Growth inhibition and changes in morphology and actin distribution in Acetabularia acetabulum by phalloidin and phalloidin derivatives

Summary.  Effects on morphology and microfilament structure caused by phalloidin, phallacidin, and some semisynthetic phalloidin derivatives were studied in vegetative cells of the green alga Acetabularia acetabulum (L.) Silva. All phalloidin derivatives (except for phalloidin itself) caused growth stop of the alga after 1 day and (except for the fluorescein-labeled phalloidin) death of the cells after 4–7 days. Hair whorl tip growth and morphology as screened by light microscopy, as well as microfilament structure in tips, suggested that growth stop is correlated with a disorganization of actin filaments similar to that recently described for jasplakinolide (H. Sawitzky, S. Liebe, J. Willingale-Theune, D. Menzel, European Journal of Cell Biology 78: 424–433, 1999). Using rabbit muscle actin as a model target protein, we found that the toxic effects in vivo did not correlate with actin affinity values, suggesting that permeation through membranes must play a role. Indeed, the most lipophilic phalloidin derivatives benzoylphalloidin and dithiolanophalloidin were the most active in causing growth stop at ca. 100 μM. In comparison to the concentration of jasplakinolide required to cause similar effects (<3 μM), the two most active phalloidin derivatives exhibited an activity ca. 30 times lower. Nonetheless, lipophilic phalloidin derivatives can be used in algae, and probably also other cells, to modulate actin dynamics in vivo. In addition, we found that the fluorescent fluorescein isothiocyanate-phalloidin is able to enter living algal cells and stains actin structures brightly. Since it does not suppress actin dynamics, we suggest fluorescein isothiocyanate-phalloidin as a tool for studying rearrangements of actin structures in live cells, e.g., by confocal laser scanning microscopy. Received November 5, 2001; accepted August 8, 2002; published online November 29, 2002     

Sperm cell surface characteristics of Plumbago zeylanica L. in relation to transport in the embryo sac

Myosin associated with the male germ cells of angiosperms interacts with actin, promoting transport of the non-motile generative and later sperm cells in the pollen tube. Myosin localizing on the sperm cell plasma membrane seems negligible in Plumbago, as reflected by the absence of: (i) anti-myosin labeling using immunoelectron microscopy, (ii) sperm motility on actin matrices, and (iii) electrophoretic movement changes after addition of antibody. Sperm cells injected directly into actively streaming Nitella internodal cells, however, follow actin bundles and their movement is sensitive to ATP and Mg²⁺. This may be based on simple charge binding since negatively charged latex beads also migrate on actin, whereas neutral or positively-charged latex beads do not. Sperm cells are negatively charged according to capillary microelectrophoresis, whereas killed sperm cells, which are positively charged do not migrate. The sperm cell that normally fertilizes the egg has a higher calculated charge (8.277 × 10³ esu/cm²) compared with the sperm cell that fuses with the central cell (6.120 × 10³ esu/cm²). Received: 15 December 1998 / Accepted: 21 January 1999     

Insulin-producing cells from human adipose tissue-derived mesenchymal stem cells detected by atomic force microscope

We successfully differentiated human adipose tissue-derived mesenchymal stem cells (haMSCs) into insulin-producing cells (IPCs) in vitro and did not use any insulin which might be absorbed by cells during in vitro culture. Expression of insulin gene was massively increased by 28,000-fold at day 12 compared with haMSCs (P < 0.05). IPCs could secrete insulin after glucose was stimulated. The higher the concentration of glucose, the more production of insulin was noted. We reported AFM images of IPCs for the first time. AFM images showed that the sizes of cells were similar to each other, and all IPC surface had a porous structure in the cytoplasm area. In sugar-free group, the size of holes was similar (diameter, 1,086.98 ± 156.70 nm; depth, 185.22 ± 52.14 nm). In higher sugar-stimulated group, there were more holes with bigger diameter and smaller depth. (diameter, 3,183.65 ± 2,229.18 nm; depth 109.42 ± 56.26 nm, P < 0.05). We found that the hole diameter and depth could change with the concentration of glucose in media. Concurrently, laser scanning confocal microscopy images indicated that cortical actin network beneath plasma membrane in IPCs was dense and continuous. After glucose stimulation, we found the actin web depolymerized and became discontinuous in IPCs. We speculated that diameter augmentation of holes located in the cytoplasm area in IPCs was one manifestation of excytosis increase.     

Jasplakinolide reversibly disrupts actin filaments in suspension-cultured tobacco BY-2 cells

Summary. Jasplakinolide is potentially a useful pharmacological tool for the study of actin organization and dynamics in living cells, since it induces actin polymerization in vitro and, unlike phalloidin, is membrane permeative. In the present work, the effect of jasplakinolide on the actin cytoskeleton of living suspension-cultured Nicotiana tabacum ‘Bright Yellow 2’ cells was investigated. Actin filaments in the living cells were disrupted by jasplakinolide. The effect of jasplakionlide on the actin cytoskeleton was concentration and time dependent. When cells were treated with a moderate concentration (150 nM) of jasplakinolide, cortical actin filaments were disrupted preferentially, whereas actin aggregated at the perinuclear region. With concentrations higher than 400 nM and exposure times longer than 30 min, actin filaments in the cell disappeared completely. The effect of jasplakinolide on the actin cytoskeleton was reversible even at high concentration. Actin bundles appeared first in the perinuclear region within 5 min, and the cortical actin array was reestablished in 15 min, suggesting that actin filaments might be organized at this region. Received July 31, 2001 Accepted December 14, 2001     

Reproduction of the red fox <Emphasis Type="Italic">Vulpes vulpes</Emphasis> in western France: does staining improve estimation of litter size from placental scar counts?

We tested a staining method on uteri for counting placental scars on red fox. We estimated reproduction parameters on 358 females collected in three study areas in western France from 1st February 2002 to 31st January 2005. Placental scars (n = 103) were described by macroscopic examinations using the following variables: (1) the width and (2) the aspect of placental scars, (3) the abundance of macrophages or the presence of blood, (4) the presence of swellings, (5) the presence and colour of a central band and (6) the presence and colour of lateral bands. A factorial correspondence analysis showed strong associations between the month when scars were examined and categories of variables. Staining on placental scars made macrophages more visible, facilitating identification of ‘active’ placental scars, i.e. related to the last pregnancy. However, distinction between placental scars due to earlier pregnancies and resorptions was not possible. The staining method used provides a standard that could be useful for obtaining comparable and repeatable results. The mean number of placental scars was 4.85 ± 1.46 (n = 103) per vixen. The mean number of embryos per vixen was 4.66 ± 1.35 (n = 68) for yearlings and 5.53 ± 1.50 (n = 96) for older females. Including percentages of barren vixens, the total population productivity was significantly smaller for yearlings (3.62 ± 1.86, n = 158) than for older females (4.28 ± 1.75, n = 186). We discuss these results in relation to fox densities, culling and food availability.     

Structure, morphology, and composition of silicon biocomposites in the palm tree Syagrus coronata (Mart.) Becc.

Summary.  Syagrus coronata is an economically important palm tree grown as an ornament, for the oil extracted from its seeds, and the wax from its leaves which has several applications in industry. Silicon biocomposites were analyzed in leaves of S. coronata. Silica bodies were found as extracellular silica masses between the hypodermal-layer cell walls and in granules present in the vacuoles of palisade cells. Scanning electron microscopy of the hypodermal layer of cells showed a collection of spherical bodies embedded in enveloping cavities that outlined the general structure of the bodies. Globular subunits with sharp edges formed the spherical bodies that ranged from 6 to 10 μm in diameter (average, 7.8 μm). X-ray microanalysis detected only silicon and oxygen homogeneously distributed throughout the bodies. Vacuoles of palisade cells contained a large number of granules ranging from 20 nm to 1.2 μm in size (average, 300 nm). Transmission electron microscopy associated with electron spectroscopic imaging and electron energy loss spectroscopy were used to determine the elemental composition of the granules. Vacuolar granules were amorphous and composed of silicon and oxygen, suggesting they consist of amorphous silica biominerals. No nitrogen, indicative of organic matter, was detected in the granules. Received November 26, 2001; accepted July 1, 2002; published online October 31, 2002 RID="*" ID="*" Correspondence and reprints: Departamento de Microbiologia Geral, Instituto de Microbiologia Professor Paulo de Góes, Centro de Ciências da Saude, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil.     

Expression of cell-cycle-related proteins and excitoxicity

Summary.  Previous work from our laboratory has suggested the functional contribution of p53 to the cascade of events triggered by excitatory amino acids and leading to cell death in primary neurons. Here we show that this paradigm can be extended to cortical neurons treated with NMDA. We found that exposure of the cells to either 300 μM or 2 mM NMDA induced an enhancement of p53 protein levels which was already significant at 60 min after the lesion, while very low staining of the protein was observed in untreated cells. The effect was time- and concentration-dependent, reaching the maximal induction at 3 h. NMDA treatment also resulted in an increase of gadd45 protein levels which was evident in both treatment at 3 h, the time when p53 was maximally induced. Our data give further evidence suggesting that a repertoire of events typical of proliferating cells is activated in degenerating neurons. Received June 29, 2001 Accepted August 6, 2001 Published online June 3, 2002     

Age and growth of Lestrolepis japonica (Aulopiformes: Paralepididae) in Kagoshima Bay,southern Japan

 Age and growth of a paralepidid, Lestrolepis japonica, were determined from sagittal otoliths of specimens collected from April 2001 to February 2002 in Kagoshima Bay, southern Japan. Marginal growth increments indicated that the annulus was formed once a year, in August and September. Four groups were recognized, having none, one, two, or three annuli, respectively. Age in months was assigned to each individual on the basis of August as the birth month. The maximum recorded age was 48 months. Growth was expressed by von Bertalanffy's equation, L _t  = 171.91{1 − exp[−0.148(t + 0.403)]}, t being the age in months and L _t the standard length (mm) at age t. Received: July 15, 2002 / Revised: November 18, 2002 / Accepted: December 10, 2002     

Induction conditions for somatic and microspore-derived structures and detection of haploid status by isozyme analysis in anther culture of caraway (Carum carvi L.)

Plant regeneration was obtained from cultured anthers and hypocotyl segments of caraway (Carum carvi L.). Microspore- and somatic tissue-derived embryos were compared by observation of the regeneration process under identical induction conditions. Fluorescent microscopy with DAPI staining showed initiation of cell divisions and formation of embryogenic callus and somatic embryos from anther sacs, with production of embryos of both microspore and somatic origin. Induction of somatic embryos from hypocotyl-derived callus was also demonstrated. Isozyme native polyacrylamide gel electrophoresis was used to identify haploids and doubled haploids, and to determine the frequency of spontaneous diploidization of regenerated plants of microspore origin. Donor plants (2n = 20) and their anther-derived derivative plants (n = 10, 2n = 20, 4n = 40) in callus stage or leafy rosette stage were compared. The esterase (EST) band patterns of regenerated plants differed from the heterozygous parental material, suggesting that the regenerated plants were microspore-derived haploid/doubled haploid plants. The similar profile of EST bands between the diploid anther-derived plants and a sample of the donor plants corresponded to a somatic regeneration pathway. Although the selected induction conditions revealed no preference for induction of microspore embryogenesis, the anther culture protocol established for caraway utilizing isozyme segregating EST loci markers is suitable for DH production.     

Migratory history of the threespine stickleback Gasterosteus aculeatus

 The migratory history of two highly divergent forms (the Japan Sea and Pacific Ocean forms) of the threespine stickleback Gasterosteus aculeatus collected from Japanese brackish water (seawater) and freshwater was studied by examining strontium (Sr) and calcium (Ca) concentrations in their otoliths using wavelength dispersive X-ray spectrometry on an electron microprobe. The Sr : Ca ratios in the otoliths changed with salinity of the habitat. The otolith Sr : Ca ratios of the freshwater resident-type samples of the Pacific Ocean form showed consistently low Sr : Ca ratios, averaging 0.85–0.96 × 10⁻³ from the core to the edge. In contrast, the otolith Sr : Ca ratios of the anadromous type of both the Japan Sea and Pacific Ocean forms fluctuated strongly along the life history transects in accordance with their migration patterns from seawater to freshwater. The higher ratios in the anadromous type, averaging 5.4 × 10⁻³, in the otolith region from the core to 200 μm, corresponded to the seagoing period, suggesting that otolith Sr : Ca ratios are affected by ambient water salinity. These findings clearly indicate that otolith Sr : Ca ratios reflect individual life histories, and that these two highly divergent forms of stickleback have a flexible migration strategy. Received: May 23, 2002 / Revised: July 29, 2002 / Accepted: August 19, 2002 Acknowledgments We are grateful to Dr. S. Mori of Gifu Keizai University, and Miss M. Yamada and Messrs. M. Kume and T. Kitamura of Hokkaido University, for their assistance in sampling. This work was supported in part by Grant-in-Aid No. 13760138 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Correspondence to:Takaomi Arai     

Actin expression in germinating seeds of Phaseolus vulgaris L.

Actin was present at very low levels in the seeds of common bean (Phaseolus vulgaris L.) compared with those from other species, and was observed mostly in the embryo. A time-course of actin expression in germinating bean seeds revealed an induced expression of both the mRNA and protein. Initially, the actin mRNA in seeds was barely detectable by northern blot analysis. However, there was a substantial increase in the expression of the actin mRNA at 24, 48 and 72 h after imbibition, compared with an internal control consisting of a late-embryogenesis-abundant (LEA) type IV gene from P. vulgaris. An increase in the amount of actin in total seed extracts that parallelled that of the mRNA was detected by western blotting starting at 24 h after imbibition. This increase was more apparent when the embryo alone was analyzed. Two-dimensional western blots initially revealed three actin isoforms with isoelectric points (pIs) of approximately 5.6, 5.7 and 5.8, the amounts of which increased within a 48-h period, when a new minor isoform of pI approximately 5.5 appeared; however, after 72 h, the pI-5.8 isoform had almost disappeared and the pI-5.5 isoform had disappeared completely, indicating that these two minor isoforms are expressed transiently. These results indicate that actin is at very low levels in the dry seed but undergoes an increased and differential expression during imbibition, an event probably required to carry out all the necessary functions for germination. Received: 21 July 1998 / Accepted: 1 September 1998     

Microtubules at wound sites of Nitella internodal cells passively co-align with actin bundles when exposed to hydrodynamic forces generated by cytoplasmic streaming

The organization of cortical microtubules at wound sites in Nitella pseudoflabellata(A. Br. & Nordst.) em. R.D.W. and N. flexilis(L.) Ag. internodal cells was examined in relation to the regeneration of actin filament bundles in order to identify the mechanisms by which microtubules are oriented. Actin bundle regrowth occurs prior to that of microtubules, so it was considered possible that microtubule alignment is actin-dependent, perhaps mediated by cross-linking proteins. In all types of wounds investigated, subcortical actin bundles regenerated parallel to the direction of cytoplasmic streaming. Microtubule orientation patterns, however, varied according to the nature of wound formation and the type of wound wall eventually produced. In chloroplast-free windows induced by blue light irradiation, microtubule orientation varied according to the size of the window. Microtubules were randomized in 10- to 30-μm-wide windows where exposure to cytoplasmic flow is minimal, but were aligned more or less parallel to regenerated actin bundles in 80- to 100-μm-wide windows. Where co-alignment between microtubules and actin bundles was obvious after fluorescence labelling, electron micrographs revealed that microtubules and actin bundles were too widely spaced to account for any cross-linkages. Furthermore, treatments that inhibited or reduced cytoplasmic streaming without altering the direction of actin bundles caused randomization of microtubules previously oriented in the streaming direction, even in the presence of taxol. When evenly flat wound walls were induced by 10⁻⁴ M chlortetracycline, microtubules were co-aligned with nearby actin bundles at the surface of the wound wall. At wounds induced by treatment with 5 × 10⁻² M CaCl₂, however, microtubules were randomly oriented and preferentially located in the narrow clefts between the wound-wall protuberances, up to several micrometers away from the actin bundles near the wound-wall tips. These results indicate that microtubules regenerated in wounds are merely co-aligned with actin filament bundles because they are passively aligned by the hydrodynamic forces created by cytoplasmic flow. Received: 4 August 1998 / Accepted: 30 January 1999     

The eggs and larvae of the giant mudskipper, <Emphasis Type="Italic">Periophthalmodon schlosseri</Emphasis>, collected from a mudflat in Penang,Malaysia

 Eggs of the giant mudskipper, Periophthalmodon schlosseri were collected from a burrow in Penang, Malaysia, in November 1998, and hatched larvae were reared in the laboratory. The eggs were demersal with adhesive filaments and elliptical in shape (0.83–1.43 mm in long-axis diameter). Newly hatched larvae (2.1–2.6 mm in notochord length) possessed a yolk sac. The number of myomeres was 10 + 17 = 27. The mouth and anus were already opened. The larvae started feeding one day after hatching and completely absorbed the yolk by the third day at a water temperature of 24.5–28.0°C. Received: April 9, 2002 / Revised: October 25, 2002 / Accepted: December 10, 2002     

Growth history and migration of the threespine stickleback <Emphasis Type="Italic">Gasterosteus aculeatus</Emphasis> in Otsuchi Bay,northeastern Japan

 Both growth and migratory history of the Pacific Ocean forms of the threespine stickleback Gasterosteus aculeatus collected in Otsuchi Bay, northeastern Japan, were examined using otolith microstructure and analysis of strontium (Sr) and calcium (Ca) concentrations with wavelength dispersive X-ray spectrometry by an electron microprobe. Age of the juveniles (21.6–25.9 mm in total length) examined ranged from 101 to 128 days (115 ± 8.5 days; mean ± SD), hatching being estimated as having occurred between March and April 2001. The Sr : Ca ratios in the otoliths changed with both ontogenic development and salinity of the habitat. The otolith Sr : Ca ratios increased gradually from 4.1 × 10⁻³ around the core to 7.5 × 10⁻³ around the edge of the otolith. The fluctuation pattern of otolith Sr : Ca ratios was different from those observed in both freshwater resident and anadromous forms in previous studies. These results suggested that the fish sampled spend their lives in the estuarine and sea environment without freshwater life after hatching. Received: June 5, 2002 / Revised: September 11, 2002 / Accepted: September 24, 2002 Acknowledgments We thank Mr. K. Morita and crews of the Otsuchi Marine Research Center, Ocean Research Institute, The University of Tokyo for their assistance in collecting specimens. This work was supported in part by Grant-in-Aid No. 13760138 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Correspondence to:Takaomi Arai     

Superoxide synthase and dismutase activity of plasma membranes from maize roots

Summary.  Superoxide synthase and superoxide dismutase activity have been monitored in isolated maize (Zea mays) root plasma membranes spectrophotometrically by determination of nitro-blue tetrazolium and cytochrome c reduction, respectively. Superoxide production was induced by NADH and NADPH, with similar kinetics and approaching saturation at 0.06 mM in the case of NADPH and 0.1 mM in the case of NADH, with rates of 18.6 ± 5.0 and 21.8 ± 7.2 nmol/min · mg of protein, respectively. These activities exhibited a broad pH optimum between pH 6.5 and 7.5. Diphenylene iodonium inhibited about 25% (10 μM DPI) and 40% (100 μM DPI) of this activity, imidazole inhibited about 20%, while KCN, a peroxidase inhibitor, did not show any significant inhibition. Superoxide-dismutating activity was shown to occur in the same isolates and depended on the quantity of plasma membrane protein present. Growth of plants on salicylic acid prior to membrane isolation induced a rise in the activity of both of the enzymes by 20–35%, suggesting their coordinated action. Received May 15, 2002; accepted September 30, 2002; published online May 21, 2003 RID="*"     

Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining

The fluorescence properties of one chemically and seven biologically produced polyhydroxyalkanoic acids were investigated as film castings and in living cells respectively after staining with Nile red. All these polyesters show a similar fluorescence behaviour, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wavelength between 570 nm and 605 nm. This could be shown by the use of two-dimensional fluorescence spectroscopy and flow cytometry. The examination of native poly(3-hydroxybutyric acid), poly(3HB), granules isolated from cells of Ralstonia eutropha H16 showed that the addition of 6.0 μg Nile red is necessary for total staining of 1.0 mg granules. The fluorescence intensity at an excitation wavelength of 550 nm and an emission wavelength of 600 nm showed high correlation to the poly(3HB) concentration of grana suspensions at different grana concentrations. These results and the staining of cell suspensions during cultivation experiments revealed that Nile red has a high potential for the quantitative determination of hydrophobic bacterial polyhydroxyalkanoic acids. Received: 13 November 1998 / Received revision: 4 February 1999 / Accepted: 12 February 1999     

Pelagic juvenile of <Emphasis Type="Italic">Halargyreus johnsonii</Emphasis> (Gadiformes: Moridae) from Suruga Bay,Japan

 A pelagic juvenile (43.0 mm standard length) of the deep-sea gadiform fish Halargyreus johnsonii was collected by a larva net towed at depths from 200 m to 90 m at Suruga Bay, Japan. The specimen had an elongate body, eyes located dorsally on the head, a depressed anal fin, small bony tubercles on the lower jaw symphysis, elongate pelvic fin rays, and a pointed caudal fin. The body was silver with numerous small black spots. The gut was filled with a copepod, Pareuchaeta russelli, that is characterized by a surface to mesopelagic distribution. Received: May 30, 2002 / Revised: December 16, 2002 / Accepted: December 24, 2002     

Effects of Contact Time, Pressure, Percent Relative Humidity (%RH), and Material Type on Listeria Biofilm Adhesive Strength at a Cellular Level Using Atomic Force Microscopy (AFM)

Whereas the transfer of Listeria from surfaces to foods and vice versa has been well documented, little is known about the mechanism of bacterial transfer. The objective of this work is to gain a better understanding of the forces involved in listerial biofilms adhesion using atomic force microscopy (AFM). L. monocytogenes Scott A was grown as biofilms on stainless steel surfaces by inoculating stainless steel coupons with Listeria and incubating the coupons for 48 h at 32 °C with a diluted 1:20 tryptic soy broth. After growth, biofilms were equilibrated over saturated salt solutions at a constant relative humidity (%RH) before measurement of adhesion forces using AFM. The effects of contact time, loading force, and biofilm relative humidity (%RH) suggested that neither contact time, loading force nor biofilm %RH had a significant effect on biofilm adhesiveness at a cellular level (P > 0.05). In a second set of experiments, the influence of material type on biofilm adhesiveness was evaluated using two different colloidal probes (SiO₂ and polyethylene). Results showed that the maximum pull-off force and retraction work needed to retract the cantilever for glass (−85.42 nN and 1.610⁻¹⁵ J, respectively) were significantly lower than those of polyethylene (−113.38 nN and 2.7 × 10^–15 J, respectively; P < 0.001). The results of this study suggest that Listeria biofilms adhere more strongly to hydrophobic surfaces than hydrophilic surfaces when measured at a cellular level. These results provide important insights that could lead to new ways to remediate and avoid listerial biofilm formation in the food industry.     

Migratory histories of three types of Cottus pollux (small-egg, middle-egg, and large-egg types) as revealed by otolith microchemistry

 Migratory histories of three types of Cottus pollux, the small-egg type (SE type), middle-egg type (ME type), and large-egg type (LE type), were studied by examining strontium (Sr) and calcium (Ca) in their otoliths with wavelength dispersive X-ray spectrometry on an electron microprobe. The Sr : Ca ratios in the otoliths changed both with ontogenetic development and with salinity of the habitat. Otolith Sr : Ca ratios of LE-type samples and the ME-type samples from the Honmyo River, Kyushu Island, showed consistently low ratios, averaging 1.8 × 10⁻³ and 2.4 × 10⁻³ from the core to the edge, respectively. In contrast, otolith Sr : Ca ratios of SE-type samples and the other four ME-type samples from Hokkaido and Honshu Islands fluctuated strongly along the life history transects in accordance with migration patterns from freshwater to the sea and vice versa. The otolith Sr : Ca ratios of SE-type samples showed low ratios from the core to a point around 15 μm, averaging 1.5 × 10⁻³, and subsequently increased sharply with a high Sr : Ca ratio phase to a point around 400 μm, averaging 5.5 × 10⁻³, and followed again a low ratio phase to the edge with averages of 3.1 × 10⁻³. Similar fluctuation patterns in otolith Sr : Ca ratios were found for the four ME-type samples. These findings clearly demonstrated that otolith Sr : Ca ratios reflected the sculpin's life histories, as being fluvial for the LE type and the Honmyo River ME type and amphidromous for the SE type and the other four populations of ME type. Received: August 1, 2002 / Revised: October 15, 2002 / Accepted: October 28, 2002 Acknowledgments We thank Dr. N. Miyazaki, University of Tokyo, for his kind guidance of our joint research. Thanks are also offered to Drs. H. Sakai, National Fisheries University, Y. Yamazaki, Toyama University, and R. Yokoyama, Hokkaido University, and Mrs. N. Okabe and Y. Suzuki of Yamagata Prefecture for their help in sample collection. This work was partly supported by a Grant-in-Aid (No. 13660171) from the Japan Ministry of Education, Science, Sports and Culture to A. Goto. Correspondence to:Akira Goto