EFFECTS OF METALS AND ORGANIC CONTAMINANTS ON THE RECOVERY OF BIOLUMINESCENCE IN THE MARINE DINOFLAGELLATE PYROCYSTIS LUNULA (DINOPHYCEAE)1

Pyrocystis lunula Schütt is a unicellular photoautotrophic dinoflagellate, commonly found in marine environments, displaying circadian‐controlled bioluminescence. Because of this species' characteristics, effects of pollutants on bioluminescence in P. lunula may make for an easy and simple bioassay that would be valuable for toxicity testing and the protection of coastal resources. This study therefore investigated the short‐term effects of metals and organic pollutants on the recovery of the bioluminescent potential in P. lunula. Recovery of bioluminescence was strongly inhibited in a dose‐dependent manner by all reference contaminants tested, the system being most sensitive to copper and cadmium (4‐h IC₅₀s 0.96 and 1.18 μM, respectively), followed by phenanthrene, lead, SDS, and nickel (4‐h IC₅₀s 1.64, 12.8, 15.6, and 73.1 μM, respectively), whereas relatively high concentrations of phenol were needed to elicit a response (4‐h IC₅₀ 1.64 mM). Except for exposure to lead and nickel, the inhibitory effects of cadmium, copper, and all organic pollutants were reversible, with P. lunula recovering 80%–100% of its bioluminescence potential after a period of 72 h in uncontaminated medium. Our results show that the restoration of bioluminescence in P. lunula is sensitive to the reference contaminants tested and obtains highly reproducible results.     

EFFECTS OF LIGHT INTENSITY ON DIVISION RATE,STIMULABLE BIOLUMINESCENCE AND CELL SIZE OF THE OCEANIC DINOFLAGELLATES DISSODINIUM LUNULA,PYROCYSTIS FUSIFORMIS AND P. NOCTILUCA12

Preadapted cultures were grown in a 12:12 LD cycle at a series of light intensities under cool-white, fluorescent lamps. Pyrocystis fusiformis Murray maintained high division rates at low light intensities at the expense of cell size. In contrast, Dissodinium lunula (Schuett) Taylor had relatively lower division rates at low light intensities with little concomitant decrease in size. The response of P. noctiluca Murray was intermediate between these two species. For all three, cell numbers did not increase above an intensity of 5–10 μEin·m^?2·sec^?1 and division rate was saturated at ca. 30, 60, and 60μEin·m^?2·sec^?1 for P. fusiformis, P. noctiluca, and D. lunula, respectively. The capacity for stimulable bioluminescence was saturated at light intensities of 0.15 μEin·m^?2·day in short-term (2-day) experiments. In cultures of P. fusiformis and P. noctiluca, maintained for at least one month at lower intensities than needed to saturate division rate, a decrease in the capacity for stimulable bioluminescence was accompanied by a reduction in cell size. Our results suggest that cell size and bioluminescent capacity may prove to be a potentially useful indication of the history of exposure of natural populations of Pyrocystis spp. to ambient intensities.     

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.     

SIMILARITIES IN THE ASEXUAL REPRODUCTION OF THE OCEANIC DINOFLAGELLATES, PYROCYSTIS FUSIFORMIS, PYROCYSTIS LUNULA, AND PYROCYSTIS NOCTILUCA1

Three cultured species of Pyrocystis (Dinoccoccales) reproduced asexually by forming 2 (or 1) aplanospores or zoospores inside the parent cell wall. In all 3 species these small reproductive cells, although they may not resemble the parent cells, swell up rapidly (～ 10 min) to the approximate size and shape of the parent cell. These swollen cells become new vegetative cells. The above asexual process is the only way by which cells numbers increase in our cultures. Pyrocystis lunula was propagated at the lunula stage of the life cycle. The nonmotile crescent-shaped cells produced reproductive cells that were Gymnodinium-shaped and had, in some cases, a trailing flagellum. With P. fusiformis and P. noctiluca, the reproductive cells were not flagellated. With P. fusiformis, these bodies had a pronounced equatorial constriction like a girdle, while in P. noctiluca the “girdle” was an inconspicuous feature if present. With P. noctiluca and P. fusiformis on a 12:12 ld cycle, reproductive cells were formed early in the dark period and they swelled up at the beginning of the light period. Reproduction of P. lunula was not well phased in our experiments, with reproductive cells developing at the end of the light period and the end of the dark period.     

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.     

LIGHT AND SCANNING ELECTRON MICROSCOPIC INVESTIGATIONS OF SEXUAL REPRODUCTION IN HYDRA CARNEA

In the freshwater hydrozoan Hydra carnea, the egg, matured and grown between the mesoglea and the epitheliomuscular cells of the ectoderm, is extruded after the emission of the second polar body. Surrounded by a clear jelly layer the egg remains attached to the polyp. Sperm which are released from the testicular sacs of male polyps are attracted by the egg. The jelly layer is penetrated only at the site of emission of the polar bodies by sperm which lack a structurally distinct acrosome. One sperm fuses with the egg at that site where the female pronucleus is found to lie close to the egg membrane. After fertilization and cleavage an acellular embryotheca is secreted by the blastomeres. These events were investigated by light and scanning electron microscopy.     

OBSERVATIONS ON THE MORPHOLOGY AND SEXUAL REPRODUCTION OF COOLIA MONOTIS (DINOPHYCEAE)1

The surface morphology of the dinoflagellate Coolia monotis Meunier was compared with the surface morphology of Ostreopsis, The apical pore of C. monotis is similar in architecture to that of Ostreopsis but considerably longer (12 μm) than in O. heptagona (8–9 μm) and O. ovata (6–7 μm). A ventral pore in C. monotis is located on the right ventral margin between apical plate l′ and precingular plate 6″ and is similar in appearance and location to the ventral pore of O. ovata. The longitudinal flagellum (20 μm) in C. monotis is longer than in O. ovata (12 μ). Although Coolia and Ostreopsis appear to be distinctly different and should remain as two separate genera, they appear to be related. Cells of C. monotis divided by binary fission. Doubling time was 3–4 days in the logarithmic phase of growth at 23°C, 12:12 h L:D, 30–90 μE-m^?2·s^?1, and a salinity of 36%. Cultures reached cell densities of 2.5 × 10³ cells·L^?1 after 15 days of growth. The sexual process in C. monotis occurred in Erdschreiber's medium when Danish soil extract was substituted with mangrove sediment extract under the culture conditions described above. Gamete fusion produced large biflagellated planozygotes (70–75 μm diam). Planozygote maturation involved cytoplasmic reorganization, loss of motility, development of a spherical shape (80–90 μm diam), and two to three orange accumulation bodies. The cells at this stage appeared to be thin-walled cysts. Further development included reorganization of cyst contents, emergence of non-motile gametes, and development of chloroplasts, sulcus, and girdle. The nucleus of the newly formed cells occupied 50% or more of the total cell volume. Meiosis occurred in the cyst, but nuclear cyclosis was not observed. Four daughter cells were produced within 36–48 h, and motile gametes developed. The gametes exhibited sexuality for 2 months and completed the sexual life cycle by going through a thin-walled cyst stage.     

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.     

SEXUAL REPRODUCTION IN THE TOXIC DINOFLAGELLATE GONYAULAX MONILATA1

The sexual cycle of Gonyaulax monilata Howell was observed in stationary cultures and in nitrogen-deficient medium. The armored, isogamous gametes fuse in a characteristic manner with cingula at oblique angles. Nuclear fusion lags slightly behind cytoplasmic fusion. The zygote enlarges for several days. The dark, double-flagellated planozygote encysts within 1–3 wk. Early hypnozygotes are round to ovoid and contain lipid and one or two large golden-yellow globules. As the hypnozygote matures, the globules become smaller and the cytoplasm darkens and pulls from the wall. All cysts examined contained only one nucleus. A very dark, uninucleate post-hypnozygotic cell escapes through an archeopyle and within 24 h divides into daughter cells which divide in 24–48 h forming a small chain. The production of thick walled zygates in culture implies that such resting stages in marine sediments could serve as a source stock for blooms. This species causes toxic red tides and the existence of benthic “seed beds” consisting of hypnozygotes is now plausible.     

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.     

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.     

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.     

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.     

ABIOTIC REGULATION OF INVESTMENT IN SEXUAL VERSUS VEGETATIVE REPRODUCTION IN THE CLONAL KELP LAMINARIA SINCLAIRII (LAMINARIALES,PHAEOPHYCEAE)1

Clonal kelp taxa may reproduce both sexually and vegetatively resulting in a potential trade‐off in the allocation of acquired carbon and nitrogen resources. Such trade‐offs may dictate a different response of clonal kelps to varying environmental conditions relative to aclonal kelp taxa. Laboratory temperature and nutrient manipulation experiments demonstrated that investment in sexual and vegetative reproduction in Laminaria sinclairii (Harv. ex Hook. f. et Harv.) Farl., C. L. Anderson et D. C. Eaton was regulated by different abiotic factors. Sorus production (investment in sexual reproduction) and blade growth were significantly higher at 12°C compared to 17°C, regardless of nutrient concentration. Net carbon storage and depletion in rhizomes were observed in the low‐ and high‐temperature treatments, respectively, suggesting that carbon stores were not responsible for increased growth. Rhizome elongation (investment in vegetative reproduction), on the other hand, was significantly higher in 12 μM NO₃^? than in 2 μM NO₃^?, irrespective of temperature. This increase in rhizome growth was concurrent with elevated rhizome percent tissue nitrogen levels also observed in treatments with higher nutrients, again indicating a growth response to treatment independent of previous nutrient stores. These results suggest that regulation of growth and investment in sexual reproduction in L. sinclairii is similar to that in aclonal kelps (i.e., warmer temperatures result in decreased reproductive output). Additionally, depletion of carbon and nitrogen from rhizomes in suboptimal conditions confirms the role of clonal kelp rhizomes in carbon and nutrient storage.     

ASEXUAL AND SEXUAL REPRODUCTION IN GONIUM QUADRATUM (CHLOROPHYTA) WITH A DISCUSSION OF PHYLOGENETIC RELATIONSHIP WITHIN THE GONIACEAE1

Morphological details of asexual and sexual reproduction in Gonium Quadratum Pringsheim ex Nozaki (Goniaceae, Chlorophyta) were observed by light microscopy, based on clonal cultured materials originating from Nepal. In asexual reproduction, the alga exhibited two different patterns of cell cleavage during formation of 8-and 16-celled daughter colonies. Sexual reproduction was heterothallic and isogamous. The gametes bore a tubular mating structure (bilateral mating papilla) at the base of the flagella, and the papillae of the two gametes. The germinating zygote gave rise to four biflagellate gone cells joined in a colony (germ colony). Possible phylogenetic relationships within the Goniaceae at the species level are outlined, mainly on the basis of reproduction characteristics.     

TIME LAPSE ANALYSES OF SEXUAL REPRODUCTION IN CLOSTERIUM EHRENBERGII (CONJUGATOPHYCEAE)1

The process of sexual differentiation was studied using heterothallic clones of Closterium ehrenbergii Meneghini. The first visible sign of sexual reproduction was agglutination of two or more cells in a group and this was followed by gametangiogenic division and conjugation of gametangial cells. Movements of gametangial cells were carefully studied. Gametangial cells occasionally participated again in gametangiogenesis instead of proceeding directly to the formation of conjugation papilla. The whole process of sexual differentiation from vegetative cell to zygospore was considered to be basically similar in both of the two closely related mating groups, A and B, of C. ehrenbergii. Nevertheless, there were some differences between the two groups in patterns of the sexual differentiation. In Group A, vegetative cell division was completely suppressed by mixing the two complementary mating type clones together into the same medium with high light illumination. This suppression was not caused by the nitrogen depletion in the medium, but by the presence of cells of opposite mating type. In Group B, vegetative cell division and sexual reproduction occurred side by side repeatedly for several days.     

SEXUAL REPRODUCTION,MATING SYSTEM,AND PROTOPLAST DYNAMICS OF SEMINAVIS (BACILLARIOPHYCEAE)1

Cell division, the mating system, and auxosporulation were studied in the marine epipelic diatom Seminavis cf. robusta Danielidis & D. G. Mann. The interphase protoplast contains two girdle‐appressed chloroplasts, each with an elongate bar‐like pyrenoid, and also a central nucleus, located in a bridge between two vacuoles. Before cell division, the chloroplasts divide transversely and translocate onto the valves. The nucleus relocates to the ventral side for mitosis. After cytokinesis and valve formation, the chloroplasts move back to the girdle, showing a constant clockwise movement relative to the epitheca of the daughter cell. Seminavis cf. robusta is dioecious, and sexual reproduction is possible once cells are less than 50 μm. In crosses of compatible clones, gametangia pair laterally, without the formation of a copulation envelope, and produce two gametes apiece. The intensity of sexualization increases as cells reduce further in size below the 50‐μm threshold. At plasmogamy, the gametangia dehisce fully and the gametes, which were morphologically and behaviorally isogamous, fuse in the space between the gametangial thecae. The auxospore forms a transverse and longitudinal perizonium. After expansion is complete, there is an unequal contraction of the protoplast within the perizonium, creating the asymmetrical shape of the vegetative cell. Apart from this last feature, almost all characteristics exhibited by the live cell and auxospores of Seminavis agree with what is found in Navicula sensu stricto, supporting the classification of both in the Naviculaceae. Haploid parthenogenesis and polyploid auxospores were found, lending support to the view that change in ploidy may be a significant mechanism in diatom evolution.     

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.     

MASS SEXUAL REPRODUCTION IN THE TOXIGENIC DIATOMS PSEUDO‐NITZSCHIA AUSTRALIS AND P. PUNGENS (BACILLARIOPHYCEAE) ON THE WASHINGTON COAST,USA1

Sexual reproduction is documented for the first time in field populations of the pennate diatoms Pseudo‐nitzschia australis Freng. and P. pungens (Grunow ex Cleve) Hasle (var. cingulata Villac and hybrids between var. cingulata and var. pungens). A bloom dominated by these species began on June 26, 2006, along Kalaloch Beach, Washington, USA, coincident with a drop in the Si(OH)₄:NO₃ ratio to below two. Multimodal size distributions were detected for both species, and synchronous auxosporulation occurred within the smallest size class during a 3‐week window. Auxospores and initial cells created a new class of large cells, and cells in the intermediate size classes increased in abundance during auxosporulation. Mating cells of both species were attached to colonies of surf‐zone diatoms. Paired gametangia, gametes, zygotes, auxospores, and large initial cells were found. Auxosporulation began first for P. pungens (June 30), apparently once a critical, high cell concentration was reached, followed by P. australis (July 5), when the total Pseudo‐nitzschia cell concentration reached 929,000 cells · L^?1. Low frequencies of auxosporulation occurred throughout the bloom but increased 4‐fold for P. australis and 3‐fold for P. pungens when macronutrients were reduced to low levels on July 11. A 2‐year life cycle was estimated for P. australis and 3 years for P. pungens, both with annual auxosporulation. Domoic acid (DA) in razor clams reached a maximum of 38 μg DA · g^?1 on July 18. A significant relationship existed between the percent of cells within the new size range and DA concentrations in razor clams on the same beach.