Abstract:

Juvenile red snapper were used to elucidate immunotoxic effects of various conditions on fish. Four treatments were used: Control group (No oil/No pathogen), Oil/No pathogen group (tPAH50 = 17.084 μg/L, No oil/Pathogen group, and Oil/Pathogen group (tPAH50 = 15.799 μg/L).

Suggested Citation:

Rodgers, Maria L. and Robert J. Griffitt. 2021. RNAseq dataset for effects of oil and pathogen exposure on red snapper. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/ZFKSBECJ

Purpose:

To understand the transcriptomic consequences of oil, pathogen, and oil+pathogen exposure on red snapper.

Data Parameters and Units:

The headers are Name, Max group mean, Log2 fold change, Fold change, P-value, FDR, p-value, Day, Water temperature (oC), pH, Dissolved oxygen (mg/L), Salinity (ppt), and Total ammonia (mg/L), Counts (transcripts per million).

Methods:

A fully factorial design was used for this experiment with four treatment groups: 1. Control (No oil/No pathogen), 2. Oil/No pathogen, 3. Pathogen only (No oil/Pathogen), and 4.Oil/Pathogen. There were four replicates per treatment, with each replicate containing three juvenile fish. All experiments were performed in triplicate test chambers with 18.5 cm × 12.5 cm dimensions and 18 L of synthetic seawater with a salinity of 30 PSU. Each day, water quality parameters including temperature, pH, dissolved oxygen (DO), salinity, and total ammonia were measured and any dead fish were removed and documented. In order to maintain optimal water quality parameters, flow-through systems were used and tanks were aerated. After one week of exposure, fish from the No oil/Pathogen and Oil/Pathogen groups were removed from their tanks and placed into new, 10 gallon tanks with 20 L of seawater inoculated with Vibrio anguillarum (ATCC 19264) for one hour. The actual concentration of V. anguillarum in exposure tanks was 7.5 × 105 CFU/mL. Fish from the other, non-pathogen challenged groups were also removed from their tanks and placed into new, 10 gallon tanks with 20 L of V. anguillarum-free seawater. Once the one hour challenge exposure window had ended, all fish were returned back to their original respective exposure tanks. One day after the challenge (day 8 from the start of the experiment), fish from each treatment were sacrificed according to approved IUCAC protocols (three from each treatment group). Blood samples were taken, and organs and carcasses were removed and placed into RNAlater (Invitrogen, Carlsbad, CA, USA) and stored at -80 °C until additional processing. For each fish, length measurements were taken using a ruler and mass was recorded with a digital scale. This same process was carried out at one additional sacrifice time points, which was 3 days post-Vibrio challenge (day 10 from the start of the experiment). Following sacrifice, total RNA was extracted using RNeasy kits (Qiagen, Hilden, Germany), and RNA quality and quantity was determined using a NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). Once samples were determined to be of appropriate quality and quantity (RIN scores of ≥6), total RNA was sent to Purdue University’s Genomics Core Facility (West Layfette, IN, USA) for cDNA library preparation and sequencing. Illumina TruSeq Stranded mRNA Sample Preparation Guide (Illumina, San Diego, USA) was used to construct polyA+ libraries, and an Illumina HiSeq 2500 was used to sequence 2 × 100 bp sequencing reads. For the HEWAF exposures, the Slick A oil used was originally obtained on July 29, 2010 from multiple skimming operations which was then placed in the hold of barge number CTC02404. From there, aliquots of oil were transferred under chain of custody (ID number SDG-10073005) to The University of Southern Mississippi on March 29, 2011 where aliquots were refrigerated and stored in amber jars until use. The method of HEWAF preparation presented herein was previously outlined by Forth et al. (2016,2017).HEWAF stock was created by using Slick A oil at a weight of 2 g in combination with 2 L of seawater (salinity 30 PSU) in a stainless steel blender (Waring, Stamford, CT, USA) for a nominal concentration of approximately 1 g/L. The mixture was blended for 30 s at low speed, and then the water/oil mixture was transferred to a separatory funnel, covered with foil, and allowed to settle for 1 h. After the settling period, the dissolved fraction was diluted to the appropriate concentrations and used in the oil-exposure treatments. Fresh HEWAF was made every other day and renewals were performed for the oil-exposure tanks every 48 h. Vibrio anguillarum was obtained from ATCC (19264) and grown to the targeted nominal density of 2.65*108 CFU/mL. At the end of the experiment, water samples were taken from all tanks and actual concentrations of V. anguillarum were determined via colony counting on agar plates.

Provenance and Historical References:

Forth, Heather P., Carys L. Mitchelmore, Jeffery M. Morris and Joshua Lipton. (2017). Characterization of oil and water accommodated fractions used to conduct aquatic toxicity testing in support of the Deepwater Horizon oil spill natural resource damage assessment. Environ Toxicol Chem, 36(6): 1450-1459. doi:10.1002/etc.3672

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