Project description:Coral bleaching occurs in response to numerous abiotic stressors, the ecologically most relevant of which is hyperthermic stress due to increasing seawater temperatures. Bleaching events can span large geographic areas and are currently a potent threat to coral reefs worldwide. Much effort has been focused on understanding the molecular and cellular events underlying bleaching, and these studies have mainly utilized heat and light stress regimes. In an effort to determine whether different stressors share common bleaching mechanisms, we used cDNA microarrays for the corals Acropora palmata and Montastraea faveolata (containing > 10,000 features) to measure differential gene expression during darkness stress. This is the first coral microarray experiment aimed at darkness stress, and the first for these species to interrogate gene expression at such a large scale. Our results reveal a striking transcriptomic response to darkness in A. palmata involving chaperone and antioxidant up-regulation, growth arrest, and metabolic modifications. As these responses were also measured during thermal stress, our results suggest that different stressors may share common bleaching mechanisms. Furthermore, our results point to ER stress as a critical cellular event involved in darkness-specific (and possibly more general) molecular bleaching mechanisms. On the other hand, we identified a meager transcriptomic response to darkness in M. faveolata where gene expression differences between host colonies and/or sampling locations were greater than differences between control and stressed fragments. To this end, we discuss the importance of factors related to host genotype, Symbiodinium genotype, and the abiotic environment that influence host gene expression and thereby can hinder an investigator’s ability to measure gene expression during a condition of interest. We employed a reference design where all control and dark-stressed samples were compared to a pooled reference aRNA sample composed of aRNA from all fragments. Since all RNA samples were compared to the reference sample, direct comparisons of gene expression across all time points and conditions can be performed.

Project description:Coral bleaching occurs in response to numerous abiotic stressors, the ecologically most relevant of which is hyperthermic stress due to increasing seawater temperatures. Bleaching events can span large geographic areas and are currently a potent threat to coral reefs worldwide. Much effort has been focused on understanding the molecular and cellular events underlying bleaching, and these studies have mainly utilized heat and light stress regimes. In an effort to determine whether different stressors share common bleaching mechanisms, we used cDNA microarrays for the corals Acropora palmata and Montastraea faveolata (containing > 10,000 features) to measure differential gene expression during darkness stress. This is the first coral microarray experiment aimed at darkness stress, and the first for these species to interrogate gene expression at such a large scale. Our results reveal a striking transcriptomic response to darkness in A. palmata involving chaperone and antioxidant up-regulation, growth arrest, and metabolic modifications. As these responses were also measured during thermal stress, our results suggest that different stressors may share common bleaching mechanisms. Furthermore, our results point to ER stress as a critical cellular event involved in darkness-specific (and possibly more general) molecular bleaching mechanisms. On the other hand, we identified a meager transcriptomic response to darkness in M. faveolata, where gene expression differences between host colonies and/or sampling locations were greater than differences between control and stressed fragments. To this end, we discuss the importance of factors related to host genotype, Symbiodinium genotype, and the abiotic environment that influence host gene expression and thereby can hinder an investigator’s ability to measure gene expression during a condition of interest. We employed a reference design where all control and dark-stressed samples were compared to a pooled reference aRNA sample composed of aRNA from all fragments. Since all RNA samples were compared to the reference sample, direct comparisons of gene expression across all time points and conditions can be performed.

Project description:The emergence of genomic tools for reef-building corals and symbiotic anemones comes at a time when alarming losses in coral cover are being observed worldwide. These tools hold great promise in elucidating novel and unforeseen cellular processes underlying the successful mutualism between corals and their algal endosymbionts (Symbiodinium spp.). Since thermal stress triggers a breakdown in the symbiosis (coral bleaching), measuring the transcriptomic response to thermal stress-induced bleaching offers an extraordinary view of the cellular processes specific to coral-algal symbioses. In the present study, we utilized a cDNA microarray containing 2,059 genes of the Caribbean Elkhorn coral Acropora palmata to identify genes differentially expressed upon thermal stress. Fragments from four separate colonies were exposed to elevated temperature (3ËC increase) for two days, and samples were frozen for microarray analysis after 24 and 48 hours. Fragments experienced a 60% reduction in algal cell density after two days. 204 genes were differentially expressed in samples collected one day after thermal stress; in samples collected after two days, 104 genes. Annotations of the differentially expressed genes indicate a conserved cellular stress response in A. palmata involving: 1) growth arrest; 2) chaperone activity; 3) nucleic acid stabilization and repair; and 4) the removal of damaged macromolecules. Other differentially expressed processes include sensory perception, metabolite transfer between host and symbiont, nitric oxide signaling, and modifications to the actin cytoskeleton and extracellular matrix. The results are also compared to those from a previous coral microarray study of thermal stress in Montastraea faveolata. The field experiment was performed at the Smithsonian Tropical Research Instituteâs Bocas del Toro field station in PanamÃ¡ during September and October 2006. Four colonies of A. palmata were sampled from two separate reefs 21 km apart (two colonies from Isla Solarte â 9o19â56.78â N and 82o12â54.65â W, and two colonies from Cayos Zapatillas â 9o15â08.79â N and 82o02â24.63â W). Each colony was broken into six fragments using a hammer and chisel. For each colony, three fragments were placed in a control aquarium, and three fragments were placed in an experimental aquarium fitted with two 200-Watt aquarium heaters, such that each colony was represented by a pair of aquaria (total of three control and three heated aquaria, all 75-liter). The three control aquaria were placed in one large fiberglass pond with continuous water flow, and the three experimental aquaria were placed in another large pond. All aquaria were exposed to shaded ambient light, and each aquarium was a closed system (but contained a pump to generate continuous water flow). Fragments were kept at a depth of ~25cm. HOBO Pendant Temperature/Light Data Loggers (Onset Corp UA-002-64) recorded temperature and light data every three minutes. These data loggers are not designed to measure photosynthetically active radiation (PAR â 400-700nm), as only ~30% of the measured light is in the range of PAR. For this reason, relative light levels in the aquaria are reported (expressed as the percentage of the average 10am to 2pm light intensity measured on a reef ~4m deep in Bocas del Toro (9o22â68.4â N and 82o18â24.6â W) during September and October 2007). Light intensity differed slightly between the four aquaria fitted with HOBOs (control aquaria 1 â 43%; control aquaria 2 â 46%; heated aquaria 1 â 35%; and heated aquaria 2 â 34% of reef light). After an acclimation period of four days at the natural temperature of the seawater system (mean temperature = 30.29Â±0.07oC), a fragment from each control and experimental aquaria was sampled (t0C and t0H). After time zero sampling, the heaters in each of the experimental aquaria were turned on. The temperatures of the experimental aquaria increased to ca. 32oC over three hours. The mean temperature of the control aquaria during the entire experiment was 29.74Â±0.03oC, and the mean temperature of the heated aquaria was 32.72Â±0.32oC. Control and experimental fragments were sampled again one day (1dC and 1dH) and two days (2dC and 2dH) after turning on the heaters. Heated fragments from one of the colonies (col3) showed extreme bleaching after one day of thermal stress. The remaining fragment of col3 was removed at this time to avoid fouling of the water due to death. Thus, there are four replicates for t0C, t0H, 1dC, and 1dH, and three replicates for 2dC and 2dH. All samples were taken at night. Fragments were frozen in liquid nitrogen. We employed a reference design where all control and heat-stressed samples were compared to a pooled reference aRNA sample composed of aRNA from the four t0C fragments. Since all RNA samples were compared to the reference sample, direct comparisons of gene expression across all time points and conditions can be performed.

Project description:Naval training exercises involving live ordnance can introduce munitions constituents (MCs) such as 1,3,5-trinitro-1,3,5 triazine (RDX) into the marine environment posing a potential environmental hazard to reef organisms, including corals. We developed a bioinformatic infrastructure and high-density microarray for a coral consortium and assessed the effects of RDX bioaccumulation on gene expression related to coral and endosymbiont health in the reef building coral (Acropora formosa). High-throughput sequencing and assembly of the transcriptomes for A. formosa and all eukaryotic endosymbionts yielded 189,616 unique sequences and 25,003 significant functional matches to protein-coding genes. Functional annotation and metabolic pathway associations were also developed. The bioinformatics base was transitioned to custom 15,000 probe microarrays that were used to assess RDX effects on gene expression in the A. formosa coral consortium. Coral fragments were exposed to RDX (0.5, 1, 2, 4, and 8 mg/L) for 5d in a controlled laboratory experiment. RDX readily accumulated into coral tissues; however, bioconcentration was minimal (bioconcentration factor = 1.09-1.50). RDX caused no significant changes in zooxanthellae tissue densities, however a significant (p<0.05) 40% increase in mucocytes was observed in the 8 mg/L exposure indicating a mucosal protective response to RDX exposure. Investigation of T-RFLP profiles indicated significant differences in bacterial community composition inhabiting the coral surface microlayer of Acropora sp. between control and RDX-exposed coral as among exposure concentrations. Differential expression of transcripts increased with increasing RDX concentration where 126, 195 and 272 transcripts were differentially expressed in the 0.5, 2.0 and 8 mg/L RDX treatments, respectively. The commonality in differentially expressed transcripts (DET) among exposure concentrations ranged from 9.9 to 29.0% where the lowest commonality was observed between the most disparate RDX exposure concentrations. Increasing RDX concentrations caused an increasing proportion of the number of transcripts differentially expressed in symbionts relative to corals. Further, a trend toward decreased transcript expression in symbionts in response to increasing RDX concentration was observed where 20.0% of differentially expressed transcripts had decreased expression at the 0.5 mg/L concentration, whereas 80.4% had decreased expression at the 8 mg/L concentration. Investigation of KEGG orthology for DET indicated potential impacts of RDX on a variety of molecular pathways, predominantly in endosymbionts compared to the coral host. Prominent effects of RDX exposure on pathways included enrichment of DET involved in carbohydrate metabolism, amino acid metabolism, energy metabolism, lipid metabolism, metabolism of cofactors and vitamins, environmental information processing and cellular processes. Fragments of the living branched coral Acropora formosa were obtained from Oceans, Reefs and Aquaria (http://www.orafarm.com). Ten gallon aquaria were used to expose 5 coral fragments to control or RDX exposure conditions (0.49, 0.93, 1.77, 3.67 and 7.18 mg/L, measured concentrations). The microarray hybridization experiment included 3 biological replicates for the 0.5, 2, and 8 mg/L RDX conditions and 4 biological replicates for the control.

Project description:The Manila clam (Ruditapes philippinarum) is a cultured bivalve species with high worldwide commercial importance. Nevertheless, diseases can cause high economical losses. For this reason, the study of immune genes in bivalve mollusks has increased in the last years. The present work describes the construction of the first R. philippinarum microarray containing immune-related hemocyte sequences and its application for the study of the gene transcription profiles of hemocytes from clams challenged with Vibrio alginolyticus through a time course. A comparative analysis of gene expression was conducted between R. philippinarum infected and non-infected by V. alginolyticus clam hemocytes. Clams (n=100) were notched in the shell next to the adductor muscles and injected with 100 Âµl of Vibrio alginolyticus, strain TA15, (10^8 UFC/ml in PBS) to mimic an intramuscular infection. Controls (n=100) were injected with 100 Âµl of PBS. After stimulation, clams were returned to the tanks and maintained at 15ÂºC until sampling at 3, 8, 24, and 72 hours after challenge Hemolymph (1 ml) was withdrawn from the adductor muscle of the clams with a 0.5mm diameter (25G) disposable needle. Hemolymph from four individuals was pooled and biological replicates were taken at each sampling point. Hemolymph was centrifuged at 4Â°C at 3000 g for 10 minutes. The pellet was resuspended in 250 Âµl of Trizol (Invitrogen). Total RNA isolation was conducted following the manufacturer's specifications in combination with the RNeasy mini kit (Qiagen) for RNA purification after DNase I treatment. Gene expression profiling was performed using an R. philippinarum oligo-DNA microarray of 13,671 probes based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA at a resolution of 5 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. Feature Extraction (FE) 9.5 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.

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Microarray studies of darkness stress and bleaching in the Caribbean corals Acropora palmata and Montastraea

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