Novel Sex Cells and Evidence for Sex Pheromones in Diatoms

Citation: Sato S, Beakes G, Idei M, Nagumo T, Mann DG ( Novel Sex Cells and Evidence for Sex Pheromones in Diatoms Shinya Sato 0 Gordon Beakes 0 Masahiko Idei 0 Tamotsu Nagumo 0 David G. Mann 0 Jean-Marc A. Lobaccaro, Clermont Universite, France 0 1 Royal Botanic Garden Edinburgh , Edinburgh , United Kingdom , 2 School of Biological Sciences, Institute of Molecular Plant Sciences, University of Edinburgh , Edinburgh , United Kingdom , 3 School of Biology, Newcastle University , Newcastle upon Tyne , United Kingdom , 4 Department of Biology, Bunkyo University , Koshigaya, Saitama , Japan , 5 Department of Biology, The Nippon Dental University , Chiyoda-ku, Tokyo , Japan 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. - Funding: This research was supported by a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science (JSPS), awarded to Shinya Sato (http://www.jsps.go.jp/english/index.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The morphology of flagella and their associated apparatus is for the most part highly conserved across each of the major groups of eukaryotes (e.g. [1]), despite huge variation in life form and life cycle. For example, the stramenopiles are a highly diverse assemblage of photosynthetic, parasitic and saprophytic organisms, including unicellular and multicellular forms [2,3,4], but the flagellate cells are of a characteristic type, being biflagellate and possessing a longer anterior flagellum with tubular mastigonemes, which produces a backward current to create thrust, and a shorter posterior flagella used as a rudder [5,6,7]. However, there are exceptions, notably in the diatoms, where only some lineages possess flagella and then only in the male gametes; furthermore these sperm have only one flagellum and even this is atypical, lacking the central pair of microtubules, i.e. there is a 9 + 0 microtubular configuration in the axoneme [8]. No posterior flagellum, not even a rudimentary one, has been found in any diatom. Historically, diatoms have been divided into two groups, centrics and pennates, based primarily on their cell wall morphology. The pennates were further subdivided into non-motile araphid and motile raphid forms [9]. Molecular phylogenies have indicated, however, that in the pennate clade the monophyletic raphids diverged from among the araphids, making the araphids paraphyletic (reviewed by [10,11,12]). With respect to sexual reproduction, all the centrics (also a paraphyletic assemblage) are oogamous, producing eggs and uniflagellate sperms, whereas pennates are generally morphologically isogamous and non-flagellate [13,14]. As an exceptional case, two araphid genera are known to exhibit anisogamy, in which compatible clones are differentiated into female and male and produce sessile eggs and smaller amoeboid male gametes, respectively [15,16], but the latter appear to be entirely without flagella. However, the timing of flagellum loss whether it occurred before the evolution of the pennate lineage or after is unknown, because sexual reproduction has been so little studied in the critical basal lineages of pennates. During a study of heterothallic sexual reproduction in an araphid pennate, Pseudostaurosira trainorii E. Morales, we found that male clones produce motile gametes. Further observation revealed that the gametes bear thread-like structures, which superficially resemble flagella. Here we report the sexual reproduction of P. trainorii with special emphasis on the behaviour and fine structure of the threads, examined using light microscopy (LM), including time lapse observations; immunofluorescence staining observed under confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM). We demonstrate that the threads do not correspond to flagella in morphology, structure or activity. We also report experimental evidence for the presence of sex pheromones that reciprocally stimulate sexualization of compatible clones. Vegetative cells Cells were rectangular in girdle view, containing one to two plate-like plastids lying along the girdle (Fig. S1A). Valves were circular to elliptical with parallel striae (30 in 10 mm) and a distinct sternum along the long axis (Figs S1B, C). The striae consisted of rows of circular to elliptical areolae, which were occluded with complex vela and extended from the valve face onto the mantle (Figs S1C, D). The valve had marginal spines between the interstriae ( = ribs, virgae) (Figs S1C, D). All these characteristics correspond to Pseudostaurosira trainorii as illustrated by Morales [17] and Morales et al. [18]. Mitotic cell division (not illustrated) was equal and followed immediately by the formation (...truncated)