Different Diets Based on Yellow Mealworm (Tenebrio molitor)—Part B: Modification of the Intestinal Inflammatory Response and the Microbiota Composition of Rainbow Trout (Oncorhynchus mykiss)

Round 1

Reviewer 1 Report

Wording of Introduction has to be improved, as there are some small mistakes and a few confusing sentences.

References 4, 5, 6 are publications about insect meals (Line 52). However, I am sure that they provide information about fish meal, it would be better to include citation(s) dealing with fish meal itself.

In part Material and Methods description of the used diets is not clear for the first insight (Lines 95-10. It is a clearer and more understandable way as they are described under the Tables: „C: control diet (no fishmeal replacement); T: 50 % fishmeal replacement with Tenebrio molitor; dT: 50 % fishmeal replacement with partially defatted Tenebrio molitor; TO1: T diet supplemented with 3.09 % of omega-3 enriched oil (EO); TO2: T diet supplemented with 7.24 % of EO. I recommend to use the latter one also in the text.

Data has to be provided about the ingredients and chemical composition of the feed. As ingredients have strong effect on results (e.g „Even though the present experiment did not reveal a severe case of inflammation, it is interesting to notice that three out of four insect-based diets (T, TO1 and TO2) had a slightly higher amount of vegetable ingredients than C, suggesting that this inhibitory effect might be considerable.”) and also included in conclusions these statements does not stand alone. Publication has to be completed with Table(s) about the proper ingredients and chemical composition of the diets.

I recommend to include the calculations of growth performance, indices etc. in part Material and Methods instead of introducing them in the footnote of Table 2.

In Table 2. FBW and DFI are missing in spite of that they are mentioned in the footnote. However, CF is shown two times.

Please use the following version of citation 1: FAO. The State of World Fisheries and Aquaculture 2022. Towards Blue Transformation. Rome, FAO. 2022  https://doi.org/10.4060/cc0463en

In the case of citation 8: a T is missing “T. O’Keefe” ….

The use of commas is not consistent in References e.g compare citations 4 and 5 to the others.

Author Response

Please, see the attachment. Answers to the comments of the reviewer have been included as statements in bold right after most of their own comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors,

I have had the opportunity to review the manuscript entitled “Different diets based on yellow mealworm (Tenebrio molitor) – Part B: modification of the intestinal inflammatory response and the microbiota composition of rainbow trout (Oncorhynchus mykiss)” (Ref. no. fishes-2340186) for Fishes.

Authors have explored the beneficial effects of insect diets in the rainbow trout. I think the manuscript deserve to be published in Fishes although there are major concerns that authors should correct and modified.

First, authors abused extremely abbreviations. This abusive use of abbreviation makes the reading of the manuscript very difficult and some parts. Some of them has not sense as they were cited once or few times (GIT – gastrointestinal tract, AA – aminoacids, etc.). I am going to recommend avoiding abbreviation except for treatments, so I think the paper will be clarified.

Text looks like different persons wrote different parts, from a very scientific and correct Material and Methods to a confusing and messy Results and Discussion.

First, treatment level description is not clear ant all. It is not clear how many treatments levels are, if there is one treatment with insect or four. If there is only one treatment with insect, why to include three controls (full fat, defatted, and supplemented with a long chain omega-3 enriched oil). Why to include several treatments instead of increasing sample size? The use of three additional control needs imperiously to be explained in detail (and it is not valid to say authors wanted to test different diets because authors should change the focus and the aim of the manuscript.

Following this argumentation, abstract needs to be rewritten urgently, as it is not informative at all, it is not clear what the aim is, it claims a hypothesis and they tested different things, except the hypothesis.

About sequence analyses and statistics:

How authors have approached Results and Discussion is incorrect. Authors have to provide first the main effects of the analyses, i.e. F, degree of freedom and P-value of treatment. Once treatment is significant related with a dependent variables, hence they can provide posthoc tests. Providing and discussing posthoc tests of non-significant main effect is wrong and has not sense.

I found some gaps in M&M about how the sequences were processed and analyzed. For instance, why had authors use Wang  et al. (2007) approach?

Wang Q.; Garrity G.M.; Tiedje J.M.; Cole J.R. Naïve bayesian classifier for rapid assignment of rRNA sequences into the new 584 bacterial taxonomy. Appl Environ Microbiol 2007, 73, 5261-7. https://doi.org/10.1128/AEM.00062-07

There is newer and more robust ways to obtain the ASV table as Deblur or DADA2. Moreover, it is not clear which kind of filtering authors have used? Have authors filtered out chloroplasts and mitochondrias? ASV without phylum assignment or even ASV not assigned to Bacteria superkingdom?

Authors have shown an important lack of knowledge of basic microbial ecology. First, alpha diversity is the diversity within a sample, while beta diversity is the difference between two communities. I strongly recommend reading Whitaker 1977.

Whittaker, R. H. (1972). Evolution and measurement of species diversity. Taxon 21, 213–251. doi: 10.2307/1218190

Therefore, ASV richness and Shannon’s and Simpson’s diversity index have to be properly written, and Shannon’s and Simpson have to be cited. The description of bacterial community in phylum and genera is not alpha diversity. It is just bacterial composition of rainbow trout gut. I think that section needs some reorganization: first alpha (showing main effect of ASV richness, and Shannon’s and Simpson’s diversity index, and afterwards, posthoc tests for those significant main effects), beta diversity and afterwards bacterial composition.

Biplots is great, but I miss two things: first, PCoA of beta diversity matrixes, and second, statistical tests. For the first issue, I recommend to perform centered-log-ratio, then to calculate Atkinson distance, and finally plot a PCoA. For the second one, authors are familiar with R, so they only need to perform some PERMANOVAS.

One important thing about statistical. I am missing too is the complete absence of Tank as a covariable. In this kind of aquaculture studies, tank resulted significant so it needs to be accounted in the analyses, for correcting for this covariation. In my modest opinion, authors should include Tank as a random covariable in all analyses.

Finally, why have authors kept some fila or some genera? How many? And which criteria? Authors are assuming (wrongly) that majority ASVs, genera of phylum are the affect taxa by treatment. What about the rest, the Rare Biosphere?

Lynch, M. D., & Neufeld, J. D. (2015). Ecology and exploration of the rare biosphere. Nature Reviews Microbiology, 13(4), 217-229.

This is a limited and wrongly way to establish association between bacteria and treatment. I recommend using ANCOM or ALDEx2 algorithms, based in log-centered ratios; or even network analyses. A weak and obsolete approach is LEFSE.

I think authors need somebody that advise them in microbial ecology.

The reading of Results and Discussion is quite difficult for both abbreviation and for the confusing discussion of none significant results as they were significant. The use of a statistical test stay that two average belongs to the same population of averages. Of course, in biology it is almost impossible that two average will be exactly the same. Saying that, it is confusing and wrong to discuss differences in a variable when the statistical test show a none-significant p-value (p > 0.005). That is the reason that the paper needs the main effect, their significance and only when they are significant, their posthoc tests.

Moreover, some interpretations are really speculative and authors have limited what their results provided. And in case of speculation, advice reader that authors are speculating.

Some sentence are categorical without a reference. I have provided some insights about sentence without references in minor concerns.

The language in this section is quite ambiguous and sometimes quite colloquial.

Minor concerns (and they are not exhaustive). Once I indicate a minor concern, check the rest of the text for similar issues (as the lack of compulsory references):

Lines 27-29: authors do not provide any evidence about how it happens. This kind of sentence sounds great but simply, it is not tested. Have authors tested this association?

Line 33-34: is it significant. If not, there is no a minor but consistent trend. There is not a trend.

Lines 46, 50: are those abbreviations necessary? IM? FM? Really?

Line 94: five in lower case.

Lines 93-102: treatment is not clear at all: C, FM, IM, EO, T, T01, T02, dT…

Line 113 and so on. Tables have to be self-informative. What does it mean “experimental diets” without reading the manuscript. Check the rest of captions (figures and tables).

Line 218-22: this paragraph needs to be completed with my above recommendation.

Lines 220-22: this sentence have to be moved to Statistical section.

Line 226: executed? Performed.

Line 226-227: delete “to compare the means, being statically different at p-Value < 0.05”. This is conventionalism and it is assume for the complete scientific community.

Line 228-230: Authors have performed a biplots using Principal Component Analysis (PCA) between diet composition and gut microbiota digesta composition at genus level.

Please delete FAs and AAs and digesta. Use the same wording in order to reduce confusion in the reader.

Lines 230-231: If authors use R, please do not Excel. It reduces credibility and is not scientifically sound.

Line 235: fro here so on, use always “significant” when there is a p-value< 0.05. And try to avoid to discuss differences in non-significant results. E.g. “T and TO2 showed a significant higher protein efficiency ratio than dT.”

Line 240: delete “It has been described that”. Avoid the use of convolvolous language. Go straight and the text will be benefit.

Lines 241-243: you cannot stay such sentence without “many” references.

Lines 251-254: therefore, they are not difference and the trend does not exist.

Line 258-259: this sentence is confusing as it starts: “Few differences”. What does it means “acid phosphatase showed lower values”? Significantly or not?

Line 263-264: Reference is missing.

About reference: probably authors should reduce the number of references in the introduction section. In some sentences, it is not necessary to cited 4-5 papers. Just with one is enough.

Line 277-279: significant or not?

Line 286: ditto. Please, provide main effects and hence, when it is significant and when not.

Line 290-291: this is an example about different writers in the text. Send this sentence to M&M and avoid to include unnecessary sentences explain here what it has been done.

Line 294-311: I do not understand pretty well this paragraph, as I do not know if authors are discussing significant or not significant results. Or what is the aim of this discussion. It looks like a complete argumentation of none-significant results.

Line 315: I would like to see such a good description in other captions.

Line 330: alpha diversity appears surprisingly here. It needs to be introduced in M&M:

Line 333: another “significant” result? Or not? Where is the main effect?

Line 337: related to this… to what?

Line 339-341: and? What authors discuss about this? Or even speculate?

Table 5 and 6: why these groups and no others?

Line 365-370: what is the difference with previous paragraph?

Line 372: second time that AA are abbreviated.

Line 386: moderate, pronounced… authors abuse of ambigous language and again, is it significant or not? I.E. Is it different of equal?

Line 418: five nouns in a row: “main distal intestine digesta microbiota genus of”. Hoiw many are main? And why those main and no others? Digesta? = gut content. Please, unify criteria between writers.

Line 424-425: unfortunate sentence. First sentence in conclusion is not a conclusion and say nothing.

Line 426: what does it mean “slightly”? it is or it is not similar. Ambigous.

Line 454: “Data Availability Statement: Data is contained within the article” is false. Data are not in the paper.

Author Response

Please, see the attachment. Answers to the comments of the reviewer have been included as statements in bold right after most of their own comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear Authors and Editors,

As for the whole publication I accept the answers and corrections of the authors.

Regarding the question below I would still recommend the presentation of the mentioned tables, but I can also accept the point of view of the Authors. Thus, I also wait for the decision of the editorial team and I will accept it in any case.

“The authors have no problem providing the required information for this manuscript as long as the editorial team agrees with the idea (it would just be “copy-pasting” some tables), but we would rather keeping the data as it is right now for the same reasons that were already mentioned. After this statement, we will await for the decision of the editorial team and the reviewer. “

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors,

I have checked authors’ responses to my comments to their manuscript entitled “Different diets based on yellow mealworm (Tenebrio molitor) – Part B: modification of the intestinal inflammatory response and the microbiota composition of rainbow trout (Oncorhynchus mykiss)” (Ref. no. fishes-2340186).

I think authors have performed a great effort and I acknowledge that. However, there are still some concerns in bacterial community analyses that they have to clarify.

The use of abbreviations is making the text hard to read. A regular reader can be lost in the abbreviations. Insect meal and Fishmeal is easier to understand bettern than IM or FM. Moreover, the first paragraph of M&M is still confusing. Treatment levels are better explained in Table footnotes than in this paragraph. Avoid also EO for algal oil. ISI appears a couple of time, is it really necessary? I do not think so. Keeping abbreviations only for treatment levels make the reading straight and clear.

I understand perfectly that bacterial community analyses are not the major point of this manuscript. However, these results are in the paper and they deserves to be properly presented. In spite of authors are aware of basic concepts in ecology microbiology, some point are not properly described.

First: lines 266-274 have to be moved to the end of pervious paragraph, because sequence analyses are not statistical analyses. Are sequences paired or authors used a single end (forward?)?

Second, I am glad authors replied me about filtering, but it is in the paper where authors have to describe. Explicitly describe what kind of filtering authors have performed (move quality filtering before DADA2), and write explicitly a line saying that chloroplasts and mitochondrias have been removed). What happened with ASVs without phylum assignation? Were they removed?

Third: after these lines, authors have to described alpha diversity index and afterwards, beta diversity index (beta diversity index are still missing in the revised version).

Fourth, let’s statistical section just for statistical analyses. Do not perform ANOSIM. Perform PERMANOVA and describe it in M&M (this analysis is missing in M&M).

About how to cite a result, explicitly say: “significant higher” or “not differed significantly”. Just saying higher does not imply that this difference is significant. And that was my major point in the previous review: it was not clear when authors were talking about significant differences and when they were discussing higher or lower, without without a significant result. I am still missing two figures including PCoA results from Weighted and Unweighted Unifrac. Finally, if authors do not want to perform beta diversity analyses, it is OK. Just keep alpha diversity analyses and change “beta diversity” into “bacterial composition”.

The reticence of authors to include TANK in the analyses could indicate a significant effect. I should avoid that suspicions including TANK as a random factor in General Linear Models. I am glad authors explain the experimental design, a really good design indeed. However, the use of different tank and random assignation can and have to be included in the statistical analyses. GLM is just a generalization of ANOVAs and Linear Regressions. And this point is relevant as the effect of tank have resulted significant in previous studies:

·       Minich, J.J.; Jantawongsri, K.; Johnston, C.; Bowie, K.; Bowman, J.; Knight, R.; Nowak, B.; Allen, E. Microbial ecology of Atlantic salmon, Salmo salar, hatcheries: Impacts of the built environment on fish mucosal microbiota. bioRxiv 2019, 1–19.

·       Rabelo-Ruiz, M., Newman-Portela, A. M., Peralta-Sánchez, J. M., Martín-Platero, A. M., Agraso, M. D. M., Bermúdez, L., ... & Martínez-Bueno, M. (2022). Beneficial Shifts in the Gut Bacterial Community of Gilthead Seabream (Sparus aurata) Juveniles Supplemented with Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO). Animals, 12(14), 1821.

Provide major effects for all ANOVA’s, including degree of freedom, F and p-values. Afterwards, authors can provide posthoc tests for significant major effects.

The criteria about keeping a % of phylum or genera have to be placed in the main manuscript. Do not skip important details, especially those that used in the analyses and results. In general, answering the reviewer is great, but these explanations have to be written down into the main text.

Beta diversity section is incorrect. Authors are mixing bacterial composition and differences in bacterial community (the proper beta diversity). Unifrac PCoA plots for beta diversity and barplots and biplots for bacterial composition.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Dear authors,

I have checked authors’ responses for this third round to their manuscript (Different diets based on yellow mealworm (Tenebrio molitor) – Part B: modification of the intestinal inflammatory response and the microbiota composition of rainbow trout (Oncorhynchus mykiss), Ref. no. fishes-2340186).

Again, I have to highlight the outstanding effort of authors improving the manuscript. I will require authors a last effort, as I would like to clarify some of my concerns.

First, it is about tank. What I was asking is precisely that authors state in Statistical Section that they could not include tank as a factor for the lack of degree of freedom. That is all. Then, readers are aware about the correct use of statistics.

About alpha, my comment is (and was) that the calculation of alpha diversity indexes have to be in statistical section.

About PCA, please clarify (for me and in the text) what authors mean with variable (line 234), and what method was employed for scaling to the same scale (centered log ratio, standardization of the relative abundance of each ASV…)

Finally, about the major effects, I am not asking to include multiple F, d.f. and P-values. What I am asking is to include is three columns from tables 2 to 6, including this requested information. I am not asking for restructuring the whole manuscript. I will try to be clearer: results for posthoc tests are only correct when the main effect is significant. Then, discuss significant differences between treatment levels when the main effect is significant.

All the best

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 4

Reviewer 2 Report

Dear authors,

I would like to congratule authors for their hard work and for addressing my suggestions and concerns.

I think the manuscript has a good quality and its results are pertinent and really interesting.

All the best