Specificity of a model cnidarian-dinoflagellate symbiosis

To understand the flexibility of symbiotic associations in coral reefs, we investigated the specificity of the Aiptasia (cf. insignis)-Symbiodinium association in the laboratory by rendering the anemones aposymbiotic and inoculating them with different isolates of SYMBIODINIUM: Infective algal symbionts were monitored over 3 months by re-isolation and identification using denaturing-gradient gel electrophoresis and sequence comparison of their amplified 18S rRNA hypervariable V1 + V2 gene region. Despite similarity in their external morphology, the algal isolates differed in their infectivity towards the host. Within days of single-isolate inoculation, aposymbiotic anemones formed associations with fresh or cultured isolates (clade B) from the anemones Aiptasia sp. or A. tagetes, respectively. They associated to a limited extent with cultured isolates (clade A) from the tridacnids Tridacna crocea or Hippopus hippopus, and not at all with a cultured isolate (clade C) from the stony coral Montipora verrucosa, nor with a free-living isolate (clade A) from subtidal sands. Aposymbiotic anemones inoculated with a mixture of all isolates had only the anemone taxon as their detectable symbionts. Re-inoculation of induced symbioses with a mixture of all isolates and incubation with wild anemones showed that the initial induced symbioses with the anemone taxon were stable. Anemones originally infected with tridacnid isolates either additionally acquired the anemone taxon or had the former outgrown by the latter. These results demonstrate the presence of a host-symbiont recognition mechanism, and possibly competition among potential algal symbionts in the Aiptasia-Symbiodinium association. Here we present a method that may be useful in monitoring the algal population dynamics in symbiotic corals in the field, along with an efficient method of rendering Aiptasia aposymbiotic for further laboratory investigation of Aiptasia-Symbiodinium 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.     

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.     

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.     

The Hydra viridis/Chlorella symbiosis. Growth and sexual differentiation in polyps without symbionts

To investigate interactions between the basal metazoan Hydra viridis and its symbiotic Chlorella algae, we generated aposymbiotic hydra lacking algae and compared them to symbiotic ones with regard to growth and sexual differentiation. Under standard feeding conditions aposymbiotic polyps proliferated similarly to symbiotic polyps. Under moderate and low feeding conditions asexual growth was reduced in polyps lacking algae, indicating that the symbionts supply nutrients to their hosts. In addition, the Chlorella symbionts had a strong influence on the sexual reproduction of Hydra viridis: in most cases female gonads were produced only when symbiotic algae were present. Spermatogenesis proceeded similarly in symbiotic and aposymbiotic polyps. Since during oogenesis symbionts are actively transferred from endodermal epithelial cells to the ectodermal oocytes, this oogenesis promoting role could indicate that the symbionts are critically involved in the control of sexual differentiation in green hydra.     

CHANGES IN THE ULTRASTRUCTURE OF SYMBIOTIC ZOOXANTHELLAE (SYMBIODINIUM SP., DINOPHYCEAE) IN FED AND STARVED SEA ANEMONES MAINTAINED UNDER HIGH AND LOW LIGHT1

The ultrastructure of symbiotic dinoflagellates (Symbiodinium sp., zooxanthellae) in the sea anemone Aiptasia pallida Verrill was examined in well-fed or starved (up to 120 days) anemones maintained under two light levels (5 and 50 μmol · m^?2· s^?1). Cell size of zooxanthellae was not affected by feeding history; however, both light and feeding history affected the relative cell volume of chloroplasts, lipids, and vacuoles. Stereological analysis of transmission electron micrographs showed that algae in low-light starved anemones had 10 times as much lipid (17.4% of cell volume) as those in well-fed anemones under the same light conditions (1.8%). The lipid content of algae from anemones in high light increased from 15.4% in well-fed anemones to 30.1% in starved anemones. The starch content of zooxanthellae in low-light anemones was law (4.1%) and not affected by feeding history, while the starch content of zooxanthellae in high-light anemones was greater (10.7%), with some differences among groups. Algal photoacclimation to low light included an increase in chloroplast relative volume from 17% (in well-fed high-light anemones) to 33% in well-fed low-light anemones. Starvation of the host resulted in a significant decrease in chloroplast volume in zooxanthellae in anemones at both light levels. Morphometry provides quantitative confirmation of biochemical and physiological data on zooxanthellae, because the changes in zooxanthellae with starvation of the host are consistent with other indicators of nutrient limitation of zooxanthellae of A. pallida held without food for long periods of time.     

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     

The role of symbiotic dinoflagellates in the temperature-induced bleaching response of the subtropical sea anemone Aiptasia pallida

Coral bleaching involves the loss of symbiotic dinoflagellates (zooxanthellae) from reef corals and other cnidarians and may be a stress response of the host, algae or both. To determine the role of zooxanthellae in the bleaching process, aposymbiotic sea anemones from Bermuda (Aiptasia pallida) were infected with symbionts from other sea anemones (Aiptasia pallida from Florida, Bartholomea annulata and Condylactis gigantea). The expulsion of algae was measured during 24-h incubations at 25, 32 and 34 degrees C. Photosynthetic rates of freshly isolated zooxanthellae were also measured at these temperatures. The C. gigantea (Cg) symbionts were expelled in higher numbers than the other algae at 32 degrees C. Photosynthesis by the Cg algae was completely inhibited at this temperature, in contrast to the other symbionts. At 34 degrees all of the symbionts had increased expulsion rates, and at this temperature only the symbionts from Florida A. pallida exhibited any photosynthesis. These results provide the first evidence that the differential release of symbionts from the same host species is related to decreased photosynthesis at elevated temperatures, and support other findings suggesting that zooxanthellae are directly affected by elevated temperatures during bleaching events.     

Ingestion,Digestion and Assimilation Efficiency of the Sea-Anemone Aiptasia pallida Fed Zooplankton Prey

Ingestion time, digestion time, and assimilation efficiency by the sea anemone Aiptasia pallida were studied in the laboratory by feeding individual anemones preweighed pellets of freeze-dried Artemia salina nauplii. There was no significant correlation between anemone size, measured as dry weight, and either ingestion time, digestion time or assimilation efficiency. Similarly, there was no significant correlation between meal size (i.e., dry weight of ingested brine shrimp pellet) and either ingestion time, digestion time or assimilation efficiency. These results suggest that, under these conditions, assimilation efficiency is unaffected by either “meal” size or anemone size.     

Light intensity as a factor in the regulation of the density of symbiotic zooxanthellae in Aiptasia tagetes (Coelenterata, Anthozoa)

Experiments designed to investigate the effect of different levels of illumination on the density of symbiotic zooxanthellae in the anemone Aiptasia tagetes (Duch. & Mich.) are described.
The anemones are found to regulate the densities of their zooxanthella populations to fixed levels which are dependent upon ambient light intensity.
Regulation is continuous and results from the interaction of increase in algal numbers, growth of host tissues, population of new host tissues by symbionts and extrusion of zooxanthellae by the host.
The nature of the process by which zooxanthellae are selected for extrusion is discussed and a scheme is outlined indicating the pathways through which the level of the algal population in Aiptasia , and other coelenterates, is believed to be controlled.     

Anemonefish depletion reduces survival,growth, reproduction and fishery productivity of mutualistic anemone–anemonefish colonies

Intimate knowledge of both partners in a mutualism is necessary to understand the ecology and evolution of each partner, and to manage human impacts that asymmetrically affect one of the partners. Although anemonefishes and their host anemones are iconic mutualists and widely sought by ornamental fisheries, the degree to which anemones depend on anemonefishes, and thus the colony-level effects of collecting anemonefishes, is not well understood. We tracked the size and abundance of anemone Entacmaea quadricolor and anemonefish Amphiprion melanopus colonies for 3 yr after none, some, or all of the resident anemonefish were experimentally removed. Total and partial removal of anemonefish had rapid and sustained negative effects on growth, reproduction and survival of anemones, as well as cascading effects on recruitment and productivity of anemonefish in the remaining colony. As predicted, total removal of anemonefish caused acute declines in size and abundance of anemones, although most anemone colonies (76 %) slowly resumed growth and reproduction after the arrival of anemonefish recruits, which subsequently grew and defended the hosts. Partial removal of anemonefish had similar but typically less severe effects on anemones. Remarkably, the colony-level effects on anemones and anemonefish were proportional to the size and number of anemonefish that were experimentally removed. In particular, anemone survival and anemonefish productivity were highest when one or more adult anemonefish remained in the colony, suggesting that adult fish not only enhanced the protection of anemones, but also increased the recruitment and/or survival of conspecifics. We conclude that the relationship between E. quadricolor and A. melanopus is not only obligate, but also demographically rigid and easily perturbed by anemonefish fisheries. Clearly, these two species must be managed together as a unit and with utmost precaution. To this end, we propose several tangible management actions that will help to minimize fishing effects.     

Impact of symbiotic algae on sea anemone metabolism: analysis by in vivo 31P nuclear magnetic resonance spectroscopy

High-field pulsed Fourier-transform nuclear magnetic resonance spectroscopy (NMR) was used to quantify the adenylate levels of sea anemones (Aiptasia pulchella) with and without symbiotic dinoflagellates (Symbiodinium sp.). Animals were fed to repletion, then starved in darkness for up to six days before collection of in vivo NMR spectra. The host adenylate ratio of ATP: (ATP + ADP) declined significantly with increasing periods of starvation in both symbiotic and aposymbiotic hosts (P less than 0.05). However, the decline in the animal adenylate ratio was significantly more rapid in animals bearing symbiotic algae (P less than 0.05). This suggests that symbiotic algae in darkness cause more rapid depletion of host energy reserves, possibly by drawing on host pools of organic substrates. In vivo NMR spectroscopy was also used to evaluate the effect on A. pulchella of photosynthesis by zooxanthellae. Symbiotic anemones were fed to repletion, then starved under high irradiance (300 to 320 mu Ein m-2 s-1) or low irradiance (70 to 80 mu Ein m-2 s-1) conditions for up to five days. The host adenylate ratio declined significantly (P less than 0.01) with starvation under both treatments, but no significant difference was detected between treatments (P greater than 0.35). Blotted wet weight of anemones under high and low irradiance declined by 50% over eight days of starvation, but there was no significant difference in the rate of weight loss by anemones in the two treatments. There results suggest that translocation of photosynthate from symbiotic zooxanthellae does not significantly affect host adenylate ratio or have a sparing effect on host biomass during starvation in this symbiotic sea anemone.     

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.     

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.     

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.     

Wolbachia sp. (Rickettsiales: Rickettsiaceae) a symbiont of the almond moth,Ephestia cautella: Ultrastructure and influence on host fertility

Wolbachia sp. is a maternally inherited symbiont of the almond moth, Ephestia cautella. It is transmitted through the cytoplasm of the egg and occurs normally in the gonads of all stages of the moth. The symbiont is responsible for reproductive cytoplasmic incompatibility between crosses of experimental laboratory strains of aposymbiotic female moths and symbiotic (normal) males. Although female moths were inseminated in laboratory tests, their eggs failed to hatch and exhibited no signs of embryonic development. The reciprocal cross, i.e., symbiotic female months × aposymbiotic males, produced normal progeny.The ultrastructure of Wolbachia was studied in sections of E. cautella larval testes. Symbionts, minute rod-shaped structures, were abundant in the cytoplasm of hypertrophied spermatids. There was no indication of deleterious influence of symbionts on sperm production or activity. Strains of Wolbachia occur in allopatric populations of insects where they may function as a genetic isolation mechanism. Microorganismal reproductive incompatibility has been suggested as a possible approach for insect control.     

Symbiotic state influences life-history strategy of a clonal cnidarian

Along the North American Pacific coast, the common intertidal sea anemone Anthopleura elegantissima engages in facultative, flexible symbioses with Symbiodinium muscatinei (a dinoflagellate) and Elliptochloris marina (a chlorophyte). Determining how symbiotic state affects host fitness is essential to understanding the ecological significance of engaging in such flexible relationships with diverse symbionts. Fitness consequences of hosting S. muscatinei, E. marina or negligible numbers of either symbiont (aposymbiosis) were investigated by measuring growth, cloning by fission and gonad development after 8.5–11 months of sustained exposure to high, moderate or low irradiance under seasonal environmental conditions. Both symbiotic state and irradiance affected host fitness, leading to divergent life-history strategies. Moderate and high irradiances led to a greater level of gonad development in individuals hosting E. marina, while high irradiance and high summer temperature promoted cloning in individuals hosting S. muscatinei and reduced fitness of aposymbiotic anemones. Associating with S. muscatinei may contribute to the success of A. elegantissima as a spatial competitor on the high shore: (i) by offsetting the costs of living under high temperature and irradiance conditions, and (ii) by promoting a high fission rate and clonal expansion. Our results suggest that basic life-history characteristics of a clonal cnidarian can be affected by the identity of the endosymbionts it hosts.     

Metal accumulation and sublethal effects in the sea anemone, Aiptasia pallida, after waterborne exposure to metal mixtures

The marine environment is subjected to contamination by a complex mixture of metals from various anthropogenic sources. Measuring the biological responses of organisms to a complex mixture of metals allows for examination of metal-specific responses in an environmentally realistic exposure scenario. To address this issue, the sea anemone, Aiptasia pallida was exposed to a control and a metal mixture (copper, zinc, nickel, and cadmium) at three exposure levels (10, 50, and 100 μg/L) for 7 days. Anemones were then transferred to metal-free seawater for an additional 7 days after the metal exposure to assess metal depuration and recovery. Metal accumulation, activity of the enzymes catalase, glutathione reductase, and carbonic anhydrase, as well as, cell density of the symbiotic zooxanthellae were measured over 14 days. Metal accumulation in A. pallida occurred in a concentration dependent manner over the 7-day exposure period. Altered enzyme activity and tentacle retraction of the host, as well as decreased zooxanthellae cell density were observed responses over the 7 days, after exposure to a metal concentration as low as 10 μg/L. Metal depuration and physiological recovery were dependent on both the metal and the exposure concentration. Understanding how A. pallida and their symbionts are affected by metal exposures in the laboratory may allow better understanding about the responses of symbiotic cnidarians in metal polluted aquatic environments.     

Cover

Loss (bleaching) of symbiotic dinoflagellates (genus Symbiodinium) from the sea anemone Aiptasia pallida caused by elevated temperatures or disruption of symbiont photosynthesis can be restored through exposure to axenic Symbiodinium cultures (top part of figure; Symbiodinium appear red due to chlorophyll autofluorescence under blue light). [Vol. 49, No. 3, pp.447–458]