Integrating invasive species risk assessment into environmental DNA metabarcoding reference libraries.

Environmental DNA (eDNA) metabarcoding has shown promise as a tool for estimating biodiversity and early detection of invasive species. In aquatic systems, advantages of this method include the ability to concurrently monitor biodiversity and detect incipient invasions simply through the collection and analysis of water samples. However, depending on the molecular markers chosen for a given study, reference libraries containing target sequences from present species may limit the usefulness of eDNA metabarcoding. To explore the extent of this issue and how it may be resolved to aid biodiversity and invasive species early detection goals, we focus on fishes in the well-studied Laurentian Great Lakes region. First, we provide a synthesis of species currently known from the region and of non-indigenous species identified as threats by international, national, regional, and introduction pathway-specific fish risk assessments. With these species lists, we then evaluate 23 primer pairs commonly used in fish eDNA metabarcoding with available databases of sequence coverage and species specificity. Finally, we identify established and potentially invasive non-indigenous fish that should be prioritized for genetic sequencing to ensure robust eDNA metabarcoding for the region. Our results should increase confidence in using eDNA metabarcoding for fisheries conservation and management in the Great Lakes region and help prioritize reference sequencing efforts. The ultimate utility of eDNA metabarcoding approaches will come when conservation management of existing fish communities is integrated with early detection efforts for invasive species surveillance to assess total fish biodiversity.

Phylogenomics of the longitarsal Colossendeidae: The evolutionary history of an Antarctic sea spider radiation.

Sea spiders (Pycnogonida) constitute a group of marine benthic arthropods that has a particularly high species diversity in the Southern Ocean. The "longitarsal" group of the sea spider family Colossendeidae is especially abundant in this region. However, this group also includes some representatives from other oceans, which raises the question where the group originates from. Therefore, we here investigated the phylogeny of the group with a hybrid enrichment approach that yielded a dataset of 1607 genes and over one million base pairs. We obtained a well-resolved phylogeny of the group, which is mostly consistent with morphological data. The data support an Antarctic origin of the longitarsal Colossendeidae and multiple dispersal events to other regions, which occurred at different timescales. This scenario is consistent with evidence found in other groups of marine invertebrates and highlights the role of the Southern Ocean as a source for non-Antarctic biota, especially of the deep sea. Our results suggest an initially slow rate of diversification followed by a more rapid radiation possibly correlated with the mid-Miocene cooling of Antarctica, similar to what is found in other taxa.

Conservation of mitochondrial genome arrangements in brittle stars (Echinodermata, Ophiuroidea).

Brittle stars are conspicuous members of benthic ecosystems, fill many ecological niches and are the most speciose of all classes of echinoderms. With high levels of biodiversity, elucidating the evolutionary history of this group is important. Understanding of higher-level relationships within Ophiuroidea has been aided by multilocus nuclear data and DNA barcoding. However, the degree of consistency between mitochondrial and nuclear data within ophiuroids remains unclear and deserves further assessment. In this study, 17 mitochondrial genomes spanning the taxonomic breadth of Ophiuroidea were utilized to explore evolutionary relationships through maximum likelihood analyses, Bayesian inference and comparative assessment of gene order. Our phylogenetic analyses, based on both nucleotide and amino acid residues, support recent findings based on multilocus nuclear data and morphology, in that the brittle star clades Ophintegrida and Euryophiurida were recovered as monophyletic with the latter comprising Euyalida, Ophiuridae and Ophiopyrgidae. Only three different arrangements of the 13 protein coding and 2 ribosomal RNA genes were observed. As expected, tRNA genes were more likely to have undergone rearrangement but the order of all 37 genes was found to be conserved in all sampled Euryalida and Ophiuridae. Both Euryalida and the clade comprised of Ophiuridae and Ophiopyrgidae, each had their own conserved rearrangement of protein coding genes and ribosomal genes, after divergence from their last common ancestor. Euryalida has a rearrangement of the two ribosomal RNA genes, rrnS and rrnL, in contrast to Ophiuridae and Ophiopyrgidae, which had an inversion of the genes nad1, nad2, and cob relative to Ophintegrida. Further, our data support the gene order found in all sampled Euryalida as the most likely ancestral order for all Ophiuroidea.

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.

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

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

The use of environmental DNA in invasive species surveillance of the Great Lakes commercial bait trade.

Over 180 non-native species have been introduced in the Laurentian Great Lakes region, many posing threats to native species and ecosystem functioning. One potential pathway for introductions is the commercial bait trade; unknowing or unconcerned anglers commonly release unused bait into aquatic systems. Previous surveillance efforts of this pathway relied on visual inspection of bait stocks in retail shops, which can be time and cost prohibitive and requires a trained individual that can rapidly and accurately identify cryptic species. Environmental DNA (eDNA) surveillance, a molecular tool that has been used for surveillance in aquatic environments, can be used to efficiently detect species at low abundances. We collected and analyzed 576 eDNA samples from 525 retail bait shops throughout the Laurentian Great Lake states. We used eDNA techniques to screen samples for multiple aquatic invasive species (AIS) that could be transported in the bait trade, including bighead (Hypophthalmichthys nobilis) and silver carp (H. molitrix), round goby (Neogobius melanostomus), tubenose goby (Proterorhinus marmoratus), Eurasian rudd (Scardinius erythrophthalmus), and goldfish (Carassius auratus). Twenty-seven samples were positive for at least one target species (4.7% of samples), and all target species were found at least once, except bighead carp. Despite current regulations, the bait trade remains a potential pathway for invasive species introductions in the Great Lakes region. Alterations to existing management strategies regarding the collection, transportation, and use of live bait are warranted, including new and updated regulations, to prevent future introductions of invasive species in the Great Lakes via the bait trade.

Quantifying environmental DNA signals for aquatic invasive species across multiple detection platforms.

The use of molecular surveillance techniques has become popular among aquatic researchers and managers due to the improved sensitivity and efficiency compared to traditional sampling methods. Rapid expansion in the use of environmental DNA (eDNA), paired with the advancement of molecular technologies, has resulted in new detection platforms and techniques. In this study we present a comparison of three eDNA surveillance platforms: traditional polymerase chain reaction (PCR), quantitative PCR (qPCR), and digital droplet PCR (ddPCR) in which water samples were collected over a 24 h time period from mesocosm experiments containing a population gradient of invasive species densities. All platforms reliably detected the presence of DNA, even at low target organism densities within the first hour. The two quantitative platforms (qPCR and ddPCR) produced similar estimates of DNA concentrations. The analyses completed with ddPCR was faster from sample collection through analyses and cost approximately half the expenditure of qPCR. Although a new platform for eDNA surveillance of aquatic species, ddPCR was consistent with more commonly used qPCR and a cost-effective means of estimating DNA concentrations. Use of ddPCR by researchers and managers should be considered in future eDNA surveillance applications.

In response to bacteria, neutrophils release extracellular vesicles capable of initiating thrombin generation through DNA-dependent and independent pathways.

Neutrophils release extracellular vesicles (EVs) and some subsets of neutrophil-derived EVs are procoagulant. In response to S. aureus, neutrophils produce EVs that associate electrostatically with neutrophil extracellular traps (NETs). DNA in NETs is procoagulant, but whether neutrophil EVs produced during bacterial challenge have similar activity is unknown. Given that EV activity is agonist- and cell-type dependent and coagulation contributes to sepsis, we hypothesized that sepsis-causing bacteria increase production of neutrophil-derived EVs, as well as EV-associated DNA, and intact EVs and DNA cause coagulation. We recovered EVs from neutrophils challenged with S. aureus (SA), S. epidermidis (SE), E. coli (EC), and P. aeruginosa (PA), and measured associated DNA and procoagulant activity. EVs from SA-challenged neutrophils (SA-EVs), which were previously characterized, displayed dose-dependent procoagulant activity as measured by thrombin generation (TG) in platelet-poor plasma. EV lysis and DNase treatment reduced TG by 90% and 37%, respectively. SE, EC, and PA also increased EV production and EV-associated extracellular DNA, and these EVs were also procoagulant. Compared to spontaneously released EVs, which demonstrated some ability to amplify Factor XII-dependent coagulation in the presence of an activator, only EVs produced in response to bacteria could initiate the pathway. SA-EVs and SE-EVs had more surface-associated DNA than EC-EVs and PA-EVs, and SA-EVs and SE-EVs contributed to initiation and amplification of TG in a DNA-dependent manner. However, DNA on EC- or PA-EVs played no role, suggesting that neutrophils release procoagulant EVs which can activate the coagulation cascade through both DNA-dependent and independent mechanisms.

Austropallenehalanychi sp. nov., a new species of sea spider (Pycnogonida, Callipallenidae) from the Ross Sea, Antarctica.

Here we present Austropallenehalanychisp. nov., a new species of pycnogonid within the family Callipallenidae (Pycnogonida), collected from the Ross Sea, Antarctica. While retaining key morphological features known for the genus Austropallene Hodgson, 1915a, the new species is distinguished from congeners by its much larger size, along with the combined absence of a denticle on the inner surface of the fixed finger of the chelifore claw along with the presence of small conical outgrowths where the fixed finger of the chelifore claw meets the movable finger on both the dorsal and ventral sides, and also the ability to fully close the chelifore claw. Additionally, the complete mitochondrial genome of A.halanychi is consistent with other members of the genus Austropallene in terms of gene order and directionality. A phylogenetic tree consisting of mitochondrial protein-coding gene data places A.halanychi as sister to Austropallenecornigera (Möbius, 1902). Additionally, a phylogenetic tree constructed using partial COI data from other callipallenids placed the new species in a clade containing the genus Austropallene. The combination of molecular data in addition to key morphological differences from similar species in the genus leaves no doubt that the new taxon is a new Antarctic species of Austropallene.

Conservation in a cup of water: estimating biodiversity and population abundance from environmental DNA.

Three mantras often guide species and ecosystem management: (i) for preventing invasions by harmful species, 'early detection and rapid response'; (ii) for conserving imperilled native species, 'protection of biodiversity hotspots'; and (iii) for assessing biosecurity risk, 'an ounce of prevention equals a pound of cure.' However, these and other management goals are elusive when traditional sampling tools (e.g. netting, traps, electrofishing, visual surveys) have poor detection limits, are too slow or are not feasible. One visionary solution is to use an organism's DNA in the environment (eDNA), rather than the organism itself, as the target of detection. In this issue of Molecular Ecology, Thomsen et al. (2012) provide new evidence demonstrating the feasibility of this approach, showing that eDNA is an accurate indicator of the presence of an impressively diverse set of six aquatic or amphibious taxa including invertebrates, amphibians, a fish and a mammal in a wide range of freshwater habitats. They are also the first to demonstrate that the abundance of eDNA, as measured by qPCR, correlates positively with population abundance estimated with traditional tools. Finally, Thomsen et al. (2012) demonstrate that next-generation sequencing of eDNA can quantify species richness. Overall, Thomsen et al. (2012) provide a revolutionary roadmap for using eDNA for detection of species, estimates of relative abundance and quantification of biodiversity.

Extracellular vesicles from A23187-treated neutrophils cause cGAS-STING-dependent IL-6 production by macrophages.

In response to several types of bacteria, as well as pharmacological agents, neutrophils produce extracellular vesicles (EVs) and release DNA in the form of neutrophil extracellular traps (NETs). However, it is unknown whether these two neutrophil products cooperate to modulate inflammation. Consistent with vital NETosis, neutrophils challenged with S. aureus, as well as those treated with A23187, released significantly more DNA relative to untreated or fMLF-treated neutrophils, with no lysis occurring for any condition. To test the hypothesis that EVs generated during NETosis caused macrophage inflammation, we isolated and characterized EVs from A23187-treated neutrophils (A23187-EVs). A23187-EVs associated with neutrophil granule proteins, histone H3, transcription factor A, mitochondrial (TFAM), and nuclear and mitochondrial DNA (mtDNA). We showed that DNA from A23187-EVs, when transfected into macrophages, led to production of IL-6 and IFN-α2, and this response was blunted by pre-treatment with the STING inhibitor H151. Next, we confirmed that A23187-EVs were engulfed by macrophages, and showed that they induced cGAS-STING-dependent IL-6 production. In contrast, neither EVs from untreated or fMLF-treated cells exhibited pro-inflammatory activity. Although detergent-mediated lysis of A23187-EVs diminished IL-6 production, removal of surface-associated DNA with DNase I treatment had no effect, and A23187-EVs did not induce IFN-α2 production. Given these unexpected results, we investigated whether macrophage mtDNA activated the cGAS-STING signaling axis. Consistent with mitochondrial outer membrane permeabilization (MOMP), a defined mechanism of mtDNA release, we observed macrophage mitochondrial membrane depolarization, a decrease in cytosolic Bax, and a decrease in mitochondrial cytochrome c, suggesting that macrophage mtDNA may initiate this EV-dependent signaling cascade. All together, these data demonstrate that A23187-EVs behave differently than transfected NET- or EV-DNA, and that neutrophil-derived EVs could be used as a model to study NF-κB-dependent STING activation.

Phylogeography and reproductive variation of the poecilogonous polychaete Boccardia proboscidea (Annelida: Spionidae) along the West Coast of North America.

The ability to produce more than one kind of offspring, or poecilogony, is a striking example of reproductive variability. Traditionally, larval nutrition has been classified as a dichotomy: if offspring obtain nutrition from their mothers (lecithotrophy), there is lower fecundity and greater chance of offspring survival than when they get their nutrition from plankton (planktotrophy). The polychaete Boccardia proboscidea (Spionidae) produces both types of embryos using three different reproductive strategies. In this study, we examined the roles of genetic history and phenotypic plasticity on explaining natural variation in B. proboscidea along the Pacific coast of the United States using two genetic mitochondrial markers, 16S rDNA and Cyt b, and common garden experiments. These data show a single North American West Coast network that is structured, geographically, by the well-documented biogeographic break near Point Conception, California. The southern group within this network covers a smaller range, but has larger haplotype diversity, than the northern group. Some individuals differing in reproductive type had the same haplotype, indicating independence of these features; however, differences between laboratory and field data suggest additional geographic variation within one of the reproductive types. Females from higher latitudes provide offspring with larger supplies of extra embryonic nutrition than females from southern latitudes. Results herein suggest that both genetic history and developmental plasticity are playing a role in the maintenance of this reproductive polymorphism.

From molecules to management: adopting DNA-based methods for monitoring biological invasions in aquatic environments.

Recent technological advances have driven rapid development of DNA-based methods designed to facilitate detection and monitoring of invasive species in aquatic environments. These tools promise to improve on traditional monitoring approaches by enhancing detection sensitivity, reducing analytical turnaround times and monitoring costs, and increasing specificity of target identifications. However, despite the promise of DNA-based monitoring methods, the adoption of these tools in decision-making frameworks remains challenging. Here, rather than explore technical aspects of method development, we examine impediments to effective translation of those methods into management contexts. In addition to surveying current use of DNA-based tools for aquatic invasive species monitoring, we explore potential sources of uncertainty associated with molecular technologies and possibilities for limiting that uncertainty and effectively communicating its implications for decision-making. We pay particular attention to the recent adoption of DNA-based methods for detection of invasive Asian carp species in the United States Great Lakes region, as this example illustrates many of the challenges associated with applying molecular tools to achieve desired management outcomes. Our goal is to provide a useful assessment of the obstacles associated with integrating DNA-based methods into aquatic invasive species management, and to offer recommendations for future efforts aimed at overcoming those obstacles.

Metagenomics of Antarctic Marine Sediment Reveals Potential for Diverse Chemolithoautotrophy.

The microbial biogeochemical processes occurring in marine sediment in Antarctica remain underexplored due to limited access. Further, these polar habitats are unique, as they are being exposed to significant changes in their climate. To explore how microbes drive biogeochemistry in these sediments, we performed a shotgun metagenomic survey of marine surficial sediment (0 to 3 cm of the seafloor) collected from 13 locations in western Antarctica and assembled 16 high-quality metagenome assembled genomes for focused interrogation of the lifestyles of some abundant lineages. We observe an abundance of genes from pathways for the utilization of reduced carbon, sulfur, and nitrogen sources. Although organotrophy is pervasive, nitrification and sulfide oxidation are the dominant lithotrophic pathways and likely fuel carbon fixation via the reverse tricarboxylic acid and Calvin cycles. Oxygen-dependent terminal oxidases are common, and genes for reduction of oxidized nitrogen are sporadically present in our samples. Our results suggest that the underlying benthic communities are well primed for the utilization of settling organic matter, which is consistent with findings from highly productive surface water. Despite the genetic potential for nitrate reduction, the net catabolic pathway in our samples remains aerobic respiration, likely coupled to the oxidation of sulfur and nitrogen imported from the highly productive Antarctic water column above. IMPORTANCE The impacts of climate change in polar regions, like Antarctica, have the potential to alter numerous ecosystems and biogeochemical cycles. Increasing temperature and freshwater runoff from melting ice can have profound impacts on the cycling of organic and inorganic nutrients between the pelagic and benthic ecosystems. Within the benthos, sediment microbial communities play a critical role in carbon mineralization and the cycles of essential nutrients like nitrogen and sulfur. Metagenomic data collected from sediment samples from the continental shelf of western Antarctica help to examine this unique system and document the metagenomic potential for lithotrophic metabolisms and the cycles of both nitrogen and sulfur, which support not only benthic microbes but also life in the pelagic zone.

Use of environmental DNA (eDNA) in streams to detect feral swine (Sus scrofa).

Invasive feral swine can damage ecosystems, disrupt plant and animal populations, and transmit diseases. Monitoring of feral swine populations requires expensive and labor-intensive techniques such as aerial surveys, field surveys for sign, trail cameras, and verifying landowner reports. Environmental DNA (eDNA) provides an alternative method for locating feral swine. To aid in detection of this harmful invasive species, a novel assay was developed incorporating molecular methods. From August 2017 to April 2018, water samples and stream data were collected along 400 m transects in two different stream types where swine DNA was artificially introduced to investigate potential factors affecting detection. A generalized linear model (family binomial) was used to characterize environmental conditions affecting swine DNA detection; detection was the dependent variable and stream measurements included stream type, distance downstream, water temperature, velocity, turbidity, discharge, and pH as independent variables. Parameters from the generalized linear model were deemed significant if 95% confidence intervals did not overlap 0. Detection probability for swine DNA negatively related to water temperature (ß =  - 0.21, 95% CI [-0.35 to -0.09]), with the highest detection probability (0.80) at 0 °C and lowest detection probability (0.05) at 17.9 °C water temperature. Results indicate that sampling for swine eDNA in free-flowing stream systems should occur at lower water temperatures to maximize detection probability. This study provides a foundation for further development of field and sampling techniques for utilizing eDNA as a viable alternative to monitoring a terrestrial invasive species in northern regions of the United States.

Effect of extracellular vesicles from S. aureus-challenged human neutrophils on macrophages.

Staphylococcus aureus enhances neutrophil extracellular vesicle (EV) production. To investigate whether S. aureus viability influences EV biogenesis, EVs were isolated from human neutrophils incubated with viable bacteria (bEVs) or heat-killed bacteria (heat-killed EVs). Protein analysis, nanoparticle tracking and transmission electron microscopy showed comparable EV production between subsets, and both viable and nonviable bacteria were also detected in respective EV subsets. As anticipated, S. aureus, as well as bEVs with viable bacteria, were proinflammatory, and killing bacteria with gentamicin reduced cytokine production to baseline levels. Although heat-killed bacteria induced macrophage IL-6 production, heat-killed EVs did not. Additionally, we found that human and bacterial DNA associated with bEVs, but not heat-killed EVs, and that the DNA association could be partially decreased by disrupting electrostatic interactions. We investigated the potential for DNA isolated from EVs (EV-DNA) or EVs to cause inflammation. Although liposomal encapsulation of EV-DNA increased IL-6 production from baseline by 7.5-fold, treatment of bEVs with DNase I had no effect on IL-6 and IL-1ß production, suggesting that the DNA did not contribute to the inflammatory response. Filtered EVs, which lacked DNA and associated bacteria, exhibited less proinflammatory activity relative to bEVs, and enhanced macrophage expression of CD86 and HLA-DR. Ultimately, we show that bEVs isolated by differential centrifugation co-purify with bacteria and DNA, and studying their concerted activity and relative contribution to immune response is important to the study of host-pathogen interactions.

Open-ocean barriers to dispersal: a test case with the Antarctic Polar Front and the ribbon worm Parborlasia corrugatus (Nemertea: Lineidae).

Open-ocean environments provide few obvious barriers to the dispersal of marine organisms. Major currents and/or environmental gradients potentially impede gene flow. One system hypothesized to form an open-ocean dispersal barrier is the Antarctic Polar Front, an area characterized by marked temperature change, deep water, and the high-flow Antarctic Circumpolar current. Despite these potential isolating factors, several invertebrate species occur in both regions, including the broadcast-spawning nemertean worm Parborlasia corrugatus. To empirically test for the presence of an open-ocean dispersal barrier, we sampled P. corrugatus and other nemerteans from southern South America, Antarctica, and the sub-Antarctic islands. Diversity was assessed by analyzing mitochondrial 16S rRNA and cytochrome c oxidase subunit I sequence data with Bayesian inference and tcs haplotype network analysis. Appropriate neutrality tests were also employed. Although our results indicate a single well-mixed lineage in Antarctica and the sub-Antarctic, no evidence for recent gene flow was detected between this population and South American P. corrugatus. Thus, even though P. corrugatus can disperse over large geographical distances, physical oceanographic barriers (i.e. Antarctic Polar Front and Antarctic Circumpolar Current) between continents have likely restricted dispersal over evolutionary time. Genetic distances and haplotype network analysis between South American and Antarctic/sub-Antarctic P. corrugatus suggest that these two populations are possibly two cryptic species.

Genomic analyses of Northern snakehead (Channa argus) populations in North America.

BACKGROUND: The introduction of northern snakehead (Channa argus; Anabantiformes: Channidae) and their subsequent expansion is one of many problematic biological invasions in the United States. This harmful aquatic invasive species has become established in various parts of the eastern United States, including the Potomac River basin, and has recently become established in the Mississippi River basin in Arkansas. Effective management of C. argus and prevention of its further spread depends upon knowledge of current population structure in the United States. METHODS: Novel methods for invasive species using whole genomic scans provide unprecedented levels of data, which are able to investigate fine scale differences between and within populations of organisms. In this study, we utilize 2b-RAD genomic sequencing to recover 1,007 single-nucleotide polymorphism (SNP) loci from genomic DNA extracted from 165 C. argus individuals: 147 individuals sampled along the East Coast of the United States and 18 individuals sampled throughout Arkansas. RESULTS: Analysis of those SNP loci help to resolve existing population structure and recover five genetically distinct populations of C. argus in the United States. Additionally, information from the SNP loci enable us to begin to calculate the long-term effective population size ranges of this harmful aquatic invasive species. We estimate long-term Ne to be 1,840,000-18,400,000 for the Upper Hudson River basin, 4,537,500-45,375,000 for the Lower Hudson River basin, 3,422,500-34,225,000 for the Potomac River basin, 2,715,000-7,150,000 for Philadelphia, and 2,580,000-25,800,000 for Arkansas populations. DISCUSSION AND CONCLUSIONS: This work provides evidence for the presence of more genetic populations than previously estimated and estimates population size, showing the invasive potential of C. argus in the United States. The valuable information gained from this study will allow effective management of the existing populations to avoid expansion and possibly enable future eradication efforts.

Investigating diversity of pathogenic microbes in commercial bait trade water.

The recreational bait trade is a potential pathway for pathogen introduction and spread when anglers dump bait shop sourced water into aquatic systems. Despite this possibility, and previous recognition of the importance of the bait trade in the spread of aquatic invasive species (AIS), to date there has been no region wide survey documenting pathogens in retail bait shops. In this study, we analyzed 96 environmental DNA samples from retail bait shops around the Great Lakes region to identify pathogens, targeting the V4 hypervariable region of the 16S rRNA gene. Additionally, we used samples from one site in Lake Michigan as a comparison to pathogen diversity and abundance in natural aquatic systems. Our results identified nine different groups of pathogens in the bait shop samples, including those that pose risks to both humans and fish species. Compared to wild sourced samples, the bait shops had higher relative abundance and greater taxonomic diversity. These findings suggest that the bait trade represents a potentially important pathway that could introduce and spread pathogens throughout the Great Lakes region. Improving pathogen screening and angler outreach should be used in combination to aid in preventing the future spread of high risk pathogens.