Zhang Shi-cui

An Estimate of Divergence Time of Parazoa and Eumetazoa and That of Cephalochordata and Vertebrata by Aldolase and Triose Phosphate Isomerase Clocks

Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined based on three different models for estimating evolutionary distances. It was shown that the evolutionary rates remain essentially unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and even between animals and plants, irrespective of the models used. Thus these enzymes might be useful molecular clocks for inferring divergence times of animal phyla. To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri, an amphioxus, have been cloned and se-quenced. Comparisons of the deduced amino acid sequences of aldolase and TPI from the freshwater sponge with known sequences revealed that the Parazoa-Eumetazoa split occurred about 940 million years ago (Ma) as determined by the average of two proteins and three models. Similarly, the aldolase and TPI clocks suggest that vertebrates and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes and protostomes.

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.

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.

Production of fertile eggs and sperms in laboratory-reared amphioxusBranchiostoma belcheri tsingtaunese

Althoug hamphioxus has been studied embryologically for over 130 years, little is known concerning the gonadal development of the laboratory-reared animal. The present study in which the amphioxus were sucessfully maintained in the laboratory for more than one year showed that they could produce fertile eggs and sperms like their wild counterparts.

In vitro study on cytotoxic effects of the organopho-sphorous pesticide profenofos on the gill cell line, FG-9307, of the flounder (Paralichihys olivaceus)

The FG-9007 cell line derived from the gill of flounderParalichthys olivaceus was used in the present study to determine the acute cytotoxic effects of the organophosphorous pesticide, profenofos. It was found that the cell growth rate was markedly reduced by profenofos at the concentrations of 2.5, 5 and 7.5 mg/L tested; and that the fine structures of the cells were also altered by profenofos, as evidenced by dilation of nuclear membranes and mitochondria cristae, and presence of enlarged lysosomes with engulfed organelles and numerous vacuoles in the cytoplasm. Probably, mitochondria, the cell energy-generating sites, are the most prominent sites of profenofos cytotoxity in the cells. This seems to be the first report of the use of marine fish cell line for evaluation of the acute in vitro cytotoxicity of organophosphorus pesticide.

CYTOTOXICITY AND GENOTOXICITY OF POLYETHYLENIMINE AND NICKEL CHLORIDE IN RED SEA BREAM (Pagrosomus major) FIN CELL LINE RSBF

A continuous marine fish cell line RSBF (i. c. Red Sea Bream Fin) was utilized to screen the cytotoxicity and genotoxicity of polyethylenimine (PEI) and nickel chloride (NiCl2) in this study on the deleterious effects of aquatic genotoxins on fish. At the 0.01 to 1 μg/ml concentration tested, PEI had acute toxicity to the treated RSBF cells (IC50=1.12, 0.92, 0.88 and 0.64 μg/ml PEI for time 0 h, 24 h, 48 h and 72 h after treatment, respectively) and markedly inhibited their proliferation in a dose-dependent manner. At the 0.001 to 5 μmol/L concentration tested, NiCl2 posed no acute toxicity but significantly stimulated their growth (107%–214% of control). Random amplified polymorphic DNA (RAPD) technique was used to detect the genotoxic effects of PEI and NiCl2 by comparing the RAPD banding patterns of the control and treated cells. RAPD analysis indicated that at the concentrations tested, PEI was more genotoxic than NiCl2 to RSBF cells; that there was a slight dose-dependent response in the genotoxic effect of PEI but not NiCl2; and that RAPD technique might provide a sensitive, non-specific genotoxic endpoint. And the potent cytotoxicity and genotoxicity of PEI on fish cells showed that we should be cautious in utilizing it as gene, vector in fish gene transfer and human gene therapy.

Development of inter-family nuclear transplant embryos by transplanting the nuclei from the loach blastulae into the non-enucleated zebrafish eggs

The developmental fate of the pronuclei in recombined embryos obtained by transplanting the donor nuclei into the non-enucleated eggs remains controversial in the case of fish. In the present study, the nuclei from the loach blastulae were transplanted into non-enucleated zebrafish eggs, the resulting 9 inter-family nuclear transplant embryos developed to larval stages. Although the development timing of the nuclear transplants resembled that of zebrafish, chromosome examination revealed that most of the recombined embryos were diploids with karyotype characteristic of loach, which was also proved by RAPD analysis. Moreover, 3 out of the 9 larval fish formed barb rudiments specific to loach. It was therefore concluded that the nuclear transplant larval fish were inter-family nucleo-cytoplasmic hybrids; and that only the donor nuclei were involved in the development of the nuclear transplant embryos, while the pronuclei in the non-enucleated eggs were likely automatically eliminated during the development.

Expression of nitric oxide synthase in the developing eye of ZebrafishDanio rerio

Expression of nitric oxide synthase (NOS) in the developing eye of zebrafish was studied by NADPH-diaphorase staining technique. NOS activity was first observed in the optic primordium and the lens placode at 5-somite stage, and remained basically unchanged up to the prim-5 stage. Upon hatching, NOS activity was nearly equally detected in the gangalion cell layer and the photoreceptor layer in the developing retina. However, it began declining in the inner plexiform layer and the inner nuclear layer at this stage. NOS activity disappeared in the lens although the anterior lens epithelium was strongly stained. Two days after hatching, NOS activity was still strong in the photoreceptor layer, but decreased markedly in the gangalion cell layer, the inner plexiform layer and the inner nuclear layer with the retinal patterning. These suggested that nitric oxide (NO), the product of NOS, is not only involved in the modulation of patterning and differentiation of the retinal cells but also in the regulation of proliferation, and differentiation of the lens fibrocytes.

Characterization of lactate dehydrogenase isozyme pattern and morphology of three marine fish cell lines

Three continuous marine fish cell lines of FG (i.e., Flounder Gill) from flounder (Paralichthys olivaceus) gill, SPH (i.e., Sea Perch Heart) from sea perch (Lateolabrax japonicus) heart and RSBF (i.e., Red Sea Bream Fin) from red sea bream (Pagrosomus major) fin, were characterized by lactate dehydrogenase (LDH) isozyme and morphological analysis. The LDH isozyme patterns of these three cell lines and their corresponding tissues of origin were investigated and compared. The results showed: (1) No difference was found in the LDH isozyme patterns of FG and flounder gill tissue. However, the LDH isozyme patterns of SPH and RSBF were significantly different from their corresponding tissues of origin; (2) LDH isozyme patterns of FG, SPH and RSBF were markedly different from each other and could serve as genetic markers for species identification and detection of cross contamination. Morphological change analysis of these three cell lines in comparison to their original tissues indicated that FG cells still appeared epithelioid without morphological transformation. However, morphological changes were found in SPH and RSBF compared to their original tissues. Therefore, the cellular morphology was still plastic in the relatively stable culture conditions, and it was possible that change of LDH patterns was related to morphological changes of fish cellsin vitro.

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.