Cnidarian Primary Cell Culture as a Tool to Investigate the Effect of Thermal Stress at Cellular Level

In the context of global change, symbiotic cnidarians are largely affected by seawater temperature elevation leading to symbiosis breakdown. This process, also called bleaching, is triggered by the dysfunction of the symbiont photosystems causing an oxidative stress and cell death to both symbiont and host cells. In our study, we wanted to elucidate the intrinsic capacity of isolated animal cells to deal with thermal stress in the absence of symbiont. In that aim, we have characterized an animal primary cell culture form regenerating tentacles of the temperate sea anemone Anemonia viridis. We first compared the potential of whole tissue tentacle or separated epidermal or gastrodermal monolayers as tissue sources to settle animal cell cultures. Interestingly, only isolated cells extracted from whole tentacles allowed establishing a viable and proliferative primary cell culture throughout 31 days. The analysis of the expression of tissue-specific and pluripotency markers defined cultivated cells as differentiated cells with gastrodermal origin. The characterization of the animal primary cell culture allowed us to submit the obtained gastrodermal cells to hyperthermal stress (+?5 and +?8 °C) during 1 and 7 days. Though cell viability was not affected at both hyperthermal stress conditions, cell growth drastically decreased. In addition, only a +?8 °C hyperthermia induced a transient increase of antioxidant defences at 1 day but no ubiquitin or carbonylation protein damages. These results demonstrated an intrinsic resistance of cnidarian gastrodermal cells to hyperthermal stress and then confirmed the role of symbionts in the hyperthermia sensitivity leading to bleaching.     

Molecular evidence of symbiotic activity between <Emphasis Type="Italic">Symbiodinium</Emphasis> and <Emphasis Type="Italic">Tridacna maxima</Emphasis> larvae

Dinoflagellates in the genus Symbiodinium (zooxanthellae) provide the photosynthesis that sustains the majority of primary production in coral reefs. They occur symbiotically with several phyla, including mollusks such as giant clams (Tridacna spp.). This mutualistic association is obligatory for the giant clams, but the exact point in which this symbiosis is established and the main translocated photosynthate are unknown. In this study, we tracked the expression of specific genes for symbiosis and glycerol synthesis during a time course experiment. Giant clam larvae were raised until 75 h post-fertilization and then infected with cultured isolates of Symbiodinium clade A3. Expression of symbiosis-specific and housekeeping genes was monitored at four time points. The expression of H⁺-ATPase, a symbiosis-specific gene in Symbiodinium, was observed at 24 h after symbiont acquisition by the clam larvae. The expression of an enzyme responsible for glycerol synthesis was also observed. Together, these results show that the symbiotic relationship was already in place 24 h after Symbiodinium acquisition, during veliger larval stage. This is the first report using a molecular symbiosis-specific marker that supports symbiotic activity between Symbiodinium and a metazoan larva of an organism that acquires symbionts horizontally. From the expression of the glycerol-synthesizing gene, it was qualitatively determined that Symbiodinium cells may produce glycerol regardless of whether they are free-living or in symbiosis.     

Metatrancriptomic analysis from the Hepatopancreas of adult white leg shrimp (<Emphasis Type="Italic">Litopenaeus vannamei)</Emphasis>

The amoeba, Mayorella viridis contains several hundred symbiotic green algae in its cytoplasm. Transmission electron microscopy revealed strong resemblance between symbiotic algae from M. viridis the symbiotic Chlorella sp. in the perialgal vacuoles of Paramecium bursaria and other ciliates. Although it is thought that the M. viridis and symbiotic algae could be model organisms for studying endosymbiosis between protists and green algae, few cell biological observations of the endosymbiosis between M. viridis and their symbiotic algae have been published. In this study, we characterized the specificity of endosymbiotic relationships between green algae and their hosts. Initially, we established stable cultures of M. viridis in KCM medium by feeding with Chlorogonium capillatum. Microscopic analyses showed that chloroplasts of symbiotic algae in M. viridis occupy approximately half of the algal cells, whereas those in P. bursaria occupy entire algal cells. The symbiotic algae in P. bursaria contain several small spherical vacuoles. The labeling of actin filaments using Acti-stain? 488 Fluorescent Phalloidin revealed no relationship between host actin filaments and symbiotic algal localization, although the host mitochondria were localized around symbiotic algae. Symbiotic algae from M. viridis could infect algae-free P. bursaria but could not support P. bursaria growth without feeding, whereas the original symbiotic algae of P. bursaria supported its growth without feeding. These data indicated the specificity of endosymbiotic algae relationships in M. viridis and P. bursaria.     

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.     

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.     

Allozyme electrophoresis as a tool for distinguishing different zooxanthellae symbiotic with giant clams

The taxonomy of zooxanthellae in marine invertebrate symbioses is not well understood owing mainly to their lack of reliable morphological differences. Nevertheless, previous work using protein and DNA electrophoreses has set the stage for advancing our taxonomic understanding of cnidarian zooxanthellae. Here we present the use of allozymes as genetic markers for distinguishing algal isolates from tridacnid hosts. Zooxanthellae from seven Tridacna and Hippopus species were isolated and maintained in axenic clonal cultures over many generations. Of 16 enzyme systems, α- and β-esterase (EST), esterase-F (EST-F), glucose phosphate isomerase (GPI), and malate dehydrogenase (MDH) were found suitable polymorphic markers of genetic differences among clonal cultures. Of 39 clonal isolates, 97% were found to be genetically distinguishable. This high extent of genetic variation in zooxanthellae within and between clam species was unexpected, and is difficult to explain based solely on the general notion of asexual reproduction in symbiotic zooxanthellae. Our results are also consistent with the occurrence of sexual reproduction in clam zooxanthellae. The close genetic similarity of the symbionts of Tridacna gigas, the largest and fastest-growing clam species, and the difficulty of initiating their clonal cultures in the given nutrient medium, compared with the symbionts of other clam species, are further indicative of possibly distinct algal symbionts in T. gigas. These findings are discussed in light of current taxonomic understanding of these organisms.     

Catalase characterization and implication in bleaching of a symbiotic sea anemone

Symbiotic cnidarians are marine invertebrates harboring photosynthesizing microalgae (named zooxanthellae), which produce great amounts of oxygen and free radicals upon illumination. Studying antioxidative balance is then crucial to understanding how symbiotic cnidarians cope with ROS production. In particular, it is suspected that oxidative stress triggers cnidarian bleaching, i.e., the expulsion of zooxanthellae from the animal host, responsible for symbiotic cnidarian mass mortality worldwide. This study therefore investigates catalase antioxidant enzymes and their role in bleaching of the temperate symbiotic sea anemone Anemonia viridis. Using specific separation of animal tissues (ectoderm and endoderm) from the symbionts (zooxanthellae), spectrophotometric assays and native PAGE revealed both tissue-specific and activity pattern distribution of two catalase electrophoretypes, E1 and E2. E1, expressed in all three tissues, presents high sensitivity to the catalase inhibitor aminotriazole (ATZ) and elevated temperatures. The ectodermal E1 form is responsible for 67% of total catalase activity. The E2 form, expressed only within zooxanthellae and their host endodermal cells, displays low sensitivity to ATZ and relative thermostability. We further cloned an ectodermal catalase, which shares 68% identity with mammalian monofunctional catalases. Last, 6 days of exposure of whole sea anemones to ATZ (0.5 mM) led to effective catalase inhibition and initiated symbiont expulsion. This demonstrates the crucial role of this enzyme in cnidarian bleaching, a phenomenon responsible for worldwide climate-change-induced mass mortalities, with catastrophic consequences for marine biodiversity.     

The Symbiotic Anthozoan: A Physiological Chimera between Alga and Animal

The symbiotic life style involves mutual ecological, physiological,structural, and molecular adaptations between the partners.In the symbiotic association between anthozoans and photosyntheticdinoflagellates (Symbiodinium spp., also called zooxanthellae),the presence of the endosymbiont in the animal cells has constrainedthe host in several ways. It adopts behaviors that optimizephotosynthesis of the zooxanthellae. The animal partner hashad to evolve the ability to absorb and concentrate dissolvedinorganic carbon from seawater in order to supply the symbiont'sphotosynthesis. Exposing itself to sunlight to illuminate itssymbionts sufficiently also subjects the host to damaging solarultraviolet radiation. Protection against this is provided bybiochemical sunscreens, including mycosporine-like amino acids,themselves produced by the symbiont and translocated to thehost. Moreover, to protect itself against oxygen produced duringalgal photosynthesis, the cnidarian host has developed certainantioxidant defenses that are unique among animals. Finally,living in nutrient-poor waters, the animal partner has developedseveral mechanisms for nitrogen assimilation and conservationsuch as the ability to absorb inorganic nitrogen, highly unusualfor a metazoan. These facts suggest a parallel evolution ofsymbiotic cnidarians and plants, in which the animal host hasadopted characteristics usually associated with phototrophicorganisms.     

Intracellular pH of symbiotic dinoflagellates

Intracellular pH (pH_i) is likely to play a key role in maintaining the functional success of cnidarian–dinoflagellate symbiosis, yet until now the pH_i of the symbiotic dinoflagellates (genus Symbiodinium) has never been quantified. Flow cytometry was used in conjunction with the ratiometric fluorescent dye BCECF to monitor changes in pH_i over a daily light/dark cycle. The pH_i of Symbiodinium type B1 freshly isolated from the model sea anemone Aiptasia pulchella was 7.25 ± 0.01 (mean ± SE) in the light and 7.10 ± 0.02 in the dark. A comparable effect of irradiance was seen across a variety of cultured Symbiodinium genotypes (types A1, B1, E1, E2, F1, and F5) which varied between pH_i 7.21–7.39 in the light and 7.06–7.14 in the dark. Of note, there was a significant genotypic difference in pH_i, irrespective of irradiance.     

Algal genotype and photoacclimatory responses of the symbiotic alga Symbiodinium in natural populations of the sea anemone Anemonia viridis

As an approach to investigate the impact of solar radiation on an alga–invertebrate symbiosis, the genetic variation and photosynthetic responses of the dinoflagellate algal symbiosis in an intertidal and a subtidal population of the sea anemone Anemonia viridis were explored. Allozyme analysis of the anemones indicated that the two populations were genetically very similar, with a Nei''s index value of genetic identity (I) of 0.998. The algae in all animals examined were identified as Symbiodinium of clade a by PCR-RFLP analysis of the small subunit ribosomal RNA gene. The symbiosis in the two populations did not differ significantly in algal population density, chlorophyll a content per algal cell or any photosynthetic parameter obtained from studies of the relationship between photosynthesis and irradiance. We conclude that there is not necessarily genetic variation or photosynthetic plasticity of the symbiotic algae in Anemonia viridis inhabiting environments characterized by the different solar irradiances of the subtidal and intertidal habitats.     

Regulation and control of intracellular algae (= zooxanthellae) in hard corals

To examine algal (= zooxanthellae) regulation and control, and the factors determining algal densities in hard corals, the zooxanthellae mitotic index and release rates were regularly determined in branch tips from a colony of a staghorn coral, Acropora formosa, recovering from a coral ''bleaching'' event (the stress-related dissociation of the coral–algal symbiosis). Mathematical models based upon density-dependent decreases in the algal division frequency and increases in algal release rates during the post-bleaching recovery period accurately predict the observed recovery period (ca. 20 weeks). The models suggest that (i) the colony recovered its algal population from the division of the remaining zooxanthellae, and (ii) the continual loss of zooxanthellae significantly slowed the recovery of the coral. Possible reasons for the ''paradoxical'' loss of healthy zooxanthellae from the bleached coral are discussed in terms of endodermal processes occurring in the recovering coral and the redistribution of newly formed zooxanthellae to aposymbiotic host cells. At a steady-state algal density of 2.1 x 10⁶ zooxanthellae cm^-2 at the end of the recovery period, the zooxanthellae would have to form a double layer of cells in the coral tissues, consistent with microscopic observations. Neighbouring colonies of A. formosa with inherently higher algal densities possess proportionately smaller zooxanthellae. Results suggest that space availability and the size of the algal symbionts determines the algal densities in the coral colonies. The large increases in the algal densities reported in corals exposed to elevated nutrient concentrations (i.e between a two- and five-fold increase in the algal standing stock) are not consistent with this theory. We suggest that increases of this magnitude are a product of the experimental conditions: reasons for this statement are discussed. We propose that the stability of the coral–algal symbiosis under non-stress conditions, and the constancy of zooxanthellae densities in corals reported across growth form, depth and geographic range, are related to space availability limiting algal densities. However, at these densities, zooxanthellae have attributes consistent with nutrient limitation.     

Symbiosis-dependent gene expression in coral–dinoflagellate association: cloning and characterization of a P-type H+-ATPase gene

We report the molecular cloning of a H⁺-ATPase in the symbiotic dinoflagellate, Symbiodinium sp. previously suggested by pharmacological studies to be involved in carbon-concentrating mechanism used by zooxanthellae when they are in symbiosis with corals. This gene encodes a protein of 975 amino acids with a calculated mass of about 105 kDa. The structure of the protein shows a typical P-type H⁺-ATPase structure (type IIIa plasma membrane H⁺-ATPases) and phylogenetic analyses show that this new proton pump groups with diatoms in the Chromoalveolates group. This Symbiodinium H⁺-ATPase is specifically expressed when zooxanthellae are engaged in a symbiotic relationship with the coral partner but not in free-living dinoflagellates. This proton pump, therefore, could be involved in the acidification of the perisymbiotic space leading to bicarbonate dehydration by carbonic anhydrase activity in order to supply inorganic carbon for photosynthesis as suggested by earlier studies. To our knowledge, this work provides the first example of a symbiosis-dependent gene in zooxanthellae and confirms the importance of H⁺-ATPase in coral–dinoflagellate symbiosis.     

Ammonium metabolism in the symbiotic sea anemone Anemonia viridis

The temperate sea anemone Anemonia viridis (Forskål) forms an endosymbiotic association with dinoflagellate algae commonly referred to as zooxanthellae. It is now well established that under appropriate environmental conditions, these associations can be autotrophic for carbon. Under such conditions, many of these symbioses, including A. viridis, not only retain excretory ammonium, but can take up ammonium added to the surrounding seawater. The flux from inorganic to organic nitrogen will be via the free amino acid pools and in A. viridis these were found to be markedly different between zooxanthellae and host with glycine and taurine dominant in the latter. When anemones were maintained with 20 M ammonium, the concentration of free amino groups increased in the zooxanthellae but appeared not to change in the host. There was no evidence that the ratio of glutamine – glutamate in zooxanthellae changed when anemones were maintained with 20 M ammonium for 47 days. These ratios imply that zooxanthellae from this temperate symbiosis may not be nitrogen-limited. GDH was detected in both zooxanthellae and host where it was most active with the coenzyme NADPH. In addition, GDH showed activity when glutamine replaced ammonium as the substrate, indicating that the host may have alternative means to assimilate ammonium. Zooxanthellae were shown to possess GOGAT activity in the presence of a ferredoxin analogue. This suggests that in vivo zooxanthellae could assimilate ammonium via the activity of GS linked with ferredoxin-dependent GOGAT. Given evidence from other studies of rapid ammonium assimilation and essential amino acid synthesis in symbiotic host tissue, it appears that the capacity of cnidarians to metabolise nitrogen may at present be underestimated.     

Body size and symbiotic status influence gonad development in <Emphasis Type="Italic">Aiptasia pallida</Emphasis> anemones

Pale anemones (Aiptasia pallida) coexist with dinoflagellates (primarily Symbiodinium minutum) in a mutualistic relationship. The purpose of this study was to investigate the role of these symbionts in gonad development of anemone hosts. Symbiotic and aposymbiotic anemones were subjected to light cycles that induced gametogenesis. These anemones were then sampled weekly for nine weeks, and gonad development was analyzed histologically. Anemone size was measured as mean body column diameter, and oocytes or sperm follicles were counted for each anemone. Generalized linear models were used to evaluate the influence of body size and symbiotic status on whether gonads were present and on the number of oocytes or sperm follicles produced. Body size predicted whether gonads were present, with larger anemones being more likely than smaller anemones to develop gonads. Both body size and symbiotic status predicted gonad size, such that larger and symbiotic anemones produced more oocytes and sperm follicles than smaller and aposymbiotic anemones. Overall, only 22 % of aposymbiotic females produced oocytes, whereas 63 % of symbiotic females produced oocytes. Similarly, 6 % of aposymbiotic males produced sperm follicles, whereas 60 % of symbiotic males produced sperm follicles. Thus, while gonads were present in 62 % of symbiotic anemones, they were present in only 11 % of aposymbiotic anemones. These results indicate that dinoflagellate symbionts influence gonad development and thus sexual maturation in both female and male Aiptasia pallida anemones. This finding substantiates and expands our current understanding of the importance of symbionts in the development and physiology of cnidarian hosts.     

The distribution of dimethylsulfoniopropionate in tropical Pacific coral reef invertebrates

Dimethylsulfoniopropionate (DMSP) is an important component of the global sulfur cycle and may be involved, via its cleavage product dimethylsulfide, in climate regulation. Although it is common in many algae, reports of DMSP in animals, particularly tropical invertebrates, are limited. This study examined the distribution of DMSP in a diverse group of coral reef invertebrates. DMSP was present in all 22 species of cnidarians and ranged from 9 to 723 μmol g⁻¹ of dry mass (DM) with a mean (± 1SD) of 110 ± 180 μmol g⁻¹ DM. It was not detected in a flatworm and an ascidian or in two of five sponges. Concentrations in sponges ranged from undetectable to 16 μmol g⁻¹ DM with a mean of 4 ± 7 μmol g⁻¹ DM. Within the cnidarians, DMSP concentrations did not differ among orders. Among cnidarian species, DMSP concentrations were correlated with symbiotic zooxanthellae densities. Within cnidarian species, DMSP concentrations of individuals were positively correlated with zooxanthellae densities in three of the four species examined. We speculate that DMSP is dietarily derived in sponges and derived from zooxanthellae in the cnidarians. The functions of DMSP in coral reef invertebrates are not known.     

Symbiosis regulation in a facultatively symbiotic temperate coral: zooxanthellae division and expulsion

Zooxanthellae mitotic index (MI) and expulsion rates were measured in the facultatively symbiotic scleractinian Astrangia poculata during winter and summer off the southern New England coast, USA. While MI was significantly higher in summer than in winter, mean expulsion rates were comparable between seasons. Corals therefore appear to allow increases in symbiont density when symbiosis is advantageous during the warm season, followed by a net reduction during the cold season when zooxanthellae may draw resources from the coral. Given previous reports that photosynthesis in A. poculata symbionts does not occur below approximately 6°C, considerable zooxanthellae division at 3°C and in darkness suggests that zooxanthellae are heterotrophic at low seasonal temperatures. Finally, examination of expulsion as a function of zooxanthellae density revealed that corals with very low zooxanthellae densities export a significantly greater proportion of their symbionts, apparently allowing them to persist in a stable azooxanthellate state.