Climate change and species facilitation affect the recruitment of macroalgal marine forests.

Marine forests are shrinking globally due to several anthropogenic impacts including climate change. Forest-forming macroalgae, such as Cystoseira s.l. species, can be particularly sensitive to environmental conditions (e.g. temperature increase, pollution or sedimentation), especially during early life stages. However, not much is known about their response to the interactive effects of ocean warming (OW) and acidification (OA). These drivers can also affect the performance and survival of crustose coralline algae, which are associated understory species likely playing a role in the recruitment of later successional species such as forest-forming macroalgae. We tested the interactive effects of elevated temperature, low pH and species facilitation on the recruitment of Cystoseira compressa. We demonstrate that the interactive effects of OW and OA negatively affect the recruitment of C. compressa and its associated coralline algae Neogoniolithon brassica-florida. The density of recruits was lower under the combinations OW and OA, while the size was negatively affected by the temperature increase but positively affected by the low pH. The results from this study show that the interactive effects of climate change and the presence of crustose coralline algae can have a negative impact on the recruitment of Cystoseira s.l. species. While new restoration techniques recently opened the door to marine forest restoration, our results show that the interactions of multiple drivers and species interactions have to be considered to achieve long-term population sustainability.

Expression patterns of sterol transporters NPC1 and NPC2 in the cnidarian-dinoflagellate symbiosis.

The symbiotic interaction between cnidarians (e.g., corals and sea anemones) and photosynthetic dinoflagellates of the genus Symbiodinium is triggered by both host-symbiont recognition processes and metabolic exchange between the 2 partners. The molecular communication is crucial for homeostatic regulation of the symbiosis, both under normal conditions and during stresses that further lead to symbiosis collapse. It is therefore important to identify and fully characterise the key players of this intimate interaction at the symbiotic interface. In this study, we determined the cellular and subcellular localization and expression of the sterol-trafficking Niemann-Pick type C proteins (NPC1 and NPC2) in the symbiotic sea anemones Anemonia viridis and Aiptasia sp. We first established that NPC1 is localised within vesicles in host tissues and to the symbiosome membranes in several anthozoan species. We demonstrated that the canonical NPC2-a protein is mainly expressed in the epidermis, whereas the NPC2-d protein is closely associated with symbiosome membranes. Furthermore, we showed that the expression of the NPC2-d protein is correlated with symbiont presence in healthy symbiotic specimens. As npc2-d is a cnidarian-specific duplicated gene, we hypothesised that it probably arose from a subfunctionalisation process that might result in a gain of function and symbiosis adaptation in anthozoans. Niemann-Pick type C proteins may be key players in a functional symbiosis and be useful tools to study host-symbiont interactions in the anthozoan-dinoflagellate association.

Chloroplast and oxygen evolution changes in Symbiodinium sp. as a response to latrunculin and butanedione monoxime treatments under various light conditions.

The actin cytoskeleton is a dynamic structure that provides an interactive platform for organelles and cellular components. It also serves as track for membranes and vesicles that move via myosin. The actin cytoskeleton of Symbiodinium is a well-organized reticular structure suggestive of multiple membrane interactions, very likely including those of the chloroplast. The Symbiodinium chloroplast membrane network is, in turn, a highly organized structure, suggestive of being under the control of an organizing network. We visualized the chloroplast membranes of cultured Symbiodinium sp. under various light conditions and observed changes dependent on illumination intensity. Since we suspected interaction between these two organelles, and we knew that the Symbiodinium actin cytoskeleton collapses upon treatment with either latrunculin B, an actin microfilament-disrupting agent, or butanedione monoxime, a myosin function inhibitor, we tested the Symbiodinium sp. oxygen evolution in their presence. Upon latrunculin B addition, the oxygen production decreased compared to non-treated cells; however, this was not observed after a 24 h latrunculin treatment. On the contrary, butanedione monoxime treatment caused a non-recoverable dysfunction of the chloroplast causing a severe loss in oxygen production even after long-term exposure. Using electron microscopy, we observed an alteration of the Symbiodinium sp. chloroplast distribution after latrunculin B treatment, with respect to untreated cells. Furthermore, a thorough disorganization of the chloroplast grana was observed after butanedione monoxime treatment. These data suggest that an actomyosin system would be important for chloroplast organization and distribution, and critical for normal photosynthetic function of Symbiodinium sp.

Are Niemann-Pick type C proteins key players in cnidarian-dinoflagellate endosymbioses?

The symbiotic interaction between cnidarians, such as corals and sea anemones, and the unicellular algae Symbiodinium is regulated by yet poorly understood cellular mechanisms, despite the ecological importance of coral reefs. These mechanisms, including host-symbiont recognition and metabolic exchange, control symbiosis stability under normal conditions, but also lead to symbiosis breakdown (bleaching) during stress. This study describes the repertoire of the sterol-trafficking proteins Niemann-Pick type C (NPC1 and NPC2) in the symbiotic sea anemone Anemonia viridis. We found one NPC1 gene in contrast to the two genes (NPC1 and NPC1L1) present in vertebrate genomes. While only one NPC2 gene is present in many metazoans, this gene has been duplicated in cnidarians, and we detected four NPC2 genes in A. viridis. However, only one gene (AvNPC2-d) was upregulated in symbiotic relative to aposymbiotic sea anemones and displayed higher expression in the gastrodermis (symbiont-containing tissue) than in the epidermis. We performed immunolabelling experiments on tentacle cross sections and demonstrated that the AvNPC2-d protein was closely associated with symbiosomes. In addition, AvNPC1 and AvNPC2-d gene expression was strongly downregulated during stress. These data suggest that AvNPC2-d is involved in both the stability and dysfunction of cnidarian-dinoflagellate symbioses.

Establishment of primary cell culture from the temperate symbiotic cnidarian, Anemonia viridis.

The temperate symbiotic sea anemone Anemonia viridis, a member of the Cnidaria phylum, is a relevant experimental model to investigate the molecular and cellular events involved in the preservation or in the rupture of the symbiosis between the animal cells and their symbiotic microalgae, commonly named zooxanthellae. In order to increase research tools for this model, we developed a primary culture from A. viridis animal cells. By adapting enzymatic dissociation protocols, we isolated animal host cells from a whole tentacle in regeneration state. Each plating resulted in a heterogeneous primary culture consisted of free zooxanthellae and many regular, small rounded and adherent cells (of 3-5 µm diameter). Molecular analyses conducted on primary cultures, maintained for 2 weeks, confirmed a specific signature of A. viridis cells. Further serial dilutions and micromanipulation allowed us to obtain homogenous primary cultures of the small rounded cells, corresponding to A. viridis "epithelial-like cells". The maintenance and the propagation over a 4 weeks period of primary cells provide, for in vitro cnidarian studies, a preliminary step for further investigations on cnidarian cellular pathways notably in regard to symbiosis interactions.

Effect of Tris-Hydroxymethyl Aminomethane on intracellular pH depends on the extracellular non-bicarbonate buffering capacity.

The effect of Tris-Hydroxymethyl Aminomethane (THAM) on intracellular pH (pHi) is unknown. We previously demonstrated that the effect of sodium bicarbonate on pHi depends on the non-bicarbonate buffering system. First, human hepatocytes from hepatocytes cell culture (HepG2) were perfused with an acidotic artificial medium containing 5-mmol/L (H5) or 30-mmol/L (H30) concentrations of 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES), a non-bicarbonate buffer. We studied the effect of THAM on the pHi in both conditions. We repeated the same protocol using an acidotic human blood with a 5% or 40% hematocrit. The pHi was measured with the pH-sensitive fluorescent dye bis-carboxyethyl carboxy-fluorescein (BCECF). Gas analysis was performed before and during the alkaline infusion. The results showed that THAM caused an intracellular alkalization that was higher when the non-bicarbonate buffer concentration was low (0.45 +/- 0.21 and 0.22 +/- 0.14 pH units with H5 and H30, respectively). A significant relationship was found between changes in pHi and changes in PCO(2). Similar results were obtained with the human blood. In conclusion, the intracellular alkalizing effect of THAM is caused by the induced decrease of PCO(2) linked to the extracellular non-bicarbonate buffer capacity: The smaller the concentration of extracellular non-bicarbonate buffer, the higher the PCO(2) decrease caused by THAM.

Characterization and variations of organic parameters in teleost fish endolymph during day-night cycle, starvation and stress conditions.

The aim of the present work was to examine the modifications of the organic composition of fish endolymph under environmental conditions (day-night cycle, starvation and Cl2-stress) known to modify otolith growth. Endolymph electrophoretic patterns were compared. An antibody raised against the trout otolith organic matrix allowed examining the variations of organic matrix precursors in the endolymph under the above conditions. Western blot analysis showed bands around 60-80 kDa. A 50% decrease of immunolabelling was observed during the night whereas increases were seen after starvation (factor 3) or stress (factor 2) suggesting that these variations could be related to the organic matrix deposit. A factor retarding in vitro CaCO3 crystallization (FRC) was shown to co-precipitate with endolymph proteins and its apparent molecular mass (determined by measuring the activity after electro elution of gel electrophoresis) was estimated around 20 kDa. The FRC activity was stable during day-night cycle whereas it decreased by 70% and nearly 100% under starvation and stress respectively. These results suggest that the FRC, although retarding in vitro crystallization, plays a major role in the process of otolith calcification and that the decreases measured after starvation and stress are responsible for the decreases of the otolith growth. The variations of these two parameters (precursors and FRC) could contribute for the changes in the microstructure of the otolith.