Exosome release by crustacean hyaline haemocytes in vitro

Exosomes are small microvesicles (40–100 nm) that are formed from the endosome to generate multi-vesicular bodies (MBVs). In mammals, exosomes play a significant role in cellular communication. Exosomes have also been reported for salmon and fruit fly but have not previously been studied in crustaceans. Therefore we undertook to study them in decapods. We chose the hyaline haemocytes of brachyuran crabs, Carcinus maenas and Hyas araneus, because these cells are abundant in haemolymph, are phagocytic and can be cultured on glass or plastic surfaces. Furthermore these cells have been observed to contain MVB-like structures that bear some resemblance to those in mammals that produce exosomes. Preliminary results show that highly enriched hyaline cells of both C. maenas and H. araneus, express actin and haemocyanin during 24 h in vitro. The presence of haemocyanin subunits is interesting because haemocyanin is a multifunctional protein that participates in immunity in crustaceans. As far as we are aware, the present study is the first to show that hyaline cells from a crustacean secrete this biologically important protein. Other proteins were found but we were unable to identify them by mass spectroscopy. Treatment of the hyaline cells in vitro with LPS at sub-lethal concentrations failed to reveal a change in the flow cytometric scatter plots or in the protein profiles on SDS-PAGE.