The specifically enhanced cellular immune responses in Pacific oyster (Crassostrea gigas) against secondary challenge with Vibrio splendidus

• The primary stimulation of V. splendidus could lead to immune priming in oyster.
• The THC increased significantly in primed oyster during infection of V. splendidus.
• The number of new generated hemocytes increased dramatically in primed oyster.
• The phagocytic rate was specifically enhanced after the secondary stimulation.
• Some phagocytosis and hematopoiesis-related genes participated in immune priming.

The increasing experimental evidences suggest that there are some forms of specific acquired immunity in invertebrates, but the underlying mechanism is not fully understood. In the present study, Pacific oyster (Crassostrea gigas) stimulated primarily by heat-killed Vibrio splendidus displayed stronger immune responses at cellular and molecular levels when they encountered the secondary challenge of live V. splendidus. The total hemocyte counts (THC) increased significantly after the primary stimulation of heat-killed V. splendidus, and it increased even higher (p < 0.01) and reached the peak earlier (at 6 h) after the secondary challenge with live V. splendidus compared with that of the primary stimulation. The number of new generated circulating hemocytes increased dramatically (p < 0.01) at 6 h after the pre-stimulated oysters received the secondary stimulation with live V. splendidus, and the phagocytic rate was also enhanced significantly (p < 0.01) at 12 h after the secondary stimulation. Meanwhile, the enhanced phagocytosis of hemocytes was highly specific for V. splendidus and they could distinguish Vibrio anguillarum, Vibrio coralliilyticus, Yarrowia lipolytica, and Micrococcus luteus efficiently. In addition, the mRNA expression of 12 candidate genes related to phagocytosis and hematopoiesis were also monitored, and the expression levels of CgIntegrin, CgPI3K (phosphatidylinositol 3-kinase), CgRho J, CgMAPKK (mitogen-activated protein kinase kinase), CgRab32, CgNADPH (nicotinamide adenine dinucleotide phosphate) oxidase, CgRunx1 and CgBMP7 (bone morphogenetic protein 7) in the hemocytes of pre-stimulated oysters after the secondary stimulation of V. splendidus were higher (p < 0.01) than that after the primary stimulation, but there was no statistically significant changes for the genes of CgPKC (protein kinase C), CgMyosin, CgActin, and CgGATA 3. These results collectively suggested that the primary stimulation of V. splendidus led to immune priming in oyster with specifically enhanced phagocytosis and rapidly promoted regeneration of circulating hemocytes when the primed oysters encountered the secondary challenge with V. splendidus.