Hypothesis Paper Volume 18 pp 327—339

Detailed flowchart of the iterative experimental strategy to delay senescence by introducing Hydra-like gene expression in rotifers (Brachionus manjavacas). Key manipulations target rotifer orthologs (primarily FoxO and stemness markers) via CRISPR. Evaluation endpoints include lifespan/mortality hazard (survival curves), fertility (fecundity curves), locomotion (swimming assays), stress resistance, and transcriptomics (pre/post similarity to Hydra stem/progenitor states; ≥0.7 correlation threshold). Neoplasia monitoring uses proliferation markers (BrdU/phospho-histone H3) and deviation from Hydra tumor profiles. Iteration decisions are guided by predefined criteria (healthspan extension, transcriptomic shift, fitness constraints). Controls: wild-type and mock-treated rotifers. The framework also tests intercellular competition and multicellular aging through integrated measurements of cellular degradation, vigor-cooperation correlation, cheater emergence (neoplasia proxies), and negative senescence potential (mortality hazard). “Hydra-like” is defined in the text.