Abstract:

To fully assess the impacts of Deepwater Horizon (DWH) oil on the vertebrate stress response and the trajectory for recovery, our research integrated molecular, cellular, gland/organ and whole animal physiological and behavioral studies using a benthic marine teleost, the Gulf toadfish, a resident of the Gulf of Mexico. These studies evaluated multiple pathways (e.g., pituitary fatigue, reduced receptor sensitivity, disruption or overstimulation of cortisol biosynthesis) and the consequence of these changes at the whole animal level (e.g., changes in carbohydrate metabolism, immune capacity and responses to natural stresses such as predation and social interaction). In this series of experiments, mRNA expression of various genes found within the hypothalamus-pituitary-interrenal (HPI) axis (i.e., the hypothalamus-POA region, pituitary, head kidney) was evaluated during short-term exposures to DWH oil. Each exposure was followed by a complementary recovery period (i.e., no oil or polycyclic aromatic hydrocarbon (PAH) exposure) to assess the recovery trajectory. These experiments were completed at Rosenstiel School of Marine and Atmospheric Science (RSMAS), University of Miami, Marine Technology & Life Sciences Seawater Complex (MTLSS) Building, room 211 between January 2018 to December 2018.

Suggested Citation:

Alloy, Matthew M., Maria C. Cartolano, Emily Milton, and Anastasiya Plotnikova. 2020. Acute exposure mRNA expression analysis. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-j532-0x61

Purpose:

The purpose of this dataset was to evaluate whether exposure to polycyclic aromatic hydrocarbons (PAHs) resulted in changes in the mRNA expression of genes important to the HPI axis as well as to measure whether there was the induction of CYP1A within the liver, which is typically associated with PAH exposure, but also within the HPI axis, which, to our knowledge, has never been tested.

Data Parameters and Units:

Fish, trial, timePoint, stresses/not stressed, replicate fish ID, hypothalamus-POA CRF mRNA expression (Ct value), hypothalamus-POA Ef1a mRNA expression (Ct value), hypothalamus-POA CYP1A mRNA expression (Ct value), pituitary Ef1a mRNA expression (Ct value), pituitary CYP1A mRNA expression (Ct value), kidney Ef1a mRNA expression (Ct value), kidney CYP1A mRNA expression (Ct value), kidney StaR mRNA expression (Ct value), kidney p450scc mRNA expression (Ct value), kidney 11B-hydroxylase mRNA expression, liver Ef1a mRNA expression (Ct value), liver CYP1A mRNA expression (Ct value). The naming convention for replicate fish ID is where A = Treatment (X = Initial sample; C = control, 0.006 ug/L sum total PAH; L = low concentration exposure, 0.009 ug/L sum total PAH; M = medium concentration exposure, 0.06 ug/L sum total PAH; H = high concentration, 2.8 ug/L sum total PAH exposure); XXX = Fish number (1-152); B = Trial # (1 or 2); C = Fish state (Not stressed [NS] or Stressed [S]); D = Time sample taken in hours (h; either 4, 8, or 24) or days (d; either 3 or 7); Z = Period sample taken (either during Exposure [E] or during Recovery [R]). Please note - cross out = Fish died during the experiment; and nd = data point not determined.

Methods:

Total RNA from hypothalamus-POA, pituitary, and the kidney was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA) and then treated with DNAse (TURBO DNA-free kit, Invitrogen). cDNA was synthesized using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems™, Foster City, CA, USA) and diluted ten-fold to quantify mRNA expression. qPCR primers for CYP1A, StAR, P450scc, and 11𝛽H were designed using sequences from degenerate primers. The reference gene used was elongation factor 1α and primers were from another study. An Mx3005P Multiple Quantitative PCR System (Stratagene, San Diego, CA, USA) was used to perform real-time quantitative polymerase chain reaction (qPCR) on the cDNA samples in duplicate using Power SYBR Green qPCR Master Mix (Applied Biosystems™) as the reporter dye.

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