Galina V. Aglyamova

Sequencing and de novo analysis of a coral larval transcriptome using 454 GSFlx

BackgroundNew methods are needed for genomic-scale analysis of emerging model organisms that exemplify important biological questions but lack fully sequenced genomes. For example, there is an urgent need to understand the potential for corals to adapt to climate change, but few molecular resources are available for studying these processes in reef-building corals. To facilitate genomics studies in corals and other non-model systems, we describe methods for transcriptome sequencing using 454, as well as strategies for assembling a useful catalog of genes from the output. We have applied these methods to sequence the transcriptome of planulae larvae from the coral Acropora millepora.ResultsMore than 600,000 reads produced in a single 454 sequencing run were assembled into ~40,000 contigs with five-fold average sequencing coverage. Based on sequence similarity with known proteins, these analyses identified ~11,000 different genes expressed in a range of conditions including thermal stress and settlement induction. Assembled sequences were annotated with gene names, conserved domains, and Gene Ontology terms. Targeted searches using these annotations identified the majority of genes associated with essential metabolic pathways and conserved signaling pathways, as well as novel candidate genes for stress-related processes. Comparisons with the genome of the anemone Nematostella vectensis revealed ~8,500 pairs of orthologs and ~100 candidate coral-specific genes. More than 30,000 SNPs were detected in the coral sequences, and a subset of these validated by re-sequencing.ConclusionThe methods described here for deep sequencing of the transcriptome should be widely applicable to generate catalogs of genes and genetic markers in emerging model organisms. Our data provide the most comprehensive sequence resource currently available for reef-building corals, and include an extensive collection of potential genetic markers for association and population connectivity studies. The characterization of the larval transcriptome for this widely-studied coral will enable research into the biological processes underlying stress responses in corals and evolutionary adaptation to global climate change.

Profiling gene expression responses of coral larvae (Acropora millepora) to elevated temperature and settlement inducers using a novel RNA‐Seq procedure

Elevated temperatures resulting from climate change pose a clear threat to reef‐building corals; however, the traits that might influence corals’ survival and dispersal during climate change remain poorly understood. Global gene expression profiling is a powerful hypothesis‐forming tool that can help elucidate these traits. Here, we applied a novel RNA‐Seq protocol to study molecular responses to heat and settlement inducers in aposymbiotic larvae of the reef‐building coral Acropora millepora. This analysis of a single full‐sibling family revealed contrasting responses between short‐ (4‐h) and long‐term (5‐day) exposures to elevated temperatures. Heat shock proteins were up‐regulated only in the short‐term treatment, while the long‐term treatment induced the down‐regulation of ribosomal proteins and up‐regulation of genes associated with ion transport and metabolism (Ca2+ and CO32−). We also profiled responses to settlement cues using a natural cue (crustose coralline algae, CCA) and a synthetic neuropeptide (GLW‐amide). Both cues resulted in metamorphosis, accompanied by differential expression of genes with known developmental roles. Some genes were regulated only by the natural cue, which may correspond to the recruitment‐associated behaviour and morphology changes that precede metamorphosis under CCA treatment, but are bypassed under GLW‐amide treatment. Validation of these expression profiles using qPCR confirmed the quantitative accuracy of our RNA‐Seq approach. Importantly, qPCR analysis of different larval families revealed extensive variation in these responses depending on genetic background, including qualitative differences (i.e. up‐regulation in one family and down‐regulation in another). Future studies of gene expression in corals will have to address this genetic variation, which could have important adaptive consequences for corals during global climate change.

Genomic determinants of coral heat tolerance across latitudes

Some like it hot Coral reefs are threatened by increasing temperatures. Acute temperature increases stress and damage corals. However, more gradual temperature changes can result in adaptation and subsequent tolerance for higher temperatures. Dixon et al. show that the heat tolerance that currently exists across coral populations from different latitudes can be inherited. Thus, natural variation in temperature tolerance may facilitate rapid adaptation among corals as our climate warms. Science, this issue p. 1460 In a warming world, existing variation in heat tolerance could help corals beat the heat. As global warming continues, reef-building corals could avoid local population declines through “genetic rescue” involving exchange of heat-tolerant genotypes across latitudes, but only if latitudinal variation in thermal tolerance is heritable. Here, we show an up–to–10-fold increase in odds of survival of coral larvae under heat stress when their parents come from a warmer lower-latitude location. Elevated thermal tolerance was associated with heritable differences in expression of oxidative, extracellular, transport, and mitochondrial functions that indicated a lack of prior stress. Moreover, two genomic regions strongly responded to selection for thermal tolerance in interlatitudinal crosses. These results demonstrate that variation in coral thermal tolerance across latitudes has a strong genetic basis and could serve as raw material for natural selection.

Evolution of Rhizaria: new insights from phylogenomic analysis of uncultivated protists.

BackgroundRecent phylogenomic analyses have revolutionized our view of eukaryote evolution by revealing unexpected relationships between and within the eukaryotic supergroups. However, for several groups of uncultivable protists, only the ribosomal RNA genes and a handful of proteins are available, often leading to unresolved evolutionary relationships. A striking example concerns the supergroup Rhizaria, which comprises several groups of uncultivable free-living protists such as radiolarians, foraminiferans and gromiids, as well as the parasitic plasmodiophorids and haplosporids. Thus far, the relationships within this supergroup have been inferred almost exclusively from rRNA, actin, and polyubiquitin genes, and remain poorly resolved. To address this, we have generated large Expressed Sequence Tag (EST) datasets for 5 species of Rhizaria belonging to 3 important groups: Acantharea (Astrolonche sp., Phyllostaurus sp.), Phytomyxea (Spongospora subterranea, Plasmodiophora brassicae) and Gromiida (Gromia sphaerica).Results167 genes were selected for phylogenetic analyses based on the representation of at least one rhizarian species for each gene. Concatenation of these genes produced a supermatrix composed of 36,735 amino acid positions, including 10 rhizarians, 9 stramenopiles, and 9 alveolates. Phylogenomic analyses of this large dataset revealed a strongly supported clade grouping Foraminifera and Acantharea. The position of this clade within Rhizaria was sensitive to the method employed and the taxon sampling: Maximum Likelihood (ML) and Bayesian analyses using empirical model of evolution favoured an early divergence, whereas the CAT model and ML analyses with fast-evolving sites or the foraminiferan species Reticulomyxa filosa removed suggested a derived position, closely related to Gromia and Phytomyxea. In contrast to what has been previously reported, our analyses also uncovered the presence of the rhizarian-specific polyubiquitin insertion in Acantharea. Finally, this work reveals another possible rhizarian signature in the 60S ribosomal protein L10a.ConclusionsOur study provides new insights into the evolution of Rhizaria based on phylogenomic analyses of ESTs from three groups of previously under-sampled protists. It was enabled through the application of a recently developed method of transcriptome analysis, requiring very small amount of starting material. Our study illustrates the potential of this method to elucidate the early evolution of eukaryotes by providing large amount of data for uncultivable free-living and parasitic protists.

Development of gene expression markers of acute heat-light stress in reef-building corals of the genus Porites.

Coral reefs are declining worldwide due to increased incidence of climate-induced coral bleaching, which will have widespread biodiversity and economic impacts. A simple method to measure the sub-bleaching level of heat-light stress experienced by corals would greatly inform reef management practices by making it possible to assess the distribution of bleaching risks among individual reef sites. Gene expression analysis based on quantitative PCR (qPCR) can be used as a diagnostic tool to determine coral condition in situ. We evaluated the expression of 13 candidate genes during heat-light stress in a common Caribbean coral Porites astreoides, and observed strong and consistent changes in gene expression in two independent experiments. Furthermore, we found that the apparent return to baseline expression levels during a recovery phase was rapid, despite visible signs of colony bleaching. We show that the response to acute heat-light stress in P. astreoides can be monitored by measuring the difference in expression of only two genes: Hsp16 and actin. We demonstrate that this assay discriminates between corals sampled from two field sites experiencing different temperatures. We also show that the assay is applicable to an Indo-Pacific congener, P. lobata, and therefore could potentially be used to diagnose acute heat-light stress on coral reefs worldwide.

Deep relationships of Rhizaria revealed by phylogenomics: a farewell to Haeckel's Radiolaria.

Rhizaria is one of the six supergroups of eukaryotes, which comprise the majority of amoeboid and skeleton-building protists living in freshwater and marine ecosystems. There is an overall lack of molecular data for the group and therefore the deep phylogeny of rhizarians is unresolved. Molecular data are particularly scarce for the clade of Retaria, which include two prominent groups of microfossils: foraminiferans and radiolarians. To fill this gap, we have produced and sequenced EST libraries for 14 rhizarian species including seven foraminiferans, Gromia and six taxa belonging to traditional Haeckels Radiolaria: Acantharea, Polycystinea, and Phaeodarea. A matrix was constructed for phylogenetic analysis based on 109 genes and a total of 56 species, of which 22 are rhizarians. Our analyses provide the first multigene evidence for branching of Phaeodarea within Cercozoa, confirming the polyphyly of Haeckels Radiolaria. It confirms the monophyly of Retaria, a clade grouping Foraminifera with other lineages of Radiolaria. However, contrary to what could be expected from morphological observations, Foraminifera do not form a sister group to radiolarians, but branch within them as sister to either Acantharea or Polycystinea depending on the multigene data set. While the monophyly of Foraminifera and Acantharea is well supported, that of Polycystinea, represented in our data by Spumellaria and Collodaria is questionable. In view of our study, Haeckels Radiolaria appears as both, a polyphyletic and paraphyletic assemblage of independent groups that should be considered as separate lineages in protist classification.

Gene Expression Associated with White Syndromes in a Reef Building Coral, Acropora Hyacinthus

BackgroundCorals are capable of launching diverse immune defenses at the site of direct contact with pathogens, but the molecular mechanisms of this activity and the colony-wide effects of such stressors remain poorly understood. Here we compared gene expression profiles in eight healthy Acropora hyacinthus colonies against eight colonies exhibiting tissue loss commonly associated with white syndromes, all collected from a natural reef environment near Palau. Two types of tissues were sampled from diseased corals: visibly affected and apparently healthy.ResultsTag-based RNA-Seq followed by weighted gene co-expression network analysis identified groups of co-regulated differentially expressed genes between all health states (disease lesion, apparently healthy tissues of diseased colonies, and fully healthy). Differences between healthy and diseased tissues indicate activation of several innate immunity and tissue repair pathways accompanied by reduced calcification and the switch towards metabolic reliance on stored lipids. Unaffected parts of diseased colonies, although displaying a trend towards these changes, were not significantly different from fully healthy samples. Still, network analysis identified a group of genes, suggestive of altered immunity state, that were specifically up-regulated in unaffected parts of diseased colonies.ConclusionsSimilarity of fully healthy samples to apparently healthy parts of diseased colonies indicates that systemic effects of white syndromes on A. hyacinthus are weak, which implies that the coral colony is largely able to sustain its physiological performance despite disease. The genes specifically up-regulated in unaffected parts of diseased colonies, instead of being the consequence of disease, might be related to the originally higher susceptibility of these colonies to naturally occurring white syndromes.

Evolutionary origins of germline segregation in Metazoa: evidence for a germ stem cell lineage in the coral Orbicella faveolata (Cnidaria, Anthozoa)

The ability to segregate a committed germ stem cell (GSC) lineage distinct from somatic cell lineages is a characteristic of bilaterian Metazoans. However, the occurrence of GSC lineage specification in basally branching Metazoan phyla, such as Cnidaria, is uncertain. Without an independently segregated GSC lineage, germ cells and their precursors must be specified throughout adulthood from continuously dividing somatic stem cells, generating the risk of propagating somatic mutations within the individual and its gametes. To address the potential for existence of a GSC lineage in Anthozoa, the sister-group to all remaining Cnidaria, we identified moderate- to high-frequency somatic mutations and their potential for gametic transfer in the long-lived coral Orbicella faveolata (Anthozoa, Cnidaria) using a 2b-RAD sequencing approach. Our results demonstrate that somatic mutations can drift to high frequencies (up to 50%) and can also generate substantial intracolonial genetic diversity. However, these somatic mutations are not transferable to gametes, signifying the potential for an independently segregated GSC lineage in O. faveolata. In conjunction with previous research on germ cell development in other basally branching Metazoan species, our results suggest that the GSC system may be a Eumetazoan characteristic that evolved in association with the emergence of greater complexity in animal body plan organization and greater specificity of stem cell functions.

Gene expression analysis of the hedgehog signaling cascade in the chick midbrain and spinal cord

The signaling molecule Sonic Hedgehog (SHH) plays a critical role in patterning the ventral midbrain of vertebrates. Our recent studies have established that the requirement for Hedgehog (HH) signaling in the chick midbrain is modulated spatially and temporally in a complex manner across the midbrain anlage. Unfortunately, the patterns of expression of downstream regulators that might modulate the HH signal in the midbrain are not currently known. To fill this gap, we have examined across time, the expression pattern of 14 genes that function in the HH signaling cascade in the midbrain and spinal cord. Our results suggest that SHH expression in the axial mesendoderm begins before the expression of known HH receptors/HH‐binding proteins (e.g., PTC1, PTC2, HHIP, BOC, MEGALIN). In the midbrain, PTC and GLI genes are expressed and then eliminated very early from the ventral midline. However, they exhibit high and persistent expression in the midbrain region circumscribing the SHH source. Intriguingly, multiple HH‐binding proteins (BOC, MEGALIN) and HH effectors (GLI1‐3, SMO, SUFU, DZIP) are expressed in the dorsal midbrain and the midbrain–hindbrain boundary. Finally, we report for the first time that IHH is expressed in intermediate regions of the spinal cord, where its expression does not overlap with that of SHH. Developmental Dynamics 236:1363–1373, 2007.

Red fluorescence in coral larvae is associated with a diapause‐like state

Effective dispersal across environmental gradients is the key to species resilience to environmental perturbation, including climate change. Coral reefs are among the most sensitive ecosystems to global warming, but factors predicting coral dispersal potential remain unknown. In a reef‐building coral Acropora millepora, larval fluorescence emerged as a possible indicator of dispersal potential since it correlates with responsiveness to a settlement cue. Here, we show that gene expression in red fluorescent larvae of A. millepora is correlated with diapause‐like characteristics highly likely to be associated with extended dispersal. We compared gene expression among three larval fluorescent morphs under three coloured light treatments. While colour morphs did not differ in their gene expression responses to light colour, red larvae demonstrated gene expression signatures of cell cycle arrest and decreased transcription accompanied by elevated ribosome production and heightened defenses against oxidative stress. A meta‐analysis revealed that this profile was highly similar to the signatures of elevated thermal tolerance in the same coral species, and moreover, functionally resembled diapause states in an insect and a nematode. Our results support a connection between red fluorescence and long‐range dispersal, which offers a new perspective on the molecular underpinnings of coral larval dispersal and the biological function of GFP‐like fluorescent proteins.