scholarly journals Scanning ferry routes: looking for eDNA traces of marine mammals and their preys

2021 ◽  
Vol 4 ◽  
Author(s):  
Elena Valsecchi
Keyword(s):  
Marine Mammals ◽  
High Throughput Sequencing ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Trophic Levels ◽  
Systematic Sampling ◽  
Marine Biodiversity ◽  
Tyrrhenian Sea ◽  
Night Time ◽  
Species Specific

Marine environmental DNA (eDNA) surveys are becoming a promising approach to monitor biodiversity status and its variation over time. However, monitoring offshore areas could be extremely costly when using dedicated vessels, beside the impossibility to sample simultaneously geographically distant (even if adjacent) areas. The unexplored possibility of availing on operating ferries as an opportunistic platform for eDNA sampling offers several advantages besides opening limitless opportunities for systematic surveys on marine biodiversity.We present the results of both metabarcoding and barcoding approaches obtained from the analysis of water samples collected on board of a ferry boat along a pilot Mediterranean route crossing the Pelagos Sanctuary for Mediterranean Marine Mammals. The recently described MarVer primer sets (12SrDNA and 16SrDNA regions), specifically designed for the simultaneous detection of marine mammals and other marine vertebrates, were employed. The High Throughput Sequencing (HTS) outcome showed that the markers successfully detected most trophic levels of vertebrate marine communities, and classes, including bony fish, rays, cetaceans and birds. Ferry-based sampling allow to collect sample at any time of the day, and we indeed found diel differences in both quantitative and qualitative distribution of read counts. For instances, we observed an increased abundance of lantern fish amplicons in night-time collect samples (50%), reflecting nocturnal migration through the water column. In general, the number of read counts was significantly higher in nocturnal samples. Such diel differences within our sample indirectly provides evidence of the efficiency of the eDNA approach to detect contemporary signals in the sampled environment. Similarly, cetaceans were detected in correspondence of visual sightings (when these occurred, supplementary samples were collected). Rare species, such as the monk seal, are difficult to be detected in metabarcoding surveys, thus we opted to side the screening of the ferry-samples with a panel of species-specific qPCR assays, which were able to detect DNA traces of the endangered pinniped in the Tuscany archipelago (Tyrrhenian Sea) long before visual observations witnessed its presence in the same area. The study demonstrates the feasibility of using commercial shipping as a platform for eDNA marine sampling without dedicated survey cruises. Commercial shipping routes have potential to act as regular systematic sampling transects which can contribute to evaluating and monitoring marine biodiversity.

scholarly journals Novel Universal Primers for Metabarcoding eDNA Surveys of Marine Mammals and Other Marine Vertebrates

2019 ◽  
Author(s):  
Elena Valsecchi ◽  
Jonas Bylemans ◽  
Simon J. Goodman ◽  
Roberto Lombardi ◽  
Ian Carr ◽  
...  
Keyword(s):  
Marine Mammals ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
Universal Primers ◽  
Marine Biodiversity ◽  
Primer Sets ◽  
Marine Megafauna

ABSTRACTMetabarcoding studies using environmental DNA (eDNA) and high throughput sequencing (HTS) are rapidly becoming an important tool for assessing and monitoring marine biodiversity, detecting invasive species, and supporting basic ecological research. Several barcode loci targeting teleost fish and elasmobranchs have previously been developed, but to date primer sets focusing on other marine megafauna, such as marine mammals have received less attention. Similarly, there have been few attempts to identify potentially ‘universal’ barcode loci which may be informative across multiple marine vertebrate Orders. Here we describe the design and validation of four new sets of primers targeting hypervariable regions of the vertebrate mitochondrial 12S and 16S rRNA genes, which have conserved priming sites across virtually all cetaceans, pinnipeds, elasmobranchs, boney fish, sea turtles and birds, and amplify fragments with consistently high levels of taxonomically diagnostic sequence variation. ‘In silico’ validation using the OBITOOLS software showed our new barcode loci outperformed most existing vertebrate barcode loci for taxon detection and resolution. We also evaluated sequence diversity and taxonomic resolution of the new barcode loci in 680 complete marine mammal mitochondrial genomes demonstrating that they are effective at resolving amplicons for most taxa to the species level. Finally, we evaluated the performance of the primer sets with eDNA samples from aquarium communities with known species composition. These new primers will potentially allow surveys of complete marine vertebrate communities in single HTS metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators.

scholarly journals Ferries and Environmental DNA: Underway Sampling From Commercial Vessels Provides New Opportunities for Systematic Genetic Surveys of Marine Biodiversity

2021 ◽  
Vol 8 ◽  
Author(s):  
Elena Valsecchi ◽  
Antonella Arcangeli ◽  
Roberto Lombardi ◽  
Elizabeth Boyse ◽  
Ian M. Carr ◽  
...  
Keyword(s):  
Environmental Dna ◽  
Marine Biodiversity ◽  
Night Time ◽  
Operational Taxonomic Units ◽  
Reasonable Cost ◽  
Temperature Ranges ◽  
June July ◽  
Species Occurrences ◽  
Marine Environmental ◽  
Sample Heterogeneity

Marine environmental DNA (eDNA) is an important tool for biodiversity research and monitoring but challenges remain in scaling surveys over large spatial areas, and increasing the frequency of sampling in remote locations at reasonable cost. Here we demonstrate the feasibility of sampling from commercial vessels (Mediterranean ferries) while underway, as a strategy to facilitate replicable, systematic marine eDNA surveys in locations that would normally be challenging and expensive for researchers to access. Sixteen eDNA samples were collected from four fixed sampling stations, and in response to four cetacean sightings, across three cruises undertaken along the 300 km ferry route between Livorno (Tuscany) and Golfo Aranci (Sardinia) in the Ligurian/Tyrrhenian Seas, June-July 2018. Using 12SrDNA and 16SrDNA metabarcoding markers, we recovered diverse marine vertebrate Molecular Operational Taxonomic Units (MOTUs) from teleost fish, elasmobranchs, and cetaceans. We detected sample heterogeneity consistent with previously known variation in species occurrences, including putative species spawning peaks associated with specific sea surface temperature ranges, and increased night time abundance of bathypelagic species known to undertake diel migrations through the water column. We suggest commercial vessel based marine eDNA sampling using the global shipping network has potential to facilitate broad-scale biodiversity monitoring in the world’s oceans.

scholarly journals Development of an eDNA metabarcoding tool for surveying the world’s largest amphibian

2021 ◽  
Author(s):  
Jie Wang ◽  
Ping Liu ◽  
Jiang Chang ◽  
Cheng Li ◽  
Feng Xie ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Survey Methods ◽  
Environmental Dna ◽  
Cost Effective ◽  
Isolated Populations ◽  
Specificity And Sensitivity ◽  
Chinese Giant Salamander ◽  
Elusive Species ◽  
Primer Sets ◽  
Species Specific

Abstract Due to the overexploitation of farming, as well as habitat loss or degradation, the wild population of Chinese giant salamander Andrias davidianus (CGS), a species with seven genetically distinct lineages, has decreased by over 80% in the past 70 years. Traditional survey methods have proven to be unsuitable for finding this rare and elusive species. We evaluated the efficacy of environmental DNA (eDNA) sampling to detect CGS indirectly from its aquatic environment. We developed several species-specific primer sets; validated their specificity and sensitivity; and assessed their utility in silico, in the laboratory, and in two field sites having released farm-bred CGSs. We detected the presence of CGS DNA by using polymerase chain reaction (PCR) and Sanger sequencing. We also sequenced an amplicon mixture of seven haplotype-represented samples using high-throughput sequencing. Our eDNA methods could detect the presence of CGS at moderate densities reported across its range, proving them as a cost-effective way to establish broad-scale patterns of occupancy for CGS. In addition, our primers enabled the detection of a mitochondrial lineage mixture or introduced individuals from geographically isolated populations of CGS.

scholarly journals Beyond traditional biodiversity fish monitoring: environmental DNA metabarcoding and simultaneous underwater visual census detect different sets of a complex fish community at a marine biodiversity hotspot

2019 ◽  
Author(s):  
Tania Valdivia-Carrillo ◽  
Axayácatl Rocha-Olivares ◽  
Héctor Reyes-Bonilla ◽  
José Francisco Domínguez-Contreras ◽  
Adrian Munguia-Vega
Keyword(s):  
Gulf Of California ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
Marine Ecology ◽  
Marine Biodiversity ◽  
Rocky Reef ◽  
Monitoring Methods ◽  
Visual Census ◽  
Traditional Methods ◽  
Underwater Visual Census

ABSTRACTSignificant advances in the study of marine fish communities have been achieved with traditional monitoring methods and recently with novel genetic approaches. eDNA metabarcoding is one of them and a powerful tool for the study of biodiversity still in continuous development. Its applicability in marine ecology and conservation studies may be gauged by comparing its results with those of traditional methods. In the present investigation, we compare results from the underwater visual census (UVC) with eDNA metabarcoding (eDNA) carried out simultaneously in 24 rocky reef sites along the Gulf of California. We developed a two-PCR library preparation protocol followed by high throughput sequencing aimed at teleost fish. Our results show that both methods had different detection capabilities, and each registered different sets of fish taxa from rocky reefs, with some overlap. In particular, eDNA identified taxa from pelagic, demersal, and estuarine habitats beyond the rocky reef itself, suggesting differences in detection mainly attributed to the transport and permanence time of the eDNA in the ocean. Overlap in the detection with both methods increased with taxonomic level. We argue that substantial gaps in sequence reference databases for teleost are at the root of major discrepancies. Our results also confirm that PCR-based eDNA metabarcoding of seawater samples does not reflect patterns in abundance and biomass of species estimated from traditional methods. We discuss how to reconcile the results of eDNA metabarcoding and traditional methods in marine hotspots.

scholarly journals Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

2017 ◽  
Author(s):  
Lynsey R. Harper ◽  
Lori Lawson Handley ◽  
Christoph Hahn ◽  
Neil Boonham ◽  
Helen C. Rees ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Detection Threshold ◽  
Environmental Dna ◽  
Cost Effective ◽  
Triturus Cristatus ◽  
Species Detection ◽  
Survey Tool ◽  
Crested Newt ◽  
Species Specific ◽  
Freshwater Monitoring

SummaryEnvironmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and ‘metabarcoding’ have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real-time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using High-Throughput Sequencing technology. With qPCR and a detection threshold of 1/12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4/12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species-specific surveys.

scholarly journals Vertical stratification of environmental DNA in the open ocean captures ecological patterns and behavior of deep-sea fishes

2021 ◽  
Author(s):  
Oriol Canals ◽  
Iñaki Mendibil ◽  
María Santos ◽  
Xabier Irigoien ◽  
Naiara Rodríguez-Ezpeleta
Keyword(s):  
Water Column ◽  
Deep Sea ◽  
Sustainable Use ◽  
Deep Ocean ◽  
Environmental Dna ◽  
Night Time ◽  
Specific Distribution ◽  
Mesopelagic Zone ◽  
Relevant Role ◽  
Species Specific

AbstractThe deep-sea remains among the most unknown ecosystems on Earth despite its relevant role in carbon sequestration and increasing threat due to interest by fishing and mining industries. This, together with the recent discovery that the upper layer of this ecosystem (mesopelagic zone) harbors about 90% of the fish biomass on Earth, claims for a deeper understanding of the deep-sea so that the foundations for a sustainable use of its resources can be established. The analysis of environmental DNA (eDNA) collected from the water column emerges as an alternative to traditional methods to acquire this elusive information, but its application to the deep ocean is still incipient. Here, we have amplified and sequenced the fish eDNA contained in vertical profile samples (from surface to 2000 m depth) collected during day and night-time throughout the Bay of Biscay. We found that eDNA-derived deep-sea fish richness and abundance follow a day-night pattern that is consistent with the diel migratory behavior of many mesopelagic species, and that eDNA can reveal species-specific distribution and movement through the water column. These results highlight the potential of eDNA-based studies to improve our knowledge on the species inhabiting the dark ocean before this still pristine ecosystem is exploited.

scholarly journals Simultaneous detection of native and invasive crayfish and Aphanomyces astaci from environmental DNA samples in a wide range of habitats in Central Europe

NeoBiota ◽  
2020 ◽  
Vol 58 ◽  
pp. 1-32 ◽  
Author(s):  
Johannes C. Rusch ◽  
Michaela Mojžišová ◽  
David A. Strand ◽  
Jitka Svobodová ◽  
Trude Vrålstad ◽  
...  
Keyword(s):  
Central Europe ◽  
Western Europe ◽  
Low Cost ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Aphanomyces Astaci ◽  
Wide Range ◽  
Species Specific ◽  
Native Crayfish ◽  
Crayfish Species

Crayfish of North American origin are amongst the most prominent high-impact invasive invertebrates in European freshwaters. They contribute to the decline of European native crayfish species by spreading the pathogen causing crayfish plague, the oomycete Aphanomyces astaci. In this study we validated the specificity of four quantitative PCR (qPCR) assays, either published or newly developed, usable for environmental DNA (eDNA) screening for widely distributed native and non-native crayfish present in Central Europe: Astacus astacus, Pacifastacus leniusculus, Faxonius limosus and Procambarus virginalis. We then conducted an eDNA monitoring survey of these crayfish as well as the crayfish plague pathogen in a wide variety of habitat types representative for Central and Western Europe. The specificity of qPCR assays was validated against an extensive collection of crayfish DNA isolates, containing most crayfish species documented from European waters. The three assays developed in this study were sufficiently species-specific, but the published assay for F. limosus displayed a weak cross-reaction with multiple other crayfish species of the family Cambaridae. In the field study, we infrequently detected eDNA of A. astaci together with the three non-native crayfish species under examination. We never detected eDNA from A. astaci together with native crayfish, but in a few locations eDNA from both native and non-native crayfish was captured, due either to passive transport of eDNA from upstream populations or co-existence in the absence of infected crayfish carriers of A. astaci. In the study, we evaluated a robust, easy-to-use and low-cost version of the eDNA sampling equipment, based mostly on items readily available in garden stores and hobby markets, for filtering relatively large (~5 l) water samples. It performed just as well as the far more expensive equipment industrially designed for eDNA water sampling, thus opening the possibility of collecting suitable eDNA samples to a wide range of stakeholders. Overall, our study confirms that eDNA-based screening for crayfish and their associated pathogen is a feasible alternative to traditional monitoring.

scholarly journals Development of a 16S metabarcoding assay for the environmental DNA (eDNA) detection of aquatic reptiles across northern Australia

2020 ◽  
Author(s):  
Katrina West ◽  
Matthew Heydenrych ◽  
Rose Lines ◽  
Tony Tucker ◽  
Sabrina Fossette ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Environmental Dna ◽  
Cost Effective ◽  
Distribution Data ◽  
Freshwater Turtles ◽  
Northern Australia ◽  
Non Invasive ◽  
Sea Snakes ◽  
Reptile Species ◽  
Species Specific

AbstractA severe lack of distribution data for aquatic reptiles in northern Australia leaves many taxa vulnerable to extirpation and extinction. Environmental DNA (eDNA) technologies offer sensitive and non-invasive genetic alternatives to trapping and visual surveys and are increasingly employed for the detection of aquatic and semi-aquatic reptiles. However, at present, these studies have largely applied species-specific primers which do not provide a cost-effective avenue for the simultaneous detection of multiple reptilian taxa. Here, we present a 16S rRNA metabarcoding assay for the broad detection of aquatic and semi-aquatic reptile species. This assay is tested on water samples collected at multiple sampling sites at two tropical locations: 12 marine/estuarine sites in Roebuck Bay, Western Australia, and 4 estuarine sites in Cooktown, Queensland, Australia. A total of nine reptile taxa were detected from 10 of the 16 sampled sites, including marine and freshwater turtles, aquatic and semi-aquatic/terrestrial snakes, and terrestrial skinks. However, inconsistencies in the detection of previously observed aquatic reptiles at our sampled sites, such as saltwater crocodile and sea snakes, indicates that further research is required to assess the reliability, strengths and limitations of eDNA methods for aquatic reptile detection before it can be integrated as a broad-scale bioassessment tool.

Species Specific Immunoassays to Measure Blood Platelet and Coagulation Activation in the Rat

1996 ◽  
Vol 76 (06) ◽  
pp. 1090-1095 ◽  
Author(s):  
C Ravanat ◽  
M Freund ◽  
S Schuhler ◽  
P Grunert ◽  
L Meyer ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Plasma Concentrations ◽  
Simultaneous Detection ◽  
Blood Platelet ◽  
Coagulation Activation ◽  
Platelet Factor ◽  
Prethrombotic State ◽  
Low Levels ◽  
Species Specific ◽  
Higher Sensitivity

SummaryThe purpose of this study was to develop specific and sensitive immunoassays to detect early indices of hypercoagulability in the rat. Rat platelet factor 4 (rPF4) and rat fibrinopeptide A (rFPA) assays, tools for the detection of activation of platelets and coagulation respectively, were designed using antibodies raised against purified rPF4 and against synthetic rFPA. The relevance of these new assays and of the commercially available ELISA kit for thrombin-antithrombin III (TAT) complexes was demonstrated in a rat model of a prethrombotic state induced by intravenous infusion of varying doses of thromboplastin (90 to 2400 μl/kg/h). In this model, the immunoassays allowed simultaneous detection of low levels of rFPA and rPF4 which were correlated with fibrinogen and platelet consumption and TAT generation and further proved to be of higher sensitivity than the classical methods of platelet count or measurement of fibrinogen levels. Plasma concentrations of rFPA, rPF4 and TAT were dependent on infusion time and thromboplastin dose, while hirudin (1 mg/kg) prevented their appearance. Thus the new specific immunoassays for rPF4 and rFPA and the commercial human TAT assay represent useful tools for pathophysiological studies or the screening of antithrombotic drugs in rats.

scholarly journals Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
Ryohei Nakao ◽  
Hideyuki Doi ◽  
Toshifumi Minamoto
Keyword(s):  
Fish Species ◽  
High Throughput Sequencing ◽  
Correlation Coefficients ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Taqman Probe ◽  
Individual Species ◽  
Natural Environments ◽  
Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

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