Effects Of Lipopolysaccharide And Dopamine On Haemocytes Of Shrimp Litopenaeus Vannamei

This paper reports the mechanism of phagocytosis and prophenoloxidase (proPO) activating system activation modulated by lipopolysaccharide (LPS) and dopamine (DA) in the White Shrimp Litopenaeus vannamei, discussed mainly the following theses:(1) Establishment of haemocyte immune activity keeping methods in vitro of Litopenaeus vannamei; (2) Haemocyte phagocytosis and their signal transduction in white shrimp Litopenaeus vannamei induced by lipopolysaccharide and dopamine; (3) Signal transduction of the prophenoloxidase activating system of Litopenaeus vannamei haemocytes.induced by lipopolysaccharide and dopamine.1. Establishment of haemocyte immune activity keeping methods in vitro of Litopenaeus vannameiThe effects of four preservative solutions (CM, AC, MBHSS, M199) on haemocyte counts, viability and exocytosis of prophenoloxidase cascade system were examined. It was founded that all four solutions have notable effects on haemocyte counts, viability, and exocytosis of prophenoloxidase (proPO) cascade system (P<0.05). During 60 minutes, the haemocyte counts, viability, serine proteinase and prophenoloxidase activity in four treatments declined gradually, while phenoloxidase activity climbed increasingly. However, the haemocyte counts, and viability in CM were significantly higher than those in MBHSS and M199, and there was no significant difference between CM and AC group during 40 min, cell viability in CM was higher than that in AC at 60 min. Both serine proteinase and prophenoloxidase activity in CM were higher than other treatments in the initial 20 min, while phenoloxidase activity was reverse. The parameters of CM group showed significantly different from those of other groups after 40 min. Meanwhile, every parameter in CM showed no significant difference during 40 min, but not at 60 min. So, it's suggested that CM buffer can be chosen as a short-term preservative solution of Litopenaeus vannamei haemocyte, and the study in vitro should be completed in 40 min.2. Haemocyte phagocytosis and their signal transduction in white shrimp Litopenaeus vannamei induced by lipopolysaccharide and dopamineEffects of bacterial lipopolysaccharide (LPS) and dopamine (DA) on phagocytic activity and signal transduction of haemocytes in white shrimp Litopenaeus vannamei were studied. The haemocytes treated with LPS (1-10 mg/L) and dopamine (5-10μmol/L) in vitro showed conspicuous dose-dependent decrease in cell count. Phagocytic activity of haemocytes was promoted by LPS and inhabited by dopamine. Meanwhile, the addition of chelerythrine (a PKA inhibitor) and genistein (a TPK inhibitor) to haemocytes can inhibit phagocytosis induced by LPS, and the inhibitory efficiency on phagocytosis was:genistein> chelerythrine. Dopamine-induced cell phagocytosis was strenghtend by H-89 (a PKA inhibitor), the two inhibitors, chelerythrine and genistein, had no impact on dopamine-induced phagocytosis.3. Signal transduction of the prophenoloxidase activating system of Litopenaeus vannamei haemocytes induced by lipopolysaccharide and dopamine.LPS and DA were shown to have a negative dose-dependent effect on total haemocyte count (THC) and different haemocyte count (DHC). After 30min incubation, THC, semi-granule count (SGC) and large granule count (LGC) in LPS and DA-treated groups all decreased significantly at concentrations higher than 1 mg ml-1 and 5μmol L-1. Both serine proteinase activity (SP) and intracellular phenoloxidase activity measured in haemocyte lysate supernatant (HLS) were significantly reduced, but extracellular phenoloxidase activity in supernatant increased significantly when haemocytes were treated by LPS and DA. The results indicated that the reduction of haemocyte counts was mainly due to the degranulation and activation of proPO system from semi-granule and large granule cells. The addition of PKC inhibitor, chelerythrine was found to have an inhibitory effect on extracelluar PO activity, but SP and intracellular PO activity increased. After TPK inhibitor, genistein being added, extracelluar PO activity was also reduced. It suggests that the activation of proPO may be regulated by PKC and TPK-related signaling pathway, but the activation of serine proteinase was only regulated by PKC pathway. Phenoloxidases induced by two different elicitors (LPS and DA) revealed the same molecular masses and high diphenolase activity by electrophoresis analysis. Two bands (526 kDa and 272 kDa) of PO activity were obtained from PAGE, while in the HLS, only one band corresponding to 272 kDa was produced, which resolved to two groups of POs,166 kDa and 126 kDa, and 78.1 kDa and 73.6 kDa respectively after SDS PAGE under non-reducing and reducing conditions, it suggests that PO in L. vannmei is an oligomer, and may shows different types in and released from cells.