Neurotoxin localization to ectodermal gland cells uncovers an alternative mechanism of venom delivery in sea anemones

Jellyfish, hydras, corals and sea anemones (phylum Cnidaria) are known for their venomous stinging cells, nematocytes, used for prey and defence. Here we show, however, that the potent Type I neurotoxin of the sea anemone Nematostella vectensis, Nv1, is confined to ectodermal gland cells rather than nematocytes. We demonstrate massive Nv1 secretion upon encounter with a crustacean prey. Concomitant discharge of nematocysts probably pierces the prey, expediting toxin penetration. Toxin efficiency in sea water is further demonstrated by the rapid paralysis of fish or crustacean larvae upon application of recombinant Nv1 into their medium. Analysis of other anemone species reveals that in Anthopleura elegantissima, Type I neurotoxins also appear in gland cells, whereas in the common species Anemonia viridis, Type I toxins are localized to both nematocytes and ectodermal gland cells. The nematocyte-based and gland cell-based envenomation mechanisms may reflect substantial differences in the ecology and feeding habits of sea anemone species. Overall, the immunolocalization of neurotoxins to gland cells changes the common view in the literature that sea anemone neurotoxins are produced and delivered only by stinging nematocytes, and raises the possibility that this toxin-secretion mechanism is an ancestral evolutionary state of the venom delivery machinery in sea anemones.     

Discharge of nematocysts isolated from aeolid nudibranchs

Nematocysts were extracted from 3 nudibranch species and one sea anemone species, and the ability of several test fluids to promote discharge was examined. Except when isolated in sodium citrate, nudibranch nematocysts did not discharge in response to any test fluids. Nudibranch nematocysts isolated in sodium citrate discharged when tested with EGTA, distilled water, and calcium-free artificial seawater, but there were differences among the 3 nudibranch species. Nematocysts isolated from one nudibranch species and nematocysts isolated from that nudibranch's sea anemone prey differed in the percentage that discharged in response to EGTA and distilled water. These results suggest that nematocysts stored by nudibranchs are altered in some way, resulting in the different discharge responses.     

Active Nematocyst Isolation Via Nudibranchs

Cnidarian venoms are potentially valuable tools for biomedical research and drug development. They are contained within nematocysts, the stinging organelles of cnidarians. Several methods exist for the isolation of nematocysts from cnidarian tissues; most are tedious and target nematocysts from specific tissues. We have discovered that the isolated active nematocyst complement (cnidome) of several sea anemone (Cnidaria: Anthozoa) species is readily accessible. These nematocysts are isolated, concentrated, and released to the aqueous environment as a by-product of aeolid nudibranch Spurilla neapolitana cultures. S. neapolitana feed on venomous sea anemones laden with stinging nematocysts. The ingested stinging organelles of several sea anemone species are effectively excreted in the nudibranch feces. We succeeded in purifying the active organelles and inducing their discharge. Thus, our current study presents the attractive possibility of using nudibranchs to produce nematocysts for the investigation of novel marine compounds.     

Identification of a new member of the GLWamide peptide family: physiological activity and cellular localization in cnidarian polyps

KPNAYKGKLPIGLWamide, a novel member of the GLWamide peptide family, was isolated from Hydra magnipapillata. The purification was monitored with a bioassay: contraction of the retractor muscle of a sea anemone, Anthopleura fuscoviridis. The new peptide, termed Hym-370, is longer than the other GLWamides previously isolated from H. magnipapillata and another sea anemone, A. elegantissima. The amino acid sequence of Hym-370 is six residues longer at its N-terminal than a putative sequence previously deduced from the cDNA encoding the precursor protein. The new longer isoform, like the shorter GLWamides, evoked concentration-dependent muscle contractions in both H. magnipapillata and A. fuscoviridis. In contrast, Hym-248, one of the shorter GLWamide peptides, specifically induced contraction of the endodermal muscles in H. magnipapillata. This is the first case in which a member of the hydra GLWamide family (Hym-GLWamides) has exhibited an activity not shared by the others. Polyclonal antibodies were raised to the common C-terminal tripeptide GLWamide and were used in immunohistochemistry to localize the GLWamides in the tissue of two species of hydra, H. magnipapillata and H. oligactis, and one species of sea anemone, A. fuscoviridis. In each case, nerve cells were specifically labeled. These results suggest that the GLWamides are ubiquitous among cnidarians and are involved in regulating the excitability of specific muscles.     

海葵神经毒素基因的克隆和序列分析

根据已发现的海葵神经毒素蛋白的两端保守氨基酸序列 ,设计简并引物 .用RT PCR方法 ,从侧花海葵 (Anthopleurasp .)触手总RNA中分离出多个神经毒素新基因 .它们分别编码 3个长度都是 4 7个氨基酸的毒素蛋白 ,它们的氨基酸序列与海葵神经毒素C(Ap C)最为相似 .新基因的发现为进一步研究其生物活性及应用打下了基础 .     

Biogeography and microhabitat variation in temperate algal-invertebrate symbioses: zooxanthellae and zoochlorellae in two Pacific intertidal sea anemones, Anthopleura elegantissima and A. xanthogrammica

Abstract. Temperate sea anemones in the genus Anthopleura are unique among cnidarians in harboring two phylogenetically distinct symbiotic algae, zooxanthellae (golden-brown dinophytes, Symbiodinium ) and zoochlorellae (green chlorophytes). To determine whether their physiological differences generate patterns in anemone habitat and biogeographic distribution, we sampled symbiotic algae in the small clonal A. elegantissima and the large solitary A. xanthogrammica at 8 field sites (and the other large solitary Anthopleura species at one site) spanning 18° of latitude along 2500 km of the Pacific coast of North America. We found that zoochlorellae predominate in low intertidal habitats and northerly latitudes and in A. xanthogrammica , while zooxanthellae constitute the majority of symbionts in high intertidal habitats and more southerly latitudes and in A. elegantissima. These data are consistent with published predictions based on photosynthetic efficiency of the two algae under varied temperature and light regimes in the laboratory. This anemone-algal system provides a potential biological signal of benthic intertidal communities' responses to El Niño events and long-term climate changes in the Pacific.     

Adaptable defense: a nudibranch mucus inhibits nematocyst discharge and changes with prey type

Nudibranchs that feed on cnidarians must defend themselves from the prey's nematocysts or risk their own injury or death. While a nudibranch's mucus has been thought to protect the animal from nematocyst discharge, an inhibition of discharge by nudibranch mucus has never been shown. The current study investigated whether mucus from the aeolid nudibranch Aeolidia papillosa would inhibit nematocyst discharge from four species of sea anemone prey. Sea anemone tentacles were contacted with mucus-coated gelatin probes, and nematocyst discharge was quantified and compared with control probes of gelatin only. Mucus from A. papillosa inhibited the discharge of nematocysts from sea anemone tentacles. This inhibition was specifically limited to the anemone species on which the nudibranch had been feeding. When the prey species was changed, the mucus changed within 2 weeks to inhibit the nematocyst discharge of the new prey species. The nudibranchs apparently produce the inhibitory mucus rather than simply becoming coated in anemone mucus during feeding. Because of the intimate association between most aeolid nudibranchs and their prey, an adaptable mucus protection could have a significant impact on the behavior, distribution, and life history of the nudibranchs.     

Internal brooding of clonal propagules by a sea anemone, Anthopleura sp.

Abstract. We investigated the seasonal prevalence of reproductive activities and of the development of brooded propagules in an intertidal sea anemone, Anthopleura sp., on the rocky shore of Mutsu Bay, in northern Japan. A monthly examination of anemones, by dissection and histological techniques, revealed no sign of gonad development, but did reveal that they produce and internally brood propagules throughout the year. Release of propagules was observed in the field. This anemone population appears to be entirely asexual and agametic, and may persist solely through clonal propagation.     

Dynamic tuning of hair bundle mechanoreceptors in a sea anemone during predation

The sea anemone Haliplanella luciae (Cnidaria, Anthozoa) detects chemical and mechanical stimuli from prey. Hair bundle mechanoreceptors on the tentacles participate in regulating discharge of microbasic p-mastigophore nematocysts. Properly stimulated hair bundles sensitize the anemone to discharge nematocysts into objects that contact the tentacles. The hair bundle mechanoreceptors are composed of stereocilia derived from a multicellular complex. This complex consists of a single sensory neuron surrounded by two to four supporting cells. The mechanoreceptor is similar in many ways to vertebrate hair cells of the acousticolateralis system. However, anemone hair bundles are adjustable in structure and responsiveness according to the activity of two different chemoreceptors. One chemoreceptor binds N -acetylated sugars and the other binds amino compounds including proline. N -acetylated sugars induce lengthening of the hair bundle and a downward shift in frequencies that elicit maximal discharge of microbasic p-mastigophore nematocysts. Furthermore, N -acetylated sugars shift maximal discharge to smaller amplitude vibrations. Thus, N -acetylated sugars likely tune hair bundles so that small, swimming zooplankton stimulate maximal discharge. Proline leaks into the seawater from the hemolymph of wounded prey. Proline induces shortening of the hair bundle and shifts maximal discharge of nematocysts to higher frequencies and to larger amplitude vibrations. Thus, proline likely tunes hair bundles so that small, wounded, prey stimulate maximal discharge of nematocysts as they struggle to escape. Thus, suitably sized prey stimulate maximal discharge of microbasic p-mastigophore nematocysts upon first contacting the anemone tentacle and again upon attempting to escape.     

Functional expression and characterization of four novel neurotoxins from sea anemone Anthopleura sp

The genes of four novel neurotoxins, named Hk2a, Hk7a, Hk8a, and Hk16a, were obtained from sea anemone Anthopleura sp. All four neurotoxins were composed of 47 amino acid residues and the variable residues among them were found in positions 14, 22, 25, and 37. To study their activities, the four toxins fused to the Escherichia coli thioredoxin were overexpressed by BL21 (DE3), cleaved off from the fusion partner, purified, and characterized with MALDI-TOF and CD assays. Contractile force studies of isolated SD atria indicated that rHk2a had the strongest and rHk7a the longest heart stimulation effect. Consequently, the Arg14, a highly conserved residue in various sea anemone neurotoxins, can be inferred to contribute to the duration but not the intensity of contraction-stimulating activity. Our work renders useful information to studies of sea anemone neurotoxins, especially to the clarification of the function of the disputative Arg14.     

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.     

Amino acid sequence of a sea anemone toxin from Parasicyonis actinostoloides

Amino-acid sequence of a toxin from sea anemone, Parasicyonis actinostoloides, is determined. The toxin consists of 31 amino acid residues and is cross-linked with four disulphide bridges. The sequence has some similarity to that of toxin III and no similarity to those of toxin I and toxin II both from sea anemone, Anemonia sulcata, or to that of Anthopleurin A from Anthopleura xanthogrammica.     

Isolation of less than Glu-Gly-Leu-Arg-Trp-NH2 (Antho-RWamide II), a novel neuropeptide from sea anemones

Using a radioimmunoassay for the peptide sequence Arg-Phe-NH2 (RFamide), a novel peptide has been purified from acetic acid extracts of the sea anemone Anthopleura elegantissima. This peptide has the structure less than Glu-Gly-Leu-Arg-Trp-NH2, and was named Antho-RWamide II. Antho-RWamide II is a neuropeptide. Its structure is closely related to an earlier characterized neuropeptide from Anthopleura less than Glu-Ser-Leu-Arg-Trp-NH2 (Antho-RWamide I).     

Biologically Active Polypeptides and Hydrolytic Enzymes in Sea Anemones of Northern Temperate Waters

The content of biologically active polypeptides in aqueous and ethanol extracts of seven sea anemone species collected near Sakhalin Island (Sea of Okhotsk) and in Posyet Bay (Sea of Japan) was analyzed. Water extracts of the sea anemone Cribrinopsis similis showed the highest hemolytic activity, while ethanol extracts proved to have toxic properties. The levels of toxic and hemolytic activity of extracts of sea anemones inhabiting northern temperate waters were 2 to 3 orders of magnitude lower, compared to tropic species. The reason for this is likely to be the differences in the habitat conditions and biological traits of these animals. The water extracts of all species possessed proteolytic, phospholipase A2, and low DNAase activities, except Actinostola sp., whose aqueous extract contained a high activity alkaline DNAase. The species studied contained a wide range of proteinase inhibitors, O-glycosyl hydrolases (glycosidases and polysaccharide hydrolases). Water extracts of C. similis and Stomphia coccinea possessed the highest laminarinase activity. High activity of N-galactopyranosidase was found in water extracts of S. coccinea and Oulactis orientalis.     

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.     

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.     

The sea anemone Calliactis tricolor and its association with the Hermit crab Dardanus venosus

The association of certain sea anemones and hermit crabs is established in different ways according to the species involved. The present study shows that the behaviour patterns of the two partners in associations between Calliactis tricolor (Lesueur) and Dardanus venosus (H. M. Edwards) in the Caribbean are similar to those seen in the Mediterranean C. parasitica and D. arrosor .
Although about half the crabs display an active behaviour pattern in laboratory trials, the anemone frequently settles on shells unaided and most C. tricolor respond to molluscan shells by clinging with their tentacles until the pedal disc can be attached. As a rule it is necessary for the anemone to relax and to cling to the shell if the crab is to be successful in transferring the anemone to its shell.
The behaviour patterns of D. venosus include a distinctive tapping of the edge of the base of C. tricolor after which the anemone is pulled or lifted off and transferred to the shell. An experimenter can also cause the anemone to relax and to detach itself by tapping the edge of the base with plastic rods after the manner of the crab.
The mechanisms by which the tentacles of Calliactis cling to, and by which the base settles upon, shells still remain to be elucidated. The participation of nematocysts in these processes could not be demonstrated in this study.
C. tricolor is found on some other pagurid and non-pagurid crabs in various localities. These associations need to be investigated fully in order that the behaviour patterns of C. tricolor may be correctly interpreted and compared with those of other species of Calliactis .     

Predation resistance and nematocyst scaling for Metridium senile and M. farcimen

Previous studies suggest that large body size reduces the risk of predation for acontiate sea anemones. For two species of Metridium, we found significant increases in the length of the acontial threads and in the mean lengths of the unfired acontial nematocyst capsules, with increasing body size. This supports the hypothesis that more damaging acontial defenses protect larger acontiate anemones from their predators. Metridium is planktivorous, and food size does not increase substantially with body size; so we expected smaller increases in nematocyst size for the feeding tentacles. In fact, scaling exponents were significantly smaller for the tentacle nematocysts than for acontial nematocysts of the same types in 3 out of 4 cases. This suggests that nematocyst scaling responds predictably to selection pressure. When specimens of the same size were compared, the non-clonal, subtidal species, M. farcimen, had significantly larger acontial nematocysts than did its clonal congener, M. senile, which lives at the upper tidal limits for major subtidal predators in the northeastern Pacific. Therefore, larger acontial nematocysts may be particularly advantageous where predation levels are high. These data demonstrate that closely related anemone species can be distinguished on the basis of ecologically and functionally relevant differences in nematocyst scaling.     

Purification and pharmacological properties of eight sea anemone toxins from Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum

Eight different polypeptide toxins from sea anemones of four different origins (Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum) have been studied. Three of these toxins are new; the purification procedure for the five other ones has been improved. Sea anemone toxins were assayed (i) for their toxicity to crabs and mice, (ii) for their affinity for the specific sea anemone toxin receptor situated on the Na+ channels of rat brain synaptosomes, and (iii) for their capacity to increase, in synergy with veratridine, the rate of 22Na+ entry into neuroblastoma cells via the Na+ channel. Some of the toxins are more active on crustaceans, whereas others are more toxic to mammals. A very good correlation exists between the toxic activity to mice, the affinity of the toxin for the Na+ channel in rat brain synaptosomes, and the stimulating effect on 22 Na+ uptake by neuroblastoma cells. The observation has also been made that the most cationic toxins are also the most active on mammals and the least active on crustaceans. Toxicities (LD50) to mice of the most active sea anemone toxins and of the most active scorpion toxins are similar, and sea anemone toxins at high enough concentrations prevent binding of scorpion toxins to their receptor. However, scorpion toxins have affinities for the Na+ channel which are approximately 60 times higher than those found for the most active sea anemone toxins. Three sea anemone toxins appear to be more interesting than toxin II from A. sulcata (the "classical" sea anemone toxin) for studies of the Na+ channel structure and mechanism when the source of the channel is of a mammalian origin. Two of these three toxins can be radiolabeled with iodine while retaining their toxic activity; they appear to be useful tools for future biochemical studies of the Na+ channel.     

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.