Detektion af miljø-DNA med ‘quantitative Polymerase Chain Reaction’ (qPCR) sammenlignet med ‘droplet digital Polymerase Chain Reaction’ (ddPCR)

Prosjektleder: Steen W. KnudsenDetektion af miljø-DNA (engelsk: ‘environmental DNA’, forkortet: ‘eDNA’) kan gøres med blandt andet qPCR og ddPCR, hvor ddPCR ved detektion af DNA fra virus eller bakterier i human patologiske studier er blevet demonstreret som værende mere præcis i forhold til qPCR-baseret detektion af eDNA. I denne undersøgelse er målet at belyse om en sådan øget præcision også er gældende for detektion af eDNA fra marine ikke-hjemmehørende arter. Her analyseres de samme 48 ekstraktioner af eDNA fra filtervandprøver, indsamlet i 2021, med både qPCR og ddPCR for tilstedeværelsen af 17 forskellige ikke-hjemmehørende marine arter. Det kan konkluderes, at for de sjældne marine NIS-arter er der med ddPCR-platform en bedre chance for at detektere meget lave niveauer af eDNA. For 17 ud af 19 afprøvede artsspecifikke detektionssystemer på ddPCR-platform var der lige så præcis detektion af eDNA som på qPCR platform, såfremt der er tilstrækkeligt høje niveauer af eDNA til stede i vandprøven. For lave niveuaer af eDNA var ddPCR-platform bedre til at vurdere usikkerhed i eDNA koncentration end qPCR. Med reduceret arbejdstid for opsætning af ddPCR i forhold til med qPCR anbefales det at bruge ddPCR for fremtidig monitorering af eDNA fra marine invasive arter, for at opnå bedre præcision og reducere arbejdstid.MiljøstyrelsenpublishedVersio

Molecular, morphological and fossil input data for inferring relationship among viviparous brotulas (Bythitidae) - Resulting in a family status change for Dinematichthyidae

This article comprise the data related to the research article (Møller et al., 2016) [1], and makes it possible to explore and reproduce the topologies that allowed [1] to infer the relationship between the families Bythitidae and Dinematichthyidae. The supplementary data holds nexus-input files for the Bayesian analysis and the ‘.xml’-input files – with and without nucleotide data – that are used in the fossil-calibrated phylogenetic analysis with a relaxed clock model. The resulting topologies are provided as ‘.new’-files together with a characters matrix file for traits to trace across the inferred phylogenies. Keywords: Bythitinae, Aphyonidae, pedomorphism, Coral reef fishes, Deepsea fishes, Cave fishe

Environmental DNA from seawater samples correlate with trawl catches of subarctic, deepwater fishes

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

Distinct latitudinal community patterns of Arctic marine vertebrates along the East Greenlandic coast detected by environmental DNA

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

Monitoring a Norwegian freshwater crayfish tragedy: eDNA snapshots of invasion, infection and extinction

1. 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. 2. 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. 3. 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. 4. 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.publishedVersio

Kortlægning af flodperlemusling i Varde Å-systemet ved brug af eDNA – en eftersøgning af nålen i høstakken

Prosjektleder: Jes J. RasmussenI dette studie blev der taget vandprøver i Varde Å, Linding Å, Ansager Å, Grindsted Å og Holme Å til kortlægning af forekomster af flodperlemusling ved brug af eDNA. Analyserne blev foretaget på en digital droplet PCR-platform, der præsterer bedre end qPCR for små bestande af sjældent forekommende arter. Der blev fundet eDNA fra flodperlemusling i Varde Å ved Varde Sommerland samt opstrøms Vagtborg. Derudover blev der fundet eDNA fra flodperlemusling i udmundingen af Ansager Å samt på fire lokaliteter i Holme Å. For Ansager Å og Holme Å er der tale om nye fund, hvor flodperlemuslingen har været antaget forsvundet. Der er således mulighed for, at der findes kildepopulationer i Holme Å, der potentielt kan danne rekrutteringsgrundlag for den nyligt restaurerede strækning fra Nordenskov til Varde Å.Aage V. Jensen Fonden, Varde kommune, MiljøstyrelsenpublishedVersio

Deep-sea sponge derived environmental DNA analysis reveals demersal fish biodiversity of a remote Arctic ecosystem

The deep-sea is vast, remote, and largely underexplored. However, methodological advances in environmental DNA (eDNA) surveys could aid in the exploration efforts, such as using sponges as natural eDNA filters for studying fish biodiversity. In this study, we analyzed the eDNA from 116 sponge tissue samples and compared these to 18 water eDNA samples and visual surveys obtained on an Arctic seamount. Across survey methods, we revealed approximately 30% of the species presumed to inhabit this area and 11 fish species were detected via sponge derived eDNA alone. These included commercially important fish such as the Greenland halibut and Atlantic mackerel. Fish eDNA detection was highly variable across sponge samples. Highest detection rates were found in sponges with low microbial activity such as those from the class Hexactinellida. The different survey methods also detected alternate fish communities, highlighted by only one species overlap between the visual surveys and the sponge eDNA samples. Therefore, we conclude that sponge eDNA can be a useful tool for surveying deep-sea demersal fish communities and it synergises with visual surveys improving overall biodiversity assessments. Datasets such as this can form comprehensive baselines on fish biodiversity across seamounts, which in turn can inform marine management and conservation practices in the regions where such surveys are undertaken.publishedVersio

Ikke-hjemmehørende arter i marine områder

Projektleder Jesper H. AndersenDenne rapport omhandler ikke-hjemmehørende arter i de danske farvande og består af to dele. Den første vedrører en opdate-ring af den nationale liste over ikke-hjemmhørende arter i de danske farvande – også kaldet NIS-listen. Den anden del er en ana-lyse af de foreliggede data og informationer om forekomst af ikke-hjemmehørende arter i de danske farvande. Datagrundlaget er væsentligt forbedret siden den første nationale NIS-liste blev udarbejdet. De fire væsentligste forbedringer er: (1) data fra Statens Naturhistoriske Museums Fiskeatlas er nu med, (2) data fra NOVANA-overvågningen omfatter nu flere år (til og med 2018), (3) data fra 2017 om forekomsten af ikke-hjemmehørende arter i 16 danske havne er tilføjet, og (4) data for 2017 og 2018, baseret på eDNA-analyser på 33 stationer i NOVANA-programmet, er nu med. Med den ny NIS-liste og den landsdæk-kende analyse foreligger der nu en opdatering af forekomster af ikke-hjemmehørende arter, herunder fisk, i de danske farvande.publishedVersio

Tekniske anvisninger for eDNA-baseret overvågning af ikke-hjemmehørende marine arter

Forskningschef Jesper H. AndersenDisse tekniske anvisninger er en revideret udgave af de ansvisninger, der tidligere er udarbejdet for Miljøstyrelsen og publiceret vinteren 2017/2018. De reviderede tekniske anvisninger indeholder en detaljeret protokol for hvordan miljø-DNA (environ-mental-DNA eller blot eDNA) kan indsamles, opbevares, oprenses og spores. Anvisningerne er delt i tre dele og omfatter (1) indsamling og filtrering af vand med instruktionerne for efterfølgende opbevaring af filterenheder, (2) ekstraktion af eDNA fra filterenheder, og (3) sporing af eDNA ved hjælp af kvantitativ PCR. Den sidste del med kvantitativ sporing forudsætter, der er udviklet og testet artsspecifikke primere og prober for korrekt sporing af eDNA stammende kun fra ikke-hjemmehørende arter.Miljøstyrelsen (MST)publishedVersio