An increasing threat to U.S. waterways is the establishment and spread of invasive and injurious fishes. A species may be designated by the U.S. Fish and Wildlife Service (USFWS) as federally “injurious” under the Lacey Act (18 U.S.C. 42) either if that species causes harm (injury) without establishing and spreading (not invasive), or if that species causes harm after establishment and spread (invasive). Species designated as injurious are prohibited from being imported, which is a highly effective way of preventing invasions by nonnative species. We developed a decision-support, risk-assessment system to aid USFWS’s prioritization of species for injuriousness. Our system is based on USFWS’s evaluation criteria of a species’ potential injuriousness and consists of a semi-quantitative, rapid-assessment procedure called the Ecological Risk Screening Summary (ERSS) and a quantitative probability network model called the Freshwater Fish Injurious Species Risk Assessment Model (FISRAM). ERSS provides information on a species’ history of invasiveness elsewhere in the world, and on its biology and ecology, potential or known effects of introduction, global and domestic distribution, and climate associations, and provides conclusions on potential risk of invasiveness. FISRAM calculates expected probability of injuriousness as a function of species potential establishment, spread, and harm, based on probable effects on native species and ecosystems, suitability of climate and habitat in introduced areas, ease of dispersal and transport, and harm to humans. FISRAM is used to assess risk probability when ERSS categorizes invasion risk as uncertain. We calibrated and updated the probability structure of FISRAM using a data set of 50 species with known invasiveness outcomes. We demonstrate the use of these two models for risk assessment and decision-support in identifying and documenting species for potential risk management actions, such as listing wildlife as injurious under the Lacey Act.

We review various applications of biocontrol for invasive fish and aquatic invertebrates. We adopt a broader definition of biocontrol that includes traditional methods like predation and physical removal (biocontrol by humans), and modern approaches like genetic engineering and use of microbes (including pathogens). While physical removal and predation (by native predators) are used relatively commonly, use of genetic technologies and microbes is in developmental stages. The two latter strategies are most advanced in case of the common carp (Cyprinus carpio), one of the world’s most invasive fish; virus release to control carp might soon occur in Australia. Drawing from empirical examples in North America, we emphasize that biocontrol strategies are most likely to be successful if they include multiple approaches that target specific behaviors or weaknesses in pests’ life histories. This is illustrated by reviewing case studies on the common carp and rusty crayfish (Orconectes rusticus) in Midwestern North America. In case of the common carp, basic research on movement patterns and recruitment bottlenecks identified a strategy where winter aggregations of adults were targeted for removal with nets, while native predators of carp eggs and larvae were instrumental in controlling carp’s reproductive success. In the case of the rusty crayfish, basic research on interactions between crayfish, habitat, and native predators identified a successful strategy of stocking selected native predators to control juvenile crayfish in conjunction with physical removal of adult crayfish using traps. We are also reviewing the case of the round goby (Neogobius melanostomus) in the Great Lakes. In this example, multiple pieces of evidence (diet, bioenergetics) illustrate how initially abundant pest was brought under control (in some areas) by several species of native predators in a large, natural ecosystem. Overall, examples of successful biocontrol of aquatic pests have been rare and have relied on physical removal and predation. We expect that new technologies (e.g. genetic technologies) will occur in the next decade but will have to clear regulatory and ethical concerns before they are applied. While developing more sophisticated control techniques, we advocate for more basic research on the life history of the pests to identify behavioral or developmental weaknesses that could be targeted with specific tools to increase chances of success while minimizing impacts on native ecosystems.

Ultraviolet radiation (UV) and total residual oxidant (TRO) treatments have been used to control microorganisms in drinking water, and are commonly used in ballast water management systems designed to reduce the transport and delivery of invasive organisms. Because treatments may not result in immediate cell death but may render organisms unable to reproduce, standard approaches to determine if a cell is living or dead (i.e., based on movement or cellular function) may not identify treated organisms. This scenario particularly applies to UV-treated cells that are rendered non-viable but still living. Thus, alternative approaches are needed to determine an organism’s viability (ability to reproduce). This study evaluated a cell proliferation assay measuring DNA concentrations in 96-well plates to determine proliferation of UV- and chlorine-treated organisms. Treatments included UV doses of 300 and 800 mWs cm^-2 or an applied ClO^- concentration of 10 mg L^-1. To determine optimal cell proliferation thresholds and detection limits, two phytoplankton species, Tetraselmis marina (UV and ClO^-) and Prorocentrum micans (ClO^-), were evaluated immediately following treatment and after 1-, 5-, and 14-day incubations in light or dark conditions. T. marina exhibited cell proliferation in all UV treatments, although no cell proliferation was detected for T. marina or P. micans in any ClO^- treatment. This type of assay allows for increased sample replication, smaller sample volumes, quicker preparation, and faster measurement than traditional growth assays. In conclusion, DNA staining assays may be useful to detect changes in cell proliferation over time in the context of ballast water testing.

Ballast water contains and transfers a variety of biological and non-biological materials between biomes. This study aimed to summarize published literature on the viruses found in ballast water, to evaluate ultrafiltration and plaque assay as methods for concentrating and enumerating viruses, and to assess the efficacy of UV irradiation in the inactivation of infectious viruses in ballast water. Previous studies reported high virus concentrations in ballast tanks, ranging from 10⁹ to 10¹⁴ viral particles/L. The ultrafiltration and plaque assay were efficient in concentrating and enumerating four types of phages: MS2, PhiX174, P22, and PP7. The UV dose (radiant fluence) necessary to achieve a 4-log reduction of the four phages ranged from 6.1 to 77.7 mJ/cm² with MS2 being the most UV-resistant phage. The results illustrated the applicability of ultrafiltration and plaque assay techniques for quantifying viruses in ballast waters and pointed to the need for additional test microorganisms for assessing ballast water quality.

Invading species are most easily eradicated or controlled if detected early and rapid action can be taken, but locating and eradicating small numbers of aquatic invaders is extremely difficult. Bighead carp (Hypophthalmichthys nobilis) and grass carp (Ctenopharyngodon idella) are Asian cyprinids that have been widely introduced and are considered undesirable and detrimental invasive species in many parts of their introduced range. The relatively novel “Judas fish” technique is used to locate aggregations of fish in the wild by strategically releasing individuals equipped with surgically implanted transmitters. However, releasing Judas fish that are capable of reproducing is problematic because it could add to the invasion problem by contributing to recruitment if not recaptured. We thus tested whether surgical ligation and division of the ductus deferens could effectively sterilize adult diploid bighead and grass carp. If effective, surgical sterilization would result in a superior Judas fish by maintaining the reproductive motivation derived from intact gonads while blocking the reproductive potential. This technique was initially successful, but ultimately most individuals were able to recanalize the surgically-severed ducts and produce milt, and were thus potentially able to reproduce during the following spawning season. Thus, ligation and division of the ductus deferens does not seem to be a viable procedure to sterilize bighead carp or grass carp for deployment as Judas fish where long term sterility of the fish is paramount in importance.

The proliferation of non-native fishes in Florida is a serious problem, and new species continue to be introduced to the state. Fishes in the Family Cichlidae have been especially successful colonizers of south Florida freshwater habitats. Herein we report a multi-agency effort to eradicate two non-native cichlid fishes in Miami, Florida (Bay Snook Petenia splendida and Blue Mbuna Labeotropheus fuelleborni). These fishes were removed before they were observed in the extensive, interconnected canal system through which they may have been able to expand throughout south Florida and access protected areas such as Everglades National Park. The study site, Pinecrest Gardens, is important because it contains remnant coastal cypress-strand habitat in an increasingly urbanized landscape that historically provided refuge to native amphidromous fishes and invertebrates. The project took considerable time (3.5 years), and we detail in this report how it evolved from a focus on isolating the non-native fishes and reducing their population sizes to an eradication. Gardens’ staff hydrologically isolated their ponds from nearby waterbodies by plugging a culvert with a solid gate. That provided the interagency team with more time to remove the potential threats. Compromises were made between fish management strategies and the Gardens’ priorities. Hurricane impacts helped shift priorities to more aggressive fish-management strategies. Cooperation among several federal and state agencies, as well as the Gardens, was key to the project’s success. We hope this effort may serve as a model for removing non-native species before they spread into ecosystems where eradication is not practical.

Loch Creran on the west coast of Scotland supports the most expansive reefs of the serpulid (Serpula vermicularis) in the world. It also supports flame shell (Limaria hians) and horse mussel (Modiolus modiolus) reefs, thereby ensuring this loch received designation as a European Special Area of Conservation in 2005. In 2015, environmental DNA of the invasive, non-native colonial tunicate Didemnum vexillum, which has a worldwide distribution, was detected in Loch Creran. Didemnum vexillum was confirmed as being present at an oyster farm in the sea loch, using the cytochrome oxidase I gene (COI), following rapid intertidal and dive surveys in early September 2016. The abundance and distribution range assessment carried out at the farm indicated an early-stage D. vexillum invasion. The follow up dive surveys and wider loch intertidal surveys carried out in 2017 and 2018 confirmed that the D. vexillum presence continues to be associated exclusively with the oyster farm. This is the first time that such a highly invasive species has been found within a protected area of global significance and it has significant biosecurity and policy implications regarding how to manage such invasive species.

With the increasing use of metagenetics for invasive species monitoring in aquatic habitats, fully assessing sample pre-processing is essential for appropriate data interpretation. This includes the impact of subsampling before DNA extraction, a common practice for processing zooplankton and other mixed samples where the volume of the sample exceeds the maximum allowed in a commercial DNA extraction kit. Our goal for this study was to assess the impact of these pre-processing methods on detecting invasive species through 1) examining operational taxonomic unit (OTU) richness assessments and 2) detecting low abundance OTUs in mixed samples. We used tagged amplicon high throughput sequencing (HTS) to amplify the mitochondrial cytochrome oxidase I (COI) gene from zooplankton samples collected from multiple bays. We then used a step-wise approach to assess the impacts of replication and homogenizing DNA extracts on the resulting OTU richness. We compared the results from 1) a single, intact subsample, 2) multiple, intact subsample replicates, 3) a single, homogenized subsample, or 4) multiple, homogenized subsample replicates. We expected OTU richness to be highest for multiple subsamples and for those homogenized, with subsequent impacts on rare OTU detection. Our results showed that homogenizing a sample prior to DNA extraction increased the number of OTUs recovered, particularly low abundance OTUs. Additionally, processing a greater number of extraction replicates increased the OTU richness of each sample, regardless of the extraction pre-processing conducted. However, with the increasing OTUs, more sequences per replicate were required to detect all OTUs. Thus, when detection of low abundance OTUs is a goal, particularly for early detection of invasive species, homogenizing multi-species samples is recommended due to the clear increase in rare OTUs detected.

Tools that bolster early detection of invasive dreissenid mussels are needed to prevent their spread across western North America. In this study, we assessed if environmental DNA (eDNA) can extend the seasonal window for dreissenid mussel early detection beyond that of plankton tows, which are limited to warmer seasons when mussel larvae are present. We focused eDNA sampling efforts at multiple sites in Tiber Reservoir (Montana) where dreissenid mussel abundance is hypothesized to be low. Samples were collected in June and October 2017, when water temperatures were cooler than thermal optima for dreissenid reproduction, and in July 2017 when water temperatures were warmer and conducive for reproduction. We detected dreissenid mussel DNA in June, July and October even though no dreissenid mussels were observed using non-molecular tools in 2017. A subset of positive and negative eDNA samples was analyzed by an independent lab and results were corroborated. We then estimated the effort needed for 95% probability detection of dreissenid DNA at each site within Tiber Reservoir and found that as many as 27, 14, and 34 samples needed to be collected in June, July and October, respectively. To further validate the utility of eDNA, we also present ancillary eDNA results from other waters in the Flathead Reservation (Montana) where dreissenid mussels have never been detected and from waters with established zebra mussel populations in the upper Mississippi River, which were sampled in the spring when water temperatures were cooler than thermal optima for dreissenid reproduction. All Flathead Reservation samples were negative for dreissenid mussel DNA, while all upper Mississippi River samples were positive. This study adds to a growing body of research that demonstrates eDNA is a highly sensitive tool for dreissenid mussel surveillance in newly invaded waters, including colder seasons when non-molecular tools are likely to be less effective or more challenging to employ.

As it produces large quantities of allergenic pollen that has a serious effect on human health Ambrosia artemisiifolia (common ragweed) is among the most noxious invasive plant species in Europe. It is most widespread in southern parts of Eastern and Central Europe and likely to spread into other regions. Thus its suppression is highly desirable. To identify species with a potential to suppress this invasive species, we carried out a growth chamber experiment to investigate the performance (height and weight) of A. artemisiifolia and 10 native Central-European species (six grasses and four herbaceous plants), growing in similar habitats and hence potential competitors of ragweed. Two factors were investigated in order to determine the processes that are likely to affect the future spread of A. artemisiifolia in Central Europe, i.e. increasing temperatures and winter treatment with salt of roads that serve as dispersal corridors. The plants were reared at five temperatures ranging from 10 to 26 °C and three levels of salinity. The height and weight of A. artemisiifolia increased with increasing temperature over the whole range of temperatures tested, with most native species growing best at 22 °C. This indicates that A. artemisiifolia will perform better in a warming climate and its spread will be facilitated by the poor performance of native species growing at suboptimal temperatures. As the largest differences in size between A. artemisiifolia and the native species were recorded at 10 and 14 °C we recommend that native seed mixtures are sown at ragweed invaded sites early in spring, or the previous autumn, to provide the competitors with a growth advantage at lower temperatures when A. artemisiifolia plants are still small and thus competitively weak. With respect to the other factor tested, A. artemisiifolia was suppressed similarly by high salinity as most of the native species tested, which indicates that the ragweed spread along roads is not primarily facilitated by its high tolerance of salinity. Different tolerances of native species to salinity indicates that this should be reflected in the selection of species for roadside seeding.

Invasive African clawed frog Xenopus laevis (Daudin, 1802) are considered a major threat to aquatic environments. Beginning in the early 1970s, invasive populations have now been established throughout much of central Chile. Between September and December 2015, we studied a population of X. laevis from a small pond in Viña del Mar, where we estimated the population size and evaluated the use of hand nets as a method of control. First, by means of a non-linear extrapolation model using the data from a single capture session of 200 min, a population size of 1,182 post-metamorphic frogs (range: 1,168–1,195 [quadratic error]) and a density of 13.7 frogs/m² (range: 13.6–13.9) of surface water were estimated. Second, based on 10 capture-and-removal sessions of 60-min and separated by approximately 2 weeks each, a total of 2,184 post-metamorphic frogs were removed, but the number of captured individuals did not significantly decrease over time. Additionally, using novel records of its occurrence, we updated the distribution range of X. laevis in Chile, estimated at 36,055 km², which is 1.7 to 3.5 times higher than previously assessed. Our results indicate that invasive X. laevis can reach extremely high densities, and removal of individuals in large numbers was not useful in reducing its abundance. This study shows that control and eradication of X. laevis from invaded areas proves extremely difficult, particularly when populations are well-established and/or expanding, as occurs in Chile. Our study reports the first record of X. laevis invading rivers of north Chile, 380 km north of previously reported. A management plan is urgently needed to prevent its further spread, and subsequent impacts on biodiversity. We propose our model and methodology as a tool to estimate and compare X. laevis densities, while suggesting the need to explore other control techniques in order to identify cost-effective strategies to contain the spread of X. laevis into new areas.

Wild boars (Sus scrofa Linnaeus, 1758), native to Eurasia and Africa, are one of the world’s most widely distributed invasive species. Their impacts on terrestrial environments have been well documented, however little is known about their effects on aquatic environments. We used standardized physical habitat surveys to compare the use of streams by invasive wild pig and native white-lipped peccaries (Tayassu pecari Link, 1795), and their effects on the physical structure from four first-order streams in the Brazilian Atlantic Forest. Two of these streams were used solely by wild pigs, while two were solely used by peccaries. Each stream was subdivided by cross-sectional transects into continuous sections, each 10-m in length, where we measured the intensity of use by each species and different variables related to the stream physical habitat. Although both species used the streams, wild pigs altered physical and environmental parameters more, and with greater intensity, than the native peccaries. Wild pigs decreased the stream bank angle and the riparian ground cover, leading to local erosion, increased fine sedimentation and wetted width, and declined stream depth. We recommend studies to evaluate the biological consequences of the alterations caused by introduced wild pigs, which should be conducted along with plans of population control in environments where the wild pig is considered an invasive species.