Environmental DNA (eDNA) from aquatic vertebrates has recently been used to estimate the presence of a species. We hypothesized that fish release DNA into the water at a rate commensurate with their biomass. Thus, the concentration of eDNA of a target species may be used to estimate the species biomass. We developed an eDNA method to estimate the biomass of common carp (Cyprinus carpio L.) using laboratory and field experiments. In the aquarium, the concentration of eDNA changed initially, but reached an equilibrium after 6 days. Temperature had no effect on eDNA concentrations in aquaria. The concentration of eDNA was positively correlated with carp biomass in both aquaria and experimental ponds. We used this method to estimate the biomass and distribution of carp in a natural freshwater lagoon. We demonstrated that the distribution of carp eDNA concentration was explained by water temperature. Our results suggest that biomass data estimated from eDNA concentration reflects the potential distribution of common carp in the natural environment. Measuring eDNA concentration offers a non-invasive, simple, and rapid method for estimating biomass. This method could inform management plans for the conservation of ecosystems.

Knowledge of the presence of an invasive species is critical to monitoring the sustainability of communities and ecosystems. Environmental DNA (eDNA), DNA fragments that are likely to be bound to organic matters in the water or in shed cells, has been used to monitor the presence of aquatic animals. Using an eDNA-based method, we estimated the presence of the invasive bluegill sunfish, Lepomis macrochirus, in 70 ponds located in seven locales on the Japanese mainland and on surrounding islands. We quantified the concentration of DNA copies in a 1 L water sample using quantitative real-time polymerase chain reaction (qPCR) with a primer/probe set. In addition, we visually observed the bluegill presence in the ponds from the shoreline. We detected bluegill eDNA in all the ponds where bluegills were observed visually and some where bluegills were not observed. Bluegills were also less prevalent on the islands than the mainland, likely owing to limited dispersal and introduction by humans. Our eDNA method simply and rapidly detects the presence of this invasive fish species with less disturbance to the environment during field surveys than traditional methods.

Biological responses to climate change have been widely documented across taxa and regions, but it remains unclear whether species are maintaining a good match between phenotype and environment, i.e. whether observed trait changes are adaptive. Here we reviewed 10,090 abstracts and extracted data from 71 studies reported in 58 relevant publications, to assess quantitatively whether phenotypic trait changes associated with climate change are adaptive in animals. A meta-analysis focussing on birds, the taxon best represented in our dataset, suggests that global warming has not systematically affected morphological traits, but has advanced phenological traits. We demonstrate that these advances are adaptive for some species, but imperfect as evidenced by the observed consistent selection for earlier timing. Application of a theoretical model indicates that the evolutionary load imposed by incomplete adaptive responses to ongoing climate change may already be threatening the persistence of species.

An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass.

Summary Environmental DNA (eDNA) analysis for detecting the presence of aquatic and terrestrial organisms is an established method, and the eDNA concentration of a species can reflect its abundance/biomass at a site. However, attempts to estimate the abundance/biomass of aquatic species using eDNA concentrations in large stream and river ecosystems have received little attention. We determined the eDNA concentration and abundance/biomass of a stream fish, Plecoglossus altivelis, by conducting a snorkelling survey in the Saba River, Japan. Furthermore, we evaluated the relationship between eDNA concentrations and the estimated abundance/biomass of P. altivelis, and determined its spatial distribution within the river. Across the three seasons from spring to autumn, we found significant correlations between the eDNA concentration of P. altivelis and its abundance/biomass at study sites within the river. We detected the eDNA at the sites where we found only feeding traces on stones (where P. altivelis was not directly observed), but not at sites without feeding traces. Additionally, we tested the optimal number of qPCR replicates needed for the eDNA evaluation of P. altivelis abundance and biomass; only a small number of replicates was required when the eDNA concentration was high. Our findings suggest that eDNA analysis is a useful tool to estimate fish abundance/biomass as well as their spatial distribution in rivers.

The field of environmental DNA (eDNA) analysis has rapidly developed over the past decade and the technique has become widely used for detecting aquatic macroorganisms in a variety of habitats. However, a variety of measurement protocols have been individually developed for different eDNA studies and this may lead to confusion for others who wish to incorporate eDNA analysis in their research. It is important therefore to synthesize the current status of—and future challenges to—the methodology of eDNA analysis. We here synthesized the protocols from total 438 published eDNA studies detecting aquatic macroorganisms were used to calculate the frequency of using each method in eDNA analysis steps. We found that the frequency of methods used converged to one or two methods for any analysis step. Furthermore, although the procedure with highest frequency is not always the best, it was shown that the eDNA collection by filtration and subsequent extraction/purification using a DNeasy Blood and Tissue DNA extraction kit (Qiagen, Hilden, Germany) or PowerWater DNA Extraction Kit (Qiagen) is the most common procedure. An understanding of the characteristics of commonly used methods can help those newly engaged in eDNA studies to understand the basic outline of eDNA analysis. Our review will be useful for the future improvement and development of analytical eDNA techniques of eDNA by sharing the recognition of methodological characteristic including advantages and disadvantages in major analytical techniques.

Objective. To examine whether depsipeptide (FK228), a histone deacetylase (HDA) inhibitor, has inhibitory effects on the proliferation of synovial fibroblasts from rheumatoid arthritis (RA) patients, and to examine the effects of systemic administration of FK228 in an animal model of arthritis.Methods. Autoantibody-mediated arthritis (AMA) was induced in 19 male DBA/1 mice (6-7 weeks old); 10 of them were treated by intravenous administration of FK228 (2.5 mg/kg), and 9 were used as controls. The effects of FK228 were examined by radiographic, histologic, and immunohistochemical analyses and arthritis scores. RA synovial fibroblasts (RASFs) were obtained at the time of joint replacement surgery. In vitro effects of FK228 on cell proliferation were assessed by MTT assay. Cell morphology was examined by light and transmission electron microscopy. The effects on the expression of the cell cycle regulators p16INK4a and p21 WAF1/Cip1 were examined by real-time polymerase chain reaction and Western blot analysis. The acetylation status of the promoter regions of p16INK4a and p21 WAF1/Cip1 were determined by chromatin immunoprecipitation assay.Results. A single intravenous injection of FK228 (2.5 mg/ml) successfully inhibited joint swelling, synovial inflammation, and subsequent bone and cartilage destruction in mice with AMA. FK228 treatment induced histone hyperacetylation in the synovial cells and decreased the levels of tumor necrosis factor ␣ and interleukin-1␤ in the synovial tissues of mice with AMA. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia, with excessive inflammatory cell infiltration in the joints, leading to erosion of the articular cartilage and marginal bone, with subsequent joint destruction (1). Despite an explosion of information over the last 2 decades, a detailed understanding of the mechanisms of synovial hyperplasia and inflammation is lacking. Recent reports have implicated rapid proliferation of synovial cells, overexpression of inflammatory genes, and impairment of apoptosis, which can allow the persistence of abnormal cells (2-4), in the disease process.Cyclin-dependent kinases (CDKs) are essential Dr.

Compound-specific stable isotope analysis (CSIA) of amino acids is a new method that enables estimates of trophic position for consumers in food webs. We examined the nitrogen isotopic composition (d 15 N) of amino acids of Japanese social insects (three bee, three wasp, and four hornet species) to evaluate the potential of CSIA of amino acids in studies of terrestrial food webs. For wasps, we also examined samples at different growth stages (ranging from egg to adult) to assess the effect of metamorphosis on CSIA estimates of trophic position. The d 15 N values of bulk tissues for Japanese social insects are only weakly correlated with the biologically expected trophic positions. In contrast, the trophic positions estimated from the d 15 N values of amino acids (yielding values of between 2.0 and 2.3 for bees, between 2.8 and 3.3 for wasps, and between 3.5 and 4.1 for hornets) are consistent with the biologically expected trophic positions for these insects (i.e., 2.0 for bees, 3.0 for wasps, and 3.0-4.0 for hornets). Although large variability is observed among the d 15 N values of individual amino acids (e.g., ranging from 3.0 to 14.9& for phenylalanine), no significant change is observed in the trophic position during wasp metamorphosis. Thus, the CSIA of amino acids is a powerful tool for investigating not only aquatic food webs but also terrestrial food webs with predatory insects.