The Deepwater Horizon oil spill in the Gulf of Mexico has made it imperative to understand the effects of crude oil exposure on marine life. This RAPID project is designed to investigate the effects of crude oil compounds on the development of fish larvae. Embryonic stages of animal development are very sensitive to environmental pollutants like those in crude oil. Fish larvae form an important component of the Gulf Coast food web, and one goal of this project is to determine how exposure to these compounds will induce developmental abnormalities that may result in severe impairment of surviving fish. The RAPID response is particularly appropriate, as the both the brood fish and their spawn have been acutely exposed to crude oil. Understanding the molecular and genetic basis of the effects of any abnormalities will help determine whether they result from molecular pathways common to other environmental pollutants or are unique to crude oil. This will be investigated using both state-of-the-art DNA sequencing gene expression technologies and bioinformatics-based gene network analysis. The implementation of these methods to study the molecular basis of the effects of crude oil exposure on embryonic development represents a major step forward in the use of technologically-advanced gene analysis tools to understand the molecular basis of interactions between organisms and their environment. This project provides training for graduate students in molecular and environmental science and the opportunity for research scientists to communicate findings to the public that concern the livelihood and welfare of American citizens.
The major contributions of this project to our understanding of the effects of crude oil exposure on vertebrate embryonic development are the recognition that relatively brief continuous crude oil exposure during vertebrate development can manifest profound developmental effects leading to larval death; that crude oil-induced defects appear to affect tissue-specific development differentially, and that crude-oil induced global gene expression changes across range across a diverse set of gene classes with unexpected changes in expression of transmembrane protein encoding genes involved in solute and ion transport. Exposure of zebrafish embryos to a specific mixture of crude oil and chemical dispersant for periods as short as 72 hours during early embryonic development resulted in highly reproducible characteristic teratogenic (embryonic malformation) effects of treated embryos. The teratogenic effects were marked predominately by edematous expansion of the pericardial sac concomitant with time-dependent deterioration of heart development leading to cessation of heart function; edematous expansion of the gut wall lining with foreshortening of the gut; the developmental restructuring and absence of formation of posterior gill arches; and deterioration of the marginal epithelium along the dorsal and ventral aspects of the developing larvae. These teratogenic effects mirrored closely the developmental defects reported previously in diverse fish embryos exposed to crude oil contaminated waters following the Exxon Valdez oil spill in Alaska and oil spills in Norwegian waters. They also mirror the developmental defects we observed upon crude oil treatment of the Gulf killifish, Fundulus grandis. Taken together, these results suggest that exposure of fish embryos to crude oil induces characteristic teratogenic effects resulting in high levels of mortality. Transcriptome analyses conducted to date have shown highly significant changes in transcript levels for a number of gene families including the expected increased transcription of cytochrome P450 and aryl hydrocarbon metabolizing gene transcripts along with the surprisingly significant changes in transcription of ABC solute carrier and G-protein coupled signaling families and other genes encoding transmembrane proteins. These results are particularly interesting because they provide the basis for a working hypothesis that the abundant hydrophobic components of crude oil perturb the physico-chemical nature of cellular cytoplasmic membranes and interfere with the normal function of transmembrane proteins in which failure of solute transport systems to properly regulate the exchange of ions and other small molecules across epithelial boundaries generate osmotic imbalances in cells and tissues. Osmotic imbalances, in turn, may result in the characteristic pericardial edema and 'blebbing' of dorsal and ventral epithelial margins in crude oil-exposed fish. Interestingly, transcriptome analysis has not demonstrated direct effects of crude oil exposure on the levels of gene expression among genes known to be involved in early cardiac development. However, additional detailed bioinformatics analysis may reveal effects on developmental gene expression that were not detectable in preliminary analyses. Although Gulf menhaden was proposed as the environmental reference species for this project, the results obtained from sampling in the Gulf of Mexico argued for the identification of an alternative environmental reference species that would be easier to sample, less likely to migrate from nearshore to offshore sites, and more amenable to laboratory study. Gulf killifish collected from crude oil contaminated sites in April 2011 have been successfully spawned naturally and in vitro. Exposure of Gulf killifish embryos in vitro to the same crude oil exposure regimen used to induce teratogenesis in zebrafish, resulted in similar high frequency teratogenic effects, which suggests that this species will be extremely useful as an environmental reference to understand both acute and chronic effects of environmental crude oil exposure. The broader impacts of the project have involved the formal training of two undergraduates, two Master’s students and a Ph.D. student. Each of the undergraduates and Master's students involved in the project have learned about experimental design, data recording and interpretation, hypothesis formulation and testing, scientific literature searches and oral presentation of scientific research. In addition, Master's students have prepared formal written proposals, research summaries, and research presentations. Fuliang Xie, a doctoral student, has learned computer algorithm design, database management, searching, and archiving. His most recently developed algorithms are being used to conduct complex time-series transcriptome comparisons among experimental and control zebrafish embryos and will have broad applicability in comparative transcriptomics. The PI has given guest lectures about the effects of crude oil exposure on embryonic fish development in ichthyology, environmental biology and graduate introduction to research courses. He has also given tutorials to undergraduates and graduate students involved in the research project and presented the results of the research at professional meetings and to local and regional environmental interest groups. Moreover, he has been featured on a PBS special report and cited in the journal Nature. The coPI's have also provided laboratory instruction to their undergraduate and graduate students both in the classroom, laboratory and field.
