A comparative study of animal erythrocyte agglutinins from marine algae

1. Fifteen marine algal species were analyzed for agglutinins to rabbit, sheep and human A, B and O blood group erythrocytes. 2. Protein extracts from all marine algae agglutinated rabbit erythrocytes, whereas twelve and five extracts agglutinated sheep and human erythrocytes, respectively. 3. The highest agglutination titers were consistently observed with rabbit erythrocytes. 4. Dictyota dichotoma strongly agglutinated human B blood group erythrocytes relative to A and O group erythrocytes. 5. Agglutination titer of rabbit erythrocytes by six algal extracts was not inhibited by mono- or polysaccharides, yet was reduced by glycoproteins.     

Analysis of the agglutinating activity from unicellular algae

Agglutinating activity often varies both between and within the algal species assayed. However, it is difficulty to interpret such variation without further analysis. We report a statistical analysis of agglutinating activities against human, cow, sheep, and pig erythrocytes, using cell extracts from 43 taxa (strains) of freshwater microalgae. Most of the extracts agglutinated erythrocytes from at least one of the sources, but pig erythrocytes appeared to be most suitable for the detection of agglutination reactions. Chlorella cell extracts preferentially agglutinated human erythrocytes, whereas extracts of other taxa were less active against mammalian erythrocytes. Cluster analysis generated four distinct subclusters of taxa, characterized by different specificities for antigens or carbohydrate receptors on the erythrocytes. Principal component analysis further separated the agglutination characteristics of Chlamydomonas from Chlorella on the first two components. Specificity for pig erythrocytes accounted for most of the clustering or grouping of algal taxa in multivariate analysis. However, clustering or grouping patterns of Chlorella species on haemagglutinating activity resembled that based on DNA sequences, revealing a possible genetic connection of agglutinins and their biochemical characteristics in algal cells. Variability of agglutination reactions among the algae investigated is simplified and interpreted most easily using multivariate analysis.     

Application of the functional-form model to the culture of seaweeds

Selecting the most appropriate species or strains is an important first step in the development of most algal cultivation systems and is usually a tedious, time-consuming, and expensive step. The functional-form model, first developed to synthesize the adaptive significance of easily assessed thallus-form attributes relative to the productivity and survival of benthic macroalgae, is applicable to the culture of seaweeds and can expedite species or strain selection. The production ecology aspects of the model are useful particularly for applications where the desired product is not species-specific, e.g., systems in which the emphasis is on algal production, such as algal biomass farms and wastewater treatment. A thallus-form with a high surface area: volume ratio is more suited for rapid production and nutrient uptake. The utility of this model to strain selection is demonstrated with the red alga Gracilaria tikvahiae, a species that has been considered a maricultural candidate for a number of utilizations. A continuum of surface area: volume ratios for eight clones of G. tikvahiae showed that this ratio decreased as morphological complexity increased and was a good predictor of both short-term photosynthesis and long-term growth rate. Clones near opposite ends of the surface area: volume ratio spectrum had significant differences for both photosynthesis and growth. Each clone of G. tikvahiae possesses concomitant combinations of benefits as well as costs, which should be carefully evaluated for the cultivation application of interest. Knowledge of functional-form relationships in seaweeds can significantly expedite their successful cultivation.     

A new screening for hemagglutinins from Vietnamese marine macroalgae

Aqueous extracts from 42 species of Vietnamese marine macroalgae, including 17 Chlorophyta, 22 Rhodophyta, and three Phaeophyta species, were examined for hemagglutination activity using native and enzyme-treated different animal and human erythrocytes. All extracts agglutinated at least one type of erythrocytes tested. Strong activity was detected in extracts from four Chlorophyta (Caulerpa serulata var. boryana, Caulerpa sertularioides f. longipes, Halimeda velasquezii, and Halimeda discoidea) and two Rhodophyta species (Gelidiella acerosa and Titanophora pulchra) with enzyme-treated rabbit and horse erythrocytes. The hemagglutinins of some active species were examined for sugar-binding specificity, pH, temperature stability, and divalent cation independency using ammonium sulfate precipitates prepared from their extracts. In a hemagglutination–inhibition test with various monosaccharides and glycoproteins, none of the hemagglutinins had affinity for monosaccharides. The activity of the hemagglutinins was inhibited by some glycoproteins tested. The inhibition profiles with glycoproteins were different depending on hemagglutinin species, suggesting the presence of lectins specific for complex N-glycans, high mannose N-glycans or O-glycans. On the other hand, the activities of almost all algal hemagglutinins were stable over a wide range of pH and temperature, and independent of the presence of divalent cations, except Gelidiopsis scoparia hemagglutinin, its activity was dependent on the presence of divalent cations. These results suggest that Vietnamese marine macroalgae may be good sources of useful lectins for many biological applications.     

Agglutination of human and animal erythrocytes in marine unicellular algae

Ethanol extracts of 12 marine unicellular algae were assayed for agglutinating activity against native and enzyme-treated human and animal erythrocytes. All of the algae assayed agglutinated at least one group of normal erythrocytes from humans. Notably, all algal extracts agglutinated erythrocytes of hemophilia patients arising from coagulation disorders. Meanwhile, all algae had a strong reaction with monkey erythrocytes, but to a lesser extent or not at all with sheep erythrocytes. Both trypsin and pronase improved the detection of most algal agglutinins and caused a drastic increase in hemagglutinating activity after treatment for 2 h or more. However, hemagglutinating activity decreased or disappeared completely when two extracts of different algal species were combined. Journal of Industrial Microbiology & Biotechnology (2000) 24, 262–266. Received 24 September 1999/ Accepted in revised form 06 January 2000     

温度对4种大型海藻氮磷吸收效率及光合生理特性的影响

研究以裂片石莼(Ulva fasciata)、肠浒苔(Ulva intestinalis)、龙须菜(Gracilaria lemaneaformis)、坛紫菜(Pyropia haitanensis)为实验材料, 分析了不同温度(15、20、25、30℃)下4种大型海藻对海水中N、P元素的吸收效率和光合特性的特点。结果显示: (1)4种大型海藻对水体N、P均有明显的吸收效果, 吸收能力高低依次为肠浒苔>裂片石莼>坛紫菜>龙须菜; (2)温度过高或过低都会限制藻类对N、P的吸收和正常生长, 同时降低4种藻的相对电子传递速率及光化学效率; (3)裂片石莼与肠浒苔的N、P吸收能力强, 且光合系统对温度耐受性高, 是实施养殖污水生物净化的良好材料; (4)4种海藻对水体中N、P营养盐的吸收在48h内基本完成, 实地应用中可考虑24—48h周期换水或采用流通循环式的培养模式, 以达到既促进藻类的生长又提高营养盐吸收效率的目的, 以避免藻体因营养缺乏引起负生长而造成二次污染。     

Investigation of Chondrus crispus as a potential source of new antifouling agents

The search for environment-friendly and non-toxic antifouling (AF) paint components has led to the investigation of natural products from seaweeds. The defence metabolites used by algae to deter unwanted epibiosis have potential for harnessing and use in AF applications. Crude algal extracts may provide a suitable mixture of compounds with AF potency. Crude ethanol extracts of the macroalgae Chondrus crispus (Rhodophyceae), from both dried and fresh sources were tested and compared using bioassays based on five marine bacterial strains, five phytoplankton strains and two macroalgae to assess the AF efficacy. Dried extract from the algae had a lower minimum inhibitory concentration at 25 μg mL⁻¹ against the growth of bacteria and phytoplankton species than that from the fresh source. Macroalgae tests indicated that the extracts had an anti-germination activity 25–50 μg mL⁻¹ against both Undaria pinnatifida and Ulva intestinalis spores. A field trial of AF paint incorporating crude extract indicated an initial AF potency lasting six weeks.     

Screening and preliminary characterization of hemagglutinins in Vietnamese marine algae

Extracts from 44 species of Vietnamese marine algae, including 15 Chlorophyta, 18 Rhodophyta and 11 Phaeophyta species, were examined for hemagglutination activity with a variety of different animal and human erythrocytes that were untreated or treated with enzymes. Almost all extracts showed activity toward at least one type of erythrocytes, although those from three Chlorophyta and two Rhodophyta species showed no hemagglutination with any type of erythrocytes examined. Strong activity was detected in extracts from two Chlorophyta (Anadyomene plicata and Avrainvillea erecta) and four Rhodophyta species (Gracilaria eucheumatoides, Gracilaria salicornia, Kappaphycus alvarezii, and Kappaphycus striatum) with enzyme-treated rabbit and sheep erythrocytes. The hemagglutinins of seven Chlorophyta and eight Rhodophyta species were examined for sugar-binding specificity, pH- and temperature-stability, and divalent cation-independency of hemagglutination using ammonium sulfate-precipitates prepared from their extracts. In a hemagglutination-inhibition test with various monosaccharides and glycoproteins, none of the hemagglutinins had affinity for monosaccharides, except the Codium arabicum and Gracilaria euchematoides hemagglutinins, whose activities were inhibited by both N-acetyl-d-galactosamine and N-acetyl-d-glucosamine. On the other hand, all of the hemagglutinins activities were inhibited by some glycoproteins. The inhibition profiles with glycoproteins were different depending on hemagglutinin species, and suggest the presence of lectins specific for high mannose N-glycans, complex N-glycans, or O-glycans. The activities of these algal hemagglutinins were stable over a wide range of pH and temperature, and independent of the presence of divalent cations. These results indicate that Vietnamese marine algae are a good source of novel and useful lectins.     

PHYLOGEOGRAPHY OF GRACILARIA TIKVAHIAE (GRACILARIACEAE,RHODOPHYTA): A STUDY OF GENETIC DISCONTINUITY IN A CONTINUOUSLY DISTRIBUTED SPECIES BASED ON MOLECULAR EVIDENCE1

Gracilaria tikvahiae, a highly morphologically variable red alga, is one of the most common species of Gracilariaceae inhabiting Atlantic estuarine environments and the Intracoastal Waterway of eastern North America. Populations of G. tikvahiae at the extremes of their geographic range (Canada and southern Mexico) are subjected to very different environmental regimes. In this study, we used two types of genetic markers, the chloroplast‐encoded rbcL and the nuclear internal transcribed spacer (ITS) region, to examine the genetic variability within G. tikvahiae, for inferring the taxonomic and phylogenetic relationships between geographically isolated populations, and to discuss its distributional information in a phylogeographic framework. Based on rbcL and ITS phylogenies, specimens from populations collected at the extreme distributional ranges reported for G. tikvahiae are indeed part of the same species; however, rbcL‐ but not ITS‐based phylogenies detected phylogenetic structure among the ten G. tikvahiae different haplotypes found in this study. The four distinct rbcL lineages were identified as 1) a Canadian–northeast U.S. lineage, 2) a southeast Florida lineage, 3) an eastern Gulf of Mexico lineage, and 4) a western Gulf of Mexico lineage. We found no evidence for the occurrence of G. tikvahiae in the Caribbean Sea. Observed phylogeographic patterns match patterns of genetic structures reported for marine animal taxa with continuous and quasicontinuous geographic distribution along the same geographic ranges.     

Lipid extracts from different algal species:1H and13C-NMR spectroscopic studies as a new tool to screen differences in the composition of fatty acids,sterols and carotenoids

One- and two-dimensional¹H- and¹³C-NMR spectra of lipid extracts fromUlva rigida, Gracilaria longa, Fucus virsoides andCodium tomentosum collected in the northern Adriatic Sea allowed screening of the content of fatty acid chains, carotenoids, free and acylated cholesterol and chlorophylls. The carotenoid-to-polyunsaturated fatty acid molar ratio was taken as a comparison parameter in samples ofUlva rigida collected in differentloci and seasons; the value was markedly higher in samples from the Lagoon of Venice than from marine coastal waters. The total cholesterol concentration was evaluated by¹H-NMR spectroscopy and similar values were found for all species. Two-dimensional heterocorrelated NMR spectroscopy was shown to give characteristic fingerprints of the lipid extracts from algal samples as regards the content in chlorophylls, unsaturated fatty acids and carotenoids.author for correspondence     

LECTIN-LIKE COMPOUNDS AND LECTIN RECEPTORS IN MARINE MICROALGAE: HEMAGGLUTINATION AND REACTIVITY WITH PURIFIED LECTINS1

We detected lectin-like compounds and lectin receptors in microalgae by hemagglutination, competitive inhibition with sugars, and reactivity with lectins isolated from other sources. Cell extracts from eight species of Dinophyceae and from one species each of Raphidophyceae and Bacillariophyceae exhibited hemagglutination toward trypsinized rabbit erythrocytes. In addition, the culture media of the dinoflagellate Alexandrium cohorticula and the raphidophyte Chattonella antiqua displayed similar hemagglutination. These activities were not inhibited by any monosaccharides or oligosaccharides tested but were inhibited by some specific glycoproteins. This suggests that the active factors were lectin-like compounds. Upon exposing intact, healthy cells of 12 species of Dinophyceae and one species each of Raphidophyceae, Cryptophyceae, Bacillariophyceae, and Chlorophyceae to lectins isolated from either macroalgae or terrestrial plants, most species were adversely affected. The negative effects included one or more of the following: impaired motility, disappearance of motility, agglutination, abnormal morphology, and cell rupture or lysis. Some species, even after freezing, thawing, and washing with saline solution, still agglutinated with macroalgal or terrestrial plant lectins. This study suggests that lectins and carbohydrate-containing lectin receptors may commonly occur on the cell surfaces of various species of microalgae.     

ARE “GREEN TIDES” HARMFUL ALGAL BLOOMS? TOXIC PROPERTIES OF WATER‐SOLUBLE EXTRACTS FROM TWO BLOOM‐FORMING MACROALGAE,ULVA FENESTRATA AND ULVARIA OBSCURA (ULVOPHYCEAE)

Toxic properties are more often associated with microalgal blooms than with macroalgal blooms, although herbivore deterrents are well known in macroalgae, and inhibition of invertebrate larval development by extracts from Ulva spp. has been suggested. This study was prompted from our observation that substantial discoloration of seawater occurred in small bays after mass desiccation-induced mortality of Ulvaria obscura (Kützing) Gayral. We examined the effects of extracts from Ulva fenestrata Postels et Ruprecht and Ulvaria obscura on Fucus gardneri Silva zygote development, growth of Ulva and Ulvaria , epiphytic algal accumulation, and oyster larval development. Fucus zygote development was inhibited by extracts from both species, although the effects of Ulvaria extracts were significantly greater. Epiphytic algal accumulation and the growth of Ulva and Ulvaria were inhibited by extracts from both species. Oyster larval development was arrested by the presence of extracts from each species. We conclude that extracts from both Ulva fenestrata and Ulvaria obscura have allelopathic properties. The effects are more widespread and occur at lower concentrations for extracts from Ulvaria than Ulva. These properties could alter competitive interactions by inhibiting germination or development of algae and invertebrates.     

Inter- and intraspecific variation of nuclear DNA reassociation kinetics in the Gracilariales (Rhodophyta)

DNA reassociation kinetics were used to determine inter- and intraspecific variation in genome organization and complexities in species ofGracilaria andGracilariopsis. Results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repeated sequences varied from 13–95%. Three geographic populations ofGracilaria tikvahiae were similar with 13–27% repeated sequences.Gracilaria sp. cultivars G-1 and G-6 with 35% and 95% repeated sequences, respectively, were distinct from each other andG. tikvahiae. No pattern of genome organization and complexity was found which permitted a distinction betweenGracilaria andGracilariopsis. Comparison of the percent of unique and repetitive sequences (U/R) indicated a wide range of ratios, withGracilaria tikvahiae populations having the highest values (2.7–7.3) andGracilaria sp. cultivar G-6,G. blodgettii andGracilariopsis lemanieformis the lowest (0.05–1.80). Unique component complexities varied one order of magnitude, from 10⁸ forGracilaria takvahiae to 10⁷ forGracilaria sp. cultivar G-6,G. blodgettii andGracilariopsis lemanieformis. Information for genome size, organization and complexity is used to develop a nuclear genome profile forGracilaria blodgettii andGracilariopsis lemanieformis which are characterized by commercial grade agars having high gel strengths (> 700 g cm^?2) and elevated melting temperatures (99 °C).     

Haemagglutinating and antibiotic activities of freshwater microalgae

We analysed the haemagglutinating activity of algal extracts from 44 species of freshwater microalgae against native and trypsin/papain-treated cow, pig, sheep, and human A-, B-, and O-type erythrocytes. Algal extracts obtained with aqueous ethanol exhibited higher haemagglutinating activity than those obtained with aqueous acetone. Most of the algal extracts agglutinated at least one of the erythrocyte types analysed. Human erythrocytes were the most sensitive of the cell types analysed. In the other species, the sensitivity of algal haemagglutinating activity for erythrocytes was pig > sheep > cow. Pre-treating erythrocytes with trypsin and papain improved the detection of most algal agglutinins and increased the haemagglutination titre; pre-treatment with papain was most effective for pig erythrocytes. Algal extracts stored at –20 °C for 4 months lost their haemagglutinating activity. Algal extracts also exhibited strong antibiotic activity against food pathogenic bacteria, especially against Bacillus. Our numerical taxonomy data showed that these microalgae might be grouped into several clusters according to their haemagglutinating activity. The detection of haemagglutinating activity may provide an efficient biochemical or physiological character to classify and differentiate microalgae. Our results suggest that freshwater microalgae might provide a potent source of haemagglutinins and antibacterial compounds for biochemical and medical studies and applications.     

A new survey of Brazilian marine algae for agglutinins

Aqueous protein extracts from 30 Brazilian marine algae were examined for haemagglutinating activity using native and enzyme-treated rabbit, chicken, sheep and human erythrocytes. Most extracts agglutinated at least one of the blood cells used. Sheep and rabbit erythrocytes were more suitable for detection of the agglutinating activity. The minimum protein concentration necessary to produce positive agglutination was usually lower with enzyme-treated erythrocytes than native ones. The five algal protein extracts showing the greatest haemagglutination titre were tested for sugar-binding specificity. Only the activity present in the green alga Cauler pacupressoides was inhibited by simple sugars and not by the glycoproteins tested. The activity of the other four extracts was inhibited by at least one of the glycoproteins utilised. This revised version was published online in August 2006 with corrections to the Cover Date.     

NITROGEN ALLOCATION AND STORAGE PATTERNS IN GRACILARIA TIKVAHIAE (RHODOPHYTA)1

Internal nitrogen pools in thalli of Gracilaria tikvahiae McLachlan were examined in three experiments as a function of total nitrogen content of the thallus, nitrogen deprivation, and nitrogen resupply. Amino acids and proteins appeared to form the major nitrogen storage pools in G. tikvahiae, while DNA appeared to be relatively unimportant in this regard. Inorganic nitrogen in the forms of NH₄⁺ and NO₃^? was found in the thalli; however, its contribution to the total nitrogen, pools was small. Within the protein pool, the phycoerythrin pigments appear important as a source of nitrogen when thalli are initially becoming nitrogen limited. In general, there was an inverse relationship between the levels of nitrogen and the carbohydrate content of the algal thalli.     

Allelochemicals produced by Caribbean macroalgae and cyanobacteria have species-specific effects on reef coral microorganisms

Coral populations have precipitously declined on Caribbean reefs while algal abundance has increased, leading to enhanced competitive damage to corals, which likely is mediated by the potent allelochemicals produced by both macroalgae and benthic cyanobacteria. Allelochemicals may affect the composition and abundance of coral-associated microorganisms that control host responses and adaptations to environmental change, including susceptibility to bacterial diseases. Here, we demonstrate that extracts of six Caribbean macroalgae and two benthic cyanobacteria have both inhibitory and stimulatory effects on bacterial taxa cultured from the surfaces of Caribbean corals, macroalgae, and corals exposed to macroalgal extracts. The growth of 54 bacterial isolates was monitored in the presence of lipophilic and hydrophilic crude extracts derived from Caribbean macroalgae and cyanobacteria using 96-well plate bioassays. All 54 bacterial cultures were identified by ribotyping. Lipophilic extracts from two species of Dictyota brown algae inhibited >50% of the reef coral bacteria assayed, and hydrophilic compounds from Dictyota menstrualis particularly inhibited Vibrio bacteria, a genus associated with several coral diseases. In contrast, both lipo- and hydrophilic extracts from 2 species of Lyngbya cyanobacteria strongly stimulated bacterial growth. The brown alga Lobophora variegata produced hydrophilic compounds with broad-spectrum antibacterial effects, which inhibited 93% of the bacterial cultures. Furthermore, bacteria cultured from different locations (corals vs. macroalgae vs. coral surfaces exposed to macroalgal extracts) responded differently to algal extracts. These results reveal that extracts from macroalgae and cyanobacteria have species-specific effects on the composition of coral-microbial assemblages, which in turn may increase coral host susceptibility to disease and result in coral mortality.     

Electron microscopy of human erythrocytes agglutinated by lectin from Codium fragile ssp. tomentosoides and pseudo-haemagglutinin from Ascophyllum nodosum

Human erythrocytes were exposed to extracts of Codium fragile ssp. tomentosoides, Ascophyllum nodosum, Dolichos biflorus seeds and a solution of purified polyphenols from Ascophyllum nodosum. In each case the erythrocytes appeared to agglutinate. Agglutinates were examined by scanning electron microscopy. Photomicrographs of the erythrocytes exposed to lectin-containing extracts of C. fragile and D. biflorus showed the typical appearance of agglutinated cells. Photomicrographs of the erythrocytes exposed to the crude extract and purified polyphenols from A. nodosum were similar to each other, but quite different in appearance to erythrocytes agglutinated by the lectins from C. fragile and D. biflorus. This evidence adds further support to the contention that lectin presence recorded in brown algal extracts is largely due to ‘pseudoagglutination’ produced by polyphenols contained in these extracts.     

NORTH CAROLINA MARINE ALGAE. IX. ONSLOWIA ENDOPHYTICA GEN. ET SP. NOV. (PHAEOPHYTA,SPHACELARIALES) AND NOTES ON OTHER NEW RECORDS FOR NORTH CAROLINA1

Four benthic algae are reported here for the first time in the North Carolina flora. The new brown algal genus and species, Onslowia endophytica Searles, is described as an endophyte of Halymenia floridana from the North Carolina continental shelf. New records of Boodleopsis pusilla and Naccaria corymbosa from North Carolina constitute range extensions of these tropical species on the American coast north from Florida. Blastophysa rhizopus, an endophyte and epiphyte known from the North Atlantic coast of Europe and America as well as the Caribbean is reported from North Carolina for the first time and in a new host, Predaea feldmannii.     

The Ecological Perspective of Microbial Communities in Two Pairs of Competitive Hawaiian Native and Invasive Macroalgae

Marine macroalgae are known to harbor large populations of microbial symbionts, and yet, microbe symbiosis in invasive macroalgae remains largely unknown. In this study, we applied molecular methods to study microbial communities associated with two invasive algae Acanthophora spicifera and Gracilaria salicornia and the two native algae Gracilaria coronopifolia and Laurencia nidifica at spatial and temporal scales in Hawaiian coral reef ecosystems. Bacterial communities of both the invasive and native macroalgae displayed little spatial and temporal variations, suggesting consistent and stable bacterial associations with these macroalgae. Results of this study identified three types of bacterial populations: nonspecific (present in both algal and water samples); algae-specific (found in all algal species); and species-specific (only found in individual species). The bacterial diversity of invasive algae was lower than that of their native counterparts at phylum and species levels. Notably, the vast majority (71 %) of bacterial communities associated with the invasive algae G. salicornia were representatives of Cyanobacteria, suggesting a potential ecological significance of symbiotic Cyanobacteria.