Complete genome sequence of a novel papillomavirus in Siamese fighting fish (Betta splendens) from Trinidad and Tobago.

Here, we announce the complete genome of a previously undescribed papillomavirus from a betta fish, Betta splendens. The genome is 5,671 bp with a GC content of 38.2%. Variants were detected in public databases. This genome is most similar to papillomaviruses that infect sea bass (52.9 % nucleotide identity).

Contaminant Exposure and Transport from Three Potential Reuse Waters within a Single Watershed.

Global demand for safe and sustainable water supplies necessitates a better understanding of contaminant exposures in potential reuse waters. In this study, we compared exposures and load contributions to surface water from the discharge of three reuse waters (wastewater effluent, urban stormwater, and agricultural runoff). Results document substantial and varying organic-chemical contribution to surface water from effluent discharges (e.g., disinfection byproducts [DBP], prescription pharmaceuticals, industrial/household chemicals), urban stormwater (e.g., polycyclic aromatic hydrocarbons, pesticides, nonprescription pharmaceuticals), and agricultural runoff (e.g., pesticides). Excluding DBPs, episodic storm-event organic concentrations and loads from urban stormwater were comparable to and often exceeded those of daily wastewater-effluent discharges. We also assessed if wastewater-effluent irrigation to corn resulted in measurable effects on organic-chemical concentrations in rain-induced agricultural runoff and harvested feedstock. Overall, the target-organic load of 491 g from wastewater-effluent irrigation to the study corn field during the 2019 growing season did not produce substantial dissolved organic-contaminant contributions in subsequent rain-induced runoff events. Out of the 140 detected organics in source wastewater-effluent irrigation, only imidacloprid and estrone had concentrations that resulted in observable differences between rain-induced agricultural runoff from the effluent-irrigated and nonirrigated corn fields. Analyses of pharmaceuticals and per-/polyfluoroalkyl substances in at-harvest corn-plant samples detected two prescription antibiotics, norfloxacin and ciprofloxacin, at concentrations of 36 and 70 ng/g, respectively, in effluent-irrigated corn-plant samples; no contaminants were detected in noneffluent irrigated corn-plant samples.

Transcriptome signatures of wastewater effluent exposure in larval zebrafish vary with seasonal mixture composition in an effluent-dominated stream.

Wastewater treatment plant (WWTP) effluent-dominated streams provide critical habitat for aquatic and terrestrial organisms but also continually expose them to complex mixtures of pharmaceuticals that can potentially impair growth, behavior, and reproduction. Currently, few biomarkers are available that relate to pharmaceutical-specific mechanisms of action. In the experiment reported in this paper, zebrafish (Danio rerio) embryos at two developmental stages were exposed to water samples from three sampling sites (0.1 km upstream of the outfall, at the effluent outfall, and 0.1 km below the outfall) during base-flow conditions from two months (January and May) of a temperate-region effluent-dominated stream containing a complex mixture of pharmaceuticals and other contaminants of emerging concern. RNA-sequencing identified potential biological impacts and biomarkers of WWTP effluent exposure that extend past traditional markers of endocrine disruption. Transcriptomics revealed changes to a wide range of biological functions and pathways including cardiac, neurological, visual, metabolic, and signaling pathways. These transcriptomic changes varied by developmental stage and displayed sensitivity to variable chemical composition and concentration of effluent, thus indicating a need for stage-specific biomarkers. Some transcripts are known to be associated with genes related to pharmaceuticals that were present in the collected samples. Although traditional biomarkers of endocrine disruption were not enriched in either month, a high estrogenicity signal was detected upstream in May and implicates the presence of unidentified chemical inputs not captured by the targeted chemical analysis. This work reveals associations between bioeffects of exposure, stage of development, and the composition of chemical mixtures in effluent-dominated surface water. The work underscores the importance of measuring effects beyond the endocrine system when assessing the impact of bioactive chemicals in WWTP effluent and identifies a need for non-targeted chemical analysis when bioeffects are not explained by the targeted analysis.

Exposure to 17α-Ethinylestradiol Results in Differential Susceptibility of Largemouth Bass (Micropterus salmoides) to Bacterial Infection.

Disease outbreaks, skin lesions, mortality events, and reproductive abnormalities have been observed in wild populations of centrarchids. The presence of estrogenic endocrine disrupting compounds (EEDCs) has been implicated as a potential causal factor for these effects. The effects of prior EEDC exposure on immune response were examined in juvenile largemouth bass (Micropterus salmoides) exposed to a potent synthetic estrogen (17α-ethinylestradiol, EE2) at a low (EE2Low, 0.87 ng/L) or high (EE2High, 9.08 ng/L) dose for 4 weeks, followed by transfer to clean water and injection with an LD40 dose of the Gram-negative bacteria Edwardsiella piscicida. Unexpectedly, this prior exposure to EE2High significantly increased survivorship at 10 d post-infection compared to solvent control or EE2Low-exposed, infected fish. Both prior exposure and infection with E. piscicida led to significantly reduced hepatic glycogen levels, indicating a stress response resulting in depletion of energy stores. Additionally, pathway analysis for liver and spleen indicated differentially expressed genes associated with immunometabolic processes in the mock-injected EE2High treatment that could underlie the observed protective effect and metabolic shift in EE2High-infected fish. Our results demonstrate that exposure to a model EEDC alters metabolism and immune function in a fish species that is ecologically and economically important in North America.

Using Biological Responses to Monitor Freshwater Post-Spill Conditions over 3 years in Blacktail Creek, North Dakota, USA.

A pipeline carrying unconventional oil and gas (OG) wastewater spilled approximately 11 million liters of wastewater into Blacktail Creek, North Dakota, USA. Flow of the mix of stream water and wastewater down the channel resulted in storage of contaminants in the hyporheic zone and along the banks, providing a long-term source of wastewater constituents to the stream. A multi-level investigation was used to assess the potential effects of oil and brine spills on aquatic life. In this study, we used a combination of experiments using a native fish species, Fathead Minnow (Pimephales promelas), field sampling of the microbial community structure, and measures of estrogenicity. The fish investigation included in situ experiments and experiments with collected site water. Estrogenicity was measured in collected site water samples, and microbial community analyses were conducted on collected sediments. During the initial post-spill investigation, February 2015, performing in situ fish bioassays was impossible because of ice conditions. However, microbial community (e.g., the presence of members of the Halomonadaceae, a family that is indicative of elevated salinity) and estrogenicity differences were compared to reference sites and point to early biological effects of the spill. We noted water column effects on in situ fish survival 6 months post-spill during June 2015. At that time, total dissolved ammonium (sum of ammonium and ammonia, TAN) was 4.41 mg NH4/L with an associated NH3 of 1.09 mg/L, a concentration greater than the water quality criteria established to protect aquatic life. Biological measurements in the sediment defined early and long-lasting effects of the spill on aquatic resources. The microbial community structure was affected during all sampling events. Therefore, sediment may act as a sink for constituents spilled and as such provide an indication of continued and cumulative effects post-spill. However, lack of later water column effects may reflect pulse hyporheic flow of ammonia from shallow ground water. Combining fish toxicological, microbial community structure and estrogenicity information provides a complete ecological investigation that defines potential influences of contaminants at organismal, population, and community levels. In general, in situ bioassays have implications for the individual survival and changes at the population level, microbial community structure defines potential changes at the community level, and estrogenicity measurements define changes at the individual and molecular level. By understanding effects at these various levels of biological organization, natural resource managers can interpret how a course of action, especially for remediation/restoration, might affect a larger group of organisms in the system. The current work also reviews potential effects of additional constituents defined during chemistry investigations on aquatic resources.

Modeling Risk Dynamics of Contaminants of Emerging Concern in a Temperate-region Wastewater Effluent-dominated Stream.

Wastewater effluent-dominated streams are becoming increasingly common worldwide, including in temperate regions, with potential impacts on ecological systems and drinking water sources. We recently quantified the occurrence/ spatiotemporal dynamics of pharmaceutical mixtures in a representative temperate-region wastewater effluent-dominated stream (Muddy Creek, Iowa) under baseflow conditions and characterized relevant fate processes. Herein, we quantified the ecological risk quotients (RQs) of 19 effluent-derived contaminants of emerging concern (CECs; including: 14 pharmaceuticals, 2 industrial chemicals, and 3 neonicotinoid insecticides) and 1 run-off-derived compound (atrazine) in the stream under baseflow conditions, and estimated the probabilistic risks of effluent-derived CECs under all-flow conditions (i.e., including runoff events) using stochastic risk modeling. We determined that 11 out of 20 CECs pose medium-to-high risks to local ecological systems (i.e., algae, invertebrates, fish) based on literature-derived acute effects under measured baseflow conditions. Stochastic risk modeling indicated decreased, but still problematic, risk of effluent-derived CECs (i.e., RQ≥0.1) under all-flow conditions when runoff events were included. Dilution of effluent-derived chemicals from storm flows thus only minimally decreased risk to aquatic biota in the effluent-dominated stream. We also modeled in-stream transport. Thirteen out of 14 pharmaceuticals persisted along the stream reach (median attenuation rate constant k<0.1 h-1) and entered the Iowa River at elevated concentrations. Predicted and measured concentrations in the drinking water treatment plant were below the human health benchmarks. This study demonstrates the application of probabilistic risk assessments for effluent-derived CECs in a representative effluent-dominated stream under variable flow conditions (when measurements are less practical) and provides an enhanced prediction tool transferable to other effluent-dominated systems.

RNA-seq reveals potential gene biomarkers in fathead minnows (Pimephales promelas) for exposure to treated wastewater effluent.

Discharged wastewater treatment plant (WWTP) effluent greatly contributes to the generation of complex mixtures of contaminants of emerging concern (CECs) in aquatic environments which often contain neuropharmaceuticals and other emerging contaminants that may impact neurological function. However, there is a paucity of knowledge on the neurological impacts of these exposures to aquatic organisms. In this study, caged fathead minnows (Pimephales promelas) were exposed in situ in a temperate-region effluent-dominated stream (i.e., Muddy Creek) in Coralville, Iowa, USA upstream and downstream of a WWTP effluent outfall. The pharmaceutical composition of Muddy Creek was recently characterized by our team and revealed many compounds there were at a low microgram to high nanogram per liter concentration. Total RNA sequencing analysis on brain tissues revealed 280 gene isoforms that were significantly differentially expressed in male fish and 293 gene isoforms in female fish between the upstream and downstream site. Only 66 (13%) of such gene isoforms overlapped amongst male and female fish, demonstrating sex-dependent impacts on neuronal gene expression. By using a systems biology approach paired with functional enrichment analyses, we identified several potential novel gene biomarkers for treated effluent exposure that could be used to expand monitoring of environmental effects with respect to complex CEC mixtures. Lastly, when comparing the results of this study to those that relied on a single-compound approach, there was relatively little overlap in terms of gene-specific effects. This discovery brings into question the application of single-compound exposures in accurately characterizing environmental risks of complex mixtures and for gene biomarker identification.

Freshwater unionid mussels threatened by predation of Round Goby (Neogobius melanostomus).

Indigenous freshwater mussels (Unionidae) are integral to riverine ecosystems, playing a pivotal role in aquatic food webs and providing ecological services. With populations on the decline worldwide, freshwater mussels are of conservation concern. In this study, we explore the propensity of the invasive Round Goby (Neogobius melanostomus) fish to prey upon indigenous freshwater mussels. First, we conducted lab experiments where Round Gobies were given the opportunity to feed on juvenile unionid mussels and macroinvertebrates, revealing rates and preferences of consumption. Several Round Gobies consumed whole freshwater mussels during these experiments, as confirmed by mussel counts and x-ray images of the fishes. Next, we investigated Round Gobies collected from stream habitats of the French Creek watershed, which is renowned for its unique and rich aquatic biodiversity. We developed a novel DNA metabarcoding method to identify the specific species of mussels consumed by Round Goby and provide a new database of DNA gene sequences for 25 indigenous unionid mussel species. Several of the fishes sampled had consumed indigenous mussels, including the Elktoe (non-endangered), Creeper (non-endangered), Long Solid (state endangered), and Rayed Bean (federally endangered) species. The invasive Round Goby poses a growing threat to unionid mussels, including species of conservation concern. The introduction of the invasive Round Goby to freshwaters of North America is shaping ecosystem transitions within the aquatic critical zone having widespread implications for conservation and management.

Draft Genome Sequence of a Novel Calicivirus from a Brown Bullhead (Ameiurus nebulosus) from Lake Memphremagog, Vermont/Quebec.

We report a draft genome sequence of a previously undescribed calicivirus from a single brown bullhead inhabiting Lake Memphremagog, Vermont/Quebec. The genome is 7,413 nucleotides long and is most similar to the Atlantic salmon calicivirus (nucleotide identity; 64.7%).

Site- and Individual-Level Contaminations Affect Infection Prevalence of an Emerging Infectious Disease of Amphibians.

Emerging infectious disease outbreaks are one of multiple stressors responsible for amphibian declines globally. In the northeastern United States, ranaviral diseases are prevalent in amphibians and other ectothermic species, but there is still uncertainty as to whether their presence is leading to population-level effects. Further, there is also uncertainty surrounding the potential interactions among disease infection prevalence in free-ranging animals and habitat degradation (co-occurrence of chemical stressors). The present study was designed to provide field-based estimates of the relationship between amphibian disease and chemical stressors. We visited 40 wetlands across three protected areas, estimated the prevalence of ranavirus among populations of larval wood frogs and spotted salamanders, and assessed chemical and biological stressors in wetland habitats and larval amphibians using a suite of selected bioassays, screening tools, and chemical analyses. Ranavirus was detected on larval amphibians from each protected area with an estimated occupancy ranging from 0.27 to 0.55. Considerable variation in ranavirus occupancy was also observed within and among each protected area. Of the stressors evaluated, ranavirus prevalence was strongly and positively related to concentrations of metalloestrogens (metals with the potential to bind to estrogen receptors) and total metals in wetland sediments and weakly and negatively related to total pesticide concentrations in larval amphibians. These results can be used by land managers to refine habitat assessments to include such environmental factors with the potential to influence disease susceptibility. Environ Toxicol Chem 2022;41:781-791. © 2022 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Tandem field and laboratory approaches to quantify attenuation mechanisms of pharmaceutical and pharmaceutical transformation products in a wastewater effluent-dominated stream.

Evolving complex mixtures of pharmaceuticals and transformation products in effluent-dominated streams pose potential impacts to aquatic species; thus, understanding the attenuation dynamics in the field and characterizing the prominent attenuation mechanisms of pharmaceuticals and their transformation products (TPs) is critical for hazard assessments. Herein, we determined the attenuation dynamics and the associated prominent mechanisms of pharmaceuticals and their corresponding TPs via a combined long-term field study and controlled laboratory experiments. For the field study, we quantified spatiotemporal exposure concentrations of five pharmaceuticals and six associated TPs in a small, temperate-region effluent-dominated stream during baseflow conditions where the wastewater plant was the main source of pharmaceuticals. We selected four sites (upstream, at, and two progressively downstream from effluent discharge) and collected water samples at 16 time points (64 samples in total, approximately twice monthly, depending on flows) for 1 year. Concurrently, we conducted photolysis, sorption, and biodegradation batch tests under controlled conditions to determine the major attenuation mechanisms. We observed 10-fold greater attenuation rates in the field compared to batch tests, demonstrating that connecting laboratory batch tests with field measurements to enhance predictive power is a critical need. Batch systems alone, often used for assessment, are useful for determining fate processes but poorly approximate in-stream attenuation kinetics. Sorption was the dominant attenuation process (t1/2<7.7 d) for 5 of 11 compounds in the batch tests, while the other compounds (n = 6) persisted in the batch tests and along the 5.1 km stream reach. In-stream parent-to-product transformation was minimal. Differential attenuation contributed to the evolving pharmaceutical mixture and created changing exposure conditions with concomitant implications for aquatic and terrestrial biota. Tandem field and laboratory characterization can better inform modeling efforts for transport and risk assessments.

Pilot-scale expanded assessment of inorganic and organic tapwater exposures and predicted effects in Puerto Rico, USA.

A pilot-scale expanded target assessment of mixtures of inorganic and organic contaminants in point-of-consumption drinking water (tapwater, TW) was conducted in Puerto Rico (PR) to continue to inform TW exposures and corresponding estimations of cumulative human-health risks across the US. In August 2018, a spatial synoptic pilot assessment of than 524 organic and 37 inorganic chemicals was conducted in 14 locations (7 home; 7 commercial) across PR. A follow-up 3-day temporal assessment of TW variability was conducted in December 2018 at two of the synoptic locations (1 home, 1 commercial) and included daily pre- and post-flush samples. Concentrations of regulated and unregulated TW contaminants were used to calculate cumulative in vitro bioactivity ratios and Hazard Indices (HI) based on existing human-health benchmarks. Synoptic results confirmed that human exposures to inorganic and organic contaminant mixtures, which are rarely monitored together in drinking water at the point of consumption, occurred across PR and consisted of elevated concentrations of inorganic contaminants (e.g., lead, copper), disinfection byproducts (DBP), and to a lesser extent per/polyfluoroalkyl substances (PFAS) and phthalates. Exceedances of human-health benchmarks in every synoptic TW sample support further investigation of the potential cumulative risk to vulnerable populations in PR and emphasize the importance of continued broad characterization of drinking-water exposures at the tap with analytical capabilities that better represent the complexity of both inorganic and organic contaminant mixtures known to occur in ambient source waters. Such health-based monitoring data are essential to support public engagement in source water sustainability and treatment and to inform consumer point-of-use treatment decision making in PR and throughout the US.

Emerging investigator series: municipal wastewater as a year-round point source of neonicotinoid insecticides that persist in an effluent-dominated stream.

Neonicotinoids in aquatic systems have been predominantly associated with agriculture, but some are increasingly being linked to municipal wastewater. Thus, the aim of this work was to understand the municipal wastewater contribution to neonicotinoids in a representative, characterized effluent-dominated temperate-region stream. Our approach was to quantify the spatiotemporal concentrations of imidacloprid, clothianidin, thiamethoxam, and transformation product imidacloprid urea: 0.1 km upstream, the municipal wastewater effluent, and 0.1 and 5.1 km downstream from the wastewater outfall (collected twice-monthly for one year under baseflow conditions). Quantified results demonstrated that wastewater effluent was a point-source of imidacloprid (consistently) and clothianidin (episodically), where chronic invertebrate exposure benchmarks were exceeded for imidacloprid (36/52 samples; 3/52 > acute exposure benchmark) and clothianidin (8/52 samples). Neonicotinoids persisted downstream where mass loads were not significantly different than those in the effluent. The combined analysis of neonicotinoid effluent concentrations, instream seasonality, and registered uses in Iowa all indicate imidacloprid, and seasonally clothianidin, were driven by wastewater effluent, whereas thiamethoxam and imidacloprid urea were primarily from upstream non-point sources (or potential in-stream transformation for imidacloprid urea). This is the first study to quantify neonicotinoid persistence in an effluent-dominated stream throughout the year-implicating wastewater effluent as a point-source for imidacloprid (year-round) and clothianidin (seasonal). These findings suggest possible overlooked neonicotinoid indoor human exposure routes with subsequent implications for instream ecotoxicological exposure.

In vitro effects-based method and water quality screening model for use in pre- and post-distribution treated waters.

Recent urban public water supply contamination events emphasize the importance of screening treated drinking water quality after distribution. In vitro bioassays, when run concurrently with analytical chemistry methods, are effective tools to evaluating the efficacy of water treatment processes and water quality. We tested 49 water samples representing the Chicago Department of Water Management service areas for estrogen, (anti)androgen, glucocorticoid receptor-activating contaminants and cytotoxicity. We present a tiered screening approach suitable to samples with anticipated low-level activity and initially tested all extracts for statistically identifiable endocrine activity; performing a secondary dilution-response analysis to determine sample EC50 and biological equivalency values (BioEq). Estrogenic activity was detected in untreated Lake Michigan intake water samples using mammalian (5/49; median: 0.21 ng E2Eq/L) and yeast cell (5/49; 1.78 ng E2Eq/L) bioassays. A highly sensitive (anti)androgenic activity bioassay was applied for the first time to water quality screening and androgenic activity was detected in untreated intake and treated pre-distribution samples (4/49; 0.93 ng DHTEq/L). No activity was identified above method detection limits in the yeast androgenic, mammalian anti-androgenic, and both glucocorticoid bioassays. Known estrogen receptor agonists were detected using HPLC/MS-MS (estrone: 0.72-1.4 ng/L; 17α-estradiol: 1.3-1.5 ng/L; 17ß-estradiol: 1.4 ng/L; equol: 8.8 ng/L), however occurrence did not correlate with estrogenic bioassay results. Many studies have applied bioassays to water quality monitoring using only relatively small samples sets often collected from surface and/or wastewater effluent. However, to realistically adapt these tools to treated water quality monitoring, water quality managers must have the capacity to screen potentially hundreds of samples in short timeframes. Therefore, we provided a tiered screening model that increased sample screening speed, without sacrificing statistical stringency, and detected estrogenic and androgenic activity only in pre-distribution Chicago area samples.

Public and private tapwater: Comparative analysis of contaminant exposure and potential risk, Cape Cod, Massachusetts, USA.

BACKGROUND: Humans are primary drivers of environmental contamination worldwide, including in drinking-water resources. In the United States (US), federal and state agencies regulate and monitor public-supply drinking water while private-supply monitoring is rare; the current lack of directly comparable information on contaminant-mixture exposures and risks between private- and public-supplies undermines tapwater (TW) consumer decision-making. METHODS: We compared private- and public-supply residential point-of-use TW at Cape Cod, Massachusetts, where both supplies share the same groundwater source. TW from 10 private- and 10 public-supply homes was analyzed for 487 organic, 38 inorganic, 8 microbial indicators, and 3 in vitro bioactivities. Concentrations were compared to existing protective health-based benchmarks, and aggregated Hazard Indices (HI) of regulated and unregulated TW contaminants were calculated along with ratios of in vitro exposure-activity cutoffs. RESULTS: Seventy organic and 28 inorganic constituents were detected in TW. Median detections were comparable, but median cumulative concentrations were substantially higher in public supply due to 6 chlorine-disinfected samples characterized by disinfection byproducts and corresponding lower heterotrophic plate counts. Public-supply applicable maximum contaminant (nitrate) and treatment action (lead and copper) levels were exceeded in private-supply TW samples only. Exceedances of health-based HI screening levels of concern were common to both TW supplies. DISCUSSION: These Cape Cod results indicate comparable cumulative human-health concerns from contaminant exposures in private- and public-supply TW in a shared source-water setting. Importantly, although this study's analytical coverage exceeds that currently feasible for water purveyors or homeowners, it nevertheless is a substantial underestimation of the full breadth of contaminant mixtures documented in the environment and potentially present in drinking water. CONCLUSION: Regardless of the supply, increased public engagement in source-water protection and drinking-water treatment, including consumer point-of-use treatment, is warranted to reduce risks associated with long-term TW contaminant exposures, especially in vulnerable populations.

Production and characterization of a mouse monoclonal antibody against smallmouth bass (Micropterus dolomieu) IgM.

A murine monoclonal antibody (mAb, IgG2a) was produced for the detection of smallmouth bass (Micropterus dolomieu) immunoglobulin (IgM). The antibody is specific for IgM heavy chain and was shown to also recognize the Ig heavy chain of largemouth bass (Micropterus salmoides) and bluegill (Lepomis macrochirus) using Western Blot analysis of plasma from 9 teleost taxa. When applied to the analysis of smallmouth bass total plasma IgM using ELISA, the mAb was found to be effective when used in an inhibition kinetic assay.

Genome Sequences of 26 White Sucker Hepatitis B Virus Isolates from White Sucker, Catostomus commersonii, Inhabiting Transboundary Waters from Alberta, Canada, to the Great Lakes, USA.

We report 26 genome sequences of the white sucker hepatitis B virus (WSHBV) from the white sucker, Catostomus commersonii The genome length ranged from 3,541 to 3,543 bp, and nucleotide identity was 96.7% or greater across genomes. This work suggests a geographical range of this virus that minimally extends from the Athabasca River, Alberta, Canada, to the Great Lakes, USA.

Phylogeographic Genetic Diversity in the White Sucker Hepatitis B Virus across the Great Lakes Region and Alberta, Canada.

Hepatitis B viruses belong to a family of circular, double-stranded DNA viruses that infect a range of organisms, with host responses that vary from mild infection to chronic infection and cancer. The white sucker hepatitis B virus (WSHBV) was first described in the white sucker (Catostomus commersonii), a freshwater teleost, and belongs to the genus Parahepadnavirus. At present, the host range of WSHBV and its impact on fish health are unknown, and neither genetic diversity nor association with fish health have been studied in any parahepadnavirus. Given the relevance of genomic diversity to disease outcome for the orthohepadnaviruses, we sought to characterize genomic variation in WSHBV and determine how it is structured among watersheds. We identified WSHBV-positive white sucker inhabiting tributaries of Lake Michigan, Lake Superior, Lake Erie (USA), and Lake Athabasca (Canada). Copy number in plasma and in liver tissue was estimated via qPCR. Templates from 27 virus-positive fish were amplified and sequenced using a primer-specific, circular long-range amplification method coupled with amplicon sequencing on the Illumina MiSeq. Phylogenetic analysis of the WSHBV genome identified phylogeographical clustering reminiscent of that observed with human hepatitis B virus genotypes. Notably, most non-synonymous substitutions were found to cluster in the pre-S/spacer overlap region, which is relevant for both viral entry and replication. The observed predominance of p1/s3 mutations in this region is indicative of adaptive change in the polymerase open reading frame (ORF), while, at the same time, the surface ORF is under purifying selection. Although the levels of variation we observed do not meet the criteria used to define sub/genotypes of human and avian hepadnaviruses, we identified geographically associated genome variation in the pre-S and spacer domain sufficient to define five WSHBV haplotypes. This study of WSHBV genetic diversity should facilitate the development of molecular markers for future identification of genotypes and provide evidence in future investigations of possible differential disease outcomes.

Monitoring wetland water quality related to livestock grazing in amphibian habitats.

Land use alteration such as livestock grazing can affect water quality in habitats of at-risk wildlife species. Data from managed wetlands are needed to understand levels of exposure for aquatic life stages and monitor grazing-related changes afield. We quantified spatial and temporal variation in water quality in wetlands occupied by threatened Oregon spotted frog (Rana pretiosa) at Klamath Marsh National Wildlife Refuge in Oregon, United States (US). We used analyses for censored data to evaluate the importance of habitat type and grazing history in predicting concentrations of nutrients, turbidity, fecal indicator bacteria (FIB; total coliforms, Escherichia coli (E. coli), and enterococci), and estrogenicity, an indicator of estrogenic activity. Nutrients (orthophosphate and ammonia) and enterococci varied over time and space, while E. coli, total coliforms, turbidity, and estrogenicity were more strongly associated with local livestock grazing metrics. Turbidity was correlated with several grazing-related constituents and may be particularly useful for monitoring water quality in landscapes with livestock use. Concentrations of orthophosphate and estrogenicity were elevated at several sites relative to published health benchmarks, and their potential effects on Rana pretiosa warrant further investigation. Our data provided an initial assessment of potential exposure of amphibians to grazing-related constituents in western US wetlands. Increased monitoring of surface water quality and amphibian population status in combination with controlled laboratory toxicity studies could help inform future research and targeted management strategies for wetlands with both grazing and amphibians of conservation concern.

Retrospective analysis of estrogenic endocrine disruption and land-use influences in the Chesapeake Bay watershed.

The Chesapeake Bay is the largest estuary in the United States and its watershed includes river drainages in six states and the District of Columbia. Sportfishing is of major economic interest, however, the rivers within the watershed provide numerous other ecological, recreational, cultural and economic benefits, as well as serving as a drinking water source for millions of people. Consequently, major fish kills and the subsequent finding of estrogenic endocrine disruption (intersex or testicular oocytes and plasma vitellogenin in male fishes) raised public and management concerns. Studies have occurred at various sites within the Bay watershed to document the extent and severity of endocrine disruption, identify risk factors and document temporal and spatial variability. Data from these focal studies, which began in 2004, were used in CART (classification and regression trees) analyses to better identify land use associations and potential management practices that influence estrogenic endocrine disruption. These analyses emphasized the importance of scale (immediate versus upstream catchment) and the complex mixtures of stressors which can contribute to surface water estrogenicity and the associated adverse effects of exposure. Both agricultural (percent cultivated, pesticide application, phytoestrogen cover crops) and developed (population density, road density, impervious surface) land cover showed positive relationships to estrogenic indicators, while percent forest and shrubs generally had a negative association. The findings can serve as a baseline for assessing ongoing restoration and management practices.