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The authors have addressed all concerns sufficiently, and I have checked that everything is included now. The manuscript is ready for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Dezene Huber, a PeerJ Section Editor covering this Section #]
Please consider the remaining revision suggestions (Reviewer 1), and also consider previous comments (Reviewer 2), which have not been properly addressed and must be considered. Please be precise in your definitions and be consistent; the reviewers have provided some valid points.
The article is written in good form. The background is well provided.
The experimental design is well thought and carried out.
The author addressed all the points of my previous review, and most of them have now been corrected. However, some minor issues remain, which I would like to highlight.
Line 271-274
You state: “The OTU 122 showed the highest relative abundance in colonies managed under the miticide-free and conventional systems, with the lowest relative abundance in the organic management system (MF vs CON: p-value = 0.30, MF vs ORG: p-value < 0.01, CON vs. ORG: p-value < 0.05).”
The problem here is that you only show the p-value, which indicates whether there is a significant difference between treatments, but does not reveal the direction of the difference (i.e., which one is higher or lower than the other). To demonstrate this, you should also report the estimate from the test.
Figure 5
I still don't understand this graph. If you describe a core taxon as present in all sites (“We defined core taxa based on the following criteria: (i) presence in 100% of sampling locations […]”), how could they show a "proportion of sites at which the taxon was present" lower than 1 (see blue bars in the upper part of the graph that do not reach 1)? If I am not understanding, please clarify the definitions.
Table 1
In the table description, you state: 'Taxonomic assignments of the 14 bacterial taxa identified as core,' but in the results section, you mention: 'The core taxa in bee bread of sampled colonies included 15 genera.' Additionally, the table itself lists 16 taxa across 16 rows (I think you repeated the Apilactobacillus kunkeei row twice). One of the taxa is Bombella apium, but I think you should stick with Bombella spp. as in the text. Please, check the table format again.
After these few points are addressed, the article will be ready to be accepted.
Self-contained with relevant results to hypotheses.
The previous recommendation was not taken into account.
The clarification regarding the origin of bee bread from honey bees is included in the article title, the short title, and is described in detail in the methodology. Repetitions in the article text can be considered unnecessary characters that contribute to the total character count of the article. The term "bee bread" is often also written as "beebread" as a single continuous term. You want to attribute the particle "bee" simultaneously to both the term "honey bee" and the term "bee bread." Therefore, in my opinion, the word "honey" is incorrectly used here as a term. I leave this for the authors to consider.
Rigorous investigation performed to a high technical & ethical standard.
The recommendation was discussed and accepted.
Conclusions are well stated, linked to original research question & limited to supporting results.
The recommendation was discussed and mutually agreed upon.
The inspected research is strong, relevant, and methodologically and statistically sound.
Please consider providing more information on the ecological setting; tidy up the R script and supplementary information and also better integrate it into the main body of the text (references). Visualisation and general images should be improved for clarity. That the microbiota is not dependent on geographical location is too strong a statement, and should be more thoroughly discussed; it also seems required to tone this done.
[# PeerJ Staff Note: It is PeerJ policy that additional references suggested during the peer-review process should *only* be included if the authors are in agreement that they are relevant and useful #]
The article is written in good form, but some improvements are advisable.
- Some references are in the numerical format, but there is no link to the bibliography. Please use the long format with the name and date of the article so it will be possible to check them. Look at lines 79, 213, 240, 366, 382, and references in Table 1.
- Check parenthesis usage in lines 67, 93, 132, 200, 213, 358, 378.
- You didn’t explain some acronyms like “CF” that you used in some result tables. Check them.
- Other minor typos will be listed at the end of the review in the line-by-line corrections.
The background is well provided. The structures and the figures/tables are of good quality and unambiguous (some comments on their content will follow). I really appreciate the sharing of the data and the R script through GitHub.
The experimental design is well thought and carried out. I just have two minor comments.
In Table S2, you thoroughly show the description of the three beekeeping management systems used in your experimental honeybee colonies. I’m wondering why you chose to use different types of hive features for the different types of treatments (e.g., comb arrangement, bottom board, inner cover). Since some of these features can influence the hive microclimate, wouldn't it have been more appropriate to standardize the hive features across all treatments?
In the Statistical Analysis section, you stated that you decided to perform a linear mixed model even though the assumption of the normality of the residual was not met, but you don’t specify which test you used to test it. I checked it in the R script, but I think it would be better to specify it in the methods. The Shapiro test is really strict, so I agree with your choice.
Bee bread bacterial alpha diversity section
The interaction of sampling location and sampling time point was a significant predictor only of Chao1, and not of species richness, as you say in lines 263-264 and as shown in Table S3. From Table S3 I can see that Treatment is marginally significant in predicting changes in the species richness.
For Figure 2, I suggest using a violin plot or showing the single data points inside the normal boxplot.
Beta diversity of the bee bread microbiota
Looking at the NMDS plots (Figure S4), it seems that a great portion of the ordination is explained by the site. I would have checked for it, even if it is already known to affect the microbiota.
I suggest using colour differentiation for the treatments to obtain a better visualisation since it is the variable you are testing. Alternatively, you can use ellipses to surround the points with the same treatment.
Responses of acidophilic bacterial taxa to miticide treatment
I tried to follow this part with the R script, but it was all messy, I suggest tidying it up.
You state that “The OTU 122 showed the highest relative abundance in colonies managed under the miticide-free and conventional systems, with the lowest relative abundance in the organic management system.”, but you didn’t show the results of the pairwise test (you talk about the Wilcox test in Materials & Methods).
Defining the core bee bread microbiota
In Line 292, you say that you found 15 genera that met the criteria to be considered “core taxa”, but in the description of Table 1 you say 14 taxa. Then, if you look at the table, there are 16 taxa listed. Also, I don’t understand why there is “NA” under the column “Genus” for the OTU 40 since there is a 100% match with Neokomagataea tanensis. I got confused. You can try to be clearer.
In the plot of Figure 5, you display the abundance and occupancy distribution of core and non-core bacterial taxa in bee bread. How is it possible that some core taxa (light blue colour bars) have a site occupancy lower than 1? You described “core taxa” as a taxa present in 100% of sampling sites.
In the description of Table S9, you state that you used post-treatment data for just 33 colonies. You didn’t explain why. Don’t you have the data for all the 36 colonies?
I believe this article presents solid work. The research questions are clear, and the methods used are appropriate for addressing them. The experiments are well-designed, properly conducted, and clearly and thoroughly described. However, I think there is still room for improvement.
As you also mention in the text, the microbial communities in bee bread can be strongly influenced by the local flora and the microorganisms associated with it. It would be interesting to have more information about the habitat of the different study sites. Are they diverse, or do they share comparable characteristics with common flora? It might be a good idea to assess the type of habitat surrounding the hives at the various sites using a land-use map to demonstrate actual variation in the sampled environments.
Other minor corrections
Line 37: spacing.
Line 83: You call L. kunkeei when you previously (line 78) used the name Apilactobacillus kunkeei. Change it to keep consistency.
Line 212: specify that you decided to keep using the linear mixed model, not just a mixed model.
Line 270: the beta symbol is not visible.
Line 276: I would say marginally significant instead of significant.
Line 352: you previously said that OUT 122 is assigned to Bombella spp., but here you say Bombella apis. Check it again.
Self-contained with relevant results to hypotheses.
Recommendations 1. Throughout the text of the article (and abstract, Table 1), check the term "honey bee bread" and replace it with the correct term "bee bread"
Recommendations 2. In line 129, you need to add the Latin name of the bee species with which the experiments were conducted. It is probably Apis mellifera L.
The rest of the Materials & Methods section is written in sufficient detail and well. Methods described with sufficient detail & information to replicate
Conclusions are well stated, linked to original research question & limited to supporting results
Recommendations 3. The Abstract and the main Сonclusion of the article contain the thesis that the microbiota is not dependent on geographical location. This is a very strong statement and may cause discussion. It should be tempered with one of the options "within our studies", "within the United States" or "within the studied locations", or other.
The inspected research is strong, relevant, and methodologically and statistically sound.
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