Gene expression of endangered coral (Orbicella spp.) in flower garden banks National Marine Sanctuary after Hurricane Harvey

About 190 km south of the Texas–Louisiana border, the East and West Flower Garden Banks (FGB) have maintained > 50% coral cover with infrequent and minor incidents of disease or bleaching since monitoring began in the 1970s. However, a mortality event, affecting 5.6 ha (2.6% of the area) of the East FGB, occurred in late July 2016 and coincided with storm-generated freshwater runoff extending offshore and over the reef system. To capture the immediate effects of storm-driven freshwater runoff on coral and symbiont physiology, we leveraged the heavy rainfall associated with Hurricane Harvey in late August 2017 by sampling FGB corals at two time points: September 2017, when surface water salinity was reduced (∼34 ppt); and 1 month later when salinity had returned to typical levels (∼36 ppt in October 2017). Tissue samples (N = 47) collected midday were immediately preserved for gene expression profiling from two congeneric coral species (Orbicella faveolata and Orbicella franksi) from the East and West FGB to determine the physiological consequences of storm-derived runoff. In the coral, differences between host species and sampling time points accounted for the majority of differentially expressed genes. Gene ontology enrichment for genes differentially expressed immediately after Hurricane Harvey indicated increases in cellular oxidative stress responses. Although tissue loss was not observed on FGB reefs following Hurricane Harvey, our results suggest that poor water quality following this storm caused FGB corals to experience sub-lethal stress. We also found dramatic expression differences across sampling time points in the coral’s algal symbiont, Breviolum minutum. Some of these differentially expressed genes may be involved in the symbionts’ response to changing environments, including a group of differentially expressed post-transcriptional RNA modification genes. In this study, we cannot disentangle the effects of reduced salinity from the collection time point, so these expression patterns could also be related to seasonality. These findings highlight the urgent need for continued monitoring of these reef systems to establish a baseline for gene expression of healthy corals in the FGB system across seasons, as well as the need for integrated solutions to manage stormwater runoff in the Gulf of Mexico.https://www.frontiersin.org/articles/10.3389/fmars.2019.00672/fullPublished versionPublished versio

Suppression Subtractive Hybridization PCR Isolation of cDNAs from a Caribbean Soft Coral

Transcriptomic studies of marine organisms are still in their infancy. A partial, subtracted expressed sequence tag (EST) library of the Caribbean octocoral Erythropodium caribaeorum and the sea fan Gorgonia ventalina has been analyzed in order to find novel genes or differences in gene expression related to potential secondary metabolite production or symbioses. This approach entails enrichment for potential non- housekeeping genes using the suppression subtractive hybridization (SSH) polymerase chain reaction (PCR) method. More than 500 expressed sequence tags (ESTs) were generated after cloning SSH products, which yielded at least 53 orthologous groups of proteins (COGs) and Pfam clusters, including transcription factors (Drosophila Big Brother), catalases, reverse transcriptases, ferritins and various hypothetical protein sequences. A total of 591 EST sequences were deposited into GenBank [dbEST: FL512138 - FL512331, and GH611838]. The results represent proof of concept for enrichment of unique transcripts over housekeeping genes, such as actin or ribosomal genes, which comprised approximately 17% of the total dataset. Due to the gene and sequence diversity of some ESTs, such sequences can find utility as molecular markers in current and future studies of this species and other soft coral biogeography, chemical ecology, phylogenetics, and evolution

On a Reef Far, Far Away: Anthropogenic Impacts Following Extreme Storms Affect Sponge Health and Bacterial Communities

Terrestrial runoff can negatively impact marine ecosystems through stressors including excess nutrients, freshwater, sediments, and contaminants. Severe storms, which are increasing with global climate change, generate massive inputs of runoff over short timescales (hours to days); such runoff impacted offshore reefs in the northwest Gulf of Mexico (NW GoM) following severe storms in 2016 and 2017. Several weeks after coastal flooding from these events, NW GoM reef corals, sponges, and other benthic invertebrates ∼185 km offshore experienced mortality (2016 only) and/or sub-lethal stress (both years). To assess the impact of storm-derived runoff on reef filter feeders, we characterized the bacterial communities of two sponges, Agelas clathrodes and Xestospongia muta, from offshore reefs during periods of sub-lethal stress and no stress over a three-year period (2016—2018). Sponge-associated and seawater-associated bacterial communities were altered during both flood years. Additionally, we found evidence of wastewater contamination (based on 16S rRNA gene libraries and quantitative PCR) in offshore sponge samples, but not in seawater samples, following these flood years. Signs of wastewater contamination were absent during the no-flood year. We show that flood events from severe storms have the capacity to reach offshore reef ecosystems and impact resident benthic organisms. Such impacts are most readily detected if baseline data on organismal physiology and associated microbiome composition are available. This highlights the need for molecular and microbial time series of benthic organisms in near- and offshore reef ecosystems, and the continued mitigation of stormwater runoff and climate change impacts

Fatty Acid Composition and Oxylipin Enzymatic Production in the Tropical Coenocytic Marine Chlorophyte Acetabularia crenulata

Acetabularia crenulata was utilized as a model to study in vitro and in vivo enzymatic oxylipin production.The fatty acid composition of A. crenulata was analyzed. Cell free extracts were derivatized by two methylation procedures, saponification and diazomethane, to compare how the two influenced fatty acid composition. Qualitatively, no significant differences were observed. The major fatty acids observed in both preparations were palmitic, palmitoleic, oleic, linoleic, linolenic and octadecatetraenoic acid. No major differences were observed in fatty acid composition of cell free extracts of alga grown in the lab and of alga collected in the wild. The fatty acid composition of chloroplasts showed the biggest difference in composition when compared to other preparations, excluding palmitoleic and octadecatetraenoic acid from the major fatty acids. The amount of C16:3 was very low in chloroplast preparations, therefore it was hypothesized that chloroplasts of A. crenulata followed the "eukaryotic" for MGDG synthesis.In general, the fatty acid composition is similar to that of other marine Chlorophytes.Cell free extracts and live cells incubated with oleic acid were shown to produce the LOX metabolites 13-HODE, 13-HOTrE and 8-HETE. 13-HODE was found to be an endogenous metabolite. The same preparations incubated with arachidonic acid synthesized 5-, 8-, 11-, 12-, and 15-HETE. In addition, cell free extracts incubated with arachidonic acid also synthesized 13-HODE and 15- HEPE. The data shows enzymatic activity of different LOX isoenzymes, with a preference for w-6 oxidation.In vitro HETE production over time displayed classic enzyme kinetics. In vivo HETE production in the dark and in the light demonstrated that some oxylipins are preferentially synthesized inside the chloroplasts while others are preferentially synthesized outside. The possible production of an LT-type molecule outside the chloroplast was proposed. A role of hydroxy fatty acids in growth was not observed.In conclusion, LOX-mediated in vitro and in vivo oxylipin production was demonstrated in A. crenulata and a possible role of these metabolites in the physiological processes of the organism was suggested

Comparison of two total RNA extraction protocols using the marine gorgonian coral Pseudopterogorgia elisabethae and its symbiont Symbiodinium sp.

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

Gene Expression of Endangered Coral (\u3ci\u3eOrbicella\u3c/i\u3e spp.) in Flower Garden Banks National Marine Sanctuary After Hurricane Harvey

About 190 km south of the Texas–Louisiana border, the East and West Flower Garden Banks (FGB) have maintained \u3e 50% coral cover with infrequent and minor incidents of disease or bleaching since monitoring began in the 1970s. However, a mortality event, affecting 5.6 ha (2.6% of the area) of the East FGB, occurred in late July 2016 and coincided with storm-generated freshwater runoff extending offshore and over the reef system. To capture the immediate effects of storm-driven freshwater runoff on coral and symbiont physiology, we leveraged the heavy rainfall associated with Hurricane Harvey in late August 2017 by sampling FGB corals at two time points: September 2017, when surface water salinity was reduced (∼34 ppt); and 1 month later when salinity had returned to typical levels (∼36 ppt in October 2017). Tissue samples (N = 47) collected midday were immediately preserved for gene expression profiling from two congeneric coral species (Orbicella faveolata and Orbicella franksi) from the East and West FGB to determine the physiological consequences of storm-derived runoff. In the coral, differences between host species and sampling time points accounted for the majority of differentially expressed genes. Gene ontology enrichment for genes differentially expressed immediately after Hurricane Harvey indicated increases in cellular oxidative stress responses. Although tissue loss was not observed on FGB reefs following Hurricane Harvey, our results suggest that poor water quality following this storm caused FGB corals to experience sub-lethal stress. We also found dramatic expression differences across sampling time points in the coral’s algal symbiont, Breviolum minutum. Some of these differentially expressed genes may be involved in the symbionts’ response to changing environments, including a group of differentially expressed post-transcriptional RNA modification genes. In this study, we cannot disentangle the effects of reduced salinity from the collection time point, so these expression patterns could also be related to seasonality. These findings highlight the urgent need for continued monitoring of these reef systems to establish a baseline for gene expression of healthy corals in the FGB system across seasons, as well as the need for integrated solutions to manage stormwater runoff in the Gulf of Mexico

Suppression subtractive hybridization PCR isolation of cDNAs from a Caribbean soft coral

Transcriptomic studies of marine organisms are still in their infancy. A partial, subtracted expressed sequence tag (EST) library of the Caribbean octocoral Erythropodium caribaeorum and the sea fan Gorgonia ventalina has been analyzed in order to find novel genes or differences in gene expression related to potential secondary metabolite production or symbioses. This approach entails enrichment for potential non-“housekeeping” genes using the suppression subtractive hybridization (SSH) polymerase chain reaction (PCR) method. More than 500 expressed sequence tags (ESTs) were generated after cloning SSH products, which yielded at least 53 orthologous groups of proteins (COGs) and Pfam clusters, including transcription factors (Drosophila Big Brother), catalases, reverse transcriptases, ferritins and various “hypothetical” protein sequences. A total of 591 EST sequences were deposited into GenBank [dbEST: FL512138 - FL512331, GH611838, and HO061755-HO062154]. The results represent proof of concept for enrichment of unique transcripts over housekeeping genes, such as actin or ribosomal genes, which comprised approximately 17% of the total dataset. Due to the gene and sequence diversity of some ESTs, such sequences can find utility as molecular markers in current and future studies of this species and other soft coral biogeography, chemical ecology, phylogenetics, and evolution