A Multi-screening Evaluation of the Nutritional and Nutraceutical Potential of the Mediterranean Jellyfish Pelagia noctiluca

The phylum Cnidaria is one of the most important contributors in providing abundance of bio- and chemodiversity. In this study, a comprehensive chemical investigation on the nutritional and nutraceutical properties of Mediterranean jellyfish Pelagia noctiluca was carried out. Also, compositional differences between male and female organisms, as well as between their main anatomical parts, namely bell and oral arms, were explored in an attempt to select the best potential sources of nutrients and/or nutraceuticals from jellyfish. With the exception of higher energy densities and total phenolic contents observed in females than males, no statistically significant differences related to the specimen’s sex were highlighted for the other compound classes. Rather, the distribution of the investigated chemical classes varied depending on the jellyfish’s body parts. In fact, crude proteins were more abundant in oral arms than bells; saturated fatty acids were more concentrated in bells than oral arms, whereas polyunsaturated fatty acids were distributed in the exact opposite way. On the other hand, major elements and trace elements demonstrated an opposite behavior, being the latter most accumulated in oral arms than bells. Additionally, important nutraceuticals, such as eicosapentaenoic and docosahexaenoic acids, and antioxidant minerals, were determined. Overall, obtained data suggest the potential employment of the Mediterranean P. noctiluca for the development of natural aquafeed and food supplements.


Introduction
The marine environment and its inhabitants are today recognized as an enormous reservoir of bioactive substances to be exploited for pharmaceutical and aquaculture applications, and as nutraceuticals as well [1][2][3][4].Nonetheless, a number of anthropogenic activities have negatively affected marine ecosystems, inhibiting the derived services, and their precious bioactive resources as well [5][6][7][8].weights (58.2 and 54.7 g, p < 0.05, and 25.7 and 31.7 g, p < 0.05, respectively) were observed.Finally in June both female and male organisms showed similar lengths, bell diameters and masses (p > 0.05) (Figure 1) Figure 1.Fresh weight (g), bell diameter (cm), and length (cm) of jellyfish samples collected per sex and month.Data are reported as mean ± standard deviation (n = 50).According to the four sampling periods, female (or male) jellyfish marked by different letters for a given biometric characteristic, differ significantly (p < 0.05 by post hoc Tukey's Honestly Significant Difference (HSD) test).According to sex specimens, female and male jellyfish marked by the asterisk for a specific parameter in the same sampling month differ significantly (p < 0.05 by post hoc Tukey's HSD test).F = female.M = male.

Gross Energy Contents
The gross energy densities of female and male jellyfish bells and oral arms obtained by bomb calorimetry are outlined in terms of Kcal 100 g −1 on a dry weight (dw) basis (Figure 2).The two-way ANOVA analysis revealed that the interaction between specimen's sex and anatomical part (independent variables) was statistically significant (p < 0.05, Table S2).Overall, statistically significant differences were found independently of the considered body part (p < 0.05), as bell was characterized by higher energy content then oral arms, both in female and male medusae.Fresh weight (g), bell diameter (cm), and length (cm) of jellyfish samples collected per sex and month.Data are reported as mean ± standard deviation (n = 50).According to the four sampling periods, female (or male) jellyfish marked by different letters for a given biometric characteristic, differ significantly (p < 0.05 by post hoc Tukey's Honestly Significant Difference (HSD) test).According to sex specimens, female and male jellyfish marked by the asterisk for a specific parameter in the same sampling month differ significantly (p < 0.05 by post hoc Tukey's HSD test).F = female.M = male.

Gross Energy Contents
The gross energy densities of female and male jellyfish bells and oral arms obtained by bomb calorimetry are outlined in terms of Kcal 100 g −1 on a dry weight (dw) basis (Figure 2).The two-way ANOVA analysis revealed that the interaction between specimen's sex and anatomical part (independent variables) was statistically significant (p < 0.05, Table S2).Overall, statistically significant differences were found independently of the considered body part (p < 0.05), as bell was characterized by higher energy content then oral arms, both in female and male medusae.
Additionally, considering the bell, statistically different energy values were detected between female and male specimens (p < 0.05).In fact, the sample from female bells showed the highest calorie value (621 Kcal 100 g −1 ), followed by the one from male bells (357 Kcal 100 g −1 ); whereas both male and female oral arms were characterized by inferior and nonsignificantly different calorie levels (174 and 151 Kcal 100 g −1 , respectively, p > 0.05).
The gross energy densities of female and male jellyfish bells and oral arms obtained by bomb calorimetry are outlined in terms of Kcal 100 g −1 on a dry weight (dw) basis (Figure 2).The two-way ANOVA analysis revealed that the interaction between specimen's sex and anatomical part (independent variables) was statistically significant (p < 0.05, Table S2).Overall, statistically significant differences were found independently of the considered body part (p < 0.05), as bell was characterized by higher energy content then oral arms, both in female and male medusae.Gross energy densities of female and male jellyfish bells and oral arms.Data are reported as Kcal 100 g −1 on a dw basis, in terms of mean ± standard deviation (n = 3).Samples marked by different letters differ significantly (p < 0.05 by post hoc Tukey's HSD test).

Crude Protein
Total nitrogen and protein contents of female and male jellyfish bells and oral arms are reported on a % dw basis by the bar graph in Figure 3.A nonstatistically significant interaction between the two independent variables (p > 0.05) was found (Table S3).Overall, investigated bells showed lower protein contents than oral arms (12.09-15.7% vs. 23.5-26.4%,p < 0.05), and nonstatistically significant differences were reported between female and male organisms (12.09-23.53% vs. 15.7-26.45%,p > 0.05).Additionally, considering the bell, statistically different energy values were detected between female and male specimens (p < 0.05).In fact, the sample from female bells showed the highest calorie value (621 Kcal 100 g −1 ), followed by the one from male bells (357 Kcal 100 g −1 ); whereas both male and female oral arms were characterized by inferior and nonsignificantly different calorie levels (174 and 151 Kcal 100 g −1 , respectively, p > 0.05).

Crude Protein
Total nitrogen and protein contents of female and male jellyfish bells and oral arms are reported on a % dw basis by the bar graph in Figure 3.A nonstatistically significant interaction between the two independent variables (p > 0.05) was found (Table S3).Overall, investigated bells showed lower protein contents than oral arms (12.09-15.7% vs. 23.5-26.4%,p < 0.05), and nonstatistically significant differences were reported between female and male organisms (12.09-23.53% vs. 15.7-26.45%,p > 0.05).

Phenolic Compounds
As mentioned in the Materials and Methods section, at an initial stage of analysis, samples underwent two different preparation procedures, based on the use of PBS and methanol, respectively.Once the method was optimized, the first procedure was preferred, due to better analytical performance in terms of speed and efficiency.The UV spectrophotometric analysis determined the total phenolic content in dried bells and oral arms of male and female organisms in terms of µg of gallic acid equivalents (GAE) g -on a dw basis (Figure 4).According to the two-way ANOVA test, the interaction of specimen's sex and anatomical part was statistically significant (p < 0.05, Table S4).In general, phenolic compounds were more abundant in oral arms rather than in bells (1892-2126 µg GAE g −1 vs. 914-1303 µg GAE g −1 , respectively, p < 0.05).Considering the same

Phenolic Compounds
As mentioned in the Materials and Methods section, at an initial stage of analysis, samples underwent two different preparation procedures, based on the use of PBS and methanol, respectively.Once the method was optimized, the first procedure was preferred, due to better analytical performance in terms of speed and efficiency.The UV spectrophotometric analysis determined the total phenolic content in dried bells and oral arms of male and female organisms in terms of µg of gallic acid equivalents (GAE) g -on a dw basis (Figure 4).According to the two-way ANOVA test, the interaction of specimen's sex and anatomical part was statistically significant (p < 0.05, Table S4).In general, phenolic compounds were more abundant in oral arms rather than in bells (1892-2126 µg GAE g −1 vs. 914-1303 µg GAE g −1 , respectively, p < 0.05).Considering the same anatomical part (bell or arms), a statistically significant difference (p < 0.05) was also determined between the phenolic contents of male and female specimens: 1303.32 µg GAE g −1 vs. 914.22µg GAE g −1 ; and 2126.56 µg GAE g −1 vs. 1892.69µg GAE g −1 , in males and females, respectively.

Fatty Acids
GC-MS analyses allowed us to determine around thirty fatty acid methyl esters (FAMEs), ranging from methyl hexanoate to methyl tetracosanoate, in dried samples from P. noctiluca (Table 1).With some exception, probably due to amounts below the detection limit, these FAMEs were common to each sample group, namely bells and oral arms of female and male specimens.
From a quantitative point of view, the main fatty acid classes varied depending on specimens' body part rather than sex.In fact, saturated fatty acids (SFAs), representing 70% ca. in bells and 65% ca. in oral arms, respectively; monounsaturated fatty acids (MUFAs), accounting for 15% ca. in all samples (p > 0.05); and polyunsaturated fatty acids (PUFAs), constituting 14% ca. in bells and 19% ca. in oral arms (p < 0.05).Only slight and nonstatistically significant differences (p > 0.05) could be attributed to the specimen's sex.For example, SFAs were slightly more abundant in male than female bells (70.6 vs. 69.5%,p > 0.05); MUFAs were slightly higher in female than male oral arms (15.8 vs. 14.6%, p > 0.05); and PUFAs content was slightly lower in male than female bells (13.8 vs. 15.4%,p > 0.05) and female than male oral arms (18.4 vs. 19.2%,p > 0.05).

Fatty Acids
GC-MS analyses allowed us to determine around thirty fatty acid methyl esters (FAMEs), ranging from methyl hexanoate to methyl tetracosanoate, in dried samples from P. noctiluca (Table 1).With some exception, probably due to amounts below the detection limit, these FAMEs were common to each sample group, namely bells and oral arms of female and male specimens.
From a quantitative point of view, the main fatty acid classes varied depending on specimens' body part rather than sex.In fact, saturated fatty acids (SFAs), representing 70% ca. in bells and 65% ca. in oral arms, respectively; monounsaturated fatty acids (MUFAs), accounting for 15% ca. in all samples (p > 0.05); and polyunsaturated fatty acids (PUFAs), constituting 14% ca. in bells and 19% ca. in oral arms (p < 0.05).Only slight and nonstatistically significant differences (p > 0.05) could be attributed to the specimen's sex.For example, SFAs were slightly more abundant in male than female bells (70.6 vs. 69.5%,p > 0.05); MUFAs were slightly higher in female than male oral arms (15.8 vs. 14.6%, p > 0.05); and PUFAs content was slightly lower in male than female bells (13.8 vs. 15.4%,p > 0.05) and female than male oral arms (18.4 vs. 19.2%,p > 0.05).
Table 1.Fatty acid methyl esters determined in the bells and oral arms of female and male specimens from Pelagia noctiluca.Data are reported on a dw basis, as average Gas Chromatography-flame ionization detector ( GC-FID) peak area percent ± standard deviation (n = 3).

Fatty Acid
Female

Major and Trace Element Profiles
The contents of four major elements (Na, Mg, K, and Ca), five essential trace elements (Fe, Cu, Zn, Mn, and Se), and five nonessential/potentially toxic trace elements (Cr, Ni, As, Cd, and Pb), were evaluated by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) in male and female jellyfish bells and oral arms, on a dw basis (Table 2).
Table 2. Elemental signatures of male and female jellyfishes' bell and oral arms revealed by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).Contents of major elements (mg 100 g −1 ) and trace elements (µg 100 g −1 ) are expressed as mean ± SD (n = 3) on a dw basis.Table S5 reports data of the validation procedure carried out by means of reference standards.The ICP-MS method showed good linearity for all the elements, with coefficients of correlation between 0.994 and 0.999.Acceptable recoveries between 93.49% (Ni) and 103.31% (Cr) were obtained.Evaluated in terms of RSD%, precision (intraday repeatability) and intermediate precision (interday repeatability), resulted to be within the range of 2.02 to 6.53%, and below 8.52%, respectively.

Bell
Major element signatures varied most in dependence of the organ rather than sex (Table 2).Overall, these metals appeared to bioaccumulate mainly in bells than the respective oral arms (p < 0.05) by the decreasing order of Na > Mg > Ca ≈ K, regardless of female and male sex (p > 0.05).As a result, Na was characterized by the highest levels (6544-8079 mg 100 g −1 for bells; 3877-3740 mg 100 g −1 for oral arms); while K was the less abundant one, showing contents inferior by one order of magnitude (196-229 mg 100 g −1 for bells; 126-143 mg 100 g −1 for oral arms) (Table 2).
Dealing with essential trace elements, they were found in the decreasing order of Fe > Cu > Zn > Mn > Se, in both male and female bells and oral arms.Fe was characterized by a behavior similar to major elements, since it was more abundant in bells (1309-1465 µg 100 g −1 ) than oral arms (854-1085 µg 100 g −1 ) (p < 0.05), regardless of specimens' sex.However, Cu, Zn, Mn, and Se completely inverted such trend, showing to be more abundant in oral arms than bells (p < 0.05).Also, Zn and Mn contents showed a slight dependence on the specimens' sex, though in a nonstatistically significant manner (p > 0.05), since males showed to most bioaccumulate such elements than females in both bell and oral arms.
Being not yet determined in any jellyfish species, Se levels ranged from 31.2 to 46.2 µg 100 g −1 in bells and from 100 to 115 µg 100 g −1 in oral arms (Table 2).
Nonessential/toxic trace elements were reported in the decreasing order of Cr ≈As > Ni > Pb > Cd.Such elements were slightly higher in oral arms than in bells (p > 0.05 in almost all cases), and, with the exception of Pb, marginally more bioaccumulated in male organisms than female ones (p > 0.05) as well.Cr and As varied respectively between 401 and 573 µg 100 g −1 and 412 and 528 µg 100 g −1 in female and male bells; while female and male oral arms reported Cr and As levels of 668 to 631 µg•100 g −1 and 690 to 663 µg•100 g −1 , respectively.Contrary to Cr and As, Pb exhibited the lowest contents in both bells (140-117 µg 100 g −1 ) and oral arms (154-132 µg 100 g −1 ), with comparable values between female and male specimens (Table 2).

Discussion
Despite of the short period (four months) of sample collection, morphometric parameters, namely body length, bell diameter, and wet mass were measured in order to integrate the knowledge about P. noctiluca in the Mediterranean Sea, particularly in the peculiar environment of the Strait of Messina.
As already reported by Sandrini et al. [66], the metabolism of P. noctiluca is directly proportional to the sea temperature, so that a temperature increase results in an increased metabolism rate and food requirement and accelerates growth.Also, enhanced food availability, in terms of zooplankton and ichthyoplankton blooms, typically occurring in the Strait of Messina during spring [67][68][69], may be responsible of the higher weights recorded in female and male organisms, in agreement with the findings reported by Rosa et al. for P. noctiluca caught in the same seawaters [70].Clearly, during March and April female organisms had the highest weight as they reached full gonads' maturity, resulting to be also sparingly bigger than males.In fact, biometrical and reproduction analyses performed by Rosa and coworkers [70] suggest that in the Strait of Messina, P. noctiluca reproduces with a maximum peak in late autumn/early winter.In this season, medusae diameter rapidly increases and most of the females are mature.They continue to grow and spawn until March-April, and in May they can reach the end of their life cycle, bringing empty, small, and slightly colored gonads.This could explain our observations on the decrease of weight during May and June in parallel to bell diameter reduction, especially in the female organisms.
As concerns bell diameter, findings of the present study are also in accordance with those reported by Rosa et al. [70]; in that case, bell diameter and water temperature were negatively correlated, providing an explanation for bell's reduction as the weather approaches the warm climate (May-June).Additionally, the mean bell's reduction recorded in May and June may be related to the presence of different age jellyfish groups, including younger (and consequently smaller) individuals, as suggested by Milisenda and colleagues [63].Even small variations of bell size had a remarkable effect on body weight, as previously reported by Lilley et al. [71], where bell diameter accounted for 98% of the variation in wet mass.
As a basic nutritional aspect, the gross energy content of P. noctiluca was first evaluated.The greater calorie values of bells compared to oral arms, both in female and male specimens, was presumably due to the presence of the high-calorie gonads.In particular, energy density of female bell exceeded the male counterpart, due to higher carbohydrate, lipid, and protein contents of eggs next to reproduction maxima period [63].The energetic value of jellyfish was barely addressed with respect to other common planktonic preys.Milisenda and coworkers carried out the energy count of P. noctiluca's gonads and oral arms, the latter resulting in similar values in male and female specimens (~52.3Kcal 100 g −1 ) [65].Doyle et al. [72] assessed the gross energy densities among several jellyfish species from Ireland (Cyanea capillata, Rhizostoma octopus and Chrysaora hysoscella) and between different tissues (bell and oral arms) within the same species as well.Besides the fact that each species was characterized by its own energy value, it was pointed out that oral arms were coherently characterized by higher gross energy contents than bells in any case.Indeed, gonads were not considered in these calorie counts, being excised from the respective bells, and separately assessed.Recently, higher gross energy densities in edible Malaysian jellyfish, such as Rhopilema esculentum, Acromitus hardenbergi, and Rhopilema hispidum, have been reported, with calorie values within in the range of 211 to 97 kcal•100 g −1 for bells and 282 to 200 kcal•100 g −1 for oral arms [38].However, as contemplated by the experimental plan, gonad-provided bells were apparently investigated in such study.
Proteins are known to constitute the largest portion of the organic matter present in jellyfish dried samples, which typically contain only lower amounts of either lipids or carbohydrates [70,71,73].Being presumably attributable to the dominant structural collagen distributed throughout the jellyfish body, the peculiar protein fraction is an appealing source of essential nutrients, thus, making such marine invertebrates potential suitable for food/feed purposes [46,74,75].Looking at P. noctiluca samples, oral arms reported higher crude protein content than bells, regardless of specimen's sex, probably not only because of the abundant structural collagen, but also due to the labile protein toxins present in the numerous nematocysts typically present in oral arms.Significant variations between the different body parts were confirmed also in C. capillata, R. octopus, and C. hysoscella, with oral arms contents (13.1-34.1%)higher than the ones detected in gonad excised bells (5.2-11.2%)[72]; and in R. esculentum, A. hardenbergi, and R. hispidum species, where bell and oral arms proteins were within the range of 19.95 and 38.12% and 33.6 and 53.8%, respectively [38].However, the qualitative and quantitative distribution of proteins both in bells and oral arms of the Mediterranean P. noctiluca should be further explored by appropriate proteomic techniques.
Polyphenols are widely acknowledged antioxidant compounds, widespread in nature and particularly concentrated in plants.When referring to polyphenols from the marine environment, literature indicates micro-and macroalgae as the main sources for such compounds [76][77][78].Nevertheless, in consideration of the scope of the present study, namely to investigate the nutritional and nutraceutical properties of the mauve stinger, the determination of the total polyphenol content was reasonably carried out.Previous reports on total polyphenols in jellyfish species are very few.Leone et al. [74,75] reported the total phenolic contents of three jellyfish species, none belonging to Pelagia genus.In two of them, ~1800-2000 µg GAE g −1 (dw) were determined.Such findings were quite in accord with the present report.The total phenolic content determined in P. noctiluca is only a preliminary and rough screening that should be followed by a targeted qualitative analysis for the identification of the specific polyphenolic structures.In fact, it has been speculated that amino acidic residues from the protein fraction could contribute to the value of the total phenolic content [74].
The results obtained from the analyses of fatty acids can be hardly compared with previous studies, since P. noctiluca results to be underinvestigated with respect to this issue.Mastronicolis et al. reported 0.19% ww of total lipids, 41.7% of which were free fatty acids [79]; whereas Cardona et al. analyzed the fresh mass of P. noctiluca obtaining a fatty acid composition roughly represented by 70% palmitic acid, 15% pentadecanoic acid, 6% EPA, and 2% DHA [80].Approximately, the fatty acid profiles here presented are in accordance with those reported for other jellyfish species, collected in the Mediterranean Sea and investigated with the same analytical procedure here applied [74,80,81].
Notoriously, essential fatty acids belonging to the ω-6 and ω-3 groups, and are significantly present in a variety of vertebrate and invertebrate marine species, and, when correctly balanced, confer a relevant nutraceutical value to the source, which they come from [82].As can be observed in Table 2, essential ω-3 fatty acids, such as EPA and DHA, and essential ω-6 fatty acids, such as linoleic acid, were determined also in the Mediterranean P. noctiluca, being more abundant in oral arms than bells.Since pioneer works on the positive correlation between essential fatty acids and human health, different schools of thought have arisen during the years, attributing a higher healthy power one time to ω6 acids, another to ω3 acids.However, in the last decades, scientists concluded that it is the balance between ω6 and ω3 that promotes normal development and preserves homeostasis.Ideally, this ratio should be 1:1, due to the observation of chronic diseases especially in those individuals having a diet deficient of ω3fatty acids and, consequently, with an unbalanced ω6/ω3 ratio [83].In this respect, all samples of P. noctiluca reported a correct balance of the two classes of fatty acids, thus confirming its nutraceutical value.In particular, bell and oral arms from female organisms showed the ratios closest to 1:1 (respectively, 0.89 and 0.80).
The screening of major and trace elements provided further insights on the nutritional and nutraceutical value of P. noctiluca.Jellyfish bioaccumulates and transfers essential minerals and trace elements from lower trophic levels to high-order fish predators, having a key role in balancing any potential nutritional shortfall of the food chain.The same applies to nonessential and potentially toxic elements, which could instead represent a threat for the health of marine ecosystems, and, not least, human consumers [84,85].To date, few published data dealt with inorganic elements in this gelatinous zooplankton, mainly to test the environmental quality of coastal systems [81,[84][85][86][87], rather than for food/feed purposes [38].
According to obtained data, jellyfish bell exhibited higher Na, Mg, K, and Ca levels than oral arms, probably because of the buffering activities carried out to maintain the osmotic balance and, thus, the floating capacity of the bell [38].Although such a mineral distribution was confirmed also by previous works on different jellyfish species from Portuguese (Catostylus tagi) and Australian (Cassiopea sp.) coasts [81,84], not comparable contents were detected, because of clear taxonomic and ecological reasons.For example, Mg in bell and oral arms belonging to C. stagi and Cassiopea sp. was equal to 328 mg 100 g −1 and 240 mg 100 g −1 [81], and 74.4-129.7 mg 100 g −1 and 73.4-125.7 mg 100 g −1 , respectively [86].Ca levels distributed between bells and oral arms within the ranges 25.8 to 46.6 mg 100 g −1 and 27.2 to 44.6 mg 100 g −1 [84] in the Australian Cassiopea sp.; whereas, in the Portoguese C. stagi, bell and oral arms showed Ca contents equal to 1026 mg 100 g −1 and 736 mg 100 g −1 [81].
Trace elements, such as Fe, Zn, Cu Mn, and Se, are well known to be both limiting nutrients and toxicants.In fact, they are essential for the metabolism of vertebrate and invertebrate marine organisms, as they constitute a variety of metalloproteins and antioxidant enzymes and play a key role in cellular detoxification activity, but, at the same time, they become toxic at high concentrations, leading to damaging oxidative processes [88].In the same way as for major elements, the comparison of trace element levels from P. noctiluca with those from other jellyfish species addressed in previous works [81,84,85] becomes challenging.In fact, an increasing discrepancy seems to appear dealing thoroughly with getting smaller contents of essential trace elements; however, similarly to P. noctiluca, also other species, such as C. stagi [81], Cassiopea sp.[84], and Cotylorhiza tuberculata [85], showed to more bioaccumulate such trace elements in oral arms than bells.These distribution patterns could be related to the metal uptake via food, occurring by oral arms directly involved in suctorial feeding, and fine filtering functions [89].Assuming that the elemental requirements of an organism, including the human being, are driven almost entirely by utility (i.e., cellular function, with shifts in biological requirements decoupled from corresponding environmental abundances), and that they should be assessed in depth case-by-case, it is reasonable to hypothesize that both essential major and trace elements found in the Mediterranean P. noctiluca, may be exploited for developing natural food and aquafeed supplements.In the latter case, other jellyfish species, such as A. aurita and Chrysaora pacifica, have already been demonstrated to support the growth and survival of specific farmed species, thanks also to a peculiar metal profile [90].
The main source of nonessential and potentially toxic elements, such as Cr, As, Ni, Pb, and Cd, typically comes from anthropogenic activities, negatively impacting the health status of marine environments.Previous works have already pointed out that different jellyfish species can bioconcentrate these harmful elements and reflect a time-integrated measured of their levels in the water, demonstrating to be useful bioindicators of coastal environments [84,85].The uptake and accumulation of nonessential/toxic elements in P. noctiluca varied, although in nonsignificant manner, between selected tissues and sex specimens.However, as the present study does not rely on an ecotoxicological basis, and no literature on the pollutant accumulation capacity of the Mediterranean P. noctiluca has been yet produced, no conclusion will be drawn about the health status of such coastal areas.Nonetheless, obtained data could be useful for stressing on the bioaccumulating capacity of jellyfish, other than constituting an input for future environmental studies on such species.
Concerning the toxicological value of P. noctiluca as aquafeed and food supplement, it is useful to mention the Commission Regulation (EU) N. 744/2012 [91] setting the limits of heavy metals in animal feeds, and the Commission Regulation (EC) N. 629/2008 [92], amending the Regulation (EC) N. 1881/2006 and fixing the maximum levels of heavy metals in food supplements.Indeed, according the EU Regulation N. 744/2012, the investigated samples were characterized by means concentrations of As and Pb (5.58 and 1.35 mg kg −1 ) well within the maximum contents set at 10 and 5 mg kg −1 for a complete feed, respectively, and at 10 and 10 mg kg −1 for a complementary feed, respectively, in every case with a moisture content of 12%.On the other hand, according the EC Regulation (EC) N. 629/2008, P. noctiluca showed mean levels of Pb and Cd (1.35 and 0.45 mg kg −1 ), well below the limits set respectively at 3.0 and 1.0 mg kg −1 .
As a result, potential aquafeed and food supplements based on P. noctiluca from the Strait of Messina would be safe for farmed fish and human consumers, in terms of toxic heavy metals.
The pioneering chemical composition data of P. noctiluca herein discussed may be a valuable starting point for acknowledging its nutritional and nutraceutical properties for food and aquafeed purposes.
Concerning food applications, it has been widely discussed the relevant value of certain jellyfish species as food for human consumption in Asian countries.Additionally, a specific protein fraction of the edible R. esculentum with high antioxidant power [93] and a new bioactive polysaccharide isolated from edible jellyfish [94] were proposed for developing novel and natural food supplements.
Regarding aquafeed field, Marques et al. recently revealed that all developmental stages of Aurelia sp. were accepted as a feed source by Sparus aurata [73]; Aurelia aurita was also demonstrated to be a valid feed source for Thamnaconus modestus, when other common preys were not visible [95], and for rearing commercial fish such as Pampus argenteus [96].Furthermore, Milisenda and colleagues proposed P. noctiluca as valuable food source for fish predators, such as Boops boops, in the Strait of Messina, due to an increased energy content during the period of gonad maturation, and to the high available biomass present during blooms [65].
Clearly, before getting into any practical application of P. noctiluca, other in vitro studies will be necessary for firstly developing the most suitable procedures of biomass processing and venom neutralization.Then, if the applications turned out to be economically and realistically feasible, further research will be required to investigate (i) the in vivo toxicity and effectiveness of food/aquafeed supplements and (ii) the potential food web cascading effects due to the harvesting of wild populations.

Sample Collection
Specimens of P. noctiluca were sampled from the Strait of Messina, South Italy.In this peculiar environment, the mauve stinger can be found in the Strait of Messina during the whole year, reaching the highest diffusion in the period of March to June.Approximately 400 adult specimens were collected from March to June 2017 (~100 organisms per month), in the coastal waters of Capo Peloro (Messina).Adult jellyfish samples were taken by means of a small net from water's edge, put in tanks filled with seawater, and immediately transported to laboratory for morphometric measurements and sex and chemical determinations.The experimental protocol was developed in accord with the ethical standards reported in the European Directive 2010/63/EU on the protection of animals used for scientific purposes [97].

Biometrics and Sex Determination
Maximum length, bell diameter, and total weight (fresh mass) of each jellyfish sample were measured with the support of a ruler and of a lab-made apparatus, shown in Figure 5.Each specimen was placed inside the most appropriate circular hole, chosen on the basis of bell diameter (Figure 5A).Oral arms were left suspended on the other side of the flat surface.Length was measured from the top of the bell to the end of the longest oral arm, along the oral-aboral axis (Figure 5B).Bell diameter was considered as the maximum distance between distal tips of opposed interradial rhopalia.Biomass (w/w) was measured after washing medusae with distilled water for salt removal.For each specimen, the sex was determined by inspection of the gonads through a stereomicroscope (Carl Zeiss Stemi SV11): male individuals presented purple follicles, while female subjects were characteristically pink-red colored, and crowded by Milisenda et al. [65].When identification of sex resulted to be challenging or uncertain, histological assessments were carried out following a standard protocol [98].
Mar. Drugs 2019, 17, x FOR PEER REVIEW 13 of 21 5A).Oral arms were left suspended on the other side of the flat surface.Length was measured from the top of the bell to the end of the longest oral arm, along the oral-aboral axis (Figure 5B).Bell diameter was considered as the maximum distance between distal tips of opposed interradial rhopalia.Biomass (w/w) was measured after washing medusae with distilled water for salt removal.
For each specimen, the sex was determined by inspection of the gonads through a stereomicroscope (Carl Zeiss Stemi SV11): male individuals presented purple follicles, while female subjects were characteristically pink-red colored, and crowded by Milisenda et al. [65].When identification of sex resulted to be challenging or uncertain, histological assessments were carried out following a standard protocol [98].

Sample Lyophilization
After biometrical measurements and sex determinations were carried out, the organisms were sacrificed separating the bells by the manubrium bearing the oral arms, with the help of a Teflon knife, avoiding metal cross-contamination.Bells and oral arms were grouped to provide four different pools (n = 200 each), as reported in Table 3.Each pool was weighed, cleansed thoroughly with distilled water, minced, and lyophilized by an Alpha 1-2/LD Plus freeze dryer (Martin Christ, Osterode, Germany), for 72 h at −55 °C using a chamber pressure of 0.110 mbar.Then, the freeze dried pools were weighed, and stored at −20 °C until use.For each pool, a moisture content of ~95% w/w and a yield of 5% w/w were assessed.

Sample Lyophilization
After biometrical measurements and sex determinations were carried out, the organisms were sacrificed separating the bells by the manubrium bearing the oral arms, with the help of a Teflon knife, avoiding metal cross-contamination.Bells and oral arms were grouped to provide four different pools (n = 200 each), as reported in Table 3.Each pool was weighed, cleansed thoroughly with distilled water, minced, and lyophilized by an Alpha 1-2/LD Plus freeze dryer (Martin Christ, Osterode, Germany), for 72 h at −55 • C using a chamber pressure of 0.110 mbar.Then, the freeze dried pools were weighed, and stored at −20 • C until use.For each pool, a moisture content of ~95% w/w and a yield of 5% w/w were assessed.

Gross Energy Assessments
To measure the gross energy content, a benchtop isoperibol calorimeter (Parr ® 6200 Oxygen Bomb Calorimeter, Parr Instrument Company, Moline, IL, USA) was employed.
Approximately 1 g of each powdered pool was placed in a 1108 model oxygen bomb.To determine the gross energy densities of bombed samples, the calorimeter chamber was previously calibrated for the heat of combustion of 1 g of benzoic acid (26.46 kJ g −1 ), under controlled and reproducible operating conditions.In fact, the known amount of heat produced by the combustion of the calibration standard determined the energy equivalent (W) per change in water temperature between initial and postcombustion of the sample (∆T).Therefore, the energy content of each sample (ES) was calculated as follows ES = W × ∆T/exact sample weight Each sample pool was run in triplicate.Results were expressed as Kcal 100 g −1 , dw.

Crude Protein
The crude protein content was determined using the AOAC Official Method 976.05 (automated Kjeldahl method) [99].Approximately 1 g of each powdered pool was separately digested by the SpeedDigester K-439 (Büchi, Switzerland) and then analyzed by the KjelMaster System K-375 (Büchi, Switzerland) and equipped with a scrubber of gases and vapors (Scrubber K-415, Büchi, Switzerland).For the calculation of the % protein in a sample, the obtained % nitrogen was multiplied by a conversion factor of 6.5.Each determination was conducted in triplicate.

Total Phenolic Content
As a preliminary part of method development, an initial group of samples was subjected to two different extraction procedures: (i) with PBS (phosphate-buffered saline), pH 3.5 and (ii) with 80% methanol [74].Aliquots (1.0 g) of each lyophilized sample pool were added with 16 mL of PBS and shaken for 2 h at 4 • C, in one case; with 16 mL of methanol and shaken for 16 h at 4 • C, in the other case.Successively, samples were homogenized at 9000 rpm and 4 • C for 30 min.To 1.0 mL of supernatant, 5.0 mL of Folin-Ciocalteau reagent and 5.0 mL of sodium carbonate (20%) were added.The resulting solution was kept in the dark for 2 h, and later analyzed at the UV-Vis spectrophotometer, model UV-2401PC (Shimadzu, Milan, Italy).The wavelength of absorbance was set at 760 nm.A 5-point calibration plot was built up by using solutions of gallic acid in methanol in the range 50 to 2000 µg mL −1 .Each point corresponded to three replicates.

Fatty Acids
The extraction of fatty acids was carried out following the procedure reported by Bligh & Dyer [100].An aliquot (10.0 mg) of each lyophilized pool was homogenized with 1.0 mL of chloroform (CHCl 3 ) and 3.0 mL of methanol (MeOH).This mixture was then added with 1.0 mL of MeOH and 1.0 mL of water, homogenized, let to settle, and filtered through paper.After settling, the filtrate was centrifuged at 3000 rpm for 15 min.Two layers were formed: the bottom one (CHCl 3 ), containing the isolated lipids, was transferred to a rotating evaporator, model P/N Hei-VAP Precision ML/G3 (Heidolph Instruments GmbH & Co., Schwabach, Germany).The upper layer (MeOH/H 2 O) was subjected again to all the steps above described in order to reach an exhaustive extraction.For the fatty acid methylation, the dried lipidic extract was recovered through addition of 1 mL hexane, then added with reagent (CH 3 OH/H 2 SO 4 , 9:1), and heated at 100 • C for 1 h.The hydrocarbon layer was collected and injected into the GC instrumentation.Qualitative analyses were carried out in a GCMS-TQ8030 (Shimadzu, Kyoto, Japan) system equipped with an AOC-20i autosampler and a capillary column Supelco SLB-IL100 (60 m × 0.25 mm, film thickness 0.20 µm).GC conditions were set as follows: injector, 280 • C; injection volume: 1.0 mL; head pressure: 26.7 kPa; carrier gas: He, at a linear velocity of 30.0 cm/s (constant); split ratio 1:100; oven temperature program: 50-280 • C at 3 • C/min, held 10 min.MS conditions: operation mode was in full scan; ion source and interface temperatures were 220 • C and 250 • C, respectively; and scan mass range was 40 to 400 m/z.For FAMEs identification, a triple means methodology was used: (i) spectral matching with Wiley and NIST databases; (ii) co-injection with standards (Supelco 37 component FAME mix, Supelco, St. Louis, MO, USA); and (iii) comparison with literature data [101].Data handling was performed by GCMS solution software.Quantitative analyses were carried out on a Master GC-DANI system, equipped with a capillary column Supelco SLB-IL100 (60 m × 0.25 mm, film thickness 0.20 µm).Oven temperature program: 120-200 • C at 1 • C min −1 (10 min).Injector and FID temperatures were respectively set at 220 and 240 • C. Carrier gas was He, at a constant linear velocity of 30.0 cm s −1 .FID conditions: sampling frequency: 25 Hz; gases: makeup (He), 25 mL min −1 ; H 2 , 40 mL min −1 ; air, 280 mL min −1 .Data were processed through the Clarity software (Dani).All determinations were run in triplicate.

Elemental Analysis
Approximately 500 mg of each lyophilized pool were digested with 10 mL of HNO 3 , exploiting a closed-vessel microwave digestion system Ethos 1 (Milestone, Bergamo, Italy) equipped with PTFE vessels.The mineralization was carried out by setting the following temperature program; 0-200 • C in 10 min (step 1), 200 • C held for 5 min (step 2), and 200-220 • C in 5 min (step 3), with a constant microwave power of 1000 W. Due to the expected very high salt concentrations, each digested pool was diluted by ultrapure water with a dilution factor of 1000, and stored at 4 • C until ICP-MS analysis.A quadrupole ICP-MS iCAP Q (ThermoScientific, Waltham, MA, USA), equipped with an ASX-520 autosampler (Cetac Technologies Inc., Omaha, NE, USA), was employed for the analytical determinations.The ICP-MS operating conditions are shown in Table 4.All samples were analyzed in triplicate, along with blanks to check for any loss or cross contamination.For quantitative purposes, the external calibration procedure was carried out with the help of multielemental standard solutions.For building up six point calibration plots, six different concentrations were evaluated per analyte, and each point resulted from triplicate extractions and analyses [102].For method validation, a linear least-square regression analysis of the calibration graphs was performed to check for the linearity between the instrumental response and the nominal concentration of each elemental standard.The intra-assay and interassay variabilities were determined by quantifying three replicates on the same day and six consecutive days, respectively.

Statistical Analysis
With the exception of biometrics, all data are reported as mean ± standard deviation of triplicate measurements.Statistical analysis was conducted by the SPSS Statistics Software v. 21.0 (SPSS Inc., Chicago, IL, USA), performing for each investigated variable a two-way analysis of variance (ANOVA) followed by the Tukey' s honestly significant difference (HSD) post-hoc test.Level of significance was set at p < 0.05

Figure 1 .
Figure1.Fresh weight (g), bell diameter (cm), and length (cm) of jellyfish samples collected per sex and month.Data are reported as mean ± standard deviation (n = 50).According to the four sampling periods, female (or male) jellyfish marked by different letters for a given biometric characteristic, differ significantly (p < 0.05 by post hoc Tukey's Honestly Significant Difference (HSD) test).According to sex specimens, female and male jellyfish marked by the asterisk for a specific parameter in the same sampling month differ significantly (p < 0.05 by post hoc Tukey's HSD test).F = female.M = male.

Figure 2 .
Figure 2. Gross energy densities of female and male jellyfish bells and oral arms.Data are reported as Kcal 100 g −1 on a dw basis, in terms of mean ± standard deviation (n = 3).Samples marked by different letters differ significantly (p < 0.05 by post hoc Tukey's HSD test).

Figure 2 .
Figure 2. Gross energy densities of female and male jellyfish bells and oral arms.Data are reported as Kcal 100 g −1 on a dw basis, in terms of mean ± standard deviation (n = 3).Samples marked by different letters differ significantly (p < 0.05 by post hoc Tukey's HSD test).

Figure 3 .
Figure 3.Total nitrogen and protein contents of female and male jellyfish bells and oral arms.Data are reported on a % dw basis, as mean ± standard deviation (n = 3).Samples marked by different letters differ significantly (p < 0.05 by post hoc Tukey's HSD test).

Figure 3 .
Figure 3.Total nitrogen and protein contents of female and male jellyfish bells and oral arms.Data are reported on a % dw basis, as mean ± standard deviation (n = 3).Samples marked by different letters differ significantly (p < 0.05 by post hoc Tukey's HSD test).

Figure 4 .
Figure 4. Total phenolic content (µg GAE g −1 ) determined in male and female jellyfishes' bell and oral arms.Data are expressed as mean ± standard deviation (n = 3), on a dw basis.Samples marked by different letter differ significantly (p < 0.05 by post hoc Tukey's HSD test).

Figure 4 .
Figure 4. Total phenolic content (µg GAE g −1 ) determined in male and female jellyfishes' bell and oral arms.Data are expressed as mean ± standard deviation (n = 3), on a dw basis.Samples marked by different letter differ significantly (p < 0.05 by post hoc Tukey's HSD test).

Figure 5 .
Figure 5. Lab-made apparatus for morphometric measurements.(A) Jellyfish were placed inside the most appropriate circular hole, chosen among different predefined measures.(B) Length was measured from the top of the bell to the end of the longest oral arm.

Figure 5 .
Figure 5. Lab-made apparatus for morphometric measurements.(A) Jellyfish were placed inside the most appropriate circular hole, chosen among different predefined measures.(B) Length was measured from the top of the bell to the end of the longest oral arm.

Table 3 .
Sample pools considered for the present study.

Table 3 .
Sample pools considered for the present study.

Table 4 .
ICP-MS operating conditions applied to elemental analysis.