Sampling and Analytical Methodology:
To investigate environmental effects on larval (prodissoconch) shell signatures, we manipulated temperature, salinity, and elemental concentration of the water surrounding developing oyster larvae. Individual tanks were set up with the following treatments: low (21°C) or high (26.5°C) temperature; low (12.5 ppt) or high (20 ppt) salinity; and ambient (no addition), mid spike (+16 ppb Mn/0.16 ppb Pb addition), or elevated spike (+32 ppb Mn/0.32 ppb Pb) in concentrations of aqueous Mn and Pb. Temperature and salinity treatments were selected based on representative high and low observations in Pamlico Sound at the time of the experiment (mid-September). Trace metal spikes were calculated to increase the ambient levels of Mn and Pb in seawater, as measured by Becker et al. (2005), by 400% and 800% for mid and elevated spike levels, respectively.
Three-day old C. virginica larvae were obtained from the University of Maryland’s Horn Point Laboratory in Cambridge, Maryland, USA. Upon arrival at the Institute of Marine sciences (IMS) in Morehead City, NC, larvae were divided equally into 2, 1.2 L aerated holding tanks filled with a 12.5 ppt seawater mix (ultrapure H2O added to filtered seawater from Bogue Sound, NC). Over the next 4 days, larvae were acclimatized, with one tank receiving a salinity increase of approximately 2 ppt per day, resulting in a final salinity of 20 ppt, while the other tank remained at 12.5 ppt.
After the acclimatization process was complete, larvae from both holding tanks, now 7 days old, were divided equally into 72 “larval homes”, with approximately 1.6 x 104 larvae per home (21.2 larvae cm-3). Larval homes were constructed from hollow PVC tubing capped on each side with nitex cloth, with a 30 μm mesh opening, to allow for the flow of water and food into the home, but prevent larvae from escaping. Homes were then placed into 24 aerated aquarium tanks (35 L), with 3 homes per tank. All tubing, PVC, air stones, and nitex were soaked in a HNO3 solution and then rinsed thoroughly with ultrapure H2O prior to its use in the experiment.
Temperatures were maintained at either high or low level by 150 W Aquatop aquarium heaters and salinity levels were established by mixing filtered seawater with ultrapure H2O until desired salinity was reached. Mn and Pb concentrations were spiked by the addition of 545 μl of Mn + 5.45 μl of Pb and 1090 μl of Mn + 10.90 μl of Pb, from1000 ppt Fisher Scientific reference standard solutions, for mid and elevated spike treatments, respectively. Water changes were conducted every other day by removing one-third (~12 L) of water from the tank and replacing it with a freshly made mix. Tanks with mid or elevated spike treatments were then re-spiked to maintain consistent trace element concentrations. Immediately following water changes, larvae were fed by depositing dilute Instant Algae Shellfish Diet 1800 (Reed Mariculture; Campbell, California, USA) into larval homes via syringe. Each treatment group was crossed, to produce a full factorial design with 12 total treatment combinations. The experiment ran for 7 days, until the larvae were 14 days old.
Dissolved oxygen, temperature and salinity were monitored daily with a HACH HQ40d dual input, multi-parameter portable water quality meter. Dissolved oxygen, pH, salinity and temperature measures remained consistent among the treatments throughout our laboratory experiments. Mean dissolved oxygen and pH were 8.68 ± 0.025 mg L-1 and 7.72 ± 0.032, respectively. Mean salinity for high and low salinity treatments were 20.7 ±0.091 ppt and 12.8± 0.120 ppt, respectively. Mean temperature for high temperature treatments was 25.7 ± 0.157 °C and 21.3 ±0.104 °C for low temperature treatments.
At the conclusion of these mesocosm incubations, larvae from each home were filtered using nitex cloth (30 μm) and then resuspended in 15 mL of water from their respective tank. A 0.5-1 mL subsample of each larval resuspension was removed and the number of whole larvae were counted. The remaining larval solution was then frozen at -23°C until sample preparation for geochemical analysis.
Frozen larvae were thawed and approximately 1000 larvae were obtained representing each replicate home. The larvae were then rinsed with ultrapure H2O and mounted as a concentrated mass on a labeled glass microscope side covered in double-sided tape. This process continued until the contents of each larval home was mounted on a slide in haphazard order (total N=72). The slides were left to dry overnight in a laminar flow hood and then stored under the hood until analysis.
Samples were analyzed using a Thermo-Fisher Element2 inductively coupled plasma mass spectrometer with a Teledyne ATLex 300si-x 193nm Excimer laser ablation unit (LA ICP-MS). To correct for mass bias and instrument drift, National Institute of Technology Standards-certified standards (Reference Material 612, 614, and 616) were run at the beginning and end of every 4 slide sequence (~140 burns). Concentrations of the following elements were quantified from laboratory larval samples: 48Ca, 55Mn, 88Sr, 138Ba, and 208Pb; and from field-collected spat: 26Mg, 48Ca, 55Mn, 63Cu, 88Sr, 118Sn, 138Ba, and 208Pb. These elements were all analyzed in low-resolution mode, and were chosen because of their previous use in uptake and tagging studies of fish otoliths and bivalve shells (Bath Martin & Thorrold 2005; Strasser et al. 2008a,b; Fodrie et al. 2011).
Larval slide-mounts from the laboratory experiment were ablated in bulk using a line transect of 150 μm with 40 μm spot size and 80% laser intensity. Isotope intensities were converted into elemental ratios (X:Ca) following Becker et al. (2007).
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