Abstract:

The goal of this project is to perform RNASeq on mucus collected from juvenile mahi-mahi (Coryphaena hippurus) following oil exposure. ~30-day old mahi-mahi was exposed to 5 or 10% HEWAF (high energy water accommodated fraction) dilutions for 48 hours. Following exposure, mucus was collected and RNASeq was performed to identify differentially expressed transcripts. In addition, water quality measurements and levels of polycyclic aromatic hydrocarbons (PAHs) were made. These transcripts could provide target genes that could be used as biomarkers for non-invasive identification of oil exposure in fish.

Suggested Citation:

Greer, J.. 2019. Transcriptional alterations in mahi-mahi (Coryphaena hippurus) mucus following oil exposure. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-m7d5-nk45

Publications:

Greer, J. B., Andrzejczyk, N. E., Mager, E. M., Stieglitz, J. D., Benetti, D., Grosell, M., & Schlenk, D. (2019). Whole-Transcriptome Sequencing of Epidermal Mucus as a Novel Method for Oil Exposure Assessment in Juvenile Mahi-Mahi (Coryphaena hippurus). Environmental Science & Technology Letters, 6(9), 538–544. doi:10.1021/acs.estlett.9b00479

Purpose:

To assess the use of mucosal RNA for biomarker identification in oil exposed mahi.

Data Parameters and Units:

Data is organized by different data types obtained from HEWAF exposures of juvenile mahi. Below are short descriptions of the data in each file: The file "Trinity.fasta" contains - Transcriptome assembly of raw reads using Trinity, after rRNA removal with sortMeRNA. The raw sequencing data can be found in the NCBI SRA database, Accession: PRJNA526742. The file "S203 Fluorescence Analysis.xlsx" contains - PAH Fluorescence measurements: Raw fluorescence measurements used to calculate initial and final PAH concentrations from each day of the exposure. --Day = Day of sample collection. A water change was performed 24 hours after the start of exposure. Day 1 encompasses the first 24 hours prior to the water change, and day 2 encompasses the final 24 hours after the water change --Sample Type = Describes if the sample was from a standard PAH solution used to generate the standard curve, or if the sample was collected from WAFs prepared for exposures. --Sample Id = identification number used to uniquely identify the sample --Nominal Dilution (%) = For standard curve samples (identified under "sample type"), describes the dilution of the PAH standard for standard curve formation. Control sample was fresh seawater. WAF dilution series with a dilution of 0 was taken from the seawater control tanks during the experiment and was not diluted. 5% and 10% WAF dilutions are samples collected from the HEWAF dilutions used for exposure. --Peak Area for Excitation Wavelength 1 = Calculated area under the curve for wavelength 1, ~224 nm. --Peak Area for Excitation Wavelength 2 = Calculated area under the curve for wavelength 2, ~258 nm. --Blank Subtracted Sum of Peak Areas 1+2 = Calculated sum of the total area under the curve for wavelengths 1 and 2, minus the sum of the areas under the curve for the control sample --Log Nominal Dilution = Log value of the nominal dilution in the standard curve samples --Log Blank Subtracted Sum of Peak Areas 1+2 = Log of the value calculated in the "Blank Subtracted Sum of Peak Areas 1+2" column. --Dilution Factor = For WAF preparations, describes if the sample was diluted prior to analysis --Measured Dilution (%) = 10^((Log blank subtracted sum of peak areas 1+2 - y-intercept) / calculated slope) * Dilution factor --Notes = For WAF preparations, describes if the sample was an initial value immediately after WAF preparation or the final value prior to the water change (day 1) or immediately prior to the end of the experiment (day 2). --Standard curve parameters = category of parameters for the standard curve --Standard curve values = values determined for each standard curve parameter The file "HEWAF stock concentrations.xls" contains - HEWAF stock concentrations and includes raw values of measured PAH concentrations in micrograms per liter (µg/L) in water samples taken from the 10% HEWAF exposure at the beginning of each day. The file "SRA_accessions.xlsx" contains sample ID and accession numbers in the NCBI Sequence Read Archive (SRA) for the raw sequencing reads. Accession (SRA accession number for each biological sample, unique to each sample), Title (Title for each sample, and includes the HEWAF percentage, followed by the biological replicate number), BioProject (Accession number for the NCBI BioProject), BioSample (Unique accession number for each biological sample in the SRA database), and SRA.filename (Filename in the SRA data for the raw sequencing data associated with each BioSample). The file “Water_quality_parameters_S203.xlsx” contains - Treatment (Describes the treatment for each exposure group. Juvenile mahi were exposed to control seawater or nominal HEWAF dilutions of 5 or 10% for 48 hours; Control, 5% HEWAF, 10% HEWAF), Replicate (Replicate tank number within each treatment; 1, 2, 3, 4), Timepoint (Describes the time from the beginning of exposure to when the water quality measurements were made. 0 h represents initial values prior to the start of exposure. A water change was performed 24 hours after the start of exposure, with water parameters taken before (23 h) and after (25 h) the water change. Measurements at 48 h were immediately prior to the experiment conclusion. ; h), Water temp (Measured water temperature in degree Celsius), pH (Measured pH, in standard units), DO (Measured dissolved oxygen in milligrams per liter; mg/L), and Salinity (Measure salinity in parts per thousand; ppt).

Methods:

HEWAFs were prepared from crude oil obtained during surface skimming (OFS) following the DWH oil spill and transferred to the University of Miami under the chain of custody. The HEWAF solutions were prepared according to established methods and were diluted to nominal concentrations of 5 or 10% using UV-sterilized seawater for testing. Juvenile mahi (age: ~28 days; mass: 4.89 g  0.14 g SEM) were placed into 10 L glass aquaria containing 8 L of either fresh seawater (control), 5% HEWAF (low), or 10% HEWAF (high) for 48 h. There were four individuals per tank, with four replicate tanks in each treatment group. An 80% water change with fresh seawater or HEWAF dilution was performed on each aquarium ~24 h after the beginning of exposure. Exposures were performed in a temperature-controlled environment at 27C with a 12:12-h light:dark photoperiod. None of the exposure concentrations elicited acute mortality. Water samples for total PAH analysis were collected from the 10% HEWAF preparation each day in 250 mL amber glass bottles with no head space, immediately stored at 4 C, and shipped overnight on ice to ALS Environmental (Kelso, WA) for analysis by gas chromatography/mass spectrometry-selective ion monitoring (GC/MS-SIM). Previously described fluorescence methods were used to calculate PAH concentrations in each treatment prior to 24 h water change, after water change, and at the end of the experiment. Water temperature, pH, dissolved oxygen (DO), and salinity were measured immediately prior to the start of exposures, before and after the 80% water changes at 24 h, and at the end of the 48 h testing period. Total ammonia was measured at the end of the testing period using a micro-modified colorimetric assay. At the end of the 48 h exposure period, fish were collected in a small net and placed in a solution of tricaine mesylate (MS-222) for ~30 s. Fish were removed from solution and dabbed dry with a paper towel, and a small weighing spatula lightly across the skin surface to collect mucus. Due to the small amounts of mucus produced by each fish, mucus from all four fish per replicate tank were pooled into one sample for a total of four samples per treatment group. Samples were flash-frozen in liquid nitrogen immediately upon collection and then stored at -20C until further processing. Total RNA was extracted from mucus samples using the RNeasy Lipid Tissue Mini Kit (Qiagen, Qiagen, Germantown, MD) following the manufacturer’s protocol. Following extraction, total RNA concentrations were quantified using an Invitrogen Qubit 4 Fluorometer and a NanoDrop ND-1000 Spectrophotometer. Finally, RNA integrity was assessed on an Agilent 2100 Bioanalyzer. Ribosomal RNA (rRNA) was removed from total RNA using the NEBNext rRNA Depletion Kit prior to library preparation. Sequencing libraries for each sample were then prepared with the NEBNext Ultra II RNA Library Prep Kit for Illumina using 500 ng of total RNA as input. Final libraries were sequenced as single-end 75 bp reads in one lane on an Illumina NextSeq500 high-throughput sequencer at the University of California, Riverside. Processing of raw sequencing reads, de novo transcriptome assembly, and differential expression analysis was performed as previously described, with slight modifications. rRNA sequences were first removed from each sample using sortmeRNA. Poor quality reads were then removed using trimmomatic and read quality assessed with FastQC. The remaining reads were used to build a de novo transcriptome with Trinity, followed by transcript quantification with RSEM and differential expression analysis with DESeq2 at p≤0.05 after Benjamini-Hochberg false discovery rate (FDR) correction. Functional annotation of differentially expressed genes was performed using Trinotate. Differentially expressed and annotated transcripts were input into Ingenuity® Pathway Analysis (IPA), along with their log-fold change values, to identify altered molecular and biological pathways (FDR-adjusted p0.05).

Individual Files: