Inhibition of Nematocyst Discharge from Pelagia noctiluca (Cnidaria: Scyphozoa)—Prevention Measures against Jellyfish Stings

Pelagia noctiluca stings are common in Mediterranean coastal areas and, although the venom is non-lethal, they are painful. Due to its high toxicity and abundance, P. noctiluca is considered a target species for the focus of research on active ingredients to reduce the symptoms of its sting. To determine the effect of 31 substances and formulations on nematocyst discharge, we performed three tests: (1) screening of per se discharge activator solutions, (2) inhibitory test with nematocyst chemical stimulation (5% acetic acid) and (3) inhibitory test quantifying the hemolytic area. Ammonia, barium chloride, bleach, scented ammonia, carbonated cola, lemon juice, sodium chloride and papain triggered nematocyst discharge. All of them were ruled out as potential inhibitors. Butylene glycol showed a reduction in nematocyst discharge, while the formulations of 10% lidocaine in ethanol, 1.5% hydroxyacetophenone in distilled water + butylene glycol, and 3% Symsitive® in butylene glycol inhibited nematocyst discharge. These last results were subsequently correlated with a significant decrease in hemolytic area in the venom assays versus seawater, a neutral solution. The presented data represent a first step in research to develop preventive products for jellyfish stings while at the same time attempting to clarify some uncertainties about the role of various topical solutions in P. noctiluca first-aid protocols.


Introduction
Jellyfish are venomous animals belonging to the Phylum Cnidaria [1,2], which is characterized by the unique presence of stinging cells termed cnidocytes [3,4]. Venom, composed of enzymes, potent pore-forming toxins and neurotoxins [2], is stored in a capsule inside the cnidocyte called the cnidocyst, along with a coiled tubule that may be armed with spines [5,6]. Cnidocytes provide a defense mechanism and are used in prey capture [7], and are one of the largest and most complex intracellular secretion products known [3]. Cnidocysts can be classified into nematocyst, spirocyst and ptychocyst categories, the nematocyst category being the most common among cnidarian specimens, with more than 30 morphological types [6,8].
Due to their toxicity, jellyfish stings have a negative impact on human health [9,10]. It is estimated that 150 million people worldwide are exposed to jellyfish annually [11], and encounters with some cubozoan species in tropical and subtropical waters can result in serious health problems (e.g., cardiopulmonary arrest) [12,13]. However, the jellyfish that inhabit the Mediterranean Sea are considered non-life-threatening species [14][15][16]. Although cases of anaphylaxis can occur [17,18], their stings generally involve skin damage and local symptoms, but can be extremely painful [9,19]. The incubation of ammonia, barium chloride, bleach, lemon juice and scented ammonia triggered massive discharge of nematocysts ( Figure 1B,C,I,K,L and Table 1). Sodium bicarbonate solutions, sodium chloride, papain, acetic acid, carbonated cola and vinegar elicited medium nematocyst discharge ( Figure 1D-H,J,M and Table 1). The rest of the compounds, including seawater ( Figure 1A), did not trigger nematocyst discharge from the tentacle after their incubation, so they were classified as neutral solutions and considered potential inhibitors (Table 1).  The incubation of ammonia, barium chloride, bleach, lemon juice and scented ammonia triggered massive discharge of nematocysts ( Figure 1B,C,I,K,L and Table 1). Sodium bicarbonate solutions, sodium chloride, papain, acetic acid, carbonated cola and vinegar elicited medium nematocyst discharge ( Figure 1D-H,J,M and Table 1). The rest of the compounds, including seawater ( Figure 1A), did not trigger nematocyst discharge from the tentacle after their incubation, so they were classified as neutral solutions and considered potential inhibitors (Table 1). To evaluate the inhibitory effect, nematocyst discharge was chemically stimulated by the application of 5% acetic acid ( Figure 2A and Table 2). After incubation and following the chemical stimulation, bromelain, choline chloride, copper gluconate, gadolinium, iodine solutions, lanthanum chloride, magnesium chloride, magnesium sulfate, distilled water, fresh water, physiological saline and urine were classified as neutral solutions (not inhibitory) ( Table 2). Nematocyst tubules were observed immediately after the 5% acetic acid application. Only isolated nematocysts were discharged in the presence of butylene glycol, so it was classified as a reducer ( Figure 2B and Table 2). After the stimulation with 5% acetic acid, the compounds of lidocaine in ethanol, 1.5% hydroxyacetophenone in distilled water + butylene glycol, and 3% Symsitive ® in butylene glycol completely inhibited nematocyst discharge and were considered inhibitor solutions ( Figure 2C-E and Table 2).
water, fresh water, physiological saline and urine were classified as neutral solutions (not inhibitory) ( Table 2). Nematocyst tubules were observed immediately after the 5% acetic acid application. Only isolated nematocysts were discharged in the presence of butylene glycol, so it was classified as a reducer ( Figure 2B and Table 2). After the stimulation with 5% acetic acid, the compounds of lidocaine in ethanol, 1.5% hydroxyacetophenone in distilled water + butylene glycol, and 3% Symsitive ® in butylene glycol completely inhibited nematocyst discharge and were considered inhibitor solutions ( Figure 2C-E and Table 2)    1 Nematocyst discharged categories: 0 = no discharge; + = low discharge; +++ = high discharge. 2 Rinse solution effect categories: Neutral = nematocysts were not activated after the first incubation with the solution but were activated by the consecutive chemical stimulation with 5% acetic acid; Reducer = nematocysts were not activated after the first incubation with the solution but isolated nematocysts were discharged after the chemical stimulation with 5% acetic acid in some areas; Inhibitor = nematocysts were not activated after the first incubation with the solution nor by the chemical stimulation with 5% acetic acid. n indicates the number of replicates.

Test 3-Venom Load
After the identification of inhibitory solutions with TSA, venom activity assays were conducted using live tentacles in ex vivo TSBAA with only the reducer and inhibitor solutions (Table 2). Seawater, a neutral solution ( Table 2) that was used as a control, obtained a hemolytic area of 60.77 ± 31.38% (Figures 3 and 4A). All the solutions identified as inhibitors in the previous assay (Section 2.2.1) decreased the hemolytic area after the sting process. The smallest hemolytic areas were obtained after incubation with 10% lidocaine in ethanol (3.30 ± 7.19%), 1.5% hydroxyacetophenone in distilled water + butylene glycol (2.48 ± 4.54%), and 3% Symsitive ® in butylene glycol (11.00 ± 17.66%) (Figures 3 and 4B,D,E). Butylene glycol obtained a value of 26.54 ± 19.64% for the hemolytic area (Figures 3 and 4C). Significant differences were found between seawater (control) and all the compounds (*** p ≤ 0.001) (ANOVA test) ( Figure 3). Statistical data can be consulted in Supplementary Material Tables S1 and S2.

Discussion
Jellyfish stings represent a human health hazard and have a negative impact on the tourism sector [9,10,18,24]. In the Mediterranean region, P. noctiluca is responsible for a high number of incidents among beachgoers [10,18,22,24]. Its high toxicity along with its wide distribution and high frequency in the basin [15,21,22] have positioned P. noctiluca as a target species for preventive measures, especially those that help to prevent and mitigate the effects of its sting [23,28,34,40,41].
In the past, a wide variety of compounds have been tested for their effectiveness in treating scyphozoan stings (Table 3). Among the most deeply rooted traditional remedies are urine, urea and ammonia solutions [9,18]. Previous preclinical tests showed inhibition of P. noctiluca nematocyst discharge in a 20% ammonia solution [40], yet the immediate and massive nematocyst discharge observed herein questions the inhibitory potential of both ammonia and scented ammonia ( Figure 1B,L and Table 1). Probably, these opposite results, as in the case of vinegar in   [28], are due to the use of different methodologies (Table 3). While the stimulation in Morabito et al. (2014) [40] was chemicalmechanical, with 20 min of incubation in a chemosensitizer compound (e.g., glutamate) followed by mechanical stimulation with gelatine-coated test probes, here we evaluated discharge using the TSA method, a widely-used technique in cnidarians to discern between activator and potential inhibitory compounds [28][29][30]36,37,42,43]. In accordance with our results, high nematocyst discharge and exacerbation of pain intensity were also identified during the ammonia test for the scyphomedusa Chrysaora quinquecirrha [37,39]. Despite promoting nematocyst discharge (Table 1), ammonia-based post-sting products expressly for treating P. noctiluca stings [41] are currently available on the market. We conclude that ammonia is not an inhibitor solution for P. noctiluca nematocysts and its use is contraindicated for the treatment of their stings. Regarding urine, in accordance with Pyo et al. (2016) [36] to Nemopilema nomurai, no nematocyst discharge was identified for P. noctiluca (Table 1), yet these results are in contrast to its effect for Cyanea capillata (Table 3).
Like ammonia solutions, vinegar or acetic acid are perceived by the public as beneficial products for the treatment of jellyfish stings. However, the stimulation of discharge and their non-inhibitory effect are well-documented for scyphozoans, including P. noctiluca [28,[36][37][38][39]. Both here and in previous studies [37,44], 5% acetic acid has been used to chemically stimulate nematocyst discharge in scyphozoans (Table 2). Other acid solutions such as carbonated cola and lemon juice also triggered discharge ( Figure 1J,K and Table 1), supporting the correlation between nematocyst discharge and extreme acidic solutions reported previously [45]. Despite the solid scientific evidence demonstrating its contraindication in scyphozoans ( Figure 1M) [28,[36][37][38][39]46], some administrations recommend the use of vinegar. For example, in the Balearic Islands, one of the major tourist destinations in Europe [47] and with a high presence of scyphozoans, the use of vinegar is still recommended to treat jellyfish stings [48]. A survey in Mallorca (Balearic Islands) [47] showed the added value of the presence of health services on beaches, but the effectiveness of such preventive measures can be diminished if the health service does not receive appropriate guidelines. In addition, first-aid protocols must be updated as research on the topic advances.
Solutions and slurry of sodium bicarbonate were strongly recommended in firstaid protocols for scyphozoan species, including those that inhabit the Mediterranean basin [39,49]. However, there is no robust scientific evidence to support this recommendation (Table 3). Morabito et al. (2014) [40] identified nematocyst discharge in P. noctiluca, which is in accordance with our results ( Figure 1D,E and Table 1). Due to the nematocyst discharge observed herein (Table 1) and the lack of clinical evidence on topical relief of jellyfish sting symptoms (Table 3), the use of sodium bicarbonate has been removed from the Spanish guidelines on Mediterranean jellyfish stings in an updated version of the first-aid protocols [16].
Anionic solutions have been observed to promote nematocyst discharge in P. noctiluca [45,50]. While anions such as I − and Cl − triggered high rates of discharge, cations such as Mg + and Ba + did not elicit nematocyst discharge. Yet, when they were added to iodine solutions discharge was inhibited [45,50]. Salleo et al. (1948) [45] reported nematocyst discharge in sodium chloride in accordance with our results ( Figure 1F and Table 1), but conflicting results have been obtained for choline chloride, barium choline and magnesium chloride (Table 3). Solutions containing lanthanum and gadolinium did not inhibit nematocyst discharge in this study as previously reported for P. noctiluca [51] (Table 2) unlike lidocaine solution (ANOVA, p ≤ 0.001) (Figures 2, 3 and 4B and Table 2) also formerly described for scyphozoans (Table 3).
Nematocyst discharge promoted by osmotic change after fresh water application is a premise in first-aid protocols, which is why it is not recommended [52,53]. Fresh water did not produce nematocyst discharge in P. noctiluca (Table 1) and it has been considered a non-activator solution in scyphozoans [36]. Distilled water is commonly used to induce the detachment of P. noctiluca cnidocytes from the epidermis, maintaining their integrity [45,54]. However, despite its neutral effect on the nematocyst discharge of P. noctiluca (Table 1), the use of fresh, tap, deionized or distilled water is not recommended due to their potential to isolate undischarged cnidocytes, since these can roll on the skin and be mechanically activated, inoculating a second venom load [28,29,42,43].
Glycols, such as butylene glycol, are used in a variety of cosmetic products [55]. Here, no discharge was observed with butylene glycol (Table 1), allowing us to validate the safe use of butylene glycol in preventive cosmetic products, in terms of non-activation of discharge, unlike products with ammonia [41] and acetic acid [56]. Moreover, butylene glycol was classified as a reducer solution (Table 2), and a decrease in hemolytic zones was later observed in TSBAA tests (ANOVA, p ≤ 0.001) ( Figures 2B, 3 and 4C). Probably, due to its viscous consistency, 100% butylene glycol wraps around the tentacle to act as a physical barrier. Lower values compared to the other formulations in TSBAA (Figure 3) are possibly due to the loss of effectiveness of the physical barrier during the sting process. When butylene glycol was dissolved in water (e.g., 50% butylene glycol in distilled water, Table 2), its inhibitor effect was lost.
Jellyfish stings cause clinical signs such as erythema, redness or papules accompanied by pain, burning sensation and itching [14,23,24]. Active ingredients that reduce skin damage and/or pain or burning sensation, as well as inhibiting nematocyst discharge provide added value in preventive products. Here, the formulation containing Symsitive ® inhibited nematocyst discharge and significantly reduced hemolytic area (ANOVA, p ≤ 0.001) (Figures 2E, 3 and 4E and Table 2). In the TSBAA test, high variability between replicates was observed for Symsitive ® (Figure 3). Discharge of nematocysts could have occurred in some areas where the product has not fully penetrated. Symsitive ® is composed of trans-4-t-butylcyclohexanol, a TRPV1 receptor antagonist [57], and pentylene glycol [55]. In the cosmetic industry, this active ingredient has been clinically proven to relieve symptoms such as erythema, stinging and burning [58,59]. An inhibitor effect was also reported for 1.5% hydroxyacetophenone in distilled water + butylene glycol (ANOVA, p ≤ 0.001) (Figures 2, 3 and 4D and Table 2), an anti-irritant used in cosmetic products [55]. Although hemolysis was not observed in most replicates of 1.5% hydroxyacetophenone in distilled water + butylene glycol, some small hemolytic areas were observed in some replicates (Figures 3 and 4D). The same occurred for lidocaine (Figures 3 and 4B). This fact could be due to the fact that the compounds did not penetrate in some areas and some discharge of nematocysts could have occurred promoted by the weight (Section 4.4.2).
For the time being, the role of Symsitive ® and hydroxyacetophenone in the modulation of P. noctiluca nematocyst discharge is unknown, and further research is needed to determine these. However, due to their inhibitor effect and decrease in hemolytic areas (ANOVA, p ≤ 0.001), both Symsitive ® and hydroxyacetophenone are considered compounds of great value for the cosmetic industry and identified as active ingredients suitable for incorporation into sunscreens and cosmetic products against jellyfish stings.

Jellyfish Cultures
Jellyfish were cultivated in Loro Parque Aquarium (Tenerife, Spain). P. noctiluca adult individuals were grown for 80 days in kreisel tanks with continuous water renewal, 33-35‰ salinity, 22 • C temperature and light cycle of 12 h light/12 h dark. Individuals were fed daily with Artemia sp. nauplii, small pieces of the jellyfish Aurelia sp., eggs of Merluccius merluccius and Acetes sp. (white prawns).
Tentacle pieces (approx. 3 cm long) of P. noctiluca from aquaculture were incubated in each reducer and inhibitor solution (Section 4.4.1) for 5 min. Subsequently, tentacles were placed on the plastic molds [61] with a weight of 0.66 g to ensure contact. After 1 min of contact, the plastic molds and the intestine sections were removed and the SRBC agarose rectangles were stored in the humidification chamber at room temperature. After 22 h, photographs were taken of the hemolytic areas which were then calculated using the Fiji version of ImageJ software [62]. The data were tested for normality and homogeneity using the stats package available as part of the R basic software platform [63]. Subsequently, an ANOVA test was performed using the aov function to test significant differences between hemolytic areas, with an additional pairwise comparison using the pairwise function to test between which groups the differences occurred. Finally, a graphical representation was performed using the ggplot2 package from the R software platform [64].

Conclusions
Pelagia noctiluca is a highly toxic jellyfish responsible for the majority of stings in Mediterranean waters, so it is considered a target species for preventive measures. Unlike lidocaine, which inhibited nematocyst discharge, solutions containing lanthanum and gadolinium were considered neutral solutions. The use of ammonia, vinegar, 5% acetic acid and baking soda, among others, is not recommended to treat P. noctiluca stings because they promote nematocyst discharge. Hydroxyacetophenone and Symsitive ® were identified as nematocyst inhibitor compounds of great value for the cosmetic industry. These active ingredients can be incorporated into sunscreens to reduce the symptoms of jellyfish stings, as well as in a rinse solution to help to remove tissue or/and residual cnidocytes after jellyfish stings.