G. La Spada

Effect of various factors on Pelagia noctiluca (Cnidaria, Scyphozoa) crude venom-induced haemolysis

The haemolytic power of isolated nematocysts from the scyphozoan Pelagia noctiluca was studied with attention to the effect of osmotic protectants as carbohydrates at different MW, cations as Mg2+, Ca2+, Ba2+,Cu2+, K+; proteases as collagenase, trypsin, alpha-chymotrypsin, papain; and antioxidants. Crude venom was at first obtained by sonication of holotrichous-isorhiza nematocysts previously isolated from oral arms of P. noctiluca and then haemolytically tested upon human erythrocytes. Osmotic protectants were effective in inhibiting the haemolytic power depending on their molecular weight so that total inhibition of crude venom-induced haemolysis was observed after PEG treatment (polyethyleneglycol 6000Da). Amongst divalent cations only Ba2+ and Cu2+ significantly inhibited the haemolytic power of crude venom. Proteases seem not to alter the haemolytic activity while antioxidant compounds only slightly reduced the haemolytic power. Such findings may suggest a pore-forming mechanism for P. noctiluca crude venom rather than an oxidative damage to the cell membrane.

Factors altering the haemolytic power of crude venom from Aiptasia mutabilis (Anthozoa) nematocysts

The effect of different agents upon the haemolytic power of Aiptasia mutabilis crude venom was studied inhuman erythrocytes to determine its toxicity and stability. Nematocysts were isolated from acontia of the Anthozoan A. mutabilis and submitted to sonication for extracting crude venom. Aliquots of venom were tested in 0.05% erythrocyte suspensions in the presence of various factors such as proteases (papain,collagenase, trypsin, alpha-chymotrypsin); cations (Ca2+, Mg2+, Ba2+, K+ and Cu2+), osmotic protectants as polyethylenglycole (PEG) of different MW and antioxidant compounds (GSH, cysteine and ascorbic acid).Results demonstrate the dose-response of the haemolytic effect of A. mutabilis. Haemolysis by the crude venom was prevented by Ca2+, Ba2+ and Cu2+ treatment, and to a minor extent by Mg2+ and K+. Papain and PEG with a molecular mass exceeding 1000 Da also prevented haemolysis. These findings are consistent with a pore-forming mechanism of crude venom in erythrocytes rather than an oxidative damage at the employed doses.

Morphological integrity and toxicological properties of Pelagia noctiluca (Scyphozoa) nematocysts

Isolated nematocysts of the Scyphozoan Pelagia noctiluca became diaphanous when incubated at low pH for 1 h or left at room temperature for a few hours. Diaphanous nematocysts were unable to undergo discharge when triggered by proper physico-chemical stimuli. The aim of the present study was to investigate the relationship between the morphological features of diaphanous nematocysts and the haemolytic power of their crude venom, obtained by sonication on ice. Nematocysts stored at−20 °C in neutral medium were used as a control. Our results show that the haemolytic power of crude venom from isolated nematocysts kept at low pH or at room temperature decreased significantly with respect to the control. From this study, it can be deduced that a neutral pH and low temperature conditions are recommended to store nematocysts properly in order to use them in toxicological investigations.

Heavy metals affect regulatory volume decrease (RVD) in nematocytes isolated from the jellyfish Pelagia noctiluca

The environmental contamination caused by heavy metals raises the question of their effect on biological systems. Among bio-indicators useful to monitor the toxicological effects of these chemicals, Cnidarians offer a unique model. Cnidarians possess highly specialized stinging cells, termed nematocytes, which respond to hyposmotic solution with well established homeostatic parameters as an acute osmotic phase (OP), leading to cell swelling, and then a slower regulatory volume decrease (RVD) phase, causing cell shrinkage. Here we report the effect of 65% artificial sea water (ASW) containing heavy metals, such as Cd, La, Co, Cu and Zn (concentrations comprised between 100 and 0.1 μM) on both OP and RVD in nematocytes isolated from the jellyfish Pelagia noctiluca by 605 mM NaSCN plus 0.01 mM Ca(2+). The exposure of the cells to Co and La inhibited RVD but not OP. However, Cu, Cd and Zn prevented the OP in a dose-dependent manner and, hence, also the detection of RVD. These results suggest that, in isolated nematocytes, heavy metal pollutants impair RVD either directly or indirectly through interference with the OP, thus negating RVD. Although further studies need to clarify the exact mechanisms whereby heavy metals exert their toxicity, it is evident that nematocytes of Cnidarians could serve as a model for ecotoxicological investigations.

Hyposmotic shock-induced discharge in acontia ofCalliactis parasitica is blocked by gadolinium

On acontia ofCalliactis parasitica it was observed that mechanical stimuli applied by a gelatin probe, a method effective in tentacles of Anthozoa, do not induce the discharge of nematocytes. Hyposmotic shock, performed by treatment with NaCl solution 35% hyposmotic with respect to sea water, induces, in the presence of Ca2+, the discharge that spreads along the acontial filament, as previously observed following treatment with SCN−. The hyposmotic shock-induced discharge is blocked by Gd3+ at a concentration of 1 μM. 10 μM Gd3+ prevents also the SCN−-induced discharge. These results suggest the presence of stretch activated cation channels either in nematocytes and/or in supporting cells as well as a possible effect of SCN− on this class of ion channels.

Discharge characteristics of nematocysts isolated from acontia ofCalliactis parasitica

Experiments were performed on specimens ofCalliactis parasitica collected near Plymouth in April 1987 and in the Mediterranean between September 1987 and April 1988. Undischarged nematocysts (basitrichous isorhizas) were separated from the acontia of by using 1M glycerol, 1M citrate, 0.5% Nonidet or 0.5% Triton X-100 as isolating agents, or by freezing the acontial tissue. The mechanisms of extrusion were not studied. The effectiveness of 50 mM thioglycolate in discharging nematocysts isolated by the above methods and suspended in Ca- and Mg-free artificial sea water (ASW) was investigated. Nematocysts extruded in glycerol were more responsive to thioglycolate than those extruded in citrate. Capsules isolated in non-ionic detergents, however, and those obtained by freezing were not discharged by thioglycolate. If capsules extruded in glycerol were suspended in ASW and this was replaced with distilled water, they did not respond to thioglycolate. The effect of distilled water was only partly reversible. It was observed that these nematocysts, unlike those ofPelagia noctiluca andAiptasia mutabilis previously investigated, did not release measurable amounts of free Ca2+ during discharge. It is suggested that if calcium is not involved in the mechanism of discharge of isolatedC. parasitica nematocysts then some other ionic species may stabilize the resting condition of these capsules.

Hemolytic effects of crude venom from Aiptasia mutabilis nematocysts

The aim of this paper was to focus on the toxinological aspects of microbasic-mastigophore nematocysts isolated from acontia of Aiptasia mutabilis, an Anthozoan collected in the Strait of Messina, by performing hemolytic assay on human, chicken, and rabbit red blood cells in suspension. The hemolytic effects of single isolated nematocysts were achieved by checking the lytic pattern after discharge. Crude extract from a population of isolated nematocysts was obtained by sonication on ice. Hemolysis induced by crude extract was detected spectrophotometrically. The results of the extraction method used show that crude venom that can be obtained is effective in inducing hemolysis of erythrocytes from the different sources. Rabbit erythrocytes in particular seem to be more sensitive to this crude venom. The hemolytic activity is dose-dependent and is inhibited if heated to 70 °C. Further investigations will better focus these results leading to venom characterization.

Effect of crude venom from nematocysts of Pelagia noctiluca (Scyphozoa) on spread discharge of acontia of Calliactis parasitica (Anthozoa)

The present investigation was aimed at assessing the toxic power of crude venom from Pelagia noctiluca nematocysts on spread discharge of in situ acontial nematocytes. Spread discharge, under 553 mM NaSCN plus 10 mM Ca2+, consists of the protrusion and firing of the nematocytes all along the acontial filament. Acontia from Calliactis parasitica were treated with the discharging solution under a constant flow as a control condition. In a separate experiment acontia were firstly treated with Pelagia noctiluca crude venom aliquots and then with the discharging solution to prove spread discharge. Crude venom blocked the discharge with a dose-dependent irreversible effect. From these results arises that crude venom of Pelagia noctiluca nematocysts can affect the spread discharge with a mechanism that is still unknown. It could be hypothesized that crude venom can block gap junctions probably involved in this cellular communication. Nevertheless either a damage of cells involved in nematocyte activation or even the inhibition of Ca2+ influx, needed for discharge triggering, could be even suggested. Other studies will verify crude venom action mechanisms and may confirm the spread discharge as an interesting biological assay aimed at toxicological investigations.

Anatomical and Physiological Characteristics of Tentacular Nematocytes Isolated by Different Methods from Aiptasia diaphana (Cnidaria: Anthozoa) in the Brackish Pond Faro (Messina, Italy)

We studied the effects of isolation on anatomical and functional characteristics of nematocytes isolated from tentacles of Aiptasia diaphana, an anthozoan living in the brackish pond Faro (Messina) where salinity can change for both climatic and anthropic reasons. The anatomical effects of isolation were investigated by scanning electron microscopy (SEM). To evaluate the functional effects, the capacity to regulate cell volume under a 35% hyposmotic shock (Regulatory Volume Decrease, RVD) was measured by image computer processing of the sagittal area. Furthermore the membrane integrity was revealed by applying Trypan blue.The isolation using a physical method was performed by heat dissociation (45δC for 20 min), while, as a chemical method, we used a treatment of tissue with SCN−. The isolated nematocytes revealed different characteristics both anatomical and physiological, although the Trypan blue test showed that in both cases the cell membrane was not damaged. In particular nematocytes isolated by heat dissociation show the mechanosensorial apparatus while those isolated by SCN− appeared to be deprived of this structure. As far as the volume regulation is concerned, the cells isolated by heat dissociation did not appear to regulate their volume until the hypotonic treatment was maintained. On the other hand the nematocytes isolated by SCN− regulate volume in hypotonic conditions.