Developing a Novel Protocol to Isolate Mitochondria from the Cnidarian Aiptasia pallida for Bioenergetic Studies

Developing a Novel Protocol to Isolate Mitochondria from the Cnidarian Aiptasia pallida for Bioenergetic Studies

Alvin Sherman Library

The symbiotic relationship between cnidarian hosts and photosynthetic dinoflagellates relies on a dynamic exchange of metabolites between key organelles including chloroplasts and mitochondria. Understanding how temperature affects this exchange is pivotal to determine its role in metabolic dysbiosis, but methodological challenges in isolating mitochondria from cnidarian tissue must be addressed. The aim of this project is to develop a reproducible homogenization and isolation protocol of mitochondria from the upside-down sea jelly Cassiopea xamachana, to be used as a model for subsequent bioenergetic studies. Mesogleal tissue from laboratory acclimated C. xamachana was finely minced, and several mechanical homogenization methods were tested to determine which approach was most efficient. Filtered seawater (FSW) and a sucrose-based Invertebrate Isolation Medium (IIM) were compared as homogenization buffers, followed by differential centrifugation. Additionally, homogenate fractions were assessed for mitochondrial function using high-resolution respirometry using a substrate-uncoupler-inhibitor-titration (SUIT) protocol at 20, 25, and 28°C. Various respiratory states were determined including photosynthetic activity, routine respiration, and electron transfer capacity at each assay temperature. All respiratory states measured increased with assay temperature, which is supported by previous findings on mitochondria from other taxonomic groups. These respiratory profiles indicated intact and coupled mitochondria with preserved membrane integrity and working electron transport. In all, these preliminary results showed that our homogenization process provides a viable method for future studies of examining the role of host mitochondria in cnidarian thermal tolerance.