Flexibility of nutritional strategies within a mutualism: food availability affects algal symbiont productivity in two congeneric sea anemone species

Mutualistic symbioses are common, especially in nutrient-poor environments where an association between hosts and symbionts can allow the symbiotic partners to persist and collectively out-compete non-symbiotic species. Usually these mutualisms are built on an intimate transfer of energy and nutrients (e.g. carbon and nitrogen) between host and symbiont. However, resource availability is not consistent, and the benefit of the symbiotic association can depend on the availability of resources to mutualists. We manipulated the diets of two temperate sea anemone species in the genus Anthopleura in the field and recorded the responses of sea anemones and algal symbionts in the family Symbiodiniaceae to our treatments. Algal symbiont density, symbiont volume and photosynthetic efficiency of symbionts responded to changes in sea anemone diet, but the responses depended on the species of sea anemone. We suggest that temperate sea anemones and their symbionts can respond to changes in anemone diet, modifying the balance between heterotrophy and autotrophy in the symbiosis. Our data support the hypothesis that symbionts are upregulated or downregulated based on food availability, allowing for a flexible nutritional strategy based on external resources.     

Localization of a symbiosis-related protein, Sym32, in the Anthopleura elegantissima-Symbiodinium muscatinei Association

Cnidarian-dinoflagellate symbioses are widespread in the marine environment. Growing concern over the health of coral reef ecosystems has revealed a fundamental lack of knowledge of how cnidarian-algal associations are regulated at the cellular and molecular level. We are interested in identifying genes that mediate interactions between the partners, and we are using the temperate sea anemone Anthopleura elegantissima as a model. We previously described a host gene, sym32, encoding a fasciclin domain protein, that is differentially expressed in symbiotic and aposymbiotic A. elegantissima. Here, we describe the subcellular localization of the sym32 protein. In aposymbiotic (symbiont-free) hosts, sym32 was located in vesicles that occur along the apical edges of gastrodermal cells. In symbiotic hosts, such vesicles were absent, but sym32 was present within the symbiosome membranes. Sym32 (or a cross-reactive protein) was also present in the accumulation bodies of the symbionts. Although the anti-sym32 antiserum was not sufficiently specific to detect the target protein in cultured Symbiodinium bermudense cells, Western blots of proteins from two Symbiodinium species revealed a protein doublet of 45 and 48 kDa, suggesting that the symbionts may also produce a fasciclin domain protein. We suggest that host sym32 is relocated from gastrodermal vesicles to the symbiosome membrane when symbionts are taken into host cells by phagocytosis.     

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.     

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.     

Comparative Lipid Profiling of the Cnidarian Aiptasia pallida and Its Dinoflagellate Symbiont

Corals and other cnidarians house photosynthetic dinoflagellate symbionts within membrane-bound compartments inside gastrodermal cells. Nutritional interchanges between the partners produce carbohydrates and lipids for metabolism, growth, energy stores, and cellular structures. Although lipids play a central role in the both the energetics and the structural/morphological features of the symbiosis, previous research has primarily focused on the fatty acid and neutral lipid composition of the host and symbiont. In this study we conducted a mass spectrometry-based survey of the lipidomic changes associated with symbiosis in the sea anemone Aiptasia pallida, an important model system for coral symbiosis. Lipid extracts from A. pallida in and out of symbiosis with its symbiont Symbiodinium were prepared and analyzed using negative-ion electrospray ionization quadrupole time-of-flight mass spectrometry. Through this analysis we have identified, by exact mass and collision-induced dissociation mass spectrometry (MS/MS), several classes of glycerophospholipids in A. pallida. Several molecular species of di-acyl phosphatidylinositol and phosphatidylserine as well as 1-alkyl, 2-acyl phosphatidylethanolamine (PE) and phosphatidycholine were identified. The 1-alkyl, 2-acyl PEs are acid sensitive suggestive that they are plasmalogen PEs possessing a double bond at the 1-position of the alkyl linked chain. In addition, we identified several molecular species of phosphonosphingolipids called ceramide aminoethylphosphonates in anemone lipid extracts by the release of a characteristic negative product ion at m/z 124.014 during MS/MS analysis. Sulfoquinovosyldiacylglycerol (SQDG), an anionic lipid often found in photosynthetic organisms, was identified as a prominent component of Symbiodinium lipid extracts. A comparison of anemone lipid profiles revealed a subset of lipids that show dramatic differences in abundance when anemones are in the symbiotic state as compared to the non-symbiotic state. The data generated in this analysis will serve as a resource to further investigate the role of lipids in symbiosis between Symbiodinium and A. pallida.     

Temperate and tropical algal-sea anemone symbioses

Abstract. In this review, we seek to develop new insights about the nature of algal‐sea anemone symbioses by comparing such associations in temperate and tropical seas. Temperate seas undergo pronounced seasonal cycles in irradiance, temperature, and nutrients, while high irradiance, high temperature, and low nutrients are seasonally far less variable in tropical seas. We compare the nature of symbiosis between sea anemones (= actinians) and zooxanthellae (Symbiodinium spp.) in both regions to test tropical paradigms against temperate examples and to identify directions for future research. Although fewer anemone species are symbiotic in temperate regions, they are locally dominant and ecologically important members of the benthic community compared to the tropics. Zooxanthella densities tend to be lower in temperate anemones, but data are limited to a few species in both temperate and tropical seas. Zooxanthella densities are far more stable over time in temperate anemones than in tropical anemones, suggesting that temperate symbioses are more resistant to fluctuations in environmental parameters such as irradiance and temperature. Light‐saturated photosynthetic rates of temperate and tropical zooxanthellae are similar, but temperate anemone hosts receive severely reduced carbon supplies from zooxanthellae during winter months when light is reduced. Symbiont transmission modes and specificity do not show any trends among anemones in tropical vs. temperate seas. Our review indicates the need for the following: (1) Investigations of other temperate and tropical symbiotic anemone species to assess the generality of trends seen in a few “model’ anemones. (2) Attention to the field ecology of temperate and tropical algal‐anemone symbioses, for example, how symbioses function under seasonally variable environmental factors and how zooxanthellae persist at high densities in darkness and winter. The greater stability of zooxanthella populations in temperate hosts may be useful to understanding tropical symbioses in which bleaching (loss of zooxanthellae) is of major concern. (3) Study of the evolutionary history of symbiosis in both temperate and tropical seas. Continued exploration of the phylogenetic relationships between host anemones and zooxanthella strains may show how and why zooxanthellae differ in anemone hosts in both environments.     

The mud flat anemone-cockle association: mutualism in the intertidal zone?

The intertidal cockle Austrovenus stutchburyi exists in a symbiotic relationship with the mud flat anemone Anthopleura aureoradiata, the latter using the shell of buried cockles as the only available hard substrate for attachment. The cockles are also host to a detrimental larval trematode Curtuteria australis that invades the bivalves through the filtration current, and here we demonstrate that the anemones significantly depress the rate by which cockles accumulate parasites in the field. Along the tidal gradient, the relative parasite load of cockles was lowest where anemones were most abundant, and the area occupied by anemones per square meter sediment surface explained 30% of the spatial variation in infection intensity. At a smaller spatial scale, parasite loads were significantly lower (34%) in cockles from patches with than without anemones at the same tidal height. A field experiment manipulating the density of anemones showed that the rate of parasite accumulation in cockles decreased with increasing anemone density, and that the generally positive relationship between infection intensity and cockle size tended to disappear in the presence of anemones. The results suggest that the anemone-cockle symbiosis is a non-obligate mutualistic relationship in which the former is provided with a suitable substrate for attachment whereas the latter obtains protection against parasitic infections.     

Interplay between Endophyte Prevalence,Effects and Transmission: Insights from a Natural Grass Population

Two main mechanisms are thought to affect the prevalence of endophyte-grass symbiosis in host populations: the mode of endophyte transmission, and the fitness differential between symbiotic and non-symbiotic plants. These mechanisms have mostly been studied in synthetic grass populations. If we are to improve our understanding of the ecological and evolutionary dynamics of such symbioses, we now need to determine the combinations of mechanisms actually operating in the wild, in populations shaped by evolutionary history. We used a demographic population modeling approach to identify the mechanisms operating in a natural stand of an intermediate population (i.e. 50% of plants symbiotic) of the native grass Festuca eskia. We recorded demographic data in the wild over a period of three years, with manipulation of the soil resources for half the population. We developed two stage-structured matrix population models. The first model concerned either symbiotic or non-symbiotic plants. The second model included both symbiotic and non-symbiotic plants and took endophyte transmission rates into account. According to our models, symbiotic had a significantly higher population growth rate than non-symbiotic plants, and endophyte prevalence was about 58%. Endophyte transmission rates were about 0.67 or 0.87, depending on the growth stage considered. In the presence of nutrient supplementation, population growth rates were still significantly higher for symbiotic than for non-symbiotic plants, but endophyte prevalence fell to 0%. At vertical transmission rates below 0.10–0.20, no symbiosis was observed. Our models showed that a positive benefit of the endophyte and vertical transmission rates of about 0.6 could lead to the coexistence of symbiotic and non-symbiotic F. eskia plants. The positive effect of the symbiont on host is not systematically associated with high transmission rates of the symbiont over short time scales, in particular following an environmental change.     

Searching for a Toxic Key to Unlock the Mystery of Anemonefish and Anemone Symbiosis

Twenty-six species of anemonefish of the genera Amphiprion and monospecific Premnas, use only 10 species of anemones as hosts in the wild (Families: Actiniidae, Stichodactylidae and Thalassianthidae). Of these 10 anemone species some are used by multiple species of anemonefish while others have only a single anemonefish symbiont. Past studies have explored the different patterns of usage between anemonefish species and anemone species; however the evolution of this relationship remains unknown and has been little studied over the past decade. Here we reopen the case, comparing the toxicity of crude venoms obtained from anemones that host anemonefish as a way to investigate why some anemone species are used as a host more than others. Specifically, for each anemone species we investigated acute toxicity using Artemia francisca (LC₅₀), haemolytic toxicity using ovine erythrocytes (EC₅₀) and neurotoxicity using shore crabs (Ozius truncatus). We found that haemolytic and neurotoxic activity varied among host anemone species. Generally anemone species that displayed greater haemolytic activity also displayed high neurotoxic activity and tend to be more toxic on average as indicated by acute lethality analysis. An overall venom toxicity ranking for each anemone species was compared with the number of anemonefish species that are known to associate with each anemone species in the wild. Interestingly, anemones with intermediate toxicity had the highest number of anemonefish associates, whereas anemones with either very low or very high toxicity had the fewest anemonefish associates. These data demonstrate that variation in toxicity among host anemone species may be important in the establishment and maintenance of anemonefish anemone symbiosis.     

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.     

Symbiosis catalyses niche expansion and diversification

Interactions between species are important catalysts of the evolutionary processes that generate the remarkable diversity of life. Symbioses, conspicuous and inherently interesting forms of species interaction, are pervasive throughout the tree of life. However, nearly all studies of the impact of species interactions on diversification have concentrated on competition and predation leaving unclear the importance of symbiotic interaction. Here, I show that, as predicted by evolutionary theories of symbiosis and diversification, multiple origins of a key innovation, symbiosis between gall-inducing insects and fungi, catalysed both expansion in resource use (niche expansion) and diversification. Symbiotic lineages have undergone a more than sevenfold expansion in the range of host-plant taxa they use relative to lineages without such fungal symbionts, as defined by the genetic distance between host plants. Furthermore, symbiotic gall-inducing insects are more than 17 times as diverse as their non-symbiotic relatives. These results demonstrate that the evolution of symbiotic interaction leads to niche expansion, which in turn catalyses diversification.     

Agonistic Behaviour and Development of Territoriality During Ontogeny of the Sea Anemone Dwelling Crab Allopetrolisthes Spinifrons (H. Milne Edwards, 1837) (Decapoda: Anomura: Porcellanidae)

Allopetrolisthes spinifrons is an ectosymbiotic crab of the sea anemone Phymactis clematis . As a consequence of low host abundance, these represent a scarce and limited resource for the crab. Additionally, the relatively small size of the sea anemone host suggests that few symbiotic crabs can cohabit on one host individual, forcing crabs to adopt a territorial behaviour. In order to examine the potential presence and ontogenetic development of territoriality, the agonistic behaviour between crabs of various ontogenetic stages (adults, juveniles, and recruits) was studied in the laboratory. Laboratory experiments demonstrated that adult or juvenile crabs aggressively defended their sea anemone hosts against adult or juvenile intruders, respectively, but both adult and juvenile crabs tolerated recruits. Adult crabs behaved indifferently towards juvenile crabs, sometimes tolerating them, sometimes expelling them. Recruits never showed agonistic behaviour among themselves. The agonistic interactions observed in the laboratory and the uniform population distribution pattern on sea anemones recently described for A . spinifrons indicate that this species exhibits territorial behaviour, which develops during ontogeny. Territoriality in this species and other symbiotic decapods may function as a density-dependent mechanism of population regulation, being mediated by the availability of hosts. Resource monopolisation behaviours may be common among other symbiotic and free-living marine invertebrates inhabiting discrete habitats that represent a limiting resource.     

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.     

Assemblage and interaction structure of the anemonefish-anemone mutualism across the Manado region of Sulawesi,Indonesia

The Manado area (Indonesia–North Sulawesi), a marine high diversity hot-spot, hosts 7 species of anemonefish (family Pomacentridae, subfamily Amphiprioniae) living in symbiosis with 9 species of sea anemones (family Stichodactylidae and Actiniidae). This high biological diversity −27% and 80%, respectively, of the total known diversity of anemonefish and sea anemones—allows us to test different hypotheses focused on the obligate mutualism between anemonefish and sea anemones. In the Manado area, species richness of anemones and anemonefish across several sites was not correlated, but all anemones contained at least one fish individual, and there was a strong positive correlation between the numbers of individual anemonefish and anemones. As expected, each fish species had a preferred anemone host; also a partial niche overlap (Pianka’s Index) was often detected. The analysis of unique species composition suggests that competition is not an important factor determining the presence or absence of particular combinations of either anemonefish or host anemones (no evidence of competitive exclusion). The NODF algorithm showed that, at both a regional and local scale, the interaction between anemonefish and host anemones is not significantly nested, as a result of a combination of local conditions with competition, forcing species that regionally are more generalist to become more specialist.     

Agonistic Behaviour and Development of Territoriality During Ontogeny of the Sea Anemone Dwelling Crab Allopetrolisthes Spinifrons (H. Milne Edwards, 1837) (Decapoda: Anomura: Porcellanidae)

Allopetrolisthes spinifrons is an ectosymbiotic crab of the sea anemone Phymactis clematis. As a consequence of low host abundance, these represent a scarce and limited resource for the crab. Additionally, the relatively small size of the sea anemone host suggests that few symbiotic crabs can cohabit on one host individual, forcing crabs to adopt a territorial behaviour. In order to examine the potential presence and ontogenetic development of territoriality, the agonistic behaviour between crabs of various ontogenetic stages (adults, juveniles, and recruits) was studied in the laboratory. Laboratory experiments demonstrated that adult or juvenile crabs aggressively defended their sea anemone hosts against adult or juvenile intruders, respectively, but both adult and juvenile crabs tolerated recruits. Adult crabs behaved indifferently towards juvenile crabs, sometimes tolerating them, sometimes expelling them. Recruits never showed agonistic behaviour among themselves. The agonistic interactions observed in the laboratory and the uniform population distribution pattern on sea anemones recently described for A. spinifrons indicate that this species exhibits territorial behaviour, which develops during ontogeny. Territoriality in this species and other symbiotic decapods may function as a density-dependent mechanism of population regulation, being mediated by the availability of hosts. Resource monopolisation behaviours may be common among other symbiotic and free-living marine invertebrates inhabiting discrete habitats that represent a limiting resource.     

Immunity and symbiosis

The invertebrate immune system, which has become a major research focus, shares basic features of innate immunity with vertebrates and men. A special feature apparently found only in invertebrates is their close association with vertically heritable symbiotic microorganisms. The validity of the simple view of symbiosis as a mutually beneficial interaction between two uneven partners mainly improving the nutritional state of the two companions has been challenged, however, as symbiotic interactions might involve more partners, and symbiotic functions of the microorganisms are much more diverse than previously assumed. Likewise, microorganisms considered to be mostly harmful to their hosts have been shown to enhance host fitness under some circumstances. The role of a symbiont itself might change between environments or life stages of the host and symbionts might have features previously thought to be specific for pathogens. Understanding symbiotic interactions requires the comprehension of the cross-talk between the symbiotic companions, and the dissection of how long-lasting infections are established without eliminating the symbiont by host immune responses. Fascinating new findings in this field revealed that symbiosis might contribute to defence against pathogens or natural enemies. New symbiont-based approaches to defeat agricultural pests or pathogen transmission by arthropod vectors are becoming conceivable.     

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.     

Apoptosis and autophagy as mechanisms of dinoflagellate symbiont release during cnidarian bleaching: every which way you lose

Cnidarian bleaching results from the breakdown in the symbiosis between the host cnidarian and its dinoflagellate symbiont. Coral bleaching in recent years has increasingly caused degradation and mortality of coral reefs on a global scale. Although much is understood about the environmental causes of bleaching, the underlying cellular mechanisms of symbiont release that drive the process are just beginning to be described. In this study, we investigated the roles of two cellular pathways, host cell apoptosis and autophagy, in the bleaching process of the symbiotic anemone Aiptasia pallida. Host cell apoptosis was experimentally manipulated using gene knockdown of an anemone caspase by RNA interference, chemical inhibition of caspase using ZVAD-fmk and an apoptosis-inducer wortmannin. Autophagy was manipulated by chemical inhibition using wortmannin or induction using rapamycin. The applications of multiple single treatments resulted in some increased bleaching in anemones under control conditions but no significant drop in bleaching in individuals subjected to a hyperthermic stress. These results indicated that no single pathway is responsible for symbiont release during bleaching. However, when multiple inhibitors were applied simultaneously to block both apoptosis and autophagy, there was a significant reduction in bleaching in heat-stressed anemones. Our results allow us to formulate a model for cellular processes involved in the control of cnidarian bleaching where apoptosis and autophagy act together in a see-saw mechanism such that if one is inhibited the other is induced. Similar interconnectivity between apoptosis and autophagy has previously been shown in vertebrates including involvement in an innate immune response to pathogens and parasites. This suggests that the bleaching response could be a modified immune response that recognizes and removes dysfunctional symbionts.