ItemDiterpene biosynthesis by the dinoflagellate symbiont of the Caribbean gorgonian Pseudopterogorgia bipinnata(Marine Ecology Progress Series, 2005-11) Santiago-Vázquez, Lory Z.Chemical analysis of the dinoflagellate symbiont of Pseudopterogorgia bipinnata (Verrill) revealed higher concentrations of kallolides and related diterpenes than are present in the intact holobiont. Further, biosynthetic studies of the dinoflagellate with 3 H-labeled geranylgeranyl diphosphate indicated that this alga is capable of kallolide biosynthesis. These data indicate that the symbiotic zooxanthellae of P, bipinnata, as we have previously shown with R elisabethae, are capable of biosynthesizing diterpenes previously ascribed solely to the host coral. Analyses of R bipinnata colonies indicate significant chemical variation within this species, and 4 distinct chemotypes have been identified. Of these, only Chernotype A was found to both contain diterpenes and be capable of diterpene biosynthesis. We analyzed small subunit rDNA restriction fragment length polymorphism patterns and internal transcribed spacer sequences of the zooxanthellae associated with the 4 chemotypes for genetic differences that could explain the chemical differences. Both studies determined that all chemotypes are predominantly populated by the symbiont Symbiodinium sp. Clade B. ItemToxin secretion in response to oxidative stress and programmed cell death in the cyanobacteria Microcystis aeruginosa from the St. Lucie River, Florida(Aquatic Toxicology, 2006) Santiago-Vázquez, Lory Z.An unprecedented bloom of the cyanobacterium Microcystis aeruginosa Kütz. occurred in the St. Lucie Estuary, FL in the summer of 2005. Samples were analyzed for toxicity by ELISA and by use of the polymerase chain reaction (PCR) with specific oligonucleotide primers for the mcyB gene that has previously been correlated with the biosynthesis of toxic microcystins. Despite the fact that secreted toxin levels were relatively low in dense natural assemblages (3.5 microg l(-1)), detectable toxin levels increased by 90% when M. aeruginosa was stressed by an increase in salinity, physical injury, application of the chemical herbicide paraquat, or UV irradiation. The application of the same stressors caused a three-fold increase in the production of H(2)O(2) when compared to non-stressed cells. The application of micromolar concentrations of H(2)O(2) induced programmed cell death (PCD) as measured by a caspase protease assay. Catalase was capable of inhibiting PCD, implicating H(2)O(2) as the inducing oxidative species. Our results indicate that physical stressors induce oxidative stress, which results in PCD and a concomitant release of toxin into the surrounding media. Remediation strategies that induce cellular stress should be approached with caution since these protocols are capable of releasing elevated levels of microcystins into the environment. ItemComparison of two total RNA extraction protocols using the marine gorgonian coral Pseudopterogorgia elisabethae and its symbiont Symbiodinium sp(Electronic Journal of Biotechnology, 2006-10) Santiago-Vázquez, Lory Z.Marine invertebrates such as soft corals are important sources of secondary metabolites with promising biomedical applications and commercial value. RNA isolation in conjunction with reverse-transcriptase polymerase chain reaction (RT-PCR) are valuable tools utilized to study the molecular elements involved in secondary metabolite production and functional genomics. Two total RNA extraction protocols were compared using fresh tissue and flash frozen preparations from the coral Pseudopterogorgia elisabethae and from its symbiont Symbiodinium sp. isolated using RNeasy minicolumns (Qiagen®) and Trizol reagent (Invitrogen®). In general, higher yields were obtained by using Trizol reagent when compared to RNeasy. No significant differences were observed in RNA yield when live or flash frozen tissue was used. However, flash frozen holobiont tissue isolated by Trizol resulted in the highest RNA yield of all preparations analyzed. To conclude, both protocols are suitable for RNA isolation. Trizol is recommended if higher yields are the primary concern, but RNeasy is recommended if time is an issue. ItemCryopreservation of the dinoflagellate symbiont of the octocoral Pseudopterogorgia elisabethae as a source of the pseudopterosins(Marine Biology, 2007) Santiago-Vázquez, Lory Z.In this paper we describe a cryopreservation protocol followed by the culture of Symbiodinium sp. isolated from the Caribbean gorgonian Pseudopterogorgia elisabethae as a potential renewable source of the dinoflagellate symbiont. Four different freezing protocols were designed: a controlled cooling device designed to cool at 1°C/min, a three-step protocol (−20°C for 2h, −70°C for 2h, liquid nitrogen-LN2), a two-step protocol (−70°C for 2h, LN2), and a one-step protocol (LN2). All cells were stored in LN2 after cryopreservation. The cryoprotective agents (CPA) used were ethanol (EtOH) and methanol (MeOH) at 10 and 20%, and seawater (FSW) was used as a control. Viability measurements using cell counts showed that all cryopreservation protocols were relatively successful, and no trends were observed regarding freezing protocol or CPA used. After 19weeks in culture the viability of samples which had high biomass was determined by the fluorescent assay CellTiter Blue™. The most viable cultures were those cryopreserved by a two-step protocol using 20% MeOH or 20% EtOH as a CPA. A genetic examination of the DNA of these samples using Symbiodinium-specific PCR primers confirmed that the composition of the culture had not changed. For the first time, we report that Symbiodinium sp. isolated from a gorgonian can be cryopreserved and subsequently cultured successfully. ItemDiversity of the bacterial communities associated with the azooxanthellate deep water octocorals Leptogorgia minimata, Iciligorgia schrammi and Swiftia exertia(Marine Biotechnology, 2007-10) Santiago-Vázquez, Lory Z.This study examined the microbiota associated with the marine azooxanthellate octocorals Leptogorgia minimata, Swiftia exertia, and Iciligorgia schrammi collected from moderate depths (45 m). Traditional aerobic plate culture, fluorescence in situ hybridization (FISH), and molecular identification of the 16S rDNA region were used for this purpose. In general, cultures were found to be selective for Gammaproteobacteria, Alphaproteobacteria, and Firmicutes. Interestingly, FISH counts for Firmicutes in the whole coral (holobiont) were near the detection limit of this assay, representing less than 6% of the total detectable microbiota in all counts. Proteobacteria, especially Alpha- and Gammaproteobacteria, made up the majority of the total microbiota in the holobionts. In addition, the absence of zooxanthellae in these three corals was confirmed by the use of polymerase chain reaction (PCR) and dinoflagellate-specific primers, and spectrophotometric chlorophyll pigment measurements. No evidence of zooxanthellae could be found in any of the corals by either of these techniques. This is the first study examining the microbiota marine octocorals, which grow at moderate depth (40 to 100 m) in the absence of direct sunlight. ItemThe diversity of the bacterial communities associated with the azooxanthellate deep water octocoral Cirrhipathes lutkeni(International Society for Microbial Ecology Journal, 2007-12) Santiago-Vázquez, Lory Z.This study examined the symbiotic microbiota of the hexacoral Cirrhipathes lutkeni using traditional plate culture, fluorescence in situ hybridization (FISH) and 16S rDNA characterization. FISH counts for the whole coral (holobiont) showed a major presence of gamma-Proteobacteria (22%) and Actinobacteria (19%), followed by alpha-Proteobacteria (14%), Firmicutes (9%), Cytophaga-Flavobacterium (7%), beta-Proteobacteria (6%) and Chloroflexi (2%). In contrast to the diversity observed by FISH, plate cultures were found to be selective for gamma-Proteobacteria (22 cultures) with the exception of an Actinobacterium. The methods employed in this study detected 76% of all microbes estimated by DAPI staining of C. lutkeni homogenates. The absence of zooxanthellae in this particular hexacoral was confirmed by PCR and spectrophotometry using fresh tissue isolated from the holobiont. This is the first study describing the microbial associations of shallow-water hexacorallia, which opens further insight into coral microbial ecology and may enhance the search for novel natural products in the near future. ItemPartial transcriptome profiles of soft corals via suppression subtractive hybridization PCR(Electronic Journal of Biotechnology, 2011) Santiago-Vázquez, Lory Z. ItemA microbial community analysis of the octocoral Eunicea fusca(Electronic Journal of Biotechnology, 2012-08) Santiago-Vázquez, Lory Z.While there is a significant and growing body of knowledge describing the microbial communities of marine invertebrates such as sponges, there are very few such studies focused on octocorals. The octocoral Eunicea fusca is common on reefs in various regions of the Caribbean and has been the subject of natural product investigations. As part of an effort to describe the microbial community associated with octocorals, a culture-independent analysis of the bacterial community of E. fusca was conducted. Specifically, a 16S rDNA clone library analysis was performed to provide baseline data. A total of 40 bacteria members from 11 groups were found. In general, Proteobacteria were the dominant group with a total of 24 species and α-Proteobacteria represented the highest percentage of bacteria associated with E. fusca (27.5%). Other prominent groups observed were Acidobacteria, Actinobacteria, Cyanobacteria, Planctomycetes, δ-Proteobacteria, Lentisphaerae and Nitrospirae. This is the first analysis of bacterial populations associated with the gorgonian E. fusca.