple comparisons was then used to test for differences in mirror zone time (a proxy for agression) among treatments where appropriate.
Lastly, video data collected during each subject’s 10 min. acclimation period were analyzed for BZT and LTZ. These data were complied across all four experiments and compared among the LD, HD, and control groups of each experiment using One-way Repeated Measures ANOVAs, with ‘BZT’ and ‘LTZ’ the dependant variables and ‘treatment’ as the independent variable. The probabilities reported for all statisical analyses are two-tailed. Standard parametric tests were applied to the data as no violations of the assumptions of normality and/or equal variance were identified. All statistical tests were conducted using SigmaStat (version 11.2).
Minor comments
Line 52 – environmental effects
done
Line 58 – fish [18],
done
Line 123 – environmental contaminants
done
Line 296 – Figure 4
changed
Table 1 – Use S, I and L abbreviations and specified the meaning in the caption
These changes have been made to Table 1 and the accompanying caption in the revised MS.
Line 320 – response
Done
Thank you for your time and efforts in reviewing the MS and for your kind words of praise and support.
Round 2
Reviewer 1 Report
Dear author,
I really appreciate your efforts to modify the paper and answering the questions in very effective way.
I think the paper can be accepted now.
Best regards,
Reviewer 3 Report
The Author has addressed all the comments. The manuscript is now ready for publication in Water.
