Bacillus subtilis Supplementation in a High-Fat Diet Modulates the Gut Microbiota and Ameliorates Hepatic Lipid Accumulation in Grass Carp (Ctenopharyngodon idella)
Round 1
Reviewer 1 Report
The MS was improved according this referee comments, thus it is relevant for pubblication in the present form. Congratulation for jour job.
Author Response
Thanks very much for your constructive comments and important support.
Reviewer 2 Report
No further revision is required.
Author Response
Thanks very much for your constructive comments and important support.
Reviewer 3 Report
The Authors of the resubmitted and renamed manuscript currently titled "Bacillus subtilis supplementation in a high-fat diet modulates gut microbiota and ameliorates hepatic lipid accumulation in grass carp (Ctenopharyngodon idella)" have applied significant changes to their paper and addressed the problems spotted by all reviewers.
I must say that the new title is much, much better than the previous one. All in all, apart from a few mistakes, there is not much else in this manuscript that would require correction. Even the linguistic aspects of the text (prefixes) have also been improved.
Specific paragraph by paragraph commentary can be seen below:
Abstract: Much improved from the previous version, quite compact but definitely sufficient.
- Line 10: Add "in" before "grass carp".
- Line 18: Italicize "B. subtilis".
Introduction: I already did not have any major objections here previously.
- Line 43: There is some little mistake here, correct it to "... causes tight junctions of the affected intestinal mucosal cells to become more permeable."
Material and Methods: The Authors have addressed all of the important issues which I had raised before, good work (especially with Table 1). There is only one mistake/ambiguity:
- Line 104: Wait a second - only 6 fish per group were measured for basic body analyses? I hope this is some kind of a mistake made during the copying of this sentence into several subsections.
Results: My concerns regarding Tables and Figures were addressed, good work once again.
Discussion and Conclusions: As previously, there was not much to address here, while the applied linguistic corrections certainly improved the content of these sections. My minor concern is that only two more citations were added, which is quite surprising given the fact, that I specifically asked to include more references.
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
the study is well constructed but authors are suggested to analyse the whole body composition as it is important
also try to make a diagram represent the mechanism of action of the used bacteria
Reviewer 2 Report
The Authors of the reviewed manuscript titled "Effect of Bacillus subtilis on the intestinal microbiota of grass carp (Ctenopharyngodon idellus) with fatty liver induced by high-fat diet" have presented a solid piece of research, which definitely deserves to be published. However, it may happen only after a significant improvement of the technical description of performed analyses and also some corrections in the Results paragraph.
Firstly, two overall remarks - I would suggest to change the species' name to Ctenopharyngodon idella throughout the manuscript, it is the most accepted form. Likewise, I would suggest to use the plural form "indices" instead of "indexes".
Specific line to line, paragraph by paragraph commentary can be found below.
Title: Italicize "Bacillus subtilis".
Abstract: This section can be improved, especially the final sentence.
Line 9: Italicize "Bacillus subtilis".
Line 17: Add "to be" before "more similar".
Lines 17-20: This last sentence mostly repeats what was already established above - I would suggest to rewrite it to be more conclusive other than just repeating the inforomation about the obtained results.
Introduction: All the necessary information has been outlined in a thorough, but clear-to-read manner. I have no major objections in regard to this paragraph.
Lines 35-38: Please add citations which describe this "newly introduced concept ...".
Line 45: Change the order to "ectopic endotoxins".
Materials and Methods: This part definitely requires several improvements and clarification of some ambiguities.
Subsection 2.2: The title needs to be upgraded - I suggest something like "Feed preparation and feeding". My main objection here is that the information about the used feeds is severely lacking - Table 1 needs to be larger and must specify at least the basic proximate composition (protein, fat, ash, fiber, moisture), and possibly also the used ingredients (FM, SBM, casein etc). Besides, how were the bacteria added to the high-fat diet? Were they inoculated on ready-to-use feed, or earlier, prior to pellet formation?
Furthermore, there are more questions to be asked:
- was the feeding regime still two meals a day, at 4% BM daily?
- was each group kept in a separate tank (and of which size)?
- were the experimental conditions as previously indicated for the "domestication period"?
- how were the fish euthanized?
Subsection 2.3: Total length or Standard length? Please add the equations for WGR and SGR (I know they are well-known, but still it is common practise to show them).
Line 98: Add "and remaining visceral organs" before "were isolated".
Subsection 2.4: Please specify the used biochemical analyzer. Was the blood isolated from all 108 fish?
Subsection 2.5: Were livers from all fish analyzed histologically? Also, please indicate how the lipid droplet measurements were performed - how many droplets/cells were measured, in how many fields of view or on which total area? How were these measurements performed (manually or automatically)? What software was used?
Subsection 2.6: Were livers from all fish analyzed for the total lipid content?
Subsection 2.8: I would suggest to separate the statistical analyses into their own subsection and to be more specific, whether all of the different analyses were all compared using ANOVA.
Results: I would suggest to indicate the n number per group in the footnotes of each table, especially if these numbers are different for every analysis (as questioned above in the commentary on the M&M section). Otherwise, this section is solid, only minor corrections are needed.
Table 3: Place the title on the top of the table.
Figure 1: I am quite sure that subpictures "b" and "c" were swapped (either swap them or swap the HFD and BS.HFD groups in the description).
Figure 2: I am not an expert in this type of analysis, but this plot lacks a proper description - what are these different colorful lines representing? Specific samples, groups? Please enhance the description of this figure.
Tables 6ab: Correct column headings to "Percent".
Figure 4: Correct "microbiota" in the description.
Discussion: Generally, the discussion is sound, but I would suggest to use more references to previous works, especially in the part describing the intestinal microbiota.
Line 286: Correct to "no difference".
Line 304: Correct to "significantly".
Conclusions: Clear and concise.
Reviewer 3 Report
The topic is interesting, the language is good but the presentation (Tables, Figures) requires some modifications (eg. Fig. 1 and Table 6)
Reviewer 4 Report
The MS submitted by Guo et al. investigates the effect of B.S. dietary administration in a NAFD model developed in grass carp on growth idexes, blood serum parameters, liver fat deposition and intestinal microbiota. The results obtained are good even if methodology arouses some perplexity. First of all, the lack of a control group for B.S. (commercial diet+B.S.) doesn't make easy the interpretation of a part of the results obtained. Not having such a control is a pity, while the significance of the results should consider this at least in the discussion section. Differently, if the authors have these results shuld include them in the MS.
Moreover, at least the proximate composition of the diet used should be included, since in experiment like this it is not enough to refer to a commercial pellet.
Also, where are the results of Soxlet extraction? Are they presented in fig.1d? If so, it should be specified in the caption and better described in the result section. Presented in this way in the fig. it seems that it is a result from istological imaging analyses (which should be added..).
The discussions on F:B ratio are relevant (lines from 325) but should be better justified and explained.
Minor comments:
ABSTRACT
-line 10: correct in "grass carp were assigned to three groups"
-line 12: correct in "after 3 weeks feeding trial"
-line 16: "while promoting growth of the host" it is not justified since no B.S. ctrl group exists
INTRO
line 25: correct "in fish"
line 35: add "in mammals"
line 41: remove dot and correct "in playing"
Lines 41-46: sentence too long
M&M
line 68: correct in "120 grass carp (60...gr) were..... and were acclimatized for two weeks...
lines 78-80: explain better metodology
line 97: correct in "specific growth rate"
line 114: correct in "cryotome" and, even if not mandatory, specify microscopy brand.
Tab2: remove and describe in the text
Overall, improve methods description.
RESULTS
- averall improve this section
Discussion
C. haematocheilus is a mullet, not a pike species
lines 273 and following: liver fat deposition is not only related to the amount of dietary lipids, but also to the lipids quality and ingredients FAs profile. See and eventually cite the following
10.1016/j.aquaculture.2021.736550;
10.3390/ani11030677
Reviewer 5 Report
Title:
1- The name of species “Bacillus subtilis” must be italic.
2- Change the title to “Bacillus subtilis supplementation in a high-fat diet modulates gut microbiota and ameliorates hepatic lipid accumulation in grass carp (Ctenopharyngodon idellus)”.
Abstract:
3- Lines 9-11: Correct this sentence to “To study the effects of Bacillus subtilis supplementation in a high-fat diet on gut microbiota and nonalcoholic fatty liver disease grass carp (Ctenopharyngodon idellus) juveniles (60±5 g) were fed three diets: a) control diet (CON), b) high-fat diet (HFD), c) high-fat diet supplemented with B. subtilis (HFD+BS).”.
4- Correct the sentence in lines 11-13 to “After eight weeks of feeding, fish growth, serum biochemical indices, total liver lipid content were measured, and gut microbiota analysis was performed by MiSeq250 high-throughput sequencing platform”.
5- The authors have not presented the results of growth and serum biochemical parameters in the abstract.
6- Line 15: Remove “effect”.
7- Line 16-17: Correct this sentence to “Moreover, B. subtilis altered the intestinal microbiota of HFD fed grass carp making it more similar to that of the control group”.
8- Lines 17-20: Restructure the sentence in these lines to “The results of this study showed that B. subtilis can modulate intestinal microbiota of grass carp fed a high-fat diet leading to improved growth, blood serum indices, and reduced lipid deposition in fish liver preventing fatty liver disease.”.
Introduction:
9- Line 25: Correct “of fish” to “in fish”.
10- Line 26: Remove “effect”.
11- Line 30: Correct “Abnormal deposition of hepatic lipid” to “Abnormal hepatic lipid accumulation”.
12- Line 47” Change “to be an important” to “as an important”.
13- Line 48: Remove “animal studies, such as”.
14- Line 61: Correct “feeds” to “feed”.
Materials and methods:
15- Line 69: Do NOT start the sentence with a number.
16- Line 70: Correct “recirculating water cultural system” to “recirculating aquaculture system (RAS)”.
17- Line 83: Do NOT start the sentence with a number.
18- Line 83 and 84: Correct “were” to “was”.
19- Lines 86-89: Correct “Feeding for 8 weeks, the growth indexes of grass carp in each group were measured; tail vein blood was drawn and serum biochemical indexes were measured; liver and intestinal contents were removed” to “Following an 8-week feeding, growth indices of grass carp in each group were measured; blood was drawn from the caudal vein for measuring serum biochemical indices; liver and intestine samples were removed”.
20- Line 94: Correct the Table caption to “Design of the three experimental diets”.
21- Line 95: Correct “indexes” to “indices”.
22- Line 96: Correct “counted” to “determined’.
23- Line 97: Correct “Special growth rate” to “specific growth rate”.
24- Lines 98-100: The authors need to specify the number of fish used for determination of HSI and VSI.
25- Line 101: Correct “indexes” to “indices”.
26- Line 102: From how many fish per tank the blood samples were collected?
27- Line 104: Correct “indexes” to “indices”.
28- Line 107: Correct “, and the kits” to “using the kits”.
29- Line 110: Correct “Grass carp liver tissues were isolated and soaked in” to “Grass carp liver samples were isolated and kept in”.
30- Line 117: Correct “frozen-dried” to “freeze-dried”.
31- Line 122: Do NOT start the sentence with a number.
Results:
32- Line 159: Correct “indexe” to “indices”.
33- Line 160: Correct “The growth indexes of grass carp were measured (Table 3).” to “The growth indices of grass carp are shown in Table 3.”
34- Line 160: Correct “indexes” to “indices”.
35- Line 162: Remove “and liver fat content”.
36- Line 163-164: Move “and the grass carp model with fatty liver induced by high-fat diet was successfully established.” to 3.3 section.
37- Line 165: Correct “and significantly lower than” to “but significantly lower than”.
38- 169: Correct “showing the suppression of B. subtilis on lipid deposition” to “showing the suppressing effect of B. subtilis on lipid deposition”.
39- Lines 172-173: Correct “small letter superscripts” to “superscript letters”.
40- Line 175: Correct “indexes” to “indices”.
41- Line 176-177: Correct “Serum biochemical indexes of grass carp were measured through biochemical analyzer (Table 4).” to “Serum biochemical indices of grass carp are presented in Table 4.”
Discussion:
42-Line 269: Use “factors” after “environmental”.
43-Lines 269-270: Correct “Many studies showed that the content and source of lipid in feed is one of the most closely related factors” to “Many studies showed that dietary lipid content and source are the most closely related factors”.
