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.     

Comparative ultrastructure of catch tentacles and feeding tentacles in the sea anemone Haliplanella

TEM observations of catch tentacles revealed that the tentacle tip epidermis is filled with two size classes of mature holotrich nematocysts and a gland cell filled with electron-dense vesicles. Vesicle production is restricted to upper-middle and tentacle tip regions, whereas holotrich development occurs in the lower-middle and tentacle base regions. Thus, catch tentacles have a maturity gradient along their length, with mature tissues concentrated at the tentacle tip. Occasional feeding tentacle cnidae (microbasic p-mastigophores and basitrichs) and mucus gland cells occur in proximal portions of catch tentacles, but are phagocytized by amoeboid granulocytes and transported to the gastrodermis for further degradation. No feeding tentacle cnidae or mucus cells occur distally in catch tentacles. Unlike catch tentacles, feeding tentacles are homogeneous in structure along their length with enidocytes containing mature spirocysts, microbasic p-mastigophore or basitrich nematocysts distributed along the epithelial surface. Cnidoblasts are recessed beneath cnidocytes, occurring along the nerve plexus. Mucus gland cells and gland cells filled with electron-dense vesicles are present in feeding tentacles, distributed at the epithelial surface. Granular phagocytes are rare in the feeding tentacle tip, but common in the tentacle base.     

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.     

The draft genome of Actinia tenebrosa reveals insights into toxin evolution

Sea anemones have a wide array of toxic compounds (peptide toxins found in their venom) which have potential uses as therapeutics. To date, the majority of studies characterizing toxins in sea anemones have been restricted to species from the superfamily, Actinioidea. No highly complete draft genomes are currently available for this superfamily, however, highlighting our limited understanding of the genes encoding toxins in this important group. Here we have sequenced, assembled, and annotated a draft genome for Actinia tenebrosa. The genome is estimated to be approximately 255 megabases, with 31,556 protein‐coding genes. Quality metrics revealed that this draft genome matches the quality and completeness of other model cnidarian genomes, including Nematostella, Hydra, and Acropora. Phylogenomic analyses revealed strong conservation of the Cnidaria and Hexacorallia core‐gene set. However, we found that lineage‐specific gene families have undergone significant expansion events compared with shared gene families. Enrichment analysis performed for both gene ontologies, and protein domains revealed that genes encoding toxins contribute to a significant proportion of the lineage‐specific genes and gene families. The results make clear that the draft genome of A. tenebrosa will provide insight into the evolution of toxins and lineage‐specific genes, and provide an important resource for the discovery of novel biological compounds.     

Hemolytic activities of stinging insect venoms

The direct hemolytic activities of the venoms from 21 species of stinging insects were determined. The activities spanned 3 1/2 orders of magnitude, ranging from a low of four to a high of 12,000 hemolytic units/mg dry venom, respectively, for the solitary wasp, Dasymutilla lepeletierii, and the social wasp, Polistes infuscatus. The latter activity is the highest reported for any insect venom and represents a level that is potentially harmful to humans stung by the wasp. The social wasps as a group generally possessed highly hemolytic venoms; the ants, poorly hemolytic venoms; and the solitary stinging species, venoms with extremely low activity. For the venoms, hemolytic activity correlated with neither lethal toxicity (LD₅₀) nor algogenicity. This finding suggests that hemolysins alone do not determine venom toxicity, and that the hemolysins of stinging insect venoms serve a variety of poorly understood roles. The range of activity of hemolysins from different venoms indicates they probably have different chemical structures and functions.     

Ingestion,Digestion and Assimilation Efficiency of the Sea-Anemone Aiptasia pallida Fed Zooplankton Prey

Ingestion time, digestion time, and assimilation efficiency by the sea anemone Aiptasia pallida were studied in the laboratory by feeding individual anemones preweighed pellets of freeze-dried Artemia salina nauplii. There was no significant correlation between anemone size, measured as dry weight, and either ingestion time, digestion time or assimilation efficiency. Similarly, there was no significant correlation between meal size (i.e., dry weight of ingested brine shrimp pellet) and either ingestion time, digestion time or assimilation efficiency. These results suggest that, under these conditions, assimilation efficiency is unaffected by either “meal” size or anemone size.     

Cloning and Characterization of the First Cnidarian ADP-Ribosylation Factor,and Its Involvement in the <Emphasis Type="Italic">Aiptasia-Symbiodinum</Emphasis> Endosymbiosis

Marine cnidarian-microalgal endosymbiosis is a form of intracellular association that contributes greatly to the high primary productivity of reefs; however, little is known about its molecular mechanisms. Since the ADP-ribosylation factor (ARF) family proteins are key regulators of host intracellular vesicle transport systems, which are critical to many endosymbiotic interactions, we set out to clone and characterize ARF proteins in the symbiotic sea anemone Aiptasia pulchella. Experiments indicated that at least 3 ARF protein classes (class I, class II and class III) were present and expressed as a single messenger RNA species in Aiptasia, with highest mRNA expression levels for apARF1, medium for apARF5, and lowest for apARF6. Quantitative analysis revealed a great reduction at both the RNA and the protein levels in apARF1, but not apARF5 and apARF6, in the symbiotic animals. The apARF1 protein was highly homologous in sequence to other known ARF1 proteins and displayed a Golgi-like localization pattern. Overall, our study identified apARF1 as a potential negative regulator of Aiptaisia-microalgal endosymbiosis.     

Behavioural plasticity in the stinging act of female ectoparasitoids

Abstract. The plasticity of the stinging behaviour of female Eupelmidae ( Eupelmus vuilleti Crawford, E. orientalis Crawford Hymenoptera: Eupelmidae) was analysed through the interrelationships of a parasitic community living on larval stage of Callosobruchus maculatus Fabricius (Coleoptera: Bruchidae). In traditional African grainstores, Eupelmus females have at their disposal all the larval stages of their host. Their exploitation is revealed by the sting observed on the host caused by the ovipositor of parasitoid females. This stinging behaviour begins immediately on the adult female's emergence, and thereafter there is a daily increase in the number of hosts stung. Stinging activity is linked to the age of the available hosts, but not all of the hosts available are exploited by the two species studied. This absence of total exploitation could indicate a threshold of activity in the stinging acts which could be in part linked to the secretion activity of the poison glands of parasitoid female.
In females of E. vuilleti and E.orientalis the stinging act is usually associated with egg-laying. The relationship between stinging and egg-laying is stronger in more advanced larval stages of the host.
In the presence of parasitized hosts, females of E. orientalis and E.vuilleti can exhibit hyperparasitism. The majority of larvae (i.e. primary parasitoid) that have been hyperparasitized have been stung and a large number of them are completely inactive. However, E. vuilleti females deposited their eggs in the available space of the pupation chamber of the host. The neonatal larvae from these scattered eggs could exploit any available host without the female having 'prepared the ground' at the time of egg-laying.
In Eupelmidae, all this happens as if there were a complementary action between the females and the neonate which could, if necessary, reinforce and/or compensate for the deficiencies in the host exploitation by the female.     

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.     

A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons

From a systematic screening of animal venoms, we isolated a new toxin (APETx2) from the sea anemone Anthopleura elegantissima, which inhibits ASIC3 homomeric channels and ASIC3-containing heteromeric channels both in heterologous expression systems and in primary cultures of rat sensory neurons. APETx2 is a 42 amino-acid peptide crosslinked by three disulfide bridges, with a structural organization similar to that of other sea anemone toxins that inhibit voltage-sensitive Na+ and K+ channels. APETx2 reversibly inhibits rat ASIC3 (IC50=63 nM), without any effect on ASIC1a, ASIC1b, and ASIC2a. APETx2 directly inhibits the ASIC3 channel by acting at its external side, and it does not modify the channel unitary conductance. APETx2 also inhibits heteromeric ASIC2b+3 current (IC50=117 nM), while it has less affinity for ASIC1b+3 (IC50=0.9 microM), ASIC1a+3 (IC50=2 microM), and no effect on the ASIC2a+3 current. The ASIC3-like current in primary cultured sensory neurons is partly and reversibly inhibited by APETx2 with an IC50 of 216 nM, probably due to the mixed inhibitions of various co-expressed ASIC3-containing channels.     

Induced response to herbivory in stinging hair traits of Japanese nettle (Urtica thunbergiana) seedlings in two subpopulations with different browsing pressures by sika deer

Evolutionary theories predict that natural selection favors inducible defense when the risk of predation is unpredictable. In this context, the magnitude of the induced defense in populations experiencing intermittent herbivory is predicted to be larger than that in populations experiencing constant herbivory when there is genetic differentiation between populations. To test this prediction, we conducted a clipping experiment to investigate induced response to shoot damage by the stinging hair traits of Japanese nettle (Urtica thunbergiana) seedlings. For this purpose, we studied two nettle subpopulations, one under constant browsing and another under intermittent browsing by sika deer in Nara Park, central Japan. The clipping experiment demonstrates that both subpopulations exhibited induced defenses in response to the clipping of the shoot apex as the number and length of stinging hairs increased after clipping. The subpopulation experiencing intermittent browsing exhibited smaller trait values and larger induced defenses, indicated by the number of stinging hairs on the upper leaf surface and the length of stinging hairs on both leaf surfaces compared with the subpopulation experiencing constant browsing. These results are consistent with the prediction and suggest that genetic differentiation of the induced defense between subpopulations is caused by adaptation to the herbivory regime. We discuss other plausible factors affecting the magnitude of the induced defense of the nettle subpopulations.     

Concerted evolution of sea anemone neurotoxin genes is revealed through analysis of the Nematostella vectensis genome

Gene families, which encode toxins, are found in many poisonous animals, yet there is limited understanding of their evolution at the nucleotide level. The release of the genome draft sequence for the sea anemone Nematostella vectensis enabled a comprehensive study of a gene family whose neurotoxin products affect voltage-gated sodium channels. All gene family members are clustered in a highly repetitive approximately 30-kb genomic region and encode a single toxin, Nv1. These genes exhibit extreme conservation at the nucleotide level which cannot be explained by purifying selection. This conservation greatly differs from the toxin gene families of other animals (e.g., snakes, scorpions, and cone snails), whose evolution was driven by diversifying selection, thereby generating a high degree of genetic diversity. The low nucleotide diversity at the Nv1 genes is reminiscent of that reported for DNA encoding ribosomal RNA (rDNA) and 2 hsp70 genes from Drosophila, which have evolved via concerted evolution. This evolutionary pattern was experimentally demonstrated in yeast rDNA and was shown to involve unequal crossing-over. Through sequence analysis of toxin genes from multiple N. vectensis populations and 2 other anemone species, Anemonia viridis and Actinia equina, we observed that the toxin genes for each sea anemone species are more similar to one another than to those of other species, suggesting they evolved by manner of concerted evolution. Furthermore, in 2 of the species (A. viridis and A. equina) we found genes that evolved under diversifying selection, suggesting that concerted evolution and accelerated evolution may occur simultaneously.     

The mitochondrial genome of a sea anemone Bolocera sp. exhibits novel genetic structures potentially involved in adaptation to the deep‐sea environment

The deep sea is one of the most extensive ecosystems on earth. Organisms living there survive in an extremely harsh environment, and their mitochondrial energy metabolism might be a result of evolution. As one of the most important organelles, mitochondria generate energy through energy metabolism and play an important role in almost all biological activities. In this study, the mitogenome of a deep‐sea sea anemone (Bolocera sp.) was sequenced and characterized. Like other metazoans, it contained 13 energy pathway protein‐coding genes and two ribosomal RNAs. However, it also exhibited some unique features: just two transfer RNA genes, two group I introns, two transposon‐like noncanonical open reading frames (ORFs), and a control region‐like (CR‐like) element. All of the mitochondrial genes were coded by the same strand (the H‐strand). The genetic order and orientation were identical to those of most sequenced actiniarians. Phylogenetic analyses showed that this species was closely related to Bolocera tuediae. Positive selection analysis showed that three residues (31 L and 42 N in ATP6, 570 S in ND5) of Bolocera sp. were positively selected sites. By comparing these features with those of shallow sea anemone species, we deduced that these novel gene features may influence the activity of mitochondrial genes. This study may provide some clues regarding the adaptation of Bolocera sp. to the deep‐sea environment.     

Losing 'Nemo': bleaching and collection appear to reduce inshore populations of anemonefishes

Surveys of anemonefishes (Amphiprioninae) were conducted on reefs in two regions of the Great Barrier Reef Marine Park with contrasting histories of disturbance to determine the degree to which spatial variation might be explained by bleaching or management status. Densities of anemonefishes were lower on reefs in the bleaching-impacted Keppel Islands than on reefs in Far North Queensland. No anemonefishes or anemones were found on or near bleached corals in the Keppel Islands. Furthermore, the highest densities of fishes were found on reefs closed to fishing and aquarium collecting in both the Keppel Islands and Far North Queensland, which suggests that collecting is compounding the effects of bleaching. These results emphasize the importance of understanding the interaction between bleaching events and anthropogenic disturbance upon commercially exploited species.     

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.     

Identification of oxalic acid and tartaric acid as major persistent pain-inducing toxins in the stinging hairs of the nettle, Urtica thunbergiana

BACKGROUND AND AIMS: Once human skin contacts stinging hairs of Urtica spp. (stinging nettles), the irritant is released and produces pain, wheals or a stinging sensation which may last for >12 h. However, the existence of pain-inducing toxins in the stinging hairs of Urtica thunbergiana has never been systematically demonstrated. Experiments were therefore conducted to identify the persistent pain-inducing agents in the stinging hairs of U. thunbergiana. METHODS: The stinging hairs of U. thunbergiana were removed and immersed in deionized water. After centrifugation, the clear supernatants were then subjected to high-performance liquid chromatography (HPLC), enzymatic analysis and/or behavioural bioassays. KEY RESULTS: The HPLC results showed that the major constituents in the stinging hairs of U. thunbergiana were histamine, oxalic acid and tartaric acid. However, the well-recognized pain-inducing agents, serotonin and formic acid, existed at a low concentration as estimated by HPLC and/or enzymatic analyses. The behavioural tests showed that 2% oxalic acid and 10% tartaric acid dramatically elicited persistent pain sensations in rats. In contrast, 10% formic acid and 2% serotonin only elicited moderate pain sensation in the first 10 min. Moreover, no significant pain-related behavioural response was observed after injecting 10% acetylcholine and histamine in rats. CONCLUSIONS: Oxalic acid and tartaric acid were identified, for the first time, as major long-lasting pain-inducing toxins in the stinging hairs of U. thunbergiana. The general view that formic acid, histamine and serotonin are the pain-inducing agents in the stinging hairs of U. dioica may require updating, since their concentrations in U. thunbergiana were too low to induce significant pain sensation in behavioural bioassays.     

A New Polypeptide Toxin from the Nematocyst Venom of an Okinawan Sea Anemone Phyllodiscus semoni (Japanese name “unbachi-isoginchaku”)

The venomous sea anemone Phyllodiscus semoni causes cases of severe stinging. We isolated Phyllodiscus semoni toxin 20A (PsTX-20A), a hemolytic and lethal polypeptide (20 kDa), from the nematocyst venom of this species for the first time. Furthermore, we sequenced the cDNA encoding PsTX-20A. The deduced amino acid sequence of PsTX-20A showed that this toxin was a new member of the actinoporin family, which consists of several cytolytic polypeptides originating from sea anemones. PsTX-20A showed lethal toxicity to the shrimp Palaemon paucidens when administered via intraperitoneal injection (LD₅₀, 50 μg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED₅₀, 80 ng/ml).