Studies On Cell Culture Of Hemocytes And Celluar Immnology Of Red Claw Crayfish, Cherax Quadricarinatus

Hemocyte plays an important part in innate immunity, and acts as a performer in cell-mediatedimmunity. The count and composition of hemocyte can change dramatically, because of complexityof environment, such as environment stress, molt, pathogenic infection, struggle, and so on. Cellculture becomes one of the key factors in studying control disease of shrimp or crayfish for further.But at present, there is lacking a stable and practical culture system in vitro, so it is difficult toresearch in their relation between pathogen and host.Crayfish becomes the first choice in cell culture and some correlation work in crustacean.Compared with shrimp, it has a bigger unit, convenient and easy to indoor farming. In this paper,the red claw crayfish, Cherax quadricarinatus were chosen to study. Hemocytes andhemopoietic tissue were also chosen to optimize culture conditions and cell culture in vitro.Meanwhile, primary cultured hemocytes and Hpt cells were used to invest WSSV how to enter intohost cells. The main purpose was to reveal which innate immune way was adopted by hemocytesand Hpt how to develop and differentiate into hemocytes.The main result of this study are as follows:(1) The hemocytes of the red claw crayfish were classified by morphologic observation intothe following types: hyalinocytes (H), semi-granulocytes (SG) and granulocytes (G). Densitygradient centrifugation with Percoll was developed to separate those three subpopulations ofhemocytes. G fractionaccumulated in the layer near thebottom of centrifugetubeafter65%Percollcentrifugation. Over99%of the cells in this fraction were Gs. After the second step of Percollcentrifugation, SG fraction were accumulated in35%gradient, comprising approximately83%SGs, while H fraction comprised more than90%Hs with SGs making up the rest;(2) Two kind of improved L-15mediums were identified as optimize culture conditions forhemocytes and Hpt cells in vitro, respectively. Results showed that the cell viabilities of Hs andSGs remarkably improved in the culture with absence of granulocytes. Hs and SGs survived up toseven days with a higher viability (70%), whereas the separated Gs survived only up to three daysin vitro with a higher viability (50%), if Gs were cultured in vitro after overnight, a higher viability (80%).(3) Results of foreign particles challenge were confirmed by electron microscope study. Gscould phagocytose the beads. Results of beads challenge showed Gs have the phagocytosis abilitywith an entire process during phagosome formation, including the following four major stages:probing, cup formation, pseudopod extension, and phagosome closure. This result also indicatedthat Gs mainly performed phagocytosis by pseudopod.(4)When E.coli was incubated with Gs, small E.coli (about0.5μm) was phagocytosed by Gsmembrane invagination, larger E.coli (>1μm) maybe macro-pinogocytosed by pseudopod, andmass bacterial was engulfed by many pseudopod;(5) With TEM, Gs were detected by WSSV. TEM observation revealed that the virus particleswere localized in the cytoplasmwith virus vesicles. At higher magnification, clusters of developingvirions and mature virions were also clearly found in the cytoplasm of Gs, even the morphogenesisof WSSV, the result indicate that WSSV can enter into Gs, but we didn’t know that whetherWSSV can replicate in Gs or not;(6) To estimate the optimal treatment concentration of two drugs, CPZ and MβCD, Trypanblue solution was used to check the cytotoxicity of the two inhibitors. By laser scan confocalmicroscope analysis (LSCM), CPZ did not inhibit clathrin-mediated endocytosis in Gs, comparedwith control experiment that was not exposed to CPZ. However, compared with control specimensthat were not exposed to MβCD, WSSV labeled with FITC were almost undetected in Gs treatedwith2mg/mlMβCD. Thus, it is concludedthatWSSV adopts thecaveolae-mediatedendocytosis toenter into Gs of crayfish.(7) WSSV can infect Hpt cells, and Virus replication take place in cell nucleus. inclusionbodies can be found in cell plasma;(8) EGFP were used to transfect by three transfection reagents, transfection efficiency ofFugene HD was close to three percent, the other two reagents were less than one percent. Theresults indicated three transfection reagents were not suitable for transfection in currentconditions;(9) The plasma membrane extended pseudopodia and surrounded the beads particles to formphagosomes, in hemocytes. However, beads also could attach and bind to the cell surface and thenwere wrapped by the invagination of the plasma membrane in Hpt cells. Maybe, those cells were the precursors of hemocytes;(10) According to solexa sequencing run,29,060,562(5.8Gb) transcriptome reads and57,884,811reads (2Gb) expression profile data were produced, as determined by GO annotationand KEGG pathway mapping, functional annotation of the Unigenes recovered diverse biologicalfunctions and processes. Transcripts putatively involved in cell growth, division, differentiationand virus replication were identified. More than400Unigenes were also detected. Our data alsocan provide the most comprehensive transcriptomic resource currently available for C.quadricarinatus. Candidate genes potentially involved in cell cycle, growth, division,differentiation were identified and worthy of further investigation.In the present study, separated granulocytes from C. quadricarinatus are used as experimentalmodel to observe the process of cellular endocytic pathways for different foreign particles. Theseresults will help to understand cellar immunity response, the relation between pathogen and host,differentiation of Hpt in other crustacean and so on.