The fate of Candida tropicalis in the black soldier fly larvae and its nutritional effect suggest indirect interactions

Lilach Ben-Mordechai; Neta Herman; Tzach Vitenberg; Sivan Margalit; Gianluca Tettamanti; Morena Casartelli; Daniele Bruno; Itai Opatovsky

doi:10.1371/journal.pone.0325056

Peer Review History

Original SubmissionJuly 29, 2024
Decision Letter - Andrey Nagdalian, Editor Dear Dr. Opatovsky, 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 Mar 06 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'. 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If your data cannot be made publicly available for ethical or legal reasons (e.g., public availability would compromise patient privacy), please explain your reasons on resubmission and your exemption request will be escalated for approval. -->?> Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: No 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: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: Questions The manuscript deals with an interesting topic concerning Candida tropicalis found in the digestive tract of Hermetia illucens. This study presents interesting findings. However, there are several major concerns that need to be addressed. Here's a list of potential limitations or areas for further investigation 1. The title uses the term "non-orthodox symbiotic" without providing a clear definition in the article. It is a highly specific term that requires a precise explanation and data confirmation. 2. There is inconsistent use of professional terminology throughout the article, such as fungi, yeast, and C. tropicalis. And the terminology for black soldier fly. The article uses "black soldier fly," "BSF," and "Hermetia illucens." Although these are all correct, the full name and abbreviation should be given at first mention, and then one term should be used consistently. 3. This article is based on the study by Kannan et al. (2023). The authors used different C. tropicalis feeding strategies for BSF larvae. However, was there a significant increase in BSF body weight after supplementing their diet with C. tropicalis? Could the authors provide supporting evidence for this effect? 4. Line 214: The meaning of significant differences between A and B was not mentioned in the article 5. Line 209-215: There is a lack of a unit for the y-axis in Figure 2. 6. How many BSF were used in Figure 2a and Figure 2b. The article doesn't clearly state the sample size and number of replications for each experiment. Small sample sizes could affect the reliability and generalizability of results. 7. How did the authors confirm that the separated gut sections were correctly identified as the anterior midgut, middle midgut, and posterior midgut? Did the authors have anatomical data or specific markers for AM, MM, and PM to validate these gut sections? 8. Line 216-221: The methodology for Figure 3 was not described in the Materials and Methods section. Please provide information about the source and purity of standards used for quantification. The fatty acid abbreviations on the x-axis in Figure 3 lack detailed explanations. Please clarify whether the y-axis represents weight percentage or molar percentage of total fatty acids. 9. Line 285-289: The conclusions drawn by Genta et al. (2009) appear to contradict the arguments presented by the authors in this paper. This discrepancy needs to be addressed and explained. 10. Line 287: Please demonstrate the relationship between lysozyme activity and the survival rate of BSF larvae. 11. The authors should clarify whether C. tropicalis enhances or suppresses the immune response in BSF larvae. Additionally, several genes associated with BSF survival have been previously identified. Could you examine and discuss the potential relationship between these known survival-related genes and the immune response observed in the discussion? Reviewer #2: The authors studied investigated the survival of C. tropicalis in the BSF gut to evaluate their ability to colonize this organ or their possible digestion by the insect. The effect of C. tropicalis on the metabolic composition and digestion process in the larvae was also analyzed and compared to the metabolic composition of the fungal biomass, and rearing substrate. Generally, the conceptualization and results of this work are interesting and the manuscript deserves consideration. However, at presented state the manuscript needs revision. The main comments and recommendations are listed below. Nutritional composition of BSFL Is changeable and can be regulated using different feed stuff. A little discussion on this aspect could improve the Introduction section. For example: doi.org/10.3920/JIFF2021.0162, 10.1093/ee/nvab135 Details for equipment and software used in the experiment should be unified: the name of manufacturer and its location should be given. Add separate subsection in section 2 with chemicals and materials. Provide information on chemicals purity and manufacturers. Tables can be modified. It is better to add separate columns with data on investigated values for experimental and control groups of BSF larvae. As suggestion, all figures can be combined to the one figure. In this regard, it will be visually comfortable for readers to compare the main results obtained. The text should be carefully checked for typos and grammatical errors. ****** 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: 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. 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Revision 1
16 Feb 2025 Author Response Reviewer #1: Questions The manuscript deals with an interesting topic concerning Candida tropicalis found in the digestive tract of Hermetia illucens. This study presents interesting findings. However, there are several major concerns that need to be addressed. Here's a list of potential limitations or areas for further investigation 1. The title uses the term "non-orthodox symbiotic" without providing a clear definition in the article. It is a highly specific term that requires a precise explanation and data confirmation. We removed this term from the title and discussion and changed it to “indirect interactions” as the effect of the C. tropicalis on the BSF seems to be through the substrate 2. There is inconsistent use of professional terminology throughout the article, such as fungi, yeast, and C. tropicalis. And the terminology for black soldier fly. The article uses "black soldier fly," "BSF," and "Hermetia illucens." Although these are all correct, the full name and abbreviation should be given at first mention, and then one term should be used consistently. We agree, we improved the term’s consistency in the manuscript by refereeing to C. tropicalis and BSF 3. This article is based on the study by Kannan et al. (2023). The authors used different C. tropicalis feeding strategies for BSF larvae. However, was there a significant increase in BSF body weight after supplementing their diet with C. tropicalis? Could the authors provide supporting evidence for this effect? Unfortunately, we did not measure the larval body mass in this experiment 4. Line 214: The meaning of significant differences between A and B was not mentioned in the article The meaning of significance was added to the figure legends 5. Line 209-215: There is a lack of a unit for the y-axis in Figure 2. The amount of DNA was compared to a relative sample, therefore it is not quantification but relative measurement. 6. How many BSF were used in Figure 2a and Figure 2b. The article doesn't clearly state the sample size and number of replications for each experiment. Small sample sizes could affect the reliability and generalizability of results. The sample sizes were added throughout the manuscript 7. How did the authors confirm that the separated gut sections were correctly identified as the anterior midgut, middle midgut, and posterior midgut? Did the authors have anatomical data or specific markers for AM, MM, and PM to validate these gut sections? The gut parts were separated according to the physiological description provided by Bonelli et al. (2019) and were ensured accurate identification of the midgut, using the pH levels known to maintained in each region (lines 128-131) 8. Line 216-221: The methodology for Figure 3 was not described in the Materials and Methods section. The methodology explained in lines 93-104 Please provide information about the source and purity of standards used for quantification. The relevant information was added The fatty acid abbreviations on the x-axis in Figure 3 lack detailed explanations. Please clarify whether the y-axis represents weight percentage or molar percentage of total fatty acids. The figure axis was changed accordingly 9. Line 285-289: The conclusions drawn by Genta et al. (2009) appear to contradict the arguments presented by the authors in this paper. This discrepancy needs to be addressed and explained. If the lysozyme afect the fungal survival, we suggest that C. tropicalis can reduce the enzymatic activity of the insect to allow fungal survival. It is describes in lines 305-308 10. Line 287: Please demonstrate the relationship between lysozyme activity and the survival rate of BSF larvae. We meant the survival of the fungi (corrected in the text) 11. The authors should clarify whether C. tropicalis enhances or suppresses the immune response in BSF larvae. The effect of Candida on the insect immune system is specific in line 307 Additionally, several genes associated with BSF survival have been previously identified. Could you examine and discuss the potential relationship between these known survival-related genes and the immune response observed in the discussion? It is not clear to us which genes that related to the survival of the BSF the reviewer suggests. The effect of the effect of fungi on the immune response in discussed in the paper by Herman et al. 2024. Reviewer #2: The authors studied investigated the survival of C. tropicalis in the BSF gut to evaluate their ability to colonize this organ or their possible digestion by the insect. The effect of C. tropicalis on the metabolic composition and digestion process in the larvae was also analyzed and compared to the metabolic composition of the fungal biomass, and rearing substrate. Generally, the conceptualization and results of this work are interesting and the manuscript deserves consideration. However, at presented state the manuscript needs revision. The main comments and recommendations are listed below. Nutritional composition of BSFL Is changeable and can be regulated using different feed stuff. A little discussion on this aspect could improve the Introduction section. For example: doi.org/10.3920/JIFF2021.0162, 10.1093/ee/nvab135 These references were added and the topic is discussed in the introduction (lines 57-59) Details for equipment and software used in the experiment should be unified: the name of manufacturer and its location should be given. Add separate subsection in section 2 with chemicals and materials. Provide information on chemicals purity and manufacturers. The information was added (not in a separate section but in the text) Tables can be modified. It is better to add separate columns with data on investigated values for experimental and control groups of BSF larvae. As the information of the LCMS contains a vast number of metabolites, usually the analysis is conducted as a comparison between the treatments and the control. Therefore the tables present only the significant difference in these comparisons As suggestion, all figures can be combined to the one figure. In this regard, it will be visually comfortable for readers to compare the main results obtained. As the figures present different analyses of different treatments we think it will be more distracting to combine them. The text should be carefully checked for typos and grammatical errors. The text was checked thoroughly ________________________________________ Attachments Attachment Submitted filename: response to reviewers.docx https://doi.org/10.1371/journal.pone.0325056.r002
Decision Letter - Andrey Nagdalian, Editor Dear Dr. Opatovsky, 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 03 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, Andrey Nagdalian Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: No Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: This manuscript provides an interesting and valuable study on the relationship between the fungus C. tropicalis and BSF. The work makes a meaningful contribution to understanding insect-microbe interactions in an economically important insect species. However, there are several points that could be improved before publication: Methodology should be clear 1. The experimental design is somewhat complex with different treatments. While Fig 1, the text could more clearly explain the rationale behind the 10-day with C. tropicalis followed by 1-day without design. What was the hypothesis behind this specific timing? 2. The authors use the 26S rRNA gene to assess the presence of C. tropicalis in the gut, but this approach may require further clarification: 1) Authors should consider using absolute quantitative PCR to establish standard curves for more accurate quantification of fungal amounts. 2) Additional methods, such as culture counts or microscopic observation, may be needed to validate the results. 3) Therefore, providing more details on how C. tropicalis was quantified would strengthen the methods section. 3. The manuscript would benefit address more information about how data normality was assessed before choosing between parametric and non-parametric tests. The evidence is insufficient to support what this study demonstrates. 4. The paper suggests C. tropicalis manipulates BSF digestive enzyme production, but the evidence is primarily correlative. Authors should either provide stronger evidence for this claim or acknowledge its speculative nature more explicitly. 5. The research data in this study is insufficient to support conclusions. The limited dataset does not provide adequate evidence to make such definitive claims about the relationship between C. tropicalis and BSF larvae. More comprehensive investigations would be needed to establish these mechanisms with certainty. Reviewer #2: The authors considered all comments and recommendations. The revised manuscript deserves further consideration by Editorial board. ****** 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: 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. https://doi.org/10.1371/journal.pone.0325056.r003
Revision 2
1 Apr 2025 Author Response 1. The experimental design is somewhat complex with different treatments. While Fig 1, the text could more clearly explain the rationale behind the 10-day with C. tropicalis followed by 1-day without design. What was the hypothesis behind this specific timing? # An additional explanation was added to the method section (lines 114-116) 2. The authors use the 26S rRNA gene to assess the presence of C. tropicalis in the gut, but this approach may require further clarification: 1) Authors should consider using absolute quantitative PCR to establish standard curves for more accurate quantification of fungal amounts. 2) Additional methods, such as culture counts or microscopic observation, may be needed to validate the results. 3) Therefore, providing more details on how C. tropicalis was quantified would strengthen the methods section. # We conducted relative quantification and the method was better described (lines 139-141). Unfortunately, we did not conduct any additional methods to support the results. 3. The manuscript would benefit address more information about how data normality was assessed before choosing between parametric and non-parametric tests. # The relevant data was included (lines 199-201) The evidence is insufficient to support what this study demonstrates. 4. The paper suggests C. tropicalis manipulates BSF digestive enzyme production, but the evidence is primarily correlative. Authors should either provide stronger evidence for this claim or acknowledge its speculative nature more explicitly. # The paragraph was changed accordingly (lines 296-309) 5. The research data in this study is insufficient to support conclusions. The limited dataset does not provide adequate evidence to make such definitive claims about the relationship between C. tropicalis and BSF larvae. More comprehensive investigations would be needed to establish these mechanisms with certainty. # A disclaimer was added in the discussion (lines 322-323) Attachments Attachment Submitted filename: Review Comments.docx https://doi.org/10.1371/journal.pone.0325056.r004
Decision Letter - Andrey Nagdalian, Editor The fate of Candida tropicalis in the black soldier fly larvae and its nutritional effect suggest indirect interactions PONE-D-24-31724R2 Dear Dr. Opatovsky, 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. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. 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, Andrey Nagdalian Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #2: Yes ****** Reviewer #2: The authors considered all comments and recommendations and decided them well. The revised manuscript deserves acceptance ****** 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 #2: No ******** https://doi.org/10.1371/journal.pone.0325056.r005
Formally Accepted
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