Shotgun analysis to identify differences in protein expression between granulocytes and hyalinocytes of the European flat oyster Ostrea edulis

Abstract

Recovery of wild populations of the European flat oyster Ostrea edulis is important for ecosystem health and conservation of this species, because native oyster populations have dramatically declined or disappeared in most European waters. Diseases have contributed to oyster decline and are important constrains for oyster recovery. Understanding oyster immune system should contribute to design effective strategies to fight oyster diseases. Haemocytes play a pivotal role in mollusc immune responses protecting from infection. Two main types of haemocytes, granulocytes and hyalinocytes, are distinguished in O. edulis. A study aiming to explore differential functions between both haemocyte types and, thus, to enrich the knowledge of Ostrea edulis immune system, was performed by comparing the proteome of the two haemolymph cell types, using a shotgun approach through liquid chromatography (LC) coupled to mass spectrometry (MS). Cells from oyster haemolymph were differentially separated by Percoll density gradient centrifugation. Shotgun LC-MS/MS performance allowed the identification of 145 proteins in hyalinocytes and 138 in the proteome of granulocytes. After a comparative analysis, 55 proteins with main roles in defence were identified, from which 28 were representative of granulocytes and 27 of hyalinocytes, plus 11 proteins shared by both cell types. Different proteins involved in signal transduction, apoptosis, oxidative response, processes related with the cytoskeleton and structure, recognition and wound healing were identified as representatives of each haemocyte type. Important signalling pathways in the immune response such as MAPK, Ras and NF-κβ seemed to be more relevant for granulocytes, while the Wnt signalling pathway, particularly relevant for wound healing, more relevant in hyalinocytes. The differences in proteins involved in recognition and in cytoskeleton and structure suggest differential specialisation in processes of phagocytosis and internalisation of pathogens between haemocyte types. Apoptosis seemed more active in granulocytes. The differences in proteins involved in oxidative response also suggest different redox processes in each cell type.

Keywords: Bivalve; Haemolymph; Immune response; LC-MS/MS; Mollusc; Proteomics.