| Cladocera,generally speaking named daphnia or water fleas,are planktonic crustaceans that belong to the Branchiopoda.They appear in high concentrations in pools, ponds,lakes,ditches,slow-moving streams and swamps where organic material is decomposing,and become especially abundant with suitable conditions in a short period of time.Daphnia carinata is a kind of familiar freshwater Cladocera in Shanghai belonging to the genus Daphnia,the family Daphniidae.It is convenient to gather and culture this species in the lab condition.In this paper,we chose D.carinata as the experimental animal.Recently,many researches have been concerntrated on mechanism of reproductive conversion,but the main researches were focused on induction of some environmental factors in reproductive conversion.As to the genes and proteins related to the reproductive conversion in Cladocera,few reports had been elucidated so far.Under the background,we hope that the analysis of express sequence tag on D.carinata may have both practical and theoretic values.In this paper,we made a preliminary study on embryonic development of the parthenogenetic egg in D.carinata.And,we constructed a cDNA library from sexual female of D.carinata to find the genes and proteins related to the reproductive conversion in Cladocera.The findings will contribute basic data regarding the artificial control of reproductive conversion,and will provide fundamental and significant information for future studies of Cladocerans' reproductive biology.The results and conclusions were listed as following:1.The morphological study on embryonic development of the parthenogenetic egg in Daphnia carinataThe embryonic development of the parthenogenetic egg(PE) in Daphnia carinata has been study morphologically in the present study.The PE is centrolecithal and its embryogenesis lasts about 45 hours at 24℃.According to the morphological and histological changes,the embryonic development of D.carinata could be divided into seven major stages,i.e.,the cleavage stage,blastula stage,gastrula stage,egg-nauplius stage,egg-metanauplius stage,embryo with eye pigments,and prepare-hatchling stage. The superficial cleavage of PE did not happen until approximately 40 minutes after egg laying.At the end of cleavage,the embryo consisted of 256 cells and reached the blastula stage at which a large part of the blastocoele was filled with yolk granules,accompanying by the formation of a thin sheet of divided cells.The cell division rate was accelerated and gastrulation took place by means of immigration of blastodermal cells at the end of this stage.Afterwards,the morphological changes in embryo development became remarkable.The egg-nauplius stage was characterized by the appearance of the rudiments of two antennae and a mandible in the head region of the embryo,while the egg-metanauplius stage characterized by the formation of the thoracic segments,thoracic limbs,carpace,and guts.At the eye pigments stage,the embryo was found presenting the eye pigmentation in the well-developed compound eyes.Simultaneously,other physiologically important tissues and organs started to develop.During the last prepare-hatching stage,the organelle structure in the embryo could be quite similar to that in the larval.The results could accumulate basic biological information for further research on embryonic development of Cladocera.2.Construction of cDNA library from sexual female of Daphnia carinata and primary analysis of its ESTsA high quality cDNA library was constructed from sexual female of D.carinata according to classical technique.The cDNA library contained 2.89×10~5pfu/ml.From this cDNA library,3456 EST sequences were obtained by randomly sequencing.A set of 1495 clusters was generated which contained 445 contigs and 1131 singletons.The nucleic acid and deduced amino acid sequences were compared with known GenBank sequences. Functional annotation found that 961 clusters showed significant homology with known genes involved in a broad range of metabolism,translation,development and reproduction.As our laboratory has obtained 3072 ESTs from parthenogenetic of D. carinata,we used IDEG6 to identify differentially expressed genes between sexual and parthenogenetic females of D.carinata.The results showed that there were 12 genes had differentially expressed.Among them,2 genes were highly expressed in parthenogenetic D.carinata,and the others were highly expressed in sexual D.carinata.Functional annotation found that 8 genes showed significant homology with known genes and 5 genes had function annotation.
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