Defense Date

12-2-2020

Document Type

Thesis

Degree Type

Master of Science

Degree Name

Marine Science

First Advisor

Joana Figueiredo, Ph.D.

Second Advisor

Esther C. Peters, Ph.D.

Third Advisor

Dorothy A. Renegar, Ph.D.

Abstract

Stony coral tissue loss disease (SCTLD) is a highly lethal coral disease that has caused a dramatic loss of coral tissue along the Florida Reef Tract and throughout the Wider Caribbean. This study seeks to understand whether programmed cell death (apoptosis) is involved in the pathology of the highly virulent SCTLD tissue loss lesion. Tissues from diseased colonies of Pseudodiploria strigosa collected in 2018 and 2020 were stained using the terminal deoxyribonucleotidyltransferase (TdT) mediated dUTP-biotin nick end labeling (TUNEL) assay to visualize areas of programmed cell death. The archived tissue samples collected in 2018 exhibited a significantly higher degree of positive staining for DNA fragmentation with high non-specific staining of cytoplasmic material, suggesting that the age of paraffin blocks (2+ years) and/or the length of fixation (100+ days) caused DNA damage that influences the samples’ reactivity to TUNEL labeling. No step of the protocol tested here (epitope retrieval method, proteinase K concentration, proteinase K incubation time, TdT enzyme concentration) significantly altered the degree of positive staining, indicating that the influence of these upstream processing steps cannot be easily remedied through staining protocol manipulation. This study highlights the limitations of archived fixed coral tissues for immunohistochemical analysis.

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