Ascidians (Tunicata) are important members of marine benthic communities, with over 3000 species described worldwide inhabiting polar, tropical, and temperate environments, either in shallow or deeper habitats (Shenkar and Swalla, 2011). Their phylogenetic relatedness to vertebrates, coupled with a somehow fast and easy development in vitro, has attracted the attention of developmental biologists since centuries (Corbo et al., 2001; Dehal et al., 2002; Imai et al., 2006). In the last decades, ascidians also generally raised additional interest as potential alternative food sources in human consumption due to the presence of compounds with high nutritive content, such as proteins, amino acids, lipids, and secondary metabolites (Zhao and Li, 2016; Palanisamy et al., 2017; Watters, 2018; Dou and Dong, 2019), and examples of wide phenotypic and environmental plasticity, which is turn reflected in a high invasive potential (Bullard and Carman, 2009; Zhan et al., 2015; Rocha et al., 2019; Atalah et al., 2021). Moreover, they are source of a variety of bioactive chemical compounds, which include peptides, alkaloids, polyethers, macrolides, terpenes, and polysulfides (Palanisamy et al., 2017; Imperatore et al., 2019). Finally, ascidians are also emerging as model organisms for several studies including immunobiology, allorecognition, angiogenesis and whole-body regeneration, and their extensive regenerative capacity is developing huge interests in regenerative medicine and ageing research (Ballarin et al., 2001; Rinkevich et al., 2007; Gasparini et al., 2008; Franchi et al., 2011; Lauzon et al., 2013; Rinkevich et al., 2013; Voskoboynik and Weissman, 2015).

Noteworthy, these variegate interests of the scientific biological community also often merge in target species. Just to mention, the Korean common sea squirt Halocynthia roretzi (Drasche, 1884), reared and exploited as a commercial seafood in Japan and Korea (Oh et al., 1997; Hirose et al., 2009), possesses lipids that positively affected the health of diabetic/obese KK-Ay mice (Mikami et al., 2010) and ingestion of its plasmalogen enhances and keeps cognitive function through induction of neuronal growth and neuroprotective effects (Watanabe et al., 2020). The clubbed tunicate Styela clava Herdman, 1881, widely cultured in Asian countries (Ko et al., 2012), is in turn not only invading the Mediterranean Sea and nearby areas (Davis and Davis, 2010), but also possesses important compounds with variegate properties, including ACE (angiotensin-converting enzyme) inhibition (Ko et al., 2012) and antioxidant, immunomodulatory, and anticancer activities (Kim, 2011; Ju et al., 2014). Finally, specimens of the Ciona intestinalis (Linnaeus, 1767) species complex, although often reported as a sea pests in several countries worldwide, leading to conspicuous economic losses (Zhan et al., 2010; Bouchemousse et al., 2016), possess abundant phospholipids and polyunsaturated fatty acids (PUFAs) with importance to human health, thus suggesting that they could become an excellent alternative source of n-3 fatty acids (Zhao et al., 2015). Moreover, natural products (NPs) derived from ascidians have also led to the production of promising drugs already marketed for the treatment of specific cancers, such as ecteinascidine 743 or Trabectedin (Yondelis^®) from Ecteinascidia turbinata Herdman, 1880 and the peptide dehydrodidemnin B or Plitidepsin (Aplidin^®) from Aplidium albicans (Milne Edwards, 1841) (Ramesh et al., 2021).

Interest about ascidians is somehow eased by their ecology, as species are often abundant in shallow waters of enclosed and semi-enclosed basins such as ports, marinas, and lagoons, where they settle on artificial substrates and form high biomasses in a short time-frame (Bullard et al., 2007; Lambert, 2009; Lambert, 2019). Although this on one hand makes them a possible threat to local communities due to physical substrate dominance and release of secondary metabolites that can harm benthic assemblages (Prado et al., 2004; Minchin and Sides, 2006; Lengyel et al., 2009; Watters, 2018), on the other it guarantees easy rearing and harvesting (Davis, 1995; Manríquez and Castilla, 2007).

In this context, investigation of compounds extracted from alien ascidians may help shedding light not only in their ecological role in localities where they are introduced, but also in a possible practical use as nutritional or bioactive compounds, thus turning a treat in a resource. Charting metabolic diversity of complex natural extracts and identify novel compounds at an early stage of the research, is high challenging. Metabolomics and dereplication (i.e., early identification of already known NPs), have become more routine in the fields of NP discovery (Caso et al., 2019), environmental research (Teta et al., 2021), and ecotoxicology (De Marco et al., 2022), and may rely upon advanced and sensitive spectroscopic techniques (NMR, mass spectrometry) (Cappello, 2020). High resolution liquid chromatography coupled with tandem mass spectrometry (LC-HRMS/MS) represents a well-suited untargeted methodology to capture the entire metabolome (i.e., the chemical profile of low molecular weight metabolites) within a heterogeneous mixture, thus enabling a deep and sensitive dereplication. Indeed, high resolution metadata and acquisition of fragmentation spectra are useful in that they provide “fingerprints” of detected metabolites, thereby facilitating their identification. Many bioinformatic tools are currently available for handling such amount of metadata and provide a bird’s eye perspective of the chemical profile of a certain extract. Among these tools, to explore large MS data sets, the Feature-Based Molecular Networking represents a suitable means for fast detection, annotation and visualization of known compounds and their novel analogues as well as for discovery of completely new NPs (Nothias et al., 2020).

Herein for the first time, as far of our knowledge, we assessed the metabolome of the colonial ascidian Botrylloides niger Herdman, 1886, a renown tropical invader which already expanded its distributional range from tropical Western Atlantic to Eastern and Western Pacific (Sheets et al., 2016; Rocha et al., 2019; Nydam et al., 2021) and is now emerging as an invasive species also in the Mediterranean Sea.

Human-mediated introduction of alien species in new biogeographic realms is a phenomenon that dates back centuries (Branch and Steffani, 2004; Provan et al., 2008). However, this seems to be furthermore amplified in the recent decades, with many ascidians spreading worldwide and interfering with native benthic communities and habitats by creating consistent environmental and even economic damages (Lambert and Lambert, 1998; Zhan et al., 2015). In the present case, when B. niger was locally discovered, it already formed large aggregates in the investigated channel, and its presence was also noted in additional lagoons of the area (Virgili et al., 2022). This suggests that the early eradication phase, often highlighted to limit the spread of alien species (Willan et al., 2000; Giakoumi et al., 2019), is already far to be potentially applied, and that local communities are presumably enriched by the presence of B. niger since at least years. Absence of field studies but mostly taxonomic impediments may be at the basis of such a result. Indeed, contrary to other localities where the presence of B. niger is well acknowledged (Rocha et al., 2012; Sheets et al., 2016; Streit et al., 2021), the arrival of this colonial ascidian in the area, but in general in the Mediterranean Sea, was presumably overlooked due to rarefaction of studies in zoology and confusion with the congeneric species B. leachii (see Griggio et al., 2014; Virgili et al., 2022). No certainties also occur regarding a possible pathway of arrival in the area. The Fusaro Lake is already known as a hub for the introduction of alien species of different phyla (e.g. Bianchi, 1983; Villani and Martinez, 1993; Crocetta et al., 2012; Crocetta et al., 2013; Hanson et al., 2013), and it also hosts a conspicuous mussel farm that may constitute the most likely source of introduction for this species in the lake. This seems to be in agreement with other studies worldwide, that suggested a species spread at a small scale based on recruitment preferences for mussel beds (Sheets et al., 2016; Rocha et al., 2019). However, another possibility is that this species is spreading locally favoured by the natural currents that exchange the lake waters through the channel, and most likely coming from the nearby Miseno Lake, an area recently acknowledged as a hotspot of non-indigenous species (NIS) ascidians, including B. niger (Virgili et al., 2022).

Although the dominance observed during sampling activities is worrying from an ecological point of view, our results confirm the interest of these alien species as they represent today a potential huge biological resource for nutritional or functional purposes, reducing, in this way, the economic losses caused by these invasive organisms.

Recent studies have shown that ascidians, in particular parts of their inner body tissues, are used to flavour foods intended for human consumption. In fact, all inner body tissues are rich in proteins, mainly collagens with a high essential amino acid index and high delicious amino acid (DAA) content. Moreover, they can supply the body with essential lipid components, including high contents of good-quality fatty acids (Zhao and Li, 2016). Lipids are a class of biomolecules involved in a huge number of different functions in biological systems, which make them fundamental for development and growth, and act as powerful signalling agents during metabolic disruption due to diseases, such as neurological disorders, autoimmune diseases, and cancer (Wymann and Schneiter, 2008; Cheng et al., 2016; Brown et al., 2017).

In this work, the employment of the Molecular Networking approach followed by a careful manual curation of HR ESI-MS² data allowed us to study the metabolome of the alien species B. niger for the first time, and shed light on the presence of novel NPs, several of them remaining still unknown. FBMN was shown to be an efficient tool for fast dereplication of complex organic mixtures and support LC-MS² data analysis for high-confidence structural prediction of detected metabolites.

Our results showed the presence of different classes of indispensable lipids, namely glycerophospholipids, glycerolipids, sphingolipids, fatty acids and derivatives, and sulfonolipids.

Among them, glycerophospholipids were the most represented lipid class, including lyso-glycerophosphocholines and lyso-phosphatidylethanolamines.

Similarly, Hou et al. (2021) recently reported the characterization of the lipid profile of C. intestinalis, H. roretzi, and S. clava, and, among the thirteen major lipid subclasses identified, glycerophospholipids and glycerolipids were the dominant components (66.30–90.60% of total lipids).

Lyso-GPCs and lyso-PEs are representatives of a class of mono-acylated/alkylated glycerophospholipids, commonly referred to as lysophospholipids, playing a key role as structural lipid constituents of cellular membranes and biological signalling molecules in eukaryotes and bacteria, including marine species. Lysophospholipids may regulate fundamental cellular functions such as cell growth, differentiation, survival, migration, adhesion, invasion, and morphogenesis, by interacting with their cognate receptors and/or modelling composition and fluidity of lipid rafts. Indeed, lysophospholipids have been reported to regulate cell motility in budding tunicates (Arai et al., 2004) as well as to enhance multicellular development in the choanoflagellate Salpingoeca rosetta Dayel et al., 2011 (Woznica et al., 2016). In the latter case, lyso-PEs and the sulfonolipids RIFs and IOR-1 are produced by the choanoflagellate endosymbiont Algoriphagus machipongonensis Alegado et al., 2013 and proposed as actors of a metabolic interplay in which multiple bacterial cues regulate the cellular growth of S. rosetta. Considering that a) lysophospholipids and sulfonolipids (IOR-1 and C15 IOR-1) co-occur in the organic extract of B. niger and that b) bacteria live in symbiosis with several Botrylloides species, the notion that a network of bacterial lipids regulate multicellular development and cell motility can be extended to B. niger, which is expected to orchestrate a complex signalling network to control seasonal dynamics of its blooms (Rinkevich et al., 1993; Oricchio and Muniz Dias, 2020; Ramalhosa et al., 2021).

To date, there has been a growing interest in studying the biological functions and metabolism of lysophospholipids, because they could represent a good starting point for the development of new therapeutics for several diseases (Varandas et al., 2019). Indeed, these molecules can affect many biological processes, such as neurogenesis, angiogenesis, wound healing, immunity, and carcinogenesis (Ishii et al., 2004).

Noteworthy, our results showed that B. niger contains also lyso-GPCs and lyso-PEs bearing PUFAs [including eicosapentaenoic acid (C20:5n-3, EPA), and docosahexaenoic acid (C22:6n-3, DHA)], which accounted for the 40% of the detected lyso-glycerophospholipids. Interestingly, glycerophospholipids enriched in PUFAs were also mainly present in the inner body tissues of different ascidians, indicating that these marine organisms could potentially be used for health-promoting food for humans (Hou et al., 2021). Marine sources containing EPA/DHA-enriched glycerophospholipids are receiving increasing attention thanks to their emerging health benefit (as reviewed in Zhang et al., 2019 and Ahmmed et al., 2020). Zhou and collaborators showed that different fatty acids composition of glycerophospholipids were involved in decreasing cognitive decline and biological damage and in brain protection, and these beneficial effects were partly enhanced in presence of EPA and DHA (Zhou et al., 2016).

In addition, unusual PUFAs ( Table 6 ) either linked to the glycerol backbone in monoacylglycerols or as free fatty acids and methyl ester derivatives, were also identified by molecular networking analysis of tandem MS data from B. niger crude extract. In tunicates unsaturated fatty acids, including PUFAs, have been shown to exert mitogenic activity and promote cell proliferation (Arai et al., 2004). Moreover, PUFAs have attracted great attention due to their enormous benefits. They can reduce or prevent the severity of several diseases, such as hyperlipidemia, diabetes, cancers, inflammation and heart and neurodegenerative diseases (reviewed in Zhang et al., 2019).

Therefore, similarly to other marine organisms, B. niger could potentially be used for health-promoting food for humans.

Dereplication of the organic extract from B. niger shed light on the presence of lyso-glycosphingolipids and free sphingoid bases. Over the years, several sphingolipids have been identified from natural sources, including microorganisms, tunicates, sponges, corals and algae. In the past, they were merely considered as components of cellular membranes, but they demonstrated to be involved in several cellular phases (Morales et al., 2007; Ponnusamy et al., 2010), and to be effective as antiproliferative drugs against different tumors (Schmelz et al., 2000; García-Barros et al., 2014; Rethna Priya et al., 2019).

Besides the presence of different lipid classes, our findings unveiled B. niger to be a source of indole and purine alkaloids. In fact, ascidians are prolific sources of nitrogenated metabolites, and more than 300 alkaloids have been reported from these organisms (Nathani et al., 2020) till now. Noteworthy, many of these have been shown to have important activities, including antimicrobial, anticancer, and antiviral, whereby several therapeutics have been isolated or inspired from tunicates-derived NPs.

Among the 12 marine-derived NPs approved by the FDA to date, 2 molecules are native to tunicates, including the alkaloid ecteinascidine (Yondelis^®) from E. turbinata and its synthetic derivative Lurbinectedin (Zepsyre^®) (Phase III) used to treat different types of cancer (Della Sala et al., 2018; McCauley et al., 2020; Ramesh et al., 2021).

Particularly, two brominated indole derivatives were identified in B. niger and predicted as 2,5,6-tribromo-1-methylgramine and its N-oxide analogue. Even if ascidians have been largely acknowledged as a reservoir of brominated alkaloids, these two metabolites have never been described in tunicates before, as being only isolated from the marine bryozoan Amathia verticillata (delle Chiaje, 1822). Particularly, 2,5,6-tribromo-1-methylgramine was found to exert antifouling activity against the barnacle Amphibalanus amphitrite (Darwin, 1854) and the blue mussel Mytilus edulis Linnaeus, 1758 (Sato and Fenical, 1983; Kon-Ya et al., 1994). In addition, three novel purine alkaloids, i.e. methyladenine derivatives, were found in the metabolome of B. niger, thus their structures remaining unsolved. Alkyl purines as well as brominated indoles play a key role in the antifouling mechanism of marine organisms. Detection of such bioactive molecules could at least partially explain the invasiveness of B. niger, which is able to prevent larvae of other marine organisms from settlement and growing on its bodies (Hiebert et al., 2019).

Indeed, by competing for space and food, a congener of B. niger, namely Botrylloides violaceus Oka, 1927, is known to displace other fouling organisms (Berman et al., 1992), including native and introduced tunicates (Dijkstra et al., 2007), bryozoans, barnacles, and mussels (Dijkstra and Harris, 2009), indicating strong competitive ability (Lambert and Lambert, 2003).

In conclusion, the alien species B. niger represents a prolific source of several valuable bioactive compounds, probably developed to adapt and compete with native species. It is also rich of a huge variety of lipid species that improve human health, including lipids rarely found in other foods as PUFAs. The presence of these molecules can undoubtedly transform this alien species from damage to the ecosystem into a precious bio-resource, easily available. Therefore, B. niger together with other invasive ascidians, although concerned from an ecological point of view, could find an important economic role to benefit society, including good opportunities in the food and pharmaceutical industry for the development of functional products based on ascidians.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

GDS, DC, and FC designed and directed the research. GDS, DC, RV, GV, VT, RT, and FC performed the experiments and analysed the data. DdP proceeded to funding acquisition. GDS, DC, and FC wrote the manuscript. All authors contributed to the editing and revision of the manuscript, and read and approved the final manuscript. All authors agree to be accountable for the content of the work.

Work supported by the following projects: (i) PO FEAMP Campania 2014–2020. DRD n.35 of 15th March 2018. Innovazione, sviluppo e sostenibilità nel settore della pesca e dell’acquacoltura per la regione Campania. Misura 2.51. WP5. Task 5.5. Presenza e distribuzione di specie non indigene del macrozoobenthos e del necton in Campania; (ii) POR Campania FESR 2014–2020. Technology Platform for Therapeutic Strategies against Cancer. Antitumor Drugs and Vaccines from the Sea (ADViSE). CUP B43D18000240007–SURF 17061BP000000011. WP1. Task 1.1. Tassonomia, distribuzione, abbondanza e stagionalità dei principali organismi macrobentonici con bioattività presenti nel Golfo di Napoli.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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