Proteomic and Small RNA Characterization of Extracellular Vesicle-enriched Particles Released from Cultured...

Extracellular vesicles and EV-enriched extracellular particles are increasingly recognized as potential mediators of intercellular communication. In coral reef ecosystems, dinoflagellate symbionts of the family Symbiodiniaceae play central roles in host metabolism, environmental acclimation, and stress responses; however, the molecular composition of extracellular particles released by cultured Symbiodiniaceae remains poorly understood. In this study, a host-isolated Symbiodiniaceae culture derived from the sea anemone Exaiptasia diaphana was established under host-free laboratory conditions for EV-enriched particle isolation and characterization. Physicochemical analysis of the recovered extracellular particle fraction showed an average diameter of 534.1 ± 63.9 nm, a polydispersity index of 0.733 ± 0.08, and an average zeta potential of - 14.57 ± 1.55 mV, indicating a heterogeneous and negatively charged extracellular particle suspension. Small RNA analysis showed that the EV-associated RNA fraction contained multiple RNA biotypes, with rRNA- and tRNA-derived reads representing the dominant annotated components, whereas miRNA-like sequences accounted for only a minor fraction of the annotated small RNA pool. Comparison against an antisense Exaiptasia diaphana CDS dataset further identified retained EV-associated small RNAs showing sequence complementarity to host coding transcripts, including cytohesin-1 and EPG5. These matches are interpreted as candidate sequence-level observations rather than evidence of functional host regulation. In parallel, miRNA-oriented analysis identified candidate miRNA-like sequences after downstream filtering. Proteomic profiling by LC-MS/MS generated 4,086 accession-level matches. After removal of uninformative entries and consolidation of duplicate protein names, 834 non-redundant named proteins were retained and classified into nine functional groups, including proteins involved in metabolism and energy production, transport and membrane trafficking, photosynthesis and chloroplast function, and signal transduction and regulation. Representative annotations included 14-3-3-related proteins, Rab/ARF family proteins, Hsp70, Hsp90, and oxygen-evolving enhancer protein. These results provide an initial qualitative molecular characterization of EV-associated molecules released by a host-isolated Symbiodiniaceae culture and serve as a baseline for future studies of algal extracellular communication and cnidarian-dinoflagellate symbiosis.