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You have dealt with the referees comments adequately. I look forward to seeing further research in this area and perhaps evidence that, as you have suggested may occur, lobster behaviour is tailored to deal with match-mismatch in ecological conditions.
# PeerJ Staff Note - this decision was reviewed and approved by Vladimir Uversky, a PeerJ Section Editor covering this Section #
Both reviewers have some further useful comments on your submission. I suggest you remove the stats from your abstract they are not really necessary there.
Please see attached review.
Please see attached review.
Please see attached review.
Please see attached review.
I have some suggestions to improve the basic reporting:
Line 58, 59: words “both” and “alike” maybe not needed
Line 62: change “as” to “are”
Line 66: remove word “also”
Line 88: I would say the word “lab” may be too colloquial
Line 102: change “we sought to test” to “we tested”
Line 150: maybe to increase succinctness remove “and placed in labeled plastic sample trays”.
Line 211: maybe remove “very”
Figure 2: please add standard error bars to the sampling points on the line graphs
Line 217: change “and these results are summarized in Tables 1 & 2” to “(Tables 1 & 2)”
Table 1: the means in this table appear to be exactly duplicating the information on Fig 2. I think the standard errors and post-hoc tests should be presented on Fig. 2 and Table 1 removed.
Table 2 legend: please mention in the 1st sentence that the values shown are P values.
Line 236: change “The main goal of this study was” to “Our main goal was”
Line 267: change “couple” to “two”
Line 272: The use of the word “real” here is a bit confusing, I would revise.
Line 305-309: this long sentence could be split in two.
Line 384: Change “dramatically” to “significantly”
Figure 2, 3: It is confusing for the reader that bar graphs were used Fig. 3 and line graphs Fig. 2, when they both show very similar time series. I would recommend for consistency to change all time series as line graphs, unless there is some specific reason its like this.
I think it would be useful for some of the discussion about pseudo-replication in the response to reviewers document to be briefly mentioned somewhere in the manuscript.
Line 102-104: The hypotheses should directly match the tests done (i.e. the actual statistical comparisons). The only factors tested here were temperature and time; no measurements of migration were made. Also, the temperatures were not changed according to any migrations, they just tested how eggs would develop from lobsters staying either inshore or offshore. So the hypotheses should state something like “It was hypothesized that different water temperatures, simulated in the laboratory to be similar to temperatures experienced by lobsters in varying coastal zones (inshore vs offshore), will affect egg biochemistry. This hypothesis was tested to gain information useful for understanding how egg development may be affected by lobsters migrating between those zones”
Line 207: the lack of significant difference in temperature between inshore and offshore water was surprising and may influence the validity of the conclusions, as this study is meant to inform about lobsters migrating between these areas to expose their eggs to different temperatures. Please provide some text about the implications of the non-significant temperature result in the discussion.
Line 324-326: I think this text needs to be clarified/corrected. I guess the “constant” temperature being referred to here is the one used in this experiment, which according to line 248 appears more optimal for growth, but here it is stated as “sub-optimal”. Also, if more protein is used for energy as stated here, then there should be less rather than more protein in the “constant” treatment which is not the case (Fig. 2).
To allow the reader to understand the conclusions better, please somewhere early in the discussion provide a clear statement about whether or not the results supported the specific hypotheses from the introduction.
Line 384-385: Please mention here why “there is still the potential”, rather than just saying that the potential exists. It seems the results may indicate that no evidence could be found to support the hypothesis that “variations in the energetics of embryogenesis influenced by the seasonal movements of some lobsters to and from these two disparate locations”, especially since these disparate locations did not even have significantly different temperatures according to my understanding (line 207). Also I think the word “are” is needed between “embryogenesis” and “influenced”.
I think a concluding sentence in the abstract stating the relevance of the results would be useful.
Line 39: add “potentially” between “thereby” and “exposing”, especially since this study did not appear to find any significant different (Line 207) between the two areas where migrations occur between.
Line 130: Here it says the “constant” treatment was 12 degrees, but in figure 1 it seems this treatment had 16 degrees. Please correct one or the other.
Line 211-217: According to how I understood this, here all the results are from the 2 factor analyses. Firstly the text here details the main effects, then results of the interactions. The interactions are all significant, which makes it redundant to mention the main effects as was done first (i.e. effects of the factors in isolation are not informative). If this understanding is correct, then the text from line 211-215 may not be needed.
Line 300-302: a reference needs to be provided for this statement.
Line 350-378: These paragraphs are on a different topic to the study and are sufficiently unrelated that they confuse the story a bit and I would say they are probably not needed, or could be reduced to a few sentences.
Both reviewers have made extensive comments and suggestions to improve the paper. I think that if you address their comments (and see my annotated pdf) this could be a good paper. You can certainly shorten it a bit by avoiding repeating data in both tables and figures (perhaps submit the data in the table as supplementary?
Please see attached pdf file.
English needs improvement.
Article structure ok but Figures 1 and 2 not needed.
Discussion should be more focused and stick to actual results.
Please see attached pdf file.
In general, the methods should be better explained.
Not sure egg volumes and biochemical content were measured at the same times, although methods suggest this was the case.
Measurements of egg biochemical content did not extend to close to time of hatching in the "inshore" and "offshore" treatments, compromising strong conclusions about the adaptive value for embryo development of thermoregulation through active movement of ovigerous females.
Please see attached pdf file.
The data appear to be sound, although some information is unspecified.
The basic reporting is well done. The writing style was a pleasure to read and the authors have done a good job with referencing the related background literature.
I will only make two points concerning the article structure:
1) I do not think the first two figures are really needed as they just show some photos of lab equipment.
2) I do not see why figure 4 shouldn't be incorporated as a line graph into figure 3, as it has the same predictor variables.
I think there are some issues with the experimental design:
1) The main issue is pseudoreplication. According to Hurlbert's review on pseudoreplication, the setup used in this experiment is classified as "isolative segregation" which is stated as one of the "various ways in which the principle of interspersion can be violated" (see Figure 1, Hurlbert, S.H., 1984. Pseudoreplication and the design of ecological field experiments. Ecological monographs, 54: 187-211). Ideally each lobster would be housed in is own separate tank and thus the eggs would be independent replicates that are randomly interspersed among other replicates. I think the authors will need to argue how their experimental setup can be valid despite the lack of interspersion of their treatments. Potentially the authors may be able to demonstrate that conditions (besides temperature) were similar among the treatments despite the fact they were isolated.
2) I had trouble following some aspects of the methods descriptions. Line 155-156 states there were 2 tanks used per treatment, and there were 3 treatments, so I was expecting 6 tanks, but line 165 states there were 4 tanks. Also, having 4 tanks does not seem to fit with having a total of 15 lobsters. Thus I was not able to work out how the experimental setup was organised, although it might become clear if the methods writing is changed/expanded and/or a diagram provided.
3) For this experimental design to work, all the treatments should have had eggs with the same level of development at the start of the experiment. From figure 3 it does seem the lipids and proteins were very similar among treatments, but in Figure 5 it seems there may have been differences in size at the 1st month. I would ask that the authors check (e.g. ANOVA) that all egg variables were statistically similar among treatments when the experiment started.
4) ANOVA assumes homogeneity of data variance, which does not appear to have been tested (e.g. Cochran's test or Levene's test). It could also be argued that as only 15 lobster were used, the sample size is quite low in which case normality of distributions will also be an important assumption to test. I would ask that the authors please address these aspects of their statistics.
5) On lines 153-155 there is a brief comment that "A subset of the eggs in each clutch were viewed under a dissecting scope and staged... These samples also served as covariates for all subsequent statistical analyses". The use of covariates is never mentioned again in the Data Analysis section nor any results concerning covariates in the results section. If covariates are used then the analysis will be an ANCOVA (not ANOVA as stated on line 223). If the analyses really did have a covariate then I would ask that there is more description of why and how this was done.
6) In the lipid and protein analyses, the constant temperature treatment was missing the last data point because the eggs hatched, and thus the design is unbalanced (i.e. May data for only 2 out of 3 treatments); was the statistical procedure used able to handle an unbalanced design? It would be my preference to have finalized the whole experiment as soon as it becomes impossible to continue getting data from one of the treatments. Overall, the fact that the design was unbalanced needs to be discussed.
7) I would say that the regression analyses (Figure 4) are not needed, as the significant effect of time and the pairwise tests in the ANOVA analyses provide all this information.
I would say that the validity of the findings is not certain at this stage due to potential issues with pseudoreplication and analysis assumptions, and the authors will need to do more convincing for the reader that their conclusions are valid.
Overall, if efforts were made to present this study in a concise way then it could have only 1 figure of results and perhaps 1 table. The discussion is well written and full of background information, but much of this is loosely related to the topic at hand and if this were reduced then the manuscript could probably only be considered as a short communication. Although it may be possible to work through most of the issues about experimental design, given that there are quite a few of them, and that the volume of results is quite low, I would be hesitant to support this manuscript without substantial and possibly fundamental changes.
here are a few further minor comments:
1) It would be very useful to see statistics tables and these will help the reader work out how the experiment was set up and the analyses done.
2) Line 221: Here you mention sampling of egg volumes was done at 5 times but Fig. 5 shows only 4 sampling times. Also on Fig. 5, why are the months different to the sampling months on the other graphs?
3) Please put SE bars on all data graphs.
4) I think all ecological laboratory studies should have at least some discussion/mention about the limitations of this approach. With enough effort it would be possible to measure lobster egg development in the field which would provide much higher quality results.
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