Abstract:
Hatchery-reared adult and juvenile oysters were placed at 0.1 m and 1.0 m above bottom at Sand Reef and Denton Reef, Mobile Bay, Alabama between May 26 and Sep 21, 2010, along with YSI sondes to measure temperature, salinity, and dissolved oxygen concentration. Oysters were sampled every two weeks for growth rate and protein analysis of heat shock proteins (Hsp 70).
Suggested Citation:
Patterson, Heather, Anne Boettcher, and Ruth H. Carmichael. 2015. Growth Rate and Heat Shock Protein Data from Oysters Under Low Dissolved Oxygen Stress. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7WS8R5S
Purpose:
The frequency and intensity of anoxic and hypoxic events are increasing worldwide, creating stress on the organisms that inhabit affected waters. To understand the effects of low dissolved oxygen stress on oysters, hatchery-reared oysters were placed in cages and deployed along with continuously recording environmental data sondes at a reef site in Mobile Bay, AL that typically experiences low oxygen conditions. Test deployments of oysters to assess expression of HSP 70 relative to environmental conditions will be useful, in addition to measuring abiotic factors, to identify appropriate sites for restoration, particularly to capture negative effects of habitat quality on biota before lethal impacts are incurred.
Data Parameters and Units:
GPS (lat., long.), Site (name), Growth Rate (millimeters per day), Temperature (degrees C), Salinity (average), Dissolved Oxygen (milligrams per liter for the day, max. and min.), Date, Hsp 70, Depth (0.1 and 1.0 meter below the surface) Oysters_DO-Biomarkers_1: GPS: A spreadsheet containing location information of oyster deployments. Site: Name of an oyster deployment location. DB pole1: Denton Reef, 0.1 meter above the sediment surface (bottom), one of two existing pilings. DB pole 2: Denton Reef, 0.1 meter above the sediment surface (bottom), other of two existing pilings. ST pole 1: Sand Reef, 1.0 meter above the sediment surface (top), one of two existing pilings. ST pole 2: Sand Reef, 1.0 meter above the sediment surface (top), other of two existing pilings. SB pole 1: Sand Reef, 0.1 meter above the sediment surface (bottom), one of two existing pilings. SB pole 2: Sand Reef, 0.1 meter above the sediment surface (bottom), other of two existing pilings. DT pole 1: Denton Reef, 1.0 meter above the sediment surface (top), one of two existing pilings. DT pole 2: Denton Reef, 1.0 meter above the sediment surface (top), other of two existing pilings. GPS N: Latitude of oyster deployment location in degrees and decimal minutes north of the equator. Oysters_DO-Biomarkers_1: Growth: A spreadsheet containing growth rates of oysters deployed at Sand Reef. Growth was measured every two weeks over 118 days. Site: Place where measured oysters were deployed. SB: Sand Reef, 0.1 meter above the sediment surface (bottom). ST: Sand Reef, 1.0 meter above the sediment surface (top). Age: Maturity of the measured oysters. Adult or Juvenile (Juvy) Date: Date of oyster collection from the field for growth measurement, mortality assessment, dry weight and protein analysis. Day: Day since the start of deployment that oysters were collected from the field for growth measurement, mortality assessment, dry weight and protein analysis. Avg: Daily mean growth rate in millimeters per day (N = up to 15). Sterr: Standard error of growth rate in millimeters per day (N = up to 15). Oysters_DO-Biomarkers_1: YSI: Temperature, salinity, and dissolved oxygen data from YSI sondes deployed at Sand Reef at the same time as oysters were deployed. Data are contained in two spreadsheets: Sand Bottom for data at 0.1 meter above the sediment surface, and Sand Top for data at 1.0 meter above the sediment surface. DO Biomarkers - Sand Bottom, Sand Top Date: Date of measurement. Day: Day since the beginning of deployment. Temp: Avg: Average temperature in Celsius for the day. Temperature was measured at 15 minute intervals. Temp: sterr: Standard error for temperature in Celsius. Salinity: Avg: Average salinity for the day. Salinity was measured at 15 minute intervals. Salinity: sterr: Standard error for salinity. DO: Avg: Average dissolved oxygen concentration in milligrams per liter for the day. Dissolved oxygen was measured at 15 minute intervals. DO: sterr: Standard error for dissolved oxygen concentration in milligrams per liter. DO: Max: Maximum dissolved oxygen concentration in milligrams per liter for the day. Dissolved oxygen was measured at 15 minute intervals. DO: Min: Minimum dissolved oxygen concentration in milligrams per liter for the day. Dissolved oxygen was measured at 15 minute intervals. Oysters_DO-Biomarkers_2: Hsp70: A series of spreadsheets containing heat shock protein data from gill, mantle, and adductor muscle tissue from juvenile and adult oysters deployed at Sand Reef. The spreadsheet "For Fig 6" refers to Denton Reef data used in Figure 6 in Patterson HK, Boettcher A, Carmichael RH (2014) Biomarkers of Dissolved Oxygen Stress in Oysters: A Tool for Restoration and Management Efforts. PLoS ONE 9(8): e104440. doi:10.1371/journal.pone.0104440. Site, Name: Name of site where oysters were deployed. Sand: Sand Reef Denton: Denton Reef Depth: Location in the water column where oysters were deployed. Bottom: 0.1 meter above sediment surface Top: 1.0 meter above sediment surface Band: Isoform of Hsp70, as a gel electrophoresis band. Hsp 69, Hsp 72, Hsp 77 Date: Date of oyster collection from the field for growth measurement, mortality assessment, dry weight and protein analysis. Rel. Intensity: Relative intensity of gel electrophoresis band to bands for actin (loading control) and anoxic treated mantle (internal standard). 2 week avg DO: Average dissolved oxygen concentration in milligrams per liter over the two week period before oyster collection.
Methods:
We deployed caged oysters at fixed heights (0.1 m (bottom) and 1.0 m (top)) above the bottom on two existing pilings at Sand Reef and Denton Reef. To capture ontogenic variation in response to low DO conditions, 50 juvenile and 45 adult hatchery-reared oysters were placed in three replicate aquaculture cages (30 cm wide x 640 cm long x 610 cm deep). Cages were cleaned and oyster survival was noted every two weeks, at which time, up to 15 individuals of each age group were collected for growth measurement, mortality assessment, dry weight and protein analysis. To test cumulative effects, oysters were deployed for 87 days, thereby allowing the oysters to be exposed to several periods of low DO concentrations. YSI 6600 data sondes were deployed to take measurements every 15 minutes, creating a 5 minute average of temperature, salinity, and DO. Sondes were deployed at the two cage depths, 1.0 m (top) and 0.1 m (bottom) off the bottom. Sondes were cleaned and calibrated ,biweekly, using the manufacturer�s standard methods for YSI 6-Series Sondes, one point saturated air method. Gill, mantle and adductor muscle tissues were dissected from sampled oysters, frozen in liquid nitrogen, and stored at -80 degrees C until analysis. Tissues were extracted in lithium dodecyl sulfate (LiDS) sample buffer (1:4 w:v) (125 mM Tris pH 6.8, 10% glycerol, 2% LiDS); samples were centrifuged at 14,000 g for 10 minutes after which the supernatant was collected and re-centrifuged for another 10 minutes. Total protein concentration was determined using the BioRad DC assay (BioRad, Hercules, CA, USA) with bovine serum albumin standards. Proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Samples of 100 mg total protein concentration were run on 8% gels at 100 V for 2.5 hours. Proteins were transferred to a nitrocellulose membrane (Amersham Hybond-ECL, GE Healthcare) at 100 V for 1 hour. Membranes were blocked for 1 hour with 50/50 Odyssey Buffer (LiCOR) and PBS (pH 7.4). Membranes were analyzed by immunoblotting with anti-HSP 70 antibodies (1:10,000, Sigma St Louis, MO), HIF (created by Thermo Fisher from Genbank sequence HM441076, 1:500 Waltham, MA) and p38 MAP kinase (1:1,000, Cell Signaling Beverly, MA). Actin (1:10,000, Neo Markers Fremont, CA) was used as a loading control, and an internal standard (anoxic treated mantle - mantle from oysters exposed to 4 days of anoxia) was run on all gels to allow between gel comparisons. Membranes were probed with GAR (1:4,000) and GAM (1:20,000) fluorescent secondary antibodies (LiCOR). Blots were viewed under infrared fluorescence (LiCOR, Lincoln, NE) and images analyzed with a LICOR Odyssey Imager and associated software (LiCOR, Lincoln, NE). Bands were quantified by drawing a box around and adjusted to the loading control (actin) and an internal standard (anoxic treated mantle). Band intensity is expressed as relative intensity.
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