Siderophore screening in marine sponge extracts using LC-HRMS and an R-based metabolomics workflow

Alejandro García Ríos; Massuo Jorge Kato; Lydia Fumiko Yamaguchi; Breno Pannia Espósito; Ailan Farid Arenas-Soto

doi:10.1371/journal.pone.0343544

Peer Review History

Original SubmissionFebruary 7, 2026
6 Apr 2026 Decision Letter - Rachid Bouharroud, Editor -->PONE-D-26-06824-->-->Siderophore identification in microorganisms associated with marine sponges by LC-HRMS and a data analytic approach in R.-->-->PLOS One Dear Dr. Arenas, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by May 21 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The conclusions must be drawn appropriately based on the data presented. --> Reviewer #1: Yes Reviewer #2: No ******** -->2. Has the statistical analysis been performed appropriately and rigorously? --> Reviewer #1: Yes Reviewer #2: Yes ****** -->3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.--> Reviewer #1: Yes Reviewer #2: Yes ****** -->4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.--> Reviewer #1: Yes Reviewer #2: No ****** -->5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)--> Reviewer #1: The topic is highly relevant, the manuscript meets the standards of scientific writing, the methodology is original and well conceived, and the results are rich and insightful I have no additional comments. Reviewer #2: The manuscript cannot be considered technically sound in its current form, and the data do not adequately support the conclusions. The study lacks sufficient transparency and detail to be reproducible, which is a core requirement for technical soundness. The manuscript has major methodological weaknesses that undermine reproducibility and rigor: Sampling design is unclear i.e. the derivation of n = 36 samples is not explained. There is no distinction between biological vs. technical replicates. In addition, there is missing metadata (sampling depth, coordinates, collection method, and permits) and the sample handling is inadequately described The experimental treatment lacks justification, i.e. the use of 200 μmol L⁻¹ iron (III) ammonium citrate is arbitrary and unreferenced. Also, the extraction protocol is inconsistent. For instance, reporting an exact recovery of 45 mL after 3 × 15 mL extractions is unrealistic. There is also missing key quantitative details: i.e. actual recovered volume , dry extract yield (mg), Final concentration (mg/mL), and injection mass for LC-MS The replication and analytical workflow unclear. For example, the relationship between 36 LC-HRMS files, samples, and replicates is not defined. Thus, the study lacks clarity on technical reproducibility. The claim of “41 identified siderophores” is overstated since the identification is based only on: Accurate mass, adducts, and retention time. Therefore, the compounds should be described as “putatively annotated siderophores”, not definitively identified. There is a high risk of false positives, especially from isobaric compounds. The conclusions are not sufficiently supported. i.e. the claim that ‘Marine sponges are reservoirs of siderophores’ is not validated because the compounds are not structurally confirmed, there is no evidence of biosynthetic origin (host vs. microbes), and no genomic (BGCs) or microbial isolation data. There weak ecological interpretation with minimal discussion on the functional roles of siderophores on sponge–microbe symbiosis and their environmental relevance. In addition, the manuscript is not consistently presented in an intelligible fashion and does not fully meet the standards of clear, formal scientific English, although the issues can be improved with careful revision. The manuscript uses overly complex and verbose language, particularly in the discussion (e.g., phrases like “The amalgamation of these analytical tools engenders…”). Such words may reduces readability and cause difficulty in communication. Scientific writing should prioritize clarity over sophistication, and many sentences would benefit from simplification. There are also multiple examples of excessively long sentences, poorly constructed statements with unclear meaning. This may significantly affects the intelligibility of the manuscript, especially in the discussion section. There are also grammar, typos, and formatting Issues. For instance, the presence of typographical errors (e.g., “smaple” instead of “sample”), and inconsistent formatting, including italics for species names and citation styles. All these issues indicate insufficient proofreading and reduce the overall professionalism of the manuscript. There also missing references in key sections such as sample description, the Iron supplementation rationale. Etc. This not only affects scientific rigor but also disrupts the logical flow and credibility of the text. ****** -->6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.--> Reviewer #1: No Reviewer #2: Yes: Huxley Makonde ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". 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Revision 1
6 May 2026 Author Response Dear Editor and reviewers, On behalf of all authors, we would like to thank you and the reviewers for the constructive feedback and the opportunity to revise our manuscript, "Siderophore identification in microorganisms associated with marine sponges by LC-HRMS and a data analytic approach in R" (PONE-S-26-08981). Below, we provide a point-by-point response to the reviewer’s comments. - Reviewer Comment: The title is misleading as the study analyzes sponge extracts, not isolated microorganisms. Suggests: "Siderophore screening in marine sponge extracts..." Response: We acknowledge the reviewer's concern regarding the biological source. Our study utilizes a metabolomics approach, aiming to capture the Siderophore landscape of the sponge holobiont (the host and its complex microbial community). To resolve any ambiguity, we change the title to: "Siderophore screening in marine sponge extracts using LC-HRMS and an R-based metabolomics workflow." - Reviewer Comment: In the introduction section, the authors should have discussed other methodologies that are used to identify and quantify the siderophores. The strengths and weaknesses of such methods should have been highlighted. Response: We added the two paragraphs below in the introduction section regarding other methods to identify Siderophores. Several methodologies have been developed for the detection, identification, and quantification of siderophores, each with distinct strengths and limitations. The Chrome Azurol S (CAS) assay is a widely used colorimetric method that detects siderophore-mediated iron chelation through a characteristic color change, providing a simple and rapid screening tool for siderophore production. However, the CAS assay is non-specific, as it cannot distinguish between different siderophore types or identify individual structures, and it is subject to interference from other iron-chelating compounds in complex matrices. Bioassays employing siderophore-utilizing indicator strains offer biological activity information and can reveal functional siderophore diversity, yet these approaches are inherently cultivation-dependent and low-throughput, limiting their applicability to environmental samples with unculturable microbiomes. Nuclear magnetic resonance (NMR) spectroscopy provides definitive structural elucidation of purified siderophores; however, NMR requires pure compounds in milligram quantities and is impractical for the analysis of complex environmental extracts where siderophores occur at low concentrations amid abundant co-extracted metabolites. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and molecular networking platforms such as the Global Natural Products Social Molecular Networking (GNPS) enable structural annotation through fragmentation pattern analysis and spectral library matching, but these approaches depend heavily on the availability of well-curated MS/MS spectral libraries, which remain limited for many siderophore classes. “Compared to the methods described above, LC-HRMS with accurate mass matching offers a complementary balance of sensitivity, throughput, and broad coverage for initial screening of complex environmental samples, while acknowledging that it does not replace definitive structural confirmation by NMR or MS/MS-based approaches.” - Reviewer Comment: Several statements have not been supported with literature (the authors should provide relevant citations for these statements). Response: We carefully reviewed the entire introduction section to ensure that relevant citations properly support all statements. In the revised manuscript, we added new references for any uncited claims. - Reviewer Comment: Unclear sample collection and experimental Design. Response: We clarified in the revised text all issues regarding sample acquisition and the inconsistency in the methodology. The following paragraph was replaced in the methodology section “Samples”. “Samples were collected from three sponge species: D. reticulatum, A. fulva, and A. viridis, along the São Paulo coast, Brazil, at depths of 3–8 m, by SCUBA diving (ICMBio permit #0289170220250814). Three independent individuals per species were sampled as biological replicates. From each individual, two separate 2-g portions of body tissue were excised from two different anatomical regions, serving as technical replicates, for a total of 18 samples (3 species × 3 individuals × 2 tissue portions). Immediately after collection, samples were flash-frozen in liquid nitrogen on board, transported on dry ice, and stored at −80 °C until extraction. Following methanol extraction of the 18 samples, the resulting extracts were each split into two aliquots: one supplemented with 200 μmol L⁻¹ Ferrous Ammonium Sulfate (FAS) (final concentration 4 μmol L⁻¹) as an iron source to promote the formation of iron adducts [22]; the control aliquot was supplemented with 2 μL of ultrapure water. This yielded a total of 36 samples for LC-HRMS injection.” - Reviewer Comment: Why was 200 µmol/L used? Why Iron(III) Ammonium Citrate (FAS)? Response: First, we corrected the substance used: Iron(III) ammonium citrate (FAS), which is not the compound used in the chelation stimulation protocol. The substance used was Ferrous Ammonium Sulfate (FAS). The concentration of 200 µmol/L was selected based on established siderophore induction and detection in-house protocols in our lab, providing sufficient iron to saturate available ligands without inducing toxicity or massive precipitation that could interfere with LC-HRMS analysis. Ferrous Ammonium Sulfate (FAS) was chosen for its high solubility in both water and organic mixtures, ensuring consistent iron availability. The fact that supplementation did not significantly alter detection suggests that the identified siderophores were already present as iron-complexes (ferri-siderophores) or were constitutively produced within the sponge environment. In this reference, “Batko, I. Z., Flannagan, R. S., Guariglia-Oropeza, V., Sheldon, J. R., & Heinrichs, D. E. (2021). Heme-Dependent Siderophore Utilization Promotes Iron-Restricted Growth of the Staphylococcus aureus hemB Small-Colony Variant. Journal of bacteriology, 203(24), e0045821. https://doi.org/10.1128/JB.00458-21” [22]. The authors use a concentration of 10 μM of FAS to complement the growth of a Staphylococcus aureus hemB Small-Colony Variant, which is a bacterium defective in growing in media with iron deficiency. Adding FAS restores bacterial growth. The final concentration of FAS used in our experiment was 4 μmol L⁻¹ since we added 2 μL of 200 μmol L⁻¹ Ferrous Ammonium Sulfate (FAS) to 98 μL of extract before LC-HRMS injection. All those quantities and concentrations were clarified in the revised manuscript. - Reviewer Comment: The mass of dried extract obtained is not reported, and the final concentration of the extract before LC-MS injection is unclear. Response: We acknowledge that this information is important for analytical reproducibility and will add it to the revised manuscript. While we did not record individual dry weights for each of the 18 biological samples (as this was not a primary objective of the study), we can provide the following details: the final reconstitution volume was 250 μL of dry methanol, and the injection volume for LC-HRMS was 5 μL per run. Based on our experience with similar marine sponge ethanol extracts processed through the same fractionation workflow, the typical dry extract yield from 2 g of wet sponge tissue after chloroform partition is in the range of 5–20 mg, corresponding to a final concentration of approximately 5 mg/mL in 250 μL methanol. We will add these estimates to the revised manuscript. We will also note that the injection of 5 μL at these concentrations is consistent with standard LC-HRMS practices for natural product screening. - Reviewer Comment: Claims of identifying specific compounds (Ferricrocin, etc.) are overinterpreted without MS/MS or standards. Response: We agree that in untargeted metabolomics, these represent putative identifications (Level 2/3 according to the Metabolomics Standards Initiative). However, we defend our current results based on our multi-orthogonal validation pipeline: Mass Accuracy: All reported features had a mass error ppm < 3. Adduct Matching: We didn't rely on a single mass; we calculated and matched three distinct iron-specific adducts: [M-2H+Fe]+, [M-H+Fe]2+, and [2M-2H+Fe]+. RT Stability: All 41 siderophores exhibited a coefficient of variation (CV) for retention time below 2%, highlighting chromatographic consistency. Visual Validation: We performed a visual inspection of all extracted ion chromatograms (EICs), accepting only those with sharp Gaussian peaks. We revised the manuscript to consistently use the term "putatively identified" or "annotated" to remain within conservative scientific boundaries. - Reviewer Comment: The manuscript concludes that marine sponges represent reservoirs of siderophores. I find this conclusion so premature since the compounds were not structurally confirmed, and the microbial source was not verified. In addition, no gene clusters or microbial isolation were performed Response: We acknowledge that our identification is at the putative annotation level (MSI Level 2) and that we did not perform microbial isolation or gene cluster analysis. However, we respectfully argue that our conclusion is not premature when considered within the context of the existing literature. The term “reservoir” in our context means a source or holder of siderophore diversity. It is well established in the literature that marine sponges harbor dense and diverse microbial communities, including bacterial taxa known to produce siderophores (e.g., Pseudomonas, Streptomyces, and various Proteobacteria) (Thomas et al., 2016, ref. [46]). The siderophores we annotated (e.g., Ferricrocin, Aeruginic acid, Madurastatin) are known products of bacterial and fungal genera commonly found in sponge microbiomes. Our study was designed as an approach, and the detection of known siderophore signatures in sponge extracts provides indirect but compelling evidence for their microbial origin within the holobiont. However, we soften the language in the Conclusions section to reflect the putative nature of the annotations and the need for future studies to confirm the microbial sources through metagenomic and/or cultivation-based approaches. - Reviewer Comment: The identified siderophores are not well discussed, and this does not bring out the significance of the study. Response. We acknowledge that the ecological significance of the individual siderophores could be discussed more thoroughly, and we expanded the Discussion section accordingly in the revised manuscript - Reviewer Comment: Long sentences (e.g., the "amalgamation" sentence), typos ("smaple"), and inconsistent reference style. Response: We appreciate the attention to those details. We simplified the complex sentences cited by the reviewer to improve readability (e.g., replacing "The amalgamation of these analytical tools engenders..." with "Integrating these tools creates an efficient workflow..."). All typos, including "smaple" and missing italics for species names, were corrected, and the reference section was strictly formatted to PLOS One requirements. We believe these clarifications, supported by the data already present in our work, address the reviewer's concerns and demonstrate the validity of our strategy for siderophore putative annotation. All changes can be observed in the revised manuscript. Likewise, the revised manuscript was suited for the PLOS ONE requirements. Sincerely, The Authors of PONE-S-26-08981 Attachments Attachment Submitted filename: Response to Reviewers.pdf https://doi.org/10.1371/journal.pone.0343544.r002
12 May 2026 Decision Letter - Rachid Bouharroud, Editor, Rachid Bouharroud, Editor <p>Siderophore screening in marine sponge extracts using LC-HRMS and an R-based metabolomics workflow. PONE-D-26-06824R1 Dear Dr. Arenas, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Rachid Bouharroud Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0343544.r003
Formally Accepted
Acceptance Letter - Rachid Bouharroud, Editor, Rachid Bouharroud, Editor PONE-D-26-06824R1 PLOS One Dear Dr. Arenas-Soto, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Rachid Bouharroud Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0343544.r004

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