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.

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.

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.

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.