Date Awarded


Document Type


Degree Name

Doctor of Philosophy (Ph.D.)


Virginia Institute of Marine Science


Michael Unger


As a way to gain better understanding of how aquatic organisms respond within their natural environment, this dissertation set up a series of controlled laboratory experiments to investigate the effects of chemical and biological stressors on the zebrafish (Danio rerio). I performed a series of studies in which animals were simultaneously exposed to the toxicant phenanthrene, and infected with the bacterium Mycobacterium marinum. Unexpectedly, we found that at a high dose of phenanthrene (284 mug L-1) results in an antagonistic relationship between stressors with dually challenged fish having significantly higher survivorship than those exposed solely to phenanthrene. Dually challenged fish also had reduced phenanthrene metabolism, suggesting higher mortality in phenanthrene only exposed fish may be due to increased toxicity from phenanthrene metabolites. We postulated metabolic reduction was due to inflammatory cytokines suppressing metabolism. However, in latter experiments using a more environmentally relevant dose of phenanthrene (157 mug L-1), dually challenged fish had significantly higher mortalities than all other treatments, and there was no disruption in phenanthrene metabolism. Our second study clearly showed an additive effect: the summation of mortalities for each stressor individually is approximate to what is observed in dually challenged fish. My third experiment was designed to better elucidate the effects of dose and exposure order on stressor interaction. I used a matrix of dose and order such that two phenanthrene doses were used (157 mug L -1 and 86 mug L-1), with a staggered order of exposure. Significantly higher mortalities in dually challenged fish for the high dose of phenanthrene versus the low dose, regardless of exposure order, demonstrated phenanthrene concentration, not exposure order is a significant factor. This study also showed activation of the cytochrome P450 pathway by phenanthrene. These studies highlight the exceptionally complex interactions between multiple stressors and how minor alterations in experimental design can produce dramatic changes in stressor interaction. I have concluded that toxicant dose plays a significant role in this interaction causing an antagonistic interaction at high concentrations; however, at lower doses an additive effect is seen. Additionally, I have shown the importance of phenanthrene metabolites in toxicity. Finally I have clearly demonstrated that phenanthrene can induce the cytochrome P450 pathway. In addition to dual-stressor experiments, I conducted a comparative study between two Mycobacterium spp.: M marinum and M. pseudoshottsii. The goal of this study was to find a bacterium to model disease recrudescence. Despite the close relationship between these two species, we observed dramatically different virulence and pathology. M. marium infected fish had <10% survivorship over a 4-week exposure; however M. pseudoshottsii infected fish had ∼98% survivorship. Additionally, M. marinum infected fish displayed a classic granulomatous inflammation with bacilli sequestered within, or in immediate proximity to, well formed granulomas. In contrast, M. pseudoshottsii infected fish displayed little granuloma formation, instead having large area of diffuse inflammation and cellular necrosis. M. pseudoshottsii were seen disseminated both extra and intra-cellularly throughout areas of inflammation, a phenomenon not seen with M. marinum. I postulate that these differences are due to a unique mycolactone toxin secreted by M. pseudoshottsii. M. ulcerans is the only other Mycobacterium spp. to produce a mycolactone toxin, and produces pathologies similar to what we observe here for M. pseudoshottsii.



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