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:Projected elevation of seawater temperatures poses a threat to the reproductive success of Caribbean reef-building corals that have planktonic development during the warmest months of the year. This study examined the transcriptomic changes that occurred during embryonic and larval development of the elkhorn coral, Acropora palmata, at a non-stressful temperature (28Â°C) and further assessed the effects of two elevated temperatures (30Â°C and 31.5Â°C) on these expression patterns. Using cDNA microarrays, we compared expression levels of 2,051 genes from early embryos and larvae at multiple developmental stages (including pre-blastula, blastula, gastrula, and planula stages) at each of the three temperatures. At 12 hours post-fertilization in 28Â°C treatments, genes involved in cell replication/cell division and transcription were up-regulated in A. palmata embryos, followed by a reduction in expression of these genes during later growth stages. From 24.5 to 131 hours post-fertilization at 28Â°C, A. palmata altered its transcriptome by up-regulating genes involved in protein synthesis and metabolism. Temperatures of 30Â°C and 31.5Â°C caused major changes to the A. palmata embryonic transcriptomes, particularly in the samples from 24.5 hpf post-fertilization, characterized by down-regulation of numerous genes involved in cell replication/cell division, metabolism, cytoskeleton, and transcription, while heat shock genes were up-regulated compared to 28Â°C treatments. These results suggest that increased temperature may cause a breakdown in proper gene expression during development in A. palmata by down-regulation of genes involved in essential cellular processes, which may lead to the abnormal development and reduced survivorship documented in other studies. Our experimental setup followed a reference design where all samples were hybridized against the same pool made up of equal amounts of RNA from all samples in the experiment. Biological duplicate samples were used for each temperature at each developmental time period. Common reference samples were labeled with Cy3 dye, while temperature samples were labeled with Cy5 dye. Microarrays for A. palmata contained 2,051 coding sequences, of which 54.3% had functional annotations as determined by homology to known genes.

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Acropora palmata

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