Challenging the status quo: Methylene blue at recommended concentrations alters metabolism in early zebrafish development

Challenging the status quo: Methylene blue at recommended concentrations alters metabolism in early zebrafish development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Challenging the status quo: Methylene blue at recommended concentrations alters metabolism in early zebrafish development Jan Mennigen, Niepukolie Nipu, Lai Wei, Hyojin Lee, Jith Thomas This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4473193/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jan, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Methylene blue (MB) is an antifungal agent widely used during critical stages of zebrafish development. Most guidelines recommend 0.00005% or 0.0001% of MB for embryo/larval rearing. The Organisation for Economic Co-operation and Development zebrafish embryo toxicity test guideline omits MB recommendations, leading to inconsistent MB use in zebrafish research. Because MB affects oxidative energy metabolism in vitro and in vivo, we investigated possible metabolic effects of recommended MB concentrations in developing zebrafish (1-5 days post-fertilization (dpf)). MB increased O2 consumption rate at 1 dpf, followed by an overall reduction in oxidative energy metabolism in post-hatch eleutheroembryos (4-5 dpf). Concomitantly, mitochondrial transcripts decreased in 1 and 4 dpf zebrafish. Our findings show that MB, at recommended husbandry concentrations, affects oxidative metabolism and can thus confound experiments. Since the zebrafish embryo/larval model is gaining traction as a high-throughput New Approach Methodology (NAM) for toxicity assessment, researchers should reconsider MB use. Biological sciences/Zoology/Animal physiology Biological sciences/Developmental biology/Disease model Full Text Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Published Journal Publication published 25 Jan, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4473193","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":308720350,"identity":"e879f3cd-5191-450d-a2a8-27c0d9db1079","order_by":0,"name":"Jan Mennigen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYBACxgYogw1EfCBZC+MMBgYJHpKsZOYhRgtz+9kDTDcq7uTx8S8+9ti2zabOnv2M4QeGGjvcDuvJS2DOOfOsmE3iWbpxbluaBA9PjrEEw7FkPH7JMWDObTuc2CZxxkwayAA6LHeDBGMDM24t/W+AWv6BtJz/Jm0J0sL/dvMPxoZ63FpmgGxpAGrh72GTZgRpkcjdBrTlMB4tbwwO5xwD2cJmJtlzLk2y58b7bxYJx47j1GLYn2P4OKfmcOL8/sPPJH6U2fCz96cl3/hQU41bSwMDwwEwSyIBSTgBUyUcyMNZ/AfwKBsFo2AUjIIRDQD0jFHaQSs0XwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4117-6654","institution":"University of Ottawa","correspondingAuthor":true,"prefix":"","firstName":"Jan","middleName":"","lastName":"Mennigen","suffix":""},{"id":308720351,"identity":"fc024f54-150b-410d-8046-c75b086da39b","order_by":1,"name":"Niepukolie Nipu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Niepukolie","middleName":"","lastName":"Nipu","suffix":""},{"id":308720352,"identity":"8a5769f1-6379-4d3d-aa9a-9dcfa37cd4cf","order_by":2,"name":"Lai Wei","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lai","middleName":"","lastName":"Wei","suffix":""},{"id":308720353,"identity":"1ca4f624-d521-47f0-a075-69fc1e614007","order_by":3,"name":"Hyojin Lee","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hyojin","middleName":"","lastName":"Lee","suffix":""},{"id":308720354,"identity":"3bff3a6b-da5d-4e9b-8a9e-64bc3363efc8","order_by":4,"name":"Jith Thomas","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jith","middleName":"","lastName":"Thomas","suffix":""}],"badges":[],"createdAt":"2024-05-24 14:55:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4473193/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4473193/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-07471-8","type":"published","date":"2025-01-25T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74719762,"identity":"05429f0f-4e2c-4ce8-85d6-0792ae8b30b2","added_by":"auto","created_at":"2025-01-25 08:07:15","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10533771,"visible":true,"origin":"","legend":"","description":"","filename":"Nipuetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4473193/v1_covered_ebae5949-941c-4280-9ece-f6287a0cd10d.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Challenging the status quo: Methylene blue at recommended concentrations alters metabolism in early zebrafish development","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4473193/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4473193/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Methylene blue (MB) is an antifungal agent widely used during critical stages of zebrafish development. 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