Reviewer Report Title: Metabolomics Investigation of Dietary Effects on Flesh Quality in Grass Carp (Ctenopharyngodon idellus) Version: Original Submission Date: 6/4/2018 Reviewer name: Tim Young, Ph.D Reviewer Comments to Author:

Abstract Background The ultrahigh density intensive farming model of grass carp (Ctenopharyngodon idellus) may elicit growth inhibition, decrease flesh quality, and increase disease susceptibility of fish. The degradation in quality and excessive fat accumulation in cultured C. idellus have long been attributed to possible alterations in the lipid metabolism of fish muscle tissues as a result of overnutrition from artificial diets. To investigate the effects of different diets on fish muscle quality, a large-scale metabolomics study was performed on 250 tails of C. idellus. Findings The experimental fish were divided into four groups based on sex and diet—female artificial feed (FAF), female grass feed, male artificial feed (MAF), and male grass feed (MGF). After a 113-day rearing period, the artificial feed (AF) group showed a significantly higher total mass of muscle fat (P < 0.01), with the FAF group being the highest. Metabolomics profiling based on liquid chromatography-mass spectrometry revealed distinctive patterns of clustering according to the four groups. Overall, artificial feeding was associated with higher concentrations of docosapentaenoic acid, dihomo-gamma-linolenic acid, and arachidonic acid, whereas grass feeding was associated with elevated n-3 unsaturated fatty acids (UFAs) such as eicosapentaenoic acid, alpha-linolenic acid, and gamma-linolenic acid. Artificial feeding also resulted in significant increased docosahexaenoic acid in MAF muscle than in MGF fish, whereas there was no significance in the comparison of female samples. Metabolic pathway analyses using both targeted and untargeted approaches consistently revealed that arachidonic acid metabolism and steroid hormone biosynthesis pathways were significantly different between AF and grass fed groups. Conclusions Our results suggest that grass is a better source of dietary fatty acid and protein when compared to artificial feed. Grass feeding could effectively lower triglycerides in serum, reduce fat accumulation, and alter lipid compositions in fish muscle by increasing the concentrations of n-3 UFAs, leading to better nutrition and health.

features which passed this previous criterion, although they could have included all data. Authors perform pairwise correlation analyses between metabolites themselves, but not between metabolite abundances and measures of flesh quality as the title suggests. This needs addressing. Additionally, discussion on the differential metabolite correlations that exist between the experimental groups is sparse.Overall, this is an interesting and relevant study, and with some minor revisions should be a valuable resource for future research in fish aquaculture, nutrition and health. Some specific comments for consideration, in no particular order of importance:1) Please clarify sample replication, as they are not currently sufficiently detailed to allow study reproduction. Two diets were tested, and three replicate pond systems per treatment group were used. However, it is not clear how the ten replicate fish muscle samples for metabolomics were obtained from this design; precisely how many fish per pond were sampled for metabolomics, and how exactly were they selected? Please provide similar information for the serum samples. 2) Please provide replication information (n = ?) in legends for all relevant figures and tables (i.e., Figs 2B & 4; Table 1 & 2). 3) Figure 2B: Please state what data represent e.g., mean +/-SD or SE.4) Were metabolite profile data normalised at all to biomass?5) Why were two different normalisation approaches used for the multivariate analyses (i.e., autoscaled for PCA and log transformed for heatmap and cluster analysis)?6) Please provide information on the exposure duration of MS-222, and to what level of anaesthesia the fish were dosed to.7) Line 271: It seems like a very large dataset of putative metabolite identifications was generated during this study, features of which may be of interest to some readers. This data is mentioned but not shown or provided -perhaps it might be useful to make it accessible. This is not essential for the manuscript, but it would make a nice addition.8) The title is misleading, and I would recommend re-wording it. I expected to see an analysis of correlations between the metabolite profiles and flesh quality. This was not the case; apart from fat content and muscle fibre thickness, other measures of fish flesh quality were not evaluated (e.g., appearance, taste, texture, gaping), and the experimental design does not appear to permit such correlations to be tested. I suggest authors make clear the difference between 'potential associations' and true 'correlations' throughout the manuscript. Indeed, a simple suggestion to make the title more reflective of the work being presented would be to just change the word 'correlations' to 'associations'. 9) Related to the previous comment, components of the discussion are speculative and would benefit from revision. For example, authors state in Lines 317-319 that the higher levels of SFA's (i.e., free [?] C20:0, C18:0 and C16:0) in GF fish would result in a crisper taste and firmer flesh (thus altering taste, texture and quality). Although a citation is provided (albeit from cow meat which may not translate to fish), these indices were not actually tested and is therefore highly speculative in my opinion for a manuscript that centres itself largely on flesh quality. The language therefore should to be toned appropriately to reflect this. The manuscript could be suggestive and build upon the novelty of hypothesis generation, rather than projected to provide conclusive evidence.10) Lines 308-309: This sentence should be re-phrased as it suggests that correlations between physiological changes and metabolic profiles were systematically studied by the authors. Unless I have missed some core information/dataset, I cannot see such correlation analyses. Also, please define 'physiological changes' if the phrase is to be used. 11) Lines 419-421: Authors state that they correlated physiological, biochemical and metabolic parameters with specific parameters of flesh quality. Please re-state. Perhaps use the term 'associated' or 'linked' rather than correlated.12) Lines 422-423: The results indicate that flavour may have been affected, or perhaps even likely. However, a flavour analysis was not performed in this study. Please re-phrase accordingly.13) Please highlight somewhere that the results of this particular study does not necessarily mean that all AF diets will perform worse than GF diets.14) Lines 438: What ratios by mass were these grasses fed to the fish? 15) Table S1: Correct the abbreviation for the 'Female-Grass feeding group' (i.e., FAF to FGF)

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