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The authors addressed all of the reviewers' comments.
[# PeerJ Staff Note - this decision was reviewed and approved by Konstantinos Kormas, a PeerJ Section Editor covering this Section #]
After the first revision the reviewers still have some concerns. I invite you to revise the manuscript and make sure you answer all of the reviewers questions. If you disagree with any request, please make sure explain why you disagree in your rebuttal letter.
The manuscript presented by Guan et. al. has been improved since last submission. Samples details and further information on the methodology were added.
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Please fix the title of Figure1. It’s not a histogram is a bar plot. Also correct this in line 191.
Line 219 - Please correct “micriobiota” to “microbiota”.
Line 188 – ‘core bacterial composition’ cannot be used here as there are no methods applied for this type of analysis here.
Table 1 might be unnecessary as all samples came from individuals in the same age and weight groups. Table could be moved to supplementary, and information could be added to the text without the need of a table.
The discussion section could still be improved. The study is descriptive of a small set of individuals, and still some not related information is discussed as mammals, obesity, Alzheimer. There is no discussion on overall rescue/captivity of wild animals.
Bacteroidetes to Firmicutes ratio related to differences in age and weight is mentioned frequently in the discussion, and the obesity discussion seems out of place. How obesity in this context is relevant?
Also, this discussion has no justification by the data presented here. In lines 259-261 authors mention differences in weight between healthy and injured krestels, however this information is again not presented properly. In Table1 a single range is used for all individuals, so this statement has no support in the data. The same is for age. For example, no attention is given by the possibility of these difference being also due to the medications the injures krestels were subjected to. Why obesity and not the use of medications are discussed?
I am not satisfied with the work the authors did based on previous comments I give them. They didn’t improve figure legends appropriately. They didn’t explain why my suggestion are bad and did not change anything and write just “Thanks for the reviewer's suggestion”. And this is not only the case of figures. I do not want to spend my time writing the same again, but I also do not think that to publish these bad legends is ok.
Most parts of the text are improved. I found out discussion and introduction ok now. Except the conclusion which is not conclusion but mainly it sounds me like aims of the manuscript, but the previous paragraph (lines 331-337) can be the conclusion and the paragraph named conclusion can be deleted (line 339-344). And from the results of this article the conclusion on line 342-344 do not make sense, because they didn’t tested the relevance of faecal samples as non-invasive method in this manuscript.
Abstract - 33-37 there should be conclusion not again aims which are in abstract also on lines 25-28
Figure 2: The picture caption should be corrected as I don't know what is meant by „species“ maybe it should be genera or taxa? In figure caption “The value represented by the color gradient is on the right of the figure.” can be deleted.
Figure 3: The image caption should be edited to make sense. What is shown is a boxplot for the diversity for two groups of samples and the significance done by the Wilcoxon rank-sum test is indicated, but the figure definitely does not show the test. Why is there now two time almost the same text?
Figure 5 species names??? species names at a certain taxonomic level, Maybe it can be something like „names of taxa at certain level“?
All figure captions fail to explain what D and H mean. It is not enough to have it in table as the figures have to be self-explanatory.
Age and weight don’t need to be in table 1 as you do not know it for individuals you can just mention the range in methods section.
Me and also the other reviewer had the same question about PCOA and MDMR. I know that there are slightly different, but still the result is almost the same so for me to make here also the metrices of weighted unifrack instead of these two makes more sense even that the results are not different. They added the PERMANOVA which is good, but why they did PERMANOVA for Bray-Curtis and not unifrac? To summarise they use different metrices for different things without any sense for me. I think that to use at least one abundance based and one presence/absence based ordination method for the result will make more sense and it is quite common. To use only one for something and for something else different is strange if they did not explain it in the manuscript.
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Also check the text for typing errors:
112 „fecal samples“ or „feces“ not „feces samples“
211 phylum level?
239 wild birds(Guan et al. 2016 - wild birds (Guan et al. 2016
Figure 2/5 diûerent „diferent“; ûgure – Figure
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While the reviewers found merit in your paper, reviewer 3 raised a concern about the similarity of this manuscript with the manuscript previously published by you in 2020. If you intend to revise this manuscript, please make sure you adequately address this concern. I also agree with the reviewers about the sampling effort. Your sample size is too small to make so many inferences. Please attempt to frame your results and conclusions, considering this limitation.
[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. #]
The article “Comparative analysis of the fecal microbiota of healthy and injured common kestrel (Falco tinnunculus) from the Beijing Raptor Rescue Center” has good structure, figures and tables are appropriate. Also, good English is used throughout, though minor issues are pointed further that would improve comprehension of the work done here.
The authors cite new information in the discussion that are not presented in the Result, or Materials and Methods, section. Lines 241- 256: There is no information on the sampled individuals besides “healthy” and “injured”, yet the authors cite age and weight range between injured and healthy krestels. Please, add a table with this information on the sampled krestels. Information like weight, sex and age should be present. Statistical comparisons between the groups, regarding this information should also be done. E.g. comparisons of means/rank-sum. And in lines 247-248 authors present the Bacteroides/Firmicutes ratio of healthy and injured groups, however this data is not presented in the Results section.
The Discussion section could be more concise. Paragraphs are long, which is not necessarily bad, however the reasoning for the ideas related are presented only at the end and are sometimes hard to follow. Briefly presenting why beforehand will improve the understanding of the conexions made with other research. Lines 297 – 313: This paragraph for example would be much more interesting to read if starting with the reason why the authors consider Spirochaetes to be relevant for monitoring of wild birds.
In addition, are there other important bacteria that could be important in monitoring diseases carried by birds also present in the sampled krestels? This should be an important information to add to this manuscript.
Is not clear if some method of normalization is used. Is proportions used here as a normalization method? This should be clearly stated in the article.
The beta diversity analysis could be better described. The authors performed two different ordinations, PCoA and NMDS, and it’s not clear on which distance these methods were applied (lines 151-152). In line 194 states that PCoA was applied to an unweighted UniFrac distance. Was this also applied to the NMDS? Also, it is not clear why use two different ordination methods, what questions does these two different methods answer?
Further explorations of the gut microbial communities composition and structure could be done by investigating the potential functions within each krestels group.Performing a functional prediction analysis, as PICRUSRt (https://pubmed.ncbi.nlm.nih.gov/30298254/), would bring interesting information into this work.
It is not clear what hypothesis test was performed on the PCoA and NMDS ordinations to state that there was no significant clustering (line 195 - 196). PERMANOVA would be appropriately used for this end.
These are some textual correctios that should be addressed:
-Line 314 – 316: “However, at the genus level, the Glutamicibacter was the only genus which significantly higher (p = 0.036) in injured common kestrel (3.337%) than in healthy group (0.002%). The genus Glutamicibacter consists of 10 genera and belongs to the family Micrococcaceae.” This should be reviewed. Glutamicibacter is a genus so it cannot consist of 10 different genera;
-Figure 1A-B y axis title “Percent” should be Proportion. Also fix it in line 180-181;
-Line 264 – 265: Organisms with relative abundances of 0.2% are not dominant organisms;
-Lines 195-196 “was observed" change to "observed";
-Line 255-256: “As the number of rescued individuals increasing, the composition of fecal microbiota in wild common kestrel would be more accurate.” First part of the sentence is confusing, could be something like: “With the increasing number of rescued individuals …“;
-Line 204-205: This sentence is unclear;
-Line 311 “disease and other infectious diseases, except the conventional trauma.”. ‘Except’ here convey a different message. ‘besides’ or ‘as well as’ should probably be more appropriate here;
-Line 228-229: Remove "had been";
-Line 236: " and Bacteroidetes” replace by "Bacteroidetes;
-Line 140 - 141 . Please, fix the way the database version is written. Is not clear SILVA(SSU132) is the version. Should be something like “SILVA (v 132)”.
This article is interesting and definitely publishable, it just needs some improvements.
The structure of the manuscript is normal and the English is at a good level, but I am not a native speaker so I cannot fully judge.
Abstract needs improvement:
28 correct “Miseq” instead of “Hiseq platform”
28-31 Here I think it may just be present "Firmicutes, Proteobacteria and Actinobacteria were the most predominant phyla in the common kestrel". Because the difference is not significant and there is very little difference in percentage especially when I saw the individual variability.
32-36 It is also included in the introduction, where it fits much more. It can be omitted here and some of the main results or conclusions given instead.
The introduction also needs improvement:
I would restructure the introduction because after the selection of the group studied, there is a section on mammals. Maybe it would be enough to switch these two parts (49-61 with 62-76).
59-61 From this sentence and paragraph (62-76) I would expect wild raptors in your study, but they are not wild because they are in a rescue center, which is also true for healthy birds, or am I wrong. Please clarify this in the introduction and add some information to the methods section. In the methods section, you could state how long the birds are at the rescue center before sampling, mention breeding conditions and add more information about the healthy birds, why they were there.
Most of the figures are relevant, but all need sufficient improvement, and I am not sure if all the figures are really necessary as they mostly show the same results in different way. All figure captions fail to explain what D and H mean. Specific comment to individual Figures:
Figure 1: The font size of the legends is very small. In the image caption can be deleted “for species” and can be added information about what the “others” mean. Is it taxa abundant less then one percent or something else?
Studying the figure in detail, I was confused that for example the genus Clostridium_sensu_stricto_1 is abundant in the two D samples (D2 and D3), but in the H vs D comparison it is more abundant in H. Also the genus Lactobacillus is most abundant in the H3 sample and very little in D1, but then it is more in the D group samples. Or I only see the genus Psychrobacter in the H1 sample and then it is abundant in the D group. I think it would be worth checking to see where the mistake was made.
Figure 2: In the picture caption, the words should be split “weightedUniFracdistance”. The font size of the legends is very small and what the “others” mean. I am not sure if it is necessary to have this figure and the figure one part A) phylum as this is almost the same just with some phylogeny tree. What clustering algorithm was used?
Figure 3: The picture caption should be corrected as I don't know what is meant by „species“ maybe it should be genera or taxa? What clustering algorithm was used?
In figure caption “The value represented by the color gradient is on the right of the figure.” can be deleted.
Figure 4: The image legend can be removed or the font size can be increased. The size of the "*" should be larger otherwise I wasn't sure what it meant until I found it in the text.
The image caption should be edited to make sense. What is shown is a boxplot for the diversity for two groups of samples and the significance done by the Wilcoxon rank-sum test is indicated, but the figure definitely does not show the test.
Figure 5: The font size of the legends is very small. I don't understand why you chose a picture showing NMDS and PCoA when it shows almost the same thing. It would be much more interesting to show PCoA with a different distance, for example weighted Unifrac. I assumed you were also using the unweighted UniFrac distance for NMDS. Why is only PCoA mentioned in the figure caption?
Figure 6: The picture caption should be corrected as I don't know what is meant by „species name“ or „species“. The font size in the figure is very small. The image caption should be edited to make sense again the Wilcoxon rank-sum test p values are shown, but the figure definitely does not show the test.
Raw data are presented correctly and can be accessed.
This is original research with an interesting group of birds that have not been studied so much before. Unfortunately, as is still quite often the case with this research, it is based on a really small number of individuals and therefore the results of the statistics, which there are not many here, have to be presented with much more caution than they actually are. The research question is well presented and has been relatively little studied to date.
I have only minor comments on the methods section:
I'm not sure how the samples were tagged because you didn't mention it. I would have expected it in one of these paragraphs 108-118, 120-128.
151 – 153 In the method section, the distance matrices for PCoA and NMDS should be given.
Why do you choose weighted Unifrac for the hierarchical clustering tree and unweighted Unifrac for PCoA? Why have you given results here for PCoA and NMDS when both are for unweighted Unifrac distance or NMDS is based on a different distance? I have no idea because it is not mentioned in the manuscript. If they are both for the same distance, why are almost similar results presented here? Perhaps using two PCOAs, with unweighted and weighted Unifrac, would be more interesting.
154 I miss the heatmap specification in the methods, and I don't think it's for species abundance, but genera.
I think this part is the biggest weakness of the paper, because it does not always take into account the relevance of the results due to the number of samples and the variability of microbiota composition between individuals.
I have a few comments on the results section:
182 – 187 Move to the next chapter beginning on line 190.
186-187 I would preferred to write here the results and not this “which indicated a similar result as that observed from the hierarchical clustering trees.”
190-192 I would expect more information than just the p value for the Wilcoxon rank-sum test.
195-196 “no significant clustering” is it tested in any way or what are the significant results based on?
197 – 201 I'm not sure if it makes sense to test this when the sample size is so small, the individual variability is so large and the results are not significant for the whole composition.
Some parts of the discussion would be more suited to the introduction of the article than the discussion (for example lines 204-210).
I would delete some parts because they are redundant here (lines 214-216).
The most relevant parts of the discussion are the following lines 217-221 and 334-340, which I would like to keep.
I would have expected the authors of the manuscript to be a bit more critical about their results. For example, when I look at the significant phyla and genera that differ between groups, they are only very poorly represented, and it is usually not even possible to verify in the figures whether they are represented by only one sample or multiple samples, so it seems to me that basing a discussion on them is misleading. Significant results may be due to very few samples in this test and so, for example, greater representation in one sample and very little in all the others may be crucial, but with a larger number of samples these taxa would not be significant. Taxa can be briefly mentioned, but it seems redundant to write long paragraphs about them. In the case of taxa, where I can look at their representation in the figures that show the individuals, it can be seen that the result is usually driven by two samples from a given group, which, however, due to the large individual variability even in healthy individuals, may not be indicative of health status.
I also find the discussion about the ratio of Firmicutes to Bacteroidetes strains redundant, given that the amount of these groups will also be affected by the amount of Proteobacteria present.
316 „genus Glutamicibacter consists of 10 genera“ can be corrected I suppose on „genus Glutamicibacter consists of 10 OTU“
In my opinion, the English language should still be improved.
The Abstract should be improved.
There is no clear hypothesis.
The authors make many inferences based on analyzes of very diverse phyla (e.g., Bacteroidetes).
The sampling effort is small to make so many inferences.
Data analysis was performed with old versions of bioinformatics tools and databases.
Many works on amplicon metagenomics in the literature better analyze and explore the results.
The work is nothing new since very similar work has already been carried out by the same authors (Guan et al., 2020).
The results are poorly discussed.
Abstract:
- Replace (50.205%), (47.985%), (1.677%), (46.116%), (42.330%) and (5.458%) by (50.20%), (47.98%), (1.68%), (46.12%), (42.33%) and (5.46%).
- Please correct the structure of the abstract and describe the objective only once.
- In the conclusion, the authors say: “The results could provide the basic data for additional research on the fecal microbiota of common kestrels and contribute to the rescue of wild raptors in the future.” How is this possible only by comparing the phyla between the two groups?
- Remove the number of samples.
- Actinobacteria, Cyanobacteria, and Bacteroidetes????
I suggest replacing some keywords. Some words are already in the title.
Introduction:
Lines 46-47: …”in recent years…” – reference from 2013?
Also, the reference Hildebrand et al. (2013) is not appropriate.
Line 48: Are the references Turnbaugh et al. (2007) and Waite & Taylor (2015) from humans and insects?
62-63: How many species? Or which?
85: Zhou et al. (2020)
99-100: Fecal samples were collected from different healthy (n = 6; H1-H6) and injured (n = 4; D1-D4) common kestrels.
103-104: analysis of uncultured bacteria
112-113: reference for the primers
131: Why did the authors not use QIIME 2?
137: Please, use Amplicon Sequence Variants (ASVs).
146: Provide references for data analysis.
146: Chao1. And Sobs?
149: R package version is too obsolete.
160: or ASVs?
169-173: Consider two digits after the comma.
179-181: What is a core microbiome? How would it be possible to access only with the data shown in figure 1?
190: Chao1
191-192: (p = 0.014, 0.025, 0.070, and 192 0.070, respectively).
195: delete “….and are shown in Fig. 5.”
200-201: I think some information is missing.
205-208: references
214-216: results
217-219: What is the difference between the present study and the one by Guan et al. 2020?
225: What are the biological functions?
248-250: As the discussion of the Bacteriodetes group is based on diet and obesity, the authors should inform the weights and diets of the two groups in the Material and Methods section.
297, 307: Spirochaetes
315-316: There is confusion.
Conclusion: The results obtained do not allow the conclusions presented by the authors.
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