Sea Anemone Bunodosoma Cangicum Research Articles

Cell volume maintenance capacity of the sea anemone Bunodosoma cangicum: the effect of copper.

Cell volume regulation is an essential strategy for the maintenance of life under unfavorable osmotic conditions. Mechanisms aimed at minimizing the physiological challenges caused by environmental changes are crucial in anisosmotic environments. However, aquatic ecosystems experience multiple stressors, including variations in salinity and heavy metal pollution. The accumulation of heavy metals in aquatic ecosystems has a significant effect on the biota, leading to impaired function. The aim of this study was to investigate the capacity of volume regulation in isolated cells of the sea anemone Bunodosoma cangicum exposed to nominal copper (Cu) concentrations of 5 and 50µg L-1, associated or not with hypoosmotic (15‰) or hyperosmotic (45‰) shock for 15min. In the absence of the metal, our results showed volume maintenance in all osmotic conditions. Our results showed that cell volume was maintained under all osmotic conditions in the absence of Cu. Similarly, no significant differences were observed in cell volumes under isosmotic and hyperosmotic conditions in the presence of both Cu concentrations. A similar homeostatic response was observed under the hypoosmotic condition with 5µg L-1 Cu. Our results showed an increase in cell volume with exposure of the cells to the hypoosmotic condition and 50µg L-1 Cu. The response could be associated with the increased bioavailability of Cu, reduced ability to resist multixenobiotics and their efflux pathways, and the impairment of water efflux in specialized transmembrane proteins. Therefore, B. cangicum pedal disk cells can tolerate osmotic variations in aquatic ecosystems. However, the capacity to regulate cell volume under hypoosmotic conditions can be affected by the presence of a metal contaminant (50µg L-1 Cu), which could be due to the inhibition of water channels.

The effects of vesicle toxin from the sea anemone Bunodosoma cangicum on the behavior of a freshwater shrimp, Palaemon argentinus, and shore crab, Neohelice granulata

Sea anemones of the genus Bunodosoma possess along their body column, longitudinally arranged brown-colored vesicles. We have shown that in B. cangicum, these warty structures contain a mixture of potent toxins. This work highlights the neuro-inhibitory effects exhibited by two decapod crustacean species exposed to the extracts from these vesicles. For this, we use the unrefined toxin in doses, exposure times, and different exposure pathways. The findings show that at least one neuro-inhibitory compound is present and remains active regardless of the exposure method or dose tested. This toxin affects neuro-motor pathways but not neuro-sensory pathways. Shrimp exposed to toxin could continue to perceive and track food pellets but could not secure and consume their ration. Of six anatomical reflexes tested under the toxin's influence, voluntary movements of the mouthparts were impacted most commonly. Interestingly, all subject animals recovered from the toxin exposure within 2 h. Finally, we propose Reflexive Action Analysis (RAMP) as a tool to evaluate the potency of other neurotoxic or neuro-inhibitory compounds in crustacea. This work is the first to show the neuro-inhibitory activity of extracts from these sea anemone columnar vesicle structures and the first to evaluate these effects using RAMP reflex analysis.

The sea anemone Bunodosoma cangicum as a potential biomonitor for microplastics contamination on the Brazilian Amazon coast

This study reports for the first time the ingestion of meso- (5.01–25 mm) and microplastics (1 μm–5 mm) by the sea anemone Bunodosoma cangicum, the most abundant actiniarian species on the Amazon coast. At three sites on the coast of Pará, Brazil, anemones were collected from beachrocks in the intertidal zone (30 at each site), measured (pedal disc diameter, mm) and weighed (wet weight, g). The contents of the gastrovascular cavity were extracted and analyzed under a stereoscope. The recovered plastic particles were characterized by Fourier Transform Infrared (FTIR) spectroscopy. Overall, 139 microplastic and 2 mesoplastic items were identified in 68 individuals (75.6%) among the 90 examined, with a mean of 1.6 (±1.5) items per individual. Plastic fibers comprised about 84% of the ingested plastics, followed by fragments (∼12%) and films (∼4%). Particle diameters ranged from 0.10 to 9.17 mm (1.57 ± 1.23 mm). A weak positive correlation was found between the weight of anemones and the number of plastic particles in the gastrovascular cavity (p = 0.03) and between the number of prey items and the number of plastic particles in the gastrovascular cavity (p < 0.01). The main polymers identified by FTIR analysis were polyethylene terephthalate (PET), polypropylene (PP), polyamide (PA), polyurethane (PU), polyethylene (PE), acrylonitrile butadiene styrene (ABS), polystyrene (PS) and rayon. Sea anemones ingested significantly more plastic debris at the most urbanized and populous sampling sites. This study provides the first evidence of microplastics contamination of marine invertebrates from the Amazon coast. Abundant species such as B. cangicum have the potential to monitor the levels of plastic contamination in the region. Our results support this potential, as the species showed a high frequency of plastic ingestion and allowed detection of plastic contamination even in the best-preserved area where anemones were collected.

Polypeptides secreted from the columnar vesicles of the sea anemone Bunodosoma cangicum and their in vivo effects on Caenorhabditis elegans.

In this study we provide new evidence that the columnar vesicles of the sea anemone Bunodosoma cangicum are toxic in vivo and contain at least two active polypeptides, a neurotoxic and an apoptosis inducing polypeptide. Here we show that it is also an effective inducer of apoptosis in vivo in the nematode Caenorhabditis elegans. In addition, the anemone peptides rapidly paralyze C. elegans, and set in motion a sequence of events that result in the complete dissolution of the internal organs in adult animals within 60 min. Nematodes that survive the toxin treatment exhibit a decreased reproductive capacity. Interestingly, adult animals appear to be much more susceptible to the effects of the toxins than larval stages, suggesting possible developmentally dependent targets of the toxins. Here we also provide chemical characterization of the compounds through chromatographic analysis and mass spectrometry. Gel filtration chromatography coupled with reverse phase HPLC shows that our partially purified extract contains at least two principle components. Additionally, MALDI-TOF mass spectrometry analysis of our extract shows three principal compounds at 814.6, 2914.1, and 4360.3 m/z plus three other minor components or fragments. Mass spectrometry analysis also indicates the presence of three disulfide bridges. Which is in agreement with other characterizations of anemone venoms.

Peripheral 5-HT3 Receptors Are Involved in the Antinociceptive Effect of Bunodosine 391.

Bunodosine 391 (BDS 391), a low molecular weight compound isolated from the sea anemone Bunodosoma cangicum, increases the nociceptive threshold and inhibits inflammatory hyperalgesia. Serotonin receptors are involved in those effects. In this study, we have expanded the characterization of the antinociceptive effect of BDS 391 demonstrating that, in rats: (a) the compound inhibits (1.2–12 ng/paw) overt pain, in the formalin test, and mechanical hyperalgesia (0.6–6.0 ng/paw) detected in a model of neuropathic pain; (b) intraplantar administration of ondansetron, a selective 5-HT3 receptor antagonist, blocks the effect of BDS 391, whereas ketanserin, a 5-HT2 receptor antagonist, partially reversed this effect, indicating the involvement of peripheral 5-HT2 and 5-HT3 receptors in BDS 391 antinociception; and (c) in binding assay studies, BDS 391 was not able to displace the selective 5-HT receptor antagonists, suggesting that this compound does not directly bind to these receptors. The effect of biguanide, a selective 5-HT3 receptor agonist, was also evaluated. The agonist inhibited the formalin’s nociceptive response, supporting an antinociceptive role for 5-HT3 receptors. Our study is the first one to show that a non-peptidic low molecular weight compound obtained from a sea anemone is able to induce antinociception and that activation of peripheral 5-HT3 receptors contributes to this effect.

Characterization of MXR activity in the sea anemone Bunodosoma cangicum exposed to copper

Transmembrane proteins of the ABC family contribute to a multiple xenobiotic resistance (MXR) phenotype in cells, driving the extrusion of toxic substances. This phenotype promotes a high degree of protection against xenobiotics. The present study provides a better understanding of the MXR activity in the podal disk cells of Bunodosoma cangicum exposed to copper, and further establishes the relationship between protein activity (measured by accumulation of rhodamine-B) and bioaccumulation of copper in these cells. Sea anemone cells were exposed for 24h to copper (0, 7.8 and 15.6μg/L) in presence and absence of MXR blocker (verapamil 50μM). Results indicate that copper exposure increases intracellular metal content when ABC proteins were blocked, causing an increase in cellular death. The present study also verified the relationship between MXR activity, ATP depletion, and general metabolic activity (by MTT). MXR activity decreased in treatment groups exposed to copper concentrations of 15.6μg/L and 10mM energy depleting potassium cyanide. Metabolic activity increased in cells exposed to 7.8μgCu/L, but 15.6μgCu/L was similar to 0 and 7.8μg/L. The presence of copper decreased the ABC proteins expression. The present study improves the knowledge of MXR in anemone cells and shows that this activity is closely associated with copper extrusion. Also, the copper exposure is able to modify the metabolic state and to lead to cytotoxicity when cells cannot defend themselves.

Who knows not where an anemone does wear his sting? Could polypeptides released from the columnar vesicles of Bunodosoma cangicum induce apoptosis in the ZF-L cell line?

We provide ultrastructural and cytological evidence that the tentacles of the sea anemone Bunodosoma cangicum does not contain cytotoxic venom. However, we show that the stimulated secretion of an apparent mixture of biomolecules containing polypeptides from the columnar vesicles of Bunodosoma cangicum is apparently a potent inducer of apoptosis in the zebrafish cell line, ZF-L. Microscopic fluorescence, cell morphology and flow cytometric assays confirm the apoptotic activity. Crude vesicle venom was partially purified by size exclusion chromatography. PAGE analysis shows that this venom contains low weight polypeptides but no measurable protein. The apoptotic activity is heat labile, and the observed peptides concurrent with this activity have a molecular weight of approximately 2000 Da. This manuscript is the first report of biologically active molecules and peptides associated with columnar vesicles of anemones, and the first to confirm that the tentacles of B. cangicum do not contain cytotoxic venom, and express spirocytes exclusively.

Influence of copper pre-exposure on biochemical responses of the sea anemone Bunodosoma cangicum to changes in oxygen availability

The influence of copper on the ability of the intertidal sea anemone Bunodosoma cangicum to cope with reactive oxygen species generation associated with changes in oxygen availability was evaluated. Sea anemones were kept under control condition or pre-exposed (96h) to dissolved copper (6.1μg±2.7μg/L) and then subjected to a 6-h period of hypoxia (0.5mg O2/L) followed by a 6-h period of re-oxygenation (7.5mg O2/L). Antioxidant capacity against peroxyl radicals (ACAP), superoxide dismutase (SOD) activity, reduced glutathione (GSH) concentration, lipid peroxidation (LPO) level, and ATP concentration were evaluated. Control sea anemones showed variations in SOD and LPO while copper pre-exposed sea anemones displayed changes in ACAP, GSH, LPO and ATP. However, no clear pattern of change over time was observed. ACAP was lower in copper pre-exposed sea anemones than in the control ones during hypoxia and recovery. SOD activity was increased during hypoxia and reduced shortly after recovery in control sea anemones. GSH concentration was higher in copper pre-exposed sea anemones than in the control ones in all experimental conditions. The LPO level increased shortly after recovery in both groups of sea anemones, being higher in control sea anemones than in copper pre-exposed ones. ATP concentration showed transient changes in copper pre-exposed sea anemones, being lower in these sea anemones than in control ones during recovery. These findings suggest that B. cangicum possess mechanisms to prevent oxidative stress generated by changes in oxygen availability associated with the tidal cycle, which can be disturbed by pre-exposure to copper.

A Gating Modulator Peptide Toxin for Shaker-Type KV1 Channels Derived from the Sea Anemone Bunodosoma cangicum

Natural peptide toxins are a rich source of channel modulators that act on the extracellular face of Kv channels, either at the external pore mouth or at the voltage-sensing domain (S3-S4 linker). As such, natural toxins provide us with highly selective and potent molecular probes to unravel the structure and function of Kv channels. Toxins binding to the external pore mouth partly or completely block the K+ permeation while those binding to the VSD inhibit channel function by modifying channel gating. The Shaker-related Kv1 channels are targeted by several peptide toxins that block the outer pore mouth but no gating modifiers that shift the voltage-dependence of channel opening have yet been identified. Furthermore, the Kv1.5 channel - an important target for the treatment of atrial fibrillation - has no known external peptide pore blockers, presumably due to the presence of a positively charged arginine residue in the outer pore mouth (R379, equivalent to Shaker T449). Bcg31.16 is a recently discovered peptide neurotoxin derived from the sea anemone Bunodosoma cangicum that inhibited several Kv1 subunits with nM potency. Bcg31.16 caused a concentration-dependent depolarizing shift in the voltage-dependence of channel opening; with 300 nM the shifts amounted to +35 mV and +12 mV for Kv1.3 and Kv1.5, respectively. The voltage-dependence of C-type inactivation displayed similar shifts, as well as the voltage-dependence of the gating kinetics. No significant effect on Kv2.1 was obtained at 1 microMolar. Thus, Bcg31.16 is a new gating modifier toxin of the Kv1 family and a novel peptide toxin to inhibit the Kv1.5 channel. (Support: FWO-G043312N to DJS and JT).

Bunodosines: novel analgesic acylamino acids from the venom of the sea anemone Bunodosoma cangicum

Sea anemones are known as a rich source of protein and peptide toxins. In contrast, however, only little is known about the non-peptidic, small molecules in the sea anemones. We have been searching for small molecule toxins in the venom of the Brazilian sea anemone, Bunodosoma cangicum, which has led to the isolation of new acylamino acids, bunodosine 391 (BDS 391, 1) and bunodosine 405 (BDS 405, 2), showing a potent analgesic activity mediated by serotonin receptors.

Bunodosine 391: An Analgesic Acylamino Acid from the Venom of the Sea Anemone Bunodosoma cangicum

A new acylamino acid, bunodosine 391 (BDS 391), was isolated from the venom of the sea anemone Bunodosoma cangicum. The structure was elucidated by spectroscopic analyses (2D NMR, ESIMS/MS) and verified by its synthesis. Intraplantar injection of BDS 391 into the hind paw of a rat induced a potent analgesic effect. This effect was not altered by naloxone (an opioid receptor antagonist), but was completely reversed by methysergide (a serotonin receptor antagonist), indicating that the effect is mediated by activation of serotonin receptors.

Proteomics of the neurotoxic fraction from the sea anemone Bunodosoma cangicum venom: Novel peptides belonging to new classes of toxins

In contrast to the many studies on the venoms of scorpions, spiders, snakes and cone snails, up to now there has been no report of the proteomic analysis of sea anemones venoms. In this work we report for the first time the peptide mass fingerprint and some novel peptides in the neurotoxic fraction (Fr III) of the sea anemone Bunodosoma cangicum venom. Fr III is neurotoxic to crabs and was purified by rp-HPLC in a C-18 column, yielding 41 fractions. By checking their molecular masses by ESI-Q-Tof and MALDI-Tof MS we found 81 components ranging from near 250 amu to approximately 6000 amu. Some of the peptidic molecules were partially sequenced through the automated Edman technique. Three of them are peptides with near 4500 amu belonging to the class of the BcIV, BDS-I, BDS-II, APETx1, APETx2 and Am-II toxins. Another three peptides represent a novel group of toxins (~ 3200 amu). A further three molecules (~∼ 4900 amu) belong to the group of type 1 sodium channel neurotoxins. When assayed over the crab leg nerve compound action potentials, one of the BcIV- and APETx-like peptides exhibits an action similar to the type 1 sodium channel toxins in this preparation, suggesting the same target in this assay. On the other hand one of the novel peptides, with 3176 amu, displayed an action similar to potassium channel blockage in this experiment. In summary, the proteomic analysis and mass fingerprint of fractions from sea anemone venoms through MS are valuable tools, allowing us to rapidly predict the occurrence of different groups of toxins and facilitating the search and characterization of novel molecules without the need of full characterization of individual components by broader assays and bioassay-guided purifications. It also shows that sea anemones employ dozens of components for prey capture and defense.

Primary structure, behavioral and electroencephalographic effects of an epileptogenic peptide from the sea anemone Bunodosoma cangicum

The primary structure of cangitoxin (CGX), a 4958 Da peptide from the sea anemone Bunodosoma cangicum, was determined: GVACRCDSDGPTVRGNSLSGTLWLTGGCPSGWHNCRGSGPFIGYCCKK. CGX contains all the 11 residues that are conserved and the 5 that are conservatively substituted within or between the type 1 and type 2 sequences of sea anemone peptides with specific action on voltage-sensitive sodium channels. Furthermore, it also has 6 identities (Asp9, Arg14, Asn16, Leu18, Trp33 and Lys48) and 1 homology (Arg36) in the 8 residues of the pharmacophore of the sea anemone ApB which are essential for interaction with mammalian sodium channels. The intrahippocampal injection of CGX induces several sequential behavioral alterations—episodes of akinesia alternating with facial automatisms and head tremor, salivation, rearing, jumping, barrel-rolling, wet dog shakes and forelimb clonic movements–and the electroencephalography analysis shows that they were followed by important seizure periods that gradually evolved to status epilepticus that lasted 8–12 h, similar to that observed in the acute phase of the pilocarpine model of epilepsy. These results suggest that CGX may be an important tool to develop a new experimental model of status epilepticus which may contribute to understanding the etiology of epilepsy and to test the effects of new antiepileptic drugs.

Identification of hemolytic and neuroactive fractions in the venom of the sea anemone Bunodosoma cangicum.

Sea anemones are a rich source of biologically active substances. In crayfish muscle fibers, Bunodosoma cangicum whole venom selectively blocks the I K(Ca) currents. In the present study, we report for the first time powerful hemolytic and neuroactive effects present in two different fractions obtained by gel-filtration chromatography from whole venom of B. cangicum. A cytolytic fraction (Bcg-2) with components of molecular mass ranging from 8 to 18 kDa elicited hemolysis of mouse erythrocytes with an EC50 = 14 microg/ml and a maximum dose of 22 microg/ml. The effects of the neuroactive fraction, Bcg-3 (2 to 5 kDa), were studied on isolated crab nerves. This fraction prolonged the compound action potentials by increasing their duration and rise time in a dose-dependent manner. This effect was evident after the washout of the preparation, suggesting the existence of a reversible substance that was initially masking the effects of an irreversible one. In order to elucidate the target of Bcg-3 action, the fraction was applied to a tetraethylammonium-pretreated preparation. An additional increase in action potential duration was observed, suggesting a blockade of a different population of K+ channels or of tetraethylammonium-insensitive channels. Also, tetrodotoxin could not block the action potentials in a Bcg-3-pretreated preparation, suggesting a possible interaction of Bcg-3 with Na+ channels. The present data suggest that B. cangicum venom contains at least two bioactive fractions whose activity on cell membranes seems to differ from the I K(Ca) blockade described previously.

Selective block of Ca(2+)-dependent K+ current in crayfish neuromuscular system and chromaffin cells by sea anemone Bunodosoma cangicum venom.

The effects of the nematocyst venom of the sea anemone Bunodosoma cangicum on depolarization-activated currents were studies in opener crayfish muscle fibers and in cultured bovine chromaffin cells. The venom selectively and reversibly blocked the Ca(2+)-dependent K+ current (IK(Ca)) present in crayfish muscle in a dose-dependent manner without affecting voltage-gated Ca2+ or K+ currents. Furthermore, the venom also reduced IK(Ca) in chromaffin cells, without modifying voltage-gated Na+, Ca2+, or K+ currents. Synaptic transmission in crayfish muscle was also affected by the venom. Repetitive excitatory and inhibitory postsynaptic currents (each associated with a presynaptic action potential) were evoked by each nerve stimulus, suggesting that presynaptic IK(Ca) may control the electrical activity of excitatory and inhibitory presynaptic fibers. We conclude that B. cangicum venom includes a toxin that selectively and reversibly blocks Ca(2+)-dependent K+ currents in crayfish muscle and in bovine chromaffin cells, and modifies excitatory and inhibitory synaptic transmission, probably abolishing a similar conductance at the presynaptic fibers.