| Innate immunity plays an important role in resistance of pathogen infection in invertebrates.Poly-?-hydroxybutyrate,the polymer of 3-hydroxybutyrate,is produced by prokaryotes as energy substance in condition of lack of nitrogen and the excess of carbon.PHB can be completely or partially degraded into 3-hydroxybutyrate in aquatic animals,which promote digestion and absorption,improve immunity and resistance to pathogen infection of the aquatic animals through a series of biological processes.Therefore,PHB has a broad application prospect as feed additive and immune stimulant applied in aquaculture.Vibrio parahaemolyticus is a key pathogenic bacteria for economically-important aquatic animals,causing great economic losses to fish and crustacean aquaculture.Being different with higher animals,crustaceans lack adaptive immune response induced by antibody and complement.Therefore,it is crucial to identify the mechanism of innate immunity on resistance against pathogen attack and disease outbreak of the crustaceans.Our previous study has shown that bacterial PHB inhibited Vibrio proliferation in the intestinal of Litopenaeus vannamei postlarvae,improved their survival and resistance against Vibrio infection.However.their cellular and molecular mechanisms have not yet clarified.Crayfish Cherax quadricarinatus and Procambarus clarkia were as used as the experimental animals,and Vibrio parahaemolyticus was used as the pathogenic bacteria in this study.The FITC-labeled PHB accumulating-Halomonas(HM-PHB)were incubated with C.quadricarinatus hemocytes in vitro and were injected into the C.quadricarinatus abdomen,respectively.The fluorescence microscopic observation on in-vitro incubated cells indicated that semi-granular cells(SGs)and granular cells(Gs)could phagocytose HM-PHB cells.And the flow cytometry analysis showed the phagocytosis rate of SGs and Gs were 9.39±0.29%and 1.92±0.38%,respectively.Transmission electron microscopic observation on HM-PHB injected hemocytes further confirmed that the phagocytosis occurred 2 h after injection of HM-PHB and bacteria was intact.Eighteen hours later,bacteria was fractured Thirty-six hours later,the cytomembrane of bacteria was disappeared and PHB particles be degraded,indicating that HM-PHB could be phagocytosed and utilized by SGs and Gs.In order to further study the effect of 3-hydroxybutyrate monomer.different concentrations of 3-hydroxybutyrate(0.0.001.0.01.0.1 and 1 mg/mL)were added to TSB medium and L-15 medium,to culture V.parahaemolyticus and hemocytes,respectively.Scanning electron microscopy showed that 1 mg/mL 3-hydroxybutyrate could reduce the growth of V.parahaemolyticus and led to cell atrophy.The flow cytometry analysis showed that 1 mg/mL 3-hydroxybutyrate increased hemocyte phagocytosis to V.parahaemolyticus,and phagocytosis rate of SGs was significantly increased when compared to Gs(P<0.05),indicating that SGs was the main effector cell to enhance the immunity.The analysis on the related immune enzyme activities in P.clarkia hemocytes indicated that 1 mg/mL 3-hydroxybutyrate could improve the activity of lysozyme and acid phosphatase.Transcriptome information showed that the injection of 3-hydroxybutyrate had an obvious effect on the biological process of microtubulin in the hemocytes of crayfish.The results implied 3-hydroxybutyrate could inhibit agglutination of haemocytes,facilitate flow of haemocytes in animals,and help continuous catch foreign pathogens.In addition,3-hydroxybutyrate also regulated many different cell metabolism associated processes,suggesting they were involved in cellular immunity.In the future,expression of the related immune genes will be studied by real time PCR,the main gene of immune regulation will be determined by RNAi and verified on cellular level.This study not only lays a foundation for exploring the cellular immune mechanism of PHB and 3-hydroxybutyrate,but also provide a theoretical basis for the application of PHB as an immune booster in aquaculture. |
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