Chemical Gradients of Plant Substrates in an Atta texana Fungus Garden

ABSTRACT Many ant species grow fungus gardens that predigest food as an essential step of the ants’ nutrient uptake. These symbiotic fungus gardens have long been studied and feature a gradient of increasing substrate degradation from top to bottom. To further facilitate the study of fungus gardens and enable the understanding of the predigestion process in more detail than currently known, we applied recent mass spectrometry-based approaches and generated a three-dimensional (3D) molecular map of an Atta texana fungus garden to reveal chemical modifications as plant substrates pass through it. The metabolomics approach presented in this study can be applied to study similar processes in natural environments to compare with lab-maintained ecosystems. IMPORTANCE The study of complex ecosystems requires an understanding of the chemical processes involving molecules from several sources. Some of the molecules present in fungus-growing ants’ symbiotic system originate from plants. To facilitate the study of fungus gardens from a chemical perspective, we provide a molecular map of an Atta texana fungus garden to reveal chemical modifications as plant substrates pass through it. The metabolomics approach presented in this study can be applied to study similar processes in natural environments.

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The manuscript "Chemical gradients of plant substrates in an Atta texana fungus garden" is an interesting article that presents a chemical perspective of fungus growing ants' symbiotic system originate from plants. It's about a detailed study based on mass spectrometry that I consider very relevant by applied several metabolomics tools to a unique dataset. The article structure is adequate, considering the extensive and complex dataset. I suggest proceeding with normalization of decimal values at m/z and mass error across the manuscript and supplementary material. Also, I would like to clarify some points: Line 310: what was the reason to use 2 μM sulfamethazine as an internal standard? Considering its structure and intrinsic chemical ionization that is completely different from the main chemical classes found in the study. Line 318-319: about the quality control solution applied, it was not clear why the author used this compound mixture. Is this related to mass error correction or retention time monitoring? I found it a little confusing in this regard. I understand that this article could be used as a guide for other ecological studies that want to apply the metabolomic tools presented.
Reviewer #2 (Comments for the Author): The manuscript "Chemical gradients of plant substrates in an Atta texana fungus garden" provides and very detailed overview of the chemical changes occurring in an A. texana fungus garden, in particular demonstrating the capacity of the fungus garden to metabolized plant substrates. This work was made possible by applying high-throughput analysis (mass spectrometry-based approaches and 3D cartographic analyses) which unable to achieve insights into the chemical transformations found in a laboratory-maintained A. texana fungus garden, modifications that were found associated with certain regions of the system. I will suggest some minor observations: 1. To make a graphical model of the main detected molecules found in the different regions of the system. For example, it is clear that flavonoids and phenolic compounds decrease from top to bottom of the fungus garden, but other compounds such as ergosterol peroxide and shingolipids increased in relative abundance across the layers being enriched in the trash. Additionally, it may be interesting to include some chemical modifications, as those shown for the bioactive molecule ginnalin A. These detected molecules and transformations could be illustrated in a graphical model that resume the main transformations across the different regions of the system.

Manuscript review mSystems00601-21 "Chemical gradients of plant substrates in an Atta texana fungus garden"
The manuscript "Chemical gradients of plant substrates in an Atta texana fungus garden" provides and very detailed overview of the chemical changes occurring in an A. texana fungus garden, in particular demonstrating the capacity of the fungus garden to metabolized plant substrates. This work was made possible by applying high-throughput analysis (mass spectrometry-based approaches and 3D cartographic analyses) which unable to achieve insights into the chemical transformations found in a laboratory-maintained A. texana fungus garden, modifications that were found associated with certain regions of the system.
The workflow proposed in this work, opens the opportunity to solve questions related to microbial -ant interactions. Specifically, to identify chemical cues associated with ant behaviors, to determine the effect of plant substrate on the type of chemical transformations and interactions with the fungal garden, to identify the effect of the fungus garden on the microbial communities inhabiting these systems.
Finally, the manuscript is very well written and results are extensively well supported by figures, tables and supplementary data.
I will suggest some minor observations: 1. To make a graphical model of the main detected molecules found in the different regions of the system. For example, it is clear that flavonoids and phenolic compounds decrease from top to bottom of the fungus garden, but other compounds such as ergosterol peroxide and shingolipids increased in relative abundance across the layers being enriched in the trash. Additionally, it may be interesting to include some chemical modifications, as those shown for the bioactive molecule ginnalin A. These detected molecules and transformations could be illustrated in a graphical model that resume the main transformations across the different regions of the system. We really appreciate your time as well as the reviewers' to read and comment on our manuscript entitled "Chemical gradients of plant substrates in an Atta texana fungus garden".
We have prepared a revised version of the manuscript based on the provided comments and suggestions. All the changes have been highlighted in the resubmission files. We have also elaborated a point-by-point response to the reviewer's comments shown below in blue.
Additionally, we modified the supplementary material for a maximum of 10 figures and included their corresponding legends (Lines 432 -523) at the end of the main text as suggested by the Editorial policy at mSystems.
We hope we addressed all the comments and look forward to the Journal's decision.

Reviewer's Comments (black) and Responses (Blue):
Reviewer #1 (Comments for the Author): The manuscript "Chemical gradients of plant substrates in an Atta texana fungus garden" is an interesting article that presents a chemical perspective of fungus growing ants' symbiotic system originate from plants.
It's about a detailed study based on mass spectrometry that I consider very relevant by applied several metabolomics tools to a unique dataset. The article structure is adequate, considering the extensive and complex dataset.
We really appreciate your comments and perception of our work, thanks.
I suggest proceeding with normalization of decimal values at m/z and mass error across the manuscript and supplementary material.
Thanks for the suggestions. For consistency, all the m/z values have been standardized to four decimals, the calc. m/z value and the mass error (ppm) were included for the identified compounds or when a molecular formula was provided (e.g. Line 91 in the highlighted version of the main text: "C 32 H 52 NO 3 (calc. m/z 498.3941, error 1.9 ppm) and C 30 H 45 O 3 (calc. m/z453.3363, error 4.9 ppm)"). These changes can be found in the highlighted version of the revised manuscript and the supplemental material (legends of supplementary figures, lines 432-523) found at the end of the main text.
Also, I would like to clarify some points: Line 310: what was the reason to use 2 μM sulfamethazine as an internal standard? Considering its structure and intrinsic chemical ionization that is completely different from the main chemical classes found in the study.
Thanks for this question. Yes, the reason we use sulfamethazine as an internal standard, which is also part of the chemical mixture we use as quality control for monitoring LC-MS runs, is part of an effort to be able to cross compare diverse datasets at a global scale. There is not a universal standard protocol in metabolomics, however we use this internal standard to monitor the analyses for retention time and mass shifts throughout runs and batches of the study. Additionally, the internal standard was used for normalizing the data based on peak area of this compound (see Materials and Methods < Statistical analysis), as it's detection is expected to be constant across entire runs under the same analytical conditions within the LC-MS system.
Line 318-319: about the quality control solution applied, it was not clear why the author used this compound mixture. Is this related to mass error correction or retention time monitoring? I found it a little confusing in this regard.
Yes. As mentioned before for the internal standard, the mixture of the six compounds (described in the Materials and Methods section) that are not related to the samples enables us to monitor the LC-MS performance across entire datasets (monitoring retention time and mass shifts) not just from this study. Then, global comparisons at the repository scale and future studies will be possible having these standard protocols.
I understand that this article could be used as a guide for other ecological studies that want to apply the metabolomic tools presented.
Yes, this is correct. The intention of having standard protocols and reproducible workflows are useful for future comparative studies at a larger scale.

Reviewer #2 (Comments for the Author):
The manuscript "Chemical gradients of plant substrates in an Atta texana fungus garden" provides and very detailed overview of the chemical changes occurring in an A. texana fungus garden, in particular demonstrating the capacity of the fungus garden to metabolized plant substrates. This work was made possible by applying high-throughput analysis (mass spectrometry-based approaches and 3D cartographic analyses) which unable to achieve insights into the chemical transformations found in a laboratory-maintained A. texana fungus garden, modifications that were found associated with certain regions of the system. I will suggest some minor observations: 1. To make a graphical model of the main detected molecules found in the different regions of the system. For example, it is clear that flavonoids and phenolic compounds decrease from top to bottom of the fungus garden, but other compounds such as ergosterol peroxide and shingolipids increased in relative abundance across the layers being enriched in the trash. Additionally, it may be interesting to include some chemical modifications, as those shown for the bioactive molecule ginnalin A. These detected molecules and transformations could be illustrated in a graphical model that resume the main transformations across the different regions of the system. This is a great suggestion and we thank the reviewer for this comment. We elaborated Figure 5, which is mentioned in the main text (Line 239 "(...) removed from the system as trash (Fig. 5)". We also included and labeled additional detected compounds and modifications (compounds 5-11) to better summarize our work as suggested by the reviewer. Figure 3 was also edited to reflect these changes in the text. The manuscript was properly adjusted to these changes and highlighted in the revised version.  Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below. Before it can be scheduled for publication, your manuscript will be checked by the mSystems senior production editor, Ellie Ghatineh, to make sure that all elements meet the technical requirements for publication. She will contact you if anything needs to be revised before copyediting and production can begin. Otherwise, you will be notified when your proofs are ready to be viewed.
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