Investigations of cellular dynamics during bleaching in the symbiotic anemone, Aiptasia pallida
Type of Degreedissertation
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The global decline of coral reefs continues at an increasing rate despite efforts to identify key cellular interactions responsible for the breakdown of these essential ecosystems. Coral bleaching involves the loss of essential, photosynthetic dinoflagellates (Symbiodinium) from host gastrodermal cells in response to temperature and/or light stress conditions. Although numerous potential cellular bleaching mechanisms have been proposed, few studies have investigated the early host stress response when symbiotic breakdown is initiated. In this investigation, both cellular and molecular techniques were employed in order to 1) carefully examine and characterize host anthozoan tissues at multiple stages of the symbiosis, including: i) healthy symbiotic, ii) several stages of active heat stress induced-bleaching, iii) aposymbiotic (symbiont-free); 2) document and describe all cellular bleaching mechanisms that occur during heat stress treatment; 3) quantify any observed cellular bleaching mechanisms in both symbiotic and aposymbiotic anemones to determine whether the response is host derived; 4) conduct a gene expression analysis on heat stressed symbiotic A. pallida anemones in order to quantify the response using RNA-Seq on an Illumina platform. First, histological and ultrastructural examinations were conducted using light and transmission electron microscopy on healthy symbiotic anemeone tentacle tissues in order to establish baseline information. A detailed ultrastructural analysis was conducted of numerous cellular regions and compared to previous cnidarian literature. This study provided an essential diagnostic analysis of normal healthy tissues that was used to assess the health condition of the anemones during the subsequent heat stress treatments. Bleaching was induced by exposing both symbiotic and/or aposymbiotic A. pallida anemones, to ~32.5oC at 120 μmols irradiance for 12 h followed by 12 h in the dark at 24 oC daily for 2 days. Samples were taken thoughout the 48 h period. Ultrastructural examination revealed numerous autophagic structures and associated cellular degradation in tentacle tissues after ~12 h of stress treatment and also after 12 h of exposure to the known autophagy inducer, rapamycin. Additionally, symbionts were observed detaching from highly degraded gastrodermal cells in an apocrine-like manner. The abundance of autophagic structures was quantified in gastrodermal and epidermal tissues of symbiotic and aposymbiotic tissues, and also in rapamycin treated tissues using ImagePro Plus 7.0 software. Results from the RNA-Seq analysis revealed the highest levels of differential gene expression in symbiotic A. pallida anemones after 3 h during a 48 h thermal stress treatment, suggesting that the gene expression profile changes in early stages of the host stress response. In addition, several key processes were identified that are involved in the host response, including stress response, protein degradation/synthesis, calcium homeostasis and others, which provides a better understanding of the genetic determinants of stress tolerance in a host anthozoan. This investigation provided the first ultrastructural evidence of host autophagic degradation during thermal stress in a cnidarian system and supports earlier suggestions that autophagy is an active cellular mechanism during early stages of bleaching.
- S. D. Hanes Dissertation-final1.pdf