Antibiotic Resistance Genes in Interconnected Surface Waters as Affected by Agricultural Activities.

Pastures have become one of the most important sources of antibiotic resistance genes (ARGs) pollution, bringing risks to human health through the environment and the food that is grown there. Another significant source of food production is greenhouse horticulture, which is typically located near pastures. Through waterways, pasture-originated ARGs may transfer to the food in greenhouses. However, how these pasture-originated ARGs spread to nearby waterways and greenhouses has been much less investigated, while this may pose risks to humans through agricultural products. We analyzed 29 ARGs related to the most used antibiotics in livestock in the Netherlands at 16 locations in an agricultural area, representing pastures, greenhouses and lakes. We found that ARGs were prevalent in all surface waters surrounding pastures and greenhouses and showed a similar composition, with sulfonamide ARGs being dominant. This indicates that both pastures and greenhouses cause antibiotic resistance pressures on neighboring waters. However, lower pressures were found in relatively larger and isolated lakes, suggesting that a larger water body or a non-agricultural green buffer zone could help reducing ARG impacts from agricultural areas. We also observed a positive relationship between the concentrations of the class 1 integron (intl1 gene)-used as a proxy for horizontal gene transfer-and ARG concentration and composition. This supports that horizontal gene transfer might play a role in dispersing ARGs through landscapes. In contrast, none of the measured four abiotic factors (phosphate, nitrate, pH and dissolved oxygen) showed any impact on ARG concentrations. ARGs from different classes co-occurred, suggesting simultaneous use of different antibiotics. Our findings help to understand the spatial patterns of ARGs, specifically the impacts of ARGs from pastures and greenhouses on each other and on nearby waterways. In this way, this study guides management aiming at reducing ARGs' risk to human health from agricultural products.

Environmental DNA methylation of Lymnaea stagnalis varies with age and is hypermethylated compared to tissue DNA.

Environmental DNA (eDNA) approaches contributing to species identifications are quickly becoming the new norm in biomonitoring and ecosystem assessments. Yet, information such as age and health state of the population, which is vital to species biomonitoring, has not been accessible from eDNA. DNA methylation has the potential to provide such information on the state of a population. Here, we measured the methylation of eDNA along with tissue DNA (tDNA) of Lymnaea stagnalis at four life stages. We demonstrate that eDNA methylation varies with age and allows distinguishing among age classes. Moreover, eDNA was globally hypermethylated in comparison to tDNA. This difference was age-specific and connected to a limited number of eDNA sites. This differential methylation pattern suggests that eDNA release with age is partially regulated through DNA methylation. Our findings help to understand mechanisms involved in eDNA release and shows the potential of eDNA methylation analysis to assess age classes. Such age class assessments will encourage future eDNA studies to assess fundamental processes of population dynamics and functioning in ecology, biodiversity conservation and impact assessments.

The particle size distribution of environmental DNA varies with species and degradation.

Environmental DNA (eDNA) analysis is frequently used as a non-invasive method to investigate species and biodiversity in ecosystems. However, such eDNA may represent both organisms currently present as well as species that released their DNA some point in the past, thereby representing a mix of current and historic biodiversity. This may lead to a false-positive detection of organisms' presence. As the eDNA particle size distribution (PSD) changes along with the decay process, it may facilitate solving the above problem. Here, we set up tank experiments with snails, zebrafish and daphnids, respectively, to monitor the change in eDNA PSD and eDNA degradation through time after removing organisms. We found that zebrafish eDNA decays more slowly for larger particle sizes. Across all species tested, the percentage of large size ranges tended to increase over time while the smaller sizes showed relatively fast decay rates. As a result, PSD changed consistently with eDNA decay, although initial PSD varied between species. In combination, we propose that eDNA PSD can be used to assess the current prevalence of organisms at an eDNA sampling location while avoiding false-positives on the presence of species. Our findings expand the applicability of eDNA for monitoring target species in freshwater ecosystems.