SEXUAL REPRODUCTION IN THE FRESHWATER DINOFLAGELLATE GLOEODINIUM MONTANUM1

The sexual life cycle of Gloeodinium montanum Klebs was examined with light and scanning electron microscopy. In unialgal cultures G. montanum divided predominantly by simple division, giving rise to two nonmotile cells. When placed in fresh medium, 2–4 biflagellated swarmers were formed from the vegetative cells. Swarmers developed directly into vegetative cells or acted as gametes. Both isogamy and anisogamy were observed. Gloeodinium montanum is homothallic. Fusion occurred in the non-motile state producing a large aplanozygote, which germinated after approximately two months to a year or more. Zygote germination liberated four aplanospores. Budding of the zygote, resulting from unequal division of the protoplast and multiple fusion attempts also were observed.     

ULTRASTRUCTURAL ASPECTS OF SEXUAL REPRODUCTION IN THE RED TIDE DINOFLAGELLATE GONYAULAX TAMARENSIS1

The marine dinoflagellate Gonyaulax tamarensis Lebour is best known for its propensity to form blooms known as red tides in coastal waters worldwide. This paper examines the sexual cycle of this organism using light and electron microscopy. Sexual reproduction begins with contact between thecate gametes which subsequently shed their thecae to fuse along their pellicular layers. Nuclear fusion occurs well after cytoplasmic fusion and is characterized by several distinctive features: a highly vesiculate nucleoplasm without microtubules; nucleoli and V-shaped chromosomes abut the nuclear envelope distal to the region of nuclear contact; and each chromosome possesses a longitudinal line, the central chromosomal axis. Fusion results in a planozygote with numerous cytoplasmic storage products and a slightly thickened layer beneath the pellicle. Subsequent loss of thecal plates and a thickening of the sub-pellicular layer results in a non-motile hypnozygote. A newly-formed hypnozygote possesses numerous minute papillae along its outer surface, formed by the up-folding of the accumulating wall layer. Maturation of the hypnozygote wall results in a smooth three-layered wall, the outermost layer of which is the pellicular layer. Hypnozygote germination produces a large quadriflagellate plan-omeiocyte with a single nucleus and thecal plates identical to vegetative cells. Two subsequent divisions, presumably meiotic, result in Jour cells morphologically identical to vegetative cells.     

THECAL ULTRASTRUCTURE OF THE TOXIC MARINE DINOFLAGELLATE GONYAULAX CATENELLA1

The toxic marine dinoflagellate Gonyaulax catenella Whedon & Kofoid was studied with scanning and transmission electron microscopy to describe the thecal morphology and to accurately define the taxonomic characters of the species. The closing platelet which lies in a U-shaped apical pore was revealed to be disassociable from a partly obscured apical platelet. Two previously unreported sulcal plates were charaterized and described. The entire complement of thecal plates numbered 33.     

INTRACELLULAR LOCALIZATION OF SAXITOXINS IN THE DINOFLAGELLATE GONYAULAX TAMARENSIS1

The potent neurotoxin saxitoxin, and possibly several of its derivatives, are localized in two types of sites within the marine dinoflagellate Gonyaulax tamarensis Lebour. Immunocytochemical techniques using a polyclonal antibody and epifluorescence microscopy demonstrate toxin localization within the nucleus as well as on the periphery of small granules thought to be starch grains. In the nuclear region, the labelling occurred on or close to the permanently-condensed chromosomes as well as in an area within the two arms of the nucleus in the vicinity of the nucleous. No binding was observed in a closely-related, non-toxic dinoflagellate. Different binding affinities were observed between the nucleus and the grains at high and low antibody dilutions. This may relate to the polyclonal nature of the antiserum and to the presence of multiple toxins within the G. tamarensis isolate studied. Mechanistic interpretations of these labelling patterns remain speculative, especially the localization of the antigen at the outer edge of starch grains, but the distinct labelling in the nuclear region suggests that saxitoxin, with its two positively charged guanidinium groups, may bind to nucleic acids or nuclear proteins in a manner analogous to the polyamines and other cations. The labelling patterns reported here suggest that the saxitoxins may not simply be secondary metabolites but instead could be important compounds involved in the structure and function of the G. tamarensis genome.     

USE OF THE CRICKET ACHETA DOMESTICA L. AS A BIOASSAY ORGANISM FOR THE TOXIC EXTRACT FROM GONYAULAX MONILATA (DINOPHYCEAE)1

A toxic extract from unialgal cultures, of Gonyaulax monilata Howell was tested for insecticidal activity on the cricket Acheta domestica L The regression line of the activity was compared with that of ouabain in this insect as well as with that of the toxic extract in mice. Although mice are approximately 2.5°as sensitive as the cricket an a body weight basis, because of the insect's smaller mass‘(1/50 that of the mouse), one can treat 26° as many crickets with the same amount of toxic material. This fact, coupled with the simple rearing requirements of the insect, makes it an ideal bioassay organism as well as a useful subject far further investigations of the physiological properties of the toxic extract and other bioactive compounds available in limited quantities.     

POTENTIAL IMPORTANCE OF BENTHIC CYSTS OF GONYAULAX TAMARENSIS AND G. EXCAVATA IN INITIATING TOXIC DINOFLAGELLATE BLOOMS1,2,3

Thick-walled, nonmotile cysts (termed hypnocysts) of two dinoflagellates were isolated from estuarine sediments in Cape Cod, Massachusetts, and germinated to produce their respective motile, thecate stages. Hypnocysts from Orleans district were identified as Gonyaulax excuvata (Braarud) Balech sensu Loeblich & Loeblich. Visually identical hypnocysts from Falmouth district were provisionally identified as Gonyaulax tamarensis Lebour. Both species were toxic. A geographic survey in September detected hypnocysts in only the sediments of locations where toxic blooms developed the preceding and following Spring. Laboratory incubation (16 C) of hypnocysts from sediment samples stored in the dark (5 C) for 6 mo initiated excystment by the temperature increase, with no appreciable effect from light regime, nutrient, or chelator concentrations. Motility of excysted germlings was optimum in highly chelated medium and in the presence of light. We conclude that hypnocysts of both tasa are important in seeding recurrent annual blooms, synchronizing early bloom development with vernal warning of seawater and increasing the geographic range of the species. We suggest that many red tides in New England and eastern Canadian waters are initiated through the displacement of motile estuarine populations into nearshore area by tidal advection and surface runoff, although the potential existence and importance of offshore cyst reservoirs cannot be discounted. Evidence is presented that hypnocysts are probable sexual zygotes whereas the thin-walled cysts readily formed in laboratory cultures (pellicle cyst) are asexual. Pellicle cysts are of limited durability, do not overwinter in nature, and therefore do not play a significant role in initiating toxic blooms.     

SEXUAL REPRODUCTION OF PERIDINIUM GATUNENSE (DINOPHYCEAE)1

Sexual reproduction was induced in the dinoflagellate Peridinium gatunense Nygaard when exponentially growing cells were inoculated into nitrogen deficient medium. Small thecate cells produced by division of vegetative cells then acted as gametes. Thecae of fusing gametes broke in the girdle region and were lost. Zygotes thus formed remained motile 3–5 days during which time they enlarged slightly with the newly formed theca becoming warty. Three to 5 days following plasmogamy the zygote became nonmotile, the protoplast contracted, and the cell wall thickened. Hypnozygotes with 4 nuclei were observed ca. 10–12 h following formation. Meiosis was inferred. Hypnozygotes germinated within 12 h of formation producing 2 vegetative cells which divided within a 24 h period. Attempts to induce sexual reproduction by inoculation of cells into media deficient in a number of basic elements other than N were unsuccessful.     

SEXUAL REPRODUCTION OF PERIDINIUM WILLEI (DINOPHYCEAE)1

Sexual reproduction was induced in the dinoflagellate Peridinium willei Huitfeld-Kass when exponentially growing cells were inoculated into nitrogen deficient medium. Small, naked vegetative cells produced by division of thecate cells acted as gametes. The zygote remained motile 13–14 days, during which time it enlarged and the theca formed became warty. Fourteen to 15 days following plasmogamy the zygote was nonmotile with the protoplast contracted. A large red oil droplet appeared and the wall thickened, becoming chitinized. Hypnozygotes with 4 nuclei were observed 7–8 wk following formation. Meiosis was inferred. The hypnozygote germinated, within 8 wk producing one post-zygotic cell retaining the red oil droplet. This cell divided within 24 h into 2 daughter cells each with a prominent red oil droplet. These daughter cells divided after 2 to 3 days into ordinary vegetative cells. Attempts to induce sexual reproduction by inoculation of cells into media deficient in a number of basic elements were unsuccessful.     

SEXUALITY AND CYST FORMATION IN PACIFIC STRAINS OF THE TOXIC D1NOFLAGELLATE GONYAULAX TAMARENSIS1,2

Sexuality has been established for a culture of Gonyaulax tarnarensis Lebour (strain NEPCC–71). The addition of a thick inoculum to a nitrogen–deprived medium results in the occurrence of anisogamous sexual fusion within the first three days in the new culture. Planozygotes, large “lumpy” cells recognizable by their four flagella, may persist up to 2 wk before forming a smooth–walled, oval hypnozygote. The latter resembles cysts released asexually by ecdysis but has a slightly thicker wall. Viable cysts resembling hypnozygotes (zygotic cysts), but with reduced photosynthetic pigmentation, have been isolated from natural murine sediments in Hidden Basin, British Columbia, and a culture (strain NEPCC–254) was initiated from excysted individuals. Zygotic cysts of NEPCC–71 remained encysted in the light at 17 C for 8 wk before excysting. The presence of a ventral pen with toxicity in the latter strain indicates that the taxonomy of G. tamarensis-like organisms is still in a stale of flux and the criteria for recognition of G. excavata (Braarud) Balech as a separate species are not satisfactory as presently formulated.     

SEXUAL REPRODUCTION OF PERIDINUM CINCTUM F. OVOPLANUM (DINOPHYCEAE)1,2

Sexual reproduction is induced in the dinoflagelate Peridinium cinctum f. ovoplanum Lindemann when exponentially growing cells are inoculated into nitrogen deficient medium. Small, naked vegetative cells, produced by division of the thecate cells, then act as gametes. The zygote remains motile for 12–13 days during which time it enlarges and the theca which it forms becomes warty. Thirteen to 14 day s following plasmogamy the zygote is nonmotile, the protoplast contracts, a large red oil droplet appears, the wall thickens and becomes chitinized. This hypnozygote germinates within 7–8 weeks at 20 c producing 1 post-zygotic cell retaining the large red oil droplet. The presence of 4 nuclei in these post-zygotic cells may be demonstrated by staining them with acetocarmine. Two of these nuclei are smaller than the other two and probably abort. One may infer that meiosis occurs immediately prior to or at the germinartion of the hypnozygote. This post-zygotic cell divides within 24 h into 2 daughter cells each with a promment red oil droplet. These daughter cells divide after 2–3 days into ordinary vegetative cells. Attempts to induce sexual reproduction by changes in temperature or light and by inoculation of cells into media deficient in a number of basic elements were unsuccessful.     

VEGETATIVE REPRODUCTION OF THE FRESHWATER DINOFLAGELLATE GLOEODINIUM MONTANUM1

The vegetative life cycle of Gloeodinium montanum Klebs was examined. In unialgal cultures G. montanum divided predominantly by binary fission once every 2-3 weeks. Nuclear division was followed by a delayed cytokinesis producing non-motile G. montanum cells. When placed in fresh media 2-4 biflagellated swarmers were formed. The swarmers, although similar in appearance to those of Hemidinium ochraceum Levander (1900), differ from that species in their dimensions. During vegetative reproduction swarmers developed directly into non-motile vegetative cells.     

CYST FORMATION IN THE RED TIDE DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE): EFFECTS OF IRON STRESS1

The toxic red tide dinoflagellate Alexandrium tamarense (Lebour) Balech (synonymous with Protogonyaulax tamarensis (Lebour) Taylor) was subjected to iron stress in batch culture over a 24-day time course. Monitoring of life history stages indicated that iron stress induced formation of both temporary (= pellicular) and resting (= hypnozygotic) cysts. Our experimental induction of sexuality appeared to be associated with iron limitation rather than the total depletion of biologically available iron. Degenerative changes in organelle (i.e. chloroplast, mitochondrion and chromosome) ultrastructure were largely restricted to pellicular cysts, suggesting that these temporary cysts were more susceptible to short-term iron stress effects than were hypnozygotes. These results are consistent with the hypothesized ecological roles of cysts in maintaining viability over brief (pellicular cysts) and extended (hypnozygotes) exposure to adverse environmental conditions.     

双角多甲藻有性生殖及厚垣合子形成条件的研究

双角多甲藻在MCV合成培养基中的最短加倍时间约6天,其有性生殖可用缺氮培养基进行诱导。批量培养的迟滞期和指数生长期末细胞的诱导效率最高,合子产率可达70％。厚垣合子形成的最适温度为19℃、pH7.4、光强5200lx。在鉴定的70个克隆藻株中,有2株为同宗配合;48株为异宗配合,其中“+”株23个,“-”株25个;其余20株未能诱导出有性生殖。合子开始萌发时,在中层壁和内层壁之间的空间出现1至多个“管状结构”,协助原生质体将合子壁顶破,外包一层薄壁的原生质体放出。     

COEXLSTENCE OF TOXIC AND NONTOXIC DINOKLAGELLAIES RESEMBLING GQNYAULAX TAMARENSIS IN NEW ENCILAND COASTAL WATERS (NW ATLANTIC)1

Two forms resembling Gonyaulax tamarrensis Lebour coexist in Maine cosat plankton; one is toxic, the other is nontoxic. At times, red patches of dinoflagellates were identified as G. tamarensis, yet only presumed to he toxic. Toxic froms were found in June, July 1975 and 1976. The nontoxic form was found in a more esraurine area, e.g., mid-July 1975, early July 1976. These two forms are not easily distinguished by conventional microncopic ohservation, nor by pigment analysis. Preliminary observations suggest that the nontoxic form is smaller than G. excavata (Braarud) Balech and tacks the “excavated” ventrat region. Characteristic G. excavata resting cysts were found in sediment from the shellfish toxic area off Newagen and Monhegan (Maine) and were not found in sediments from areas where the nontoxic form bloomed. There appear to be a minimum of two G. tamarensis-like organisms. Full systematic treatment of these must await further information.     

IMMUNOLOCALIZATION OF NITRATE REDUCTASE IN THE MARINE DINOFLAGELLATE GONYAULAX POLYEDRA (PYRROPHYTA)1

A polyclonal antibody, raised against nitrate reductase (NR) purified from the photosynthetic dinoflagellate Gonyaulax polyedra Stein, was used as a probe in immunogold-labeling experiments on thin sections prepared from cells harvested both during day and night phases. Previous experiments have shown that both NR activity and the amount of immunoreactive NR in cell extracts is greater when day-phase cells are examined, and this property was exploited as an internal control for the cytochemical labeling. We observed that in day-phase cells, chloroplasts contained approximately three times more gold particles than night-phase cells (highly significant difference; P < 0.0001), whereas cytoplasmic labeling levels remained relatively level between day and night. We conclude from the diurnal difference in labeling that our antibody faithfully reflects the distribution of NR in Gonyaulax cells. Thus, as in to some other higher plants and green algae, Gonyaulax compartmentalizes active NR in its chloroplasts.     

NUTRITIONAL REQUIREMENTS FOR SEXUAL REPRODUCTION IN MESOTAENIUM KRAMSTAI (CHLOROPHYTA) 1

Optimum conditions for conjugation in the heterothallic saccoderm desmid Mesotaenium kramstai Lemmer-mann have been determined. In culture, cells acquired the ability to form gamete pairs just prior to the onset of stationary phase after sufficient nitrate had been depleted from the medium. The appearance of potential gametes was delayed by increasing the concentration of KNO₃ When cells of both mating types were harvested from 15 to 18 day old cultures, washed, resuspended in fresh medium, and mixed, approximately 50 percent of the cells paired (measured three days after mixing) in a medium containing 0.13 mM or less KNO₃. At greater concentrations, fewer pairs formed; no pairs formed in medium containing 0.5 mM KNO₃. Conjugation was not inhibited by other macronutrients. Calcium and magnesium were essential for maximum conjugation. Although Ca²⁺ and Mg²⁺ contentrations of 0.05 mM and 0. I mM, respectively, were sufficient for optimum growth, maximum conjugation required more than 10 times these values. Few gamete pairs formed when either Ca²⁺ or Mg²⁺ was omitted from the medium, no pairing occurred when both Ca²⁺ and Mg²⁺ were omitted.     

EVIDENCE FOR SEXUAL REPRODUCTION IN THE PRIMITIVE GREEN ALGA NEPHROSELMIS OLIVACEA (PRASINOPHYCEAE)1

Evidence for a specialized sexual process in Nephroselmis olivacea Stein is presented. This alga is a member of the Prasinophyceae, which is regarded by some as the most primitive Class of green plants. N. olivacea has a heterothallic type of mating system. Plus and minus gametes were morphologically similar but showed different behaviors during the mating process. The minus gamete settled to the substratum, attaching by its ventral side. The plus gamete attached to the dorsal side of the minus gamete by the region near the flagellar bases of the plus gamete. The mature zygotes were spherical and strongly adhered to the substratum. After zygote germination, two biflagellate daughter cells, each with two pyrenoids were liberated. These cells divided, resulting in four vegetative cells, each with a single pyrenoid.     

SEXUAL REPRODUCTION IN THE DINOFLAGELLATE NOCTILUCA MILIARIS SURIRAY1,2

The existence of sexuality in dinoflagellates has long been controversial. Events described as the differentiation and release of isogametes and sexual fusion in cultures of Noctiluca miliaris were studied with the light microscope. In cultures fed. twice weekly with Dunaliella sp., kept under a 12-hr day, 12-hr night light regime at 25–30 ft-c and held at 18 C, certain vegetative cells, termed gametocyte mother cells, were observed to undergo what is interpreted as meiosis. This was followed by several synchronous mitotic divisions occurring at 45-min to 1-hr intervals and. resulting in the production of up to 1024 mature uniflagellated isogametes attached on the surface of the gametocyte mother cell body. After several hours gametes now seen swimming freely in the media were observed to fuse and form zygotes. One zygote subsequently became vacuolated and eventually differentiated into a small but reasonably typical vegetative cell. The life cycle of N. miliaris appears thus to be of the diplontic type. The taxonomic relationships between Noctiluca and other mesocaryotic dinoflagellates are discussed.     

SEXUAL REPRODUCTION IN STEPHANODISCUS NIAGARAE (BACILLARIOPHYTA)1

We observed sexual reproduction in a clonal culture of Stephanodiscus niagarae Ehrenb. and used light and scanning electron microscopy to absent flagellated male cells, auxospore growth, initial valve structure and production, and subsequent daughter cell division. Free auxospores were spherical and nonsiliceous throughout growth, producing hemispherical initial valves devoid of spines and with nonfasciculate striae. Pregametangial cells averaged 43% of the diameter of the daughter cell population and were 1/9 the biovolume of initial, cells. This paper is the first confirmed report of sexual reproduction in S. niagarae, although it appears that specimens of Actinocyclus niagarae H. L. Smith, described from Lake Erie in 1878, are actually initial valves of S. niagarae.