Galleria mellonella as a Novel Model for Mitochondrial Respirometry Analysis

Mitochondria play a central role in cellular energy metabolism and signaling pathways, making them crucial targets in light-based therapeutic approaches such as photobiomodulation (PBM) and photodynamic therapy (PDT). While PBM aims to stimulate local metabolic activity, PDT relies on light activation of a photosensitizer to generate cytotoxic reactive oxygen species, leading to cell death. Despite the widespread use of both techniques, the precise impact of irradiation on mitochondrial bioenergetics is still not fully elucidated. To bridge this gap, our research investigates the effects of light–mitochondria interactions using the alternative animal model Galleria mellonella, which offers advantages such as low costs, an innate immune system analogous to vertebrates, and no ethical committee restrictions. However, since mitochondrial activity has not been previously characterized via respirometry assays in this organism, standardized respirometry protocols currently do not exist. This work focuses on the establishment and comparative evaluation of different experimental approaches for performing respirometry assays, including various mitochondrial isolation protocols via differential centrifugation as well as tissue respirometry methods using biopsy preparations. Furthermore, standardized parameters are being established to enable precise and reproducible evaluation of respiratory states, coupling efficiency, and electron transport system capacity under both baseline and irradiated conditions. In parallel, methodologies to enable the photosensitizer to properly interact with the epidermal cells of G. mellonella, overcoming the cuticle barrier without causing damage to the underlying tissue or impairing mitochondrial function, were also tested. These ongoing methodological advances represent an essential step toward elucidating the cellular mechanisms underlying PDT and PBM. Ultimately, by establishing and validating standardized mitochondrial respirometry in G. mellonella, this work pioneers the application of a novel invertebrate model for bioenergetics research, opening new avenues for scalable and ethically sound screening of light-based therapies.