Otoliths of Caspian gobies (Teleostei: Gobiidae): Morphological diversity and phylogenetic implications.

Otoliths (ear stones) of the inner ears of teleost fishes, which develop independently from the skeleton and are functionally associated with hearing and the sense of equilibrium, have significantly contributed to contemporary understanding of teleost fish systematics and evolutionary diversity. The sagittal otolith is of particular interest, since it often possesses distinctive morphological features that differ significantly among species, and have been shown to be species- and genus-specific, making it an informative taxonomic tool for ichthyologists. The otolith morphology of the Caspian Sea gobiids has not been thoroughly studied yet, with data available for only a few species. The aim of the present paper is to examine the qualitative and quantitative taxonomic and phylogenetic information in the sagittal otoliths of these species. A total of 118 otoliths representing 30 gobiid species (including 53.5% of the Caspian gobiofauna) in three gobiid lineages (i.e., Gobius, Pomatoschistus, and Acanthogobius) and 11 genera (i.e., all Ponto-Caspian gobiid genera except Babka) were analysed at taxonomic levels using an integrated descriptive and morphometric approach. The results indicated high taxonomic efficiency of otolith morphology and morphometry at taxonomic levels for the Ponto-Caspian gobiids. Our qualitative and quantitative otolith data also (i) support the monophyly of neogobiin gobies, (ii) along with other morphological and ecological data, offer a new perspective on the systematics of Neogobius bathybius, (iii) suggest the reassignment of Hyrcanogobius bergi to the genus Knipowitschia, and (iv) question the phylogenetic integrity of the four phenotypic groups previously defined in the tadpole-goby genus Benthophilus; however, more studies are needed to complete these evaluations and confirm our otolith study findings.

Gobies (Teleostei: Gobiidae) of the oldest and deepest Caspian Sea sub-basin: an evidence-based annotated checklist and a key for species identification.

An evidence-based annotated checklist of gobiid species (Teleostei: Gobiidae) inhabiting the South Caspian Sea and its catchment area (i.e., the South Caspian Sea sub-basin) is compiled. The South Caspian Sea sub-basin gobiofauna currently comprises 38 confirmed species in 11 genera (i.e., 88.4% of the Caspian gobiofauna); the most diverse genus is Benthophilus (16 species, 42.1%), followed by Ponticola (seven species, 18.4%), and Neogobius (four species, 10.5%). Ten species (26.3%) are endemic to the South Caspian Sea sub-basin, another 21 species (55.3%) are endemic in the Caspian Sea basin as a whole, six (15.8%) are native to the Ponto-Caspian region, and one species (2.6%) is exotic. According to the current IUCN Red List, 24 species (64.9%) are listed as being of "Least Concern", eight species (21.6%) are "Data Deficient", and five species (13.5%) as "Not Evaluated". Similar numbers of species are confirmed to inhabit the South Caspian Sea sub-basin waters of the three countries that border it: Iran harbors 25 species (nine genera), Azerbaijan has 28 species (10 genera), and Turkmenistan has 26 species (10 genera). The greatest known diversity of Benthophilus in South Caspian waters occurs in Azerbaijan and Turkmenistan (11 species each), whereas Iranian waters harbor seven species. In comparison, Iran, with six out of eight species (75%), has the greatest diversity of Ponticola known from the Caspian Sea basin. Species richness and endemism of the Caspian Sea gobiid-fauna varies considerably with latitude: the North, Middle and South sub-basins respectively harbor 21, 31, and 37 native species, of which 0, 3, and 10 species are endemic in that sub-basin alone. The high species diversity and endemism of Gobiidae in the South Caspian Sea sub-basin may have resulted from: (i) greater ecological diversity compared to the northern Caspian Sea marine areas (e.g., water depths) that may have led to differential niche adaptation and adaptive radiation in the Benthophilus-Anatirostrum species flock, (ii) lower historical extinction rate compared to Caspian higher latitudes, which had greater exposure to the Pleistocene's extreme climatic changes, (iii) geological history of freshwater habitats in the South Caspian Sea sub-basin that set the speciation and evolutionary stage for the genus Ponticola during these Pleistocene climatic oscillations, (iv) presently less limiting conditions compared to the North Caspian Sea, i.e., higher present winter minimum of water temperature and higher salinity, and (v) Iranian freshwater abundance, variability and habitat diversity. Contemporary gobiid diversity and endemism in the Caspian Sea basin suggests two higher-priority conservation areas: (i) freshwater habitats of the South Caspian Sea region in Iran and Azerbaijan, and (ii) shallow coastal and deep waters of the South and Middle Caspian Sea sub-basins. An identification key is provided for the updated gobiid species from the South Caspian Sea sub-basin.

Genomic and immunogenic changes of Piscine novirhabdovirus (Viral Hemorrhagic Septicemia Virus) over its evolutionary history in the Laurentian Great Lakes.

A unique and highly virulent subgenogroup (-IVb) of Piscine novirhabdovirus, also known as Viral Hemorrhagic Septicemia Virus (VHSV), suddenly appeared in the Laurentian Great Lakes, causing large mortality outbreaks in 2005 and 2006, and affecting >32 freshwater fish species. Periods of apparent dormancy have punctuated smaller and more geographically-restricted outbreaks in 2007, 2008, and 2017. In this study, we conduct the largest whole genome sequencing analysis of VHSV-IVb to date, evaluating its evolutionary changes from 48 isolates in relation to immunogenicity in cell culture. Our investigation compares genomic and genetic variation, selection, and rates of sequence changes in VHSV-IVb, in relation to other VHSV genogroups (VHSV-I, VHSV-II, VHSV-III, and VHSV-IVa) and with other Novirhabdoviruses. Results show that the VHSV-IVb isolates we sequenced contain 253 SNPs (2.3% of the total 11,158 nucleotides) across their entire genomes, with 85 (33.6%) of them being non-synonymous. The most substitutions occurred in the non-coding region (NCDS; 4.3%), followed by the Nv- (3.8%), and M- (2.8%) genes. Proportionally more M-gene substitutions encoded amino acid changes (52.9%), followed by the Nv- (50.0%), G- (48.6%), N- (35.7%) and L- (23.1%) genes. Among VHSV genogroups and subgenogroups, VHSV-IVa from the northeastern Pacific Ocean has shown the fastest substitution rate (2.01x10-3), followed by VHSV-IVb (6.64x10-5) and by the VHSV-I, -II and-III genogroups from Europe (4.09x10-5). A 2016 gizzard shad (Dorosoma cepedianum) from Lake Erie possessed the most divergent VHSV-IVb sequence. The in vitro immunogenicity analysis of that sample displayed reduced virulence (as did the other samples from 2016), in comparison to the original VHSV-IVb isolate (which had been traced back to 2003, as an origin date). The 2016 isolates that we tested induced milder impacts on fish host cell innate antiviral responses, suggesting altered phenotypic effects. In conclusion, our overall findings indicate that VHSV-IVb has undergone continued sequence change and a trend to lower virulence over its evolutionary history (2003 through present-day), which may facilitate its long-term persistence in fish host populations.

Strategic considerations for invasive species managers in the utilization of environmental DNA (eDNA): steps for incorporating this powerful surveillance tool.

Invasive species surveillance programs can utilize environmental DNA sampling and analysis to provide information on the presence of invasive species. Wider utilization of eDNA techniques for invasive species surveillance may be warranted. This paper covers topics directed towards invasive species managers and eDNA practitioners working at the intersection of eDNA techniques and invasive species surveillance. It provides background information on the utility of eDNA for invasive species management and points to various examples of its use across federal and international programs. It provides information on 1) why an invasive species manager should consider using eDNA, 2) deciding if eDNA can help with the manager's surveillance needs, 3) important components to operational implementation, and 4) a high-level overview of the technical steps necessary for eDNA analysis. The goal of this paper is to assist invasive species managers in deciding if, when, and how to use eDNA for surveillance. If eDNA use is elected, the paper provides guidance on steps to ensure a clear understanding of the strengths and limitation of the methods and how results can be best utilized in the context of invasive species surveillance.

Intra-Annual and Interannual Dynamics of Evaporation Over Western Lake Erie.

Evaporation (E) is a critical component of the water and energy budget in lake systems yet is challenging to quantify directly and continuously. We examined the magnitude and changes of E and its drivers over Lake Erie-the shallowest and most southern lake of the Laurentian Great Lakes. We deployed two eddy-covariance tower sites in the western Lake Erie Basin-one located nearshore (CB) and one offshore (LI)-from September 2011 through May 2016. Monthly E varied from 5 to 120 mm, with maximum E occurring in August-October. The annual E was 635 ± 42 (±SD) mm at CB and 604 ± 32 mm at LI. Mean winter (October-March) E was 189 ± 61 mm at CB and 178 ± 25 mm at LI, accounting for 29.8% and 29.4% of annual E. Mean daily E was 1.8 mm during the coldest month (January) and 7.4 mm in the warmest month (July). Monthly E exhibited a strong positive linear relationship to the product of wind speed and vapor pressure deficit. Pronounced seasonal patterns in surface energy fluxes were observed with a 2-month lag in E from R n, due to the lake's heat storage. This lag was shorter than reports regarding other Great Lakes. Difference in E between the offshore and nearshore sites reflected within-lake spatial heterogeneity, likely attributable to climatic and bathymetric differences between them. These findings suggest that predictive models need to consider lake-specific heat storage and spatial heterogeneity in order to accurately simulate lake E and its seasonal dynamics.

Evolutionary trajectory of fish Piscine novirhabdovirus (=Viral Hemorrhagic Septicemia Virus) across its Laurentian Great Lakes history: Spatial and temporal diversification.

Piscine novirhabdovirus = Viral Hemorrhagic Septicemia Virus (VHSV) first appeared in the Laurentian Great Lakes with large outbreaks from 2005 to 2006, as a new and novel RNA rhabdovirus subgenogroup (IVb) that killed >30 fish species. Interlude periods punctuated smaller more localized outbreaks in 2007, 2010, and 2017, although some fishes tested positive in the intervals. There have not been reports of outbreaks or positives from 2018, 2019, or 2020. Here, we employ a combined population genetics and phylogenetic approach to evaluate spatial and temporal evolutionary trajectory on its G-gene sequence variation, in comparison with whole-genome sequences (11,083 bp) from a subset of 44 individual isolates (including 40 newly sequenced ones). Our results show that IVb (N = 184 individual fish isolates) diversified into 36 G-gene haplotypes from 2003 to 2017, stemming from two originals ("a" and "b"). G-gene haplotypes "a" and "b" differed by just one synonymous single-nucleotide polymorphism (SNP) substitution, remained the most abundant until 2011, then disappeared. Group "a" descendants (14 haplotypes) remained most prevalent in the Upper and Central Great Lakes, with eight (51%) having nonsynonymous substitutions. Group "b" descendants primarily have occurred in the Lower Great Lakes, including 22 haplotypes, of which 15 (68%) contained nonsynonymous changes. Evolutionary patterns of the whole-genome sequences (which had 34 haplotypes among 44 isolates) appear congruent with those from the G-gene. Virus populations significantly diverged among the Upper, Central, and Lower Great Lakes, diversifying over time. Spatial divergence was apparent in the overall patterns of nucleotide substitutions, while amino acid changes increased temporally. VHSV-IVb thus significantly differentiated across its less than two decades in the Great Lakes, accompanied by declining outbreaks and virulence. Continuing diversification likely allowed the virus to persist at low levels in resident fish populations, and may facilitate its potential for further and future spread to new habitats and nonacclimated hosts.

Invasion genetics from eDNA and thousands of larvae: A targeted metabarcoding assay that distinguishes species and population variation of zebra and quagga mussels.

Identifying species and population genetic compositions of biological invasions at early life stages and/or from environmental (e)DNA using targeted high-throughput sequencing (HTS) metabarcode assays offers powerful and cost-effective means for early detection, analysis of spread patterns, and evaluating population changes. The present study develops, tests, and applies this method with a targeted sequence assay designed to simultaneously identify and distinguish between the closely related invasive Eurasian zebra and quagga mussels (Dreissena polymorpha and D. rostriformis) and their relatives and discern their respective population genetic patterns. Invasions of these dreissenid mussel species have markedly changed freshwater ecosystems throughout North America and Europe, exerting severe ecological and economic damage. Their planktonic early life stages (eggs and larvae) are morphologically indistinguishable, yet each species exerts differential ecological effects, with the quagga often outcompeting the zebra mussel as adults. Our targeted assay analyzes genetic variation from a diagnostic sequence region of the mitochondrial (mt)DNA cytochrome oxidase I (COI) gene, to assess temporal and spatial inter- and intra-specific genetic variability. The assay facilitates analysis of environmental (e)DNA from water, early life stages from thousands of individuals, and simultaneous analysis of 50-100 tagged field-collected samples. Experiments evaluated its accuracy and performance using: (a) mock laboratory communities containing known DNA quantities per taxon, (b) aquaria with mixed-species/haplotype compositions of adults, and (c) field-collected water and plankton versus traditional sampling of adult communities. Results delineated species compositions, relative abundances, and population-level diversity differences among ecosystems, habitats, time series, and life stages from two allopatric concurrent invasions in the Great Lakes (Lake Erie) and the Hudson River, which had separate founding histories. Findings demonstrate application of this targeted assay and our approach to accurately and simultaneously discern species- and population-level differences across spatial and temporal scales, facilitating early detection and ecological understanding of biological invasions.

Invasion genetics of the silver carp Hypophthalmichthys molitrix across North America: Differentiation of fronts, introgression, and eDNA metabarcode detection.

In the 1970s, the introduced silver carp Hypophthalmichthys molitrix (which is indigenous to eastern Asia) escaped from southern U.S. aquaculture to spread throughout the Mississippi River basin, and since has steadily moved northward. This large, prolific filter-feeder reduces food availability for other fishes. It now has reached the threshold of the Laurentian Great Lakes, where it likely will significantly impact food chains and fisheries. Our study evaluates population genetic variability and differentiation of the silver carp using 10 nuclear DNA microsatellite loci, and sequences of two mitochondrial genes-cytochrome b and cytochrome c oxidase subunit 1, along with the nuclear ribosomal protein S7 gene intron 1. We analyze population samples from: two primary Great Lakes' invasion fronts (at the Illinois River outside of Chicago, IL in Lake Michigan and in the Wabash River, which leads into the Maumee River and western Lake Erie), the original establishment "core" in the Lower Mississippi River, and expansion areas in the Upper Mississippi and Missouri rivers. We analyze and compare our results with bighead and other invasive carps, and cyprinid relatives. Results reveal that the silver carp invasion possesses moderate levels of genetic diversity, with more mtDNA haplotypes and unique microsatellite alleles in the "core" Lower Mississippi River population, which also diverges the most. The two invasion fronts also significantly genetically differ. About 3% of individuals (including all populations except the Illinois River) contain a unique and very divergent mtDNA haplotype, which likely stems from historic introgression in Asia with female largescale silver carp H. harmandi. The nuclear microsatellites and S7 sequences of the introgressed individuals do not differ from silver carp and are very distant from bighead carp. These sequence variation data are employed to design and evaluate a targeted high-throughput metabarcoding sequence assay that identifies and distinguishes among species of invasive carps (i.e., silver, bighead, grass, black, and common carps, along with goldfish), as well as native cyprinids, using cytochrome b. Our assay further differentiates among selected silver carp haplotypes (including between H. molitrix and H. harmandi), for use in population genetics and future analyses of spread pathways. We test and evaluate this assay on environmental (e)DNA water samples from 48 bait shops in the Great Lakes' region (along the Lake Erie, Lake St. Clair, and Wabash River watersheds), using positive and negative controls and custom bioinformatic processing. Test results discern silver carp eDNA in four of the shops-three in Lake Erie and one in the Wabash River watershed-and bighead carp from one of the same Lake Erie venues, suggesting that retailers (who often source from established southerly populations) comprise another introduction vector. Our overall findings thus provide key population genetic and phylogenetic data for understanding and tracing introductions, vectors, and spread pathways for silver carp, their variants, and their relatives.

Effects of cortisol and lipopolysaccharide on expression of select growth-, stress- and immune-related genes in rainbow trout liver.

Many studies have shown that stress-induced cortisol levels negatively influence growth and immunity in finfish. Despite this knowledge, few studies have assessed the direct effects of cortisol on liver immune function. Using real-time PCR, the expression of three cortisol-responsive genes (GR: glucocorticoid receptor, IGF-1: insulin-like growth factor-I and SOCS-1: suppressor of cytokine signaling-I), genes involved with innate and adaptive immunity (IL-1ß: interleukin-1 beta, IgM: immunoglobin-M and Lyz: lysozyme), and liver-specific antimicrobial peptides (hepcidin and LEAP-2A: liver-expressed antimicrobial peptide-2A) was studied in vitro using rainbow trout liver slices. The abundances of GR, SOCS-1 and IGF-1 mRNAs were suppressed by cortisol treatment. Abundance of IL-1ß mRNA was upregulated by LPS and suppressed by cortisol treatment in a time-dependent manner. While abundance of IgM mRNA was suppressed by cortisol treatment and stimulated by LPS, there were no effects of cortisol or LPS on abundance of Lyz mRNA. Abundance of hepcidin and LEAP-2A mRNA levels were suppressed by cortisol treatment and stimulated by LPS. These results demonstrate that cortisol directly suppresses abundance of GR, IGF-1, IL-1ß, IgM, hepcidin, LEAP-2A and SOCS-1 mRNA transcripts in the rainbow trout liver. We report for the first time, a suppressive effect of cortisol (within 8 h of treatment) on hepcidin and LEAP-2A mRNAs in rainbow trout liver, which suggests that acute stress may negatively affect liver immune function in rainbow trout.

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription.

Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression.IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.

Early detection monitoring for aquatic non-indigenous species: Optimizing surveillance, incorporating advanced technologies, and identifying research needs.

Following decades of ecologic and economic impacts from a growing list of nonindigenous and invasive species, government and management entities are committing to systematic early- detection monitoring (EDM). This has reinvigorated investment in the science underpinning such monitoring, as well as the need to convey that science in practical terms to those tasked with EDM implementation. Using the context of nonindigenous species in the North American Great Lakes, this article summarizes the current scientific tools and knowledge - including limitations, research needs, and likely future developments - relevant to various aspects of planning and conducting comprehensive EDM. We begin with the scope of the effort, contrasting target-species with broad-spectrum monitoring, reviewing information to support prioritization based on species and locations, and exploring the challenge of moving beyond individual surveys towards a coordinated monitoring network. Next, we discuss survey design, including effort to expend and its allocation over space and time. A section on sample collection and analysis overviews the merits of collecting actual organisms versus shed DNA, reviews the capabilities and limitations of identification by morphology, DNA target markers, or DNA barcoding, and examines best practices for sample handling and data verification. We end with a section addressing the analysis of monitoring data, including methods to evaluate survey performance and characterize and communicate uncertainty. Although the body of science supporting EDM implementation is already substantial, research and information needs (many already actively being addressed) include: better data to support risk assessments that guide choice of taxa and locations to monitor; improved understanding of spatiotemporal scales for sample collection; further development of DNA target markers, reference barcodes, genomic workflows, and synergies between DNA-based and morphology-based taxonomy; and tools and information management systems for better evaluating and communicating survey outcomes and uncertainty.

Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes.

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.

Genetic patterns across an invasion's history: a test of change versus stasis for the Eurasian round goby in North America.

Biological invasions comprise accidental evolutionary experiments, whose genetic compositions underlie relative success, spread and persistence in new habitats. However, little is known about whether, or how, their population genetic patterns change temporally and/or spatially across the invasion's history. Theory predicts that most would undergo founder effect, exhibit low genetic divergence across the new range and gain variation over time via new arriving propagules. To test these predictions, we analyse population genetic diversity and divergence patterns of the Eurasian round goby Neogobius melanostomus across the two decades of its North American invasion in the Laurentian Great Lakes, comparing results from 13 nuclear DNA microsatellite loci and mitochondrial DNA cytochrome b sequences. We test whether 'genetic stasis', 'genetic replacement' and/or 'genetic supplement' scenarios have occurred at the invasion's core and expansion sites, in comparison with its primary native source population in the Dnieper River, Black Sea. Results reveal pronounced genetic divergence across the exotic range, with population areas remaining genetically distinct and statistically consistent across two decades, supporting 'genetic stasis' and 'founder takes most'. The original genotypes continue to predominate, whose high population growth likely outpaced the relative success of later arrivals. The original invasion core has stayed the most similar to the native source. Secondary expansion sites indicate slight allelic composition convergence towards the core population over time, attributable to some early 'genetic supplementation'. The geographic and temporal coverage of this investigation offers a rare opportunity to discern population dynamics over time and space in context of invasion genetic theory vs. reality.

Gene Diversification of an Emerging Pathogen: A Decade of Mutation in a Novel Fish Viral Hemorrhagic Septicemia (VHS) Substrain since Its First Appearance in the Laurentian Great Lakes.

Viral Hemorrhagic Septicemia virus (VHSv) is an RNA rhabdovirus, which causes one of the world's most serious fish diseases, infecting >80 freshwater and marine species across the Northern Hemisphere. A new, novel, and especially virulent substrain-VHSv-IVb-first appeared in the Laurentian Great Lakes about a decade ago, resulting in massive fish kills. It rapidly spread and has genetically diversified. This study analyzes temporal and spatial mutational patterns of VHSv-IVb across the Great Lakes for the novel non-virion (Nv) gene that is unique to this group of novirhabdoviruses, in relation to its glycoprotein (G), phosphoprotein (P), and matrix (M) genes. Results show that the Nv-gene has been evolving the fastest (k = 2.0 x 10-3 substitutions/site/year), with the G-gene at ~1/7 that rate (k = 2.8 x 10-4). Most (all but one) of the 12 unique Nv- haplotypes identified encode different amino acids, totaling 26 changes. Among the 12 corresponding G-gene haplotypes, seven vary in amino acids with eight total changes. The P- and M- genes are more evolutionarily conserved, evolving at just ~1/15 (k = 1.2 x 10-4) of the Nv-gene's rate. The 12 isolates contained four P-gene haplotypes with two amino acid changes, and six M-gene haplotypes with three amino acid differences. Patterns of evolutionary changes coincided among the genes for some of the isolates, but appeared independent in others. New viral variants were discovered following the large 2006 outbreak; such differentiation may have been in response to fish populations developing resistance, meriting further investigation. Two 2012 variants were isolated by us from central Lake Erie fish that lacked classic VHSv symptoms, having genetically distinctive Nv-, G-, and M-gene sequences (with one of them also differing in its P-gene); they differ from each other by a G-gene amino acid change and also differ from all other isolates by a shared Nv-gene amino acid change. Such rapid evolutionary differentiation may allow new viral variants to evade fish host recognition and immune responses, facilitating long-time persistence along with expansion to new geographic areas.

A population genetic window into the past and future of the walleye Sander vitreus: relation to historic walleye and the extinct "blue pike" S. v. "glaucus".

BACKGROUND: Conserving genetic diversity and local adaptations are management priorities for wild populations of exploited species, which increasingly are subject to climate change, habitat loss, and pollution. These constitute growing concerns for the walleye Sander vitreus, an ecologically and economically valuable North American temperate fish with large Laurentian Great Lakes' fisheries. This study compares genetic diversity and divergence patterns across its widespread native range using mitochondrial (mt) DNA control region sequences and nine nuclear DNA microsatellite (µsat) loci, examining historic and contemporary influences. We analyze the genetic and morphological characters of a putative endemic variant- "blue pike" S. v. "glaucus" -described from Lakes Erie and Ontario, which became extinct. Walleye with turquoise-colored mucus also are evaluated, since some have questioned whether these are related to the "blue pike". RESULTS: Walleye populations are distinguished by considerable genetic divergence (mean FST mtDNA = 0.32 ± 0.01, µsat = 0.13 ± 0.00) and substantial diversity across their range (mean heterozygosity mtDNA = 0.53 ± 0.02, µsat = 0.68 ± 0.03). Southern populations markedly differ, possessing unique haplotypes and alleles, especially the Ohio/New River population that houses the oldest haplotype and has the most pronounced divergence. Northern formerly glaciated populations have greatest diversity in Lake Erie (mean heterozygosity mtDNA = 0.79 ± 0.00, µsat = 0.72 ± 0.01). Genetic diversity was much less in the historic Lake Erie samples from 1923-1949 (mean heterozygosity mtDNA = 0.05 ± 0.01, µsat = 0.47 ± 0.06) than today. The historic "blue pike" had no unique haplotypes/alleles and there is no evidence that it comprised a separate taxon from walleye. Turquoise mucus walleye also show no genetic differentiation from other sympatric walleye and no correspondence to the "blue pike". CONCLUSIONS: Contemporary walleye populations possess high levels of genetic diversity and divergence, despite habitat degradation and exploitation. Genetic and previously published tagging data indicate that natal homing and spawning site philopatry led to population structure. Population patterns were shaped by climate change and drainage connections, with northern ones tracing to post-glacial recolonization. Southerly populations possess unique alleles and may provide an important genetic reservoir. Allelic frequencies of Lake Erie walleye from ~70-90 years ago significantly differed from those today, suggesting population recovery after extensive habitat loss, pollution, and exploitation. The historic "blue pike" is indistinguishable from walleye, indicating that taxonomic designation is not warranted.

Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay.

Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R(2) = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/10(6) actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics.

A new StaRT-PCR approach to detect and quantify fish Viral Hemorrhagic Septicemia virus (VHSv): enhanced quality control with internal standards.

Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world's most important finfish diseases, killing >80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR(®) green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR(®) green real time qRT-PCR tests, and 47-70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0×10(0)-1.2×10(5) VHSv/10(6)actb1 molecules in wild caught fishes and 1.0×10(0)-8.4×10(5) molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean=1.9×10(4)) than those without (1.1×10(3)) (p<0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv.

Waterscape genetics of the yellow perch (Perca flavescens): patterns across large connected ecosystems and isolated relict populations.

Comparisons of a species' genetic diversity and divergence patterns across large connected populations vs. isolated relict areas provide important data for understanding potential response to global warming, habitat alterations and other perturbations. Aquatic taxa offer ideal case studies for interpreting these patterns, because their dispersal and gene flow often are constrained through narrow connectivity channels that have changed over geological time and/or from contemporary anthropogenic perturbations. Our research objective is to better understand the interplay between historic influences and modern-day factors (fishery exploitation, stocking supplementation and habitat loss) in shaping population genetic patterns of the yellow perch Perca flavescens (Percidae: Teleostei) across its native North American range. We employ a modified landscape genetics approach, analysing sequences from the entire mitochondrial DNA control region and 15 nuclear DNA microsatellite loci of 664 spawning adults from 24 populations. Results support that perch from primary glacial refugium areas (Missourian, Mississippian and Atlantic) founded contemporary northern populations. Genetic diversity today is highest in southern (never glaciated) populations and also is appreciable in northern areas that were founded from multiple refugia. Divergence is greater among isolated populations, both north and south; the southern Gulf Coast relict populations are the most divergent, reflecting their long history of isolation. Understanding the influence of past and current waterway connections on the genetic structure of yellow perch populations may help us to assess the roles of ongoing climate change and habitat disruptions towards conserving aquatic biodiversity.

Evolution and biogeography of an emerging quasispecies: diversity patterns of the fish Viral Hemorrhagic Septicemia virus (VHSv).

Viral Hemorrhagic Septicemia virus (VHSv) is an RNA rhabdovirus that causes one of the most important finfish diseases, affecting over 70 marine and freshwater species. It was discovered in European cultured fish in 1938 and since has been described across the Northern Hemisphere. Four strains and several substrains have been hypothesized, whose phylogenetic relationships and evolutionary radiation are evaluated here in light of a quasispecies model, including an in-depth analysis of the novel and especially virulent new substrain (IVb) that first appeared in the North American Laurentian Great Lakes in 2003. We analyze the evolutionary patterns, genetic diversity, and biogeography of VHSv using all available RNA sequences from the glycoprotein (G), nucleoprotein (N), and non-virion (Nv) genes, with Maximum Likelihood and bayesian approaches. Results indicate that the G gene evolves at an estimated rate of µ=2.58×10(-4) nucleotide substitutions per site per year, the N gene at µ=4.26×10(-4), and Nv fastest at µ=1.25×10(-3). Phylogenetic trees from the three genes largely are congruent, distinguishing strains I-IV as reciprocally monophyletic with high bootstrap and posterior probability support. VHSv appears to have originated from a marine ancestor in the North Atlantic Ocean, diverging into two primary clades: strain IV in North America (the Northwestern Atlantic Ocean), and strains I-III in the Northeastern Atlantic region (Europe). Strain II may comprise the basal group of the latter clade and diverged in Baltic Sea estuarine waters; strains I and III appear to be sister groups (according to the G and Nv genes), with the former mostly in European freshwaters and the latter in North Sea marine/estuarine waters. Strain IV is differentiated into three monophyletic substrains, with IVa infecting Northeastern Pacific salmonids and many marine fishes (with 44 unique G gene haplotypes), IVb endemic to the freshwater Great Lakes (11 haplotypes), and a newly-designated IVc in marine/estuarine North Atlantic waters (five haplotypes). Two separate substrains independently appeared in the Northwestern Pacific region (Asia) in 1996, with Ib originating from the west and IVa from the east. Our results depict an evolutionary history of relatively rapid population diversifications in star-like patterns, following a quasispecies model. This study provides a baseline for future tracking of VHSv spread and interpreting its evolutionary diversification pathways.

Microsatellite loci for dreissenid mussels (Mollusca: Bivalvia: Dreissenidae) and relatives: markers for assessing exotic and native populations.

We developed and tested 14 new polymorphic microsatellite loci for dreissenid mussels, including the two species that have invaded many freshwater habitats in Eurasia and North America, where they cause serious industrial fouling damage and ecological alterations. These new loci will aid our understanding of their genetic patterns in invasive populations as well as throughout their native Ponto-Caspian distributions. Eight new loci for the zebra mussel Dreissena polymorpha polymorpha and six for the quagga mussel D. rostriformis bugensis were compared with new results from six previously published loci to generate a robust molecular toolkit for dreissenid mussels and their relatives. Taxa tested include D. p. polymorpha, D. r. bugensis, D. r. grimmi, D. presbensis, the 'living fossil'Congeria kusceri, and the dark false mussel Mytilopsis leucophaeata (the latter also is invasive). Overall, most of the 24 zebra mussel (N = 583) and 13 quagga mussel (N = 269) population samples conformed to Hardy-Weinberg equilibrium expectations for the new loci following sequential Bonferroni correction. The 11 loci (eight new, three previously published) evaluated for D. p. polymorpha averaged 35.1 alleles and 0.72 mean observed heterozygosity per locus, and 25.3 and 0.75 for the nine loci (six new, three previously published) developed for D. r. bugensis. All but three of these loci successfully amplified the other species of Dreissena, and all but one also amplified Congeria and Mytilopsis. All species and populations tested were significantly divergent using the microsatellite data, with neighbour-joining trees reflecting their evolutionary relationships; our results reveal broad utility for resolving their biogeographic, evolutionary, population and ecological patterns.