Lab study on the effect of temperature and pCO2 on mussel byssal attachment (thread number) with mussels collected in May 2012 from Argyle Creek, San Juan Island, WA (48.52 ̊ N, 123.01 ̊ W)

These data were used in a structural analysis study to evaluate how pCO2 and an additional stressor, elevated temperature, influences byssal thread quality and production. Mussels (M. trossulus) were collected in May 2012 from Argyle Creek, San Juan Island, WA (48.52 ̊ N, 123.01 ̊ W) and held in a mesh box submerged under the dock at Friday Harbor Laboratories (FHL), San Juan Island, WA for up to 14 d. Mussels were placed in controlled temperature and pCO2 treatments in the Ocean Acidification Experimental Laboratory (OAEL), then newly produced threads were counted and pulled to failure to determine byssus strength.

Mussels were acclimated to their treatment temperatures in ambient pH (~7.8) over 9 d, ramping temperature up no more than 2˚C per day, and fed a maintenance level of Shellfish Diet 1800 (6 g l-1 day-1, Reed Mariculture, Campell, CA, USA).
The twelve independent temperature x pCO2 treatments spanned the range of local marine conditions (Newcomb, 2015;George et al., 2019;temperature at 10˚C, 18˚C, or 25˚C andpCO2 at 400, 750, 1200, or 2500 µatm). Each mussel was trimmed of external byssus before placement in an experimental treatment for 3 d, sufficient time to produce new mature byssal threads (Bell & Gosline 1996) while minimizing the effect of treatment on mussel condition. Mussels were starved during the 3 d trials to minimize changes in chamber water chemistry due to food addition and to reduce fouling. Three trials were conducted in succession to replicate treatments over time, increasing sample size (n=8 x 3) for each temperature*pCO2 treatment.
At the end of each trial, mussels and the rocks to which they had attached with byssal threads were removed from the chambers. The entire byssus was dissected from each mussel and stored air-dried for up to 20 days. Byssus was rehydrated in seawater prior to testing, a method that does not alter the mechanical properties of the byssal threads (Brazee, 2004). The number of byssal threads each mussel produced was counted, and one thread was haphazardly chosen for mechanical testing following the procedure of Bell & Gosline (1996). Briefly, an individual thread was clamped with submersible pneumatic grips on either end by holding the proximal byssal stem between cardstock with cyanoacrylate glue and affixing the distal plaque with attached rock to an aluminum T-bar with epoxy. An Instron 5565 tensometer (Norwood MA, USA), extended the thread at a rate of 10 mm min-1 in a temperature-controlled water bath (3130-100 BioPuls Bath, Instron, Norwood, MA, USA) until failure. The tensometer measured force (±10-3 N) and extension (± 10-3 mm) at 10 Hz. Tests were performed in seawater with a pH of 7.8 and the relevant treatment temperature.
Pull to failure mechanical tests provided estimates of thread breaking force, yield force, extensibility, initial stiffness and failure location (Bell & Gosline 1996). Yield, due to quasiplastic deformation in the distal region, was identified as the point where the initial slope of the force-extension curve decreased by 40%. Extensibility was calculated by dividing thread extension at failure by initial length and initial stiffness was determined from the initial slope of the force extension curve. The location of failure (proximal, plaque, and/or distal region) was noted and threads were retested to quantify the breaking force of each remaining region. Tests that broke at the grips were considered underestimates and were discarded.
The cross-sectional area of the proximal region was measured to evaluate morphological differences among treatments. The elliptical area was estimated from measures of the major and minor axes (+ 1 um using a dissecting microscope (Brazee & Carrington 2006). Proximal breaking stress (N mm-2), a material property, was calculated as proximal breaking force divided by proximal area. Thread surface structure was examined using a scanning electron microscope (FEI Sirion XL30 SEM, Hillsboro, OR).
Whole mussel attachment strength was estimated using two mathematical models developed by Bell & Gosline (1996). Each model assumes a mussel is anchored with a constant thread number (n=50) arranged in a circle. The normal model estimates dislodgment force perpendicular to the substrate (e.g, lift); all threads are engaged and extend until they reach their maximum force. The parallel model estimates dislodgement force for an animal pulled parallel to the substrate (e.g., drag); threads on the upstream side are the first in tension, yield and extend until they reach maximum force and break, while more threads are recruited into tension until they have all broken. Additionally, we modified each model to incorporate the variation in thread production across treatments. Because thread production was measured for only three days, treatment means were scaled to a maximum value of 50 threads.

Instrument Description
Testing systems that are used to test a wide range of materials in tension or compression.
[ The overall focus of the project is to determine how ocean acidification affects the integrity of biomaterials and how these effects in turn alter interactions among members of marine communities. The research plan emphasizes an ecomaterial approach; a team of biomaterials and ecomechanics experts will apply their unique perspective to detail how different combinations of environmental conditions affect the structural integrity and ecological performance of organisms. The study targets a diversity of ecologically important taxa, including bivalves, snails, crustaceans, and seaweeds, thereby providing insight into the range of possible biological responses to future changes in climate conditions. The proposal will enhance our understanding of the ecological consequences of climate change, a significant societal problem. Each of the study systems has broader impacts in fields beyond ecomechanics. Engineers are particularly interested in biomaterials and in each system there are materials with commercial potential. The project will integrate research and education by supporting doctoral student dissertation research, providing undergraduate research opportunities via three training programs at FHL, and summer internships for talented high school students, recruited from the FHL Science Outreach Program. The participation of underrepresented groups will be broadened by actively recruiting URM and female students.
Results will be disseminated in a variety of forums, including peer-reviewed scientific publications, undergraduate and graduate course material, service learning activities in K-8 classrooms, demonstrations at FHL's annual Open House, and columns for a popular science magazine. [