Abstract:
Past studies in several species of teleost fish have predicted cholesterol biosynthetic pathways are likely impacted by crude oil exposure. The goal of this project is to determine if crude oil exposure impacts gene expression in this pathway and quantify changes in total cholesterol in the exposed red drum (Sciaenops ocellatus) larvae. In this experiment we utilize Enzyme Linked Absorbance Assays, Real-time PCR, and Immunohistochemistry techniques. The generated dataset includes: Assay results reported as total cholesterol normalized to total protein for each sample; relative change in gene expression; immunohistochemistry staining measurements along with raw images; Total PAH in water samples analysis. The dataset consists of 2 main folders. The folder named “Reddrum_Cholesterol_Version2_Updated_2021_10_1” is the updated and final version.
This dataset supports the publication: McGruer, V., Khursigara, A. J., Magnuson, J. T., Esbaugh, A. J., Greer, J. B., & Schlenk, D. (2021). Exposure to Deepwater Horizon crude oil increases free cholesterol in larval red drum (Sciaenops ocellatus). Aquatic Toxicology, 241, 105988. https://doi.org/10.1016/j.aquatox.2021.105988.
Suggested Citation:
McGruer, Victoria and Daniel Schlenk. 2020. Impacts of oil exposure on cholesterol synthesis in embryonic red drum (Sciaenops ocellatus). Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/BCA0N51T
Data Parameters and Units:
Data is organized by the different data types obtained from High Energy Water Accommodated Fraction (HEWAF) Exposures to Red drum (Sciaenops ocellatus) embryos. The following abbreviations are used for the file and folders.
Gene_expression = gene expression measured in 72 hours post fertilization (hpf) larval homogenates
Cholesterol_RD = Total cholesterol normalized to total protein in 72 hours post fertilization (hpf) larval homogenates
PericardialArea = Pericardial area measured in 72 hours post fertilization (hpf) larvae
Survival_RD = Survival of 72 hours post fertilization (hpf) larval following treatment
Water_Quality_RD = Water Quality measurements recorded throughout treatments
Water_chemistry2017 = Summary of individual PAH measurements in water samples taken at the beginning of the exposure initiated on 10/24/2017
Water_chemistry2018 = Summary of individual PAH measurements in water samples taken at the beginning of the exposure initiated on 11/13/2018
Water_chemistry2019 = Summary of individual PAH measurements in water samples taken at the beginning of the exposure initiated on 07/17/2019
2020_RedDrum_FilipinStaining = raw and summarized data from image analysis of filipin stained 72 hpf red drum larvae
Below are short descriptions of the data in each folder or file.
Title meanings & acronym definitions:
Cholesterol_RD = Total cholesterol levels normalized to total protein in whole 72 hours post fertilization (hpf) larval homogenates after HEWAF exposure
--Treatment (sumPAH50) = Describes the treatment for each exposure group. Embryos were exposed to either a seawater control or a percent oil from the surface (OFS) HEWAF. OFS HEWAF exposures are reported as the percent OFS and sumPAH50 in micrograms per liter (µg/L). sumPAH50 values represent the sum of 50 PAH analytes, selected by the EPA based on individual toxicity and concentration. These values are calculated from the raw analytical data presented in the file titled "Water_chemistry2018" from a sample taken at the beginning of the exposure. These final calculations are summarized in "Water_chemistry2018"
--Replicate = biological replicate number within each exposure group
--Total Cholesterol (µM) = total cholesterol reported in micromolar, measured in each homogenized biological replicate for larvae exposed to seawater control or HEWAF
--Total Protien (µg/mL)= total protein content reported in micrograms per milliliter sample in each homogenized biological replicate for larvae exposed to seawater control or HEWAF
--Total Cholesterol normalized to total protein (µM)/((µg/mL)) = total cholesterol in each biological replicate divided by total protein measured in the same replicate, reported as micromolar cholesterol divided by micrograms per milliliter protien
Survival_RD = Percent red drum larvae survival following seawater control or HEWAF exposure
--Nominal Treatment = Describes the treatment for each exposure group. Embryos were exposed to either a seawater control or a percent oil from the surface (OFS) HEWAF. Nominal treatment OFS HEWAF exposures are reported as "Seawater Control" or the percent OFS.
--sumPAH50 (µg/L) = sumPAH50 in micrograms per liter (µg/L). sumPAH50 values represent the sum of 50 PAH analytes, selected by the EPA based on individual toxicity and concentration. These values are calculated from the raw analytical data presented in the files titled "Water_chemistry2017", "Water_chemistry2018", and "Water_chemistry2019" from samples taken at the beginning of each exposure. These final calculations are summarized in "Water_chemistry2017", "Water_chemistry2018", and "Water_chemistry2019".
--Start Date = Date the exposures were initiated
--End Date = Date the exposures were terminated
--Number at test start = total number of individual red drum embryos each replicate at the start of the exposure
--Number observed treatment mortalities = number of red drum embryos which died during the treatment due to the treatment
--Number observed alive = total number of individual red drum larvae in each replicate alive after exposure, at 72 hours post fertilization
Gene_expression = Relative gene expression of HMGCR, SQLE, and FDFT1 following in red drum larval homogenates following 72 hours post fertilization OFS HEWAF exposure; each target gene was targeted on two separate plates (plate 1 and plate 2). Two housekeeping genes were used for normalization and EF-a was targeted on plate 1, Beta-actin was targeted on plate 2.
--HMGCR = 3-Hydroxy-3-Methylglutaryl-CoA Reductase
--FDFT1 = farnesyl-diphosphate farnesyltransferase 1
--SQLE = squalene epoxidase
--EF-a = elongation factor 1-alpha
--beta_actin = beta-actin
--Treatment (sumPAH50) = Describes the treatment for each exposure group. Embryos were exposed to either a seawater control or a percent oil from the surface (OFS) HEWAF. HEWAF exposures are reported as the percent OFS and in parentheses sumPAH50 in micrograms per liter (µg/L). sumPAH50 values represent the sum of 50 PAH analytes, selected by the EPA based on individual toxicity and concentration. These values are summarized in the files "Water_chemistry2017", "Water_chemistry2018", and "Water_chemistry2019".
--EF-a CT plate 1 = average cycle threshold (CT)of three technical replicates for EF-a in each biological replicate; EF-a was the house keeping gene targeted on the first qPCR plate run (plate 1)
--Beta_actin CT plate 2 = average cycle threshold of three technical replicates for beta_actin in each biological replicate; beta_actin was the house keeping gene targeted on the second qPCR plate run (plate 2)
--FDFT1 CT plate 1= average cycle threshold of three technical replicates for FDFT1 in each biological replicate on plate 1
--FDFT1 CT plate 2= average cycle threshold of three technical replicates for FDFT1 in each biological replicate on plate 2
--Average FDFT1 CT (plate 1 and plate 2) = the geometric mean of the FDFT1 cycle threshold values from plate 1 and plate 2
--Average housekeeping CT (EF-a and beta-actin) = the geometric mean of the EF-a CT values from plate 1 and beta-actin CT values from plate 2 for each sample
--Average CT FDFT1 = average cycle threshold in the seawater controls of FDFT1
--Average CT housekeeping gene = average CT values of average housekeeping gene in the seawater controls
--FDFT1 fold change = expression fold change of FDFT1 for each biological replicate as calculated through according to the 2-deltadeltaCT method
--Average FDFT1 fold change = average fold change of FDFT1 in each exposure group
-- Average 2-deltadeltaCt (control)= average fold change of FDFT1 in control group
-- Normalized 2-deltadeltaCt FDFT1 = fold change of each sample normalized to the average fold change of the control
--HMGCR CT plate 1= average cycle threshold of three technical replicates for HMGCR in each biological replicate on plate 1
--HMGCR CT plate 2= average cycle threshold of three technical replicates for HMGCR in each biological replicate on plate 2
--Average HMGCR CT (plate 1 and plate 2) = the geometric mean of the HMGCR cycle threshold values from plate 1 and plate 2
--Average CT HMGCR = average cycle threshold in the seawater controls of HMGCR
--HMGCR fold change = expression fold change of HMGCR for each biological replicate as calculated through according to the 2-deltadeltaCT method
--Average HMGCR fold change = average fold change of HMGCR in each exposure group
-- Average 2-deltadeltaCt (control)= average fold change of HMGCR in control group
-- Normalized 2-deltadeltaCt HMGCR = fold change of each sample normalized to the average fold change of the control
--SQLE CT plate 1= average cycle threshold of three technical replicates for SQLE in each biological replicate on plate 1
--SQLE CT plate 2= average cycle threshold of three technical replicates for SQLE in each biological replicate on plate 2
--Average SQLE CT (plate 1 and plate 2) = the geometric mean of the SQLE cycle threshold values from plate 1 and plate 2
--Average CT SQLE = average cycle threshold in the seawater controls of SQLE
-- SQLE fold change = expression fold change of SQLE for each biological replicate as calculated through according to the 2-deltadeltaCT method
--Average SQLE fold change = average fold change of SQLE in each exposure group
-- Average 2-deltadeltaCt (control)= average fold change of SQLE in control group
-- Normalized 2-deltadeltaCt SQLE = fold change of each sample normalized to the average fold change of the control
Water_chemistry2017 = summary of individual PAH measurements in water samples taken at the beginning of the exposures initiated on 10/24/2017. SumPAH values represent the sum of 50 PAH analytes, selected by the EPA based on individual toxicity and concentration.
--Nominal treatment = either seawater control treatment or a percet oil from the surface (OFS) HEWAF.
--Date water sample collected = date the water sample was collected. Samples were collected on the first day of red drum exposure, immediately following initiation of treatment
--Units = units of the measurements reported in the results column. Micrograms per liter = µg/L
--Component = individual PAH measured in the water sample
--Detection limit = limit of analytical detection. If results do not exceed this value they are reported as Non-detect (ND).
Water_chemistry2018 = summary of individual PAH measurements in water samples taken at the beginning of the exposures initiated on 11/13/2018. SumPAH values represent the sum of 50 PAH analytes, selected by the EPA based on individual toxicity and concentration.
--Nominal treatment = either seawater control treatment or a percet oil from the surface (OFS) HEWAF.
--Date water sample collected = date the water sample was collected. Samples were collected on the first day of red drum exposure, immediately following initiation of treatment
--Units = units of the measurements reported in the results column. Micrograms per liter = µg/L
--Component = individual PAH measured in the water sample
--Detection limit = limit of analytical detection. If results do not exceed this value they are reported as Non-detect (ND).
Water_chemistry2019 = summary of individual PAH measurements in water samples taken at the beginning of the exposures initiated on 7/17/2019. SumPAH values represent the sum of 50 PAH analytes, selected by the EPA based on individual toxicity and concentration.
--Nominal treatment = either seawater control treatment or a percet oil from the surface (OFS) HEWAF.
--Date water sample collected = date the water sample was collected. Samples were collected on the first day of red drum exposure, immediately following initiation of treatment
--Units = units of the measurements reported in the results column. Micrograms per liter = µg/L
--Component = individual PAH measured in the water sample
--Detection limit = limit of analytical detection. If results do not exceed this value they are reported as Non-detect (ND).
Water_Quality_RD = Water Quality measurements recorded throughout treatments
--Nominal treatment = either seawater control treatment or a percet oil from the surface (OFS) HEWAF.
--Period = What period of time was the sample collected during. 0 H = between 0 hours post fertilization (hpf) and 24 hpf; 24 H = between 24 hpf and 48 hpf; 72 H = between 48 hpf and 72 hpf
--Water temp. (C) = water temperature recorded for each exposure group in degrees Celsius.
-- pH (S.U.) = pH recorded for each exposure group (S.U. = standard units)
-- D.O. (mg/L) = Dissolved oxygen recorded in each exposure group, measured in milligrams per liter
-- Salinity (ppt) = Salinity recorded in each exposure group, reported as parts per thousand
PericardialArea = Pericardial area measured in 72 hours post fertilization (hpf) larvae
--Nominal treatment = either seawater control treatment or a percet oil from the surface (OFS) HEWAF.
--sumPAH50 (µg/L) = sumPAH50 in micrograms per liter (µg/L). sumPAH50 values represent the sum of 50 PAH analytes, selected by the EPA based on individual toxicity and concentration. These values are calculated from the raw analytical data presented in the files titled "Water_chemistry2017", "Water_chemistry2018", and "Water_chemistry2019" from samples taken at the beginning of each exposure. These final calculations are summarized in "Water_chemistry2017", "Water_chemistry2018", and "Water_chemistry2019".
--Pericardial area (µm^2) = 2D measurement of pericardial area from still frame images of 72 hour post fertilization red drum larvae following treatment
2020_RedDrum_FilipinStaining = raw and summarized data from image analysis of filipin stained 72 hpf red drum larvae
NA = not applicable for the given sample
--measurement = ImageJ was used to analyze fluorescence images of laterally oriented 72 hpf red drum larvae. A value of "full" indicates that the region of interest measured is an outline of full red drum larval body. "Background" indicated that the values are from a background measurement taken in the image. Three background measurements were recorded per image.
--sumPAH50 (µg/L) = sumPAH50 in micrograms per liter (µg/L). sumPAH50 values represent the sum of 50 PAH analytes, selected by the EPA based on individual toxicity and concentration. These values are calculated from the raw analytical data presented in the files titled "Water_chemistry2017", "Water_chemistry2018", and "Water_chemistry2019" from samples taken at the beginning of each exposure. These final calculations are summarized in "Water_chemistry2017", "Water_chemistry2018", and "Water_chemistry2019".
--Nominal treatment = either seawater control treatment or a percet oil from the surface (OFS) HEWAF.
-- Sample = sample ID
--Area (mm^2) = area of the region of interest in millimeters squared
--mean = mean gray value within the region of interest
--stdev =Standard deviation of the gray values used to generate the mean gray value
--mode= - Most frequently occurring gray value within the selection.
--min = minimum gray value within the selection
--max = maximum gray value within the selection
--IntDen = the product of the area and mean gray value
-- Median = the median value of the pixels in the selection
-- RawIntDen = the sum of the gray values of the pixels in the selection
-- avg.background = average background (mean gray value) of three background measurements
--area.bkg = area of the region of ingerest *avg.background
--TCF = IntDen minus area.bkg
-- Corr_mean = mean - avg.background
--avg_bkg_rawintden = average background RawIntDen of three background measurements
--corr_rawintden = RawIntDen minus avg_bkg_rawintden
--raw_int_den.area = corr_rawintden divided by Area
--Corr_mean_normArea = Corr_mean divided by Area
Methods:
Dataset description: Preparation of Water Accommodated Fractions: -- Naturally weathered oil from the surface (OFS) was used to prepare all HEWAFs (high-energy water-accommodated fraction) for this experiment. HEWAFs were prepared on the day of the start of the exposure with a loading rate of 1 gram of oil per 1 liter of 1μm filtered, UV-sterilized, seawater. The mixture was then blended at low speed in a Waring CB15 blender for 30 seconds and immediately transferred to a glass separatory funnel. The mixture was allowed to settle for 1 hour before the lower 90% of the fluid was drained and determined to be the 100% HEWAF. The 100% HEWAF was then diluted to nominal concentrations which are recorded as a percent OFS HEWAF with UV-sterilized seawater. Experimental Animals: --Red drum embryos were collected from broodstock tanks at the Texas Parks and Wildlife Department-CCA Marine Development Center in Corpus Christi, Texas, and transported under constant aeration to the University of Texas Marine Science Institute. Embryos were subsequently treated with formalin (1ppt) during aeration to remove any bacteria. Embryos were then rinsed with sterilized seawater and checked for buoyancy and coloration to assess viability using a Nikon SMZ800N microscope. Spawns with low fertilization rates or poor egg quality were not used. Embryonic exposures: --Embryos were transferred at 12 hours post fertilization to 1L glass beakers where the exposures were conducted. Each beaker contained UV-sterilized seawater for either seawater controls or OFS HEWAF dilutions for treatment exposures with approximately 40 embryos per beaker. Exposures took place in an environmental chamber with temperature and light control (photoperiod: 14 Light: 10 Dark; temperature: 25 degrees Celsius). Temperature, pH, dissolved oxygen, salinity and ammonia were monitored daily. At 72 hpf larvae from each replicate beaker were collected, snap-frozen in liquid nitrogen and stored at -80 degrees Celsius for later analysis. A subset of 72 hpf larvae were anesthetized was anesthetized using 250 mg L−1 MS222 (buffered with 500 mg L−1 NaHCO3). Individuals were then mounted in left lateral view on 3% methylcellulose in a petri dish for image collection and morphological analysis. Water chemistry analysis: --A subsample of the initial diluted HEWAF was collected in a 250 mL amber bottle and stored at 4 °Celsius until it was analyzed by ALS Environmental (Kelso, WA) for PAHs using gas chromatography/mass spectrometry−selective ion monitoring (GC/MS−SIM; based on U.S. Environmental Protection Agency method 8270D). Reported ∑PAH values represent the sum of 50 PAH analytes, selected by the EPA based on individual toxicity and concentration. Total cholesterol quantification: --Total cholesterol was determined in whole fish homogenates using the “Total Cholesterol Assay Kit (Colorimetric)” from Cell Biolabs, Inc. (San Diego, CA) following the manufacturer’s “tissue lysates” protocol. Three biological replicates were analyzed per treatment. Standard curves were prepared and used to calculate total cholesterol (µM) in each sample. Total cholesterol was subsequently normalized to the protein concentration (µg/mL) in each sample, determined using a Pierce BCA Protein Assay Kit. RNA isolation and Real-Time PCR (qPCR): --To describe relative expression of genes in the cholesterol biosynthetic pathway either three biological replicates, containing approximately 40 embryos each, were collected per treatment for qPCR analysis. Total RNA was extracted, and RNA quality and quantity were determined using a Nanodrop. cDNA synthesis was conducted using 1µg of RNA. --qPCR reactions were carried out using SsoAdvancedTM Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on the BioRad CFX Connect instrument (Hercules, CA). Thermocycling conditions used for qPCR analysis of all genes were as follows: 95 °Celsius for 5 minutes, followed by 40 cycles of 95 °Celsius for 10 seconds and 60 °Celsius for 30 seconds. Three technical replicates were performed for each biological replicate. Following amplification, qPCR products were separated on a 1.2% agarose gel to confirm product specificity. Data was normalized using elongation factor 1-alpha, and mRNA levels were determined according to the 2−ΔΔCT method. Morphological assessment: --At 72 hpf a subset of larvae was anesthetized using 250 mg L−1 MS222 (buffered with 500 mg L−1 NaHCO3). Individuals were then mounted in left lateral view on 3% methylcellulose in a petri dish for image collection. All images were collected using a Nikon SMZ800N microscope and a Nikon Digital Sight DS U-3 instrument and associated software. Still frames were imported into ImageJ to quantify individual pericardial area.
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