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Remote polar and deepwater fish faunas are under pressure from ongoing climate change and increasing fishing effort. However, these fish communities are difficult to monitor for logistic and financial reasons. Currently, monitoring of marine fishes largely relies on invasive techniques such as bottom trawling, and on official reporting of global catches, which can be unreliable. Thus, there is need for alternative and non-invasive techniques for qualitative and quantitative oceanic fish surveys. Here we report environmental DNA (eDNA) metabarcoding of seawater samples from continental slope depths in Southwest Greenland. We collected seawater samples at depths of 188-918 m and compared seawater eDNA to catch data from trawling. We used Illumina sequencing of PCR products to demonstrate that eDNA reads show equivalence to fishing catch data obtained from trawling. Twenty-six families were found with both trawling and eDNA, while three families were found only with eDNA and two families were found only with trawling. Key commercial fish species for Greenland were the most abundant species in both eDNA reads and biomass catch, and interpolation of eDNA abundances between sampling sites showed good correspondence with catch sizes. Environmental DNA sequence reads from the fish assemblages correlated with biomass and abundance data obtained from trawling. Interestingly, the Greenland shark (Somniosus microcephalus) showed high abundance of eDNA reads despite only a single specimen being caught, demonstrating the relevance of the eDNA approach for large species that can probably avoid bottom trawls in most cases. Quantitative detection of marine fish using eDNA remains to be tested further to ascertain whether this technique is able to yield credible results for routine application in fisheries. Nevertheless, our study demonstrates that eDNA reads can be used as a qualitative and quantitative proxy for marine fish assemblages in deepwater oceanic habitats. This relates directly to applied fisheries as well as to monitoring effects of ongoing climate change on marine biodiversity-especially in polar ecosystems.

Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. Furthermore, misidentification of species represents a major problem. Here, we investigate the potential of using metabarcoding of environmental DNA (eDNA) obtained directly from seawater samples to account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (Sardina pilchardus). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days. Although further studies are needed to validate the eDNA approach in varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of marine biodiversity and resources.

Environmental DNA (eDNA) extracted from water samples has recently shown potential as a valuable source of population genetic information for aquatic macroorganisms. This approach offers several potential advantages compared with conventional tissue‐based methods, including the fact that eDNA sampling is noninvasive and generally more cost‐efficient. Currently, eDNA approaches have been limited to single‐marker studies of mitochondrial DNA (mtDNA), and the relationship between eDNA haplotype composition and true haplotype composition still needs to be thoroughly verified. This will require testing of bioinformatic and statistical software to correct for erroneous sequences, as well as biases and random variation in relative sequence abundances. However, eDNA‐based population genetic methods have far‐reaching potential for both basic and applied research. In this paper, we present a brief overview of the achievements of eDNA‐based population genetics to date, and outline the prospects for future developments in the field, including the estimation of nuclear DNA (nuDNA) variation and epigenetic information. We discuss the challenges associated with eDNA samples as opposed to those of individual tissue samples and assess whether eDNA might offer additional types of information unobtainable with tissue samples. Lastly, we provide recommendations for determining whether an eDNA approach would be a useful and suitable choice in different research settings. We limit our discussion largely to contemporary aquatic systems, but the advantages, challenges, and perspectives can to a large degree be generalized to eDNA studies with a different spatial and temporal focus.

Aim: Greenland is one of the places on Earth where the effects of climate change are most evident. The retreat of sea ice has made East Greenland more accessible for longer periods during the year. East Greenland fjords have been notoriously difficult to study due to their remoteness, dense sea ice conditions and lack of infrastructure. As a result, biological monitoring across latitudinal gradients is scarce in East Greenland and relies on sporadic research cruises and trawl data from commercial vessels. We here aim to investigate the transition in fish and marine mammal communities from South to Northeast Greenland using environmental DNA (eDNA). Location: South to Northeast Greenland. Methods: We investigated the transition in fish and marine mammal communities from South to Northeast Greenland using eDNA metabarcoding of seawater samples. We included both surface and mesopelagic samples, collected over approximately 2400 km waterway distance, by sampling from Cape Farewell to Ella Island in August 2021. Results: We demonstrate a clear transition in biological communities from south to northeast, with detected fish and mammal species matching known distributions. Samples from the southern areas were dominated by capelin (Mallotus villosus) and redfish (Sebastes), whereas northeastern samples were dominated by polar cod (Boreogadus saida), sculpins (Myoxocephalus) and ringed seal (Pusa hispida). We provide newly generated 12S rRNA barcodes from 87 fish species, bringing the public DNA database closer to full taxonomic coverage for Greenlandic fish species for this locus. Main Conclusions: Our results demonstrate that eDNA sampling can detect latitudinal shifts in marine biological communities of the Arctic region, which can supplement traditional fish surveys in understanding species distributions and community compositions of marine vertebrates. Importantly, sampling of eDNA can be a feasible approach for detecting northward range expansions in remote areas as climate change progresses.

For several hundred years freshwater crayfish (Crustacea-Decapoda-Astacidea) have played an important ecological, cultural and culinary role in Scandinavia. However, many native populations of noble crayfish Astacus astacus have faced major declines during the last century, largely resulting from human assisted expansion of non-indigenous signal crayfish Pacifastacus leniusculus that carry and transmit the crayfish plague pathogen. In Denmark, also the non-indigenous narrow-clawed crayfish Astacus leptodactylus has expanded due to anthropogenic activities. Knowledge about crayfish distribution and early detection of non-indigenous and invasive species are crucial elements in successful conservation of indigenous crayfish. The use of environmental DNA (eDNA) extracted from water samples is a promising new tool for early and non-invasive detection of species in aquatic environments. In the present study, we have developed and tested quantitative PCR (qPCR) assays for species-specific detection and quantification of the three above mentioned crayfish species on the basis of mitochondrial cytochrome oxidase 1 (mtDNA-CO1), including separate assays for two clades of A. leptodactylus. The limit of detection (LOD) was experimentally established as 5 copies/PCR with two different approaches, and the limit of quantification (LOQ) were determined to 5 and 10 copies/PCR, respectively, depending on chosen approach. The assays detected crayfish in natural freshwater ecosystems with known populations of all three species, and show promising potentials for future monitoring of A. astacus, P. leniusculus and A. leptodactylus. However, the assays need further validation with data 1) comparing traditional and eDNA based estimates of abundance, and 2) representing a broader geographical range for the involved crayfish species.

The European noble crayfish Astacus astacus is threatened by crayfish plague caused by the oomycete Aphanomyces astaci, which is spread by the invasive North American crayfish (e.g. signal crayfish Pacifastacus leniusculus). Surveillance of crayfish plague status in Norway has traditionally relied on the monitoring survival of cage‐held noble crayfish, a method of ethical concern. Additionally, trapping is used in crayfish population surveillance. Here, we test whether environmental DNA (eDNA) monitoring could provide a suitable alternative to the cage method, and a supplement to trapping. We took advantage of an emerging crayfish plague outbreak in a Norwegian watercourse following illegal introduction of disease‐carrying signal crayfish, and initiated simultaneous eDNA monitoring and cage‐based surveillance, supplemented with trapping. A total of 304 water samples were filtered from several sampling stations over a 4‐year period. eDNA data (species‐specific quantitative real‐time PCR [qPCR]) for the presence of A. astaci, noble and signal crayfish within the water samples were compared to cage mortality and trapping. This is the first study comparing eDNA monitoring and cage surveillance during a natural crayfish plague outbreak. We show that eDNA monitoring corresponds well with the biological status measured in terms of crayfish mortality and trapping results. eDNA analysis also reveals the presence of A. astaci in the water up to 2.5 weeks in advance of the cage method. Estimates of A. astaci and noble crayfish eDNA concentrations increased markedly during mortality and vanished quickly thereafter. eDNA provides a snapshot of the presence, absence or disappearance of crayfish regardless of season, and constitutes a valuable supplement to the trapping method that relies on season and legislation. Synthesis and applications. Simultaneous eDNA monitoring of Aphanomyces astaci (crayfish plague) and relevant native and invasive freshwater crayfish species is well‐suited for early warning of invasion or infection, risk assessments, habitat evaluation and surveillance regarding pathogen and invasive/native crayfish status. This non‐invasive, animal welfare friendly method excludes the need for cage‐held susceptible crayfish in disease monitoring. Furthermore, eDNA monitoring is less likely to spread A. astaci than traditional methods. This study resulted in the implementation of eDNA monitoring for Norwegian crayfish plague and crayfish surveillance programmes, and we believe other countries could improve management strategies for freshwater crayfish using a similar approach. Sammendrag Europeisk edelkreps (Astacus astacus) trues av krepsepest som forårsakes av eggsporesoppen Aphanomyces astaci. Smitten spres av fremmed nordamerikansk ferskvannskreps (f.eks. signalkreps; Pacifastacus leniusculus). Overvåking av krepsepest i Norge har tradisjonelt basert seg på burforsøk, en etisk problematisk metode hvor dødelighet hos edelkreps i bur overvåkes ved relevante lokaliteter. Overvåking av edelkrepsbestander blir gjort ved bruk av teiner. Vi har testet om overvåking basert på innsamling av miljø‐DNA (eDNA) kan være et egnet alternativ til burforsøk, og et supplement til teinefangst. Etter en ulovlig introduksjon av smittebærende signalkreps i en innsjø med edelkreps, utnyttet vi vissheten om et kommende krepsepestutbrudd til å initiere eDNA‐overvåking og burforsøk samtidig, supplert med teinefangst. Tilsammen ble 304 vannprøver filtrert fra ulike prøvetakingsstasjoner over en fire‐års periode. eDNA data (arts‐spesifikk qPCR) for tilstedeværelse av A. astaci, edelkreps og signalkreps i vannprøver ble sammenlignet med dødelighet i burforsøk og teinefangst. Dette er den første studien som sammenligner eDNA‐overvåkning og burforsøk under et naturlig krepsepestutbrudd. Vi viser at eDNA‐overvåking korresponderer godt med biologisk status målt i form av dødelighet hos burkreps og resultater fra teinefangst. eDNA‐analyser avslører også tilstedeværelsen av A. astaci smittestoff i vannet opptil 2,5 uker før edelkreps dør i burforsøk. Mengdeestimater av eDNA fra A. astaci og edelkreps i vannet økte markant under dødelighet, og forsvant deretter raskt. Uansett årstid gir eDNA et øyeblikksbilde av tilstedeværelse, fravær eller bortfall av edelkreps, og utgjør derfor også et verdifullt supplement til teinefiske, som avhenger av sesong og nasjonal lovgivning. Syntese og bruksområder. Parallell eDNA‐overvåkning av Aphanomyces astaci (krepsepest agens) og relevante stedegne og fremmede arter av ferskvannskreps er velegnet for tidlig varsling av invasjon og smitte, risikovurderinger, evaluering av habitatstatus, og overvåking av status for smittestoff og fremmed/stedegen ferskvannskreps. Metoden er dyrevelferdsvennlig, og utelukker behovet for burforsøk med levende kreps i sykdomsovervåking. Videre gir eDNA‐overvåkning mindre sannsynlighet for å spre Aphanomyces astaci smitte enn tradisjonelle metoder. Denne studien har bidratt til å implementere eDNA‐overvåking i norsk overvåkning av krepsepest, edelkreps og signalkreps, og vi tror at andre land også kan forbedre sine forvaltningsstrategier for ferskvannskreps ved hjelp av en lignende tilnærming. Simultaneous eDNA monitoring of Aphanomyces astaci (crayfish plague) and relevant native and invasive freshwater crayfish species is well‐suited for early warning of invasion or infection, risk assessments, habitat evaluation and surveillance regarding pathogen and invasive/native crayfish status. This non‐invasive, animal welfare friendly method excludes the need for cage‐held susceptible crayfish in disease monitoring. Furthermore, eDNA monitoring is less likely to spread A. astaci than traditional methods. This study resulted in the implementation of eDNA monitoring for Norwegian crayfish plague and crayfish surveillance programmes, and we believe other countries could improve management strategies for freshwater crayfish using a similar approach.

Temporal variation in eDNA signals is increasingly explored for understanding community ecology in aquatic habitats. Seasonal changes have been addressed using eDNA sampling, but very little is known regarding short-term temporal variation that spans hours to days. To address this, we filtered marine water samples from a single coastal site in Denmark every hour for 32 h. We used metabarcoding to target both fish and broader eukaryote diversity and evaluated temporal changes in this marine community. Results revealed variation in fish species richness (15–27) and eukaryote class richness (35–64) across the 32 h of sampling, and we further evaluated sampling efforts needed to reach different levels of diversity saturation. Relative read frequency data for both fish and eukaryotes indicated a clear diel change in community composition, with different communities detected during daylight versus dark hours. The abundance signals in our data reflected biological variation rather than stochastic variation, since replicates taken at the same hour were more similar to each other than those taken at different hours. Our compositional results indicated a dynamic community, rather than a static pool of eDNA—even across a few hours. The fish data showed a daily pattern of relative species abundances, and the uncoupling of fish and broader eukaryote data suggest that variation in eDNA profiles across a single day can provide valuable information reflecting diel changes, at least for highly mobile organism groups. However, our results also point to several pitfalls in current eDNA experimental design, in which samples are taken over large areas without relative time-consistency or short-term replication. Our findings shed new light on short-term variation in coastal eDNA and have wide implications for experimental study design and for incorporating temporality into project conceptualization for future aquatic biodiversity monitoring.

Background: Buoyancy and balance are important parameters for slow-moving, low-metabolic, aquatic organisms. The extant coelacanths have among the lowest metabolic rates of any living vertebrate and can aford little energy to keep station. Previous observations on living coelacanths support the hypothesis that the coelacanth is neutrally buoyant and in close-to-perfect hydrostatic balance. However, precise measurements of buoyancy and balance at diferent depths have never been made. Results: Here we show, using non-invasive imaging, that buoyancy of the coelacanth closely matches its depth distribution. We found that the lipid-flled fatty organ is well suited to support neutral buoyancy, and due to a closeto-perfect hydrostatic balance, simple maneuvers of fns can cause a considerable shift in torque around the pitch axis allowing the coelacanth to assume diferent body orientations with little physical efort. Conclusions: Our results demonstrate a close match between tissue composition, depth range and behavior, and our collection-based approach could be used to predict depth range of less well-studied coelacanth life stages as well as of deep sea fshes in general.

Wildlife tracking devices are key in obtaining detailed insights on movement, animal migration, natal dispersal, home-ranges, resource use and group dynamics of free-roaming animals. Despite a wide use of such devices, tracking for entire lifetimes is still a considerable challenge for most animals, mainly due to technological limitations. Deploying battery powered wildlife tags on smaller animals is limited by the mass of the devices. Micro-sized devices with solar panels sometimes solve this challenge, however, nocturnal species or animals living under low light conditions render solar cells all but useless. For larger animals, where battery weight can be higher, battery longevity becomes the main challenge. Several studies have proposed solutions to these limitations, including harvesting thermal and kinetic energy on animals. However, these concepts are limited by size and weight. In this study, we used a small, lightweight kinetic energy harvesting unit as the power source for a custom wildlife tracking device to investigate its suitability for lifetime animal tracking. We integrated a Kinetron MSG32 microgenerator and a state-of-the-art lithium-ion capacitor (LIC) into a custom GPS-enabled tracking device that is capable of remotely transmitting data via the Sigfox ‘Internet of Things’ network. Prototypes were tested on domestic dog (n = 4), wild-roaming Exmoor pony (n = 1) and wisent (n = 1). One of the domestic dogs generated up to 10.04 joules of energy in a day, while the Exmoor pony and wisent generated on average 0.69 joules and 2.38 joules per day, respectively. Our results show a significant difference in energy generation between animal species and mounting method, but also highlight the potential for this technology to be a meaningful advancement in ecological research requiring lifetime tracking of animals. The design of the Kinefox is provided open source.

Stoat (Mustela erminea) and weasel (M. nivalis) are hard to monitor as they are elusive of nature and leave few identifying marks in their surroundings. Stoat and weasel are both fully protected in Denmark and are thought to be widely distributed throughout the country. Despite this stoat and weasel were listed on the Danish Red List as Near Threatened in 2019, as their densities and population trends are unknown. Using a modified novel camera trapping device, the Double-Mostela, a wooden box comprising a tracking tunnel and two camera traps, we attempted to obtain density estimates based on identification of individual stoats and weasels. We deployed camera traps both inside Double-Mostela traps and externally in three different study areas in northern Zealand, Denmark, and tested commercial, American scent-based lures to attract stoat and weasel. We obtained very low seasonal trapping rates of weasel in two study areas, but in one study area, we obtained a seasonal trapping rate of stoat larger compared to another study using the Mostela. In one study area, both species were absent. We observed no effect of scent-based lures in attracting small mustelids compared to non-bait traps. Potential reasons behind low capture rates of weasel and stoat are suboptimal habitat placement and timing of deployment of the DoubleMostelas, land-use changes over the last 200 years, predation from larger predators, as well as unintended secondary poisoning with rodenticides. Due to the scarcity of weasel and stoat captures, we were unable to make density estimates based on identification of individuals; however, we identified potential features that could be used for identification and density estimates with more captures.