Carotenoid dynamics and lipid droplet containing astaxanthin in response to light in the green alga Haematococcus pluvialis.

The unicellular green alga Haematococcus pluvialis accumulates large amounts of the red ketocarotenoid astaxanthin to protect against environmental stresses. Haematococcus cells that accumulate astaxanthin in the central part (green-red cyst cells) respond rapidly to intense light by distributing astaxanthin diffusively to the peripheral part of the cell within 10 min after irradiation. This response is reversible: when astaxanthin-diffused cells were placed in the dark, astaxanthin was redistributed to the center of the cell. Although Haematococcus possesses several pigments other that astaxanthin, the subcellular distribution and content of each pigment remain unknown. Here, we analyzed the subcellular dynamics and localization of major pigments such as astaxanthin, ß-carotene, lutein, and chlorophylls under light irradiation using time-lapse and label-free hyperspectral imaging analysis. Fluorescence microscopy and freeze-fracture transmission electron microscopy showed that, preceding/following exposure to light, astaxanthin colocalized with lipid droplets, which moved from the center to the periphery through pathways in a chloroplast. This study revealed that photoresponse dynamics differed between astaxanthin and other pigments (chlorophylls, lutein, and ß-carotene), and that only astaxanthin freely migrates from the center to the periphery of the cell through a large, spherical, cytoplasm-encapsulating chloroplast as a lipid droplet. We consider this to be the Haematococcus light-protection mechanism.

Gonadal Morphology and Gametogenesis in Japanese Red Coral Corallium japonicum (Octocorallia: Alcyonacea) Collected off Cape Ashizuri, Japan.

Colonies of the Japanese red coral Corallium japonicum Kishinouye, 1903 collected off Cape Ashizuri, Japan were gonochoric and produced gonads in siphonozooids annually, mainly during the spring season. Polyp anatomy, gonadal morphology and gametogenesis in this species were revealed by light and electron microscopy. A siphonozooid had a pharynx with a prominent siphonoglyph and eight mesenteries: two sulcal, two asulcal, and four lateral. A rudimentary retractor was found on one side of each mesoglea of these mesenteries. The retractor arrangement in the siphonozooid was reverse of what was described in the autozooids of octocorals. Gonads initiated as small protrusions on the mesenteries, except in the asulcal ones, and even at an incipient stage they were covered with a sac-shaped thin layer of mesoglea, which was continuous with the mesoglea of mesenteries. Gastrodermis enveloped the complete outer surface of the thin layer of mesoglea throughout gametogenesis in both oocytes and sperm cysts. Oocytes produced many microvilli on their cortical surfaces beneath the thin layer of mesoglea concomitantly with the accumulation of lipid globules in the cells, whereas in sperm cysts spermatocytes and spermatids increased in number without microvilli production, followed by synchronous spermiogenesis involving remarkable changes in the shape and position of organelles. Based on the comparison of patterns in gonadal development between octocorals including C. japonicum, hexacorals and scyphozoans, octocoral and stauromedusa species may be characterized by the fact that gametogenesis never occurs in the matrix of mesoglea, but rather exclusively within the thin sac of mesoglea surrounded by gastrodermis.

Identification of homogeneously staining regions by G-banding and chromosome microdissection, and FISH marker selection using human Alu sequence primers in a scleractinian coral Coelastrea aspera Verrill, 1866 (Cnidaria).

Karyotype analysis was performed on the scleractinian coral Coelastrea aspera Verrill, 1866, commonly found along temperate coasts in Japan (30-35°N) and in coastal waters in the Indian and Pacific oceans. G-banding of Coelastrea aspera was successfully performed, although the banding pattern was not as clear as that in mammals. The karyogram clearly revealed that this coral had a homogeneously staining region (hsr) in chromosome 11. This hsr consisted of ribosomal RNA (rRNA) related genes, which was demonstrated by fluorescence in situ hybridization (FISH) with probes generated using 28S ribosomal DNA (rDNA) primers and those generated through chromosome microdissection. In addition, we conducted silver-stained nucleolus organizer region (Ag-NOR) analysis and found Ag depositions in the interphase nuclei but not on rRNA gene loci and hsr(s) in the mitotic stage. The hsr of this coral was observed in approximately 50% of the metaphase spreads analyzed. This may explain the diversity of coral rDNA based on the molecular study of sequence analysis. Furthermore, it was discovered that human telomere and Alu repeated sequences were present in this Coelastrea aspera. Probes derived from human Alu sequences are expected to play an important role in the classification of corals. Overall, our data can be of great value in discriminating among scleractinian coral species and understanding their genetics, including chromosomal evolution.

Ubiquitous distribution of helmchrome in phototactic swarmers of the stramenopiles.

Most swarmers (swimming cells) of the stramenopile group, ranging from unicellular protist to giant kelps (brown algae), have two heterogeneous flagella: a long anterior flagellum (AF) and a relatively shorter posterior flagellum (PF). These flagellated cells often exhibit phototaxis upon light stimulation, although the mechanism by which how the phototactic response is regulated remains largely unknown. A flavoprotein concentrating at the paraflagellar body (PFB) on the basal part of the PF, which can emit green autofluorescence under blue light irradiance, has been proposed as a possible blue light photoreceptor for brown algal phototaxis although the nature of the flavoprotein still remains elusive. Recently, we identified helmchrome as a PF-specific flavoprotein protein in a LC-MS/MS-based proteomics study of brown algal flagella (Fu et al. 2014). To verify the conservation of helmchrome, in the present study, the absence or presence and the localization of helmchrome in swarmers of various algal species were investigated. The results showed that helmchrome was only detected in phototactic swarmers but not the non-phototactic ones of the stramenopile group. Electron microscopy further revealed that the helmchrome detectable swarmers bear a conserved PFB-eyespot complex, which may serve as structural basis for light sensing. It is speculated that all three conserved properties: helmchrome, the PFB structure, and the eyespot apparatus, will be essential parts for phototaxis of stramenopile swarmers.

Molecular cytogenetic analysis of the scleractinian coral Acropora solitaryensis Veron & Wallace 1984.

We performed a molecular cytogenetic investigation of the scleractinian coral Acropora solitaryensis, which is dominant in the temperate region of Japan (30-35°N). Molecular cytogenetic analysis, using fluorescence in situ hybridization (FISH), was carried out for karyotyping and gene mapping. We propose the karyotype of this coral (2n = 30) based on C-banding and FISH analyses. FISH mapping of the rRNA gene was carried out with a probe generated by PCR amplification using rRNA gene primers. Furthermore, the telomeres and centromeres of all chromosomes were visualized using FISH. By comparative genomic hybridization using DNA from sperm and unfertilized eggs of this coral, we offer evidence suggesting the existence of sex chromosomes in this species. Collectively, these data advance our understanding of coral genetics.

Five Cyanophora (Cyanophorales, Glaucophyta) species delineated based on morphological and molecular data.

Cyanophora is an important glaucophyte genus of unicellular biflagellates that may have retained ancestral features of photosynthetic eukaryotes. The nuclear genome of Cyanophora was recently sequenced, but taxonomic studies of more than two strains are lacking for this genus. Furthermore, no study has used molecular methods to taxonomically delineate Cyanophora species. Here, we delimited the species of Cyanophora using light and electron microscopy, combined with molecular data from several globally distributed strains, including one newly established. Using a light microscope, we identified two distinct morphological groups: one with ovoid to ellipsoidal vegetative cells and another with dorsoventrally flattened or broad, bean-shaped vegetative cells containing duplicated plastids. Our light and scanning electron microscopy clearly distinguished three species with ovoid to ellipsoidal cells (C. paradoxa Korshikov, C. cuspidata Tos.Takah. & Nozaki sp. nov., and C. kugrensii Tos.Takah. & Nozaki sp. nov.) and two species with broad, bean-shaped cells (C. biloba Kugrens, B.L.Clay, C.J.Mey. & R.E.Lee and C. sudae Tos.Takah. & Nozaki sp. nov.) based on differences in cell shape and surface ornamentations of the vegetative cells under the field-emission scanning electron microscope. Molecular phylogenetic analyses of P700 chl a apoprotein A2 (psaB) genes and internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA), as well as a comparison of secondary structures of nuclear rDNA ITS-2 and genetic distances of psaB genes, supported the delineation of five morphological species of Cyanophora.

Inducible growth mode switches influence Valonia rhizoid differentiation.

Cell differentiation and cell type commitment are an integral part of plant growth and development. Investigations on how environmental conditions affect the formation of shoots, roots, and rhizoids can help illustrate how plants determine cell fate and overall morphology. In this study, we evaluated the role of substratum and light on rhizoid differentiation in the coenocytic green alga, Valonia aegagropila. Elongating rhizoids displayed varying growth modes and cell shape upon exposure to different substrata and light conditions. It was found that soft substrata and dark incubation promoted rhizoid elongation via tip growth while subsequent exposure to light prevented tip growth and instead induced swelling in the apical region of rhizoids. Swelling was accompanied by the accumulation of protoplasm in the rhizoid tip through expansion of the cell wall and uninhibited cytoplasmic streaming. Subsequent diffuse growth led to the transformation from slender, rod-shaped rhizoids into spherical thallus-like structures that required photosynthesis. Further manipulation of light regimes caused vacillating cell growth redirections. An elongating V. aegagropila rhizoid cell thus appears capable of growth mode switching that is regulated by immediate environmental conditions thereby influencing ultimate cell shape and function. This is the first description of inducible, multiple growth mode shifts in a single intact plant cell that directly impact its differentiation.

Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae).

Plasmodesmata are intercellular bridges that directly connect the cytoplasm of neighboring cells and play a crucial role in cell-to-cell communication and cell development in multicellular plants. Although brown algae (Phaeophyceae, Heterokontophyta) are phylogenetically distant to land plants, they nevertheless possess a complex multicellular organization that includes plasmodesmata. In this study, the ultrastructure and formation of plasmodesmata in the brown alga Dictyota dichotoma were studied using transmission electron microscopy and electron tomography with rapid freezing and freeze substitution. D. dichotoma possesses plasma membrane-lined, simple plasmodesmata without internal endoplasmic reticulum (desmotubule). This structure differs from those in land plants. Plasmodesmata were clustered in regions with thin cell walls and formed pit fields. Fine proteinaceous "internal bridges" were observed in the cavity. Ultrastructural observations of cytokinesis in D. dichotoma showed that plasmodesmata formation began at an early stage of cell division with the formation of tubular pre-plasmodesmata within membranous sacs of the cytokinetic diaphragm. Clusters of pre-plasmodesmata formed the future pit field. As cytokinesis proceeded, electron-dense material extended from the outer surface of the mid region of the pre-plasmodesmata and finally formed the nascent cell wall. From these results, we suggest that pre-plasmodesmata are associated with cell wall development during cytokinesis in D. dichotoma.

EFFECTS OF HIGH PRESSURE IN THE ARMORED DINOFLAGELLATE SCRIPPSIELLA HEXAPRAECINGULA (PERIDINIALES, DINOPHYCEAE): CHANGES IN THECAL PLATE PATTERN AND MICROTUBULE ASSEMBLY(1).

The possible role of cortical microtubules in dinoflagellates was studied using high-pressure treatments applied to nonmotile cells (just after ecdysis) of Scrippsiella hexapraecingula T. Horig. et Chihara. Whereas considerable disorganization of cortical microtubules was observed when cells were exposed to high-pressure treatments of 98 MPa or more for 5-15 min, they were mostly intact in cells exposed to a pressure of <98 MPa for 5 min. After nonmotile cells were exposed to high-pressure treatments sufficient to disorganize the cortical microtubules, they produced new motile cells with thecal plate patterns that differed considerably from the pattern known for this species. Increasing the intensity of high pressure applied to nonmotile cells resulted in an increase in the number of cells that exhibited disorganized cortical microtubules as well as a change in their thecal plate pattern, suggesting that high pressure disorganizes cortical microtubules leading to a change in the thecal plate pattern.

Ultrafine cellulose fibers produced by Asaia bogorensis, an acetic acid bacterium.

The ability to synthesize cellulose by Asaia bogorensis, a member of the acetic acid bacteria, was studied in two substrains, AJ and JCM. Although both strains have identical 16S rDNA sequence, only the AJ strain formed a solid pellicle at the air-liquid interface in static culture medium, and we analyzed this pellicle using a variety of techniques. In the presence of cellulase, glucose and cellobiose were released from the pellicle suggesting that it is made of cellulose. Field emission electron microscopy allowed the visualization of a 3D knitted structure with ultrafine microfibrils (approximately 5-20 nm in width) in cellulose from A. bogorensis compared with the 40-100 nm wide microfibrils observed in cellulose isolated from Gluconacetobacter xylinus, suggesting differences in the mechanism of cellulose biosynthesis or organization of cellulose synthesizing sites in these two related bacterial species. Identifying these differences will lead to a better understanding of cellulose biosynthesis in bacteria.

Membrane fusion process and assembly of cell wall during cytokinesis in the brown alga, Silvetia babingtonii (Fucales, Phaeophyceae).

During cytokinesis in brown algal cells, Golgi-derived vesicles (GVs) and flat cisternae (FCs) are involved in building the new cell partition membrane. In this study, we followed the membrane fusion process in Silvetia babingtonii zygotes using electron microscopy together with rapid freezing and freeze substitution. After mitosis, many FCs were formed around endoplasmic reticulum clusters and these then spread toward the future cytokinetic plane. Actin depolymerization using latrunculin B prevented the appearance of the FCs. Fusion of GVs to FCs resulted in structures that were thicker and more elongated (EFCs; expanded flat cisternae). Some complicated membranous structures (MN; membranous network) were formed by interconnection of EFCs and following the arrival of additional GVs. The MN grew into membranous sacs (MSs) as gaps between the MNs disappeared. The MSs were observed in patches along the cytokinetic plane. Neighboring MSs were united to form the new cell partition membrane. An immunocytochemical analysis indicated that fucoidan was synthesized in Golgi bodies and transported by vesicles to the future cytokinetic plane, where the vesicles fused with the FCs. Alginate was not detected until the MS phase. Incubation of sections with cellulase-gold showed that the cellulose content of the new cross wall was not comparable to that of the parent cell wall.

Morphogenesis in giant-celled algae.

The giant-celled algae, which consist of cells reaching millimeters in size, some even centimeters, exhibit unique cell architecture and physiological characteristics. Their cells display a variety of morphogenetic phenomena, that is, growth, division, differentiation, and reproductive cell formation, as well as wound-healing responses. Studies using immunofluorescence microscopy and pharmacological approaches have shown that microtubules and/or actin filaments are involved in many of these events through the generation of intracellular movement of cell components or entire protoplasmic contents and the spatial control of cell activities in specific areas of the giant cells. A number of environmental factors including physical stimuli, such as light and gravity, invoke localized but also generalized cellular reactions. These have been extensively investigated to understand the regulation of morphogenesis, in particular addressing cytoskeletal and endomembrane dynamics, electrophysiological elements affecting ion fluxes, and the synthesis and mechanical properties of the cell wall. Some of the regulatory pathways involve signal transduction and hormonal control, as in other organisms. The giant unicellular green alga Acetabularia, which has proven its usefulness as an experimental model in early amputation/grafting experiments, will potentially once again serve as a useful model organism for studying the role of gene expression in orchestrating cellular morphogenesis.

Relationship between body temperature and ovarian cycle in Asian and African elephants.

The aim of the present study was to investigate whether changes in body temperature are related to the ovarian cycle in elephants. Rectal, tongue or fecal temperature was measured for 2 Asian and 5 African elephants using an electric thermometer. Evaluation of ovarian cycles was based on the changes in serum or fecal progestin. The mean +/- SD values of the rectal, tongue, and fecal temperatures were 36.3 +/- 0.3 (2 Asian), 36.2 +/- 0.5 (1 African) and 36.5 +/- 0.3 C (4 African), respectively; the fecal temperature was the highest of the 3 temperatures (P<0.01). The longitudinal changes in body temperatures correlated with the ovarian cycle, with higher temperatures occurring during the luteal phase. The fecal temperatures of one acyclic African elephant did not change cyclically. These results suggest that measurement of body temperature can be used to easily evaluate the ovarian cyclicity of an individual animal, although it might not be able to determine the ovarian cycle length.

Cell wall extensibility during branch formation in the xanthophycean alga Vaucheria terrestris.

In the tip-growing filamentous cell of the xanthophycean alga Vaucheria terrestris sensu Götz, a new growing tip develops in the non-growing, cylindrical region of the cell that was exposed by local illumination. The present study examined changes in the strength and extensibility of the cell wall of the new growing tip and in the matrix components of the inner surface of the cell wall. The internal pressure required to rupture the cell walls decreased remarkably during the early to middle stages of growing tip development, but the cell wall hardly extended before rupture. In contrast, during the middle and late stages of development, cell walls were extended by internal pressure. Atomic force microscopy revealed that protease-resistant, fine granular matrix components were present only at the apical portion of a normal growing tip, and were absent in the non-growing cylindrical region. In the early and middle stages of new growing tip development, these matrix components appeared in the cell walls in patches. These results suggest that first cell wall strength decreases and then cell wall extensibility increases in the development of new growing tips, and that protease-resistant, fine granular matrix components may be involved in rendering a cell wall extensible.

Fine structure of cell wall surfaces in the giant-cellular xanthophycean alga Vaucheria terrestris.

The mechanical strength of cell walls in the tip-growing cells of Vaucheria terrestris is weakened by treatment with proteolytic enzymes. To clarify the morphological characteristics of the components maintaining cell wall strength, the fine structures of the cell walls, with and without protease treatment, were observed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Observations indicated that cellulose microfibrils were arranged in random directions and overlapped each other. Most of the microfibrils observed in the inner surface of the cell wall were embedded in amorphous materials, whereas in the outer surface of the cell wall, microfibrils were partially covered by amorphous materials. The matrix components embedding and covering microfibrils were almost completely removed by protease treatment, revealing layers of naked microfibrils deposited deeply in the cell wall. Topographic data taken from AFM observations provided some additional information that could not be obtained by TEM, including more detailed images of the granular surface textures of the matrix components and the detection of microfibrils in the interior of the cell wall. In addition, quantitative AFM data of local surface heights enabled us to draw three-dimensional renderings and to quantitatively estimate the extent of the exposure of microfibrils by the enzymatic treatment.

Annual changes in testicular size and serum and fecal testosterone concentrations in male bharals, Pseudois nayaur.

The serum and fecal testosterone (T) concentrations and testicular sizes of two male bharals (Pseudois nayaur) were determined for approximately one year. The profiles of the fecal T concentrations showed a similar tendency as the profiles of serum T concentrations, and there was a significant correlation between serum and fecal T concentrations (r=0.72). T concentrations rose drastically in October and decreased gradually until January. The maximum testicular size was observed between November and January. Semen collected between December and January was excellent in quality and comparable to domestic sheep and goats. The active periods of the testes were synchronized with the early breeding season of females.

Characterization of the reproductive cycle in female bharals (Pseudois nayaur) based on the changes in serum progesterone concentrations and parturition season.

The purpose of present study was to determine annual changes in serum progesterone (P(4)) concentrations and to clarify basic reproductive characteristics, such as breeding season, estrous cycle, and puberty in female bharals (Pseudois nayaur). Blood was collected from 9 female bharals once or twice weekly for approximately one year. Serum P(4) concentrations were determined by radioimmunoassay. Serum P(4) concentrations showed remarkable and cyclic changes between November/December (winter) and May/June (late spring). The mean estrous cycle was 24.9 +/- 0.5 days. Chasing insistently to other females and discharge of mucus from the vulva were observed around the time when the serum P(4) concentrations began to increase. The chasing behavior and discharge of mucus were considered to be external indicators of estrus in female bharals. Serum P(4) concentrations of a pregnant female had non-cyclic changes, and the values remained high. In this study, all 37 deliveries were between April and September, and about 70% of these were concentrated in May and June. The conception month determined on the day of birth was between October and April for all animals, and the most common month was in December (54%). This month corresponded to an early stage of the period when the serum P(4) concentrations changed cyclically. These results indicate that many female bharals become pregnant at the beginning of the breeding seasons and, if they do not become pregnant, the estrous cycle, about 25 days in length, is repeated.

Extensibility of isolated cell walls in the giant tip-growing cells of the xanthophycean alga Vaucheria terrestris.

Apical cell wall fragments isolated from the giant-cellular xanthophycean alga Vaucheria terrestris sensu Götz were inflated with silicone oil by applying internal pressure ranging from 0.1 to 0.7 MPa, and the time-course of cell wall deformation was recorded and analyzed by videomicroscopy. Cell wall extensibility in the tip-growing region was estimated by the pressure required for cell wall extension, the amount of total extension until cell wall rupture and the rate of cell wall extension. Apical cell walls exhibited gradual extension, or creep, during inflation, which was eventually followed by rupture at the apical portion, whereas no appreciable extension was found in the cylindrical basal portion of the cell wall fragment. Besides the largest extension observed around the tip, substantial extension was also observed along the subapical region of the cell wall. The wall extensibility was dependent on the buffer pH used for infiltration before inflation. The optimum pH for the extension was about 8.0, but the cell wall was much less extensible after infiltration with an acidic buffer. Cell wall extensibility was dependent on the pH of the buffer used before inflation, regardless of that used in the previous infiltration. Moreover, pretreatment of the cell wall with a protease caused considerable loosening of cell walls, but affected the pH dependence of cell wall extensibility little. These results indicate that the extensibility of the cell walls in the giant tip-growing cells of the alga is distinct from that of plant cells that exhibit "acid growth" in its dependence on environmental pH and the role of cell wall proteins.

Schizocladia ischiensis: a new filamentous marine chromophyte belonging to a new class, Schizocladiophyceae.

A new marine filamentous chromophyte Schizocladia ischiensis sp. nov. is described from Naples, Italy, and a new class, Schizocladiophyceae, is proposed to accommodate the species based on morphology, photosynthetic pigment analysis, and rbcL and 18S rRNA gene sequences. The vegetative thallus is composed of branched filaments, 3-7 microm in diameter, containing one to two light brown parietal plastids. Cell walls are composed of layered fibers containing alginates, but lacking cellulose. Plastids are of the typical chromophyte type, containing chlorophylls a and c, and abundant fucoxanthin. Zoospores are formed by direct transformation of vegetative cells or through a process including a multinucleated cell stage. Zoospores are teardrop-shaped with a longer anterior flagellum with tubular mastigonemes and a shorter smooth posterior flagellum with a basal swelling. Flagella have a single basal plate and multi-gyred transitional helix distal to the basal plate. Each zoospore has an eyespot. Phylogenetic analyses using rbcL and 18S rDNA sequences suggest the closest phylogenetic relationship with Phaeophyceae, and then with Xanthophyceae and Phaeothamniophyceae. Nevertheless, Schizocladia differs from Phaeophyceae in some essential features (i.e. cell wall lacking cellulose and plasmodesmata, presence of flagellar transitional helix). Therefore, an independent class Schizocladiophyceae is proposed to accommodate this new taxon.