SPECTRAL CHARACTERIZATION OF NEW CHLOROPHYLL C PIGMENTS ISOLATED FROM EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY1

Pigment extracts from the prymnesiophyte Emiliania huxleyi (Lohm.) Hay & Mohler (CCMP 370) were analyzed using a new chromatographic method based on the use of polymeric octadecylsilica columns, which split both the chlorophyll c₃ peak and phytol-substituted chlorophyll c-like peak into two fractions. The pigments corresponding to each of these fractions were isolated. The spectra and chromatographic behavior suggest that chlorophyll c₃ from E. huxleyi is a mixture of two different compounds whereas the splitting of the phytol-substituted c-like pigment could be the result of the equilibrium of two forms showing the same absorption spectrum. The method allowed, as well, the detection of magnesium 3,8-divinylpheoporphy-rin a, monomethyl ester in the prymnesiophytes E. huxleyi (CCMP 370) and Prymnesium parvum Carter (CCMP 708) and the chrysophyte Pelagococcus subviridis Norris (CCMP 1429).     

STEROLS OF DICTYOCHA FIBULA (CHRYSOPHYCEAE) AND OLISTHODISCUS LUTEUS (RAPHIDOPHYCEAE)1

Sterols from cultured Dictyocha fibula Ehrenberg and Olisthodiscus luteus Carter were extracted and identified for comparison with sterols from other Chromophycota species. Although orientation at C-24 was not absolutely determined, the sterols of Dictyocha, a silicoflagellale, appeared to consist of only 24-methyl-22-dehydrocholesterol and 24-methylenecholesterol, a sterol composition not known in any other alga. This is in keeping with the taxonomic isolation Dictyocha has among algae. On the other hand, Olisthodiscus luteus contained 24-ethylcholesterol, 24-ethylcholestanol, 24-methylcholesterol, and cholesterol. This composition is in accord with sterols of members of the Xanthophyceae and Raphidophyceae.     

PIGMENTS OF THE DINOFLAGELLATE PERIDINIUM BALTICUM AND ITS PHOTOSYNTHETIC ENDOSYMBIONT1

An examination of the pigments of the binucleate dinoflagellate Peridinium balticum (Levander) Lemmerman revealed the presence of chlorophylls a, c₁ and c₂ and the carotenoids: fucoxanthin (most abundant), diadinoxanthin, diatoxanthin, an unidentified fucoxanthin-like xanthophyll, β-carotene, γ-carotene and astaxanthin. A comparison of the pigments of P. balticum and P. foliaceum (Stein) Biecheler, also a binucleate dinoflagellate, demonstrated similar compositions. However P. balticum lacked the β-carotene precursors (e.g. phytoene) which accumulated outside the chloroplast in P. foliaceum. This study indicates that P. balticum and P. foliaceum are closely related; each species is a heterotrophic dinoflagellate with a photosynthetic endosymbiont taxonomically affiliated with the Chrysophyta (Chrysophyceae or Bacillariophyceae).     

EFFECT OF LOW TEMPERATURE ON THE STEREOISOMER COMPOSITION OF β-CAROTENE IN THE HALOTOLERANT ALGA DUNALIELLA BARDAWIL (CHLOROPHYTA)1

Dunaliella bardawil Ben-Amotz & Avron, a β-carotene-accumulating halotolerant alga, was analyzed for the effect of growth temperatures on its pigment content and on the stereoisomeric composition of β-carotene by the use of advanced liquid chromatography and photodiode array detection. Decreasing culture temperature from 30° to 10°C increased the β-carotene content twofold and the ratio of 9-cis to all-trans β-carotene fourfold, with no significant changes in the other cell pigments. The variation of total β-carotene content by temperature was correlated with the integral irradiance received by the algal culture during a cell division cycle, whereas the 9-cis stereoisomer increased over the amount expected by that integration. The massive accumulation of 9-cisβ-carotene within the β-carotene globules is interpreted as indicating that the oily 9-cis stereoisomer protects against the crystallization of all-trans β-carotene at low temperatures.     

STORAGE AND STRUCTURAL PRODUCTS FORMED DURING PHOTOSYNTHESIS IN THE SIPHONOUS ALGA CAULERPA SIMPLICIUSCULA(CHLOROPHYCEAE)1

The distribution of radioactivity into storage and intermediary metabolites during photosynthesis over periods of up to three hours was followed in the siphonous green alga Caulerpa simpliciuscula C. Ag. After the first hour, almost all the carbon assimilated was recovered as insoluble 1,4-α-glucan. There was rapid movement of carbon into insoluble 1,3-β-glucans during the first 20 min of photosynthesis, but little additional carbon moved into these compounds after that time. This contrasted with the movement of carbon into the soluble 1,3-β-glucan fraction, which continued for 2 h. Sucrose accumulated very little ¹⁴C during the entire period of photosynthesis. There was a very slow transfer of ¹⁴C into lipid throughout the period but the rate was linear. Protein labelling showed a lag of 2 h before it reached the same rate of ¹⁴C accumulation as shown by the lipid initially. It is suggested that the distribution of radioactivity can be explained in part by proposing that the tissue is in the latter stages of recovery from the wounding which look place at the time of dividing the thallus into sections.     

THE OCCURRENCE OF CHLOROPHYLLS C1 AND C2 IN THE CHRYSOPHYCEAE1

We analyzed 34 strains representing 25 species of Chrysophyceae for chlorophylls c₁ and c₂ using thin-layer chromatography. Most organisms had both chlorophylls c₁ and c₂ in addition to chlorophyll a but 17 strains of 9 species of Synura and Mallomonas possessed only chlorophylls a and c₁. These are the first chlorophyll c-bearing algae which lack chlorophyll c₂. We postulate that at least some of the silica-scaled algae including Mallomonas and Synura may be distinct from other Chrysophyceae based upon pigmentation and other characters described in the literature.     

COMPARATIVE EFFECTS OF LIGHT ON PIGMENTS OF TWO STRAINS OF EMILIANIA HUXLEYI (HAPTOPHYTA)1

Calcifying and a noncalcifying strains of Emiliania huxleyi were cultured in nutrient replete turbidostats under a photon flux density (PFD) gradient from 50 to 600 μmol E·m^?2·s^?1. For both strains, growth was PFD‐saturated at 300 μmol E·m^?2·s^?1. The strains, although with clearly different physiological properties due to the presence or absence of calcification, showed the same trends and magnitude of change in their pigment compliment as a function of PFD. Light‐controlled pigment composition and the trends of change in pigment composition were identical in both strains. Fucoxanthin (Fuco) was the major carotenoid in the calcifying strain, while in the noncalcifying strain this role was assumed by 19′ hexanoyloxyfucoxanthin (19 Hex). The photoprotective pigments and 19 Hex, normalized to chl a, increased with increasing light, while chl a content per cell and chl c's and Fuco, normalized to chl a, decreased with increasing PFD. The sum of all carotenoids normalized to chl a was remarkably similar in all PFDs used. Collectively, our results suggest that 19 Hex was synthesized from Fuco with light as a modulating factor and that the total amount of carotenoids is strain‐specific and synthesized/catabolized in tandem with chl a to a genetically predefined level independent of PFD.     

ULTRASTRUCTURE OF CYST FORMATION IN OCHROMONAS TUBERCULATA (CHRYSOPHYCEAE)1

The cysts (statospores) of Ochromonas tuberculata Hibberd are produced within a cytoplasmic silica deposition vesicle (SDV) whose membrane (silicalemma) appears to be formed by the coalescence of golgi vesicles. Silica is first deposited as small nodules and the collar and spines develop by centrifugal growth only after a complete but still thin wall has been laid down. Small vesicles appear to be attached to the SDV only in the region overlying the developing collar; a cap of radially arranged, moderately electron-dense material occurs at the tip of the growing spines. The cyst pore is formed at the anterior end of the flagellate cell, by lack of silica deposition over a small region of the SDV and rupture of the SDV and other membranes crossing this region. When the cyst wall is complete, an extracystic plug is formed in the pore, resulting in the loss of some extracystic cytoplasm and the plasmalemma, and the inner SDV membrane becomes the functional plasmalemma. The plug develops first by coalescence with the cell membrane of golgi-derived vesicles containing dense but apparently nonsiliceous spicules surrounded by amorphous material. During later stages of plug formation only fibrous material is deposited, some of which may be extruded through the pore forcing out some of the spiculate component. Scanning electron micrographs of the mature wall show it is smooth except for the concentrically wrinkled inner face of the flared collar and that the real pore diameter is only ca. half that of the collar. At germination the plug completely disappears in an unknown way and a single cell, similar to a normal vegetative cell emerges through the pore. Chrysophycean cyst formation generally resembles cell wall formation in diatoms, but differs in some details.     

ULTRASTRUCTURE OF THE GLAND CELLS OF THE RED ALGA ASPARAGOPSIS ARMATA (BONNEMAISONIACEAE)1

Localization of natural products in the gland cells of the tetrasporophyte of Asparagopsis armata Harvey was examined using light microscopy, epifluorescence microscopy, and TEM. A. armata produces a range of halogenated metabolites that deter herbivores and inhibit bacterial fouling. The halogenated metabolites accumulate as a refractile inclusion inside specialized gland cells and this inclusion was no longer produced when the alga was cultured without bromine. Gland cells are formed soon after the apical division and can occupy a large portion of the algal volume, up to 10% of some parts of the filament. TEM was carried out on cryofixed and freeze‐substituted samples. Ultrastructure studies revealed that gland cells are positioned inside the pericentral cell, originating from the axial cell wall. The refractile inclusion of these gland cells is comprised of numerous electron‐translucent vacuoles enclosed by an electron‐opaque matrix. Some contents of the inclusion autofluoresced under UV excitation by epifluorescence microscopy. Light microscopy further revealed that stalk‐like structures connected the gland cell to the outer wall of the pericentral cell. These stalk‐like structures may provide the mechanism for metabolite transfer to the algal surface. Gland cell walls are relatively thin, which in turn would aid the transfer of metabolites to the stalk‐like structure. These features of the gland cells provide essential clues to the production and storage of the halogenated metabolites in A. armata and offer new insights into a possible mechanism for their release.     

TWO SNOW SPECIES OF THE QUADRIFLAGELLATE GREEN ALGA CHLAINOMONAS (CHLOROPHYTA,VOLVOCALES): ULTRASTRUCTURE AND PHYLOGENETIC POSITION WITHIN THE CHLOROMONAS CLADE1

The quadriflagellate snow alga Chlainomonas Christen, distributed in New Zealand and North America, has several unusual structural attributes. A process assumed to be cytokinesis involves extrusion of protoplasm from the parent through a narrow canal, C. kolii (J. T. Hardy et Curl) Hoham produces a net‐like outer envelope rather than a cell wall, and the flagellar basal apparatus of Chlainomonas consists of two semi‐independent pairs of basal bodies. Structural connections between basal body pairs appear minimal, but a connecting system different from that observed in other genera exists within each pair. Phylogenetic analysis using rbcL sequences places Chlainomonas in the Chloromonas clade, other known members of which are all biflagellate. Chlainomonas is split into two robust lineages, with New Zealand collections sharing an origin with northern North American collections. Although the quadriflagellate condition is regarded as ancestral in the Chlorophyceae, we speculate—based on ultrastructural and molecular data presented here—that Chlainomonas represents a derived form that has arisen from fusion of two ancestral biflagellate cells. Other explanations (for example, that Chlainomonas represents a diploid form of a biflagellate species) are remotely possible but are presently at odds with extensive observations of field material. Improvements in techniques for experimental manipulation of these sensitive cryophiles will be required to fully characterize their structure and progress our understanding of their biology.     

IMPROVED DETECTION AND CHARACTERIZATION OF FUCOXANTHIN‐TYPE CAROTENOIDS: NOVEL PIGMENTS IN EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

A new atmospheric pressure chemical ionization mass spectrometry (APCI‐LC/MS) method improved detection and aided characterization of fucoxanthin related carotenoids, revealing the coccolithophorid Emiliania huxleyi (Lohm.) Hay et Mohler (strain MBA 92, Plymouth) to contain a wider range of acyloxyfucoxanthins than reported previously. The diversity is confirmed as arising from differences in the length of the alkanoic acid substituent esterified at position C‐19′. Acyloxyfucoxanthins with substituents of between four and eight carbons at the C‐19′ position have been detected in a culture of Emiliania huxleyi, where previously only 19′‐butanoyloxyfucoxanthin and 19′‐hexanoyloxyfucoxanthin have been reported in the literature. Novel fucoxanthinol derivatives were also found. The detection of these novel carotenoids in Emiliania huxleyi permits detailed studies of the impact of environmental factors on individual components of the complex pool of fucoxanthin‐type carotenoids in this organism.     

ULTRASTRUCTURE OF VEGETATIVE ORGANIZATION AND CELL DIVISION IN THE UNICELLULAR RED ALGA DIXONIELLA GRISEA GEN. NOV. (RHODOPHYTA) AND A CONSIDERATION OF THE GENUS RHODELLA1

This study suggests that the genus Rhodella be restricted to that set of features currently observed only in Rhodella maculata Evans and Rhodella violacea (Korn-mann) Wehrmeyer, that a new genus Dixoniella be established to accommodate the unicellular red alga, Rhodella grisea (Geitler) Fresnel, Billard, Hindák et Pekár-ková, and that Rhodella cyanea Billard et Fresnel be further studied for probable reclassification. These conclusions are based on ultrastructural comparisons of Dixoniella grisea with published information on the genus Rhodella. The presence of thylakoids in the pyrenoid, a peripheral encircling thylakoid in the chloroplast, a dictyosome/nuclear envelope association, and the lack of a specialized pyrenoid/nucleus association in D. grisea separate this alga from the genus Rhodella. Cell division in D. grisea is not demonstrably different from that in Rhodella, although the unusually well-defined material of the presumptive microtubule organizing center (MTOC) made it possible to follow the development and behavior of the MTOC to a greater degree than in previously studied red algal cells. The surprising amount of conformity in cell division characters between D. grisea and the genus Rhodella prompted a comparison of cell division characteristics in all red algal unicells studied to date. All unicells show a remarkable degree of similarity except for differences in interzonal spindle length, dissimilarities in size of the nucleus-associated organelle (NAO), and the unusual NAO of Porphyridium purpureum (Bory) Drew et Ross.     

MITOSIS AND CELL DIVISION IN HYDRURUS FOETIDUS (CHRYSOPHYCEAE)1

Mitosis and cell division have been examined ultrastructurally in the vegetative cells of Hydrurus foetidus (Vill) Trev. and found to resemble that of Ochromonas in two important aspects. First, the rhizoplast acts as the spindle organizing body and second, the spindle elongates considerably during anaphase. It differs from Ochromonas in that there is no movement of the basal bodies and flagella towards the poles. Moreover, the nuclear envelope remains relatively intact throughout early stages of mitosis, with gaps developing at the poles during prophase to permit entry of spindle microtubules. Disruption of the nuclear envelope does not occur in the equatorial plane until late anaphase. The spindle persists into telophase and is bent towards the posterior of the cell by the ingrowing edge of the cleavage furrow. Persistence of the spindle and lack of Ochromoms-type cell elongation may be related to the constricting presence of the sheath during cell division—a completely different strategy to that adopted by the green algae under conditions of similar constraint.     

EFFECT OF NITROGEN STARVATION ON OPTICAL PROPERTIES,PIGMENTS, AND ARACHIDONIC ACID CONTENT OF THE UNICELLULAR GREEN ALGA PARIETOCHLORIS INCISA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

Spectral properties of cell suspensions, individual cells, and extracts of the unicellular green alga Parietochloris incisa (Reisigl) Shin Watan. grown under low light were studied. Long‐term nitrogen (N) deprivation resulted in a decrease of chloroplast volume, appearance of numerous large cytoplasmic oil bodies, and the deposition of triacylglycerols with a high proportion of arachidonic acid. Chlorophylls a and b underwent a synchronous decline, whereas carotenoids (Car) showed a relative increase. Simultaneously, significant qualitative changes in the spectral properties of P. incisa individual cells, cell extracts, and cell suspensions were observed. To a large extent, the spectral changes observed in cell suspension could be attributed to a decrease in overall pigment content, leading to a gradual weakening of the so‐called package effect and accumulation of additional amounts of Car over chl, most probably, in oil bodies. Several optical characteristics of cell suspensions could serve as sensitive indicators of N‐deficiency in P. incisa. Furthermore, the absorption ratios, A₄₇₆/A₆₇₆ and A₆₅₀/A_676, showed close correlations with the Car‐to‐chl ratio and relative arachidonic acid (AA) content, respectively. The latter makes it possible to suggest that the increase in AA percentage in P. incisa proceeds in parallel with a decrease in cell chl content, accounting for the weakening of the package effect. N‐replenishment resulted in complete recovery of cell optical properties. The possible significance of the changes in cell ultrastructure, pigments, lipids, and optical properties is discussed with special reference to the ability of algae to adapt to and survive under conditions of long‐term nutrient deficiency.     

Validation of a high-performance liquid chromatographic-mass spectrometric method for the measurement of 5-chloro-2′,3′-dideoxy-3′-fluorouridine (935U83) in human plasma

An isocratic reversed-phase LC-MS method for measuring concentrations of 5-chloro-2′,3′-dideoxy-3′-fluorouridine (935U83; I) directly and its 5′-glucuronide metabolite (5-chloro-2′,3′-dideoxy-5′-O-β- -glucopyranuronosyl-3′-fluorouridine) indirectly in human plasma was developed, validated, and applied to a Phase I clinical study. The pyrimidine nucleoside, I, was extracted from human plasma by using anionic solid-phase extraction. The concentration of the glucuronide conjugate was determined from the difference between the molar concentration of I in a sample hydrolyzed with β-glucuronidase and the nonhydrolyzed sample. Recovery of I from human plasma averaged 90%. The bias of the assay for I ranged from −5.5 to 7.1% during the validation and from −6.0 to 1.4% during application of the assay to the Phase I single-dose escalation study. The intra- and inter-day precision was less than 8% for I and its glucuronide conjugate. The lower and upper limits of quantitation for a 50-μl sample were 4 ng/ml and 3000 ng/ml, respectively. No significant endogenous interferences were noted in human plasma obtained from drug-free volunteers nor from predose samples of HIV-infected patients.     

TWO NEW SPECIES OF PSEUDOKEPHYRION (CHRYSOPHYCEAE) IN THE PLANKTON OF A LAKE1

Two new species of Pseudokephyrion are described from Little Aurora Lake, Ontario, Canada. The distribution of both P. taeniatum sp. nov. and P. auroreum sp. nov. is restricted, for reasons which are not clear; neither species was found in samples from lakes adjacent to Little Aurora Lake. All lakes in the vicinity are very dilute, oligotrophic and acidic.     

PHOTOSYNTHETIC ACTION SPECTRUM OF THE COCCOLITHOPHORID,EMILIANIA HUXLEYI (HAPTOPHYCEAE): 19′ HEXANOYLOXYFUCOXANTHIN AS ANTENNA PIGMENT1,2

Action spectra for modulated O₂ production were compared with cell absorbance in Emiliania huxleyi (Lohm.) Hay and Mohler, a marine coccolithophorid in which 19′ hexanoytoxyfucoxanthin is the major carotenoid of the photosynthetic apparatus, Photosynthetic effectiveness (action/absorption) was high throughout the spectral interval, 500–550 nm, where 19′ hexanoyloxyfucoxanthin accounted for most of the photon capture. 19′ hexanoyloxyfucoxanthin is thus considered to be an efficient antenna pigment for photosynthesis and to be preferentially associated with photosystem II. It may be a quantitatively significant light harvesting pigment for some algae of the marine environment.     

CHLOROPLAST GENETIC TOOL FOR THE GREEN MICROALGAE HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE,VOLVOCALES)1

At present, there is strong commercial demand for recombinant proteins, such as antigens, antibodies, biopharmaceuticals, and industrial enzymes, which cannot be fulfilled by existing procedures. Thus, an intensive search for alternative models that may provide efficiency, safety, and quality control is being undertaken by a number of laboratories around the world. The chloroplast of the eukaryotic microalgae Haematococcus pluvialis Flotow has arisen as a candidate for a novel expression platform for recombinant protein production. However, there are important drawbacks that need to be resolved before it can become such a system. The most significant of these are chloroplast genome characterizations, and the development of chloroplast transformation vectors based upon specific endogenous promoters and on homologous targeting regions. In this study, we report the identification and characterization of endogenous chloroplast sequences for use as genetic tools for the construction of H. pluvialis specific expression vectors to efficiently transform the chloroplast of this microalga via microprojectile bombardment. As a consequence, H. pluvialis shows promise as a platform for expressing recombinant proteins for biotechnological applications, for instance, the development of oral vaccines for aquaculture.     

THE ULTRASTRUCTURE OF SCENEDESMUS (CHLOROPHYCEAE). II. CELL DIVISION AND COLONY FORMATION1

Cell division in Scenedesmus is fairly typical of other chlorococcalean genera. The closed spindle has centrioles at polar fenestrae and apparently a series of nuclear divisions precedes cytokinesis. The phycoplast system of cytokinetic microtubules predicts the path of cleavage furrows whose mode of formation is obscure. Before and during cell division, the endoplasmic reticulum invariably accumulates granular material which later, during cytokinesis, appears to he secreted via the golgi bodies. Similar dense granular material then at accumulates outside the forming daughter cells but inside the parental wall, as the latter begins eroding away. By the end of colony formation, the cellulosic parental wall has disappeared, leaving its outer sheath and attached ornamentative features (spines, combs, reticulate or warty layer, etc.) intact as a “ghost.” The spines and combs of new colonies appear to condense out of the extracellular aggregate; their precise mode of formation is obscure. As they form, the daughter cells, having become rearranged within the parental wall, stick to one another apparently at specific sites on their outer surface. A trilaminar (sporopollenin-containing) layer arises first in each cell at these adhesive sites and immediately afterwards, dense material aggregates between the adjacent layers to give rise to the coenobial adhesive. Plaques of the trilaminar layer later appear over the rest of the cell's surface; they grow and fuse so that eventually each cell is enclosed by one continuous Trilaminar Sheath (TLS). While the plaques are forming, another dense layer materializes around the whole coenobium. Depending on the species, this layer turns into either the warty layer, in which instance it is applied directly on to the surface of the TLS except near the coenobial adhesive, or else it becomes the reticulate layer, in which instance it remains entirely separate from the TLS, soon acquiring the complex system of propping spikelets which suspend it from the coenobial surface. When fully farmed, the daughter coenobium is tightly compressed within the parental TLS, with its spines folded lengthwise along the daughter cells. Release of the colony follows a quite explosive rupturing of the parental TLS, and immediately upon release, the daughter colony flattens out and erects its spines.     

CELL DIVISION AND COLONY FORMATION IN THE GREEN ALGA COELASTRUM (CHLOROCOCCALES)1

Multinucleate cells of Coelastrum undergo precisely directed cytokinesis, guided by phycoplast microtubules, to form a number of uninucleate daughter cells which subsequently adhere to form characteristically patterned aggregates. As there is no movement of the daughter cells relative to one another before their adhesion, the disposition of cells in daughter colonies reflects the pattern of cytokinesis of parent cells. Centrioles lie at the poles of the mitotic nuclei which are partially enclosed by a perinuclear envelope of endoplasmic reticulum. The centrioles disappear at the time of cytokinesis of the parental cell and apparently reform de novo once the daughter cells have acquired a cell wall following their adhesion. The trilaminar layer of cell wall, often termed the pectic layer, does not stain with ruthenium red and resists acetolysis suggesting that it contains sporopollenin rather than pectin.