Capture And Identification Of Nematocysts From The Giant Jellyfish In Chinese East Coast And Analysis Of Their Venoms

Increased frequency and magnitude of jellyfish blooms worldwide,together with increased exposure of tourists and sea-workers to nematocysts’envenomation,brings broad implications to health and economical development.Jellyfish belong to the phylum Cnidaria.They generally refer to the umbrellashaped gelatinous zooplanktons and their sizes,shapes,and habitats are diverse.In jellyfish,nematocysts densely covere the surfaces of tentacles,upon physical contact,the capsules of the nematocysts fire a barbed arrow-like tubule at high velocity to deliver the venom。The nematocyst’s venom contains a complex mixture of polypeptides and proteins.Nematocyst firing normally induces local symptoms,but cardiovascular or neurological complications can also occur.Hemolysis is a frequent effect of cnidarian stinging,and this dangerous condition is known to be caused by several toxins and can sometimes be lethal.At present,the bulk of data concerning hemolytic cnidarian venoms comes from the study of benthic species,such as sea anemones and soft corals,and hemolytic factors were also found in venoms of several siphonophore,cubozoan and scyphozoan jellyfish.Nematocyst type and quantity are substantially different in various species of jellyfish,even in the same kind of jellyfish,they also have abvious changes during the development and regeneration.Objectives:Cyanea capillata and Nemopilema nomurai both belong to Scyphozoa,and they are the most common types of outbreak in the Chinese southeast sea areas,Thus the identification of each nematocyst of these jellyfish,comparative analyse of the venoms from different jellyfish or different nematocyst and purification of the cardiovascular and hemolysis toxins will be critical to elucidate the mechanism of action of their venoms and develop therapeutic approaches for jellyfish stings.Methods:1.Nematocyst preparation:As nematocysts and other structural components are different in size,density and sedimentation coefficient,and all types of nematocysts are different from each other as well,we isolated the undischarged nematocysts from the tissue debris and discharged nematocysts by gradual sieving and density gradient centrifugation.2.Preparation and comparative analyses of nematocyst venom:the venom was obtained from the nematocystys by ultrasonic crush at 0-4°C,then the lysate was centrifuged at 10000×g for 30 min at 4°C.After determining the protein concentration as well as SDS-PAGE,the hemolytic and cardiovascular activity of Cyanea capillata nematocyst venom（CNV）,Nemopilema nomurai nematocyst venom（NNV）,O-isorhiza nematocyst venom（ONV）and Anisorhizas nematocyst venom（ANV）were analysed,respectively.3.Purification of cardiovascular and hemolysis toxins from NNV:A size-exclusion chromatography（Sephadex G-200）and HPLC,combining with MALDI-TOF mass spectrometry analysis,were utilized for the purification of NNV after screening all the fractions basing on the measurement ofhemolytic and cardiotoxic activity and SDS-PAGE analyses.Results:1.More than 6.59×10⁶ nematocysts,calculated by cell counting method,were captured from about 100 g of jellyfish tentacle.Here we examined the morphological characters of nematocysts from Cyanea capillata and Nemopilema nomurai by light microscopy.Four types of nematocyst had been identified in Cyanea capillata,including:（1）microbasic mastigophores;（2）Heterotrichous microbasic birhopaloids/euryteles;（3）A-isorhizas（homotrichous-isorhizas）and（4）O-isorhizas,and other four types in Nemopilema nomurai,including:（1）O-isorhizas;（2）Heterotrichous microbasic birhopaloids/euryteles;（3）A-isorhizas and（4）Anisorhizas.Furthermore,this technique allowed the emission of approximately 85%of these undischarged nematocysts when the environmental osmotic pressure changed markedly by adding de ionized water.2.We acheived the nematocyst venoms without contamination with tentacular tissue debris and other components.NNV showed a strong lethal cardiovascular activity and hemolytic activity,while such activities of CNV were weaker.In different types of Nemopilema nomurai nematocysts（N3）,ONV totally contains the component of cardiovascular lethal activity,while ANV does not contain any component of lethal activity.However,both ONV and ANV contain hemolytic activity.3.After purification,we altogether achieved 11 fractions,among which two components were identified as cardiovascular toxin F and hemolytic toxin D.The cardiovascular activity of toxin F was significantly higher than that of NNV,while the hemolytic activity of toxin D was slightly lower than that of NNV.Conclusions:The method we reported is an improved technique that maximizes the harvest of intact nematocysts from jellyfish tentacles.The hemolytic activity of NNV was 10 to 20 times more than that of CNV and the lethal activity was 20 to 50 times more than CNV,while the lethal activity of purified cardiovascular toxin F was likewise 5 to 10 times more than that of NNV.In different types of Nemopilema nomurai nematocysts,ONV totally contains the component of cardiovascular lethal activity,while ANV does not contain any component of lethal activity,indicating that the severest cardiovascular lethal activity located in O-isorhizas that may be the feeding nematocysts of Nemopilema nomurai.In Nemopilema nomurai,the cardiovascular lethal activity of NNV was independent of its hemolytic activity,though the hemolytic fraction might be lethal as well.Two components identified as cardiovascular toxin F and hemolytic toxin D were obtained from NNV,and the cardiovascular activity of toxin F was significantly higher than that of NNV.