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.     

Population genetic data of a model symbiotic cnidarian system reveal remarkable symbiotic specificity and vectored introductions across ocean basins

The Aiptasia–Symbiodinium symbiosis is a promising model for experimental studies of cnidarian–dinoflagellate associations, yet relatively little is known regarding the genetic diversity of either symbiotic partner. To address this, we collected Aiptasia from 16 localities throughout the world and examined the genetic diversity of both anemones and their endosymbionts. Based on newly developed SCAR markers, Aiptasia consisted of two genetically distinct populations: one Aiptasia lineage from Florida and a second network of Aiptasia genotypes found at other localities. These populations did not conform to the distributions of described Aiptasia species, suggesting that taxonomic re‐evaluation is needed in the light of molecular genetics. Associations with Symbiodinium further demonstrated the distinctions among Aiptasia populations. According to 18S RFLP, ITS2‐DGGE and microsatellite flanker region sequencing, Florida anemones engaged in diverse symbioses predominantly with members of Symbiodinium Clades A and B, but also C, whereas anemones from elsewhere harboured only S. minutum within Clade B. Symbiodinium minutum apparently does not form a stable symbiosis with other hosts, which implies a highly specific symbiosis. Fine‐scale differences among S. minutum populations were quantified using six microsatellite loci. Populations of S. minutum had low genotypic diversity and high clonality (R = 0.14). Furthermore, minimal population structure was observed among regions and ocean basins, due to allele and genotype sharing. The lack of genetic structure and low genotypic diversity suggest recent vectoring of Aiptasia and S. minutum across localities. This first ever molecular‐genetic study of a globally distributed cnidarian and its Symbiodinium assemblages reveals host–symbiont specificity and widely distributed populations in an important model system.     

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.     

Evidence for a role of protein phosphorylation in the maintenance of the cnidarian–algal symbiosis

The endosymbiotic relationship between cnidarians and photosynthetic dinoflagellate algae provides the foundation of coral reef ecosystems. This essential interaction is globally threatened by anthropogenic disturbance. As such, it is important to understand the molecular mechanisms underpinning the cnidarian–algal association. Here we investigated phosphorylation‐mediated protein signalling as a mechanism of regulation of the cnidarian–algal interaction, and we report on the generation of the first phosphoproteome for the coral model system Aiptasia. Mass spectrometry‐based phosphoproteomics using data‐independent acquisition allowed consistent quantification of over 3,000 phosphopeptides totalling more than 1,600 phosphoproteins across aposymbiotic (symbiont‐free) and symbiotic anemones. Comparison of the symbiotic states showed distinct phosphoproteomic profiles attributable to the differential phosphorylation of 539 proteins that cover a broad range of functions, from receptors to structural and signal transduction proteins. A subsequent pathway enrichment analysis identified the processes of “protein digestion and absorption,” “carbohydrate metabolism,” and “protein folding, sorting and degradation,” and highlighted differential phosphorylation of the “phospholipase D signalling pathway” and “protein processing in the endoplasmic reticulum.” Targeted phosphorylation of the phospholipase D signalling pathway suggests control of glutamate vesicle trafficking across symbiotic compartments, and phosphorylation of the endoplasmic reticulum machinery suggests recycling of symbiosome‐associated proteins. Our study shows for the first time that changes in the phosphorylation status of proteins between aposymbiotic and symbiotic Aiptasia anemones may play a role in the regulation of the cnidarian–algal symbiosis. This is the first phosphoproteomic study of a cnidarian–algal symbiotic association as well as the first application of quantification by data‐independent acquisition in the coral field.     

Isolation of clonal axenic strains of the symbiotic dinoflagellate Symbiodinium and their growth and host specificity1

The cnidarian‐dinoflagellate mutualism is integral to the survival of the coral‐reef ecosystem. Despite the enormous ecological and economic importance of corals, their cellular and molecular biology and the ways in which they respond to environmental change are still poorly understood. We have been developing a proxy system for examining the coral mutualism in which the dinoflagellate symbiont Symbiodinium is introduced into a clonal population of the host Aiptasia, a small sea anemone closely related to corals. To further develop the tools for this system, we generated five clonal, axenic strains of Symbiodinium and verified the lack of contaminants by growth on rich medium, microscopic examination, and PCR analysis. These strains were assigned to clades A (two strains), B, E, and F based on their chloroplast 23S rDNA sequences. Growth studies in liquid cultures showed that the clade B strain and one of the clade A strains were able to grow photoautotrophically (in light with no fixed carbon), mixotrophically (in light with fixed carbon), or heterotrophically (in dark with fixed carbon). The clade E strain, thought to be free‐living, was able to grow photoautotrophically but not heterotrophically. Infection of an aposymbiotic Aiptasia host with the axenic strains showed consistent patterns of specificity, with only the clade B and one of the clade A strains able to successfully establish symbiosis. Overall, the Aiptasia‐Symbiodinium association represents an important model system for dissecting aspects of the physiology and cellular and molecular biology of cnidarian‐dinoflagellate mutualism and exploring issues that bear directly on coral bleaching.     

Impact of Menthol on Growth and Photosynthetic Function of Breviolum Minutum (Dinoflagellata,Dinophyceae, Symbiodiniaceae) and Interactions with its Aiptasia Host

Environmental change, including global warming and chemical pollution, can compromise cnidarian‐(e.g., coral‐) dinoflagellate symbioses and cause coral bleaching. Understanding the mechanisms that regulate these symbioses will inform strategies for sustaining healthy coral–reef communities. A model system for corals is the symbiosis between the sea anemone Exaiptasia pallida (common name Aiptasia) and its dinoflagellate partners (family Symbiodiniaceae). To complement existing studies of the interactions between these organisms, we examined the impact of menthol, a reagent often used to render cnidarians aposymbiotic, on the dinoflagellate Breviolum minutum, both in culture and in hospite. In both environments, the growth and photosynthesis of this alga were compromised at either 100 or 300 µM menthol. We observed reduction in PSII and PSI functions, the abundances of reaction‐center proteins, and, at 300 µM menthol, of total cellular proteins. Interestingly, for free‐living algae exposed to 100 µM menthol, an initial decline in growth, photosynthetic activities, pigmentation, and protein abundances reversed after 5–15 d, eventually approaching control levels. This behavior was observed in cells maintained in continuous light, but not in cells experiencing a light–dark regimen, suggesting that B. minutum can detoxify menthol or acclimate and repair damaged photosynthetic complexes in a light‐ and/or energy‐dependent manner. Extended exposures of cultured algae to 300 µM menthol ultimately resulted in algal death. Most symbiotic anemones were also unable to survive this menthol concentration for 30 d. Additionally, cells impaired for photosynthesis by pre‐treatment with 300 µM menthol exhibited reduced efficiency in re‐populating the anemone host.     

Protection of host anemones by snapping shrimps: a case for symbiotic mutualism?

The sea anemone Bartholomea annulata is an ecologically important member of Caribbean coral reefs which host a variety of symbiotic crustacean associates. Crustacean exosymbionts typically gain protection from predation by dwelling with anemones. Concurrently, some symbionts may provide protection to their host by defending against anemone predators such as the predatory fireworm, Hermodice carunculata, which can severely damage or completely devour prey anemones. Herein we show through both field and laboratory studies that anemones hosting the symbiotic alpheid shrimp Alpheus armatus are significantly less likely to sustain damage by H. carunculata than anemones without this shrimp. Our results suggest that the association between A. armatus and B. annulata, although complex because of the numerous symbionts involved, may be closer to mutualism on the symbiotic continuum.     

Long-term laboratory culture of symbiotic coral juveniles and their use in eco-toxicological study

Reef-building (or hermatypic) corals live in mutualistic symbiosis with the dinoflagellates Symbiodinium spp. (Alveolata, Dinophyceae, Gymnodiniales), and contribute to the accretion of coral reefs. Due to the difficulty in culturing them in laboratories, these ecologically important cnidarians have not been characterized extensively in physiological, biochemical, molecular and toxicological experiments. The present study was conducted to develop a model symbiosis system for long-term experimental analyses of a symbiotic coral. Aposymbiotic (symbiont-free) juveniles of the hermatypic coral Acropora tenuis were infected with three Symbiodinium strains, and the resulting symbiotic corals were examined for growth and maintenance of the symbiosis for approx. three months. Of the tested Symbiodinium cell lines, CCMP2467 (clade A1) inhabited the host the most densely, and the population in hospite did not decline over the period of three months in laboratory culture. The CCMP2467-inhabited juveniles outgrew the populations infected with the other two strains and aposymbiotic specimens. The A. tenuis juveniles in symbiosis with CCMP2467 cells were used in eco-toxicological tests to study long-term effects of two commonly used biocides (tributyltin-chloride and diuron). Delay in growth was observed after exposing the symbiotic juveniles to the two chemicals for approx. 50 days at the nominal concentrations of 0.4 and 1 μg/L, respectively.     

Effects of anemonefish on giant sea anemones: expansion behavior,growth, and survival

The symbiosis between giant sea anemones and anemonefish on coral reefs is well known, but little information exists on impacts of this interaction on the sea anemone host. On a coral reef at Eilat, northern Red Sea, individuals of the sea anemone Entacmaea quadricolor that possessed endemic anemonefish Amphiprion bicinctus expanded their tentacles significantly more frequently than did those lacking anemonefish. When anemonefish were experimentally removed, sea anemone hosts contracted partially. Within 1–4 h in most cases, individuals of the butterflyfish Chaetodon fasciatus arrived and attacked the sea anemones, causing them to contract completely into reef holes. Upon the experimental return of anemonefish, the anemone hosts re-expanded. The long-term growth rate and survival of the sea anemones depended on the size and number of their anemonefish. Over several years, sea anemones possessing small or no fish exhibited negative growth (shrinkage) and eventually disappeared, while those with at least one large fish survived and grew. We conclude that host sea anemones sense the presence of symbiotic anemonefish via chemical and/or mechanical cues, and react by altering their expansion behavior. Host sea anemones that lack anemonefish large enough to defend them against predation may remain contracted in reef holes, unable to feed or expose their tentacles for photosynthesis, resulting in their shrinkage and eventual death.     

Predation on symbiont sea anemones by their host hermit crab Dardanus pedunculatus

Feeding by host hermit crabs Dardanus pedunculatus on their symbiotic sea anemones Calliactis polypus was investigated using animals collected at Shirahama, Wakayama Prefecture, Japan. In the first experiment, changes in the number of sea anemones on hermit crab shells were recorded in single‐and double‐crab trials without food and single‐crab trials with food. The number of sea anemones significantly decreased under starved conditions. The extent of this decrease per single hermit crab was higher in the double‐crab trials than in the single‐crab trials. Direct observations and video recordings showed that hermit crabs occasionally removed sea anemones from their own shells, and also from partners’ shells in the double‐crab trials, and consumed them. In the second experiment, fed and unfed hermit crabs with or without sea anemones were examined for body weight changes. Fed hermit crabs gained weight whereas unfed hermit crabs lost it. The degree of weight loss in unfed hermit crabs was significantly higher in those without sea anemones, which indicates some value of the latter as food. We offer some speculations on the course of development of this symbiosis, with predation on sea anemones having played an important initial role.     

Lotus hosts delimit the mutualism–parasitism continuum of Bradyrhizobium

Symbioses are modelled as evolutionarily and ecologically variable with fitness outcomes for hosts shifting on a continuum from mutualism to parasitism. In a classic example, rhizobia fix atmospheric nitrogen for legume hosts in exchange for photosynthetic carbon. Rhizobial infection often enhances legume growth, but hosts also incur interaction costs because of root tissues and or metabolites needed to support symbionts in planta. Rhizobia exhibit genetic variation in symbiotic effectiveness, and ecological changes in light or mineral nitrogen availability can also alter the benefits of rhizobial infection for hosts. The net effects of symbiosis thus can range from mutualistic to parasitic in a context‐dependent manner. We tested the extent of the mutualism–parasitism continuum in the legume–rhizobium symbiosis and the degree to which host investment can shape its limits. We infected Lotus strigosus with sympatric Bradyrhizobium genotypes that vary in symbiotic effectiveness. Inoculations occurred under different mineral nitrogen and light regimes spanning ecologically relevant ranges. Net growth benefits of Bradyrhizobium infection varied for Lotus and were reduced or eliminated dependent on Bradyrhizobium genotype, mineral nitrogen and light availability. But we did not detect parasitism. Lotus proportionally reduced investment in Bradyrhizobium as net benefit from infection decreased. Lotus control occurred primarily after infection, via fine‐scale modulation of nodule growth, as opposed to control over initial nodulation. Our results show how divestment of symbiosis by Lotus can prevent shifts to parasitism.     

Comparative Proteomics of Symbiotic and Aposymbiotic Juvenile Soft Corals

The symbiotic association between corals and photosynthetic unicellular algae is of great importance in coral reef ecosystems. The study of symbiotic relationships is multidisciplinary and involves research in phylogeny, physiology, biochemistry, and ecology. An intriguing phase in each symbiotic relationship is its initiation, in which the partners interact for the first time. The examination of this phase in coral–algae symbiosis from a molecular point of view is still at an early stage. In the present study we used 2-dimensional polyacrylamide gel electrophoresis to compare patterns of proteins synthesized in symbiotic and aposymbiotic primary polyps of the Red Sea soft coral Heteroxenia fuscescens. This is the first work to search for symbiosis-specific proteins during the natural onset of symbiosis in early host ontogeny. The protein profiles reveal changes in the host soft coral proteome through development, but surprisingly virtually no changes in the host proteome as a function of symbiotic state.