Masahiko Idei

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.

MATING SYSTEM, SEXUAL REPRODUCTION, AND AUXOSPORULATION IN THE ANOMALOUS RAPHID DIATOM EUNOTIA (BACILLARIOPHYTA)1

The diatom genus Eunotia is unusual among raphid diatoms in having a raphe system consisting of two short slits that are not integrated into the primary pattern center. This and other characteristics, particularly the presence of rimoportulae, are consistent with the hypothesis that Eunotia is a basal lineage within the raphid group. We studied auxosporulation in E. bilunaris (Ehrenberg) Mills and E. tropica Hustedt for comparison with other raphid pennate diatoms and with araphid pennates; E. bilunaris was studied in parental and F1 generations. Like araphid pennates, E. bilunaris and E. tropica are heterothallic. Clones of the same mating type did not interact sexually, and intraclonal sexual reproduction was absent or very rare. Clones retained the same sex throughout the life cycle, as shown by experiments using abrupt size reduction to produce clones of similar age but different size and using subclones derived from a single initial cell within six mitotic generations. Unlike in araphid pennate diatoms, in the Eunotia species the gametes are not visibly or behaviorally differentiated. Gametogenesis is merogenous, because the gametangium formed a supernumerary cell as well as a single gametic cell, both undergoing meiosis II to form a surviving functional nucleus and a nucleus that quickly degenerated. Plasmogamy is via papillae that grew out toward each other from the ends of the gametangia to create a copulation canal. After plasmogamy, the gametes moves bodily into the copulation canal, producing an elongate zygote, which expands to form a curved sausage‐like auxospore.

Proposals for a terminology for diatom sexual reproduction, auxospores and resting stages

The past few decades have brought about a significant expansion in our understanding of the diatom life cycle, particularly its sexual part. Presented here is a set of proposals for the terminology of processes and structures associated with sexual reproduction and for the resting stages of diatoms, some of which have at times been confused with each other. The proposals fill the void present in widely used publications offering standardized terminology related to diatom frustule micro-architecture.

Fine-structure of the vegetative frustule, perizonium and initial valve of Achnanthes yaquinensis (Bacillariophyta)

This study clarifies the fine structure of the vegetative and initial valves of Achnanthes yaquinensis and briefly compares them to other Achnanthes species. It also elucidates the structure of the perizonium, based on auxospore development in short-term cultures. The araphid valve has marginal ridges and terminal spines that allow connecting valves to form a chain. The terminal spines develop from the rapheless sternum. The complete cingulum consists of 3–5 split bands with two rows of areolae. These features can be used to discriminate species within the genus. Sexual reproduction is isogamous with two mother cells producing two auxospores, which are enclosed in mucilage. The perizonium develops on one side of an auxospore only, comprising one large central longitudinal band and four closed bands. There are no transverse perizonial bands. The raphid valve of the initial cell forms first, underneath the longitudinal perizonium, followed by the araphid valve, which is not covered by any perizonial bands. The araphid valve of the initial cells lacks a marginal spine, and the rapheless sternum lies more centrally than in the vegetative cell. The relationship of the genus Achnanthes to other monoraphid diatoms is discussed briefly.

Successive observations on the fertilization of a centric diatomMelosira moniliformis var.octagona

The details of fertilization in the centric diatom,Melosira moniliformis var.octagona, were examined by light microscopy with a video system. The sperm penetrated into the egg cell through a temporary opening of the oogonium. The flagellum of the sperm was brought into the egg cell and it remained active for a few minutes. Three observations on the behavior of the sperm and egg cell during fertilization are shown.

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.

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.

Fine structure of two Hygropetra species, Hygropetra gelasina sp. nov. and Hygropetra balfouriana (Bacillariophyceae), and the taxonomic position of the genus with special reference to Frankophila

A small diatom, Hygropetra gelasina sp. nov., obtained from wet moss is described. This diatom is similar to Hygropetra balfouriana (Grunow ex Cleve) Krammer & Lange‐Bertalot, which was found in the same moss sample. Fine structural observations revealed that H. gelasina has a reduced raphe slit and depressions along the margin of the axial area, at the proximal ends of the striae. Both species are characterized by multiple rows of areolae in each stria and a hexagonal pattern of sub‐pores similar to that in Pinnularia, but differing in the position of the areola occlusions or hymenes, which are internal in Hygropetra. Comparison with Frankophila, which shares the characteristics of reduced raphe slits and areola structure with Hygropetra, provides a reference for future taxonomic study of these related genera.

Sexual reproduction and auxospore structure in Diploneis papula (Bacillariophyta)

Idei M., Sato S., Watanabe T., Nagumo T. and Mann D.G. 2013. Sexual reproduction and auxospore structure in Diploneis papula (Bacillariophyta). Phycologia 52: 295–308. DOI: 10.2216/12–021.1 We give a detailed account of sexual reproduction and auxospore development in the diatom genus Diploneis, principally from clonal cultures of the marine benthic Diploneis papula. Sexual reproduction of D. papula was apparently homothallic. After pairing side to side, cells entered meiosis, and each gametangium produced two gametes. Fertilization was physiologically anisogamous, and both gametes of one gametangium were active and those of the other gametangium passive so that the two zygotes were formed within or close to the ‘passive’ gametangium. Each gamete contained a single chloroplast. The zygote became surrounded by delicate incunabula that contained circular or elliptical scales; this confirmed recent observations that scales (or apparently homologous strips or plates) are quite commonly formed by the zygotes of raphid diatoms. Subsequently, a robust transverse perizonium was built up as the auxospore expanded, and the perizonium was comprised of a closed primary band at the centre and open secondary bands towards each pole. The ends of the secondary bands, which met along one side of the auxospore to form a suture as in other pennate diatoms, were curved inwards towards the centre of the auxospore. Novel features were the extension of the transverse perizonium to cover the tips of the auxospores (in other diatoms it stopped short of the poles, leaving an apical dome covered only by the incunabula) and a differentiation of the ends of the transverse bands into truncated and prolonged variants, which alternated along the suture. The longitudinal perizonium possessed the same highly conserved configuration as in other pennate diatoms, with a wide bifacial central band flanked by two other bands, which here differed in shape and structure. All the perizonial bands were fimbriate. The possible homology of the longitudinal perizonium to the thecae of vegetative cells was discussed. During formation of the initial cell, the protoplast contracted away from the perizonium, but the latter nevertheless moulded the outline shape of the initial valves, which were more strongly constricted than preauxospore cells and gametangia. Other Diploneis species produced either one or two gametes per gametangium.

Three new species of freshwater Diploneis from Japan

Three new Diploneis species are described from freshwater lakes in Japan. A complex wall structure is demonstrated in D. aokiensis sp. nov. and D. yamanakaensis sp. nov., where the basic alternation of transapical ribs (virgae) and striae is overlain by a system of secondary branches and struts supporting the external perforate surface. Diploneis linearifera sp. nov. possesses remarkable external slits, distributed irregularly around the margin of the valve and near the raphe. The grooves in the outer layer of the valve wall, which do not penetrate to the valve interior, have no known parallel in other diatoms but may be related to the angular holes noted previously in D. finnica (Ehrenberg) Cleve and D. marginestriata Cleve.