Epigenetic, transcriptional and phenotypic responses in Daphnia magna exposed to low-level ionizing radiation.

Ionizing radiation is known to induce oxidative stress and DNA damage as well as epigenetic effects in aquatic organisms. Epigenetic changes can be part of the adaptive responses to protect organisms from radiation-induced damage, or act as drivers of toxicity pathways leading to adverse effects. To investigate the potential roles of epigenetic mechanisms in low-dose ionizing radiation-induced stress responses, an ecologically relevant crustacean, adult Daphnia magna were chronically exposed to low and medium level external 60Co gamma radiation ranging from 0.4, 1, 4, 10, and 40 mGy/h for seven days. Biological effects at the molecular (global DNA methylation, histone modification, gene expression), cellular (reactive oxygen species formation), tissue/organ (ovary, gut and epidermal histology) and organismal (fecundity) levels were investigated using a suite of effect assessment tools. The results showed an increase in global DNA methylation associated with loci-specific alterations of histone H3K9 methylation and acetylation, and downregulation of genes involved in DNA methylation, one-carbon metabolism, antioxidant defense, DNA repair, apoptosis, calcium signaling and endocrine regulation of development and reproduction. Temporal changes of reactive oxygen species (ROS) formation were also observed with an apparent transition from ROS suppression to induction from 2 to 7 days after gamma exposure. The cumulative fecundity, however, was not significantly changed by the gamma exposure. On the basis of the new experimental evidence and existing knowledge, a hypothetical model was proposed to provide in-depth mechanistic understanding of the roles of epigenetic mechanisms in low dose ionizing radiation induced stress responses in D. magna.

Gamma irradiation during gametogenesis in young adult zebrafish causes persistent genotoxicity and adverse reproductive effects.

The biological effects of gamma radiation may exert damage beyond that of the individual through its deleterious effects on reproductive function. Impaired reproductive performance can result in reduced population size over consecutive generations. In a continued effort to investigate reproductive and heritable effects of ionizing radiation, we recently demonstrated adverse effects and genomic instability in progeny of parents exposed to gamma radiation. In the present study, genotoxicity and effects on the reproduction following subchronic exposure during a gametogenesis cycle to 60Co gamma radiation (27 days, 8.7 and 53 mGy/h, total doses 5.2 and 31 Gy) were investigated in the adult wild-type zebrafish (Danio rerio). A significant reduction in embryo production was observed one month after exposure in the 53 mGy/h exposure group compared to control and 8.7 mGy/h. One year later, embryo production was significantly lower in the 53 mGy/h group compared only to control, with observed sterility, accompanied by a regression of reproductive organs in 100% of the fish 1.5 years after exposure. Histopathological examinations revealed no significant changes in the testis in the 8.7 mGy/h group, while in 62.5% of females exposed to this dose rate the oogenesis was found to be only at the early previtellogenic stage. The DNA damage determined in whole blood, 1.5 years after irradiation, using a high throughput Comet assay, was significantly higher in the exposed groups (1.2 and 3-fold increase in 8.7 and 53 mGy/h females respectively; 3-fold and 2-fold increase in 8.7 and 53 mGy/h males respectively) compared to controls. A significantly higher number of micronuclei (4-5%) was found in erythrocytes of both the 8.7 and 53 mGy/h fish compared to controls. This study shows that gamma radiation at a dose rate of ≥ 8.7 mGy/h during gametogenesis causes adverse reproductive effects and persistent genotoxicity (DNA damage and increased micronuclei) in adult zebrafish.

Parental gamma irradiation induces reprotoxic effects accompanied by genomic instability in zebrafish (Danio rerio) embryos.

Gamma radiation represents a potential health risk to aquatic and terrestrial biota, due to its ability to ionize atoms and molecules in living tissues. The effects of exposure to 60Co gamma radiation in zebrafish (Danio rerio) were studied during two sensitive life stages: gametogenesis (F0: 53 and 8.7mGy/h for 27 days, total doses 31 and 5.2Gy) and embryogenesis (9.6mGy/h for 65h; total dose 0.62Gy). Progeny of F0 exposed to 53mGy/h showed 100% mortality occurring at the gastrulation stage corresponding to 8h post fertilization (hpf). Control and F0 fish exposed to 8.7mGy/h were used to create four lines in the first filial generation (F1): control, G line (irradiated during parental gametogenesis), E line (irradiated during embryogenesis) and GE line (irradiated during parental gametogenesis and embryogenesis). A statistically significant cumulative mortality of GE larva (9.3%) compared to controls was found at 96 hpf. E line embryos hatched significantly earlier compared to controls, G and GE (48-72 hpf). The deformity frequency was higher in G and GE, but not E line compared to controls at 72 hpf. One month after parental irradiation, the formation of reactive oxygen species (ROS) was increased in the G line, but did not significantly differ from controls one year after parental irradiation, while at the same time point it was significantly increased in the directly exposed E and GE lines from 60 to 120 hpf. Lipid peroxidation (LPO) was significantly increased in the G line one year after parental irradiation, while significant increase in DNA damage was detected in both the G and GE compared to controls and E line at 72 hpf. Radiation-induced bystander effects, triggered by culture media from tissue explants and observed as influx of Ca2+ ions through the cellular membrane of the reporter cells, were significantly increased in 72 hpf G line progeny one month after irradiation of the parents. One year after parental irradiation, the bystander effects were increased in the E line compared to controls, but not in progeny of irradiated parents (G and GE lines). Overall, this study showed that irradiation of parents can result in multigenerational oxidative stress and genomic instability in irradiated (GE) and non-irradiated (G) progeny of irradiated parents, including increases in ROS formation, LPO, DNA damage and bystander effects. The results therefore highlight the necessity for multi- and transgenerational studies to assess the environmental impact of gamma radiation.

Parental exposure to natural mixtures of persistent organic pollutants (POP) induced changes in transcription of apoptosis-related genes in offspring zebrafish embryos.

Apoptosis is an integral element of development that may also be initiated by environmental contaminants. The aim of the present study was to assess potential changes in the regulation of apoptotic genes in zebrafish embryos following parental exposure to two natural mixtures of persistent organic pollutants (POP). The mixture from Lake Mjøsa contained exceptionally high concentrations of polybrominated diphenyl ethers (PBDE), as well as relatively high levels of polychlorinated biphenyls (PCB) and dichlorodiphenyltrichloroethane (DDT). The mixture from Lake Losna contained background concentrations of POP. Genes involved in the apoptotic machinery were screened for their expression profile at four time points during embryonic development. Thirteen and 15 genes involved in apoptosis were found to be significantly upregulated in the high-exposure and background exposure groups, respectively, compared with controls. Modulation of apoptotic genes was restricted only to the first time point, which corresponds with the blastula stage. Although there were substantial differences in POP concentrations between mixtures, genes underlying the apoptosis process showed almost similar responses to the two mixtures. In both exposure groups the main executors of apoptosis p53, casp 2, casp 6, cassp 8, and BAX displayed upregulation compared to controls, suggesting that these POP induce apoptosis via a p53-dependent mechanism. Upregulation of genes that play a critical role in apoptosis suggests that disturbance of normal apoptotic signaling during gametogenesis and embryogenesis may be one of the central mechanisms involved in adverse reproductive effects produced by POP in zebrafish.

Endocrine effects of real-life mixtures of persistent organic pollutants (POP) in experimental models and wild fish.

A series of studies have assessed the occurrence, levels, and potential adverse effects of persistent organic pollutants (POP) in fish from Lake Mjøsa. In this lake, high levels of various POP were detected in biota. Fish from the nearby Lake Losna contain background levels of POP and served as reference (controls) in these studies. Significantly higher prevalence of mycobacteriosis and pathological changes were documented in burbot (Lota lota) from Mjøsa compared to burbot from Losna. Further, transcriptional profiling identified changes in gene expression in burbot from Mjøsa compared to burbot from Losna associated with drug metabolism enzymes and oxidative stress. POP extracted from burbot liver oil from the two lakes was used to expose zebrafish (Danio rerio) during two consecutive generations. During both generations, POP mixtures from both lakes increased the rate of mortality, induced earlier onset of puberty, and skewed sex ratio toward males. However, opposite effects on weight gain were found in exposure groups compared to controls during the two generations. Exposure to POP from both lakes was associated with suppression of ovarian follicle development. Analyses of genome-wide transcription profiling identified functional networks of genes associated with weight homeostasis, steroid hormone functions, and insulin signaling. In human cell studies using adrenocortical H295R and primary porcine theca and granulosa cells, exposure to lake extracts from both populations modulated steroid hormone production with significant difference from controls. The results suggest that POP from both lakes may possess the potential to induce endocrine disruption and may adversely affect health in wild fish.

The specific alteration of histone methylation profiles by DZNep during early zebrafish development.

Early embryo development constitutes a unique opportunity to study acquisition of epigenetic marks, including histone methylation. This study investigates the in vivo function and specificity of 3-deazaneplanocin A (DZNep), a promising anti-cancer drug that targets polycomb complex genes. One- to two-cell stage embryos were cultured with DZNep, and subsequently evaluated at the post-mid blastula transition stage for H3K27me3, H3K4me3 and H3K9me3 occupancy and enrichment at promoters using ChIP-chip microarrays. DZNep affected promoter enrichment of H3K27me3 and H3K9me3, whereas H3K4me3 remained stable. Interestingly, DZNep induced a loss of H3K27me3 and H3K9me3 from a substantial number of promoters but did not prevent de novo acquisition of these marks on others, indicating gene-specific targeting of its action. Loss/gain of H3K27me3 on promoters did not result in changes in gene expression levels until 24h post-fertilization. In contrast, genes gaining H3K9me3 displayed strong and constant down-regulation upon DZNep treatment. H3K9me3 enrichment on these gene promoters was observed not only in the proximal area as expected, but also over the transcription start site. Altered H3K9me3 profiles were associated with severe neuronal and cranial phenotypes at day 4-5 post-fertilization. Thus, DZNep was shown to affect enrichment patterns of H3K27me3 and H3K9me3 at promoters in a gene-specific manner.

Zebrafish mRNA sequencing deciphers novelties in transcriptome dynamics during maternal to zygotic transition.

Maternally deposited mRNAs direct early development before the initiation of zygotic transcription during mid-blastula transition (MBT). To study mechanisms regulating this developmental event in zebrafish, we applied mRNA deep sequencing technology and generated comprehensive information and valuable resources on transcriptome dynamics during early embryonic (egg to early gastrulation) stages. Genome-wide transcriptome analysis documented at least 8000 maternal genes and identified the earliest cohort of zygotic transcripts. We determined expression levels of maternal and zygotic transcripts with the highest resolution possible using mRNA-seq and clustered them based on their expression pattern. We unravel delayed polyadenylation in a large cohort of maternal transcripts prior to the MBT for the first time in zebrafish. Blocking polyadenylation of these transcripts confirms their role in regulating development from the MBT onward. Our study also identified a large number of novel transcribed regions in annotated and unannotated regions of the genome, which will facilitate reannotation of the zebrafish genome. We also identified splice variants with an estimated frequency of 50%-60%. Taken together, our data constitute a useful genomic information and valuable transcriptome resource for gene discovery and for understanding the mechanisms of early embryogenesis in zebrafish.

Differential transcript isoform usage pre- and post-zygotic genome activation in zebrafish.

BACKGROUND: Zebrafish embryos are transcriptionally silent until activation of the zygotic genome during the 10th cell cycle. Onset of transcription is followed by cellular and morphological changes involving cell speciation and gastrulation. Previous genome-wide surveys of transcriptional changes only assessed gene expression levels; however, recent studies have shown the necessity to map isoform-specific transcriptional changes. Here, we perform isoform discovery and quantification on transcriptome sequences from before and after zebrafish zygotic genome activation (ZGA). RESULTS: We identify novel isoforms and isoform switches during ZGA for genes related to cell adhesion, pluripotency and DNA methylation. Isoform switching events include alternative splicing and changes in transcriptional start sites and in 3' untranslated regions. New isoforms are identified even for well-characterized genes such as pou5f1, sall4 and dnmt1. Genes involved in cell-cell interactions such as f11r and magi1 display isoform switches with alterations of coding sequences. We also detect over 1000 transcripts that acquire a longer 3' terminal exon when transcribed by the zygote compared to their maternal transcript counterparts. ChIP-sequencing data mapped onto skipped exon events reveal a correlation between histone H3K36 trimethylation peaks and skipped exons, suggesting epigenetic marks being part of alternative splicing regulation. CONCLUSIONS: The novel isoforms and isoform switches reported here include regulators of transcriptional, cellular and morphological changes taking place around ZGA. Our data display an array of isoform-related functional changes and represent a valuable resource complementary to existing early embryo transcriptomes.

Epigenetic complexity during the zebrafish mid-blastula transition.

The zebrafish developmental transcription program is determined by temporal post-translational histone modifications established in a step-wise and combinatorial manner on specific promoters around the time of zygotic genome activation (ZGA). Here, we characterize this increasing epigenetic complexity before, during and after ZGA. H3K4me3/H3K27me3 co-enrichment prevails over H3K4me3/H3K9me3 at the time of ZGA. Whereas most H3K4me3-marked promoters are devoid of transcriptionally repressive H3K9me3 or H3K27me3, the latter marks rarely occur in absence of H3K4me3. On co-enriched genomic regions, H3K4me3 and H3K27me3 can overlap regardless of H3K9me3 enrichment, but H3K4me3 and H3K9me3 are mutually exclusive. H3K4me3 and H3K9me3 may however overlap only when H3K27me3 also marks the overlapping domain, suggesting that H3K27me3 may modulate chromatin states. On metagenes, H3K27me3 enrichment correlates with local alteration in H3K4me3 density, and co-enrichment in H3K9me3 is linked to alterations in both H3K27me3 and H3K4me3 profiles. This suggests physical proximity of these marks and supports a view of existence of bi- or tri-valent chromatin domains. Thus enrichment in trimethylated H3K9 or H3K27 is associated with local remodeling of chromatin manifested by changes in H3K4me3 density. We propose that metagenes can provide information on the multivalency of chromatin sates.

Accumulation and effects of natural mixtures of persistent organic pollutants (POP) in Zebrafish after two generations of exposure.

Effects of exposure to environmentally realistic mixtures of persistent organic pollutants (POP) harvested from aquatic ecosystems in Norway were studied in an in vivo zebrafish model. POP were extracted from burbot (Lota lota) liver from two separate lakes, Lake Losna and Lake Mjøsa, and exposed to zebrafish through the diet in a two-generation study. Effects on survival, growth, sex ratio, and timing of puberty were investigated. In addition, the biomarkers 7-ethoxyresorufin O-deethylase (EROD) and vitellogenin (Vtg) were measured. The ratios of contaminant levels in extracts collected from Lake Mjøsa:Lake Losna were 6, 10, and 270 for polychlorinated biphenyls (PCB), dichlorodiphenyltrichloroethanes (DDT), and polybrominated diphenyl ethers (PBDE), respectively. The concentration range of POP measured in zebrafish was lower than in burbot originating from Lake Mjøsa, but comparable to concentrations previously reported in humans and wildlife. The results showed that exposure to environmentally realistic mixtures of POP exerted a negative effect on survival of fish in both generations. The marked drop in survival during 9-20 days post fertilization (dpf) suggested that this period may be a critical window for development. In both generations an earlier onset of puberty was observed and a higher proportion of males than females was noted in exposed fish compared to controls. Suprising effects of exposure were found on body weight. In the first generation (F(0)), body weight was significantly higher in both exposure groups compared to controls, while in the next generation (F(1)) the same exposures were associated with a decrease in body weight. Zebrafish exposed to relatively low quantities of POP showed a significant induction of biomarkers (EROD and Vtg), while fish exposed to higher exposure doses did not demonstrate induction.

Altered non-coding RNA expression profile in F1 progeny 1 year after parental irradiation is linked to adverse effects in zebrafish.

Gamma radiation produces DNA instability and impaired phenotype. Previously, we observed negative effects on phenotype, DNA methylation, and gene expression profiles, in offspring of zebrafish exposed to gamma radiation during gametogenesis. We hypothesize that previously observed effects are accompanied with changes in the expression profile of non-coding RNAs, inherited by next generations. Non-coding RNA expression profile was analysed in F1 offspring (5.5 h post-fertilization) by high-throughput sequencing 1 year after parental irradiation (8.7 mGy/h, 5.2 Gy total dose). Using our previous F1-γ genome-wide gene expression data (GSE98539), hundreds of mRNAs were predicted as targets of differentially expressed (DE) miRNAs, involved in pathways such as insulin receptor, NFkB and PTEN signalling, linking to apoptosis and cancer. snRNAs belonging to the five major spliceosomal snRNAs were down-regulated in the F1-γ group, Indicating transcriptional and post-transcriptional alterations. In addition, DEpiRNA clusters were associated to 9 transposable elements (TEs) (LTR, LINE, and TIR) (p = 0.0024), probable as a response to the activation of these TEs. Moreover, the expression of the lincRNAs malat-1, and several others was altered in the offspring F1, in concordance with previously observed phenotypical alterations. In conclusion, our results demonstrate diverse gamma radiation-induced alterations in the ncRNA profiles of F1 offspring observable 1 year after parental irradiation.

A Systematic Analysis of Metal and Metalloid Concentrations in Eight Zebrafish Recirculating Water Systems.

Metals and metalloids are integral to biological processes and play key roles in physiology and metabolism. Nonetheless, overexposure to some metals or lack of others can lead to serious health consequences. In this study, eight zebrafish facilities collaborated to generate a multielement analysis of their centralized recirculating water systems. We report a first set of average concentrations for 46 elements detected in zebrafish facilities. Our results help to establish an initial baseline for trouble-shooting purposes, and in general for safe ranges of metal concentrations in recirculating water systems, supporting reproducible scientific research outcomes with zebrafish.

Natural mixtures of persistent organic pollutants (POP) increase weight gain, advance puberty, and induce changes in gene expression associated with steroid hormones and obesity in female zebrafish.

In the present study, developmental and reproductive effects of lifelong exposure to environmental relevant concentrations of two natural mixtures of persistent organic pollutants (POP) were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot (Lota lota) liver originating from freshwater systems in Norway: one mixture with high levels and one mixture with background levels of polybrominated diphenyl ethers (PBDE), polychlorinated biphenyls (PCB), and dichlorodiphenyltrichloroethane metabolites (DDT). The concentration of POP measured in the zebrafish ranged from levels detected in wild fish from Lake Mjøsa to concentrations reported in human and wildlife populations, indicating that the experimental fish were exposed to concentrations comparable with wild fish. Phenotypic effects observed in both exposure groups included earlier onset of puberty, increased male/female sex ratio, and differences in body weight at 5 mo of age. Interestingly, genome-wide transcription profiling showed changes in regulation of genes involved in endocrine signaling and growth. The transcriptomics changes include key regulator genes for steroid hormone functions (ncoa3), and growth (c/ebp, ncoa3). The effects observed in the experimental zebrafish model raise the question whether chemical pollution represents a risk to reproductive health of wild fish inhabitating the freshwater system.

Zebrafish: Housing and husbandry recommendations.

This article provides recommendations for the care of laboratory zebrafish (Danio rerio) as part of the further implementation of Annex A to the European Convention on the protection of vertebrate animals used for experimental and other scientific purposes, EU Commission Recommendation 2007/526/EC and the fulfilment of Article 33 of EU Directive 2010/63, both concerning the housing and care of experimental animals. The recommendations provide guidance on best practices and ranges of husbandry parameters within which zebrafish welfare, as well as reproducibility of experimental procedures, are assured. Husbandry procedures found today in zebrafish facilities are numerous. While the vast majority of these practices are perfectly acceptable in terms of zebrafish physiology and welfare, the reproducibility of experimental results could be improved by further standardisation of husbandry procedures and exchange of husbandry information between laboratories. Standardisation protocols providing ranges of husbandry parameters are likely to be more successful and appropriate than the implementation of a set of fixed guidance values neglecting the empirically successful daily routines of many facilities and will better reflect the wide range of environmental parameters that characterise the natural habitats occupied by zebrafish. A joint working group on zebrafish housing and husbandry recommendations, with members of the European Society for Fish Models in Biology and Medicine (EUFishBioMed) and of the Federation of European Laboratory Animal Science Associations (FELASA) has been given a mandate to provide guidelines based on a FELASA list of parameters, 'Terms of Reference'.

Inhibition of methyltransferase activity of enhancer of zeste 2 leads to enhanced lipid accumulation and altered chromatin status in zebrafish.

BACKGROUND: Recent studies indicate that exposure to environmental chemicals may increase susceptibility to developing metabolic diseases. This susceptibility may in part be caused by changes to the epigenetic landscape which consequently affect gene expression and lead to changes in lipid metabolism. The epigenetic modifier enhancer of zeste 2 (Ezh2) is a histone H3K27 methyltransferase implicated to play a role in lipid metabolism and adipogenesis. In this study, we used the zebrafish (Danio rerio) to investigate the role of Ezh2 on lipid metabolism and chromatin status following developmental exposure to the Ezh1/2 inhibitor PF-06726304 acetate. We used the environmental chemical tributyltin (TBT) as a positive control, as this chemical is known to act on lipid metabolism via EZH-mediated pathways in mammals. RESULTS: Zebrafish embryos (0-5 days post-fertilization, dpf) exposed to non-toxic concentrations of PF-06726304 acetate (5 µM) and TBT (1 nM) exhibited increased lipid accumulation. Changes in chromatin were analyzed by the assay for transposase-accessible chromatin sequencing (ATAC-seq) at 50% epiboly (5.5 hpf). We observed 349 altered chromatin regions, predominantly located at H3K27me3 loci and mostly more open chromatin in the exposed samples. Genes associated to these loci were linked to metabolic pathways. In addition, a selection of genes involved in lipid homeostasis, adipogenesis and genes specifically targeted by PF-06726304 acetate via altered chromatin accessibility were differentially expressed after TBT and PF-06726304 acetate exposure at 5 dpf, but not at 50% epiboly stage. One gene, cebpa, did not show a change in chromatin, but did show a change in gene expression at 5 dpf. Interestingly, underlying H3K27me3 marks were significantly decreased at this locus at 50% epiboly. CONCLUSIONS: Here, we show for the first time the applicability of ATAC-seq as a tool to investigate toxicological responses in zebrafish. Our analysis indicates that Ezh2 inhibition leads to a partial primed state of chromatin linked to metabolic pathways which results in gene expression changes later in development, leading to enhanced lipid accumulation. Although ATAC-seq seems promising, our in-depth assessment of the cebpa locus indicates that we need to consider underlying epigenetic marks as well.

Transcriptional regulation in liver and testis associated with developmental and reproductive effects in male zebrafish exposed to natural mixtures of persistent organic pollutants (POP).

Persistent organic pollutants (POP) occur as mixtures in nature and it is difficult to predict the toxicity of such mixtures based on knowledge about toxicity and mechanisms of action for single compounds. The present knowledge on the combined toxic effects and modes of actions of exposure to mixtures is limited. Thus, the scientifically based hazard and risk assessment of POP requires analytical and toxicological data from studies with environmental mixtures of POP. The application of genome wide transcription profiling in toxicology, in combination with classical endpoints, will improve the current understanding of the mechanisms of toxic processes. Furthermore, gene expression data may be useful in establishing new hypothesis and discovering new biomarkers for known toxicity as well as not yet recognized toxicity endpoints. In the present study, developmental and reproductive effects of lifelong exposure to environmental relevant concentrations of two natural mixtures of POP were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot (Lota lota) liver originating from freshwater systems in Norway: one mixture with high levels and one mixture with background levels of polybrominated diphenyl ethers (PBD), polychlorinated biphenyls (PCB), and DDT. The concentration of POP in the zebrafish ranged from levels detected in wild fish from Lake Mjøsa, to concentrations reported in human and wildlife populations. Phenotypic effects observed in both exposure groups included (1) reduced survival, (2) earlier onset of puberty, (3) increased male/female sex ratio, and (4) differences in body weight at 5 mo of age. Interestingly, genome-wide transcription profiling showed changes in regulation of genes involved in endocrine signaling and growth. The transcriptomics changes included (1) key regulator genes for steroid and thyroid hormone functions (cga, ncoa3), (2) insulin signaling and metabolic homeostasis (pik3r1, pfkfb3, ptb1), and (3) p53 activation (mdm4). The effects observed in the experimental zebrafish model raise the question of whether chemical pollution represents a risk to the reproductive health of wild fish inhabiting the freshwater system.

The Nordic Zebrafish and Husbandry Meeting 2018.

The Nordic zebrafish and husbandry meeting took place at Karolinska Institutet in Stockholm, November 7-9, 2018. More than 120 scientists from Europe joined this meeting, which also attracted world-leading keynote speakers such as Zoltan Varga, Didier Stainier, and Hernán Lopez-Schier. The meeting comprised both scientific as well as zebrafish husbandry and animal welfare sessions. This combination led to fruitful discussions, new collaborations as well as in the formation of a working group that will review and compile evidence-based husbandry guidelines for the local authorities. The success of this meeting emphasizes in general that smaller local conferences provide an excellent platform to establish local networks, to build up and share local infrastructures as well as to provide knowledge and help to peer researchers.

Epigenetic, transcriptional and phenotypic responses in two generations of Daphnia magna exposed to the DNA methylation inhibitor 5-azacytidine.

The water flea Daphnia magna is a keystone species in freshwater ecosystems and has been widely used as a model organism in environmental ecotoxicology. This aquatic crustacean is sensitive to environmental stressors and displays considerable plasticity in adapting to changing environmental conditions. Part of this plasticity may be due to epigenetic regulation of gene expression, including changes to DNA methylation and histone modifications. Because of the generally hypomethylated genome of this species, we hypothesized that the histone code may have an essential role in the epigenetic control and that histone modifications might be an early marker for stress. This study aims to characterize the epigenetic, transcriptional and phenotypic responses and their causal linkages in directly exposed adult (F0) Daphnia and peritoneal exposed neonates (F1) after a chronic (7-day) exposure to a sublethal concentration (10 mg/l) of 5-azacytidine, a well-studied vertebrate DNA methylation inhibitor. Exposure of the F0 generation significantly reduced the cumulative fecundity, accompanied with differential expression of genes in the one-carbon-cycle metabolic pathway. In the epigenome of the F0 generation, a decrease in global DNA methylation, but no significant changes on H3K4me3 or H3K27me3, were observed. In the F1 offspring generation, changes in gene expression, a significant reduction in global DNA methylation and changes in histone modifications were identified. The results indicate that exposure during adulthood may result in more pronounced effects on early development in the offspring generation, though interpretation of the data should be carefully done since both the exposure regime and developmental period is different in the two generations examined. The obtained results improve our understanding of crustacean epigenetics and the tools developed may promote use of epigenetic markers in hazard assessment of environmental stressors.

Gamma radiation induces locus specific changes to histone modification enrichment in zebrafish and Atlantic salmon.

Ionizing radiation is a recognized genotoxic agent, however, little is known about the role of the functional form of DNA in these processes. Post translational modifications on histone proteins control the organization of chromatin and hence control transcriptional responses that ultimately affect the phenotype. The purpose of this study was to investigate effects on chromatin caused by ionizing radiation in fish. Direct exposure of zebrafish (Danio rerio) embryos to gamma radiation (10.9 mGy/h for 3h) induced hyper-enrichment of H3K4me3 at the genes hnf4a, gmnn and vegfab. A similar relative hyper-enrichment was seen at the hnf4a loci of irradiated Atlantic salmon (Salmo salar) embryos (30 mGy/h for 10 days). At the selected genes in ovaries of adult zebrafish irradiated during gametogenesis (8.7 and 53 mGy/h for 27 days), a reduced enrichment of H3K4me3 was observed, which was correlated with reduced levels of histone H3 was observed. F1 embryos of the exposed parents showed hyper-methylation of H3K4me3, H3K9me3 and H3K27me3 on the same three loci, while these differences were almost negligible in F2 embryos. Our results from three selected loci suggest that ionizing radiation can affect chromatin structure and organization, and that these changes can be detected in F1 offspring, but not in subsequent generations.

Growth enhancement of shrimp (Litopenaeus schmitti) after transfer of tilapia growth hormone gene.

Electroporation of Litopenaeus schmitti embryos was used to transfer the pE300tiGH15 plasmid that contains the tilapia growth hormone gene (tiGH) complexed with a nuclear localization signal peptide into the zygotes. The gene construct was detected in 35 (36%) of the 98 larvae screened by PCR and Southern blot analyses. Western blot analyses revealed that 34% of the screened larvae expressed a single tiGH-specific band with the expected molecular mass (23.1 kDa). The development index and larval length indicated a significant growth enhancement from day 3 on after electroporation, with an average of 32% of the growth enhancement. To our knowledge, this is the first report on gene transfer enhanced growth in crustaceans.