Sea Anemone Anemonia Research Articles

Temperature induction of viruses in symbiotic dinoflagellates

Bleaching manifests itself as a loss of symbiotic dinoflagellates (zooxanthellae) and/or chlorophyll from a variety of symbiotic hosts, including corals and sea anemones. Bleaching is known to result from a range of environmental stresses, the most significant of which is elevated tempera- ture; how these stresses elicit a bleaching response is cur- rently the focus of intense research. One consequence of environmental stress that has yet to be considered is viral attack. Here, we have isolated a transferable infectious agent believed to be a virus, from zooxanthellae of the temperate sea anemone Anemonia viridis. The infectious agent is induced by elevated temperature. Once induced, the filter- able agent can be further propagated without heat induction, thus fulfilling Koch's postulates. We propose that zooxanthel- lae harbor a latent viral infection that is induced by exposure to elevated temperatures. If such a mechanism also operates in the zooxanthellae harbored by reef corals, and these viruses kill the symbionts, then this could contribute to tem- perature-induced bleaching.

Cracks in the beta-can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria).

We characterize two green fluorescent proteins (GFPs), an orange fluorescent protein, and a nonfluorescent red protein isolated from the sea anemone Anemonia sulcata. The orange fluorescent protein and the red protein seem to represent two different states of the same protein. Furthermore, we describe the cloning of a GFP and a nonfluorescent red protein. Both proteins are homologous to the GFP from Aequorea victoria. The red protein is significantly smaller than other GFP homologues, and the formation of a closed GFP-like beta-can is not possible. Nevertheless, the primary structure of the red protein carries all features necessary for orange fluorescence. We discuss a type of beta-can that could be formed in a multimerization process.

Localisation of antioxidant enzymes in the cnidarians Anemonia viridis and Goniopora stokesi

Antioxidant enzymes scavenge to protect cells against destructive oxyradicals. In excess, reactive radicals can have wide-reaching consequences, including lipid peroxidation, protein degradation and DNA damage, resulting in tissue damage and cell death. Recently, oxyradicals have been implicated in coral-bleaching, but only the activities of antioxidant enzymes in the host and endosymbiotic algae have been reported. To locate the potential cellular targets of excess oxyradicals in cnidarians, it is important to establish in which cell areas these enzymes are active. Using immunocytochemical techniques, the antioxidant enzymes superoxide dismutase [SOD], catalase [CAT] and glutathione peroxidase [GPX] were localised in the sea anemone Anemonia viridis (Forskal) and the coral Goniopora stokesi (Edwards & Haime). Using affinity-purified primary antibodies and transmission electron microscopy, antioxidant enzymes were found predominantly associated with granulated vesicles, accumulation bodies of endosymbiotic algae and cnida. SOD and CAT gold-labelling was localised in all forms of cnida, with SOD being particularly pronounced on the ruptured threads and shafts of b-mastigophores in A. viridis, possibly suggesting that the b-mastigophore had undergone autolysis and required SOD to prevent damage to host cells. The presence of both SOD and CAT in the accumulation body of endosymbiotic algae is consistent with the hypothesised role of these bodies in digestion and cell-ageing. CAT was also found in isolated electron-dense bodies, often near microvillous borders in G. stokesi. Similar bodies were recorded in A. viridis, but contained GPX rather than CAT. GPX was also present in symbiotic algae, where it was associated with electron-dense bodies.

Natural Animal Coloration Can Be Determined by a Nonfluorescent Green Fluorescent Protein Homolog

It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.

Biometric investigations on the cnidae of the Aegean colour morphs of Anemonia viridis

The sea anemone Anemonia viridis is one of the most common species of the North Aegean Sea. The variety in colour morphs exhibited by this species, causes problems to taxonomy and ecology, and suggests that A. viridis should be subdivided into two distinct species according to the colour of the individuals. The approach used in this study was based on the biometrical cnidae characteristics of the two colour morphs (rustica and smaragdina), as it is known that cnidae biometry has been used as a taxonomic tool for the phylum Cnidaria. More than 20,000 nematocysts were measured to try and find a correlation between these measurements and some body parameters indicative of the maturity grade of the individuals, as potential taxonomic characteristics. Stable cnidae characteristics of the two different colour morphs were compared. However, they differed only in the biometry of the tentacles' a-basitrichs. This slight difference does not support the proposal to subdivide Anemonia viridis into two different species.

Primary site and initial products of ammonium assimilation in the symbiotic sea anemone Anemonia viridis

Invertebrates containing endosymbiotic dinoflagellate algae (zooxanthellae) retain excretory nitrogen, and many are able to take up ammonium from the surrounding seawater. However, the site of assimilation and role of nitrogen recycling between symbiont and host remains unclear. In the present study, ammonium uptake by the symbiotic sea anemone Anemonia viridis (Forskal) was examined by following the pathway of assimilation using 15N-enriched ammonium. Since zooxanthellae became enriched with 15N from ammonium at up to 17 times the rate of the host, they appear to be the primary site of assimilation. In the light, the rate of zooxanthellae enrichment at 20 M was twice that at 10 M, whereas the rate of host enrichment was not significantly affected by ammonium concentration. When anemones were incubated with [15N]ammonium in the dark, after 12 h without light the rate of enrichment was lowered in both zooxanthellae and host. However, while the enrichment of the host was significantly reduced when the light level was lowered from 300 to 150 μmol photons m−2 s−1, zooxanthellae enrichment was unchanged. Low molecular weight material from the zooxanthellae became enriched at 20 times the rate of that from the host, and enrichment was detected in the amino acids glutamate, glutamine, aspartate, alanine, glycine, phenylalanine, threonine, valine, tyrosine, and leucine from zooxanthellae. In the zooxanthellae, amino acids accounted for 65% of the total enrichment of low molecular weight material. Of the amino acids detected in zooxanthellae, over 90% of the enrichment was accounted for by glutamate, glutamine and aspartate. The enrichment of the amide group of glutamine was greater than that of the amine group of glutamate or glutamine, consistent with the glutamine synthetase/glutamine 2-oxoglutarate amidotransferase cycle as the mechanism of ammonium assimilation. To examine the flux of 15N from zooxanthellae to host, anemones were pulse-labelled with [15N]ammonium and then transferred to an unlabelled chase. Over a 2 h period there was no evidence for a flux of nitrogen from zooxanthellae to host. However, during the chase period, the enrichment of low molecular weight material declined and that of high molecular weight material increased in both zooxanthellae and host, indicating that protein was synthesized using 15N from ammonium in both components of the symbiosis. Again by using a pulse-chase system, it was found that glutamate was metabolised most rapidly by zooxanthellae, followed by (in order of decreasing rate of turnover) aspartate, alanine, glycine and valine (no data are available for glutamine). Unlike these amino acids, nitrogen was transferred to the essential amino acids phenylalanine and threonine, increasing their enrichment during the chase period. While recycled nitrogen is clearly important to this symbiosis, the mechanism by which it is cycled remains to be resolved.

An all-electron calculation of an antihypertensive protein with the Gaussian-based density functional method

An all-electron calculation of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata was carried out using the density functional method based on Gaussian-type orbitals with workstations. Our computer program ProteinDF coded by the object-oriented language C++, was originally designed for all-electron calculations of proteins and controlled in parallel with high efficiency. The numbers of residues, atoms, orbitals and auxiliary functions of the calculated protein were 43, 629, 3615 and 6545, respectively. This Letter reports timing data of the computation and some calculated electronic properties. Our program will open the door for advanced studies of larger proteins.

Effect of Deltamethrin Treatment on Lepidopteran Larvae Infected with Baculoviruses Expressing Insect-Selective Toxins μ-Aga-IV, As II, or Sh 1

Abstract Modification of sodium channels by pyrethroids is promoted by channel opening. The insect-specific neurotoxins μ-Aga-IV from the spider Agelenopsis aperta, As II from the sea anemone Anemonia sulcata, and Sh I from the sea anemone Stichadactyla helianthus all increase sodium channel opening and so we tested each of them in combination with deltamethrin for synergistic insecticidal activity. We found that As II increased the insecticidal activity of deltamethrin against the blowfly Lucilia sericata. We also examined the effect of deltamethrin on the infectivity and virulence of Autographa californica nuclear polyhedrosis virus (AcMNPV) and recombinant toxin-expressing viruses in both dose-dependent and time-mortality studies. The recombinant viruses expressed chimeric synthetic genes for μ-Aga-IV, As II, or Sh I (genes mag4, sat2, and ssh1, respectively). When Trichoplusia ni larvae were infected with a LC 20 of the recombinant viruses and treated with a LC 20 of deltamethrin in diet incorporation bioassays, the doses were strictly additive. A cooperative decrease in mortality rate was observed in droplet feeding bioassays when a low dose (LC 20 ) of deltamethrin and high doses of toxin-expressing viruses were fed to larvae but this effect was host-dependent. In T. ni, only wild-type AcMNPV and a recombinant expressing mag4 under very late viral promoter control exhibited reduced times in controlling host feeding and mortality in the presence of deltamethrin. For Heliothis virescens, larvae infected with sat2 -expressing viruses displayed shorter feeding times in the presence of deltamethrin than those infected with virus alone but only at very high virus doses. H. virescens larvae infected with wild-type AcMNPV and a mag4 -expressing recombinant had shorter feeding times and also shorter effective and lethal times in the presence of deltamethrin. Our results indicate that coapplication of this pyrethroid will confer little or no advantage in field applications of recombinant baculovirus pesticides and that application of pyrethroids will probably have no adverse affect on recombinant virus efficacy unless it deters larval feeding and virus acquisition.

Effects of Simultaneous Expression of Two Sodium Channel Toxin Genes on the Properties of Baculoviruses as Biopesticides

Abstract Previously, we have described the properties of recombinant baculoviruses expressing three chimeric genes, mag4, sat2, and ssh1, that encode secretable insect selective sodium channel toxins, μ-Aga-IV from the spider Agelenopsis aperta, As II from the sea anemone Anemonia sulcata, and Sh I from the sea anemone Stichadactyla helianthus, respectively. We now show that μ-Aga-IV and As II act at distinct sites on voltage-sensitive sodium channels of insects and synergistically promote channel opening. We also show that these toxins have synergistic insecticidal activity against the blowfly Lucilia sericata and the fall armyworm Spodoptera frugiperda. To determine if toxin synergy also occurs in the context of virus replication, we inserted the chimeric toxin genes into nonessential sites of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome under the control of either a modified promoter, P synXIV , or an insect derived promoter, P hsp70 . Comparative analyses showed that viruses expressing toxin genes under the control of the P hsp70 promoter were more effective as biopesticides than under the control of P synXIV . Two toxins, μ-Aga-IV and As II, exerted the most potent effects in S. frugiperda and Trichoplusia ni larvae, respectively. A virus simultaneously expressing two P hsp70 -promoted toxin genes, mag4 and sat2, exhibited properties similar to the two viruses expressing each of the toxin genes individually except that larval feeding time (FT 50 ) was reduced an additional 10%, indicating a small advantage to coproducing synergistic toxins.

Diffusional permeability of dissolved inorganic carbon through the isolated oral epithelial layers of the sea anemone, Anemonia viridis

In a recent report, Bénazet-Tambutté et al. (1996a) showed that the major source of dissolved inorganic carbon for photosynthesis of the sea anemone Anemonia viridis was external seawater and a minor source was the internal medium of the tentacles. In order to determine the mechanism of DIC transfer from the external seawater to the endodermal cells, we explored the different mechanisms potentially involved in this transfer: transepithelial passive diffusion of DIC across the oral epithelial layers, renewal of coelenteric fluid by the mouth or transepithelial active transport. In addition, we also studied the role of expansion/contraction of the tentacle in the transepithelial transport of DIC. We investigated diffusion of DIC, using isolated perfused tentacles, by measuring transepithelial H 14CO 3 flux. We found that the unidirectional transport of DIC was light-sensitive: the transepithelial flux was about three-fold lower in light than in dark conditions. The flux was insensitive to the anion transport inhibitor, DIDS, and to the carbonic anhydrase inhibitor, Diamox. It was directly proportional to the concentration of bicarbonate in the surrounding seawater. These results suggest that this flux is simply driven by diffusion via a paracellular pathway. Use of inhibitors and inside-out tentacles showed that the transepithelial flux measured in the dark was depressed in the light due to symbiont photosynthesis which diverts about 15% of the DIC flux. Consequently, the passive diffusional flux of DIC can account for less than 1/6 of the dissolved inorganic carbon used for symbiont photosynthesis under saturating light conditions. Expansion/contraction state of the tentacles did not significantly modify the permeability coefficient ( P d=2.28×10 −5 and 2.50×10 −5 cm s −1 respectively for expanded and contracted tentacles) in spite of a change of tissue thickness of a factor of about 3. This result suggests that the mesoglea could represent a significant barrier to transepithelial diffusion of molecules, due to change in its water content level upon contraction. Measurement of water intake by whole sea anemones, using 14C-Dextran, showed that the renewal of the coelenteric fluid which bathes the dinoflagellate-containing endodermal cells cannot provide DIC to symbiont photosynthesis. Therefore it is concluded that a carrier-mediated DIC transporter located on the ectodermal host cells, supplies the bulk of dissolved inorganic carbon used for symbiont photosynthesis.

Functional polarity of the tentacle of the sea anemone Anemonia viridis: role in inorganic carbon acquisition.

The oral epithelial layers of anthozoans have a polarized morphology: photosynthetic endosymbionts live within endodermal cells facing the coelenteric cavity and are separated from the external seawater by the ectodermal layer and the mesoglea. To study if this morphology plays a role in the supply of inorganic carbon for symbiont photosynthesis, we measured the change in pH and the rate of OH- (H+) fluxes induced by each cell layer on a tentacle of the sea anemone Anemonia viridis. Light-induced pH increase of the medium bathing the endodermal layers led to the generation of a transepithelial pH gradient of approximately 0.8 pH units across the tentacle, whereas darkness induced acidification of this medium. The light-induced pH change was associated with an increase of total alkalinity. Only the endodermal layer was able to induce a net OH- secretion (H+ absorption). The light-induced OH- secretion by the endodermal cell layer was dependent on the presence of HCO3- in the compartment facing the ectoderm and was sensitive to several inhibitors of ion transport. [14C] HCO3- incorporation into photosynthates confirmed the ectodermal supply, the extent of which varied from 25 to > 90%, according to HCO3- availability. Our results suggest that the light-induced OH- secretion by the endodermal cell layer followed the polarized transport of HCO3- and its subsequent decarboxylation within the endodermal cell layer. This polarity may play a significant role both in inorganic carbon absorption and in the control of light-enhanced calcification in scleractinian corals.

Algal genotype and photoacclimatory responses of the symbiotic alga Symbiodinium in natural populations of the sea anemone Anemonia viridis

As an approach to investigate the impact of solar radiation on an alga–invertebrate symbiosis, the genetic variation and photosynthetic responses of the dinoflagellate algal symbiosis in an intertidal and a subtidal population of the sea anemone Anemonia viridis were explored. Allozyme analysis of the anemones indicated that the two populations were genetically very similar, with a Nei's index value of genetic identity (I) of 0.998. The algae in all animals examined were identified as Symbiodinium of clade a by PCR-RFLP analysis of the small subunit ribosomal RNA gene. The symbiosis in the two populations did not differ significantly in algal population density, chlorophyll a content per algal cell or any photosynthetic parameter obtained from studies of the relationship between photosynthesis and irradiance. We conclude that there is not necessarily genetic variation or photosynthetic plasticity of the symbiotic algae in Anemonia viridis inhabiting environments characterized by the different solar irradiances of the subtidal and intertidal habitats.

Novel polypeptide toxins with crab lethality from the sea anemone Anemonia erythraea

The sea anemone Anemonia erythraea was found to contain polypeptide toxins with crab lethality as well as hemolysins. Three polypeptide toxins (AETX I, II and III) were isolated by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. A geographic variation in toxin composition was suggested. The LD 50 against crabs of AETX I, II and III were estimated to be 2.2, 0.53 and 0.28 μg/kg, respectively, but none of the toxins showed lethality in mice. The amino acid sequences of the three toxins were deduced from sequencings of the whole molecules and their enzymatic fragments. Amino acid analyses and molecular mass determinations supported the accuracy of the deduced sequences. AETX I, comprising 47 amino acid residues including 6 half-Cys residues, is an analog of sea anemone type 1 toxins. On the other hand, AETX II and III, which are highly homologous with each other, are quite distinct from the known sea anemone polypeptide toxins in that they are composed of 59 residues including 10 half-Cys residues. Interestingly, both toxins have sequence similarities with neurotoxins isolated from the Brazilian `armed' spider Phoneutria nigriventer.

Novel polypeptide toxins with crab lethality in the sea anemone Anemonia erythraea

Novel polypeptide toxins with crab lethality in the sea anemone Anemonia erythraea

A simple biochemical method in the search for bioactive polypeptides in a sea anemone ( Anemonia sulcata)

The sea anemone Anemonia sulcata is a well-known natural source of supply of biologically active polypeptides. So far, five toxins, ATX I, II, III, IV and AS V, several polyvalent protease inhibitors, an elastase inhibitor, two blood pressure-depressive polypeptides and very recently peptides that inhibit competitively the binding of 125I-dendrotoxin to rat brain membranes and block the voltage-sensitive K + channels, have been isolated from it. The sea anemone toxins (especially toxin II of A. sulcata, ATX II) are very important tools in neurophysiological and pharmacological research, and their structure-function relationship has been investigated. Because of the great scientific value of the sea anemone toxins a simplification of their purification procedure was elaborated.

Kalicludines and kaliseptine: Two different classes of sea anemone toxins for voltage-sensitive K + channels

New peptides have been isolated from the sea anemone Anemonia sulcata which inhibit competitively the binding of 125I-dendrotoxin I (a classical ligand for K+ channel) to rat brain membranes and behave as blockers of voltage-sensitive K+ channels. Sea anemone kalicludines are 58-59-amino acid peptides cross-linked with three disulfide bridges. They are structurally homologous both to dendrotoxins which are snake venom toxins and to the basic pancreatic trypsin inhibitor (Kunitz inhibitor) and have the unique property of expressing both the function of dendrotoxins in blocking voltage-sensitive K+ channels and the function of the Kunitz inhibitor in inhibiting trypsin. Kaliseptine is another structural class of peptide comprising 36 amino acids with no sequence homology with kalicludines or with dendrotoxins. In spite of this structural difference, it binds to the same receptor site as dendrotoxin and kalicludines and is as efficient as a K+ channel inhibitor as the most potent kalicludine.

The sea anemone Anemonia sulcata is a rich source of biologically active polypeptides

The sea anemone Anemonia sulcata is a rich source of biologically active polypeptides

Kalicludines and Kaliseptine: TWO DIFFERENT CLASSES OF SEA ANEMONE TOXINS FOR VOLTAGE-SENSITIVE K+ CHANNELS

New peptides have been isolated from the sea anemone Anemonia sulcata which inhibit competitively the binding of 125I-dendrotoxin I (a classical ligand for K+ channel) to rat brain membranes and behave as blockers of voltage-sensitive K+ channels. Sea anemone kalicludines are 58-59-amino acid peptides cross-linked with three disulfide bridges. They are structurally homologous both to dendrotoxins which are snake venom toxins and to the basic pancreatic trypsin inhibitor (Kunitz inhibitor) and have the unique property of expressing both the function of dendrotoxins in blocking voltage-sensitive K+ channels and the function of the Kunitz inhibitor in inhibiting trypsin. Kaliseptine is another structural class of peptide comprising 36 amino acids with no sequence homology with kalicludines or with dendrotoxins. In spite of this structural difference, it binds to the same receptor site as dendrotoxin and kalicludines and is as efficient as a K+ channel inhibitor as the most potent kalicludine.

Symbiont photosynthesis increases both respiration and photosynthesis in the symbiotic sea anemone Anemonia viridis

Dark respiration of the symbiotic sea anemone Anemonia viridis (Forskal) was observed to increase by ∼34% when anemones were exposed to hyperoxic sea water (150% oxygen saturation) overnight, and by 39% after exposure to 6 h in the light at a saturating irradiance of 300 μE m-2 s-1 at normoxia (100% oxygen saturation). No increase due to light stimulation was observed in aposymbiotic control anemones. In darkness, the oxygen concentration of the coelenteric fluid was hypoxic. However, within 10 min of anemones being illuminated, coelenteric fluid was hyperoxic, and it remained elevated throughout a 12 h light period. When measured over a 24 h period (12 h light: 12 h dark), the dark respiration rate increased gradually over the first 6 h of the light period until it was 35% above the dark night-time resting rate. It remained elevated throughout the remaining light period and for 2 h into the following dark period, after which it fell back to the resting rate. Gross photosynthesis (Pgross) increased significantly when anemones were exposed to either hyperoxia (150% oxygen saturation) or 300 μE m-2 s-1 at normoxia. This increase was not observed when symbiotic anemones were illuminated at a low-light intensity of 100 μE m-2 s-1. The results of this study suggest that respiration in the dark is limited by oxygen diffusion and that normal respiration is restored in the daytime by utilisation of the oxygen released by photosynthesis. Furthermore, it appears that the increased respiration following exposure to high-light intensities provides a CO2-rich intracellular environment which further enhances the photosynthetic rate of the zooxanthellae.

Three-dimensional structure in solution of neurotoxin III from the sea anemone Anemonia sulcata.

The three-dimensional structure in aqueous solution of the 27-residue polypeptide neurotoxin Anemonia sulcata toxin III (ATX III) has been determined from 1H NMR data. As ATX III self-associates in the millimolar concentration range, causing a marked concentration dependence for the chemical shifts of several residues [Norton, R. S., Cross, K., Braach-Maksvytis, V., & Wachter, E. (1993) Biochem. J. 293, 545-551], it was necessary to record NOESY spectra over a range of concentrations in order to eliminate any intermolecular interactions from the NOE restraint set. The pairings of the six half-cystine residues were also unknown and had to be determined (as 3-17, 4-11, and 6-22) from preliminary structure calculations performed using both upper bound distance restraints from NOESY data and a substantial number of lower bound restraints inferred from the absence of NOESY cross-peaks. Final structures were determined, using the program X-PLOR, from interproton distance restraints inferred from NOEs, backbone and side chain dihedral angle restraints from spin-spin coupling measurements, and a smaller number of lower bound restraints. Stereospecific assignments for 11 beta-methylene pairs were also included. The final set of 28 structures had an average pairwise RMS difference of 1.32 A over the backbone heavy atoms (N, C alpha, and C) and 2.18 A over all heavy atoms. For the well-defined region encompassing residues 3-22, the corresponding values were 0.62 and 1.28 A, respectively. ATX III adopts a compact structure containing four reverse turns (a distorted type I beta-turn at residues 6-9, a type I beta-turn at residues 8-11, and inverse gamma-turns at residues 12-14 and 15-17) and two other chain reversals, but no regular alpha-helix or beta-sheet. Several of the residues most affected by aggregation are located on the surface of the molecule, forming a hydrophobic patch which may constitute part of the sodium channel binding surface. Possible relationships between the structure of ATX III and those of other sea anemone toxins that interact with the same site on the voltage-gated sodium channel are considered.