The Immunity Features And The Underlying Anti-bacterial Mechanism Of Fish Erythrocyte And Shrimp Hemocyte

Fish and shrimp are important economic cultured animals in China.The studies of the fundamental immunity of the fish and shrimps are important for the effective prevention and control of the animal diseases.Erythrocyte and hemocytes are the main vehicles for oxygen transport of fish and shrimp,respectively.In these cells,oxygen is mainly carried and transported to various tissues by the respiratory proteins.Hemoglobin(Hb)and hemocyanin(Hc)is the respiratory protein of fish and shrimp,respectively.In addition to their function in respiration,the immune and anti-infection properties of the respiratory proteins have received increasing attention in recent years.In this report,grass carp(Ctenopharygodon idella),Red swamp crayfish(Procambarus clarkia),and white shrimp(Penaeus vannamei)were selected as our models to the characterize the immunity features and the anti-bacterial mechanism underlying the fish erythrocytes and shrimp hemocytes.The main results are as follows: 1.The phagocytic activity of grass carp erythrocyte against bacteria.The erythrocytes of grass carp were incubated with Staphyloccocus aureus and Areomona hydrophila which were labeled with fluoresceine isothiocyanate(FITC).By fluorescence microscopy,the results showed that RBCs were able to adhere to the two bacterial species.By scanning electron microscopy,the results showed that RBCs were not only able to adhered to the pathogens,but also could capture the bacteria by forming fibrils through filamentous pseudopods.Under confocal microscope,transmission electron microscope and scanning electron microscope,RBCs have been found to have phagocytic activity against the two bacterial species.2.The mechanism underlying the anti-bacterial features of grass carp erythrocyte.By incubating RBCs with A.hydrophila,we observed that the bacteria could cause the hemolysis of RBCs.On the other hand,the RBCs exhibited extensive killing effects on bacteria with the up-regulation of the reactive oxygen species(ROS)and high valent iron cation(HVIC).To explore the origin of ROS,hemoglobin was separated and purified,and it was found that Hb was rapidly auto-oxidized in air and produced high levels of ROS and HVIC.We found that RBCs also expressed NADPH enzyme,however,it did not take part in the regulation of the expression of ROS.Furthermore,bacterial-derived proteases and Lipopolysaccharides(LPS)could synergistically promote the Hb hydrolysis,subsequently promoted the generation of ROS which showed a strong killing effect on bacteria.3.The mechanism underlying the clearance of the released Hb by the macrophage of grass carp.The infection of the bacteria could cause the oxidation of Hb and generation of high level of ROS.However,the high level of ROS can cause damage to the fish tissues,therefore,the surplus ROS must be cleared.To address the clearance mechanism,the Hb and RBCs was injected into grass carp,respectively.The antioxidative enzymes and the amount of hemoglobin in the sera were rapidly up-regulated,indicating that the sera were associated with the clearance of the surplus Hb.Furthermore,the surplus Hb could cause damage of the head kidney tissue.It has been shown that macrophages in the head kidney can engulf Hb and promotes the release of many cytokines.In our In vitro experiments,macrophages could engulf hemoglobin,subsequently,Hb released high levels of ROS which promoted the apoptosis of the macrophage.In order to figure out the underlying mechanism,50 ?g/ml and 2 mg/ml of hemoglobin was incubated with the macrophages for 12 h,thereafter,the treated macrophages were subjected to transcriptomic analysis.The results showed that there were totally 2853 differential expressed genes(DEGs)(with 1800 up-regulated and 1053 down-regulated genes)and 3582 DEGs(with 2171 up-regulated and 1411 down-regulated genes),respectively.Among these DEGs,there were 30 and 29 cytokine genes,respectively,including chemokines and inflammatory factors,etc.The expression of these cytokines gene were confirmed by qRT-PCR assay.Taken together,macrophage could clear the surplus Hb and the Hb promoted the apoptosis released cytokines of macrophages.4.The mechanism underlying the anti-bacterial infection of hemocyanin(Hc)of red swamp crayfish.The challenge of Staphylococcus aureus and Vibrio parahaemolyticus resulted in the significant up-regulation of Hc mRNA levels,indicating that Hc might be involved in the immune response of red swamp crayfish during the bacterial challenge.To decipher the underlying mechanism,the full-length Hc(PcHMC)was purified from the hemocyte of the crayfish,while the other three GST-fused sub-domains of Hc(rPcHMC1-T,rPc HMC1-C and rPcHMC1-N)were expressed and purified from E.coli.The bacterial binding assay showed that the full-length Hc and the three sub-domains of Hc were able to bind to the bacteria with high affinity.In addition,they all were able to bind to the LPS from the bacteria.The agglutination assay showed that the agglutination ability of the four proteins depended on the bacterial species.In the case of S.aureus,PcHMC,rPcHMC1-T,and rPcHMC1-C all had stronger agglutination ability than that of rPcHMC1-N.By contrast,in the case of V.parahaemolyticus,they all showed similar growth inhibition.In phenoloxidase(PO)assay,the PO released by the stimulation of rPcHMC1-T was much greater than those of other two domains.By the phagocytosis experiments,except rPcHMC1-T,the other 3 proteins were able to promote the phagocytosis of hemocytes.In summary,Hc of the red swamp crayfish was able to bind,agglutinate and inhibit the growth of the bacteria.They could inhibit the infection of bacteria via release of PO by Hc and promote the the phagocytosis of hemocytes.The activities of the 3 sub-domains of Hc depended on the species of bacteria.5.The mechanism underlying the anti-Vibrio parahaemolyticus infection of hemocyte from white shrimp.The PBS with or without V.parahaemolyticus was injected into white shrimp,respectively.12 h post of the injection,the hemocytes of the treated white shrimp were collected and were subjected to transcriptomic analysis.Totally 2258 DEGs were obtained,with 1017 up-regulated genes and 1241 down-regulated genes in V.parahaemolyticus treated group.Among the DEGs,5 genes named MNK1/2 MAP kinase interacting serine(MNK),C-type lectin 3(CTL3),scavenger receptor B1(SR),gamma-interferon-inducible lysosomal thiol reductase(GILT),and anti-lipopolysaccharide factor-like protein(ALFP)might directly participate in the immune responses.Therefore,these 5 genes were chosen for further studies.These 5 genes were knock-downed by specific dsRNAs and the results showed that they had involved in the clearance of the V.parahaemolyticus.