Project summary: Ants are social insects that live in colonies of morphologically and physiologically different individuals that are essentially identical genetically, making ants an attractive system to study epigenetic phenomena. Ant colonies contain many workers that perform most tasks but do not lay eggs, while queens are solely responsible for reproduction. Remarkably, queens live up to 10X longer than workers, in sharp contrast with most animals in which high reproduction leads to shortened lifespan. The jumping ant Harpegnathos saltator exhibits a high degree of aging plasticity: In the absence of the queen, some workers can become pseudo-queens called gamergates. Gamergates dramatically change their behavior, produce eggs, reconfigure their brain and most dramatically, have a 5X lifespan extension. Remarkably, when placed in the presence of a genuine queen, gamergates transition back into workers with an accompanying shortened lifespan. We established Harpegnathos as a model system that can be manipulated with CRISPR/Cas9, providing a unique opportunity to study the molecular mechanisms that control aging, as well as the crosstalk between aging and reproduction. Using a combination of transcriptomics as well as both ex vivo and in vivo pharmacological manipulations, we discovered that gamergates have an elevated production of Insulin accompanied by differential regulation of the two branches of the Insulin signaling pathway (IIS) in target tissues. The MAPK branch of IIS is activated in the gamergate fat body and ovary, while the AKT branch is repressed by extracellular “anti-Insulin” proteins, ImpL2. As MAPK activity is required for egg-laying, we hypothesize that repression of the AKT branch contributes to the dramatically extended longevity in gamergates. We now propose to investigate the molecular mechanism of ImpL2 function and test its role in aging. First, we will identify the source and organismal distribution of ImpL2, and then experimentally modulate its levels and mutate ImpL2 to examine the effect(s) on IIS and increased metabolism for egg formation. Furthermore, we will explore the molecular interactions of ImpL2 and the mechanisms that lead to the specific inhibition of the AKT (but not MAPK) IIS pathway. Next, we will test the effect of ImpL2 on aging in manipulated animals, assessing a panel of aging biomarkers and extend our investigations to another anti-Insulin protein, ALS. Moreover, tissue- specific manipulation of ImpL2 expression in Drosophila will address its potentially conserved effect on reproduction and lifespan in a powerful model system. Finally, we will extend our study to the brain remodeling events that accompany and orchestrate the social transition. We will perform single-cell mRNA sequencing of the different social groups/ages to survey age-associated changes in the Harpegnathos brain and identify candidate regulators responsible for delayed aging in gamergates. We will exploit the transcriptomic data...