Physiological Stress Responses in Amphibian Larvae to Multiple Stressors Reveal Marked Anthropogenic Effects even below Lethal Levels

Natural and anthropogenic disturbances cause profound alterations in organisms, inducing physiological adjustments to avoid, reduce, or remedy the impact of disturbances. In vertebrates, the stress response is regulated via neuroendocrine pathways, including the hypothalamic-pituitary-interrenal axis that regulates the secretion of glucocorticoids. Glucocorticoids have cascading effects on multiple physiological pathways, affecting the metabolic rate, reactive oxygen species production, or immune system. Determining the extent to which natural and anthropogenic environmental factors induce stress responses in vertebrates is of great importance in ecology and conservation biology. Here we study the physiological stress response in spadefoot toad tadpoles (Pelobates cultripes) against three levels of a series of natural and anthropogenic stressors common to many aquatic systems: salinity (0, 6, and 9 ppt), herbicide (0, 1, and 2 mg/L acid equivalent of glyphosate), water acidity (pH 4.5, 7.0, and 9.5), predators (absent, native, and invasive), and temperature (217, 257, and 29 degrees C). The physiological stress response was assessed examining corticosterone levels, standard metabolic rate, activity of antioxidant enzymes, oxidative cellular damage in lipids, and immunological status. We found that common stressors substantially altered the physiological state of tadpoles. In particular, salinity and herbicides cause dramatic physiological changes in tadpoles. Moreover, tadpoles reduced corticosterone levels in the presence of natural predators but did not do so against invasive predators, indicating a lack of innate recognition. Corticosterone and the antioxidant enzyme glutathione reductase were the most sensitive parameters to stress in this study. Anthropogenic perturbations of aquatic systems pose serious threats to larval amphibians even at nonlethal concentrations, judging from the marked physiological stress responses generated, and reveal the importance of incorporating physiological information onto conservation, ecological, and evolutionary studies.Peer Reviewe

Mechanisms and costs of developmental plasticity in amphibian larvae

Programa de Doctorado en Estudios MedioambientalesNature is complex and organisms commonly need to rapidly be able to detect and respond to environmental inputs in order to increase their survival odds. The ability of a given genotype to alter its morphology, behavior or development against changing environments is known as phenotypic plasticity, which is adaptive when the induced phenotypes confer increased fitness in the altered environment. Adaptive plasticity favors phenotypic diversity and increases population viability, as well as facilitates the maintenance of genetic variation reducing the severity of bottleneck events during rapid environmental change, and also by shielding genetic variants from selection. In particular, phenotypic plasticity is essential for species with low vagility and high philopatry, as is the case of most amphibian species. The life-cycle of amphibians is often a complex one that includes an aquatic larval stage that gives rise to a terrestrial stage through metamorphosis. Metamorphosis is thus a key ontogenetic switch point that entails vast anatomical, physiological and ecological changes in the organism. The timing and body condition at which metamorphosis occurs largely determine the likelihood of survival in larval and juvenile amphibians. Growth and development are remarkably decoupled over long periods of the amphibian larval ontogeny. This allows amphibian larvae to grow without advancing in development under benign conditions of high food availability, reduced competition, and abundant water, or else accelerate development at the expense of truncating growth when conditions worsen, as when at risk of pond drying. Such fine-tuning of growth and development relies on the ability of amphibian larvae to sense their environment and is regulated by neuroendocrine pathways, which can activate/repress multiple metabolic cascades, which in turn can involve short and long-term consequences for body condition and even life span. The main objective of this thesis is to understand the physiological mechanisms enabling developmental and growth plasticity in amphibians, and their consequences. Firstly, several simultaneous experiments were conducted to study the physiological alterations inflicted by common potential external stressors on amphibian larvae. In particular, we studied the effect of varying levels of salinity, herbicide, water pH, types of predators, and temperature in spadefoot toad larvae (Pelobates cultripes) evaluating their changes in corticosterone level, metabolic rate, activity of various antioxidant enzymes, lipid peroxidation, and immune state. Most of the studied levels factors caused some physiological imbalances in tadpoles, although high levels of salinity and herbicide caused the most dramatic physiological alterations. Tadpoles showed decreased levels of corticosterone when exposed to native predators, congruent with the common reduction in foraging and metabolic activity in the presence of predators. Interestingly, however, tadpoles did not reduce corticosterone in the presence of invasive predators, indicating a lack of innate recognition. Furthermore, corticosterone and the antioxidant enzyme glutathione reductase were the most sensitive parameters against the studied factors, and hence good candidates for further use in physiological monitoring of natural populations. Pond drying and predators are two of the main risks for amphibian larvae in temporary ponds. Physiological consequences of plastic responses in tadpoles after being exposed to both factors may allow us to understand short-term consequences of the developmental alterations induced, as well as to predict their long-term effects. We crossed the presence/absence of predators with permanent and drop-down water levels in a 2x2 experimental design simulating natural conditions in large outdoor mesocosms to test for induced changes in life-history traits, and also assessed their effects on fat reserves, oxidative stress, and telomere length of the surviving Pelobates cultripes juveniles that survived to both risks. Tadpoles accelerated their development in response to decreased water level, but at the expense of metamorphosing smaller and greatly depleting their fat reserves. Cellular oxidative stress due to developmental acceleration was successfully buffered by increased antioxidant enzyme activity, and telomere length remained unchanged. On the other hand, predators greatly reduced larval density, which relaxed competition and allowed survivors to develop fast, grow bigger and accumulate fat. However, tadpoles showed signs of oxidative stress and experienced telomere shortening. Telomere length is reduced during cell replication, and telomere shortening is known to be associated with senescence and reduced life span. Therefore, regarding body size and fat reserves at metamorphosis, developmental acceleration in response to pond drying compromises short-term survival, although its consequences are reversible in the long run. In turn, oxidative stress and telomere shortening due to rapid growth when tadpoles survived predators are likely reduce their long-term survival. Even within species, organisms do not respond phenotypically to the same extent against environmental inputs. In fact, natural populations often harbor substantial variation in their degree of phenotypic plasticity. Theoretical studies suggest that the evolution of plasticity is limited by costs of maintaining the machinery needed to detect and respond to environmental cues. However, only a few studies have empirically detected maintenance costs of plasticity. In the third chapter of this thesis, we tested for physiological costs of maintaining developmental, growth, and morphological plasticity in Pelobates cultripes larvae in response to both pond drying and predators. For this purpose, we first determined the degree of developmental plasticity of a total of twenty families (sibships) from various populations in response to these two environmental factors. Simultaneously, we assessed among families variation in body mass, fat reserves, metabolic rate, antioxidant enzyme activities, lipid peroxidation, reduced/oxidized glutathione, and immune state under benign control conditions. We tested the existence of costs of plasticity by testing for an association between the degree of plasticity of each family and the molecular markers of physiological stress. We found maintenance costs of plasticity associated to the degree of developmental and growth plasticity induced by predators, in terms of increased glutathione reductase activity and granulocyte to lymphocyte ratio, respectively. Morphological plasticity in response to both pond drying and predators were also linked to levels of antioxidant enzymes, lipid peroxidation, immune state or growth. Also, we detected trade-offs between the developmental responses of larvae to each factor so that genotypes (families) that readily accelerated development in response to pond drying were not effective in delaying metamorphosis in the presence of predators. Such a trade-off suggests possible constrains on the evolution of adaptive plasticity to conflicting environmental stimuli. Environmental heterogeneity can affect the degree of adaptive plasticity, which may also imply short and long-term consequences. In amphibians, the ability to accelerate development commonly results adaptive, since larvae usually inhabit pools in which water availability is seasonal and heterogeneous. Adaptive developmental plasticity is expected to evolve despite its possible costs when environmental heterogeneity precludes a single phenotype to maximize fitness across all conditions. Swedish Rana temporaria island populations show marked differences among populations in developmental rate and their responsiveness to pond drying so that populations with more variable durations in pond hydroperiod tend to show higher levels of developmental plasticity. In the fourth chapter, we study the physiological mechanisms and consequences of divergent developmental plasticity among some of these R. temporaria populations. We exposed larvae from six populations from three different island habitat types differing in their pool drying regime to simulated desiccation to determine their developmental plasticity. Populations from islands with only ephemeral pools showed higher developmental plasticity than populations from islands with permanent or long-lasting ponds. Individuals from islands with ephemeral ponds experience physiological alterations indicative of physiological costs of increased plasticity, such as altered catalase and glutathione reductase activities, and reduced telomere length. Elevated antioxidant activities indicate metabolic costs associated to increased developmental plasticity which may also compromise the health and lifespan of individuals and the viability of those populations, as shortened telomeres suggested. During the course of this thesis, we have also evaluated the suitability of some methodological aspects. (I) We evaluated the performance of three commonly used procedures for corticosterone determination using Xenopus laevis tadpoles: radioimmunoassay (RIA) in whole-body homogenates, enzyme immunoassay (EIA) on whole-body, and EIA on plasma. Each procedure presented advantages and disadvantages regarding sensitivity, the use of radioactivity, sampling size, or handling time. RIA is preferred in small-bodied animals from which blood cannot be obtained. EIA in plasma resulted a good non-radioactive alternative when blood sampling is possible. EIA on whole-body homogenates was the less sensitive procedure, although it may be a non-radioactive useful alternative to assess qualitative changes in corticosterone in small individuals when considerable differences are expected. (II) Immune response in amphibians has been commonly evaluated through indirect methods like phytohemagglutinin (PHA) injections or by direct like cell counts from blood smears. Here, we validated immunological evaluations in amphibians by means of flow cytometry. The immunological state of Pelobates cultripes tadpoles were experimentally altered by exposing them to exogenous corticosterone. Then, leukocyte proportions were quantified through both blood smears and flow cytometry. Both techniques showed similar patterns of leukocyte proportions. Once validated, flow cytometry also allowed quantification of changes in absolute number of leukocytes. The suitability of both techniques attending to accuracy, body size requirements, or the useful in field studies was also discussed. The results obtained in this thesis highlight the key role of physiological mechanisms in amphibian larvae plasticity and in its evolution. Therefore, for a holistic knowledge of ecological and evolutionary process results essential to understand the physiology underlying them.Universidad Pablo de Olavide. Departamento de Biología Molecular e Ingeniería BioquímicaPostprin

Telomeres and anthropogenic disturbances in wildlife: a systematic review and meta-analysis

Human-driven environmental changes are affecting wildlife across the globe. These challenges do not influence species or populations to the same extent and therefore, a comprehensive evaluation of organismal health is needed to determine their ultimate impact. Evidence suggests that telomeres (the terminal chromosomal regions) are sensitive to environmental conditions and have been posited as a surrogate for animal health and fitness. Evaluation of their use in an applied ecological context is still scarce. Here, using information from molecular and occupational biomedical studies, we aim to deliver ecologists and evolutionary biologists an accessible synthesis of the links between human disturbances and telomere length. In addition, we perform a systematic review and meta-analysis on studies measuring telomere length in wild/wild-derived animals facing anthropogenic disturbances. Despite the relatively small number of studies to date, our meta-analysis revealed a significant small negative association between disturbances and telomere length (-0.092 [-0.153, -0.030]; n= 28; k=159). Yet, our systematic review suggests that the use of telomeres as a biomarker to understand the anthropogenic impact on wildlife is limited. We propose some research avenues that will help to broadly evaluate their suitability: i) further causal studies on the link between human disturbances and telomeres; ii) investigating the organismal implications, in terms of fitness and performance, of a given telomere length in anthropogenically-disturbed scenarios; iii) better understanding of the underlying mechanisms of telomere dynamics. Future studies in these facets will help to ultimately determine their role as markers of health and fitness in wildlife facing anthropogenic disturbances

Telomeres in a spatial context: a tool for understanding ageing pattern variation in wild populations

Ageing refers to the loss of organismal functionality with age, a process that is characterised by decreased reproduction and survival probability. In natural populations, it is expected that environmental conditions influence an individual's ageing trajectory. Understanding the role of environmental heterogeneity on ageing variation could provide critical insights into population resilience and species distribution but remains overlooked. Telomeres, the end cap of chromosomes, are a promising integrative physiological marker of an individual's health and a possible proxy to aid the understanding of variation in ageing trajectories. Here, we review the existing information on telomere length and its dynamics in wild populations distributed across spatial scales. Despite a relative scarcity of information, there is evidence for divergence in telomere length between populations facing contrasting environments. Nonetheless, a higher spatial resolution and temporal replication are needed to fully understand the role that environmental conditions play on telomere length variation. Since most of the existing studies are correlational, future field and laboratory experiments are required. For the first time, we demonstrate the use of population telomere data to predict species habitat suitability through species distribution models (SDMs). This represents a promising new research area in the study of ageing pattern variation in wild populations. Furthermore, the inclusion of telomere data in future physiological SDMs may improve our understanding of species distribution and population resilience. However, the use of telomeres within this context could be limited if no previous knowledge on the relevance of telomeres as markers of health and survival at the species level is available. Finally, we suggest some key practical and theoretical considerations that, ideally, future studies combining biogeographic and telomere data should pay attention

Ionizing radiation and melanism in Chornobyl tree frogs

Human actions are altering ecosystems worldwide. Among human-released pollutants, ionizing radiation arises as a rare but potentially devastating threat to natural systems. The Chornobyl accident (1986) represents the largest release of radioactive material to the environment. Our aim was to examine how exposure to radiation from the Chornobyl accident influences dorsal skin coloration of Eastern tree frog (Hyla orientalis) males sampled across a wide gradient of radioactive contamination in northern Ukraine. We assessed the relationship between skin frog coloration (which can act as a protective mechanism against ionizing radiation), radiation conditions and oxidative stress levels. Skin coloration was darker in localities closest to areas with high radiation levels at the time of the accident, whereas current radiation levels seemed not to influence skin coloration in Chornobyl tree frogs. Tree frogs living within the Chornobyl Exclusion Zone had a remarkably darker dorsal skin coloration than frogs from outside the Zone. The maintenance of dark skin coloration was not linked to physiological costs in terms of frog body condition or oxidative status, and we did not detect short-term changes in frog coloration. Dark coloration is known to protect against different sources of radiation by neutralizing free radicals and reducing DNA damage, and, particularly melanin pigmentation has been proposed as a buffering mechanism against ionizing radiation. Our results suggest that exposure to high levels of ionizing radiation, likely at the time of the accident, may have been selected for darker coloration in Chornobyl tree frogs. Further studies are needed to determine the underlying mechanisms and evolutionary consequences of the patterns found here

The impact of chemical pollution across major life transitions: a meta-analysis on oxidative stress in amphibians

Among human actions threatening biodiversity, the release of anthropogenic chemical pollutants which have become ubiquitous in the environment, is a major concern. Chemical pollution can induce damage to macromolecules by causing the overproduction of reactive oxygen species, affecting the redox balance of animals. In species undergoing metamorphosis (i.e. the vast majority of the extant animal species), antioxidant responses to chemical pollution may differ between pre- and post-metamorphic stages. Here, we meta-analysed (N = 104 studies, k = 2283 estimates) the impact of chemical pollution on redox balance across the three major amphibian life stages (embryo, tadpole, adult). Before metamorphosis, embryos did not experience any redox change while tadpoles activate their antioxidant pathways and do not show increased oxidative damage from pollutants. Tadpoles may have evolved stronger defences against pollutants to reach post-metamorphic life stages. In contrast, post-metamorphic individuals show only weak antioxidant responses and marked oxidative damage in lipids. The type of pollutant (i.e. organic versus inorganic) has contrasting effects across amphibian life stages. Our findings show a divergent evolution of the redox balance in response to pollutants across life transitions of metamorphosing amphibians, most probably a consequence of differences in the ecological and developmental processes of each life stage

Assessment of exposure to ionizing radiation in Chernobyl tree frogs (Hyla orientalis)

Ionizing radiation can damage organic molecules, causing detrimental effects on human and wildlife health. The accident at the Chernobyl nuclear power plant (1986) represents the largest release of radioactive material to the environment. An accurate estimation of the current exposure to radiation in wildlife, often reduced to ambient dose rate assessments, is crucial to understand the long-term impact of radiation on living organisms. Here, we present an evaluation of the sources and variation of current exposure to radiation in breeding Eastern tree frogs (Hyla orientalis) males living in the Chernobyl Exclusion Zone. Total absorbed dose rates in H. orientalis were highly variable, although generally below widely used thresholds considered harmful for animal health. Internal exposure was the main source of absorbed dose rate (81% on average), with 90Sr being the main contributor (78% of total dose rate, on average). These results highlight the importance of assessing both internal and external exposure levels in order to perform a robust evaluation of the exposure to radiation in wildlife. Further studies incorporating life-history, ecological, and evolutionary traits are needed to fully evaluate the effects that these exposure levels can have in amphibians and other taxa inhabiting radio-contaminated environments

Eucalypt leaf litter impairs growth and development of amphibian larvae, inhibits their antipredator responses and alters their physiology

Consequences of human actions like global warming, spread of exotic species or resource consumption are pushing species to extinction. Even species considered to be at low extinction risk often show signs of local declines. Here, we evaluate the impact of eucalypt plantations, the best-known exotic tree species worldwide and its interaction with temperature and predators on amphibian development, growth, antipredator responses and physiology. For this purpose, we applied a fully factorial experiment crossing two types of leaf litter (native oak or eucalypt), two temperatures (15 and 20°C) and presence/absence of native predators. We found that leachates of eucalypt leaf litter reduced amphibian development and growth, compromised their antipredator responses and altered their metabolic rate. Increased temperature itself also posed serious alterations on development, growth, antioxidant ability and the immune status of tadpoles. However, the combined effects of eucalypt leaf litter and increased temperature were additive, not synergistic. Therefore, we show that non-lethal levels of a globally spread disruptor such as leachates from eucalypt leaf litter can seriously impact the life history and physiology of native amphibian populations. This study highlights the need to evaluate the status of wild populations exposed to human activities even if not at an obvious immediate risk of extinction, based on reliable stress markers, in order to anticipate demographic declines that may be hard to reverse once started. Replacing eucalypt plantations with native trees in protected areas would help improving the health of local amphibian larvae. In zones of economic interest, we would recommend providing patches of native vegetation around ponds and removing eucalypt leaf litter from pond basins during their dry phase.This study was funded by Ministerio de Economía y Competitividad (grant CGL2014−59206-P). P.B. and M.I.C. were supported by fellowships F.P.U.-AP2010-5373 and 2012-04148 from Ministerio de Educación

Análisis genético preliminar para el estudio de las interacciones intraespecíficas de oso pardo de la Cordillera Cantábrica

XIV Congreso de la Sociedad Española para la Conservación y Estudio de los Mamíferos (SECEM), Jaca, 5-8 diciembre de 2019.El rascado en los árboles juega un papel fundamental en la comunicación intraespecífica de los osos pardos (Ursus arctos), cuyas áreas de campeo a menudo se encuentran solapadas. A través de estos mensajes, los osos son capaces de reconocer a todos los individuos de su zona sin necesidad de interacciones directas. Se ha demostrado que los osos pardos invierten una parte de su tiempo en la búsqueda activa de las señales químicas dejadas por los conspecíficos como parte esencial de su red de comunicación. El presente trabajo busca descubrir: (a) la red social existente en torno a los árboles de rascado (o árboles de marcaje) del occidente de la Cordillera Cantábrica (n= 18 árboles de rascado); (b) los patrones de marcaje a lo largo de todo el año (n= 32 árboles de rascado); así como(c) entender dinámicas de envejecimiento en la población sujeta a estudio, mediante la toma de muestras de pelo depositado en trampas no lesivas, situadas en Asturias y León, tras el rascado. La recogida de los pelos se ha realizado mensualmente durante un período de un año, por lo que disponemos de un volumen total de 216 muestras, cada una de ellas con material de distintos ejemplares de oso. Se ha realizado la extracción genética de cada pelo con un total de ocho folículos pilosos por muestra, de forma que la posibilidad de error disminuya al 5% y se obtenga una concentración de ADN nuclear suficiente para su identificación. Para la determinación del individuo, sexo y rango de edad se han escogido siete marcadores microsatélites, descritos como los más informativos para este fin. La población occidental es la que cuenta con el mayor número de individuos de la población pero, en base a los resultados preliminares y debido a la limitada variabilidad genética es necesario determinar si es suficiente para realizar una correcta identificación individual. Datos preliminares muestran que los patrones anuales en el rascado se han estudiado como una variable en torno a la época del año, siendo los meses de abril a septiembre de los que mayor cantidad de pelo se obtiene, así como en los meses en torno a la hibernación cuando se denota una reducción en el comportamiento de rascado coincidente con su disminución de actividad

Predator-induced physiological responses in tadpoles challenged with herbicide pollution

Predators induce plastic responses in multiple prey taxa, ranging from morphological to behavioral or physiological changes. In amphibians, tadpoles activate plastic responses to reduce predation risk by reducing their activity rate and altering their morphology, specifically tail depth and pigmentation. Furthermore, there is now evidence that tadpoles' defenses are modified when predators combine with other stressful factors such as pollutants or competitors, but our knowledge on the physiological responses underlying these responses is still scarce. Here we study physiological responses in Pelobates cultripes tadpoles exposed to a natural predator (larvae of the aquatic beetle Dytiscus circumflexus), non-lethal concentrations of herbicide (glyphosate, 0.5 mg/L and 1.0 mg/L) or both factors combined. We measured corticosterone levels, standard metabolic rate, oxidative damage (TBARS) and activity of antioxidant enzymes, and immune response (via leukocyte count). Tadpoles reduced their corticosterone concentration by ca. 24% in the presence of predator cues, whereas corticosterone did not change in the presence of glyphosate. Two enzymes involved in antioxidant response also decreased in the presence of predators (14.7% and 13.2% respectively) but not to glyphosate. Herbicide, however, increased the number of neutrophils and reduced that of lymphocytes, and had an interaction effect with predator presence. Standard metabolic rate did not vary across treatments in our experiment. Thus we show a marked physiological response to the presence of predators but little evidence for interaction between predators and low levels of herbicide. Multiple assessment of the physiological state of animals is important to understand the basis and consequences of phenotypic plasticityPeer reviewe