Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
  • Problem Description
• Data Files
• Supplemental Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

Blue mussels (Mytilus edulis Linneaus, 1758) were collected from Black Point in Narragansett Bay, RI (41° 24′ 4.4964″ N, 71° 27′ 43.8228″ W) and transported in chilled coolers to the lab at Carleton College in Nothfield, MN, USA. Mussels were acclimated in recirculating seawater (Instant Ocean, Blacksburg, VA, USA) for 2-3 weeks at 14°C and fed commercial Shellfish Diet 1800 (Reed Mariculture, Campbell, CA, USA) at a rate of 5% dry mussel tissue weight day^-1.

72 adult mussels [initial shell length = 60.11 ± 0.58 mm (± SE)] were divided into four treatment groups and placed into glass aquaria (50 × 25 × 31 cm) filled with 10 L of artificial seawater. Tanks were aerated and temperatures maintained using submersible heating elements (Biotherm 1000 W Titanium Heating Element, Blueline Aquatics, San Antonio, TX, USA). Temperature loggers (HOBO Pro v2; Onset Computer Corporation, Pocasset, MA;  temperature sensor precision = 0.01°C. ) measured water temperature in each tank every 10 s. Thermal conditions included three stable temperature treatments (14.96 ± 0.14°C, 20.51 ± 0.49°C and, 24.99 ± 0.62°C; mean ± standard deviation) and one fluctuating temperature treatment alternating between 15°C and 25°C every 6 hours (20.15 ± 4.67°C). All mussels were fed commercial Shellfish Diet 1800 (Reed Mariculture, Campbell, CA, USA) at a rate of 5% dry mussel tissue weight. day^-1.

For each of the four temperature treatments, three replicate mussels were sampled on six consecutive days to extract RNA from gill lamellae tissue (20 mg). Gene expression was halted immediately by immersing each mussel in liquid nitrogen. Tissue was manually homogenized using needle syringes (19 gauge; Becton Dickinson, Franklin Lakes, NJ) and RNA was extracted using Monarch® Total RNA Miniprep Kits according to the manufacturer’s protocol (New England Biolabs, Ipswich, MA). After RNA extraction, each sample concentration, integrity, and quality were assessed using both a Qubit 4 Fluorometer using Qubit™ RNA Broad Range Assay Kits and Qubit™ RNA IQ Assay Kits (Thermo Scientific, Wilmington, DE, USA) and a Nanodrop One UV-Vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA).  

Primers for heat-shock protein 70 (hsp70), NADH-ubiquinone oxidoreductase (NADH), and elongation factor 1 alpha (ELF-a) genes were derived from previous sequences reported for Mytilus edulis (sequence information provided below). Forward and reverse primers were designed using Primer3web (v4.1.0) and synthesized by Integrated DNA Technologies (Coralville, IA, USA). We quantified gene expression via one-step quantitative reverse transcription (Luna Universal One-Step RT-qPCR Kit, New England Biolabs, Ipswich, MA) using a QuantStudio 5 qPCR machine (Thermo Scientific, Wilmington, DE, USA). The PCR thermal program consisted of initial denaturation (95°C, 1 minute) followed by 40 cycles of denaturation (95°C, 10 seconds) and extension (60°C, 30 seconds). A melt curve was run to confirm that the products were indeed a single amplicon. For each biological replicate (e.g., each of the 69 mussels sampled), 3 technical replicates were run. C_q values for technical replicates were within 0.5 cycles (Nolan et al., 2006). Expression levels were standardized to elf-a reference gene expression. Amplification data was used to estimate relative expression (RQ = 2-∆∆CT). For every temperature treatment, differences in relative gene expression were analyzed between each day compared to day 1 with unpaired Student’s t-tests. Analyses were conducted using MATLAB R2024a (MathWorks, Natick, MA, USA).

Species             Target    Forward primer (5'-3')                                     Reverse primer (5'-3')                                    size (bp)      Genbank_Accession #

Mytilus edulis  HSP70  AGA CAC AGG GCG TAC AAG AA          ATG CTG CCA AGA ACC AAG TG           259               HBDQ01298109.1 (BioProject: PRJEB41447)

Mytilus edulis  ELF-a    CAG GAG ACA ATG TTG GTT TCA A      AAA TTC ATC AAA TCT GGG GAT G     328               AY580270.1

Organism identifiers:

Taxonomic name used in metadata, common name, Life Science Identifier (LSID) 
"Mytilus edulis ( Linneaus, 1758)", blue mussel, urn:lsid:marinespecies.org:taxname:140480

Expression levels were standardized to a housekeeper gene, elf-a. Amplification data were used to estimate relative expression between temperature treatments. For every temperature treatment, differences in relative gene expression were analyzed between each day compared to day 1 and reported as Relative Quantification (RQ) = 2-∆∆CT.  Analyses were conducted using the Relative Quantification app on the ThermoConnect platform (https://www.thermofisher.com/us/en/home/digital-science/thermo-fisher-connect.html).

* Data table within submitted file "Mytilus_edulis_hsp70.csv" was imported into the BCO-DMO data system for this dataset. Values "nd" imported as missing data values.   Table will appear as Data File: 953794_v1_mussel-hsp70.csv (along with other download format options).

Missing Data Identifiers:
* In the BCO-DMO data system missing data identifiers are displayed according to the format of data you access. For example, in csv files it will be blank (null) values. In Matlab .mat files it will be NaN values. When viewing data online at BCO-DMO, the missing value will be shown as blank (null) values.

* Column names adjusted to conform to BCO-DMO naming conventions designed to support broad re-use by a variety of research tools and scripting languages. [Only numbers, letters, and underscores.  Can not start with a number]

* Date converted to ISO 8601 format

* LSIDs added for taxonomic names used in the metadata using the world register of marine species on 2025-02-18. Exact match to name used.

* Geospaital bounds added for organism source location in RI as indicated was the relevant location for this dataset. "Lab experiment at Carleton College in Northfild, MN conducted with organisms collected at Black Point in Narragansett Bay, RI "
41° 24′ 4.4964″ N, 71° 27′ 43.8228″ W converted to decimal degrees for entry: 41.401249,-71.462173

NSF Award Abstract:
The project investigates how the metabolic activity of dense aggregations of marine organisms alter the water chemistry of their interstitial spaces, and how these microscale alterations feedback to affect the organisms’ interactions in coastal ecosystems. The research team focuses on bivalve mussels, foundation species that form dense ‘beds’ typically known for facilitating other species by ameliorating harsh flow conditions. This ability can become a liability, however, if flow is not sufficient to flush the interstitial spaces and steep, metabolically-driven concentration gradients develop. The research evaluates whether corrosive chemical microclimates (such as low oxygen or low pH) are most extreme in low flow, high temperature conditions, especially for dense aggregations of mussels with large biomass and/or high respiration rates, and if they negatively impact mussel beds and the diverse biological communities they support. The research addresses a global societal concern, the impact of anthropogenic climate change on coastal marine ecosystems, and has potential applications to aquaculture and biofouling industries by informing adaptation strategies to “future-proof” mussel farms in the face of climate change and improved antifouling practices for ships, moorings, and industrial cooling systems. The project forges new collaborations with investigators from three campuses and integrates research and education through interdisciplinary training of a diverse group of graduate, undergraduate and high school students. STEM education and environmental stewardship is promoted by the development of a K-12 level science curriculum module and a hand’s-on public exhibit of bivalve biology at a local shellfish farm. Research findings are disseminated in a variety of forums, including peer-reviewed scientific publications and research presentations at regional, national and international meetings.

The research team develops a framework that links environmental conditions measured at a coarse scale (100m-100km; e.g., most environmental observatories) and ecological processes at the organismal scale (1 cm – 10 m). Specifically, the project investigates how aggregations of foundation species impact flow through interstitial spaces, and how this ultimately impacts water chemistry immediately adjacent to the organisms. The research focuses on mytilid mussels, with the expectation that the aggregation alters the flow and chemical transport in two ways, one by creating a physical resistance, which reduces the exchange, and the other by enhancing the exchange due to their incurrent/excurrent pumping. These metabolically-driven feedbacks are expected to be strongest in densely packed, high biomass aggregations and under certain ambient environmental conditions, namely low flow and elevated temperature, and can lead to a range of negative ecological impacts that could not be predicted directly from coarse scale measures of ambient seawater chemistry or temperature. The team develops computational fluid dynamic (CFD) models to predict interstitial flows and concentration gradients of dissolved oxygen and pH within mussel beds. The CFD model incorporates mussel behavior and physiological activity (filtration, gaping, respiration) based on published values as well as new empirical work. Model predictions are compared to flow and concentration gradients measured in mussel aggregations in the laboratory and field. Finally, the team conducts several short-term experiments to quantify some of the potential negative ecological impacts of corrosive interstitial water chemistry on mussel aggregations, such as reduced growth, increased dislodgement, increased predation risk, and reduced biodiversity. Because the model is based on fluid dynamic principles and functional traits, the framework is readily adaptable to other species that form dense assemblages, thereby providing a useful tool for predicting the ability of foundation species to persist and provide desirable ecosystem services under current and future multidimensional climate scenarios.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.