Geology Reports⌕ Search

Geology topics

Luke R. Iwanowicz

Publications and source records attributed to Luke R. Iwanowicz.

At least 19 recordsLinked to original sources
USGS2025

Novel adomaviruses associated with blotchy bass syndrome in black basses ( Micropterus spp. )

Black bass ( Micropterus spp. ) are the most important warmwater game fishes in the United States. They have high socioeconomic and recreational value and support an important aquaculture industry. Since 2008, fisheries managers have been reporting the observation of hyperpigmented melanistic lesions (HPMLs) on smallmouth bass ( M. dolomieu ) in different ecoregions of the United States. Similar HPMLs have been observed in largemouth bass ( M. nigricans ) since the 1980’s. Here, we report the association between novel adomaviruses and the hallmark blotchy clinical presentation of hyperpigmented lesions on the skin of smallmouth and largemouth black bass. The two adomaviruses are structurally and phylogenetically similar but share only 68.0% identity at aligned nucleotide sites and each has been found in only one host species to date. The manifestation of this skin disease appears to be seasonal (observed between the fall and spring) in both species, primarily affects adults and is of unknown health consequence. Although the significance of infection to fish health remains unclear, understanding the disease ecology of these viruses can inform biosecurity and the interjurisdictional movement of individuals. Moreover, as hyperpigmentation in other fish species is often idiopathic, our findings reframe perspectives for future investigations into this clinical presentation in other species.

Michigan, Pennsylvania, Texas, Vermont, Virginia↗
USGS2025

Identical sequence types of Yersinia ruckeri associated with lethal disease in wild-caught invasive Blue Catfish and cultured hybrid catfish (Channel Catfish ♀ × Blue Catfish ♂) from disparate aquatic ecosystems

Objective The Blue Catfish Ictalurus furcatus is commonly raised in warmwater aquaculture in the United States to produce Channel Catfish I. punctatus × Blue Catfish hybrids. It is also a prominent aquatic invasive species of concern in the mid-Atlantic region of the United States. Here, Yersina ruckeri was isolated from moribund Blue Catfish and hybrid catfish from disparate regions of the USA. The goal of the research here was to compare these Y. ruckeri strains to each other and other known strains for which adequate sequence data was available. In addition, we sought to determine if the strain from Blue Catfish was pathogenic to Rainbow Trout Oncorhynchus mykiss . Methods Moribund hybrid catfish from culture ponds in Mississippi were processed for diagnostic evaluation in March 2016. In April 2022, a moribund Blue Catfish specimen was collected from a tributary of the Nanticoke River in Maryland. Bacterial isolates were identified and characterized using biochemical tests, antimicrobial sensitivity screening, serotyping, and complete or partial genome sequencing. Disease pathology was described via histology. The isolate from Blue Catfish was used in challenge experiments to determine if it was pathogenic to Rainbow Trout. Multilocus sequencing typing was conducted using the PubMLST database. Results Biochemical testing was consistent with Y. ruckeri . A draft genome of the Y. ruckeri isolate was assembled based on Oxford Nanopore Technology sequencing and identified a single genomic replicon (3,791,418 bp) consistent in size to other Y. ruckeri genomes and a pLT plasmid (60, 933 bp). The challenge study demonstrated no significant virulence of this isolate for Rainbow Trout ( Y. ruckeri ). This isolate was most similar to other strains isolated from ictalurids. Notably, the gyrase B gene from this isolate was identical to that of archived strains isolated from moribund Mississippi hybrid catfish aquaculture during 2016 and these isolates share identical PubMLST sequence type profiles. Similarly, they shared a pLT plasmid that differed by only 6 bp. This plasmid has never been reported from trout isolates and appears to be unique to ictalurids. Conclusions Analyses here provide preliminary genetic evidence that geographically distant (Maryland and Mississippi, USA) isolates of Y. ruckeri from ictalurids are genetically similar to each other and Y. ruckeri (strain SC09) that infects ictalurids in China. This strain is not a biothreat to Rainbow Trout at typical culture temperatures.

Journal of Aquatic Animal Health↗
USGS2025

Assessing microplastics, per- and polyfluoroalkyl substances (PFAS), and other contaminants of global concern in wadable agricultural streams

Microplastics, per- and polyfluoroalkyl substances (PFAS), antibiotic resistance genes (ARGs), pharmaceuticals and personal care products (PPCPs), and pesticides may lead to unintended environmental contamination through many pathways in multiple matrices. This statewide, multi-matrix study of contaminants of global concern (CGCs) in agricultural streams across Iowa (United States) is the first to examine multiple CGCs in water, bed sediment, and fish to understand their occurrence in small streams located in regions of intense agriculture activity. Iowa plays a pivotal role in agriculture, with more than 85% of Iowa’s landscape devoted to agriculture making it an ideal location for determining the prevalence of CGCs to provide critical baseline exposure data. Fifteen sites were sampled across a range of predominant land uses (e.g., poultry, swine); all sites had detections of microplastics in all matrices. Concentrations of PFAS varied but were detected in water and sediment; all fish had detections of perfluorooctanesulfonate (PFOS), a type of PFAS. More than 50% of water and bed sediment samples had detections of ARGs. The most frequently detected PPCP was metformin. No sites had a cumulative exposure activity ratio greater than 1.0 for chemical exposures; 13 sites were above the 0.001 precautionary threshold. Toxicity quotients calculated using Aquatic Life Benchmarks were below the 0.1 moderate risk threshold for chemical exposures for all but one site. For fish, all sites exceeded the moderate and high-risk thresholds proposed for microplastic particles for food dilution (both chronic and acute exposures) and all sites exceeded the microplastic moderate threshold proposed for chronic tissue translocation, and two sites exceeded the threshold for acute tissue translocation.

Iowa↗
USGS2025

Novel adomaviruses associated with blotchy bass syndrome in black basses (Micropterus spp.)

Black bass ( Micropterus spp. ) are the most important warmwater game fishes in the United States. They have high socioeconomic and recreational value and support an important aquaculture industry. Since 2008, fisheries managers have been reporting the observation of hyperpigmented melanistic lesions (HPMLs) on smallmouth bass ( M. dolomieu ) in different ecoregions of the United States. Similar HPMLs have been observed in largemouth bass ( M. nigricans ) since the 1980’s. Here, we report a close association between novel adomaviruses and the hallmark blotchy clinical presentation of hyperpigmented lesions on the skin smallmouth and largemouth black bass and provide evidence that satisfies Rivers’ postulates. The two adomaviruses are structurally and phylogenetically similar but share only 68.0% identity at aligned nucleotide sites and each has been found in only one host species to date. The manifestation of this skin disease appears to be seasonal in both species, primarily affects adults and is of unknown health consequence. Although the significance of infection to fish health remains unclear, understanding the disease ecology of these can inform biosecurity and the interjurisdictional movement of individuals. Moreover, as hyperpigmentation in other fish species is often idiopathic, our findings reframe perspectives for future investigations into this clinical presentation in other species.

BioRxiv↗
USGS2024

Discovery and genomic characterization of a novel hepadnavirus from asymptomatic anadromous alewife (Alosa pseudoharengus)

The alewife ( Alosa pseudoharengus) is an anadromous herring that inhabits waters of northeastern North America. This prey species is a critical forage for piscivorous birds, mammals, and fishes in estuarine and oceanic ecosystems. During a discovery project tailored to identify potentially emerging pathogens of this species, we obtained the full genome of a novel hepadnavirus (ApHBV) from clinically normal alewives collected from the Maurice River, Great Egg Harbor River, and Delaware River in New Jersey, USA during 2015–2018. This previously undescribed hepadnavirus contained a circular DNA genome of 3146 nucleotides. Phylogenetic analysis of the polymerase protein placed this virus in the clade of metahepadnaviruses (family: Hepadnaviridae ; genus: Metahepadnavirus ). There was no evidence of pathology in the internal organs of infected fish and virions were not observed in liver tissues by electron microscopy. We developed a Taqman-based quantitative (qPCR) assay and screened 182 individuals collected between 2015 and 2018 and detected additional qPCR positives (n = 6). An additional complete genome was obtained in 2018 and it has 99.4% genome nucleotide identity to the first virus. Single-nucleotide polymorphisms were observed between the two genomes, including 7/9 and 12/8 synonymous vs nonsynonymous mutations across the polymerase and surface proteins, respectively. While there was no evidence that this virus was associated with disease in this species, alewives are migratory interjurisdictional fishes of management concern. Identification of microbial agents using de novo sequencing and other advanced technologies is a critical aspect of understanding disease ecology for informed population management.

New Jersey↗
USGS2024

Seasonal mortality of Wild Atlantic Menhaden (Brevoortia tyrannus) is caused by a virulent clone of Vibrio (Listonella) anguillarum; Implications for biosecurity along the Atlantic Coastal United States

Atlantic menhaden are a highly migratory marine species in the Eastern United States that suffer from seasonal chronic mortality. Affected fish show neurologic signs referred to as spinning disease, including circling at the surface and erratic corkscrew swimming before death. We investigated three similar menhaden mortality events consistent with spinning disease in coastal New Jersey and New York between 2020 and 2021 to understand the cause. A unique strain of Vibrio (Listonella) anguillarum (serogroup O3) was detected regularly in high loads, particularly in the brains of moribund fish, by both metagenomics and bacterial isolation. The most common histopathological changes in moribund fish were hemorrhagic meningitis, encephalitis, pyknosis, and karyorrhexis of hematopoietic tissues in the kidney and spleen. Whole genome sequencing of isolates from moribund fish representing a wide spatial and temporal range showed that they were nearly identical clones, suggesting it to be a pathogenic strain circulating in the population. Though V. anguillarum is believed to be the main pathogen associated with spinning disease and mortality, Yersinia ruckeri (serotype O1) was isolated from smaller numbers of fish. Considering the highly migratory nature of Atlantic menhaden throughout the eastern United States and their use as bait for other fisheries, these findings identify potential biosecurity challenges that should be considered in Atlantic salmon aquaculture, fisheries, and emerging marine aquaculture in the region.

Transboundary and Emerging Diseases↗
USGS2024

Immunomodulation in adult largemouth bass (Micropterus salmoides) exposed to a model estrogen or mixture of endocrine disrupting contaminants during early gonadal recrudescence

Disease outbreaks, skin lesions, fish kill events, and reproductive abnormalities have been observed in wild populations of Centrarchids in watersheds throughout the United States. Occurrence of synthetic and natural hormones from wastewater treatment plants and livestock operations, pesticides from agricultural land use, and phytoestrogens have been implicated as potential causes of these adverse effects. Our objective was to investigate possible immunomodulation in adult largemouth bass ( Micropterus salmoides ) in response to a seasonal exposure to environmentally relevant contaminants in outdoor experimental ponds. Exposures included 17α-ethinylestradiol (EE2; 3.6 ng/L) or a binary mixture of endocrine-active substances commonly detected in surface waters, estrone (E1; 85.6 ng/L) and atrazine (ATR; 5.4 µg/L). The 4-month exposure was conducted from July to November. Functional immune responses of anterior kidney-derived leukocytes were evaluated in December in the week following the end of the dosing period, and in the following April, four months after dosing ended and just prior to spawning. Concentrations of EE2 and E1 in the ponds fell below detectable levels in December, but detectable concentrations of ATR (2.9 µg/L) persisted at least through May. For each sampling time, anterior kidney leukocytes were isolated and grown in primary culture for the assessment of zymosan-stimulated respiratory burst and lectin-stimulated mitogenic responses. We observed seasonal differences in respiratory burst stimulation over time and treatment with a significantly greater response in April relative to December. Respiratory burst activity was also significantly greater in April for fish exposed to the E1+ATR relative to control. In April, prior to spawning, we observed a significantly dampened mitogenic response to PHAP (a T cell mitogen) and LPS (a B cell mitogen) in the EE2 treatment relative to control fish. There were no significant differences in mitogenic responses or respiratory burst between sexes. However, there was significantly higher alternative complement pathway hemolytic activity in males compared to females in both the control and E1+ATR treatment groups. Our results demonstrate that environmentally relevant concentrations of contaminants can alter immune function in a socioeconomically important fish species.

Comparative Immunology Reports↗
USGS2023

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.

Oklahoma↗
USGS2023

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.

Iowa↗
USGS2022

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 (EE2 Low , 0.87 ng/L) or high (EE2 High , 9.08 ng/L) dose for 4 weeks, followed by transfer to clean water and injection with an LD 40 dose of the Gram-negative bacteria Edwardsiella piscicida . Unexpectedly, this prior exposure to EE2 High significantly increased survivorship at 10 d post-infection compared to solvent control or EE2 Low -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 EE2 High treatment that could underlie the observed protective effect and metabolic shift in EE2 High -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.

Environmental Science and Technology↗
USGS2022

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 NH 4 /L with an associated NH 3 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.

North Dakota↗
USGS2022

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.

Maryland, New York, Pennsylvania, West Virginia↗
USGS2022

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 current 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. Estimated ranavirus occupancy varied among the three protected areas and ranged from 0.27 to 0.55 with considerable variation within 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.

Maryland, Massachusetts↗
USGS2022

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.

Iowa↗
USGS2021

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.

Alberta, Michigan, Minnesota, Ohio, Wisconsin↗