Biological Weighting Functions for Evaluating the Role of Sunlight-Induced Inactivation of Coliphages at Selected Beaches and Nearby Tributaries.

Coliphages can indicate contamination of recreational waters and previous studies show that sunlight is important in altering densities of coliphages, other indicator microorganisms, and pathogens in aquatic environments. Here, we report on laboratory studies of light-induced inactivation of two coliphage groups-male-specific (F+) and somatic coliphage-under various conditions in phosphate-buffered water (PBW). Strains isolated from wastewater treatment facilities and laboratory strains (MS2 and phiX174 coliphages) were evaluated. Inactivation rates were determined in a series of irradiations using simulated solar radiation passed through light filters that blocked different parts of the ultraviolet spectral region. Inactivation rates and spectral irradiance from these experiments were then analyzed to develop biological weighting functions (BWFs) for the light-induced inactivation. BWFs were used to model the inactivation of coliphages over a range of conditions in aquatic environments that included two beach sites in Lake Michigan and one in Lake Erie. For example, modeled effects of sunlight attenuation, using UV absorption data from the three Great Lakes beach sites, inferred that direct photoinactivation rate constants, averaged over a one-meter water column in swimmable areas, were reduced 2- to 5-fold, compared to near-surface rate constants.

Using Integrated Environmental Modeling to Assess Sources of Microbial Contamination in Mixed-Use Watersheds.

Microbial fate and transport in watersheds should include a microbial source apportionment analysis that estimates the importance of each source, relative to each other and in combination, by capturing their impacts spatially and temporally under various scenarios. A loosely configured software infrastructure was used in microbial source-to-receptor modeling by focusing on animal- and human-impacted mixed-use watersheds. Components include data collection software, a microbial source module that determines loading rates from different sources, a watershed model, an inverse model for calibrating flows and microbial densities, tabular and graphical viewers, software to convert output to different formats, and a model for calculating risk from pathogen exposure. The system automates, as much as possible, the manual process of accessing and retrieving data and completes input data files of the models. The workflow considers land-applied manure from domestic animals on undeveloped areas; direct shedding (excretion) on undeveloped lands by domestic animals and wildlife; pastureland, cropland, forest, and urban or engineered areas; sources that directly release to streams from leaking septic systems; and shedding by domestic animals directly to streams. The infrastructure also considers point sources from regulated discharges. An application is presented on a real-world watershed and helps answer questions such as: What are the major microbial sources? What practices contribute to contamination at the receptor location? What land-use types influence contamination at the receptor location? and Under what conditions do these sources manifest themselves? This research aims to improve our understanding of processes related to pathogen and indicator dynamics in mixed-use watershed systems.

Capturing Microbial Sources Distributed in a Mixed-use Watershed within an Integrated Environmental Modeling Workflow.

Many watershed models simulate overland and instream microbial fate and transport, but few provide loading rates on land surfaces and point sources to the waterbody network. This paper describes the underlying equations for microbial loading rates associated with 1) land-applied manure on undeveloped areas from domestic animals; 2) direct shedding (excretion) on undeveloped lands by domestic animals and wildlife; 3) urban or engineered areas; and 4) point sources that directly discharge to streams from septic systems and shedding by domestic animals. A microbial source module, which houses these formulations, is part of a workflow containing multiple models and databases that form a loosely configured modeling infrastructure which supports watershed-scale microbial source-to-receptor modeling by focusing on animal- and human-impacted catchments. A hypothetical application - accessing, retrieving, and using real-world data - demonstrates how the infrastructure can automate many of the manual steps associated with a standard watershed assessment, culminating in calibrated flow and microbial densities at the watershed's pour point.

Rainfall-induced release of microbes from manure: model development, parameter estimation, and uncertainty evaluation on small plots.

A series of simulated rainfall-runoff experiments with applications of different manure types (cattle solid pats, poultry dry litter, swine slurry) was conducted across four seasons on a field containing 36 plots (0.75 × 2 m each), resulting in 144 rainfall-runoff events. Simulating time-varying release of Escherichia coli, enterococci, and fecal coliforms from manures applied at typical agronomic rates evaluated the efficacy of the Bradford-Schijven model modified by adding terms for release efficiency and transportation loss. Two complementary, parallel approaches were used to calibrate the model and estimate microbial release parameters. The first was a four-step sequential procedure using the inverse model PEST, which provides appropriate initial parameter values. The second utilized a PEST/bootstrap procedure to estimate average parameters across plots, manure age, and microbe, and to provide parameter distributions. The experiment determined that manure age, microbe, and season had no clear relationship to the release curve. Cattle solid pats released microbes at a different, slower rate than did poultry dry litter or swine slurry, which had very similar release patterns. These findings were consistent with other published results for both bench- and field-scale, suggesting the modified Bradford-Schijven model can be applied to microbial release from manure.

Decay of fecal indicator bacterial populations and bovine-associated source-tracking markers in freshly deposited cow pats.

Understanding the survival of fecal indicator bacteria (FIB) and microbial source-tracking (MST) markers is critical to developing pathogen fate and transport models. Although pathogen survival in water microcosms and manure-amended soils is well documented, little is known about their survival in intact cow pats deposited on pastures. We conducted a study to determine decay rates of fecal indicator bacteria (Escherichia coli and enterococci) and bovine-associated MST markers (CowM3, Rum-2-bac, and GenBac) in 18 freshly deposited cattle feces from three farms in northern Georgia. Samples were randomly assigned to shaded or unshaded treatment in order to determine the effects of sunlight, moisture, and temperature on decay rates. A general linear model (GLM) framework was used to determine decay rates. Shading significantly decreased the decay rate of the E. coli population (P < 0.0001), with a rate of -0.176 day(-1) for the shaded treatment and -0.297 day(-1) for the unshaded treatment. Shading had no significant effect on decay rates of enterococci, CowM3, Rum-2-bac, and GenBac (P > 0.05). In addition, E. coli populations showed a significant growth rate (0.881 day(-1)) in the unshaded samples during the first 5 days after deposition. UV-B was the most important parameter explaining the decay rate of E. coli populations. A comparison of the decay behaviors among all markers indicated that enterococcus concentrations exhibit a better correlation with the MST markers than E. coli concentrations. Our results indicate that bovine-associated MST markers can survive in cow pats for at least 1 month after excretion, and although their decay dynamic differs from the decay dynamic of E. coli populations, they seem to be reliable markers to use in combination with enterococci to monitor fecal pollution from pasture lands.

Temporal and spatial relationships of CrAssphage and enteric viral and bacterial pathogens in wastewater in North Carolina.

Enteric disease remains one of the most common concerns for public health, particularly when it results from human exposure to surface and recreational waters contaminated with wastewater. Characterizing the temporal and spatial variation of enteric pathogens prevalent in wastewater is critical to develop approaches to mitigate their distribution in the environment. In this study, we aim to characterize pathogen variability and test the applicability of the human-associated wastewater indicator crAssphage as an indicator of enteric viral and bacterial pathogens. We conducted weekly samplings for 14 months from four wastewater treatment plants in North Carolina, USA. Untreated wastewater samples were processed using hollow fiber ultrafiltration, followed by secondary concentration methods. Adenovirus, norovirus, enterovirus, Salmonella, Shiga toxin 2 (stx2), Campylobacter, and crAssphage were measured by quantitative polymerase chain reaction (qPCR) and reverse transcriptase (rt)-qPCR. Our results revealed significant correlations between crAssphage and human adenovirus, enterovirus, norovirus, Salmonella, and Campylobacter (p<0.01). Pathogens and crAssphage concentrations in untreated wastewater showed distinct seasonal patterns, with peak concentrations of crAssphage and viral pathogens in fall and winter, while bacterial pathogens showed peaked concentrations in either winter (Campylobacter), fall (Salmonella), or summer (stx2). This study enhances the understanding of crAssphage as an alternative molecular indicator for both bacterial and viral pathogens. The findings of this study can also inform microbial modeling efforts for the prediction of the impact of wastewater pathogens on surface waters due to increased flooding events and wastewater overflows associated with climate change.

Influence of inorganic ions on aggregation and adsorption behaviors of human adenovirus.

In this study, we investigated the influence of inorganic ions on the aggregation and deposition (adsorption) behavior of human adenovirus (HAdV). Experiments were conducted to determine the surface charge and size of HAdV and viral adsorption capacity of sand in different salt conditions. The interfacial potential energy was calculated using extended Derjaguin and Landau, Verwey and Overbeek (XDLVO) and steric hindrance theories to interpret the experimental results. Results showed that different compositions of inorganic ions have minimal effect on varying the iso-electric point pH (pH(iep)) of HAdV (ranging from 3.5 to 4.0). Divalent cations neutralized/shielded virus surface charge much more effectively than monovalent cations at pH above pH(iep). Consequently, at neutral pH the presence of divalent cations enhanced the aggregation of HAdV as well as its adsorption to sand. Aggregation and adsorption behaviors generally agreed with XDLVO theory; however, in the case of minimal electrostatic repulsion, steric force by virus' fibers can increase the energy barrier and distance of secondary minimum, resulting in limited aggregation and deposition. Overall, our results indicated that subsurface water with low hardness residing in sandy soils may have a higher potential of being contaminated by HAdV.

Sources and Drivers of ARGs in Urban Streams in Atlanta, Georgia, USA.

The spread of antibiotic resistance genes (ARGs) in the aquatic environment is an emerging concern in the interest of protecting public health. Stemming the environmental dissemination of ARGs will require a better understanding of the sources and drivers of ARGs in the water environment. In this study, we used direct measurement of sewage-associated molecular markers, the class 1 integron gene, standard water quality parameters, and watershed characteristics to evaluate the sources and drivers of ARGs in an urban watershed impacted by a gradient of human activities. Quantitative polymerase chain reaction (qPCR) was used to quantify the abundance of the sewage-associated HF183, the E. coli fecal indicator, class 1 integron gene (int1), and the ARGs sulI, sulII, tetW, tetM, ampC, and blaSHV in stream water samples collected from the Proctor Creek watershed in Atlanta, Georgia. Our findings show that ARGs were widely distributed, with detection frequencies of 96% (sulI and sulII), 82% (tetW and tetM), and 49% (ampC and blaSHV). All the ARGs were positively and significantly correlated (r > 0.5) with the HF183 and E. coli markers. Non-linear machine learning models developed using generalized boosting show that more than 70% of the variation in ARG loads in the watershed could be explained by fecal source loading, with other factors such as class 1 integron, which is associated with acquired antibiotic resistance, and environmental factors contributing < 30% to ARG variation. These results suggest that input from fecal sources is a more critical driver of ARG dissemination than environmental stressors or horizontal gene transfer in aquatic environments highly impacted by anthropogenic pollution. Finally, our results provide local watershed managers and stakeholders with information to mitigate the burden of ARGs and fecal bacteria in urban streams.

Characterizing temporal variability in streams supports nutrient indicator development using diatom and bacterial DNA metabarcoding.

Interest in developing periphytic diatom and bacterial indicators of nutrient effects continues to grow in support of the assessment and management of stream ecosystems and their watersheds. However, temporal variability could confound relationships between indicators and nutrients, subsequently affecting assessment outcomes. To document how temporal variability affects measures of diatom and bacterial assemblages obtained from DNA metabarcoding, we conducted weekly periphyton and nutrient sampling from July to October 2016 in 25 streams in a 1293 km2 mixed land use watershed. Measures of both diatom and bacterial assemblages were strongly associated with the percent agriculture in upstream watersheds and total phosphorus (TP) and total nitrogen (TN) concentrations. Temporal variability in TP and TN concentrations increased with greater amounts of agriculture in watersheds, but overall diatom and bacterial assemblage variability within sites-measured as mean distance among samples to corresponding site centroids in ordination space-remained consistent. This consistency was due in part to offsets between decreasing variability in relative abundances of taxa typical of low nutrient conditions and increasing variability in those typical of high nutrient conditions as mean concentrations of TP and TN increased within sites. Weekly low and high nutrient diatom and bacterial metrics were more strongly correlated with site mean nutrient concentrations over the sampling period than with same day measurements and more strongly correlated with TP than with TN. Correlations with TP concentrations were consistently strong throughout the study except briefly following two major precipitation events. Following these events, biotic relationships with TP reestablished within one to three weeks. Collectively, these results can strengthen interpretations of survey results and inform monitoring strategies and decision making. These findings have direct applications for improving the use of diatoms and bacteria, and the use of DNA metabarcoding, in monitoring programs and stream site assessments.

Developing Indicators of Nutrient Pollution in Streams Using 16S rRNA Gene Metabarcoding of Periphyton-Associated Bacteria.

Indicators based on nutrient-biota relationships in streams can inform water quality restoration and protection programs. Bacterial assemblages could be particularly useful indicators of nutrient effects because they are species-rich, important contributors to ecosystem processes in streams, and responsive to rapidly changing conditions. Here, we sampled 25 streams weekly (12-14 times each) and used 16S rRNA gene metabarcoding of periphyton-associated bacteria to quantify the effects of total phosphorus (TP) and total nitrogen (TN). Threshold indicator taxa analysis identified assemblage-level changes and amplicon sequence variants (ASVs) that increased or decreased with increasing TP and TN concentrations (i.e., low P, high P, low N, and high N ASVs). Boosted regression trees confirmed that relative abundances of gene sequence reads for these four indicator groups were associated with nutrient concentrations. Gradient forest analysis complemented these results by using multiple predictors and random forest models for each ASV to identify portions of TP and TN gradients at which the greatest changes in assemblage structure occurred. Synthesized statistical results showed bacterial assemblage structure began changing at 24 µg TP/L with the greatest changes occurring from 110 to 195 µg/L. Changes in the bacterial assemblages associated with TN gradually occurred from 275 to 855 µg/L. Taxonomic and phylogenetic analyses showed that low nutrient ASVs were commonly Firmicutes, Verrucomicrobiota, Flavobacteriales, and Caulobacterales, Pseudomonadales, and Rhodobacterales of Proteobacteria, whereas other groups, such as Chitinophagales of Bacteroidota, and Burkholderiales, Rhizobiales, Sphingomonadales, and Steroidobacterales of Proteobacteria comprised the high nutrient ASVs. Overall, the responses of bacterial ASV indicators in this study highlight the utility of metabarcoding periphyton-associated bacteria for quantifying biotic responses to nutrient inputs in streams.

Interlaboratory performance and quantitative PCR data acceptance metrics for NIST SRM® 2917.

Surface water quality quantitative polymerase chain reaction (qPCR) technologies are expanding from a subject of research to routine environmental and public health laboratory testing. Readily available, reliable reference material is needed to interpret qPCR measurements, particularly across laboratories. Standard Reference Material® 2917 (NIST SRM® 2917) is a DNA plasmid construct that functions with multiple water quality qPCR assays allowing for estimation of total fecal pollution and identification of key fecal sources. This study investigates SRM 2917 interlaboratory performance based on repeated measures of 12 qPCR assays by 14 laboratories (n = 1008 instrument runs). Using a Bayesian approach, single-instrument run data are combined to generate assay-specific global calibration models allowing for characterization of within- and between-lab variability. Comparable data sets generated by two additional laboratories are used to assess new SRM 2917 data acceptance metrics. SRM 2917 allows for reproducible single-instrument run calibration models across laboratories, regardless of qPCR assay. In addition, global models offer multiple data acceptance metric options that future users can employ to minimize variability, improve comparability of data across laboratories, and increase confidence in qPCR measurements.

Feral swine as sources of fecal contamination in recreational waters.

Recreational waters are primary attractions at many national and state parks where feral swine populations are established, and thus are possible hotspots for visitor exposure to feral swine contaminants. Microbial source tracking (MST) was used to determine spatial and temporal patterns of fecal contamination in Congaree National Park (CONG) in South Carolina, U.S.A., which has an established population of feral swine and is a popular destination for water-based recreation. Water samples were collected between December 2017 and June 2019 from 18 surface water sites distributed throughout CONG. Host specific MST markers included human (HF183), swine (Pig2Bac), ruminant (Rum2Bac), cow (CowM3), chicken (CL), and a marker for shiga toxin producing Escherichia coli (STEC; stx2). Water samples were also screened for culturable Escherichia coli (E. coli) as part of a citizen science program. Neither the cow nor chicken MST markers were detected during the study. The human marker was predominantly detected at boundary sites or could be attributed to upstream sources. However, several detections within CONG without concurrent detections at upstream external sites suggested occasional internal contamination from humans. The swine marker was the most frequently detected of all MST markers, and was present at sites located both internal and external to the Park. Swine MST marker concentrations ≥ 43 gene copies/mL were associated with culturable E. coli concentrations greater than the U.S. Environmental Protection Agency beach action value for recreational waters. None of the MST markers showed a strong association with detection of the pathogenic marker (stx2). Limited information about the health risk from exposure to fecal contamination from non-human sources hampers interpretation of the human health implications.

Sediment and fecal indicator bacteria loading in a mixed land use watershed: Contributions from suspended sediment and bedload transport.

Overland transport of fecal bacteria in water and their resuspension from bed sediments are important transport mechanisms that help explain the transport of enteric pathogens in watersheds. In this study, multiyear monitoring along with regression relationships between sediment and fecal indicator bacteria (FIB) were used to investigate annual loading in the South Fork Broad River watershed, located in northeastern Georgia, USA. Suspended transport was the dominant transport mechanism contributing to in-stream total annual loads for sediment (81.4-98.1%) and FIB (>98%). Annual bedload transport of FIB was small and Escherichia coli (up to 1.8%) contributed more to annual bedload FIB than enterococci (≤0.03%). Bedload contributions of FIB increased with the duration of critical discharge exceedance, indicating a prolonged risk of exposure to enteric pathogens during extended periods of high flows, which is important during major storm events. The risk of exposure to enteric pathogens through pathways such as recreational use and drinking water treatment could be much greater because fecal bacteria are released from sediment during higher flows and dominantly transported in suspension when bedload are not actively moving. Therefore, the combined contribution of fecal bacteria from overland and bedload-associated transport should be considered in risk assessments. Discharge, bedload, and FIB data collected over 2 yr in this study can supplement future hydrologic modeling and microbial risk assessment modeling efforts.

Dietary flexibility aids Asian earthworm invasion in North American forests.

On a local scale, invasiveness of introduced species and invasibility of habitats together determine invasion success. A key issue in invasion ecology has been how to quantify the contribution of species invasiveness and habitat invasibility separately. Conventional approaches, such as comparing the differences in traits and/or impacts of species between native and/or invaded ranges, do not determine the extent to which the performance of invaders is due to either the effects of species traits or habitat characteristics. Here we explore the interaction between two of the most widespread earthworm invaders in the world (Asian Amynthas agrestis and European Lumbricus rubellus) and study the effects of species invasiveness and habitat invasibility separately through an alternative approach of "third habitat" in Tennessee, USA. We propose that feeding behaviors of earthworms will be critical to invasion success because trophic ecology of invasive animals plays a key role in the invasion process. We found that (1) the biomass and isotopic abundances (delta13C and delta15N) of A. agrestis were not impacted by either direct effects of L. rubellus competition or indirect effects of L. rubellus-preconditioned habitat; (2) A. agrestis disrupted the relationship between L. rubellus and soil microorganisms and consequently hindered litter consumption by L. rubellus; and (3) compared to L. rubellus, A. agrestis shifted its diet more readily to consume more litter, more soil gram-positive (G+) bacteria (which may be important for litter digestion), and more non-microbial soil fauna when soil microorganisms were depleted. In conclusion, A. agrestis showed strong invasiveness through its dietary flexibility through diet shifting and superior feeding behavior and its indirectly negative effect of habitat invasibility on L. rubellus via changes in the soil microorganism community. In such context, our results expand on the resource fluctuation hypothesis and support the superior competitor hypothesis. This work presents additional approaches in invasion ecology, provides some new dimensions for further research, and contributes to a greater understanding of the importance of interactions between multiple invading species.

Microbial source tracking (MST) in Chattahoochee River National Recreation Area: Seasonal and precipitation trends in MST marker concentrations, and associations with E. coli levels, pathogenic marker presence, and land use.

Escherichia coli levels in recreational waters are often used to predict when fecal-associated pathogen levels are a human health risk. The reach of the Chattahoochee River that flows through the Chattahoochee River National Recreation Area (CRNRA), located in the Atlanta-metropolitan area, is a popular recreation area that frequently exceeds the U.S. Environmental Protection Agency beach action value (BAV) for E. coli. A BacteriALERT program has been implemented to provide real-time E. coli estimates in the reach and notify the public of potentially harmful levels of fecal-associated pathogens as indicated by surrogate models based on real-time turbidity measurements from continuous water quality monitoring stations. However, E. coli does not provide information about the sources of fecal contamination and its accuracy as a human health indicator is questionable when sources of contamination are non-human. The objectives of our study were to investigate, within the Park and surrounding watersheds, seasonal and precipitation-related patterns in microbial source tracking marker concentrations of possible sources (human, dog, and ruminant), assess correlations between source contamination levels and culturable E. coli levels, determine which sources best explained model-based E. coli estimates above the BAV and detection of esp2 (a marker for the esp gene associated with pathogenic strains of Enterococcus faecium and Enterococcus faecalis), and investigate associations between source contamination levels and land use features. Three BacteriALERT sites on the Chattahoochee River were sampled six times per season in the winter and summer from December 2015 through September 2017, and 11 additional stream sites (synoptic sites) from the CRNRA watershed were sampled once per season. Samples were screened with microbial source tracking (MST) quantitative PCR (qPCR) markers for humans (HF183 Taqman), dogs (DogBact), and ruminants (Rum2Bac), the esp2 qPCR marker, and culturable E. coli. At the BacteriALERT sites, HF183 Taqman concentrations were higher under wet conditions DogBact concentrations were greater in the winter and under wet conditions, and Rum2Bac concentrations were comparatively low throughout the study with no difference across seasons or precipitation conditions. Concentrations of HF183 Taqman, DogBact, and Rum2Bac were positively correlated with culturable E. coli concentrations; however, DogBact had the largest R2 value among the three markers, and the forward stepwise regression indicated it was the best predictor of culturable E. coli concentrations at the BacteriALERT sites. Recursive partitioning indicated that BAV exceedances of model-based E. coli estimates were best explained by DogBact concentrations ≥3 gene copies per mL (CN/mL). Detections of esp2 at BacteriALERT sites were best explained by DogBact concentrations ≥11 CN/mL, while detections of esp2 at synoptic sites were best explained by HF183 Taqman ≥29 CN/mL. At the synoptic sites, HF183 Taqman levels were associated with wastewater treatment plant density. However, this relationship was driven primarily by a single site, suggesting possible conveyance issues in that catchment. esp2 detections at synoptic sites were positively associated with development within a 2-km radius and negatively associated with development within the catchment, suggesting multiple sources of esp2 in the watershed. DogBact and Rum2Bac were not associated with the land use features included in our analyses. Implications for Park management include: 1) fecal contamination levels were highest during wet conditions and in the off season when fewer visitors are expected to be participating in water-based recreation, 2) dogs are likely contributors to fecal contamination in the CRNRA and may be sources of pathogenic bacteria indicating further investigation of the origins of this contamination may be warranted as would be research to understand the human health risks from exposure to dog fecal contamination, and 3) high levels of the human marker at one site in the CRNRA watershed suggests more extensive monitoring in that catchment may locate the origin of human fecal contamination detected during this study.

Evaluation of the soil and water assessment tool (SWAT) for simulating E. coli concentrations at the watershed-scale.

Water quality management at the watershed level requires a framework to identify sources, apportion water quality risks and develop mitigation strategies to reduce health risks. Watershed-scale models have been used as a support tool to understand the sources, fate and transport of fecal bacteria and pathogens in the environment. The Soil and Water Assessment Tool (SWAT) model was applied in this study to understand the sources and drivers of microbial water quality in the Clouds Creek watershed in Georgia, USA. A criterion to evaluate the performance of the SWAT bacterial model was also developed in this study using the Nash-Sutcliffe Efficiency (NSE) performance measure. The SWAT model was successfully calibrated and validated for flow with Nash-Sutcliffe Efficiency (NSE) of 0.81 and 0.55, respectively. Escherichia coli (E. coli) predictions were good with NSE of 0.32 and 0.34 for the calibration and validation timeframes, respectively. Based on the criteria developed in this study, SWAT bacterial model for E. coli and fecal coliform can be judged as "satisfactory" when NSE > 0.20. The contribution of sources followed this order: in-stream cattle manure deposition > cattle manure application > poultry manure application > septic systems > wildlife manure, suggesting that a reduction in livestock access to streams would be the most effective approach to reduce fecal bacterial loads in this watershed and others impacted by fecal contamination. Finally, our results suggest that the SWAT model is capable of simulating E. coli dynamics in the Clouds Creek watershed and can provide insights into source impacts for risk management.

Resilience of aquatic systems: Review and management implications.

Our understanding of how ecosystems function has changed from an equilibria-based view to one that recognizes the dynamic, fluctuating, nonlinear nature of aquatic systems. This current understanding requires that we manage systems for resilience. In this review, we examine how resilience has been defined, measured and applied in aquatic systems, and more broadly, in the socioecological systems in which they are embedded. Our review reveals the importance of managing stressors adversely impacting aquatic system resilience, as well as understanding the environmental and climatic cycles and changes impacting aquatic resources. Aquatic resilience may be enhanced by maintaining and enhancing habitat connectivity as well as functional redundancy and physical and biological diversity. Resilience in aquatic socioecological system may be enhanced by understanding and fostering linkages between the social and ecological subsystems, promoting equity among stakeholders, and understanding how the system is impacted by factors within and outside the area of immediate interest. Management for resilience requires implementation of adaptive and preferably collaborative management. Implementation of adaptive management for resilience will require an effective monitoring framework to detect key changes in the coupled socioecological system. Research is needed to (1) develop sensitive indicators and monitoring designs, (2) disentangle complex multi-scalar interactions and feedbacks, and (3) generalize lessons learned across aquatic ecosystems and apply them in new contexts.

Modeling the photoinactivation and transport of somatic and F-specific coliphages at a Great Lakes beach.

Fecal indicator organisms (FIOs), such as Escherichia coli and enterococci, are often used as surrogates of contamination in the context of beach management; however, bacteriophages may be more reliable indicators than FIO due to their similarity to viral pathogens in terms of size and persistence in the environment. In the past, mechanistic modeling of environmental contamination has focused on FIOs, with virus and bacteriophage modeling efforts remaining limited. In this paper, we describe the development and application of a fate and transport model of somatic and F-specific coliphages for the Washington Park beach in Lake Michigan, which is affected by riverine outputs from the nearby Trail Creek. A three-dimensional model of coliphage transport and photoinactivation was tested and compared with a previously reported E. coli fate and transport model. The light-based inactivation of the phages was modeled using organism-specific action spectra. Results indicate that the coliphage models outperformed the E. coli model in terms of reliably predicting observed E. coli/coliphage concentrations at the beach. This is possibly due to the presence of additional E. coli sources that were not accounted for in the modeling. The coliphage models can be used to test hypotheses about potential sources and their behavior and for predictive modeling.

Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix).

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

Temporal assessment of the impact of exposure to cow feces in two watersheds by multiple host-specific PCR assays.

Exposure to feces in two watersheds with different management histories was assessed by tracking cattle feces bacterial populations using multiple host-specific PCR assays. In addition, environmental factors affecting the occurrence of these markers were identified. Each assay was performed using DNA extracts from water and sediment samples collected from a watershed directly impacted by cattle fecal pollution (WS1) and from a watershed impacted only through runoff (WS2). In WS1, the ruminant-specific Bacteroidales 16S rRNA gene marker CF128F was detected in 65% of the water samples, while the non-16S rRNA gene markers Bac1, Bac2, and Bac5 were found in 32 to 37% of the water samples. In contrast, all source-specific markers were detected in less than 6% of the water samples from WS2. Binary logistic regressions (BLRs) revealed that the occurrence of Bac32F and CF128F was significantly correlated with season as a temporal factor and watershed as a site factor. BLRs also indicated that the dynamics of fecal-source-tracking markers correlated with the density of a traditional fecal indicator (P < 0.001). Overall, our results suggest that a combination of 16S rRNA gene and non-16S rRNA gene markers provides a higher level of confidence for tracking unknown sources of fecal pollution in environmental samples. This study also provided practical insights for implementation of microbial source-tracking practices to determine sources of fecal pollution and the influence of environmental variables on the occurrence of source-specific markers.