Physiological changes of a green alga (Micractinium sp.) involved in an early-stage of association with Tetrahymena thermophila during 5-year microcosm culture

Endosymbioses between phototrophic algae and heterotrophic organisms are an important symbiotic association in that this association connects photo- and heterotrophic metabolism, and therefore, affects energy/matter pathways and cycling in the ecosystem. However, little is known about the early processes of evolution of an endosymbiotic association between previously non-associated organisms. In previous studies, we analyzed an early process of the evolution of an endosymbiotic association between an alga and a ciliate by using a long-term culture of an experimental model ecosystem (CET microcosm) composed of a green alga (Micractinium sp.), a bacterium (Escherichia coli), and a ciliate (Tetrahymena thermophila). The results revealed that an algal type, isolated from 5-year cultures of the microcosm, prolonged the longevity of the ancestral and derived clones of T. thermophila in the absence of bacteria, suggesting that a cooperative algal phenotype that benefited the ciliate had evolved in the microcosm. Here, we investigated the physiological changes of the derived Micractinium clones that benefited Tetrahymena, focusing on the release of carbohydrates by and abundance of photopigments in the ancestral and 2 derived algal clones (SC10-2 and SC9-1) isolated from inside Tetrahymena cells. Analyses using HPLC revealed that the algal isolates released glycerol and sucrose at higher concentrations per cell and also contained higher levels of photopigments per cell at pH 7.2, in comparison with the ancestral strain. These phenotypic characters were considered responsible for the increased longevity of Tetrahymena cells, and thus supported the cooperator alga hypothesis.     

Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day^?1) and yield (60 μg chlorophyll/ml culture) than in pure cultures (0.4 day^?1 and 10 μg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.     

Azolla-Anabaena Relationships: VII. Distribution of Ammonia-assimilating Enzymes, Protein, and Chlorophyll between Host and Symbiont

The N₂-fixing Azolla-Anabaena symbiotic association is characterized in regard to individual host and symbiont contributions to its total chlorophyll, protein, and levels of ammonia-assimilating enzymes. The phycocyanin content of the association and the isolated blue-green algal symbiont was used as a standard for this characterization. Phycocyanin was measured by absorption and fluorescence emission spectroscopy. The phycocyanin content and total phycobilin complement of the symbiotic algae were distinct from those of Anabaena cylindrica and a free-living isolate of the Azolla endophyte. The algal symbiont accounted for less than 20% of the association's chlorophyll and protein. Acetylene reduction rates in the association (based solely on the amount of algal chlorophyll) were 30 to 50% higher than those attained when the symbiont was isolated directly from the fern. More than 75% of the association's glutamate dehydrogenase and glutamine synthetase activities are contributed by the host plant. The specific activity of glutamate dehydrogenase is greater than that of glutamine synthetase in the association and individual partners. Both the host and symbiont have glutamate synthase activity. The net distribution of these enzymes is discussed in regard to the probable roles of the host and symbiont in the assimilation of ammonia resulting from N₂ fixation by the symbiont.     

Two species of Halimeda, a calcifying genus of tropical macroalgae, are robust to epiphytism by cyanobacteria

Cyanobacteria, an increasingly important epiphyte on macroalgae and seagrass, have been shown to have strong effects on its hosts; this association has been identified as a driving mechanism that maintains algal blooms on coral reefs. We examined both the costs and benefits of epiphytism on 2 algal congeners of Halimeda (H. tuna and H. opuntia), both of which are abundant members of tropical reef communities in the Caribbean. To evaluate potential benefits of an associational defense as well as costs to growth, we manipulated herbivore access to (uncaged/caged) and cyanobacteria presence on (epiphytized/cleaned) 2 species of Halimeda on shallow patch reefs in Belize and measured change in branch length and segment number after 10 (H. tuna) and 5 (H. opuntia) days. Cyanobacterial epiphytes did not serve as an associational defense from herbivory as there were no differences between caged and uncaged treatments for either response variable. The presence of cyanobacterial epiphytes did not affect the growth of branches or net generation of new segments, demonstrating there was also no cost to growth. The robustness of both species of Halimeda to epiphytism contrasts strongly with recent research that found strong effects of epiphytes on several other species of tropical algae. Our results may be attributed to the unique characteristics of Halimeda, a heavily physically and chemically defended algal genus, and the shallow nature of the patch reefs reducing the potential for significant light limitation. These findings suggest that close interactions such as epiphytism may not be as generalizable as originally assumed; studies must consider differences among host species, as this may lead to a better understanding of community-wide effects.     

Lytic Organisms and Photooxidative Effects: Influence on Blue-Green Algae (Cyanobacteria) in Lake Mendota, Wisconsin

The effects of exposure to high light intensities on blue-green algal (cyanobacterial) populations were examined in Lake Mendota, Wis. The algal populations were shown to be susceptible to inhibition of photosynthetic activity and pigment bleaching as a result of exposure. These effects generally influence only a small percentage of the lake population and thus are probably not important in causing major declines in chlorophyll a. Lytic organisms were shown to increase in numbers in the lake in response to the seasonal development of blue-green algae, reaching values of greater than 1,000 plaque-forming units per ml in midsummer. Both bacteria and protozoa were observed in plaque zones, but it could not be determined whether these lytic organisms had a major role in algal biomass declines.     

Preliminary Raman microscopic analyses of a lichen encrustation involved in the biodeterioration of renaissance frescoes in Central Italy

Raman microscopic analysis has proved effective in the interpretation and characterization of both the physical and chemical nature of the lichen/substratum system. In the case of Dirina massiliensis forma sorediata, a lichen causing extensive damage to Italian frescoes, the lichen-generated encrustation has been proved to be composed essentially of calcium oxalate. This particular biodeterioration process may be slowed down, or indeed inhibited, by the presence of certain compounds, as yet undertermined, used in the preparation of surfaces and the painting of frescoes. From a conservator's point of view, Raman microscopic analysis uses microscopic samples in the nanogram-picogram range and is non-destructive of the sample.     

A study of the fungi occurring on 15th century frescoes in Florence,Italy

The role of fungi in the biodeterioration of frescoes has been investigated by means of transparent adhesive tape and by swabbing the surface of selected areas as well as by a study of the materials used in restoration. In the Ognissanti church at Florence a large fungal population was found on two 15th century frescoes, one by Botticelli of ‘St Augustine’ and the other by Ghirlandaio of ‘St Jerome’. Twenty-three species were identified, 15 on the Botticelli where the majority of the colony forming units (cfu) were Penicillium brevi-compactum Dierckx (61%) and P. purpurogenum Stoll (18%), and 13 on the Ghirlandaio where Aspergillus versicolor (Vuill.) Tiraboschi was the most common (74%), together with Cladosporium sphaerospermum Penz. (22%). The materials often used in restoration namely calcium caseinate, masonite and animal glue were tested and found to be suitable substrates for species of Aspergillus and Penicillium but not for Cladosporium cladosporioides and C. sphaerospermum.     

Group foraging by a coral reef fish: a mechanism for gaining access to defended resources

Solitary blue tang surgeonfish, Acanthurus coeruleus, are virtually excluded from feeding on algal mats defended by the dusky damselfish, Stegastes dorsopunicans. Foraging in groups enables blue tangs to overwhelm the defences of the damselfish and to feed in their territories. The rate of biting on algal mats by individual participants is positively correlated with group size, probably because individuals in large groups suffer attacks from damselfish less frequently. Experimental reduction of the density of damselfish on a small patch reef resulted in feeding rates by solitary surgeonfish equal to those of participants in large groups. This demonstrates that the positive association between foraging group size and participant bite rates on defended algal mats is due to the presence of the damselfish rather than to a reduction in time spent watching for predators. Overwhelming territory defenders by aggregating in groups is a habit widespread among fishes, suggesting that effective defence of a valuable resource may promote the evolution of a social behaviour pattern which permits access to an otherwise unobtainable resource.     

Degradation of agarophytic red algal cell wall components by new crude enzyme preparations

Several new crude enzyme preparations were isolated from a marine association of the agarolytic bacterium Cytophaga diffluens and the infusorium Uronema marinum, an axenic culture of Cytophaga diffluens, some species of land micro- and macromycetes adapted to assimilate red algal biomass and from the marine mollusc Littorina littorea. Fungal and mollusc enzyme preparations were shown to have cellulase, xylanase, protease and agarase activities. Fungal agarase activity was revealed only after 3–4 passages of the culture on the medium containing algal biomass. Enzyme preparations from the association and the pure bacterial culture growing on the medium with bactoagar as the sole carbon source contained only agarase activity. The maximum specific agarase activity was found in a preparation from the marine association. The preparations obtained can be used for isolating protoplasts and single cells from red seaweed thalli. This revised version was published online in June 2006 with corrections to the Cover Date.     

The impact of associated bacteria on morphology and physiology of the dinoflagellate Alexandrium tamarense

Despite their potential impact on phytoplankton dynamics and biogeochemical cycles, biological associations between algae and bacteria are still poorly understood. The aim of the present work was to characterize the influence of bacteria on the growth and function of the dinoflagellate Alexandrium tamarense. Axenic microalgal cultures were inoculated with a microbial community and the resulting cultures were monitored over a 15-month period, in order to allow for the establishment of specific algal–bacterial associations. Algal cells maintained in these new mixed cultures first experienced a period of growth inhibition. After several months, algal growth and cell volume increased, and indicators of photosynthetic function also improved. Our results suggest that community assembly processes facilitated the development of mutualistic relationships between A. tamarense cells and bacteria. These interactions had beneficial effects on the alga that may be only partly explained by mixotrophy of A. tamarense cells. The potential role of organic exudates in the establishment of these algal–bacterial associations is discussed. The present results do not support a role for algal–bacterial interactions in dinoflagellate toxin synthesis. However, variations observed in the toxin profile of A. tamarense cells during culture experiments give new clues for the understanding of biosynthetic pathways of saxitoxin, a potent phycotoxin.     

Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization

Prorocentrum is a common dinoflagellate genus along the Chinese seacoast, which frequently causes harmful algal blooms. Efforts to understand and prevent blooms caused by these harmful species require the development of methods for rapid and precise identification and quantification so that an adequate early warning of harmful algal blooms may be given. Here, we report the development and application of rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection of Prorocentrum micans. The hypervariable D1–D2 regions of a large subunit rDNA of a strain isolated from East China Sea identified as P. micans were first sequenced to design species-specific probes. Analysis of sequences identified as P. micans and deposited in GenBank revealed significant base differences among them and phylogenetic analyses revealed multiple clades within the taxon P. micans. Thus, it is likely that more than one taxonomic and genetically distinct entity has been identified as P. micans, if not misidentified. A series of probes were identified to one of these clades and tested for their specificity. Second, whole cell in situ hybridization procedures were established and the optimal probes were screened among the candidate probes. Next, cross-reactivity was performed to test the specificity of the probes and the detection reliability under various culture conditions, including different nutrient levels, temperatures, and light intensities. Finally, an improved protocol for natural samples was applied to the field material. The designed rRNA-targeted probe was specific, showing no cross-reactivity with other microalgae. The optimized detection protocol could be completed within 1.5 h. All target cells were speculated to be identified during all stages of their whole growth cycle under different culture conditions because the difference in fluorescence intensities throughout the experiment was not significant (p?>?0.05). The cell densities determined by FISH and light microscopy (LM) were comparable, without any significant difference (p?>?0.05) between them. In general, the established FISH probe was promising for specific, rapid, precise detection of a selected set of P. micans in natural samples and served as a good detection model for other Prorocentrum in the future.     

Evidence for the presence of non-receptor protein tyrosine kinases in algal cells

Two orders of green alga (Cladophorales and Charales) were investigated for the presence of protein tyrosine kinase activity. Proteins of 70 and 85 kDa were found to be tyrosine phosphorylated in Cladophora fracta, with an additional phosphorylated band evident at the 120-kDa region in Chara vulgaris, suggestive of the presence of putative tyrosine kinase activity in these algal species. A 70-kDa protein was immunoprecipitated from both species using a polyclonal antibody against non-receptor protein tyrosine kinase Syk. The protein was found to be phosphorylated on tyrosine, which was prevented upon pretreatment of algal cells with piceatannol. The extent of phosphorylation directly correlated with algal growth, suggesting a link between Syk kinase activity and growth signaling. These observations supported the presence of Syk-like kinase in the green algal species, which could have critical role in the algal growth and development.     

Flight paths of night-flying moths to light

The terminal portion of the flight paths of the corn earworm moth, Heliothis zea, to blacklights were studied on a moth flight pond and in the field. It was found that moths did not spiral into a lamp or fly directly at the light, but flew towards a region next to the lamp. It is suggested that near-the-light moths perceive a dark Mach band next to the lamp and fly towards this region in an effort to escape.     

Spatial associations among palatable and unpalatable macroalgae: A test of associational resistance with a herbivorous amphipod

Associational resistance is the process by which plants may gain protection from spatial associations with neighbouring plants. We tested whether association with an unpalatable alga, Dictyopteris acrostichoides, affects the abundance and colonisation behaviour of the herbivorous amphipod Peramphithoe parmerong on its preferred host alga Sargassum linearifolium. Despite predictions, natural densities on S. linearifolium when surrounded by D. acrostichoides were higher than on isolated individuals of S. linearifolium. Colonisation experiments in the laboratory and the field tested the hypotheses that the observed variation in field abundance with algal neighbourhood was due to variation in the size of habitat patches, physical obstruction of host finding by D. acrostichoides and variation in the relative abundance of S. linearifolium and D. acrostichoides. None of these possible mechanisms was found to significantly alter rates of amphipod colonisation on the scales of individuals selecting among algal pieces in the laboratory or among habitat patches in the field. The failure of colonisation processes to explain observed variation in natural amphipod densities suggests that post-colonisation processes such as survival or emigration may vary with the spatial associations among algae.     

Lageniastrum macrosporae (fossil Volvocales, Lageniastraceae nov. fam.), an endophyte in megaspores from the Carboniferous of the French Massif Central

Sublagenicula nuda lycophyte megaspores from the upper Visean of central France frequently host a colonial green alga as an endophyte. This association was first recorded more than 100 years ago by the French paleobotanist B. Renault, who introduced the name Lageniastrum macrosporae for the alga. However, the biological significance of the discovery was not fully assessed until recently. The L. macrosporae-S. nuda association represents the oldest compelling fossil evidence for algal endophytes in land plants, and the only example to date of an alga residing in the interior of spores of vascular cryptogams. Here we present a detailed re-evaluation and photographic documentation of the surviving original specimens from the Visean of Combres/Lay and Esnost. Moreover, a newly discovered specimen from the Stephanian of central France represents the first record of this association from the Upper Carboniferous. An emended diagnosis for L. macrosporae Renault, 1896 is provided, and a lectotype and paralectotype are designated. Although L. macrosporae displays a striking suite of morphological characters found in members of the extant chlorophyte family Volvocaceae (especially the genus Volvox), the peculiar biology of the fossil necessitates establishment of a new family, for which the name Lageniastraceae is proposed. Considerations of the adaptive advantages for the alga of occupying the interior of megaspores are offered. These include the possible effectiveness of the spores in protection during periods of desiccation and against plankton-feeding animals, and use of the spores as potential vectors for dispersal from one isolated body of water to another by spore-feeding animals.     

Nature and Role of Bacterial Contaminants in Mass Cultures of Thermophilic Chlorella pyrenoidosa

A study was made of bacterial contaminants isolated from an algal mass-culture unit. The study was performed specifically to determine the dependence of the size of bacterial population on algal density and the nature of any association of the contaminants with the algal cell. Growth of the bacterial contaminants on standard medium was also investigated. An estimate was made of the O₂ uptake of the bacterial population under normal operating conditions of the algal massculture system. Viable numbers of bacteria tended to increase with increased algal density. Bacteria were found imbedded in the surface of algal cells when the cultures of algae were characterized by subnormal rates of growth and photosynthetic gas exchange. Bacterial isolates failed to grow in standard medium alone, thus implying a dependency of bacterial growth on material(s) produced by the algae. A slight inhibitory effect on algal growth was noted in the case of two of three of the bacterial isolates. Manometric studies demonstrated that the bacterial population normally found in the algal cultures did not appreciably effect total gas exchange.     

Photobiont selectivity for lichens and evidence for a possible glacial refugium in the Ross Sea Region, Antarctica

Lichens are a symbiosis consisting of heterotrophic, fungal (mycobiont) and photosynthetic algal or cyanobacterial (photobiont) components. We examined photobiont sequences from lichens in the Ross Sea Region of Antarctica using the internal transcribed spacer region of ribosomal DNA and tested the hypothesis that lichens from this extreme environment would demonstrate low selectivity in their choice of photobionts. Sequence data from three targeted lichen species (Buellia frigida, Umbilicaria aprina and Umbilicaria decussata) showed that all three were associated with a common algal haplotype (an unnamed Trebouxia species) which was present in all taxa and at all sites, suggesting lower selectivity. However, there was also association with unique, local photobionts as well as evidence for species-specific selection. For example, the cosmopolitan U. decussata was associated with two photobiont species, Trebouxia jamesii and an unnamed species. The most commonly collected lichen (B. frigida) had its highest photobiont haplotype diversity in the Dry Valley region, which may have served as a refugium during glacial periods. We conclude that even in these extreme environments, photobiont selectivity still has an influence on the successful colonisation of lichens. However, the level of selectivity is variable among species and may be related to the ability of some (e.g. B. frigida) to colonise a wider range of habitats.     

Effects of the Caribbean threespot damselfish,Stegastes Planifrons (Cuvier), on algal lawn composition

Algal lawn communities within territories of herbivorous threespot damselfish, Stegastes planifrons (Cuvier) were studied in a shallow back-reef environment at La Parguera, Puerto Rico. Caging exclusion experiments, monitoring of non-manipulated lawns and gut content analyses were used to determine the effect of S. planifrons on algal species composition of the lawn. An average of 40 algal species per sampling period were found in naturally occuring lawns, with a total of 53 species being recorded throughout the course of study. Examination of algal species found in damselfish guts and comparison with natural lawns indicate that damselfish feed primarily on their algal lawns and graze the algae present in proportion to abundance. Caged algal lawns showed significant decline in algal species number and abundance over time. Non-selective feeding by Stegastes planifrons within their territorial lawns appears to result in an “intermediate disturbance” which maintains a greater algal species richness than is found in their absence.     

Interspecific biodiversity enhances biomass and lipid productivity of microalgae as biofuel feedstock

Large improvements in biomass and lipid production are required to make massive scale algal biodiesel production an economic reality. The application of the biodiversity strategy to enhance algal biomass as biofuel feedstock is little. The algal diversity was manipulated in this study to investigate the effects of a combination of biodiversity complementarity and a new medium consisting of seawater and agricultural fertilizer on lipid productivity. The algae diverse community includes two strains of Dunaliella salina (Dunaliella salina 19/30 and 19/18) and three species of Nannochloropsis (Nannochloropsis oculata, Nannochloropsis salina, and Nannochloropsis gaditana). The results showed that the most diverse community (5 species) produced an average of sixfold more biomass in the new medium than did the standard f/2 culture medium. The most diverse polyculture had the highest growth rate (1.01 day^?1), biomass (1.2 g L^?1), and lipid productivity (0.31 g L^?1 day^?1). The assessment of algal polycultures relative to monocultures is particularly interesting and novel for this biofuel field, and the observations that these polycultures resulted in significant lipid productivity improvements are very useful addition to the biofuel research. The possible mechanism (resource diversity) to explain the synergy in mixed cultures warrants further investigation.