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Microbial colonization and competition on the marine alga Ulva australis.

Pseudalteromonas tunicata and Roseobacter gallaeciensis are biofilm-forming marine bacteria that are often found in association with the surface of the green alga Ulva australis. They are thought to benefit the plant host by producing inhibitory compounds that are active against common fouling organisms. We investigated factors that influence the ability of P. tunicata and R. gallaeciensis to attach to and colonize the plant surface and also the competitive interactions that occur between these organisms and other isolates from U. australis during biofilm formation on the plant surface. A surprisingly high number of P. tunicata cells, at least 10(8) cells ml(-1), were required for colonization and establishment of a population of cells that persists on axenic surfaces of U. australis. Factors that enhanced colonization of P. tunicata included inoculation in the dark and pregrowth of inocula in medium containing cellobiose as the sole carbon source (cellulose is a major surface polymer of U. australis). It was also found that P. tunicata requires the presence of a mixed microbial community to colonize effectively. In contrast, R. gallaeciensis effectively colonized the plant surface under all conditions tested. Studies of competitive interactions on the plant surface revealed that P. tunicata was numerically dominant compared with all other bacterial isolates tested (except R. gallaeciensis), and this dominance was linked to production of the antibacterial protein AlpP. Generally, P. tunicata was able to coexist with competing strains, and each strain existed as microcolonies in spatially segregated regions of the plant. R. gallaeciensis was numerically dominant compared with all strains tested and was able to invade and disperse preestablished biofilms. This study highlighted the fact that microbial colonization of U. australis surfaces is a dynamic process and demonstrated the differences in colonization strategies exhibited by the epiphytic bacteria P. tunicata and R. gallaeciensis.

Bacterial Adhesion↗

Positive selection and relaxed purifying selection contribute to rapid evolution of sex-biased genes in green seaweed Ulva.

BACKGROUND: The evolution of differences in gamete size and number between sexes is a cornerstone of sexual selection theories. The green macroalga Ulva, with incipient anisogamy and parthenogenetic gametes, provides a unique system to investigate theoretical predictions regarding the evolutionary pressures that drive the transition from isogamy to anisogamy, particularly in relation to gamete size differentiation and sexual selection. Its minimal gamete dimorphism and facultative parthenogenesis enable a rare window into early evolutionary steps toward anisogamy. RESULTS: By analyzing the expression profiles of sex-biased genes (SBGs) during gametogenesis, we found that SBGs evolve faster than unbiased genes, driven by higher rates of non-synonymous substitution (dN), indicating that SBGs are under stronger selective pressures. Mating type minus-biased genes (mt-BGs) exhibit higher dN/dS values than mating type plus-biased genes (mt+BGs), suggesting stronger selective pressures on mt-BGs, although this difference was not statistically significant (P = 0.08). Using branch-site and RELAX models, we found positive selection and relaxed purifying selection acting on a significant proportion of SBGs, particularly those associated with flagella function. CONCLUSIONS: This study highlights the selective pressures shaping anisogamy and provides insights into the molecular mechanisms underlying its evolution. The faster evolution of SBGs, particularly mt-BGs, and the positive selection on genes associated with motility, such as those related to flagella function, suggest the importance of enhanced gamete motility in the transition to anisogamy. These findings contribute to our understanding of sexual selection and the evolutionary forces that drive the differentiation of gamete size and number between sexes.

Selection, Genetic↗

Selective toxic effects of polyunsaturated fatty acids derived from Ulva fasciata on red tide phyotoplankter species.

Alpha-linolenic acid and linoleic acid isolated from Ulva fasciata showed toxic effects on red tide phytoplankters in a concentration-dependent manner. Among six species tested, raphidophycean flagellate Heterosigma akashiwo was the most susceptible to these fatty acids, and 50% lethal concentrations (LC50) of alpha-linolenic acid and linoleic acid were estimated to be 0.58 and 1.91 microg/ml respectively, whereas dinoflagellate Gymnodinium impudicum and Heterocapsa circularisquama were highly resistant and no significant toxic effects were observed up to 1,000 microg/ml. Both fatty acids were less toxic to fish (devil stinger), zooplankters (brine shrimp and rotifer), and mammalian cell lines (U937, HeLa, Vero, and CHO cells) than H. akashiwo.

Animals↗

Isolation and structure determination of algicidal compounds from Ulva fasciata.

Thirty-seven species of seaweeds including 10 Chlorophyta, 13 Phaeophyta, and 14 Rhodophyta collected from the coast of Nagasaki Prefecture, Japan, were screened for algicidal activity against the red-tide phytoplankton Heterosigma akashiwo. The green alga Ulva fasciata (Ulvaceae, Chlorophyta) showed the strongest algicidal activity among the seaweeds tested. Bioassay-guided fractionation of the methanol extract of U. fasciata led to isolation of three algicidal compounds whose structures were determined to be hexadeca-4,7,10,13-tetraenoic acid (HDTA), octadeca-6,9,12,15-tetraenoic acid (ODTA), and alpha-linolenic acid on the basis of spectroscopic information. These polyunsaturated fatty acids (PUFAs) showed potent algicidal activity against H. akashiwo (LC(50) 1.35 microg/ml, 0.83 microg/ml, and 1.13 microg/ml for HDTA, ODTA, and alpha-linolenic acid, respectively), and the result demonstrated the potential of these PUFAs for practical harmful algal bloom control.

Chlorophyta↗

Fouling coverage of a green tide alga, Ulva pertusa on some antifouling test surfaces exposed to Ayagin harbor waters, east coast of South Korea.

Toxic antifouling chemicals released into the seawaters leads to marine environmental degradation. In order to identify a nontoxic antifoulant, an assessment of antifouling (AF) efficacy of some AF candidates was made at Ayagin harbor, east coast of South Korea. In this static panel study conducted during October 2000-March 2001, some commercial antifoulants, seaweed and seagrass extracts were screened. On panel surfaces coated with a seaweed extract, Ishige okamurae exhibited effective AF activity. Ulva pertusa was encountered as a 'monospecific' fouler with fairly high fouling coverage on many of the test panel surfaces. In recent years the increased influx of inorganic pollutants in the coastal waters causes exorbitant growth of fouling marine algae found all along the Korean peninsula. Especially, a cosmopolitan ship fouling alga U. pertusa occur with high abundance. It was largely suggested that the proposed international ban on the toxic antifoulant tributyltin (TBT) had significant effect on the 'green tide' phenomenon occurring in different parts of the world. However, it appears that Korean scenario of 'green tide' is a localized. Antifouling efficacy of some AF coatings and fouling coverage of a green tide alga, U. pertusa are discussed.

Animals↗

[Effects of Ulva pertusa and Gracilaria lemaneiformis on growth of Heterosigma akashiwo (Raphidophyceae) in co-culture].

We studied the effects of fresh tissue and culture medium filtrate of two species of macroalgae, Ulva pertusa (Chlorophyta) and Gracilaria lemaneiformis (Rhodophyta) on growth of Heterosigma akashiwo (Raphidophyceae) in co-culture. Both U. pertusa and G. lemaneiformis, and especially their fresh tissues, significantly impede the growth of H. akashiwo. Carbonate limitations and the presence of environment bacteria are not necessary for the negative effects of macroalgal on H. akashiwo. The simultaneous nutrient assays show that nitrate and phosphate are almost exhausted in the G. lemaneiformis co-culture system, but remain at acceptable levels in the U. pertusa system, when all cells of H. akashiwo are completely dead. When f/2 medium is supplied daily to G. lemaneiformis culture, the growth of H. akashiwo is greatly inhibited but not completely terminated. Furthermore, different amounts of fresh seaweed tissue, and culture medium filtrate prepared from different macroalgal concentrations are analyzed to determine their effects on the growth of H. akashiwo. The results show a positive correlation between the initial macroalgal concentration and the negative effects they exert on the co-cultured microalgae. Results suggest that the allelopathic effects of U. pertusa may be essential for negative effects on H. akashiwo; however, the combined roles of allelopathy and nutrient competition may be responsible for the negative effect of G. lemaneiformis the release of allelochemicals by U. pertusa.

Biodegradation, Environmental↗

[Purification of plastocynin from Ulva pertusa by column chromatography and analysis of its N-terminal amino acid sequence].

Protein plastocyanin from a green alga, Ulva pertusa, has been purified. Samples were homogenized in 0.02 mol/L phosphate buffer (pH 7.2) and then centrifuged to remove debris and subjected to ammonium sulfate fractionation (40%-80% saturation). Ion exchange column chromatography with DEAE-Sepharose Fast Flow and gel filtration column chromatography with Sephadex G-75 were then employed for further purification of plastocyanin. Three peaks, A, B and C, were eluted with 0.01 mol/L phosphate buffer, containing a NaCl linear gradient from 0 to 1.0 mol/L at the flow rate of 32 mL/h through DEAE-Sepharose chromatography. The protein fractions containing the plastocyanin were then purified further with Sephardex G-75 column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophores (SDS-PAGE) is analysis indicates that the protein was purified to homogeneity and its relative molecular mass is 10,000. N-terminal amino acid sequence was used to identify the protein. The protein was transblotted to PVDF membrane and N-terminal amino acid sequence was performed via Edman degradation with an automated amino acid sequencer. The 20 N-terminal amino acid residues are AAIVKLGPDDGSLAFVPSKI, which share 85% homology with the 20 N-terminal amino acid sequence of U. prolifera and U. arasakii, and share 90% homology with the ones of U. pertusa formerly reported.

Chlorophyta↗

Biologically active steroid from the green alga Ulva lactuca.

The marine green alga, Ulva lactuca, was shown to contain 3-O-beta-D glucopyranosyl-stigmasta-5,25-dien. The structure of the compound was established on the basis of its spectroscopic data and it was extracted for the first time from this alga. The topical antiinflammatory activity of this compound was examined using the mouse ear oedema assay as an experimental model of topical inflammation. Also, the antimicrobial activity of the isolated compound was tested against 10 various microorganisms (G+, G-, fungi and yeast strains).

Administration, Topical↗

Cardiac inhibitory action of constituents of the marine green alga Ulva pertusa.

As part of a search for substances from marine organisms, which exhibit inotropic effects, the chemical constituents of the marine green alga, Ulva pertusa were investigated. The fractionated extract was tested for inotropic effects on the isolated guinea pig atria. The aqueous layer obtained from the acetone extract of fresh algae was concentrated, and the residue was extracted with methanol. The methanolic extract was fractionated by chromatography using mixtures of aqueous methanol. Elution with 50% aqueous methanol afforded material that had a significant negative inotropic effect. Further purification of this material by high-performance liquid chromatography (HPLC) using methanol-water (2:5) afforded crystalline adenosine, which was shown to be the active substance of U. pertusa, causing a negative inotropic action.

Adenosine↗

Metal accumulation in a biological indicator (Ulva rigida) from the lagoon of Venice (Italy).

Ulva rigida (C. Agardht) was collected from the Palude della Rosa (lagoon of Venice), a particular area characterized by a salinity gradient, from December 1991 to June 1993. Metal contents (Al, Mn, Fe, Cu, Zn, Cr, Co, Ni, Cd, and Pb) were determined by atomic absorption spectrophotometry and statistically analysed for differences between months and sampling sites. The mean seasonal variability is very significant (p < 0.001) for all studied metals. Seasonal trends are comparable for groups of metals according to possible functional similarities. A significant positive correlation was calculated for the pairs Fe-Al and Fe-Pb. Mechanisms regulating metal uptake are discussed, particularly the role of Fe and its linear relationship with Al. Metal concentrations in relation to salinity also were studied. Larger amounts of Fe, Zn, and Cd accumulated in correspondence of lower salinity, may be owing to both higher metal activity and burden of bioavailable metals in freshwater flowing into the Palude. Calculation of concentration factor (CF) indicates that U. rigida specifically accumulates Fe. CF linearly correlates with dissolved metal concentrations, suggesting that U. rigida is a good bioindicator of metal bioavailability, although seasonal active accumulation occurs in some cases (Mn). Metal concentrations determined in this macroalga support the conclusion that the studied area is rich in bioavailable Fe and poor in toxic metals. In any case, a great affinity for Al in U. rigida may be proposed.

Chlorophyta↗

Observations of mechanisms of attachment in the green alga Ulva mutabilis Føyn. An ultrastructural and light microscopical study of zygotes and rhizoids.

The development of the rhizoid cells of the green alga Ulva mutabilis was investigated at the ultrastructural level paying special attention to the mechanism of attachment of the plant. Cytochemical data concerning the initial settling of the early zygote are also given. On the basis of histochemical staining and enzyme treatment it is concluded that the adhesive material secreted by the rhizoid cells is chemically different from that secreted by the zygote during the initial settling of the alga.

Cell Division↗

Requisite Morphologic Interaction for Attachment between Ulva pertusa (Chlorophyta) and Symbiotic Bacteria.

: In order to understand the morphogenesis-inducing mechanism of Ulva pertusa by symbiotic bacteria, we observed the requisite conditions of bacteria for attachment to U. pertusa for algal morphogenesis. Non-morphogenesis-inducing bacterial mutants derived by ultraviolet irradiation did not attach onto the surface of this alga. Scanning electron microscopic observation during the process of morphogenesis in U. pertusa revealed a network-like structure formed on the algal surface within 1 week after application of bacteria. The bacteria attached onto the alga after 2 weeks of incubation. After this attachment process the morphologic change was observed in U. pertusa.

Journal Article↗

Application of the sequential elution technique to determine Cd and Cu cellular location in Ulva lactuca Linnaeus.

The sequential elution technique, used to determine the cellular location of metals in nonvascular plants, was adapted to the seaweed Ulva lactuca Linnaeus for the heavy metals Cu and Cd. The optimum conditions for the extraction of extracellular Cu and Cd were determined using specimens previously loaded with these metals. The best extracellular metal extraction media and the number of extracellular metal extraction steps obtained were the following: Two steps (1 h + 40 min) in a filtered sea water-20 mM Ni solution for Cd and two steps in a filtered sea water-50 mM Co solution for Cu. To find these extraction conditions the extracellular metal extraction capacity of the different media used as well as the alteration provoked in plasmalemma permeability were compared. The concentrations of the incubation solutions of Cd and Cu that allowed reliable comparisons between the different extracellular metal extraction media used were previously determined.

Cadmium↗

Non-photosynthetic enhancement of growth by high CO2 level in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta).

The effects of increased CO2 levels (10,000 microl l(-1)) in cultures of the green nitrophilic macroalga Ulva rigida C. Agardh were tested under conditions of N saturation and N limitation, using nitrate as the only N source. Enrichment with CO2 enhanced growth, while net photosynthesis, gross photosynthesis, dark respiration rates and soluble protein content decreased. The internal C pool remained constant at high CO2, while the assimilated C that was released to the external medium was less than half the values obtained under ambient CO2 levels. This higher retention of C provided the source for extra biomass production under N saturation. In N-sufficient thalli, nitrate-uptake rate and the activity of nitrate reductase (EC 1.6.6.1) increased under high CO2 levels. This did not affect the N content or the internal C:N balance, implying that the extra N-assimilation capacity led to the production of new biomass in proportion to C. Growth enhancement by increased level of CO2 was entirely dependent on the enhancement effect of CO2 on N-assimilation rates. The increase in nitrate reductase activity at high CO2 was not related to soluble carbohydrates or internal C. This indicates that the regulation of N assimilation by CO2 in U. rigida might involve a different pathway from that proposed for higher plants. The role of organic C release as an effective regulatory mechanism maintaining the internal C:N balance in response to different CO2 levels is discussed.

Algal Proteins↗

Cell-wall polysaccharides from the marine green alga Ulva "rigida" (Ulvales, Chlorophyta)--NMR analysis of ulvan oligosaccharides.

In order to obtain information on sugar sequences in the water soluble polysaccharides from Ulva "rigida", oligosaccharides were purified by anion exchange and gel permeation chromatography from the partial acid hydrolysate of the native or desulfated ulvan. The chemical structure and sugar sequence of these oligomers were determined by 1H and 13C NMR spectroscopy to be alpha-L-rhamnosyl (1-->4) D-xylose, beta-D-glucuronosyluronic acid (1-->2)-alpha-L-rhamnosyl (1-->4) D-xylose, beta-D-glucuronosyluronic acid (1-->4) L-rhamnose 3 sulfate, beta-D-glucuronosyluronic acid (1-->4) [beta-D-glucuronosyluronic acid (1-->2)] L-rhamnose and beta-D-glucuronosyluronic acid (1-->4) [beta-D-glucuronosyluronic acid (1-->2)] alpha-L-rhamnosyl (1-->4) D-xylose. The sugar linkages and particularly the branching and the sulfate position were in total agreement with previous chemical results.

Carbohydrate Conformation↗

Seasonal and local variation of Cr, Ni and Co concentrations in Ulva rigida C. Agardh and Enteromorpha linza (Linnaeus) from Thermaikos Gulf, Greece.

Cobalt, nickel and chromium concentrations were determined in Enteromorpha linza and Ulva rigida in seawater and sediment after seasonal collection from Thermaikos Gulf, Greece. Industrial waste and sewage is poured into this Gulf. The relative abundance of metals in the seaweeds and seawater decreased in the order Ni > Cr > Co, whereas in the sediment the order was Cr > Ni > Co. The two seaweeds bioaccumulated similar metal concentrations. The seasonal variation of metals in the two algae was not significant, but seemed to depend on the growth dynamics, the age of the tissue, the metal concentrations in the environment and the abiotic factors (such as salinity and temperature). Only Cr concentrations in E. linza showed significant local variation which depended on the area into which the industrial and domestic wastes were discharged and on their transport by sea currents. Nickel and chromium concentrations in U. rigida and Cr concentrations in E. linza displayed significant positive correlation with their concentrations in the seawater. Concentrations of the three metals in the seaweeds and seawater of Thermaikos Gulf generally exceeded the 'background' levels.

Journal Article↗

Fine chemical structure analysis of oligosaccharides produced by an ulvan-lyase degradation of the water-soluble cell-wall polysaccharides from Ulva sp, (Ulvales, Chlorophyta).

A marine bacterium degrading the water-soluble cell wall polysaccharides from Ulva sp. (ulvan) has been isolated. The good correlation between ulvan degradation monitored by reducing-power, UV absorbance and viscosimetry, indicated that the crude enzymatic extract contains essentially an endo-ulvan lyase activity. This activity was rapidly inhibited by the reaction products which consisted of a series of ulvanobiouronic acid A 3-sulfate [-->4)-beta-D-GlcpA-(1-->4)-alpha-L-Rhap 3-sulfate-(1-->]n with 4-deoxy-L-threo-hex-4-enopyranosiduronic acid at the non-reducing end. Other deviant repeating structures with beta-D-Xylp or alpha-IdopA replacing beta-D-GlcpA in the repeating ulvanobiouronic acid disaccharide and the presence of two consecutive (1-->4) linked beta-D-Glc pA demonstrated the great variability and complexity of ulvan chemical structure.

Carbohydrate Conformation↗

NMR spectroscopic characterisation of oligosaccharides from two Ulva rigida ulvan samples (Ulvales, Chlorophyta) degraded by a lyase.

The chemical structure and the sequence of repeating units in ulvans of similar compositions from two different Ulva rigida samples collected in the Canary Islands and in Brittany were studied after ulvan-lyase degradation and NMR spectroscopic analysis of the reaction products. Both ulvans were composed of ulvanobiuronic acid 3-sulfate type A [-->4)-beta-D-GlcA-(1-->4)-alpha-L-Rha 3-sulfate-(1-->] (symbolised as A3s) and contained disaccharides composed of [-->4)-beta-D-Xyl-(1-->4)-alpha-L-Rha 3-sulfate-(1-->] and [-->4)-beta-D-Xyl 2-sulfate-(1-->4)-alpha-L-Rha 3-sulfate], respectively referred to as ulvanobiose 3-sulfate (U3s) and ulvanobiose 2',3-disulfate (U2's,3s). In the Canary Islands sample, these U3s and U2's,3s occurred dispersed among A3s sequences and as short blocks of two or three units. In contrast, in the Brittany samples, these units were dispersed among A3s structures and next to A3s units branched at O-2 of alpha-L-Rha 3-sulfate by a terminal beta-D-GlcA and symbolised as A2g,3s. However, more complex structures are likely to occur in the enzyme resistant fraction remaining from this ulvan. An average structure sequence of these two ulvans was proposed. The transposition of the 13C NMR data of the new identified structures to the parent polysaccharides was not possible, probably due to the different sequence distributions affecting the carbons chemical shifts.

Carbohydrate Sequence↗