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Title:  Unraveling the gut microbiota of Tibetan chickens: insights into highland 2 adaptation and ecological advantages 

Reviewed by:  Xuyu Liu, Marcus Lee, Clarence Sim, Hashmath Fatimah, Samantha Quah, Sreelakshmi Cheruvalli, Karaiyagowder Govindarajan Deenadayalan, Nicole Loh, Zhiqi Tang, Viduthalai Rasheedkhan Regina  

Singapore Centre for Environmental Life Science Engineering, Nanyang Technological University, SBS-B2N-27, 60 Nanyang Drive, Singapore 637551, Singapore  

Summary:  

The paper discussed the effects of altitude on the gut microbiota of different chickens, namely Tibetan chickens and Qingyuan chicken. The authors seek to understand the role of gut microbiome in high-altitude animals in withstanding harsh conditions by investigating and quantifying the effects of altitude on chicken’s cecal microbiome using multi-omics, which included meta-transcriptomics and metabolomics, and have attempted to explain the disappearance of valuable traits of Tibetan chickens living in the plain. The research topic is interesting and we found that some, aspects of the methodology and may lack sufficient explanation, which led us to raise some questions that could be useful for the authors to add value to the presented research.  

Overall, the discussion section appears lengthy. While the authors have done a good job in collecting all the relevant literature leading to results interpretation, the section could be written in a concise way to have a better readability. Emphasis on explaining data and highlighting key or important observations of this research work accompanied by relevant literature would have been a better approach. In addition, certain information can be omitted altogether (e.g. lines 268-271) as these are now considered common knowledge by biologists familiar in the field. 

Introduction 

The introduction section requires more clarity to better understand the motivation of the study, and certain specific information to explain what has been understood in this field.   

Lines 74-76 It would be useful to outline or propose background biochemical mechanisms that support adaptive advantages in terms of energy utilization and immune systems of Tibetan chicken populations so that readers could understand why is this important. Further elaboration as to how these mechanisms relate to the results obtained can be substantiated in the Discussion section. 

Line 84-88 The authors tried to establish a correlation between the effects of high altitude with gut microbiome and the example given is Tibetan pigs, as wells as people of Tibetan and Han ethnicity. It is understandable that since there are not prior studies on chickens or other birds (if there are they can also be used), however the wording of the first example may be misleading, as it is not clear if the pigs are of different breed living in different altitude or the same breed living in different altitude. Also, it will strengthen their argument if they could provide a summary of the study and explain how this study relates to the current study. For the Han and Tibetan example, it will be great to consider other factors, such as diet, that could influence the gut microbiome composition, as the two has distinct diets due to difference in ethnicity. Naturally a question arises as how diet is not a cofounding variable when drawing parallels. The authors may add some justification to this. In addition, it will provide more insights if the authors could note down the genus/strain that contribute to the distinct metabolic profile of different population. 

Lines 93-97 Please briefly mention how SCFAs afford increased host immunity. The authors mentioned that SCFA has a role to play in regulating immune functions. Is succinate a key metabolite responsible for supporting the role of SCFAs in immune regulation? It will be great if they could provide an example or two, to describe how specific SCFA could regulate immunity and through what mechanism.  

Line 99-102 Same comment as above for LCA & DCA 

Lines 102-104 It is not entirely clear from the writing that why should the relationship between gut microbiota, health and highland adaption be investigated further. Perhaps more information is needed to address why these three factors are important in studying the mechanism of highland animals. 

Methods 

Essential information on the selection criteria for chickens, hosting conditions and diet of chickens was not provided. It was assumed that the gut microbiome of Tibetan chickens at different locations have similar gut microbiome profiles. Understand that the authors are looking into cecal microbiome, thus might be difficult to access prior the study to establish a baseline. However, more justification is needed from the authors on why they assume the gut microbiome profile of the chicken are similar or else this will weaken their findings. Alternatively, if no baseline could be established, please refer this as a study design limitation. 

Lines 148-149 The mentioning of In-Tc, Ex-Tc and QY abbreviations should be made earlier in the paragraph. 

Lines 164-165 What might be some of the more significant or interested genes responsible for this metabolism? 

Line 364 onwards The normalisation method for meta-transcriptomics is normally used in single cell transcriptomics. Please confirm if this is appropriately prescribed for processing data as complex as the chicken gut microbiome. 

Lines 374-375 while the authors have discarded the rRNA reads, would it help deriving taxonomy information from the rRNA reads? Are there limitations in performing this analysis? Please include normalisation information on all metabolomics data.  

Results 

In general, we suggest a better presentation strategy for the figures. For example, in the metatranscriptomics and metabolomics data, it is currently confusing and difficult to interpret by readers unfamiliar with select omics technologies. 

Lines 236-238 is it not very clear from the paper that how increase in ratio of B/F is determined, is it via CFU or other means.  

Note on Figures 2-6. We think that the figures can be presented with better illustrations. Currently, it is challenging for the reader to understand clearly as to why certain experiments were performed and for what purpose. In figure 2A, no significance label on the graphs was shown although it is significant. 

Discussion 

Line 254-256 Information on the type of feeding and hosting conditions of chickens in different groups were not provided in the methods section, as such the evidence is not enough to reasonably conclude if the difference in microbiome was contributed by altitude only or if diet played a significant role.  

Line 273-277 These points could be expanded on. Please clarify how increased DEG and employing fatty/saccharide pathways would have contributed to efficient energy utilization. 

Lines 324-327 In the previous sentence, line 324-324 authors say that B/F cannot be used as an indicator for gut health but in line 327, they authors mentioned that B/F can act as a marker for immunological activity. This is unclear and may need further justification. 

Minor comments:  

In general, please check for inconsistencies in all figures with respect to labelling. 

Fig 3 captions. There are 2 ‘B’s. ‘C’ is missing. 

Fig 5. Inconsistent switch to lower case in captions to: (a)… (b)… (c)….  

Table S1/S2 appears to be omitted. 

Please spell out short forms e.g. ORF, bp, N50, COG etc, in full at some point in the appropriate sections. 

Competing interests

The authors declare that they have no competing interests.