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...     

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.     

Sequential biochemical and morphological events during assembly of the fertilization membrane of the sea urchin.

The fertilization membrane of Strongylocentrotus purpuratus undergoes changes in morphology, solubility, and permeability during the process of hardening. As the fertilization membrane elevates from the egg surface, it retains casts of the tips of the microvillous processes of the plasma membrane. The dome-shaped microvillar casts become angular at the same time that the fertilization membrane becomes resistant to solubilization in mercaptan solutions. 2-4 min after this morphological and chemical transition, the fertilization membrane becomes impermeable to the lectin conconavalin A, as monitored by binding of 125I- or fluorescein-labeled concanavalin A. Glycine ethyl ester inhibits the changes in morphology, solubility, and permeability, whereas sodium sulfite inhibits only the permeability block and resistance to solubilization by mercaptans. Parthenogenetic activation with the divalent ionophore, A23187, elicits fertilization membrane elevation more rapidly than does activation by fertilization; however, the morphological and permeability changes characteristic of hardening proceed more slowly. Elevation and hardening of the fertilization membrane thus appear to be discrete, multiple-step assembly processes that occur in fixed sequence, with kinetics that are affected by the mechanism of cortical granule exocytosis.     

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.     

Regulatory volume decrease in nematocytes isolated from acontia of Aiptasia diaphana.

Regulatory volume decrease (RVD) and the mechanisms of its regulation were investigated in microbasic mastigophore nematocytes isolated from the acontia of Aiptasia diaphana (Coelenterates, Cnidaria), a marine species that can be exposed to considerable changes in osmotic pressure. Exposure of isolated cells to a 35% hypoosmotic shock lead to the expected osmotic swelling followed by a rapid RVD. RVD was blocked if Ca2+ influx was prevented either by applying a Ca2+-free medium or by treating the cells with Gd3+. Furthermore, the calmodulin action inhibitor trifluoperazine (TFP), prevented RVD and also caused a larger swelling than that induced by preventing Ca2+ influx. Treatment of nematocytes with quinine completely blocked the RVD. Such an effect was prevented by gramicidine. A partial inhibition of RVD was caused by treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). It is concluded that: i) the nematocytes regulate volume under hypoosmotic shock; ii) the regulatory mechanisms consist mainly in increased conductance to K+, and consequently, of Cl-, and, to a lesser extent, in H+/K+-Cl-/HCO3- exchange, and iii) the ionic fluxes are triggered by increased [Ca2+]i with the possible involvement of calmodulin.     

ApRab3, a biosynthetic Rab protein,accumulates on the maturing phagosomes and symbiosomes in the tropical sea anemone,Aiptasia pulchella

Symbiosome biogenesis and function are central to the endosymbiotic interaction between symbiotic dinoflagellates and their host cnidarians. To understand these important organelles, we have been conducting studies to identify and characterize symbiosome-associated proteins of the Rab family, key regulatory components of vesicular trafficking and membrane fusion in eukaryotic cells. Our prior studies have implicated three endocytic Rab proteins in the regulation of symbiosome biogenesis. Here, we show that ApRab3 is a new member of the Rab3 subfamily, associating with symbiosomes and accumulating on the maturing phagosomes in the A. pulchella digestive cells. ApRab3 is 78% identical to human Rab3C, and contains all Rab 3-specific signature motifs. EGFP-ApRab3-labeled vesicular structures tended to either align along the cell peripheral, or aggregate at one side of the nucleus. ApRab3 specifically co-distributed with the TGN marker, WGA, but not other organelle-specific markers tested. Immunofluorescence staining with a specific peptide antibody showed similar results. Significantly, an expression of a constitutively active mutant caused the enlargement and random dispersion of EGFP-ApRab3-decorated compartments in PC12 cells. Together, these data suggest that ApRab3 is a new member of the Rab3 subfamily, participating in the biosynthetic trafficking pathway, and symbiosome biogenesis involves an interaction with ApRab3-positive vesicles.     

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.     

Hyperthermic stress-induced increase in the expression of glutamate-cysteine ligase and glutathione levels in the symbiotic sea anemone Aiptasia pallida

Hyperthermic stress is known to trigger the loss of unicellular algae from a number of symbiotic cnidarians, a phenomenon commonly referred to as bleaching. Oxidative and nitrosative stress have been suggested to play a major role during the process of bleaching, however the underlying molecular mechanisms are still poorly understood. In animals, the intracellular tripeptide glutathione (GSH) is involved in antioxidant defense, redox homeostasis and intracellular redox signaling. Therefore, we tested the hypothesis that hyperthermal stress-induced bleaching in Aiptasia pallida, a model for symbiotic cnidarians, results in increased levels of GSH synthesis. We report the cDNA sequence and functional analysis of the catalytic subunit of glutamate-cysteine ligase (GCLC), which catalyzes the rate-limiting step in GSH biosynthesis. In a time-series experiment, both GCLC gene expression and total GSH levels increased 4- and 1.5-fold, respectively, in response to hyperthermal stress. These results suggest that hyperthermal stress triggers adaptive increases in intracellular GSH biosynthesis in cnidarians as a protective response to oxidative/nitrosative stress. Our results show the conserved function of GCLC and GSH across animals while placing a new perspective on the role of GSH in redox signaling during cnidarian bleaching.     

Sequential 1H-NMR assignments and secondary structure of the sea anemone polypeptide anthopleurin-A

The sequence-specific assignment of resonances in the 500-MHz 1H-NMR spectrum of a cardioactive sea anemone polypeptide, anthopleurin-A, is described. The assignment procedure involved analysis of two-dimensional phase-sensitive multiple-quantum-filtered, double-quantum, homonuclear Hartmann-Hahn and nuclear Overhauser effect spectra. Using sequential information, specific assignments have been made for resonances arising from all 49 amino acid residues. Resonances arising from a number of residues in a minor conformer present in solution are also assigned. These results greatly extend previous resonance assignments made from spectra acquired at 300 MHz [Gooley, P. R. and Norton, R. S. (1985) Eur. J. Biochem. 153, 529-539] and provide the basis for a more accurate definition of the conformation of anthopleurin-A in aqueous solution. The secondary structure includes a four-stranded antiparallel beta-sheet encompassing residues 2-4, 21-23, 34-36 and 45-49, and possibly a beta-bulge located at Ser-19 and Gly-20. A type II beta-turn is formed by residues 30-33. These structural elements also occur within other related sea anemone polypeptides, but the conformation of the small loop region containing Pro-41 appears to be unique to anthopleurin-A.     

Oxidative stress and apoptotic events during thermal stress in the symbiotic sea anemone, Anemonia viridis

Symbiosis between cnidarian and photosynthetic protists is widely distributed over temperate and tropical seas. These symbioses can periodically breakdown, a phenomenon known as cnidarian bleaching. This event can be irreversible for some associations subjected to acute and/or prolonged environmental disturbances, and leads to the death of the animal host. During bleaching, oxidative stress has been described previously as acting at molecular level and apoptosis is suggested to be one of the mechanisms involved. We focused our study on the role of apoptosis in bleaching via oxidative stress in the association between the sea anemone Anemonia viridis and the dinoflagellates Symbiodinium species. Characterization of caspase-like enzymes were conducted at the biochemical and molecular level to confirm the presence of a caspase-dependent apoptotic phenomenon in the cnidarian host. We provide evidence of oxidative stress followed by induction of caspase-like activity in animal host cells after an elevated temperature stress, suggesting the concomitant action of these components in bleaching.     

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.     

Nervous control of light responses in the sea anemone, Calamactis praelongus.

1. The burrowing sea anemone, Calamactis praelongus, responds to light with local, non-nervous contractions of the column. There are also more extensive responses of the column and retractor muscles co-ordinated by nerve net pulses (NNP's) under pacemaker control. 2. NNP's occur in at least two types of bursts and in sequences which sometimes indicate a rotating site of pulse initiation. 3. Light-evoked NNP sequences can be tape recorded and used later to drive a stimulator to reproduce the original sequences in the same or different anemones, evoking muscular responses which approximate the originals. This technique separates the pacemaker-directed component of the light response from the local effects of light stimulation. 4. Isolated circular and parietal muscles contract slowly when stimulated by light or excited indirectly by NNP's. Retractor muscles are insensitive to light but produce rapid contractions when excited by closely spaced light-evoked NNP's. 5. A model for light responses is proposed which incorporates the characteristics of isolated muscles and intact anemones.     

Purification, sequence, and pharmacological properties of sea anemone toxins from Radianthus paumotensis. A new class of sea anemone toxins acting on the sodium channel

Four new toxins have been isolated from the sea anemone Radianthus paumotensis: RpI, RpII, RpIII, and RpIV. They are polypeptides comprised of 48 or 49 amino acids; the sequence of RpII has been determined. Toxicities of these toxins in mice and crabs are similar to those of the other known sea anemone toxins, but they fall into a different immunochemically defined class. The sequence of RpII shows close similarities with the N-terminal end (up to residue 20) of the previously sequenced long sea anemone toxins, but most of the remaining part of the molecule is completely different. Like the other sea anemone toxins, Radianthus toxins are active on sodium channels; they slow down the inactivation process. Through their Na+ channel action, Radianthus toxins stimulate Na+ influx into tetrodotoxin-sensitive neuroblastoma cells and tetrodotoxin-resistant rat skeletal myoblasts. The efficiency of the toxins is similar in the two cellular systems. In that respect, Radianthus toxins behave much more like scorpion neurotoxins than sea anemone toxins from Anemonia sulcata or Anthopleura xanthogrammica. In binding experiments to synaptosomal Na+ channels, Radianthus toxins compete with toxin II from the scorpion Androctonus australis but not with toxins II and V from Anemonia sulcata.     

Characterization of outer arm dynein in sea anemone, Anthopleura midori.

Outer arm dynein was purified from sperm flagella of a sea anemone, Anthopleura midori, and its biochemical and biophysical properties were characterized. The dynein, obtained at a 20S ATPase peak by sucrose density gradient centrifugation, consisted of two heavy chains, three intermediate chains, and seven light chains. The specific ATPase activity of dynein was 1.3 micromol Pi/mg/min. Four polypeptides (296, 296, 225, and 206 kDa) were formed by UV cleavage at 365 nm of dynein in the presence of vanadate and ATP. In addition, negatively stained images of dynein molecules and the hook-shaped image of the outer arm of the flagella indicated that sea anemone outer arm dynein is two-headed. In contrast to protist dyneins, which are three-headed, outer arm dyneins of flagella and cilia in multicellular animals are two-headed molecules corresponding to the two heavy chains. Phylogenetic considerations were made concerning the diversity of outer arm dyneins.     

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.     

Genetics and asexual reproduction of the sea anemone Metridium senile.

1. Metridium senile was studied for phosphohexose-isomerase variation at three locations on Cape Cod, Massachusetts: Woods Hole, Cape Cod Canal, and Barnstable Town Boat Harbor. 2. All three locations exhibited significant polymorphism for PHI. 3. Mapping of individual polyps was performed at Barnstable to analyze spatial distributions of clones and genotypes. 4. In Barnstable, PHI does not depart significantly from Hardy-Weinberg expectations at the time of establishment of new polyps, and establishment of larvae is spatially random with respect to PHI genotype. 5. Asexual reproduction was uses as a meausre of the relative success of different PHI genotypes. There are indications that not all genotypes are equally likely to produce large clones. 6. There is significant heterogeneity among the three locations with respect to PHI genotype frequencies, suggesting that there may be geographical differentiation of the populations. 7. Sessile, asexual organisms provide powerful tools for examining the dynamic aspects of genetic structure in natural populations.