**3. 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 [67–69], may be responsible of the higher weights recorded in female and male organisms, in agreemen<sup>t</sup> 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] sugges<sup>t</sup> 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 (~52.3 Kcal 100 g<sup>−</sup>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<sup>−</sup><sup>1</sup> for bells and 282 to 200 kcal·100 g<sup>−</sup><sup>1</sup> 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 [76–78]. 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<sup>−</sup><sup>1</sup> (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 ω3- fatty 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 [81,84–87], 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<sup>−</sup><sup>1</sup> and 240 mg 100 g<sup>−</sup><sup>1</sup> [81], and 74.4–129.7 mg 100 g<sup>−</sup><sup>1</sup> and 73.4–125.7 mg 100 g<sup>−</sup>1, respectively [86]. Ca levels distributed between bells and oral arms within the ranges 25.8 to 46.6 mg 100 g<sup>−</sup><sup>1</sup> and 27.2 to 44.6 mg 100 g<sup>−</sup><sup>1</sup> [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<sup>−</sup><sup>1</sup> and 736 mg 100 g<sup>−</sup><sup>1</sup> [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 ye<sup>t</sup> 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−<sup>1</sup> for a complete feed, respectively, and at 10 and 10 mg kg−<sup>1</sup> 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.

### **4. Materials and Methods**
