Using environmental DNA for detection of Batrachochytrium salamandrivorans in natural water

Rapid, early, and reliable detection of invasive pathogenic microorganisms is essential in order to either predict or delineate an outbreak, and monitor appropriate mitigation measures. The chytrid fungus Batrachochytrium salamandrivorans is expanding in Europe, and infection with this fungus may cause massive mortality in urodelans (salamanders and newts). In this study, we designed and validated species‐specific primers and a probe for detection of B. salamandrivorans in water. In a garden pond in close proximity to the B. salamandrivorans index site in the Netherlands, B. salamandrivorans‐infected newts had been detected in 2015 and have been monitored since. In 2016 and 2017, no B. salamandrivorans was detected at this site, but in 2018 B. salamandrivorans flared up in this isolated pond which allowed validation of the technique in situ. We here present the development of an environmental DNA technique that successfully detects B. salamandrivorans DNA in natural waterbodies even at low concentrations. This technique may be further validated to play a role in B. salamandrivorans range delineation and surveillance in both natural waterbodies and in captive collections

Batrachochytrium dendrobatidis in amphibians from the Po River Delta, Northern Italy

Batrachochytrium dendrobatidis is a pathogen infecting amphibians at the global scale and causing their decline, but knowledge of the distribution of this pathogen is far from complete. We sampled amphibians from three species (Hyla intermedia, Rana dalmatina and Pelophylax synklepton esculentus) to evaluate whether B. dendrobatidis infects amphibians in the Po River Delta Natural Park, Northern Italy. We detected the pathogen in one population of P. sk. esculentus (prevalence: 0.33). These findings expand the known distribution of B. dendrobatidis in Italy and add further concern to the conservation of amphibians in this area

Estimating the extended and hidden species diversity from environmental DNA in hyper-diverse regions

Species inventories are the building blocks of our assessment of biodiversity patterns and human impact. Yet, historical inventories based on visual observations are often incomplete, impairing subsequent analyses of ecological mechanisms, extinction risk and management success. Environmental DNA (eDNA) metabarcoding is an emerging tool that can provide wider biodiversity assessments than classical visual-based surveys. However, eDNA-based inventories remain limited by sampling effort and reference database incompleteness. In this study, we propose a new framework coupling eDNA surveys and sampling-theory methods to estimate species richness in under-sampled and hyper-diverse regions where some species remain absent from the checklist or undetected by visual surveys. We applied this framework to the coastal fish diversity in the heart of the coral triangle, the richest marine biodiversity hotspot worldwide. Combining data from 279 underwater visual censuses, 92 eDNA samples and an extensive custom genetic reference database, we show that eDNA metabarcoding recorded 196 putative species not detected by underwater visual census including 37 species absent from the regional checklist. We provide an updated checklist of marine fishes in the ‘Raja Ampat Bird's Head Peninsula' ecoregion with 2534 species including 1761 confirmed and 773 highly probable presences. The Chao lower-bound diversity estimator, based on the incidence of rare species, shows that the region potentially hosts an additional 123 fish species, including pelagic, cryptobenthic and vulnerable species. The extended and hidden biodiversity along with their asymptotic estimates highlight the ability of eDNA to expand regional inventories and species distributions to better guide conservation strategies

Sturgeons in large rivers: detecting the near‑extinct needles in a haystack via eDNA metabarcoding from water samples

Sturgeon populations are declining worldwide and are the target of extensive conservation efforts. Addressed in several pieces of legislation, sturgeons have received considerable attention as flagship or umbrella species. Despite the need for a better understanding of the distribution and population status, the use of traditional sampling methods failed in the past, thereby hampering reliable assessments, a prerequisite for conservation. Here, we describe the development and application of an environmental DNA (eDNA) metabarcoding approach for detecting rare sturgeons in large rivers. Exemplarily, we developed a reference database for five native Danube sturgeons (Acipenser stellatus, Acipenser gueldenstaedtii, Acipenser ruthenus, Acipenser nudiventris, and Huso huso) and two nonnative species (Acipenser baerii and Acipenser transmontanus), assessed these ex situ, and used eDNA as a detection tool along the entire length of the Danube (Europe, ~ 2850 km) and major tributaries. In ex situ analyses, all assays yielded positive amplifications for the assessed sturgeon species. In the Danube, the presence of A. ruthenus was confirmed at 14 of 29 sites (48.3%), and in 2 of 18 tributary sites (11.1%), providing the first comprehensive large-scale biogeographical snapshot of this species. Relative number of reads assigned to A. ruthenus varied between 0 and 2.5%, with sites registering positive detections being clustered in 3 sections of the Danube. Our findings enabled us to confirm the advantages of eDNA monitoring over traditional sampling methods for comprehensive whole-river snapshot studies of sturgeons conducted on a large geographical scale, and therefore we consider it to be a promising approach for application in conservation measures, fisheries management, scientific studies, and adaptive management plans for sturgeons on a global scale

Persistence of Environmental DNA in Freshwater Ecosystems

The precise knowledge of species distribution is a key step in conservation biology. However, species detection can be extremely difficult in many environments, specific life stages and in populations at very low density. The aim of this study was to improve the knowledge on DNA persistence in water in order to confirm the presence of the focus species in freshwater ecosystems. Aquatic vertebrates (fish: Siberian sturgeon and amphibian: Bullfrog tadpoles) were used as target species. In control conditions (tanks) and in the field (ponds), the DNA detectability decreases with time after the removal of the species source of DNA. DNA was detectable for less than one month in both conditions. The density of individuals also influences the dynamics of DNA detectability in water samples. The dynamics of detectability reflects the persistence of DNA fragments in freshwater ecosystems. The short time persistence of detectable amounts of DNA opens perspectives in conservation biology, by allowing access to the presence or absence of species e.g. rare, secretive, potentially invasive, or at low density. This knowledge of DNA persistence will greatly influence planning of biodiversity inventories and biosecurity surveys

Cross-ocean patterns and processes in fish biodiversity on coral reefs through the lens of eDNA metabarcoding

Increasing speed and magnitude of global change threaten the world's biodiversity and particularly coral reef fishes. A better understanding of large-scale patterns and processes on coral reefs is essential to prevent fish biodiversity decline but it requires new monitoring approaches. Here, we use environmental DNA metabarcoding to reconstruct well-known patterns of fish biodiversity on coral reefs and uncover hidden patterns on these highly diverse and threatened ecosystems. We analysed 226 environmental DNA (eDNA) seawater samples from 100 stations in five tropical regions (Caribbean, Central and Southwest Pacific, Coral Triangle and Western Indian Ocean) and compared those to 2047 underwater visual censuses from the Reef Life Survey in 1224 stations. Environmental DNA reveals a higher (16%) fish biodiversity, with 2650 taxa, and 25% more families than underwater visual surveys. By identifying more pelagic, reef-associated and crypto-benthic species, eDNA offers a fresh view on assembly rules across spatial scales. Nevertheless, the reef life survey identified more species than eDNA in 47 shared families, which can be due to incomplete sequence assignment, possibly combined with incomplete detection in the environment, for some species. Combining eDNA metabarcoding and extensive visual census offers novel insights on the spatial organization of the richest marine ecosystems

The PREDICTS database: a global database of how local terrestrial biodiversity responds to human impacts

Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species’ threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project – and avert – future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local-scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups – including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems – www.predicts.org.uk). We make site-level summary data available alongside this article. The full database will be publicly available in 2015

Biodiversity decline and inventory : the role of amphibian diseases and use of environmental DNA.

Depuis plusieurs décennies, un déclin important de la biodiversité est observé à l'échelle mondiale. Les amphibiens constituent aujourd'hui le groupe le plus vulnérable sur la planète. Près d'un tiers des espèces recensées dans le monde est à ce jour menacé d'extinction. Dans le cadre de ce doctorat, nous nous sommes intéressés dans un premier temps à l'émergence d'une maladie infectieuse des amphibiens, la chytridiomycose, provoquée par le champignon pathogène Batrachochytrium dendrobatidis (Bd). Nous avons étudié la répartition actuelle de ce champignon en France, démontré son impact sur la batrachofaune locale et mis en évidence le rôle de la Grenouille taureau (Lithobates catesbeianus) comme vecteur de transmission du pathogène. Afin de limiter la dissémination de Bd, nous avons également élaboré deux protocoles d'hygiène à mettre en oeuvre lors d'interventions sur le terrain ou lors d'utilisations d'amphibiens en laboratoire. Dans une seconde partie, nous avons développé une nouvelle méthode d'inventaire de la biodiversité basée sur la détection de l'ADN environnemental (ADNe). Nous avons mis en évidence que la persistance de l'ADNe dans un écosystème d'eau douce était d'environ 15 jours et que cette méthode innovante permettait d'améliorer la détection d'espèces exotiques envahissantes, comme la Grenouille taureau. Nous avons ensuite développé cette approche pour le suivi d'autres groupes taxonomiques (poissons, macro-invertébrés, chiroptères, etc.), dans des milieux différents et en utilisant notamment les technologies de séquençage nouvelle génération.Since several decades, a significant decline in biodiversity is observed worldwide. Amphibians are now the most vulnerable group on the planet. Nearly a third of known species in the world is today threatened of extinction. Among many causes, diseases appear as an emerging threat worldwide. As part of this PhD, we were interested at first to the emergence of an infectious disease of amphibians, chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). We studied the current distribution of this fungus mostly in France, showed the impact on local batrachofauna and highlighted the role of the Bullfrog (Lithobates catesbeianus) as a vector for transmission of the pathogen. To limit the spread of Bd, we also developed two hygiene protocols to implement during field trips or use of amphibians in the laboratory. In the second part of this thesis, we have developed a new method of biodiversity inventory based on the detection of environmental DNA (eDNA). We have shown that the persistence of vertebrates (fish and amphibian) eDNA in freshwater ecosystems was about 15 days and that this innovative method greatly improves the detection of invasive alien species, such as Bullfrog. We are then developed this approach for monitoring other taxonomic groups (fish, macro-invertebrates, bats, etc...), in various environments, taking advantage of bio-technological developments such as next generation DNA sequencing

Déclin et inventaire de la biodiversité (les maladies des amphibiens et la méthode de l'ADN environnemental)

Depuis plusieurs décennies, un déclin important de la biodiversité est observé à l'échelle mondiale. Les amphibiens constituent aujourd'hui le groupe le plus vulnérable sur la planète. Près d'un tiers des espèces recensées dans le monde est à ce jour menacé d'extinction. Dans le cadre de ce doctorat, nous nous sommes intéressés dans un premier temps à l'émergence d'une maladie infectieuse des amphibiens, la chytridiomycose, provoquée par le champignon pathogène Batrachochytrium dendrobatidis (Bd). Nous avons étudié la répartition actuelle de ce champignon en France, démontré son impact sur la batrachofaune locale et mis en évidence le rôle de la Grenouille taureau (Lithobates catesbeianus) comme vecteur de transmission du pathogène. Afin de limiter la dissémination de Bd, nous avons également élaboré deux protocoles d'hygiène à mettre en oeuvre lors d'interventions sur le terrain ou lors d'utilisations d'amphibiens en laboratoire. Dans une seconde partie, nous avons développé une nouvelle méthode d'inventaire de la biodiversité basée sur la détection de l'ADN environnemental (ADNe). Nous avons mis en évidence que la persistance de l'ADNe dans un écosystème d'eau douce était d'environ 15 jours et que cette méthode innovante permettait d'améliorer la détection d'espèces exotiques envahissantes, comme la Grenouille taureau. Nous avons ensuite développé cette approche pour le suivi d'autres groupes taxonomiques (poissons, macro-invertébrés, chiroptères, etc.), dans des milieux différents et en utilisant notamment les technologies de séquençage nouvelle génération.Since several decades, a significant decline in biodiversity is observed worldwide. Amphibians are now the most vulnerable group on the planet. Nearly a third of known species in the world is today threatened of extinction. Among many causes, diseases appear as an emerging threat worldwide. As part of this PhD, we were interested at first to the emergence of an infectious disease of amphibians, chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). We studied the current distribution of this fungus mostly in France, showed the impact on local batrachofauna and highlighted the role of the Bullfrog (Lithobates catesbeianus) as a vector for transmission of the pathogen. To limit the spread of Bd, we also developed two hygiene protocols to implement during field trips or use of amphibians in the laboratory. In the second part of this thesis, we have developed a new method of biodiversity inventory based on the detection of environmental DNA (eDNA). We have shown that the persistence of vertebrates (fish and amphibian) eDNA in freshwater ecosystems was about 15 days and that this innovative method greatly improves the detection of invasive alien species, such as Bullfrog. We are then developed this approach for monitoring other taxonomic groups (fish, macro-invertebrates, bats, etc...), in various environments, taking advantage of bio-technological developments such as next generation DNA sequencing.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF