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.

Water availability and seasonality shape elemental stoichiometry across space and time.

The interaction of climate change and increasing anthropogenic water withdrawals is anticipated to alter surface water availability and the transport of carbon (C), nitrogen (N), and phosphorus (P) in river networks. But how changes to river flow will alter the balance, or stoichiometry, of these fluxes is unknown. The Lower Flint River Basin (LFRB) is part of an interstate watershed relied upon by several million people for diverse ecosystem services, including seasonal crop irrigation, municipal drinking water access, and public recreation. Recently, increased water demand compounded with intensified droughts have caused historically perennial streams in the LFRB to cease flowing, increasing ecosystem vulnerability. Our objectives were to quantify how riverine dissolved C:N:P varies spatially and seasonally and determine how monthly stoichiometric fluxes varied with overall water availability in a major tributary of LFRB. We used a long-term record (21-29 years) of solute water chemistry (dissolved organic carbon, nitrate/nitrite, ammonia, and soluble reactive phosphorus) paired with long-term stream discharge data across six sites within a single LFRB watershed. We found spatial and seasonal differences in soluble nutrient concentrations and stoichiometry attributable to groundwater connections, the presence of a major floodplain wetland, and flow conditions. Further, we showed that water availability, as indicated by the Palmer Drought Severity Index (PDSI), strongly predicted stoichiometry with generally lower C:N and C:P and higher N:P fluxes during periods of low water availability (PDSI < -4). These patterns suggest there may be long-term and significant changes to stream ecosystem function as water availability is being dramatically altered by human demand with consequential impacts on solute transport, in-stream processing, and stoichiometric ratios.

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.

Preparing Aquatic Research for an Extreme Future: Call for Improved Definitions and Responsive, Multidisciplinary Approaches.

Extreme events have increased in frequency globally, with a simultaneous surge in scientific interest about their ecological responses, particularly in sensitive freshwater, coastal, and marine ecosystems. We synthesized observational studies of extreme events in these aquatic ecosystems, finding that many studies do not use consistent definitions of extreme events. Furthermore, many studies do not capture ecological responses across the full spatial scale of the events. In contrast, sampling often extends across longer temporal scales than the event itself, highlighting the usefulness of long-term monitoring. Many ecological studies of extreme events measure biological responses but exclude chemical and physical responses, underscoring the need for integrative and multidisciplinary approaches. To advance extreme event research, we suggest prioritizing pre- and postevent data collection, including leveraging long-term monitoring; making intersite and cross-scale comparisons; adopting novel empirical and statistical approaches; and developing funding streams to support flexible and responsive data collection.

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.

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.

Revealing biogeochemical signatures of Arctic landscapes with river chemistry.

Riverine fluxes of carbon and inorganic nutrients are increasing in virtually all large permafrost-affected rivers, indicating major shifts in Arctic landscapes. However, it is currently difficult to identify what is causing these changes in nutrient processing and flux because most long-term records of Arctic river chemistry are from small, headwater catchments draining <200 km2 or from large rivers draining >100,000 km2. The interactions of nutrient sources and sinks across these scales are what ultimately control solute flux to the Arctic Ocean. In this context, we performed spatially-distributed sampling of 120 subcatchments nested within three Arctic watersheds spanning alpine, tundra, and glacial-lake landscapes in Alaska. We found that the dominant spatial scales controlling organic carbon and major nutrient concentrations was 3-30 km2, indicating a continuum of diffuse and discrete sourcing and processing dynamics. These patterns were consistent seasonally, suggesting that relatively fine-scale landscape patches drive solute generation in this region of the Arctic. These network-scale empirical frameworks could guide and benchmark future Earth system models seeking to represent lateral and longitudinal solute transport in rapidly changing Arctic landscapes.

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.

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.

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.

Influence of Stream Bottom Substrate on Retention and Transport of Vertebrate Environmental DNA.

While environmental DNA (eDNA) is now being regularly used to detect rare and elusive species, detection in lotic environments comes with a caveat: The species being detected is likely some distance upstream from the point of sampling. Here, we conduct a series of seminatural stream experiments to test the sensitivity of new digital droplet PCR (ddPCR) to detect low concentrations of eDNA in a lotic system, measure the residence time of eDNA compared to a conservative tracer, and we model the transport of eDNA in this system. We found that while ddPCR improves our sensitivity of detection, the residence time and transport of eDNA does not follow the same dynamics as the conservative tracer and necessitates a more stochastic framework for modeling eDNA transport. There was no evidence for differences in the transport of eDNA due to substrate type. The relatively large amount of unexplained variability in eDNA transport reveals the need for uncovering mechanisms and processes by which eDNA is transported downstream leading to species detections, particularly when inferences are to be made in natural systems where eDNA is being used for conservation management.

Modelling the transport of environmental DNA through a porous substrate using continuous flow-through column experiments.

Detecting environmental DNA (eDNA) in water samples is a powerful tool in determining the presence of rare aquatic species. However, many open questions remain as to how biological and physical conditions in flowing waters influence eDNA. Motivated by what one might find in a stream/river benthos we conducted experiments in continuous flow columns packed with porous substrates to explore eDNA transport and ask whether substrate type and the presence of colonized biofilms plays an important role for eDNA retention. To interpret our data, and for modelling purposes, we began with the assumption that eDNA could be treated as a classical tracer. Comparing our experimental data with traditional transport models, we found that eDNA behaves anomalously, displaying characteristics of a heterogeneous, polydisperse substance with particle-like behaviour that can be filtered by the substrate. Columns were quickly flushed of suspended eDNA particles while a significant amount of particles never made it through and were retained in the column, as calculated from a mass balance. Suspended eDNA was exported through the column, regardless of biofilm colonization. Our results indicate that the variable particle size of eDNA results in stochastic retention, release and transport, which may influence the interpretation eDNA detection in biological systems.