Exploratory study on the impact of Ganoderma australe extract on gut microbiota and immune gene expression in honey bees exposed to Vairimorpha ceranae

Sarah Zuern; Bella Romero; Carlos Spichiger; Leandro Ortiz; Alejandro Jerez; Esteban Basoalto; Max Emil Schön; Sigisfredo Garnica

doi:10.1371/journal.pone.0344173

Peer Review History

Original SubmissionJuly 21, 2025
13 Oct 2025 Decision Letter - Kai Wang, Editor Dear Dr. Garnica, 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 Nov 27 2025 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. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Kai Wang Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. 3. Thank you for stating the following financial disclosure: “Funding for this project was made possible by project the Applied Research and Innovation 2023 grant INID210009. ME. Schön would like to thank I. Schlichting and the Max Planck Society for their support.” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. 5. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. [Note: HTML markup is below. Please do not edit.] Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Partly ******** 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 Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ******** Reviewer #1: After I looked through the manuscript titled “Exploratory study on the impact of Ganoderma australe extract on gut microbiota and immune gene expression in honey bees exposed to Nosema ceranae”; Number PONE-D-25-39736. The manuscript looks fair to me; however, the manuscript needs to be revised as a major revision. Please see the comments below. Abstract: Looks good to me; however, I suggest the authors add the spore load (number of spores per bee). Keywords: looks good to me. Introduction: Page 11: lines 35: “xxxx first described in the Asian honey bee (Apis ceranae)” Please replace Apis ceranae with Apis cerana Materials and methods: Page 14, Lines 119-120: “The fungal material was macerated using a sterile pistil and 300 μl buffer was added.” Please provide what buffer the authors used for this one. Line 122: “xxx of 5 min each in a Mini-BeadBeater-16 at 3450 rpm.” Please convert 3450 rpm to xxx g xg (RCF) = (RPM)2 x radius* x 1.118 x 10-5 The distance of particles from the center of rotation (centrifuge radius), unit in centrimeter (cm) Please also provide the model and brand of the centrifuge the authors used. Page 15, Lines 139-140: “Fungal culture was grown for three weeks in laboratory conditions.” Please provide the actual laboratory condition (e.g., temperature, nutrients, humidity, sterile environment. Line 147: “using high-performance liquid chromatography (HPLC)” Please provide the model, brand, and the condition, e.g., Detector type, xxx nm; the Column? (e.g., Cxx, xx mm × xx mm, x µm column), temperature xx °C; the mobile phase consisted of Solvent x: Solvent y The flow rate of xx mL/min in xx mode; The injection volume was xx µL. The software name used for data acquisition and analysis. Line 151-153: “ Only hives that were vigorous and showed no symptoms of any disease were used in the experiments” I am wondering how the authors ensure that those five colonies are free of pathogens. Please provide the methods the authors used to confirm that they are disease-free colonies. Page 16, Line 170-171: “On the 7th day post-emergence five treatment groups were infected with Nosema ceranae.” Please provide the dose of Nosema ceranae, how many spores per bee the authors used for this experiment. Page 17, Lines 198-200: “Seven days after emergence, honey bees from the treatment groups CN, HAN, GBN-1, GBN-2, and GBN-3 were infected with N. ceranae spores. Honey bees were fed two times a 2 ml spore solution. Once the total infection dose was consumed,” What exactly number of N. ceranae spores used for each group? 2x 106 spores per group? By group feeding? If so, please add more detail. Lines 201-202: “xxx five honey bees of each cage were fed individually 10 µl of N. ceranae spore solution using a micropipette according to the protocol described by Williams et al. [54].” How many spores were in 10 µl that the authors fed each bee? Results: Page 21, line 317: “xxx GanoBee at 1.5% concentration was reduced by 0.1 g/cm³ and 0.2 g/cm³, “ I suggest the authors provide the unit of concentration as g/ml instead of g/cm3 Line 321: “Effects of different diets and N. ceranae infection on thexxx “ I suggest the authors replace N. ceranae with Nosema ceranae since it is a subtopic Page 22, Line 343-344: “Effects of different diets and N. ceranae infection on the gut bacterial microbiota ” Please replace N. ceranae with Nosema ceranae Figure 3: Please provide a better solution for the graphs and figure legend of all statistics Figures 5 and 6: Please provide the different letters showing the statistical difference in the data in Figure 5 and 6 instead of or Discussion: I suggest that the authors improve the discussion part by making it shorter, emphasizing, and clarifying the scientific contribution of this paper. References: Please take care of scientific names, make sure they are in italic form. Supporting information: Please take care of scientific names, make sure they are in italic form. Reviewer #2: 1. The Nosema ceranae is now considered as Vairimorpha ceranae, therefore, the author should correct this genus everywhere in this manuscript. 2. The major concern of this manuscript is the author mentioned the establishment of N. ceranae infection appeared limited, likely due to low spore viability. In my view point, the microsporidia infection in honey bee should be the basic experiment of this study, which the author should overcome this problem before they trying to publish this paper, otherwise the V. ceranae infection test should be removed in this manuscript and focus on the Ganoderma australe extract on gut microbiota and immune gene expression in honey bees 3. Table 1: The standard V. ceranae infection process should observe more than 2-weeks (14~21days) and therefore can see the pathogenesis of V. ceranae. 4. The flowchart of bioinformatics could be submitted to GitHub system ****** 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: Yes: Guntima Suwannapong Reviewer #2: No ******** [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". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: No-1-Reviewer comments-Plos One-AUG-08-2025.docx Attachment Submitted filename: No-1-Reviewer comments-Plos One-AUG-08-2025.pdf https://doi.org/10.1371/journal.pone.0344173.r001
Revision 1
11 Dec 2025 Author Response PONE-D-25-39736 Exploratory study on the impact of Ganoderma australe extract on gut microbiota and immune gene expression in honey bees exposed to Nosema ceranae In the following pages, we give the details of all changes/improvements to the revised version of our manuscript. We have improved our manuscript (incl. figures) following the PLOS ONE staff’ recommendations and reviewers (Please see “Point-by-point response to reviewers”). Sincerely, Sigisfredo Garnica PONE-D-25-39736 Exploratory study on the impact of Ganoderma australe extract on gut microbiota and immune gene expression in honey bees exposed to Nosema ceranae PLOS ONE Dear Dr. Garnica, 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 Nov 27 2025 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. Please include the following items when submitting your revised manuscript: • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Kai Wang Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf We have revised our manuscript to meet PLOS ONE's style requirements, including file naming. 2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. The ORCID iD for the corresponding author was validated in Editorial Manager. 3. Thank you for stating the following financial disclosure: “Funding for this project was made possible by project the Applied Research and Innovation 2023 grant INID210009. ME. Schön would like to thank I. Schlichting and the Max Planck Society for their support.” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Yes, effectively the funders had no role. Please include the following sentence: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 4. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. Please for Data Availability change to “Raw sequencing data files are available from the Sequence Read Archive (SRA) under BioProject number PRJNA1347411 (http://www.ncbi.nlm.nih.gov/bioproject/1347411).” 5. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. There are no such comments from the reviewers. [Note: HTML markup is below. Please do not edit.] Reviewer's Responses to Questions Point-by-point response to reviewers. 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: After I looked through the manuscript titled “Exploratory study on the impact of Ganoderma australe extract on gut microbiota and immune gene expression in honey bees exposed to Nosema ceranae”; Number PONE-D-25-39736. The manuscript looks fair to me; however, the manuscript needs to be revised as a major revision. Please see the comments below. Abstract: Looks good to me; however, I suggest the authors add the spore load (number of spores per bee). We introduced the information suggested by the reviewer into the abstract. Introduction: Page 11: lines 35: “xxxx first described in the Asian honey bee (Apis ceranae)” Please replace Apis ceranae with Apis cerana We replaced Apis ceranae with Apis cerana. Materials and methods: Page 14, Lines 119-120: “The fungal material was macerated using a sterile pistil and 300 μl buffer was added.” Please provide what buffer the authors used for this one. Now we provide the Buffer used. Line 122: “xxx of 5 min each in a Mini-BeadBeater-16 at 3450 rpm.” Please convert 3450 rpm to xxx g xg (RCF) = (RPM)2 x radius* x 1.118 x 10-5 The distance of particles from the center of rotation (centrifuge radius), unit in centrimeter (cm) The shaking motion of the Mini-BeadBeater-16 involves a "throw" or vial displacement, which is specified as 2.22 cm. This value represents the distance the vials move during agitation, not a radius of the device itself. Please also provide the model and brand of the centrifuge the authors used. We provide the model and brand: Mini-BeadBeater-16 Model670EUR, BioSpec Products, Inc., USA. Page 15, Lines 139-140: “Fungal culture was grown for three weeks in laboratory conditions.” Please provide the actual laboratory condition (e.g., temperature, nutrients, humidity, sterile environment. We added a sentence characterizing the laboratory conditions as follows: Sterile environment, around 23°C room temperature and 49% humidity. Line 147: “using high-performance liquid chromatography (HPLC)” Please provide the model, brand, and the condition, e.g., Detector type, xxx nm; the Column? (e.g., Cxx, xx mm × xx mm, x µm column), temperature xx °C; the mobile phase consisted of Solvent x: Solvent y The flow rate of xx mL/min in xx mode; The injection volume was xx µL. The software name used for data acquisition and analysis. The following information was introduced into the main manuscript text: Ganoderic acids were detected with a high-performance liquid chromatography (HPLC) high-performance liquid chromatography (HPLC) analysis using an Agilent 1200 Series system equipped with a diode array detector (MWD 61365 D, Agilent). The analytical procedure was modified from Liu et al. [49]. The detection wavelength was set at 250 nm. Separation was achieved on an Agilent Zorbax 300SB column (5 µm, 4.6 mm × 250 mm), maintained at 30°C. The mobile phase consisted of a gradient elution using (A) acetonitrile and (B) 0.1% acetic acid in water (v/v). The injection volume was 20 µL, and the flow rate was set to 1.0 mL/min in continuous mode. Data acquisition and analysis were conducted using Agilent ChemStation Software. Line 151-153: “Only hives that were vigorous and showed no symptoms of any disease were used in the experiments” I am wondering how the authors ensure that those five colonies are free of pathogens. Please provide the methods the authors used to confirm that they are disease-free colonies. Now we detailed the methods used to indicate that they are disease-free colonies. Page 16, Line 170-171: “On the 7th day post-emergence five treatment groups were infected with Nosema ceranae.” Please provide the dose of Nosema ceranae, how many spores per bee the authors used for this experiment. The concentration solution used was 1 x 106 spores per ml. We introduced this information into the text. Page 17, Lines 198-200: “Seven days after emergence, honey bees from the treatment groups CN, HAN, GBN-1, GBN-2, and GBN-3 were infected with N. ceranae spores. Honey bees were fed two times a 2 ml spore solution. Once the total infection dose was consumed,” What exactly number of N. ceranae spores used for each group? 2x 106 spores per group? By group feeding? If so, please add more detail. Similarly, we used the concentration solution of spores per group as previously described. Lines 201-202: “xxx five honey bees of each cage were fed individually 10 µl of N. ceranae spore solution using a micropipette according to the protocol described by Williams et al. [54].” How many spores were in 10 µl that the authors fed each bee? We used the concentration solution of 1 x 106 spores per ml. Results: Page 21, line 317: “xxx GanoBee at 1.5% concentration was reduced by 0.1 g/cm³ and 0.2 g/cm³, “ I suggest the authors provide the unit of concentration as g/ml instead of g/cm3 We replaced 0.1 g/cm³ and 0.2 g/cm³ with 0.1 g/mL and 0.2 g/mL, respectively. Line 321: “Effects of different diets and N. ceranae infection on thexxx “ I suggest the authors replace N. ceranae with Nosema ceranae since it is a subtopic Replaced V. ceranae with Vairimorpha ceranae. Page 22, Line 343-344: “Effects of different diets and N. ceranae infection on the gut bacterial microbiota ” Please replace N. ceranae with Nosema ceranae Replaced V. ceranae with Vairimorpha ceranae. Figure 3: Please provide a better solution for the graphs and figure legend of all statistics Improved Figures 5 and 6: Please provide the different letters showing the statistical difference in the data in Figure 5 and 6 instead of or ** Now we use different letters showing the statistical difference in the data in Figure 5 and 6, respectively. Discussion: I suggest that the authors improve the discussion part by making it shorter, emphasizing, and clarifying the scientific contribution of this paper. Following the reviewer’s suggestions, we have shortened the discussion, focusing and clarifying the contribution of our work. References: Please take care of scientific names, make sure they are in italic form. Supporting information: Please take care of scientific names, make sure they are in italic form. We used Zotero to manage the references in our manuscript. All citations were manually reviewed and adjusted as needed. We also verified the reviewer’s suggested references throughout our the Supporting Information files. Reviewer #2: 1. The Nosema ceranae is now considered as Vairimorpha ceranae, therefore, the author should correct this genus everywhere in this manuscript. The reviewer is right, revising recently published taxonomic papers and the databases like GenBank, Mycobank, and Index Fungorum use Vairimorpha as valid genus and not Nosema. Therefore, we corrected the genus name across the complete manuscript. 2. The major concern of this manuscript is the author mentioned the establishment of N. ceranae infection appeared limited, likely due to low spore viability. In my view point, the microsporidia infection in honey bee should be the basic experiment of this study, which the author should overcome this problem before they trying to publish this paper, otherwise the V. ceranae infection test should be removed in this manuscript and focus on the Ganoderma australe extract on gut microbiota and immune gene expression in honey bees. We appreciate the reviewer’s valuable comment. As indicated, the establishment of Vairimorpha ceranae infection appeared limited, likely due to low spore viability. However, since the funding source required us to obtain experimental results during the spring–summer season of the project, we were unable to conduct preliminary infection trials with honey bees prior to initiating the assays. In addition, we did not have sufficient resources to acquire intestinal epithelial cell cultures of honey bees for in vitro assays to characterize the infective capacity of V. ceranae spores. Although the infection level was lower than expected (despite the inoculum being obtained from colonies heavily affected by nosemosis), the presence of the causal agent of the disease still produced measurable effects on the immune system and bacteriome compared with non-infected honey bees. Therefore, we consider that the infection component remains relevant to the interpretation of the physiological responses observed in this study. 3. Table 1: The standard V. ceranae infection process should observe more than 2-weeks (14~21days) and therefore can see the pathogenesis of V. ceranae. Previous studies investigating the honey bee immune system, such as Glacinic et al. (2017), quantified gene expression levels up to 12 days post-infection, while Aufauvre et al. (2014) collected samples at 1 and 7 days after treatment application. Both studies were published in PLOS ONE. The following Table summarizes some additional publications and the corresponding observation periods used in each study: Reference Journal Summary Antúnez et al. (2009) “Immune suppression in the honey bee (Apis mellifera) following infection by N. ceranae” Environmental Microbiology The RNA expression levels of seven genes were determined 4 and 7 days after N. ceranae infection. Antúnez et al. (2013) “Differential expression of vitellogenin in honey bees (Apis mellifera) with different degrees of N. ceranae infection” Journal of Apicultural Research Newly emerged bees from both colonies were artificially infected with N. ceranae and seven days after infection Attachments Attachment Submitted filename: PONE-S-25-47835_Rebuttal Letter.docx https://doi.org/10.1371/journal.pone.0344173.r002
19 Jan 2026 Decision Letter - Kai Wang, Editor Exploratory study on the impact of Ganoderma australe extract on gut microbiota and immune gene expression in honey bees exposed to Vairimorpha ceranae PLOS One Dear Dr. Garnica, 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 Feb 24 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. Please include the following items when submitting your revised manuscript: A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Kai Wang Academic Editor PLOS One Journal Requirements: 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewer's Responses to Questions Comments to the Author Reviewer #1: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: After I looked through the manuscript titled “Exploratory study on the impact of Ganoderma australe extract on gut microbiota and immune gene expression in honey bees exposed to Vairimorpha ceranae”. The manuscript looks good to me; however, the manuscript needs to be revised as a minor revision. Please see the comments below. Abstract: Looks good to me; however, I suggest the authors replace honeybee with honey bee throughout the manuscript. Comment 1: Line 8-9 “…..honey bees subjected to experimental exposure to V. ceranae 1 x 106 spores/ml.” I suggest the author provide the spore load (number of spores per bee) instead of the number of spores per ml. Introduction: Overall, the introduction looks good to me. Materials and methods: The authors used the standard methods and showed scientific rigor. However, some minor concerns as follows. Comment2: Page 18, lines 102-115: Fruiting bodies collection and pure cultures Please provide the conditions, such as temperature, humidity, and period for the culture. Comment 3: Page 18, line 124: Molecular identification of fungal isolates. Please use the xg instead of “at 3450 rpm” Comment 4: Page 20, lines 140-156: Fungal extract Please provide the internal standard for HPLC that the authors used for analysis. Comment5: line 210: Five bees of each cage were fed individually (10 µl), and lines 212-213, then further fed by group feeding? (2 mL per cage?). Please provide when group feeding was provided. Results: Please provide a better resolution of all figures. Discussion: Looks pretty good and make senses to me. ****** 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 ******** [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". If this link does not appear, there are no attachment files.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. Attachments Attachment Submitted filename: No-1-Reviewer comments-Plose One-GS.pdf https://doi.org/10.1371/journal.pone.0344173.r003
Revision 2
9 Feb 2026 Author Response Reviewer #1: After I looked through the manuscript titled “Exploratory study on the impact of Ganoderma australe extract on gut microbiota and immune gene expression in honey bees exposed to Vairimorpha ceranae”. The manuscript looks good to me; however, the manuscript needs to be revised as a minor revision. Please see the comments below. Abstract: Looks good to me; however, I suggest the authors replace honeybee with honey bee throughout the manuscript. Changed throughout the manuscript. Comment 1: Line 8-9 “…..honey bees subjected to experimental exposure to V. ceranae 1 x 106 spores/ml.” I suggest the author provide the spore load (number of spores per bee) instead of the number of spores per ml. 1 × 10⁴ spores per bee. This load was administered by individual feeding. Introduction: Overall, the introduction looks good to me. Fine Materials and methods: The authors used the standard methods and showed scientific rigor. However, some minor concerns as follows. Comment2: Page 18, lines 102-115: Fruiting bodies collection and pure cultures Please provide the conditions, such as temperature, humidity, and period for the culture. We provided now the culture conditions, including temperature, humidity, and duration; in our study, cultures were maintained at 23°C and 60% humidity for a maximum of 30 days. Comment 3: Page 18, line 124: Molecular identification of fungal isolates. Please use the xg instead of “at 3450 rpm” We have now changed the unit to ×g, which in our study corresponds to a centrifugal force of 253 × g. Comment 4: Page 20, lines 140-156: Fungal extract Please provide the internal standard for HPLC that the authors used for analysis. A ganoderic acid A standard (CAS No. 81907-62-2) was used at a concentration of 50 µg/mL and dissolved in 100% acetonitrile. The standard was purchased from Sigma-Aldrich (Chile). Comment 5: line 210: Five bees of each cage were fed individually (10 µl), and lines 212-213, then further fed by group feeding? (2 mL per cage?). Please provide when group feeding was provided. Improved Results: Please provide a better resolution of all figures. All figures were newly generated to ensure high resolution suitable for publication in PLOS ONE. Discussion: Looks pretty good and make senses to me. 7. 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 [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". If this link does not appear, there are no attachment files.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. Attachments Attachment Submitted filename: PONE-D-25-39736R1 Rebuttal Letter.docx https://doi.org/10.1371/journal.pone.0344173.r004
17 Feb 2026 Decision Letter - Kai Wang, Editor Exploratory study on the impact of Ganoderma australe extract on gut microbiota and immune gene expression in honey bees exposed to Vairimorpha ceranae PONE-D-25-39736R2 Dear Dr. Garnica, 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, Kai Wang Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0344173.r005
Formally Accepted
Acceptance Letter - Kai Wang, Editor PONE-D-25-39736R2 PLOS One Dear Dr. Garnica, 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. Kai Wang Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0344173.r006

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