Novel Sex Cells and Evidence for Sex Pheromones in Diatoms

BACKGROUND: Diatoms belong to the stramenopiles, one of the largest groups of eukaryotes, which are primarily characterized by a presence of an anterior flagellum with tubular mastigonemes and usually a second, smooth flagellum. Based on cell wall morphology, diatoms have historically been divided into centrics and pennates, of which only the former have flagella and only on the sperm. Molecular phylogenies show the pennates to have evolved from among the centrics. However, the timing of flagellum loss--whether before the evolution of the pennate lineage or after--is unknown, because sexual reproduction has been so little studied in the 'araphid' basal pennate lineages, to which Pseudostaurosira belongs. METHODS/PRINCIPAL FINDING: Sexual reproduction of an araphid pennate, Pseudostaurosira trainorii, was studied with light microscopy (including time lapse observations and immunofluorescence staining observed under confocal scanning laser microscopy) and SEM. We show that the species produces motile male gametes. Motility is mostly associated with the extrusion and retrieval of microtubule-based 'threads', which are structures hitherto unknown in stramenopiles, their number varying from one to three per cell. We also report experimental evidence for sex pheromones that reciprocally stimulate sexualization of compatible clones and orientate motility of the male gametes after an initial 'random walk'. CONCLUSIONS/SIGNIFICANCE: The threads superficially resemble flagella, in that both are produced by male gametes and contain microtubules. However, one striking difference is that threads cannot beat or undulate and have no motility of their own, and they do not bear mastigonemes. Threads are sticky and catch and draw objects, including eggs. The motility conferred by the threads is probably crucial for sexual reproduction of P. trainorii, because this diatom is non-motile in its vegetative stage but obligately outbreeding. Our pheromone experiments are the first studies in which gametogenesis has been induced in diatoms by cell-free exudates, opening new possibilities for molecular 'dissection' of sexualization

Impurity emission characteristics of long pulse discharges in Large Helical Device

Line spectra from intrinsic impurity ions have been monitored during the three kinds of long-pulse discharges (ICH, ECH, NBI). Constant emission from the iron impurity shows no preferential accumulation of iron ion during the long-pulse operations. Stable Doppler ion temperature has been also measured from Fe XX, C V and C III spectra

Recent Results from LHD Experiment with Emphasis on Relation to Theory from Experimentalist’s View

he Large Helical Device (LHD) has been extending an operational regime of net-current free plasmas towardsthe fusion relevant condition with taking advantage of a net current-free heliotron concept and employing a superconducting coil system. Heating capability has exceeded 10 MW and the central ion and electron temperatureshave reached 7 and 10 keV, respectively. The maximum value of β and pulse length have been extended to 3.2% and 150 s, respectively. Many encouraging physical findings have been obtained. Topics from recent experiments, which should be emphasized from the aspect of theoretical approaches, are reviewed. Those are (1) Prominent features in the inward shifted configuration, i.e., mitigation of an ideal interchange mode in the configuration with magnetic hill, and confinement improvement due to suppression of both anomalous and neoclassical transport, (2) Demonstration ofbifurcation of radial electric field and associated formation of an internal transport barrier, and (3) Dynamics of magnetic islands and clarification of the role of separatrix

Gametogenesis and auxospore development in Actinocyclus (Bacillariophyta).

cGametogenesis and auxospore development have been studied in detail in surprisingly few centric diatoms. We studied the development of sperm, eggs and auxospores in Actinocyclus sp., a radially symmetrical freshwater diatom collected from Japan, using LM and electron microscopy of living cultures and thin sections. Actinocyclus represents a deep branch of the 'radial centric' diatoms and should therefore contribute useful insights into the evolution of sexual reproduction in diatoms. Spermatogenesis was examined by LM and SEM and involved the formation of two spermatogonia (sperm mother-cells) in each spermatogonangium through an equal mitotic division. The spermatogonia produced a reduced 'lid' valve, resembling a large flat scale with irregular radial thickenings. Sperm formation was merogenous, producing four sperm per spermatogonium, which were released by dehiscence of the 'lid' valve. The sperm were spindle-shaped with numerous surface globules and, as usual for diatoms, the single anterior flagellum bore mastigonemes. One egg cell was produced per oogonium. Immature eggs produced a thin layer of circular silica scales before fertilization, while the eggs were still contained within the oogonium. Sperm were attracted in large numbers to each egg and were apparently able to contact the egg surface via a gap formed between the long hypotheca and shorter epitheca of the oogonium and a small underlying hole in the scale-case. Auxospores expanded isodiametrically and many new scales were added to its envelope during expansion. Finally, new slightly-domed initial valves were produced at right angles to the oogonium axis, after a strong contraction of the cell away from the auxospore wall. At different stages, Golgi bodies were associated with chloroplasts or mitochondria, contrasting with the constancy of Golgi-ER-mitochondrion (G-ER-M) units in some other centric diatoms, which has been suggested to have phylogenetic significance. Electron-dense bodies in the vacuole of Actinocyclus are probably acidocalcisomes containing polyphosphate

Spermatocyte after meiosis II in <i>Actinocyclus</i>.

<p>A, B, E–G, LM. Figs. C, D, H–K, SEM. A–D. <b>A</b>. Cell with four projections (arrows), each bearing a single flagellum. <b>B</b>. DAPI-stained cell with four separated nuclei. <b>C</b>. Cell with four flagella extending from four projections (arrows). <b>D</b>. Enlargement of a projection with a flagellum with mastigonemes. Note narrower posterior end. <b>E</b>. Final stage of spermatogenesis: sperms separating from the cell (e.g. at arrow). <b>F</b>. Sperm with almost fully elongated flagellum being pinched off from the cell. <b>G</b>. Enlargement of F. <b>H</b>. Four separated sperms (arrows) and a large residual body in a spermatogonium. <b>I</b>. DAPI-stained cell at a similar stage to H, with four sperms in a spermatogonium. <b>J</b>. Spermatogonium with a residual body and uniflagellate sperms (arrowheads) and an apparently fragmentary lid valve around the margin (arrows). <b>K</b>. A spermatogonium with a partly detached lid valve and sperm inside (arrow). All scale bars = 10 µm except Figs. D (2 µm) and G (5 µm).</p

Sperm morphology and flagellar fine-structure in <i>Actinocyclus</i>.

<p>A, B, J, LM. C–I, SEM. <b>A, B</b>. Phase contrast and DAPI images of a sperm showing long anterior uniflagellate and an elongate nucleus occupying most of the sperm ‘body’. Scale bars = 10 µm. <b>C</b>. Two sperm: both are elongate but one is more pointed than the other. Note that one sperm bears a swelling near the tip of the flagellum. Scale bar = 10 µm. <b>D, E</b>. Enlargement of the sperm shown in Fig. 29. Scale bars = 2 µm. <b>F</b>. Enlargement of the swollen portion of one flagellum shown in Fig. 4C: note also the line of mastigonemes (pointing towards the viewer). Scale bar = 1 µm. <b>G</b>. Detail of mastigonemes. Scale bar = 1 µm. <b>H</b>. Detail of mastigonemes lined in a row at both sides of the flagellum. Scale bar = 1 µm. <b>I</b>. Detail of mastigonemes, showing that their tips (arrows) appear branched, because they bear terminal filaments (arrows). Scale bar = 1 µm. <b>J</b>. Many sperms crowding around a broken egg (center). Scale bar = 50 µm.</p

Spermatocyte after meiosis I in <i>Actinocyclus</i>.

<p>A, B, E, F, LM. Figs. C, D, G, H, SEM. A–D. <b>A</b>. Cell with paired flagella (arrows) associated with each nucleus. <b>B</b>. DAPI-stained cell with two separated nuclei. <b>C</b>. Spermatocyte with peripheral vesicles and elongating paired flagella on each side, which have reached about 1/3 of their final length. <b>D</b>. Enlargement of basal area of the paired flagella with mastigonemes. <b>E–H</b>. Spermatocyte during meiosis II. <b>E</b>. Cell undergoing nuclear division with separated flagella (arrows). <b>F</b>. DAPI-stained cell showing dividing nuclei (the median vertical line in the left-hand nucleus marks the position of the spindle). <b>G</b>. Cell with separated flagella on either side of two flat projections where the nuclei are located. <b>H</b>. Enlargement of a flat projection, with flagella extending out on either side. Scale bars = 10 µm except D (1 µm) and H (5 µm).</p

Young auxospore in <i>Actinocyclus</i>.

<p>A, C, LM. B, E–G, TEM. D, SEM. <b>A</b>. A young, partly expanded auxospore, still associated with the oogonium hypotheca and epitheca. Scale bar = 20 µm. <b>B</b>. A thin section of the auxospore illustrated in <b>A</b>, showing organelles arranged peripherally and a large central vacuole. Scale bar = 10 µm. <b>C</b>. A young auxospore showing peripheral chloroplasts and a single nucleus (arrow). Scale bar = 20 µm. <b>D</b>. A young auxospore completely covered by a layer of scales. Scale bar = 20 µm. <b>E</b>. Another thin section of the auxospore shown in Fig. 8A, passing through the centre of nucleus. Note that the nucleus is bigger than that of oogonium. Scale bar = 10 µm. <b>F</b>. Close association of a mitochondrion and a Golgi body producing vesicles with electron-dense contents. Scale bar = 1 µm. <b>G</b>. Scales with a central annulus and branching radial ribs. Scale bar = 1 µm.</p