Dataset: Mussel gene expression

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 and Mytilus californianus (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
"Mytilus californianus", California mussel,  urn:lsid:marinespecies.org:taxname:367837