Dietary restriction (DR) extends life span across taxa. Despite considerable research, universal mechanisms of DR have not been identified, limiting its translational potential. Guided by the conviction that DR evolved as an adaptive, pro-longevity physiological response to food scarcity, biomedical science has interpreted DR as an activator of pro-longevity molecular pathways. Current evolutionary theory predicts that organisms invest in their soma during DR, and thus when resource availability improves, should outcompete rich-fed controls in survival and/or reproduction. Testing this prediction in Drosophila melanogaster (N > 66,000 across 11 genotypes), our experiments revealed substantial, unexpected mortality costs when flies returned to a rich diet following DR. The physiological effects of DR should therefore not be interpreted as intrinsically pro-longevity, acting via somatic maintenance. We suggest DR could alternatively be considered an escape from costs incurred under nutrient-rich conditions, in addition to costs associated with DR.

14Dietary restriction (DR) consistently and universally extends health-and lifespan across 15 taxa. Despite efforts to uncover the mechanisms underpinning DR -and ultimately translate 16 its beneficial outcomes to humans -precise and universal mechanisms have not been 17 identified. In biomedical science, the effects of DR are interpreted as regulating pro-longevity 18 molecular pathways. This reasoning is guided by the conviction that DR evolved as an 19 adaptive, pro-longevity physiological response to restricted food intake. Current evolutionary 20 theory states that organisms should invest in their soma more heavily during periods of DR, 21 and, when their resource availability improves, should outcompete age-matched rich-fed 22 controls in survival and/or reproduction. Here we present a formal test of these key 23 predictions utilising a large-scale demographic approach detailing mortality and fecundity in 24 Drosophila melanogaster fed alternating dietary regimes (N > 66,000 flies across 11 genetic 25 lines) . Our experiments reveal surprising and substantial mortality costs when returning to a 26 rich diet after periods of DR. These results suggest the effects of DR are not necessarily 27 intrinsically pro-longevity and could be considered an escape from costs incurred under 28 nutrient-rich conditions, in addition to novel, discrete costs associated with restricting dietary 29 protein. These insights question the relevance of current evolutionary explanations of DR in 30 interpreting the mechanistic basis of dietary restriction. 31

12Research on the mechanisms of ageing has identified ways via which lifespan can 13 be extended in model organisms, increasing the potential for translation of these 14 findings to our own species. However, the large majority of research on animal 15 models involves dietary, genetic or pharmacological treatments throughout life -16 limiting translational potential and ignoring age-dependent effects. Previously, we 17 have suggested using demographic meta-analysis that reduced mTor signalling has 18 the potential to instantly rejuvenate. We have now tested this prediction 19 experimentally using large-scale demographic data (N > 10,000) combined with 20 conditional knockdown of mTor in Drosophila melanogaster. Indeed, reduced mTor 21 decreased mortality rate when applied during old age. Interestingly, we found that 22 transient treatment during early adult life had long-lasting benefits. Age-dependent 23 deep-RNAseq indicated that these effects arose from distinct physiology and 24 implicate alternative splicing as a potential mechanism for the long-lasting benefits of 25 transient mTor reduction. These findings suggest that reducing mTor short term or 26 during old age could be used to combat ageing. In addition, our findings suggest that 27 the results from experimental research on mTor signalling, and potentially other 28 mechanisms of ageing, that employ life-long interventions are likely to be a complex 29 composite of age-dependent effects that counteract or enhance each other. 30 157 effects cox proportional hazard models correcting for cage effects (see methods 14,31 ). B) Transient 158 mTor knockdown (maximum induction of GeneSwitch at blue dotted line) resulted in a modest 159 increase in mortality during early life, but subsequently resulted in a sustained mortality reduction 160 throughout life (P < 0.0001). Similar effects were seen when mTor was knocked down continuously. 161The red dotted line now indicated when mTor in the transient treatment is back to control levels. 162These experiments were all ran together at the same time but are split in two panels for graphical 163 purposes. 342