Dispersal potential of the invasive green alga Codium fragile ssp. fragile

Since its introduction in 1989, the invasive green alga Codium fragile ssp. fragile (formerly Codium fragile ssp. tomentosoides) has spread rapidly in Atlantic Canada. Although its spread has likely been facilitated by human transport, C. fragile possesses diverse modes of natural dispersal. In addition to parthenogenetically developing swarmer cells, it can propagate through the release of vegetative buds, thallus fragments, and entire, dislodged thalli. We examined the natural dispersal potential of these propagules using a combination of field and laboratory experiments. Vegetative buds were most abundant on thalli in late August, coinciding with increasing late summer wave activity. At two locations within two field sites, we examined the effects of tidal state, wave action and topography on the dispersal of fragments and intact thalli. Over a 4-hour period, retention rate was generally lower and dispersal distance of retained propagules higher: during flood than ebb tide; at the more wave-exposed locations; and for fragments than thalli. Dispersal direction corresponded with topography or flood tide currents at certain locations, but was often bi-directional or random, suggesting an added role of wave action. Over periods of weeks, the retention rate of marked detrital thalli was negatively related to the magnitude of wave activity. In the laboratory, settling rate was negatively related to propagule length, and large buds had higher critical shear erosional velocities than either small buds or fragments. Our results indicate that large propagules of C. fragile, such as intact adult thalli, have a generally low dispersal potential (meters), but may be transported longer distances (kilometers) during storms or when positively buoyant. Fragments and buds are regularly transported 10's of meters at a time at average flow velocities. However, they also become resuspended in average flows, and probably disperse longer distances over multiple resuspension events, or when positively buoyant. Once settled, the smallest propagules may be less easily resuspended or transported along the bottom as they become entrapped by small-scale topographic features or turf algae. The wide variety of propagules produced by C. fragile and the variable distances over which different propagule types may be transported give C. fragile the advantage of both short- and long-distance dispersal, and have likely played a role in the invasive success of this alga.     

Thrombolytic,anticoagulant and antiplatelet activities of codiase,a bi-functional fibrinolytic enzyme from Codium fragile

Thrombosis is a leading cause of morbidity and mortality throughout the world. Thrombolytic agents are important for both the prevention and treatment of thrombosis. In this study, codiase, a new bi-functional fibrinolytic serine protease having thrombolytic, anticoagulant, and antiplatelet activities was purified from marine green alga, Codium fragile. The molecular weight of the enzyme was estimated to be 48.9 kDa by SDS-PAGE, and mass spectrometry. Fibrin zymography analysis showed an active band with similar molecular weight. The N-terminal sequence was found to be APKASTDQTLPL, which is different from that of other known fibrinolytic enzymes. Codiase displayed maximum activity at 30 °C and pH 6.0, and the activity was inhibited by Zn²⁺ and Fe²⁺. Moreover, the enzyme activity was strongly inhibited by serine protease inhibitor such as PMSF. Codiase exhibited high specificity for the substrate S-2288, and the K_m and V_max values for this substrate were found to be 0.24 mM and 79 U/ml respectively. Fibrin plate assays revealed that it was able to hydrolyze fibrin clot either directly or by activation of plasminogen. Codiase effectively hydrolyzed fibrin and fibrinogen, preferentially degrading α- and Aα chains, followed by γ–γ, and γ-chains. However, it provoked slower degradation of Bβ and β-chains. The structural change of fibrin clot and fibrinogen by codiase was also detected by FTIR-ATR spectroscopy analysis. In vitro and in vivo studies revealed that codiase reduces thrombosis in concentration-dependent manner. Codiase was found to prolong activated partial thromboplastin time (APTT), and prothrombin time (PT). PFA-100 studies showed that codiase prolonged the closure time (CT) of citrated whole human blood. These favorable antithrombotic profiles together with its anticoagulant and platelet disaggregation properties, and lack of toxicity to mice and NIH-3T3 cells, make it a potential agent for thrombolytic therapy.     

Grazing of the invasive alga Codium fragile ssp. tomentosoides by the common periwinkle Littorina littorea: Effects of thallus size, age and condition

We examined the potential of herbivory by the common periwinkle Littorina littorea to limit recruitment and vegetative re-growth of the invasive green alga Codium fragile ssp. tomentosoides in a series of manipulative field experiments in tidepools on a wave-exposed rocky shore in Nova Scotia, Canada. Snails were excluded or included from circular plots (14 to 20 cm diameter) with cages to compare growth and survival of C. fragile against procedural (partial cages) or natural (uncaged) control plots. Our results show that L. littorea may restrict growth and survival of C. fragile by grazing new recruits (< 2 cm thallus length), fronds of adult thalli that are bleached and necrotic, and residual holdfasts (< 2 mm thickness) of detached thalli (artificially severed to mimic wave dislodgement). Once recruits grow beyond a critical size (~ 3 cm), or damaged tissues or holdfasts regenerate, grazing of C. fragile by L. littorea appears to be limited and ineffectual. Our experimental results corroborate correlative evidence from previous studies that herbivory by L. littorea limits the abundance of C. fragile in tidepools on the Atlantic coast of Nova Scotia, particularly pools in the high intertidal zone where these snails are abundant. Lower on the shore, littorinid grazing and physical stressors that render algae more vulnerable to grazers (e.g. UV radiation and freezing) are less intensive, and probably have less of a regulatory effect on populations of C. fragile.     

Antiangiogenic properties of a sulfated galactan isolated from a marine green alga, Codium cylindricum

An anticoagulant sulfated galactan isolated from the marine green alga, Codium cylindricum, was shown to have antiangiogeinic activity. This galactan suppressed microvessel formation in an ex vivo serum-free matrix culture model using rat aortic ring. It also inhibited human umbilical vein endothelial cells (HUVEC) tube formation on reconstituted basement membrane gel. These results show the value of algal sulfated galactans in the design of antiangiogenic agents.     

The structure of a sulfated galactan from Porphyra haitanensis and its in vivo antioxidant activity

The sulfated galactan fraction F1 isolated from the red seaweed, Porphyra haitanensis, showed typical porphyran structure. It has a linear backbone of alternating 3-linked beta-D-galactosyl units and 4-linked alpha-L-galactosyl 6-sulfate and 3,6-anhydro-alpha-L-galactosyl units. The L-residues are mainly composed of alpha-L-galactosyl 6-sulfate units, and the 3,6-anhydrogalactosyl units are minor. Partial methylation occurred at the C-6 position of the D-galactosyl units and at the C-2 position of the 3,6-anhydro-alpha-L-galactosyl units. Intraperitoneal administration of F1 significantly decreased the lipid peroxidation in aging mice. F1 treatment increased the total antioxidant capacity and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in aging mice. The results indicated that F1 had significant in vivo antioxidant activity.     

Structural analysis and antiviral activity of a sulfated galactan from the red seaweed Schizymenia binderi (Gigartinales, Rhodophyta)

Aqueous extraction of gametophytic Schizymenia binderi afforded a polysaccharide composed of galactose and sulfate groups in a molar ratio of 1.0:0.89 together with uronic acids (6.8 wt%) and minor amounts of other neutral sugars. Alkali-treatment of the polysaccharide afforded a polysaccharide devoid of 3,6-anhydrogalactose. 13C NMR spectroscopy of the desulfated alkali-treated polysaccharide showed a backbone structure of alternating 3-linked beta-D-galactopyranosyl and 4-linked alpha-galactopyranosyl units that are predominantly of the D-configuration and partly of the L-configuration. Methylation, ethylation and NMR spectroscopic studies of the alkali-treated polysaccharide indicated that the sulfate groups are located mainly at positions O-2 of 3-linked beta-D-galactopyranosyl residue and at position O-3 of 4-linked-alpha-galactopyranosyl residues, the latter is partially glycosylated at position O-2. The sulfated galactan from S. binderi exhibited highly selective antiviral activity against Herpes simplex virus types 1 and 2, with selectivity indices (ratio cytotoxicity/antiviral activity) >1000 for all assayed virus strains. This compound was shown to interfere with the initial adsorption of viruses to cells.     

Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum

An anticoagulant was isolated from a marine green alga, Codium cylindricum. The anticoagulant was composed mainly of galactose with a small amount of glucose, and was highly sulfated (13.1% as SO Na). The anticoagulant properties of the purified anticoagulant were compared with that of heparin by assays of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using normal human plasma. The anticoagulant showed similar activities with heparin, however, weaker than heparin. On the other hand, the anticoagulant did not affect PT even at the concentration at which APTT and TT were strongly prolonged. The anticoagulant did not potentiate antithrombin III (AT III) and heparin cofactor II (HC II), thus the anticoagulant mechanism would be different from that of other anticoagulants isolated so far from the genus Codium.     

Fractionation of sulfated galactan from the red alga Botryocladia occidentalis separates its anticoagulant and anti-SARS-CoV-2 properties

Sulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation. The lowest MW fraction was found to be primarily composed of octasaccharides of monosulfated monosaccharides. Unlike heparin or native BoSG, we found that hydrolyzed BoSG products had weak anticoagulant activities as seen by aPTT and inhibitory assays using purified cofactors. In contrast, lower MW BoSG-derivatives retained anti-SARS-CoV-2 activity using SARS-CoV-2 spike (S)-protein pseudotyped lentivirus vector in HEK-293T-hACE2 cells monitored by GFP. Surface plasmon resonance confirmed that longer chains are necessary for BoSG to interact with coagulation cofactors but is not required for interactions with certain S-protein variants. We observed distinct affinities of BoSG derivatives for the S-proteins of different SARS-CoV-2 strains, including WT, N501Y (Alpha), K417T/E484K/N501Y (Gamma), and L542R (Delta) mutants, and stronger affinity for the N501Y-containing variants. Docking of the four possible monosulfated BoSG disaccharides in interactions with the N501Y mutant S-protein predicted potential binding poses of the BoSG constructs and favorable binding in close proximity to the 501Y residue. Our results demonstrate that depolymerization and fractionation of BoSG are an effective strategy to segregate its anticoagulant property from its anti-SARS-CoV-2 action.     

Inhibitory effects of leachate from Eupatorium adenophorum on germination and growth of Amaranthus retroflexus and Chenopodium glaucum

Amaranthus retroflexus L. and Chenopodium glaucum L. are two widely distributed destructive weeds. Their strong adaptability and massive seed production make them the hardest weeds to deal with. This present study intended to investigate the effect of leachate from Eupatorium adenophorum on the growth of these weeds and explore the potential to develop an environmental friendly strategy to use the leachate to control the weeds. Seeds of A. retroflexus L. and C. glaucum L. were soaked in solutions containing 0%, 0.6%, 1.25%, 2.5%, and 5% leachate from E. adenophorum leaves. A. retroflexus and C. glaucum seedlings grown in pots were sprayed with leachate solutions in the same concentration range. The effects of these leachate solutions on membrane permeability and germination of seeds, and growth and physiological characteristics of the seedlings were investigated. The highest concentration of leachate (5%) caused significant damage to the cell membrane of seeds of both weed species, whereas lower concentrations (0.6%) promoted repair of the membrane system, as reflected by higher and lower than control in relative conductivity (RC), respectively. Different concentrations of leachate showed distinct allelopathic inhibitory effects on the two weed species; lower concentrations showed weak inhibitory or even positive effects, whereas higher concentrations showed stronger inhibitory effects. Higher concentrations of leachate (2.5% and 5%) delayed germination and significantly decreased the emergence rate of the seeds, survival rate, and dry matter accumulation of the seedlings. When treated by 5% leachate, the emergence date of A. retroflexus was delayed by 3.6 d, emergence rate of the seeds and survival rate was 69.1% and 70.6% of the control, respectively, seedling dry matter was 48.6% less than the control; In the case of C. glaucum, the emergence date was delayed by 2.7 d, emergence rate of the seeds and survival rate was 45.1% and 58.6% of the control, respectively, seedling dry matter was 44.7% less than the control. There were significant interactions among the different concentrations of leachate and the length of treatment period with respect to activities of antioxidant enzymes, malondialdehyde (MDA) contents, and chlorophyll contents. Seedlings treated with 0.6%, 1.25%, or 2.5% leachate solution for 24–72 h showed increased superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities. When seedlings were treated with leachate solutions for 96 h, antioxidant enzyme activities and chlorophyll content decreased in A. retroflexus, but only CAT activity decreased in C. glaucum. When seedlings of the two weed species were treated with 5% leachate solution, CAT activity and chlorophyll content decreased and MDA content gradually increased with longer treatment times (from 24 to 96 h). The two weed species showed different allelopathic responses to E. adenophorum; A. retroflexus was more sensitive than C. glaucum. Based on the investigation, it could be speculated that the delayed germination and low germination rate of the weeds after treatment by leachate could be due to the fact that leachate damaged the membrane system of the seeds. By delaying germination, lowering the germination rate of the weeds and inhibiting seedling growth, leachate from E. adenophorum could provide an effective way of controlling the weeds.     

Chemotaxonomy of New Zealand red algae in the family Gigartinaceae (Rhodophyta) based on galactan structures from the tetrasporophyte life-stage

The identification of the polysaccharides from tetrasporophytic plants of nine endemic New Zealand species belonging to the Gigartinaceae, ‘Gigartina’ ancistroclada, ‘G.’ grandifida, Gigartina dilatata, G. divaricata, G. macrocarpa, G. marginifera, G. pachymenioides, G. sp. ‘Lindauer 164’ and Sarcothalia livida using infra-red spectroscopy in conjunction with constituent sugar and glycosyl linkage/substitution analysis is reported. All nine species contain galactans with structures consistent with λ-type carrageenans. Differences in the structures of the galactans in these and a further six previously studied species indicate chemotaxonomically distinct groupings that correspond to Sarcothalia, ‘Sarcothalia’ and Gigartina genera plus some outliers. These distinct, chemotaxonomic groupings are aligned to those determined by rbcL sequence analysis reported in the literature.     

JM47, a cyclic tetrapeptide HC-toxin analogue from a marine Fusarium species

The known metabolite, enniatin B, and a cyclic tetrapeptide, JM47, which is a new natural product, were extracted from brown rice cultures of a marine fungus, identified as a Fusarium species, isolated from the marine alga Codium fragile. NMR studies, including 15N HMQC and 15N HMBC, established the structure of JM47 as cyclo(Ala-Ala-Aoh-Pro), where Aoh is the amino acid, (2S,9S)-2-amino-8-oxo-9-hydroxydecanoic acid. The absolute stereochemistry of the Aoh side chain carbinol centre was determined using Mosher ester methodology. Analysis of NOESY data assisted by molecular modelling revealed an alternating L-, D-, L-, D-configuration for the tetrapeptide core. The absolute stereochemistry of the core was determined by acidic hydrolysis and chiral TLC analysis of the proline residue. JM47 belongs to the HC-toxin family of cyclic tetrapeptides which possess a 2-amino-8-oxo-9,10-epoxydecanoic acid residue in place of the Aoh unit. This is the first report of an analogue of HC-toxin from a marine Fusarium species.     

Effect of dietary history and algal traits on feeding rate and food preference in the green sea urchin Strongylocentrotus droebachiensis

Feeding behaviour is influenced by a variety of factors, including nutritional requirements, the quality of available foods, and environmental conditions. We examined the effect of two factors, food morphology and dietary history, on the feeding rate and preference of the sea urchin Strongylocentrotus droebachiensis. Standardizing food shape and structure did not alter urchins' expected preference for the native kelp Laminaria longicruris over the invasive alga Codium fragile ssp. tomentosoides. However, when foods containing L. longicuris were shaped to mimic the algae, the C. fragile mimic was consumed more rapidly than the kelp mimic. Dietary history had no effect on single diet feeding rate. Urchins feeding on C. fragile consistently consumed twice as much (by mass) as those fed kelp, regardless of their previous diet. Despite higher feeding rates on C. fragile, urchins feeding on this alga were unable to compensate for its low energetic content and ingested less energy. Dietary history had a short-term effect on food preference, with urchins tending to prefer less familiar foods. Our findings suggest that urchins feed on C. fragile at a high rate, due to ease of handling and/or compensatory feeding, and that they do not a have strict preference hierarchy. Rather, food choice appears to reflect active maintenance of a mixed diet.     

Characterization of a 3-linked galactan from pinus radiata callus cells

A polysaccharide was obtained in the neutral fraction of a hot water extract (100°) of cell walls, by DEAE ion exchange chromatography, from callus of Pinus radiata cells. The polysaccharide was found by sugar analysis to be composed predominantly of galactose, and methylation analysis showed it to be 3-linked. The anomeric configuration was not determined.     

New 19-oxygenated and 4-methylated steroids from the Formosan soft coral Nephthea chabroli

Chemical investigation of the Formosan soft coral Nephthea chabroli resulted in the isolation of four new 19-oxygenated steroids, nebrosteroids I-L (1-4), together with a new 4α-methylated steroid, nebrosteroid M (5). The molecular structures of these isolated metabolites were elucidated on the basis of extensive spectroscopic analysis and by comparison of the data with those of related metabolites. Compounds 1-5 were evaluated for anti-inflammatory activity using RAW 264.7 macrophages.     

Chemical compositions,antimicrobial and herbicidal effects of essential oils isolated from Turkish Tanacetum aucheranum and Tanacetum chiliophyllum var. chiliophyllum

The chemical composition of essential oils isolated from the aerial parts by hydrodistillation of Turkish Tanacetum aucheranum and Tanacetum chiliophyllum var. chiliophyllum were analyzed by GC–MS. The oils contain similar major components. The major components of T. aucheranum oil were 1,8-cineole (23.8%), camphor (11.6%), terpinen-4-ol (7.2%), α-terpineol (6.5%), borneol (3.8%), (E)-thujone (3.2%), epi-α-cadinol (3.1%), and artemisia ketone (3.0%). Camphor (17.9%), 1,8-cineole (16.6%) and borneol (15.4%) were found to be predominant constituents in the oil of T. chiliophyllum. It is interesting to find that ester derivatives of dihydro-α-cyclogeranic acid (2,2,6-trimethylcyclohexylcarboxylate), dihydro-α-cyclogeranyl hexanoate (10.1%), dihydro-α-cyclogeranyl pentanoate (3.0%), dihydro-α-cyclogeranyl butanoate (2.1%) and dihydro-α-cyclogeranyl propionate (1.2%) are firstly found as chemotaxonomically important components in T. chiliophyllum oil. From these, dihydro-α-cyclogeranyl hexanoate was isolated on silica gel column chromatography and its structure was confirmed by spectroscopic methods. This is the first report on the occurrence of ester derivatives of dihydro-α-cyclogeranic acid in essential oils of Tanacetum species. The oils were also characterized to have relatively high amounts of oxygenated monoterpenes. Results of the antifungal testing by microbial growth inhibition assays showed that the oils completely inhibit the growth of 30 phytopathogenic fungi. However, their growth inhibition effects were lower than commercial benomyl. The oils tested for antibacterial activity against 33 bacterial strains showed a considerable antibacterial activity over a wide spectrum. Herbicidal effects of the oils on seed germination of Amaranthus retroflexus, Chenopodium album and Rumex crispus were also determined and the oils completely inhibited the seed germination and seedling growth of the plants.     

Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum

An anticoagulant was isolated from a marine green alga, Codium cylindricum. The anticoagulant was composed mainly of galactose with a small amount of glucose, and was highly sulfated (13.1% as SO3Na). The anticoagulant properties of the purified anticoagulant were compared with that of heparin by assays of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using normal human plasma. The anticoagulant showed similar activities with heparin, however, weaker than heparin. On the other hand, the anticoagulant did not affect PT even at the concentration at which APTT and TT were strongly prolonged. The anticoagulant did not potentiate antithrombin III (AT III) and heparin cofactor II (HC II), thus the anticoagulant mechanism would be different from that of other anticoagulants isolated so far from the genus Codium.     

Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis

Background

The polysaccharide of culture medium from Arthrospira platensis was extracted by ultrafiltration, partially characterized and assayed for anticoagulant activity.

Methods

The crude polysaccharidic fraction was fractionated by anion exchange chromatography on DEAE-cellulose, subjected to acetate cellulose electrophoresis and characterized by physicochemical procedures. The anticoagulant effect of the ultrafiltrated polysaccharide was checked by several coagulation tests.

Results

Anion exchange chromatography revealed in the whole ultrafiltrated polysaccharidic fraction the occurrence of a sulfated spirulan-like component designated PUF2. The average molecular weight of PUF2 was determined by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) and viscosimetry and was 199 kDa and the sulfate content was 20% weight/dry weight. The physicochemical characterization indicated the occurrence of rhamnose (49.7%), galacturonic and glucuronic acid (32% of total sugar). The anticoagulant effect of this sulfated polysaccharide was mainly due to the potentiation of thrombin inhibition by heparin cofactor II and was 4-times higher than that of the porcine dermatan sulfate whereas it had no effect on anti-Xa activity.

Conclusions

An ultrafiltrated sulfated polysaccharide, likely a calcium spirulan was obtained from the culture medium of A. platensis and showed an anticoagulant activity mediated by heparin cofactor II.

General significance

Old culture medium of A. platensis may represent an important source for the spirulan-like PUF2 which was endowed with potentially useful anticoagulant properties whereas its obtention by ultrafiltration may represent an extraction procedure of interest.