eDNA-based detection reveals invasion risks of a biofouling bivalve in the world's largest water diversion project.

Environmental DNA (eDNA) has increasingly been used to detect rare species (e.g., newly introduced nonindigenous species) in both terrestrial and aquatic ecosystems, often with distinct advantages over traditional methods. However, whether water eDNA signals can be used to inform invasion risks remains debatable owing to inherent uncertainties associated with the methods used and the varying conditions among study systems. Here, we sampled eDNA from canals of the central route of the South-to-North Water Diversion Project (hereafter SNWDP) in China to investigate eDNA distribution and efficacy to inform invasion risks in a unique lotic system. We first conducted a total of 16 monthly surveys in this system (two sites in the source reservoir and four sites in the main canal) to test if eDNA could be applied to detect an invasive, biofouling bivalve, the golden mussel Limnoperna fortunei. Second, we initiated a one-time survey in a sub-canal of the SNWDP using refined sampling (12 sites in ~22 km canal) and considered a few environmental predictors. We found that detection of target eDNA in the main canal was achieved up to 1100 km from the putative source population but was restricted to the warmer months (May-November). Detection probability exhibited a significant positive relationship with average daily minimum air temperature and with water temperature, consistent with the expected spawning season. eDNA concentration in the main canal generally fluctuated across months and sites and was generally higher in warmer months. Golden mussel eDNA concentration in the sub-canal decreased significantly with distance from the source and with increasing water temperature and became almost undetectable at ~22 km distance. Given the enormity of the SNWDP, golden mussels may eventually expand their distribution in the main canal, with established "bridgehead" populations facilitating further spread. Our findings suggest an elevated invasion risk of golden mussels in the SNWDP in warm months, highlighting the critical period for spread and, possibly, management.

An overlooked source of false positives in eDNA-based biodiversity assessment and management.

Biodiversity conservation and management in urban aquatic ecosystems is crucial to human welfare, and environmental DNA (eDNA)-based methods have become popular in biodiversity assessment. Here we report a highly overlooked source of significant false positives for eDNA-based biodiversity assessment in urban aquatic ecosystems supplied with treated wastewater - eDNA pollution originating from treated wastewater represents a noteworthy source of false positives. To investigate whether eDNA pollution is specific to a certain treatment or prevalent across methods employed by wastewater treatment plants, we conducted tests on effluent treated using three different secondary processes, both before and after upgrades to tertiary treatment. We metabarcoded eDNA collected from effluent immediately after full treatment and detected diverse native and non-native, commercial and ornamental fishes (48 taxa) across all treatment processes before and after upgrades. Thus, eDNA pollution occurred irrespective of the treatment processes applied. Release of eDNA pollution into natural aquatic ecosystems could translate into false positives for eDNA-based analysis. We discuss and propose technical solutions to minimize these false positives in environmental nucleic acid-based biodiversity assessments and conservation programs.

Phytosphingosine inhibits cell proliferation by damaging DNA in human cell lines.

Harmful cyanobacterial blooms have caused numerous biosecurity incidents owing to the production of hazardous secondary metabolites such as microcystin. Additionally, cyanobacteria also release many other components that have not been explored. We identified compounds of a toxic mixture exudated from a dominant, blooming species, Microcystis aeruginosa, and found that phytosphingosine (PHS) was one of the bioactive components. Since PHS exhibited toxicity and is deemed a hazardous substance by the European Chemicals Agency, we hypothesized that PHS is a potentially toxic compound in M. aeruginosa exudates. However, the mechanisms of PHS ecotoxicity remain unclear. We assessed the cytotoxicity of PHS using an in vitro cell model in eight human cell lines and observed that the nasopharyngeal carcinoma cell line CNE2 was the most sensitive. We exposed CNE2 cells to 0-25 µmol/L PHS for 24 hr to explore its toxicity and mechanism. PHS exposure resulted in abnormal nuclear morphology, micronuclei, and DNA damage. Moreover, PHS significantly inhibited cell proliferation and arrested cell cycle at S phase. The results of Western blot suggested that PHS increased the expression of DNA damage-related proteins (ATM, p-P53 and P21) and decreased the expression of S phase-related proteins (CDK2, CyclinA2 and CyclinE1), indicating the toxicological mechanism of PHS on CNE2 cells. These data provide evidence that PHS has genetic toxicity and inhibits cell proliferation by damaging DNA. Our study provides evidence that PHS inhibits cell proliferation by damaging DNA. While additional work is required, we propose that PHS been considered as a potentially toxic component in MaE in addition to other well-characterized secondary compounds.

Complementary genomic and epigenomic adaptation to environmental heterogeneity.

While adaptation is commonly thought to result from selection on DNA sequence-based variation, recent studies have highlighted an analogous epigenetic component as well. However, the relative roles of these mechanisms in facilitating population persistence under environmental heterogeneity remain unclear. To address the underlying genetic and epigenetic mechanisms and their relationship during environmental adaptation, we screened the genomes and epigenomes of nine global populations of a predominately sessile marine invasive tunicate, Botryllus schlosseri. We detected clear population differentiation at the genetic and epigenetic levels. Patterns of genetic and epigenetic structure were significantly influenced by local environmental variables. Among these variables, minimum annual sea surface temperature was identified as the top explanatory variable for both genetic and epigenetic variation. However, patterns of population structure driven by genetic and epigenetic variation were somewhat distinct, suggesting possible autonomy of epigenetic variation. We found both shared and specific genes and biological pathways among genetic and epigenetic loci associated with environmental factors, consistent with complementary and independent contributions of genetic and epigenetic variation to environmental adaptation in this system. Collectively, these mechanisms may facilitate population persistence under environmental change and sustain successful invasions across novel environments.

Abnormal neurobehavior in fish early life stages after exposure to cyanobacterial exudates.

Cyanobacterial harmful algal blooms (cHABs) pose a risk to exposed aquatic and terrestrial species. Numerous studies have addressed effects of single toxins while much less attention has been devoted to mixtures of cHAB metabolites that are continually released by living cyanobacteria. Neuro-impairment associated with cHABs has been reported in fish, though the mechanism remains unclear. Here we exposed embryos of Sinocyclocheilus grahami, an endangered fish, to Microcystis aeruginosa exudates (MaE) to evaluate neurotoxicity and the toxicity mechanism(s). We found that MaE affected embryonic development by increasing malformation and mortality rates and decreasing the fertilization rate. MaE also inhibited fish neurobehavior including touch response, social frequency, swimming distance, and aggravated light-stimulation response. Neurobehavior suppression resulted from a decrease in excitatory neurotransmitters acetylcholine and dopamine, even though receptors increased. MaE also affected gene and protein expression of neurotransmitters, synthetic and/or degrading enzymes, and receptors. Our findings shed light on specific mechanisms by which MaE induces neurotoxicity in early life stages in fish and contributes to improvement of the conservation strategy for this species.

Drivers of future alien species impacts: An expert-based assessment.

Understanding the likely future impacts of biological invasions is crucial yet highly challenging given the multiple relevant environmental, socio-economic and societal contexts and drivers. In the absence of quantitative models, methods based on expert knowledge are the best option for assessing future invasion trajectories. Here, we present an expert assessment of the drivers of potential alien species impacts under contrasting scenarios and socioecological contexts through the mid-21st century. Based on responses from 36 experts in biological invasions, moderate (20%-30%) increases in invasions, compared to the current conditions, are expected to cause major impacts on biodiversity in most socioecological contexts. Three main drivers of biological invasions-transport, climate change and socio-economic change-were predicted to significantly affect future impacts of alien species on biodiversity even under a best-case scenario. Other drivers (e.g. human demography and migration in tropical and subtropical regions) were also of high importance in specific global contexts (e.g. for individual taxonomic groups or biomes). We show that some best-case scenarios can substantially reduce potential future impacts of biological invasions. However, rapid and comprehensive actions are necessary to use this potential and achieve the goals of the Post-2020 Framework of the Convention on Biological Diversity.

Non-native species spread in a complex network: the interaction of global transport and local population dynamics determines invasion success.

The number of released individuals, which is a component of propagule pressure, is considered to be a major driver for the establishment success of non-native species. However, propagule pressure is often assumed to result from single or few release events, which does not necessarily apply to the frequent releases of invertebrates or other taxa through global transport. For instance, the high intensity of global shipping may result in frequent releases of large numbers of individuals, and the complexity of shipping dynamics impedes predictions of invasion dynamics. Here, we present a mathematical model for the spread of planktonic organisms by global shipping, using the history of movements by 33 566 ships among 1477 ports to simulate population dynamics for the comb jelly Mnemiopsis leidyi as a case study. The degree of propagule pressure at one site resulted from the coincident arrival of individuals from other sites with native or non-native populations. Key to sequential spread in European waters was a readily available source of propagules and a suitable recipient environment. These propagules were derived from previously introduced 'bridgehead' populations supplemented with those from native sources. Invasion success is therefore determined by the complex interaction of global shipping and local population dynamics. The general findings probably hold true for the spread of species in other complex systems, such as insects or plant seeds exchanged via commercial trade or transport.

Human-mediated and natural dispersal of an invasive fish in the eastern Great Lakes.

The globally invasive Round Goby (Neogobius melanostomus) was introduced to the Great Lakes around 1990, spreading widely and becoming the dominant benthic fish in many areas. The speed and scope of this invasion is remarkable and calls into question conventional secondary spread models and scenarios. We utilized nine microsatellites to identify large-scale genetic structure in Round Goby populations in the eastern Great Lakes, and assessed the role of colonization vs. secondary transport and dispersal in developing this structure. We identified three clusters, corresponding with Lake Huron, eastern Lake Erie, and western Lake Erie plus eastern Lake Ontario, along with three highly divergent populations. Bottleneck analysis identified founder effects in two divergent populations. Regression analyses of isolation by distance and allelic richness vs. distance from the initial invasion site were consistent with limited migration. However, some populations in eastern Lake Erie and Lake Ontario showed anomalously low genetic distance from the original site of colonization, consistent with secondary transport of large numbers of individuals via ballast water. We conclude that genetic structure of Round Goby in the Great Lakes principally resulted from long-distance secondary transport via ballast water with additional movement of individual via bait buckets and natural dispersal. The success of Round Gobies represents an interesting model for colonization characterization; however, those same attributes present significant challenges for conservation and fisheries management. Current management likely prevents many new species from arriving in the Great Lakes, but fails to address the transport of species within the lakes after they arrive; this is an issue of clear and pressing importance.

Beyond propagule pressure: importance of selection during the transport stage of biological invasions.

Biological invasions are largely considered to be a "numbers game", wherein the larger the introduction effort, the greater the probability that an introduced population will become established. However, conditions during transport - an early stage of the invasion - can be particularly harsh, thereby greatly reducing the size of a population available to establish in a new region. Some successful non-indigenous species are more tolerant of environmental and anthropogenic stressors than related native species, possibly stemming from selection (ie survival of only pre-adapted individuals for particular environmental conditions) during the invasion process. By reviewing current literature concerning population genetics and consequences of selection on population fitness, we propose that selection acting on transported populations can facilitate local adaptation, which may result in a greater likelihood of invasion than predicted by propagule pressure alone. Specifically, we suggest that detailed surveys should be conducted to determine interactions between molecular mechanisms and demographic factors, given that current management strategies may underestimate invasion risk.

Molecular Insights Into the Ctenophore Genus Beroe in Europe: New Species, Spreading Invaders.

The genus Beroe Browne, 1756 (Ctenophora, Beroidae) occurs worldwide, with 25 currently-described species. Because the genus is poorly studied, the definitive number of species is uncertain. Recently, a possible new Beroe species was suggested based on internal transcribed spacer 1 (ITS1) sequences from samples collected in Svalbard, Norway. Another species, Beroe ovata, was introduced to Europe from North America, initially in the Black Sea and subsequently (and possibly secondarily) into the Mediterranean and Baltic Seas. In areas where ctenophores have been introduced, they have often had significant detrimental ecological effects. The potential for other cryptic and/or undescribed Beroe species and history of spread of some species in the genus give reason for additional study. When alive, morphological hallmarks may be challenging to spot and photograph owing to the animals' transparency and near-constant motion. We sampled and analyzed 109 putative Beroe specimens from Europe, using morphological and molecular approaches. DNA analyses were conducted using cytochrome oxidase 1 and internal transcribed spacer sequences and, together with published sequences from GenBank, phylogenetic relationships of the genus were explored. Our study suggests the presence of at least 5 genetic lineages of Beroe in Europe, of which 3 could be assigned to known species: Beroe gracilis Künne 1939; Beroe cucumis Fabricius, 1780; and Beroe ovata sensu Mayer, 1912. The other 2 lineages (here provisionally named Beroe "norvegica" and Beroe "anatoliensis") did not clearly coincide with any known species and might therefore reflect new species, but confirmation of this requires further study.

Ships' Ballast Water Treatment by Chlorination Can Generate Toxic Trihalomethanes.

The International Maritime Organization (IMO) will enforce a new abundance-based performance standard for ballast water in September, 2017. Strong oxidants, like chlorine, have been proposed as a method for achieving this standard. However chlorine treatment of ballast water can produce hazardous trihalomethanes. We assessed maximum trihalomethane production from one chlorine dose for three types of ballast water (fresh, brackish and marine) and three levels of total organic carbon (TOC) concentration (natural, filtered, enhanced). While the current standard test considers a 5 day voyage, there is a high possibility of shorter trips and sudden change of plans that will release treated waters in the environment. Water source and TOC significantly affected trihalomethane production, with the highest amounts generated in brackish waters and enhanced TOC concentration. The concentration of brominated trihalomethanes increased from background levels and was highest in brackish water, followed by marine and fresh water.

On the context-dependent scaling of consumer feeding rates.

The stability of consumer-resource systems can depend on the form of feeding interactions (i.e. functional responses). Size-based models predict interactions - and thus stability - based on consumer-resource size ratios. However, little is known about how interaction contexts (e.g. simple or complex habitats) might alter scaling relationships. Addressing this, we experimentally measured interactions between a large size range of aquatic predators (4-6400 mg over 1347 feeding trials) and an invasive prey that transitions among habitats: from the water column (3D interactions) to simple and complex benthic substrates (2D interactions). Simple and complex substrates mediated successive reductions in capture rates - particularly around the unimodal optimum - and promoted prey population stability in model simulations. Many real consumer-resource systems transition between 2D and 3D interactions, and along complexity gradients. Thus, Context-Dependent Scaling (CDS) of feeding interactions could represent an unrecognised aspect of food webs, and quantifying the extent of CDS might enhance predictive ecology.

Assessing introduction risk using species' rank-abundance distributions.

Mixed-species assemblages are often unintentionally introduced into new ecosystems. Analysing how assemblage structure varies during transport may provide insights into how introduction risk changes before propagules are released. Characterization of introduction risk is typically based on assessments of colonization pressure (CP, the number of species transported) and total propagule pressure (total PP, the total abundance of propagules released) associated with an invasion vector. Generally, invasion potential following introduction increases with greater CP or total PP. Here, we extend these assessments using rank-abundance distributions to examine how CP : total PP relationships change temporally in ballast water of ocean-going ships. Rank-abundance distributions and CP : total PP patterns varied widely between trans-Atlantic and trans-Pacific voyages, with the latter appearing to pose a much lower risk than the former. Responses also differed by taxonomic group, with invertebrates experiencing losses mainly in total PP, while diatoms and dinoflagellates sustained losses mainly in CP. In certain cases, open-ocean ballast water exchange appeared to increase introduction risk by uptake of new species or supplementation of existing ones. Our study demonstrates that rank-abundance distributions provide new insights into the utility of CP and PP in characterizing introduction risk.

Fortune favours the bold: a higher predator reduces the impact of a native but not an invasive intermediate predator.

Emergent multiple predator effects (MPEs) might radically alter predictions of predatory impact that are based solely on the impact of individuals. In the context of biological invasions, determining if and how the individual-level impacts of invasive predators relates to their impacts in multiple-individual situations will inform understanding of how such impacts might propagate through recipient communities. Here, we use functional responses (the relationship between prey consumption rate and prey density) to compare the impacts of the invasive freshwater mysid crustacean Hemimysis anomala with a native counterpart Mysis salemaai when feeding on basal cladoceran prey (i) as individuals, (ii) in conspecific groups and (iii) in conspecific groups in the presence of a higher fish predator, Gasterosteus aculeatus. In the absence of the higher predator, the invader consumed significantly more basal prey than the native, and consumption was additive for both mysid species - that is, group consumption was predictable from individual-level consumption. Invaders and natives were themselves equally susceptible to predation when feeding with the higher fish predator, but an MPE occurred only between the natives and higher predator, where consumption of basal prey was significantly reduced. In contrast, consumption by the invaders and higher predator remained additive. The presence of a higher predator serves to exacerbate the existing difference in individual-level consumption between invasive and native mysids. We attribute the mechanism responsible for the MPE associated with the native to a trait-mediated indirect interaction, and further suggest that the relative indifference to predator threat on the part of the invader contributes to its success and impacts within invaded communities.

Non-microcystin extracellular metabolites of Microcystis aeruginosa impair viability and reproductive gene expression in rainbow trout cell lines.

Microcystis aeruginosa is a ubiquitous freshwater cyanobacterium best known for producing hepatotoxic microcystins; however, this common bloom-forming species also produces myriad biologically active and potentially deleterious other metabolites. Our understanding of the effects of these non-microcystin metabolites on fish is limited. In this study, we evaluated cytotoxicity of extracellular metabolites harvested from both microcystin-producing (MC+) and non-producing (MC-) strains of M. aeruginosa on rainbow trout (Oncorhynchus mykiss) cell lines derived from tissues of the brain, pituitary, heart, gonads, gills, skin, liver, and milt. We also examined the influence of M. aeruginosa exudates (MaE) on the expression of critical reproduction-related genes using the same cell lines. We found that exudates of the MC- M. aeruginosa strain significantly reduced viability in RTBrain, RTgill-W1, and RT-milt5 cell lines and induced significant cellular stress and/or injury in six of the eight cell lines-highlighting potential target tissues of cyanobacterial cytotoxic effects. Observed sublethal consequences of Microcystis bloom exposure occurred with both MC+ and MC- strains' exudates and significantly altered expression of developmental and sex steroidogenic genes. Collectively, our results emphasize the contributions of non-MC metabolites to toxicity of Microcystis-dominated algal blooms and the need to integrate the full diversity of M. aeruginosa compounds-beyond microcystins-into ecotoxicological risk assessments.

Assessment of estrogenic potential from exudates of microcystin-producing and non-microcystin-producing Microcystis by metabolomics, machine learning and E-screen assay.

Cyanobacterial blooms, often dominated by Microcystis aeruginosa, are capable of producing estrogenic effects. It is important to identify specific estrogenic compounds produced by cyanobacteria, though this can prove challenging owing to the complexity of exudate mixtures. In this study, we used untargeted metabolomics to compare components of exudates from microcystin-producing and non-microcystin-producing M. aeruginosa strains that differed with respect to their ability to produce microcystins, and across two growth phases. We identified 416 chemicals and found that the two strains produced similar components, mainly organoheterocyclic compounds (20.2%), organic acids and derivatives (17.3%), phenylpropanoids and polyketides (12.7%), benzenoids (12.0%), lipids and lipid-like molecules (11.5%), and organic oxygen compounds (10.1%). We then predicted estrogenic compounds from this group using random forest machine learning. Six compounds (daidzin, biochanin A, phenylethylamine, rhein, o-Cresol, and arbutin) belonging to phenylpropanoids and polyketides (3), benzenoids (2), and organic oxygen compound (1) were tested and exhibited estrogenic potency based upon the E-screen assay. This study confirmed that both Microcystis strains produce exudates that contain compounds with estrogenic properties, a growing concern in cyanobacteria management.

Can a shift in dominant species of Microcystis alter growth and reproduction of waterfleas?

The bloom-forming species Microcystis wesenbergii and M. aeruginosa occur in many lakes globally, and may exhibit alternating blooms both spatially and temporally. As environmental changes increase, cyanobacteria bloom in more and more lakes and are often dominated by M. wesenbergii. The adverse impact of M. aeruginosa on co-existing organisms including zooplanktonic species has been well-studied, whereas studies of M. wesenbergii are limited. To compare effects of these two species on zooplankton, we explored effects of exudates from different strains of microcystin-producing M. aeruginosa (Ma905 and Ma526) and non-microcystin-producing M. wesenbergii (Mw908 and Mw929), on reproduction by the model zooplankter Daphnia magna in both chronic and acute exposure experiments. Specifically, we tested physiological, biochemical, molecular and transcriptomic characteristics of D. magna exposed to Microcystis exudates. We observed that body length and egg and offspring number of the daphnid increased in all treatments. Among the four strains tested, Ma526 enhanced the size of the first brood, as well as total egg and offspring number. Microcystis exudates stimulated expression of specific genes that induced ecdysone, juvenile hormone, triacylglycerol and vitellogenin biosynthesis, which, in turn, enhanced egg and offspring production of D. magna. Even though all strains of Microcystis affected growth and reproduction, large numbers of downregulated genes involving many essential pathways indicated that the Ma905 strain might contemporaneously induce damage in D. magna. Our study highlights the necessity of including M. wesenbergii into the ecological risk evaluation of cyanobacteria blooms, and emphasizes that consequences to zooplankton may not be clear-cut when assessments are based upon production of microcystins alone.

A non-microcystin-producing Microcystis wesenbergii strain alters fish food intake by disturbing neuro-endocrine appetite regulation.

Cyanobacterial harmful algal blooms (cHABs) are pervasive sources of stress resulting in neurotoxicity in fish. A member of the widely distributed Microcystis genus of bloom-forming cyanobacteria, Microcystis wesenbergii can be found in many freshwater lakes, including Dianchi Lake (China), where it has become one of the dominant contributors to the lake's recurrent blooms. However, unlike its more well-known counterpart M. aeruginosa, the effects of dense non-microcystin-containing M. wesenbergii blooms are seldom studied. The disturbance of appetite regulation and feeding behaviour can have downstream effects on the growth of teleost fish, posing a significant challenge to aquaculture and conservation efforts. Here we examined the effects of M. wesenbergii blooms on the food intake of Acrossocheilus yunnanensis, a native cyprinid in southern China. This fish species has disappeared in Dianchi Lake, and its reintroduction might be negatively affected by the presence of this newly-dominant Microcystis species. We co-cultured juvenile A. yunnanensis with a non-microcystin-producing strain of M. wesenbergii at initial densities between 5 × 104 and 1 × 106 cells/mL and monitored fish feeding behaviour and changes in neurotransmitter and hormone protein levels. High-density M. wesenbergii cultures increased the feeding rate of co-cultured fish, elevating concentrations of appetite-stimulating signalling molecules (Agouti-related protein and γ-aminobutyric acid), while decreasing inhibitory ones (POMC). These changes coincided with histopathological alterations and reduced somatic indices in brain and intestinal tissues. Given this potential for detrimental effects and dysregulation of food intake, further studies are necessary to determine the impacts of chronic exposure of M. wesenbergii in wild fish.

Is a lower-toxicity strain of Microcystis aeruginosa really less toxic?

Some well-known hazards of blooming cyanobacteria are caused by toxic metabolites such as microcystins (MCs), though many other bioactive chemicals of unknown toxicity are present in their exudates. It is also unclear whether toxicity of cyanobacterial cells depends on growth phases in the life cycle. In this study, we compared toxicity to Daphnia magna of Microcystis aeruginosa - a common cyanobacterial species - exudates (MaE) from two MC-producing strains over both exponential growth and stationary phases in acute and chronic experiments. Specifically, we assessed mitochondrial dysfunction, oxidative stress and lipid peroxidation, and filtering activity and heartbeat rate of Daphnia exposed to MaE. All MaE treatments induced common characteristics of Microcystis toxicity including disorder in the mitochondrial membrane and aberrant heart rate. MaE from cells at stationary growth phase were more toxic than those at exponential phase. Surprisingly, the MC-lower strain had higher toxicity than MC-higher one. Microcystis at different stage of blooms may differentially affect waterfleas owing to variable MaE-induced physiological dysfunction, abundance and grazing rate. Our study suggested that Microcystis strains with lower microcystin-producing ability might release other detrimental chemicals and should not be ignored in harmful bloom monitoring.

Corrigendum: Comparative metabolomic analysis of exudates of high-microcystin-producing and low-microcystin-producing Microcystis aeruginosa strains.

[This corrects the article DOI: 10.3389/fmicb.2022.1075621.].