Administration of the Potential Probiotic Paenibacillus ehimensis NPUST1 Enhances Expression of Indicator Genes Associated with Nutrient Metabolism, Growth and Innate Immunity against Aeromonas hydrophila and Streptococcus indie Infections in Zebrafish (Danio rerio)
Round 1
Reviewer 1 Report
The criteria for the selection of the potential probiotics and their importance and future perspectives in aquaculture are well-known to all those who involve in fish farming and aquafeed manufacturers. Therefore, this manuscript is in the area of interest for publishing in Fishes. However, I spent a lot of time to revise the manuscript as much as I could and the authors should address the flaws and also they must provide further investigations (additional experiments needed) in the next round as a follow:
- Title: add the word “Potential Probiotic”. Clarify which pathogen in the title.
- L22-23: with draw the sentence about the definition of zebrafish, please.
- L24: It is necessary to mention the bacterial accession no. or strain no. in the parenthesis.
- L29: Only use abbreviations in the abstract section that will be repeated again in this section, otherwise only the full form is sufficient.
- L29-35: As the authors mentioned in the title, the results of “Nutrient Utilization and Innate Immune parameters” are missed.
- Again L29-35: It makes more sense for readers to know how much these parameters have changed compared to the control fish.
- Again L29-35: The results of the challenge experiments were missed.
- L36-37: Please rephrase the sentence in terms of word choice and grammatical aspects.
- L38: Try to use keywords that are not in the title and then, re-order according to their importance and/or alphabetic order.
- L43: any citation(s)?
- L108-124: Remove it. Because it is not really necessary to declare this info.
- L129: what is AB strain?
- L130: One weeks is not enough for fish acclimation under experimental environment, but at least two weeks is well. What is your justification(s)?
- L134: In my point of view, supplementing commercial diets with additives is feasible for fish farmers and acceptable. But what is the justification of the authors to ensure the commercial feed is free form probiotics and other additives/supplements that can interfere with the results? Because the formulation of commercial aquafeed is secret.
- L138-139: It is not accepted. The authors should provide all the info about the bacteria. e.g. Where isolated from which organism what is the strain no. or accession no.
- L140: stored at -20 for how long? Why the authors used 20% glycerol to preserve?
- L158: be careful about using superscript for counting bacteria throughout the manuscript.
- L172: Please clarify here that what was the selection criterion for this particular doses?
- L180: What is the initial weight of fish? How many fish in total?
- L182: what was the criterion for the feeding rate? why the authors did not use apparent satiety? Was there any attempt to collect extra feed and feces during the feeding trail?
- How many fish from each treatment were used for this analysis?
- L194-238: Summarize them by notifying the relevant citations
- L241: How they isolated and distinguish the liver samples from zebrafish?
- L256: The information on the LD50 should be provided in the supplementary file.
- L261: according to the number of treatments and small statistical population, why authors select Tukey's multiple comparison test as a post-hoc? What about normality test?
- L263: Why the authors select the Kaplan‒Meier method for presenting the results of the challenge experiments
- In all figs and tables, provide full name the potential bacteria + accession no or strain no.
- Figures 3 and 4: Normally, the gene expression level is about 1 for the control group. But the control group levels are lower than 1. Please clarify.
- Figure 5: The authors mentioned that they used Kaplan‒Meier method, but they used simple the cumulative mortality method. Solve this contradiction.
- L360: Please provide full name of the bacteria
- L443: the authors should prove a comprehensive discussion on the disease resistance.
- L450: This is a big claim by conducting one research with limited treatments and short period of time.
Critical questions/concerns/comments:
1) The study suffers from a dose study to find the optimal dietary level of P. ehimensis in zebrafish.
2) Any idea of counting P. ehimensis in the diets after preparation? Finally is the viability of the probiotics is controlled after drying.
3) As the authors say, some of the studied parameters were improved in the treated fish compared to the control fish. However, this is correct when occurs some circumstance altering the normal conditions, especially in the inflammatory cytokines. If the fish of the control group are normal (as it is desirable) any deviation of the values registered in the parameters of experimental groups cannot be considered beneficial. In fact, these determinations would make sense and might be done for the fish challenged with the pathogens in which the increased of cytokines could afford protection against the pathogen. Do you have any justification(s)?
4) In this study, the authors used fixed rates as a feeding strategy instead of apparent satiation. Eating rate during a fixed-portion meal does not represent the palatability of the diets enriched with P. ehimensis.
5) The information about growth performance must be provided in the next round.
6) There is no data no the levels of the serum immune parameters and relying only on the related gene expression is not adequate. Therefore, please revise the title.
7) The probiotic abilities must be tested to consider it as a potential probiotic and there are series of experiments suggested by FAD/WHO for any strain to be suggested as probiotic like “Ability to survive under simulated gastrointestinal tract conditions, Bile salt tolerance, Antagonistic activity, Assessment of antibiotic susceptibility, Antioxidative activity against α-diphenyl-α-picrylhydrazyl (DPPH) free radicals, Screening for lipase and bile salt hydrolase enzyme (Bsh) activity, Haemolytic and gelatinase activity, Determination of cell surface hydrophobicity, etc.” I can understand that at this stage it is not easy and possible to perform all experiments and prove its probiotic nature in one study. But, it is very important to perform some of the suggested experiments to reach a strong hypothesis that P. ehimensis is safe and effective as a potential probiotic.
Author Response
We thank the Reviewer for the comments. The Reviewers’ comments have been helpful in improving the quality of our manuscript. The responses to review comments are listed one by one. Please find attached. Thank you.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The study demonstrated the dietary supplementation of probiotics on modulating digestive enzymes, innate-immune-related gene expression, and resistance to pathogen challenge in the zebrafish model. Overall, the manuscript is well written, but some questions need to be addressed before considering for publication.
Since the effect of the probiotic used in this study had been tested in Nile tilapia, Why the study is conducted in zebrafish? Finally, the probiotic will be utilized and beneficial in aquaculture, so it is better to study the effect of dietary supplementation of probiotics in the target species.
In Fig 5, how many fish were tested? The detail of the experiment must be clearly explained in the materials and methods.
Check all scientific names (both in the main text and reference) must be italic.
Author Response
We thank the Reviewer for the comments. The Reviewers’ comments have been helpful in improving the quality of our manuscript. The responses to reviewer comments are listed one by one. Please find attached.
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript entitled: “Administration of the probiotic Paenibacillus ehimensis improves nutrient utilization and innate immunity against pathogen infections in zebrafish (Danio rerio)”, describes the possible use of the mentioned probiotic to increases the resistance of zebrafish, used as fish model, against bacterial infections. Even the manuscript subject is quite interesting, there are some minor aspects that must be clarified.
- Introduction: I have found the introduction quite long, being included in this section information that it is not needed. For instance, from lane 77 to102 the authors describe the effects of bacteriocins; however, the production of bacteriocins by Paenibacillus ehimensis is not investigated in the present article, being the aim of previous articles of the authors.
- Line 146 to 156, English language should be revised throughout the descriptions of the enzymatical assays in vitro. For instance, line 148: “indicated the bacteria with protease activity”, it should be replaced by bacterial protease activity. The description of the control conditions used are not described in the text, this information should be included. Furthermore, the measure of these activities in vitro is quite subjective. Is it not possible to quantified these bacterial activities in the same way as it has been done for digestive enzymes in zebrafish intestines?.
- Antibiotic susceptibility of the probiotic. Which is the finality to perform this assay?. No discussion of these results has been included in the manuscript. Furthermore, to determine if a bacteria is resistant or not to an antibiotic, it is necessary to measure the halo diameter and compare to the standards for each antibiotic, these data are not presented in the manuscript. In the result, the authors afirm that the bacteria is sensitive (or has lack of resistance) to all the antibiotics tested, how is this possible if the inhibition halos have not been measured? even more, for the sulfadimethoxine, the inhibition halo is not observed.
- Line 178-179, this part is confusing, and initially seems that E1 and E2 are control group when they are the experimental ones. Please rewrite this part to clarify that there are three groups, one control and two experimental groups.
- The epigraph 3.1. from results should be divided into two, one corresponding to the in vitro assays and another one to the in vivo.
Author Response
We thank the Reviewer for the comments. The Reviewers’ comments have been helpful in improving the quality of our manuscript. The response to reviewer comments are listed one by one. Please find the attached, Thank you
Author Response File: Author Response.pdf
Reviewer 4 Report
fishes-1946182 “Administration of the Probiotic Paenibacillus ehimensis Improves Nutrient Utilization and Innate Immunity Against Pathogen Infections in Zebrafish (Danio rerio)”.
GENERAL COMMENT:
The work entitled “Administration of the Probiotic Paenibacillus ehimensis Improves Nutrient Utilization and Innate Immunity Against Pathogen Infections in Zebrafish (Danio rerio)” is a good work; the subject of the study is original and interesting.
This study investigated the ability of probiotics to produce hydrolytic enzymes and the effect of probiotics on nutrient metabolism, immunity and disease resistance in zebrafish (Danio rerio).
The introduction is exhaustive.
Central argument is supported by evidence and analysis.
The methodology described by the author is accurate.
The discussion needs to be expanded
This work is a good work; in my opinion it needs some minor changes, I require a Minor Revision.
DETAILED COMMENT:
· Title
-The title is adequate.
· Abstract
-The abstract is well structured and the objective of the study is clearly described.
Keywords are adequate.
· Introduction
The introduction section is exhaustive.
· Materials and Methods
The section is well written and accurate.
· Results
This section is accurate and detailed
· Discussion
The discussion section needs to be expanded.
· Conclusions
Conclusion are adequate.
· Tables and figures
Tables and Figures are clear and understandable.
· References
The references are adequate.
Author Response
Thank you for reviewing the manuscript and recognizing our study.
Round 2
Reviewer 1 Report
Good luck
