M. californianus Conrad, 1837 were obtained from Cattle Point on San Juan Island, WA (48°26'59.6811"N, 122°57'51.9358"W). M. trossulus and M. galloprovincialis were obtained from a commercial supplier (Penn Cove Shellfish, Coupeville, WA, USA; 48°13'06.5579" N, 122°42'25.4331"W). The mussels were maintained in seatables (66 × 135 × 32 cm) with a constant flow of seawater at the Friday Harbor Laboratories (48°32'45.7249" N, 123°00'46.9262"W). Mussels were fed twice daily with a total of approximately 10 mL of Shellfish Diet 1800 TM (Reed Mariculture, Campbell, CA, USA).
Aquatic respiration rates were measured in a closed, recirculating flow chamber of 1-liter capacity. The transparent acrylic test chamber (15 cm × 5 cm × 5 cm, L × W × H) was connected to a submersible pump (360 GPH, Model 25D, Rule Industries, Gloucester, MA, USA) via PVC pipe (25.4 mm i.d.) and custom machined high-density polyethylene connector fittings. The water-tight test chamber was filled with artificial seawater (32 PSU; Instant Ocean; Spectrum Brands, Blacksburg, VA) and submersed in a temperature-controlled water bath (50 L). The water bath's temperature was maintained using a programmable, recirculating water heater/chiller (±0.005°C; AP07R-20-A11B, Polyscience, Niles, IL, USA). Trials were run at water velocities were estimated along the centerline of the testing chamber by tracking the displacement of glass microbeads at each flow setting (mean particle diameter 9 μm, density 2.0 g cm−3; Potters Industries, Malvern, PA, USA).
Oxygen concentrations were measured with non-intrusive fiber-optic fluorescence-based optodes and sensor spots on the inner surface of the chamber (FireSting Pro; OXSP5 and TPSP5 spots, Pyroscience, Aachen, DEU). Samples were recorded at a rate of 1 Hz and drift of the O2 probe was negligible (e.g., <0.1% over 2 h at 20°C).
Each trial involved positioning individual mussels along the centerline of the chamber and subsequently measuring the organism's oxygen usage at stable temperatures of 5, 11, 17, 23, and 29°C. Trials were run for 2 h, ensuring that a stable rate of decline could be identified. After each trial, all soft tissues were dried for 72 hours at 60°C (Lindeberg/Blue M Vacuum Oven; ThermoScientific Inc., Waltham, MA, USA), and weighed using an analytical balance (0.001 g; PA153 Pioneer Analytical Balance; Ohaus Corp., Pine Brook, NJ, USA).
