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Environmental DNA (eDNA) analysis has successfully detected organisms in various aquatic environments. However, there is little basic information on eDNA, including the eDNA shedding and degradation processes. This study focused on water temperature and fish biomass and showed that eDNA shedding, degradation, and size distribution varied depending on water temperature and fish biomass. The tank experiments consisted of four temperature levels and three fish biomass levels. The total eDNA and size‐fractioned eDNA from Japanese Jack Mackerels (Trachurus japonicus) were quantified before and after removing the fish. The results showed that the eDNA shedding rate increased at higher water temperature and larger fish biomass, and the eDNA decay rate also increased at higher temperature and fish biomass. In addition, the small‐sized eDNA fractions were proportionally larger at higher temperatures, and these proportions varied among fish biomass. After removing the fish from the tanks, the percentage of eDNA temporally decreased when the eDNA size fraction was >10 µm, while the smaller size fractions increased. These results have the potential to make the use of eDNA analysis more widespread in the future. This study showed that the Japanese Jack Mackerel eDNA shedding rate increased at higher water temperatures and larger fish biomass, and the most supported model for the eDNA decay curves included both temperature and fish density as explanatory variables. In addition, eDNA size distribution varied depending on temperature, fish biomass, and time passage.

Environmental DNA (eDNA) analysis is a rapid, cost‐effective, non‐invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species‐specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real‐time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high‐throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species‐specific surveys. Environmental DNA (eDNA) metabarcoding has enormous potential for community biodiversity assessment but the detection sensitivity of this tool for single species, particularly rare species, within communities is relatively unexplored. We compared targeted real‐time quantitative PCR (qPCR) and eDNA metabarcoding for great crested newt (Triturus cristatus) detection across 532 ponds in the UK and found metabarcoding was less sensitive than qPCR depending on detection thresholds applied. However, sequence read count was correlated with number of positive qPCR replicates, cost of both methods was comparable, and metabarcoding revealed a variety of aquatic and terrestrial fauna alongside great crested newt, thus metabarcoding can be used for initial survey of water bodies to better inform species‐specific survey.

Background and aims Overwintering is a critical part of the annual cycle of animals living at high latitudes, and selection of overwintering sites (hibernacula) is important to population persistence. Identifying the overwintering sites of aquatic species is challenging in areas where water bodies are frozen for significant parts of the year. We tested whether environmental DNA (eDNA) approaches could help to locate them. Materials and methods We conducted environmental DNA surveys of underwater overwintering sites of the northern map turtle (Graptemys geographica), a species of conservation concern in Canada. We collected water samples under the ice in winter across a mid‐sized temperate lake and used quantitative PCR with a species‐specific probe to quantify concentrations of map turtle eDNA. Results and discussion We found localized eDNA signals consistent with known overwintering sites and one previously suspected site. The latter was further confirmed using underwater remote operated vehicle (ROV) visual surveys. Conclusions Our study confirms that eDNA can offer insights on a critical part of the annual cycle of aquatic species, for which we know very little. We test whether winter environmental DNA surveys of a medium‐sized freshwater lake could help us to locate underwater overwintering sites of the northern map turtle, a species of conservation concern in Canada. Using under‐ice winter water sampling across a mid‐sized temperate lake and quantitative PCR with a species‐specific probe, we find localized eDNA signals consistent with a known overwintering site detected using radiotelemetry, or a previously suspected site, which was further confirmed using an underwater drone.

Environmental DNA (eDNA) analysis methods have been developed to detect organism distribution and abundance/biomass in various environments. eDNA degradation is critical for eDNA evaluation. However, the dynamics and mechanisms of eDNA degradation are largely unknown, especially when considering different eDNA sources, for example, cells and fragmental DNA. We experimentally evaluated the degradation rates of eDNA derived from multiple sources, including fragmental DNA (internal PCR control [IPC]), free cells (from Oncorhynchus kisutch), and resident species. We conducted the experiment with pond and seawater to evaluate the differences between freshwater and marine habitats. We quantified the eDNA copies of free cells, fragmental DNA, and resident species (Cyprinus carpio in the pond and Trachurus japonicus in the sea). We found that eDNA derived from both cells and fragmental DNA decreased exponentially in both the sea and pond samples. The degradation of eDNA from resident species showed similar behavior to the cell‐derived eDNA. We evaluated three degradation models with different assumptions and degradation steps and found that a simple exponential model was effective in most cases. Our findings on cell‐ and fragmental DNA‐derived eDNA provide fundamental information about the eDNA degradation process and can be applied to quantify eDNA behavior in natural environments. We conducted the experiments for degradation of eDNA from cell and fragmental DNA. eDNA from inhabiting species showed similar behavior to that derived from cells. A simple exponential model was mostly useful to evaluate eDNA degradation.

Background  Environmental DNA (eDNA) analysis has been recently applied to the surveillance of species distribution and composition in aquatic ecosystems. However, most eDNA studies have used mitochondrial DNA markers, and those using nuclear DNA markers are quite scarce. Moreover, although some studies reported the availability of nuclear DNA markers for eDNA analyses, the characteristics and dynamics of nuclear environmental DNA (nu‐eDNA) of macro‐organisms remain unknown. Herein, we re‐analyzed eDNA samples described in a previously published paper to investigate the shedding and decay rates of nu‐eDNA from Japanese Jack Mackerel (Trachurus japonicus) and compared them to those of mt‐eDNA (mitochondrial environmental DNA). Materials & Methods Tank experiments consisting of 12 combinations of four temperatures and three fish biomass levels were performed, and four tank replicates were prepared for each treatment level. Before and after removing the fish from the tanks, we sampled rearing water over time to quantify nu‐eDNA copy numbers. Results & Discussion Model fitting to eDNA decay curves demonstrated that nu‐eDNA decay rates increased in higher water temperature and with larger fish biomass. The estimated shedding rates of nu‐eDNA also increased with higher temperature and larger biomass. These results were generally consistent with those of mt‐eDNA. Moreover, the ratio of mt‐eDNA to nu‐eDNA shedding and concentration decreased with larger fish biomass levels, which implied that these values may be among the potential indices for estimating the age and body size of organisms from environmental samples. Our findings contribute to the understanding of eDNA characteristics and dynamics between different DNA markers and may help us to interpret future results of eDNA surveillance. We estimated the shedding and decay rates of nu‐eDNA from Japanese Jack Mackerel. Both of them were facilitated with higher water temperature and larger fish biomass. In addition, the ratio of mt‐eDNA to nu‐eDNA decreased with larger fish biomass.

1. Vertical differentiation in root placement is one of the potential mechanisms of plant niche differentiation. It can be due to the remarkable plasticity of roots in response to nutrients and neighbours, but most data on it come from pot or garden experiments. The roles of vertical differentiation and of plasticity in it in the field are thus not well known. 2. We examined species-specific root vertical distribution in a montane grassland using quantitative real-time PCR. We asked whether individual species differ in their rooting depths, whether such differences are associated with above-ground functional traits (such as height or specific leaf area), and whether they respond to the presence of a competitor. This response was assessed by comparison of species-specific vertical profiles between control plots and plots where the dominant species, Festuca rubra, had been removed. 3. Vertical profiles of individual species varied considerably, from species with most root biomass concentrated in the uppermost (<2 cm) soil layer, through species with uniform vertical distribution, to a species with roots predominantly below 8 cm (Nardus stricta). Species at the fast end of the plant economy spectrum were more likely to place their roots in the uppermost layers. Grassland species, thus, exploit different parts of the below-ground resources in spite of their short stature, minor differences in height above-ground and shallow soil. 4. While below-ground and above-ground biomasses of most species were higher in the removal plots, species rooting patterns did not change in response to the removal. The interspecific differences in vertical profiles were thus due to species' innate differences, not to plastic responses to the presence of the dominant species. 5. Synthesis. The findings imply that vertical root differentiation in the field is strong and can contribute to niche differentiation. However, the role of root plasticity in natural systems may be considerably weaker than in artificial systems with few species and strong nutrient gradients. This absence of the plastic response in the field is likely to be due to a fairly homogeneous distribution of nutrients in the soil and to the predominantly symmetric nature of below-ground competition.

ABSTRACT Background DNA double‐strand breaks (DSBs) are the most lethal and dangerous type of lesions with significant implications for both cellular function and organismal health. The number of DSBs (NDSBs) across the genome reflects DNA damage severity. However, current quantification methods mainly rely on next‐generation sequencing, which is laborious and expensive. This study aims to provide a simple, low‐cost, and high‐throughput standard curve‐based method for quantifying genome‐wide DSBs. Method Genomic DNA from human, mouse, Arabidopsis, Saccharomyces cerevisiae, and Escherichia coli was digested by seven blunt‐end restriction enzymes to generate DSB standards. Theoretical NDSBs for each standard were calculated based on restriction site frequency. Ligation‐mediated quantitative PCR (LM‐qPCR) was performed to obtain the Ct values, which were plotted against log‐transformed NDSBs to construct standard curves. Method reliability was assessed by comparing results with neutral single‐cell gel electrophoresis and γ‐H2AX flow cytometry. Results All genomes were successfully digested by seven blunt‐end restriction enzymes to produce standard DSB fragments. Standard curves demonstrated high linearity (R2 > 0.95), with intra‐ and inter‐assay coefficients of variation of 1.101% and 2.528%, respectively. The detection limit was below 100 DSBs. Quantification results strongly correlated with traditional DSB detection methods (|r| > 0.9). Conclusion This standard curve‐based method enables accurate, reproducible quantification of genome‐wide DSBs in various organisms. It is simple, low‐cost, and easily standardized, offering a promising tool for applications in genotoxicity testing, environmental exposure monitoring, and DNA damage research. This study presents a simple and cost‐effective method for quantifying DNA double‐strand breaks (DSBs) using standard curves based on Ct values from LM‐qPCR and the theoretical number of DSBs from enzyme‐digested DNA standards. Its simplicity, affordability, and high‐throughput capability make it a valuable tool for genotoxicity testing, environmental monitoring, and DNA damage studies across diverse organisms.

ABSTRACT Environmental DNA (eDNA) analysis is effective for non‐invasive biodiversity monitoring, as it reveals species distribution and abundance without ecosystem disruption. Four key steps in eDNA analysis include water preservation, DNA capture, DNA extraction, and detection. Among these, the capture of eDNA has attracted significant research interest due to the variability of water samples. Although various methods for eDNA concentration have been developed, including filtration using disc or cartridge filters and passive samplers, no single method is universally applicable because of the variabilities of eDNA distribution and water characteristics, including turbidity levels. Therefore, the development of alternative eDNA concentration methods is important for advancing eDNA research. This study introduces QuickConc, a novel nucleic acid capture method that combines an enhancer of benzalkonium chloride with dispersed silica glass fibers, allowing better binding between nucleic acids and glass fibers. Our results indicate that this approach enhances eDNA capture and extraction efficiency by likely improving the interaction between glass fibers and eDNA. We tested QuickConc in three environments using qPCR and metabarcoding. QuickConc yielded 1.3–3 times more total eDNA compared to glass fiber filtration and Sterivex methods under our experimental settings. Species‐specific qPCR results showed that QuickConc detected 2–10 times higher copy numbers compared to the other two methods. Metabarcoding analyses using the MiFish method revealed that the number of fish species detected in river water was higher with QuickConc compared to other methods, while in sea water, the number of fish species detected was similar to the glass fiber filtration and Sterivex methods. QuickConc offers new options for eDNA analysis for biodiversity monitoring and conservation strategies. Environmental DNA (eDNA) analysis allows non‐invasive monitoring of biodiversity, but effective eDNA capture remains challenging due to variable water characteristics. This study introduces QuickConc, a novel method combining benzalkonium chloride and silica glass fibers, which improved eDNA binding, capture, and extraction efficiency across river, sea, and pond environments. QuickConc offers new options for eDNA analysis for biodiversity monitoring and conservation strategies.

Reduced oxygen availability is not only associated with flooding, but occurs also during growth and development. It is largely unknown how hypoxia is perceived and what signaling cascade is involved in activating adaptive responses. We analysed the expression of over 1900 transcription factors (TFs) and 180 microRNA primary transcripts (pri-miRNAs) in Arabidopsis roots exposed to different hypoxic conditions by means of quantitative PCR. We also analysed the promoters of genes induced by hypoxia with respect to over-represented DNA elements that can act as potential TF binding sites and their in vivo interaction was verified. We identified various subsets of TFs that responded differentially through time and in an oxygen concentration-dependent manner. The regulatory potential of selected TFs and their predicted DNA binding elements was validated. Although the expression of pri-miRNAs was differentially regulated under hypoxia, only one corresponding mature miRNA changed accordingly. Putative target transcripts of the miRNAs were not significantly affected. Our results show that the regulation of hypoxia-induced genes is controlled via simultaneous interaction of various combinations of TFs. Under anoxic conditions, an additional set of TFs is induced. Regulation of gene expression via miRNAs appears to play a minor role during hypoxia.