Mechanisms and costs of developmental plasticity in amphibian larvae

Resumen

Nature 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.