Ammonium effects on oxidative stress, telomere length, and locomotion across life stages of an anuran from habitats with contrasting land-use histories.

Understanding the mechanistic implications behind wildlife responses to global changes is a central topic in eco-evolutionary research. In particular, anthropic pollution is known to impact wild populations across the globe, which may have even stronger consequences for species with complex life cycles. Among vertebrates, amphibians represent a paradigmatic example of metamorphosis, and their characteristics make them highly vulnerable to pollution. Here, we tested for differences in the redox status, telomere length, and locomotor performance across life stages of green frogs (Pelophylax perezi) from agrosystem and natural habitats, both constitutively and in response to an experimental ammonium exposure (10 mg/L). We found that larvae from the agrosystem constitutively showed an enhanced redox status (better antioxidant balance against H2O2, lower lipid peroxidation) but shorter telomeres as compared to larvae from the natural environment. The larval redox response to ammonium was, overall, similar in both habitats. In contrast, after metamorphosis, the redox status of individuals from the natural habitat seemed to cope better with ammonium exposure (denoted by lower lipid peroxidation), and differences between habitats in telomere length were no longer present. Intriguingly, while the swimming performance of larvae did not correlate with individual's physiology, metamorphs with lower glutathione reductase activity and longer telomeres had a better jumping performance. This may suggest that locomotor performance is both traded off with the production of reactive oxygen species and potentiated directly by longer telomeres or indirectly by the mechanisms that buffer their shortening. Overall, our study suggests that contrasting land-use histories can drive divergence in physiological pathways linked to individual health and lifespan. Since this pattern was life-stage dependent, divergent habitat conditions can have contrasting implications across the ontogenetic development of species with complex life cycles.

Long-term sex-dependent inflammatory response of adult frogs to ammonium exposure during the larval stage.

Among others, the global change involves a worldwide increase in cropland area, with the concomitant rise in nitrogenous fertilizer supplementation and species range alterations, including parasites and pathogens. As most animals rely on their immune systems against these infectious agents, studying the potential effects of nitrogenous compounds on animal immune response is vital to understand their susceptibility to infections under these altered circumstances. Being subjected to an alarming process of global declines, amphibians are the object of particular attention, given their sensitivity to these compounds, especially to ammonium. Moreover, whereas adults can actively avoid polluted patches, larvae are confined within their waterbodies, thus exposed to contaminants in it. In this work, we test whether chronic exposure to a sublethal dose of ammonium during the larval stage of Pelophylax perezi frogs, released from all contamination after metamorphosis, leads to impaired inflammatory response to phytohemagglutinin in adults. We also test whether such a response differs between agrosystem individuals as compared with conspecifics from natural habitats. We found negative carryover effects of chronic exposure of larvae to ammonium on adult inflammatory response, which could imply a greater susceptibility to pathogens and parasites. However, this damage is only true for males, which, according to the immunocompetence handicap hypothesis, could be a consequence of a testosterone-triggered impairment of male immune function. In disagreement with our prediction, however, we detected no differences in the inflammatory response of agrosystem frogs to phytohemagglutinin as compared with natural habitat conspecifics.

Carryover effects of chronic exposure to ammonium during the larval stage on post-metamorphic frogs.

Water contamination poses an important challenge to aquatic fauna, including well-documented effects on amphibian larvae. However, little is known about how contamination during the larval stages may affect post-metamorphic phases, or whether resistance may have evolved in some populations. In this work, we tested the hypothesis that chronic exposure to ammonium (a common contaminant in agroecosystems with confirmed effects on anuran tadpoles) during the larval stage of Pelophylax perezi frogs would affect growth and locomotor performance of metamorph, juvenile, subadult and adult stages. We also predicted that the effects of ammonium would be milder in offspring originated from parental agroecosystem frogs than those originating from forests. We compared tadpoles from both habitats either reared in untreated water or chronically exposed to ammonium. We found that exposure to ammonium during the larval stage inflicted effects on morphology (different measures of body size) and swimming speed after metamorphosis until adulthood. However, these effects were not always consistent through post-metamorphic stages and the effects differed as a function of treatment and habitat. In adults, body size and condition were greater in non-ammonium and ammonium exposed individuals, respectively. These differences were not detectable in metamorphs, for which only ammonium-exposed individuals from agroecosystem showed reduced body size in intermediate post-metamorphic stages. In turn, treatment reduced jumping distance only in agroecosystem adults, subadults and juveniles, which was opposite to the trend observed just after metamorphosis. These changes of patterns throughout the ontogeny of P. perezi could be due to processes such as compensatory growth, delayed energy costs derived from it, or early sexual differences that could be present even before they can be accounted for. In summary, this study suggests that exposure to ammonium during larval stages can result in diverse biological and long-term outcomes in later life stages.

Direct and indirect effects of chronic exposure to ammonium on anuran larvae survivorship, morphology, and swimming speed.

Several constituents of the current global change are usually deemed accountable for the worldwide declines of amphibian populations. Among these, water contamination poses a major threat, especially to larval stages, which are unable to escape a polluted water body. This problem is remarkable in agrosystems, one of the main sources of water pollution and whose area is forecasted to increase in the forthcoming decades. However, pollutants represent a selective pressure that may result in tolerance in affected areas. In this work, we tested whether chronic exposure to a sublethal concentration of ammonium (10 mg/L), one of the most frequent agrochemicals, affects differently hatching success, survivorship, morphology and swimming performance of Pelophylax perezi tadpoles from agrosystem and pine grove habitats. Ammonium diminished survivorship at the earliest stages after hatching. Thus, lower density was a by-product of exposure to ammonium. Higher density slowed down development, reduced snout-vent length, and had a sharper negative effect on body mass and tail length and depth of ammonium treated individuals with respect to the control. In turn, ammonium accelerated development and increased body mass, SVL, and tail length and depth. These effects did not depend on provenance habitat. However, only pine grove tadpoles' swimming speed was negatively affected by ammonium, which supports the hypothesis that agrosystem tadpoles are more tolerant to ammonium. Finally, corroborating previous findings, tadpoles with larger bodies and tails were faster swimmers, whereas proportionally more massive individuals were slower, and tail depth was unrelated to swimming speed.