HYPERMETHYLATION AT CPG-MOTIFS IN THE DINOFLAGELLATES AMPHIDINIUM CARTERAE (DINOPHYCEAE) AND SYMBIODINIUM MICROADRIATICUM (DINOPHYCEAE): EVIDENCE FROM RESTRICTION ANALYSES, 5-AZACYTIDINE AND ETHIONINE TREATMENT

Because of their peculiar chromatin features and phylogenetic position, dinoflagellates are potentially uniquely informative with respect to possible roles and evolution of DNA methylation systems. Here we report that DNA from Amphidinium carterae Hulburt and Symbiodinium microadriaticum Freudenthal was not significantly digested by a range of CpG methylation-sensitive endonucleases. Corresponding methylation-insensitive isoschizomers cleaved all DNA preparations. Treatment with the methylation inhibitors 5-azacytidine and ethionine resulted in major increases in digestibility of dinoflagellate DNA by CpG methylation-sensitive enzymes. The 5-azacytidine effect was not confined to heterochromatin. Our studies indicate that at least in some dinoflagellates, a high proportion of CpG motifs and a significant number of CpNpG motifs are normally methylated, implying methyltransferase substrate specificities similar to those of higher plants.     

NITRATE REDUCTASE ACTIVITY OF AMPHIDINIUM CARTERI AND CACHONINA NIEI (DINOPHYCEAE) IN BATCH CULTURE: DIEL PERIODICITY AND EFFECTS OF LIGHT INTENSITY AND AMMONIA1,2

The activity of extracted NADH-NO₃^? reductase was measured in the marine dinoflagellates Amphidinium carteri Hulburt and Cachonina niei Loeblich. Its activity showed a diel periodicity and was ca. twice as great at midday as at midnight. The enzyme activity was unstable, with an in vitro half-life of 2–3 h. Values of enzyme activity were low or undetectable during lag phase but paralleled the instantaneous growth rate value during log phase. Nitrate reductase activity was not found in the stationary phase of growth, but additions of NO₃^? resulted in enzyme activity after 24h. When A. carteri was exposed to a series of light intensities for several weeks, the division rate and enzyme activity increased with increasing light intensity up to saturating intensities. In 6 h exposures, enzyme activity decreased with decreasing light intensities below light intensities saturating division rate. Additions of NH₄⁺ (0.5–50 μm) to A. carteri cultures decreased the amount of extractable enzyme. The in vitro activity was not inhibited by similar NH⁺₄ concentrations.     

AMPHIDINIUM CRYOPHILUM SP. NOV. (DINOPHYCEAE) A NEW FRESHWATER DINOFLAGELLATE. II. ULTRASTRUCTURE1

The dinoflagellate Amphidinium cryophilum sp. nov. is one of the few gymnodinians to be studied at the ultrastructural level. It resembles other dinoflagellates in the structure of the nucleus, trichocysts, storage materials, flagella, mitochondria, and microbodies. Other features of A. cryophilum less commonly observed in related organisms include a network of small interconnected vesicles, a system of large, peripheral vacuoles, chloroplasts bound by two rather than three membranes, an accumulation body, thylakoid-associated plastoglobuli, a vesiculated nuclear envelope, a complex tubular pusule, striated flagellar collars, collared pits, and a peduncle. The occurrence of a peduncle, a structure implicated in phagotrophy, in this autotrophic organism is noteworthy. The ultrastructure of the peduncle of A. cryophilum differs significantly from that reported in another dinoflagellate.     

RECYCLING OF METABOLIZED IRON BY THE MARINE DINOFLAGELLATE AMPHIDINIUM CARTERAE1

Senescent, iron-limited cultures of Amphidinium carterae Hulburt were used to detect the presence of iron made available for phytoplankton growth by recycling of metabolized iron from disrupted cells. Both physical disruption of cells and physical disruption plus exposure to a low pH were tested. The test cultures remained viable over long periods of time, and were stimulated to full growth by the addition of Fe, but not by the addition of any other nutrients. Simple physical disruption of cells caused only a very slow release of available Fe, while disruption of cells plus exposure to a low pH resulted in a rapid release of available Fe. It is suggested that the digestive processes of herbivores are instrumental in the rapid regeneration of Fe as a nutrient available for phytoplankton growth.     

THE MICROTUBULAR CYTOSKELETON OF AMPHIDINIUM RHYNCHOCEPHALUM (DINOPHYCEAE)1

The sub-thecal microtubular cytoskeleton of Amphidinium rhynchocephalum Anissimowa was investigated using indirect immunofluorescence microscopy and transmission electron microscopy. The majority of sub-thecal microtubules are longitudinally oriented and radiate from one of two sub-thecal transverse microtubular bands that lie adjacent to the anterior and posterior edge of the cingulum.Both transverse bands consist of 3–5 microtubules and are loop shaped with one end adjacent to the cell's right edge of the sulcus and the other end adjacent to the fibrous ventral ridge. The posterior transverse microtubular band (PTB) defines the posterior edge of the cingulum and gives rise to numerous posteriorly directed longitudinal microtubular bundles that consist of 1–3 microtubules per bundle. These bundles end at the posterior end of the cell. The PTB also gives rise to the cingular longitudinal microtubules that underlie the cingular groove and terminate at the anterior transverse microtubular band (ATB). The ATB defines the anterior edge of the cingulum and loops around the base of the epicone. This band gives rise to anteriorly directed longitudinal microtubular bundles that terminate in the small epicone of the cell. The longitudinal microtubular root of the flagellar apparatus is directed posteriorly and lies immediately beneath the theca but is distinct from the subthecal microtubule system. A narrow fibrous ridge is ventrally located to the cell's left between the exit apertures of the transverse and longitudinal flagella. In this position, the ventral ridge lies between and also connects with the anterior and posterior transverse microtubular bands. The ventral ridge is also associated with three microtubules that are distinct from other cytoskeletal microtubules. Our results demonstrate that the majority of sub-thecal microtubules originate from one of two microtubular bands associated with the cingulum. The possible role of the fibrous ventral ridge and its associated microtubules is also discussed.     

PHAGOTROPHY IN THE FRESHWATER,PHOTOSYNTHETIC DINOFLAGELLATE AMPHIDINIUM CRYOPHILUM1

The cold-water, photosynthetic dinoflagellate Amphidinium cryophilum Wedemayer, Wilcox & Graham feeds phagotrophically on other dinoflagellate species. Food is ingested through a feeding tube, termed here the “phagopod,” which extends from the antapex. The peduncle of this organism plays no observable role in the feeding process. The phagopod is essentially a hollow cylinder composed electron-opaque material that is possibly deposited on a membrane. No Amphidinium cytoplasmic components, including microtubules or other cytoskeletal elements, were observed in the phagopod. Prefeeding cells aggregate, in small clumps near prey organisms with their phagopods extended. Eventually some cells commence feeding, first inserting the phagopod through the prey cell-covering and then slowly, over a period of 10 min or more, drawing cytoplasm through the phagopod and into a nascent food vacuole. Both light and electron microscopy suggest that one or more prey cell amphiesmal membranes remain intact during the feeding process. Upon completion of feeding, the Amphidinium cell swims off with a prominent food vacuole in the hypocone, leaving at least part of the phagopod attached to the prey cell. Phagotrophy in A. cryophilum seems to vary with light intensity. At low light intensities, cells feed phagotrophically and are nearly colorless, whereas at high light levels they feed much less frequently, if at all, and are brightly pigmented.     

AMPHIDINIUM REVISITED. II. RESOLVING SPECIES BOUNDARIES IN THE AMPHIDINIUM OPERCULATUM SPECIES COMPLEX (DINOPHYCEAE), INCLUDING THE DESCRIPTIONS OF AMPHIDINIUM TRULLA SP. NOV. AND AMPHIDINIUM GIBBOSUM. COMB. NOV.1

Amphidinium operculatum Claparède et Lachmann, the type species of the dinoflagellate genus Amphidinium, has long had an uncertain identity. It has been considered to be either difficult to distinguish from other similar species or a morphologically variable species itself. This has led to the hypothesis that A. operculatum represents a “species complex.” Recently, the problem of distinguishing A. operculatum from similar species has become particularly acute, because several morphologically similar species have been found to produce bioactive compounds of potential interest to the pharmaceutical industry. In this study, we cultured and examined existing cultures of several species of Amphidinium, most of which have been previously identified as A. operculatum or as species considered by some to be synonyms or varieties of A. operculatum. Thirty strains were examined using comparative LM, SEM, and partial large subunit (LSU) rDNA sequence data. Through morphological and molecular phylogenetic analyses, six distinct species were identified, including Amphidinium trulla sp. nov. and Amphidinium gibbosum comb. nov. Amphidinium operculatum was redescribed based on four cultures. Genetic variability within the examined Amphidinium species varied greatly. There was little difference among strains in partial LSU rDNA for most species, but strains of A. carterae and A. massartii Biencheler differed by as much as 4%. In both A. carterae and A. massartii, three distinct genotypes based on partial LSU rDNA were found, but no morphological differences among strains could be observed using LM or SEM. In the case of A. carterae, no biogeographically related molecular differences were found.     

EFFECT OF LIGHT HISTORY ON THE ULTRASTRUCTURE AND PHYSIOLOGY OF PROROCENTRUM MARIAE-LEBOURIAE (DINOPHYCEAE)1

Ultrastructural and physiological responses of Prorocentrum mariae-lebouriae (Parke & Ballantine) Faust are reported for cultures maintained at growth irradiances (I_g) ranging from 20.6 to 0.3 E m^?2.d^?1 and following downward shifts in light intensity. We tested the hypothesis that Prorocentrum grown under light regimes that elicit different responses in photosynthesis and pigmentation exhibit distinctive cell ultrastructures. Prorocentrum from high-light conditions had high saturation intensities for photosynthesis (I_k) and low levels of Chl a, Chl c and peridinin-cell^?1 These cultures were morphologically distinguished by a large starch volume fraction (V_v), small chloroplast V_v and fewer thylakoids lamella^?1. I_k values were lower and pigment concentrations higher in low-light treatments, and cells showed reduced starch V_v, large chloroplast V_v, and higher numbers of thylakoids · lamella ^?1. Cells grown under extremely low-light conditions appeared stressed as indicated by the absence of starch reserves and the presence of large vacuoles within the cytoplasm. Results for presence of large vacuoles within the cytoplasm. Results for quantiative electron microscopy, photosynthesis-irradiance (P-I) relations and cell pigmentation indicate that photoadaptation in P. mariae-lebouriae involves a strategy that encompasses changes in both the “size” and “number” of photosynthetic units.     

AMPHIDINIUM CRYOPHILUM SP. NOV. (DINOPHYCEAE) A NEW FRESHWATER DINOFLAGELLATE. I. SPECIES DESCRIPTION USING LIGHT AND SCANNING ELECTRON MICROSCOPY1

Amphidinium cryophilum sp. nov. was found in the fall of 1979 in a small pond near Madison, Wisconsin. During the ensuing winter, it became the dominant phytoplankter. Cell numbers remained high despite a thick layer of ice and snow. After the ice melted in the spring the organism disappeared from plankton samples. A successful culture of A. cryophilum was established only when isolates were incubated at 5–7° C. It is compared with two morphologically similar species, A. amphidinioides (Geitler) Schiller and Gymnodinium inversum Nygaard. Amphidinium cryophilum is distinguished from the former by its pigmentation (golden-yellow vs. blue-green), the location of the cingulum, and its lack of an eyespot. It differs from the latter in cell shape, the route of the sulcus and position of the nucleus.     

EFFECT OF NITRATE CONCENTRATION ON THE RELATIONSHIP BETWEEN PHOTOSYNTHETIC OXYGEN EVOLUTION AND ELECTRON TRANSPORT RATE IN ULVA RIGIDA (CHLOROPHYTA)1

The electron transport rate (ETR) versus gross photosynthesis (GPS) relationship varies as a function of species, temperature, irradiance, and inorganic carbon levels, but less is known about the effect of nitrogen supply on this relationship. The objective of this study was to evaluate the effect of nitrate concentration on the ETR versus GPS relationship in Ulva rigida C. Agardh from the Mediterranean Sea. Chlorophyll content and tissue absorptance increased 2‐fold as nitrate in the media increased from 0 to 50 μM. Whereas internal N content increases 3‐fold at 50 μM, internal C increased slightly. Oxygen evolution and ETR, evaluated as in vivo chl fluorescence using pulse amplitude modulated fluorometry, in general saturated at irradiances above 100 μmol photons·m^?2·s^?1. Both maximum ETR and GPS values increased as nitrate concentration increased. In general, the ETR versus GPS relationship showed a linear response to increasing nitrate with little variance of the data. This relationship, however, became more variable at high irradiances and high nitrate concentrations. The ETR/GPS ratio was close to the theoretical value of 4 at low nitrate concentrations, and the ratio decreased exponentially when nitrate concentration in the media increased. The variations of ETR/GPS under different inorganic nitrogen supply are discussed in terms of the effect of nitrate on the photosynthesis and respiration relationship.     

COMPARATIVE ANALYSES OF THE DINOFLAGELLATE FLAGELLAR APPARATUS. III. FREEZE SUBSTITUTION OF AMPHIDINIUM RHYNCHOCEPHALUM1

The flagellar apparatus of the marine dinoflagellate Amphidinium rhynchocephalum Anissimowa was examined using the techniques of rapid freezing/freeze substitution and serial thin section three dimensional reconstruction. The flagellar apparatus is composed of two basal bodies that are offset from one another and lie at an angle of approximately 150° The transverse basal body is associated with two individual microtubules that extend from the proximal end of the basal body toward the flagellar opening. One of these microtubules is closely appressed to a striated fibrous root that also extends from the proximal base of the transverse basal body. The longitudinal basal body is associated with a nine member microtubular root that extends from the proximal end of the basal body toward the posterior of the cell. The longitudinal microtubular root and the transverse striated fiber are connected by a striated connective fiber. In addition to the microtubules associated with the transverse and longitudinal basal bodies, a group of microtubules originates adjacent to one of the transverse flagellar roots and extends into the cytoplasm. Vesicular channels extend from the flagellar openings to the region of the basal bodies where they expand to encompass the various connective structures of the flagellar apparatus. The possible function and evolutionary importance of these structures is discussed.     

光下无CO_2气体处理对叶片光合强度的影响

小麦等C_3植物的叶片在光下经无CO_2或低CO_2气体处理后,通入高CO_2气体,光合强度出现“升、降、升”的波动,而玉米等C_4植物无此现象。不同植物的光合波动幅度不同。强光、高CO_2、低O_2等能缩短第一次光合上升时间,增大光合下降幅度;而低CO_2、高O_2等则减少光合下降幅度。此现象与RuBP及ATP的含量变化有关。     

COLONIZATION OF THE EPINEUSTON BY CYSTODINIUM BATAVIENSE(DINOPHYCEAE): BEHAVIOR OF THE ZOOSPORE1

Cystodinium bataviense Klebs is the first dinoflagellate observed to exhibit specialized zoospore behavior which results in colonization of the epineuston. The zoospore: (1) is strongly phototactic; (2) changes shape rapidly upon release; (3) possesses at least two types of swimming behavior, including a “stop mechanism”; and, (4) sheds its theca as the new cell wall asymmetrically elongates into the immobile vegetative stage. These features working in concert facilitate the entrance of Cystodinium into the epineuston. Detailed observations of zoospore morphology and analysis of its behavior are used as new characters to further delimit C. bataviense. Since vegetative morphology has proven unsatisfactory in circumscribing other Cystodinium species, the study of variation in zoospore characters will help to clarify taxonomic units within the genus.     

EFFECT OF MALTOSE RELEASE ON UPTAKE AND ASSIMILATION OF AMMONIUM BY SYMBIOTIC CHLORELLA (CHLOROPHYTA)1

When incubated at pH 4–5, Chlorella freshly isolated from symbiosis with Hydra viridissima PALLAS 1766 (green hydra) release large amounts of photosynthetically fixed carbon in the form of maltose, and assimilation of inorganic N is inhibited. Physiological responses to N starvation of the cultured 3N813A strain of maltose-releasing Chlorella differed from those caused by 48 h of maltose release induced by low pH. N starvation increased rates of ammonium assimilation at pH 7.0 in light or darkness, and ammonium assimilation in darkness stimulated cell respiration. In contrast, cells pretreated at pH 5.0 to induce maltose release were unable to take up ammonium at pH 7.0 unless supplied with an external carbon source such as bicarbonate, acetate, or succinate, and rates of uptake were similar to control cells. Freshly isolated symbionts displayed a similar dependency. Rates of ammonium uptake by cells pretreated at pH 5.0 were reduced in darkness and did not stimulate cell respiration. N-starved cells supplied with ammonium also showed a large short-term increase in glutamine pools at the expense of glutamate, as might be expected if large amounts of ammonium were rapidly assimilated via glutamine synthetase/glutamate synthase, whereas after long-term maltose release cells showed only a small increase in glutamine when supplied with ammonium. Furthermore, maltose release caused a fall in pool sizes of a number of amino acids, including glutamine and glutamate, and also caused a decrease in pool sizes of 2-oxoglutarate and phospho-enol-pyruvate, which are required for ammonium assimilation into amino acids. Cells stimulated to synthesize and release maltose may be unable to assimilate ammonium and synthesize amino acids because of diversion of fixed carbon from N metabolism. We estimate that 40–50% affixed C is required for maximal maltose synthesis, whereas up to 30% fixed C is required for ammonium assimilation. These results are discussed in the context of host regulation of symbiotic algal growth.     

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.     

BIOCHEMICAL COMPOSITION AND METABOLIC ACTIVITY OF SCRIPPSIELLA TROCHOIDEA (DINOPHYCEAE) RESTING CYSTS1

The composition and metabolic activity of cysts of the marine dinoflagellate Scrippsiella trochoidea (Stein) Loeblich were examined during dormancy, quiescence, and germination. On a per cell basis, newly formed cysts contained an order of magnitude more carbohydrate but significantly less protein and chlorophyll a than did exponentially growing vegetative cells. Loss of lipid and carbohydrate from cysts during the initial dormancy period reflected a respiration rate estimated to be 10% of the respiratory activity in vegetative cells. Among older, quiescent cysts the calculated respiration rate decreased further to approximately 1.5% of the vegetative rate and appeared to proceed largely at the expense of carbohydrate reserves. These estimated rates of respiration were in good agreement with direct measurements of cyst oxygen consumption. The transfer of quiescent cysts to conditions permissive for germination resulted in a rapid increase in respiration rate, as evidenced by carbohydrate loss and O₂ consumption. The increased respiratory activity was followed by an increase in protein content and, later, by an increase in chlorophyll a content and photosynthetic capacity. Just prior to germination the P/R ratio became greater than 1, and the estimated chlorophyll-specific photosynthetic activity reached 75% of the rate in vegetative cells. Complete restoration of photosynthetic and respiratory capacity apparently was not achieved until after excystment. These data confirm the common assumption that dinoflagellate cysts represent true “resting” cells, containing extensive energy reserves and displaying greatly reduced metabolic activity.     

5℃夜间低温对红树幼苗光合速率和蒸腾速率的影响

5℃夜间低温处理温室栽培红海榄（Rhizophora stylosa和银叶树（Heritiera littoralis)幼苗,白天20℃室温分别恢复1h和4h,测定功能叶的净光合速率、气孔导度、胞间CO2浓度、蒸腾速率和叶绿素含量。结果表明：夜间低温明显降低红海榄和银叶树的净光合速率、气孔导度、蒸腾速率和水分利用率,促进胞间CO2浓度增加,而叶绿素含量变化不大。白天空温恢复时间增长,净光合速率、气孔导率和蒸腾速率回升,胞间CO2浓度下降,红树幼苗对低温有一定的适用能力。低温处理2d,红海榄叶净光合速率的抑制程度大于1d处理,而银叶树叶净光合速率的抑制程度则有所减轻。两种红树叶气孔导度与净光合速率和蒸腾速率均呈线性关系,气孔导度是控制叶片光合成和蒸腾水分丢失动态平衡的主要因素。