Date of Award

Spring 3-13-2017

Degree Type

Dissertation

Degree Name

PhD Molecular Bioscience

Department

Biology

Advisor

Carolyn S. Bentivegna, Ph.D.

Committee Member

Jane L Ko, Ph.D.

Committee Member

Wyatt R. Murphy Jr., Ph.D.

Committee Member

Marian Glenn, Ph.D.

Committee Member

Daniel B. Nichols, Ph.D.

Keywords

De novo assembly, Fundulus heteroclitus, NGS, transcriptomics, RNA-seq

Abstract

The killifish, Fundulus heteroclitus, is a common fish model in aquatic toxicology. However, little is known in this organism about how endocrine disrupting compounds, (EDCs) including polycyclic aromatic hydrocarbons (PAHs), impact the reproductive status from a molecular standpoint. The objective of this project is to apply high throughput sequencing to F. heteroclitus testes to examine the molecular mechanisms that impair reproduction when exposed to crude oil. First, a crude oil exposure experiment was conducted. Following exposure, semi-quantitative PCR was performed to detect changes in gene expression of the following genes known to respond to EDCs: gonadal aromatase (CYP19a), vitellogenin (VTG), and cytochrome P450 1A (CYP1A). Excitation-Emission matrix (EEM) spectroscopy was performed to verify PAH exposure in the gonads. Killifish that demonstrated endocrine disruption along with verified PAH exposure were selected for sequencing. Illumina NextSeq 500 technology was applied to three experimental groups to determine genes and pathways turned on during sexual activation and disrupted by crude oil exposure: 1) an exposed spawning male gavaged with crude oil collected from the DeepWater Horizon oil rig, 2) a control spawning male gavaged with fish oil, and 3) a control non-spawning male. The de novo assembler, Bridger, was used to assemble the sequence reads. The Trinotate pipeline was used to annotate the resulting genes and determine differential gene expression among the three transcriptomes. The Trinotate annotation and differential expression analysis was validated by qPCR of select genes. Heatmaps displayed genes that were turned on, off, or had at minimum a two-fold change in expression. The Cytoscape plug-in ClueGO created a functionally organized GO/pathway term network to visualize gene interactions. Genes found within the “Regulation of Androgen Receptor Signaling Pathway” node was shown to be impacted by GO terms associated with “Response to Heat” and “Apoptosis”. Candidate biomarkers were found associated with apoptosis (EP300; histone acetyltransferase p300, SIRT1; Sirtuin 1 and SMARCA4; Transcription activator BRG1), impaired spermatogenesis (SMARCA4 and DNAJA1; heat shock protein family (Hsp40) member A1,) and suppressed androgen receptor transcriptional activation (HDAC6; (Histone deacetylase 6 and PTGES3; Prostaglandin E Synthase 3). Overall, this research is the first to assign functional annotations to the testes transcriptomes in killifish, resulting in the most comprehensive reproductive information available for the species to date. This transcriptomic data can provide the ground work for studying the killifish’s population dynamics, biomonitoring, and reproductive health. Additionally, this data can be utilized in comparative studies in other fish models to further enhance breeding programs and evolutionary studies.

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