Studies On Hemocytes And Their Immunological Charaterization Of Cristaria Plicata Leach

In order to understand morphological features and classification of hemocytes of C.plicata,four methods were used to study the morphological features of hemocytes of C.plicata,such as living-cell survey technique,Wrights staining technique, electron microscopy and flow cytometry.Two types of hemocytes could be identified, granulocytes and agranulocytes,according to presence or absence of granules in the cytoplasm.Granulocytes are easy to distinguish from other hemocytes for a positive feature,the possession of cytoplasmic granules while agranulocytes have few or a few electron-dense cytoplasm granules.In addition,according to the diameter of granulocytes and granular size,granulocytes were further divided into two categories; large granulocytes and small granulocytes.In accordance with the diameter of agranulocytes or N/C ratio,agranulocytes were further classified to two categories; hyalinocytes and lymphoid hemocytes.The mean concentration of the hemocytes of C.plicata was about (2.37±0.51)×106 cell ml^-1from 18 to 25 centimeters of the shell length.Large granulocytes press many large electron-dense cytoplasmic granules,they are the largest in diameter,13.18±1.20μm,the diameter of nuclear is 4.63±0.55μm,the N/C is 0.35±0.03.Granulocytes were acidophils after Wrights staining.Granulocytes are about(4.47±0.12)% in the hemocyte population.Small granulocytes have lots of small electron-dense cytoplasmic granules,the diameter is 9.11±1.11μm,the nuclear diameter is 3.58±1.11μm,and the N/C is 0.39±0.09.Small granulocytes had two tinctorial properties,acidophils and basophils,after Wrights staining.These cells are the most percentage in hemocyte population,about(56.50±1.41)%.Hyalinocytes have few cytoplasmic granules,about 7.56±0.77μm in diameter,the nuclear diameter is 3.53±0.57μm,and the N/C is large,0.47±0.08.Hyalinocytes also had two tinctorial properties,acidophils and basophils after Wrights staining.Hyalinocytes are about (35.80±2.11)% in hemocyte population.Llymphoid hemocytes have no granules in cytoplasm,are the smallest in diameter,4.68±0.43μm,the nuclear diameter is 3.73±0.05μm,and the N/C ratio is the largest,0.80±0.07.Lymphoid hemocytes were only basophils after Wrights staining.These are the least percentage in hemocyte population,about(2.96±0.23)%.After observed the sections of gill,mantle,foot,water pipe,liver,kidney,heart, male and female sexual gland,the distributions of hemocytes are different in tissues. Large granulocytes,small granulocytes and hyalinocytes were easily found in many tissues,but lymphoid hemocytes were difficult to find in many tissues.The influence of temperature on the total hemocyte count(THC)of C.plicata is very obviously.THC descended with the temperature rose before 15℃,the lowest value of THC is at 15℃,(1.9±0.59)×10⁶/ml,but after 15℃,THC grandually ascended with temperature rose.The highest value of THC was(2.62±0.6)×106/ml at 30℃.The changing trend of the number of small granulocytes and hyalinocytes with temperature were the same as the change of THC,but the changing trend of the number of large granulocytes and lymphoid hemocytes with temperature were different.On base of the results of hemocytes phagocytized yeasts and Bacillus subtilis, we can find that four types of hemocytes had activity to phagocytized bacterium out body.The phagocytic activity of small granulocytes is the stongest,the next is large granulocytes,and then is hyalinocytes,and the last is lymphoid hemocytes.The phagocytosis test is performed at 10℃,20℃,30℃and 37℃respectively,it is obviously that temperature has evident infections to hemocytes phagocytized yeasts and B.subtilis.Phagocytic ratio and phagocytic index of hemocytes increased with temperature rose.When temperature arrived 30℃,phagocytic ratio and phagocytic index were maximal value.When temperature rose again,phagocytic ratio and phagocytic index be begin to descended,when temperature arrived 37℃,the values were minimal value.Through electron microscope observed,it is different that the number of bacterium was phagocytized by various hemocytes.After hemocytes phagocytized bacterium,second lysosome and residual body could be observed.The test of capsulation of C.plicata stimulated by eggs from Unionicala arcuata found that;there is a round or oval capsule around eggs.It could observe that there were a lot of hemocytes aggregated around the capsule,such large granulocytes and small granulocytes,which become flat,spread pseudopodium and connect with each other.Through the electron microscope,it could observe that pathologic changes took place in those tissues which had capsules.Tissue cells packed seriously by eggs, hemocytes became flat and spread many long pseudopodiums.We found that large granulocytes released large granules to the capsules.It observed that five tissues of C.plianat,such as gill,mantle,water pipe,foot and a labial palp,had many eggs from Unionicala arcuata parasitic,through observed five tissues' sections.The number of eggs in five tissues carried out G-test(G =160.8975>χ²*0.001=11.345,df=4 P<0.01),it showed that the distributions of eggs in five tissues were remarkable different.