Molecular analysis of a fungal disease in the habitat-forming brown macroalga Phyllospora comosa (Fucales) along a latitudinal gradient.

Infectious diseases affecting habitat-forming species can have significant impacts on population dynamics and alter the structure and functioning of marine ecosystems. Recently, a fungal infection was described as the causative agent of necrotic lesions on the stipe of the forest-forming macroalga Phyllospora comosa, a disease named "stipe rot" (SR). Here, we developed a quantitative PCR (qPCR) method for rapid detection and quantification of this pathogen, which was applied to evaluate the level of SR infection in eight P. comosa populations spanning the entire latitudinal distribution of this species along southeastern Australia. We also investigated the relationship between the abundance and prevalence of Stipe Rot Fungus (SRF) and potential host chemical defenses as well as its relationship with morphological and ecophysiological traits of P. comosa. qPCR estimates of SRF abundance reflected the levels of infection estimated by visual assessment, with higher numbers of SRF copies being observed in individuals showing high or intermediate levels of visual symptoms of SR. Concordance of conventional PCR and visual assessments was 92 and 94%, respectively, compared to qPCR detection. SRF prevalence was positively related to fucoxanthin content and herbivory, but not significant related to other traits measured (phlorotannin content, total length, thallus diameter, stipe width, number of branches, frond width, fouling, bleaching, gender, and photosynthetic efficiency). These results provide confidence for previous reports of this disease based upon visual assessments only, contribute to the development of monitoring and conservation strategies for safeguarding P. comosa forests, and generate insights into potential factors influencing host-pathogen interactions in this system.

Additional evidence of the trypanocidal action of (-)-elatol on amastigote forms through the involvement of reactive oxygen species.

Chagas' disease, a vector-transmitted infectious disease, is caused by the protozoa parasite Trypanosoma cruzi. Drugs that are currently available for the treatment of this disease are unsatisfactory, making the search for new chemotherapeutic agents a priority. We recently described the trypanocidal action of (-)-elatol, extracted from the macroalga Laurencia dendroidea. However, nothing has been described about the mechanism of action of this compound on amastigotes that are involved in the chronic phase of Chagas' disease. The goal of the present study was to evaluate the effect of (-)-elatol on the formation of superoxide anions (O2•-), DNA fragmentation, and autophagy in amastigotes of T. cruzi to elucidate the possible mechanism of the trypanocidal action of (-)-elatol. Treatment of the amastigotes with (-)-elatol increased the formation of O2•- at all concentrations of (-)-elatol assayed compared with untreated parasites. Increased fluorescence was observed in parasites treated with (-)-elatol, indicating DNA fragmentation and the formation of autophagic compartments. The results suggest that the trypanocidal action of (-)-elatol might involve the induction of the autophagic and apoptotic death pathways triggered by an imbalance of the parasite's redox metabolism.

Trypanocidal action of (-)-elatol involves an oxidative stress triggered by mitochondria dysfunction.

Natural compounds have shown good potential for the discovery of new chemotherapeutics for the treatment of Chagas' disease. Recently, our group reported the effective trypanocidal activity of (-)-elatol, extracted from the red macroalgae Laurencia dendroidea present in the Brazilian coast against Trypanosoma cruzi. However, the mechanism of action of this compound has remained unclear. There are only hypotheses concerning its action on mitochondrial function. Here, we further investigated the mechanisms of action of (-)-elatol on trypomastigotes of T. cruzi. For this, we evaluated some biochemical alterations in trypomastigotes treated with (-)-elatol. Our results show that (-)-elatol induced depolarization of the mitochondrial membrane, an increase in the formation of mitochondrial superoxide anion and loss of cell membrane and DNA integrity. Additionally, (-)-elatol induced formation of autophagic vacuoles and a decrease in cell volume. All together, these results suggest that the trypanocidal action of (-)-elatol involves multiple events and mitochondria might be the initial target organelle. Our hypothesis is that the mitochondrial dysfunction leads to an increase of ROS production through the electron transport chain, which affects cell membrane and DNA integrity leading to different types of parasite death.

Chemical defense in developmental stages and adult of the sea star Echinaster (Othilia) brasiliensis.

To date, evidence regarding the performance of secondary metabolites from larval stages of sea stars as an anti-predation defense relates only to a few species/specimens from a few geographic ranges. Unfortunately, this hinders a comprehensive global understanding of this inter-specific predator-prey interaction. Here, we present laboratory experimental evidence of chemical defense action in the early developmental stages and adults of the sea star Echinaster (Othilia) brasiliensis from Brazil against sympatric and allopatric invertebrate consumers. Blastulae, early and late brachiolarias of E. (O.) brasiliensis were not consumed by the sympatric and allopatric crabs Mithraculus forceps. Blastulae were also avoided by the sympatric and allopatric individuals of the anemone Anemonia sargassensis, but not the larval stages. Extracts from embryos (blastula) and brachiolarias of E. (O.) brasiliensis from one sampled population (João Fernandes beach) significantly inhibited the consumption by sympatric M. forceps, but not by allopatric crabs and A. sargassensi anemone. In this same site, extracts from adults E. (O.) brasiliensis significantly inhibited the consumption by sympatric and allopatric specimens of the crab in a range of concentrations. Whereas equivalent extract concentrations of E. (O.) brasiliensis from other population (Itaipu beach)inhibited the predation by allopatric M. forceps, while sympatric individuals of this crab avoided the only the higher level tested. Then, early stages and adult specimens of E. (O.) brasiliensis can be chemically defended against consumers, but this action is quite variable, depending on the type (anemone or crab) and the origin of the consumer (sympatric or allopatric).

Effect of elatol, isolated from red seaweed Laurencia dendroidea, on Leishmania amazonensis.

In the present study, we investigated the antileishmanial activity of sesquiterpene elatol, the major constituent of the Brazilian red seaweed Laurencia dendroidea (Hudson) J.V. Lamouroux, against L. amazonensis. Elatol after 72 h of treatment, showed an IC(50) of 4.0 µM and 0.45 µM for promastigote and intracellular amastigote forms of L. amazonensis, respectively. By scanning and transmission electron microscopy, parasites treated with elatol revealed notable changes compared with control cells, including: pronounced swelling of the mitochondrion; appearance of concentric membrane structures inside the organelle; destabilization of the plasma membrane; and formation of membrane structures, apparently an extension of the endoplasmic reticulum, which is suggestive of an autophagic process. A cytotoxicity assay showed that the action of the isolated compound is more specific for protozoa, and it is not toxic to macrophages. Our studies indicated that elatol is a potent antiproliferative agent against promastigote and intracellular amastigote forms, and may have important advantages for the development of new anti-leishamanial chemotherapies.

New Ecological Role of Seaweed Secondary Metabolites as Autotoxic and Allelopathic.

Allelopathy and autotoxicity are well-known biological processes in angiosperms but are very little explored or even unknown in seaweeds. In this study, extract and major pure compounds from two distinct populations of the red seaweed Laurencia dendroidea were investigated to evaluate the effect of autotoxicity through auto- and crossed experiments under laboratory conditions, using chlorophyll fluorescence imaging to measure inhibition of photosynthesis (ΦPSII) as a variable response. Individuals of L. dendroidea from Azeda beach were inhibited by their own extract (IC50 = 219 µg/ml) and the major compound elatol (IC50 = 87 µg/ml); both chemicals also inhibited this seaweed species from Forno beach (IC50 = 194 µg/ml for the extract and IC50 = 277 µg/ml for elatol). By contrast, the extract of L. dendroidea from Forno and its major compound obtusol showed no inhibitory effect in individuals of both populations; but obtusol was insoluble to be tested at higher concentrations, which could be active as observed for elatol. The Azeda population displayed higher susceptibility to the Azeda extract and to elatol, manifested on the first day, unlike Forno individuals, in which the effect was only detected on the second day; and inhibition of ΦPSII was more pronounced at apical than basal portions of the thalli of L. dendroidea. This first finding of seaweed autotoxicity and allelopathic effects revealed the potential of the chemistry of secondary metabolites for intra- and inter-populational interactions, and for structuring seaweed populations.

Molecular Mechanisms for Microbe Recognition and Defense by the Red Seaweed Laurencia dendroidea.

The ability to recognize and respond to the presence of microbes is an essential strategy for seaweeds to survive in the marine environment, but understanding of molecular seaweed-microbe interactions is limited. Laurencia dendroidea clones were inoculated with the marine bacterium Vibrio madracius. The seaweed RNA was sequenced, providing an unprecedentedly high coverage of the transcriptome of Laurencia, and the gene expression levels were compared between control and inoculated samples after 24, 48, and 72 h. Transcriptomic changes in L. dendroidea in the presence of V. madracius include the upregulation of genes that participate in signaling pathways described here for the first time as a response of seaweeds to microbes. Genes coding for defense-related transcription activators, reactive oxygen species metabolism, terpene biosynthesis, and energy conversion pathways were upregulated in inoculated samples of L. dendroidea, indicating an integrated defensive system in seaweeds. This report contributes significantly to the current knowledge about the molecular mechanisms involved in the highly dynamic seaweed-bacterium interactions. IMPORTANCE Marine bacteria are part of the healthy microbiota associated with seaweeds, but some species, such as Vibrio spp., are frequently associated with disease outbreaks, especially in economically valuable cultures. In this context, the ability of seaweeds to recognize microbes and, when necessary, activate defense mechanisms is essential for their survival. However, studies dedicated to understanding the molecular components of the immune response in seaweeds are rare and restricted to indirect stimulus. This work provides an unprecedentedly large-scale evaluation of the transcriptional changes involved in microbe recognition, cellular signaling, and defense in the red seaweed Laurencia dendroidea in response to the marine bacterium Vibrio madracius. By expanding knowledge about seaweed-bacterium interactions and about the integrated defensive system in seaweeds, this work offers the basis for the development of tools to increase the resistance of cultured seaweeds to bacterial infections.

Quantitative GC-ECD analysis of halogenated metabolites: determination of surface and within-thallus elatol of Laurencia obtusa.

Information on natural concentrations or variability of secondary metabolites in marine organisms may be important both to ecological/evolutionary and applied approaches. A gas chromatographic procedure with an electron capture detector (GC-ECD) was developed to quantify the sesquiterpenoid elatol at the surface and within-thalli of 70 specimens of the red seaweed Laurencia obtusa. The concentration of elatol was highest within-thalli [9.89 mg g(-1) of L. obtusa, dry weight (d.w.)], compared to lower values found at the surface [0.006 mg g(-1) of L.obtusa (d.w.), or 0.5-10.0 ng cm(-2)]. This method provides a rapid and inexpensive quantification of small quantities of elatol, and probably may also be used to quantify other halogenated compounds usually found in red seaweeds.