Interaction with refuse piles is associated with co-occurrence of core gut microbiota in workers of the ant Aphaenogaster picea

  1. Alison Pagalilauan
  2. Christina Pavloudi
  3. Santiago Meneses Ospina
  4. Adam Smith
  5. Jimmy Saw

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Abstract

Comparing the diversity of gut microbiota between and within social insect colonies can illustrate interactions between bacterial community composition and host behavior. In many eusocial insect species, different workers exhibit different task behaviors. Evidence of compositional differences between core microbiota in different worker types could suggest a microbial association with division of labor among workers. Here we present the core microbiota of Aphaenogaster picea ant workers with different task behaviors. The genus Aphaenogaster is abundant worldwide, yet the associated microbiota of this group is unstudied. Bacterial communities from A. picea gut samples in this study consist of 19 phyla, dominated by Proteobacteria, Cyanobacteria, and Firmicutes. Analysis of 16S rRNA gene sequences reveals distinct similarity clustering of A. picea gut bacterial communities in workers that have more interactions with the refuse piles. Though gut bacterial communities of nurse and foraging ants are similar in overall composition and structure, the worker groups differ in relative abundances of dominant taxa. Gut bacterial communities from ants that have more interactions with refuse piles are dominated by ASVs associated with Entomoplasmataceae. Interaction with fecal matter via refuse piles seems to have the greatest impact on microbial taxa distribution, and this effect appears to be independent of worker type. This is the first report surveying the gut microbiome community composition of Aphaenogaster ants.

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  1. Access Microbiology

    While the revisions have improved the manuscript, some more amendments are necessary, in particular the description of the methodology and some of the figures need some further work. For details please refer to the comments raised by the reviewer.

  2. Access Microbiology

    Comments to Author

    This manuscript is much improved from the initial submission. In particular, the introduction more accurately places the work in the context of previous research. The methods and results could still use further specification and the figures improvement. See below for specific comments. Line 260 - Listing all packages at the end of the methods rather than pairing each with the specific analysis used puts the burden on the reader to disentangle how this software was used. The authors should more clearly explain how analyses are paired with specific software and any choices that have the potential to influence the results. Even with firsthand familiarity with most of these packages, I don't think I could effectively recreate the exact analyses or figures presented. Line 254 - The methods are not adequately justified or explained in detail. For example, why was this phylum singled out for more detailed phylogenetic analysis? I am certain there was good reason (antibiotic potential is mentioned in the introduction), but again, this feels like the authors are leaving this detective work up to the reader. The methods would read much more clearly if the authors critically evaluated their own descriptions - are we doing the work for the reader or are we providing the minimum acceptable standard for total information? For each method in the statistics section, it would be helpful if the readers understood what was done, how it was done, why it was done. Some of this could be accomplished by rearranging current content and phrasing, but in general I don't think these methods for microbiome composition allow for effective reproducibility. Line 870 - I'm not exactly sure how this figure was created, as the methods didn't adequately describe it. Line 865 - This figure is very hard to read, and I am not sure it is necessary for the point being made. Consider a log scale and moving to the supplement. Line 337 - Along with the vague descriptions of these methods, the descriptions of results also read as though they are "by eye". If multivariate dispersion is of interest, then quantify this and measure if the samples are more tightly clustered. Line 329 - Again here, the results aren't clearly articulating what standard is being applied for the statement that a microbe is a "driver" or not. Fig S1 - This color scale is uninterpretable. I sympathize with the dilemma of how to display so many categories, but this is supplemental, so space isn't an issue. Perhaps unstacking bars and labelling taxa would help if this level is important to visualize. Also, is this relative abundance or raw ASV abundance? It may be mislabeled.

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  3. Version published to 10.1099/acmi.0.000832.v2 on Access Microbiology
  4. Access Microbiology

    This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The work presented is relevant and timely, however both reviewers have highlighted a number of important points. Please make sure you address these, especially regarding the methodology used, and further analyses regarding compositional differences and the suggestion of discussing a few aspects in more depths.

  5. Access Microbiology

    Comments to Author

    Overall, the study's introduction needs to be substantially revised to better cover work performed in other systems and tone down claims of novelty. As more of a recommendation based on making the study broadly interesting, the notion of constraints on co-evolution between microbes and their multi-caste hosts is interesting and has received little attention. The methods are incomplete, see specific comments below. The investigation of which microbes were driving differences was essentially left out of the methods, but there are some good avenues to improve that portion of the paper with further analysis. Overall, this was an interesting study, but the paper here is not yet appropriate for publication as more work is needed. I emphasize the "yet" as I think it clearly could be given some changes to the introduction, methods, and results. I encourage the authors to continue working on this paper as it is a very interesting topic and the result they have sheds light on a poorly understood aspect of social insect microbiomes. Specific comments Title: The"driver" implies that cause was established, which certainly is not the case given the observational nature of the study. Line 45 - "metabolic driver" is a little confusing here. Line 50 - This seems a strange way to propose this relationship and a bit circular. There are lots of things to cover in this intro, but consider more clear distinctions and more exhaustive list here comparing the causes and consequences of any differences in microbiome observed between sub-castes. For example, review any differences found among castes, review consequences of differences, whether they may advantage the colony, etc. Line 66 - Outside of ants, this introduction should really incorporate things learned from honeybees regarding hygiene, sub-caste roles, and the microbiome. This has to be the best studied example among social insects. Line 80 - double check this citation [12], it seems like something else was intended. Line 81 - this notion of benefit comes up here again and I think is contributing to a broader mischaracterization for how microbiome associations (particularly among social insects) would be expected to evolve. There are constraints on differences among castes and the mechanisms of inheritance that don't allow this sort of highly parallelized co-evolution (i.e. different castes evolving with their own highly tailored microbiota). I think this type of research presented here is needed for exactly this reason, so it is a bit unfortunate the authors ignore this (at least up until this point). Line 97 - This is a strong statement and not an accurate portrayal of previous work. See many reviews and studies, but just a few: Zientz, E., Feldhaar, H., Stoll, S. et al. Insights into the microbial world associated with ants. Arch Microbiol 184, 199-206 (2005). Russell JA, Moreau CS, Goldman-Huertas B, Fujiwara M, Lohman DJ, Pierce NE. Bacterial gut symbionts are tightly linked with the evolution of herbivory in ants. Proc Natl Acad Sci U S A. 2009 Dec 15;106(50):21236-41. doi: 10.1073/pnas.0907926106. Russell, Jacob A., Jon G. Sanders, and Corrie S. Moreau. "Hotspots for symbiosis: function, evolution, and specificity of ant-microbe associations from trunk to tips of the ant phylogeny (Hymenoptera: Formicidae)." Myrmecological News 24.21 (2017): 43-69. Line 117 - This is cited accurately as, but perhaps an unfortunate phrasing by the authors in the original article cited in [23]. Instead of playing up the novelty repeatedly, it would be more helpful to review what is known from the few systems where this has been extensively studied (termites and bees). Line 120 - There have been a few of these predictions, which may well be founded in previous results and theory, but those results were not outlined in this introduction effectively, so they often read as being off-the-cuff and unfounded. Line 132 - Another sentence about collection protocol would help here and also specific lat/lon and elevation. Line 136 - manufacturer info would be helpful, concentration of sucrose, etc. Line 156 - Were these done with software? What software? It is challenging to review this manuscript with fundamental methodological information missing. Line 529 - this figure is challenging to read with this color palette. I would suggest restricting the colors to just the top 5 phyla and bulking everything else together. This could be included as a supplemental figure, but seems to obscure most of the points made in the paper because of the number of colors. Line 283 - Why are there so many cyanobacteria? This seems odd for an ant gut. Line 310 - this isn't really focused on beta diversity. Perhaps multivariate compositional analyses would be a more appropriate name. Line 310 - It would be great to look into which bacterial taxa are driving the main compositional differences observed between the tight cluster of fecal associated workers and all the other individual ants. Lot's of approaches could be used including DESeq2. Regardless of the choice, it seems like this portion of the analysis is not yet complete. There also seem to be results introduced in the discussion, with the relative abundance of phyla being discussed there but not in the results. Results below the level of phyla would help consider what the differences might mean. Perhaps run DESeq2 on ASV data aggregated at the genus level? I would try a few things, but regardless, additional analysis is needed here. Line 552 - this analysis was not described in the methods or results section.

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Poor

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  6. Access Microbiology

    Comments to Author

    Dear Editor and Authors, Thank you very much for the invitation; it was a pleasure to review the manuscript. The study titled "Interaction with refuse piles drives co-occurrence of core gut microbiota in workers of the ant Aphaenogaster picea" aims to investigate the bacterial community for the genus for the first time and how they differentiate with different behavioral tasks. This study is extremely relevant, and I suggest only a few changes that I believe can be easily implemented to improve the robustness of the manuscript. Here are my main concerns: -This species belongs to a species complex group - How did the authors reach the taxonomic decision that this is indeed Aphaenogaster picea? I believe you could add more details on how this taxonomic decision was reached. Also, was this material deposited in any entomological collection? It would be interesting to highlight this in the manuscript, as well as the voucher in case this group of ants undergoes any taxonomic review. -Authors should include negative controls from DNA extraction to sequencing the 16S rRNA amplicon. Negative controls are necessary since contamination of the samples could occur during the natural process of data acquisition. Without negative controls, we cannot rely on the results of the bacterial analysis. Additionally, I recommend using the Decontam package (a new R package) to provide statistical power to deal with contaminants (https://benjjneb.github.io/decontam/vignettes/decontam_intro.html). -I also suggest avoiding the term "microbiome." Although this term is excellent for outreach, it can signify other techniques and create confusion. I recommend replacing it with "bacterial community" for clarity. -The manuscript only has about 40 references. I suggest expanding the discussion and comparing the main results with other studies already conducted in the field. Adding more papers that have already been conducted in myrmecology and comparing them with the results presented here could greatly improve the manuscript. Minor: L75: Talking about A. rudis here can be confusing because you haven't mentioned the species complex problem yet. I suggest elaborating a bit more before it's still relevant to talk about A. rudis here. You will talk about it later, so I suggest restructuring this part. L120: Please add some reference about trophallaxis here. L149: Please provide more details about the paint marking you used for this. Ants are very sensitive to smell, so I am wondering if this could impact the results. L187: Change to: The total of 20… L243: movement? L273: This is not common for an ant queen, right? Did you think this is common for this species? L285: Can you rewrite this, so it does not start the sentence with a number? L286: Awesome. Could you provide stats here that support this statement? L287: Did you run some stats to see if there is a core? In Qiime 2, I know that you need to run these stats and confirm a core of 50% or 40%, etc. If you did this, could you describe it in the methods sections and provide more details here? L288: Why did you focus on these bacteria? Did I miss something before? If you are interested in this one, it would be nice to know more about this in the introduction. L289: Reading this data made me think about the three different colonies. Did you see if there is a difference among them? Previous studies showed that belonging to a specific colony could impact the bacterial community. I found later that you mentioned this. Maybe it is worth talking about this before? L326: This is a lot of figures. Could you add 5 or 6 to the supplementary material and combine Figure 7 with one of those as a panel figure? L332: My suggestion is to start by saying why this study is important and provide a summary of the main outcomes. The first two sentences of your discussion are not selling your manuscript and the work that you have done well. L346: I do not agree. Besides Army ants, they are in Atta, Daceton, etc... L357: Yeah... sequencing the diet! It would be cool to see this. L368: What about Wolbachia? Did you find it? Also common in ants. L376: Add "(mammal)" Figure 5: NMDS - you need to show the stress level. You can also add the stats in the Figure too. Also, "none" on the label seems odd. Is there a better term?

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  7. Version published to 10.1099/acmi.0.000832.v1 on Access Microbiology