Comparison Of Hemolymph And Extrapallial Fluid On Cytology And Transcriptome In Pinctada Martensii

Optical and transmission electron microscopical studies were carried out to characterizethe haemocytes and extrapallial fluid cell of the pearl oyster Pinctada martensii. Andtranscriptome libraries of haemocytes and extrapallial fluid cell were constructed,respectively, then sequenced by RNA-high-throughput sequencing approach withbioinformatics analysis.According to the structural characteristics of cell morphology, haemocytes of Pinctadamartensii were divided into four categories: Lymphoid haemocyte, Large granularhaemocyte, Small granule haemocyte and Agranule haemocyte. Lymphoid haemocyteaccounts about24.7%, the average size of the cell is4.13μm, and no granules in thecytoplasm. Large granule haemocyte accounts about25%, the average size of the cell is6.40μm, granules in the cell were stained blue-purple by Wright–Giemsa. Small granulehaemocyte accounts about3.7%, the average size of the cell is4.35μm, granules filledwith the cytoplasm, and stained bright red by Wright–Giemsa. Agranule haemocyteaccounts about46.6%, the average size of the cell is6.24μm, and no granules in cells.Transmission electron microscopy (TEM) observation of haemocytes showed that: there isa small amount of mitochondria and small vacuoles in the cell, cytoplasm is uniform;Mitochondria, small cavity and high electron-dense granules were found in Large granularhaemocyte, rich endoplasmic reticulum and ribosome were found, too; There are somevacuoles and high electron-dense granules in the Small granular cell. A few small vacuoleswere found in the Small granule haemocyte, but no high electron-dense granules in thecell.According to the structural characteristics of cell morphology, extracellular fluid cells ofPinctada martensii were divided into five categories: Lymphoid haemocyte, Large granularhaemocyte, Small granule haemocyte, Agranule haemocyte and Giant cells. Lymphoidhaemocyte account for about18.8%of the total cells, the average size of the cell is4.10μm, and no granules were found in the cytoplasm. Large granular haemocyte account forabout19.0%of the total cells, the average size of the cell is6.44μm, and there are lots ofgranules in the cell, and stained blue-purple by Wright–Giemsa. Small granule haemocyteaccount for about4.4%of the total cells, the average size of the cell is4.33μm, the cellnucleus surrounded by granules, and granules stained bright red by Wright–Giemsa.Agranule haemocyte accounted for about24.1%of the total number of cells, the average size of the cell is6.08μm, and no granules were found in cytoplasm. Giant cells accountfor about33.7%of the total cells, the average size of the cell is11.09μm, cell surface werestained blue-purple by HE, a large number of granules in the cells were eosinophilic.Transmission electron microscopy (TEM) observation of extracellular fluid cells showedthat: the nucleus of Lymphoid haemocyte occupies most of the cell space, and there is asmall amount of mitochondria and small vacuoles in the cell, but high electron-densegranules were not found in the cell, cytoplasm is uniform; Mitochondria, small cavity，high electron-dense granules, were found in the Large granular haemocyte. There are somevacuoles and high electron-dense granules in the Small granular cell. A few small vacuoleswere found in the Small granule haemocyte, but no high electron-dense granules in the cell.Rich mitochondria, granules and high electron-dense granules were found in the Giantcells, and we found that some secretory vesicles were budding.Extracellular fluid cell is similar to haemocytes in the structural characteristics, wespeculated that some of extracellular fluid cells (Lymphoid haemocyte, Large granularhaemocyte, Small granule haemocyte, Agranule haemocyte) may come from haemocytes.9994differential expression genes were screened from transcriptomes of haemocytesand extracellular fluid cell. These genes were performed GO functional significantlyenriched analysis and KEGG pathway enrichment analysis. We found that most KEGGsignificantly enriched pathways and GO significantly enriched pathways in the haemocytesinvolved in migration-related activities when the haemocyte suffer from external stimuli,presume that haemocyte play an important role in stress response. Meanwhile, a largenumber of highly expressed genes of mineralization were found in the extracellular fluidcells, suggesting that the extracellular fluid cells may be involved in the process of shellformation.The findings further deepened understanding of extracellular fluid cells in origin andfunctions. Meanwhile, the discovery in the study will facilitate studies in mineralizedresearch of pearl formation in Pinctada martensii.