Masanori Okauchi

Culture and development of electrically fused protoplasts from red marine algae, Porphyra yezoensis and P. suborbiculata

Abstract The electrically fused products of protoplasts from a wild type and a mutant strain of Porphyra yezoensis were cultured in a modified agar medium, named “soft agarose two-layer medium” for 40 days, and then transferred and cultured subsequently in a liquid medium. In the soft agarose two-layer medium, fused products could be distinguished easily from unfused protoplasts. The survival rate of fused products in the soft agarose two-layer medium was considerably higher than that of those in the liquid medium. However, growth rate of multi-cellular bodies which developed from fused protoplasts was higher when protoplasts were moved from the two-layer medium to liquid medium. The fused products repeated cell divisions and formed irregular-shaped multi-cellular bodies. After 2 months of culture, a large number of cells in these cellular bodies differentiated into monospores which then germinated and grew to young thalli. Eventually, four out of approximately 180 fused protoplasts developed to young thalli. Morphologically hybrid-like thalli were obtained in an electrofusion experiment with protoplasts from Porphyra suborbiculata and a mutant strain of P. yezoensis , which suggested that genetic recombination would be possible between these two species.

Origin of adult-type pigment cells forming the asymmetric pigment pattern, in Japanese flounder (Paralichthys olivaceus)

The flatfish‐specific asymmetric pigment pattern depends on the asymmetric appearance of adult‐type pigment cells after the late metamorphic stages. To understand the mechanism enabling the formation of this asymmetric pattern, we investigated the behavior of pigment cell latent precursors in postembryonic Japanese flounder, Paralichthys olivaceus, by analysis of the expression patterns of pigment lineage markers (colony stimulating factor 1 receptor, dopachrome tautomerase, kit) and the DiI (DiO) labeling test for latent precursors. We found that, throughout the larval stages, pigment cell latent precursors were predominantly localized along the dorsal and ventral margins of the flank symmetrically and migrated continuously from these regions to the lateral sides symmetrically, and after late metamorphic stages these precursors differentiated into adult‐type pigment cells on the lateral side asymmetrically. We conclude that adult‐type pigment cells that form the asymmetric pigment pattern are continuously derived from the dorsal and ventral margins of the flank during larval development. Developmental Dynamics 239:3147–3162, 2010.

Effects of cell wall-lytic enzymes on the electrofusion efficiency of protoplasts from Porphyra yezoensis

Abstract The electrofusion efficiency of protoplasts of the marine alga Porphyra yezoensis U. was investigated for the effects of cell wall-lytic enzymes. Fusion products between wild (brown color) and mutant (green color) type protoplasts and between pairs of protoplasts of wild type were visually identified and used to determine the fusion efficiency. Cell wall digestions were carried out in solutions consisting of two kinds of enzymes. One was derived from the marine bacterium Pseudomonas sp. and the other was from one of the following sources: abalones Natohaliotis discus (AAP), top shells Turbo cornutus (TSAP), sea hares Aplysia kurodai (SHAP) or sea urchins Anthocidaris crassispine (SUAP). Both AAP and TSAP, either one combined with bacterial crude enzymes, showed approximately similar effects on the protoplast fusion. Under these enzyme treatments, a maximum of 15 to 20% of the isolated protoplasts formed hybrids after exposure to electric pulses. However, their fusion activities were quite unstable and the fusion rates declined immediately. Thus, fusion rates were only a few percent even when protoplast isolation reached maximum. In the case of the combined SUAP and bacterial enzymes, optimal fusion rate of protoplasts also was observed at approximately 20% but protoplasts could form hybrids with relatively high rates (more than 10%) for at least 2 h during enzyme treatment. Conversely, little or no fusion product was obtained from the protoplasts prepared with the combined SHAP and bacterial enzyme or with the combination of partially purified enzyme from abalone and bacterial, although protoplast production was effective in these solutions. Consequently, the combination of SUAP and bacterial crude enzyme was the most efficient enzyme system for protoplast electrofusion among all the systems examined. In addition, study of the survival rates of the algal protoplasts suggested that the electrofusion efficiency would be largely influenced by the viability of protoplasts during treatment with the wall-lytic enzymes.

Factors affecting the electrofusion efficiency ofPorphyra protoplasts

The effects of various factors on the electrofusion efficiencies ofPorphyra protoplasts were investigated. These factors were protoplast stabilizing reagents, divalent cations, membrane digestive enzymes and cold storage of the protoplasts. Fusion efficiencies were dependent on the concentrations of reagents used to adjust the osmotic pressure of the medium. With mannitol or sorbitol the maximum fusion efficiency (approximately 16%) was observed at concentrations of 0.6 to 0.7 M; glucose was less effective. Brief treatment of the protoplasts with pronase stimulated electrofusion, whereas treatment with proteinase K, trypsin, phospholipase C or lipase repressed fusion. The addition of Ca2+ at 10-5 to 10-4 M in the protoplast medium enhanced the fusion efficiency to approximately four times that of the non-treated control. Sr2+ and Co2+ also stimulated electrofusion, but less effectively than Ca2+. The fusion capacity of the protoplasts remained stable for about 3 h when kept on ice, but decreased gradually when left at room temperate.

Induction of ambicoloration by exogenous cortisol during metamorphosis of spotted halibut Verasper variegatus

Cortisol, the main glucocorticoid in fish, increases during flatfish metamorphosis and peaks before the surge of thyroxine. A large body of evidence indicates the essential role of thyroxine in flatfish metamorphosis, whereas information on cortisol is limited. We administered cortisol to spotted halibut Verasper variegatus larvae in order to examine the effect on pigmentation during metamorphosis. Administration of 10 μg cortisol per mL of water from before the onset of metamorphosis (stage E) to metamorphic climax (stage G) induced the development of adult type pigment cells on the blind side of the metamorphosed juveniles and increased the occurrence of ambicolored juveniles. When 10 μg/mL cortisol was administered during stage D, stages E-F, stage G or stage H, only the administration during stages E-F induced the development of adult type pigment cells on the blind side. In addition, the expression of the gene dopachrome tautomerase (dct), a marker of melanoblasts, was enhanced at Stage E by cortisol administration. These results clearly indicated, for the first time, the enhancement of pigmentation by exogenous high-dose cortisol. Since endogenous cortisol is secreted in response to various kinds of stress in rearing conditions, these results indicate a possible influence of stress conditions in the occurrence of ambicoloration in flatfish.