From Blue to Pink: Resazurin as a High-Throughput Proxy for Metabolic Rate in Oysters

Metabolic rate assays are critical tools for assessing organismal stress and resilience, yet their widespread application in aquaculture and ecological monitoring is limited. Improving these assays is essential for hatchery managers, farmers, and scientists seeking to identify resilient stocks and monitor stress in shellfish populations. Resazurin, a redox-sensitive dye commonly used in cell viability assays, offers a promising, high-throughput assay for metabolic rate assessment, but its application at the whole-organism level remains under explored. This study evaluates the efficacy of a resazurin-based metabolic assay in oysters (Crassostrea gigas and Crassostrea virginica) through four experimental approaches: (1) adaptation of the resazurin assay to measure oyster metabolism, (2) examination of temperature effects on oyster metabolism, (3) characterization of acute thermal stress responses, (4) examination of genetic variability in metabolism, and (5) correlations between metabolism and predicted performance in a selective breeding case study. Our findings confirm that resazurin fluorescence is correlated with oxygen consumption, validating its use as a measure of metabolism. Thermal performance assays reveal expected metabolic responses to temperature, including identification of optima and tipping points where metabolic stimulation shifts to depression under temperature stress. Acute thermal stress experiments demonstrate that oysters exhibiting greater metabolic depression are more likely to survive, supporting metabolism as a predictor of mortality. Further, genetic variation in stress responses is detected as family-level variation in metabolism. Metabolism of 50 families (C. virginica) selectively bred for performance in varying environments was measured and significantly correlated with predicted performance. By establishing resazurin as an additional reliable and scalable method for metabolic assessment, this study lays the groundwork for its broader adoption in aquaculture and conservation. Implementing this approach may provide a tool to enhance stock selection, improve hatchery management practices, and support adaptive strategies in the face of climate variability and increased environmental stress in coastal oceans.