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
0%
Research Effort Categories
Basic
0%
Applied
50%
Developmental
50%
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