Morphological plasticity in response to salinity change in the euryhaline diatom Pleurosira laevis (Bacillariophyta).

Pleurosira laevis is a salt-tolerant diatom distributed around the world. The valve of P. laevis has distinct structures called ocelli, which are sharply defined areas with fine, densely packed pores. Two formae of this diatom, P. laevis f. laevis and P. laevis f. polymorpha, are distinguished from each other by their flat or dome-shaped valve faces and degree of elevation of the ocelli, respectively. In this study, we established 4 strains of P. laevis isolated from freshwaters or coastal areas in Japan and the United States, and tracked the formation of newly formed valves with the fluorescent SDV-specific dye PDMPO in culture under several salinity conditions. The result clearly demonstrated the morphological plasticity of the valves, controlled by environmental salinity. The laevis form and polymorpha form valves were produced at salinities of 2 and 7, respectively. The salinity thresholds dictating the morphological plasticity of the valve were consistent in all 4 strains. A similar morphology to the polymorpha form was reproduced in a freshwater medium with the addition of sorbitol, suggesting that osmotic pressure plays a key role in this morphological plasticity. The highly reproducible and easily manipulated change in morphology makes this diatom an ideal model for lab experiments focusing on the molecular and genetic factors involved with valve morphogenesis.

Valve morphogenesis in Diploneis smithii (Bacillariophyta).

Diploneis species have perhaps the most complex valve structure among pennate diatoms. The development of this structure was studied in Diploneis smithii and begins with the formation of a primary band, which then develops secondary arms at both poles and the center, as in the classic Chiappino-Volcani model of raphid diatom ontogeny. Spine-like projections grow out from the primary band and secondary arms to establish the transapical ribs (virgae) of the mature valve and themselves develop spines, which are spaced first oppositely and then alternately and fuse with each other to delimit the stria pores. Subsequently, new pattern and structures develop both externally (formation of bifurcating projections that fuse to delimit the outer, sieve-like layer of the valve) and internally (growth and fusion of flanges from the first-formed ribs to create the longitudinal canals and deposition of a hymenate strip over the internal face of each stria). Comparisons are made with morphogenesis in other diatoms. Diploneis smithii ontogeny suggests how very slight developmental changes might have created the very variable external morphology of Diploneis species. It also indicates that the longitudinal canals of Diploneis and Fallacia have different origins, since the porous external wall is not formed as a unilaterally attached flap in Diploneis and the canal is internal to the first-formed rib-stria system in Diploneis, but external to it in Fallacia.

Spermatogenesis and auxospore structure in the multipolar centric diatom Hydrosera.

Spermatogenesis and auxospore development were studied in the freshwater centric diatom Hydrosera triquetra. Spermatogenesis was unusual, lacking depauperating cell divisions within the spermatogonangium. Instead, a series of mitoses occurred within an undivided cell to produce a multinucleate plasmodium with peripheral nuclei, which then underwent meiosis. 32 or 64 sperm budded off from the plasmodium leaving a large residual cell containing all the chloroplasts. Similar development apparently occurs in Pleurosira, Aulacodiscus, and Guinardia, these being so distantly related that independent evolution of plasmodial spermatogenesis seems likely. After presumed fertilization, the Hydrosera egg cell expanded distally to form a triangular end part. However, unlike in other triangular diatoms (Lithodesmium, Triceratium), the development of triradiate symmetry was not controlled by the "canonical" method of a perizonium that constrains expansion to small terminal areas of the auxospore wall. Instead, the auxospore wall lacked a perizonium and possessed only scales and a dense mat of thin, apparently entangled strips of imperforate silica. No such structures have been reported from any other centric diatoms, the closest analogs being instead the incunabular strips of some raphid diatoms (Nitzschia and Pinnularia). Whether these silica structures are formed by the normal method (intracellular deposition within a silica deposition vesicle) is unknown. As well as being more rounded than vegetative cells, the initial cell is aberrant in its structure, since it has a less polarized distribution of the "triptych" pores characteristic of the species.

Sperm ultrastructure in the diatoms Melosira and Thalassiosira and the significance of the 9 + 0 configuration.

The most complete account to date of the ultrastructure of flagellate cells in diatoms is given for the sperm of Thalassiosira lacustris and Melosira moniliformis var. octogona, based on serial sections. The sperm are uniflagellate, with no trace of a second basal body, and possess a 9 + 0 axoneme. The significance of the 9 + 0 configuration is discussed: lack of the central pair microtubules and radial spokes does not compromise the mastigoneme-bearing flagellum's capacity to perform planar beats and thrust reversal and may perhaps be related to sensory/secretory function of the sperm flagellum during plasmogamy. The basal bodies of diatoms are confirmed to contain doublets rather than triplets, which may correlate with the absence of some centriolar proteins found in most cells producing active flagella. Whereas Melosira possesses a normal cartwheel structure in the long basal body, no such structure is present in Thalassiosira, which instead possesses 'intercalary fibres' linking the basal body doublets. No transitional helices or transitional plates are present in either species studied. Cones of microtubules are associated with the basal body and partially enclose the nucleus in M. moniliformis and T. lacustris. They do not appear to be true microtubular roots and may arise through transformation of the meiosis II spindle. A close association between cone microtubules and tubules containing mastigonemes may indicate a function in intracellular mastigoneme transport. No correlation can yet be detected between methods of spermatogenesis and phylogeny in diatoms, contrary to previous suggestions.

OVERLOOKED HETEROPOLARITY IN SURIRELLA CF. FASTUOSA (BACILLARIOPHYTA) AND RELATIONSHIPS BETWEEN VALVE MORPHOGENESIS AND AUXOSPORE DEVELOPMENT.

Surirella cf. fastuosa is an apparently isopolar elliptic marine raphid diatom. We observed cells before and after sexual reproduction in monoclonal cultures using light and scanning electron microscopy (LM and SEM). After sexual reproduction cells were approximately twice as large as before, in valve length and width. The stria and infundibula densities were stable during the life cycle. Subtle morphological differences were detectable between the two poles of the frustule. One pole (pole A) was characterized by endings of the external raphe fissure that turned toward the valve face, continuity of the domed wall of the raphe canal externally, an elliptic chamber visible internally, a shallow nick in the interior of the valvocopula. The other pole (pole B) was with the following: straight endings of the external raphe fissures, a dent in the domed wall of the raphe canal externally, a double chamber internally, presence of the open ends of the valvocopula nearby, a deep nick in the valvocopula. Furthermore, at pole A virgae developed at an early stage in morphogenesis, whereas at pole B they were not formed. In the auxospores, pole A was situated beneath the primary transverse perizonial band. Pole A is suggested to be homologous with the head pole in heteropolar Surirella and is the "protopole" likely equivalent to the central nodule in naviculoid taxa. Pole B is homologous with the foot pole in heteropolar Surirella and is the "synaptic pole" formed by fusion of components equivalent to both poles of naviculoid taxa.

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.

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.