Genome sequence and cell biological toolbox of the highly regenerative, coenocytic green feather alga Bryopsis.

Green feather algae (Bryopsidales) undergo a unique life cycle in which a single cell repeatedly executes nuclear division without cytokinesis, resulting in the development of a thallus (>100 mm) with characteristic morphology called coenocyte. Bryopsis is a representative coenocytic alga that has exceptionally high regeneration ability: extruded cytoplasm aggregates rapidly in seawater, leading to the formation of protoplasts. However, the genetic basis of the unique cell biology of Bryopsis remains poorly understood. Here, we present a high-quality assembly and annotation of the nuclear genome of Bryopsis sp. (90.7 Mbp, 27 contigs, N50 = 6.7 Mbp, 14 034 protein-coding genes). Comparative genomic analyses indicate that the genes encoding BPL-1/Bryohealin, the aggregation-promoting lectin, are heavily duplicated in Bryopsis, whereas homologous genes are absent in other ulvophyceans, suggesting the basis of regeneration capability of Bryopsis. Bryopsis sp. possesses >30 kinesins but only a single myosin, which differs from other green algae that have multiple types of myosin genes. Consistent with this biased motor toolkit, we observed that the bidirectional motility of chloroplasts in the cytoplasm was dependent on microtubules but not actin in Bryopsis sp. Most genes required for cytokinesis in plants are present in Bryopsis, including those in the SNARE or kinesin superfamily. Nevertheless, a kinesin crucial for cytokinesis initiation in plants (NACK/Kinesin-7II) is hardly expressed in the coenocytic part of the thallus, possibly underlying the lack of cytokinesis in this portion. The present genome sequence lays the foundation for experimental biology in coenocytic macroalgae.

Mebamamide C, a deoxy analogue of mebamamides in Bryopsis marine green algae and Elysia sacoglossan mollusks.

Kahalalides, originally isolated from the sacoglossan mollusk Elysia rufescens, have been found in various Elysia and Bryopsis species, with over 20 variants identified to date. These compounds are biosynthesized by Candidatus Endobryopsis kahalalidefaciens within Bryopsis species. In this study, we report the isolation and structural determination of a new cyclic depsipeptide, mebamamide C (1), from Bryopsis sp. The planar structure was determined by spectroscopic data analyses, and the absolute configurations were determined using Marfey's method and modified Mosher's method. Additionally, our study explores the chemical relationship between Bryopsis algae and Elysia mollusks. The individual chemical profiles of these marine organisms highlight a fascinating aspect of marine chemical ecology. The distinct, species-specific chemical profiles observed in Elysia species imply the possibility of a symbiotic relationship with the kahalalide-producing bacteria.

A Novel Anticancer Peptide Derived from Bryopsis plumosa Regulates Proliferation and Invasion in Non-Small Cell Lung Cancer Cells.

The discovery of new highly effective anticancer drugs with few side effects is a challenge for drug development research. Natural or synthetic anticancer peptides (ACPs) represent a new generation of anticancer agents with high selectivity and specificity. The rapid emergence of chemoradiation-resistant lung cancer has necessitated the discovery of novel anticancer agents as alternatives to conventional therapeutics. In this study, we synthesized a peptide containing 22 amino acids and characterized it as a novel ACP (MP06) derived from green sea algae, Bryopsis plumosa. Using the ACP database, MP06 was predicted to possess an alpha-helical secondary structure and functionality. The anti-proliferative and apoptotic effects of the MP06, determined using the cytotoxicity assay and Annexin V/propidium iodide staining kit, were significantly higher in non-small-cell lung cancer (NSCLC) cells than in non-cancerous lung cells. We confirmed that MP06 suppressed cellular migration and invasion and inhibited the expression of N-cadherin and vimentin, the markers of epithelial-mesenchymal transition. Moreover, MP06 effectively reduced the metastasis of tumor xenografts in zebrafish embryos. In conclusion, we suggest considering MP06 as a novel candidate for the development of new anticancer drugs functioning via the ERK signaling pathway.

Anticancer Activity of Mannose-Specific Lectin, BPL2, from Marine Green Alga Bryopsis plumosa.

Lectin is a carbohydrate-binding protein that recognizes specific cells by binding to cell-surface polysaccharides. Tumor cells generally show various glycosylation patterns, making them distinguishable from non-cancerous cells. Consequently, lectin has been suggested as a good anticancer agent. Herein, the anticancer activity of Bryopsis plumosa lectins (BPL1, BPL2, and BPL3) was screened and tested against lung cancer cell lines (A549, H460, and H1299). BPL2 showed high anticancer activity compared to BPL1 and BPL3. Cell viability was dependent on BPL2 concentration and incubation time. The IC50 value for lung cancer cells was 50 µg/mL after 24 h of incubation in BPL2 containing medium; however, BPL2 (50 µg/mL) showed weak toxicity in non-cancerous cells (MRC5). BPL2 affected cancer cell growth while non-cancerous cells were less affected. Further, BPL2 (20 µg/mL) inhibited cancer cell invasion and migration (rates were ˂20%). BPL2 induced the downregulation of epithelial-to-mesenchymal transition-related genes (Zeb1, vimentin, and Twist). Co-treatment with BPL2 and gefitinib (10 µg/mL and 10 µM, respectively) showed a synergistic effect compared with monotherapy. BPL2 or gefitinib monotherapy resulted in approximately 90% and 70% cell viability, respectively, with concomitant treatment showing 40% cell viability. Overall, BPL2 can be considered a good candidate for development into an anticancer agent.

A siphonous morphology affects light-harvesting modulation in the intertidal green macroalga Bryopsis corticulans (Ulvophyceae).

MAIN CONCLUSION: The macroalga Bryopsis corticulans relies on a sustained protective NPQ and a peculiar body architecture to efficiently adapt to the extreme light changes of intertidal shores. During low tides, intertidal algae experience prolonged high light stress. Efficient dissipation of excess light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is therefore required to avoid photodamage. Light-harvesting regulation was studied in the intertidal macroalga Bryopsis corticulans, during high light and air exposure. Photosynthetic capacity and NPQ kinetics were assessed in different filament layers of the algal tufts and in intact chloroplasts to unravel the nature of NPQ in this siphonous green alga. We found that the morphology and pigment composition of the B. corticulans body provides functional segregation between surface sunlit filaments (protective state) and those that are underneath and undergo severe light attenuation (light-harvesting state). In the surface filaments, very high and sustained NPQ gradually formed. NPQ induction was triggered by the formation of transthylakoid proton gradient and independent of the xanthophyll cycle. PsbS and LHCSR proteins seem not to be active in the NPQ mechanism activated by this alga. Our results show that B. corticulans endures excess light energy pressure through a sustained protective NPQ, not related to photodamage, as revealed by the unusually quick restoration of photosystem II (PSII) function in the dark. This might suggest either the occurrence of transient PSII photoinactivation or a fast rate of PSII repair cycle.

Functional Expression and Characterization of the Recombinant N-Acetyl-Glucosamine/N-Acetyl-Galactosamine-Specific Marine Algal Lectin BPL3.

Lectins, characterized by their carbohydrate-binding ability, have extensive practical applications. However, their industrial use is limited due to impurity. Thus, quality-controlled production of recombinant lectin is necessary. In this study, the algal lectin BPL3 (Bryopsis plumosa lectin 3) was successfully produced using a bacterial expression system, BL21(DE3), with an artificial repeated structure (dimeric construct). Recombinant dimeric BPL3 (rD2BPL3) was confirmed by LC-MS/MS spectrometry. Expression efficiency was greater for the construct with the repeat structure (rD2BPL3) than the monomeric form (rD1BPL3). Optimal conditions for expression were 1 mM IPTG at 20 °C. Recombinant lectin was purified under denaturing conditions and refolded by the flash dilution method. Recombinant BPL3 was solubilized in 1× PBS containing 2 M urea. rD2BPL3 showed strong hemagglutination activity using human erythrocyte. rD2BPL3 had a similar sugar specificity to that of the native protein, i.e., to N-acetyl-glucosamine (GlcNAc) and N-acetyl-galactosamine (GalNAc). Glycan array results showed that recombinant BPL3 and native BPL3 exhibited different binding properties. Both showed weak binding activity to α-Man-Sp. Native BPL3 showed strong binding specificity to the alpha conformation of amino sugars, and rD2BPL3 had binding activity to the beta conformation. The process developed in this study was suitable for the quality-controlled large-scale production of recombinant lectins.

Prey species and abundance affect growth and photosynthetic performance of the polyphagous sea slug Elysia crispata.

Some sacoglossan sea slugs steal functional macroalgal chloroplasts (kleptoplasts). In this study, we investigated the effects of algal prey species and abundance on the growth and photosynthetic capacity of the tropical polyphagous sea slug Elysia crispata. Recently hatched sea slugs fed and acquired chloroplasts from the macroalga Bryopsis plumosa, but not from Acetabularia acetabulum. However, adult sea slugs were able to switch diet to A. acetabulum, rapidly replacing the great majority of the original kleptoplasts. When fed with B. plumosa, higher feeding frequency resulted in significantly higher growth and kleptoplast photosynthetic yield, as well as a slower relative decrease in these parameters upon starvation. Longevity of A. acetabulum-derived chloroplasts in E. crispata was over twofold that of B. plumosa. Furthermore, significantly lower relative weight loss under starvation was observed in sea slugs previously fed on A. acetabulum than on B. plumosa. This study shows that functionality and longevity of kleptoplasts in photosynthetic sea slugs depend on the origin of the plastids. Furthermore, we have identified A. acetabulum as a donor of photosynthetically efficient chloroplasts common to highly specialized monophagous and polyphagous sea slugs capable of long-term retention, which opens new experimental routes to unravel the unsolved mysteries of kleptoplasty.

Recent advances and limitations in the application of kahalalides for the control of cancer.

Since the discovery of the kahalalide family of marine depsipeptides in 1993, considerable work has been done to develop these compounds as new and biologically distinct anti-cancer agents. Clinical trials and laboratory research have yielded a wealth of data that indicates tolerance of kahalalides in healthy cells and selective activity against diseased cells. Currently, two molecules have attracted the greates level of attention, kahalalide F (KF) and isokahalalide F (isoKF, Irvalec, PM 02734, elisidepsin). Both compounds were originally isolated from the sarcoglossan mollusk Elysia rufescens but due to distinct structural characteristics it has been hypothesized and recently shown that the ultimate origin of the molecules is microbial. The search for their true source has been a subject of considerable research in the anticipation of finding new analogs and a culturable expression system that can produce sufficient material through fermentation to be industrially relevant.

The complete mitochondrial genome sequence of Bryopsis plumosa.

In 2015, a novel macroalgae began to bloom in Qinhuangdao on the coast of the Bohai Sea, Northern China. Bryopsis plumosa, a green macroalga, is one of the causal species of the macroalgae blooms, which have severely affected the environment and ecosystem on the coast of Qinhuangdao. In the present study, we sequenced the mitochondrial genome of B. plumosa for the first time (GenBank accession number MN853874). It was found that the ring-shaped genome was made up of 110,912 base pairs, including 29 protein-coding genes, 23 tRNAs, and 2 rRNAs. Phylogenetic analysis showed B. plumosa had close genetic relationships with algae Ulva prolifera, Ulva linza, and Ulva flexuosa. The present data will provide important information on the phylogenetics and molecular evolution of Bryopsis species.

Induction of Recombinant Lectin Expression by an Artificially Constructed Tandem Repeat Structure: A Case Study Using Bryopsis plumosa Mannose-Binding Lectin.

Lectin is an important protein in medical and pharmacological applications. Impurities in lectin derived from natural sources and the generation of inactive proteins by recombinant technology are major obstacles for the use of lectins. Expressing recombinant lectin with a tandem repeat structure can potentially overcome these problems, but few studies have systematically examined this possibility. This was investigated in the present study using three distinct forms of recombinant mannose-binding lectin from Bryopsis plumosa (BPL2)-i.e., the monomer (rD1BPL2), as well as the dimer (rD2BPL2), and tetramer (rD4BPL2) arranged as tandem repeats. The concentration of the inducer molecule isopropyl ß-D-1-thiogalactopyranoside and the induction time had no effect on the efficiency of the expression of each construct. Of the tested constructs, only rD4BPL2 showed hemagglutination activity towards horse erythrocytes; the activity of towards the former was 64 times higher than that of native BPL2. Recombinant and native BPL2 showed differences in carbohydrate specificity; the activity of rD4BPL2 was inhibited by the glycoprotein fetuin, whereas that of native BPL2 was also inhibited by d-mannose. Our results indicate that expression as tandem repeat sequences can increase the efficiency of lectin production on a large scale using a bacterial expression system.

Larvicidal activity, inhibition effect on development, histopathological alteration and morphological aberration induced by seaweed extracts in Aedes aegypti (Diptera: Culicidae).

OBJECTIVE: To investigate the larvicidal activity, inhibition effect on development, histopathological alteration and morphological aberration induced by the extracts derived from seaweeds Bryopsis pennata (B. pennata), Sargassum binderi (S. binderi) and Padina australis in Aedes aegypti (Ae. aegypti) larvae and to characterize the phytochemical components of the three seaweeds. METHODS: Larvicidal activity of the seaweeds towards the larvae of Ae. aegypti was determined according to WHO. The inhibition effect of seaweeds was assessed by determining the mortality, adult emergence rate, larval and pupa duration of the treated larvae. Histopathological effect on midgut epithelium of larvae and morphological aberration induced by the methanol extracts were examined. Phytochemical analysis was done to determine the presence of alkaloids, saponins, steroids and terpenoids in the seaweeds. RESULTS: Chloroform partition of B. pennata extract exhibited the strongest larvicidal activity (LC50 = 82.55 µg/mL), followed by methanol extract of B. pennata (LC50 = 160.07 µg/mL) and chloroform partition of S. binderi extract (LC50 = 192.43 µg/mL). The methanol extract of S. binderi exhibited the strongest effect on prolongation of larval period (1.5-fold longer as compared to control) and resulted in strongest inhibition effect in adult emergence (98.67%). The histopathological study showed that larvae treated with seaweed extracts had cytopathological alteration of the midgut epithelium. The morphological observation revealed that the anal papillae and terminal spiracles of larvae were the common sites of aberrations. CONCLUSIONS: The study provided information on various effects of seaweed extracts on Ae. aegypti. Further investigation on identifying the active compounds and their mechanisms of action is recommended.

GAMETE RELEASE IS INCREASED BY CALM CONDITIONS IN THE COENOCYTIC GREEN ALGA BRYOPSIS (CHLOROPHYTA).

Water motion did not inhibit gamete release in cultures of the coenocytic green alga Bryopsis plumosa (Hudson) C. Agardh; however, the number of gametangia that released gametes increased significantly under transiently calm conditions. This stimulatory effect of calm conditions in the laboratory was found in isolates from two different areas of the Maine coast. The isolates were all monoecious, but strong differences in levels of fertilization, numbers of male and female gametes remaining following fertilization, and levels of polygamy (=zygotes formed by fusions of more than two gametes) were observed among isolates. These data support the hypothesis that organisms with external fertilization are able to sense and respond to water motion in ways that favor reproductive success.

CHARACTERIZATION OF CELL WALL POLYSACCHARIDES OF THE COENCOCYTIC GREEN SEAWEED BRYOPSIS PLUMOSA (BRYOPSIDACEAE, CHLOROPHYTA) FROM THE ARGENTINE COAST(1).

Bryopsis sp. from a restricted area of the rocky shore of Mar del Plata (Argentina) on the Atlantic coast was identified as Bryopsis plumosa (Hudson) C. Agardh (Bryopsidales, Chlorophyta) based on morphological characters and rbcL and tufA DNA barcodes. To analyze the cell wall polysaccharides of this seaweed, the major room temperature (B1) and 90°C (X1) water extracts were studied. By linkage analysis and NMR spectroscopy, the structure of a sulfated galactan was determined, and putative sulfated rhamnan structures and furanosidic nonsulfated arabinan structures were also found. By anion exchange chromatography of X1, a fraction (F4), comprising a sulfated galactan as major structure was isolated. Structural analysis showed a linear backbone constituted of 3-linked ß-d-galactose units, partially sulfated on C-6 and partially substituted with pyruvic acid forming an acetal linked to O-4 and O-6. This galactan has common structural features with those of green seaweeds of the genus Codium (Bryopsidales, Chlorophyta), but some important differences were also found. This is the first report about the structure of the water-soluble polysaccharides biosynthesized by seaweeds of the genus Bryopsis. These sulfated galactans and rhamnans were in situ localized mostly in two layers, one close to the plasma membrane and the other close to the apoplast, leaving a middle amorphous, unstained cell wall zone. In addition, fibrillar polysaccharides, comprising (1→3)-ß-d-xylans and cellulose, were obtained by treatment of the residue from the water extractions with an LiCl/DMSO solution at high temperature. These polymers were also localized in a bilayer arrangement.

MOLECULAR CHARACTERIZATION OF THE LECTIN, BRYOHEALIN, INVOLVED IN PROTOPLAST REGENERATION OF THE MARINE ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA)(1).

When a coenocytic cell of the green alga Bryopsis plumosa (Hudson) C. Agardh was cut open and the cell contents expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. This process was mediated by a lectin, Bryohealin. The full sequence of the cDNA encoding Bryohealin was obtained, which consisted of 1,101 base pairs (bp), with 24 bp of 5' untranslated region (UTR) and 201 bp of 3' UTR. It had an open reading frame (ORF) of 771 bp encoding 257 amino acid residues. A signal peptide consisted of 22 amino acids presented before the start codon of Bryohealin, indicating that this lectin was a vacuolar (storage) protein. The C-terminal sequence of Bryohealin was composed of antibiotic domains, suggesting that this lectin could perform two functions: (i) aggregation of cell organelles in seawater and (ii) protection from bacterial contamination for successful protoplast regeneration. The BLAST search result showed that Bryohealin had little sequence homology with any known plant lectins, but rather resembled animal lectins with fucolectin domains. The expression of recombinant Bryohealin (rBryohealin) was obtained in the Escherichia coli system.

Participação de um agente adrenérgico não peptídico na ação inotrópica positiva da fração polar da Bryopsis pennata / Participation of a non-peptide adrenergic agent on positive inotropic effect of the polar fraction of Bryopsis pennata

As algas marinhas representam uma rica fonte de compostos bioativos, algumas delas precursoras de ferramentas farmacológicas e de substâncias potencialmente úteis para o desenvolvimento de novos fármacos. A macroalga Bryopsis pennata, Cloroficea pertencente à ordem Caulerpales, sin. Bryopsidales é uma espécie tropical encontrada em diversos costões rochosos. Essa espécie produz uma defesa química tóxica para organismos herbívoros e potencial de se tornar invasiva e dominante em condições ambientais favoráveis. Este trabalho é uma investigação do efeito cardiotônico da fração polar da B. pennata .O cultivo, em ambiente controlado destituído de contaminantes, foi realizado no intuito de comparar seus efeitos com os efeitos da alga coletada. O efeito cardiotônico cronotrópico positivo em tiras ventriculares de anuros ficou fortemente evidenciado nos testes com frações polares de algas coletadas e cultivadas, não apresentando diferenças significativas entre as frações. O propranolol antagonizou o efeito cardiotônico e enzimas endopeptidases não reagiram com a fração polar da B. pennata. Testes bioquímicos demonstraram que a fração com efeito cardiotônico é de caráter ácido e apresenta peso molecular menor que 10.000 daltons.

Marine algae are a rich source of bioactive compounds and some of them have shown to be useful for the development of new pharmacological tools and medicines. Bryopsis pennata, (Clorofícea, Caulerpales, sin. Bryopsidales) is a marine algae that can be found in the Southeastern Brazilian coast and elsewhere. The species produces a toxic chemical defense to the herbivorous organisms and possesses a potential to become invasive and dominant in favorable environmental conditions.The present study is about the investigation of the cardiotonic effect of the polar extract of B. pennata. The cultive in laboratory without any contaminants was made to compare studies performed with the collected algae extracts and the ones cultivated effect. The cardiotonic effect in the anurou’s ventricular strips was strongly unequivocal when it was tested with the polar extracts from the collected and cultivated algae. The propranolol antagonized polar effects of these algae and the endopeptidase enzyme did not change the effects of polar extracts of Bryopsis pennata. Biochemical tests showed that the polar fraction presenting the inotropic cardiac activity weighs less than 10.000 Daltons and has an acid character.