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Dear Dr Höjesjö,
Thanks for resubmitting your manuscript and clarifying any remaining issues. The manuscript is now ready for acceptance.
Dear Dr Höjesjö,
The prior reviewers have now commented on your revised manuscript, and you will see that changes are still requested. The reviewers' comments are appended below.
With respect to my own comments on your original manuscript, in the methods section, please write the full model in the as yijk = u + effecti + effectj, etc.
Overall I found the reporting to be OK in this manuscript.
If found not major flaws in the experimental design. Can you please describe the location where the chemical cue is injected into the experimental tank.
It is difficult to assess the conclusions and implications of this paper. While I do not disagree with the authors that chemical cue do not appear to incite any behavioral responses equal to, or greater, than those of the visual cue, and there do not appear to be any additive effects, the data is not overwhelmingly supportive. In this study the authors used a series of metrics to assess the behavioral and physiological reactions to visual, chemical and combined cues. The first metric was the position of the fish relative to dominant fish tank. The most supportive data in this paper was the fact that both the visual and combined cues moved closer to the dominant tank compared to the control and chemical cue alone. First, it seems counterintuitive to me that the submissive fish would move closer to the dominant fish, however the authors claim this may be inspection behavior. Secondly, in the methods section it is unclear where the chemical cue was pumped into the experimental tank. Based on figure 1 it appears that the chemical cue was pumped into the side of the tank. If this is the case then one wouldn’t expect a fish to move toward the dominants fish’s tank. For chemical cues a better metric would be movement toward the source of the cue. This effectively biases the data to look for patterns which should appear in the visual studies and not appear in the chemical studies. Additionally, these behaviors broke down on the second and third observations.
The second metric was the activity of the fish. My first issue is that this data was collected as categorical data yet appeared to be averaged and treated as if it was continuous data. Secondly, there were no significant trends in this data set to support the hypothesis. Which argues the justification of its inclusion into this manuscript?
The final metric was the heart rate data. Based on the statistics, the authors mentioned that there was no significant difference between treatments over time, in observation 1. They did find evidence of a bradycardic response in the visual and combined cues, however when looking at the data this bradycardic response in the visual treatment is small and similar observations similar or greater were made prior to treatment. Which begs the question, was this a physiological reaction to the treatment or natural variability? Additionally figure 6 shows that there was this bradycardic response in males over all treatments and none in females over all treatment. Additionally, as with the other metrics, these patterns were not observed in subsequent observations. In the discussion (line 364-365) that patterns in this study were not similar to those in previous studies.
Overall, I find this research to be interesting and potentially relevant. However I am unassured as to the power of the conclusions based on these tenuous results.
Additional modifications
Line 143-144: “If such behaviors or injuries had been observed, the trials would have been stopped immediately”. This sentence is unnecessary since you previously statement that no such events occurred.
I would recommend moving figure 5 up to figure 3 to follow the order of the figures listed in the paper.
In the heart rate section, can you please give the sample sizes of the different treatments.
Also can you provide a sentence as to how the heart rate was calculated? Was it simply the number of beats in 10 seconds x 6 or was time between beats calculated?
Lines 310-311: I am not sure that I agree with the conclusion that a “strong” bradycardia response was observed after the visual treatment, given that similar responses were observed prior to the treatment and elsewhere in the dataset.
The authors have addressed roughly 55% of my concerns and chose not to modify their MS in relation to other 45%, providing various reasoning. I suggest that they continue to address the remaining 45% of items in my original review.
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Please address all my concerns
This is a really interesting study and it looks rather well designed. I have a few major comments. First of all, please have an English speaker correct the manuscript, in particular the material and methods and the results section. Some parts really need to be rewritten.
Please include line and page numbers in your submission.
Please move a large part of the introduction to the discussion.
In the methods section, write the full model as yijk = u + effecti + effectj, etc.
The results section is rather confusing. The Dyadic contest is really conducted only for determining which fish is dominant and which fish is submissive. The real test which thus appears in the results section is the visual and chemical exposure test. This test is repeated 4 times. In the results section you start at observation 1, however, this is the first repeat, right? What about the initial observation (Obs 0)?
I cannot understand what you did with the activity vs position index. Please explain this clearly.
I particularly am not convinced about your experimental set-up with respect to the sexes. How are they distributed over the 4 treatments? How are they distributed within confrontation? Is it always male against male, female against female, or male against female? The sex obviously influences your data so this must be very important.
Numbering should be with points separating decimals, not commas.
Fishes can only refer to fish species in plural, not to multiple individuals. The plural is ‘fish’.
In your discussion: why would fish move closer to the dominant fish?
Please see if you can reduce the number of figures.
I thought that this was a well-written paper. The text was clear and easy to understand. The introduction put the work in context with previous research and provided a basis for the current study. Overall, there are no basic reporting issues which I believe would prevent this paper from being published.
Overall, the experimental design was relatively good. The basic tank set-up and fish handling appeared logical and unbiased. The measurement system appeared sufficient. I had an issue with the position indexing system that was used. I found the explanation of the position index to confusing. Is this simply a coordinate system? I am assuming that it is one-dimensional since the data looks one dimensional. What are the values that were recorded, one through nine with nine being the front of the tank? Why was an real unit based system (cm or m) not used?
Another point of confusion had to do with the descriptions of the three observation periods. For the three testings (4,6 and 28 hrs) were the animals placed in the tank a time zero, then the treatment started (i.e. chemicals introduced, divider lifted) at each time or was the treatment started at 4 hours then the behaviors measured at 4,6 and 28 hrs? Can you please clarify.
In general I found the conclusions derived in this paper to be acceptable. As for the results section, I found this section to be more qualitative and data deficient then it need to be. The only data presented was the output of the statistics. In this study the significance level was set at 0.1, which while acceptable, appeared to be chosen due to the abundance of statistics which were above 0.05. This gave the general impression that while the trends and patterns may be present, they may not be strong or the sample size maybe too low. This ultimately leads to some reservations about the strength of the conclusions.
Below are a series of small changes to the text.
Introduction
Page 5, last paragraph, first line: “chemically” should be changed to “chemical”
Methods
Paragraph 1, sentence 2: change “app” to ~ or approx.
In the Dyadic contest section you refer to the experimental tank have a front and rear section. In the subsequent section you refer to section 1 and 2, and right and left. I would recommend using the same terminology to reduce confusion.
Visual and chemical exposure section, paragraph 2, sentence 2: replace “eg.” with “e.g.”
Results
Can you please put some data relevant to what size these fish were (i.e. mean +- sd) beyond ~one year old. I think that it might be useful for repeatability.
Page 14, Heart rate section: fix "bradychardiac" to bradycardiac" in both paragraphs.
Why are the error bars only on one side of the line?
Discussion
Page 17: “off course” should be “of course”
Page 17 end of second paragraph: fix “bradychardiac” to “bradycardiac”
Page 19, sentence 5 change section to “moved closer to opponents
no concerns
please see further
please see further
The study is clearly explained and reasonably designed, and makes interesting observations. However, I have several technical, but essential concerns [so I will focus on concerns, because all other aspects in this study are fine]:
Abstract - please remove the word "though" (not a common scientific word)
Intro - please explain what resource holding potential is (unclear to no-specialists)
Line 5 - "potential" is used twice on the same line
Intro - the notion that winners always remain winners is not always true, and hardly generalizable. Anyone who run chronic social defeat in mice knows that the status during paired confrontations can flip several times
ALSO - I am surprised to see a clear winner-loser distinction. Social defet with other species shows that some subjects (as many as 30% in some cohorts) are consistently giving a 'tie'
Intro is generally lengthy and hard to follow. Multiple species discussed here, and this does not help to send a clear message.
Intro - please clarify why this particular species was used. What is special about it? What was the rationale to use it?
ALSO - please explain 9at the end of Intro) why this study is important/needed/worth attention
Methods - please mention sex, and #males/#females here
Methods - I am not sure how long the confrontations lasted. Was not a standard duration used? If not, why?
Methods - it is totally unclear how dominant status was determined.
Methods - "we believe" is not a scientific language
Statistics - it is unclear which exactly ANOVA was used, what were the factors, and which post-hoc tests were used. Also, why P<0.1 (and not more conventional <0.5) was used?
Results - how can the sex of 2 fish not be determined, and what happened to these fish?
Discussion - "contrasting evidence" or "conflicting evidence"?
The entire Discussion section is way too long and unfocused. Multiple species are compared, and this may not always be necessary. I suggest putting all species in a table, which will be easy to assess, without too many words
Discussion - I am unclear about the idea that females are bolder because they better memorize the competitiveness of the opponent. I would say the opposite - poor memory and inability to remember prior failures - will also result in a bolder phenotype
Conclusion - the authors have studied only one species, but conclusion is generalized to all salmonids. This is not supported by these data.
ALSO - please make a stong statement explaining (or reiterating) why this study is important/interesting/necessary/valuable/useful
Figures - 8 figures is just way too many. Please reduce to 4
All figures with line diagrams are hard to read because most lines overlap, and so do the SEM bars.
ALSO - there are no asterisks in the figures, so it is totally unclear what is different from what
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