Dataset: Oyster Extrapallial Fluid

Prior to the start of the experiment, extrapallial fluid extraction ports were inserted into the shells of all oysters. To do this, a 3-mm hole was drilled into the right valve of each oyster. This hole was drilled approximately 1 cm from the umbo along the central band. Drill sites were flushed with seawater during drilling to minimize friction and overheating. A lear-lock was sealed into place using epoxy, and capped to create an extraction port. Following this procedure, oysters were returned to their sea tables for at least 7 days prior to being assigned to an experimental tank.

Extrapallial fluid was extracted immediately prior to tissue sampling. Oysters were removed from their tanks and patted dry before extrapallial fluid pH was measured. The cap was removed from the port, and all extrapallial fluid was extracted using a syringe. The port was then re-sealed and oysters were returned to their treatment tanks.

Extrapallial fluid pH was immediately measured following extraction using an Orion 91'10DJWP Double Junction Micro pH probe standardized with pH 7 and 10 NBS buffers. Extrapallial fluid samples were stored and refrigerated in 2 mL microcentrifuge tubes for later analysis of DIC. DIC was analyzed on a subset of individuals using an Apollo SciTech AS-C2 dissolved inorganic carbon analyzer standardized against a Dickson CRM. The salinity of the extrapallial fluid was measured using a Mettler Toledo InLab Expert Pro-ISM conductivity probe standardized against a Dickson CRM.

Twenty nine oysters were excluded from tissue sampling and set aside for measurements of respiration rate and EPF chemistry at the end of the experimental period. Respiration rate was measured by placing oysters in sealed air-tight containers (height = 70 mm, diameter = 130 mm, volume = 1100 mL) filled with air-equilibrated seawater. A PreSens SP-PSt-NAU oxygen sensor dot was glued to the inside of the lid of each container to record oxygen concentration during measurements. An egg-crate platform inside each container separated the oyster from a stir-bar. Containers were sealed underwater to remove air bubbles. Containers were placed in a temperature-controlled water bath held at treatment temperature. This water bath was placed on top of six stir plates, so that six replicate measurements could be done simultaneously (5 oysters, and one negative control containing just seawater). Stir plates were set so that stir bars rotated at a speed of 136 - 150 resolutions per minute. Oysters were left for an acclimation time of 15 minutes, after which an air saturation reading was obtained every 15 minutes using a PreSens Fibox 4 oxygen meter. Three readings were obtained per container at each time point and an average value was taken. Respiration measurements were conducted until oxygen percentage had declined from 15% of the initial oxygen content recorded.

To understand rates of EPF recharge, the EPF was re-extracted from these 29 oysters at one, two, four, eight, twelve and twenty-four hour intervals. The order of these time periods was randomized to minimize the coupled effects of time-period and any sequential stress caused by re-extracting the EPF.

Problem Report: DIC could not be measured for all oysters, as measurement requires at least 0.5 mL, and some oysters did not contain enough extrapallial fluid.