| Ciona intestinalis, the representative species of Urochordata, belongs to a critical taxonomic position between invertebrates and vertebrates. It was regarded as a "biology's rising star" and was also one of the best experimental system for evolutionary and comparative immunology. In this paper, methodology for artificial reproduction and whole-life-span cultivation of Ciona intestinalis was presented. Taking this animal as material, morpho-functinal characterization of haemocytes and immune responses of haemocytes to bacterial challenge was studied. The results showed that: C. intestinalis larvae were cultured with the diet combination of dry spirulina, egg yolk,Dicrateria sp., edible yeast and weaning diet for shrimp and grew up to average 59mm and matured with a surviving rate of 85% after 2 months. A whole-life-expansion laboratory culture of C. intestinalis under controlled conditions was accomplished. 8 types of blood cells, i.e. stem cell like haemocytes, hyaline amoebocytes, granular amoebocytes, refractile cells, signet ring cells, pigment cells, morula cells and compartment cells were characterized using cytochemical assay, cytoenzymatic assay, immunocytochemical assay, ultrastructural assay and phagocytosis methods. There are some similar morphological and functional traits between: Ciona stem cell like haemocytes and vertebrate hematopoietic stem cell, Ciona hyaline amoebocytes and vertebrate macrophage cells, Ciona granular amoebocytes and vertebrate granular cells, which may indicate that haemocytes of C.intestinalis have some primordial traits of vertebrate lymphocytes. However, absence of some fundamental elements of adaptive immunity made them differ from vertebrate lymphocytes. Enhanced-green-fluorescence-protein (EGFP)-expressing Escherichia coli is a good tool for studies of host-bacteria bilateral effect. Taking EGFP-expressing E.coli as infectious source, we found total haemocytes counts(THC) of Ciona intestinalis changed significantly. At 5min after infection, THC decreased 36.6% and at 3h THC reached the minimum. At 24h, THC climbed notably and at 96h recovered to normal level. Hyaline amoebocytes could phagocytose E.coli 5min post infection and excrete lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for humoural immunity while stem cell like haemocytes remained intact during infection. After bacteria injection, the amount of stem cell like haemocytes increased firstly till peaked at 24h and then decreased till resumed at 96h; with the THC increasing, stem cell like haemocytes could be witnessed division and proliferation under microscope, which explained why the THC could recover in 12-96h. Thus, it was proved that haemocytes of C. intestinalis could proliferate and mature in circulating system. In this study, a small portion of hyaline amoebocytes and granular amoebocytes died through apoptosis due to bacteria infection. Some hyaline amoebocytes exhibited at early apoptosis stage 1h after infection. Granular amoebocytes degranulated and many vesicles presented inside the cytoplasm and typical apoptosis body emerged. A few of the infected haemocytes were witnessed undergoing DNA damage after V.anguillarum infection disclosed using single cell gel electrophoresis method. It was also revealed that haemocytes experienced apoptosis to various extents both infected by E.coli and V.anuillarum at 1h, 3h, 6h and 12h by terminal-deoxynucleotidyl transferase mediated nick end labeling assay. An obvious apoptosis peak was also observed in infected haemocytes samples using flow ctometry. In conclusion, apoptosis was an important immune response of ascidian haemocytes to bacterial infection, which was the first time that validated apoptosis existing in invertebratehaemocytes after bacteria challenge to our best knowledge.
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