Daniela Bueno Sudatti

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 IC50 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.

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.

TRANSPORT AND DEFENSIVE ROLE OF ELATOL AT THE SURFACE OF THE RED SEAWEED LAURENCIA OBTUSA (CERAMIALES, RHODOPHYTA) 1

Natural within‐thallus concentrations of elatol produced by Laurencia obtusa (Huds.) J. V. Lamour. inhibit herbivory and prevent fouling. However, elatol occurs in larger amounts within the thallus compared with the quantities from the surface of this alga. We evaluated whether the surface elatol concentrations inhibit both herbivory and fouling and whether the content of corps en cerise can be transferred to the external cell walls. Surface elatol concentrations did not inhibit herbivory by sea urchins, settlement of barnacle larvae, or mussel attachment. Evidence of a connection between the corps en cerise, where elatol is probably stored, and the cell wall of L. obtusa was based on channel‐like membranous connections that transport vesicles from the corps to the cell wall region. Therefore, L. obtusa presents a specific process of chemical transport between the cell storage structures and the plant surface. We hypothesized that if high amounts of elatol are capable of inhibiting herbivory and fouling, if the tested organisms are ecologically relevant, and if elatol really occurs on the surface of L. obtusa and this seaweed can transport this compound to its surface, the low natural concentration of defensive chemicals on the surface of L. obtusa is probably not absolute but may be variable according to environmental conditions. We also hypothesized that herbivory and fouling would not exert the same selective force for the production of defensive chemicals on L. obtusa’s surface since the low concentrations of elatol were inefficient to inhibit either processes or distinguish selective pressures.

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.

Inter- and intrapopulation variation in the defensive chemistry of the red seaweed Laurencia dendroidea (Ceramiales, Rhodophyta)

Oliveira A.S., Sudatti D.B., Fujii M.T., Rodrigues S.V. and Pereira R.C. 2013. Inter- and intrapopulation variation in the defensive chemistry of the red seaweed Laurencia dendroidea (Ceramiales, Rhodophyta). Phycologia 52: 130–136. DOI: 10.2216/12-058.1 Variation in the production of defensive chemicals by seaweeds is an important feature to understand the ecological and evolutionary aspects of the predator–prey interactions in marine systems. In this study, we evaluated the inter- and intrapopulation variations in the amount of elatol, which is a sesquiterpene produced as a chemical defence in specimens from four populations of the red seaweed Laurencia dendroidea along the Brazilian coast. The concentration of elatol varied from 2 to 10 times among the L. dendroidea individuals but was approximately three orders of magnitude broader among the populations from 0.001% to 1.24% of the dry weight. The specimens collected at lower latitudes produced low amounts of defences, which did not support the hypothesis that chemical defences increase with a decrease in latitude. When cultivated under distinct temperatures, L. dendroidea specimens produced more defences (= elatol) under high temperatures (25°C). Although we observed a genotypic relevance for chemical defence, the environmental differences along the Brazilian coast support the hypothesis that the variability observed in elatol production by L. dendroidea was most likely influenced by small-scale changes in environmental factors.

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.

Chemical Defense as a Condition-Dependent Trait in Harvestmen

The expression of costly traits often depends on the amount of food available to the individuals. Chemical defenses are costly, thus their production should be condition-dependent. Here, we tested the hypothesis that an increase in food availability and an acetate-supplemented diet will increase the production of chemical defenses by the harvestman Magnispina neptunus, which releases alkylated benzoquinones biosynthesized using acetate as a precursor. We manipulated the diet of the individuals and created four experimental groups: well-fed with acetate, well-fed without acetate, poorly-fed with acetate, and poorly-fed without acetate. Well-fed individuals produced secretions with higher mass and concentration of benzoquinones than poorly-fed individuals, but we detected no significant effect of the acetate supplement. Thus, the production of benzoquinones is condition-dependent, and even short periods of dietary restriction may make individuals more vulnerable to predators, imposing fitness consequences to chemically-protected arthropods that biosynthesize their own defensive compounds.

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

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. ABSTRACT 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.