ANTINEOPLASTIC ACTIVITY OF CULTURED BLUE-GREEN ALGAE (CYANOPHYTA)1

A large-scale screening program was initiated to evaluate laboratory-cultured blue-green algae (cyanobacteria) as a source of novel antineoplastic agents. Approximately 1000 cyanophyte strains from diverse habitats were cultured to provide extracts for testing. The screening program identified the families Scytonemataceae and Stigonemataceae as prolific producers of novel cytotoxic compounds. Rates of rediscovery of known compounds were relatively low.     

ANTIVIRAL ACTIVITY OF CULTURED BLUE-GREEN ALGAE (CYANOPHYTA)1

Lipophilic and hydrophilic extracts from approximately 600 strains of cultured cyanophytes, representing some 300 species, were examined for antiviral activity against three pathogenic viruses. Approximately 10% of the cultures produced substances that caused significant reduction in cytopathic effect normally associated with viral infection. The screening program identified the order Chroococcales as commonly producing antiviral agents.     

AKINETE DIFFERENTIATION IN ANABAENA CIRCINALIS (CYANOPHYTA)1

Three lines of evidence established conclusively that phosphorus limitation triggered akinetes to differentiate in Anabaena circinalis Rabenhorst. First, akinetes differentiated when phosphorus was limited, but not when nitrogen, inorganic carbon, iron, trace elements, or light were limited, or when dissolved oxygen concentration was increased. In the phosphorus limitation experiment, akinetes appeared first in the 0 mg P-L^?1 cultures, and the higher the initial concentration of phosphorus was, the longer it took for akinetes to differentiate. Second, akinete differentiation commenced when Q_p fell to the same critical concentration in all cultures. The critical Q_p for akinete differentiation in A. circinalis was 0.3-0.45 pg P·cell^?1, and there was no significant difference between cultures grown with 0.6, 0.2, 0.06, or 0 mg P · L^?1 (F= 5.48, of = 3, P > 0.05). Similarly, there were no significant differences between P cultures in internal cellular soluble reactive phosphorus (SRP) concentration (F= 0.63, df = 3, P > 0.05) or external SRP per cell in the medium (F= 5.16, df= 3, P > 0.05) when akinete differentiation commenced. Both were between 0.01 and 0.07 pg SRP-cell^?1. A thorough literature search indicates that this information has not been reported previously. The third line of evidence came from electron micrographs, which illustrated that polyphosphate was present in trichomes prior to akinete differentiation but was absent in trichomes with akinetes indicating that phosphorus reserves were depleted when akinetes differentiated. Lipid globules (carbon reserve) and cyanophycin granules (nitrogen reserve) increased in number in trichomes with akinetes, compared to trichomes without akinetes. Thus, the ratio of internal P:C:N was different in trichomes with akinetes compared to trichomes without akinetes and may be important in activating akinete-differentiating genes.     

OCCURRENCE OF THREE NITZSCHIA (BACILLARIOPHYCEAE) TAXA WITHIN COLONIES OF TUBE-FORMING DIATOMS1

In benthic samples from the unchannelized Missouri River, the diatoms Nitzschia dissipata (Kütz.) Grun., N. filiformis (W.Sm.) Schütt and N. pseudofonticola Hust. were observed within the mucilage tubes of four tube-forming diatoms: Cymbella prostrata (Berk.) Cl., C. prostrata var. auerswaldii (Rabh.) Reim., Navicula tripunctata var. schizonemoides (V.H.) Patr., and Nitzschia filiformis. Microscopical observations of live and preserved specimens indicated that “invasion” by Nitzschia occurred primarily in older tubes. Data are presented on the environmental conditions in which the tube-formers and their cohabitants have been found.     

DNA POLYMORPHISM WITHIN THE WH7803 SEROGROUP OF MARINE SYNECHOCOCCUS SPP. (CYANOBACTERIA)1,2

Genetic differences among ten strains of chroococcoid cyanobacteria (Synechococcus spp.) were identified by Southern blot hybridization. Data on shared number of restriction fragment length polymorphisms were used to identify the pattern and degree of genetic relatedness among the strains by two different methods of phylogenetic analysis. All the marine strains in the study contained phycoerythrin (PE) and cross-reacted with antisera directed against strain WH7803. Five contained a PE composed of phycourobilin (PUB) and phycoerythrobilin (PEB) Chromophores, and three contained a PE composed of only PEB chromophores. Two freshwater strains which do not contain PE and do not cross-react with the anti-WH7803 serum were included in the study for comparison. Dollo Parsimony analysis and cluster analysis showed that the WH7803 serogroup includes at least four widely separated genetic lineages. Strains within each lineages were closely related but the differences between lineages were as great as those between any of the marine lineages and the freshwater lineage. Strains cultured simultaneously from the same water mass were associated with different lineages. Thus, we conclude that natural assemblages of marine. Synechococcus are, at least occasionally, composed of individuals as genetically distinct from each other as members of different species or genera in other taxa.     

DEPRESSION OF MASTIGOCLADUS LAMINOSUS (CYANOPHYTA) NITROGENASE ACTIVITY UNDER NORMAL SUNLIGHT INTENSITIES1

The effect of light intensity (PAR) on the nitrogenase activity of Mastigocladus laminosus Cohn was studied by the acetylene reduction technique. Benthic mat from a thermal stream, Hot River, in Yellowstone National Park was used in both experimental and in situ incubations. This hot spring maintained a mean pH of 7.0, was essentially isothermal (ca. 50°C), and had virtually no upstream to downstream physicochemical gradients (P > 0.05). Two surveys of the stream showed that nitrogenase of the M. laminosus mat was significantly more active (P > 0.02) under low light intensities than under high intensities, 252 and 712 μE · m^?2· s^?1, respectively. Maximum activity of Hot River Mastigocladus (268 nmol C₂H₄· mg Chl a^?1· h^?1) occurred at 50% full midday light intensities; the rates at low light (mean = 247 nmol C₂H₄· mg Chl a^?1· h^?1) were significantly (P > 0.001) greater than those at high light (mean = 106). The results indicate that M. laminosus nitrogenase activity is low light adapted and suggest that the temporal pattern for nitrogen fixation might be significantly different from that of thermophilic Calothrix.     

PROPERTIES OF MICROCYSTIS AERUGINOSA AND M. FLOS-AQUAE (CYANOPHYTA) IN CULTURE: TAXONOMIC IMPLICATIONS1

Cultures were cloned from a sample containing Microcystis aeruginosa, M. flos-aquae and a few morphological intermediates. The M. aeruginosa cultures remained distinct from the M. flos-aquae cultures in (a) cell size, (b) cell aggregation pattern, (c) width of the mucilage surrounding the multicellular colonies, (d) sharpness of the mucilage boundary, (e) efect of 0.1–1.0 μM calcium chloride on the disaggregation of multicellular colonies, (f) frequency of mucilage mutants and (g) colony morphology on agar media. No M. flos-aquae culture produced morphs resembling M. aeruginosa, inconsistent with proposals that M. flos-aquae is a developmental stage or environmentally-induced variant of M. aeruginosa. After longterm cultivation, but not soon after origanal isolation, several M. aeruginosa cultures contained mutants with diminished mucilage production and an altered colony shape.     

GENETIC DIVERSITY WITHIN BALTIC SEA POPULATIONS OF NODULARIA (CYANOBACTERIA)1

The determination of the genetic structure of microbial populations has, until recently, required the establishment of many independent clonal cultures for genotypic analysis. In such studies it has been necessary to assume that isolates able to grow in laboratory culture are representative of the full range of diversity within the natural population. In order to test this assumption we used the polymerase chain reaction (PCR) to amplify the intergenic spacer region of the Phycocyanin operon (PC-IGS) from filaments of Nodularia taken both from clonal cultures and from natural populations in the Baltic Sea. Analysis of the nucleotide sequences revealed more variation among 16 cultured isolates than within 23 single filaments sampled from a natural population. As a means of rapidly determining population genetic structure we designed and used mixtures of allele-specific amplification primers in diagnostic PCRs to identify which PC-IGS allele was present in single filaments from natural cyanobacterial assemblages. Using this method, we determined the PC-IGS genotype of 156 filaments from 9 sampling stations throughout the central basin of the Baltic Sea in July 1996. Our results show that two distinct genotypes of Nodularia are present in the population at all stations. Although the two types were present in approximately equal numbers, they were not distributed uniformly.     

MOLECULAR AND MORPHOLOGICAL CHARACTERIZATION OF TEN POLAR AND NEAR‐POLAR STRAINS WITHIN THE OSCILLATORIALES (CYANOBACTERIA)1

An approximately 1400‐bp region of the 16S rRNA gene was sequenced for 10 polar or near‐polar strains putatively placed in the Oscillatorialean genera Oscillatoria, Phormidium, and Lyngbya obtained from the University of Toronto Culture Collection to assess phylogenetic relationships. The strains were also examined for thylakoid structure and cell division type with TEM as well as traditional morphology with LM. Phylogenetic trees constructed using parsimony, distance, and maximum likelihood methods were similar in topology. If the original epithets applied to the sequenced strains (both polar and those from GenBank) were used, it was clear that taxa were not monophyletic. However, using the revised taxonomic system of Anagnostidis and Komárek, we were able to reassign these strains to their current correct taxa (species, genus, and family). When these assignments were made, it was determined that the molecular sequence data analyses were congruent with morphology and ultrastructure. Nine of the polar strains were found to be new species, and eight were described as such: Arthronema gygaxiana Casamatta et Johansen sp. nov., Pseudanabaena tremula Johansen et Casamatta sp. nov., Leptolyngbya angustata Casamatta et Johansen sp. nov., Phormidium lumbricale Johansen et Casamatta sp. nov., Microcoleus glaciei Johansen et Casamatta sp. nov., Microcoleus rushforthii Johansen et Casamatta sp. nov., Microcoleus antarcticus Casamatta et Johansen sp. nov., Microcoleus acremannii Casamatta et Johansen sp. nov. Some genera (Leptolyngbya and Microcoleus) were clearly not monophyletic and require future revision.     

PHYLOGENETIC RELATIONSHIPS WITHIN THE GENUS HYPNEA (GIGARTINALES,RHODOPHYTA), WITH A DESCRIPTION OF H. CAESPITOSA SP. NOV.1

Species discrimination within the gigartinalean red algal genus Hypnea has been controversial. To help resolve the controversy and explore phylogeny within the genus, we determined rbcL sequences from 30 specimens of 23 species within the genus, cox1 from 22 specimens of 10 species, and psaA from 16 species. We describe H. caespitosa as a new species characterized by a relatively slender main axis; a pulvinate growth habit with entangled, anastomosing, and subulate uppermost branches; and unilaterally borne tetrasporangial sori. The new species occurs in the warm waters of Malaysia, the Philippines, and Singapore. The phylogenetic trees of rbcL, psaA, and cox1 sequences showed a distant relationship of H. caespitosa to H. pannosa J. Agardh from Baja California and the marked differentiation from other similar species. The rbcL + psaA tree supported monophyly of the genus with high bootstrap values and posterior probabilities. The analysis revealed three clades within the genus, corresponding to three sections, namely, Virgatae, Spinuligerae, and Pulvinatae first recognized by J. G. Agardh. Exceptions were H. japonica T. Tanaka in Pulvinatae and H. spinella (C. Agardh) Kütz. in Spinuligerae.     

FACTORS INFLUENCING THE TOXICITY AND ANIMAL SUSCEPTIBILITY OF ANABAENA FLOS-AQUAE (CYANOPHYTA) BLOOMS1

Biological factors have been found which can cause variable toxicity of colony and clonal isolates of Anabaena flos-aquae (Lyngb.) de Bréb. when cultured in the laboratory. These factors help to explain some of the variable toxicity of and animal susceptibility to A. flos-aquae blooms in nature. Two bacteria in the Enterobacteriaceae isolated from a toxic waterbloom, depressed toxin production in selected bacteria-free toxic clones of A. flos-aquae. These toxin-depressing bacteria decreased culture toxicity 3-fold from 80 to 240 mg/kg (intra-peritoneal in male mice). Many colony isolates from a toxic bloom had minimum lethal dosages (LD_min) greater than 240 mg/kg. This was because they were composed of mixtures of toxic and nontoxic filaments. The oral LD_min of the toxin from A. flos-aquae clonal isolate NRC-44-1 varied significantly for 6 different animal species. Using these oral LD_min it is estimated that a surface-concentrated bloom of toxic A. flos-aquae, having a biomass density of 20 mg/ml dry weight, would cause death of ducks or calves when 20 ml/kg was consumed whereas a monogastric animal such as a rat would require 80 ml/kg.     

FLUCTUATING ALGAL FOOD POPULATIONS AND THE OCCURRENCE OF LESSER FLAMINGOS (PHOENICONAIAS MINOR) IN THREE KENYAN RIFT VALLEY LAKES1

The last two decades have witnessed increasing episodes of lesser flamingo die‐offs in East Africa. Based on data on phytoplankton composition, biomass, and flamingo population density in three alkaline‐saline lakes of Kenya (Bogoria, Nakuru, and Oloidien) in 2001–2010, this study explored the link between sudden flamingo deaths and fluctuations in algal food quantity and quality. The phytoplankton biomass ranged from 13 to 768 mg · L^?1. Similarly, flamingo numbers varied widely from <1,000 to >500,000 individuals in the study lakes. The dominance of the cyanobacterium Arthrospira fusiformis (Woron.) Komárek et J. W. G. Lund was interrupted at irregular intervals in each lake and replaced partly by populations of different species of the nostocalean Anabaenopsis or by the picoplanktonic chlorophyte Picocystis salinarum Lewin. The populations of Anabaenopsis have the potential of blocking the flamingo food filtration system with their large and slimy colonies; moreover, they are able to produce cyanotoxins. Estimates of flamingo populations suggest that low flamingo numbers coincided with periods of low algal food quantity and/or poor quality. A food deficit can be theorized to have two effects on the flamingos: (i) it weakens them to the point of being susceptible to attacks of infective diseases, such as the ones caused by Mycobacterium avium and Pseudomonas aeruginosa, and (ii) it predisposes them to poisoning by cyanotoxins and pollutants, by reducing their capacity to handle toxic substances. This study therefore concludes that the challenges facing the flamingos are associated with changes in their environment, which affect food and water supply.     

RELATIONSHIP BETWEEN PHOTOSYNTHETIC OXYGEN CYCLING AND CARBON ASSIMILATION IN SYNECHOCOCCUS WH7803 (CYANOPHYTA)1

Mass spectrometric analysis of oxygen uptake and evolution in the light by marine Synechococcus WH7803 indicated that the respiration rate was near zero at low irradiance levels but increased significantly at high irradiances. The light intensity (I_r) at which oxygen uptake began to increase with increasing light intensity depended on the growth irradiance of the culture. In each case, I_r coincided with the minimum light intensity for saturation of carbon assimilation (I_k). At irradiances >I_r, net oxygen evolution rates paralleled carbon assimilation rates. Oxygen uptake at high light intensities was inhibited by DCMU, indicating that oxygen uptake was due to Mehler reaction activity. The onset of Mehler activity at I_k supports the idea that oxygen becomes an alternative sink for electrons from photosystem I when NADPH turnover is limited by the capacity of the dark reactions to utilize reductant.     

PROTEIN AND AMINO ACID COMPOSITION OF THE OBLIGATE HALOPHILE APHANOTHECE HALOPHYTICA (CYANOPHYTA)1

Total protein was determined for cells of Aphanothece halophytica Fremy harvested during early log, mid-log and linear growth phases in media containing 1, 2, and 3 M NaCl. Cells grown in medium containing 1 M NaCl showed a progressive increase in protein content up to a maximum of 76% of dry weight (linear phase). Total protein also increased in cells grown in 2 M NaCl. medium (56.5–72.0%). Cells grown in 3 M NaCl medium showed a progressive decrease in total protein (59.9–43%). Although amounts of protein varied, the percentages of the respective amino acids of hydrolyzed bulk protein were consistent to within 1% for linear phase cells grown in 1, 2, and 3 M NaCl cultures. Percentages of acidic amino acids were 2.3–2.6 times greater than those of the basic amino acids. The amino acid composition of phycocyanin was similar to that of bulk protein. Free amino acids varied with both age of the culture and the concentration of NaCl. The high quantity and quality of the protein observed suggest that A. halophytica might be a useful food organism.     

INFLUENCE OF NITROGEN SOURCE ON MORPHOLOGY OF RIVULARIACEAE (CYANOPHYTA)1

Thirty-four heterocyst-producing strains of Rivulariaceae (29 Calothrix, 1 Dichothrix, 2 Gloeotrichia, 2 Rivularia), which produced tapered trichomes in medium minus combined nitrogen, were grown in the presence of nitrate. One strain was unchanged in morphology under this condition. The remaining 33 strains developed trichomes lacking heterocysts. In 19 strains almost all the trichomes became untapered and in the other 14, similar untapered trichomes were produced, but also many tapered trichomes resembling Homoeothrix or Hammatoidea. Similar results were obtained when representative strains were incubated with ammonia as the source of combined N. Only five strains formed colorless hairs in the control medium (minus combined N). The presence of combined N did not diminish hair development in the two strains which had only a few short hairs, but hair frequency and length were both reduced considerably in the three strains with many long hairs in the control medium. Two strains of the non-heterocystous genus Homoeothrix were incubated in medium without combined N. Neither strain showed any growth or heterocyst development, indicating that neither is simply a growth form of a heterocystous genus.     

EFFECT OF EDTA ADDITIONS ON NATURAL TRICHODESMIUM SPP. (CYANOPHYTA) POPULATIONS1

Despite nearly two decades of intensive research, many questions regarding the physiology and ecology of the marine, non‐heterocystous cyanobacterium, Trichodesmium, remain unresolved. We note here the effect of EDTA (ethylenediaminetetraacetate) on N₂ fixation by Trichodesmium, and the use of EDTA as a means of extending the viability of natural Trichodesmium spp. populations. We examined nitrogenase activity (NA) as a function of EDTA concentration, time of collection, light level, and iron addition. Samples collected early in the day and treated with EDTA maintain a steady rate of activity for hours longer than controls. Furthermore, samples preincubated through the night with EDTA were active the next morning, compared with controls that were inactive. The discovery that (10–50 μM) low concentrations of EDTA prolong the duration of NA of Trichodesmium during experimental manipulations without affecting the rate of acetylene reduction allows for longer term manipulative experiments to be conducted.     

POLYPHYLETIC ORIGIN OF THE DICTYOSPHAERIUM MORPHOTYPE WITHIN CHLORELLACEAE (TREBOUXIOPHYCEAE)1

The green algal Dictyosphaerium morphotype is characterized by spherical or oval cells connected by gelatinized strands to microscopic colonies, which are covered by prominent mucilaginous envelopes. Combined SSU and ITS rRNA gene sequence analyses revealed that this morphotype evolved independently both in the Chlorella and Parachlorella clades of the Chlorellaceae. It was shown that strains exhibiting the morphology of the type species Dictyosphaerium ehrenbergianum Nägeli established a sister lineage to Parachlorella. The strain D. ehrenbergianum CCAP 222/1A was designated as an authentic strain for establishing the epitype of the genus Dictyosphaerium. The comparison of this strain with the authentic strain of Parachlorella beijerinckii Krienitz, E. Hegewald, Hepperle, V. Huss, T. Rohr et M. Wolf (SAG 2046) showed considerable differences in the secondary structure of the ITS region. Within the whole ITS‐1 and ITS‐2 region, 27 compensatory base changes (CBCs) were recognized. In the conserved Helix III of the ITS‐2, five CBCs/HemiCBCs were detected. This is a conclusive argument for separation of these two species. The clear definition of Dictyosphaerium is intended to be the necessary starting point of taxonomic reevaluation of Dictyosphaerium‐like algae within different evolutionary lineages of the Chlorellaceae.