Effects of microbial-derived long-chain polyunsaturated fatty acids from Aurantiochytrium limacinum BCC52274 and Mortierella sp. on growth and immunity in Litopenaeus vannamei post-larvae

Virak Visudtiphole; Panida Unagul; Sage Chaiyapechara; Waraporn Jangsutthivorawat; Metavee Phromson; Siriporn Tala; Pacharawan Deenarn; Punsa Tobwor; Pisut Yotbuntueng; Surasak Jiemsup; Suganya Yongkiettrakul; Looksorn Koichai; Wananit Wimuttisuk

doi:10.1371/journal.pone.0329358

Peer Review History

Original SubmissionJune 10, 2025
Decision Letter - Lee Seong, Editor Dear Dr. Wimuttisuk, 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 Aug 11 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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We look forward to receiving your revised manuscript. Kind regards, Lee Seong 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. Thank you for stating the following financial disclosure: “This research has received funding support from the Functional Ingredient & Food Innovation Program, National Center for Genetic Engineering and Biotechnology [grant number P19-51807] to VV and the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation [grant number B05F640184] to WW.” 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. 3. 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.] 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: Yes Reviewer #2: Yes ******** 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: Dear Author Thank you for your good manuscript I accept it only with minor change please add new references and revised again discussion .and short material and methods and explain more result and add main idea Reviewer #2: This study provides valuable insights into sustainable alternatives to fish oil in aquaculture feed by evaluating the efficacy of Aurantiochytrium limacinum BCC52274 (AL) and Mortierella sp. oil (ARASCO) as sources of long-chain polyunsaturated fatty acids (LC-PUFAs) for Pacific white shrimp (Litopenaeus vannamei) post-larvae. The experimental design, which involved Artemia enrichment with varying DHA:ARA ratios, effectively demonstrated that different LC-PUFA profiles can be strategically applied to promote either growth or immune enhancement in shrimp. Notably, Group A (DHA:ARA 100:0) achieved the highest biomass and body weight, while Group D (DHA:ARA 25:75) exhibited elevated prophenoloxidase II expression and increased production of anti-inflammatory eicosanoids. Furthermore, several enriched groups showed a reduced viral load following WSSV infection, highlighting the immunoprotective potential of these dietary supplements. Overall, the findings support the viability of AL and ARASCO as sustainable, functional replacements for traditional marine-derived fatty acids in shrimp aquaculture. The manuscript is well-written and well-structured, with only a few minor comments for consideration. Minor Comments: 1. Lines 211–217: Please specify the sample size and the age of the post-larvae used for RNA extraction. 2. The concentration of RNA used for cDNA synthesis should be mentioned. 3. Vibrio harveyi is not mentioned in the abstract and should be included. 4. The discussion section would benefit from a deeper analysis of the findings in the context of existing literature, or by proposing hypotheses where data are currently lacking. ****** 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 https://doi.org/10.1371/journal.pone.0329358.r001
Revision 1
14 Jul 2025 Author Response 1. 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'. Answer: We have provided a rebuttal letter (this document), a marked-up copy of our manuscript, and an unmarked version of the revised manuscript based on the journal’s instructions. 2. 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. Answer: We have made clarification regarding the financial disclosure in the second-to-last paragraph of the updated cover letter (yellow highlight). Should you have any more questions, please let us know. Journal Requirements: When submitting your revision, we need you to address these additional requirements. 3. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Answer: We have adjusted the manuscript so that it meets PLOS ONE’s style requirements in the revised version of the manuscript. 2. Thank you for stating the following financial disclosure: “This research has received funding support from the Functional Ingredient & Food Innovation Program, National Center for Genetic Engineering and Biotechnology [grant number P19-51807] to VV and the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation [grant number B05F640184] to WW.” 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. Answer: We have added the suggested sentence regarding the roles of the funders into the updated cover letter. Please change the online submission form on our behalf. 3. 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. Answer: We have rechecked all references by going through the journal’s database, google scholars, as well as databases for the retracted publications. However, we were unable to identify the retracted paper. Reviewer Comments to the Author Reviewer #1: Dear Author Thank you for your good manuscript I accept it only with minor change please add new references and revised again discussion .and short material and methods and explain more result and add main idea Answer: We thank Reviewer #1 for your kind suggestions. As there was no specific instruction given in the reviewers’ response by PLOS ONE (which reference to add and which part of the discussion to be revised), we have improved our manuscript as follows: 1) Added new references can be found in line 416-419, 440-449, 479-481, and 496-498. 2) Revised the discussions: The discussion was revised to correct grammatical errors as well as the added discussion on the main idea (line 424-435) as well as more discussion based on the added new references shown in 1). 3) Short materials and methods: We have shortened the materials and methods as follows. 3.1) Under the section pathogen challenge, we have removed “to assess the effect of Artemia enrichment on the immunological phenotype of the PL in response to bacterial and viral infection, PL19 were challenged with Vibrio harveyi or white spot syndrome virus (WSSV). Details of the challenge are as follows." 3.2) Under the Quantitative real-time PCR analysis to detect WSSV copy number, we have removed the following sentences. “The water volume in each tank was adjusted from 150 L to 180 L on day 13 of the experiment to account for the larger size of the shrimp.” “As WSSV infection in PLs typically does not result in mortality [33,34], the effect of feed supplementation on the WSSV-infected PLs was evaluated by determining the WSSV copy number using a qPCR analysis.” “The extracted DNA was spectrophotometrically quantified (OD260) and qualified (OD260/OD280 ~ 1.6-1.9)” and “Oligonucleotide primers were obtained from Integrated DNA Technologies in South Korea.” 4) Explain more results: We have added an explanation in the result section in line 280-282. 5) Added the main idea: A proposed model was added in line 410 to 435 as well as the added Conclusion section (line 507-518) Reviewer #2: This study provides valuable insights into sustainable alternatives to fish oil in aquaculture feed by evaluating the efficacy of Aurantiochytrium limacinum BCC52274 (AL) and Mortierella sp. oil (ARASCO) as sources of long-chain polyunsaturated fatty acids (LC-PUFAs) for Pacific white shrimp (Litopenaeus vannamei) post-larvae. The experimental design, which involved Artemia enrichment with varying DHA:ARA ratios, effectively demonstrated that different LC-PUFA profiles can be strategically applied to promote either growth or immune enhancement in shrimp. Notably, Group A (DHA:ARA 100:0) achieved the highest biomass and body weight, while Group D (DHA:ARA 25:75) exhibited elevated prophenoloxidase II expression and increased production of anti-inflammatory eicosanoids. Furthermore, several enriched groups showed a reduced viral load following WSSV infection, highlighting the immunoprotective potential of these dietary supplements. Overall, the findings support the viability of AL and ARASCO as sustainable, functional replacements for traditional marine-derived fatty acids in shrimp aquaculture. The manuscript is well-written and well-structured, with only a few minor comments for consideration. Answer: We thank the reviewer for your kind comments. We have clarified the manuscript based on the comments provided in the following section. Minor Comments: 1. Lines 211–217: Please specify the sample size and the age of the post-larvae used for RNA extraction. Answer: PL18 (N = 30) were used for RNA extraction to determine the impact of DHA:ARA supplementation on shrimp immune genes. We have added this information to line 217. For the consistency of the manuscript, we have also added the information regarding the PL sample size in each rearing tank (N = 15,000) in line 139-140 and the number of PL used to determine WSSV copy number (N = 35) in line 173. 2. The concentration of RNA used for cDNA synthesis should be mentioned. Answer: The author has added the concentration of RNA used for the cDNA synthesis in lines 222 along with the amounts of cDNA used in each real-time PCR reaction in lines 229. 3. Vibrio harveyi is not mentioned in the abstract and should be included. Answer: The authors have included a sentence regarding the V. harveyi experiment in the abstract (lines 48-49). 4. The discussion section would benefit from a deeper analysis of the findings in the context of existing literature, or by proposing hypotheses where data are currently lacking. Answer: We have added a proposed model and more discussion in line 419-431, 490-496, and 500-511. Attachments Attachment Submitted filename: A rebuttal letter.docx https://doi.org/10.1371/journal.pone.0329358.r002
Decision Letter - Lee Seong, Editor Effects of microbial-derived long-chain polyunsaturated fatty acids from Aurantiochytrium limacinum BCC52274 and Mortierella sp. on growth and immunity in Litopenaeus vannamei post-larvae PONE-D-25-31399R1 Dear Dr. Wimuttisuk, 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, Lee Seong Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0329358.r003

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