High-Temperature Stress Induces Autophagy in Rainbow Trout Skeletal Muscle

Round 1

Reviewer 1 Report

Molina et al. analyze the impact of the increase in temperature on the rainbow trout's skeletal muscle. They study the alterations by studying different parameters such as quantifying different stress-related compounds and enzymes, oxidative stress, modulation of RNA levels by RNA-seq, and the changes of LC3 by western blot. From the data, the authors conclude that the autophagy is activated.

The introduction is informative. However, it should be included additional information about climate change. The authors mention that it is expected an increase of 1-4 ºC by the end of the century, but they have designed an experiment with a 5 ºC increase. It should be put in context so the reader can understand the relevance of the work concerning climate change. Why is a 5 ºC while a maximum 4 ºC is expected? In addition, it is a mean increase in the Earth's temperature, but is it the same in water as in the atmosphere? In addition, information about the regular habitat of the species should be included. Although it is a model organism, the reader should have information about the habitat where it is found in the wild. Finally, using "high temperature" for the treatment could confuse the reader. Five degrees increase is not a high temperature since other authors' articles indicate that it has been measured at 19 ºC in certain marine regions near the Chilean coast during summer (Dettleff et al., 2020). Therefore, using thermal exposure more than high-temperature treatment would be adequate.

The material and methods describe them adequately. However, clarifications are needed. Which was the producer of the commercial pellets to feed the animals? In addition, was the water circulating? What were the culture conditions (water composition, oxygenation, …)? The animals used were juveniles? It is not specified the time of acclimation before the experiment, so the stage of the animals should be indicated because it has relevance because of the different sensitivity to the stress of juveniles and adults. The experimental design involves exposure for four days after reaching the temperature. Why was selected this time? To euthanize the animals, which was the anesthetic used?

Cayman has more than one Elisa kit to detect cortisol, so the catalog number should be included. Similarly, Abcam has different glucose kits, so the catalog number should be included. Including the catalog number would also be convenient for the kits used in DNA and protein oxidative damage.

Was the EZNA Total RNA Kit I or the EZNA Total RNA Kit II used for RNA extraction? The transcriptome accession number should be included to allow other researchers access.

Concerning Real-Time PCR, which was the efficiency of the primer sets used? In addition, were the reference genes tested to confirm the adequacy (using any of the tools available such as GeNorm, NormFinder, …)?

Which was the protease inhibitor cocktail? Homemade? Commercial? Which was the antibody dilution in each case? Was the film scanning done using a scanner or taking a picture?

In the statistical analysis, were the data tested to confirm that they follow a normal distribution and the homogeneity of variances?

The results are well described. Unfortunately, the bar graphics of Figures 1 and 2 have no sense. Providing the dots and removing the bars is more informative since they confuse the observer. It is striking to observe that some genes (hspa1l, hspa4, hsp90ab1) which code for heat shock proteins are increased, but there is no indication in the KEGG pathways of heat shock response. Instead, they are assigned to other processes, including response to infection. Also, it is striking to observe that atp2a1 is downregulated while atp2a2 is upregulated. Analyzing them by Real-Time PCR to confirm the data would be interesting.

The discussion fits the results. However, it is striking that it is not mentioned at any moment that the temperature increase also has relevance in the basal metabolism, increasing it on ectotherms. In addition, it is relevant when juveniles are used since they suffer a development that can be interfered with by the temperature increase. Since physiology is altered, should be mention anything about it. Furthermore, the juvenile is more sensitive to stress than the adult, justifying some differences observed between the literature and the results obtained in this article. The thermal response can involve different steps and is also affected by the treatment used, so it should be considered to get a clear picture of the results obtained. The physiological alterations in acute response and chronic response to thermal stress differ. It would be interesting to have more than a one-time point to elucidate the modulation of the genes, which can also respond to an acclimation process. Finally, although climate change is mentioned in the introduction, it is not considered at any moment in the discussion or the conclusions. Which would be the relevance of the results obtained in surviving climate change?

Author Response

We would like to thank to the reviewer for providing positive comments of our manuscript. After a revision of the manuscript based on the comments of all the reviewers, we have made the final modifications to address all concerns, comments and suggestions expressed. English language edition was made (certificate attached)

Molina et al. analyze the impact of the increase in temperature on the rainbow trout's skeletal muscle. They study the alterations by studying different parameters such as quantifying different stress-related compounds and enzymes, oxidative stress, modulation of RNA levels by RNA-seq, and the changes of LC3 by western blot. From the data, the authors conclude that the autophagy is activated.

The introduction is informative. However, it should be included additional information about climate change. The authors mention that it is expected an increase of 1-4 ºC by the end of the century, but they have designed an experiment with a 5 ºC increase. It should be put in context so the reader can understand the relevance of the work concerning climate change.  Why is a 5 ºC while a maximum 4 ºC is expected? In addition, it is a mean increase in the Earth's temperature, but is it the same in water as in the atmosphere? In addition, information about the regular habitat of the species should be included. Although it is a model organism, the reader should have information about the habitat where it is found in the wild. Finally, using "high temperature" for the treatment could confuse the reader. Five degrees increase is not a high temperature since other authors' articles indicate that it has been measured at 19 ºC in certain marine regions near the Chilean coast during summer (Dettleff et al., 2020). Therefore, using thermal exposure more than high-temperature treatment would be adequate.

Response: We incorporated the required information in the new version of the manuscript (lines 47-53). The species under study is relevant to national aquaculture. Although this specie is found in natural habitats is considered invasive. The purpose of this study is to provide information of its adaptive capacity under farming conditions, not in natural habitats. We chose a difference of 5° between the control and stress conditions, for comparison purposes. The objective is to compare the adaptive response of rainbow trout with red cusk eel (a farmed marine species), using the same stress protocol.

The material and methods describe them adequately. However, clarifications are needed. Response: We incorporated all the required information in the new version of the manuscript

Which was the producer of the commercial pellets to feed the animals? (see line 98)

In addition, was the water circulating? What were the culture conditions (water composition, oxygenation, …)? The animals used were juveniles?  (see lines 94 to 97)

It is not specified the time of acclimation before the experiment, so the stage of the animals should be indicated because it has relevance because of the different sensitivity to the stress of juveniles and adults.  (see line 99)

The experimental design involves exposure for four days after reaching the temperature. Why was selected this time? To euthanize the animals, which was the anesthetic used? (see line 100 and lines 106-107)

Cayman has more than one Elisa kit to detect cortisol, so the catalog number should be included. Similarly, Abcam has different glucose kits, so the catalog number should be included. Including the catalog number would also be convenient for the kits used in DNA and protein oxidative damage. (see lines 110 to 123)

Was the EZNA Total RNA Kit I or the EZNA Total RNA Kit II used for RNA extraction? The transcriptome accession number should be included to allow other researchers access. (see line 127 and lines 380 to 384)

Concerning Real-Time PCR, which was the efficiency of the primer sets used? (see Suplementary table 1) . In addition, were the reference genes tested to confirm the adequacy (using any of the tools available such as GeNorm, NormFinder, …)? (see line 157)

Which was the protease inhibitor cocktail? Homemade? Commercial? Which was the antibody dilution in each case? Was the film scanning done using a scanner or taking a picture? (see lines 161 to 173)

In the statistical analysis, were the data tested to confirm that they follow a normal distribution and the homogeneity of variances?  Response: In the statistical analysis, we do not analyze the normal distribution of the population, considering that the chosen individuals come from the same progeny, ensuring homogeneity in their genetic background.

The results are well described. Unfortunately, the bar graphics of Figures 1 and 2 have no sense. Providing the dots and removing the bars is more informative since they confuse the observer. Response: The figure 1 and 2 were adjusted according to the reviewer's requirement (lines 188 and 198)

 It is striking to observe that some genes (hspa1l, hspa4, hsp90ab1) which code for heat shock proteins are increased, but there is no indication in the KEGG pathways of heat shock response. Instead, they are assigned to other processes, including response to infection. Also, it is striking to observe that atp2a1 is downregulated while atp2a2 is upregulated. Analyzing them by Real-Time PCR to confirm the data would be interesting.

Response: The observations made by the reviewer are very interesting. We appreciate the detail in the review, and we will consider it for future work and analysis.

The discussion fits the results. However, it is striking that it is not mentioned at any moment that the temperature increase also has relevance in the basal metabolism, increasing it on ectotherms. In addition, it is relevant when juveniles are used since they suffer a development that can be interfered with by the temperature increase. Since physiology is altered, should be mention anything about it. Furthermore, the juvenile is more sensitive to stress than the adult, justifying some differences observed between the literature and the results obtained in this article. The thermal response can involve different steps and is also affected by the treatment used, so it should be considered to get a clear picture of the results obtained. The physiological alterations in acute response and chronic response to thermal stress differ. It would be interesting to have more than a one-time point to elucidate the modulation of the genes, which can also respond to an acclimation process. Finally, although climate change is mentioned in the introduction, it is not considered at any moment in the discussion or the conclusions. Which would be the relevance of the results obtained in surviving climate change?

Response: We incorporated the required information in the revised discussion of the manuscript (see lines 254 to 261 and 308 to 321)

Reviewer 2 Report

Dear Editors,

Dear Authors,

The manuscript entitled: “High-Temperature Stress Induces Autophagy in Rainbow Trout Skeletal Muscle” represents interesting study that have potential to significantly improve our understanding the processes responsible for muscle autophagy and its association with heat stress. The reviewed study evaluated the effects of high-temperature stress on global gene expression in the skeletal muscle of rainbow trout to comprehend the impact of temperature stress on skeletal muscle and predict the influence of global warming on fish's adaptive capability during their development. The manuscript, however, was not well written, considering substantive content and language fluency. Abstract and Discussion chapters are not much informative.The manuscript bears numerous flaws and major improvements are required. All remarks about this have been placed in the attached file.

In conclusion, I do not recommend the manuscript for the publication in the present form and major revision should be made by authors. After that the manuscript should be evaluated again. All remarks, questions and fixes were placed in the attached pdf file (yellow highlights contain fixes and sentence suggestions, while red highlights contain comments and questions).

Thank you for another interesting manuscript that I could review!

Comments for author File: Comments.pdf

Dear Editors,

Dear Authors,

The manuscript entitled: “High-Temperature Stress Induces Autophagy in Rainbow Trout Skeletal Muscle” represents interesting study that have potential to significantly improve our understanding the processes responsible for muscle autophagy and its association with heat stress. The reviewed study evaluated the effects of high-temperature stress on global gene expression in the skeletal muscle of rainbow trout to comprehend the impact of temperature stress on skeletal muscle and predict the influence of global warming on fish's adaptive capability during their development. The manuscript, however, was not well written, considering substantive content and language fluency. Abstract and Discussion chapters are not much informative.The manuscript bears numerous flaws and major improvements are required. All remarks about this have been placed in the attached file.

In conclusion, I do not recommend the manuscript for the publication in the present form and major revision should be made by authors. After that the manuscript should be evaluated again. All remarks, questions and fixes were placed in the attached pdf file (yellow highlights contain fixes and sentence suggestions, while red highlights contain comments and questions).

Thank you for another interesting manuscript that I could review!

Author Response

We thank the reviewer for the supportive comments and major corrections. We believe we have addressed all the reviewer’s concerns, making significant improvements to the manuscript. English language edition was included (certificate attached).

The manuscript entitled: “High-Temperature Stress Induces Autophagy in Rainbow Trout Skeletal Muscle” represents interesting study that have potential to significantly improve our understanding the processes responsible for muscle autophagy and its association with heat stress. The reviewed study evaluated the effects of high-temperature stress on global gene expression in the skeletal muscle of rainbow trout to comprehend the impact of temperature stress on skeletal muscle and predict the influence of global warming on fish's adaptive capability during their development. The manuscript, however, was not well written, considering substantive content and language fluency. Abstract and Discussion chapters are not much informative.The manuscript bears numerous flaws and major improvements are required. All remarks about this have been placed in the attached file. In conclusion, I do not recommend the manuscript for the publication in the present form and major revision should be made by authors. After that the manuscript should be evaluated again. All remarks, questions and fixes were placed in the attached pdf file (yellow highlights contain fixes and sentence suggestions, while red highlights contain comments and questions). Discussion chapter is very shore and almost nothing say about genetic mechanisms responsible for muscle autophagy. Should be significantly revised. There is a lot information that rather belong to other chapters.

Response: We appreciate the detailed work done by reviewer 2. We include the many comments and corrections in the new version. The discussion was revised and expanded.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Dear Editors,

Dear Authors,

The manuscript entitled: “High-Temperature Stress Induces Autophagy in Rainbow Trout Skeletal Muscle” has been significantly improved. All my remarks and questions were fully addressed. Language presentation has been very well improved. Therefore, I have to say that manuscript is ready for publication without any doubts.

Some few minor fixes were placed in the attached pdf file.

It is very interesting and neat study – one of the best written papers I have reviewed. Congratulations!

Comments for author File: Comments.pdf