Cnidaria include the most venomous animals of the world, among them Physalia physalis that is high abundant in the North Portugal. The venom of P. physalis is a complex mixture of bioactive substances that are only partially characterized. The study of the jellyfish venom requires the release of venom from the interior of the nematocyst or from the tissue samples. However, the methods for stimulation or inactivation nematocytes lack consensus with a gap in successful methods employed the discharge of the nematocysts, mostly due to the differences observed among species. We tested a range of physical and chemical approaches regarding nematocyte stimulation, including different chemical solvents, ionic solutions and electrical stimulation and we analyzed nematocyst discharge under light microscopy. Venoms released in each situation were analyzed for protein profile and enzymatic activities. Artemia was used as a tool for the preliminary tests of toxicity, paralyzes and modification on the nociception.
According to our observations, nematocyst discharge is not stimulated by seawater; 5% acetic acid solution induces 15 to 30% nematocyst discharge after 30 min exposure; 100% ethanol induces 40 to 60%; 0.9% NaCl for 30 min results in 90% discharge; 0.3M KCl solution induces 80% discharge in less than 15 minutes. Remarkably, it was observed, for the first time, that electrical stimulation with 8 V pulse for 30 seconds of a tentacle immersed in 0.9% NaCl resulted in 80% discharge.
The SDS-page profile of venom proteins released after stimulation with ethanol and ionic solutions showed a similar pattern with a broad MW distribution range and thicker bands at 55 kDa, 40 kDa and 20 kDa. These mixtures had reveals high proteolytic activities that are inhibited differently by PMSF, phenanthroline and EDTA, thus indicating the presence of serine- and metallo- proteases.
Toxicity tests using hatched nauplii revealed a dose-dependent effect on mortality with an LD90 of 17 mg/ml and an LD50 of 7.2 mg/ml. The paralysis was observed 2 hours after exposition whereas, the loose of light nociception was registered after 1 hour of exposition. To be noticed, the venom obtained by stimulation with ethanol loses enzymatic activity, but still can kill and paralyze hatched nauplii.
The present study provides methodologies for venom extraction prior to proteomic analysis and toxicity assays. They also provide useful to understanding on nematocysts stimulation, which is essential for the first rescue into accidental contact, preventing the discharge of nematocysts that may be adhering to the skin.
