Population declines of a widespread amphibian in agricultural landscapes.

Modern agricultural practices are suspected to play a major role in the ongoing erosion of biodiversity. In order to assess whether this biodiversity loss is linked to past habitat modifications (e.g. land consolidation) or to current consequences of modern agriculture (e.g. use of agrochemicals), it remains essential to monitor species that have persisted in agricultural landscapes to date. In this study, we assessed the presence, abundance and recent population trends of one such species, the spined toad (Bufo spinosus) along a gradient of habitats from preserved (forests) to highly agricultural sites in rural Western France. Our results showed that both presence and abundance of spined toads were markedly lower in reproductive ponds surrounded by intensive agriculture. The most salient result of our study is the ongoing decline of this species in farmland habitats. Indeed, this result suggests that unknown factors are currently affecting a widespread terrestrial amphibian previously thought to persist in agricultural landscapes. These factors have recently induced strong population declines over the course of a few years. Future investigations are required to identify these factors at a time when anthropogenic activities are currently leading to unprecedented rates of biodiversity loss.

Colouration matters in dull toads: ultraviolet adornment for ladies and agrochemicals fading effects.

Integument colouration can influence many aspects of fitness, and is under strong sexual selection. Amphibians often express sexual dichromatism, and ultra-violet (UV) colouration is usually biased toward males as a sexual signal. As an honest signal, colouration is related to several individual traits, but can also be related to environmental factors such as anthropogenic pollutants, to which amphibians are highly sensitive. In this study, we investigated sexual dichromatism and UV reflectance covering a large visual spectrum (wavelength ranging from 300 to 700 nm) on different body areas (throat, ventral and dorsal areas), in a widespread amphibian species, the spiny toad (Bufo spinosus). Then, we tested the impact of chronic exposure to two widespread herbicides (glyphosate's primary metabolite [AMPA] and Nicosulfuron) on their colouration. We found a strong but unexpected sexual dichromatism with females reflecting more in the UV spectrum (throat and ventral area) than males, suggesting these body parts might be critical in intra-specific signalling. Females with higher ventral UV reflectance were in better body condition, suggesting an honest signal role of UV reflectance which could influence male choice. Throat colouration was further differentially influenced by agrochemicals according to sexes. In AMPA-exposed males, throat was more saturated in yellow-orange than in control males, and Nicosulfuron exposure decreased the throat's reflectance hue in females, which can bear consequences on mate attractiveness. Future studies need to investigate the underlying mechanisms that are altered by agrochemical exposure.

What are the contributions of maternal and paternal traits to fecundity and offspring development? A case study in an amphibian species, the spined toad Bufo spinosus.

Assessing the determinants of reproductive success is critical but often complicated because of complex interactions between parental traits and environmental conditions occurring during several stages of a reproductive event. Here, we used a simplified ecological situation-an amphibian species lacking post-oviposition parental care-and a laboratory approach to investigate the relationships between parental (both maternal and paternal) phenotypes (body size and condition) and reproductive success (fecundity, egg size, embryonic and larval duration, larval and metamorphic morphology). We found significant effects of maternal phenotype on fecundity, hatching success, and tadpole size, as well as on the duration of larval development. Interestingly, and more surprisingly, we also found a potential contribution of the paternal phenotype occurring during early (embryonic development duration) offspring development. Although our study focused on life-history traits such as body size and development duration, additional mechanisms involving physiological costs of development may well mediate the relationships between parental phenotypes and offspring development. Future studies are required to decipher the mechanisms underlying our findings in order to clarify the mechanistic basis of the links between parental phenotypes and offspring development.

Exposure to Low Concentrations of AMPA Influences Morphology and Decreases Survival During Larval Development in a Widespread Amphibian Species.

Glyphosate's primary metabolite, AMPA (aminomethylphosphonic acid), is one of the most widely detected anthropogenic substance in surface waters worldwide. However, ecotoxicological studies on the potential effects of this metabolite at environmental concentrations on wildlife are scarce. Yet, due to its chemical properties, AMPA is likely to affect non-target species. In this study, we investigated sublethal effects of environmental concentrations of AMPA on the larval development of a widespread amphibian species, the spined toad Bufo spinosus. We performed a factorial experiment to study the effect of concentration and the timing of exposure (during embryonic development, larval development or both) to AMPA on the morphology, rate of development and survival of tadpoles. AMPA and timing of exposure interactively affected tadpole size (individuals exposed to AMPA after hatching were transitorily smaller, while individuals exposed to AMPA before hatching were longer), but not duration of development. Most of these effects were linked to exposure during embryonic development. Such effects in individuals exposed during embryonic development solely were long-lasting and persisted until the latest larval stages. Finally, we found that exposure to AMPA after hatching (during the larval stage) increased mortality. Exposure to low environmental concentrations of AMPA could have long-lasting consequences on fitness and population persistence. These findings are especially important to take into account at a time when multiple threats can interact to affect wildlife.

Exposure, but not timing of exposure, to a sulfonylurea herbicide alters larval development and behaviour in an amphibian species.

Environmental contamination is one of the major causes of biodiversity loss. Wetlands are particularly susceptible to contamination and species inhabiting these habitats are subjected to pollutants during sensitive phases of their development. In this study, tadpoles of a widespread amphibian, the spined toad (Bufo spinosus), were exposed to environmental concentrations of nicosulfuron (0 µg/L; 0.15 ± 0.05 µg/L and 0.83 ± 0.04 µg/L), a sulfonylurea herbicide, during different phases of development. Tadpoles were exposed during embryonic (12.98 ± 0.90 days) or larval development (93.74± 0.85 days), or throughout both phases, and we quantified development duration, morphological traits and behavioural features as responses to exposure. Developing tadpoles exposed to nicosulfuron were larger, but with smaller body, and had shorter but wider tail muscles. They were also more active and swam faster than control tadpoles and showed diverging patterns of behavioural complexity. We showed that higher concentrations had greater effects on individuals than lower concentrations, but the timing of nicosulfuron exposure did not influence the metrics studied: Exposure to nicosulfuron triggered similar effects irrespective of the developmental stages at which exposure occurred. These results further indicate that transient exposure (e.g., during embryonic development) can induce long-lasting effects throughout larval development to metamorphosis. Our study confirms that contaminants at environmental concentrations can have strong consequences on non-target organisms. Our results emphasize the need for regulation agencies and policy makers to consider sublethal concentrations of sulfonulyrea herbicides, such as nicosulfuron, as a minimum threshold in their recommendations.

Nicosulfuron, a sulfonylurea herbicide, alters embryonic development and oxidative status of hatchlings at environmental concentrations in an amphibian species.

The widespread use of agrochemicals for controlling pests and diseases of crops is recognized as a main threat to biodiversity. Sulfonylurea herbicides are being increasingly used and display low levels of degradation in water which suggest that they might affect non-target organisms. In a common garden experiment, eggs of a widespread amphibian (Bufo spinosus) were exposed to sublethal environmentally relevant concentrations of a widely used sulfonylurea herbicide, nicosulfuron, during the whole embryonic development. We assessed development-related traits (i.e., development duration, hatching success, hatchling size and occurrence of malformation) as well as antioxidant markers in response to contamination (i.e., SOD, GPx, catalase, thiols and relevant ratios thereof). We found that sublethal concentrations of nicosulfuron increased embryonic development duration, increased hatchling size and tended to increase malformations. Embryos exposed to nicosulfuron displayed decreased thiols and increased catalase activity suggesting alteration of oxidative status. We did not find any effect of nicosulfuron on SOD and GPx levels. Interestingly, higher catalase activity was linked to higher proportion of malformed individuals, suggesting that exposure to nicosulfuron induced teratogenic effects. Our results suggest that alteration of antioxidant levels might be one physiological mechanism through which nicosulfuron might cause detrimental effects on amphibian embryos. Sublethal effects of pesticides at environmentally relevant concentrations have been overlooked and require further investigations, especially in non-target taxa occurring in agricultural landscapes.

Aminomethylphosphonic acid (AMPA) alters oxidative status during embryonic development in an amphibian species.

Glyphosate's primary metabolite (aminomethylphosphonic acid, AMPA) is known to alter embryonic development at environmentally relevant concentrations in amphibians. However, we have limited understanding of the physiological mechanisms through which AMPA affects organisms. In this study, we tested whether alteration of the oxidative status is one mechanism through which AMPA affects organism performance. To this end, we analysed several oxidative status markers in hatchling tadpoles that were exposed to sublethal concentrations of AMPA during embryonic development (~16 days). We compared the influence of environmentally relevant concentrations of AMPA (from 0.07 to 3.57 µg l-1) on the relation between developmental traits (i.e, embryonic development duration, embryonic mortality and hatchling size) and oxidative status markers known to alter homeostasis when unbalanced (superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), thiols and ratios thereof). We included measures of telomere length as an indicator of physiological state. We found that AMPA concentrations induce non-monotonic effects on some oxidative status markers with hatchlings displaying elevated antioxidant responses (elevated thiols and unbalanced SOD/(GPx + CAT) ratio). The lack of effect of AMPA on the relation between developmental traits, oxidative status and telomere length suggests that selective mortality of embryos susceptible to oxidative stress may have occurred prior to hatching in individuals less resistant to AMPA which display lower hatching success. Future studies are required to disentangle whether oxidative unbalance is a cause or a consequence of AMPA exposition. This study highlights the need to investigate effects of the metabolites of contaminants at environmental concentrations to comprehensively assess impacts of anthropogenic contamination on wildlife.

Getting ready for a long bath: skin permeability decreases prior to aquatic breeding in male toads.

Vertebrate skin regulates exchanges between the organism and its environment and notably plays a fundamental role in regulating water fluxes. Dynamic changes of skin resistance to water fluxes are expected to occur in species that regularly shift between habitat types especially if these habitats differ in their hydric properties (e.g., terrestrial versus aquatic). We investigated changes of skin permeability using a study system (terrestrial toads) where reproduction induces a transition from terrestrial to freshwater habitats and a prolonged immersion that can last several weeks in males. In this system, the simultaneity between skin shedding and the onset of breeding suggests that the production of new integument layers prior to immersion for reproduction may regulate water influxes. We found that the skin permeability of male toads decreases significantly prior to breeding, suggesting that skin shedding at the onset of breeding regulates water fluxes to alleviate osmotic costs of immersion during reproduction. The continued decrease of skin permeability detected during breeding suggests that additional mechanisms interact with skin structure to further decrease permeability to water during a prolonged immersion. Future studies are required to assess whether changes in skin permeability to water tradeoffs with other skin characteristics (gas exchanges) relevant to aquatic breeding amphibians.

Aminomethylphosphonic acid alters amphibian embryonic development at environmental concentrations.

Despite intense societal and scientific debates regarding glyphosate toxicity, it remains the most widely used herbicide. The primary metabolite of glyphosate, AMPA (aminomethylphosphonic acid), is the main contaminant detected in surface waters worldwide, both because of the extensive use of glyphosate and because of other widespread sources of AMPA (i.e., industrial detergents). Studies on potential effects of glyphosate using environmentally relevant concentrations of AMPA on non-target wildlife species are lacking. We experimentally tested the effects of AMPA on embryonic development in a common European toad at concentrations spanning the range found in natural water bodies (from 0.07 to 3.57 µg l-1). Our experimental concentrations of AMPA were 100-6000 times lower than official Predicted-No-Effect-Concentrations. We found that these low-level concentrations of AMPA decreased embryonic survival, increased development duration and influenced hatchling morphology. Response patterns were more complex than classical linear concentration-response relationships, as concentration responses were nonmonotonic, with greater effects at low-concentrations of AMPA than at high levels. Based on our results we recommend that investigators focus not only on effects of "parent compounds," but also their metabolites at environmentally relevant concentrations in order to comprehensively assess impacts of anthropogenic contaminants on the environment.

Osmotic 'cost' of reproduction in breeding male toads.

Shifts between habitats during reproduction can induce costs that are independent of the reproductive effort and that often apply to both sexes. Such shifts can also illustrate physiological costs complementary to those involving energetic currencies. In this study, we investigated osmotic consequences of reproduction in a context where reproduction induces a shift from terrestrial habitats to freshwater environments. During reproduction, toads migrate to breeding ponds where males remain for several weeks, while females leave shortly after egg-laying. We assessed plasma osmolality of male spined toads during the whole reproductive period (approx. 30 days) in conjunction with markers of individual condition. We found that osmolality decreases during the protracted period of immersion in freshwater during reproduction, presumably through water influx as indicated by body mass changes. Hormonal markers of metabolism and sexual activity were positively correlated with osmolality. Recent research has highlighted hydric 'costs' of reproduction when access to water is limited. Our study adds to this growing field of investigation, yet with an opposite perspective, where water availability linked to reproduction provokes hyperhydration rather than dehydration.