**3. Discussion**

The apparent increase of global jellyfish abundance in coastal marine ecosystems has recently attracted scientific interest for the potential impacts on human activities and ecosystem functioning. Also, the possible use of jellyfish biomass as a source of energy and bioactive compounds useful for pharmaceutical and nutritional applications has been suggested [30,31]. In this context, understanding the biological mechanisms underlying jellyfish outbreaks is crucial to predict and/or mitigate impacts of recurrent bloom events. The occurrence of jellyfish outbreaks is also directly linked to the reproductive success. The present paper represents the first insight into the biochemical composition of ovaries and the lysozyme antibacterial activity associated with oocytes of the barrel jellyfish *R. pulmo* in order to investigate aspects related to the mechanisms boosting the success of sexual reproduction.

In *R. pulmo*, more than 90% of the ovary volume is composed of water in accordance with previous studies on *R. octopus* [32] and other scyphozoan jellyfish gonads (e.g., *Cyanea capillata*, *Chrysaora hysoscella*, and *Pelagia nocticula* [5,32]). The organic matter in *R. pulmo* ovaries represented 60% of dry weight and was composed mainly of proteins, lipids, and low content of carbohydrates, corroborating the general trend in gonadal composition recorded in other scyphozoans, including *P. nocticula* [5,32]. In particular, proteins were twofold concentrated in *R. pulmo* female gonads compared to *C. capillata* and threefold compared to *R. octopus* gonads [32]. The biochemical composition of *R. pulmo* ovaries reflects the composition of the entire jellyfish in which the organic content is mainly represented by protein, followed by lipid and carbohydrate fractions [21].

The 1H NMR characterization of both lipid and aqueous extracts of *R. pulmo* female gonads provided advanced information on the chemistry of this jellyfish compartment. As known [22,33], the untargeted 1H NMR-based metabolomic approach could be used to provide simultaneous determination of the end products of metabolism, such as small-molecular-weight molecules in solution [24]. In the present study, many 1H NMR signals of the aqueous extract were due to free amino acids, such as leucine, isoleucine, valine, threonine (essential amino acids) and alanine, glycine, proline, and glutamate, representing also the dominant amino acids in the gonads of the edible Asiatic jellyfish *Rhopilema esculentum* [34] and other edible jellyfish species [35]. In the aromatic region, 1H NMR analysis revealed also the presence of tyrosine, which is considered an essential amino acid for humans, useful during stages of human prenatal development [36]. Alanine, glycine, and glutamate are also implicated in other physiological processes and on account of these features are considered useful as antioxidant constituents in foodstuffs and beverages [37]. Thus, the *R. pulmo* ovaries could

represent a source of amino acids exploitable for nutraceutical and pharmaceutical applications as well as a source of proteins for the development of innovative dietary supplements for fish nourishment. In this framework indeed, considering the growing cost of fish feed worldwide, each innovative natural resource as a potential ingredient in their preparation must be considered. Furthermore, the aqueous extract was characterized by the presence of compounds with important metabolic roles in marine invertebrates [23,38,39], such as the osmolytes trimethylamine *N-*oxide (TMAO), betaine, taurine, and homarine. Interestingly, in the colonial hydropolyp *Hydractinia echinata* (Hydrozoa), it was observed that betaine, homarine, and trigonelline, when present simultaneously, play a regulatory role during the development and prevent the onset of metamorphosis [40,41]. On account of the high amount evidenced in *R. pulmo* gonads, it is likely that, as in the case of *H. echinata*, also in the here-investigated jellyfish, these compounds could be involved in maintaining the larval state until an appropriate signal allows metamorphosis. Moreover, TMAO is an osmolyte that commonly occurs in several marine animals and it has been found to neutralize the effects of hydrostatic pressure on cnidarian, fish, and mammalian [42]. The densities differ widely among habitats, species, and season and ontogeny within species A relationship exists between the concentration of TMAO (and betaine) in muscle tissue and lipid. At present, we do not rule out the possibility that, in *R. pulmo* ovaries, TMAO plays a role for its protein-stabilizing attributes as already hypothesized for several marine organisms [43].

In the female gonads of *R. pulmo*, a higher content of total lipids was also recorded in comparison with the total jellyfish [21]. As reported in literature [21,44], the lipid composition of jellyfish can be considerably influenced by several external factors such as diet, size, and age of organism. The lipid NMR analysis of *R. pulmo* gonads showed the presence of different lipid classes. As already observed in the whole jellyfish [21], the analysis of *R. pulmo* ovaries confirmed the presence of a high content of ω-3 PUFAs. In the gonads of marine invertebrates, there is a notable richness of PUFAs, particularly 20:5 ω-3 and 22:6 ω-3 [45]. PUFAs actively participate in gonad maturation, egg quality [46], and larval growth of fish [47]. In the common octopus, *Octopus vulgaris*, PUFAs can improve membrane fluidity and flexibility of spermatozoa membrane, and are actively implicated in the regulation of cellular movement, gonadal metabolism of lipids, and fusion capacity [48]. In crustaceans and mollusks, PUFAs not only determine hatching and growth [49] but also play an important role in metabolism processes, like production of prostaglandins and hormones, and regulate ionic fluxes [50]. Among the categories of fatty acids (FAs) of *R. pulmo* ovaries, the two signals of EPA and DHA were also revealed. Noteworthy, these categories of fatty acids were already recorded in other jellyfish, including *Aurelia* sp., whose fatty acid profiles were broadly similar to 16:0, EPA, 18:0, AA, and DHA as the five main components accounting for around 66% of the total FAs. Furthermore, EPA and DHA have been detected in different species of *Aurelia* jellyfish [44,51]. The presence of ω-3 PUFAs, mainly DHA and EPA, in the gonads of *R. pulmo* suggests their potential exploitation as sources of these compounds for the application in the pharmaceutical field. It is well known indeed that ω-3 PUFAs, DHA and EPA, possess antioxidant and anti-inflammatory properties useful for potential treatment strategies for mental health and neuro-inflammation-induced memory deficits [52,53]. Moreover, taking into account that diets for fish are usually enriched with additional supplements of EPA and DHA, the gonads of *R. pulmo* could furnish these essential FAs to be added in the production of fish feed.

An antibacterial lysozyme-like activity was detected in the oocyte lysate of *R. pulmo*. As in the majority of scyphozoans, also in *R. pulmo*, reproduction is external; thus, an antibacterial activity may prevent eggs and embryos from being overgrown and killed by pathogenic bacteria. To our knowledge, this is the first record of an antimicrobial activity in jellyfish eggs. Many marine taxa synthesize specific metabolites to protect themselves against the settlement and growth of microbial agents. For example, surface attachment and growth of several marine bacteria are inhibited by the extracts from the eggs of several coral species [54]; potentially pathogenic bacteria, including a *Vibrio* sp., are subjected to the toxic action induced by the extracts of various developmental stages of the soft coral, *Parerythropodum fulvum* [55]. Gunthorpe and Cameron [56,57] found that in some species of soft corals, the extracts exerted an antibiotic activity negatively related to the presence of immature gonads, suggesting that reproductive status represented a significant predictor of antimicrobial activity [56]. A similar phenomenon was found in the octocoral *Lobophytum compactum*; indeed, antimicrobial diterpenes were selectively included into egg lipid material [58] detectable in adults before spawning and absent afterwards. Moreover, the extracts of the damselfish *Pomacentrus mollucensis* eggs is defended chemically [59]. In addition, egg extracts of the coral species *Montipora digitata* are able to produce growth inhibition of *Escherischia coli* [60]. Among marine invertebrates, also in echinoderms, characterized by external fertilization, the eggs and larvae from the regular echinoid *Paracentrotus lividus* exert an antibacterial lysozyme-like activity [16]. Lysozyme-like proteins have also already been evidenced in other cnidarians [61,62]. Regarded as the best and most active enzyme involved in the innate immunity [63], lysozyme is a glycoside hydrolase whose constitutive levels defend the organism from pathogenic bacteria present in the surrounding environment and regulate natural symbiotic microflora. Besides antimicrobial activity, lysozymes play a role in digestion, antiviral, anti-inflammatory, and antitumor activities, taking part in the innate immunity as first defensive line [64]. It is well known that lysozyme activity is affected by various factors such as temperature, pH, and salts [65,66]. In the case of *R. pulmo* egg lysate, the highest lysozyme-like activity is detected at pH 4 and the ionic strength 0.175. A similar result was already obtained from egg lysate of the sea star *Marhasterias glacialis* (maximum of activity at pH 4.2 and the ionic strength 0.175) [16]. Further studies will be undertaken to assess whether the category of lysozyme involved in egg protection is the same in marine invertebrates as well as to estimate the antibacterial activity against other living microorganisms besides *Micrococcus luteus.* At this stage, we only focused on the occurrence of a lysozyme-like activity in the whole oocyte lysate. However, to further elucidate the mechanisms related to *R. pulmo* egg defenses, it is necessary to perform the isolation, purification, and quantification of the effectors of such antimicrobial activity which will be carried out in the near future.

The evidence of a lysozyme-like activity in *R. pulmo* oocyte lysate suggests that this species may also represent a new and exciting resource for the extraction of potent antibacterial agents and encourages the potential use of the jellyfish for lysozyme-based preparations in pharmaceutical research. Currently, lysozyme is used for pharmaceutical preparations due to the therapeutic effectiveness of lysozyme based not only on its ability to control the growth of bacteria but also to modulate the immune responses of the host. Moreover, the treatment with lysozyme leads to a regression in the growth of some tumor cells [67]. Lysozyme can be also used in the treatment of a wide range of infections in humans since it has no toxic effect on humans and thus it is a good candidate for the use of epidermal and cosmetic formulations. Finally, considering that controlling bacterial infections is currently one of the main problems of aquaculture on an industrial scale, lysozyme is attracting the interest of researchers for its potential applications in aquaculture.

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

### *4.1. Sample Collection and Preparation*

Sixty specimens of *R. pulmo* adult medusae (umbrella diameter > 25 cm at sexual maturity; Basso, personal observations) were sampled at the Ginosa Marina (Ionian Sea 40◦25.7 N, 16◦53.1 E; Italy) throughout 2017 with a 1 cm mesh hand net from a boat. Immediately after sampling, jellyfish were transported into the laboratory and washed with filter-sterilized seawater (0.2 μm, Millipore) to remove the mucus layer produced during transport. Ovaries appeared from pink to orange, with easily distinguishable eggs. When gender determination was uncertain visually, a small piece of gonad tissue was removed and examined under the stereomicroscope. The ovaries were carefully dissected with microscissors at the stereomicroscope to avoid loss of gonadic tissue or accidental inclusion of subumbrellar or exumbrellar tissues and mature eggs were collected from a number of mature gonads. Each gonad was then divided in two aliquots. The first aliquot was frozen at −80 ◦C in liquid nitrogen to be lyophilized and then employed for the biochemical and NMR analyses; the second aliquot was

employed to obtain the mature eggs. In particular, the eggs were obtained in pasteurized seawater (PSW) by placing ovaries on four layers of gauze. Eggs were allowed to settle and, after removal of the supernatant, were resuspended in sufficient PSW to obtain a 10% (*v*/*v*) suspension. After that, the eggs were gently swirled and then centrifuged at 12,000 g for 30 min [68]. The resulting supernatant (oocyte lysate) was dialyzed against distilled water, then lyophilized, and then concentrated 10-fold in PSW and used to evaluate the lysozyme-like antibacterial activity.
