Dataset: Mussel Adhesive Plaques - pH and oxygen fluctuations

Adult mussels (Mytilus trossulus, Gould 1850) were gathered from the top of aquaculture rope lines at the Penn Cove Shellfish hatchery, Quilcene Bay, Quilcene, Washington, USA (47°47’48.0” N, 122°51”16.6” W) during the winter of 2015 (November 2015 to February 2016), transported on ice to the laboratory, and kept in 50 L aquaria. Aquaria typically contained 20-30 mussels and were filled with 0.2 µm filtered, UV-sterilized seawater, with constant aeration. Mussels were in the laboratory for no longer than three weeks and were fed Shellfish Diet 1800 (Reed Mariculture, Campbell, CA) up to 5% of their wet tissue mass day-1, dispensed at a concentration of 2000 algal cells ml-1, a diet that has been shown to maintain body weight for up to one month (unpublished data). After a week of acclimation, mussels either produced threads that were included in plaque-curing experiments or the animal itself was included in a thread production assay.

Byssal threads were collected in the laboratory by securing mussels to mica plates with rubber bands, orienting the valve opening towards the substrate and allowing them to attach under seawater conditions that mimicked those found in the open-ocean (pH ca. 8, O2 ca. 8.5 mg L-1, Sal ca. 30, T ca. 9°C). After four hours, threads were separated from each animal at the shell margin by cutting the proximal region of each thread, preserving the attachment with each plate. Plates with attached threads were then incubated in seawater treatments, using only plates from mussels that made three or more attachments. After incubation, plates were removed from seawater, dried, and stored for up to two weeks before mechanical testing was performed.

To determine whether rare, extreme excursions in pH and dissolved oxygen can directly affect the plaque-curing process, plaques were aged to maturity in fluctuating seawater treatments that mimicked the magnitude and duration of the ‘worst-case’ scenario, as defined by the most extreme excursion observed in field measurements (pH <5.0 or O2 <2 mg L-1, for 5 days). Mica plates with freshly attached (ca. 4 hrs after deposition) threads were haphazardly assigned to one of five experimental treatments, controlled using the pH and oxygen-stat system previously described. Threads aged in the first two experiments experienced constant dissolved oxygen (ca. 8 mg L-1), temperature (ca. 9ᵒC), and salinity conditions (ca. 29), while also being subjected to an excursion in seawater pH (pH ca. 5.0) after either 1 (Exp2) or 8 (Exp3) days. The second two experiments mimicked the conditions of the first, except that seawater pH was maintained at ca. 8.0 throughout and threads were exposed to hypoxia excursions (O2 <2 mg L-1) either at 1 (Exp4) or 8 (exp5) days into the experiment. pH and oxygen excursions were maintained for 5 days, after which conditions returned to a baseline that represented open-ocean conditions (pH ca. 8.0, O2 ca. 8.5, T ca. 9ᵒC, Sal ca. 29). A subset of plates was removed within each experiment after either 3, 5, 8, 12, or 20 days and stored dry for up to two weeks before mechanical testing was performed. A control treatment (Exp1) wherein open-ocean conditions were maintained for 20 days was also performed with the same sampling regime.

Byssal thread production during acidification and hypoxia excursions was investigated by placing mussels secured to mica plates in one of five pH treatments (Exp6; pH target = 5.0, 6.0, 7.0, 7.5, or 8.0) or one of two dissolved oxygen treatments (Exp7; O2 target = <2.0 or >8.0 mg L-1) for seven days. pH treatments were maintained using a pH-stat system similar to the one described in O’Donnell et al. (2013). Briefly, seawater pH (NBS) and temperature (°C) were measured with a Honeywell Durafet III pH electrode and monitored with a Honeywell UDA2182 analyzer that controlled the operation of a solenoid valve. The solenoid value regulated the flow of CO2 into the aerator of each tank. Using a PID loop, the analyzer tailored a CO2:air mixture by controlling the proportional operation of the valve, using pH as the response variable. Dissolved oxygen treatments were accomplished in a similar way by equipping the analyzer with a Honeywell DL5000 equilibrium oxygen probe (accuracy ± 0.1) and replacing the CO2 cylinder with N2 gas. The salinity in each treatment was monitored with a Honeywell DL4000 conductivity cell (accuracy ± 1), which was also monitored by the analyzer. pH, oxygen, temperature, and salinity were logged every 10 minutes using a 4-20 mA data logger. Any pre-existing byssal threads were removed from each mussel, by cutting threads in the proximal region at the shell margin, prior to being placed in a treatment. Once in a treatment, a subset of mussels (ca. 20) were removed at 1, 3, 5, and 7 days, counting the number of new threads each mussel produced.

Plaque attachment strength was determined by gripping the distal region of each byssal thread and pulling perpendicular (90ᵒ) to the substrate until failure, following the protocol of George & Carrington (2018). This testing angle was chosen for  its reproducibility;  it should be noted that the contact angle of the thread with the plaque varies and threads are rarely brought into tension fully perpendicular to the substrate (Desmond et al. 2015). Plaques were rehydrated in their respective seawater treatments prior to mechanical testing for more than 5 minutes. The thread distal region was gripped with a hemostat ca. 1 mm above the plaque-thread junction, and force was recorded using a 10 N digital force gauge (OMEGA, Stamford, CT, USA; accuracy ± 0.01 N) attached to a motor-driven testing frame. Threads were pulled at an extension of 10 mm min-1 until plaque failure (the distal region is much stronger than the plaque; Bell & Gosline 1996) and force (N) were recorded at 20 Hz. The adhesion strength (kPa) of each plaque was determined by normalizing the maximum force required to dislodge each plaque by the attachment planform area (mm2), measured by tracing the outline of each plaque from above using a dissection scope with accompanying AmScope MU1000 camera (Irvine, CA, USA) and AmScope X imaging software prior to testing (Burkett et al. 2009). The mean adhesion strength of 3-5 plaques is reported for each mussel.

In an effort to link observed differences in plaque adhesion with the failure mechanics of the adhesive, the failure mode of each plaque was also scored visually during mechanical testing following Young & Crisp (1982) as outlined in George & Carrington (2018). Briefly, plaques were binned within three failure types: adhesive, peeling, or tearing. In the case of adhesive failure, plaques detached from the substrate in a single, swift, plunger like motion. Peeling failure was characterized by a detachment beginning at a location along the perimeter of the plaque, propagating from one side of the structure to the other. Tearing failure was evident when a portion of the plaque remained attached to the substrate after the test was completed, or the thread became dislodged from the attachment plaque at the thread-plaque junction.

Detailed methods and results are provided in George et al. (in press).