Impact of Global Warming on Kryal Fauna: Thermal Tolerance Response of Diamesa steinboecki (Goetghebuer, 1933; Chironomidae)
Round 1
Reviewer 1 Report
The manuscript is dealing with important topic such as climate change and the possibility of organisms to adapt, particularly in the high altitude habitats.
Experiment was well designed, although it would be interesting to see how do larvae cope with prolonged stress, not just an hour.
Overall, I believe that the researchers studying kryal fauna and habitats, climate change, aquatic Diptera, as well as HSP and gene expression, will find these results useful and interesting.
The text of the manuscript has several issues that need to be addressed:
Uniform use and writing of the abbreviations for proteins and genes. There are too many inconsistencies.
Genus and species names are often not written in italic - that has to be corrected.
References - format has to done according to the journal propositions.
Also, the Figure 2. "Whisker boxes are shown. The number of larvae for each box is three. The horizontal line within the box indicates the median...." You can hardly see the box, let alone the median.
Some of the sentences have been unclear, or wrong word or verb was used.
Additional references are needed in the Discussion.
All other comments are given in the revised manuscript.
Comments for author File: 
Comments.pdf
Author Response
(Comments and Suggestions for Authors)
Firstly, the manuscript has been peer reviewed by a professional English proofreading service, Proof-Reading.service.com.
The manuscript is dealing with important topic such as climate change and the possibility of organisms to adapt, particularly in the high altitude habitats.
Thank you to the revisor for recognize the interest of this research.
Experiment was well designed, although it would be interesting to see how do larvae cope with prolonged stress, not just an hour.
We agree with the reviewer, however, this work was a first step approach, from these results we are thinking to design future assays in this line.
Overall, I believe that the researchers studying kryal fauna and habitats, climate change, aquatic Diptera, as well as HSP and gene expression, will find these results useful and interesting.
The text of the manuscript has several issues that need to be addressed:
Uniform use and writing of the abbreviations for proteins and genes. There are too many inconsistencies.
We apologize for these and we have checked all the text and eliminated these inconsistences and improved the final version.
Genus and species names are often not written in italic - that has to be corrected.
We have observed that once the text was transferred to the template, a large part of the species names did not maintain their initial format, so it has been revised and all of them are in italics.
References - format has to done according to the journal propositions.
We have checked the journal guideline and we have modified the references according to its rules.
Also, the Figure 2. "Whisker boxes are shown. The number of larvae for each box is three. The horizontal line within the box indicates the median...." You can hardly see the box, let alone the median.
We have tried to modify the figures, but the average is even weaker, so we have modified the quality but not the final format.
Some of the sentences have been unclear, or wrong word or verb was used.
The entire manuscript has been reviewed and improved , and professional service has reviewed the manuscript. Additional references are needed in the Discussion.
Some references have been included in the discussion to improve it.
The pdf is sent attached with the answers to all the changes and indications of the reviewer as well as the article including all the changes of each reviewer.
Reviewer 2 Report
The present paper reports on experiments in which 4th instar larvae of the ice fly Diamesa steinboecki, an inhabitant of very cold glacial waters, were exposed to shock treatment in the laboratory. The expression of four genes known to be respond to thermal stress were also evaluated in the larvae. Larvae exposed to high temperatures for an hour were able to tolerate much higher temperatures than expected, whereas the genetic studies provided mixed reactions. These were relatively simple studies and essentially preliminary in nature and the authors are rightly cautious about their interpretation. Nevertheless, the findings will be of interest to entomologists and stream ecologists working on alpine freshwater systems.
In the last few decades there has been considerable interest in alpine and glacial river systems and the field has advanced considerably over that period of time. Many ecologists have lumped Diamesa species at the generic level and work such as the present study show the importance of understanding the biology of individual species. A recent paper on the taxonomy of the D. steinboecki group (Makarchenko et al. 2022 Zootaxa 5125) would be worth citing and would seem to confirm that the Italian alps species is indeed steinboecki.
The paper has a good introduction and a comprehensive abstract. The methods are generally well described and expressed, and the limited statistics used are appropriate. Results are clearly presented in text and graphical form. The discussion largely comprises comparisons with other findings on temperature responses and thermal gene expression, and I wonder if it could be expanded to include some comment/speculation on why D. steinboecki might have such strong high temperature tolerance. What is known about the evolution and historical biogeography of the Diamesinae? Could the high tolerance of this species be something that has been retained from ancestors that lived in more benign climates, despite their now being confined to cold kryal habitats?
The paper seems adequately referenced although perhaps some of the fundamental papers describing glacial stream communities could be added to the introduction. For example, early papers by Milner and by Ward for example. See Freshwater Biology 32 (2) 1994. The reference list also needs attention to standardize the format of papers included. All species and generic names in the titles of papers need to be in italics.
My main concern with the manuscript is that much of the written text would benefit from considerable improvement in the English expression to make it both more readable and grammatically correct.
Specific points
Names of insects (genus and species, including genus when used alone) should be in italics throughout the paper.
Authorities and dates are given for some species names but not others. Be consistent, one way or the other.
Line 44. You say Diamesa species are the sole colonizers of kryal habitats. I think you will find other taxa have been reported in the literature. Certainly, Diamesa is dominant. I would like to see kryal defined here, especially for non-freshwater ecologists.
Line 58. How is D. steinboecki being used as a bioindicator of climate change? By its absence from places where it was known to occur?
Line 105. I assume B. antarctica is Belgica antarctica as written inline 125. Give full name on first mention.
Line 110. Does ‘on the trail’ refer to the path from the collecting site to the car? Clarify.
Line 150. Was a substrate provided for the larvae to settle on?
Figure 1. What are the error bars? State in the caption.
Line 230. By temperature variation do you mean temperature increase?
Author Response
Reviewer 2:
Comments and Suggestions for Authors
The present paper reports on experiments in which 4th instar larvae of the ice fly Diamesa steinboecki, an inhabitant of very cold glacial waters, were exposed to shock treatment in the laboratory. The expression of four genes known to be respond to thermal stress were also evaluated in the larvae. Larvae exposed to high temperatures for an hour were able to tolerate much higher temperatures than expected, whereas the genetic studies provided mixed reactions. These were relatively simple studies and essentially preliminary in nature and the authors are rightly cautious about their interpretation. Nevertheless, the findings will be of interest to entomologists and stream ecologists working on alpine freshwater systems.
We want to thank the reviewer for his/her good assessment of our work and, as he/she well emphasizes, it is a preliminary study but it can open different paths for future work, due to the great importance of these organisms to know the impact of climate change in extreme habitats.
In the last few decades there has been considerable interest in alpine and glacial river systems and the field has advanced considerably over that period of time. Many ecologists have lumped Diamesa species at the generic level and work such as the present study show the importance of understanding the biology of individual species. A recent paper on the taxonomy of the D. steinboecki group (Makarchenko et al. 2022 Zootaxa 5125) would be worth citing and would seem to confirm that the Italian alps species is indeed steinboecki.
This reference is really interesting and we agree to include it in the manuscript, in the introduction section.
The paper has a good introduction and a comprehensive abstract. The methods are generally well described and expressed, and the limited statistics used are appropriate. Results are clearly presented in text and graphical form. The discussion largely comprises comparisons with other findings on temperature responses and thermal gene expression, and I wonder if it could be expanded to include some comment/speculation on why D. steinboecki might have such strong high temperature tolerance. What is known about the evolution and historical biogeography of the Diamesinae? Could the high tolerance of this species be something that has been retained from ancestors that lived in more benign climates, despite their now being confined to cold kryal habitats?
It is arduous speculate on this topic, Diamesinae is a subfamily of cold adapted taxa and within species, Diamesa steinboecki is the best cold-adpated in the Alps (Lencioni et al. 2021). No references are available in literature on heat tolerance for these taxa, apart from studies cited in this ms.
Lencioni V, Rodriguez-Prieto A., Allegrucci G. (2021) Congruence between molecular and morphological systematics of Alpine non-biting midges (Chironomidae, Diamesinae). ZOOLOGICA SCRIPTA, 50(4), 455-472. https://doi.org/10.1111/zsc.12480
The paper seems adequately referenced although perhaps some of the fundamental papers describing glacial stream communities could be added to the introduction. For example, early papers by Milner and by Ward for example. See Freshwater Biology 32 (2) 1994. The reference list also needs attention to standardize the format of papers included. All species and generic names in the titles of papers need to be in italics.
A reference on macroinvertebrate community was added:
Milner, A. M., Brittain, J. E., Castella, E. & Petts, G. E., 2001: Trends of macroinvertebrate community structure in glacial fed streams in relation to environmental conditions: a synthesis. – Freshwater Biology 46: 1833–1848.
All the reference list has been checked and improved.
My main concern with the manuscript is that much of the written text would benefit from considerable improvement in the English expression to make it both more readable and grammatically correct.
The entire manuscript has been reviewed by a proofreading service, so the certificate is attached.
Specific points
Names of insects (genus and species, including genus when used alone) should be in italics throughout the paper.
We have reviewed and modified these mistakes.
Authorities and dates are given for some species names but not others. Be consistent, one way or the other.
We have reviewed this issue to be consistency along the text.
Line 44. You say Diamesa species are the sole colonizers of kryal habitats. I think you will find other taxa have been reported in the literature. Certainly, Diamesa is dominant. I would like to see kryal defined here, especially for non-freshwater ecologists.
From lines 53-55 kryal habitat features are described. In the uppermost sectors of kryal (eu and metakryal), i.e. in the bedieres and in the first few hundred meters downstream of the glacier front, Diamesa can be the only colonizer. Others might be oligochaetes but not always present.
Line 58. How is D. steinboecki being used as a bioindicator of climate change? By its absence from places where it was known to occur?
- steinboecki could be a novelty and interesting climate change biomarker due to the fact that this species lives in a very specific temperature regime (2-4 °C), being very dependent on the temperature variations as poikilotherm organisms. So the continuous increase in temperature over the last decades has generated a decrease of this species in these environments in which previously it was the main colonize as observed in the article of Lencioni et al., 2022..
Lencioni, V., Stella, E., Grazia, M., & Bellin, A. (2022). Science of the Total Environment On the delay between water temperature and invertebrate community response to warming climate. Science of the Total Environment, 837(April), 155759. https://doi.org/10.1016/j.scitotenv.2022.155759
Line 105. I assume B. antarctica is Belgica antarctica as written inline 125. Give full name on first mention.
Done
Line 110. Does ‘on the trail’ refer to the path from the collecting site to the car? Clarify.
In this case, the trail is the route from the sampling area to the car since the larvae were collected in glacier-fed streams at high altitudes, so it must be accessed using a cable-car and walking. We have clarified this issue in the manuscript.
Line 150. Was a substrate provided for the larvae to settle on?
No substrate was employed during the acclimatization or subsequent exposures.
Figure 1. What are the error bars? State in the caption.
The error bars correspond to the standard deviation
Line 230. By temperature variation do you mean temperature increase?
As poikilotherm organisms is dependent on the surrounding temperature, in this context in this case the variation refers to an increase in said temperature.
Author Response File: Author Response.pdf
Reviewer 3 Report
my comments are directly in pdf and docx
Comments for author File: Comments.zip
Author Response
All the responses to the reviewer are included in the attached pdf, as well as the supplementary material with the corrections and the final version of the manuscript with all the changes applied according to the suggestions of the 3 reviewers.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
I don't have any further comments.
