High natural gene expression variation in the reef-building coral Acropora millepora: Potential for acclimative and adaptive plasticity
ABSTRACT: This experiment assessed the natural gene expression variation present between colonies of the Indo-Pacific reef-building coral Acropora millepora, and additionally explored whether gene expression differed between two different intron haplotypes according to intron 4-500 in a carbonic anhydrase homolog. This study found no correspondence between host genotype and transcriptional state, but found significant intercolony variation, detecting 488 representing unique genes or 17% of the total genes analyzed. Such transcriptomic variation could be the basis upon which natural selection can act. Underlying variation could potentially allow reef corals to respond to different environments. Whether this source of variation and the genetic responses of corals and its symbionts will allow coral reefs to cope to the rapid pace of global change remains unknown. A. millepora colonies were brought to a common garden in the reef lagoon, i.e. under the same environmental conditions. This common garden combined with acclimatization removes environmental effects on the physiology of the coral colonies. For the comparison of the two intron haplotypes, we applied a multiple dye-swap microarray design for the two groups of coral colonies (N=3 per group) defined based on the two genotypes resolved with the use of intron 4-500 (Fig. 1). To also examine the intra-haplotype variation we added a loop design nested to the above multiple dye-swap design, where three samples per colony were included. Colonies 1, 2, and 3 are of intron 4-500 haplotype 1; colonies 4, 5, and 6 are haplotype 2.
Project description:Thermal history plays a role in the response of corals to subsequent heat stress. Prior heat stress can have a profound impact on later thermal tolerance, but the mechanism for this plasticity is not clear. The understanding of gene expression changes behind physiological acclimatization is critical in forecasts of coral health in impending climate change scenarios. Acropora millepora fragments were preconditioned to sublethal bleaching threshold stress for a period of 10 days; this prestress conferred bleaching resistance in subsequent thermal challenge, in which non-preconditioned coral bleached. Using microarrays, we analyze the transcriptomes of the coral host, comparing the bleaching-resistant preconditioned treatment to non-preconditioned and control treatments. This experiment compared host gene expression of Acropora millepora across control, non-preconditioned, and preconditioned treatments. Fragments were sampled prior to preconditioning (Day 4), following 10 days of thermal preconditioning (Day 20), and after two (Day 23), four (Day 25), and eight days (Day 29) of 31°C thermal challenge. The analysis implements 45 arrays, representing 5 sampling points of three treatments (n=3).
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 (3˚C 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 Institute’s Bocas del Toro field station in Panamá during September and October 2006. Four colonies of A. palmata were sampled from two separate reefs 21 km apart (two colonies from Isla Solarte – 9o19’56.78” N and 82o12’54.65” W, and two colonies from Cayos Zapatillas – 9o15’08.79” N and 82o02’24.63” 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 – 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 (9o22’68.4” N and 82o18’24.6” W) during September and October 2007). Light intensity differed slightly between the four aquaria fitted with HOBOs (control aquaria 1 – 43%; control aquaria 2 – 46%; heated aquaria 1 – 35%; and heated aquaria 2 – 34% of reef light). After an acclimation period of four days at the natural temperature of the seawater system (mean temperature = 30.29±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 29.74±0.03oC, and the mean temperature of the heated aquaria was 32.72±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:Publication Abstract: As climate changes, sea surface temperature anomalies that negatively impact coral reef organisms continue to increase in frequency and intensity. Yet, despite widespread coral mortality, genetic diversity remains high even in those coral species listed as threatened. While this is good news in many ways it presents a challenge for the development of biomarkers that can identify resilient or vulnerable genotypes. Taking advantage of three coral restoration nurseries in Florida that serve as long-term common garden experiments, we exposed over thirty genetically distinct Acropora cervicornis colonies to hot and cold temperature shocks seasonally and measured pooled gene expression responses using RNAseq. Targeting a subset of twenty genes, we designed a high-throughput qPCR array to quantify expression in all individuals separately under each treatment with the goal of identifying predictive and/or diagnostic thermal stress biomarkers. We observed extensive transcriptional variation in the population, suggesting abundant raw material is available for adaptation via natural selection. However, this high variation made it difficult to correlate gene expression changes with colony performance metrics such as growth, mortality, and bleaching susceptibility. Nevertheless, we identified several promising diagnostic biomarkers for acute thermal stress that may improve coral restoration and climate change mitigation efforts in the future. Overall design: This project studied the effects of thermal stress on gene expression in the Staghorn coral (Acropora cervicornis). At four seasonal time points, tissue from at least ten colonies at three nurseries were exposed to three temperature treatments for one hour then preserved in RNALater. RNA was extracted with TRI Reagent then cleaned with a Qiagen RNEasy kit. A subset of samples were chosen and pooled for RNA-Seq. The subset included genotypes from 2 of the 3 nurseries (Miami and Lower Keys), 2 of the 4 seasonal collections (Summer 1 and Winter 1), and all 3 temperature treatments (ambient, hot, and cold), resulting in 12 libraries. For each library, RNA from 7 of the 10+ genotypes at each nursery were pooled (84 samples total). Following the manufacturer’s protocol, 150 bp single reads were generated from oligo(dT)-selected total RNA using the Illumina TruSeq Stranded mRNA HT Kit. mRNA sequencing libraries were individually barcoded, multiplexed in equal quantities, and run on three lanes of the Illumina HiSeq 2000 platform. The data were demultiplexed into 12 individual library files.
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:Despite their early evolutionary divergence, reef-building corals exhibit complex circadian responses to diurnal, lunar and annual changes in the conditions around them. Understanding circadian regulation in reef-building corals is, however, complicated by the presence of photosynthetic endosymbionts that have a profound physiochemical influence on the intracellular environment. How corals tune their animal-based clock machinery to respond to external cues while at the same time responding to internal physiological changes imposed by the symbiont is not clear. We explore this issue using microarray analysis to dissect genes governed directly by the circadian machinery from those responding indirectly as a consequence of changing internal oxygen tensions. Three coral colonies were sampled at 4 hr intervals during two consecutive days under an ambient light/dark (LD) cycle and under constant darkness (DD). In total 72 arrays were hybridized, as each array represented a sample from a treatment and a time point (n=3).
Project description:A mutualistic relationship between reef-building corals and endosymbiotic algae (Symbiodinium spp.) forms the basis for the existence of coral reefs. Genotyping tools for Symbiodinium spp. have added a new level of complexity to studies concerning cnidarian growth, nutrient acquisition, and stress. For example, the response of the coral holobiont to thermal stress is connected to the host-Symbiodinium genotypic combination, as different partnerships can have different bleaching susceptibilities. If, and to what extent, differences in algal symbiont clade contents can exert effects on the coral host transcriptome is currently unknown. In this study, we monitored algal physiological parameters and profiled the coral host transcriptional responses in acclimated, thermally stressed, and recovered coral fragments using a custom cDNA gene expression microarray. Combining these analyses with results from algal and host genotyping revealed a striking symbiont effect on both the acclimated coral host transcriptome and the magnitude of the thermal stress response. This is the first study that links coral host transcriptomic patterns to the clade content of their algal symbiont community. Our data provide a critical step to elucidating the molecular basis of the apparent variability seen among different coral-algal partnerships. Sample Collection and Tank Experiment. On July 31 2007, six replicate fragments (9.5 ± 3.5cm2) were collected using hammer and chisel from the upper sun-exposed surface of five different, healthy-looking Montastraea faveolata colonies near Puerto Morelos, Quintana Roo, México (20o52’28.77”N and 86o51’04.53”W). The fragments were transported to the institute within 1 h and divided evenly between two 50 L aquaria (i.e., 3 fragments from each colony were placed into each tank) that received a constant flow of seawater (0.6 L/min). Each aquarium was fit with a water pump connected to a spray-bar to provide constant water movement and aeration. Both aquaria were placed in a common pond with flowing water to buffer diurnal temperature fluctuations, and both aquaria were acclimated to the same shaded ambient light condition. All coral fragments were mounted on plasticene and kept at a depth of 7 cm. From 10 to 19 August 2007, both aquaria received an average water temperature of 27.9 ± 0.6oC (as recorded by HOBO Light/Temperature Data Loggers, Onset Corp.). Beginning on 11 August, dark-adapted maximum quantum yields for charge separation (Fv/Fm) were measured at dusk for all coral fragments using a DIVING-Pulse Amplitude Modulated (PAM) fluorometer (Walz, Germany). Photosynthetically active radiation (PAR) was measured at noon and averaged 318 ± 129 µmol m-2 s-1. From 20 to 21 August 2007, all coral fragments were brought inside during the passage of Hurricane Dean. On 22 August, the experiment was reconstituted, and acclimation continued on 23 August and lasted until 1 September. During this time, both aquaria received a mean water temperature of 28.5 ± 0.8oC, and mean PAR of 371 ± 169 µmol m-2 s-1. On the night of 1 September, control time point samples from five different colonies were collected from each tank (Figure 1), before one 200-Watt aquarium heater was turned on in the treatment aquarium. A second heater was turned on 3 days later. During the thermal stress experiment, the control aquarium received mean water temperature of 28.8 ± 1.2oC; the heated aquarium, 31.5 ± 1.1oC. During the thermal stress experiment, tanks received mean PAR of 420 ± 152 µmol m-2 s-1. On 11 September, and 19 October, one sample each from five different colonies was collected from each tank for the bleaching and recovery time point, respectively. Microarray Gene Expression Analysis of Coral Host Gene Expression. For the biologically replicated time series experiment, a pool of RNA from all control tank fragments was used as a reference RNA sample, against which each sample from the treatment tank was competitively hybridized. All control and heat-stressed samples were competitively hybridized against the reference RNA. Dye swaps were not performed, as any dye bias present is equal in all comparisons to the reference. Note: Samples VALUEs used for statistical analysis of differential expression. GSE15253_shini_cbr_matrix2_norm.txt used for hierarchical clustering.
Project description:Given the overwhelming evidence that symbiont genotypes differentially affect host processes such as growth, bleaching susceptibility, and nutrient acquisition, we set out to measure gene expression differences in fragments of Montastraea faveolata harboring two different clades of Symbiodinium. On the reefs near Puerto Morelos, México, colonies of M. faveolata are known to shift algal symbiont clade with depth, often associating with clade A at the top, clade B in the middle, and clade C near the bottom of the colony. By measuring photosynthetic efficiency and gene expression in control and heat-stressed fragments containing either clade B, clade C, or a mix of both, we found that: 1) the algal response to thermal stress is due to both host and algal factors; 2) fragments of M. faveolata express different genes in response to sub-bleaching thermal stress depending on algal genotype; 3) the overall effect of heat stress on coral gene expression is less significant than the effect of housing different zooxanthellae types. Overall, we present convincing evidence that different Symbiodinium clades may be functionally distinct, which in turn, greatly influences host gene expression. Six fragments (9.5 ± 3.5cm2) from the top (2.7m), middle (3.7m), and bottom (5.2m) of one massive colony of Montastraea faveolata were collected with a hammer and chisel at “La Bocana” reef near Puerto Morelos, Quintana Roo, Mexico on 31 July 2007. The fragments were divided evenly between two aquaria (50L) that received a constant flow of seawater (~0.64L/min). Each aquarium was fit with a water pump connected to a spray-bar to provide constant water movement and aeration. Both aquaria were placed in a common pond with flowing water to buffer diurnal temperature fluctuations, and both aquaria were exposed to shaded ambient light. All coral fragments were mounted on plasticene and kept at a depth of ~7cm. From 10 to 19 August 2007 (acclimation #1), both aquaria received an average water temperature of 27.9 ± 0.6oC (as recorded by HOBO Light/Temperature Data Loggers by Onset Corp.). Effective quantum yield (ΔF/Fm’) measurements were taken at noon, and maximum quantum yield (Fv/Fm) measurements were taken at dusk for all 18 coral fragments using a DIVING-PAM (Walz) beginning on 11 August. Photosynthetically active radiation (PAR) was measured at noon and averaged 318 ± 129 umol/m2/sec. From 20 to 21 August 2007, all coral fragments were brought inside during the passage of Hurricane Dean. On 22 August, the experiment was reconstituted, and a second acclimation period began on 23 August and lasted until 1 September. During this time, both aquaria received water at an average temperature of 28.5 ± 0.8oC, and PAR at an average of 371 ± 169 umol/m2/sec. On the night of 1 September, one 200-Watt aquarium heater was turned on in the treatment aquarium, and a second heater was turned on 3 days later. During the thermal stress experiment, the control aquarium received an average water temperature of 28.8 ± 1.2oC; the heated aquarium, 31.5 ± 1.1oC. PAR present during the thermal stress experiment averaged 420 ± 152 uE. On the night of 7 September, all fragments were frozen in liquid nitrogen. Eighteen total microarray hybridizations were performed. A reference RNA sample was created by pooling RNA from all 18 prep’s. All control and heat-stressed samples were competitively hybridized against the reference RNA to the M. faveolata microarray (1,310 features).
Project description:The metabolic bases of the interaction between the coral Acropora millepora and its dinoflagellate symbiont were investigated by comparing gene expression levels under light and dark conditions at the whole transcriptome level. Among the differentially expressed genes identified, a suite of genes involved in cholesterol transport was found to be up-regulated under light conditions, confirming the significance of this compound in the coral symbiosis. Although ion transporters likely to have roles in calcification were not differentially expressed in this study, expression levels of many genes associated with skeletal organic matrix composition and organization were higher in light conditions. This implies that the rate of organic matrix synthesis is one factor limiting calcification at night. Thus, LEC during the day is likely to be a consequence of increases in both matrix synthesis and the supply of precursor molecules as a result of photosynthetic activity. Branch tips from three adult colonies of Acropora millepora were sampled at midday and midnight
Project description:The potential to adapt to a changing climate depends in part upon the standing genetic variation present in wild populations. In corals, the dispersive larval phase is particularly vulnerable to the effects of environmental stress. Larval survival and response to stress during dispersal and settlement will play a key role in the persistence of coral populations. To test the hypothesis that larval transcription profiles reflect population specific responses to thermal stress, symbiont-free gametes of the scleractinian coral Montastraea faveolata were collected from Florida and Mexico and raised under normal and elevated temperatures. These populations have been shown to exchange larvae frequently enough to prevent significant differentiation of neutral loci. Differences among thousands of genes were simultaneously characterized using microarrays, allowing investigation of gene expression patterns among wild populations under stressful environmental conditions. Results show site-specific signatures of gene expression in larvae of a reef-building coral from different parts of its range (despite low genetic divergence), and reveal both local and general components of stress response during later stages of larval development. These results provide evidence of site-specific variation in the face of gene flow, which may represent functional genetic variation in different subpopulations, and support the idea that coral host genomes may indeed house the adaptive potential needed to deal with changing environmental conditions. The experimental setup followed a reference design, i.e. all samples were hybridized against the same pool made up of equal amounts of RNA from all samples collected in Mexico. For samples from Mexico we used three technical replicates for each treatment temperature, for samples from Florida three biological replicates were used for each treatment temperature, except for the high temperature samples at day two where only two replicates were available due to high larval mortality at that temperature. Common reference samples were labeled with Cy3, temperature treatment samples with Cy5. Microarrays for M. faveolata contained 1,314 coding sequences, of which 43% had functional annotations as determined by homology to known genes.
Project description:A thermal stress experiment on Heron Island (Great Barrier Reef) which involved a slow ramp in temperature over one week and then sampling after 4 days at 32 degrees was completed. Control samples were maintained at 27C. The idea of the experiment was to study bleaching from a single cell perspective and thus look at cell condition (both animal and host) in symbio (inside the host tissue) and compare it with the physiological/macromolecular composition of the expelled symbionts (dinoflagellates). Are the cells expelled from the coral host during thermal stress are a result of host stress or algae stress. We took samples for proteomics from the extracted endoderm cells (in symbio) and also of expelled cells. Samples were collected as the symbio left the host. These were flash frozen. Are the symbiont cells expelled from the coral host during thermal stress are a result of host stress or algae stress?