Is dimethylsulfoniopropionate (DMSP) produced by the symbionts or the host in an anemone–zooxanthella symbiosis?

Many groups of tropical cnidarians including scleractinian corals, octocorals, corallimorphs, and anemones contain the tertiary sulfonium compound dimethylsulfoniopropionate (DMSP). It is not known if the compound is synthesized by the animals, their microalgal symbionts, or derived through their diet. We determined the source of the DMSP in several species of tropical and temperate anemones using three approaches: (1) conducting comparative measurements of DMSP in aposymbiotic and zooxanthellate anemones of three species that harbor zooxanthellae, and similar measurements in one species that can harbor both zooxanthellae and zoochlorellae, (2) manipulating the presence or absence of zooxanthellae by inoculating juvenile aposymbiotic anemones (Aiptasia pallida) with their symbiont, Symbiodinium bermudense, and (3) manipulating the numbers of S. bermudense by growing aposymbiotic and zooxanthellate A. pallida in the light and the dark. DMSP was present in zooxanthellate anemones in concentrations of 3.4–15 μmol g⁻¹ fresh mass (FM). In aposymbiotic Aiptasia spp. and Anthopleura elegantissima that lacked large numbers of zooxanthellae, concentrations ranged from being undetectable to 0.43 μmol g⁻¹ FM. When aposymbiotic A. pallida were inoculated with zooxanthellae, concentrations of DMSP were an average of 4.24 μmol g⁻¹ FM after 5 weeks; DMSP was undetectable in uninoculated control animals. Aposymbiotic anemones maintained in the light or the dark for 6 weeks contained no DMSP or zooxanthellae. Zooxanthellate anemones in the light contained five times as many zooxanthellae and approximately 7.5 times as much DMSP as zooxanthellate anemones maintained in the dark. Taken together, these data show that the zooxanthellae are the sole source of DMSP in A. pallida. The trends in DMSP concentrations in other species of zooxanthellate anemones suggest that this phenomenon is not limited to A. pallida but may be more generally true for other anemones or even other cnidarians hosting species of Symbiodinium. Communicated by Biology Editor Dr. Michael Lesser     

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.     

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.     

Copper accumulation and oxidative stress in the sea anemone, Aiptasia pallida, after waterborne copper exposure

Copper is a common marine pollutant yet its effects on symbiotic cnidarians are largely understudied. To further understand the impact of elevated copper concentrations on marine symbiotic organisms, toxicity tests were conducted using the model sea anemone, Aiptasia pallida, with and without its zooxanthellae symbiont. Symbiotic and aposymbiotic A. pallida were exposed to sublethal copper concentrations (0, 5, 15, and 50 µg/L) for 7 d and copper accumulation, behavior, and the activity of the oxidative stress enzymes, superoxide dismutase (SOD), and catalase (CAT) were measured. Additionally, acute 96-h toxicity tests were conducted to determine LC₅₀ values of the organisms after copper exposure. Both symbiotic and aposymbiotic A. pallida rapidly accumulated copper in a time and dose dependent manner. However, higher copper concentrations accumulated in the aposymbiotic as compared to the symbiotic A. pallida. In response to the highest two copper exposures (15 and 50 µg/L) symbiotic A. pallida upregulated CAT activity to combat the damaging effects of hydrogen peroxide. Contrary to these results, SOD activity significantly decreased during the highest copper exposure, when compared to controls. CAT activity was not detected and SOD was substantially (> 10 fold) reduced in aposymbiotic A. pallida, suggesting that the zooxanthellae are associated with the oxidative stress response. Copper exposure as low as 5 µg/L caused tentacle retraction and increased mucus production in both symbiotic and aposymbiotic anemones. The LC₅₀ values for symbiotic and aposymbiotic A. pallida exposed to copper for 96 h were 148 µg/L (95% confidence interval = 126.4, 173.8) and 206 µg/L (95% confidence interval = 175.2, 242.2), respectively. Understanding the varying responses of symbiotic and aposymbiotic A. pallida to copper stress may advance our comprehension of the functional roles of zooxanthellae and host. Although the mechanism of copper toxicity has not been fully elucidated, it is clear that A. pallida accumulate copper and are sensitive, as effects were detected at environmentally relevant copper concentrations. Likewise, A. pallida may be useful in biomonitoring copper polluted environments.     

Vibrio communities in scleractinian corals differ according to health status and geographic location in the Mediterranean Sea

The increase in seawater temperature associated with global warming is a significant threat to coral health and is linked to increasing mass mortality events and Vibrio-related coral diseases. In the Mediterranean Sea, the endemic Cladocora caespitosa and the invasive species Oculina patagonica are the main scleractinian corals affected by mass mortalities. In this study, culturable Vibrio spp. assemblages associated with healthy and unhealthy colonies of these two shallow coral species were characterized to assess the presence of Vibrio pathogens in tissue necrosis. Vibrio communities associated with O. patagonica and C. caespitosa showed geographical differences, although these became more homogeneous in unhealthy specimens of both species. Furthermore, the number of recovered Vibrio specimens was more than five times higher in unhealthy than in healthy corals. Within these culturable vibrios, the known pathogens Vibrio mediterranei and Vibrio coralliilyticus were present in unhealthy colonies of both coral species in the two localities, suggesting that they could play a role in the health status of C. caespitosa and thus act as generalist pathogens in Mediterranean corals. Nonetheless, a clonal type of V. coralliilyticus detected in C. caespitosa was not associated with disease signs, suggesting that this species could encompass assemblages with different levels of virulence.     

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.     

Molecular signatures of host specificity linked to habitat specialization in Exaiptasia sea anemones

Rising ocean temperatures associated with global climate change induce breakdown of the symbiosis between coelenterates and photosynthetic microalgae of the genus Symbiodinium. Association with more thermotolerant partners could contribute to resilience, but the genetic mechanisms controlling specificity of hosts for particular Symbiodinium types are poorly known. Here, we characterize wild populations of a sea anemone laboratory model system for anthozoan symbiosis, from contrasting environments in Caribbean Panama. Patterns of anemone abundance and symbiont diversity were consistent with specialization of holobionts for particular habitats, with Exaiptasia pallida/S. minutum (ITS2 type B1) abundant on vertical substrate in thermally stable, shaded environments but E. brasiliensis/Symbiodinium sp. (ITS2 clade A) more common in shallow areas subject to high temperature and irradiance. Population genomic sequencing revealed a novel E. pallida population from the Bocas del Toro Archipelago that only harbors S. minutum. Loci most strongly associated with divergence of the Bocas‐specific population were enriched in genes with putative roles in cnidarian symbiosis, including activators of the complement pathway of the innate immune system, thrombospondin‐type‐1 repeat domain proteins, and coordinators of endocytic recycling. Our findings underscore the importance of unmasking cryptic diversity in natural populations and the role of long‐term evolutionary history in mediating interactions with Symbiodinium.     

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.     

Ability of Monaco shrimp Lysmata seticaudata (Decapoda: Hippolytidae) to control the pest glass anemone Aiptasia pallida (Actiniaria: Aiptasidae)

The present work highlights the ability of wild and cultured Monaco shrimp Lysmata seticaudata to control the glass anemone Aiptasia pallida. Starved shrimp ingested the highest percentages of glass anemones [85.7% and 89.3% for wild (W) and cultured (C) shrimp, respectively]. The absence of symbiotic zooxanthellae in glass anemones did not influence the shrimps feeding rate, with shrimp offered aposymbiotic anemones displaying the same feeding percentages (67.3% and 70.7% for W and C shrimp, respectively) as those offered sea anemones with symbiotic zooxanthellae (70.0% and 74.4% for W and C shrimp, respectively). Shrimp offered larger sea anemones had the lowest feeding percentages (33.0% and 36.3% for W and C shrimp, respectively), along with shrimp offered an alternative food (27.3% and 36.0% for W and C shrimp, respectively). There were no significant differences in the percentage of glass anemones ingested by cultured and wild Monaco shrimp in the same feeding trial.     

Draft genomes of the corallimorpharians Amplexidiscus fenestrafer and Discosoma sp.

Corallimorpharia are the closest noncalcifying relatives of reef‐building corals. Aside from their popularity among aquarium hobbyists, their evolutionary position between the Actiniaria (sea anemones) and the Scleractinia (hard corals) makes them ideal candidates for comparative studies aiming at understanding the evolution of hexacorallian orders in general and reef‐building corals in particular. Here we have sequenced and assembled two draft genomes for the Corallimorpharia species Amplexidiscus fenestrafer and Discosoma sp. The draft genomes encompass 370 and 445 Mbp, respectively, and encode for 21,372 and 23,199 genes. To facilitate future studies using these resources, we provide annotations for the predicted gene models—not only at gene level, by annotating gene models with the function of the best‐matching homologue, and GO terms when available; but also at protein domain level, where gene function can be better verified through the conservation of the sequence and order of protein domains. Further, we provide an online platform ( http://corallimorpharia.reefgenomics.org ), which includes a blast interface and a genome browser to facilitate the use of these resources. We believe that these two genomes are important resources for future studies on hexacorallian systematics and the evolutionary basis of their specific traits such as the symbiotic relationship with dinoflagellates of the genus Symbiodinium or the evolution of calcification in reef‐building corals.     

Mucus antigenicity in sea anemones and corals

The antigenicities of external mucus from the sea anemones Stoichactis haddoni, Radianthus ritteri and Gyrostoma hertwigi and from the coral Trachyphyllia geoffroyi were compared. Marked differences were found between species but not within species. The differences suggested that mucus composition might be species specific, and hence that it might be one of the factors used by sea anemones and corals in the recognition of foreign anthozoan species.     

Responses of coral‐associated bacterial communities to heat stress differ with Symbiodinium type on the same coral host

This study compared the effect of heat stress on coral‐associated bacterial communities among juveniles of the coral, Acropora tenuis, hosting different Symbiodinium types. In comparison to a control temperature treatment (28 °C), we documented dramatic changes in bacterial associates on juvenile corals harbouring ITS 1 type D Symbiodinium when placed in a high (32 °C) temperature treatment. In particular, there was a marked increase in the number of retrieved Vibrio affiliated sequences, which coincided with a 44% decline in the photochemical efficiency of the D‐juveniles. Interestingly, these Vibrio sequences affiliated most closely with the coral pathogen, Vibrio coralliilyticus, which has been implicated in some coral disease outbreaks. In contrast, A. tenuis hosting ITS 1 type C1 Symbiodinium did not exhibit major bacterial shifts in the elevated temperature treatment, indicating a more stable bacterial community during thermal stress; concomitantly a decline (10%) in photochemical efficiency was minimal for this group. D juveniles that had been exposed to moderately elevated sea temperatures (30 °C) in the field before being placed in the control temperature treatment displayed a decrease in the number of Vibrio affiliated sequences and bacterial profiles shifted to become more similar to profiles of corals harbouring type C1 Symbiodinium. In combination, these results demonstrate that thermal stress can result in shifts in coral‐associated bacterial communities, which may lead to deteriorating coral health. The lower resilience of A. tenuis to thermal stress when harbouring Symbiodinium D highlights the importance of inter‐kingdom interactions among the coral host, dinoflagellate endosymbiont and bacterial associates for coral health and resilience.     

Comparative gene expression in the symbiotic and aposymbiotic Aiptasia pulchella by expressed sequence tag analysis

Intracellular symbiotic relationships are prevalent between cnidarians, such as corals and sea anemones, and the photosynthetic dinoflagellate symbionts. However, there is little understanding about how the genes express when the symbiotic relationship is set up. To characterize genes involved in this association, the endosymbiosis between sea anemone, Aiptasia pulchella, and dinoflagellate zooxanthellae, Symbiodinium spp., was employed as a model. Two complementary DNA (cDNA) libraries were constructed from RNA isolated from symbiotic and aposymbiotic A. pulchella. Using single-pass sequencing of cDNA clones, a total of 870 expressed sequence tags (ESTs) clones were generated from the two libraries: 474 from symbiotic animal and 396 from aposymbiotic animal. The initial ESTs consisted of 143 clusters and 231 singletons. A BLASTX search revealed that 147 unique genes had similarities with protein sequences available from databases; 120 of these clones were categorized according to their putative function. However, many ESTs could not assign functionally. The putative roles of some of the identified genes relative to endosymbiosis were discussed. This is the first report of the use of EST analysis to examine the gene expression in symbiotic and aposymbiotic states of the cnidarians. The systematic analysis of EST from this study provides a useful database for future investigations of the molecular mechanisms involved in algal-cnidarian symbiosis.     

Cloning and Use of a Coral 36B4 Gene to Study the Differential Expression of Coral Genes Between Light and Dark Conditions

This paper aims to validate reference genes for gene expression studies between light and dark conditions in the scleractinian coral Stylophora pistillata for future gene expression studies of the “light-enhanced calcification” phenomenon. For this purpose, we cloned, sequenced, and characterized a candidate reference gene, the 36B4 gene from the coral S. pistillata, and validated 36B4 and β-actin as reference genes. To illustrate the future applications of these reference genes, we tested the dark and light expression of two photosynthetic genes (Rubisco and D1 protein of the photosystem II) and two genes encoding proteins involved in calcium transport for coral calcification (a calcium ATPase and a calcium channel). Results show that both photosynthetic genes are enhanced during the light when standardized against 36B4 and β-actin, whereas the two genes encoding proteins involved in calcium transport are not differentially expressed between light and dark conditions. The characterization of a coral 36B4 and the establishment of such valid reference genes will be useful for future gene expression studies between diverse conditions (aposymbiotic/symbiotic, stress/control, light/dark conditions) in scleractinian corals. Nucleotide sequence of the coral 36B4 gene cloned in this study is available in the Genbank database under the accession number EU069460.