Profiling sex-biased gene expression during parthenogenetic reproduction in Daphnia pulex

<p>Abstract</p> <p>Background</p> <p>Sexual reproduction is a core biological function that is conserved throughout eukaryotic evolution, yet breeding systems are extremely variable. Genome-wide comparative studies can be effectively used to identify genes and regulatory patterns that are constrained to preserve core functions from those that may help to account for the diversity of animal reproductive strategies. We use a custom microarray to investigate gene expression in males and two reproductive stages of females in the crustacean <it>Daphnia pulex</it>. Most <it>Daphnia </it>species reproduce by cyclical parthenogenesis, alternating between sexual and clonal reproduction. Both sex determination and the switch in their mode of reproduction is environmentally induced, making <it>Daphnia </it>an interesting comparative system for the study of sex-biased and reproductive genes.</p> <p>Results</p> <p>Patterns of gene expression in females and males reveal that 50% of assayed transcripts show some degree of sex-bias. Female-biased transcription is enriched for translation, metabolic and regulatory genes associated with development. Male-biased expression is enriched for cuticle and protease function. Comparison with well studied arthropods such as <it>Drosophila melanogaster </it>and <it>Anopheles gambiae </it>suggests that female-biased patterns tend to be conserved, whereas male-biased genes are evolving faster in <it>D. pulex</it>. These findings are based on the proportion of female-biased, male-biased, and unbiased genes that share sequence similarity with proteins in other animal genomes.</p> <p>Conclusion</p> <p>Some transcriptional differences between males and females appear to be conserved across Arthropoda, including the rapid evolution of male-biased genes which is observed in insects and now in a crustacean. Yet, novel patterns of male-biased gene expression are also uncovered. This study is an important first step towards a detailed understanding of the genetic basis and evolution of parthenogenesis, environmental sex determination, and adaptation to aquatic environments.</p

Genomic data integration for ecological and evolutionary traits in non-model organisms

Why is it needed to develop system biology initiatives such as ENCODE on non-model organisms

Sampling Daphnia's expressed genes: preservation, expansion and invention of crustacean genes with reference to insect genomes

<p>Abstract</p> <p>Background</p> <p>Functional and comparative studies of insect genomes have shed light on the complement of genes, which in part, account for shared morphologies, developmental programs and life-histories. Contrasting the gene inventories of insects to those of the nematodes provides insight into the genomic changes responsible for their diversification. However, nematodes have weak relationships to insects, as each belongs to separate animal phyla. A better outgroup to distinguish lineage specific novelties would include other members of Arthropoda. For example, crustaceans are close allies to the insects (together forming Pancrustacea) and their fascinating aquatic lifestyle provides an important comparison for understanding the genetic basis of adaptations to life on land versus life in water.</p> <p>Results</p> <p>This study reports on the first characterization of cDNA libraries and sequences for the model crustacean <it>Daphnia pulex</it>. We analyzed 1,546 ESTs of which 1,414 represent approximately 787 nuclear genes, by measuring their sequence similarities with insect and nematode proteomes. The provisional annotation of genes is supported by expression data from microarray studies described in companion papers. Loci expected to be shared between crustaceans and insects because of their mutual biological features are identified, including genes for reproduction, regulation and cellular processes. We identify genes that are likely derived within Pancrustacea or lost within the nematodes. Moreover, lineage specific gene family expansions are identified, which suggest certain biological demands associated with their ecological setting. In particular, up to seven distinct ferritin loci are found in <it>Daphnia </it>compared to three in most insects. Finally, a substantial fraction of the sampled gene transcripts shares no sequence similarity with those from other arthropods. Genes functioning during development and reproduction are comparatively well conserved between crustaceans and insects. By contrast, genes that were responsive to environmental conditions (metal stress) and not sex-biased included the greatest proportion of genes with no matches to insect proteomes.</p> <p>Conclusion</p> <p>This study along with associated microarray experiments are the initial steps in a coordinated effort by the <it>Daphnia </it>Genomics Consortium to build the necessary genomic platform needed to discover genes that account for the phenotypic diversity within the genus and to gain new insights into crustacean biology. This effort will soon include the first crustacean genome sequence.</p

A comprehensive epigenomic analysis of phenotypically distinguishable, genetically identical female and male Daphnia pulex

Background Daphnia species reproduce by cyclic parthenogenesis involving both sexual and asexual reproduction. The sex of the offspring is environmentally determined and mediated via endocrine signalling by the mother. Interestingly, male and female Daphnia can be genetically identical, yet display large differences in behaviour, morphology, lifespan and metabolic activity. Our goal was to integrate multiple omics datasets, including gene expression, splicing, histone modification and DNA methylation data generated from genetically identical female and male Daphnia pulex under controlled laboratory settings with the aim of achieving a better understanding of the underlying epigenetic factors that may contribute to the phenotypic differences observed between the two genders. Results In this study we demonstrate that gene expression level is positively correlated with increased DNA methylation, and histone H3 trimethylation at lysine 4 (H3K4me3) at predicted promoter regions. Conversely, elevated histone H3 trimethylation at lysine 27 (H3K27me3), distributed across the entire transcript length, is negatively correlated with gene expression level. Interestingly, male Daphnia are dominated with epigenetic modifications that globally promote elevated gene expression, while female Daphnia are dominated with epigenetic modifications that reduce gene expression globally. For examples, CpG methylation (positively correlated with gene expression level) is significantly higher in almost all differentially methylated sites in male compared to female Daphnia. Furthermore, H3K4me3 modifications are higher in male compared to female Daphnia in more than 3/4 of the differentially regulated promoters. On the other hand, H3K27me3 is higher in female compared to male Daphnia in more than 5/6 of differentially modified sites. However, both sexes demonstrate roughly equal number of genes that are up-regulated in one gender compared to the other sex. Since, gene expression analyses typically assume that most genes are expressed at equal level among samples and different conditions, and thus cannot detect global changes affecting most genes. Conclusions The epigenetic differences between male and female in Daphnia pulex are vast and dominated by changes that promote elevated gene expression in male Daphnia. Furthermore, the differences observed in both gene expression changes and epigenetic modifications between the genders relate to pathways that are physiologically relevant to the observed phenotypic differences.Peer reviewe

Predator-induced defences in Daphnia pulex: Selection and evaluation of internal reference genes for gene expression studies with real-time PCR

<p>Abstract</p> <p>Background</p> <p>The planktonic microcrustacean <it>Daphnia pulex </it>is among the best-studied animals in ecological, toxicological and evolutionary research. One aspect that has sustained interest in the study system is the ability of <it>D. pulex </it>to develop inducible defence structures when exposed to predators, such as the phantom midge larvae <it>Chaoborus</it>. The available draft genome sequence for <it>D. pulex </it>is accelerating research to identify genes that confer plastic phenotypes that are regularly cued by environmental stimuli. Yet for quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data.</p> <p>Results</p> <p>In this study, we tested six candidate reference genes for normalizing transcription levels of <it>D. pulex </it>genes; alpha tubulin (aTub), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box binding protein (Tbp) syntaxin 16 (Stx16), X-box binding protein 1 (Xbp1) and CAPON, a protein associated with the neuronal nitric oxide synthase, were selected on the basis of an earlier study and from microarray studies. One additional gene, a matrix metalloproteinase (MMP), was tested to validate its transcriptional response to <it>Chaoborus</it>, which was earlier observed in a microarray study. The transcription profiles of these seven genes were assessed by qRT-PCR from RNA of juvenile <it>D. pulex </it>that showed induced defences in comparison to untreated control animals. We tested the individual suitability of genes for expression normalization using the programs geNorm, NormFinder and BestKeeper. Intriguingly, Xbp1, Tbp, CAPON and Stx16 were selected as ideal reference genes. Analyses on the relative expression level using the software REST showed that both classical housekeeping candidate genes (aTub and GAPDH) were significantly downregulated, whereas the MMP gene was shown to be significantly upregulated, as predicted. aTub is a particularly ill suited reference gene because five copies are found in the <it>D. pulex </it>genome sequence. When applying aTub for expression normalization Xbp1 and Tbp are falsely reported as significantly upregulated.</p> <p>Conclusions</p> <p>Our results suggest that the genes Xbp1, Tbp, CAPON and Stx16 are suitable reference genes for accurate normalization in qRT-PCR studies using <it>Chaoborus</it>-induced <it>D. pulex </it>specimens. Furthermore, our study underscores the importance of verifying the expression stability of putative reference genes for normalization of expression levels.</p

Pattern of DNA methylation in daphnia : evolutionary perspective

DNA methylation is an evolutionary ancient epigenetic modification that is phylogenetically widespread. Comparative studies of the methylome across a diverse range of non-conventional and conventional model organisms is expected to help reveal how the landscape of DNA methylation and its functions have evolved. Here, we explore the DNA methylation profile of two species of the crustacean Daphnia using whole genome bisulfite sequencing. We then compare our data with the methylomes of two insects and two mammals to achieve a better understanding of the function of DNA methylation in Daphnia. Using RNA-sequencing data for all six species, we investigate the correlation between DNA methylation and gene expression. DNA methylation in Daphnia is mainly enriched within the coding regions of genes, with the highest methylation levels observed at exons 2-4. In contrast, vertebrate genomes are globally methylated, and increase towards the highest methylation levels observed at exon 2, and maintained across the rest of the gene body. Although DNA methylation patterns differ among all species, their methylation profiles share a bimodal distribution across the genomes. Genes with low levels of CpG methylation and gene expression are mainly enriched for species specific genes. In contrast, genes associated with high methylated CpG sites are highly transcribed and evolutionary conserved across all species. Finally, the positive correlation between internal exons and gene expression potentially points to an evolutionary conserved mechanism, whereas the negative regulation of gene expression via methylation of promoters and exon 1 is potentially a secondary mechanism that has been evolved in vertebrates

Functional genomics of acclimation and adaptation in response to thermal stress in Daphnia

BACKGROUND: Gene expression regulation is one of the fundamental mechanisms of phenotypic plasticity and is expected to respond to selection in conditions favoring phenotypic response. The observation that many organisms increase their stress tolerance after acclimation to moderate levels of stress is an example of plasticity which has been long hypothesized to be based on adaptive changes in gene expression. We report genome-wide patterns of gene expression in two heat-tolerant and two heat-sensitive parthenogenetic clones of the zooplankton crustacean Daphnia pulex exposed for three generations to either optimal (18°C) or substressful (28°C) temperature. RESULTS: A large number of genes responded to temperature and many demonstrated a significant genotype-by-environment (GxE) interaction. Among genes with a significant GxE there were approximately equally frequent instances of canalization, i.e. stronger plasticity in heat-sensitive than in heat-tolerant clones, and of enhancement of plasticity along the evolutionary vector toward heat tolerance. The strongest response observed is the across-the-board down-regulation of a variety of genes occurring in heat-tolerant, but not in heat-sensitive clones. This response is particularly obvious among genes involved in core metabolic pathways and those responsible for transcription, translation and DNA repair. CONCLUSIONS: The observed down-regulation of metabolism, consistent with previous findings in yeast and Drosophila, may reflect a general compensatory stress response. The associated down-regulation of DNA repair pathways potentially creates a trade-off between short-term benefits of survival at high temperature and long-term costs of accelerated mutation accumulation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-859) contains supplementary material, which is available to authorized users

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

Background: New 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\ud 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.\ud Results: More 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\ud 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.\ud Conclusion: The 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\ud corals, and include an extensive collection of potential genetic markers for association and population connectivity studies. The characterization of the larval transcriptome for this widelystudied coral will enable research into the biological processes underlying stress responses in corals\ud and evolutionary adaptation to global climate change