Erin Macro

Date of Award

12-2012

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Marine Biology

Advisor

Paul Rawson

Second Committee Member

Rebecca Van Beneden

Third Committee Member

Ian Bricknell

Abstract

Roseovarius Oyster Disease (ROD) involves the colonization of the inner shell of the Eastern Oyster (Crassostrea virginica) by the marine alpha-proteobacterium Roseovarius crassostreae. The ensuing disease can result in upwards of 90% mortality in hatchery-raised juvenile (< 25 mm shell length) oysters. Symptoms of ROD include heavy, brown ring deposits of conchiolin on the surface of the shell, as well as uneven valve growth and shell curvature. Although the bacterium does not invade oyster tissue or digestive tract, fluorescent localization of an R. crassostreae biofilm on the inner shells of ROD-affected oysters, and toxic effects of R. crassotreae extracellular products on hemocytes and larval oysters suggest that a chemical warfare takes place between the R. crassostreae biofilm and the oyster host. In this thesis, I cultured R. crassostreae under laboratory conditions and analyzed the affects of physical growth condition, temperature, culture volume, and iron limitation on bacterial growth and bacterial extracellular protein (ECP) production. Culture growth rate increased with increasing temperature. Iron limitation resulted in slower growth but had no affect on maximum culture density. Iron limited cells produced a greater volume of ECPs, especially at low cell density. ECPs from liquid and solid-phase cultures were analyzed by LC-tandem mass spectrometry. The resulting protein sequences had high similarity to proteins in other bacterial species that act as virulence factors in other hosts, including metal transporters (2), proteases (2) and a surface-associated GroEL chaperonin. I also exposed live oysters to ECPs of live R. crassostreae cells by embedding a filter capsule into the oyster shell. I sampled extrapallial fluid from oysters pre-exposure and post-exposure and analyzed the relative expression of the oyster defense protein dominin in addition to the extent of brown shell deposition as a function of bacterial presence. The injury response due to capsule embedment masked any potential response by oysters to R. crassostreae ECPs.

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