Monitoring of environmental DNA from nonindigenous species of algae, dinoflagellates and animals in the North East Atlantic.

Monitoring the distribution of marine nonindigenous species is a challenging task. To support this monitoring, we developed and validated the specificity of 12 primer-probe assays for detection of environmental DNA (eDNA) from marine species, all nonindigenous to Europe. The species include sturgeons, a Pacific red algae, oyster thief, a freshwater hydroid from the Black Sea, Chinese mitten crab, Pacific oyster, warty comb jelly, sand gaper, round goby, pink salmon, rainbow trout and North American mud crab. We tested all assays in the laboratory, on DNA extracted from both the target and non-target species to ensure that they only amplified DNA from the intended species. Subsequently, all assays were used to analyse water samples collected at 16 different harbours across two different seasons during 2017. We also included six previously published assays targeting eDNA from goldfish, European carp, two species of dinoflagellates of the genera Karenia and Prorocentrum, two species of the heterokont flagellate genus Pseudochattonella. Conventional monitoring was carried out alongside eDNA sampling but with only one sampling event over the one year. Because eDNA was relatively fast and easy to collect compared to conventional sampling, we sampled eDNA twice during 2017, which showed seasonal changes in the distribution of nonindigenous species. Comparing eDNA levels with salinity gradients did not show any correlation. A significant correlation was observed between number of species detected with conventional monitoring methods and number of species found using eDNA at each location. This supports the use of eDNA for surveillance of the distribution of marine nonindigenous species, where the speed and relative easy sampling in the field combined with fast molecular analysis may provide advantages compared to conventional monitoring methods. Prior validation of assays increases taxonomic precision, and laboratorial setup facilitates analysis of multiple samples simultaneously. The specific eDNA assays presented here can be implemented directly in monitoring programmes across Europe and potentially worldwide to infer a more precise picture of the dynamics in the distribution of marine nonindigenous species.

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.

Integrative assessment of low-dose gamma radiation effects on Daphnia magna reproduction: Toxicity pathway assembly and AOP development.

High energy gamma radiation is potentially hazardous to organisms, including aquatic invertebrates. Although extensively studied in a number of invertebrate species, knowledge on effects induced by gamma radiation is to a large extent limited to the induction of oxidative stress and DNA damage at the molecular/cellular level, or survival, growth and reproduction at the organismal level. As the knowledge of causal relationships between effects occurring at different levels of biological organization is scarce, the ability to provide mechanistic explanation for observed adverse effects is limited, and thus development of Adverse Outcome Pathways (AOPs) and larger scale implementation into next generation hazard and risk predictions is restricted. The present study was therefore conducted to assess the effects of high-energy gamma radiation from cobalt-60 across multiple levels of biological organization (i.e., molecular, cellular, tissue, organ and individual) and characterize the major toxicity pathways leading to impaired reproduction in the model freshwater crustacean Daphnia magna (water flea). Following gamma exposure, a number of bioassays were integrated to measure relevant toxicological endpoints such as gene expression, reactive oxygen species (ROS), lipid peroxidation (LPO), neutral lipid storage, adenosine triphosphate (ATP) content, apoptosis, ovary histology and reproduction. A non-monotonic pattern was consistently observed across the levels of biological organization, albeit with some variation at the lower end of the dose-rate scale, indicating a complex response to radiation doses. By integrating results from different bioassays, a novel pathway network describing the key toxicity pathways involved in the reproductive effects of gamma radiation were proposed, such as DNA damage-oocyte apoptosis pathway, LPO-ATP depletion pathway, calcium influx-endocrine disruption pathway and DNA hypermethylation pathway. Three novel AOPs were proposed for oxidative stressor-mediated excessive ROS formation leading to reproductive effect, and thus introducing the world's first AOPs for non-chemical stressors in aquatic invertebrates.

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.

Detection of an invasive aquatic plant in natural water bodies using environmental DNA.

The ability to detect founding populations of invasive species or rare species with low number of individuals is important for aquatic ecosystem management. Traditional approaches use historical data, knowledge of the species' ecology and time-consuming surveys. Within the past decade, environmental DNA (eDNA) has emerged as a powerful additional tracking tool. While much work has been done with animals, comparatively very little has been done with aquatic plants. Here we investigated the transportation and seasonal changes in eDNA concentrations for an invasive aquatic species, Elodea canadensis, in Norway. A specific probe assay was developed using chloroplast DNA to study the fate of the targeted eDNA through space and time. The spatial study used a known source of Elodea canadensis within Lake Nordbytjern 400 m away from the lake outlet flowing into the stream Tveia. The rate of disappearance of E. canadensis eDNA was an order of magnitude loss over about 230 m in the lake and 1550 m in the stream. The time series study was performed monthly from May to October in lake Steinsfjorden harbouring E. canadensis, showing that eDNA concentrations varied by up to three orders of magnitude, peaking during fall. In both studies, the presence of suspended clay or turbidity for some samples did not hamper eDNA analysis. This study shows how efficient eDNA tools may be for tracking aquatic plants in the environment and provides key spatial and temporal information on the fate of eDNA.

Rapid expansion of the invasive oyster Crassostrea gigas at its northern distribution limit in Europe: Naturally dispersed or introduced?

The Pacific oyster, Crassostrea gigas, was introduced to Europe for aquaculture purposes, and has had a rapid and unforeseen northward expansion in northern Europe. The recent dramatic increase in number of C. gigas populations along the species' northern distribution limit has questioned the efficiency of Skagerrak as a dispersal barrier for transport and survival of larvae. We investigated the genetic connectivity and possible spreading patterns between Pacific oyster populations on the southern Norwegian coast (4 localities) and Swedish and Danish populations by means of DNA microsatellite analysis of adult oysters, and by simulating larvae drift. In the simulations we used a 3D oceanographic model to explore the influence of recent climate change (1990-2010) on development, survival, and successful spreading of Danish and Swedish Pacific oyster larvae to Norwegian coastal waters. The simulations indicated adequate temperature conditions for development, survival, and settlement of larvae across the Skagerrak in warm years since 2000. However, microsatellite genotyping revealed genetic differences between the Norwegian populations, and between the Norwegian populations and the Swedish and Danish populations, the latter two populations being more similar. This patchwork pattern of genetic dissimilarity among the Norwegian populations points towards multiple local introduction routes rather than the commonly assumed unidirectional entry of larvae drifted from Denmark and Sweden. Alternative origins of introduction and implications for management, such as forecasting and possible mitigation actions, are discussed.

Genomics and the challenging translation into conservation practice.

The global loss of biodiversity continues at an alarming rate. Genomic approaches have been suggested as a promising tool for conservation practice as scaling up to genome-wide data can improve traditional conservation genetic inferences and provide qualitatively novel insights. However, the generation of genomic data and subsequent analyses and interpretations remain challenging and largely confined to academic research in ecology and evolution. This generates a gap between basic research and applicable solutions for conservation managers faced with multifaceted problems. Before the real-world conservation potential of genomic research can be realized, we suggest that current infrastructures need to be modified, methods must mature, analytical pipelines need to be developed, and successful case studies must be disseminated to practitioners.