Optimizing detectability of the endangered fan mussel using eDNA and ddPCR.

Spatial and temporal monitoring of species threatened with extinction is of critical importance for conservation and ecosystem management. In the Mediterranean coast, the fan mussel (Pinna nobilis) is listed as critically endangered after suffering from a mass mortality event since 2016, leading to 100% mortality in most marine populations. Conventional monitoring for this macroinvertebrate is done using scuba, which is challenging in dense meadows or with low visibility. Here we developed an environmental DNA assay targeting the fan mussel and assessed the influence of several environmental parameters on the species detectability in situ. We developed and tested an eDNA molecular marker and collected 48 water samples in two sites at the Thau lagoon (France) with distinct fan mussel density, depths and during two seasons (summer and autumn). Our marker can amplify fan mussel DNA but lacks specificity since it also amplifies a conspecific species (Pinna rudis). We successfully amplified fan mussel DNA from in situ samples with 46 positive samples (out of 48) using ddPCR, although the DNA concentrations measured were low over almost all samples. Deeper sampling depth slightly increased DNA concentrations, but no seasonal effect was found. We highlight a putative spawning event on a single summer day with much higher DNA concentration compared to all other samples. We present an eDNA molecular assay able to detect the endangered fan mussel and provide guidelines to optimize the sampling protocol to maximize detectability. Effective and non-invasive monitoring tools for endangered species are promising to monitor remaining populations and have the potential of ecological restoration or habitat recolonization following a mass mortality event.

Drivers of litter ingestion by sea turtles: Three decades of empirical data collected in Atlantic Europe and the Mediterranean.

Sea turtles are considered as bio-indicators for monitoring the efficiency of restoration measures to reduce marine litter impacts on health. However, the lack of extended and standardised empirical data has prevented the accurate analysis of the factors influencing litter ingestion and the relationships with individual health. Historic data collected from 1988 and standard data collected from 2016 were harmonised to enable such analyses on necropsied loggerhead turtles (Caretta caretta) in eight Mediterranean and North-East Atlantic countries. Litter was found in 69.24 % of the 1121 individuals, mostly single-use and fishing-related plastics. Spatial location, sex and life history stage explained a minor part of litter ingestion. While no relationships with health could be detected, indicating that all individuals can be integrated as bio-indicators, the mechanistic models published in literature suggest that the high proportion of plastics in the digestive contents (38.77 % per individual) could have long-term repercussions on population dynamics.

Landscape epidemiology of Batrachochytrium salamandrivorans: reconciling data limitations and conservation urgency.

Starting in 2010, rapid fire salamander (Salamandra salamandra) population declines in northwestern Europe heralded the emergence of Batrachochytrium salamandrivorans (Bsal), a salamander-pathogenic chytrid fungus. Bsal poses an imminent threat to global salamander diversity owing to its wide host range, high pathogenicity, and long-term persistence in ecosystems. While there is a pressing need to develop further research and conservation actions, data limitations inherent to recent pathogen emergence obscure necessary insights into Bsal disease ecology. Here, we use a hierarchical modeling framework to describe Bsal landscape epidemiology of outbreak sites in light of these methodological challenges. Using model selection and machine learning, we find that Bsal presence is associated with humid and relatively cool, stable climates. Outbreaks are generally located in areas characterized by low landscape heterogeneity and low steepness of slope. We further find an association between Bsal presence and high trail density, suggesting that human-mediated spread may increase risk for spillover between populations. We then use distribution modeling to show that favorable conditions occur in lowlands influenced by the North Sea, where increased survey effort is needed to determine how Bsal impacts local newt populations, but also in hill- and mountain ranges in northeastern France and the lower half of Germany. Finally, connectivity analyses suggest that these hill- and mountain ranges may act as stepping stones for further spread southward. Our results provide initial insight into regional environmental conditions underlying Bsal epizootics, present updated invasibility predictions for northwestern Europe, and lead us to discuss a wide variety of potential survey and research actions needed to advance future conservation and mitigation efforts.

Using Boops boops (osteichthyes) to assess microplastic ingestion in the Mediterranean Sea.

This study assesses microplastic ingestion in Boops boops at different geographical areas in the Mediterranean Sea. A total of 884 fish were caught at 20 coastal sites in Spain, France, Italy and Greece and analyzed using a common methodological protocol. Microplastics were found in 46.8% of the sampled fish, with an average number of items per individual of 1.17 ± 0.07. Filaments were the predominant shape type, while polyethylene and polypropylene were indicated by FTIR as the most common polymer types of ingested microplastics. The frequency of occurrence, as well as the abundance and proportion of types (size, shape, color and polymer) of ingested microplastics, varied among geographical areas. The spatial heterogeneity of the abundance of ingested microplastics was mainly related to the degree of coastal anthropogenic pressure at the sampling sites. Our findings further support the suitability of B. boops as bioindicator of microplastic pollution in the Mediterranean Sea.

Slow life-history strategies are associated with negligible actuarial senescence in western Palaearctic salamanders.

Actuarial senescence has been viewed for a long time as an inevitable and uniform process. However, the work on senescence has mainly focused on endotherms with deterministic growth and low regeneration capacity during the adult stage, leading to a strong taxonomic bias in the study of ageing. Recent studies have highlighted that senescence could indeed display highly variable trajectories that correlate with species life-history traits. Slow life histories and indeterminate growth seem to be associated with weak and late senescence. Furthermore, high regenerative abilities could lead to negligible senescence in ectotherms. However, demographic data for species that would allow testing of these hypotheses are scarce. Here, we investigated senescence patterns in 'true salamanders' from the western Palaearctic. Our results showed that salamanders have slow life histories and that they experience negligible senescence. This pattern was consistent at both intra- and interspecific levels, suggesting that the absence of senescence may be a phylogenetically conserved trait. The regenerative capacities of salamanders, in combination with other physiological and developmental features such as an indeterminate growth and a low metabolic rate, probably explain why these small ectotherms have lifespans similar to that of large endotherms and, in contrast with most amniotes, undergo negligible senescence. Our study seriously challenges the idea that senescence is a ubiquitous phenomenon in the tree of life.

Data Collection on Marine Litter Ingestion in Sea Turtles and Thresholds for Good Environmental Status.

The following protocol is intended to respond to the requirements set by the European Union's Marine Strategy Framework Directives (MSFD) for the D10C3 Criteria reported in the Commission Decision (EU), related to the amount of litter ingested by marine animals. Standardized methodologies for extracting litter items ingested from dead sea turtles along with guidelines on data analysis are provided. The protocol starts with the collection of dead sea turtles and classification of samples according to the decomposition status. Turtle necropsy must be performed in authorized centers and the protocol described here explains the best procedure for gastrointestinal (GI) tract isolation. The three parts of the GI (esophagus, stomach, intestine) should be separated, opened lengthways and contents filtered using a 1 mm mesh sieve. The article describes the classification and quantification of ingested litter, classifying GI contents into seven different categories of marine litter and two categories of natural remains. The quantity of ingested litter should be reported as total dry mass (weight in grams, with two decimal places) and abundance (number of items). The protocol proposes two possible scenarios to achieve the Good Environmental Status (GES). First: "There should be less than X% of sea turtles having Y g or more plastic in the GI in samples of 50-100 dead turtles from each sub-region", where Y is the average weight of plastic ingested and X% is the percentage of sea turtles with more weight (in grams) of plastic than Y. The second one, which considers the food remain versus plastic as a proxy of individual health, is: "There should be less than X% of sea turtles having more weight of plastic (in grams) than food remains in the GI in samples of 50-100 dead turtles from each sub-region".

eDNA Increases the Detectability of Ranavirus Infection in an Alpine Amphibian Population.

The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can also infect other hosts than amphibian species. Diagnostic techniques allowing the detection of the pathogen outside the period of host die-off would thus be of particular use. In this study, we tested a method using environmental DNA (eDNA) on a population of common frogs (Rana temporaria) known to be affected by a Ranavirus in the southern Alps in France. In six sampling sessions between June and September (the species' activity period), we collected tissue samples from dead and live frogs (adults and tadpoles), as well as insects (aquatic and terrestrial), sediment, and water. At the beginning of the breeding season in June, one adult was found dead; at the end of July, a mass mortality of tadpoles was observed. The viral DNA was detected in both adults and tadpoles (dead or alive) and in water samples, but it was not detected in insects or sediment. In live frog specimens, the virus was detected from June to September and in water samples from August to September. Dead tadpoles that tested positive for Ranavirus were observed only on one date (at the end of July). Our results indicate that eDNA can be an effective alternative to tissue/specimen sampling and can detect Ranavirus presence outside die-offs. Another advantage is that the collection of water samples can be performed by most field technicians. This study confirms that the use of eDNA can increase the performance and accuracy of wildlife health status monitoring and thus contribute to more effective surveillance programs.

Determining suitable fish to monitor plastic ingestion trends in the Mediterranean Sea.

The presence of marine litter is a complex, yet persistent, threat to the health and biodiversity of the marine environment, and plastic is the most abundant, and ubiquitous type of marine litter. To monitor the level of plastic waste in an area, and the prospect of it entering the food chain, bioindicator species are used extensively throughout Northern European Seas, however due to their distribution ranges many are not applicable to the Mediterranean Sea. Guidance published for the Marine Strategy Framework Directive suggests that the contents of fish stomachs may be analyzed to determine trends of marine plastic ingestion. In order to equate transnational trends in marine plastic ingestion, the use of standardized fish species that widely occur throughout the basin is favoured, however for the Mediterranean Sea, specific species are not listed. Here we propose a methodology to assess how effective Mediterranean fish species, that are known to have ingested marine plastic, are as bioindicators. A new Bioindicator Index (BI) was established by incorporating several parameters considered important for bioindicators. These parameters included species distribution throughout the Mediterranean basin, several life history traits, the commercial value of each species, and the occurrence of marine litter in their gut contents. By collecting existing data for Mediterranean fish, ranked scores were assigned to each trait and an average value (BI value) was calculated for each species. Based on their habitat preferences, Engraulis encrasicolus (pelagic), Boops boops (benthopelagic), three species of Myctophidae (Hygophum benoiti, Myctophum punctatum and Electrona risso) (mesopelagic), Mullus barbatus barbartus (demersal) and Chelidonichthys lucerna (benthic), were identified as currently, the most suitable fish for monitoring the ingestion of marine plastics throughout the Mediterranean basin. The use of standardized indicator species will ensure coherence in the reporting of marine litter ingestion trends throughout the Mediterranean Sea.

Recent Asian origin of chytrid fungi causing global amphibian declines.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide.

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi.

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

Does habitat unpredictability promote the evolution of a colonizer syndrome in amphibian metapopulations?

Dispersal is a central component of life history evolution. An increasing number of studies suggest that spatiotemporally variable environments may promote the evolution of "dispersal syndromes," consisting of covariation patterns between dispersal and morphological, physiological, behavioral, and life history traits. At the interspecific scale, the "colonizer syndrome" appears to be one of the most frequently recorded associations between dispersal and life history traits, linking a high dispersal rate, high fecundity, and a short lifespan as systematically combined adaptations in spatiotemporally varying environments. However, few studies have highlighted the existence of a "colonizer syndrome" at the intraspecific scale, and none have investigated how different degrees of habitat stochasticity might shape covariation patterns between dispersal and life history traits. In this study, we examined this issue in free-ranging metapopulations of the yellow-bellied toad (Bombina variegata) using capture-recapture data. Combining the results of this study with another recent study, we found that a high dispersal rate, high fecundity, and a short lifespan are associated in metapopulations experiencing unpredictable environments. In contrast, a very low dispersal rate (close to zero), low fecundity and a long lifespan are associated in metapopulations occupying predictable environments. We discuss these results as well as their demographic and evolutionary consequences.

Contrasting patterns of environmental fluctuation contribute to divergent life histories among amphibian populations.

Because it modulates the fitness returns of possible options of energy expenditure at each ontogenetic stage, environmental stochasticity is usually considered a selective force in driving or constraining possible life histories. Divergent regimes of environmental fluctuation experienced by populations are expected to generate differences in the resource allocation schedule between survival and reproductive effort and outputs. To our knowledge, no study has previously examined how different regimes of stochastic variation in environmental conditions could result in changes in both the temporal variation and mean of demographic parameters, which could then lead to intraspecific variation along the slow-fast continuum of life history tactics. To investigate these issues, we used capture-recapture data collected on five populations of a long-lived amphibian (Bombina variegata) experiencing two distinct levels of stochastic environmental variation: (1) constant availability of breeding sites in space and time (predictable environment), and (2) variable spatio-temporal availability of breeding sites (unpredictable environment). We found that female breeding propensity varied more from year to year in unpredictable than in predictable environments. Although females in unpredictable environments produced on average more viable offspring per year, offspring production was more variable between years. Survival at each ontogenetic stage was slightly lower and varied significantly more from year to year in unpredictable environments. Taken together, these results confirm that increased environmental stochasticity can modify the resource allocation schedule between survival and reproductive effort and outputs and may lead to intraspecific variation along the slow-fast continuum of life history tactics.

Demographic responses to weather fluctuations are context dependent in a long-lived amphibian.

Weather fluctuations have been demonstrated to affect demographic traits in many species. In long-lived organisms, their impact on adult survival might be buffered by the evolution of traits that reduce variation in interannual adult survival. For example, skipping breeding is an effective behavioral mechanism that may limit yearly variation in adult survival when harsh weather conditions occur; however, this in turn would likely lead to strong variation in recruitment. Yet, only a few studies to date have examined the impact of weather variation on survival, recruitment and breeding probability simultaneously in different populations of the same species. To fill this gap, we studied the impact of spring temperatures and spring rainfall on survival, on reproductive skipping behavior and on recruitment in five populations of a long-lived amphibian, the yellow-bellied toad (Bombina variegata). Based on capture-recapture data, our findings demonstrate that survival depends on interactions between age, population and weather variation. Varying weather conditions in the spring result in strong variation in the survival of immature toads, whereas they have little effect on adult toads. Breeding probability depends on both the individual's previous reproductive status and on the weather conditions during the current breeding season, leading to high interannual variation in recruitment. Crucially, we found that the impact of weather variation on demographic traits is largely context dependent and may thus differ sharply between populations. Our results suggest that studies predicting the impact of climate change on population dynamics should be taken with caution when the relationship between climate and demographic traits is established using only one population or few populations. We therefore highly recommend further research that includes surveys replicated in a substantial number of populations to account for context-dependent variation in demographic processes.

RANAVIRUS CAUSES MASS DIE-OFFS OF ALPINE AMPHIBIANS IN THE SOUTHWESTERN ALPS, FRANCE.

Pathogenic fungi and viruses cause mortality outbreaks in wild amphibians worldwide. In the summer of 2012, dead tadpoles and adults of the European common frog Rana temporaria were reported in alpine lakes in the southwestern Alps (Mercantour National Park, France). A preliminary investigation using molecular diagnostic techniques identified a Ranavirus as the potential pathogenic agent. Three mortality events were recorded in the park, and samples were collected. The amphibian chytrid fungus Batrachochytrium dendrobatidis was not detected in any of the dead adult and juvenile frogs sampled (n=16) whereas all specimens were positive for a Ranavirus. The genome sequence of this Ranavirus was identical to previously published sequences of the common midwife toad virus (CMTV), a Ranavirus that has been associated with amphibian mortalities throughout Europe. We cultured virus from the organs of the dead common frogs and infecting adult male common frogs collected in another alpine region where no frog mortality had been observed. The experimentally infected frogs suffered 100% mortality (n=10). The alpine die-off is the first CMTV outbreak associated with mass mortality in wild amphibians in France. We describe the lesions observed and summarize amphibian populations affected by Ranaviruses in Europe. In addition, we discuss the ecologic specificities of mountain amphibians that may contribute to increasing their risk of exposure to and transmission of Ranaviruses.

Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding.

Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present in the environment. In this study, we tested whether an environmental DNA (eDNA) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, in vitro and in situ. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 (CI = 0.90-0.99) vs. 0.58 (CI = 0.50-0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA-based approach has the potential to become the next-generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems.

To breed or not to breed: past reproductive status and environmental cues drive current breeding decisions in a long-lived amphibian.

Iteroparity is an adaptive response to uncertainty in reproductive success. However, spreading reproductive success over multiple reproduction events during a lifetime is constrained by adult mortality and the stochasticity associated with interactions between external factors and physiological states. The acquisition of information about environmental conditions during the growth of progeny and sufficient resources during the non-reproductive period are key factors for breeding success. Consequently, we hypothesized that long-lived animals may skip a breeding opportunity when information about unfavourable environmental conditions is available. In addition, nutritional constraints could prevent an animal from replenishing its reserves sufficiently to invest in the current breeding period. We investigated these questions using capture-recapture data from a 5-year study on a large population of yellow-bellied toads in a forest in north-eastern France. We took advantage of various advances in multi-state capture-recapture models (e.g. unobservable states and mixture models) to test our hypotheses. Our results show that the combined effects of rainfall deficit and the breeding/non-breeding state of individuals during the past breeding season affect breeding probability during the following breeding opportunity. We also found that females breed less frequently than males, suggesting that the overall energy cost of reproduction differs between genders. Finally, the results indicate that toad survival appears to be negatively influenced by rainfall deficits. We discuss the yellow-bellied toad's reproductive behaviour in term of bet-hedging strategy and life history trait evolution.

Toxicokinetic of benzo[a]pyrene and fipronil in female green frogs (Pelophylax kl. esculentus).

A general consensus that an increased logK(ow) led to an increase in xenobiotic uptake and bioaccumulation is accepted. In this study we compared the toxicokinetics of two chemically different xenobiotics, i.e. benzo[a]pyrene and fipronil in female green frogs. Surprisingly, the uptake rates and the bioconcentration factors (BCF) of the two contaminants were not predicted by their logK(ow). The uptake rates obtained were of the same order of magnitude for the two contaminants and the BCFs measured for fipronil were about 3-fold higher than those obtained for benzo[a]pyrene. Fipronil appeared to be more recalcitrant than benzo[a]pyrene to detoxification processes leading to the accumulation of sulfone-fipronil especially in the ovaries. This phenomenon may explain reproductive influence of this contaminant described in other studies. Detoxification processes, including metabolism and the excretion of pollutants, are of importance when considering their persistence in aquatic organisms and trying to quantify their risks.

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.

Intra-specific variation in nitrate tolerance in tadpoles of the Natterjack toad.

Anthropogenic sources of nitrogen that pollute bodies of water can have toxic and sub-lethal effects on amphibians. It has been hypothesized that such exposure may promote local adaptation, that is, selection for higher tolerance in individuals in populations exposed to pollutants. We tested this hypothesis with respect to the Natterjack toad (Bufo calamita Laurenti, 1768), by comparing the nitrate dose response of tadpoles from eight populations (doses: 0, 50, 100, 500 and 1000 mg/l nitrate) from relatively unpolluted and intensively farmed environments. We evaluated the effect of nitrate exposure by observing the behavior (movements) of tadpoles exposed to different concentrations of nitrates. Exposure to high nitrate levels did not cause tadpole mortality in the populations used in our experiments; however, we did observe changes in activity for all populations, with these changes being either dose-related responses (decreased activity after exposure to 500 or 1000 mg/l), or more complex responses (increased activity when exposed to 50 or 100 mg/l nitrate, followed by decreased activity at higher concentrations). Natterjack toad tadpoles exhibited variable behavioural responses among the tested populations. Although these populations were selected on the basis of their potential agrochemical contamination, the observed variation in population tolerance was not related to the parameters used to estimate this contamination in these breeding sites. Possible explanations for this apparent lack of local adaptation in B. calamita tadpoles include inadequate estimates of the toads' actual nitrate exposure in the field, and the biological characteristics of B. calamita, which may limit the effects of exposure or favor phenotypic plasticity.

Using amphibians in laboratory studies: precautions against the emerging infectious disease chytridiomycosis.

The African clawed frog Xenopus laevis is by far the most widely used amphibian species in laboratories. In the wild, X. laevis is an asymptomatic carrier of an emerging infectious disease called chytridiomycosis. The vector is the chytrid fungus Batrachochytrium dendrobatidis (Bd), which has devastating effects on wild amphibian populations around the world. The impact of Bd on the metabolism of X. laevis has not been comprehended yet. However, even if asymptomatic, an infection is likely to affect the individual's physiology, immunology, development, reproduction and overall response to stress from a purely medical point of view, which will introduce noise and therefore increase variance within experimental groups of X. laevis. This could have implications on the scientific results from studies using this species. Here, we review the current knowledge on treatments of infected amphibians and propose a hygiene protocol adapted to laboratory populations and amphibian husbandry. Following the presented sanitation guidelines could further prevent the spread of Bd and probably of other amphibian pathogens. The sanitation guidelines will help to reduce the impact of amphibian husbandry on natural populations and must be considered a crucial contribution to amphibian conservation, as today 32% of all amphibians are considered threatened.