Synergistic Effect of Proteinase Activity by Purification and Identification of Toxic Protease From Nemopilema nomurai

Scyphozoan Nemopilema nomurai envenomation is an unresolved threat to human health in Asian waters. Nemopilema nomurai venom metalloproteinases show important toxicities in skin damage and inflammation, but there is still no purified protein for further studies. In this study, high proteinase activity fractions in tentacle autolysis were isolated by ammonium sulfate precipitation, DEAE Sepharose Fast Flow, and Superdex 75 chromatography successively. Purification was guided by azocasein hydrolysis activity and SDS-PAGE. The final products were analyzed by LC-MS/MS. Four elution peaks purified by Superdex 75 chromatography had multiple protein bands but did not show proteinase activity. These fractions would recover proteinase activity after mixing again. Regulation mechanisms were speculated as binding metalloproteinase regulator or disaggregating metalloproteinase inhibitor by LC-MS/MS analysis. For the first time, a synergistic effect in N. nomurai proteinase activity was found in the purification process.

Refinement and Neutralization Evaluation of the F(ab’)2 Type of Antivenom against the Deadly Jellyfish Nemopilema nomurai Toxins

Jellyfish stings threaten people’s health and even life in coastal areas worldwide. Nemopilema nomurai is one of the most dangerous jellyfish in the East Asian Marginal Seas, which not only stings hundreds of thousands of people every year but also is assumed to be responsible for most deaths by jellyfish stings in China. However, there is no effective first-aid drug, such as antivenoms, for the treatment of severe stings by N. nomurai to date. In this study, we prepared a N. nomurai antiserum from rabbits using inactivated N. nomurai toxins (NnTXs) and isolated the IgG type of antivenom (IgG-AntiNnTXs) from the antiserum. Subsequently, IgG-AntiNnTXs were refined with multiple optimizations to remove Fc fragments. Finally, the F(ab’)2 type of antivenom (F(ab’)2-AntiNnTXs) was purified using Superdex 200 and protein A columns. The neutralization efficacy of both types of antivenom was analyzed in vitro and in vivo, and the results showed that both IgG and F(ab’)2 types of antivenom have some neutralization effect on the metalloproteinase activity of NnTXs in vitro and could also decrease the mortality of mice in the first 4 h after injection. This study provides some useful information for the development of an effective antivenom for N. nomurai stings in the future.

Field Experiment Effect on Citrus Spider Mite Panonychus citri of Venom from Jellyfish Nemopilema nomurai: The Potential Use of Jellyfish in Agriculture

Jellyfish are rich in resources and widely distributed along coastal areas. As a potential approach to respond to jellyfish blooms, the use of jellyfish-derived products is increasing. The citrus spider mite (Panonychus citri) is one of the key citrus pests, negatively impacting the quality and quantity of oranges. Due to the resistance and residue of chemical acaricides, it is important to seek natural substitutes that are environmentally friendly. The field efficacy of the venom from the jellyfish Nemopilema nomurai against P. citri was assayed in a citrus garden. The frozen N. nomurai tentacles were sonicated in different buffers to isolate the venom. The venom isolated by PBS buffer (10 mM, pH 6.0) had the strongest acaricidal activity of the four samples, and the corrected field efficacy 7 days after treatment was up to 95.21%. This study demonstrated that jellyfish has potential use in agriculture.

Contrasting Effects of Regional and Local Climate on the Interannual Variability and Phenology of the Scyphozoan, Aurelia coerulea and Nemopilema nomurai in the Korean Peninsula

The East Asian marginal seas are among the most productive fisheries grounds. However, in recent decades they experienced massive proliferations of jellyfish that pose vast challenges for the management of harvested fish stocks. In the Korean Peninsula, the common bloom-formers Scyphozoan species Aurelia coerulea and Nemopilema nomurai are of major concern due to their detrimental effects on coastal socio-ecological systems. Here, we used pluriannual field observations spanning over 14 years to test the extent of climate influence on the interannual variability and bloom dynamics of A. coerulea and N. nomurai. To depict climate-jellyfish interactions we assessed partitioning effects, direct/indirect links, and the relative importance of hydroclimate forces on the variability of these species. We show that jellyfish interannual patterns and bloom dynamics are shaped by forces playing out at disparate scales. While abundance changes and earlier blooms of A. coerulea were driven by local environmental conditions, N. nomurai interannual patterns and bloom dynamics were linked with regional climate processes. Our results provide a synoptic picture of cascading effects from large scale climate to jellyfish dynamics in the Korean Peninsula that may affect fisheries sustainability due to the prominent detrimental impact these species have in the region.

Proteomic Changes during the Dermal Toxicity Induced by Nemopilema nomurai Jellyfish Venom in HaCaT Human Keratinocyte

Jellyfish venom is well known for its local skin toxicities and various lethal accidents. The main symptoms of local jellyfish envenomation include skin lesions, burning, prickling, stinging pain, red, brown, or purplish tracks on the skin, itching, and swelling, leading to dermonecrosis and scar formation. However, the molecular mechanism behind the action of jellyfish venom on human skin cells is rarely understood. In the present study, we have treated the human HaCaT keratinocyte with Nemopilema nomurai jellyfish venom (NnV) to study detailed mechanisms of actions behind the skin symptoms after jellyfish envenomation. Using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/MS), cellular changes at proteome level were examined. The treatment of NnV resulted in the decrease of HaCaT cell viability in a concentration-dependent manner. Using NnV (at IC50), the proteome level alterations were determined at 12 h and 24 h after the venom treatment. Briefly, 70 protein spots with significant quantitative changes were picked from the gels for MALDI-TOF/MS. In total, 44 differentially abundant proteins were successfully identified, among which 19 proteins were increased, whereas 25 proteins were decreased in the abundance levels comparing with their respective control spots. DAPs involved in cell survival and development (e.g., Plasminogen, Vinculin, EMILIN-1, Basonuclin2, Focal adhesion kinase 1, FAM83B, Peroxisome proliferator-activated receptor-gamma co-activator 1-alpha) decreased their expression, whereas stress or immune response-related proteins (e.g., Toll-like receptor 4, Aminopeptidase N, MKL/Myocardin-like protein 1, hypoxia up-regulated protein 1, Heat shock protein 105 kDa, Ephrin type-A receptor 1, with some protease (or peptidase) enzymes) were up-regulated. In conclusion, the present findings may exhibit some possible key players during skin damage and suggest therapeutic strategies for preventing jellyfish envenomation.

Density Estimation of Nemopilema nomurai (Scyphozoa, Rhizostomeae) Using a Drone

Research to understand the distribution and density of jellyfish is actively being conducted using training ships, but this is hindered by the high cost of manpower and the limitations of the irradiation area. Unmanned aerial vehicles (UAVs or drones), however, provide cost-effective means for assessing marine animal populations. Therefore, we tested the application of UAVs in estimating jellyfish density and probed the altitude-dependent suitability of these devices. We analyzed images obtained by a drone as well as by manual counting and used ImageJ to measure the density of Nemopilema nomurai off Sang-Chuja Island, Jeju, South Korea. Analysis of the image obtained at altitudes of 5–120 m allowed for the identification of 2–173 individuals, while 1.49–9.09 individuals were identified per 100 m2. Jellyfish density data measured by manual count and by ImageJ did not show any difference below 90 m; however, a difference was presented at altitudes of 100 m (98%) and 120 m (95%). These results demonstrate the potential of drones for jellyfish monitoring and recommend an optimal altitude for observation.