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Publication Date

2009-01-01

Availability

UM campus only

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Marine Biology and Fisheries (Marine)

Date of Defense

2009-06-09

First Committee Member

Andrew Baker - Committee Chair

Second Committee Member

Pat Gibbs - Committee Member

Third Committee Member

Mike Schmale - Committee Member

Fourth Committee Member

Larry Brand - Committee Member

Fifth Committee Member

Akira Chiba - Outside Committee Member

Abstract

Symbiotic dinoflagellates in the genus Symbiodinium are critical to the success of scleractinian reef corals in shallow tropical seas. These symbionts are commonly isolated from hosts and cultured separately in liquid media (f/2 or ASP8a), but initial isolations can be prone to abundant contaminants that can persist long-term in culture. To help remove these contaminants, I developed a solid growth substrate composed of 1% f/2 medium in agar, supplemented with a variety of antibiotics, to help isolate individual clones and establish new ?axenic? cultures. I found that an antibiotic cocktail of kanamycin (50 µg/mL), ampicillin (100 µg/mL) and streptomycin (50 µg/mL) was the most effective at eliminating visual signs of contamination without apparent harm to a variety of Symbiodinium in culture. Photophysiological measurements of Symbiodinium grown on f/2 agar plates, taken using an Imaging Pulse Amplitude Modulated (I-PAM) fluorometer, were comparable with those grown in liquid f/2, both with and without antibiotics. Eight types of Symbiodinium in clades A-D grown on f/2 agar plates at low irradiance (19-46 µmol photons m-2 s-1) were exposed to higher irradiance conditions (50-90 µmol photons m-2s-1) for 13 days and their photosynthetic efficiencies (Fv/Fm) were compared using the I-PAM. There were significant differences among and within clades, except for two types in clade C (C2 and C3) which did not perform differently from eachother. All types showed reduced Fv/Fm after 12 days in higher light. Type D1a showed high mortality after 13 days of higher light stress. Finally, preliminary work to fluorescently label Symbiodinium determined that available vital stains were not generally practical for symbiosis studies. Attempts to transform Symbiodinium with a variety of plasmids containing fluorescent reporters and/or genes for antibiotic resistance were not successful, but did provide a baseline for future work. In summary, Symbiodinium cultures grown on solid substrates supplemented with antibiotics are useful for: (1) isolating individual cells or clones for subsequent applications and establishing and maintaining ?axenic? cultures that are free of observable contaminants; (2) directly comparing the photophysiology of different cultures using an I-PAM fluorometer; (3) quantifying cells on agar plates using the I-PAM and (4) selecting possible transgenic symbionts for symbiosis studies.

Keywords

Transformations; Coral Physiology; Symbiodinium Culturing; F/2 Agar Plates; KAS Agar Plates; Imaging Pulse Amplitude Modulated Fluorometer

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