Induction of ectodermal cells from vegetal-endodermal blastomeres of amphioxus (Branchiostoma belcheri tsingtaunese) embryos by the calcium ionophore A23187

Timing of vegetal-endodermal cell determination in amphioxus embryos remains uncertain. We tentatively tested effects of A23187, the calcium ionophore, on the development of vegetal blastomeres isolated at the 16-cell stage. It was found that when vegetal blastomeres committed to endoderm were treated with A23187 prior to gastrulation, they were transformed into ectodermal cells as evidenced by the cell morphology and function characteristic of epidermis. However, the developmental fate of the same blastomeres untreated or treated with DMSO at the same stage or of those treated with A23187 after gastrulation remained unchanged. Thus, vegetal-endodermal cells in amphioxus embryos are not irreversibly determined before the gastrula stage, and artificial increase in intracelluar Ca²⁺ concentration can induce transdetermination of the predetermined endodermal cells into ectodermal cells. Project 39470091 supported by NSFC and partly supported by Shandong Natural Science Foundation, Grant No.93D0140.     

Cytoplasmic regionalization in starfish oocyte occurrence and localization of cytoplasmic determinants responsible for the formation of archenteron and primary mesenchyme in starfish (asterias amurensis) oocytes

Starfish oocytes with intact germinal vesicles (GVs) were cut along desired planes with glass needles or ligated using silk thread loops into two parts and allowed to mature in vitro, and inseminated. The experimental results showed that (1) only the parts with GVs or partial GV contents (PGVCs) cleaved, those without any GV materials did not; but nucleated and non-nucleated fragments cut from mature eggs were able to divide; (2) the development of animal parts of oocytes containing GVs or PGVCs was like that of animal fragments of matured oocytes with female pronuclei; most of them gave rise to permanent blastulae, and just a few formed ectodermal vesicles with a little primary mesenchyme; (3) a large part of vegetal fragments with GVs or PGVCs, and the vegetal parts of mature eggs without female pronuclei developed into small but normal embryos; (4) the fragments containing GVs or PGVCs obtained from the oocytes along a plane parallel to the animal-vegetal (A-V) axis developed as normally as the halves (with or without female pronuclei) severed from mature eggs along the same axis. Based on the data above, it was concluded that (1) the non-chromatin materials in the oocyte GVs are indispensable for successful fertilization and cleavage of starfish eggs; (2) some factor (s) located asymmetrically in the vegetal hemispheres of starfish oocytes is (are) responsible for formation of the archenteron and primary mesenchyme. It is evident from the above findings that the oocyte cytoplasm of the starfish had already regionalized before the GV break-down. Contribution No. 1722 from the Institute of Oceanology, Academia Sinica     

外源视黄酸对草鱼早期胚胎发育的影响

以 10 - 6 M和 10 - 5 M的视黄酸体外处理草鱼 ( Ctenopharyngodon idellus)早期胚胎 ,结果发现其影响了胚胎的形态发育 ,影响其中轴器官的形成和神经系统的分化 ,使胚胎前部结构全部或部分缺失 ,由此诱导的神经器官 ,如眼、听囊、嗅囊等缺失或不分化 ,胚胎的尾部缩短 ;同时也减少了色素细胞的形成。并且这种畸形程度随着处理浓度加大 ,时间加长而加剧。但在原肠中期以后处理的胚胎 ,神经系统发育基本正常 ,因此说明视黄酸并不抑制原肠作用和随后的神经诱导。它可能是通过抑制某些基因的表达从而影响神经外胚层的区域性分化。     

Initiation of primary cell culture from amphioxus Branchiostoma belcheri tsingtauense

Amphioxus, a cephalochordate, is an important model fish for studies in evolution and comparative biology. A successful cell culture from amphioxus tissues in vitro would help understanding some basic issues. To determine the optimal culture conditions for proliferation of amphioxus cells, primary cultures were initiated from buccal cirri, tail, gill, gut and metapleural fold of amphioxus Branchiostoma belcheri tsingtauense. The media tested were L-15, F-12, M 199, MEM, DMEM, PRMI 1640 and LDF, each was supplemented with 20% fetal bovine serum. The optimal conditions include tail tissue cultured in L-15 or F-12 with supplement of 20% FBS and 1.5% NaCl at about 25°C.     

LOCALIZATION OF A PUTATIVE EPIBOLY-DETERMINING FACTOR IN OOCYTES OF THE GOLDFISH (CARASSIUS AURATUS)

It is unknown whether cytoplasmic determinants in goldfish eggs are asymmetrically localized beforematuration. In this study fully grown goldfish oocytes with infact germinal vesicles (GVs) were ligated withbaby hair loops along desired planes into two parts, mature in vitro, and inseminated. Embryos developedfrom the animal halves with GV of oocytes ligated along the equator un unable to undergoepiboly, while those developed from the GV-containing parts of oocytes ligated along the animal-vegetal axiswere able to. Also, embryos developed from the animal halves obtained from fertilized eggs could undergo epiboly. From there results, it was postulated that the goldfish's premature oocyte possessed a putativefactor in the vegetal hemisphere which was responsible for epiboly of the embryonic cells.     

MORPHOLOGICAL AND FUNCTIONAL STUDIES ON THE EPIDERMAL CELLS OF AMPHIOXUS (BRANCHIOSTOMA BELCHERI TSINGTAUENSE) AT DIFFERENT DEVELOPMENTAL STAGES

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Observation and analysis of morphology abnormalities in development of Oryzias melastigma embryos

Fish embryos are widely used as models in toxicology,drug development,and human disease research because of their high sensitivity,observability,and operability,providing the basis for an in-depth understanding of the embryogenesis.Increasing studies have indicated that birth defects are hereditary.In this study,we used Oryzias melastigma as a model to conduct a study of 185-day embryogenesis and observed self-induced non-pathological abnormal embryogenesis.O.melastigma experienced pre-puberty stage,adolescence stage,and senescence stage,and individuals produced up to 102 eggs per day.However,the fecundity was markedly reduced during the senescent stage.During the active egg and blastodisc stages,pseudo-fertilization and pseudo-blastocysts were observed.During cleavage at the 4-to 32-cell stages,we observed blastomeres separation or dislocation.Excessively separated blastomeres formed double blastoderms,eventually resulting in conjoined twins.During the blastula stage,we observed abnormally increased cell volume,narrowed and elongated blasto cysts,and ab normally coated blastoderms.At the organogenesis stage,we observed abnormal numbers of Kupffer's vesicles and conjoined twins.Abnormality in the location and number of oil droplets were observed in various development stages.Abnormal development was more commonly observed in fertilized eggs produced by broodstock in prepuberty or senescence stages,which is probably related to the age of fish and the egg quality.This study can provide the materials for comparative analysis in toxicological and molecular studies of O.melastigma,and may provide evidence for other economic fish that produce sticky eggs.     

Initiation of primary cell culture from amphioxus <Emphasis Type="Italic">Branchiostoma belcheri tsingtauense</Emphasis>

Amphioxus, a cephalochordate, is an important model fish for studies in evolution and comparative biology. A successful cell culture from amphioxus tissues in vitro would help understanding some basic issues. To determine the optimal culture conditions for proliferation of amphioxus cells, primary cultures were initiated from buccal cirri, tail, gill, gut and metapleural fold of amphioxus Branchiostoma belcheri tsingtauense. The media tested were L-15, F-12, M 199, MEM, DMEM, PRMI 1640 and LDF, each was supplemented with 20% fetal bovine serum. The optimal conditions include tail tissue cultured in L-15 or F-12 with supplement of 20% FBS and 1.5% NaCl at about 25°C. Supported by Doctoral Initial Fund of Ludong University (No.43304)     

Developmental expression pattern of calmodulin gene in amphioxus Branchiostoma belcheri tsingtauense

We investigated the developmental expression pattern of AmphiCaM in cephalochordate amphioxus (Branchiostoma belcheri tsingtauense). We cultured and sampled the animals at different developmental stages (eggs and larvae), and used in-situ hybridization and northern blotting to document the spatial and temporal changes in AmphiCaM expression. The alimentary tract dominates the development from the late neurula stage to the adult stage. AmphiCaM expression increased significantly in the alimentary tract during the late neurula stage and remained elevated in the adults. Our results indicate that AmphiCaM is involved in the differentiation of the alimentary tract in amphioxus; and furthermore, provide an insight into the change in function of CaM genes during evolution.     

Morphological and functional studies on the epidermal cells of amphioxus (Branchiostoma belcheri tsingtauense) at different developmental stages

Epidermal cells of amphioxus at different developmental stages were investigated by electron microscopy and colloidal carbon tracing experiments. Amphioxus epidermal cells showed different ultrastructural characteristics at larval and adult stages. The epidermal cells at all larval stages studied (24–96 h) had numerous vesicles containing electron dense materials in their apical cytoplasm. In tracing experiments, carbon particles were found in apical vesicles and interoellular spaces. Under scanning electron microscope, many crater-like protrusions were observed on the surface of the cells. These results indicated that amphioxus larval epidermal cells may be capable of endocytosis. The epidermal cells of 3-month and adult amphioxus were obviously secretory ones characterized by well-developed peripheral filaments, a prominent Golgi apparatus and abundant apical secretory vesicles. This study also showed that adult amphioxus body surface mucus contained lectin that could agglutinate human red blood cells. The authors propose that the epidermal cells of amphioxus larva and adult may contribute to the immune defense of the amimal by different means. Project 3860811 supported by NSFC and study also supported by the Shandong Natural Science Foundation (Grant No. 92D1144).     

Interplay between amphioxus complement with sea bass macrophages: Opsonic activity of amphioxus humoral fluids

Previous studies have shown the existence of a complement system in the amphioxus Branchiostoma japonicum. However, whether it has an opsonic activity similar to that of vertebrates remains unknown. We demonstrated that the humoral fluid (HF) of amphioxus promoted the phagocytosis of yeast cells with sea bass (Lateolabrax japonicus) macrophages, whereas the C3-depleted and heated HF significantly lost the phagocytosis-promoting capacity. In addition, the precipitation of factor B (Bf) led to a marked loss of opsonic activity. Moreover, C3 fragments in the HF were found to bind to yeast cell surfaces. The results indicate that the amphioxus complement system is an important element involved in the opsonic activity, which promotes the sea bass macrophage phagocytosis by tagging yeast cells with C3 fragments via the activation of alternative complement pathway.     

The reaction time of organ-forming substance in goldfish EGG and its relationship with mesodermal formation

Fertilized goldfish eggs were dechorionated with a pair of forceps and were cut off along or a little above the equator into animal and vegetative parts at desired stages with a glass needle or ligated into two connected fragments before cleavage with baby hair loop. Some of the ligated eggs were detached by further fastening soon after ligation, and some released later at different stages (2-cell, 16-cell, 128-cell, 512-cell, mid-blastula) to let the organ-forming substance (OFS) enter the blastoderm. The cholinesterase (ChE) in the resulting embryos was assayed. The results are as follows. 1. All the 142 embryos developed from the animal hemispheres cut off or ligated off before cleavage gave rise to hyperblastula in which no ChE activity was observed. 2. All 50 embryos obtained from animal halves isolated at the 8-cell stage produced ChE. 3. Embryos developed from the eggs released before the 512-cell stage formed ChE, but the later the releasing of the hair knots, the smaller the number of ChE-producing embryos. 4. After the 512-cell stage (excluding this stage), neither ChE nor tissue differentiation occurred in the embryos developed from the unfastened eggs though their OFS flow was set free. Since ChE is thought to be a muscle-specific enzyme in the early developmental stage, it is concluded that the OFS in goldfish egg appears to be indispensable for the establishment of the mesoderm. Contribution No. 1507 from the Institute of Oceanology, Academia Sinica     

Artificial culture of amphioxus (Branchiostoma belcheri tsingtauense)

Mature amphioxus (Branchiostoma belcheri tsingtauense) collected by trawl in June, 1994 from Qingdao Shazikou sea area bottom sands, were cultured under controlled indoor conditions, where they spawned and their eggs were fertilized and hatched into larvae which gradually settled at the bottom and dug into the sands after 40-50 days culture in a water trough outdoors. The total survival rate of the larvae(LSR)was 5.5% before they went into the sands, 1.9% in 4 months, 0.7% in 10 months. The survival rate of the young fish which had dug into the sands (YSR) was 35.6% in 4 months, and 12.6% in 10 months. The amphioxus number tended to be constant from the 11th month on. In the first five months after incubation, the amphioxus body length increased by an average of about 1.5 mm a month, and about 0.4 mm a month from Dec. to May of next year. The maximum length after ten months was 24 mm; the average was about 11 mm. After two years culture in the water trough, the maximum length could reach 34-35 mm, when the amphioxus gonads began to develop.     

Localization of a putative epiboly-determining factor in oocytes of the goldfish (Carassius auratus)

It is unknown whether cytoplasmic determinants in goldfish eggs are asymetrically localized before maturation. In this study fully grown goldfish oocytes with intact germinal vesiles (GVs) were ligated with baby hair loops along desired planes into two parts, matured in vitro, and inseminated. Embryos developed from the animal halves with GV of oocytes ligated along the equator were unable to undergo epiboly, while those developed from the GV-containing parts of oocytes ligated along the animal-vegetal axis were able to. Also, embryos developed from the animal halves obtained from fertilized eggs could undergo epiboly. From these results, it was postulated that the goldfish's premature oocyte possessed a putative factor in the vegetal hemisphere which was responsible for epiboly of the embryonic cells. This work was supported by the NSFC and the “Climb Plan B” (PD-B6-1-1) of the Chinese Commission of Science and Technology. Contribution No. 2915 from the Institute of Oceanology, Chinese Academy of Sciences.     

波纹龙虾胚胎的离体培养及发育观察

对波纹龙虾不同发育时期的胚胎进行离体培养和显微观察。结果表明,初始培育的离体胚胎愈接近孵化期,其孵化率愈高(最高达80.0%);波纹龙虾胚胎发育经历受精卵、卵裂期、囊胚期、原肠期、中眼色素形成期、复眼色素形成期、心跳期、破膜前期和出膜期;胚胎在发育过程中颜色变化:橙红→深橙红→砖红→灰白→接近透明。     

PRODUCTION OF FERTILE EGGS AND SPERMS IN LABORATORY-REARED AMPHIOXUS BRAN- CHIOSTOMA BELCHERI TSINGTAUNESE

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Laboratory observation on spawning,fecundity and larval development of amphioxus (Branchiostoma belcheri Tsingtaunese)

Although amphioxus is widespread in temperate and tropical seas, its population is diminishing because of environmental pollution. To keep the population of this evolutionarily important animal from diminishing, study on its reproduction and development is necessary. The main findings in this study on the spawning and fecundity of the amphioxus reared in laboratory and its larval development are as follows. 1. Water temperature markedly affected the spawning. It spawned only when water temperature reached 21°C. 2. Spawning of the amphioxus in laboratory was markedly extended. Initially, the amphioxus spawned at about 7:00 PM, but spawning time was postponed as spawning days went on. 3. The number of eggs produced by a female ranged from 1400 to 12800, average of 5800. This also represents the fecundity of the amphioxus because it shedded all eggs within the ovary at a time. 4. During the first few months of life of the amphioxus, its growth rate changed seasonally. The growth rate in summer and fall was greater than that in winter. 5. The pelagic larva became a benthic adult after 50 days. 6. The amphioxus reared in laboratory from fertilized eggs could produce fertile eggs and sperms. These findings can be a foundation for measures to address the problem of diminishing amphioxus population. Contribution No. 2274 from the Institute of Oceanology, Chinese Academy of Sciences This work was supported by CNNSF, and Chinese Academy of Sciences.     

Antioxidant enzyme activities in different genders and tissues of amphioxus Branchiostoma belcheri tsingtauense

Information regarding antioxidant enzymes in amphioxus remains lacking, and this study was carried out to examine the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in different genders and tissues of amphioxus Branchiostoma belcheri tsingtauense. Results show that (1) CuZn-SOD, CAT and GPX activities in the whole amphioxus B. belcheri tsing- tauense were basically at the same levels in male and female amphioxus, whereas both T-SOD and Mn-SOD activities in male amphioxus were significantly higher than that in the female (P<0.05); (2) The testis had significantly higher T-SOD and CuZn-SOD activities than the ovary (P<0.05); (3) CuZn-SOD activity was undetectable in the guts of male and female amphioxus; (4) For both male and female am- phioxus, the activities of CAT and GPX in the gonads including testis and ovary were the lowest (P<0.05) among the tissues examined; (5) The gut and gill had the same level GPX activities while the gut had a higher CAT activity; (6) There was no clear difference in CAT and GPX activities in the corresponding tissues between male and female amphioxus. The study on SOD, CAT and GPX activities in different genders and tissues of the protochordate provides data for future comparison of amphioxus antioxidant enzymes with those of invertebrates and vertebrates.     

Chromosome Behavior of Heat Shock Induced Triploid in Fenneropenaeus Chinensis

INTRODUCTIONTriploidanimalsareusefulbecausetheyaresterile ,characterizedbypositivegrowthinthere productiveseason ,usefulfortesting ,forintroductionofnon nativespecies ,andforprotectionofde velopedstrains.Theinductionoftriploidyhadbeenreportedinmanyaquaculturespecies .Triploidshavebeensuccessfullyinducedinshrimpusingtemperatureshockorchemicalshock (CB ,6 DMAP)in 4species :Sicyoniaingentis (Xiangetal.,1 991 ) ,Fenneropenaeuschinensis (Xiangetal.,1 992 ;Daietal.,1 993;Baoetal.,1 993;Li…     

ULTRASTRUCTURAL OBSERVATIONS ON SPOROGENESIS OF MYXOBOLUS KWANGSIENSIS HSIEH, GONG & XIAO FROM THE KIDNEY OF DACE LABEO ROHITA

The early stages and capsulogenetic stages of sporogenesis of Myxobolus kwangsiensis were studied indetail by electron microscopy. M. kwangsiensis is an intrasellular myxsporen infecting the epithelial cellsof kidney tubules of young dace Labeo rohita. The M. kwangsiensis sporogenesis presents the typical featuresof myxosporea. The initial stages are multinuceate primary cells, with one primary cell typicallyenclosing one secondary cell. The secondary cells are formed directly by cytokinesis inside the primary cells. Theprimary cell participates in the formation of the secondary cell nucleus. The nuclear division of primarycell is asynchronous by amitosis. The parasitic organisms may degenerate inside the epithelial cells at anystage. The modes of sporoblast formation , the typical features of myxosporea and the cause of its degradationare discussed.