Transcriptome-wide genotype-phenotype associations in Daphnia in a predation risk environment.

Phenotypic variation plays an important role in how species cope with environmental challenges. Pinpointing which genes and genomic regions are underlying phenotypic variability thus helps to understand the processes of acclimation and adaptation. We used Daphnia as a system to identify candidates playing a role in phenotypic variation related to a predation risk environment with a genome-wide association approach. Furthermore, a gene co-expression network analysis allowed identifying clusters of co-expressed genes which correlated to life history traits. To enhance the understanding of the functional roles of the transcripts, we identified orthologs and paralogs from related species and used ontologies to annotate the candidates of interest. Our study revealed that only one life history trait and two morphometric traits have a genetic association in the presence of predation risk (fish kairomones), whereas most genotype-phenotype associations were detected in a genotype-environment interaction analysis for reproduction-related phenotypic traits. The gene co-expression network analysis identified a total of 44 modules, of which one module correlated to another life history trait namely the 'total number of broods'. The combined use of gene co-expression network and transcriptome-wide association analysis allowed the identification of 131 candidate transcripts associated with life history traits in Daphnia galeata. These results lay the ground for targeted studies to further understand phenotypic variability in this species.

Insights into the genetic basis of predator-induced response in Daphnia galeata.

Phenotypic plastic responses allow organisms to rapidly adjust when facing environmental challenges-these responses comprise morphological, behavioral but also life-history changes. Alteration of life-history traits when exposed to predation risk have been reported often in the ecological and genomic model organism Daphnia. However, the molecular basis of this response is not well understood, especially in the context of fish predation. Here, we characterized the transcriptional profiles of two Daphnia galeata clonal lines with opposed life histories when exposed to fish kairomones. First, we conducted a differential gene expression, identifying a total of 125 candidate transcripts involved in the predator-induced response, uncovering substantial intraspecific variation. Second, we applied a gene coexpression network analysis to find clusters of tightly linked transcripts revealing the functional relations of transcripts underlying the predator-induced response. Our results showed that transcripts involved in remodeling of the cuticle, growth, and digestion correlated with the response to environmental change in D. galeata. Furthermore, we used an orthology-based approach to gain functional information for transcripts lacking gene ontology (GO) information, as well as insights into the evolutionary conservation of transcripts. We could show that our candidate transcripts have orthologs in other Daphnia species but almost none in other arthropods. The unique combination of methods allowed us to identify candidate transcripts, their putative functions, and evolutionary history associated with predator-induced responses in Daphnia. Our study opens up to the question as to whether the same molecular signature is associated with fish kairomones-mediated life-history changes in other Daphnia species.

Author Correction: Daphnia stressor database: Taking advantage of a decade of Daphnia '-omics' data for gene annotation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Daphnia stressor database: Taking advantage of a decade of Daphnia '-omics' data for gene annotation.

Gene expression patterns help to measure and characterize the effect of environmental perturbations at the cellular and organism-level. Complicating interpretation is the presence of uncharacterized or "hypothetical" gene functions for a large percentage of genomes. This is particularly evident in Daphnia genomes, which contains many regions coding for "hypothetical proteins" and are significantly divergent from many of the available arthropod model species, but might be ecologically important. In the present study, we developed a gene expression database, the Daphnia stressor database (http://www.daphnia-stressordb.uni-hamburg.de/dsdbstart.php), built from 90 published studies on Daphnia gene expression. Using a comparative genomics approach, we used the database to annotate D. galeata transcripts. The extensive body of literature available for Daphnia species allowed to associate stressors with gene expression patterns. We believe that our stressor based annotation strategy allows for better understanding and interpretation of the functional role of the understudied hypothetical or uncharacterized Daphnia genes, thereby increasing our understanding of Daphnia's genetic and phenotypic variability.

Intraspecific phenotypic variation in life history traits of Daphnia galeata populations in response to fish kairomones.

Phenotypic plasticity is the ability of a genotype to produce different phenotypes depending on the environment. It has an influence on the adaptive potential to environmental change and the capability to adapt locally. Adaptation to environmental change happens at the population level, thereby contributing to genotypic and phenotypic variation within a species. Predation is an important ecological factor structuring communities and maintaining species diversity. Prey developed different strategies to reduce their vulnerability to predators by changing their behaviour, their morphology or their life history. Predator-induced life history responses in Daphnia have been investigated for decades, but intra-and inter-population variability was rarely addressed explicitly. We addressed this issue by conducting a common garden experiment with 24 clonal lines of European Daphnia galeata originating from four populations, each represented by six clonal lines. We recorded life history traits in the absence and presence of fish kairomones. Additionally, we looked at the shape of experimental individuals by conducting a geometric morphometric analysis, thus assessing predator-induced morphometric changes. Our data revealed high intraspecific phenotypic variation within and between four D. galeata populations, the potential to locally adapt to a vertebrate predator regime as well as an effect of the fish kairomones on morphology of D. galeata.

Current European Labyrinthula zosterae are not virulent and modulate seagrass (Zostera marina) defense gene expression.

Pro- and eukaryotic microbes associated with multi-cellular organisms are receiving increasing attention as a driving factor in ecosystems. Endophytes in plants can change host performance by altering nutrient uptake, secondary metabolite production or defense mechanisms. Recent studies detected widespread prevalence of Labyrinthula zosterae in European Zostera marina meadows, a protist that allegedly caused a massive amphi-Atlantic seagrass die-off event in the 1930's, while showing only limited virulence today. As a limiting factor for pathogenicity, we investigated genotype × genotype interactions of host and pathogen from different regions (10-100 km-scale) through reciprocal infection. Although the endophyte rapidly infected Z. marina, we found little evidence that Z. marina was negatively impacted by L. zosterae. Instead Z. marina showed enhanced leaf growth and kept endophyte abundance low. Moreover, we found almost no interaction of protist × eelgrass-origin on different parameters of L. zosterae virulence/Z. marina performance, and also no increase in mortality after experimental infection. In a target gene approach, we identified a significant down-regulation in the expression of 6/11 genes from the defense cascade of Z. marina after real-time quantitative PCR, revealing strong immune modulation of the host's defense by a potential parasite for the first time in a marine plant. Nevertheless, one gene involved in phenol synthesis was strongly up-regulated, indicating that Z. marina plants were probably able to control the level of infection. There was no change in expression in a general stress indicator gene (HSP70). Mean L. zosterae abundances decreased below 10% after 16 days of experimental runtime. We conclude that under non-stress conditions L. zosterae infection in the study region is not associated with substantial virulence.

Quantitative PCR reveals strong spatial and temporal variation of the wasting disease pathogen, Labyrinthula zosterae in northern European eelgrass (Zostera marina) beds.

Seagrass beds are the foundation species of functionally important coastal ecosystems worldwide. The world's largest losses of the widespread seagrass Zostera marina (eelgrass) have been reported as a consequence of wasting disease, an infection with the endophytic protist Labyrinthula zosterae. During one of the most extended epidemics in the marine realm, ∼90% of East and Western Atlantic eelgrass beds died-off between 1932 and 1934. Today, small outbreaks continue to be reported, but the current extent of L. zosterae in European meadows is completely unknown. In this study we quantify the abundance and prevalence of the wasting disease pathogen among 19 Z. marina populations in northern European coastal waters, using quantitative PCR (QPCR) with primers targeting a species specific portion of the internally transcribed spacer (ITS1) of L. zosterae. Spatially, we found marked variation among sites with abundances varying between 0 and 126 cells mg(-1) Z. marina dry weight (mean: 5.7 L. zosterae cells mg(-1) Z. marina dry weight ±1.9 SE) and prevalences ranged from 0-88.9%. Temporarily, abundances varied between 0 and 271 cells mg(-1) Z. marina dry weight (mean: 8.5±2.6 SE), while prevalences ranged from zero in winter and early spring to 96% in summer. Field concentrations accessed via bulk DNA extraction and subsequent QPCR correlated well with prevalence data estimated via isolation and cultivation from live plant tissue. L. zosterae was not only detectable in black lesions, a sign of Labyrinthula-induced necrosis, but also occurred in green, apparently healthy tissue. We conclude that L. zosterae infection is common (84% infected populations) in (northern) European eelgrass populations with highest abundances during the summer months. In the light of global climate change and increasing rate of marine diseases our data provide a baseline for further studies on the causes of pathogenic outbreaks of L. zosterae.

A quantitative real-time polymerase chain reaction assay for the seagrass pathogen Labyrinthula zosterae.

The protist Labyrinthula zosterae (Phylum Bigyra, sensu Tsui et al. 2009) has been identified as a causative agent of wasting disease in eelgrass (Zostera marina), of which the most intense outbreak led to the destruction of 90% of eelgrass beds in eastern North America and western Europe in the 1930s. Outbreaks still occur today, albeit at a smaller scale. Traditionally, L. zosterae has been quantified by measuring the necrotic area of Z. marina leaf tissue. This indirect method can however only lead to a very rough estimate of pathogen load. Here, we present a quantitative real-time polymerase chain reaction (qPCR) approach to directly detect and quantify L. zosterae in eelgrass tissue. Based on the internal transcribed spacer (ITS) sequences of rRNA genes, species-specific primers were designed. Using our qPCR, we were able to quantify accurately and specifically L. zosterae load both from culture and eelgrass leaves using material from Europe and North America. Our detection limit was less than one L. zosterae cell. Our results demonstrate the potential of this qPCR assay to provide rapid, accurate and sensitive molecular identification and quantification of L. zosterae. In view of declining seagrass populations worldwide, this method will provide a valuable tool for seagrass ecologists and conservation projects.