Sexual auxosporulation of the marine diatom Navicula directa var. directa

Sexual auxosporulation was observed in a mixed culture of two strains of Navicula directa var. directa. Two gametes did not re‐arrange in their gametangium and each adhered to the inner surfaces of the gametangial theca. Each of the two gametes of one gametangium fused with a gamete of the other gametangium iso‐gamously. As a result, two zygotes and hence two auxo‐spores were produced per paired gametangia. As the gametangial thecae kept close to the gametes during fusion, the zygote became associated with two different thecae. The presence of type IB2a of Geitler's (1973) system was confirmed by the present observations.     

THE ULTRASTRUCTURE OF DERBESIA TENUISSIMA (DE NOTARIS) CROUAN. I. ORGANIZATION OF THE GAMETOPHYTE PROTOPLAST,GAMETANGIUM, AND GAMETANGIAL PORE1,2,3

The fine structure of vegetative and reproductive gametophytes of Derbesia tenuissima is described. Development of the gametangium and release of the gametes progress as follows: (1) In initial stages of gametangium formation, prior to 24 hr before gamete release, there is an accumulation and proliferation of nuclei, chloroplasts, and other organelles. (2) This is followed by separation of the gametangium from the rest of the plant by a gametangial membrane; segregation of organelles into gametes has begun by 12 hr before release and the process is completed by 2.5 hr before release. (3) Enzymatic wall dissolution of the pore area occurs between 2.5 and, 12 hr before normal lights-on time. (4) The release mechanism appears to be an instantaneous light-induced increase in lurgor pressure rupturing the weakened pore area, of the wall and causing a forcible expulsion of the gametes. (5) Following release, the pore is sealed by organellar debris and the gametangial membrane. Additional wall layers are presumed to be laid down internal to the plugged pore by the vegetative protoplasm which migrates into the area.     

Cytology of oomycetes

Evidence is presented for gametangial meiosis in six Oomycetes. Particular attention is drawn to late meiotic prophase stages (pachytene-diplotene) in which chromomeres and paired homologues can be seen. One of these Oomycetes has been shown to have an abortive meiosis. Multiple chromosome associations (quadrivalent configurations, with some trivalent and univalent configurations) at first metaphase with unequal divisions at first telophase or second metaphase suggest that autopolyploidy is to be found in several species. Allopolyploidy is suspected at intermediate levels of ploidy. Basic chromosome numbers of n=3 for Achlya and n=10 for Pythium are proposed.     

Auxosporulation of Licmophora communis (Bacillariophyta) and a review of mating systems and sexual reproduction in araphid pennate diatoms

The auxosporulation of Licmophora communis is allogamous and dioecious. Pairing between sessile, shortstalked cells of compatible clones is followed by meiosis and gametogenesis, to form two gametes in each gametangium. The behavior of the gametes differs between the gametangia. In the male gametangium, the gametes detach from the frustule, round up, and migrate out of the gametangium after its dehiscence at the broader, unattached pole. In the female gametangium, both gametes remain attached to the adjacent theca over almost their whole length and do not move. Plasmogamy therefore occurs within the female gametangium and this is where the zygotes are formed and remain. After fertilization, the zygotes detach from the thecae of the female gametangia, contract, and become ellipsoidal, before expanding parallel to the apical axis of the gametangium. We review the types of auxosporulation in other pennate diatoms and the systems used for classifying these. Dioecy and cis‐type anisogamy (in which one gametangium produces active gametes and the other produces passive gametes), as in L. communis, are probably primitive within the pennate group (although there is no information on the AsterionellopsisRhaphoneis clade). However, size can also be restored in various araphid pennates by allogamous sexual reproduction involving the formation of only one gamete per gametangium, or in rare cases by automixis or (apparently) vegetative enlargement.     

Improved mating efficiency inAcetabularia acetabulum: recovery of 48–100% of the expected zygotes

Summary We have improved zygote recovery 11–1,000 fold by optimizing the physiology of gamete release and mating inAcetabularia acetabulum. Gamete release was affected by agar purity, concentration, and volume/gametangial pair. Cold pre-treatment of gametangia (14–30 d at 10°C in the dark) synchronized subsequent gamete release at 21°C in the light. Cold pre-treatment was nearly twice as effective in synchronizing subsequent gamete release when intact, gametangia-bearing caps rather than isolated gametangia were pretreated. Synchronizing gamete release doubled mating efficiency. In a wild-type laboratory strain ofA. acetabulum, there were 1,561±207 gametes/gametangium which had half-lives of 14.5 d in 0.1% seawater-agar. We recovered 48–93% of the expected numbers of zygotes from a mass mating of 8 to 1,226 gametangia and 11–128% of the expected numbers of zygotes from mating single gametangial pairs: the large range in the calculated mating efficiency may be attributable to the variation in the numbers of gametes made per gametangium. Zygote recovery from single gametangial pairs was highly dependent on the volume of mating matrix. In addition, most zygotes recovered were unattached to any other zygotes in the subsequent generation (> 95% single cells from matings of 1–500 gametangial pairs). Our improvements in mating conditions and zygote recovery (1) have facilitated cell manipulation and culture ofA. acetabulum in the laboratory; and (2) have made controlled crosses for selection and genetic analysis of mutants feasible. These advances have removed a major barrier to genetic analysis of development inAcetabularia.Abbreviations LB Luria-Bertani bacteriological broth - SE standard error of the mean - T_g agar gelling temperatures - DAPI 4,6-diamidino-2-phenylindole     

Oosporogenesis by Lagenidium giganteum: induction and maturation are regulated by calcium and calmodulin

Induction and maturation of the sexual stage (oospores) of the facultative mosquito parasite Lagenidium giganteum (Oomycetes: Lagenidiales) are complex developmental processes regulated by calcium-dependent events. Use of developmentally synchronized cultures of L. giganteum allowed stage-specific disruption of calcium metabolism. A calcium chelator (EGTA), an ionophore (chlortetracycline), and inhibitors of the calcium-binding protein calmodulin (dibucaine, trifluoperazine, chlorpromazine) disrupted several discrete developmental steps associated with oosporogenesis: induction of antheridia, gametangial fusion, meiosis, oospore wall formation, and subsequent spore maturation. Extracellular calcium is necessary for oosporogenesis to proceed normally and under some conditions magnesium has a synergistic effect with calcium on oospore induction. Results are discussed in relation to calcium mediation of fusion events in a number of model membrane and biological systems.     

CULTURE STUDIES ON THE MARINE GREEN ALGA HALICYSTIS PARVULA—DERBESIA TENUISSIMA. II. SYNCHRONY AND PERIODICITY IN GAMETE FORMATION AND RELEASE

Unialgal cultures of the macroscopic, vesicular, coenocytic gametophyte (Halicystis parvula Schmitz) of Derbesia tenuissima (DeNotaris) Crouan fr. were grown under various environmental regimes to elucidate the cytology of gamete formation and the factors controlling synchronous gamete formation and release. No synchrony of nuclear division was observed in vegetative plants or during the early stages of gamete formation. In the later stages of gamete formation in plants in a light-dark cycle, nuclear divisions within any gametangium were synchronous, and the stages of gamete formation were synchronous for the population. This synchrony was not as great for plants in continuous light. Gametes of plants in a light-dark cycle were released explosively immediately following the dark-to-light transition. Release was random and much less forceful for plants in continuous light. After a certain stage of gamete formation, gamete release was timed to occur after a particular interval of darkness, but release could be triggered by light during the last portion of this interval. The length of the dark interval was shorter for male plants than for females, but the period of light sensitivity was longer for females. Formation of gametangia by series of isolated plants was also synchronous and sometimes periodic under certain conditions. Intervals between gametangia on the same plant varied from 2 to 14 days but were usually 4 or 5 days (unlike plants in nature, which show a bi- or tri-weekly periodicity). Male and female plants did not differ in synchrony or periodicity. Different media affected the number of gametangia formed over a period of time but not the synchrony of formation. Under some conditions changing the medium had a stimulating or synchronizing effect. Non-repeated temperature changes also synchronized gamete formation. Optimum temperature for continued gamete formation was about 21 C. Regular daily light and temperature variation together maintained synchronous and periodic gamete formation in populations of isolated plants. Reproduction diminished and became less synchronous at constant temperature either in continuous light or under a light-dark schedule, although in the light-dark regime steps in the formation of any given gametangium remained synchronous with the light-dark cycle. Length of times between gametangial formation on individual plants showed a tendency to occur in multiples of the usual period lengths; e.g., plants sometimes tend to “skip” intervals, thus maintaining the synchrony of the population. These results suggest that interaction between daily environmental cycles and an endogenous physiological cycle may maintain periodic reproduction.     

SEXUAL REPRODUCTION IN NAVICULA CRYPTOCEPHALA (BACILLARIOPHYCEAE)1

Homothallic sexual reproduction and auxosporulation were studied in monoclonal cultures and seminatural populations of the freshwater epipelic diatom Navicula cryptocephala Kütz. Gametangia paired via the girdle, one gamete was formed per gametangium (and hence one zygote per pair of gametangia), and gamete fusion took place without the formation of any copulation envelope or copulation canal. Superfluous nuclei from meiosis survived unusually long, so that gametes and young zygotes were probably functionally polyploid; later, all but two haploid nuclei degenerated. Expanded auxospores had a swollen center, but during formation of the initial valves, the auxospore contracted away from the perizonium to produce linear‐lanceolate valves. The pattern of reproductive behavior found in N. cryptocephala can be classified as type IIA2a auxosporulation in Geitler's system. The same type of zygote and auxospore formation seen in clonal cultures was observed in seminatural material from four lakes in Scotland and the Czech Republic. Variation in nuclear structure and auxosporulation in the N. cryptocephala species complex is discussed, as is the evolution of type II auxosporulation (one zygote per pair of gametangia) from type I auxosporulation (two zygotes per pair). The penalty of smaller numbers of zygote produced in type II may be outweighed by formation of larger auxospores (prolonging the vegetative phase) or more vigorous auxospores. The variation present among members of the N. cryptocephala complex indicates that biogeographical analyses based on use of the name N. cryptocephala, as performed recently to support the ubiquity hypothesis of protist distributions, are almost meaningless.     

A Virus Infection in the Marine Brown Alga Ectocarpus siliculosus (Phaeophyceae)

Laboratory cultures of Ectocarpus siliculosus originating from New Zealand showed a defect in gametangium formation. Nuclear divisions in gametangium initials are not followed by cell wall formation. In the resulting multinucleate cells nuclear DNA increases dramatically, and nuclear membranes disintegrate. Eventually, the entire structure is filled with hexagonal particles of approximately 130 nm diameter. These were isolated and shown by EM to consist of a dense core surrounded by a 3-layered shell. They are released into the culture medium when the host cells burst. Ectocarpus gametes from healthy cultures could be infected by these particles. The resulting partheno-sporophytes developed pathological symptoms, suggesting that the particles are viruses. The expression of the defect is temperature dependent. At 10°C all gametangia are abnormal, while between 15 and 20 °C defective and normal gametangia and gametes are formed on the same plant. Partheno-sporophytes developing from such gametes carry the viral particles expressed in deformed unilocular and plurilocular sporangia.     

Mal3, the Schizosaccharomyces pombe homolog of EB1, is required for karyogamy and for promoting oscillatory nuclear movement during meiosis

Two successive rounds of chromosome segregation following a single round of DNA replication enable the production of haploid gametes during meiosis. In the fission yeast Schizosaccharomyces pombe, karyogamy is the process where the nuclei from 2 haploid cells fuse to create a diploid nucleus, which then undergoes meiosis to produce 4 haploid spores. By screening a collection of S. pombe deletion strains, we found that the deletion of 2 genes, mal3 and mto1, leads to the production of asci containing up to 8 spores. Here, we show that Mal3, the fission yeast member of the EB1 family of conserved microtubule plus-end tracking proteins, is required for karyogamy, oscillatory nuclear movement, and proper segregation of chromosomes during meiosis. In the absence of Mal3, meiosis frequently initiates before the completion of karyogamy, thus producing up to 8 nuclei in a single ascus. Our results provide new evidence that fission yeast can initiate meiosis prior to completing karyogamy.     

Production of anisogametes and gamete motility dimorphism in Monostroma angicava

 The reproductive strategy of a marine alga with a heteromorphic biphasic life cycle was studied by analyzing various sexual reproductive characters in light of the evolution of anisogamy. Gametophytes of Monostroma angicava were dioecious and their gametes were slightly anisogamous. Volume of gametangium, density of gametangia and area of mature gametangial parts on each gametophyte did not differ from male to female. Therefore, the reproductive biomass investment for gamete production was considered to be the same for each sex. Anisogamy in this alga appeared to be derived from the difference in the number of cell divisions during gametogenesis, because the majority of male gametangia each produced 64 (2⁶) gametes and the female produced 32 (2⁵) gametes. This corresponded with measurements of cell size in male and female gametes. Further, the sex ratio was 1:1 for sexually mature plants sampled at Charatsunai. Therefore, it was suggested that in the field twice as many male gametes are released as female gametes. Liberated gametes of both sexes showed positive phototaxis. The swimming velocity of freshly liberated male gametes was a little higher than that of female gametes. Male gametes had the potential to swim for ca. 72 h and female gametes for ca. 84 h. The difference in gamete motility between the two sexes seemed to be related to cell size. Planozygotes were negatively phototactic and swam more rapidly than gametes of either sex. Received: 5 March 1997 / Revision accepted: 18 July 1997     

Chromosomal Evidence on the Sporogony of Amblyospora californica (Microspora: Amblyosporidae) in Culex tarsalis (Diptera: Culicidae)

ABSTRACT. Amblyospora californica is a polymorphic, eukaryotic microsporidian. Three types of sporogony producing three types of spores occur in male larvae and female adults of its mosquito host, Culex tarsalis , and an alternate copepod host, Acanthocyclops vernalis. Development of A. californica in male larvae includes merogony and sporogony. Karyogamy and meiosis was observed in sporogony in male larvae but not in the female adult or in the copepod. Chromosomal evidence showed that sporogony included two consecutive meiotic divisions and a subsequent mitosis forming an octosporont, ultimately containing eight haploid, uninucleate mature spores. In this species, the haploid number of chromosomes is nine. Macrosporoblasts and macrospores, containing 1, 2 or more nuclei, can be seen in infected male larvae. The stage of sporogony in which cytokinesis was arrested seems to determine the number of nuclei. Those with only one nucleus, we believe are due to failed nuclear division at meiosis. Although A. californica displayed a process of karyogamy and meiosis similar to that of the species from Cx. salinarius , they may not be the same species because of the difference in their chromosome numbers.     

CYTOLOGICAL STUDIES IN LACCARIA (AGARICALES). I. MEIOSIS AND POSTMEIOTIC MITOSIS

In the genus Laccaria, basidial formation, dikaryotic basidia, karyogamy, and meiosis were generally similar to structures and phenomena reported for other Agaricales. The haploid nuclei of dikaryotic basidia resided side by side in the basidium prior to karyogamy. Following karyogamy a single nucleolus was observed in L. montana (four-spored species); several nucleoli remained in the nucleus of L. tortilis (two-spored species). The haploid number of chromosomes for L. montana appeared to be n = 9. Postmeiotic mitosis typically occurred in the basidiospores resulting in binucleate basidiospores for four-spored species and quadrinucleate basidiospores for two-spored species. Postmeiotic mitosis sometimes occurred in the sterigmata and basidia proper. In instances where postmeiotic mitosis occurred in basidia, mature basidiospores were not formed and the basidia were collapsed, and contained up to eight nuclei.     

Evidence for Meiosis in the Testate Amoeba Arcella

ABSTRACT. Electron microscopic studies of Arcella vulgaris have revealed a sexual process occurring in cysts. The evidence includes: I) synaplonemal complexes in prophasic nuclei. 2) two consecutive divisions different from vegetative mitosis, 3) degeneration of certain products of these nuclear divisions, and 4) fusion of nuclei indicating karyogamy. The process is apparently autogamy preceded by a two-division meiosis. The testate amoebae can no longer be considered asexual.     

Life cycle and mode of infection of Leptolegnia chapmanii (Oomycetes) parasitizing Aedes aegypti

The life cycle and mode of infection of mosquito larvae by Leptolegnia chapmanii (Oomycetes: Saprolegniales) were determined. The life cycle is typical of saprolegniaceous fungi, as the species is dimorphic producing diplanetic biflagellate zoospores. Sexual reproduction is by means of gametangial contact and results in the production of a characteristic papillate oogonium containing a subcentric or eccentric oospore. L. chapmanii is capable of infecting Aedes aegypti larvae both by germination of encysted secondary zoospores on the exterior cuticle and by germination of ingested zoospore cysts in the larval midgut. Once the fungus is established in the host, the disease, a coelomomycosis, is fatal. The encystment pattern of secondary zoospores on the larval cuticle appcars preferential. Scanning electron microscopy indicates that mechanical pressure is not the sole force utilized by the fungus for cuticle penetration.     

Life Cycle of Culicosporella lunata (Hazard & Savage, 1970) Weiser, 1977 (Microspora) as Revealed in the Light Microscope with a Redescription of the Genus and Species1

Light microscopy studies of Culicosporella lunata (Hazard & Savage), a parasite of the mosquito Culex pilosus (Dyar & Knab), revealed two sporogonial sequences. One sequence begins with diplokaryotic meronts that undergo repeated nuclear divisions to produce sporogonial plasmodia with nuclei in diplokaryotic arrangement. These plasmodia form rosette-like clusters of sporoblasts during incomplete cytokinesis and, eventually, binucleate spores. These spores initiate infections in healthy larvae when they ingest spores. The second sequence begins with diplokaryotic meronts that undergo karyogamy and meiosis to form Thelohania-like sporonts and haploid spores. Anomalies are often observed in these sporonts which result in aberrant spores, usually fewer than eight, in an accessory (pansporoblastic) membrane. Normal haploid spores are morphologically similar to those of species of Amblyospora. The genus and the type species are redefined based on new information presented here and it and the type species are placed in the family Amblyosporidae.     

Nuclear behaviour during ascus formation in Saccharomyces rosei (Guilliermond) Lodder et Kreger-van Rij

Stained cells of Saccharomyces rosei prepared from 4 to 10-day-old cultures were studied under the light microscope. Mitotic and meiotic divisions involving a ring-like structure as well as preceding and subsequent stages were observed. Cells presenting supernumerary mitoses in a varying number were frequent. These mitoses, having terminated their multiplication activity, suspended the process shortly before its conclusion and, in a development which was identical at all, assumed a curious arrangement forming a mitoses-ring. Meiosis-buds were detected. These especial buds, where karyogamy and meiosis took place, resulted from the development of the mitoses-ring, whose mitoses upon resuming their activity moved toward the cell wall giving rise to the appearance of these appendices. Each one of these buds received the corresponding pair of daughter nuclei, diploidization occurring subsequently. Meiosis was usually processed in a single bud (effective-meiosis-bud) and all four meiotic nuclei migrated to the mother cell, and gave rise to a tetra-nucleate spore or binucleate spores if two were formed.Other modalities of sporulation were observed. These may result either from the association of two cells, in which one assumed the function of meiosis-bud (false-meiosis-bud), or from a cell association in which this function was performed for several linearly arranged cells forming a protuberance.Conjugation between mother cell and an attached bud, or between independent cells, was not observed.     

AN ELECTRON MICROSCOPE STUDY OF GAMETE RELEASE AND SETTLING IN THE COMPLANATE FORM OF SCYTOSIPHON (SCYTOSIPHONACEAE,PHAEOPHYTA)1

This paper describes the ultrastructural characteristics of gametes and their liberation from the gametangia in Scytosiphon sp., a brown alga showing only slight sexual differentiation. Both male and female gametes are released initially into a central cavity which forms in the gametangial sorus by the extensive dissolution of the internal cell walls. Scytosiphon sp. gametes possess structural features in common with the zoids of other species of brown algae. Gamete fine structure is discussed in relation to cell function. After release from the gametangial sorus, female gametes can be distinguished from males by the presence of a large number of Golgi-derived vesicles with electron dense cores. It is possible that these vesicles contain the sex attractant compound. When gametes settle they become spherical, the flagella and eyespot are withdrawn into the cell and adhesive material, apparently originating from the activity of the Golgi body, appears on the surface of the cell.     

Patterns of sexual reproduction in diatoms

Sexual reproduction takes many forms within the diatoms. The variation has been classified by several authors, but in most cases the distinctions between their main categories have depended on the number of gametes produced per gametangium (and thus on how many zygotes per pair of copulating cells), and upon whether fusion is oogamous, anisogamous or isogamous. These classifications are not themselves an adequate basis for taxonomic comparison, which should be based on individual characteristics of the sexual process. Diatoms seem to be primitively oogamous. In araphid pennate diatoms and some raphid diatoms the gametes and gametangia are morphologically alike but physiologically distinct; one gametangium produces active gametes and the other passive ones. This may be the primitive condition in pennate diatoms, providing a link to the oogamy of centrics via the morphological anisogamy of Rhabdonema Kütz.     

Cytological studies of the two-spotted spider mite Tetranychus urticae Koch (Tetranychidae,trombidiformes). I: Meiosis in eggs

Meiosis in eggs of Tetranychus urticae Koch is described. The two maturation divisions result in (a) a haploid female pronucleus consisting of three karyomeres; — (b) a divided first polar body in which the chromosomes change into karyomeres; — (c) a second polar body, entering a new mitosis which is blocked in metaphase. Irradiation of adult females produced chromosome fragments in the meiotic divisions. The fragments behave as intact chromosomes which proves that during meiosis a diffuse kinetochore is present. The meiotic divisions show the cytologically characteristic features of an inverted meiosis. The presence of such a meiosis is corroborated by observations on eggs heterozygous for chromosome mutations. In both maturation divisions the chromosomes are orientated equatorially. It is suggested that the equatorial orientation is brought about by chiasmata having terminalized to both ends of the dyads. It is argued that in organisms with holokinetic chromosomes during meiosis an axial orientation of the bivalents does not necessarily imply a normal meiosis but can also imply an inverted meiosis.This work was carried out with financial aid of the Institute for Atomic Sciences in Agriculture and the Ministry of Public Health and Environment.