Cultural conditions for forming and stabilizing protoplasts of Streptomyces griseus and S. venezuelae were studied by reference to the number of protoplasts formed, leakage from protoplasts and reversion rate. Effective formation and stabilization of the protoplasts was accomplished by using a hypertonic medium containing 10 m~-MgCl, and 25 m~-CaCl,. Electron microscopy showed that the ])rotoplasts of 5'. griseus, when prepared in the above medium, formed vesicles on the cytoplasmic membrane or out of the protoplasts, but when prepared in a medium with 3 m~-MgCl, and 3 mM-CaCI, they provided few such vesicles.The reversion of protoplasts to the normal filamentous state was examined by the growth on various synthetic agar media. A high reversion rate was obtained by incubating the protoplasts on a hypertonic agar medium containing 20 or 50 mM-MgCl,, 50 or 20 m~-CaCl,, 0.44 or 0-22 mM-phosphate and 0.01 % Casamino acids. Combinations of appropriate concentrations of MgCl, and CaC1, were significantly effective in the reversion as well as in the stability of protoplasts. The concentration of phosphate should be adjusted to 0.44 or 0-22 mM. Casamino acids enhanced both growth and reversion rates.The reversion of protoplasts of Streptomyces griseus was followed by using phase-contrast microscopy. The protoplasts incubated on medium R I enlarged to about 40 ,um and then generated filamentous hyphae from the periphery.

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The first morphological observation of a euryalid brittle star, Asteronyx loveni, using non-destructive X-ray micro-computed tomography (µCT) was performed. The body of euryalids is covered by thick skin, and it is very difficult to observe the ossicles without dissolving the skin. Computed tomography with micrometer resolution (approximately 4.5–15.4 µm) was used to construct 3D images of skeletal ossicles and soft tissues in the ophiuroid’s body. Shape and positional arrangement of taxonomically important ossicles were clearly observed without any damage to the body. Detailed pathways inside the vertebral ossicles, lateral arm plates, and arm spines for passage of nerves and water vascular structures were observed. Inter-vertebral muscles were also observed. Forms and 3D arrangements of many important taxonomical characters of the euryalids were scrutinized by µCT in high enough resolution for taxonomic description of ophiuroids.

In 1974, we1} reported on the culture media and conditions for preparing stable protoplasts of streptomycetes and the procedures for reverting them at high frequency to a filamentous state by plating the surface of the synthetic agar media (Rl and R2). This method, sometimes with minor modifications, has been employed by many researchers on protoplast fusion2~7) or genetic transformation,8~n) including recombinant DNAexperiments, in streptomycetes.However, it has been found that the regeneration frequency varies according to the species used and that high regeneration frequency is limited to a narrow range of streptomycete species. Therefore, we have worked out a new regeneration method which is applicable to some regeneration-negative species or mutants, and this is the subject of the present communication. R. H. Baltz, independently, has reported on the conditions for effective regeneration of streptomycete protoplasts at the International Fermentation Symposium held in London, Canada, in July 1980. The effective conditions he reported were quite similar to the those we had found at that time, viz., a) to overlay soft agar together with protoplasts over the basal agar layer of medium R2; b) to dry the surface of the underlayer of the agar plates before plating protoplasts; and c) to incubate the protoplasts at a lower temperature for regeneration. The emphasis in the present paper is on a newly established medium and the procedure for, regeneration which differs from that reported by Baltz, as well as on the effect of temperature on the stability of protoplasts.Streptomyces kasugaensis MB 273-18a (Atp~Amy" Arg~) were used in most of the experiments. To prepare the protoplasts, cells from a slant culture were shakecultured at 28°C for 2 days in mediumS.1} The culture was transferred with 2% inoculum size to fresh medium S supplemented with glycine (0.4%, this value differed depending on the species used1}), and shake-cultured for 26hr. Cells from 8ml culture were harvested by centrifugation, and washed with 0.35m sucrose solution. The washed cells were suspended in 8ml of medium P3 consisting of70 mM NaCl, 5 mM MgCl2, 5 him CaCl^, 0.5 m sucrose and 0.025 m TES buffer (TV-tris (hydroxymethyl)-methyl-2-aminoethanesulfonic acid, pH 7.2). Then lysozyme was added to a concentration of 0.3~1.0mg/ml, followed by incubation at 28°C for 30~60min. Since the cells of S. kasugaensis grow as short hyphae resembling bacilli in liquid medium, the filtration procedure1> was inadequate to remove the intact cells. The lysozymetreated cell suspension was therefore centrifuged at 200x0 for lOmin to remove the intact cells, and the turbid supernatant containing the protoplasts was centrifuged again at 500 x g for lOmin. The protoplasts precipitated were gently suspended in 8ml of medium PWP consisting of 70mM NaCl, 10mM MgCl2, 20mM CaCl2, 0.5m sucrose and 0.025m TES buffer (pH 7.2). After treatment with lysozyme, centrifugation and resuspension of protoplasts were carried out as soon as possible, because the protoplasts were unstab...

The genus Astrodia (Echinodermata: Ophiuroidea: Asteronychidae) was revised based on 34 specimens including two syntypes of Asteronyx excavata, two syntypes of Asteronyx plana and two syntypes of Ophiocreas abyssicola. We proposed a transfer of Ophiocreas abyssicola (Euryalidae) to the genus Astrodia. Consequently, Astrodia now includes four species, Astrodia abyssicola, A. excavata, A. plana and A. tenuispina. These four species are all redescribed. A taxonomic key to the species of the genus Astrodia is also provided.