Lichens are defined as symbiotic associations composed of a fungal partner, the mycobiont, and one or more photosynthetic partners, the photobiont (1). A currently employed method for the identification of photobionts is the culture of photobiont from the lichen, but this method employs a labor intensive and long cultivation period, thus identification has been neglected. Out of the approximatelyl4,000 lichen described, only about 4% oflichen photobionts have been identified to species (1). In this study we investigated the feasibility of developing a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) identification system for rapidly identifying lichen algae of the genus Trebouxia (In this study we consider Pseudo-Trebouxia part of the genus Trebouxia). DNA was isolated and purified from cultures of each Trebouxia species. A 1300 hp fragment of the 5' region of the nuclear-encoded large subunit (26S) ribosomal RNA genes was amplified by PCR (2). This 5'region of the 26S region is considered to be a byper-variable region because it differs amongst Trebouxia (2) making it a good candidate for RFLP. The sequences were then analyzed with restriction analysis software to determine restriction maps and individual virtual RFLP patterns. Patterns were constructed using the program SPR Opt (SNP and PCR-RFLP Optimization) allowing each Trebouxia species to be identified by a distinctive restriction pattern. We were unable to validate the key due to contamination of materials, which lead to inconclusive data. Future experiments aim to validate the key by comparing the virtual RFLP patterns to the actual patterns obtained for each type culture of each species.

The Marine sponge Cinachyrella sp used in this study are commonly found in offshore South Florida and Caribbean waters and appeared to be resilient in closed system aquaculture. Marine sponges host diverse bacterial symbionts that are distinct compared to bacteria found in ambient seawater, however the roles of a large fraction of the bacterial community in marine sponges are unknown. Comparison of symbiotic to aposymbiotic (bacteria-free) sponges could provide information about interactions (metabolic and physiologic) between the bacteria and sponge. In this study, a single Cinachyrella kuekenthali individual was subsectioned into explants (N=240) in order to provide identical bacterial communities to perform comparative studies. Presence of photosymbionts was also analyzed by characterizing bacterial communities from varying light and dark conditions. Tools for characterization included transmission electron microscopy (TEM) and 16S rRNA sequence analysis obtained from Illumina Miseq. High throughput DNA sequencing revealed bacterial taxa belonging to phyla Thaumarchaeota, Chloroflexi, Nitrospira, Acidobacteria and Verrucomicrobia persist in the explants. This study also demonstrated that antibiotics (Ampicillin, Tetracycline, Penicillin-Streptomycin and combination of all) can alter the bacterial community in the marine sponge C. kuekenthali explants in vitro. Bacterial communities of explants treated with different antibiotics were statistically (Unifrac and Bray-Curtis analysis) different from controls (p-value < 0.001, R2=41%). Penicillin-streptomycin and cocktail of antibiotics treatment contributed to the highest difference in the bacterial communities. Also, bacterial communities of explants at difference time points treated with corresponding antibiotics were also statistically significant (p-value<0.05, R2=15%). TEM observations of denatured nucleic acid and osmotic lysis of bacteria, due to the effect of antibiotics were observed, creating a LMA mesohyl. However light versus dark conditions did not produce any statistically significant difference in beta diversity between bacterial communities. These interdisciplinary results indicate that while individual bacterial symbiont taxa may persist after community disruption, significant changes in the overall composition of the bacterial symbiont population can be created