Alternative Feed Raw Materials Modulate Intestinal Microbiota and Its Relationship with Digestibility in Yellowtail Kingfish Seriola lalandi

Round 1

Reviewer 1 Report

fishes-745203

Unfortunately I could not find the tables and figures on the manuscript PDF file, but this is not crucial for me to evaluate this work.

This work should have been combined with the preceding work, Dam et al. (2019) and published as one work.  In fact, the authors repeated in the current work the experimental design in Materials and Methods and used again the tables of the diet formulation and diet nutrient composition presented in Dam et al. (2019).

I agree with the finding that the ingredients affected the microbiota community of the intestine.  However, the authors merely elucidated the microbiota composition and did not quantify the amounts of intestinal microbiota.  In addition, several statements in Discussion do not seem to be rational.  For example, the authors attributed the cause of changes in the microbiota community in fish fed CGM and/or BLM to antinutrients (L218-L219), amino acid imbalance (L184-L185, L217-L218) and carbohydrate (L219, L237).  However, corn is well known to have less antinutrients than soybean meal, soybean protein is deficient in methionine, and WH (and probably FBM, too) has much more carbohydrate than CGM.  Protease inhibitors are denatured during heat processing in ingredient production and diet preparation.  Taken together, it could not be concluded from the current results that the intestinal microbiota per se affected the digestibility.

I am quite wondering why the authors selected in this work only 8 kind of ingredients from 14 ingredients tested in Dam et al. (2019).  Two PBM were evaluated in the current work whereas only one of two SPC was evaluated.  Should both two SPC have been evaluated in the current work since the coefficients of crude protein digestibility between the two SPC were quite different.

I don’t think the digestibility coefficient data presented in Dam et al. (2019) are reliable due to several reasons.  It is well known that digestibility value determined by stripping method is underestimated since fish still undergo digestion and absorption.  Of course, it is also known that digestibility coefficient determined by fecal collection sometimes cause overestimation.  However, the general trend observed in Dam et al. (2019) values do not seem to coincide with the previously reported trends.  For example, Dam et al. (2019) observed very high digestibility coefficient of crude protein for MM (correctly this ingredient is meat and bone meal, MBM, due to its low crude protein content) despite of the very high ash content that adversely affects protein digestibility.  In addition, the digestibility coefficient of dry matter for MM (69.6%) does not agree with the sum of digestible crude protein (439 g/kg) and digestible crude fat (51 g/kg), and also for SPC1 (dry matter digestibility 15.8% vs. digestible crude protein 418 g/kg).

L73 gilthead sea bream instead of red sea bream

L132, L233 Gut microbiota metabolize bile acids into secondary bile acids rather than synthesize bile acids.

L156-157, L165 Did the authors examine the microbiota in the distal intestinal content rather than the distal intestinal mucosa (L318-L319)?

L157, L318 scraping instead of scarping?

L160 …among the experimental diet groups?

L186 …excessive undigested protein content in the gut digesta in these groups?

L264 glycoses is not an enzyme.

L313-L321 When were the fish sampled after the last meal?  Were the same fish used for fecal collection used in the current work?

L369-L371 Ingredients and diets are contaminated by various microbiota during there storage.  In addition, is an ingredient from animal origin naturally contaminated by microbiota?  Hence, is it essential to examine the microbiota community in the diets?  Is there a possibility that a gut microbiota composition reflected that of a given diet?

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Before going into further details, correct me if I am wrong but the major flaw is that the pdf does not contain any of the figures or tables mentioned in the manuscript. That being said, the authors need to resubmit the proper manuscript form, including the figures and tables. I believe that this is just an omission by the authors and it will be soon fixed but unfortunately in the current format I could not combine the results section with the corresponding graphic representations. This is also the reason that I chose not to answer some of the bullets points above, I will do when I get the figures and tables.

Meanwhile, you could consider as well the following comments and specific points in order to make an overall rebuttal of the paper.

Overall, the paper has a nice structure and it could be a useful contribution to the current literature on the hot topic of replacement sources for aquaculture nutrition. I think that the authors included a wide variety of different treatments and the information obtained on data microbiome worth sharing. Please find some more specific points below:

Line 30, 101, 139 etc.: Have you any correlations about fish health as well? I am asking because it seems that potential pathogenic bacteria such as vibrios or Clostridium are positively correlated with digestibility. These bacteria can potentially cause a problem even to the animal’s survival and it would definitely be an issue for the producer to know that a particular diet will increase the presence of eg. vibrios in the system.

Line 103: whereas in trial 2.

Line 108: what are the differences between PBM 1 and PBM 2?

Line 113: LEfSe is explained in line 387 but better be placed here istead.

Line 125: citation for PICRUSt.

Line 126: Explain NST here instead of line 402.

Line 129: were significantly different

Line 131: pathway functions instead of ¨abundant pathway function¨

Line 133: abundant instead of ¨abundance¨

Line 162-163: remove Ramirez and Romero

Line 165-168: I feel that the explanations of the authors here is not clear. I would recommend a more elaborate argumentation for the comparison of the contradictory previous reports.

Line 173: members

Line 173: against pathogen??

Line 180-181: correct the citations here

Line 195: I would recommend a strong rebuttal here. Vibrios are definitely not beneficial bacteria. Frankly, when vibrios come to play, they are usually notoriously known bacteria i.e. V. anguillarum, V. vulnificus, V. harveyi, V. parahaemolyticus, V. alginolyticus etc. responsible for heavy losses and diseases in aquaculture eg. hemorrhagic septicemia. It is though indeed a very diverse genus, hence I would not exclude the possibility some of them to function as contributors to nutritional breakdown. However, that would demand further investigation and results in order to support their allegedly beneficial role.

Line 203: fix citation

Line 205: I agree but maybe you can take a look here https://onlinelibrary.wiley.com/doi/full/10.1111/jfd.12981, maybe that could also compensate for some of the potential side effect by the increase in vibrios. This is just a speculation though.

Line 228: fix citation

Line 236: restructure the sentence

Line 242-247: very interesting finding!

Line 258-260: the sensitivity of this point was raised in a comment above. Eg. in citation 75 it is mentioned ¨Pathogenic Vibrios commonly infect larvae and can cause sudden and significant mortalities. However, it has been hypothesised that many Vibrio species are not true pathogens, but in fact opportunistic pathogens whose virulence is accentuated under intensive aquaculture conditions¨.

In our case we are talking about aquaculture, intensive livestock production so it might be an issue, we need to evaluate the risks.

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

This is a very interesting research, the topic is relatively new and several research programs are actually investigating effects of alternative raw materials on fish gut microbiota, considered as playing a crucial role in fish health and on ingredient digestibility. Authors investigated a high number of ingredients alternative to fish meal, methods are innovative and statistical analysis is complete and reliable. In general, the mauscript is very clear and results are supported by adequate bibliography.

I have just some minor suggestions, listed below:

Abstract

Line 24. Don’t cite previous studies in the abstract.

Line 26. “The most abundant” instead of “The most abudance”

Line 26. “phylum” instead of “phyla”.

Line 31. Add “,” between “correlated” and “whereas”

Line 31. “Ralstonia genus was” instead of “Ralstonia genus were”

Line 31. “This study demonstrated that the…” instead of “We demonstrated the”

Line 32. “Instrument” instead of “strategy”

Introduction

Line 39. “Aquatic food products” instead of “”marine products”

Line 40-41: The sentence “which recently estimated that aquaculture supplies about 47% of food fish in the world” should be rephrased as follows: “supplying about 47% of aquatic food for human consumption”.

Line 46-47: The sentence “Alternative feed raw materials can be generally divided into those of plant and land-animal origin” should be rephrased as follows: “Alternative feed raw materials can be for most derived from plants or from land-animals”.

Line 55: Rephrase as follows “… amino acids. However, in some regions, such as Europe, there has been limited use…”

Line 73: Replace “red sea bream, Sparus aurata” with “gilthead seabream, Sparus aurata”

Line 82: Please specify with alternative feed raw materials in the aims of the study.

Results

Line 103: Please add a “,” between “trial 2” and “whereas”.

Line 127-130: The sentence doesn’t make sense, please control and rephrase.

Line 133: “More abundant” instead of “more abundance”

Line 141: “genera” instead of “genus”

Discussion

Line 143: Use “fish meal” instead of abbreviations. Avoid abbreviations through all the discussion (e.g., YTK).

Line 165-167: The English of this sentence should be revised, because it’s not clear enough what the authors would say.

Line 172: “Members” instead of “member”.

Line 183: “the relatively high abundance” instead of “the high relative abundance”

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors clearly state in the rebuttal (Response 4) that “we did not find any significant differences in the microbiota community between the two of them (=SPC)”.  This means that the microbiota community does not affect, or is not correlate with, the digestibility coefficients of SPC protein that are quite different between the two SPC.

The differences in apparent protein digestibility coefficients between two SPC might be due to the palatability of the test diets, although actual feed intake data are not presented in Dam et al. (2019).  When feed intake is reduced, the effect of relative contribution of endogeneous losses increases, leading to a reduction of apparent protein digestibility coefficient.  Blood meal is known to be a good source of histidine and iron but also known as unpalatable at a high inclusion, and less than 5% inclusion is recommended.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

I think that the authors have done a very nice rebuttal of the manuscript and they have addressed all my points.

I would like to mention that their responses 3 and 5 should be incorporated in the main text as well. Also, figures 5a and 5b might fit better in the supplementary information.

Other than that I think this is a very valuable work that would greatly contribute to the current literature.

Author Response

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Author Response File: Author Response.docx