To answer the questions indicated above, extensive data were collected in two separated experiments. In the first experiment, we quantified the variation in telomere length (a parameter related to cellular ageing rate and known to be influenced by environmental harshness) and citrate synthase activity (a marker of mitochondrial volume, also linked to ageing), across larval and metamorphic stages (five sampling points covering the main life transitions in amphibians, i.e. from early larval stage to 2-year-old adults) and in five different tissues (tail muscle, hindlimb muscle, heart, gut, and liver) of the African clawed frog Xenopus laevis exposed to two different temperatures (19ºC and 23ºC). In the second experiment, we collected additional information on telomere length variation across four developmental stages, and we investigated the variation in telomerase expression (the enzyme responsible for telomere maintenance) in three different tissues (hindlimb muscle, liver, gut), and we quantified the immune status of individuals by measuring the proportion of white blood cells, in frogs facing simulated heat waves at pre and post metamorphic stages. Overall, we found that telomere length changes across metamorphic transitions, but these changes seemed to be linked to the degree of transformation undergone by each tissue. While gut telomeres showed a huge variation through metamorphosis, telomere length in other tissues (in hindlimb muscle and liver) shortened with age, or experienced negligible changes across the studied life stages (in tail muscle and heart). Also, during pre- and metamorphic life stages, we observed a strong negative relationship between telomere length and organismal growth, which highlights the relevance of metamorphosis in shaping the relationship between ageing mechanisms and life-history traits. The activity of citrate synthase varied between tissues and across life stages. Citrate synthase activity was higher in heart and muscle compared with gut and liver. Whereas the activity of citrate synthase increased from larva to adult in heart and muscle, it only increased through metamorphosis in gut, and remained unaltered in liver. These results suggest that differences in tissue development shape the required volume of mitochondria. Preliminary data also suggest that the expression of telomerase is tissue and developmental stage dependent in Xenopus laevis. Finally, we found that the neutrophil-to-lymphocyte ratio was higher in 70-day than in 100-day individuals, likely indicating that the immune response is higher immediately after metamorphosis. Regarding the impact of temperature, we found that higher temperature induced subtle but quantifiable changes in telomere length in some tissues of pre-metamorphic individuals. Unexpectedly, telomeres in gut and hindlimb muscle were longer in larvae raised under the higher temperature, but this effect disappeared later in life. The combined exposure to higher temperature at the larva and juvenile stage and at the juvenile stage did not influence telomere dynamics of post-metamorphic frogs, thus only temperature changes before metamorphosis seem to affect telomeres in this amphibian species. Preliminary data suggest that temperature conditions have a small effect on telomerase expression or citrate synthase. Finally, we found that high temperature before metamorphosis induced lower neutrophil-to-lymphocyte ratios in fully formed frogs (i.e. 70 and 100-day-old individuals), which suggests that moderate heat waves may not be detrimental but enhance the immune machinery of frogs.
