Impacts of Long-Term Exposure to Ocean Acidification and Warming on Three-Spined Stickleback (Gasterosteus aculeatus) Growth and Reproduction

Round 1

Reviewer 1 Report

This study aimed to look at the impact of ocean warming and acidification on growth and reproduction of the three-spined stickleback. The authors provided an interesting and important study, on long term exposure of the species to the stressor in different stages (juvenile and adult) and with a lot of different outputs. However the experimental design appears to not have been the more adequate, and that should be mention and discussed in the discussion. The discussion could also become stronger with if more examples and bibliography are added and explained. I believe this study meet the standards to publication, but only after addressing some issues regarding methodology and discussion clarity.

I have made some observations and specific comments below which I hope will help the authors to improve the clarity and quality of this study.

Abstract

L33-35 – The authors state the findings clearly indicate impacts of combine acidification and warming on the mortality, growth and reproduction, however in line 22 they state no size and mass differences were found.

The authors finishing line on long term impacts of acidification and warming on fish, should be specified to the species study as these findings cannot be inferred for any teleost fish.

Introduction:

Overall the introduction, browses over different subjects relevant to the study, however it feels too focus on the big picture and less focus on the more specific knowledge, needed to frame this study. The style of how the introduction is written varies from author to author, but it usually follows an inverted pyramid structure, where you first talk about the general subjects (like OA and OW), and then start to specify, for example to studies on similar species (temperate or tropical), or in similar habitats, or other more specific subjects related to your study.

L56-57 – The authors should add bibliography to support the statement

L64-67 – The authors should add bibliography after each process impacted by warming to support their statement e.g. “Warming is known to impact also fish sexual maturation” (REF) “…steroidogenesis” (Ref), etc.

L74-78 – Overall the authors tend to put all the references at the end of the sentences, instead of inserting the references for the statements, as they are writing them. I would advise they correct this, as it better to support their statements if they insert in the adequate place e.g. “The end result could represent an impact on key  physiological functions (basal metabolism, growth, reproduction)” (REF) “… behaviour (swimming activity, foraging, etc.)” (REF) “…and ultimately increase the vulnerability of the species” (REF).

L81-84 – Same problem as already mention regarding the placement of the references.

 L89-90 – The authors call out the need to diversify the species studied under OA and OW, which is in fact important, to have a better understanding of the effects of these stressors on fish species. However they proceed to present their study on the three-spined stickleback, which is a heavily used model, as they also acknowledge. This lessen their previous statement (about the use of more varied species), and make it seem out of place. I would suggest they move these considerations to the discussion where they can show, that although they used a heavily studied species, it is important to also study different, less used species.

L117 “growth by estimationing” correct to estimating

Materials and Methods

An overall critique to the study is the design. The authors state throughout the manuscript that the multi-stressors, long term studies are important to enable a more realistic prediction of climate change. Which is in fact true, although not always possible, and does not demerit short term studies or their results. But multi-stressors experimental designs should always consider the individual parameters to be tested in their design (a cross factorial design). In the present study the “Current scenario” tests temperature and pH at their present values, and “RCP8.5 scenario” tests temperature and pH for this scenario predictions for 2100. To assure that the results are from the interaction of temperature and pH, the authors should also have tested a warming scenario (where the temperature should be in the same value as the RCP8.5 scenario, but the pH was in the current scenario) and an acidification scenario (where the pH should be in the same value as the RCP8.5 scenario, but the temperature was in the current scenario). This would guarantee that the authors knew if only one stressor was influencing the experiment, or if it was in fact the interaction of both factors.

L127 “where obtained” correct to were

L132-133- The authors should specify which salinity the fish were in after the increase of 5‰ of salinity per week. They should also specify the age of the juveniles at the start of the experiment, and if the juveniles had different ages, different sizes and were randomly distributed in the tank, as this can impact the results.

It would also be valuable to share more studies where freshwater three-spined stickleback were acclimated to marine water environments. Is this a common practice in this species? Do study results differed between populations acclimated to marine water environments and populations original from marine waters?

L134-137 – The authors should expand a little more on the feeding process, explaining how did they define the fish were satiated, as well as how did they recorded the food intake: was it by observation, did they weight the food that entered the tanks and the food left on the tanks, etc?

L135 “The quantity given were” correct to was

L138 – Each experimental tank had 557 fish, please specify in the text if it was 557 per tank or in total

Figure 1 The authors should colour correct the graphic, or change the colour of the lines, so the line for the Current scenario it is not faded, as it is right now the line for Current scenario it is not readable. I would suggest using black and grey, or colours.

L175-177 - The explanation of the process is confused, try using collected instead of captured, and adding in the end that they were returned to their tanks, or something similar, the use of the terms captured and released may not be the most correct here.

L178 - How did the sedated fish recover? Considering you were sedating fish every two months, and you have probably sedated the same fish more than once, this should be discuss or at least mentioned, as it can influence the fish physiology, if you have studies that show it does not, please refer them in the manuscript.

L187 – If thermal growth only uses the mass of the fish in the beginning and the end of the experience, why did the authors measured size and weight every two months? If it was to use for a different analysis that is not understandable in the text.

L192 – Why was the final sampling of only 33 fish per scenario? The authors should explain it in the manuscript.

The statistical analysis chapter it is confusing, and it would benefit the reader if the authors try to describe the statistics used more straightforward. As they used a lot of different statistical approaches for the different outputs tested, it could be easier the use of a table, or a simplification in the text. For example, were the measurements of size and weight (taken every two months) used in the analysis? If so did the authors consider they have possible measure the same individual fish more than once, and there for used repeated measures in the statistics?

Results:

The results section presents valid information, however the presentation of the results in itself feels chaotic as the authors have, figures, tables, supplementary material and statistical data in the text (with the name of each test they use before the actual statistics). This makes the results harder to read, and since the authors already explained in the Statistical Analysis chapter all the statistical approach I would suggest they only report the value of the test and the p-value in the text (if the authors want to present all the statistics in the manuscript, maybe it is better if they were featured in the supplementary material).

For homogeneity I suggest all the labels where the graphs have the same pattern, to present the same text. Example: In figure 3 the authors say “Stickleback muscle lipid storage after 7 months under Current (in light grey) and RCP8.5 396 (in dark grey) scenario for female (white stripes) and male (no stripes)”, which is easy to understand, but in figure 4 for the same type of boxplots (for different results) they state “Light grey boxes refer to Current scenario and dark grey ones to RCP8.5 scenario. White stripes refer to female and their absence for male”. I would recommend standardizing your figure and table labels as it is easier for the reader to follow.

Figure 2 – The label of the figure should not have information for the indexes calculated or statistical information that does not regard the figure.

L463-464 – The authors don’t specify in what of the scenario the eggs were smaller

Discussion:

Overall the discussion is very broaden and presents valid and important points. There is however a lack of bibliography on similar studies for the authors to compare their results.

As mentioned before, the experimental design using only current scenario and RCP8.5 scenario lacks testing just the effects warming and acidification, with the RCP8.5 scenario values, by itself. This is important as it helps understanding which stressors are actually affecting the fish. For example if warming by itself impacts the fish, but acidification by itself does not, it is possible that in the interaction scenario, acidification also does not impact (in any direction) the fish, and it is still warming that is impacting the fish in the interaction scenario. This is important to be discussed by the authors, when they stated “…In our experimental scenario, the obtained results suggested that the combination of warming and acidification could increase the vulnerability of sticklebacks.”, and also throughout the discussion when considering the interaction of the stressors was what affected or not the fish.

The impact of using freshwater populations should be address, if it does not affect the study should be demonstrated with different studies, but should definitely be address here in the discussion and in the methods and possible also talked in the introduction, if it is relevant.

L514-516 – The authors’ observations about food intake are valid but they also should consider the size and age of the fish in the beginning of the experiment, and refer it in methods. If the fish had different sizes and weights in the beginning of the experience and were not randomly distributed in the tanks that can have an effect on their size and weight later on.

L565 – The authors should place the references after “stickleback” and then expand on what other species (with the references after each species), and in what conditions. This is important because not all warming studies conditions can be compared, and the authors should give examples of other studies that are relevant for the species and the habitat studied.

L608 – “The neuroendocrine control of reproduction appeared to be only slightly affected by the RCP8.5 scenario” Slightly affected it is not a statistical term, and should not be used to discuss results. The authors can talk and discuss trends but not assume any impact from it.

L653 – The authors should use “suggest” and not “evidence”

Regarding the phenotypic variation paragraph I suggest the authors take a different approach on it. As it is, the author’s statements on phenotypic plasticity use few or no references to back the sentences. For example: “Most of environmental reduction in phenotype variations is observed in wild population that face whole-life exposure to the changing environmental conditions.”

Papers like: Barbasch et al. Substantial plasticity of reproduction and parental care in response to local resource availability in a wild clownfish population; Seebacher et al. Physiological plasticity increases resilience of ectothermic animals to climate change; Gunderson et al. Plasticity in thermal tolerance has limited potential to buffer ectotherms from global warming; Bernal et al. Plasticity to ocean warming is influenced by transgenerational, reproductive, and developmental exposure in a coral reef fish; among others can help the authors in this part of the discussion.

Although phenotypic plasticity usually enhances and optimises organisms’ performances to environmental change, the authors should not use strong words as “indicate” or “evidence”, as they can’t ensure their results show phenotypic variation in the RCP8.5 scenario. Moreso the authors should also address developmental plasticity - that happens when organisms are exposed to changes during their development, and it is pertinent for their study.

Conclusion:

L678-681 - The authors should rephrase this last part, as they did a multi-stressor experiment, but not a cross factorial one, that would ensure to understand the effects of single stressors or the interaction of stressors.

Minor editing of English language required

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Warming and ocean acidification are a recognised threat to marine ecosystems as demonstrated by many worldwide works on this issue, hence any work on this topic is highly interesting since they always provide new points of view and, in addition, help to establish baselines for future studies. In general, the topic is properly introduced, the experimental design and statistical analyses seem correct and the results and discussion are adequate.

Some observations are as follows, and I hope that these help to improve the ms:

-. I would recommend the use of the scientific name in the title (even if it appears in keywords). The use of the common name is usual among Anglo-Saxons but it can lead to errors, especially taking into account that this species fish has different common names in English. In the same sense, when the scientific name of the species is cited for the first time in the main text, it is also advisable to write the author's name. In this case, Linnaeus, 1758.

-. Although I think it does not imply changes in the results obtained, perhaps it would be advisable to update to the new scenarios established in the new 2022 IPCC report. Now they are more pessimistic and five scenarios have been established, so that the one applied in this study could be the SSP3-7.0 scenario.

-. One question about the experimental design: was a single tank used per condition or were replicates established? The replication of the different conditions is important to avoid bias in the results due to causes external to the experimental conditions. This needs to be clarified.

-. Figure 2 caption: lines 366-379 “At the last sampling day,…p-value=0.77)” is main text.

-. Line 65: delete “;” after H2CO3.

-. Line 292: “Mai” is “May”.

-. Line 326: “Wilcox” is “Wilcoxon”.

-. Table 3 caption: Maybe it's better to use "asterisk" instead of "star"

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors revision of the manuscript increased its clarity and quality. The authors addressed all the questions and suggestions and I believe the manuscript meets the standards for publication. Down below I suggest just some minor changes to add to the manuscript

L118-120 and L127-129 – the authors repeat the same sentence in both paragraphs, it can be used just in the second paragraph.

L416 - the authors should add “significantly” to the text to emphasized the statistical significance

Table 3 - the authors should add the information that asterisk means a significant result

L470 - the authors should add “significantly higher” to the text to emphasized the statistical significance

L487 - the authors should add “significantly lower” to the text to emphasized the statistical significance

L586-587  - “Moreover, and in contrast to many other studies, sticklebacks were not exposed to constant temperatures during this experiment “ If the authors could give the references to 1 or 2 studies where the sticklebacks were exposed to constant temperatures, I believe the manuscript would be more complete.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf