Methods: Novel use of mitochondrial function to optimize the permeabilization of crustacean gill types

While gills are the primary interface between the environment and bioenergetic responses, crustacean gill mitochondrial studies remain scarce. Their gill lamellae are thicker and less permeable than teleost gills, requiring harsher permeabilization. Yet saponin, commonly used for tissue permeabilization, can impair mitochondrial function. In osmo-regulating decapods, anterior (purely respiratory) and posterior (osmoregulatory) gills differ in ultrastructure and composition; raising the question of whether exposure to effective saponin treatment, compromises the balance between tissue permeabilization and maintained mitochondrial function. We used high-resolution respirometry, for both gill types, with different saponin treatments to measure their impact on key markers of mitochondrial function. Three saponin concentrations at two incubation times were tested to identify permeabilization protocols that maintained mitochondrial function. Optimal conditions differed between gill types: anterior gills required longer incubation times and higher concentrations, whereas posterior gills performed best under intermediate conditions. For the first time, measures of mitochondrial function post-permeabilization were used to identify optimal protocols for both crustacean gill types, paving the way for robust bioenergetic and ecophysiological investigations.