Dear authors, 
 
I apologize again for the late reply and I thank you for your hard work answering the reviewers’ and my comments. The abstract and introduction now read very well. I do not think there is a need for another round of reviews but I feel that there are still few modifications that need to be done on the manuscript before recommending it. After these last modifications, I would be happy to write a recommendation. Please see in the document attached some specific comments.

Best wishes,

Angélique Gobet

Dear Mr Katsoulis-Dimitriou and co-authors,
 
Thank you for submitting your manuscript for a recommendation from PCI microbiology. I apologize for the time it took to hand in the recommendation. Please see below some comments and suggestions from Dr Liu, an anonymous reviewer, and I. Please address them as much as you can and I look forward to receiving a revised version of the manuscript.
 
General comments
Overall, this is an interesting study with a good potential to better understand the effect of algal-based diets on the gut microbiota of the sea bream. However, I feel that the manuscript needs some work and rewriting before publication.
 
In the introduction, some rearrangements may be needed but the information is there. In the materials and methods, more details are needed to understand better what has been done in the study and for it to be reproducible. More details on the interpretation of the results are also needed, please spend more time on describing the figures. Also, for each paragraph in the results, one sentence to introduce the content would help the reading and the description of the figures and tables must be much more detailed by giving values. In the discussion, the authors may also consider that the diet likely comes along with its own microbiota, for instance see some literature on the phycosphere.
 
Throughout the manuscript, please verify that all taxonomic names are in italic. Also, the manuscript may be carefully re-read by the authors to rewrite several sentences to ease the reading.
 
Specific comments
Abstract
To introduce the aim of the study, I would add one or two sentences of context.
L23: There is a typing mistake: “Mic**rochloropsis”.
L25-27: As a teaser to the audience, the authors may add some details on the differences between FO and the other diets, maybe the differences in taxonomic composition?
L28: There is a typing mistake: “suggest*ed*”, description of results should be in the past tense.
L30: Please be more specific on the importance of fucose. Is fucose a storage carbohydrate? Part of the cell wall? Part of a specific metabolism?
L34-35: Give some names of the genera potentially beneficial?
 
 
Introduction
In the introduction, the authors may consider putting the 2nd paragraph first, and the first as second which would more smoothly introduce the third paragraph.
L47: Rewriting suggestion: “…fish stocks and thus reducing their sustainability.”.
L47-48: Please replace “For this” by “As a result”.
L50-51: Please rephrase: “Among these alternatives, microalgae are suitable and sustainable…”. Also add some details/examples about the promising results and do not forget about oomycetes.
L52: Please rephrase: “In *reared* fish,…”.
L56: I would remove “in”.
L60: I believe you can talk about dysbiosis here. There are several articles/reviews on the topic: https://onlinelibrary.wiley.com/doi/full/10.1111/raq.12862
https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2020.00114/full
L63-66: These sentences may be kept at the end of the introduction before presenting the objectives of the study.
L91: Please replace “However” by “To our knowledge”.
 
 
Materials and methods
L102-105: The authors may add more background on the way the fishes were reared in the feeding trial and also for instance, their age, sex, the feeding diet during the trial, their health state. This would help understand how homogeneous the chosen specimens were before the current study.
L105-106: Following the previous comment, details on the composition/homogeneity of each group may be informative here.
L115-116: ten individuals were randomly collected from each dietary group but at the beginning of the paragraph it is said that 40 specimens were put into 4 groups. So, if I understand this well, all fishes were collected for each diet, right?
L116: The concentration of the anesthetic may be added here.
L118: There is a typing mistake, please replace “its” by “each”.
L124: There is a typing mistake, please add “gene” in “16S rRNA gene amplicon sequencing”. Please check the article for this mistake throughout the article.
L126: If the sequencing platform gave the information, maybe add at least the Tm of the PCR cycle.
L129-130: A couple of lines indicating what the Mothur MiSeq SOP is doing may help the reader.
L131: There is a typing mistake, please replace “() with PRJNA1068122 BioProject accession number” by “under the BioProject accession number PRJNA1068122”.
L133: Were sequence identified as non-bacterial groups removed? (e.g. chloroplasts, mitochondria, eukaryotes). Please specify.
L133-135: Here, add some details on the parameters chosen to use blastn and also the accession numbers of the closest relatives should be written somewhere (as supplementary information for instance?).
L136: Add the version of PAST. Also add details on the table used for further calculations, as well as on the alpha diversity and multivariate analyses done.
L137-138: Add the version of PICRUSt2.
L143: There is a typing mistake, please replace “have” by “had”.
 
 
Results
L149-151: a table with the number of sequences in each sample for each step of the Mothur procedure would help checking for the quality of the sequencing and it would illustrate the results in the sentence.
L151-153: Details are needed on the way the dataset was normalized, on the reason of the choice of 2859 reads per sample, and why two samples were removed.
L154-155: Do you mean taxonomically assigned?
L156: Please check the literature on Taxa_S, Shannon_H, Simpson_1-D and Chao-1 indices as they are not all indices. Taxa_S is probably OTU richness and Chao 1 is an estimator of the diversity (it calculates the potentially missed diversity using the presence of singletons and doubletons). Please rephrase the text accordingly.
L161-162: More information is needed here or in the methods part to explain why the Bray-Curtis index and PERMANOVA were used here.
L164-165: Please give details on the statistical test used + the P value for each comparison.
L168: There is a typing mistake, please replace “is” by “was”.
L174-182: In this paragraph, the authors may add the correspondence of shared/unique OTUs in read numbers. It usually gives interesting information on the number of reads actually shared between conditions.
This is a proposition but, to help compare the microbiota composition between the 4 diets, the authors could add the taxonomic composition of the shared/unique OTUs to the figure. This could be complementary to the paragraph “Most important OTUs” L205-221.
L175: Please do not start a sentence by a number
L183-194: Please verify that all taxonomic names are in italic.
L183-184: Please rephrase, it seems that there is a word missing.
L188: Please explain what means this ratio.
L195-L204: Please give values to the description of the results.
L210: Please replace: “was among the most dominant* OTUs of* FO…”.
L218-220: Please rephrase.
L221: Please rewrite: “being common *to* FO-MI and FO-PI.”.
L222-230: Please give values to describe the figures and tables cited in the paragraph.
 
 
Discussion
L241: Please correct: “the Shannon index ** decreased in fish fed *with* the Schizochytrium…”.
L242: Please correct: “but ** slightly increased in fish fed *with* the…”.
L243-249: Please rewrite this sentence in shorter sentences.
L249: “In our results*,* the”.
L249-251: Proposition to rephrase the sentence: “Compared to the control (FO), the diet containing M. gaditana (MI) showed lower alpha diversity indices, but this was not statistically significant.”.
L255: Please correct: “also showed vari*ying*”.
L257: I would be careful comparing Chao indices as the calculation depends on the way the original dataset was treated, i.e. whether rare OTUs such as singletons and doubletons were removed or not. 
L270-276: The authors may also consider that the diet likely comes along with its own microbiota (metabarcoding the diet would have given extra information on this, maybe to consider for a next experiment).
L296: Please correct: “p*h*ylogenetically”.
L324-325: Please correct and maybe rewrite this way to be more careful : “search of the KEGG database confirm*ed* that most of the important bacterial genera found in *are affiliated to genomes with* five enzymes to carry out the L-fucose degradation.”.
L342-343: If the data are available, why not showing them?
L343-344: Please give some possible perspective, do the authors have specific experiments in mind to complete the study?
L349: Please correct: “inclusion *were*s inferred”.
L333-344: To conclude on this paragraph, the authors may add that the putative underexpression of peptidoglycan synthesis with the 3 diets compared to FO is likely representative of the higher presence of gram negative strains.
 
 
References
Please read references carefully, especially taxonomic names that are not written conventionally (upper/lower cases, italic).
 
 
Figures and tables
Table 1: Replace “OTUs” by “number of OTUs”. Please check the literature on Taxa_S, Shannon_H, Simpson_1-D and Chao-1 indices as they are not all indices and then please rephrase the text legend accordingly.
 
Figure 1: Please rephrase « The MI samples have the largest range, PI does not coincide with FO and SP.”. Give details of the diet acronyms in the legend. Here, it seems that the 10 samples per diet (as written in the methods part) were not considered, were some samples missed (in addition to the two lower than 2859 reads)?
 
Figure 2: Please rephrase “Relative abundances were calculated adding the relative abundance (calculated based on the average samples reads) of all OTUs belonging to each family.”. Give details of the diet acronyms in the legend.
Please add the corresponding abundance table as supplementary data, it will help reading the relative abundances.
 
Figure 3: Give details of the diet acronyms in the legend.
 
Figure 4: Give details of the diet acronyms in the legend. Please describe the dark blue plots.
 
Figure 5: Give details of the diet acronyms in the legend.
 
 
Supplementary tables and figures
Please give details of the diet acronyms in the legends. 
Table S3: Please explain the column “No. of dominant OTUs”.
 
Table S4: Please give the accession numbers of the closest relatives.
 
Table S6: Please explain what means “x” and “-“.
 
Figure S1: The “a” and “b” are missing on the figure. Do the percentages indicate the proportion in number of OTU or in number of reads?
 
Figure S2: The title may be removed as the description is already in the text legend. % may be removed in the axis title “Relative abundance”. Please explain in the methods part in the main text how the ratio has been calculated, I do not understand. If the representation in the figure comes from the calculation of a ratio I wonder if the obtained results should still be in %? Please add the corresponding table as supplementary data, it will help reading the data.