Lab study on the effect of dissolved oxygen, salinity, and temperature on mussel adhesive plaques with mussels collected from Penn Cove Shellfish in Coupeville, Washington from 2015-12-01 to 2016-02-29 (NCEI Accession 0291525)

Acquisition Description:
Adult mussels (Mytilus trossulus, Gould 1850; ~4-6 cm shell length) were collected from aquaculture lines at Penn Cove Shellfish’s mussel aquaculture operation located in Quilcene Bay, Quilcene, Washington, USA (47°47’42.0” N, 122°51”10.8” W) during the winter of 2016 (December-February). Mussel were kept in 50 L aquaria for up to two weeks, filled with 0.2 µm filtered seawater and fed Shellfish Diet 1800 (Reed Mariculture, Campbell, CA) up to 5% of wet tissue mass day-1 at an algal concentration of 2000 cells ml-1. Mussels were allowed to attach to mica sheets over the course of 4 hours. Byssal threads were cut away from the animal at the proximal region’s interface with the shell. Threads from individuals that made less than three attachments were not included in a treatment group. Mica sheets with plaque attachments were stored dry at room temperature (~21°C, ~30-40% RH) for up to two weeks and then moved into treatment conditions.

Mussels were secured to mica plates with rubber bands and allowed to produce byssal threads for up to four hours in typical open-ocean seawater conditions (pH ~ 8.1, T ~ 10ᵒC, Sal ~ 31 PSU, O2 ~ 8 mg L-1), after which threads were cut away from the animal in the proximal region of the thread (at the shell margin). Only mussels that produced three or more attachments were included in a treatment group. A subset of threads was tested immediately, serving as a 4 hour, ‘freshly made’ control. The remainder of mica plates with attached threads were placed in one of four treatments (‘control’, ‘oxygen’, ‘temperature’, or ‘salinity’) and allowed to mature for 12 days, removing a subset of plates at 3, 5, 8, and 12 days in some cases.

Seawater treatments were designed to mimic open-ocean conditions in all ways but one, pushing either temperature, dissolved oxygen, or salinity to the most extreme values seen in estuarine systems that are metabolically driven by the local biota (Lowe et al. 2019). A hypoxia treatment (‘oxygen’; O2 <2 mg L-1) was achieved through the injection of N2 gas into a 3 L container, using an aerator. The dissolved oxygen concentration of seawater treatments was monitored in real-time with a DirectLine DL5000 equilibrium probe (accuracy ± 1%) attached to a UDA2182 analyzer (Honeywell, Fort Washington, PA), which controlled the injection of N2 by dynamically opening a solenoid valve in-line with a nitrogen gas cylinder. A high temperature treatment (‘temperature’; T = 30°C) was achieved using a 500-Watt titanium aquarium heater and accompanying PID controller (Aquatop Aquatic Supplies, Brea, CA). A low salinity treatment (‘salinity’; <1 PSU) was achieved by placing plaques in deionized water. Seawater pH and temperature were monitored in each treatment with a Honeywell Durafet III pH electrode (Martz et al. 2010; accuracy ± 0.01), while salinity was monitored with a DL4000 conductivity cell (accuracy ± 1 PSU).

The adhesion strength of individual plaques was determined by gripping each byssal thread in the distal region, 1 mm away from the adhesive plaque, and pulling perpendicular to the substrate until failure using a tensometer (George and Carrington, 2018). Adhesion strength (kPa) was calculated as the maximum of the force extension curve (N), normalized by the planform area of the attachment plaque measured in mm2 (Burkett et al. 2009). The adhesion strength for 3-5 plaques were averaged and reported as a single value for each mussel. During mechanical testing, the failure mode of each plaque was also visually scored as an adhesive, peeling, or tearing failure, as outlined by Young and Crisp (1982). Adhesive failure occurred when a plaque disengaged from a surface uniformly at the adhesive-substrate interface, while a peeling failure characteristically began at a single point along the outer edge of the plaque, propagating to the rest of the structure. A tearing failure was evident when a portion of the adhesive remained attached to the surface after the test had completed.

The stiffness of the plaque cuticle was determined by following the protocol outlined by George and Carrington (2018). Briefly, stiffness (DMT modulus) was measured using a Dimension ICON atomic force microscope (AFM), fitted with a ScanAsyst-Air probe with a silicon-nitride tip (Bruker, Billerica, MA). Prior to testing, plaques were rinsed with DI water and allowed to dry for 5 minutes. Efforts were taken to probe smooth patches away from the thread-plaque junction, avoiding the innervating roots of the thread. DMT modulus (GPa) was calculated as the slope of the force curve during tip-sample separation. To obtain a representative stiffness of the cuticle, DMT modulus was averaged over a 10 nm2 scan area, with a sampling rate of 512 per line. DMT Modulus was calibrated against a fused silica standard (Veeco, Plainview, NY). Multiple locations (3-5) were scanned for each plaque and then averaged.

In preparation for amino acid (AA) analysis, adhesive plaques were collected from three different seawater treatments (4 hours and 12 days in open-ocean conditions; 12 days in nitrogen infused seawater) and stored in nitrogen flushed microfuge tubes at -80ᵒC for up to 4 weeks. Acid hydrolysis was then performed in vacuo at 110°C for 48 hours in 6M HCl, with 5% phenol added to preserve DOPA residues. The hydrolysate of each plaque was flash evaporated against DI water and methanol, dissolving the precipitate in 0.02 M HCl. 100 µl of the mixture was then analyzed using an amino acid analyzer system based on ninhydrin-based chemistry (Hitachi L-8900; Tokyo, Japan). A typical spectrum obtained from the analyzer with identified peaks is presented in Figure 6a. The integral of each amino acid peak was divided by the integral of all peaks to determine the relative molar concentration of each amino acid, normalizing against a background of 0.02 M HCl and subtracting the ammonia peak.

Detailed methods and results are provided in George et al., 2018.