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.     

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.     

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.     

Immunocytochemical evidence for biogenic amines and immunogold labeling of serotonergic synapses in tentacles of Aiptasia pallida (Cnidaria, Anthozoa)

Abstract. Evidence for classical neurotransmitters in sea anemones remains controversial. We used high performance liquid chromatography with electrochemical detection (HPLC-EC) and electron microscopical imunocytochemistry to determine the presence of serotonin and precursor synthetic enzymes of other biogenic amines in tentacles of the sea anemone Aiptasia pallida. Using HPLC-EC we found dopamine and serotonin (5-hydroxytryptamine, 5-HT) in both tentacles and whole animal homogenates. Antibodies to tyrosine hydroxylase, dopamine β-hydroxylase, phenylethanolamine N-methyltransferase, and 5-HT were used with the peroxidase-antiperoxidase method to reveal positive immunoreactivity to these substances in neurons of tentacles. Immunogold labeling of serial thin sections with the anti-5–HT antibody revealed reactive products in synaptic vesicles at interneuronal, neuromuscular, and neurospirocyte synapses. These results suggest that both catecholamine and indolamine neurotransmitters occur in sea anemones in addition to the neuropeptide Antho-RFamide, indicating the presence of multiple types of transmitter substances in an early nervous system.     

Tropomyosin isoforms present in the sea anemone,Anthopleura japonica (Anthozoa,Cnidaria)

Five isoforms of tropomyosin, designated as TMa, TMb, TMc, TMd, and TMe, were detected in the sea anemone, Anthopleura japonica. The apparent molecular weights of these isoforms were estimated to be approximately 30 kD to 37.5 kD, and their pI values were approximately 4.55 (TMa and TMb) and 4.65 (TMc, TMd, and TMe). Although sea anemone tropomyosin isoforms have the ability to bind to rabbit skeletal muscle actin, they preferably bind to actin at higher concentrations of Mg(2+) (10-20 mM) and slightly lower pH (6.2-7.2) than those used in conventional conditions. Antigenic properties of sea anemone tropomyosin seemed to be considerably specific to each isoform. Distribution of tropomyosin isoforms in the sea anemone body was somewhat portion-specific. TMa, TMb, and TMe were detected similarly in the extracts from tentacle, oral disc, column, mouth, and pedal disc. Although TMc and TMd were detected abundantly in the tentacle extract and moderately in the column and mouth extracts, these components were not contained in the pedal disc extract and detected only faintly in the oral disc extract.     

Cnidae in the sea anemone Sagartiogeton viduatus (Muller, 1776) (Cnidaria,Anthozoa); A comparison to cnidae in the sea anemone Metridium senile (Linnaeus, 1761) (Cnidaria,Anthozoa)

The cnidom of the sea anemone Sagartiogeton viduatus (Muller, 1776) is described from interference‐contrast light micrographs (LMs) and scanning electron micrographs (SEMs). Special attention is given to nematocyst maturation, including the differentiation of the shaft into proximal and main regions as helical folding of the shaft wall proceeds. Comparisons are made with Metridium senile (Linnaeus, 1761), whose cnidom, with a few exceptions, is closely similar to that of S. viduatus. The two anemones possess b‐ and p‐mastigophores, p‐amastigophores, isorhizas and spirocysts. Although the majority of cnidae in S. viduatus is smaller than corresponding ones in M. senile, they are grouped into the same size classes as those of M. senile, namely small, medium and large. The main differences from M. senile cnidae are the followings: (1) Large acontia p‐amastigophores are the largest nematocysts in S. viduatus. (2) They are noticeably larger than the large acontia b‐mastigophores, and (3) they are separated from the p‐amastigophores of M. senile by the sinusoid pattern of their U‐shaped capsular matrix. (4) The large acontia b‐mastigophores are microbasic and not mesobasic as in M. Senile, and (5) they do not produce darts. (6) Another difference from M. senile is the absence of catch‐tentacle isorhizas.     

Calcium and cytoskeleton signaling during cell volume regulation in isolated nematocytes of Aiptasia mutabilis (Cnidaria: Anthozoa)

Cell volume regulation has not been completely clarified in Coelenterates. The present investigation focuses on cell volume regulation under anisosmotic conditions, both hyposmotic and hypertonic, and on the underlying signals in nematocytes isolated from the Coelenterate Aiptasia mutabilis living in sea water. Nematocytes, once isolated from acontia, that were submitted to either hyposmotic (35%) and hypertonic shock (45%) show RVD and RVI capabilities, respectively. In order to ascertain the role of Ca2+ in triggering such regulatory mechanisms and the possible involvement of cytoskeleton components, tests were performed by employing either Ca2+ free conditions, Gd3+ as Ca2+ channel blockers, TFP as calmodulin inhibitor, colchicine as microtubule inhibitor and cytochalasin B as microfilament polymerization inhibitor. Results show that isolated nematocytes of A. mutabilis can regulate their volume upon both hyposmotic and hypertonic challenge. Ca2+ both from external medium and from internal stores is needed to perform RVD mechanisms, whereas, intracellular Ca2+ seems to be mainly involved in RVI. Moreover cytoskeletal components may play an important role since a significant RVD and RVI inhibition was observed in treated cells. On the basis of our observations further studies are warranted to further verify the role of signals, including phosphatases and phosphorylases, in cell volume regulation of primitive eukaryotic cells.     

An ultrastructural study of the establishement of the testicular cysts during spermatogenesis in the sea anemone Actinia fragacea (Cnidaria: Anthozoa)

Spermatogenesis in the sea anemone Actinia fragacea takes place in numerous testicular cysts located in the mesoglea of the gonads. Prospermatogonia arise among the bases of the gonadal epithelial cells bordering the mesoglea, and later migrate into the mesoglea to establish the cysts. The prospermatogonia arise singly, but soon most are found as small groups within the endoderm. They are small cells, 6–7 μm in diameter, and have relatively large nuclei with a single nucleolus. Their cytoplasm is dense, and contains dense bodies and nuage material as well as Golgi, mitochondria, and individual cisternae of endoplasmic reticulum. Each prospermatogonium bears a flagellum, originating in a groove or channel in the cytoplasm. A small proportion of prospermatogonia enter the mesoglea singly, but most migrate as elongate groups or “slugs” of cells. As they enter, the groups often become constricted into hour-glass shapes, and they become covered by the endodermal basal lamina. During the later stages of entry, the last part of the group to enter retains contact with the bases of the epithelial cells, which are dragged into the mesoglea behind the germ cells. This contact between germ cells and endoderm persists throughout spermatogenesis and prevents closure of the mesoglea behind the group. The endodermal cells involved begin specialization to form the trophonema. Once entry is complete, the groups enlarge rapidly to form the testicular cysts. A small number of germ cells appear to remain behind in the endoderm after most have entered the mesoglea, and the possible significance of these cells is discussed.     

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.     

Gametogenic cycles and reproduction in the beadlet sea anemone Actinia equina (Cnidaria: Anthozoa)

Sexual Actinia equina have been maintained in the laboratory. Monthly biopsies show that these animals have annual gametogenic cycles. Regular samples from several natural populations suggest that animals in the wild show similar gametogenic periodicity. In the laboratory, males release sperm, but females brood young, at least for a few weeks. Members of these broods are released sporadically, usually after a water change. Males and non-sexual adults kept in isolation for over a year release a few juveniles during and at the end of that time. These young are likely to be asexually reproduced. There appears to be geographical variation of anemone size and in the frequency of sexual individuals in populations round the British Coast, so that modes of reproduction may vary.     

Ultrastructure of nematocyst discharge in catch tentacles of the sea anemone Haliplanella luciae (Cnidaria: Anthozoa)

The mature nematocyst lies just beneath the cnidodyte plasma membrane. A microtubule array surrounds the nematocyst capsule just beneath the capsule tip. We propose that the array helps to hold the capsule at the cnidocyte cell surface until discharge. The undischarged capsule tip is sealed by three apical flaps, joined together along complex radial seams. The seams are filled with subunits that appear to bind the flaps together. Upon discharge, the flaps separate along the radial seams to permit thread eversion. The everted thread is lined on both sides by subunits that are stained by antimonate, indicating that they bind calcium. We suggest that, together, the subunits hold the uneverted thread in its folded and coiled configuration. Thread eversion would follow subunit uncoupling. The capsule and thread interiors of partially discharged nematocysts are stained by antimonate. In contrast, the capsule and thread interiors of fully discharged nematocysts are not stained by antimonate. Thus, nematocyst calcium might be injected into the target tissue where it is presumed to act in conjunction with nematocyst venom to promote cell death.     

Modes of reproduction in sea anemones (Cnidaria,Anthozoa)

The data on different modes of reproduction in sea anemones are generalized. These animals can reproduce sexually in an ordinary way or by parthenogenesis. Asexual reproduction occurs in various forms, such as transverse and longitudinal fission, pedal laceration, or autotomy of tentacles. Specific features of different variants of sexual and asexual reproduction and their combinations in sea anemones from different habitats of the World Ocean are discussed.     

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.     

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.     

Nematocysts of sea anemones (Actiniaria,Ceriantharia and Corallimorpharia: Cnidaria): nomenclature

A brief historical review of nematocyst terminology is given and three nomenclatural problems are discussed. It is proposed to combine the terms initiated by Weill (1934) with those of Schmidt (1969). A new mesobasic grade, intermediate between microbasic and macrobasic is proposed for amastigophores and p-mastigophores possessing a short Faltstück. A more liberal interpretation of Weill's (1934) terminology for nematocysts than that proposed by Cutress (1955) is suggested in respect of microbasic amastigophores and p-mastigophores. Basitrichs and b-mastigophores continue to be recognized as separate categories.deceased     

A new species of sea anemone (Cnidaria: Anthozoa: Actiniaria) from Manus Basin hydrothermal vents, South-western Pacific

During the BIOACCESS Japanese cruises (1996 & 1998), active hydrothermalism and associated vent fauna were studied on the South-eastern Rift of Manus Basin (South-western Pacific). In the PACMANUS vent field, a conspicuous vent fauna was sampled, including an actinostolid sea anemone (Actiniaria) belonging to an undescribed genus and species. Pacmanactis hashimotoi gen. et spec. nov. is here described, and represents the 9th sea anemone reported from hydrothermal vents.     

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.     

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.     

The genetic structure of the rare lagoonal sea anemone,Nematostella vectensis Stephenson (Cnidaria; Anthozoa) in the United Kingdom based on RAPD analysis

The sea anemone Nematostella vectensis occurs in lagoons in the United States and along the southern and eastern coasts of the United Kingdom. In the United Kingdom it is considered rare and is threatened, principally through the destruction of lagoonal habitat. Random amplified polymorphic DNA (RAPD) data from populations across most of the rane of N. vectensis in the United Kingdom revealed that 61% of individuals had an identical genotype, the frequency of which varied from 0.01 to 1.00. These data provide strong evidence for predominantly clonal reproduction and for the existence of a 'general-purpose genotype' in the UK populations. Alternatively, the low levels of genetic variation observed in some N. vectensis populations may have resulted if they were founded from very few successful individuals from the United States. Analysis of molecular variance (amova) showed significant genetic differentiation between lagoons with no large-scale pattern of geographical variation. This result is consistent with occasional passive or anthropogenic dispersal of low numbers of individuals between lagoons followed by asexual proliferation of immigrants. Transplantation of individuals of the predominant (general-purpose) genotype, for conservation purposes, will probably stand a good chance of survival given its prevalence throughout the United Kingdom.