Research On The Ontogenesis Of Granulocytes In Developing Larvae And Cross-Reactivity With Haemocytes Of Other Five Bivalve Species By Monoclonal Antibody Against Granulocytes Of Scallop (Chlamys Farreri)

In this dissertation, the monoclonal antibody (MAb)6C6, specific forgranulocytes of scallop Chlamys farreri, was selected from our monoclonal antibodylibrary for studying the ontogenesis and distribution of C. farreri granulocytes indeveloping larvae and analyzing the cross-reactivity to hemocytes of other fivebivalve species. The main aim of this dissertation is to accumulate data for studyingthe ontogenesis, differentiation and immune function of hemocytes in shellfish. Thisdissertation includes the following three parts:1Characterization of monoclonal antibodyUsing methods of indirect immunofluorescence assay (IIFA), flow cytometricimmunofluorescence assay (FCIFA), immuno-electron microscopy (IEM) andimmunohistochemistry (IHC), MAb6C6specific for the granulocytes of scallop C.farreri was screened out from our monoclonal antibody library. IIFA and FCIFAanalysis indicated that MAb6C6was particularly positive to granulocytes of C.farreri with a positive rate of53±2.5%in granulocytes and6±2.3%in hyalinocytes.Immuno-electron microscopic obsetvation showed that, MAb6C6was mainlycombined to the cytoplasmic granules of granulocytes. The specificity of Mab6C6was also identified using IHC, the results indicated that MAb6C6showed highspecifictiy to the granulocytes with no cross-reactivity to other cells and tissues.Therefore, Mab6C6was employed as a specific probe to investigate the distributionand ontogenesis of granulocytes in C. farreri.2Ontogenesis and distribution of granulocytes in developing larvae of scallop(Chlamys farreri) studied by monoclonal antibody In this part, MAb6C6was employed to study the ontogenesis and distribution ofgranulocytes in C. farreri larvae including trochophora, D-shaped larvae,umbo-veliger larvae and creeping larvae using hematoxylin-eosin staining andimmunohistochemistry. The results showed that the positive signals were firstdetected in D-shaped larval stage, which was about28h~4days after fertilisation.Granulocytes were mostly observed in connective tissues in velum, digestive gland,and stomach, with a diameter of3~5μm. However, positive granulocytes withdistinguishable morphology were widely observed in umbo-veliger larval stage,which was about4~5days away from fertilisation. In umbo-veliger larval stage,granulocytes, were mostly localised in velum, stomach, digestive gland andesophagus, with a diameter of3~5μm, and often presented as cell clusters.Afterwards, in creeping larvae stage, about two weeks after fertilisation, the size andcount of positive granulocytes greatly increased, additionally, the localised sites werewidespread. Conclusively, granulocytes first appeared in D-shaped larval stage, andwere distributed in tissues and organs including velum, stomach, digestive gland etc,which are associated with feeding, transport and digestion. Moreover, the absence ofgranulocytes in early larval stages may partly attribute to the immature immunesystem in these stages.3Cross-reactivity of a monoclonal antibody against granulocytes of scallop(Chlamys farreri) with haemocytes of other five bivalve speciesIn this part, MAb6C6was employed to analyze cross-reactivity to haemocytesamong bay scallop Argoecten irradians, Japanese scallop Patinopecten yessoensis,blue mussel Mytilus edulis, Pacific oyster Crassostrea gigas and subcrenated arkScapharca subcrenata using methods of dot blotting (DB), IIFA and FCIFA. Theresults of DB showed that, MAb6C6was positive against haemocytes of A. irradians,P. yessoensis and C. gigas, and the same results were also found in IIFA, moreover,FCIFA showed that in A. irradians, P. yessoensis and C. gigas, MAb6C6had apositive rate of15±2.5%,12±2.1%and19±2.1%, respectively. In addition, Mab6C6was not against hyalinocytes of A. irradians, P. yessoensis and C. gigas andhaemocytes of R. philippinarum and M. edulis. The antigenic similarity among granulocytes of A. irradians, P. yessoensis and C. gigas suggested the closerrelationship of scallops and oyster.