A METHOD OF OBTAINING AXENIC CULTURES OF TRENTEPOHLIA SPP. (CHLOROPHYTA)1

Axenic cultures of Trentepohlia species are necessary for the study of growth and hysiological characters of the algae. We describe the use of a Sherman micromanipulator to isolate filaments from samples of T. aurea and T. odorata collected from their natural habitats. These filaments were then used as inocula for the establishment of axenic cultures. In the case of T. aurea, further treatment with lactic acid was necessary.     

BRIEF INCUBATION OF GAMETANGIA-BEARING CAPS IN ANTIBIOTICS ELIMINATES BRANCHING IN PROGENY OF ACETABULARIA ACETABULUM (CHLOROPHYTA)1

Branching of the stalk of Acetabularia acetabulum L. (Silva) was investigated by inbreeding and by a brief treatment of gametangia with a variety of antibiotics. The position of the branch along the stalk varied, implying that branching was not restricted to any one time in development (base is oldest and apex is youngest). The branching phenotype was not inherited in Mendelian fashion. Although three microscopic structures (“bubbles,”“pustules,” and “scars”) occurred on the stalks of cells that had branched, these structures were not statistically correlated with branching in the population (n=699 cells). However, brief treatment of gametangia with a new antibiotic mixture did eliminate all macro- and microscopic structures associated with branching of the stalk in the subsequent generation. We could not fulfill Koch's postulates or provide clear evidence for the pathogenic nature of cell branching. Our brief antibiotic treatment of gametangaa of Acetabularia acetabulum was rapid, had no adverse effects, and virtually eliminated branching (and any potential pathogens) from laboratory cultures in the subsequent generations. Our method allows biochemical and molecular analyses to proceed uncomplicated by the possible presence of other organisms and provides a clean baseline for the future selection of mutations that may induce heritable branching.     

VEGETATIVE GROWTH OF ACETABULARIA ACETABULUM (CHLOROPHYTA): STRUCTURAL EVIDENCE FOR JUVENILE AND ADULT PHASES IN DEVELOPMENT1

We characterized vegetative development in two inbred cell lines of Acetabularia acetabulum (L.) Silva. Cell growth occurred at the apex and by elongation of older interwhorls throughout vegetative development. Although cell length and hairs per whorl increased regularly during development, interwhorl length, hair persistence on the stalk, and complexity of each whorl (degree of branching of whorl hairs) showed sharp discontinuities during development in both cell lines. The first (earliest) discontinuity, formation of a short interwhorl, was the sixth interwhorl made in all cells. Even though cell line Aa1055 was twice the height ofAa4010 when mature, cells in both lines were 0.8–1.0 cm tall after formation of the short interwhorl. The second discontinuity, increases in hair persistence on the stalk and complexity of each whorl of hairs, began shortly before cap initiation. We propose the following nomenclature: 1) that slower growth before formation of the short interwhorl be called “juvenile”; 2) that more rapid growth after formation of the short interwhorl be called “adult”; and 3) that adult growth be separated into “early” and “late” phases by the discontinuities in whorl hair characteristics. The proposed developmental phases (juvenile, early adult, and late adult) are temporally sequential and spatially stacked.     

A METHOD FOR OBTAINING AXENIC ALGAL CULTURES USING THE ANTIBIOTIC CEFOTAXIME WITH EMPHASIS ON CLADOPHOROPSIS MEMBRANACEA (CHLOROPHYTA)1

Axenic cultures of the tropical green seaweed Cladophoropsis membranacea (C. Agardh) Boergesen were obtained by cutting 2-mm apical tips from fast-growing unialgal filaments and incubating them in 1/2PES containing 100 μg · mL^?1 cefotaxime. After 1 week, apical tips were cut from the newly grown plantlets, washed through a series of sterile drops of seawater, and incubated in sterile 1/2PES. Plantlets were screened for the presence of bacteria by incubating droplets of the culture medium on peptone agar plates and by examining DAPI-stained filaments. Ninety to one hundred percent of the plantlets obtained with this treatment were axenic. Cefotaxime was also effective against cyanobacteria but of only limited value in rhodophytes.     

A NEW,ARTIFICIAL SEA WATER THAT FACILITATES GROWTH OF LARGE NUMBERS OF CELLS OF ACETABULARIA ACETABULUM (CHLOROPHYTA) AND REDUCES THE LABOR INHERENT IN CELL CULTURE1

We designed a new, artificial seawater (Ace 25) in order to grow bulk cultures of Acetabularia acetabulum (L.) Silva with a minimum of labor. To this end, we modified the traditional recipe for cell growth (Müller's medium as modified by Schweiger et al.) We eliminated five of the inorganic chemicals and determined the optimum concentration for 16 of the remaining 18 inorganic chemicals from modified Müller's seawater. Ace 25 enables growth of A. acetabulum from the beginning of the juvenile phase through gametangial formation in 11 weeks at high cell densities without medium replenishment. This represents a 98% reduction in the seawater volume required to mature each cell, a 30–40% reduction of the duration of the life cycle, an estimated 80% reduction in labor, and a 50–95% reduction in the space required for culturing A. acetabulum as compared with traditional procedures. These improvements may facilitate studies that require large numbers of cells such as population studies, genetics, and biochemistry, contribute to understanding the nutritional requirements of marine algae, and extend the use of this cell to those who lack the space or manpower to grow the cells in the traditional manner.     

PHYSIOLOGICAL FACTORS THAT AID DIFFERENTIATION OF ZYGOTES AND EARLY JUVENILES OF ACETABULARIA ACETABULUM (CHLOROPHYTA)

To improve our understanding of early development and to enable standard genetic analyses in Acetabularia acetabulum (L.) Silva, we examined some physiological factors that impact differentiation of young diploid plants. Eighty percent of zygotes differentiated within 6–7 days when they were inoculated at high densities (≥5,000 zygotes·mL⁻¹), and zygote differentiation was independent of the photon flux density of cool white light. In contrast, 90% of juveniles differentiated within 25 days when they were inoculated at medium densities (<1,000 individuals·mL⁻¹) and required >250 μmol·m⁻²·s⁻¹ cool white light. Both zygotes and juveniles differentiated best at an alkaline pH (7.96), when the temperature was between 18.6° and 23.5° C, and when the external potassium concentration was 1–10 mM. Compared to prior publications, adjustment of these parameters reduced the time to differentiation by 25% (from 8 to 6 days) and more than tripled the percentage of zygotes that developed into healthy juveniles (from 23% to ∼80%).     

TIMING AND LIGHT REGULATION OF APICAL MORPHOGENESIS DURING REPRODUCTIVE DEVELOPMENT IN WILD-TYPE POPULATIONS OF ACETABULARIA ACETABULUM (CHLOROPHYCEAE)

In the giant unicellular green alga, Acetabularia acetabulum (L.) Silva, development is altered by light. For example, blue light induces the vegetative apex to produce whorls of hairs that encircle the stalk and, later, blue light may trigger reproductive onset. The two goals of this study were to determine when changes in apical shape occur during formation of the reproductive structure, or "cap," and to determine which of these differentiation events require light. The first visible indication of cap initiation was a rounded swelling of the apex, which we call a knob-shaped apex (time = 0 hours). Subsequent changes in shape were a hyaline, knob-shaped apex, reached by 50% of the population 3 h later, and the formation of a whorl of unilobed chambers at 16 h. These chambers became bilobed at 33 h and trilobed at 34 h. Successive sets of cap hairs grew from protuberances found on the surface of the uppermost lobes of the chambers (superior corona). After knob, the remainder of cap formation was largely independent of light. However, the initiation of each set of cap hairs required light. If a recently initiated cap was amputated, the individual recapitulated development, repeating a portion of vegetative morphogenesis (i.e. it made whorls of sterile hairs) before initiating a new cap. The developmental sequence between amputation and initiation of a new cap required light. A model for light-regulated changes in shape at the apex of Acetabularia acetabulum, which integrates whorl and cap formation and encompasses both vegetative and reproductive development of this organism, is presented.     

BACTERIA THAT INDUCE MORPHOGENESIS IN ULVA PERTUSA (CHLOROPHYTA) GROWN UNDER AXENIC CONDITIONS1

Marine foliaceous green macroalgae such as Ulva lose their typical morphology when cultured aseptically in defined synthetic media. However, after reinfection by certain marine bacteria (isolated from unialgal cultures of Ulva pertusa Kjellman), the organisms regain their typical foliaceous or tubular morphology. To investigate the morphogenesis (MG) induced in U. pertusa by bacteria, we isolated and identified bacteria with MG activity on U. pertusa and studied the distribution of such bacteria in seawater and on various marine macroalgae. We isolated 1555 bacterial strains from 18 species of marine macroalgae (six Chlorophyta, five Phaeophyta, and seven Rhodophyta), from seawater and from sediment collected at the beach at Omaezaki, Shizuoka Prefecture; Japan. Of these, 676 bacterial strains (43.5%) showed MG activity. They were classified into six bacterial groups, Flavobacterium, Vibrio, Pseudomonas, Deleya, Escherichia, and gram-positive cocci. These bacteria were ubiquitous among the samples and were not specific to U. pertusa. Several plant growth regulators had no MG activity. Filter-sterilized supernatants of culture media of MG-active bacteria strains did not induce MG. Cocultivation of Ulva with active bacterial strains is so far the only way to induce the MG effect, which suggests that for MG direct contact between Ulva and the bacterial strain is necessary.     

PARTICIPATION OF EXTRACELLULAR NUCLEOTIDES IN THE WOUND RESPONSE OF DASYCLADUS VERMICULARIS AND ACETABULARIA ACETABULUM (DASYCLADALES,CHLOROPHYTA)1

As assayed by fluorescent reporter dyes, nitric oxide (NO) and H₂O₂, two downstream signaling agents induced by wounding in the alga Dasycladus vermicularis (Scop.) Krasser, can also be induced in unwounded Dasycladus cells by μM Adenosine 5′[γ‐thio]triphosphate (ATPγS) and Adenosine 5′‐[β‐thio]diphosphate (ADPβS), but not by Adenosine 5′‐O‐thiomonophosphate (AMPS). These nucleotide‐induced responses are blocked by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), an antagonist of animal purinoceptors, and by adenosine, a feed‐back inhibitor of extracellular nucleotide responses in animals. Similar nucleotide‐ and nucleotide‐antagonist responses were observed in Acetabularia acetabulum (L.) P. C. Silva. Significant levels of ATP released from Dasycladus cells were measured at wound sites by a sensitive luciferin‐luciferase assay. Additionally, the normal wound‐induced production of NO and H₂O₂ in Dasycladus can be blocked by pretreating the cells with PPADS. Our results indicate that nucleotides released from wounds can serve as a signal to trigger wound responses in algae, and that coordinated signaling between extracellular nucleotides and the NO pathway may have been established early during the evolution of plants.     

PRODUCTION OF AXENIC CULTURES OF MICROMONAS PUSILLA (PRASINOPHYCEAE) USING ANTIBIOTIC 1

Bacteria-free cultures of the prasinophyte Micromonas pusilla (Butcher) Monton and Parke, UTEX LB 991, were produced by intially determining the effects of several antibiotics on the growth of this alga and then using a combination of these antibiotics to eliminate associated bacteria, Micrononas pusilla was resistant ot penicillin G, neomycin, gentamicin and streptomycin at bactericidal concentrations but sensitive to chloramphenicol and polymixin B. Passage of M. pusilla through the sequence of antibioties penicillin G → neomycin → gentamician → kanamycin resulted in an axenic culture of M, pusilla this method should be suitable for producing axenic culture of other strains of M. pusilla.     

EFFECTS OF LOW NITROGEN LEVELS AND VARIOUS NITROGEN SOURCES ON GROWTH AND WHORL DEVELOPMENT IN ACETABULARIA (CHLOROPHYTA)1

Nitrate, ammonia, urea, and glycine were compared as nitrogen sources for Acetabularia mediterranea. Cells grew normally in media containing nitrate or urea, while cells did not grow at all when the same amount of N was supplied as ammonium ion. The utilization of glycine remains questionable. Cells in medium without added N (NDM) increased in length and some formed reproductive caps. The whorls of vegetative cells showed considerable hypertrophy in NDM and in glycine. This hypertrophy was due to the elongation of only the first-(a₁) and second- (a₂) order articles. When cut, the basal portion of cells without added N regenerated new apices with whorls. The development of these whorls was inversely proportional to the NO₂ concentration. Analyses showed that the intracellular nitrogen pool in young cells and regenerating bases was very small, about 1/10 of that of fully grown cells. Therefore we suggest that trace amounts of N contaminants in the medium supported growth and development, the uptake of which was facilitated by the hypertrophied whorls, under N-limited conditions.     

THE LACK OF CHLOROPLAST DNA IN ACETABULARIA MEDITERRANEA (ACETABULUM) (CHLOROPHYCEAE): A REINVESTIGATION1

Three features of chloroplast DNA (cpDNA) in plastids isolated from Acetabularia mediterranea (acetabulum) were analyzed after staining the organelles with the fluorochrome 4′6-diamidino-2-phenyl indole (DAPI): (1) number of chloroplasts exhibiting DNA fluorescence, (2) number of nucleoids per plastid, and (3) nucleoid morphology. In vegetative Acetabularia cells only half of the total chloroplast population comprising several millions displayed the whitish-blue fluorescence of the DNA/DAPI complex. This percentage remained stable independent of whether cells were grown in supplemented natural sea water or enriched synthetic sea water. A single nucleoid, widely differing in size and morphology among the organelles, was characteristic of 76–81% of chloroplasts with DNA. Less than 20% contained two nucleoids, and in rare cases three or four nucleoids were present. The pattern of nucleoid numbers followed a Poisson distribution in one experiment, if calculated with the intrinsic mean of the observed data. In two other experiments, however, a significant difference existed between observed and expected values for a Poisson distribution according to the Chisquared test. After secondary enlargement of portions of the negatives, the nucleoids’substructure was disclosed and found to consist of brightly fluorescent spots interspersed by unstained regions The lack of cpDNA in Acetabularia cells appears to be brought about by (1) the polarized pattern of growth and translation confined to the apical region of the single cell and (2) the cpDNA arrangement in a single nucleoid acentrically located in the organelle. A scheme for the evolution of a chloroplast population having plastids without DNA is proposed. In theory the lack of cpDNA could arise in each plant, since chloroplasts never evolved a mitotic-like spindle to ensure the equal distribution of genetic material. The different nucleoid arrangement in most other plants, however, efficiently counteracts this ‘carelessness of nature’     

KINETICS OF EXTRACELLULAR RELEASE IN AXENIC ALGAE AND IN MIXED ALGAL-BACTERIAL CULTURES: SIGNIFICANCE IN ESTIMATION OF TOTAL (GROSS) PHYTOPLANKTON EXCRETION RATES1

In axenic Chlorella pyrenoidosa Chick cultures, extracellular release was linear with time, but plateau-type curves were obtained in cultures with added bacteria. Initial rates of excretion were identical in both, systems. Kinetics of extracellular release in axenic Anabaena flos-aquae (Lyng.) Bréb. cultures were more complex than in Chlorella but the initial excretion rates were identical in axenic and mixed algal-bacterial cultures. In lakewater, extracellular release kinetics resemble the pattern in mixed Chlorella-bacteria cultures. An explanation is an initial lag in bacterial utilization of algal extracellular products. As a result, both in situ and in the laboratory, consecutive short, experiments give higher excretion rates than single long incubations. It is suggested that the former are close to total or gross extracellular release rates whereas the latter give net values, detecting only substances not, removed by heterotrophs.     

18S rDNA AND EVOLUTION IN THE DASYCLADALES (CHLOROPHYTA): MODERN LIVING FOSSILS1

Phylogenetic relationships were inferred from parsimony and distance analyses of nuclear small-subunit ribosomal DNA sequences taken from 14 species representing 8 of the 11 extant genera in the Dasycladales. Of 1733 aligned positions, 412 (23.8%) were variable and 251 (61%) of those were phylogenetically informative within the Dasycladales. Secondary structure was analyzed and taken into account during all phases of data analysis. Robustness of the trees was assessed using bootstrap analysis and g₁ statistics of tree-length decay. Strongly supported branches were robust to all methods of analysis regardless of weighting schemes used. The secondary structure of the 18S within the Dasycladales agrees with that of other green algae with the exception of a shared deletion in stemloop E10-1 (ca. 13 nucleotides long), which provides additional support for the uniqueness of this monophyletic group. A molecular clock was calibrated from the dasyclad fossil record and suggests a radiation of the Acetabulariaceae at 120 ± 30 million years (Ma) ago and the Dasycladaceae 215 ± 40 Ma ago. The split of the two lineages from a shared ancestor is estimated at 265 ± 50 Ma ago. Within the Dasycladaceae, Neomeris and Cymopolia are sister taxa, as are Batophora and Chlorocladus. Bornetella groups with the Neomeris and Cymopolia clade in 78% of the bootstrap replicates. Relationships among the Acetabulariaceae show that Acetabularia and Polyphysa do not form monophyletic groups as presently circumscribed. No evidence indicates that Acicularia is the oldest genus. Halicoryne, Chalmasia, and Dasycladus were not included in the analysis. Molecular data provide afresh background perspective from which to discuss the evolution of one of the most ancient lineages of green plants.     

DISJUNCT DISTRIBUTION OF THE XANTHOPHYLL LOROXANTHIN IN THE GREEN ALGAE (CHLOROPHYTA)1

Thirty-five green algal species, representing 17 orders in 5 classes, were examined for the presence of the xanthophyll loroxanthin by reversed-phase high-performance liquid chromatography. Of these, 16 possessed loroxanthin, including at least one member of each class of green algae. The distribution of loroxanthin as established by this and previous studies is disjunct within the Chlorophyta and does not appear to have any taxonomic significance. Most of the green algae examined possessed one or more xanthophyll pigments that have not been found in land plants.     

POLYMORPHIC BEHAVIOR OF ULVA LACTUCA (CHLOROPHYTA) IN AXENIC CULTURE. I. OCCURRENCE OF ENTEROMORPHA-LIKE PLANTS IN HAPLOID CLONES1

An isolate of Ulva lactuca L. was brought into axenic culture in both defined synthetic medium and enriched seawater. Haploid clones were established and followed through several generations in both media. Plants possessing distromatic, partially distromatic or completely tubular blades in cross sections, as well as individuals that were completely distromatic in one area of the blade and tubular in another all developed from the same swarmer population. Variations in basal area morphology also occurred. Progeny from swarmers of any of these morphological types showed a similar mixture of morphological variation, indicating non-mutational variability in phenotypic expression of the blade and basal areas. A mechanism by which such variation may occur without mutation is discussed.     

A NOVEL TECHNIQUE FOR PREPARATION OF AXENIC CULTURES OF SYMBIODINIUM (PYRROPHYTA) THROUGH SELECTIVE DIGESTION BY AMOEBAE1

The marine amoeba Trichosphaerium Am-I-7 was used as a tool for preparing unialgal axenic cultures of nondigestible Symbiodinium and Porphyridium species. The resistance of these unicellular algae to the amoebal digestive enzymes, and the differential digestion of bacteria, protozoans, and other algae, resulted in cleansed cells of Symbiodinium and Porphyridium that remained in the amoebal food vacuoles. During multiple fission, the amoeba evacuated its food vacuoles and released the trapped and intact algae, which were then successfully cultured. This method of cleaning was especially useful with algal species that were sensitive to antibiotics or other germicidal agents.     

DIFFERENTIATION OF ULVA MUTABILIS (CHLOROPHYTA) GAMETANGIA AND GAMETE RELEASE ARE CONTROLLED BY EXTRACELLULAR INHIBITORS1

Blade cells of Ulva mutabilis Føyn (Chlorophyta) excrete regulatory factors into their cell walls and into the environment. These factors are essential for the maintenance of the vegetative state. “Sporulation inhibitor-1a” (SI-1a) is a glycoprotein that was isolated from the culture medium of axenic Ulva growing as an undifferentiated callus. This protein was unusually stable to denaturing treatments and showed an extremely high apparent molecular mass (M_r) of 1–4 × 10⁷ daltons estimated by size exclusion chromatography. The glycosylation was not essential for activity. SI-1a suppressed gametogenesis completely at concentrations lower than 10^?14 M. When Ulva developed normally in the presence of their symbiotic bacteria, smaller forms of SI-1 accumulated in the medium (10⁴–10⁶ daltons). Sporulation inhibitors of the same size spectrum and with similar properties were also extracted from crude cell walls of nonaxenic Ulva. A class of different nonprotein sporulation inhibitors (SI-2) of very low M_r and yet unknown structure was isolated from the inner space between the two blade cell layers. Excretion of all SI-1 forms decreased with maturation of the thallus, whereas the overall concentration of SI-2 in the thallus stayed constant throughout the life cycle. The SI-2 affected different Ulva species whereas SI-1 was species-specific. Gametogenesis was induced upon removal of both Sporulation inhibitors from small single-layered fragments of mature blades. After a “determination phase” of 23–46 h, dependent on the time of induction within the cell cycle, the cells became irreversibly committed to differentiation and were no longer susceptible to SI-1 or SI-2. Subsequently, during a 28-h “differentiation phase,” 16 progametes were formed by synchronous genome doublings and cell divisions and differentiated into mature gametes. These became motile and were released from the gametangia when the concentration of a “swarming inhibitor” of low M_r, excreted mainly during the “determination phase,” declined below a threshold concentration. The biochemical properties of these regulatory factors and their effects on gametogenesis and gamete release are described.     

MOLECULAR DELINEATION OF SPECIES AND SYNGENS IN VOLVOCACEAN GREEN ALGAE (CHLOROPHYTA)1

Two species of the colonial green flagellate family Volvocaceae are worldwide in distribution yet exhibit contrasting species structure. Geographically disparate isolates of Gonium pectorale Mueller can interbreed while isolates of Pandorina morum Bory behave quite differently. More than 20 sexually isolated subpopulations occur within this species; these have been termed “syngens” (sensu Sonneborn). Because prezygotic barriers to mating cause intersyngen pairings to fail, breeding analyses cannot be used to estimate genetic relatedness among the syngens of P. morum. DNA comparisons provide an alternative method of assessing genetic relatedness. We compared the nucleotide sequence of the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat among clones of P. morum and of G. pectorale. Members of syngens of P. morum with distribution restricted to one small geographical area show great similarity. Likewise, members of any syngen of worldwide distribution show near uniformity, even those from different continents. However, the ITS sequence of each syngen differs from that of other syngens. In contrast, G. pectorale, which has an ITS region that is remarkably uniform throughout the world, appears to consist of a single syngen within North America and Europe by mating tests. The molecular data are in complete conformity with previous syngen assignment. Because the latter is based on mating affinity, with two complementary mating types per syngen, the evolution of new mating type pairs appears to be the basis of microevolution in these algae. We infer that either P. morum is a more ancient species than G. pectorale or that P. morum has a less stable genome. In either case, the biogeographic distribution of certain syngens may reflect climatological changes of the past.