An oocyte-yolk protein was purified by double-step chromatography from amphioxus ovaries. The purified protein appeared to exist as a homodimer of approximately 320kDa in native polyacrylamide gel electrophoresis(PAGE), and was reduced to a single monomer of approximately 160 kDa in sodium dodecyl sulfate-PAGE(SDS-PAGE). The protein was characterized as a phospholipoglycoprotein by native PAGE and staining of gels for phosphorus with methyl green, for lipids with oil red O and Sudan black B, and for carbohydrates using periodic acid Schiff reagent. In addition, the amino acid composition of the oocyte-yolk protein was generally similar to that of vitellogenins(Vgs) isolated from different phyla of animals including both vertebrates and invertebrates. The purified phospholipoglycoprotein is thus considered as putative amphioxus Vg.

A simple and convenient protocol for the cryopreservation of the flounder (Paralichthys olivaceus) sperm was established for "on the spot" cryopreservation of large quantities of semen. The use of three cryoprotectants, dimethyl sulphoxide (DMSO), glycerol (Gly) and methanol was tested in the method. The percentage of motile sperm present in semen after it had been frozen and thawed in the presence of DMSO, Gly or methanol was 60.5±3.6, 79.17±4.5 and 13.25±4.7%, respectively. The fertilization rates of this sperm were 67.06±15.1, 76.20±10.0 and 44.93±22.6%, while the hatching rates of eggs fertilized with this sperm were 37:40 8:3, 48:18 25:7 and 23:35±0.08%, respectively. It was found that Gly and DMSO were better cryoprotectants than methanol, with Gly giving the best overall results. Under scanning electron microscopy, it could be seen that while the majority of the frozen-thawed sperm remained morphologically normal, some exhibited lost or dilated mitochondria, swollen mid-pieces, broken tails, or damaged cell membrane, which probably caused the decrease in motility and fertility of the frozen–thawed sperm.

Phenoloxidase (PO) has been shown to be present in both invertebrates and verstebrates, yet little is known about the PO in Branchiostoma belcherisi tsingtauense. The present study demonstrated that the PO activity existedhistochemically the epithelil cells of the gill bar and intetne of adult animal, producing brown to black melanin deposits. Ultrastructural examination revealed that PO reaction products were localixed in the cytoplasmic granules of the epidermal cells, the epithlial cells of gill bar and intestine and most of them were electron-dense and homogenous. Frequently, the PO reaction products were also observed in the secondarylysosomes. The mucus form the body surface had little antibacterial activity against Escherichia coli, but it enhancedthe antibacterial activity of L-dopa and the enhancement was suppresed by PTUm a PO specific inhibiotor. This indicates that the active material inhibiting the E. coli growth in the mucus is PO.

The presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase ( proPO). The optimum temperature for activation of the proPO ranged from 30℃ to 35℃, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, zymosan and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl-p'-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.

The development of phenoloxidase during amphioxus embryogenesis was spectrophotometrically and histochemically studied for the first time in the present study. It was found that (1) PO activity initially appeared in the general ectoderm including the neural ectoderm and the epidermal ectoderm at the early neurula stage but not in the mesoderm or the endoderm, and (2) PO activity disappeared in the neural plate cells but remained unchanged in the epidermal cells when the neural plate was morphologically quite distinct from the rest of the ectoderm. It is apparent that PO could serve as a marker enzyme for differentiation of the neural ectoderm from the epidermal ectoderm during embryonic development of amphioxus.

FG-9307, a cell line derived from a gill of the flounder, Paralichthys olivaceus, was used to determine the cytotoxic effects of the organophosphorus (OP) pesticide parathion. Cytotoxicity was measured by three endpoint systems: neutral red (NR) uptake assay, tetrazolium (MTT) assay and cell protein assay. The lowest concentration of parathion tested (1 mg/ml) was toxic and there was no significant difference in cytotoxic effects among the three assays. The FG-9307 cell line is a suitable bioindicator for the screening of the acute toxicities of parathion. The fine structures of the cells were also studied. Ultrastructures were markedly altered by parathion, as evidenced by dilation of nuclear membranes and mitochondrial cristae and by the presence of lysosomes with engulfed particles. With the increase of the parathion concentration, the damage degree of the cellular structures was more serious. At the highest concentration tested (15 mg/ml), there were few visible organelles, although such changes in cell morphology were not observed under a light microscope. Apparently, this is the unnoted report of marine fish cell line used for the evaluation of the acute in vitro cytotoxicity of parathion.

Amphioxus HMGB gene, AmphiHMGB, encoding a high mobility group (HMG) protein was identified from the gut cDNA library of Branchiostoma belcheri tsingtauense. Primary structural examination indicates that it is a member of HMGB family. Molecular phylogenetic analysis suggests that AmphiHMGB represents the archetype of chordate HMGB genes including HMGB1, HMGB2 and HMGB3 lthough it may have lost its C-terminal domain during evolution. Northern blotting revealed that AmphiHMGB expression was restricted mainly to guts in the adult, and AmphiHMGB transcripts were in greater abundance in gastrula and neurula though they were markedly low in 1-day larva.

Chromosome banding study remains lacking in amphioxus heretofore. This paper reports the C-band pattern of the metaphase chromosomes of amphioxus Branchiostoma belcheri tsingtauense, the first description of banded karyotype in cephalochordate. The C-banding showed that around 50% of the second chromosome pair examined was heteromorphic, while the remaining ones were homomorphic. In contrast, the other 17 pairs were all homomorphic. These broadly support our previous suggestion that the second chromosome pair is a pair of sex-chromosomes. The C-banding stained positively about 54.3% of the chromosome surface. Most of the centromeric and terminal regions in many chromosomes had positive C-bands, and some interstitial C-bands were also observed in some chromosomes. It seems clear that chromosomes of amphioxus are highly heterochromatinized. In addition, silver staining revealed a single pair of nucleolar organizer regions (NORs) located in the telomeric regions of mediumsized chromosomes. It is suggested that the number and location of NORs in amphioxus represent the primitive condition for chordates.

In thiss tudy, a member of the MyoD gene family, AmphiMDF, wasis olated from the embryos of amphioxusby degenerate PCR, followed by rapid amplification of cDNA ends (RACE). Southern blot analysis confirmed that only a single myogenic bHLH gene waspres ent in the genome of amphioxus Branchiostoma belcheri tsingtauense. Sequence and phylogenetic analyses indicated that AmphiMDF falls at the base of its vertebrate homologs. The amino acid sequence of AmphiMDF was almost equally similar to those of the four clusters of the vertebrate MyoD family. This suggests that AmphiMDF is not only the sister but also the archetype of the vertebrate myogenic bHLH genes. The scenarios to explain the origin of the vertebrate MyoD gene family from the ancestral myogenic bHLH gene like AmphiMDF are also discussed.

The humoral fluid of Branchiostoma belcheri tsingtauense was examined for the presence of complement-like activity. The humoral fluid showed hemolytic activity for rabbit erythrocytes and those from species representing mammals, birds, amphibians and fish, but not sensitized sheep rythrocytes. There was no relationship between phylogeny of the target erythrocytes and degree of hemolysis. The hemolytic activity was optimally assayed at 20℃, at pH 7.5, and in the presence of 10mM Mg2+. The hemolytic activity was Mg2+ -dependent and heat-sensitive, and was abrogated by treatment with rabbit anti-human C3 serum, zymosan, methylamine, hydrazine, and henylmethylenesulfonyl fluoride. In addition, Western blotting and titration by turbidimetric immunoassay (TIA) revealed that amphioxus humoral fluid contained C3 component, and its oncentration is about 1.17mg/ml, which is comparable to C3 concentration in human or dog sera. These suggest that the hemolytic activities displayed by amphioxus humoral fluid appear to represent the vertebrate complement system probably operating via the alternative pathway.