Differential susceptibility of distinct parts of the aquatic plant Nymphoides humboldtiana to herbivory supports the optimal defense theory.

PREMISE: The optimal defense theory (ODT) predicts that the allocation of chemical defenses in plants will be concentrated in parts or tissues that are of higher fitness value for the individuals that produce them. Chemicals are known to be allocated to certain parts of aquatic plants, and the morphological architecture of Nymphoides humboldtiana, a species that exposes its parts to different environmental factors and consumers, may be an excellent model to evaluate within-plant susceptibility to consumers according to the ODT. METHODS: Under laboratory experimental conditions, we evaluated the defensive properties of extracts from vegetative (leaves, rhizomes, roots) and reproductive (long stem internodes, flowers, fruits) parts of N. humboldtiana against consumption by the generalist herbivorous gastropod Biomphalaria glabrata. Extracts were also subjected to chemical analysis by high-performance liquid chromatography, principal component analysis, and analysis of their relationships to defensive actions. RESULTS: Extracts of all vegetative and internode (reproductive) parts of N. humboldtiana exhibited defensive properties against B. glabrata, but the long stem internodes exhibited the highest percentage of inhibition. Chemical profiles of these parts were qualitatively and quantitatively different, but a major unidentified compound is presumably responsible for the higher defensive property found in internodes. CONCLUSIONS: Our results support the ODT, since chemical defense was more effective in long stem internodes, which have a high fitness value for N. humboldtiana to keep the flowers emerged on the water surface in response to the rapid and dynamic changes in water levels typical of freshwater environments.

Bromopyrrole Alkaloid Inhibitors of the Proteasome Isolated from a Dictyonella sp. Marine Sponge Collected at the Amazon River Mouth.

The new pyrrole-imidazole and pyrrole-guanidine alkaloids 4-debromooroidin (1), 4-debromougibohlin (2), 5-debromougibohlin (3), and 5-bromopalau'amine (4), along with the known hymenidin (5) and (+)-monobromoisophakellin (6), have been isolated from a Dictyonella sp. marine sponge, collected at the Amazon River mouth. The bromine-substitution pattern observed for compounds 1, 2 and 4 is unusual among bromopyrrole alkaloids isolated from marine sponges. The 20S proteasome inhibitory activities of compounds 1-6 have been recorded, with 5-bromopalau'amine (4) being the most active in this series.

Diversity of Secondary Metabolites in the Liverwort Syzygiella rubricaulis (Nees) Stephani (Jamesoniellaceae, Marchantiophyta) from Neotropical High Mountains.

In order to evaluate the chemical diversity of Syzygiella rubricaulis (Nees) Stephani, a species with a disjunct distribution in the neotropical high mountains, a phytochemical study was carried out with samples from 12 different populations of different altitudes from four South American countries. The chemical profiles of lipophilic extracts were analyzed by GC/MS for each population and 50 different compounds were found with the predominance and richness of sesquiterpenes. The majority of the compounds were found only in one population and the total number of substances ranged from 1 to 15 among the populations, but these numbers were not correlated with altitude, and characterize each population as distinct, based on similarity analysis. The qualitative and quantitative variations of metabolites found are a response to different conditions, under which they live, mostly likely altitudinal conditions. Further studies on the quantification of these chemicals may provide information on their ecological roles and importance for the distribution of S. rubricaulis at different altitudes. Despite the known richness of secondary metabolites produced by bryophytes, they are still poorly explored in the context of the ecological expressions.

Microbial community diversity and physical-chemical features of the Southwestern Atlantic Ocean.

Microbial oceanography studies have demonstrated the central role of microbes in functioning and nutrient cycling of the global ocean. Most of these former studies including at Southwestern Atlantic Ocean (SAO) focused on surface seawater and benthic organisms (e.g., coral reefs and sponges). This is the first metagenomic study of the SAO. The SAO harbors a great microbial diversity and marine life (e.g., coral reefs and rhodolith beds). The aim of this study was to characterize the microbial community diversity of the SAO along the depth continuum and different water masses by means of metagenomic, physical-chemical and biological analyses. The microbial community abundance and diversity appear to be strongly influenced by the temperature, dissolved organic carbon, and depth, and three groups were defined [1. surface waters; 2. sub-superficial chlorophyll maximum (SCM) (48-82 m) and 3. deep waters (236-1,200 m)] according to the microbial composition. The microbial communities of deep water masses [South Atlantic Central water, Antarctic Intermediate water and Upper Circumpolar Deep water] are highly similar. Of the 421,418 predicted genes for SAO metagenomes, 36.7 % had no homologous hits against 17,451,486 sequences from the North Atlantic, South Atlantic, North Pacific, South Pacific and Indian Oceans. From these unique genes from the SAO, only 6.64 % had hits against the NCBI non-redundant protein database. SAO microbial communities share genes with the global ocean in at least 70 cellular functions; however, more than a third of predicted SAO genes represent a unique gene pool in global ocean. This study was the first attempt to characterize the taxonomic and functional community diversity of different water masses at SAO and compare it with the microbial community diversity of the global ocean, and SAO had a significant portion of endemic gene diversity. Microbial communities of deep water masses (236-1,200 m) are highly similar, suggesting that these water masses have very similar microbiological attributes, despite the common knowledge that water masses determine prokaryotic community and are barriers to microbial dispersal. The present study also shows that SCM is a clearly differentiated layer within Tropical waters with higher abundance of phototrophic microbes and microbial diversity.

Role of ABC Proteins in Secondary Metabolism and Immune (=Defensive) Response in Seaweeds.

Laurencia seaweed species synthesize a broad range of secondary metabolites, mainly terpenes (e.g., elatol), exhibiting diverse ecological roles, such as defense against fouling and herbivores. Recently, an intricate cellular machinery was described concerning terpenes biosynthetic pathways, storage inside corps en cerise (CC), and regulated exocytosis in these species. But for seaweeds in general, the proteins involved in transmembrane transport of secondary metabolites remain unknown. Assays with Rhodamine-123 and cyclosporine A (CSA) revealed the presence of ABC transporters in CC membrane of Laurencia dendroidea. In vivo incubation assays with CSA resulted in CC morphological changes, reduced intracellular elatol concentrations, and increased biofouling cover on the seaweed surface. Cultivation assays in the presence of a marine pathogenic bacteria induced the expression of ABC proteins belonging to the subfamilies ABCB, ABCD, ABCF, and ABCG. The latter subfamily is known to be associated with the transport of plant terpenes. Our results shed new light on the role of ABC proteins in key mechanisms of the defensive system in seaweeds against fouling and herbivory.

The effects of the diterpenes isolated from the Brazilian brown algae Dictyota pfaffii and Dictyota menstrualis against the herpes simplex type-1 replicative cycle.

We describe in this paper that the diterpenes 8,10,18-trihydroxy-2,6-dolabelladiene ( 1) and (6 R)-6-hydroxydichotoma-4,14-diene-1,17-dial ( 2), isolated from the marine algae DICTYOTA PFAFFII and D. MENSTRUALIS, respectively, inhibited HSV-1 infection in Vero cells. We initially observed that compounds 1 and 2 inhibited HSV-1 replication in a dose-dependent manner, resulting in EC (50) values of 5.10 and 5.90 microM, respectively, for a multiplicity of infection (MOI) of 5. Moreover, the concentration required to inhibit HSV-1 replication was not cytotoxic, resulting in good selective index (SI) values. Next, we found that compound 1 sustained its anti-herpetic activity even when added to HSV-1-infected cells at 6 h after infection, while compound 2 sustained its activity for up to 3 h after infection, suggesting that these compounds inhibit initial events during HSV-1 replication. We also observed that both compounds were incapable of impairing HSV-1 adsorption and penetration. In addition, the tested molecules could decrease the contents of some HSV-1 early proteins, such as UL-8, RL-1, UL-12, UL-30 and UL-9. Our results suggest that the structures of compounds 1 and 2, Brazilian brown algae diterpenes, might be promising for future antiviral design.

Induction of halogenated vesicle transport in cells of the red seaweed Laurencia obtusa.

In clones of the red alga Laurencia obtusa, the frequency of vesicle transport from corps en cerise (CC) to the cell wall region was evaluated in response to differences in temperature, irradiance, desiccation, bacterial fouling, and bromine (Br) availability. In addition, the morphology of the corps en cerise was analyzed. Traffic of vesicles was induced by exposing L. obtusa to low temperatures and variations in irradiance. It was also verified that bacterial fouling induced vesicle traffic. Under high temperatures and desiccation, the membranous tubular connections were lost and transport of vesicles was not seen. The morphology of the corps en cerise varied according to the availability of Br in seawater. Exocytosis of secondary metabolites by L. obtusa was shown to vary in relation to temperature, irradiance, desiccation and bacterial fouling. The data suggest that the transport of vesicles in L. obtusa may be related to the inhibition of the microfouling community on the algal surface.

The sea-hare Aplysia brasiliana promotes induction in chemical defense in the seaweed Laurencia dendroidea and in their congeneric neighbors.

Inducible chemical defenses are more common in temperate seaweeds than tropical ones, and are directly detected by increase of chemical contents, or indirectly by differential consumption of live seaweed tissues or artificial food with algal extracts by herbivores. In general, seaweed-induced chemical defense occur between 11 and 20 days after both simulated/artificial or direct herbivory. Here, we used experimental procedures to assess induced chemical defense in the tropical red seaweed Laurencia dendroidea as response to direct grazing, chemical cues from grazed conspecific neighbors and only presence of herbivores. Chemical defenses were analyzed by detecting the palatability of artificial food containing L. dendroidea extracts offered to Aplysia brasiliana and by comparative analyses of extracts from this seaweed by Gas Chromatography/Mass Spectroscopy, as well as metabolomic data analysis by Principal Component Analysis. Our results revealed that direct grazing by A. brasiliana induced a rapid (after 48 h) response among individuals of L. dendroidea, as did waterborne chemical cues from grazed conspecifics, but the presence of sea hare alone did not elicit a response. Increased resistance to grazing was accompanied by significative changes in sesquiterpene metabolomic chemical profile, revealing that induced defense: may be more widespread among seaweeds, independent of latitude; can involve changes in other classes of substances besides phlorotannins or not only the increase in the content of a single compound; and may be a rapid and ecologically coherent response to consumers. In addition, the importance of incorporating the metabolomic approach when examining inducible chemical defense in seaweeds is also emphasized.

Effects of different biotic substrata on mussel attachment.

Surface colonization by invertebrates can be stimulated or inhibited by cues produced by biofilms, conspecifics or other macroorganisms. To study the effects of living substrata on the attachment of the brown mussel, Perna perna, two different approaches were employed: (1) mussels were distributed in sets of Petri dishes consisting of one sterile set (controls), three sets in which marine biofilms were allowed to develop in aquaria for 1, 7 or 15 days and another set that had been immersed in a natural marine environment for 1-day. There was no significant effect of biofilms on attachment, suggesting that neither age nor the source of the biofilm influenced attachment. (2) Mussels were suspended over PVC panels (controls) and over panels on which Balanus trigonus (Crustacea), Schizoporella errata (Bryozoa), Symplegma rubra or Didemnum speciosum (Ascidiacea) were present. Attachment was significantly higher on the controls and on B. trigonus than on colonial taxa such as S. rubra, S. errata and D. speciosum, probably due to antifouling defenses of these species. The results show that the composition of the biological substratum is an important factor affecting mussel behavior.

Induction of attachment of the Mussel Perna perna by natural products from the brown seaweed Stypopodium zonale.

Marine invertebrates settle, attach, and/or metamorphose in response to signals from several sources, including seaweeds. In response to the aquaculture challenge of producing constant numbers of juveniles from cultured species, natural inducers have been screened for their ability to improve those processes. However, few chemical inducers of attachment of invertebrates have been identified, and even less of these were secondary metabolites. The goal of this work was to isolate the natural products responsible for induction activity using bioassay-guided fractionation of the organic extract of the brown seaweed Stypopodium zonale and the attachment of juveniles of the common brown mussel, Perna perna, as a model. The meroditerpene epitaondiol, identified by comparison of spectral data with the literature, promoted as much as 4.7 times more mussel attachment compared to controls at the natural concentration found in this alga (0.041% of the crude extract or 0.012% of algal dry weight). This is the first report showing that a seaweed produces terpenoid compounds as cues for invertebrate attachment, and future studies evaluating this action on settlement of mussels in the field are expected to improve aquaculture technology by increasing mussel spat production.

The effect of epibionts on the susceptibility of the red seaweed Cryptonemia seminervis to herbivory and fouling.

Epibiosis or fouling on living organisms can have direct and indirect detrimental effects, in particular on photosynthetic organisms such as seaweeds. It thus seems reasonable to hypothesize that macroalgae have been selected for the presence or induction of antifouling (AF) defences. The red seaweed Cryptonemia seminervis is usually found in nature with an elevated cover of epibionts. To assess the effect of epibiosis on the susceptibility of this seaweed to herbivory and fouling, the abundance of fouling was evaluated and compared to herbivore consumption (by amphipods and sea urchins) of fouled (bryozoan and sponge) and non-fouled C. seminervis. Attachment of the mussel Perna perna to surfaces treated with extracts from seaweeds with and without epibionts was also assessed. Epibiosis corresponded to ca. 51% of the blade surface of C. seminervis, sometimes covering as much as 90% and up to 51% of the thallus weight, encompassing mainly the bryozoan Membranipora membranacea and an unidentified sponge. Algae colonized by M. membranacea were preferred compared to algae devoid of epibionts, a 'shared doom' effect, either by the amphipod Elasmopus brasiliensis or by the urchin Lytechinus variegatus (p < 0.01). Sponge epibiosis also increased consumption by both herbivores (p < 0.001), suggesting that epibionts may act as lures to herbivores, attracting consumers that otherwise would not feed significantly on the seaweed. Foods containing extracts from fouled C. seminervis were preferred by urchins over the alga devoid of epibionts. However, extracts from fouled alga inhibited mussel attachment when compared to epibiont-free alga. Differences might be a direct detrimental effect of the presence of epibionts. On the other hand, epibiosis may induce the production of AF defences in C. seminervis.

Long-term effects of competition and environmental drivers on the growth of the endangered coral Mussismilia braziliensis (Verril, 1867).

Most coral reefs have recently experienced acute changes in benthic community structure, generally involving dominance shifts from slow-growing hard corals to fast-growing benthic invertebrates and fleshy photosynthesizers. Besides overfishing, increased nutrification and sedimentation are important drivers of this process, which is well documented at landscape scales in the Caribbean and in the Indo-Pacific. However, small-scale processes that occur at the level of individual organisms remain poorly explored. In addition, the generality of coral reef decline models still needs to be verified on the vast realm of turbid-zone reefs. Here, we documented the outcome of interactions between an endangered Brazilian-endemic coral (Mussismilia braziliensis) and its most abundant contacting organisms (turf, cyanobacteria, corals, crustose coralline algae and foliose macroalgae). Our study was based on a long (2006-2016) series of high resolution data (fixed photoquadrats) acquired along a cross-shelf gradient that includes coastal unprotected reefs and offshore protected sites. The study region (Abrolhos Bank) comprises the largest and richest coralline complex in the South Atlantic, and a foremost example of a turbid-zone reef system with low diversity and expressive coral cover. Coral growth was significantly different between reefs. Coral-algae contacts predominated inshore, while cyanobacteria and turf contacts dominated offshore. An overall trend in positive coral growth was detected from 2009 onward in the inshore reef, whereas retraction in live coral tissue was observed offshore during this period. Turbidity (+) and cyanobacteria (-) were the best predictors of coral growth. Complimentary incubation experiments, in which treatments of Symbiodinium spp. from M. braziliensis colonies were subjected to cyanobacterial exudates, showed a negative effect of the exudate on the symbionts, demonstrating that cyanobacteria play an important role in coral tissue necrosis. Negative effects of cyanobacteria on living coral tissue may remain undetected from percent cover estimates gathered at larger spatial scales, as these ephemeral organisms tend to be rapidly replaced by longer-living macroalgae, or complex turf-like consortia. The cross-shelf trend of decreasing turbidity and macroalgae abundance suggests either a direct positive effect of turbidity on coral growth, or an indirect effect related to the higher inshore cover of foliose macroalgae, constraining cyanobacterial abundance. It is unclear whether the higher inshore macroalgal abundance (10-20% of reef cover) is a stable phase related to a long-standing high turbidity background, or a contemporary response to anthropogenic stress. Our results challenge the idea that high macroalgal cover is always associated with compromised coral health, as the baselines for turbid zone reefs may derive sharply from those of coral-dominated reefs that dwell under oligotrophic conditions.

Antipredator defense and phenotypic plasticity of sclerites from Renilla muelleri, a tropical sea pansy.

Calcified sclerites are common in many benthic marine invertebrates, and despite their widespread occurrence, little is known about their ecological roles. Previous studies suggested that the sclerite composition of coral colonies may be altered in response to environmental cues such as predation and water motion. Furthermore, larger sclerites are thought to be more effective than small ones in deterring predators, while small sclerites may provide greater stiffness and resistance to deformation. The present study compared the length of the sclerites of the sea pansy Renilla muelleri from three depths in Guanabara Bay in southeastern Brazil. Our results show that sclerites are larger in deep-water specimens than in those from shallow water. Field assays were conducted in which sclerites from sea pansies at three depths were incorporated into artificial foods and offered to a natural assemblage of fish. These assays demonstrate that sclerites from R. muelleri from all three depths significantly reduced consumption by generalist carnivorous fishes. We conclude that R. muelleri uses skeletal elements not only to give the body its form but also as a defense against biotic threats.

Alpha-amylase inhibitors from Brazilian seaweeds and their hypoglycemic potential.

The acetone crude extract of Lobophora variegata did not show any alpha-amylase inhibition activity. On the contrary, acetone crude extract of both Spatoglossum schroederi and Caulerpa racemosa inhibited the enzyme activity, with an ED(50) of 0.58 and 0.09 mg/ml.

Structuring effects of chemicals from the sea fan Phyllogorgia dilatata on benthic communities.

Despite advances in understanding the ecological functions of secondary metabolites from marine organisms, there has been little focus on the influence of chemically-defended species at the community level. Several compounds have been isolated from the gorgonian octocoral Phyllogorgia dilatata, a conspicuous species that forms dense canopies on rocky reefs of northern Rio de Janeiro State, Brazil. Manipulative experiments were performed to study: (1) the effects of live colonies of P. dilatata (physical presence and chemistry) on recruitment of sympatric benthic organisms; (2) the allelopathic effects of its chemicals on competitors; and (3) chemotactic responses of the non-indigenous brittle star, Ophiothela mirabilis. Early establishment of benthic species was influenced on substrates around live P. dilatata colonies and some effects could be attributed to the gorgonian's secondary metabolites.In addition, the gorgonian chemicals also exerted an allelopathic effect on the sympatric zoanthid Palythoa caribaeorum, and positive chemotaxis upon O. mirabilis. These results indicate multiple ecological roles of a chemically-defended gorgonian on settlement, sympatric competitors, and non-indigenous species.

An extensive reef system at the Amazon River mouth.

Large rivers create major gaps in reef distribution along tropical shelves. The Amazon River represents 20% of the global riverine discharge to the ocean, generating up to a 1.3 × 10(6)-km(2) plume, and extensive muddy bottoms in the equatorial margin of South America. As a result, a wide area of the tropical North Atlantic is heavily affected in terms of salinity, pH, light penetration, and sedimentation. Such unfavorable conditions were thought to imprint a major gap in Western Atlantic reefs. We present an extensive carbonate system off the Amazon mouth, underneath the river plume. Significant carbonate sedimentation occurred during lowstand sea level, and still occurs in the outer shelf, resulting in complex hard-bottom topography. A permanent near-bottom wedge of ocean water, together with the seasonal nature of the plume's eastward retroflection, conditions the existence of this extensive (~9500 km(2)) hard-bottom mosaic. The Amazon reefs transition from accretive to erosional structures and encompass extensive rhodolith beds. Carbonate structures function as a connectivity corridor for wide depth-ranging reef-associated species, being heavily colonized by large sponges and other structure-forming filter feeders that dwell under low light and high levels of particulates. The oxycline between the plume and subplume is associated with chemoautotrophic and anaerobic microbial metabolisms. The system described here provides several insights about the responses of tropical reefs to suboptimal and marginal reef-building conditions, which are accelerating worldwide due to global changes.

Glycolipids from seaweeds and their potential biotechnological applications.

Marine macroalgae, or seaweeds, are a formidable source of natural compounds with diverse biological activities. In the last five decades it has been estimated that more than 3000 natural compounds were discovered from these organisms. The great majority of the published works have focused on terpenoids. In comparison, glycolipids are a neglected class of macroalgal secondary metabolites therefore remaining as a largely unknown reservoir of molecular diversity. Nevertheless, the interest regarding these compounds has been growing fast in the last decades as activities of ecological or pharmaceutical interest have been highlighted. This paper will review recent work regarding isolation and structural characterization of glycolipids from seaweeds and their prospective biological activities.

Diversity and antimicrobial potential of culturable heterotrophic bacteria associated with the endemic marine sponge Arenosclera brasiliensis.

Marine sponges are the oldest Metazoa, very often presenting a complex microbial consortium. Such is the case of the marine sponge Arenosclera brasiliensis, endemic to Rio de Janeiro State, Brazil. In this investigation we characterized the diversity of some of the culturable heterotrophic bacteria living in association with A. brasiliensis and determined their antimicrobial activity. The genera Endozoicomonas (N = 32), Bacillus (N = 26), Shewanella (N = 17), Pseudovibrio (N = 12), and Ruegeria (N = 8) were dominant among the recovered isolates, corresponding to 97% of all isolates. Approximately one third of the isolates living in association with A. brasiliensis produced antibiotics that inhibited the growth of Bacillus subtilis, suggesting that bacteria associated with this sponge play a role in its health.

Clathriamide, an hexapeptide isolated from the marine sponge Clathria (Clathria) nicoleae

ABSTRACT Chemical investigation of the aqueous fraction of the ethanol extract from the Brazilian endemic marine sponge Clathria (Clathria) nicoleae Vieira de Barros, Santos & Pinheiro, 2013, Microcionidae, sampled from a 55 m deep rhodolith bed at the Amazon River mouth, led to the isolation of a new hexapeptide, clathriamide (1). HP-20 resin was used to capture compound 1 from the aqueous fraction, which was purified by additional chromatographic steps. The absolute configuration of the amino acids of 1 was determined by advanced Marfey's analysis using 5-fluoro-2,4-dinitrophenyl-Nα-L-tryptophanamide. The amino acid derivatives analyzed by ultra-performance liquid chromatography coupled to a mass spectrometry using a C8 column enabled a good chromatographic resolution of L-Ile and L-allo-Ile, previously unfeasible using C18 column.

Traffic of secondary metabolites to cell surface in the red alga Laurencia dendroidea depends on a two-step transport by the cytoskeleton.

In Laurencia dendroidea, halogenated secondary metabolites are primarily located in the vacuole named the corps en cerise (CC). For chemical defence at the surface level, these metabolites are intracellularly mobilised through vesicle transport from the CC to the cell periphery for posterior exocytosis of these chemicals. The cell structures involved in this specific vesicle traffic as well as the cellular structures related to the positioning and anchoring of the CC within the cell are not well known. Here, we aimed to investigate the role of cytoskeletal elements in both processes. Cellular and molecular assays were conducted to i) determine the ultrastructural apparatus involved in the vesicle traffic, ii) localise cytoskeletal filaments, iii) evaluate the role of different cytoskeletal filaments in the vesicle transport, iv) identify the cytoskeletal filaments responsible for the positioning and anchoring of the CC, and v) identify the transcripts related to cytoskeletal activity and vesicle transport. Our results show that microfilaments are found within the connections linking the CC to the cell periphery, playing an essential role in the vesicle traffic at these connections, which means a first step of the secondary metabolites transport to the cell surface. After that, the microtubules work in the positioning of the vesicles along the cell periphery towards specific regions where exocytosis takes place, which corresponds to the second step of the secondary metabolites transport to the cell surface. In addition, microtubules are involved in anchoring and positioning the CC to the cell periphery. Transcriptomic analysis revealed the expression of genes coding for actin filaments, microtubules, motor proteins and cytoskeletal accessory proteins. Genes related to vesicle traffic, exocytosis and membrane recycling were also identified. Our findings show, for the first time, that actin microfilaments and microtubules play an underlying cellular role in the chemical defence of red algae.