In this study, the investigators determined how respiration by newly hatched larvae of the porcelain crab (Petrolisthes cinctipes) cultured under current (385 uatm) and future predicted (1000 uatm) levels of atmospheric pCO2 differed when exposed to low, normal, and high salinity conditions.

Detailed methodology and results are described in following publication:
Miller, S.H., S. Zarate, E.H. Smith, B. Gaylord, J.D. Hosfelt, and T.M. Hill. 2014. Effect of elevated pCO2 on metabolic responses of porcelain crab (Petrolisthes cinctipes) larvae exposed to subsequent salinity stress. PLoS One 9: e109167, doi:10.1371/journal.pone.0109167

Briefly (excerpted from above):
Fifty ovigerous Petrolisthes cinctipes were collected from Shell Beach (38 degrees 25.033' N, 123 degrees 06.350' W) and Twin Coves (38 degrees 27.490' N, 123 degrees 08.621' W), California. Crabs were held in individual mesh containers in flow-through seawater at Bodega Marine Laboratory for 2–14 days. Containers were checked daily for hatched larvae, and newly released individuals from multiple broods were transferred in mixed batches of 75 to replicate culture jars. These jars contained 0.45 um filtered seawater bubbled with prescribed pCO2 levels to maintain carbonate chemistry and water movement, and were held at a constant temperature and normal salinity. Not all females released larvae on the same night, which allowed culture start dates to be staggered and respirometry trials to be run on sequential days using larvae of the same age.

Larvae were cultured at 14 degrees C under two pCO2 conditions: 385 uatm (ambient) and 1000 uatm (elevated). Larvae were held at a density of 75 larvae per 3 L, and were fed newly hatched Artemia sp. nauplii at a density of 1 nauplius/ml. The water in each culture jar was changed every two days. During water changes, dead larvae and uneaten Artemia sp. were removed, and water exiting the jars was sampled for carbonate chemistry adhering to recommended practices. The investigators measured pH (total scale) using a glass electrode (Accumet Excel XL60; Thermo Fisher Scientific, Waltham, Massachusetts, USA) and repeated analysis of certified TRIS reference material (measured in millivolts; batch no. 8; A. Dickson, Scripps Institute of Oceanography). Total alkalinity (TA) was measured by autotitration (Metrohm 809; Metrohm, Herisau, Switzerland), using the same certified reference material. Dissolved inorganic carbon (DIC) samples were processed using coulometric titration at the Monterey Bay Aquarium Research Institute (MBARI).

Respirometry trials were conducted in seawater at ambient pCO2 using a dissolved oxygen meter (model 781b Strathkelvin Instruments Ltd., Glasgow, UK) with a Clark-type microcathode polarographic electrode with a 22 micron diameter platinum cathode and silver/silver chloride anode connected by a buffered potassium chloride electrolyte solution (model 1302). Data were transmitted via a data interface unit to a computer and the oxygen consumption rates were monitored using the Strathkelvin 949 Oxygen System (Version 2.2, Strathkelvin Instruments Ltd., Glasgow, UK).

The electrode was placed in a PVC holder with double O rings to seal the chamber. The holder and electrode were inserted into a test chamber (26.34 mm x 47.09 mm ID) holding 25 ml of test solution. The test chamber was maintained at a temperature of 14 degrees C. For each new trial, the electrode was calibrated using 2% sodium sulfite and air-saturated seawater to determine zero and saturated oxygen values, respectively. Before each series of experiments, a control chamber with no larvae was monitored to establish background microbial oxygen consumption rates, which were then subtracted from the values obtained during trial runs. During each trial, eight first-stage larvae were removed from the culturing system and placed in the chamber for respirometry analysis, and visual analysis indicated that their activity was sufficient to maintain flow over the surface of the electrode. Seawater oxygen saturation state was measured every two minutes for one hour, though the investigators only analyzed data from the start of the trial until the oxygen in the test chamber reached 5 mg/L to eliminate potential effects of hypoxia in the chamber on larval respiration. Trials with larvae from each pCO2 treatment were run sequentially in pairs (i.e., one trial with larvae from ambient pCO2 conditions was followed by one trial with larvae from elevated pCO2 conditions), and 13 pairs of trials were run at salinities of 34 over a period of 14 days.

Distilled water was added to filtered, ambient-pCO2 seawater (salinity 34) to make salinity 22 solution, and artificial seawater (Instant Ocean, Spectrum Brands, Inc.) was added to make salinity 40 solution. This procedure created two altered salinity treatments for testing effects of salinity stress on larvae drawn from each of the preceding pCO2 treatments. Larvae were allowed to acclimatize in treatment solutions for ten minutes prior to respirometry trials to attenuate a startle response, and the system was calibrated before each trial. Paired trials (4 pairs at salinity 22 and 3 pairs at salinity 40) with larvae from each pCO2 treatment were run sequentially over a period of seven days.

crab_treatment.csv