| The scallop Chlamys farreri is one of the important economic mollusc speciescultured widely in China. However, the frequently occurrence of infectious disease inscallop aquaculture, especially during the larviculture stage has been threatening thesustainable development of the industry seriously. A better understanding of theontogenesis of immune system in scallop C. farreri and the maternal immunity atearly life stage is not only enriching the knowledge of mollusc developmentalimmunology, but also helping us to find available way for disease management duringlarviculture. In the present study, using molecular biological and immunologicalmethods, hematopoietic transcription factors were identified and functional analyzed,and the expression pattern of these transcription factors and immune factor duringscallop ontogenesis was examined to explore the ontogeny of immune system.Moreover, the maternal immunity of scallop and its trans-generational effect wasinvestigated to explore their protective role at early life stage.1. Identification and functional analysis of hematopoietic transcription factorsin scallopThree hematopoietic transcription factor genes CfRunt, CfCBFβ and CfGATAwere cloned from scallop C. farreri, which contains conserved runt homology domain,core binding factor beta domain and zinc finger GATA domain respectively,belonging to the runx, CBFβ and GATA123family. The recombinant protein ofCfRunt and CfGATA could bind to the DNA motif specifically, while CfCBFβ couldsignificantly increase the DNA binding activity of CfRunt. After Vibrio anguillarumstimulation, the number of circulating hemocytes was significant decreased to thelowest level at6h, and has no significant difference with that of control until48h,whereas the mRNA expression of CfRunt and CfCBFβ was significantly up-regulatedat6-48h or6-96h respectively, and the expression of CfGATA was dramaticallydown-regulated between6-12h. In vivo RNA interference of CfRunt and CfGATAcould induce the remarkable decreases of hemocytes renew rate and circulatinghemocytes number in scallop. Moreover, after the RNA interference of CfRunt, thecontinual LPS stimulation could up-regulate the expression of CfRunt, and induce the increases of hemocytes renew rate and circulating hemocytes number. In contrast,LPS stimulation in CfGATA interference group could not induce significant changesof CfGATA expression, as well as hemocytes renew rate and circulating hemocytesnumber. The results indicated that hematopoietic transcription factor CfRunt, CfCBFβand CfGATA play essential role in regulating hemocyte production.2. The expression of hematopoietic transcription factors during ontogenesis ofscallop and its response to bacterial challengeDuring the early developmental stages of scallop, both mRNA expression levelsof CfRunt and CfCBFβ increased significantly at32-cell stage and D-hinged larvaestage, also at late veliger larval stage for CfCBFβ, while CfGATA expression wasreached to the highest level at gastrula. Further whole mount embryoimmunofluorescence assay showed that CfRunt protein was first expressed in thewhole embryo at32-cell stage, then its distribution was specialized with development,locating mainly in the vegetal pole cells of morula, mesoderm of gastrula and ventralprototroch of trochophore subsequently. At early trochophore stage, CfRunt, CfCBFβand CfGATA were all appeared symmetrically on the basal side of ventral prototrochof larvae as the bilateral cells. Since the bilateral cells of CfRunt and CfGATA wereshifted to the basal side of larvae velum at D-hinged larval stage, the expression ofCfCBFβ on velum was disappeared; however a new saccular area appeared in thedorsal posterior of larvae with strong expression of the three hematopoietictranscription factors. Subsequently, in mid-veliger larvae, the expression of CfRuntand CfGATA on velum was also disappeared, and all of the hematopoietictranscription factors were expressed remarkably in the saccular area. In addition, themRNA expression of CfRunt and CfGATA at late trochophore, D-hinged and veligerlarval stages was significantly up-regulated at6,12or24h after V. anguillarumstimulation respectively, while the expression of CfCBFβ was significantly down-regulated and up-regulated at D-hinged and veliger larval stages after12h V.anguillarum stimulation. The results indicated that the scallop might experience twowaves of hematopoiesis during ontogenesis, which occurred at ventral prototroch oftrochophore as the bilateral cells and the saccular area in the dorsal posterior ofveliger larvae resceptively. 3. The expression of immune factors during ontogenesis of scallop and itsresponse to bacterial challengeThe mRNA expression of PRR (CfPGRP-S1, CfLGBP, CfLec-1and CfLec-3)and immune effectors (CfLYZ and CfLBP/BPI) was dramatically increased attrochophore or D-hinged larvae respectively, and then kept at a high expression level,whereas the mRNA expression of CfCu/Zn-SOD and CfCAT was significantlyincreased until early veliger larval stage. Consistently, the whole mount embryoimmunofluorescence assay showed that all of the PRR and immune effectors was firstappeared symmetrically in the bilateral cells of prototroch in trochophore or the velumin D-hinged larvae. However, the distribution of these immune factors was differentafter early veliger larvae. PRR and CfLBP/BPI was mainly located in the velum,mouth, esophagus or stomach region of early and mid-veliger larvae, while CfLYZ,CfCAT and CfCu/Zn-SOD mainly located in the digestive gland, stomach or velumregion. After V. anguillarum stimulation, the mRNA expression of immune effectorswas significantly up-regulated at6or12h in trochophore respectively. In D-hingedlarvae, the mRNA expression of immune factors was significantly up-regulated ordown-regulated after stimulation except CfCAT, while all expression of the immunefactors was up-regulated at6or12h at late veliger larval stage. The results suggestedthat the immune system might firstly formed in the bilateral cells of ventral prototrochof trochophore, developed gradually at D-hinged larvae, and then matured at earlyveliger larvae when it possessed functional immune response.4.Maternal transfer of immune factors and its trans-generational effect inscallopIn the eggs of scallop, the mRNA transcript of CfCu/Zn-SOD was higher thanother immune factors, such as CfLBP/BPI, CfLGBP, CfLec-3and CfLYZ, which wasextremely lower in eggs. In contrast, the protein level of those immune factors wasdectected highly except CfLec-3, while the protein level of CfCu/Zn-SOD was alsothe highest. The protein extract of scallop eggs exhibited remarkable agglutinationactivity and bactericidal effect against gram-negative bacteria Escherichia coli and V.anguillarum, and fungi P. pastoris, but gram-positive bacteria Staphyloccocus aureus.Furthermore, the protein contents of LBP/BPI, LYZ and Cu/Zn-SOD increasedsignificantly in the offspring when female scallop was stimulated with heat-killed V.anguillarum, also a significant enhancement of the Cu/Zn-SOD and antibacterialactivity was observed in the offspring from maternal immune stimulated mother scallops at eggs,4-cell and trochophore stages. Moreover, the mortality of offspringfrom the maternal immune stimulated mother scallops was significantly lower thanthat of control in trochophore and D-hinged larvae after bacterial challenge. Theresults suggested that the maternal factors could be transferred to offspring in scallop,and the maternal immunity have the trans-generational effect which played importantrole in protecting the offspring against pathogens.In summary, it could be considered that with two waves of hematopoiesis, theimmune system of scallop was formed firstly at trochophore stage, and functionalmatured after early veliger larval stage. Maternal immunity was available to protectthe offspring from invading pathogens before the maturation of immune system.These data provided useful evidences for the further study on the regulationmechanism of immune system ontogenesis, as well as enriched our knowledge todevelop new strategy for scallop aquaculture. |
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