The Study On Mechanism Of Phagocytosis And Apoptosis Of Haemocyte In Eriocheir Sinensis

Eriocheir sinensis was an important economic crustacean species in China. However, the intensive farming and environmental deterioration have frequently induced infectious diseases through the pathogens like Aeromonas hydrophila, Vibrio parahemolyticus, EsRV905 and EsRNV, which resulted in significant economic loss. Due to the lack of specific immunity, crabs solely prevents itself from the pathogenic infection by the innate immune system including phagocytosis, melanization, nodulation, encapsulation and the release of relative bactericidal substances. In order to reduce the outbreak of disease during E. sinensis breeding, it is essential to know the immune mechanisms of E. sinensis haemocytes against pathogen. In this paper, we characterised the different types of haemocytes of E. sinensis and determined the in vivo phagocytic ability of different haemocyte types. Besides, we investigate the possible toxicity mechanism of lipopolysaccharide (LPS) on E. sinensis haemocytes apoptosis and the immune response of relative genes peroxinectin and mitogen-activated protein kinase (MAPK) during the phagocytosis and haemocytes apoptosis. Main results are listed below:1. Classification and phagocytosis of circulating haemocytes of E. sinensis.Based on the phase contrast microscope, transmission electron microscope and cell staining techniques, the haemocytes of E. sinensis were divided into three types: hyalinocytes, semigranulocytes and granulocytes. Hyalinocytes approximately accounted for 55.0% and 59.8% of total circulating haemocytes in the female and male E. sinensis, respectively. They were the most abundant circulating haemocytes with round, oval or irregular shapes. Semigranulocytes represented approximately 24.9% and 21.1% of the THC in the female and male E. sinensis, respectively. These haemocytes, with a spindle or ovoid shape, contained numerous small round granules and a few large refractile granules. Granulocytes represented approximately 20.1% and 19.2% of the THC in the female and male E. sinensis, respectively. These haemocytes, with a round, spindle or ovoid shape, was filled with numerous large round refractile granules. The hyalinocytes were demonstrated to be the most avid phagocytic haemocytes, accounting for approximately 88.7% of the total phagocytes. The haemocyte-containing granules displayed limited phagocytic ability, with approximately 5.0% of granulocytes and 6.3% of semigranulocytes displaying positive phagocytic ability against the invading polystyrene beads in vivo. The phagocytic ratio of hyalinocytes, semigranulocytes and granulocytes were 6.0,5.7 and 5.9, respectively.2. Effect of lipopolysaccharide on the haemocyte apoptosis off. sinensisThe mechanism of LPS on apoptosis of E. sinensis haemocytes was investigated by transmission electron microscope, DNA ladder, flow cytometry and laser confocal microscopy. After injection with Aeromonas hydrophila, Bacillus subtilis and LPS, all three types of haemocytes experienced dramatic decline and then rapid recovery to their initial levels, and the injection of high concentration of LPS and A. hydrophila resulted in more serious loss and slower recovery of haemocytes. It was shown that LPS induced serious damage on the DNA and morphological change of haemocytes, which resulted in obvious haemocytes apoptosis. The ratio of apoptotic haemocytes increased with time and the concentration of LPS, and most apoptotic haemocytes were considered to be late apoptotic cells. ROS production observed by bright fluorescent signal was detected within haemocytes, especially in granules-containing haemocytes. Significant outburst of ROS production was observed in LPS treated haemocytes followed by a drastic decline. These results indicated that LPS would induce oxidative stress on haemocytes from E. sinensis, and cause ROS burst, DNA damage and subsequently apoptosis. The process of ROS-mediated apoptosis might be one of the potential toxicity mechanisms of LPS on crustacean haemocytes.3. Immune response of E. sinensis peroxinectin (EsPX) during haemocytes phagocytosisEsPX contained an open reading frame of 2325 bp encoding a polypeptide of 775 amino acids with a signal peptide of 16 amino acids. The predicted molecular mass of the mature EsPX protein is 85.2 kDa, with an estimated pI of 6.91. EsPX is composed of a C terminal peroxidase domain and a N terminal integrin binding KGD motif. RT-PCR and Real-time PCR revealed that the EsPX was distributed in haemocytes, gonad, hepatopancreas, gill, muscle, heart, intestine and stomach, especially in muscle, haemocytes and stomach. Furthermore, the EsPX was verified to be located in hyalinocytes, semigranulocytes and granulocytes, and was distributed throughout the cytoplasm and nucleus, especial in cytoplasm. After injected with beads, LPS and A. hydrophila, the EsPX mRNA expression was significantly up-regulated in vivo and restored to its initial level within 32 h. In the in vitro experiment, the stimulation of LPS and beads also induced a prominent boost of EsPX followed by remarkable release of the incremental EsPX into the extracellular matrix, and the additive beads was found to be engulfed by the haemocytes, which was considered to be relative to the release of the incremental EsPX.4. Immune response of MAPK of E. sinensis during haemocytes apoptosisThe full-length cDNA sequence of EsMAPK was 2455 bp and encoding 365 amino acids. The predicted molecular mass of the mature EsMAPK protein is 42.4 kDa, with an estimated pI of 5.93. EsMAPK owned a Catalytic domain of Serine/Threonine protein kinases and shared highly similarities (95%) with other crustaceans MAPK. EsMAPK was distributed in haemocytes, gonad, hepatopancreas, gill, muscle, heart, intestine and stomach, especially in hepatopancreas. Furthermore, the EsMAPK was verified to be located in hyalinocytes, semigranulocytes and granulocytes, and was distributed throughout the cytoplasm. After injected with LPS and A. hydrophila, the EsMAPK mRNA expression was significantly up-regulated and down-regulated in vivo, respectively. In the in vitro experiment, the stimulation of LPS and A. hydrophila induced a prominent boost of EsMAPK protein within haemocytes accompanying with melanization and haemocytes degranulation. These findings suggested that the expression of EsMAPK was susceptible to exterior stimulation.