Age-dependent physiological responses and longevity in three Drosophila species with contrasting lifespans exposed to low temperature and constant darkness.

Aging is influenced by both genetic and environmental factors, yet comparative studies across species with different natural lifespans remain limited. We examined how reduced ambient temperature (18 °C vs 25 °C) and constant darkness, compared with a 12 h light/12 h dark cycle (DD vs LD), affect lifespan, age-related physiological traits, and gene expression in three Drosophila species with contrasting natural lifespans and climatic adaptations (tropical, short‑lived D. kikkawai, tropical, intermediate‑lived D. melanogaster, and temperate, long‑lived D. virilis). Low temperature extended lifespan in all three species, with the largest relative gains occurring in the shortest‑lived D. kikkawai males, yet the longest absolute lifespans were consistently attained by the long‑lived D. virilis. Constant darkness moderately increased lifespan at 25 °C, particularly in males, but its combination with low temperature became antagonistic in D. virilis, revealing that the interaction between photoperiod and temperature depends on both baseline longevity and sex. Longer lifespan correlated with lower metabolic rate, greater body mass, and sustained late‑life activity. Gene expression analyses in D. melanogaster revealed that low temperature induced a youthful metabolic and immune profile, whereas DD often counteracted these changes. Our findings do not support a simple inverse or direct relationship between baseline longevity and geroprotective efficacy. Instead, these results suggest that the responses to low temperature and darkness are species‑ and sex‑specific and reflect each species' evolutionary and ecological background.