Separation Of Crayfish Hemocyte Subpopulations And Investigation Of Their Function

Crustaceans lack of acquired immunity and rely mainly on innate immunity to eliminate pathogens.Hemocytes play important roles in the immune defense of crustaceans.Crayfish hemocytes can be classified into granular cells(GCs),semigranular cells(SGCs)and hyaline cells(HCs).GCs and SGCs are the dominant cell types in the circulation.Nowadays,the function of different hemocytes subpopulations is unclear.Separation of different hemocyte subpopulations is essential for the understanding of their function.In this study,we developed a method to separate the GC and SGC of C.quadricarinatus by using discontinuous Percoll density gradient centrifugation.The dilution buffer and the combination of density gradient were optimized.We found that density gradient consisted of 20 %,65 % and 100 % Percoll provided the best result.After centrifugation at 500 ×g for 20 min,the hemocytes were separated into two layers,corresponding to SGC and GC respectively.Flow cytometry analysis showed that the purities of both fractions were >95% and the cell mortalities were <5 %.The isolated cells were in good status and could be readily used for further analysis.the proPO system resides mainly in granules of GCs.The release of the proPO system from GCs is useful for the elimination of invading pathogens.SGCs also contain cytoplasmic granules,but the degranulation of SGC is still unknown.In this study,GCs and SGCs were isolated,purified and adhered cultured.The culture supernatant and thecells were collected for SDS-PAGE analysis separately.The results showed that SGC and GC both underwent exocytosis,but their proteins released kinetics were different.Moreover,the type and amount of the proteins they released were different.Next,to explore the roles of SGCs and GCs in virus defends,crayfish were infected with three WSSV strains isolated in distinct virulent(high-virulent WSSV-CN01,moderate-virulent WSSV-CN02,and low-virulent WSSV-CN03).The effects caused by different WSSV strains in different hemocyte subpopulations were compared.We found that all three WSSV strains could induce hemocytes apoptosis,leading to the cliff-like drop of hemocyte number in the last stage of infection.SGCs seemed to be more sensitive to WSSV than GCs in our experiment.The number of SGCs decreased more rapidly than that of GCs.It results in significant increase of the GC % in the infected animals.This variation trends of immune parameters in three WSSV treated groups are similar,but the high-virulent strains and moderate-virulent strains caused more seriously variation than low-virulent strains.Three WSSV strains had similar proliferation kinetics in crayfish,suggesting that the difference in virulence is not due to the difference in viral replication efficiency.These results showed by above studies are useful for us to understand the functions of different hemocytes subpopulations in the immune system of crayfish,and lay a foundation for further study of the immune defense mechanism and disease control of crayfish.