Project Overview

Summary:

Dr. Robert Griffitt at University of Southern Mississippi’s Division of Coastal Sciences, School of Ocean Science and Technology, was awarded an RFP-VI grant at $756,953 to conduct the RFP-VI project titled, “Integrating Teleost Transcriptomes to Identify Ecologically Meaningful Responses Following Oil Exposure”. The project consisted of 1 other institution (Purdue University), 1 principal investigator (Griffitt), 1 co-PI (Dr. Maria Sepulveda); 1 post-doc (Maria Rodgers); 2 PhD students (Elizabeth Allmon, Elizabeth Jones); and 1 Masters degree students (Maggie Wigren).

     We proposed to carry out a large-scale meta-analysis of global transcriptional profiles from multiple species exposed to oil. The goal of this project was to leverage the considerable resources expended by GoMRI and the NRDA process to identify 1) conserved patterns of response across multiple species that will allow for generalizable hypotheses to be drawn about the effects of oil to GoM fish species, 2) identification of subtle effects resulting from sublethal exposure to oil that may have implications for future fisheries management practices, and 3) identification of novel hypotheses regarding the effects of oil on ecologically important fish species in the Gulf of Mexico.

    We propose three Aims that will guide us in our investigations. Aim 1. Identification of conserved, ecologically meaningful, transcriptional responses in teleosts following oil exposure. Aim 2. Interrogating fish transcriptomes to identify synergistic effects of environmental stressors. Aim 3. Evaluating multiple teleost transcriptomes for evidence of impairment of basic molecular functions resulting from oil exposure.

     The resources available for this project are substantial. As part of their GoMRI-II funded project Griffitt and Sepulveda produced 78 RNAseq libraries (36 from Cyprinodon variegatus, 42 from Fundulus grandis) that cover multiple exposure conditions and age classes in fully balanced designs. Further, PI Griffitt has 52 southern flounder RNAseq libraries from NRDA funded work that have not been fully analyzed. PI Griffitt is also in possession of tissue samples taken from three ecologically important pelagic species (Red snapper, Atlantic croaker, and Red drum) that we propose to sequence as well (24 from each, 72 additional RNAseq libraries). These contain both lab-exposed and field collected samples, and so represent, in combination with the GoMRI funded datasets, a very fertile area of investigation. This is obviously a substantial dataset (130 complete RNAseq libraries), and an in-depth analysis of all possible comparisons was not possible in the duration of the previous projects. We propose to have a significant and in-depth bioinformatics analysis of the collected samples that will allow us to maximize the data yield and knowledge gained from the resources already invested in this research.

Research Highlights

      Dr. Griffitt’s research resulted in 3 peer-reviewed publications, 2 book chapters, and 15 conference presentations to date. In addition, 9 datasets were submitted to the GoMRI Information and Data Cooperative (GRIIDC), which are available to the public. One MS student (Maggie Wigren, Purdue University) and one PhD student (Elizabeth Jones, USM) completed her research under this grant. An additional 3 papers are currently under review with 6-8 papers being analyzed and written now. Significant outcomes of their research (all related to GoMRI Research Theme 3) are highlighted below.  The table below highlights all of the RNA and microbiome samples that were sequenced or analyzed.

-Transient exposure to oil during development of sheepshead minnow (Cyprinodon variegatus) alters DNA methyl transferase enzyme expression and suggests a mechanism underlying PAH-induced hypomethylation noted in previous studies

- The combination of oil and hypoxia resulted in far greater impacts to methylation than either stressor individually, indicating the two act synergistically to exert methylation changes

-We noted a significant association between differentially methylated regions of the genome and differential gene expression, suggesting that methylation plays an important role in regulating the transcriptional responses that mitigate the effects of these stressors

-The promoter of the gene slc2a1 exhibited decreased methylation in both the oil and oil plus hypoxia groups, and was associated with increased mRNA expression

-Sheepshead minnow transitioning from embryos to hatchlings suffer minimal transcriptomic consequences to immune genes and pathways under oil, hypoxia, and oil+hypoxia exposure conditions

-Sheepshead minnow transitioning from hatchlings to larvae experienced serious transcriptomic immune disruption after oil exposure (via enrichment of many immune-related genes and 43 immune-related pathways)

-Sheepshead minnow transitioning from hatchlings to larvae experienced suppression of immune-related genes and pathways after exposure to hypoxia alone, but not when exposed to hypoxia and oil

-In early life stage F. grandis, exposure to hypoxia alters immune-related gene expression under 3 ppt salinity conditions, but has little effect under 30 ppt salinity conditions

-In comparing early life stage F. grandis to C. variegatus, we noted a unique signature of immune-related genes for each species with only two immune-related pathways in common, one of which showed oppositional responses (LPS/IL-1 Mediated Inhibition of RXR Function; activated in F. grandis and suppressed in C. variegatus

-We revealed that cellular immune responses in F. grandis are significantly altered by HEWAF exposure, but that these responses are not significantly altered in C. variegatus, suggesting that transcriptional immune responses of at this early life stage are more sensitive to HEWAF in F. grandis than in C. variegatus

-While F. grandis and C. variegatus inhabit similar areas and share similar life history traits, they are not necessarily interchangeable models for toxicology research, especially in the realm of transcriptional immune responses

-Exposure of F. grandis to oil and abiotic stressors (high temperature, salinity and hypoxia) impacts signaling pathways associated with cardiovascular function; specifically, combined exposures appear to strongly impact the development of the systemic vasculature. 

-Chronic (35-day) oiled sediment exposure in southern flounder (Paralichthys lethostigma) tend to impact thyroid hormone metabolism-related pathways in gill tissue and cholesterol biosynthesis and immune-related pathways in the liver

-Allowing for a 30-day recovery period after 35 days of oiled sediment exposure increases the number of differentially expressed genes in both gill and liver tissue

-Cellular and humoral immune responses are altered in liver tissue following 30 days of recovery after oiled sediment exposure potentially indicating that the immune system is attempting to respond to damage caused by oiled sediment exposure

-P. lethostigma exposed to 57.432 g/kg (tPAH₅₀) of oiled sediment for 8 days experienced significantly higher mortality than those exposed to 28.501 g/kg (tPAH₅₀) of oiled sediment and control fish

- P. lethostigma exposed to 57.432 g/kg (tPAH₅₀) of oiled sediment for 8 days and challenged with Vibrio anguillarum for 1 hour (on day 7 of exposure) experienced significantly higher mortality than those exposed to just 57.432 g/kg (tPAH₅₀) of oiled sediment, just V. anguillarum, or control fish

- We characterized the gut microbiome (16S rRNA) of Cyprinodon variegatus after fish were exposed to oil (tPAH = 80.99 ± 12.5 µg/L) for 7 days. The most significant finding was that known hydrocarbon degrading taxa (order Pseudomonadales, family Paenibacillaceae, and Pseudomonas pachastrellae) were discriminant of oil-exposed communities and were absent in controls.

 Proposal Abstract - RFP-VI Robert Griffitt