Suspended Materials Affect Particle Size Distribution and Removal of Environmental DNA in Flowing Waters.

Environmental DNA (eDNA) in aquatic systems is a complex mixture that includes dissolved DNA, intracellular DNA, and particle-adsorbed DNA. Information about the various components of eDNA and their relative proportions could be used to discern target organism abundance and location. However, a limited knowledge of eDNA adsorption dynamics and interactions with other materials hinders these applications. To address this gap, we used recirculating stream mesocosms to investigate the impact of suspended materials (fine particulate organic matter, plankton, clay, and titanium dioxide) on the eDNA concentration and particle size distribution (PSD) from two fish species in flowing water. Our findings revealed that eDNA rapidly adsorbs to other materials in the water column, affecting its concentration and PSD. Nonetheless, only particulate organic matter affected eDNA removal rate after 30 h. Moreover, we observed that the removal of larger eDNA components (≥10 µm) was more strongly influenced by physical processes, whereas the removal of smaller eDNA components was driven by biological degradation. This disparity in removal mechanisms between larger and smaller eDNA components could explain changes in eDNA composition over time and space, which have implications for modeling the spatial distribution and abundance of target species and optimizing eDNA detection in high turbidity systems.

Environmental DNA (eDNA) removal rates in streams differ by particle size under varying substrate and light conditions.

The use of environmental DNA (eDNA) as a sampling tool offers insights into the detection of invasive and/or rare aquatic species and enables biodiversity assessment without traditional sampling approaches, which are often labor-intensive. However, our understanding of the environmental factors that impact eDNA removal (i.e., how rapidly eDNA is removed from the water column by the combination of decay and physical removal) in flowing waters is limited. This limitation constrains predictions about the location and density of target organisms after positive detection. To address this question, we spiked Common Carp (Cyprinus carpio) eDNA into recirculating mesocosms (n = 24) under varying light (shaded versus open) and benthic substrate conditions (no substrate, bare substrate, and biofilm-colonized substrate). We then collected water samples from each mesocosm at four time points (40 min, 6 h, 18 h, and 48 h), and sequentially filtered the samples through 10, 1.0, and 0.2 µm filters to quantify removal rates for different eDNA particle sizes under varying light and substrate conditions. Combining all size classes, total eDNA removal rates were higher for mesocosms with biofilm-colonized substrate compared to those with no substrate or bare (i.e., no biofilm) substrate, which is consistent with previous findings linking biofilm colonization with increased eDNA removal and degradation. Additionally, when biofilm was present, light availability increased eDNA removal; eDNA levels fell below detection after 6-18 h for open mesocosms versus 18-48 h for shaded mesocosms. Among size classes, larger particles (>10 µm) were removed faster than small particles (1.0-0.2 µm). These results suggest that changes in the distribution of eDNA size classes over time (e.g., with downstream transport) and with differing environmental conditions could be used to predict the location of target organisms in flowing waters, which will advance the use of eDNA as a tool for species monitoring and management.

Persistence of Antibiotic Resistance Genes Varies with Particle Size and Substrate Conditions in Recirculating Streams.

Antibiotic resistance (AR) determinants are enriched in animal manures, a significant portion of which is land-applied as a soil amendment or as fertilizer, leading to potential AR runoff and microbial pollution in adjacent surface waters. To effectively inform AR monitoring and mitigation efforts, a thorough understanding and description of the persistence and transport of manure-derived AR in flowing waters are needed. We used experimental recirculating mesocosms to assess water-column removal rates of antibiotic resistance genes (ARGs) originating from a cow manure slurry collected from a dairy farm. We quantified the effect of three benthic (i.e., bottom) substrate variations and particle sizes of manure slurry on water column removal rates. Overall, we observed variation in ARG behavior across substrate treatments and particle sizes. For ARGs associated with small particles, removal rates were higher in mesocosms with a substrate. tetW was typically removed at the highest rates across particle size and treatment, followed by ermB and blaTEM. Our data suggests that both substrate character and particle size exert control on the fate and transport of ARGs in surface waters, laying the foundation for future research in this area to establish a predictive framework for AR persistence and fate in flowing waters.

Particle size influences decay rates of environmental DNA in aquatic systems.

Environmental DNA (eDNA) analysis is a powerful tool for remote detection of target organisms. However, obtaining quantitative and longitudinal information from eDNA data is challenging, requiring a deep understanding of eDNA ecology. Notably, if the various size components of eDNA decay at different rates, and we can separate them within a sample, their changing proportions could be used to obtain longitudinal dynamics information on targets. To test this possibility, we conducted an aquatic mesocosm experiment in which we separated fish-derived eDNA components using sequential filtration to evaluate the decay rate and changing proportion of various eDNA particle sizes over time. We then fit four alternative mathematical decay models to the data, building towards a predictive framework to interpret eDNA data from various particle sizes. We found that medium-sized particles (1-10 µm) decayed more slowly than other size classes (i.e., <1 and > 10 µm), and thus made up an increasing proportion of eDNA particles over time. We also observed distinct eDNA particle size distribution (PSD) between our Common carp and Rainbow trout samples, suggesting that target-specific assays are required to determine starting eDNA PSDs. Additionally, we found evidence that different sizes of eDNA particles do not decay independently, with particle size conversion replenishing smaller particles over time. Nonetheless, a parsimonious mathematical model where particle sizes decay independently best explained the data. Given these results, we suggest a framework to discern target distance and abundance with eDNA data by applying sequential filtration, which theoretically has both metabarcoding and single-target applications.

Novel Field-Based Protein Detection Method Using Light Transmission Spectroscopy and Antibody Functionalized Gold Nanoparticles.

Protein detection is a universal tool critical to many applications in medicine, agriculture, and biotechnology. We developed a novel protein detection method combining light transmission spectroscopy and particle-size analysis of gold nanospheres monovalently functionalized with polyclonal antibodies and applied it to an emerging challenge for such technologies─the monitoring of environmental proteins (eProteins) present in natural aquatic systems. These are an underreported source of pollution and include the pseudopersistent Cry toxins that enter aquatic ecosystems from surrounding genetically engineered crops. The assay is capable of detecting proteins in complex matrices, such as water samples collected in the field, making it a competitive assay for eProtein detection. It is sensitive, reaching 1.25 ng mL-1, and we demonstrate its application to the detection of Cry1Ab from subsurface tile-drain and streamwater samples from agricultural waterways. The assay can also be quickly adapted for other protein detection applications in the future.

Extending vegetative cover with cover crops influenced phosphorus loss from an agricultural watershed.

Excess phosphorus (P) from agriculture is a leading cause of harmful and nuisance algal blooms in many freshwater ecosystems. Throughout much of the midwestern United States, extensive networks of subsurface tile drains remove excess water from fields and allow for productive agriculture. This enhanced drainage also facilitates the transport of P, particularly soluble reactive phosphorus (SRP), to adjacent streams and ditches, with harmful consequences. Thus, reducing SRP loss from tile-drained cropland is a major focus of regional and national efforts to curb eutrophication and algal blooms. The planting of cover crops after crop harvest is a conservation practice that has the potential to increase retention of fertilizer nutrients in watersheds by extending the growing season and limiting bare ground in the fallow season; however, the effect of cover crops on SRP loss is inconsistent at the field-scale and unknown at the watershed-scale. In this study, we conducted a large-scale manipulation of land cover in a small, agricultural watershed by planting cover crops on >60% of croppable acres for six years and examining changes in SRP loss through tile drains and at the watershed outlet. We found reduced median SRP loss from tiles with cover crops compared to those without cover crops, particularly during periods of critical export from January to June. Variation in tile discharge influenced SRP loss, but relationships were generally weaker in tiles with cover crops (i.e., decoupled) compared to tiles without cover crops. At the watershed outlet, SRP yield was highly variable over all seasons and years, which complicated efforts to detect a significant effect of changing land cover on SRP export to downstream systems. Yet, watershed-scale planting of cover crops slowed cumulative SRP losses and reduced SRP export during extreme events. Overall, this study demonstrates the potential for cover crops to alter patterns of SRP loss at both the field- and watershed-scale.

Characterizing bioavailable phosphorus concentrations in an agricultural stream during hydrologic and streambed disturbances.

In freshwater ecosystems, phosphorus (P) is often considered a growth-limiting nutrient. The use of fertilizers on agricultural fields has led to runoff-driven increases in P availability in streams, and the subsequent eutrophication of downstream ecosystems. Isolated storms and periodic streambed dredging are examples of two common disturbances that contribute dissolved and particulate P to agricultural streams, which can be quantified as soluble reactive P (SRP) using the molybdate-blue method on filtered water samples, or total P (TP) measured using digestions on unfiltered water reflecting all forms of P. While SRP is often considered an approximation of bioavailable P (BAP), research has shown that this is not always the case. Current methods used to estimate BAP do not account for the role of biology (e.g., NaOH extractions) or require specialized platforms (e.g., algal bioassays). Here, in addition to routine analysis of SRP and TP, we used a novel yeast-based bioassay with unfiltered sample water to estimate BAP concentrations during two storms (top 80% and > 95% flow quantiles), and downstream of a reach where management-associated dredging disturbed the streambed. We found that the BAP concentrations were often greater than SRP, suggesting that SRP is not fully representative of P bioavailability. The SRP concentrations were similarly elevated during the two storms, but remained consistently low during streambed disturbance. In contrast, turbidity and TP were elevated during all events. The BAP concentrations were significantly related to turbidity during all disturbance events, but with TP only during storms. The novel yeast assay suggests that BAP export can exceed SRP, particularly when streams are not in equilibrium, such as the rising limb of storms or during active dredging.

Fate of Environmental Proteins (eProteins) from Genetically Engineered Crops in Streams is Controlled by Water pH and Ecosystem Metabolism.

Environmental proteins (eProteins), such as Cry proteins associated with genetically engineered (GE) organisms, are present in ecosystems worldwide, but only rarely reach concentrations with detectable ecosystem-level impacts. Despite their ubiquity, the degradation and fate of Cry and other eProteins are mostly unknown. Here, we report the results of an experiment where we added Cry proteins leached from GE Bt maize to a suite of 19 recirculating experimental streams. We found that Cry exhibited a biphasic degradation with an initial phase of rapid and variable degradation within 1 h, followed by a slow and steady phase of degradation with traces of protein persisting after 48 h. The initial degradation was correlated with heterotrophic respiration and water column dissolved oxygen, confirming a previously documented association with stream metabolism. However, protein degradation persisted even with no biofilm and was faster at a more acidic pH, suggesting that water chemistry is also a critical factor in both degradation and subsequent detection. We suggest that Cry, as well as other eProteins, will have a rapid degradation caused by denaturation of proteins and pH changes, which confirms that the detection of Cry proteins in natural streams must be the result of steady and consistent leaching into the environment.

Effect of winter cover crops on soil nutrients in two row-cropped watersheds in Indiana.

The midwestern United States is a highly productive agricultural region, and extended crop-free periods in winter/spring can result in nitrogen (N) and phosphorus (P) losses to waterways that degrade downstream water quality. Planting winter cover crops can improve soil health while reducing nutrient leaching from farm fields during the fallow period. In this study, we used linear mixed effects models and multivariate statistics to determine the effect of cover crops on soil nutrients by comparing fields with cover crops (n = 9) versus those without (n = 6) in two Indiana agricultural watersheds: the Shatto Ditch Watershed, which had >60% of croppable acres in winter cover crops, and the Kirkpatrick Ditch Watershed, which had ∼20%. We found that cover crops decreased soil nitrate-N by >50% and that the magnitude of reduction was related to the amount of cover crop biomass. In contrast, cover crops had variable effects on water extractable P and Mehlich III soil test P. Finally, cover crop biomass significantly increased soil N mineralization and nitrification rates, demonstrating that cover crops have the potential to supply bioavailable N to cash crop after termination. Our study showed that widespread implementation of winter cover crops holds considerable promise for reducing nutrient loss and improving soil health. The degree to which these results are generalizable across other systems depends on factors such as climate, soil characteristics, and past and current agronomic practices.

Seasonal evaluation of biotic and abiotic factors suggests phosphorus retention in constructed floodplains in three agricultural streams.

Floodplain restoration constructed via the two-stage ditch in agricultural streams has the potential to enhance nutrient retention and prevent the eutrophication of downstream ecosystems. Identifying the role of biotic and abiotic factors influencing soluble reactive phosphorus (SRP) retention in floodplains is of interest given that changing redox conditions associated with floodplain inundation can result in a release of geochemically sorbed SRP to the water column. In three agricultural waterways (Indiana, USA), we conducted seasonal measurements of a suite of biogeochemical pools (total P, bioavailable P and Fe) and processes (SRP flux and microbial respiration) from multiple floodplain transects, along with their adjacent stream sediments, to determine the role of biotic and abiotic processes on floodplain SRP retention or release. Across floodplain soils, organic matter explained a significant amount of variation in soil respiration, and SRP flux from the water column to the floodplain soils was driven by the molar ratio of Fe: P, with values >6 indicating potential SRP sorption due to increased available sorption sites. We developed a mass balance model at a single site to relate seasonal floodplain processes with water column SRP export, above and below the study reach, using measurements in this study combined with data from the literature. Grab sample data suggest that the reach retained 26% of incoming SRP, which the mass balance model attributed to seasonal synergy between plant assimilation in spring and summer (removing P from floodplain soils) and abiotic P sorption during winter and spring inundation (adding SRP to the floodplain). Retention of SRP was higher in floodplain soils compared to stream sediments based on the modeled SRP budget. Thus, we suggest that these constructed floodplains will maximize SRP retention from the water column if they inundate regularly, have floodplain soils with Fe:P > 3-6, and that promote sustained plant life.

Declines in an abundant aquatic insect, the burrowing mayfly, across major North American waterways.

Seasonal animal movement among disparate habitats is a fundamental mechanism by which energy, nutrients, and biomass are transported across ecotones. A dramatic example of such exchange is the annual emergence of mayfly swarms from freshwater benthic habitats, but their characterization at macroscales has remained impossible. We analyzed radar observations of mayfly emergence flights to quantify long-term changes in annual biomass transport along the Upper Mississippi River and Western Lake Erie Basin. A single emergence event can produce 87.9 billion mayflies, releasing 3,078.6 tons of biomass into the airspace over several hours, but in recent years, production across both waterways has declined by over 50%. As a primary prey source in aquatic and terrestrial ecosystems, these declines will impact higher trophic levels and environmental nutrient cycling.

Transport and instream removal of the Cry1Ab protein from genetically engineered maize is mediated by biofilms in experimental streams.

The majority of maize planted in the US is genetically-engineered to express insecticidal properties, including Cry1Ab protein, which is designed to resist the European maize borer (Ostrinia nubilalis). After crop harvest, these proteins can be leached into adjacent streams from crop detritus left on fields. The environmental fate of Cry1Ab proteins in aquatic habitats is not well known. From June-November, we performed monthly short-term additions of leached Cry1Ab into four experimental streams with varying benthic substrate to estimate Cry1Ab transport and removal. At the start of the experiments, when rocks were bare, we found no evidence of Cry1Ab removal from the water column, but uptake steadily increased as biofilm colonized the stream substrate. Overall, Cry1Ab uptake was strongly predicted by measures of biofilm accumulation, including algal chlorophyll a and percent cover of filamentous algae. Average Cry1Ab uptake velocity (vf = 0.059 ± 0.009 mm s-1) was comparable to previously reported uptake of labile dissolved organic carbon (DOC; mean vf = 0.04 ± 0.008 mm s-1). Although Cry1Ab has been shown to rapidly degrade in stream water, benthic biofilms may decrease the distance proteins are transported in lotic systems. These results emphasize that once the Cry1Ab protein is leached, subsequent detection and transport through agricultural waterways is dependent on the structure and biology of receiving stream ecosystems.

Rapid quantitative protein detection by light transmission spectroscopy.

Rapid, sensitive, and quantitative protein detection is critical for many applications in medicine, environmental monitoring, and the food industry. Advancements in detection of proteins include the use of antigen-antibody binding; however, many current methods are time-consuming and have limiting factors such as low sensitivity and the inability to provide absolute values. We present a new high-throughput method for protein detection using light transmission spectroscopy (LTS), which can quantify and size nanoparticles in fluid suspension. LTS can quantify proteins directly and target specific proteins through antigen-antibody binding. This work shows that LTS can distinguish between and quantify bovine serum albumin, its antibody, and the BSA + Ab complex and determine BSA protein concentrations down to 5 µg/mL. We use both Mie and discrete dipole approximation models to provide geometric insight into the binding process.

Microplastic deposition velocity in streams follows patterns for naturally occurring allochthonous particles.

Accumulation of plastic litter is accelerating worldwide. Rivers are a source of microplastic (i.e., particles <5 mm) to oceans, but few measurements of microplastic retention in rivers exist. We adapted spiraling metrics used to measure particulate organic matter transport to quantify microplastic deposition using an outdoor experimental stream. We conducted replicated pulse releases of three common microplastics: polypropylene pellets, polystyrene fragments, and acrylic fibers, repeating measurements using particles with and without biofilms. Depositional velocity (vdep; mm/s) patterns followed expectations based on density and biofilm 'stickiness', where vdep was highest for fragments, intermediate for fibers, and lowest for pellets, with biofilm colonization generally increasing vdep. Comparing microplastic vdep to values for natural particles (e.g., fine and coarse particulate organic matter) showed that particle diameter was positively related to vdep and negatively related to the ratio of vdep to settling velocity (i.e., sinking rate in standing water). Thus, microplastic vdep in rivers can be quantified with the same methods and follows the same patterns as natural particles. These data are the first measurements of microplastic deposition in rivers, and directly inform models of microplastic transport at the landscape scale, making a key contribution to research on the global ecology of plastic waste.

Water Flow and Biofilm Cover Influence Environmental DNA Detection in Recirculating Streams.

The increasing use of environmental DNA (eDNA) for determination of species presence in aquatic ecosystems is an invaluable technique for both ecology as a field and for the management of aquatic ecosystems. We examined the degradation dynamics of fish eDNA using an experimental array of recirculating streams, also using a "nested" primer assay to estimate degradation among eDNA fragment sizes. We introduced eDNA into streams with a range of water velocities (0.1-0.8 m s-1) and substrate biofilm coverage (0-100%) and monitored eDNA concentrations over time (∼10 d) to assess how biophysical conditions influence eDNA persistence. We found that the presence of biofilm significantly increased initial decay rates relative to previous studies conducted in nonflowing microcosms, suggesting important differences in detection and persistence in lentic vs lotic systems. Lastly, by using a nested primer assay that targeted different size eDNA fragments, we found that fragment size altered both the estimated rate constant coefficients, as well as eDNA detectability over time. Larger fragments (>600 bp) were quickly degraded, while shorter fragments (<100 bp) remained detectable for the entirety of the experiment. When using eDNA as a stream monitoring tool, understanding environmental factors controlling eDNA degradation will be critical for optimizing eDNA sampling strategies.

Drivers of nitrogen transfer in stream food webs across continents.

Studies of trophic-level material and energy transfers are central to ecology. The use of isotopic tracers has now made it possible to measure trophic transfer efficiencies of important nutrients and to better understand how these materials move through food webs. We analyzed data from thirteen 15 N-ammonium tracer addition experiments to quantify N transfer from basal resources to animals in headwater streams with varying physical, chemical, and biological features. N transfer efficiencies from primary uptake compartments (PUCs; heterotrophic microorganisms and primary producers) to primary consumers was lower (mean 11.5%, range <1% to 43%) than N transfer efficiencies from primary consumers to predators (mean 80%, range 5% to >100%). Total N transferred (as a rate) was greater in streams with open compared to closed canopies and overall N transfer efficiency generally followed a similar pattern, although was not statistically significant. We used principal component analysis to condense a suite of site characteristics into two environmental components. Total N uptake rates among trophic levels were best predicted by the component that was correlated with latitude, DIN:SRP, GPP:ER, and percent canopy cover. N transfer efficiency did not respond consistently to environmental variables. Our results suggest that canopy cover influences N movement through stream food webs because light availability and primary production facilitate N transfer to higher trophic levels.

Controls on eDNA movement in streams: Transport, Retention, and Resuspension.

Advances in detection of genetic material from species in aquatic ecosystems, including environmental DNA (eDNA), have improved species monitoring and management. eDNA from target species can readily move in streams and rivers and the goal is to measure it, and with that infer where and how abundant species are, adding great value to delimiting species invasions, monitoring and protecting rare species, and estimating biodiversity. To date, we lack an integrated framework that identifies environmental factors that control eDNA movement in realistic, complex, and heterogeneous flowing waters. To this end, using an empirical approach and a simple conceptual model, we propose a framework of how eDNA is transported, retained, and resuspended in stream systems. Such an understanding of eDNA dispersal in streams will be essential for designing optimized sampling protocols and subsequently estimating biomass or organismal abundance. We also discuss guiding principles for more effective use of eDNA methods, highlighting the necessity of understanding these parameters for use in future predictive modeling of eDNA transport.

Role of surface and subsurface processes in scaling N2O emissions along riverine networks.

Riverine environments, such as streams and rivers, have been reported as sources of the potent greenhouse gas nitrous oxide ([Formula: see text]) to the atmosphere mainly via microbially mediated denitrification. Our limited understanding of the relative roles of the near-surface streambed sediment (hyporheic zone), benthic, and water column zones in controlling [Formula: see text] production precludes predictions of [Formula: see text] emissions along riverine networks. Here, we analyze [Formula: see text] emissions from streams and rivers worldwide of different sizes, morphology, land cover, biomes, and climatic conditions. We show that the primary source of [Formula: see text] emissions varies with stream and river size and shifts from the hyporheic-benthic zone in headwater streams to the benthic-water column zone in rivers. This analysis reveals that [Formula: see text] production is bounded between two [Formula: see text] emission potentials: the upper [Formula: see text] emission potential results from production within the benthic-hyporheic zone, and the lower [Formula: see text] emission potential reflects the production within the benthic-water column zone. By understanding the scaling nature of [Formula: see text] production along riverine networks, our framework facilitates predictions of riverine [Formula: see text] emissions globally using widely accessible chemical and hydromorphological datasets and thus, quantifies the effect of human activity and natural processes on [Formula: see text] production.

Occurrence, leaching, and degradation of Cry1Ab protein from transgenic maize detritus in agricultural streams.

The insecticidal Cry1Ab protein expressed by transgenic (Bt) maize can enter adjacent water bodies via multiple pathways, but its fate in stream ecosystems is not as well studied as in terrestrial systems. In this study, we used a combination of field sampling and laboratory experiments to examine the occurrence, leaching, and degradation of soluble Cry1Ab protein derived from Bt maize in agricultural streams. We surveyed 11 agricultural streams in northwestern Indiana, USA, on 6 dates that encompassed the growing season, crop harvest, and snowmelt/spring flooding, and detected Cry1Ab protein in the water column and in flowing subsurface tile drains at concentrations of 3-60ng/L. In a series of laboratory experiments, submerged Bt maize leaves leached Cry1Ab into stream water with 1% of the protein remaining in leaves after 70d. Laboratory experiments suggested that dissolved Cry1Ab protein degraded rapidly in microcosms containing water-column microorganisms, and light did not enhance breakdown by stimulating assimilatory uptake of the protein by autotrophs. The common detection of Cry1Ab protein in streams sampled across an agricultural landscape, combined with laboratory studies showing rapid leaching and degradation, suggests that Cry1Ab may be pseudo-persistent at the watershed scale due to the multiple input pathways from the surrounding terrestrial environment.