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.     

Knockdown of actin and caspase gene expression by RNA interference in the symbiotic anemone Aiptasia pallida

Since the discovery of the ancient eukaryotic process of RNA-mediated gene silencing, the reverse-genetics technique RNA interference (RNAi) has increasingly been used to examine gene function in vertebrate and invertebrate systems. In this study, we report on the use of RNAi, adapted from studies on animal model systems, to manipulate gene expression in a symbiotic marine cnidarian. We describe gene knockdown of actin and of acasp--a cysteine protease, or caspase--in the symbiotic sea anemone Aiptasia pallida. Knockdown was assessed qualitatively with in situ hybridizations for both genes. Quantitative PCR and caspase activity assays were used as a quantitative measure of knockdown for acasp.     

Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida

The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [³H]arginine to [³H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor N^G-methyl- -arginine ( -NMA), but not the arginase inhibitors -valine and -ornithine. NOS activity was predominantly cytosolic, and was characterised by a K_m for arginine of 19.05 μM and a V_max of 2.96 pmol/min per μg protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems.     

Photo-movement in the sea anemone Aiptasia influenced by light quality and symbiotic association

Coral Reefs - The relationship between cnidarians and their micro-algal symbionts is crucial for normal animal function and the formation of coral reefs. We used the sea anemone Exaiptasia pallida...     

Cloning and sequencing of cDNA encoding glutamine synthetase from the sea anemone Aiptasia pallida

Glutamine synthetase (GS) catalyzes the addition of ammonium to glutamic acid to form glutamine and plays a crucial role in the nitrogen assimilation of the sea anemone Aiptasia pallida and its endosymbiotic algae. We describe the cDNA cloning and sequence analysis of GS mRNA from A. pallida based on polymerase chain reaction (PCR) technology that employed a combination of degenerate and A. pallida-specific primers. The sequenced cDNA approximates 1620 nucleotides and is characterized by an open reading frame of 1107 nucleotides that encodes a protein of 369 amino acid residues. Comparisons of the deduced sea anemone GS protein to a wide range of species demonstrated greatest amino acid sequence identity to sea urchin GS (66%) and least identity to green algae GS (51%). The sequenced cDNA can be used in future research to study GS gene expression in A. pallida.     

Neuroglandular synapses in the pharynx of the sea anemone Aiptasia pallida (Cnidaria, Anthozoa)

Scanning and transmission electron microscopy of the pharynx of the sea anemone Aiptasia pallida revealed a heavily ciliated epidermis and two types of gland cells not known previously to be innervated. By tracing serial cross sections of the pharynx, we located and characterized two types of neuroglandular synapses (i.e., those having clear vesicles and those with dense-cored vesicles). The diameters of the vesicles at each synapse were averaged; clear vesicles ranged from 70 to 103 nm in diameter and were observed at synapses to both mucous and zymogenic gland cells. Dense-cored vesicles ranged from 53 to 85 nm in diameter and were observed at synapses to two mucous gland cells. One mucous gland cell had three neuroglandular synapses, one with clear vesicles and two with dense-cored vesicles. The occurrence of either clear or dense-cored vesicles at neuroglandular synapses suggests that at least two types of neurotransmitter substances control the secretion of mucus in the sea anemone pharynx. To date, only clear vesicles have been observed at a neurozymogenic gland cell synapse in the pharynx. No evidence of immunoreactivity to phenylethanolamine-N-methyl transferase was observed at neuroglandular synapses, suggesting that adrenaline is not a transmitter in the pharynx of A. pallida.     

Different nematocytes have different synapses in the sea anemone Aiptasia pallida (Cnidaria,Anthozoa)

Sea anemones feed by discharging nematocysts into their prey, but the pathway for control of nematocyst discharge is unknown. The purpose of this study was to investigate the ultrastructural evidence of neuro-nematocyte synapses and to determine the types of synaptic vesicles present at different kinds of nematocyst-containing cells. The tip and middle of tentacles from small specimens of Aiptasia pallida were prepared for electron microscopy and serial micrographs were examined. We found clear vesicles in synapses on mastigophore-containing nematocytes and dense-cored vesicles in synapses on basitrich-containing nematocytes and on one cnidoblast with a developing nematocyst. In addition, we found reciprocal neuro-neuronal and sequential neuro-neuro-nematocyte synapses in which dense-cored vesicles were present. It was concluded that : (1) neuro-nematocyte synapses are present in sea anemones, (2) different kinds of synaptic vesicles are present at cells containing different types of nematocysts, (3) synapses are present on cnidoblasts before the developing nematocyst can be identified and these synapses may have a trophic influence on nematocyst differentiation, and (4) both reciprocal and sequential synapses are present at the nematocyte, suggesting a complex pathway for neural control of nematocyst discharge. J. Morphol. 238:53–62, 1998. © 1998 Wiley-Liss, Inc.     

Host autophagic degradation and associated symbiont loss in response to heat stress in the symbiotic anemone,Aiptasia pallida

Coral bleaching involves the loss of essential photosynthetic dinoflagellates (Symbiodinium sp.) from host gastrodermal cells in response to temperature or light stress. Although numerous potential cellular bleaching mechanisms have been proposed, there remains much uncertainty regarding which cellular events occur during early breakdown of the host–dinoflagellate symbiosis. In this study, transmission electron microscopy was used to conduct a detailed examination of symbiotic tissues of the tropical anemone Aiptasia pallida during early stages of host stress. Bleaching was induced by exposing specimens to a stress treatment of 32.5±0.5°C at 140±7 μ mol photons m^?2 s^?1 light intensity for 12 h, followed by 12 h at 24±1°C in darkness, repeated over a 48 h period. Ultrastructural examinations revealed numerous dense autophagic structures and associated cellular degradation in tentacle tissues after ~12 h of the stress treatment. Anemones treated with rapamycin, a known autophagy inducer, exhibited the same ultrastructural characteristics as heat‐stressed tissues, confirming that the structures observed during heat stress treatment were autophagic. In addition, symbionts appeared to be expelled from host cells via an apocrine‐like detachment mechanism from the apical ends of autophagic gastrodermal cells. This study provides the first ultrastructural evidence of host autophagic degradation during thermal stress in a cnidarian system and also supports earlier suggestions that autophagy is an active cellular mechanism during early stages of bleaching.     

Ultrastructural evidence for two-cell and three-cell neural pathways in the tentacle epidermis of the sea anemone Aiptasia pallida.

Sensory and ganglion cells in the tentacle epidermis of the sea anemone Aiptasia pallida were traced in serial transmission electron micrographs to their synaptic contacts on other cells. Sensory cell synapses were found on spirocytes, muscle cells, and ganglion cells. Ganglion cells, in turn, synapsed on sensory cells, spirocytes, muscle cells, and other neurons and formed en passant axo-axonal synapses. Axonal synapses on nematocytes and gland cells were not traced to their cells of origin, i.e., identified sensory or ganglion cells. Direct synaptic contacts of sensory cells with spirocytes and sensory cells with muscle cells suggest a local two-cell pathway for spirocyst discharge and muscle cell contraction, whereas interjection of a ganglion cell between the sensory and effector cells creates a local three-cell pathway. The network of ganglion cells and their processes allows for a through-conduction system that is interconnected by chemical synapses. Although the sea anemone nervous system is more complex than that of Hydra, it has similar two-cell and three-cell effector pathways that may function in local responses to tentacle contact with food.     

Ultrastructure of neuro‐spirocyte synapses in the sea anemone Aiptasia pallida (Cnidaria,Anthozoa, Zoantharia)

Using transmission electron microscopy of serially sectioned tentacles from the sea anemone Aiptasia pallida, we located and characterized two types of neuro‐spirocyte synapses. Clear vesicles were observed at 10 synapses and dense‐cored vesicles at five synapses. The diameters of vesicles at each neuro‐spirocyte synapse were averaged; clear vesicles ranged from 49–89 nm in diameter, whereas the dense‐cored vesicles ranged from 97–120 nm in diameter. One sequential pair of synapses included a neuro‐spirocyte synapse with clear vesicles (81 nm) and a neuro‐neuronal synapse with dense‐cored vesicles (168 nm). A second synapse on the same cell had dense‐cored vesicles (103 nm). An Antho‐RFamide‐labeled ganglion cell and three different neurites were observed adjacent to spirocytes, but no neuro‐spirocyte synapses were present. Many of the spirocytes also were immunoreactive to Antho‐RFamide. The presence of sequential neuro‐neuro‐spirocyte synapses suggests that synaptic modulation may be involved in the neural control of spirocyst discharge. The occurrence of either dense‐cored or clear vesicles at neuro‐spirocyte synapses suggests that at least two types of neurotransmitter substances control the discharge of spirocysts in sea anemones. J. Morphol. 241:165–173, 1999. © 1999 Wiley‐Liss, Inc.     

Cytotoxicity of the nematocyst venom from the sea anemone Aiptasia mutabilis

Crude extracts of the coelenterate Aiptasia mutabilis (Anthozoa, Aiptasiidae) nematocysts have been tested for their cytotoxicity of Vero and HEp-2 cells monolayers. The results indicate that the nematocyte venom contains one or more toxins with an extremely powerful cytolytic activity. An extract containing the equivalent of as little as 0.6 nematocysts/microL is sufficient to induce significant cellular necrosis, and IC50 can be estimated to be ca. 2 nematocysts/microL on Vero cells. These values are 1-2 orders of magnitude lower than those reported so far for other sea anemone venoms. The extreme potency is accompanied by poor stability of the venom that is readily inactivated by moderate heat and by buffers at non-neutral pH values. The extract is unstable even when kept for short times at 4 degrees C, or after storage at -20 degrees C. Separation of crude venom by affinity chromatography on ConA-Sepharose allowed us to identify two main components with molecular masses of 95 and 31 kDa, respectively, as responsible for the cytolytic properties of A. mutabilis nematocyst extract.     

Photobiology of natural populations of zooxanthellae from the sea anemone Aiptasia pallida: assessment of the host's role in protection against ultraviolet radiation

Natural populations of the sea anemone Aiptasia pallida containing endosymbiotic dinoflagellates were acclimated to different irradiance regimes, with and without ultraviolet radiation (UV). They showed a compensatory response in the amount of chlorophyll and the activities of enzymes responsible for detoxifying active species of oxygen that are produced by the interaction between visible or ultraviolet radiation and photosynthetically produced oxygen. Protection from active species of oxygen is essential to prevent the photooxidation of chlorophyll and the concomitant loss of productivity. Bulk analyses of chlorophyll showed differences between the populations exposed to varying irradiance regimes, but revealed no significant independent effect of UV. However, analysis by flow cytometry of the individual cells from treated populations did detect statistically significant differences in cell size and the amount of chlorophyll fluorescence per cell, which could be attributed to treatment with ultraviolet radiation. With flow cytometry we are able to detect the population variability that is undetectable by bulk measurements which is important in assessing the effects of environmental parameters in both symbiotic and free-living microalgae. Research using simultaneous measurements by flow cytometry could add considerable insight into the population dynamics of both zooxanthellae and host cells.     

Ammonium metabolism in the symbiotic sea anemone Anemonia viridis

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

Cyclophilin and the regulation of symbiosis in Aiptasia pallida

The sea anemone Aiptasia pallida, symbiotic with intracellular dinoflagellates, expresses a peptydyl-prolyl cis-trans isomerase (PPIase) belonging to the conserved family of cytosolic cyclophilins (ApCypA). Protein extracts from A. pallida exhibited PPIase activity. Given the high degree of conservation of ApCypA and its known function in the cellular stress response, we hypothesized that it plays a similar role in the cnidarian-dinoflagellate symbiosis. To explore its role, we inhibited the activity of cyclophilin with cyclosporin A (CsA). CsA effectively inhibited the PPIase activity of protein extracts from symbiotic A. pallida. CsA also induced the dose-dependent release of symbiotic algae from host tissues (bleaching). Laser scanning confocal microscopy using superoxide and nitric oxide-sensitive fluorescent dyes on live specimens of A. pallida revealed that CsA strongly induced the production of these known mediators of bleaching. We tested whether the CsA-sensitive isomerase activity is important for maintaining the activity of the antioxidant enzyme superoxide dismutase (SOD). SOD activity of protein extracts was not affected by pre-incubation with CsA in vitro.     

Catalase characterization and implication in bleaching of a symbiotic sea anemone

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

Programmed cell death and cell necrosis activity during hyperthermic stress-induced bleaching of the symbiotic sea anemone Aiptasia sp.

Different cell death pathways were investigated during bleaching in the sea anemone Aiptasia sp. in response to hyperthermic treatment. Using a suite of techniques, (haematoxylin and eosin staining of paraffin wax-embedded tissue sections, in-situ end labelling (ISEL) of fragmented DNA, agarose gel electrophoresis electron microscopy) both necrotic and programmed cell death (PCD) activity were indicated. After a treatment period of 4 days, the host endoderm tissues underwent necrotic cell death. This was indicated by widespread cellular degradation, dilation of cell cytoplasm and organelles, cell swelling and rupture, irregular pyknotic condensation of nuclear chromatin, and abundant cell debris. Host cell necrosis was associated with the release of zooxanthellae with a normal, healthy appearance into the coelenteron. Longer periods of hyperthermic treatment (7 days) were correlated with further animal cell degradation and the in-situ degradation of zooxanthellae remaining within the degraded endoderm. Within the same degraded endoderm tissue, the degradation of zooxanthellae resulted from two forms of cell death occurring simultaneously, which were identified as programmed cell death and cell necrosis. Programmed cell death of zooxanthellae was characterised by condensation of the cytoplasm and organelles, cell shrinkage, formation of accumulation bodies at the periphery of the cell wall, and DNA fragmentation. Cell necrosis of zooxanthellae was characterised by dilation of the cytoplasm and organelles, cell swelling and lysis, dispersion of cell component debris, and DNA fragmentation. The existence of a programmed cell death pathway within zooxanthellae is important to the understanding of coral bleaching events, raising interesting questions regarding the evolution of this process and the activation of the cellular trigger mechanisms involved.     

Characterization of nematocyst proteins from the sea anemones Aiptasia pallida and Pachycerianthus torreyi (Cnidaria: Anthozoa)

Nematocysts from the anemones Aiptasia pallida and Pachycerianthus torreyi were investigated. SDS-polyacrylamide electrophoresis of solubilized Aiptasia nematocysts revealed one major protein band (mol. wt 31,800) and several minor components. Coelectrophoresed whole venom contained numerous protein components, of which a major one appeared to be identical to the major nematocyst protein. Nematocyst capsules and everted threads from both species contained levels of glycine and proline-hydroxyproline characteristic of vertebrate collagens. Cysteine was present in significant amounts. Aiptasia whole venom contained high levels of glutamic acid and/or glutamine (71%) with no detectable cysteine or proline-hydroxyproline. The 31,800-dalton venom protein possessed only glycine (80%) and glutamyl and/or glutaminyl (20%) residues.     

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.