Source: PRESIDENT AND TRUSTEES OF COLBY COLLEGE, THE submitted to NRP
DEVELOPMENT AND APPLICATION OF ENVIRONMENTAL RNA (ERNA) TOOLS TO ENSURE SUCCESSFUL WILD SEED RECRUITMENT FOR BLUE MUSSEL AQUACULTURE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032385
Grant No.
2024-68008-42648
Cumulative Award Amt.
$300,000.00
Proposal No.
2023-09601
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2026
Grant Year
2024
Program Code
[A1701]- Critical Agricultural Research and Extension: CARE
Recipient Organization
PRESIDENT AND TRUSTEES OF COLBY COLLEGE, THE
4120 MAYFLOWER HL
WATERVILLE,ME 049018841
Performing Department
(N/A)
Non Technical Summary
The success of the U.S. blue mussel aquaculture industry is dependent on the ability to reliably capture healthy wild larvae ('seed') from the ocean. In recent years, however, mussel larval supply has become increasingly unpredictable, leading to seed collection failures and thus considerable financial and production hardships for mussel farmers. In this work, we will develop novel technologies that will transform farmers' ability to monitor wild mussel larvae at their farms or seed collection sites. To accomplish this, our objectives are to: (1) develop rapid and user-friendly molecular tools designed to detect live mussel larvae in water samples, (2) partner with Maine's mussel farmers to collect commercially relevant samples and to test these tools on operational mussel farms, and (3) disseminate our resulting methods, sampling kits, assays, and early data to the U.S. shellfish aquaculture community to facilitate their immediate use. Through the incorporation of research, education, and extension activities, we will deliver science-based knowledge and field-validated technologies to end users (e.g., mussel farmers and other stakeholders), allowing them to more accurately predict when and where wild mussel seed will be in the water column to ultimately enhance domestic mussel production. The tools developed through this work will mitigate stakeholder productivity losses as a result of shifting mussel population dynamics and seed availability, permit growers to make data-informed decisions about lease-site applications and/or investment in shellfish hatcheries, significantly reduce mussel larvabiofouling removal efforts and costs for non-mussel farmers, and optimize sustainable protein production to meet the projected food demands of a rising population.
Animal Health Component
50%
Research Effort Categories
Basic
0%
Applied
50%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3043724108050%
3063724105010%
3070811108040%
Knowledge Area
304 - Animal Genome; 306 - Environmental Stress in Animals; 307 - Animal Management Systems;

Subject Of Investigation
3724 - Clams and mussels; 0811 - Shellfish;

Field Of Science
1080 - Genetics; 1050 - Developmental biology;
Goals / Objectives
Our overarching goal is to work with mussel farmers to develop, test, and implement cutting-edge environmental RNA (eRNA) toolkits that will transform blue mussel farmers' ability to accurately monitor live mussel seed in the water column in near-real-time. Our integrated research, extension, and education program will be accomplished through the following objectives:Objective 1: Build upon our preliminary work to develop rapid and user-friendly environmental RNA (eRNA) assays designed to detect live mussel larvae in water samplesObjective 2: Partner with Maine's mussel farmers to collect commercially relevant samples and to test the efficacy and ease of use of these assays on operational mussel farmsObjective 3: Disseminate our resulting methods, sampling kits, assays, and early data to the U.S.shellfish aquaculture community to facilitate their immediate use
Project Methods
Objective 1: Build upon our preliminary work to develop rapid and user-friendly environmental RNA (eRNA) assays designed to detect live mussel larvae in water samplesObj. 1a. Development of seed (pediveliger)-specific RT-qPCR assays:Identify and select top 10 candidate genes with the highest pediveliger-specific expression for RT-qPCR assay design using mussel developmental transcriptomics dataDesign RT-qPCR primer/probe sets for selected candidate genesTest specificity of RT-qPCR assays against RNA from the major mussel developmental stages (gametes - adult) and non-target bivalve speciesClone amplicons from promising assays, confirm sequences via Sanger sequencing, and order appropriate Taqman probeCreate standards by linearizing and serially diluting clone plasmidsTest the limit of detection (LoD) for assays at both the mRNA transcript and larval levelCq values will be converted to mRNA transcript copy numbers to investigate the relationship between mRNA transcript copy number and larva abundanceObj. 1b. Conversion of larva-specific and pediveliger-specific assays from RT-qPCR to RT-LAMP:Design RT-LAMP primer sets using mRNA sequences from final RT-qPCR assays developed in Obj. 1aTest specificity of RT-LAMP assays against RNA from the major mussel developmental stages (gametes - adult) and non-target bivalve speciesClone RT-LAMP amplicons from promising assays and confirm sequences via Sanger sequencingCreate standards by linearizing and serially diluting clone plasmidsTest the limit of detection (LoD) for assays at both the mRNA transcript and larval levelTest how larval concentration relates to color and reaction durationLoD will be determined by identifying the lowest concentration that maintains a 95% positive detection rateVisual (coarse) color assignment of RT-LAMP assay results will be determined by independent viewers blind to reaction compositionPrecise color assignment will be conducted via photography and ImageJ and/or Photoshop based on saturation, hue, and reference colorsRegression analysis will be used to relate color change (e.g., saturation or hue) and time-to-color-change to sample concentration to develop coarse and precise larval quantification guidelinesDevelop user-friendly color code quantification system for growersObjective 2: Partner with Maine's mussel farmers to collect commercially relevant samples and to test the efficacy and ease of use of these assays on operational mussel farmsObj. 2a. Collection of farm relevant field samples in collaboration with Bangs Island Mussels:Collect monthly water/plankton tow samples from two local mussel aquaculture farm (Bangs Island Mussels) lease sites, collect eRNA subsample via filtration, and preserve eRNAExtract eRNA from filtersTest eRNA samples against assays developed in Objective 1Quantify bivalve larvae for each sample via microscopyCollect environmental data (sea surface temperature, salinity, dissolved O2, seawater pH, pCO2 data, chlorophyll a, dissolved inorganic carbon, and total alkalinity)Compare assay results (copy number and time-to-color-change) to the number of bivalve larvae in corresponding plankton samples, with the expectation that eRNA signal will correlate with the number of bivalve larvaeCompare oceanographic data to the timing and concentration of mussel larvae to determine which environmental parameters correlate with larval timing and abundanceObj. 2b. Testing the efficacy and ease of implementation of assays on an operational mussel farm (Bangs Island Mussels):Work directly with Bangs Island Mussels crew to better understand their seed monitoring and collection methodsCollaboratively develop a field collection and sample testing protocol that can be easily incorporated into routine work on the mussel farmConduct monthly sampling on one mussel aquaculture lease site, while Bangs Island Mussels crew conducts sampling on a second siteOptimize the methodology and toolkits to ensure the greatest ease-of-use while maintaining reliability of the assay resultsWork with Bangs Island Mussels to design and initiate a long-term seed monitoring program that will permit them to track the mussel seed set throughout the yearEfforts:Objective 3: Disseminate our resulting methods, sampling kits, assays, and early data to the U.S. shellfish aquaculture community to facilitate their immediate usePublish the final methodology and case studies in peer-reviewed journals of professional societiesDesign and print pamphlets that provide descriptions of our toolkits, example assay results, and infographics demonstrating their useHost all relevant project activities, outputs, and training videos through the Bigelow Center for Seafood Solutions website and Bigelow's YouTube channelPresent results and conduct in-person toolkit demonstrations at scientific conferences, trade shows, and exposOrganize "farm days" to demonstrate toolkits at closely partnering shellfish farms (e.g., Mook Sea Farm)Design and teach a Colby College Capstone course within the Environmental Studies program that incorporates our eRNA tools and sampling methodologiesTo gauge interest in our toolkits and evaluate the efficacy of our educational efforts, we will record statistics for demo events, presentations, pamphlets/infographics distributed, event registration numbers, Colby student enrollment, and webpage/post engagementEvaluation:To evaluate progress toward achieving project objectives, Bruesewitz (PD) and the Advisory Board, consisting of stakeholders that are regional and national experts in shellfish hatcheries, wild harvest, and aquaculture, will track project milestones to ensure all aspects of the project are on track for successful conclusion. This will be accomplished through annual hybrid meetings consisting of the PD, Advisory Board, and all project investigators, with videoconferencing tools (e.g., Zoom) available for those that require remote participation. As necessary, the Advisory Board will also meet quarterly with coPDs to evaluate the quality of the project, ensure that measurable and defined outcomes are produced, assess the potential impacts of the work, and discuss progress towards milestones.Milestone 1: Prototype toolkit for validation on farmsSuccess indicators: Number of primer sets tested; evidence that LoD for RT-qPCR and/or RT-LAMP assays is below that of anticipated larval densities in situ; confidence that kits detect only certain larval stages of blue musselsMilestone 2: Optimized toolkits for disseminationSuccess indicators: Duration of testing kit use becomes speedier and is faster, cheaper, and more accurate than traditional plankton enumeration efforts; the results are comparable with partner scientist efforts; farmers expressing increased likelihood of implementing toolkits for in-house monitoring effortsMilestone 3: Widespread dissemination through extension and education activitiesSuccess indicators: Number of peer-reviewed publications and professional presentations; number of undergraduate students engaged in coursework and internships; instances where kit details were requested or demonstrations provided, virtually or in-person; geographic distribution or shellfishery type of engaged stakeholders

Progress 07/01/24 to 06/30/25

Outputs
Target Audience: Maine Aquaculture Association (MAA) Aquaculture producers Bangs Island Mussels Pemaquid Mussel Farms Mook Sea Farms Downeast Institute Summer Independent undergraduate intern from Colby College (1 student) Senior Capstone students at Colby College (12 undergraduate students) Ocean Studies Department at Maine Maritime Academy (~100 students and faculty) Attendees of the 2025 Maine Fishermen's Forum (~2,500 attendees) Attendees of the 2025 World Aquaculture Society Meeting (~4,000 attendees) The Bigelow Laboratory for Ocean Sciences Community Changes/Problems: The Project Director, Denise Bruesewitz, recently took on a new larger admin role at Colby College that is taking up a significant portion of her time. We have had to readjust our timeline accordingly to accommodate. Our main project partner at Bangs Island Mussels is taking a parental leave of absence, which will require us to adjust our timeline to accommodate. The Zymo DNA/RNA Shield DirectDetect preservative has not worked as expected with our colorimetric RT-LAMP assays. We have been in contact with the manufacturer about this issue regarding a solution and alternatives, and have established contacts to help us move forward. Our industry partners (Bangs Island Mussels) have informed us that it might be more useful to have an assay that can detect trochophores (the first larval stage) rather than the pediveligers (the last larval stage), which would allow them to have more advanced notice for seed collection planning and preparation. Therefore, we are also in the early stages of developing trochophore-specific assays. What opportunities for training and professional development has the project provided? Expanding data management and sampling capabilities with our aquaculture collaborators (Bangs Island Mussels) Colby College undergraduate intern Ernst mentored this intern as part of Bigelow's 10-week summer REU program Ernst worked closely with this intern to design and conduct an experiment associated with the work in this award, after which the intern publicly presented the results as a poster at Bigelow Laboratory The intern also plans to present this work at the upcoming 2026 Northeast Aquaculture Conference and Exposition (NACE) meeting in Portland, ME Maine Maritime Academy Ocean Studies Seminar Series Ernst was invited to speak to the Ocean Studies department, where he presented the ongoing work from this award Bigelow Laboratory for Ocean Sciences Seminar Series Ernst presented ongoing work from this award to the Bigelow Laboratory community Promotion The work in this award has directly led to Ernst's promotion to Research Scientist at Bigelow Laboratory Blue Venture Investment Summit Ernst and Price attended the 2024 Blue Venture Investment Summit in Portland, ME for networking opportunities and to share preliminary results from this award with stakeholders World Wildlife Foundation (WWF) eDNA team Price and Ernst established WWF eDNA contacts in Washington DC to explore potential future eRNA collaborations We also provided training regarding the utility of eRNA tools World Wildlife Foundation (WWF) Aquaculture team Ernst attended a musselinformation exchange trip to New Zealand and established new connections within the New Zealand mussel aquaculture space Colby College Senior Capstone course Ernst expanded his proficiency as an education through teaching a Colby College Senior Capstone course that focused on eDNA applications for water quality monitoring in Maine How have the results been disseminated to communities of interest? Maine Fishermen's Forum (~2,500 attendees) Ernst conducted a real-time demonstration of the mussel-larvae-specific RT-LAMP assays on Shellfish Focus Day 2025 World Aquaculture Society Meeting (~4,000 attendees) Ernst gave 2 oral presentations on the work conducted through this award, including a talk in the 'Mussels' session and a talk in the 'eDNA in Shellfisheries' session Institutional Maine Aquaculture Association membership We (Bigelow) are now official paying members of the MAA, which represents nearly 200 farms and more than 700 farmers, and plan to add project updates to the private news letters that go out to constituents World Wildlife Foundation (WWF) New Zealand Trip Ernst disseminated information regarding the eRNA technologies being developed and optimized through this award with mussel researchers and aquaculturists in New Zealand, as well as other attendees from Maine comprised of members of the Department of Marine Resources, mussel farmers, and other stakeholders Bigelow Laboratory Winter Webinar Series Ernst presented on the work resulting from this award to Bigelow Laboratory supporters and donors Colby College Senior Capstone Course Ernst educated 12 undergraduate students on eDNA and eRNA technologies and applications, including extensive hands-on field and laboratory training What do you plan to do during the next reporting period to accomplish the goals? Trochophore stage tool development Our aquaculture collaborators (Bangs Island Mussels) have expressed an interest in tools that are capable of detecting the earliest larval stage (trochophore) for blue mussels to facilitate seed collection planning We plan to leverage our existing RNA-seq data to develop and implement these assays Field sampling and eRNA toolkit optimization We will continue our field sampling efforts through the end of the award We will work closely with our collaborators (Bangs Island Mussels) to optimize both our current field sampling methods and eRNA toolkits This will be accomplished through farmers testing our methodologies and kits and providing us with feedback regarding ease-of-use and potential issues they encounter Test field samples with eRNA assays We will test all collected field samples with both our qPCR and LAMP eRNA assays Confirm effectiveness of assays via sequencing To confirm the effectiveness of our tools in mixed plankton field samples, we are planning to take a sequencing approach (metabarcoding) to identify the composition of organisms in a subset of samples Outreach and dissemination of results We will present at the 2026 Ocean Sciences Meeting We will present at the 2026 Northeast Aquaculture Conference and Exposition (NACE) We will conduct another demo of our toolkits at the 2026 Maine Fishermen's Forum We will develop media for the broad dissemination of the capabilities of and methodologies for our eRNA toolkits Teach a Colby College Senior Capstone Course focused on eDNA and eRNA technologies in Fall 2025 We plan to publish the results of our work in a peer-review journal

Impacts
What was accomplished under these goals? There is considerable interest in expanding U.S. blue mussel production capacity, which will decrease the mussels' contribution to the U.S. seafood trade deficit (>$100 million) and provide an abundance of job opportunities throughout the working waterfront. However, mussel aquaculturists in the northeast have reported failures to collect wild larvae (essential for mussel production) for at least a decade. These failures are due to the changing seasonality of mussel spawning and settlement, which is hypothesized to result from warming surface ocean waters and corresponding alterations in precipitation and thermal patterns of the Gulf of Maine, one of the fastest warming bodies of ocean water on the planet. This project aims to develop and implement monitoring tools that will overcome the unpredictability of mussel larvae collection and consequently enhance domestic mussel production. These toolswill: (1) mitigate stakeholder productivity losses as a result of shifting natal shellfish population dynamics and seed availability; (2) permit U.S. growers to make data-informed decisions about lease-site applications and/or investment in shellfish hatcheries; and (3) optimize sustainable protein production to meet the projected food demands of a rising population. Moreover, the use of the novel technologies developed from this work will ultimately catalyze rural economic development and promote animal welfare and a thriving working waterfront in response toincreased focus on sustainability. Objective 1: Build upon our preliminary work to develop rapid and user-friendly environmental RNA (eRNA) assays designed to detect live mussel larvae in water samples Major activities completed / experiments conducted: Completed analyses with updated chromosome-level blue mussel genome assembly to identify new promising biomarkers for designing assays Developed and validated quantitative PCR (qPCR) assays and more user-friendly LAMP assays that have a simple color change result and allow for the specific detection of blue mussel larvae Data collected: mRNA sequences for genes exhibiting stage-specific expression patterns and associated gene expression data across mussel developmental stages DNA sequences and protocols for the qPCR and LAMP assays described above Results from qPCR and LAMP tests on mussel hatchery samples and non-mussel species Results for initial sensitivity tests for each assay Summary statistics and discussion of results: 20+ assays tested for functionality and specificity qPCR: Developed 5+ assays specific to blue mussel larval stages LAMP: Developed 3 assays specific to blue mussel larval stages, which correspond to a subset of the qPCR assays and allow for direct comparison The results from all qPCR assays were converted to mRNA transcript copy numbers, which allowed us to compare copy numbers to mussel larva abundance. In general, there is a linear relationship between mRNA transcript copy number and larval abundance for each of our assays. The results from our LAMP assays show a similar relationship as our qPCR assays. Testing for these assays is ongoing. Objective 2: Partner with Maine's mussel farmers to collect commercially relevant samples and to test the efficacy and ease of use of these assays on operational mussel farms Major activities completed / experiments conducted: Conducted field sampling cruises at two aquaculture lease sites from 7/1/24-present Developed accessible protocols for field sampling using plankton tow net and water pump methodologies Quantified bivalve larvae for each sampling event Developed a data sharing plan and live shared database with industry partners (Bangs Island Mussels) Extracted most field samples collected from aquaculture leases Conducted initial field sample testing with qPCR assays and LAMP assays developed in Objective 1 Conducted initial testing of samples preserved with different DNA/RNA preservative types Data collected: Ethanol-preserved plankton subsamples from two mussel aquaculture leases from 7/1/24-present DNA and RNA extracts from plankton subsamples from 7/1/24-present Environmental data from each sampling day Initial results from a subset of the field sample RNA extracts Bivalve larval counts for each field sampling event Summary statistics and discussion of results: 17 field cruises to sample at two aquaculture leases, resulting in 112 preserved plankton samples 71 DNA and RNA extractions from field samples 80+ environmental datasets corresponding to each site on each collection day, comprising a time-series of oceanographic data 2 time-series datasets for larva-specific qPCR assays, 1 for each lease site 2 time-series datasets for bivalve larval counts, 1 for each lease site Objective 3: Disseminate our resulting methods, sampling kits, assays, and early data to the U.S.shellfish aquaculture community to facilitate their immediate use Major activities completed / experiments conducted: Conducted a demonstration of our LAMP assays and sampling kits at the Shellfish Day of the 2025 Maine Fishermen's Forum, an annual trade show and seminar event that brings together Maine's fishermen, sea farmers, gear suppliers, state and federal scientists and regulators, and other stakeholders Presented results at 2025 World Aquaculture Society Meeting through two oral presentations Taught eDNA-based Colby College Senior Capstone course, which incorporated several methods and techniques used in our eRNA sampling and testing protocols Gave an invited oral presentation to the Maine Maritime Academy (MMA) Ocean Studies Department Mentored a summer REU student intern from Colby College in an independent research project closely related to the work conducted in this award Data collected: Number of 2025 Maine Fishermen's Forum attendees Number of 2025 World Aquaculture Society Meeting attendees Number of students taught through Colby College Senior Capstone course Number of attendees present at MMA Ocean Studies Seminar Number of students mentored through summer REU internship at Bigelow Summary statistics and discussion of results: Colby College summer independent undergraduate intern: 1 student mentored Senior Capstone students at Colby College taught: 12 students Ocean Studies Department Seminar Series at Maine Maritime Academy: ~100 students and faculty in attendance 2025 Maine Fishermen's Forum toolkit demonstration: ~2,500 total in attendance 2025 World Aquaculture Society Meeting presentation: ~4,000 total in attendance

Publications

  • Type: Other Status: Other Year Published: 2025 Citation: Ernst, D. A. Shining a light into the larval black box: Environmental RNA (eRNA) tools for understanding blue mussel larval ecology. 2025. Maine Maritime Academy Ocean Studies Seminar Series. Castine, ME USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Ernst, D. A., Beal, B. F., Grey, E. K., Salter, B., Whitney, L. P., Price, N. N. Characterizing the developmental transcriptome and methylome of the blue mussel Mytilus edulis. 2025. World Aquaculture Society Meeting. New Orleans, LA USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Ernst, D. A. Accessible molecular tools for detecting mussel seed. 2025. Maine Fishermen's Forum. Rockport, ME USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Ernst, D. A., Beal, B. F., Grey, Whitney, L. P., Price, N. N. Elucidating mussel larval dynamics in Casco Bay, Maine with user-friendly, eRNA-based tools. 2025. World Aquaculture Society Meeting. New Orleans, LA USA.