The activation properties of in situ nematocytes of Pelagia noctiluca (Scyphozoa) were investigated by physical contact with a gelatin probe that, besides stimulating the nematocyte battery, retains the discharged nematocysts, thereby allowing a quantitative evaluation of the response. In oral arms previously treated with 2 mmol l−1 La3+ the discharge was inhibited. This result confirms the Ca2+-dependence of nematocyte activation. A similar inhibitory effect was induced by treatment with 20 μmol l−1 Gd3+, a powerful blocker of mechanosensitive ion channels. It is therefore proposed that Ca2+-permeable mechanosensitive channels are involved in the activation of nematocytes. 50 μmol l−1 Gd3+ added to the gelatin probe was effective in otherwise untreated oral arms. This result suggests that Gd3+ could be useful in preventing stings from harmful Cnidaria.
Nematocytes are the stinging cells of Cnidaria, used for defence and for prey capture. They contain a capsule, termed the nematocyst, that, given the correct stimulus, everts a long tubule (filament) through which the venomous content of the capsule is injected into the foreign integument. Such a response is called nematocyst discharge. Nematocyte excitation requires both mechanical and chemical stimuli (Pantin, 1942). Thorington and Hessinger (1988) and Giebel et al. (1988) identified two classes of chemoreceptors in the tentacles of Aiptasia pallida, one sensitive to low molecular weight amino compounds and the other to N-acetylated sugars. The chemoreceptive sites seem to be located on the supporting cells surrounding the nematocyte rather than on the nematocyte itself (Watson and Hessinger, 1989). Watson and Hessinger (1989, 1991) have proposed that both contact-sensitive and vibration-sensitive mechanoreceptors are present in sea anemones and that the chemoreceptors sensitize the former and tune the latter towards lower frequencies.
The transduction mechanisms activated by mechanical and chemical stimuli remain uncertain because the small size of most nematocytes is not favourable for experiments. The electrophysiological studies performed by Anderson and McKay (1987) on Chrysaora quinquecirrha, Cladonema sp. and Physalia physalis revealed that discharge is not triggered by membrane depolarization. Furthermore, isolated nematocytes do not respond well to stimuli in vitro. Santoro and Salleo (1991a, b) and Salleo et al. (1993) observed that in situ nematocytes of acontia of Aiptasia mutabilis and Calliactis parasitica do not discharge in Ca2+-free media, but are able to discharge at Ca2+ concentrations of 0.1–30 mmol l−1, and that the Ca2+ channel blockers La3+, Cd2+ and Co2+ prevent discharge. It was therefore concluded that an inflow of Ca2+ could couple the stimulus to the discharge response. Recently, Watson and Hessinger (1992) have suggested that cyclic AMP could be the second messenger in the supporting cells involved in this coupling.
As far as mechanoreception is concerned, a particular class of ion channel, the stretch-activated (SA) channels (Morris, 1990), is expected to be involved. Besides mechanoreception, these channels are involved in cell volume regulation (Filipovic and Sackin, 1991; McCarty and O’Neil, 1992). Among the various subtypes of SA channels, the cationic subtype has been most extensively investigated. A powerful and specific blocking effect of gadolinium on cationic SA channels has been described by Yang and Sachs (1989). This lanthanide is effective at micromolar concentration and has the further advantage of not permeating the cell. The SA channels have been investigated in various taxa, from procaryotes to mammals, with the notable exception of coelenterates. We reasoned that, since mechanoreceptors must be stimulated to trigger a Ca2+-dependent discharge of nematocytes, a blocking effect of Gd3+ could suggest a role for SA channels in the discharge process. Since most investigations on the activation properties of in situ nematocytes have been performed on Anthozoa, the present experiments were performed on Pelagia noctiluca (Scyphozoa), a species in which the properties of in situ nematocyte discharge have not previously been investigated.
Materials and methods
The experiments were performed on 40 specimens of Pelagia noctiluca Forskål collected in the Straits of Messina, Italy. The animals were placed immediately in 20 l tanks filled with sea water maintained at 20–22 °C. Sea water was replaced after 24 h. All tests were performed within 48 h of collection. Only specimens that showed active swimming movements were used.
Experiments were carried out on excised oral arms; these were placed in Sylgard-coated Petri dishes, rinsed repeatedly with artificial sea water (ASW) and fixed to the Sylgard base at both ends with two Opuntia spines. The ASW had the following composition (in mmol l−1): NaCl, 520; KCl, 9.7; MgCl2, 42.6; CaCl2, 10, and was buffered at pH 7.65 with 5 mmol l−1 imidazole. The composition of ASW was derived from that described by McKay and Anderson (1988), except for the lack of sulphate, which was omitted to avoid any possible interaction with the lanthanides, and a higher NaCl concentration to reach the osmotic pressure of sea water in the Straits of Messina. To test whether the nematocyst discharge in this species is a Ca2+-dependent process, as it is in Anthozoa (Santoro and Salleo, 1991a,b), the blocking effect of 2 mmol l−1 LaCl3 was investigated. GdCl3 was added to ASW at a concentration of 20 μmol l−1 to test the effectiveness of this lanthanide in blocking discharge. The excised oral arms were incubated for 30 min either in ASW or in ASW containing the blocker. Physical contact with a gelatin probe, as proposed by Giebel et al. (1988), was used for stimulating the nematocytes. The gelatin (Sigma, type B) was dissolved in ASW at a concentration of 30 % (w/v). One end of a nylon wire was dipped in gelatin to form a bead approximately 200 μm in diameter. The probe was operated with a micromanipulator under a microscope. Single nematocyte batteries, easily identified by their colour, were touched once for about 2 s with the gelatin bead, into which the tubules of the discharged nematocysts penetrated. The number of discharged nematocysts of the holotrichous isorhiza type was estimated by the following method (Giebel et al. 1988). Immediately after each test, the probe was placed in a microtitration well containing 80 μl of 1 % Trizyme (Amway Products), a mixture of a proteolytic enzyme and detergent (a generous gift from Dr D. A. Hessinger). After 4 h at room temperature, the gelatin was completely hydrolyzed and the discharged holotrichous isorhiza nematocysts released into the titration well were counted under an inverted microscope. In each test, the response of 10–15 nematocyte batteries was investigated by using one probe for each battery. Eight tests on different animals on different days were performed for each experimental condition. Control tests (N=21) were generally performed on oral arms excised from the same animal that was used for the experiments. The number of nematocysts discharged from single batteries in each oral arm was averaged. The frequency distribution of the mean discharge response observed in each test is shown in Fig. 1.
To test the rapidity of action of Gd3+ in blocking the activation of nematocytes, the oral arms were incubated in control ASW and stimulated with a probe constructed from a gelatin solution made in ASW containing either 20 μmol l−1 (N=5) or 50 μmol l−1 (N=10) GdCl3. In this case, therefore, the blocker was present only in the test probe.
Contact with the gelatin probe did not induce discharge of all the holotrichous isorhiza nematocysts present in each battery (20–60), because of the round shape of the probe and the differing degrees of maturation of the cells. The mean number of nematocysts discharged in each oral arm varied to a certain extent (Fig. 1). Nevertheless, the frequency distribution of the mean discharge response in control tests differs clearly from that of the experimental ones. The mean of means for single control tests was 16±1.4 (± S.E.M.).
The results of various blocking experiments are summarized in Fig. 2 as mean of means for each experimental condition. In the oral arms incubated in ASW containing 2 mmol l−1 La3+, discharge occurred only occasionally, so that the mean number of discharged nematocysts was only 1.2±0.4 (Fig. 2B). The difference from the control value is highly significant using a t-test (P<0.001).
The direct incubation of oral arms in ASW containing 20 μmol l−1 Gd3+ reduced the responsiveness of nematocytes to contact with the gelatin probe (Fig. 2C). The mean number of nematocysts discharged in the batteries on the oral arms treated with Gd3+ was 2.8±0.7, a value that also differed significantly from the control (P<0.001).
20 μmol l−1 Gd3+ added to the gelatin probe was found to be completely ineffective in preventing the discharge in untreated oral arms (Fig. 2D). However, 50 μmol l−1 Gd3+ in the gelatin probe exerted a significant inhibitory effect on the discharge (Fig. 2E). The mean number of discharged nematocysts (2.5±0.7) was also significantly different from the control value (P<0.001).
The method for quantitative assessment of nematocyst discharge proposed by Giebel et al. (1988) for tentacles of Anthozoa was also found to be suitable for the nematocyte batteries on the oral arms of Pelagia noctiluca. Only the excised oral arms could be used in this experiment because of the spontaneous swimming movements of the animals.
The inhibitory effect of La3+ on the nematocyst discharge is in agreement with results obtained by Santoro and Salleo (1991a,b) on in situ nematocytes of the acontia of Aiptasia mutabilis and Calliactis parasitica. Such a result suggests that the Ca2+-dependence of the discharge process could be a general aspect of this physiological function in Cnidaria. This would be expected if the discharge were an exocytotic process (Lubbock et al. 1981). The discharge of few nematocytes in some batteries probably results from an incomplete diffusion of the blockers.
The effectiveness of micromolar Gd3+ in blocking nematocyst discharge is striking. This lanthanide is considered to be a powerful blocker of SA cationic channels (Yang and Sachs, 1989; McCarty and O’Neil, 1992), although the mechanism of its action is still uncertain. It is therefore likely that its inhibitory effect on nematocyst discharge is exerted via SA channels that are expected to operate in contact mechanoreceptors. If Gd3+ inhibits the discharge by acting on SA channels, it could provide a new tool for investigating mechanoreception in detail in Cnidaria.
The main result of the present investigation is that 50 μmol l−1 Gd3+ applied through a gelatin probe, in the absence of any previous treatment of the tissue, prevents discharge of nematocyte batteries in the oral arms of Pelagia noctiluca. This result suggests that an amount of Gd3+ sufficient to block the discharge diffuses from the gelatin to the batteries during the brief time required for stimulus–activation coupling. Besides confirming that Gd3+ acts at very low concentrations in this system, this result could have an important practical application in preventing stings from those Cnidaria harmful to humans.