Lifespan is modulated by both genetic and environmental factors. Dietary nutrients are among the most potent environmental factors in modulating healthspan and lifespan. Dietary restriction (DR) without malnutrition has been shown to promote healthspan and lifespan in numerous species. Nutraceuticals are rich in phytochemicals that possess diverse bioactivities and exert numerous health benefits, including anti-aging effects. Whether and how dietary nutrients influence the prolongevity effects of aging interventions remains elusive. Considering diverse dietary customs among human populations in different geographic regions, it is important to assess the effectiveness of any aging intervention using pharmaceutical and nutraceutical compounds under different dietary conditions in humans. To this end, we have investigated the interactions between nutraceuticals and dietary macronutrients on healthspan and lifespan using two fly models, the Mexican fruit fly (Mexfly) and Drosophila. First, we designed a nutritional geometry approach to determine the interplay among dietary fat, sugar, protein and freeze-dried aai pulp in modulating lifespan and reproductive output in the Mexfly. Aai is a fruit indigenous to the Amazon River region and contains numerous phytochemicals that have anti-oxidant, anti-inflammation, anti-cancer and anti-cardiovascular disease properties. We found that aai supplementation promoted survival, but decreased lifetime reproductive output in Mexflies on diets with high fat and high sugar but not those on other diets. This work was published in Experimental Gerontology (2012). Second, we determine the interaction of resveratrol with dietary sugar, protein and fat on lifespan in Drosophila. Resveratrol is a polyphenolic compound that has been shown to extend lifespan in different organisms. However, several lines of evidence have indicated that resveratrol is not capable of extending lifespan in yeast, worms and flies under any culture conditions. We postulated that the prolongevity effect of resveratrol may depend on dietary composition, and designed a nutritional geometry study to test this hypothesis. We found that resveratrol supplementation did not extend the lifespan of flies fed a standard, restricted or high sugar-low protein diet. However, resveratrol did extend the lifespan of flies fed a low sugar-high protein or a high-fat diet. Lifespan extension by resveratrol was associated with downregulation of genes involved in insulin-like signaling and oxidative stress response. This work was published in AGE 2011. Our aai and resveratrol studies in two different fly species reveal the context-dependent effect of nutraceuticals on lifespan and stress the importance of diet composition in implementing effective aging interventions. One of the challenges in implementing effective aging interventions is the requirement of long-term intervention. Therefore, it is important to determine the age period critical for any pharmaceutical or nutraceutical agents to achieve desired prolongevity effects in humans. Identification of such critical age periods will significantly reduce the cost and potential side-effects of the often long-term interventions. We conducted two studies to address the temporal effects of aging interventions using nutraceuticals. First, based on our previous findings that an oregano-cranberry mixture (OC) can extend the lifespan of the mexfly, we determined the effect of short-term OC supplementation on lifespan. We found that only short-term OC supplementation at the middle age period was sufficient to extend lifespan in Mexflies. This work was published in AGE (2011). Second, we investigated the effect of short-term aai supplementation on promoting survival and reducing oxidative damage in sod1 knockdown flies, which have high levels of oxidative damage. We have previously shown that lifetime supplementation of aai can extend the lifespan of sod1 knockdown flies, which was published in Experimental Gerontology (2010). We have now demonstrated that aai supplementation, initiated at the time when the mortality rate of flies begins to accelerate, is sufficient to promote survival, reduce oxidative damage and increase reproductive output in sod1 knockdown flies. This work was published in AGE (2012). The findings from both the OC and aai studies suggest that short-term supplementation of nutraceuticals can be effective in promoting healthy aging. In summary, we have determined the impact of dietary nutrients on lifespan and critical molecular pathways in several fly lines, specifically the Mexfly and Drosophila. More importantly, we have demonstrated that the prolongevity effects of nutraceuticals in both Mexfly and Drosophila models are observed when initiated in middle aged subjects. Our future research will be directed to understand the molecular mechanisms underlying the interplay between nutraceuticals, dietary nutrients and the timing of these interventions. These studies should provide insight into the mechanisms underlying lifespan modulation by dietary nutrients and their interactions with genetic and non-genetic factors. This project should advance the objectives of the Laboratory of Experimental Gerontology and the National Institute on Aging, and provide valuable information for understanding human aging and more importantly for developing efficient aging intervention strategies in humans, including the timing of those interventions during the lifespan.