Gene Body Methylation Patterns in Daphnia Are Associated with Gene Family Size.

The relation between gene body methylation and gene function remains elusive. Yet, our understanding of this relationship can contribute significant knowledge on how and why organisms target specific gene bodies for methylation. Here, we studied gene body methylation patterns in two Daphnia species. We observed both highly methylated genes and genes devoid of methylation in a background of low global methylation levels. A small but highly significant number of genes was highly methylated in both species. Remarkably, functional analyses indicate that variation in methylation within and between Daphnia species is primarily targeted to small gene families whereas large gene families tend to lack variation. The degree of sequence similarity could not explain the observed pattern. Furthermore, a significant negative correlation between gene family size and the degree of methylation suggests that gene body methylation may help regulate gene family expansion and functional diversification of gene families leading to phenotypic variation.

The Equine Embryo Influences Immune-Related Gene Expression in the Oviduct.

Although the equine oviduct clearly affects early embryo development and the selective transport of equine embryos through the oviduct indicates a reciprocal interaction, the influence of the embryo on gene expression in the oviduct remains to be determined in the horse. The aim of this study was to examine this by means of RNA sequencing. Four days after ovulation, epithelial cells ipsilateral and contralateral to the ovulation side from five cyclic and five pregnant mares were collected from the oviduct. RNA was extracted, samples were sequenced, and data analysis was performed to determine differentially expressed genes (DEGs) (P value ≤0.05 and absolute fold change ≥2) and to provide functional interpretation. A total of 10 743 transcripts were identified and 253 genes were found to be upregulated and 108 to be downregulated in the pregnant ipsilateral oviduct when compared to the cyclic ipsilateral oviduct. Comparison of the ipsilateral and the contralateral oviduct indicated 164 DEGs in pregnant mares and 77 DEGs in cyclic mares. Enriched functional categories were detected only in the comparison of pregnant and cyclic ipsilateral oviducts and showed that the equine embryo affects the expression of immune response-related genes in the oviduct, with marked upregulation of interferon-associated genes. This research represents the foundation for further assessment of the role of specific genes in the early embryo-maternal dialogue of the horse.

Suboptimal culture conditions induce more deviations in gene expression in male than female bovine blastocysts.

BACKGROUND: Since the development of in vitro embryo production in cattle, different supplements have been added to culture media to support embryo development, with serum being the most popular. However, the addition of serum during embryo culture can induce high birthweights and low viability in calves (Large Offspring Syndrome). Analysis of global gene expression in bovine embryos produced under different conditions can provide valuable information to optimize culture media for in vitro embryo production. RESULTS: We used RNA sequencing to examine the effect of in vitro embryo production, in either serum-containing or serum-free media, on the global gene expression pattern of individual bovine blastocysts. Compared to in vivo derived embryos, embryos produced in serum-containing medium had five times more differentially expressed genes than embryos produced in serum-free conditions (1109 vs. 207). Importantly, in vitro production in the presence of serum appeared to have a different impact on the embryos according to their sex, with male embryos having three times more genes differentially expressed than their female counterparts (1283 vs. 456). On the contrary, male and female embryos produced in serum-free conditions showed the same number (191 vs. 192) of genes expressed differentially; however, only 44 of those genes were common in both comparisons. The pathways affected by in vitro production differed depending on the type of supplementation. For example, embryos produced in serum-containing conditions had a lower expression of genes related to metabolism while embryos produced in serum-free conditions showed aberrations in genes involved in lipid metabolism. CONCLUSIONS: Serum supplementation had a major impact on the gene expression pattern of embryos, with male embryos being the most affected. The transcriptome of embryos produced in serum-free conditions showed a greater resemblance to that of in vivo derived embryos, although genes involved in lipid metabolism were altered. Male embryos appeared to be most affected by suboptimal in vitro culture, i.e. in the presence of serum.

In vivo study of Dicer-2-mediated immune response of the small interfering RNA pathway upon systemic infections of virulent and avirulent viruses in Bombus terrestris.

Recent studies suggest a potent role of the small interfering RNA (siRNA) pathway in the control of bee viruses and its usefulness to tackle these viral diseases. However, the involvement of the siRNA pathway in the defense against different bee viruses is still poorly understood. Therefore, in this report, we comprehensively analyzed the response of the siRNA pathway in bumblebees of Bombus terrestris to systemic infections of the virulent Israeli acute paralysis virus (IAPV) and the avirulent slow bee paralysis virus (SBPV). Our results showed that IAPV and SBPV infections induced the expression of Dicer-2. IAPV infections also triggered the production of predominantly 22 nt-long virus-derived siRNAs (vsiRNAs). Intriguingly, these 22 nt-long vsiRNAs showed a high proportion of antigenomic IAPV sequences. Conversely, these predominantly 22 nt-long vsiRNAs of SBPV were not detected in SBPV infected bees. Furthermore, an "RNAi-of-RNAi" experiment on Dicer-2 did not result in altered genome copy numbers of IAPV (n = 17-18) and also not of SBPV (n = 11-12). Based on these results, we can speculate about the importance of the siRNA pathway in bumblebees for the antiviral response. During infection of IAPV, this pathway is probably recruited but it might be insufficient to control viral infection in our experimental setup. The host can control SBPV infection, but aside from the induction of Dicer-2 expression, no further evidence of the antiviral activity of the siRNA pathway was observed. This report may also enhance the current understanding of the siRNA pathway in the innate immunity of non-model insects upon different viral infections.

Conserved transcriptional responses to cyanobacterial stressors are mediated by alternate regulation of paralogous genes in Daphnia.

Despite a significant increase in genomic data, our knowledge of gene functions and their transcriptional responses to environmental stimuli remains limited. Here, we use the model keystone species Daphnia pulex to study environmental responses of genes in the context of their gene family history to better understand the relationship between genome structure and gene function in response to environmental stimuli. Daphnia were exposed to five different treatments, each consisting of a diet supplemented with one of five cyanobacterial species, and a control treatment consisting of a diet of only green algae. Differential gene expression profiles of Daphnia exposed to each of these five cyanobacterial species showed that genes with known functions are more likely to be shared by different expression profiles, whereas genes specific to the lineage of Daphnia are more likely to be unique to a given expression profile. Furthermore, while only a small number of nonlineage-specific genes were conserved across treatment type, there was a high degree of overlap in expression profiles at the functional level. The conservation of functional responses across the different cyanobacterial treatments can be attributed to the treatment-specific expression of different paralogous genes within the same gene family. Comparison with available gene expression data in the literature suggests differences in nutritional composition in diets with cyanobacterial species compared to diets of green algae as a primary driver for cyanobacterial effects on Daphnia. We conclude that conserved functional responses in Daphnia across different cyanobacterial treatments are mediated through alternate regulation of paralogous gene families.

Global cytosine methylation in Daphnia magna depends on genotype, environment, and their interaction.

The authors characterized global cytosine methylation levels in 2 different genotypes of the ecotoxicological model organism Daphnia magna after exposure to a wide array of biotic and abiotic environmental stressors. The present study aimed to improve the authors' understanding of the role of cytosine methylation in the organism's response to environmental conditions. The authors observed a significant genotype effect, an environment effect, and a genotype × environment effect. In particular, global cytosine methylation levels were significantly altered after exposure to Triops predation cues, Microcystis, and sodium chloride compared with control conditions. Significant differences between the 2 genotypes were observed when animals were exposed to Triops predation cues, Microcystis, Cryptomonas, and sodium chloride. Despite the low global methylation rate under control conditions (0.49-0.52%), global cytosine methylation levels upon exposure to Triops demonstrated a 5-fold difference between the genotypes (0.21% vs 1.02%). No effects were found in response to arsenic, cadmium, fish, lead, pH of 5.5, pH of 8, temperature, hypoxia, and white fat cell disease. The authors' results point to the potential role of epigenetic effects under changing environmental conditions such as predation (i.e., Triops), diet (i.e., Cryptomonas and Microcystis), and salinity. The results of the present study indicate that, despite global cytosine methylation levels being low, epigenetic effects may be important in environmental studies on Daphnia.

Genome-wide transcription profiles reveal genotype-dependent responses of biological pathways and gene-families in Daphnia exposed to single and mixed stressors.

The present study investigated the possibilities and limitations of implementing a genome-wide transcription-based approach that takes into account genetic and environmental variation to better understand the response of natural populations to stressors. When exposing two different Daphnia pulex genotypes (a cadmium-sensitive and a cadmium-tolerant one) to cadmium, the toxic cyanobacteria Microcystis aeruginosa, and their mixture, we found that observations at the transcriptomic level do not always explain observations at a higher level (growth, reproduction). For example, although cadmium elicited an adverse effect at the organismal level, almost no genes were differentially expressed after cadmium exposure. In addition, we identified oxidative stress and polyunsaturated fatty acid metabolism-related pathways, as well as trypsin and neurexin IV gene-families as candidates for the underlying causes of genotypic differences in tolerance to Microcystis. Furthermore, the whole-genome transcriptomic data of a stressor mixture allowed a better understanding of mixture responses by evaluating interactions between two stressors at the gene-expression level against the independent action baseline model. This approach has indicated that ubiquinone pathway and the MAPK serine-threonine protein kinase and collagens gene-families were enriched with genes showing an interactive effect in expression response to exposure to the mixture of the stressors, while transcription and translation-related pathways and gene-families were mostly related with genotypic differences in interactive responses to this mixture. Collectively, our results indicate that the methods we employed may improve further characterization of the possibilities and limitations of transcriptomics approaches in the adverse outcome pathway framework and in predictions of multistressor effects on natural populations.

An approach to assess the regulatory relevance of microevolutionary effects in ecological risk assessment of chemicals: a case study with cadmium.

The authors suggest an approach to assess the regulatory relevance of microevolutionary effects of chemicals based on a comparison of concentrations at which microevolutionary effects have been reported in the literature and conventionally derived ecotoxicological threshold concentrations. The authors found reports of microevolutionary effects of cadmium in freshwater organisms at hardness-normalized concentrations between 0.5 µg Cd L(-1) and 6290 µg Cd L(-1) (normalized to a hardness of 50 mg CaCO3 L(-1)). These concentrations were at least 1.5 times higher than the hardness-normalized hazardous concentration for 5% of the organisms of 0.34 µg Cd L(-1). This suggests that there is no immediate need to consider microevolutionary effects of Cd in environmental risk assessments of freshwater environments. However, some other aspects should be kept in mind as well. First, microevolutionary effects have so far only been investigated at few, relatively high concentrations of Cd and not encompassing the 5% hazardous concentration. Second, different types of microevolutionary effects or investigated ecotoxicological end points may influence the conclusions of the suggested comparative approach. Finally, factors influencing the bioavailability of Cd were not commonly reported in the literature, which made normalization of concentrations at which evolutionary effects occurred impossible and affected the number of studies that could be evaluated in the suggested approach.

An investigation of the inter-clonal variation of the interactive effects of cadmium and Microcystis aeruginosa on the reproductive performance of Daphnia magna.

Interactive effects between chemical and natural stressors as well as genetically determined variation in stress tolerance among individuals may complicate risk assessment and management of chemical pollutants in natural ecosystems. Although genetic variation in tolerance to single stressors has been described extensively, genetic variation in interactive effects between two stressors has only rarely been investigated. Here, we examined the interactive effects between a chemical stressor (Cd) and a natural stressor (the cyanobacteria Microcystis aeruginosa) on the reproduction of Daphnia magna in 20 genetically different clones using a full-factorial experimental design and with the independent action model of joint stressor action as the reference theoretical framework. Across all clones, the reduction of 21-day reproduction compared to the control treatment (no Cd, no M. aeruginosa) ranged from -10% to 98% following Cd exposure alone, from 44% to 89% for Microcystis exposure alone, and from 61% to 98% after exposure to Cd+Microcystis combined. Three-way ANOVA on log-transformed reproduction data of all clones together did not detect a statistically significant Cd×Microcystis interaction term (F-test, p=0.11), meaning that on average both stressors do not interact in inhibiting reproductive performance of D. magna. This finding contrasted expectations based on some known shared mechanisms of toxicity of Cd and Microcystis and therefore cautions against making predictions of interactive chemical+natural stressor effects from incomplete knowledge on affected biological processes and pathways. Further, still based on three-way ANOVA, we did not find statistically significant clone×Cd×Microcystis interaction when data for all clones were analyzed together (F-test, p=0.07), suggesting no inter-clonal variation of the interactive effect between Cd and Microcystis. However, when the same data were quantitatively analyzed on a clone-by-clone scale, we found a relatively wide range of deviations between observed and IA-model-predicted reproduction in combined Cd+Microcystis treatments (both in direction and magnitude), suggesting some biological significance of inter-clonal variation of interactive effects. In one of the twenty clones this deviation was statistically significant (two-way ANOVA, F-test, p=0.005), indicating an interactive Cd×Microcystis effect in this clone. Together, these two observations caution against the extrapolation of conclusions about mixed stressor data obtained with single clones to the level of the entire species and to the level of natural, genetically diverse populations.

Interactive effects of a bacterial parasite and the insecticide carbaryl to life-history and physiology of two Daphnia magna clones differing in carbaryl sensitivity.

Natural and chemical stressors occur simultaneously in the aquatic environment. Their combined effects on biota are usually difficult to predict from their individual effects due to interactions between the different stressors. Several recent studies have suggested that synergistic effects of multiple stressors on organisms may be more common at high compared to low overall levels of stress. In this study, we used a three-way full factorial design to investigate whether interactive effects between a natural stressor, the bacterial parasite Pasteuria ramosa, and a chemical stressor, the insecticide carbaryl, were different between two genetically distinct clones of Daphnia magna that strongly differ in their sensitivity to carbaryl. Interactive effects on various life-history and physiological endpoints were assessed as significant deviations from the reference Independent Action (IA) model, which was implemented by testing the significance of the two-way carbaryl×parasite interaction term in two-way ANOVA's on log-transformed observational data for each clone separately. Interactive effects (and thus significant deviations from IA) were detected in both the carbaryl-sensitive clone (on survival, early reproduction and growth) and in the non-sensitive clone (on growth, electron transport activity and prophenoloxidase activity). No interactions were found for maturation rate, filtration rate, and energy reserve fractions (carbohydrate, protein, lipid). Furthermore, only antagonistic interactions were detected in the non-sensitive clone, while only synergistic interactions were observed in the carbaryl sensitive clone. Our data clearly show that there are genetically determined differences in the interactive effects following combined exposure to carbaryl and Pasteuria in D. magna.

Identification of pathways, gene networks, and paralogous gene families in Daphnia pulex responding to exposure to the toxic cyanobacterium Microcystis aeruginosa.

Although cyanobacteria produce a wide range of natural toxins that impact aquatic organisms, food webs, and water quality, the mechanisms of toxicity are still insufficiently understood. Here, we implemented a whole-genome expression microarray to identify pathways, gene networks, and paralogous gene families responsive to Microcystis stress in Daphnia pulex . Therefore, neonates of a sensitive isolate were given a diet contaminated with Microcystis to contrast with those given a control diet for 16 days. The microarray revealed 2247 differentially expressed (DE) genes (7.6% of the array) in response to Microcystis , of which 17% are lineage-specific (i.e., these genes have no detectable homology to any other gene in currently available databases) and 49% are gene duplicates (paralogues). We identified four pathways/gene networks and eight paralogous gene families affected by Microcystis . Differential regulation of the ribosome, including three paralogous gene families encoding 40S, 60S, and mitochondrial ribosomal proteins, suggests an impact of Microcystis on protein synthesis of D. pulex . In addition, differential regulation of the oxidative phosphorylation pathway (including the NADH:ubquinone oxidoreductase gene family) and the trypsin paralogous gene family (a major component of the digestive system in D. pulex ) could explain why fitness is reduced based on energy budget considerations.