Flow Cytometry Optimisation for the Morphological, and Functional Characterisation of Haemocytes for the Silver-Lipped Pearl Oyster Pinctada Maxima

Annie J Macnab

By the early 1900’s the pearling town of Broome, had become one of the world’s most important pearling centres. Western Australia’s pearling industry is focused on the production of the South Sea pearls from the Silver-lipped pearl oyster P.maxima. Once before all pearls were found in the wild but most are now from cultured. Severe mortalities occurred in 2006 and affected the shell, and quality and size of the pearl of the oysters, the cause of the mortality/ morbidity is unknown but it is suspected that bacteria Vibrio sp. might play a role. However, research conducted has yet to determine the cause of the problem. As the cause remains unknown, it is vital to understand the role and functions of the immune cells, the haemocytes that defend the oyster against potential invaders. Better understanding the immune function of haemocytes in P.maxima could improve subsequent management of pearl oyster farming. We investigated the immune functions and morphology of haemocytes of the P. maxima oyster using flow cytometry in combination with light microscopy. A flow cytometry protocol was developed based on the optimum concentration of dyes, threshold, plate type, incubation temperature and incubation time to improve the sensitivity and specificity of the flow cytometric analyses in P. maxima. Two types of haemocyte populations were identified in the haemolymph including granulocytes and hyalinocytes. Hyalinocytes were able to be categorised into further sub-populations of large and small hyalinocytes. Large hyalinocytes were the largest and most abundant cells within the haemolymph, whilst granulocytes were intermediate-sized cells containing large quantities of granules in their cytoplasm, and were the least numerous. Small hyalinocytes were the smallest cells and were distinguished by their size, and the absence of granules from their cytoplasm. Flow cytometry revealed that large hyalinocytes are most active in the oxidative activity and mitochondrial activity of cells. The Granulocytes revealed the highest lysosomal activity and biogenesis. In contrast small hyalinocytes, were involved to a lesser extent than the large hyalinocytes and granulocytes in the oxidative activity as well as the mitochondrial and lysosomal activity.

Flow Cytometry Optimisation for the Morphological, and Functional Characterisation of Haemocytes for the Silver-Lipped Pearl Oyster Pinctada Maxima

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