Studies On The Novel Immunomodulators And Purification And Characterization Of Phenoloxidase Of Chinese Mitten Crab Eriocheir Sinensis

The current research advancement on immune defense mechanism of crustacean was reviewed based on the data that have been issued in domestic and overseas journals.Effects of levamisole(LMS),rice bran polysaccharide(RBP),Chinese yam polysaccharide(CYP) and CpG oligodeoxynucleotide(ODN) on immune response and disease resistance of Chinese mitten crab Eriocheir sinensis,signal transduction of the prophenoloxidase activating system of haemocytes of E.Sinensis triggered by CpG oligodeoxynucleotides, and purification and biochemical characterization of phenoloxidase from E.sinensis were studied for the first time in this paper.The results are summarized as follows:(1) Effects of levamisole on immune response and disease resistance of E.sinensisIn order to determine the immunomodulatory effect of the dietary intake of levamisole on the E.sinensis,the crabs were fed diets containing 0(control),100,200 and 300 mg levamisole kg^-1 dry diet for 7 days.The total haemocyte count(THC),differential haemocyte count(DHC),phagocytic activities,respiratory burst(release of superoxide anion),phenoloxidase(PO) and lysozyme(LSZ) activities were examined at 0,2,4,6,and 8 weeks after administration of levamisole.Crabs were challenged at 1.2×10⁷ colony forming units(cfu) kg^-1 crab weight with a virulent strain of Aeromonas hydrophila (CL99920) of E.sinensis at 4 weeks after administration of levamisole,and mortalities were recorded over a 10-day period.The results demonstrate that crab treated with levamisole showed significantly higher THC,the amount of hyaline cells(HC),phagocytic percentage(PP),respiratory burst,PO and LSZ activities than those of the control group (P＜0.05).No significant differences were observed in the amount of granular cells(GC) and phagocytic index(PI) of haemocyte among the crabs fed with diets containing 0 (control) and those fed with diets at 100,200 or 300 mg levamisole kg^-1 dry die(P＞0.05). Furthermore,the levamisole treated E.sinensis were the more resistant.It was concluded that E.sinensis were fed with diets at 100,200 or 300 mg levamisole kg^-1 dry die showed increased immune ability as well as resistance to A.hydrophila infection.Under the experimental condition,the optimum dose of dietary intake of levamisole should be 200 mg levamisole kg^-1 dry diet.(3) Regulation of two plant polysaccharides on the immune response of E.sinensisIn order to determine the immunomodulatory effect of the dietary intake of RBP or CYP in plant polysaccharides on the E.sinensis,the crabs were fed diets containing 0(control), 1.0,2.0 and 4.0 g polysaccharides kg^-1 dry diet for 7 consecutive days,alternated with 7 days without polysaccharides throughout a 6-weeks test period.A series of immune parameters of E.sinensis were examined at 1,2,4 and 6 weeks after the experiment started. Crabs were challenged at 1.2×10⁷ cfu kg^-1 crab weight with a virulent strain of A. hydrophila(CL99920) of E.sinensis at 2 weeks after the experiment started,and mortalities were recorded over a 7-day period.The results showed that the THC and the amount of HC; the phagocytic activities of hacmocytcs;the polysaccharidcs-stimulated PO(POs) activities, total PO(PO_T) activities in haemocyte lysate supematant(HLS);the SOD,ACP,ALP and antibacterial activities in serum of E.sinensis fed with diets containing 2.0,4.0 g RBP kg^-1 or 1.0,2.0 g CYP kg^-1 were higher than those of the control group(P＜0.05).The effect of RBP or CYP on LSZ activity in serum of E.sinensis was irregular.Furthermore,the two plant polysaccharides treated E.sinensis were the more resistant.It was concluded that E. sinensis that were fed with diets at 2.0,4.0 g RBP kg^-1 or 1.0,2.0 CYP kg^-1 dry die showed increased immune ability as well as resistance to A.hydrophila infection.(3) Regulation of CpG oHgodeoxynucleotides on the immune response of E.sinensisThe immunomodulatory effects of two ODNs on the E.sinensis were studied when the crabs were coelom injected individually with ODN-2006(containing 3 CpG motifs) or ODN-R(containing 1 inverted CpG motif) at 0(control),1,5 and 25μg kg^-1 weight(0.1 mL crab^-1),and the crabs in another group were injected individually with saline of an equal volume was also set as a control.A series of immune parameters of E.sinensis were examined at 0,1,3,5,and 8 days after administration of the ODNs or saline.Crabs were challenged at 1.2×10⁷ cfu kg^-1 crab weight with a virulent strain of A.hydrophila (CL99920) of E.sinensis at 3 days after administration of the ODNs or saline,and mortalities were recorded over a 7-day period.The results showed that the THC,the phagocytic activities and respiratory burst of haemocytes,the PO,SOD,ACP,ALP, homolysin and antibacterial activities in serum of E.sinensis injected with ODN-2006 at 25μg kg^-1 weight were higher than those of the control group(P＜0.05).However,No significant differences were observed in the DHC,POD and LSZ activities in serum of E. sinensis among the crabs injected with ODN-2006 at 0(control),1,5,25μg kg^-1 weight and injected with saline.Furthermore,the crabs injected with ODN-2006 at 25μg kg^-1 weight were the more resistant.Surprisingly,the PO activities in serum of E.sinensis that injected with ODN-R at 25μg kg^-1 weight were higher than the control group(P＜0.05).It was concluded that ODN-2006 could enhance the non-specific immune response and resistance to potential infection of E.sinensis.(4) Signal transduction of the prophenoloxidase activating system of haemocytes of E. sinensis triggered by CpG oligodeoxynucleotidesEffects of ODN-2006 on the PO activities of haemocytes of E.sinensis were studied when haemocytes were separately treated in vitro with ODN-2006 at 0(control),5,10,15, 20 and 25μg mL^-1 for 30 min.The results showed that the intracellular and extracellular stimulated PO(PO_s) activities and extracellular total PO(PO_T) activities of haemocytes treated with ODN-2006 at 10μg mL^-1 or more were higher than control group(P＜0.05), but the intracellular PO_T activities were decreased,and that the increase or decrease of the intra- and extracellular PO activities of haemocytes was a dose-dependent following the addition of the ODN-2006.These results suggested that the degranulation of haemocytes of E.sinensis could be stimulated by ODN-2006,but new proPO were not synthesized under the test conditions.In an attempt to determine which signal transduction pathway is involved in the activation of the proPO system,haemocytes were treated in vitro with 10μg mL^-1 of ODN-2006 and a suitable concentration activators or inhibitors of specific signalling components by alone or together for 30 min.And then,the intra- and extracellular PO activities of these haemocytes were measured.The results showed that there was an increase in both intra- and extracellular PO_T activities of haemocytes treated with sodium fluoride(a G-protein activator);the intracellular PO_T activities of haemocytes were increased and the extracellular PO activities of haemocytes were decreased when 8-bromo-cAMP(a phosphokinase A(PKA) activator) or caffeine(a phosphodiesterase inhibitor) or chelerythrine(a PKC inhibitor) was added to haemocytes,in contrast,the intracellular PO_T activities of haemocytes were decreased and the extracellular PO activities of haemocytes were increased when haemocytes were treated with phorbol-12-myristate-13-acetate (PMA;a phosphokinase C(PKC) activator).On the other hand,the intra- and extracellular PO_s activities and extracellular PO_T activities of haemocytes were increased and the intracellular PO_T activities of haemocytes were increased when haemocytes were treated with genistein(an inhibitor of protein tyrosine kinase).When haemocytes were treated with chelerythrine and ODN-2006 or PMA together,the increase of the ODN-2006 or PMA-induced intra- and extracellular PO_s activities and extracellular PO_T activities of haemocytes were greatly decreased.In addition,sodium fluoride or PMA or genistein with ODN-2006 on the degranulation of haemocytes of E.sinensis have a synergistic action.Furthermore,the 8-bromo-cAMP or caffeine or chelerythrine induced inhibition on the degranulation of haemocytes could be partially relieved by ODN-2006. These results suggest that the activation of the proPO system of haemocytes of E.sinensis, including degranulation and PO activities,are induced by ODN-2006 via a G-protein mediated PKC-activating signaling pathway,but negatively regulated via the tyrosine kinase pathway.(5) Purification and biochemical characterization of phenoloxidase from E.sinensisPO from HLS of E.sinensis was purified by gel-filtration and ion-exchange chromatography,and characterized in terms of its molecular mass and biochemical and enzymatic properties by using L-dihydroxyphenylalanine(L-DOPA) as the specific substrate.It was found that proPO,isolated as a monomeric protein,had a molecular mass of 76.4 kDa,and a 70.1 kDa PO molecule was often contained in preparations.The PO activities showed optimal temperature of 40℃,optimal pH of 7.0,and an apparent K_m value of 2.63 mmol L^-1 on L-DOPA,and 6.25 mmol L^-1 on catechol.The PO was extremely sensitive to 1-phenyl-2-thiourea,very sensitive to thio urea,cysteine,ascorbic acid and benzoic acid.Based on its inhibition characteristics and the substrate affinity,the PO was classified as a kind of o-diphenoloxidase.The PO was very sensitive to trypsin-specific inhibitors SBTI,but almost not affected by trypsin-specific inhibitor TLCK at all,indicates that it is like a tyrosine-type protease.Besides,the PO was strongly inhibited by diethyldithiocarbamate(DETC),ethylenediaminetetraacetic acid(EDTA),Cu²⁺,Zn²⁺ and Ca²⁺.The results on DETC,EDTA,and some metal ions,combined with the perfect recovery effect of Cu²⁺ on DETC,EDTA-inhibited PO activities,indicate that Eriociteir PO is most probably a copper-containing metalloenzyme.