Tbx 4 and Tbx 5 gene expression associated with appendage development and its relationship with the absence of the pelvic fin in Pampus argenteus

The Tbx family is first known through the study of their functions in the body and limbs,and its members Tbx4 and Tbx5 genes are important factors in determining the characteristics of the appendages.Pampus argenteus is one of the important economical marine fishes widely distributed in offshore areas.Therefore,it is necessary to study the role of Tbx family genes in the deletion of pelvic fin in P.argenteus.In this study,we cloned Tbx4 and Tbx5 cDNA sequence of P.argenteus(GenBank:MH709128 and MH712458).The Western blot and real time PCR were used to detect the expressions of Tbx4 and Tbx5 in different developmental stages and tissues of P.argenteus.In addition,whole-mount in-situ hybridization was used to study the localization of Tbx4 and Tbx5 genes in different developmental stages of P.argenteus.Results show that the translation of Tbx4 mRNA was inhibited during the critical period of pelvic fin development.Among different tissues,Tbx4 protein levels were the lowest in the abdominal epithelium,and even lower than that in the pectoral fin,suggesting that the protein expression was also inhibited in the abdominal epithelium of adult P.argenteus.Therefore,the results indicated that upstream genes regulation led to the key stage-specific and low expression of Tbx4 during pelvic fin development and in the abdominal epithelium.

Expression and localization of vitellogenin genes(VTG)and receptor(VGR)in the gonad development of silver pomfret Pampus argenteus

Vitellogenesis is the main event of oocyte growth in oviparous animals,which is mainly manifested by the accumulation of vitellogenin(VTG).The accumulation of vitellogenin depends mainly on the absorption of exogenous vitellogenin,which enters oocyte through endocytosis mediated by its receptor(VGR).We investigated the expression and localization of VTG and VGR during gonad development of Pampus argenteus.The qPCR results show that vtgs were not expressed in male fish,but in the ovary and liver of female fish;the expression levels went up at first and then down.The expression levels of vgr in the testis were low and only 1%-3%of that in ovary.ELISA results show that during the ovarian development of P.argenteus,VTG in liver,serum,and ovary all showed a trend from increasing to decreasing.However,VTG in liver peaked in StageⅣ,and in serum and ovary peaked in Stage V,reflecting changes in the characteristics of VTG in the liver(synthesis),blood(transport),and ovaries(accumulation).During gonad development,VGR in the ovaries first increased and then decreased,reaching a peak in Stage V,in contrast to vgr mRNA expression.The VGR content in the testis was extremely low and stable,consistent with vgr mRNA.Immunohistochemistry results show that the location and intensity of VTG and VGR positive signals were synchronized with the changes of their protein content,which revealed that VTG was mainly synthesized in the liver cytoplasm,secreted into the blood,and transported to ovary in StageⅢ.VGR is highly expressed in oocytes in StageⅡ.In StageⅢ,a large amount of VTG reaches the ovary,when VGR begins to translate and is subsequently transported to the plasma membrane of the oocyte.Therefore,the positive signal of VGR was stronger near the plasma membrane of oocytes in StagesⅠandⅡ.By using qPCR,ELISA,and immunohistochemistry,the synthesis,transport,and accumulation of vitellogenin were elucidated and the mechanism of its endocytosis on egg membrane mediated by VTG during the development of P.argenteus was revealed preliminarily.

Life cycle of Craspedacusta sowerbyi xinyangensis

To explore the life cycle of Craspedacusta,the authors collected male and female specimens of the Craspedacusta sowerbyi xinyangensis in a small fire-fighting pond in Ningbo,Zhejiang Province in July,2005 and 2006.The development of C.sowerbyi xinyangensis was studied from zygote to medusa by means of light microscopy and digital camera.The zygotes of C.sowerbyi xinyangensis are globular and smooth(90-105 μm diameter)and have an equal,total cleavage to the two-cell stage 15 min after fertilization.The embryos enter the four-cell stage after another 15 min and become multicellular embryos after 3h 15min.At this stage the embryos have a diameter similar to fertilized eggs but have uneven surfaces that are distinct from the smooth surfaces of the uncleaved zygotes.Solid gastrulae are formed 7 h after fertilization.These are spherical planulae with short surface cilia that begin to swim in slow clockwise circles.After 12 h,they lose their cilia,cease swimming and become elongated planulae with one end larger than the other.Rod-like planulae,similar in thickness at both ends,are formed after an additional 7 h.After 4 days,the planulae develop into tiny polyps having two germ layers and a gastrovascular cavity.The polyp mouth is 50-62 μm in diameter,lacking tentacles but having nematocysts around the mouth.Planulae become mature polyps after 10 days(15 days after fertilization).Medusa buds(45-88 μm diameter)are formed by polyp budding,which soon become free-living medusae with 8 tentacles(380-620 μm diameters).Sometimes,the movement of frustules,which are formed by the polyps and similar to planulae in morphology can also be observed.