The functional role of Daphnia in the host-pathogen interaction of crayfish and the crayfish plague disease agent (Aphanomyces astaci).

Pathogen spores have been recognized as prey with implications for resource dynamics, energy transfer and disease transmission. In aquatic ecosystems, filter-feeders are able to consume such motile forms of pathogens that can cause severe disease in susceptible hosts. The interactions between European crayfish and the crayfish plague pathogen Aphanomyces astaci are of particular conservation interest. In this study, we aim to evaluate the ecological interactions between Ap. astaci, its host Astacus astacus and individuals of the genus Daphnia, filter-feeding planktonic crustaceans. Our focus was on the consumption of the motile zoospores by Daphnia individuals, but we also considered the potential of Daphnia as non-target hosts. We conducted a series of infection and life-history experiments with Ap. astaci, three Daphnia species (D. magna, D. galeata, and D. pulex) and the noble crayfish As. astacus. We did not observe any lethal effects in the infection experiments involving Ap. astaci and Daphnia. Only D. pulex showed differences in some life-history traits. The feeding experiment using the motile zoospores of Ap. astaci as alternative food source or as supplement to different amounts of algal food revealed their nutritional value: D. magna individuals survived, grew, and reproduced on a zoospore diet alone. When zoospores were supplemented to the regular algal diet, all life-history parameters have been significantly improved. However, this successful consumption of zoospores did not result in a reduced mortality of the susceptible crayfish As. astacus during the infection experiment. Nevertheless, the pathogen load of Ap. astaci in the tissues of As. astacus was significantly reduced as a consequence of the feeding activity of Daphnia. Our results indicate that an abundant filter-feeding community can reduce the amount of infective zoospores in the water body and thus be beneficial to susceptible crayfish hosts, potentially acting as a general buffer against zoospore-transmitted diseases in lentic waters.

Low temperatures lead to higher toxicity of the fungicide folpet to larval stages of Rana temporaria and Bufotes viridis.

Pesticides are one of the main drivers of the worldwide amphibian decline. Their actual toxicity depends on a number of factors, like the species in focus or the developmental stage of exposed individuals. As ectothermic species, the metabolism of amphibians is influenced by ambient temperature. Therefore, temperature also affects metabolic rates and thus processes that might enhance or reduce toxic effects. Studies about the interactive effect of temperature and toxicity on amphibians are rare and deliver contrasting results. To investigate the temperature-dependent pesticide sensitivity of larvae of two European species we conducted acute toxicity tests for the viticultural fungicide Folpan® 500 SC with the active ingredient folpet at different temperatures (6°C, 11°C, 16°C, 21°C, 26°C). Sensitivity of Rana temporaria and Bufotes viridis was highly affected by temperature: early larvae (Gosner stage 20) were about twice more sensitive to Folpan® 500 SC at 6°C compared to 21°C. Next to temperature, species and developmental stage of larvae had an effect on sensitivity. The most sensitive individuals (early stages of R. temporaria at 6°C) were 14.5 times more sensitive than the least sensitive ones (early stages of B. viridis at 26°C). Our results raise concerns about typical ecotoxicological studies with amphibians that are often conducted at temperatures between 15°C and 20°C. We suggest that future test designs should be performed at temperatures that reflect the temperature range amphibians are exposed to in their natural habitats. Variations in the sensitivity due to temperature should also be considered as an uncertainty factor in upcoming environmental risk assessments for amphibians.

Pesticide exposure affects reproductive capacity of common toads (Bufo bufo) in a viticultural landscape.

Amphibian populations are declining worldwide at alarming rates. Among the large variety of contributing stressors, chemical pollutants like pesticides have been identified as a major factor for this decline. Besides direct effects on aquatic and terrestrial amphibian stages, sublethal effects like impairments in reproduction can affect a population. Therefore, we investigated the reproductive capacity of common toads (Bufo bufo) in the pesticide-intensive viticultural landscape of Palatinate in Southwest Germany along a pesticide gradient. In a semi-field study, we captured reproductively active common toad pairs of five breeding ponds with different pesticide contamination level and kept them in a net cage until spawning. Toads from more contaminated ponds showed an increased fecundity (more eggs) but decreased fertilization rates (fewer hatching tadpoles) as well as lower survival rates and reduced size in Gosner stage 25, suggesting that the higher exposed populations suffer from long-term reproductive impairments. In combination with acute toxicity effects, the detected sublethal effects, which are mostly not addressed in the ecological risk assessment of pesticides, pose a serious threat on amphibian populations in agricultural landscapes.

Avoidance behavior of juvenile common toads (Bufo bufo) in response to surface contamination by different pesticides.

Most agricultural soils are expected to be contaminated with agricultural chemicals. As the exposure to pesticides can have adverse effects on non-target organisms, avoiding contaminated areas would be advantageous on an individual level, but could lead to a chemical landscape fragmentation with disadvantages on the metapopulation level. We investigated the avoidance behavior of juvenile common toads (Bufo bufo) in response to seven pesticide formulations commonly used in German vineyards. We used test arenas filled with silica sand and oversprayed half of each with different pesticide formulations. We placed a toad in the middle of an arena, filmed its behavior over 24 hours, calculated the proportion of time a toad spent on the contaminated side and compared it to a random side choice. We found evidence for the avoidance of the folpet formulation Folpan® 500 SC, the metrafenone formulation Vivando® and the glyphosate formulation Taifun® forte at maximum recommended field rates for vine and a trend for avoidance of Wettable Sulphur Stulln (sulphur). No avoidance was observed when testing Folpan® 80 WDG (folpet), Funguran® progress (copper hydroxide), SpinTorTM (spinosad), or 10% of the maximum field rate of any formulation tested. In the choice-tests in which we observed an avoidance, toads also showed higher activity on the contaminated side of the arena. As video analysis with tracking software is not always feasible, we further tested the effect of reducing the sampling interval for manual data analyses. We showed that one data point every 15 or 60 minutes results in a risk of overlooking a weak avoidance behavior, but still allows to verify the absence/presence of an avoidance for six out of seven formulations. Our findings are important for an upcoming pesticide risk assessment for amphibians and could be a template for future standardized tests.

Potential pesticide exposure during the post-breeding migration of the common toad (Bufo bufo) in a vineyard dominated landscape.

Two important drivers of the global amphibian decline are habitat destruction due to an intensification of farming and a related increase of pesticide applications. Recent studies have shown that there might be an underestimated risk of pesticides on terrestrial amphibians. However, there are too few data on the terrestrial habitat use of amphibians in agricultural landscapes to estimate the exposure risk. To fill this knowledge gap, we used telemetry to investigate the post-breeding migration of 51 common toads (Bufo bufo) from a breeding pond in a vineyard-dominated landscape in Southern Palatinate (Germany). We expected most toads to migrate to the nearby Palatinate Forest as a terrestrial habitat. However, only four individuals reached the forest, suggesting that a part of the population is inhabiting the agricultural landscape over large parts of the year. Individuals were also found directly in the vineyards (15% of all relocations), but 23% less often than expected from a random choice and therefore tend to avoid vineyards as terrestrial habitat. To estimate a possible spatial-temporal overlap of toad migration and pesticide application, we combined telemetry data with information about pesticide applications from local wine growers. Seven individuals had a high probability (>75%) of being directly exposed to a pesticide application. Taking spray drift and the half-life values of applied pesticides into account, the number of toads potentially exposed raised to 15 individuals. We estimated that, on a single day up to 24% of the whole breeding population came in contact with pesticides, resulting in a high overall exposure risk. Pesticides can have negative effects on amphibians, and toads try to avoid vineyards as habitats. Therefore, we conclude that a heterogeneous cultural landscape, with buffer strips around ponds, uncultivated patches and migration corridors, might be the best management measure for sustaining amphibians in the agricultural landscape.

Validating buccal swabbing as a minimal-invasive method to detect pesticide exposure in squamate reptiles.

The use of enzymatic biomarkers constitutes a widely used approach in ecotoxicology. However, standard sampling procedures are invasive, requiring tissue, organ or blood extraction. This leads to concerns regarding conservation practice, animal welfare and restrictions in study design. New techniques are needed to avoid these problems, but still generate reliable data. Here, we aimed at validating the use of buccal swabs as a reliable method to detect pesticide exposure in reptiles. Common wall lizards (Podarcis muralis) were divided into control, dermal and oral treatment groups and exposed to different pesticide formulations. Subsequently, buccal swabs were taken and enzymatic activity was analyzed. We were able to confirm the suitability of the method to detect effects of pesticide exposure on the enzymatic level. While exposure to the formulation Roundup Ultramax® didn't match when compared to effects previously observed in situ when compared to other glyphosate based formulations, effects could still be detected. This can be seen as a strong indicator that the active ingredient of a formulation may not always be the mian driver for ecotoxicological effectsat the enzymatic level. At the same time, exposure towards the single formulation Vivando® didn't result in any effects. However, individuals residing in agricultural landscapes will mostly be exposed to pesticide mixes containing different formulations. Our results strongly advocate that buccal swabbing is a reliable minimal invasive method to generate samples for detecting effects of pesticide exposure in reptiles. Due to its easy handling, we believe it will provide new opportunities concerning study designs.

Amphibian population genetics in agricultural landscapes: does viniculture drive the population structuring of the European common frog (Rana temporaria)?

Amphibian populations have been declining globally over the past decades. The intensification of agriculture, habitat loss, fragmentation of populations and toxic substances in the environment are considered as driving factors for this decline. Today, about 50% of the area of Germany is used for agriculture and is inhabited by a diverse variety of 20 amphibian species. Of these, 19 are exhibiting declining populations. Due to the protection status of native amphibian species, it is important to evaluate the effect of land use and associated stressors (such as road mortality and pesticide toxicity) on the genetic population structure of amphibians in agricultural landscapes. We investigated the effects of viniculture on the genetic differentiation of European common frog (Rana temporaria) populations in Southern Palatinate (Germany). We analyzed microsatellite data of ten loci from ten breeding pond populations located within viniculture landscape and in the adjacent forest block and compared these results with a previously developed landscape permeability model. We tested for significant correlation of genetic population differentiation and landscape elements, including land use as well as roads and their associated traffic intensity, to explain the genetic structure in the study area. Genetic differentiation among forest populations was significantly lower (median pairwise FST = 0.0041 at 5.39 km to 0.0159 at 9.40 km distance) than between viniculture populations (median pairwise FST = 0.0215 at 2.34 km to 0.0987 at 2.39 km distance). Our analyses rejected isolation by distance based on roads and associated traffic intensity as the sole explanation of the genetic differentiation and suggest that the viniculture landscape has to be considered as a limiting barrier for R. temporaria migration, partially confirming the isolation of breeding ponds predicted by the landscape permeability model. Therefore, arable land may act as a sink habitat, inhibiting genetic exchange and causing genetic differentiation of pond populations in agricultural areas. In viniculture, pesticides could be a driving factor for the observed genetic impoverishment, since pesticides are more frequently applied than any other management measure and can be highly toxic for terrestrial life stages of amphibians.