Comparative Analysis of Metabolites between Different Altitude Schizothorax nukiangensis (Cyprinidae, Schizothoracine) on the Qinghai-Tibet Plateau in Nujiang River
Round 1
Reviewer 1 Report
In the manuscript entitled "Comparative Analysis of metabolites between different altitude Schizothorax nukiangensis (Cyprinidae, Schizothoracine) on 3 the Qinghai-Tibet Plateau in Nujiang River", Xu et al., conducted metabolism comparison between six fish along Nujiang River in south west China. They identified several genes enriched into clusters that are related to oxidative stress, energy metabolism, carbohydrate metabolism and lipid metabolism, which may explain the fish response to high altitude environment adaptations. In general, the view point is clear and the language is fluent, it is well appropriate for acceptance.
Author Response
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This study analyzed the metabolites of Schizothorax nukiangensis located in two altitudes from Nujiang River. Overall, the design of this study is sound, and results are reliable. In the discussion part, it is suggested that the author make a citation according to the research findings and compare the previous research results, especially the findings in freshwater fish. For this version of the discussion, it is recommended to cite similar or comparable species. The references are too old. It is recommended to use the references of the last 10 years.After revision, this manuscript can be considered for publication.
Some specific comments:
Line 11: “sample collection and metabolomics studies were apllied in this study.” Please revise this part as “sample collection” is not your research method.
Line 41-43: “relatively little research has been conducted on animals, , particularly fish along an altitude gradient”, lines 70-72: “To our knowledge, no study has investigated the effect of altitude on the metabolome of schiz-othoracinae fish.”, they are not good reason to conduct this study.
Line 85: why use 150 g fish in this study.
Line 150: “measure” or “compare”?
Lines 154-159: please add the standard deviation to each parameter.
Lines 162-163: “suggested that altitude affects the metabolite accumulations in S. nukiangensis liver.” Delete this sentence, be aware that only describe your results in the Results section.
Line 171: “received” or “found” or “observed”?
Line 174: “were interestingly found and quite different in two groups.” Is there any significant difference? Such description is not suitable in the result section.
Line 201: delete “And”.
Lines 248-250: “In high-altitude S. nukiangensis, the decreased glycerophospholipid metabolites including 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, 1-Stearoyl-sn-glycerol-3-phosphocholine, 1-Myristoyl-sn-glycero-3-phosphocholine, 1-Palmitoyl-sn-glycero-3-phosphocholine were indicated. ”, please rewrite this part, this description is controversial.
Lines 257-258: “Based on these finding, we speculated that oxidative stress was occurred seriously in high-altitude fish.” The evidence for this inference is insufficient.
Line 258: change “occured” to “occurred”. Change “oxidative” to “Oxidative”.
Lines 288-289: “D-mannose-6-phosphate is formed by the hexokinase-derived phosphorylation of mannose [54]. ” this process was observed in what kind of animals?
Author Response
Response to Reviewer 2 Comments
Point 1: Line 11: “sample collection and metabolomics studies were apllied in this study.” Please revise this part as “sample collection” is not your research method.
Response 1: Thank you for your suggestion. We have removed “sample collection” in this sentence according to the your suggestion in manuscript.
Point 2: Line 41-43: “relatively little research has been conducted on animals, , particularly fish along an altitude gradient”, lines 70-72: “To our knowledge, no study has investigated the effect of altitude on the metabolome of schiz-othoracinae fish.”, they are not good reason to conduct this study.
Response 2: We have made correction according to your suggestion in manuscript.
Point 3: Line 85: why use 150 g fish in this study.
Response 3: Thank you very much for your question. In this study, 6 fishes with similar size were selected for further metabolomics analysis. The value of 150 g was determined for two reasons. Firstly, the number of S. nukiangensis at two sites weighing around 150 g was sufficient for tissue dissection and subsequent research. Secondly, sexual maturity occurred in 150 g S. nukiangensis. Consequently, our research revealed changes in the metabolites of sexually mature fish residing in environments of different altitudes.
Point 4: Line 150: “measure” or “compare”?
Response 4: We have made correction according to your suggestion in manuscript.
Point 5: Lines 154-159: please add the standard deviation to each parameter.
Response 5: Thank you for your suggestion. We have added the standard deviation to each parameter.
Point 6: Lines 162-163: “suggested that altitude affects the metabolite accumulations in S. nukiangensis liver.” Delete this sentence, be aware that only describe your results in the Results section.
Response 6: We have removed this sentence in the manuscript.
Point 7: Line 171: “received” or “found” or “observed”?
Response 7: We have made correction according to your suggestion. We have replaced “received” by “observed” in the manuscript.
Point 8: Line 174: “were interestingly found and quite different in two groups.” Is there any significant difference? Such description is not suitable in the result section.
Response 8: Thank you for your suggestion. We have made correction in manuscript.
Point 9: Line 201: delete “And”.
Response 9: We have removed “And” according to your suggestion.
Point 10: Lines 248-250: “In high-altitude S. nukiangensis, the decreased glycerophospholipid metabolites including 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, 1-Stearoyl-sn-glycerol-3-phosphocholine, 1-Myristoyl-sn-glycero-3-phosphocholine, 1-Palmitoyl-sn-glycero-3-phosphocholine were indicated. ”, please rewrite this part, this description is controversial.
Response 10: Thank you for your suggestion. We have improved writing this sentence in manuscript.
Point 11: Lines 257-258: “Based on these finding, we speculated that oxidative stress was occurred seriously in high-altitude fish.” The evidence for this inference is insufficient.
Response 11: Thank you for your suggestion. Oxidative stress in fish can be directly affected by a multitude of stressors such as temperature fluctuations, UV-exposure and pollution [1][2]. According to the transcriptomic analysis of S. nukiangensis in different altitude environment, we found the the upexpressed genes mainly distributed in DNA repair and preoxisome pathway. We concluded that high UV-exposure and low temperature in high altitude environment could increase the oxidative stress in S. nukiangensis. Changes in the structure and function of cellular membranes in fish subjected to oxidative stress may account for the low glycerophospholipids contents.
Point 12: Line 258: change “occured” to “occurred”. Change “oxidative” to “Oxidative”.
Response 12: These two errors have been corrected in the manuscript.
Point 13: Lines 288-289: “D-mannose-6-phosphate is formed by the hexokinase-derived phosphorylation of mannose [54]. ” this process was observed in what kind of animals?
Response 13: Thank you for your question. D-Mannose 6-phosphate is a part of the fructose and mannose metabolic pathway. The first step of the metabolism of d-mannose is the phosphorylation of d-mannose to D-mannose-6- phosphate using hexokinase and adenosine triphosphate (ATP) as cofactors [3]. Within the cell, mannose is phosphorylated by hexokinase (HK) to produce mannose-6-phosphate. These studies about mannose metabolism were mainly focus on humans, mice and zebrafish [4]. Due to the fact that zebrafish and S. nukiangensis belong to the same family, we hypothesized that their mannose metabolism is comparable. Ultimately, we appreciate your suggestions tremendously.
References
[1] Birnie-Gauvin, K.; Costantini, D.; Cooke, S.J.; Willmore, W.G.; A comparative and evolutionary approach to oxidative stress in fish: a review. Fish Fish. 2017, 18, 928-942.
[2] Hurem, S.; Fraser, T.W.K.; Gomes, T.; Mayer, I.; Christensen, T. Sub-lethal UV radiation during early life stages alters the behaviour, heart rate and oxidative stress parameters in zebrafish (Danio rerio). Ecotox. Environ. Safe. 2018, 166, 359-365.
[3] Sharma, V.; Ichikawa, M.; Freeze, H.H. Mannose metabolism: more than meets the eye. Biochem. Bioph. Res. Co. 2014, 453, 220-228.
[4] Hu, X.; Shi, Y.; Zhang, P.; Miao, M.; Zhang, T.; Jiang, B. D-Mannose: Properties, production, and applications: An overview. Compr. Rev. Food Sci. F. 2016, 15, 773-785.
Author Response File: Author Response.pdf
Reviewer 3 Report
This is a very interesting and thought-provoking paper. The Schizothoracine fish display a great range of adaptations to high altitudes, and recent studies of their metabolomes are few (if any). This study has disclosed some very interesting and probably significant differences between high altitude and low altitude individuals of one species. The higher ones are challenged by pollutants, but more interesting are the low levels of omega-3 fatty acids, making one wonder about the effects on the physiology of these fish. This is a valuable study that could be foundational to a range of studies of fatty acids and other metabolites in fish at different altitudes.
I am not competent to evaluate the methods (I don't do this kind of analysis) but the explanation is certainly detailed and clear.
There is very minor copyediting to do. I note the following:
Line 44, “twenty-one century” should be “twenty-first century”
66, “primitive grades” should be “primitive-grade”
208, rephrase as follows: …“is among the omnivore and partial-carnivore fish, and chironomids are the major food”…
218 “an negative” should be “a negative”
220 “rough markers” should be “a rough marker”
233-237, really interesting. Presumably foods are the source of these fatty acids. The chironomids must be getting them somehow. Or…?
293, “by different diet” should be “a different diet.”
314 “flow” should be “flowed”
There are some other very minor things. All in all a good study.
Author Response
Response to Reviewer 3 Comments
Point 1: Line 44, “twenty-one century” should be “twenty-first century”
Response 1: We have made correction according to your suggestion in the manuscript.
Point 2: 66, “primitive grades” should be “primitive-grade”
Response 2: We have made correction in manuscript.
Point 3: 208, rephrase as follows: …“is among the omnivore and partial-carnivore fish, and chironomids are the major food”…
Response 3: Thank you for your suggestion. We have improved writing this sentence in manuscript.
Point 4: 218 “an negative” should be “a negative”
Response 4: We have made correction according to your suggestion in the manuscript.
Point 5: 220 “rough markers” should be “a rough marker”
Response 5: We have improved writing this sentence in the manuscript.
Point 6: 233-237, really interesting. Presumably foods are the source of these fatty acids. The chironomids must be getting them somehow. Or…?
Response 6: Thank you for your question. Oleic acid content in liver was related to the higher content of oleic acid in the diet [1]. Myristoleic acid is mostly derived from plants, specifically the seed oil of Myristicaceae species, and is present in all eukaryotic organisms [2]. These two monoenoic acids were mainly from food for S. nukiangensis. S. nukiangensis is primarily fed by chironomids. The food of chironomid is mainly natural detritus, which include quite diverse dietary sources [3]. Consequently, we inferred that these two fatty acids in S. nukiangensis liver were mostly related to the dietary amount of chironomid larvae. Ultimately, we appreciate your suggestions tremendously.
Point 7: 293, “by different diet” should be “a different diet.”
Response 7: We have improved writing this sentence in manuscript.
Point 8: 314 “flow” should be “flowed”
Response 8: We have improved writing this sentence in manuscript.
Reference
[1] Ruiz-Gutiérrez,V.; Pérez-Espinosa, A.; Vázquez, C.M.; Santa-María, C. Effects of dietary fats (fish, olive and high-oleic-acid sunflower oils) on lipid composition and antioxidant enzymes in rat liver. B J. Nutr. 1999, 82, 233-241.
[2] Choi,Y.K.; Kang, J.I.; Hyun, J.W.; Koh, Y.S.; Kang, J.H.; Hyun, C.G.; Yoon, K.S.; Lee, K.S.; Lee, K.S.; Kim, T.Y.; Yoo, E.S.; Kang, H.K. Myristoleic acid promotes anagen signaling by autophagy through activating Wnt/β-catenin and ERK pathways in dermal papilla cells. Biomol. Ther. 2021, 29,
[3] De Haas,E.M.; Wagner, C.; Koelmans, A.A.; Kraak, M.H.S.; Admiraal, W. Habitat selection by chironomid larvae: fast growth requires fast food. Anim. Ecol. 2006, 75, 148-155.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The authors have addressed all the comments in the revised manuscript.
