Organisms react to environmental changes through plastic responses that often involve physiological alterations with the potential to modify life-history traits and fitness. Environmentally

induced shifts in growth and development in species with complex life cycles determine the timing of transitions between subsequent life stages, as well as body condition at transformation,

which greatly determine survival at later stages. Here we show that spadefoot toad larvae surviving pond drying and predators experienced marked alterations in growth and development, and in

their fat reserves, oxidative stress, and relative telomere length. Tadpoles accelerated development but reduced growth and consumed more fat reserves when facing pond drying. However,

survival odds, but showed signs of oxidative stress and had shorter telomeres. Developmental acceleration and enhanced growth thus seemed to have different physiological consequences:

reduced fat bodies and body size compromise short-term survival, but are reversible in the long run, whereas telomere shortening is non-reversible and could reduce long-term survival.

Selection in heterogeneous environments often results in the evolution of adaptive phenotypic plasticity1, 2. However, phenotypic alteration is often costly, costs referred to as ‘production

costs’3. Known production costs involve physiological alterations such as immune state suppression or increased metabolism, which result in changes in life-history traits and ultimately

affect fitness4. Understanding the physiological effects of plastic alterations of the phenotype will help us assess their short-term and long-term consequences4, 5.

In vertebrates, stress responses to environmental challenges are orchestrated via activation of neuroendocrine pathways, being the hypothalamic-pituitary-adrenal (HPA) axis the most studied.

HPA-axis activation induces the mobilization of metabolites6 but may result in immunological imbalances7 that produce profound alterations in developmental and growth rates8, 9. Such

environmentally induced alterations of growth and development are particularly relevant in species with complex life cycles10 because the timing of transitions between life stages and body

condition at transformation greatly determine survival at later stages11. Accelerating or slowing development and growth have marked consequences across most taxa, ranging from changes in

protein turnover within tissues12, to allometric changes in body shape and degree of ossification13, to variation in fat storage14. From a molecular perspective, enhanced developmental and

growth rates may result in two major physiological alterations with extensive consequences for fitness: telomere shortening and oxidative stress15, 16.

Telomeres are non-coding repetitive terminal regions of the chromosomes specialized in chromosome protection from deterioration, or from fusion with other chromosomes17. Telomere sequences

are restored via reverse transcriptase telomerase that adds telomeric repeats (TTAGGG in vertebrates) to 3′ overhang. Critical telomere shortness stops cell division and initiates a state of

replicative senescence leading to programmed cell death18. Telomere shortening is thought of as an internal clock that could potentially be used for estimating chronological age in the

wild, although telomere length can vary at different rates over different ontogenetic stages across species. Patterns of telomere length variation over time can be rather complex, as for

example in the edible dormouse (Glis glis), a hibernating rodent, where telomeres elongate from ages 6 to 9 years19, much like in humans telomeres elongate during the development of stem

cells, B cells or some tumor cells20,21,22. Therefore, spontaneous ontogenetic shifts from telomere elongation phases to shortening phases, combined with environmentally induced alterations

of the rate of telomere shortening loosen the link between biological and chronological ageing15, 16. Interestingly, the fact that telomere shortening is susceptible to physiological

adjustments due to the environmental conditions experienced provides the means to evaluate the relative costs and trade-offs of phenotypic responses to environmental challenges, especially

in early ontogenetic stages16, 23.

Developmental acceleration and enhanced growth have been shown to cause telomere shortening as observed in vitro in rat pancreatic islets24 probably as a consequence of multiple cell

divisions, a phenomenon referred to as the ‘end replication problem’25, and of oxidative damage accumulated over the cellular lifespan26, 27. Accelerated development and enhanced growth, as

well as acute episodes of environmental stress, produce excess reactive oxygen substances (ROS) that can result in severe oxidative stress and damage cell structures such as telomere

sequences5. Both reduced telomere length and increased oxidative stress seem to play a key role in ageing and are good predictors of individual lifespan5, 28 although further evidences are

still needed29.

Most systems studied so far in the context of the interplay of oxidative stress and telomere shortening as a consequence of environmentally induced phenotypic responses have focused on taxa

in which growth and development tended to be rather correlated, such as mammals (mainly humans26) and birds30. However, amphibian larvae have been much less studied in this context despite

being an ideal system for evaluating the consequences of developmental and growth plasticity separately. The development of most amphibian species include abrupt ontogenetic switch points in

which timing is usually highly plastic since larvae readily modify their activity, morphology, differentiation rate, and growth rate in response to environmental cues31, 32. Two main

environmental hazards for amphibian larvae are predators and pond drying33. Tadpoles are capable of detecting and responding plastically to both risks. However, responses against pond drying

and predators seem to be opposite in many respects. Thus, amphibian larvae accelerate development and decrease growth under pond drying conditions34. In contrast, predators induce reduced

activity and metabolism of amphibian larvae35. In addition, predators directly reduce larval density hence relaxing competition and allowing the surviving larvae to reach metamorphosis

faster and at a larger size36. Analysing the physiological consequences of changes in growth and development in amphibian larvae is important to understand both short-term and long-term

carry-over effects of adaptive plastic responses.

Here we evaluate the effects of altered developmental and growth rates in western spadefoot toad tadpoles (Pelobates cultripes) as a consequence of pond drying and presence of freely roaming

predators. We examined the physiological consequences of such alterations in development and growth on the surviving larvae of each treatment in terms of their fat body content, oxidative

stress, and telomere length after metamorphosis. We expected tadpoles to accelerate development in response to pond drying, but at the expense of metamorphosing at a smaller size and with

reduced fat reserves. We also hypothesized that pond drying would involve telomere shortening and oxidative damage as a consequence of the increased metabolic effort required for

developmental acceleration. Similarly, we expected reduced larval density due to predation to result in lower competition, hence providing better growing conditions for the surviving larvae.

Therefore, we expected individuals surviving predators to have a larger mass at metamorphosis, and more abundant fat reserves. In terms of oxidative stress, high resource availability could

entail increased metabolism and ROS production, which would have to be balanced with increased antioxidant enzyme activity. Fast growing individuals would be expected to have undergone a

greater number of rounds of cell replication, resulting in shortened telomeres.

During the experiment 324 individuals survived and completed metamorphosis out of the initial 960. Of these, 79% kept color markings (VIE tags, see Methods) when they reached metamorphic

climax (Gosner stage 42) and could therefore be assigned to sibship.

Pond drying significantly reduced tadpole survival by 41.88% (df = 1, 959; χ2 = 12.005, p