The influence of light quality on the development of the brown algae Petalonia and Scytosiphon

Petalonia fascia (O. F. Müll.) Kuntze, P. zosterifolia (Reinke) Kuntze and Scytosiphon lomentaria (Lyngb.) J. Ag. have been cultured in white, blue and red light of equal quantum irradiance at 15°C. Hairs, knot-filaments and Ralfsia-like crusts were formed only in blue light, whereas the prostrate system of red-grown plants consisted entirely of sparingly-branched and mostly uniseriate filaments. The production of erect thalli from the prostrate system was controlled or stimulated by red light, but these erect thalli became fertile only in the presence of blue light. All three species exhibited all of these types of response, although specific differences in the degree of certain types of response were observed.     

Comparison of the long-wave chlorophyll fluorescence in various green and blue-green algae and diatoms

Two types of long-wave fluorescence bands with similar band shape occur at room temperature in various algae: F_II700 and F_I715. F_II700 occurs in a limited number of algae, follows PS II transients, increases with culture age and is moderately increased by cooling to 83 K. F_I715 occurs in most algae, especially Anabaena, but much less in most diatoms and Tribonema. It does not follow PS II transients, does not increase with culture age and is much increased by cooling to 83 K.An interpretation for the characteristics of F_II700 and F_I715 is given.     

Taxonomic identification of algae (morphological and molecular): species concepts,methodologies, and their implications for ecological bioassessment

Algal taxonomy is a key discipline in phycology and is critical for algal genetics, physiology, ecology, applied phycology, and particularly bioassessment. Taxonomic identification is the most common analysis and hypothesis‐testing endeavor in science. Errors of identification are often related to the inherent problem of small organisms with morphologies that are difficult to distinguish without research‐grade microscopes and taxonomic expertise in phycology. Proposed molecular approaches for taxonomic identification from environmental samples promise rapid, potentially inexpensive, and more thorough culture‐independent identification of all algal species present in a sample of interest. Molecular identification has been used in biodiversity and conservation, but it also has great potential for applications in bioassessment. Comparisons of morphological and molecular identification of benthic algal communities are improved by the identification of more taxa; however, automated identification technology does not allow for the simultaneous analysis of thousands of samples. Currently, morphological identification is used to verify molecular taxonomic identities, but with the increased number of taxa verified in algal gene libraries, molecular identification will become a universal tool in biological studies. Thus, in this report, successful application of molecular techniques related to algal bioassessment is discussed.     

The vertical zonation of adpressed diatoms and other epiphytic algae on Phragmites australis

The seasonal colonization and vertical zonation of adpressed and upright algae on Phragmites stems in Lake Belau, Germany, was investigated from November 1988 to October 1991. During the autumns and winters from 1988 to 1990 the dominant adpressed species included the diatoms Epithemia adnata, E. sorex, E. turgida and Rhopalodia gibba, while during the same seasons in 1990 and 1991, Cocconeis placentula var. euglypta was abundant. The upright diatoms were most abundant on the middle sections of the stems, whereas those epiphytes with most of their cell surfaces directly in contact with the substratum constituted the greatest part of the biomass on the lowest sections of the stems. Epithemia spp. and R. gibba attained their highest abundances near the sediment as well as buried beneath the most dense periphyton aggregations, while there were dense populations of C. placentula var. euglypta on the entire surface of the stems. Cocconeis is able to tolerate higher light intensities, with photosynthetically active radiation (PAR) values averaging 38?μmol?m^?2?s^?1, than Epithemia and Rhopalodia, which settle at a significantly lower PAR (26 and 20?μmol?m^?2?s^?1, respectively). The change in dominance among the adpressed species from Epithemia and Rhopalodia to Cocconeis can be explained by changes in the grazing habits of the snail Acroloxus lacustris.     

Subunit organization of PSI particles from brown algae and diatoms: polypeptide and pigment analysis

P700 enriched fractions were isolated from two brown algae and one diatom using sucrose density centrifugation after digitinin solubilization. They had a Chl a/P700 ratio of about 250 to 375 according to the species, they were enriched in long-wavelength absorbing Chl a and exhibited a fluorescence emission maximum at 77 K near 720 nm. They all presented a major polypeptide component at 66±2 kDa, but their polypeptide composition was rather complex and somewhat different from one species to another. Further solubilization with dodecylmaltoside of those native PSI particles allowed the separation of two or three fractions. The lightest, xanthophyll-rich, fraction was identified to be a light-harvesting complex. It contained no P700 and had a major polypeptide of molecular weight near 20 kDa (at the same molecular weight than the respective LH native fraction of each species) and exhibited a 77 K peak fluorescence emission at 685 nm. The other fractions were enriched in P700 and almost entirely depleted in xanthophylls. When two of them are present, they both exhibited a major polypeptide at 66±2 kDa and were totally devoid of the LH polypeptide, but the two fractions widely differed one from another in the abundance and molecular weight of the other polypeptide components. The most purified of these two fractions presented a composition similar to PSI core complex from green plants.Abbreviations LH light-harvesting - LHCII light-harvesting complex II of green plants - P700 reaction center chlorophyll of PSI     

An optimized protocol for the production of high purity maltose

An economical protocol, which is simple, rapid and reproducible for the production of maltose by enzymatic hydrolysis of tapioca starch, has been optimized. The protocol involves liquefaction of 35% (w/w) tapioca starch by bacterial -amylase at 78±2°C to 3 to 5% (w/w) reducing sugars, followed by maximal (85±3% w/w maltose equivalent) saccharification with barley -amylase and pullulanase at 50°C for 24 to 30 h. The post-saccharification recovery protocol comprised decolourization by charcoal, de-dextrinization by denatured spirit precipitation, de-ionization by passage through cation and anion exchangers and dehydration by vacuum drying. A white crystalline maltose powder was obtained with specifications comparable to commercial high purity maltose. The protocol yields at least 60% (w/w) recovery of maltose and is suitable for use by the pharmaceutical industry. The protocol is unique in that it utilizes cheap and easily hydrolysed tapioca starch, leaves no mother liquor, enabling higher recovery of maltose, and allows almost quantitative recovery of limit maltodextrins, a value-added marketable by-product.     

An effective DNA extraction protocol for brown algae

Successful extraction of total DNA from brown algae, which are generally polysaccharide and polyphenol rich, is often problematic using current methods. Persistent polysaccharide and polyphenolic compounds can hinder further application of modern molecular techniques requisite to molecular‐based evolutionary studies. Our broad and long‐term research goals with fucalean taxa, especially Sargassum, and problems with existing DNA extraction methods were an impetus to develop a reliable DNA extraction method. Initial research established hexadecyltrimethylammonium bromide (CTAB) based total‐DNA methods as the most viable for further empirical development. Several constituents effective at either complexing secondary compounds or creating a reductive extraction environment were increased in concentration or added to the extraction buffer. These seemingly minor changes resulted in the creation of a highly reductive extraction buffer and effective total‐ DNA harvesting technique. We detail these modifications and demonstrate the reliability of the modified protocol with a variety of brown algae and tissue preservation methods. Such DNA is shown to be suitable for a variety of molecular techniques.     

Biomass, species composition and diversity of epipelic algae in mire pools

The biomass, species composition and diversity of epipelic algae in two small pools of contrasting physicochemical characteristics in Miyatoko Mire were studied in 1992 (pool 3 =site B4 and pool 50 =site D2). A total of 93 species and 67 species of epipelic algae occurred at sites B4 and D2, respectively. Considerable differences were observed between the two sites in the seasonal fluctuations of species number, biomass and dominant species. At site B4, little changed with species number during April–August and markedly increased in October, while biomass was largest in April and gradually decreased during June–October. Diatoms and desmids occupied 33–82% and 15–63% of total algal biomass, respectively. At site D2, species number and biomass were small in April just after the snow-thaw, and increased in June and decreased in August and October. Diatoms occupied 90–98% of total algal biomass. The species diversity was always higher at site D2 than B4. As a result of analyses of water chemistry in the two pools, pool B4 can be recognized as a habitat experiencing high disturbance frequency. It is predicted that pools experiencing frequent disturbance will have less epipelic algal biomass and diversity.     

Use of algae and other plants for monitoring rivers

Abstract A brief account is given of the methods developed for use of algae, bryophytes and angiosperms to monitor rivers and streams. The methods fall broadly into those based on features of populations of representative species, those based on part or all of the whole photosynthetic community and those based on various types of bioassay and ecotoxicological test. The methodology for using bioaccumulation of heavy metals, insecticides and other organic molecules is well developed and has been applied widely in western Europe for practical purposes. Coarse filamentous algae and bryophytes are especially useful, but some angiosperms are suitable, provided general information about bioaccumulation in the particular species is available; it is difficult to use periphyton. Other methods based on species include measurement of genetic tolerance, physiological approaches (chlorophyll: phaeophytin ratio, tissue N: P ratio and surface phosphatase assays) and observations on cyanobacterial and eukaryotic algal morphology. Among community-based methods for monitoring, measurement of biomass is used widely for phytoplankton, but seems of little use for benthos. The use of indices based on benthic communities, especially diatoms, has, however, been applied widely. The earlier indices based on diversity have been replaced almost entirely by ones integrating ecological information from component species. In addition, the authors have started to produce a diatom-based parallel to the macroinvertebrate-based RIVPACS by obtaining floristic data from ‘clean’ sites. Bioassays and ecological tests, mainly using algae or Lemna, are often carried out together with the other methods.     

Toxic Pseudo-nitzschia multistriata (Bacillariophyceae) from the Gulf of Naples: morphology,toxin analysis and phylogenetic relationships with other Pseudo-nitzschia species

The genus Pseudo-nitzschia includes several species capable of producing domoic acid, the causative agent of Amnesic Shellfish Poisoning. Some of these species have been recorded frequently in the Gulf of Naples. For one of the species, P. multistriata, which has been recurrently found in our sampling area since 1995, this is the first report for European waters. Here we provide further details on the fine structure of this species. Pseudo-nitzschia multistriata was the only one found to produce domoic acid among all the Pseudo-nitzschia species from the Gulf of Naples, and this finding raises the number of potentially toxic species in this genus to nine. Phylogenetic relationships among several Pseudo-nitzschia species were assessed using the hypervariable domains (D1–D3) of the large subunit (LSU) rDNA. The match between the phylogeny obtained and important taxonomic characters used in this genus are discussed. Results show that P. multistriata clusters with wider species lacking a central larger interspace in the raphe. Close genetic relationships were determined between P. fraudulenta and P. subfraudulenta, and between P. pungens and P. multiseries. Genetic differences among these pairs of species are comparable to those among isolates of P. pseudodelicatissima from the Gulf of Naples, indicating high intraspecific genetic diversity of Pseudo-nitzschia species in the relatively conserved LSU region. This could explain the problematic results obtained when testing a match between species-specific Pseudo-nitzschia LSU probes and our sequences.     

Studies on marine algae of the British Isles. 10. The genus Rhodymenia

Antarcticothamnion polysporum gen. et sp. nov. (Rhodophyceae: Ceramiaceae) is described from the South Shetland Is. and the Antarctic Peninsula. It differs from all previously described Ceramiaceae in vegetative structure: an indeterminate apex, which divides by alternating oblique septa, produces simple alternate-distichous determinate branches, while indeterminate branches are initiated on main axes in a verticillate arrangement. Reproductive structures are borne on modified indeterminate branch systems. Sporangia are polyhedrally divided. Spermatangia are formed in loose heads. Procarps are borne near the apex, but not always on the subapical cell, of a fertile branch. Two auxiliary cells may participate in the formation of a carposporophyte. A fusion cell is lacking and most cells of the gonimoblast develop into carposporangia, which are binucleate.

The suite of morphological characters exhibited by Antarcticothamnion sets this genus sufficiently apart from all previously described Ceramiaceae to warrant placement in its own tribe, Antarcticothamnieae trib. nov. It shares important features, however, with Callithamnieae, Compsothamnieae, and Ptiloteae.

Apical division is reviewed in the various tribes of Ceramiaceae. It is concluded that a pattern of alternating oblique septa is correlated with transitory or chronic structural imbalance resulting from a particular combination of timing and spatial sequence in the initiation and development of branches. The distribution of oblique apical division within Ceramiaceae is strongly correlated with features generally considered to be important at the level of tribe.

Phylogenetic relationships within Ptiloteae, a tribe characterized inter alia by oblique apical division, are suggested from an analysis of branching patterns. The validity of Falklandiella as a genus distinct from Dasyptilon is emphasized. Gymnothamnion and Tokidaea are removed from Ptiloteae, but without being assigned to another tribe. Tanakaella is removed from Sphondylothamnieae to Compsothamnieae, while Mazoyerella is removed from Compsothamnieae to Spermothamnieae.

Spongoclonium orthocladum A. et E. S. Gepp is discussed as a possible species of Antarcticothamnion, while an undescribed alga from the South Orkney Is. is definitely indicated as a second species of the genus.     

An optimized protocol for the in vitro generation and functional analysis of human PD1/PD-L1 signal

Programmed cell death-1 (PD1) is an inhibitory receptor expressed on the activated T and B cells. Binding of PD1 to its ligands, PD-L1 and PD-L2 has led to deliver an inhibitory signal into the activated T cells. Recently, blocking PD1/PD-L1 pathway has emerged as a new treatment paradigm across a broad spectrum of malignancies. Remarkable clinical responses of monoclonal antibodies specific for PD-1 or its ligands in patients with many different types of cancer, attracted several pharmaceutical companies and researchers to investigate the agents that block PD1/PD-L1 signal. The safety and efficacy of the agents are needed to examine in the preclinical studies. In this study, we optimized a facile and cost-effective protocol for in vitro generation and functional analysis of human PD1/PD-L1 pathway. Activation of CD8?+?CD279?+?T cell was performed by anti-CD3 and D28 antibodies and the recombinant PD-L1 was used for inactivation of T cells through PD1/PD-L1 pathway. In this protocol, T-cell cytokine production (IL-2 and IFN-γ) and proliferation assay confirmed that a measurable PD1/PD-L1 signal was generated. We expected that in vitro PD1/PD-L1 signal that has been optimized in this study will serve as a valuable protocol for preclinical studies involving PD1/PD-L1 pathway.     

An optimized protocol for isolation of soluble proteins from microalgae for two-dimensional gel electrophoresis analysis

Two-dimensional gel electrophoresis (2-DE)is a core proteomic technique to studyprotein expression and function in livingorganisms. Although it has been extensivelyused for investigation of bacterial, yeast,animal and plant tissue cells, there islimited information about the use of 2-DEin microalgal research. In this study, anumber of key chemical reagents, includingacetone, trichloroacetic acid, urea,thiourea, dithiothreitol, and tributylphosphine, were quantitatively evaluatedfor 2-DE of green microalgae, using Haematococcus pluvialis as a model system.The goal was to maximize the number andstaining intensity of protein spots whileminimizing streaking and smearing on thesecond dimensional SDS gel. Compared tonon-frozen immobilized pH gradients (IPG)strips, freezing of the IPG strips at –20 ^°C after isoelectric focusing (IEF)enhanced protein resolubilization andtransfer into the SDS gel, and thusimproved resolution while eliminatingvertical point streaking on the SDS gel. Itwas also confirmed that manipulation ofsample loading capacity is a simple,effective purification strategy forselective investigation of the proteins ofinterest and of varying abundances. Theprotocol was also successfully applied toprofiling protein expression in H.pluvialis under external stressconditions, indicating its potentialusefulness in further proteomics studies ofthis organism and related species.     

Is genus or species identification of periphytic diatoms required to determine the impacts of river regulation?

As periphytic diatoms are widely used for biological assessment of rivers, a comparison was made of the use of information at genus or species levels to test the effects of river regulation in eastern Australia. 74 species from 30 genera were recorded from 10 regulated and 13 unregulated sites. The interpretation of the effects of eight dams and weirs on the periphytic diatom assemblages gave broadly similar results whichever taxonomic level was used. The similarity of the efficiency of using data at either taxonomic level is probably related to the fact that the majority of genera have few species. It is suggested that the small number of species within each genus effectively reduces the chances of differing species responses to environmental disturbance negating the response at the genus level. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fourteen FITC-conjugated lectins as a tool for the recognition and differentiation of some harmful algae in Chinese coastal waters

In order to test the use of lectins as a tool for the differentiation of harmful algal species, 13 species and 23 strains of algae were tested with 14 fluorescein isothiocyanate (FITC)-conjugated lectins, and the results examined using flow cytometry (FCM), epifluorescence microscopy (EFM) and spectrofluorometry (SFM). The lectin probes SBA, WGA, GSL I, DBA and PHA-E could distinguish between morphologically similar Gymnodinium-like species, such as Karenia mikimotoi (GMDH01), Takayama pulchellum (TPXM01) and Gymnodinium sp. (GspXM01), by their different binding activities. With the precise quantitative measurements of binding obtained using SFM and FCM, lectins appeared to be useful in distinguishing different strains of the same species. The results also showed that PHA-E could differentiate Alexandrium tamarense (ATDH04) from other strains of this species, and SJA could distinguish A. tamarense (ATMJ02) from other strains of this species (including ATMJ01). Similarly, PNA could identify A. tamarense (ATDH01, 02, 03); UEA I could recognize A. tamarense (ATCI01-JN, ATCI01); and RCA120 could differentiate Alexandrium sp (AspGX01) from strain AspGX02, which was shown to produce different levels of paralytic shellfish poisoning toxin. Lectin probes could also bind these target cells in mixed algal samples. Positive cells identified by FCM were clearer than negative cells thus, in EFM, both GspXM01 and TPXM01 labeled with a WGA lectin probe could be distinguished from target cells of K. mikimotoi, Prorocentrum donghaiense and P. minimum (PMDH01, PMXM01) in mixed algal samples. FCM, EFM and SFM analysis could clearly distinguish lectin-probe-bound cells from negative cells in culture.     

Relationships between lakewater pH and sedimentary diatoms in the Matamek Watershed,Northeastern Quebec,Canada

Twenty lakes in the Matamek and nearby watersheds were sampled in the summer of 1983 for water chemistry and surficial sediments. Thirty-two physical and chemical variables, including pH, alkalinity, cations and metal concentrations were measured on samples from the epilimnion and hypolimnion of each lake. In three lakes, two to four replicate cores were collected to estimate spatial variability of the sediment flora.All lakes were acidic (pH 4.59 to 5.80), highly colored and poorly buffered. Aluminum and magnesium concentrations reached 494 and 70 µg l^-1, respectively. The pH of the lakes appears to be declining, as indicated by a comparison of our results with those from a survey done in 1970. An empirical chemical model based on the alkalinity/sulfate ratio and the regression of pH on calcium also indicated that these lakes may be undergoing acidification.Analyses of the diatom flora of the surficial sediments showed strong dominance of 6 species that cooccurred with a large number of rare species (in all, 229 taxa were found). Variability among samples within a lake was as high as among lakes. Calculation of Nygaard's alpha index for each sample and the regression of its log-transformed value on surface pH yielded a relationship that was significantly from other published models. The regression model was applied to a down-core analysis of the diatom flora of the sediments of two lakes. Although the large confidence intervals on pH values predicted by the model obscured any evidence of pH change with sediment depth, there was a significant increase of acidophilous and a decrease of circumneutral species over time, suggesting that a change in the flora, possibly correlated with pH, is taking place.In making comparisons among lakes, the surficial-sediment flora did not provide clear evidence of a relationship with the pH of surface water. Indices computed from the surficial-sediment flora are apparently insensitive to differences in pH over a narrow range, particularly when the lakes being compared are similar chemically. Nygaard's alpha index is shown to be unduly sensitive to outliers. The currently accepted assumption of a progressive linear change in communities may be inadequate for the quantification of acidification processes. Other models, derived from catastrophe theory, may prove more fruitful.     

A CARD-FISH protocol for the identification and enumeration of epiphytic bacteria on marine algae

A CARD-FISH protocol was developed and applied to analyse surface-associated bacteria on the marine algae Ulva lactuca, Delisea pulchra, Corallina officinalis, Amphiroa anceps, Porphyra sp. and Sargassum linearifolium. The combination of Alexa(546)-labelled tyramide as the reporter molecule with SYBR Green II counterstain allowed for superior detection of the hybridised probe fluorescence against plant tissue from which pigment autofluorescence has been reduced.     

Functional studies of CpSRP54 in diatoms show that the mechanism of thylakoid protein insertion differs from that in plants and green algae

The chloroplast signal recognition particle 54 kDa (CpSRP54) protein is a member of the CpSRP pathway known to target proteins to thylakoid membranes in plants and green algae. Loss of CpSRP54 in the marine diatom Phaeodactylum tricornutum lowers the accumulation of a selection of chloroplast-encoded subunits of photosynthetic complexes, indicating a role in the co-translational part of the CpSRP pathway. In contrast to plants and green algae, absence of CpSRP54 does not have a negative effect on the content of light-harvesting antenna complex proteins and pigments in P. tricornutum, indicating that the diatom CpSRP54 protein has not evolved to function in the post-translational part of the CpSRP pathway. Cpsrp54 KO mutants display altered photophysiological responses, with a stronger induction of photoprotective mechanisms and lower growth rates compared to wild type when exposed to increased light intensities. Nonetheless, their phenotype is relatively mild, thanks to the activation of mechanisms alleviating the loss of CpSRP54, involving upregulation of chaperones. We conclude that plants, green algae, and diatoms have evolved differences in the pathways for co-translational and post-translational insertion of proteins into the thylakoid membranes.