Effects of symbiotic population impairment on microbiome composition and longevity of Bactrocera dorsalis

In order to understand the role of symbionts for their insect hosts, it is customary to treat them with antibiotics or to sterilize eggs (treatments), resulting in aposymbiotic and axenic insects, respectively. Such axenic insects can then be compared with untreated controls. Fruit flies often bear complex communities which are greatly reduced by such treatments. However, the bacterial community is not completely eliminated. Here, we examine the effect of these procedures on the structure of the remaining bacterial communities in Bactrocera dorsalis (Diptera: Tephritidae) and on the insect longevity. The antibiotics (Norfloxacin and Ceftazedime) were administered to 1-day-old adult flies through sugar meal for 7 days, and eggs were surface sterilized and dechorionated to produce axenic lines. The flies were starved of protein before they were offered full diets or diets containing non-essential amino acids only. Antibiotic and egg disinfection treatments resulted in a significant reduction of the vast majority of gut bacterial populations, especially Proteobacteria, Firmicutes and Bacteroidetes. On the other hand, antibiotic allowed the persistence of Actinobacteria, Cyanobacteria and Acidobacteria populations. In untreated control flies, longevity was extended irrespective of diet quality in comparison to treated flies. Conversely, when gut bacteria were largely reduced (aposymbiotic and axenic flies), longevity was reduced in the non-essential amino acids diet treatment versus slightly improved in the presence of a protein diet. We discuss these results in an ecological–life-history perspective.


1.
Reorganizing the methods section, not necessarily shortening it, making a figure and explicitly indicating the number of replicates for each analysis would help to understand the method section more easily. 2. Figure 2 could be presented as supplemental materials as rarefaction curves are part of the data management prior to statistical analysis. 3.
The table in supplemental data is unreadable. The authors should consider dispatching the different taxonomy levels in different columns. The meaning of each color used in the table should be indicated in the table caption. A heatmap would be more appropriate than a table. 4.
The idea of core bacterial community (figure S1) is very interesting and would highlight the point about resistant bacteria. The authors should consider discussing this point and adding a table to present the taxonomy of the 47 OTUs. Is one of these OTUs dominant bacteria in term of abundance? 5.
The dataset is rich but the discussion relies on the phyla that were detected. Proteobacteria is such a large phylum so hypothesizing about their role in the communities is a bit daring considering that species belonging to the same genus do not necessarily have the same function. The discussion would be more interesting if some results were discussed at the genus level though it is still hypothesis. 6.
In the table in supplemental data, it seems that bacteria were identified at the species level, how is it possible when the authors used primers for 16S rRNA? 7.
Also, the authors mentioned in the introduction that the fly microbiota is very rich and diverse but only a couple of genera was mentioned and only in supplemental data. 8.
The authors should consider mentioning the number of OTUs and genera (and name) that disappeared from the communities of antibiotic and dechorionization-treated flies.¨ 9.
The authors should choose between the word "microbiome" and the word "microbiota" and use the same word throughout the whole manuscript. Same comment for "egg-sterilization" and "dechorionization". 10.
The authors should revise the way they wrote the tests results (F value, df and P value, there is one value of each per factor in each ANOVA, there is 1 ANOVA per alpha diversity index or the size of the community). P values from multiple comparisons could appear in figures and table as letters (commonly used in paper to indicate significant differences between modalities within one factor). 11.
It is now frequent for authors of a paper on microbiota to make the raw sequences available on a platform (e.g. ENA), do the authors plan to do this? 12.
The authors should consider discussing the bacteria resistant to antibiotics (they should be able to find papers on insect on this topic). Can bacteria "resist" dechorionization too? 13.
The authors should also consider discussing the nutrition role of B. dorsalis bacteria (if some are known in the literature) in order to explain the longevity results and identifying if these bacteria were present in symbiotic flies and absent from antibiotic/dechorionized flies.

Minor comments 14.
L60: what does "reduced the midgut microbiota" mean? Is it a decrease of diversity, of the abundance of certain bacteria or is it the number of bacteria that disappeared? L71: in the sentence presenting the aim of the study, please add the Latin name of the studied insect to make it clear because several species were mentioned in the introduction 17.
L96: please briefly describe the different diets composition (maybe in a table in supplemental data?). What is a full diet? It is also unclear which diets are fed to the flies in the different experiments/analyses. 18.
L106: the fact that the insects were not fed for 2 days of antibiotics might not be enough as effects of antibiotics could be observed after two generations in adult flies (Ourry et al 2020). That could be something to discuss in the discussion, also in regard of antibiotic-resistant bacteria. 19.
L114: the method section about the Validation of egg sterility could be a supplemental data and the authors could mention in the results section that egg sterility was checked and obtained. 20.
L116: please mention the concerned regions of the 16 rRNA gene 21.
L133: what is the point of measuring the gut bacterial community size with qPCR? Is it to obtain an absolute abundance in contrast to the relative abundance obtained after sequencing?

22.
L152: what about the sugar diet? Why was the sugar diet excluded from the longevity assay? Did the authors make sure that a change in diet (from sugar to a full diet) did not decrease/increase the performance of symbiotic flies? 23.
L210: Did the mortality data respect the normality? 24.
L221: which distance matrix was used? Bray Curtis dissimilarity matrix is often used. 25. L225: is it really possible to obtain mean values for the beta diversity, especially when using a distance matrix? 26.
L240: why did the authors use 5 indices for the alpha diversity? It is expected that all of them are discussed in the discussion. The most common ones are the number of observed OTUs (absent from the indices), Shannon and Simpson. 27.
L240-250: in this paragraph, the F and P values are not correctly associated to each index. The way it is written, it could be interpreted as p value from multiple comparisons (Tukey test) between the 3 treatments and not ANOVA results. For each index ANOVA, there should be one F value, 1 df (with a value of 2 because there are 3 treatments) and 1 p value. P values to compare two treatments do not come from ANOVA but Tukey test. 28.
The authors should consider adding letters in table 1 to indicate significant differences between symbiotic, aposymbiotic and axenic flies for each alpha diversity index.

29.
L258: in the section title, do the authors mean CFU and not OTUs? Because OTUs are not mentioned in this section. 30.
L260: how can CFU be obtained when a qPCR analysis was performed? Shouldn't it be a quantity of DNA instead of CFU? 31.
L260-266: same comments on the test values. There should be 1 ANOVA and thus 1 F value, 1 df and 1 P value. 32. Figure S2: three (and not two) statistical results should be indicated in the figure, between symbiotic and aposymbiotic, between symbiotic and axenic, and between aposymbiotic and axenic. 33.
L267: same comments on ANOVA. For bacterial relative abundance at different taxonomy levels, the authors should consider a Generalized Linear Model (poisson family and log link). 34.
L304: usually the results about community structure (i.e. beta diversity) are presented after, or sometimes before, the alpha diversity results (i.e. richness and diversity indices), and not at the end of the results section 35.
L305: what is the MRPP analysis? Is it a statistical analysis commonly performed on beta diversity? Again, why are there so many p values? What are the values for the test that determine whether the treatment factor has a significant effect or not on the beta diversity (i.e. community structure)? 36.
L319: that is the correct way of writing the test result, but the result about the sex factor is missing. 37.
L323-327: these test results should be indicated in the figure 5 as letters. 38.
L335-337: is the reduction due to the egg sterilization consistent with the literature? Only the antibiotic treatment was discussed in the sentence. 39.
L348: could the difference be due to different sequencing methods (technology, primers?) or due to the origin (location and host plant) of the insect population (i.e. where it was sampled in the field)? 40. L355: could it be possible to discuss bacterial functions at the genus level instead? Especially since Proteobacteria is a very large and diverse phylum. 41.
L361-362: is it a result consistent with the literature? 42.
L363-364: any explanation/hypothesis of why there was not difference between the antibiotics and egg sterilization treatments? 43.
L365: can this result be discussed at the genus level? There are some papers that discussed about an increase of bacterial abundance after an antibiotic treatments and about bacteria being antibiotic-resistant. That could also be worth mentioning in this paper. 44.
L380: are there any bacterial genera or species in B. dorsalis which are known to provide nutrients to their insect host and that were present in the bacterial communities of symbiotic flies and absent from the communities of antibiotic-treated and egg-sterilized flies of the present study? That could be interesting to discuss. 45.
L384: "This result suggests that the antibiotic and the dechorionization may have directly affected the flies" What do the authors mean by directly? Are they refereeing to the toxicity of antibiotics and the negative effect of dechorionization or to the effect of each method on the insect microbiota (which correspond to an indirect effect in the latter case)? The antibiotic toxicity and negative effect of dechorionization should be discussed. 46.
L385: The absence of difference in the longevity of aposymbiotic and axenic flies may come from the disappearance of bacteria beneficial to the host nutrition (see comments L380) 47.
L387-388: How can "Actinobacteria, Cyanobacteria and Acidobacteria […] have no potentials to extend fly longevity" when no bacteria from these phyla nor their functions were discussed priory? 48.
L396: "horizontally transmitted", that is a new idea that was not discussed at all in the discussion. Do the authors mean contamination?

49.
L400: the sentence is a bit too daring. The decrease of longevity can either be due to the reduction of bacterial diversity but also to the negative effects of the antibiotic and dechorionization treatments. 50.
What could be a concrete perspective of this work?
Review form: Reviewer 2 Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Comments to the Author(s)
Many experimental studies on model organisms use axenic individuals or treatment with antibiotics to study the role of the gut microbiota on various host fitness parameters. If done appropriately, axenic individuals should, as per definition, be germ free. Antibiotic treatment, on the other hand, does not eliminate bacteria but reduces bacterial abundance and changes the microbial composition dramatically. I therefore think that experimental studies to compare axenic individuals with those treated by antibiotics potentially could be relevant and of interest to a general audience. Especially, if the magnitude and direction of changes to the gut microbial composition differs predictably between the two methods.
Unfortunately, this manuscript makes no effort to generalize the findings from the model species used. Especially the Discussion clearly does not place the findings of this study into the context of current knowledge in this field of research as very few references are included in the Discussion. Also, it is unclear what the implications of this study will be to the field. How do the findings affect future work on model organisms that aim to modify/eliminate gut bacteria? The study is therefore currently a very specific study on a specific model organism, which I believe misses the scope of this journal, and probably belongs in a more specialized journal with a narrower scope.

Other comments
In the Introduction, it should be made more clear what the purpose of the study is to the overall research topic. Why was this study conducted, and how does this model system related to other model systems?
Line 70-71: You obviously expect the treatments to differ, so maybe say here that the magnitude and direction of changes remain unknown in most cases, and that it is the objective of your study to outline if the two ways of reducing gut bacteria produce predictable differences in bacterial composition.
Not necessary to summarize results in the Introduction. Instead, produce firm hypotheses/predictions that the experimental work was built upon.
Please provide an overview of the diets used. Although this is based on previously published work, it would be very helpful to see the diets briefly outlined here.
Line 396: "or they were horizontally transmitted to the host". This could be very important to the conclusions of your study. Yet it only appears towards the end of the conclusion. Did you describe bacteria from the diets and/or the environment?
Decision letter (RSOS-210267.R0) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Dr Akami: I write you in regards to manuscript RSOS-210267 entitled "Effects of symbiotic population impairment on microbiome composition and fly longevity of Bactrocera dorsalis" which you submitted to Royal Society Open Science.
While both reviewers found data of interest within the paper, they both raise a number of substantive concerns with the manuscript, including the premise of the paper, data interpretation and the context of the work with respect to developments in the wider field. In view of the comments of the reviewers and editors, found at the bottom of this letter, your manuscript has been rejected for publication.
Thank you for considering Royal Society Open Science for the publication of your research. I hope this decision will not discourage you from submitting manuscripts in the future. The reviewers felt that the paper contained much valuable results; however these were not fully explored and taken advantage of. Both reviewers had difficulty with the premise of the paper, data interpretation and how it was presented in the broader field. The study aimed at assessing the effect of two methods, commonly used to remove symbionts (i.e. antibiotics and dechorionization) on fruit flies bacterial communities and at evaluating the longevity of the treated (i.e. aposymbiotic and axenic) and untreated (i.e. symbiotic) adult flies when exposed to different diets. The authors have found that both treatments have a similar effect: they decreased but not eliminated the bacterial diversity and changed community composition compared to the untreated flies. Symbiotic flies lived longer than the aposymbiotic and axenic ones, but flies of each treatment tended to live longer under full diet compared to diet with non-essential amino acids. The strengths of the study is the comparison of two methods that remove insect symbiont, their effects on the insect bacterial communities (not often studied for this purpose) using a metabarcoding approach, and the link between the treatments and the diets since some bacteria play a role in their host nutrition by providing essential nutrients. The limits of the study are that i) the data are underexploited; ii) the link between treatments and diet is under-developed; iii) the important results are not well-highlighted despite the study being an interesting subject, making the discussion a bit dull to read; iv) the way statistics are presented is confusing (see comments); v) the methods section is long and confusing because in the end I was still unsure of which and how many samples were used for which treatment, diet and experiment (see comments).
Major comments 1. Reorganizing the methods section, not necessarily shortening it, making a figure and explicitly indicating the number of replicates for each analysis would help to understand the method section more easily. 2. Figure 2 could be presented as supplemental materials as rarefaction curves are part of the data management prior to statistical analysis. 3. The 5. The dataset is rich but the discussion relies on the phyla that were detected. Proteobacteria is such a large phylum so hypothesizing about their role in the communities is a bit daring considering that species belonging to the same genus do not necessarily have the same function. The discussion would be more interesting if some results were discussed at the genus level though it is still hypothesis. 6. In the table in supplemental data, it seems that bacteria were identified at the species level, how is it possible when the authors used primers for 16S rRNA? 7. Also, the authors mentioned in the introduction that the fly microbiota is very rich and diverse but only a couple of genera was mentioned and only in supplemental data. 8. The authors should consider mentioning the number of OTUs and genera (and name) that disappeared from the communities of antibiotic and dechorionization-treated flies.¨ 9. The authors should choose between the word "microbiome" and the word "microbiota" and use the same word throughout the whole manuscript. Same comment for "egg-sterilization" and "dechorionization". 10. The authors should revise the way they wrote the tests results (F value, df and P value, there is one value of each per factor in each ANOVA, there is 1 ANOVA per alpha diversity index or the size of the community). P values from multiple comparisons could appear in figures and table as letters (commonly used in paper to indicate significant differences between modalities within one factor). 11. It is now frequent for authors of a paper on microbiota to make the raw sequences available on a platform (e.g. ENA), do the authors plan to do this? 12. The authors should consider discussing the bacteria resistant to antibiotics (they should be able to find papers on insect on this topic). Can bacteria "resist" dechorionization too   Figure S2: three (and not two) statistical results should be indicated in the figure, between symbiotic and aposymbiotic, between symbiotic and axenic, and between aposymbiotic and axenic. 33 There are some papers that discussed about an increase of bacterial abundance after an antibiotic treatments and about bacteria being antibiotic-resistant. That could also be worth mentioning in this paper. 44. L380: are there any bacterial genera or species in B. dorsalis which are known to provide nutrients to their insect host and that were present in the bacterial communities of symbiotic flies and absent from the communities of antibiotic-treated and egg-sterilized flies of the present study? That could be interesting to discuss. 45. L384: "This result suggests that the antibiotic and the dechorionization may have directly affected the flies" What do the authors mean by directly? Are they refereeing to the toxicity of antibiotics and the negative effect of dechorionization or to the effect of each method on the insect microbiota (which correspond to an indirect effect in the latter case)? The antibiotic toxicity and negative effect of dechorionization should be discussed. 46. L385: The absence of difference in the longevity of aposymbiotic and axenic flies may come from the disappearance of bacteria beneficial to the host nutrition (see comments L380) 47. L387-388: How can "Actinobacteria, Cyanobacteria and Acidobacteria […] have no potentials to extend fly longevity" when no bacteria from these phyla nor their functions were discussed priory? 48. L396: "horizontally transmitted", that is a new idea that was not discussed at all in the discussion. Do the authors mean contamination? 49. L400: the sentence is a bit too daring. The decrease of longevity can either be due to the reduction of bacterial diversity but also to the negative effects of the antibiotic and dechorionization treatments. 50. What could be a concrete perspective of this work?
Reviewer: 2 Comments to the Author(s) Many experimental studies on model organisms use axenic individuals or treatment with antibiotics to study the role of the gut microbiota on various host fitness parameters. If done appropriately, axenic individuals should, as per definition, be germ free. Antibiotic treatment, on the other hand, does not eliminate bacteria but reduces bacterial abundance and changes the microbial composition dramatically. I therefore think that experimental studies to compare axenic individuals with those treated by antibiotics potentially could be relevant and of interest to a general audience. Especially, if the magnitude and direction of changes to the gut microbial composition differs predictably between the two methods.
Unfortunately, this manuscript makes no effort to generalize the findings from the model species used. Especially the Discussion clearly does not place the findings of this study into the context of current knowledge in this field of research as very few references are included in the Discussion. Also, it is unclear what the implications of this study will be to the field. How do the findings affect future work on model organisms that aim to modify/eliminate gut bacteria? The study is therefore currently a very specific study on a specific model organism, which I believe misses the scope of this journal, and probably belongs in a more specialized journal with a narrower scope.

Other comments
In the Introduction, it should be made more clear what the purpose of the study is to the overall research topic. Why was this study conducted, and how does this model system related to other model systems?
Line 70-71: You obviously expect the treatments to differ, so maybe say here that the magnitude and direction of changes remain unknown in most cases, and that it is the objective of your study to outline if the two ways of reducing gut bacteria produce predictable differences in bacterial composition.
Not necessary to summarize results in the Introduction. Instead, produce firm hypotheses/predictions that the experimental work was built upon.
Please provide an overview of the diets used. Although this is based on previously published work, it would be very helpful to see the diets briefly outlined here.

RSOS-211104.R0
Review form: Reviewer 3 Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation?
Accept as is

Comments to the Author(s)
I find that all the major and minor comments raised in the framework of the previous revision have been adequately addressed.
There are still a couple of typos that could be easily fixed: -in the conclusions the sentence at line 509: "These procedures resulted in significant reduction of the abundant gut bacterial populations in untreated flies" doesn't seem right as referring to the treated flies and not to the untreated.
-in the methods, line 123: "Symbiotic and aposymbiotic flies were produced from the wild larvae as mentioned above", shoud rather be "as mentioned below" Decision letter (RSOS-211104.R0) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Dr Akami, I am pleased to inform you that your manuscript entitled "Effects of symbiotic population impairment on microbiome composition and longevity of Bactrocera dorsalis" is now accepted for publication in Royal Society Open Science.
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On behalf of the Editors of Royal Society Open Science, thank you for your support of the journal and we look forward to your continued contributions to Royal Society Open Science. Reviewer comments to Author: Reviewer: 3 Comments to the Author(s) I find that all the major and minor comments raised in the framework of the previous revision have been adequately addressed.
There are still a couple of typos that could be easily fixed: -in the conclusions the sentence at line 509: "These procedures resulted in significant reduction of the abundant gut bacterial populations in untreated flies" doesn't seem right as referring to the treated flies and not to the untreated.
-in the methods, line 123: "Symbiotic and aposymbiotic flies were produced from the wild larvae as mentioned above", shoud rather be "as mentioned below" 2. Figure 2 could be presented as supplemental materials as rarefaction curves are part of the data management prior to statistical analysis.
Reply: Done. Please see Figure S1, supplementary (ESM_1). Thank you. What is a full diet? It is also unclear which diets are fed to the flies in the different experiments/analyses.

L133
: what is the point of measuring the gut bacterial community size with qPCR? Is it to obtain an absolute abundance in contrast to the relative abundance obtained after sequencing?
Reply: Yes, the rational of measuring the gut bacterial community size with qPCR is to evaluate the absolute abundance of the bacterial communities in host fly in comparison to the relative abundance obtained after sequencing.

L152
: what about the sugar diet? Why was the sugar diet excluded from the longevity assay? Did the authors make sure that a change in diet (from sugar to a full diet) did not decrease/increase the performance of symbiotic flies?

Reviewer: 2 (Comments to the Author(s)
Many experimental studies on model organisms use axenic individuals or treatment with antibiotics to study the role of the gut microbiota on various host fitness parameters. If done appropriately, axenic individuals should, as per definition, be germ free. Antibiotic treatment, on the other hand, does not eliminate bacteria but reduces bacterial abundance and changes the microbial composition dramatically. I therefore think that experimental studies to compare axenic individuals with those treated by antibiotics potentially could be relevant and of interest to a general audience. Especially, if the magnitude and direction of changes to the gut microbial composition differs predictably between the two methods.
Reply: Thank you for this interesting and faithful highlight, worth to be used as flagship of this manuscript.
Unfortunately, this manuscript makes no effort to generalize the findings from the model species used.
Especially the Discussion clearly does not place the findings of this study into the context of current knowledge in this field of research as very few references are included in the Discussion. Also, it is unclear what the implications of this study will be to the field. How do the findings affect future work on model organisms that aim to modify/eliminate gut bacteria? The study is therefore currently a very specific study on a specific model organism, which I believe misses the scope of this journal, and probably belongs in a more specialized journal with a narrower scope.

Reply:
The study prospects have been broadened. Please see the revised versions of the introduction and Discussion sections.