Marine Natural Products from Tunicates and Associated Microbes: History
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Contributor:
Chatragadda Ramesh
,
Bhushan Rao Tulasi
,
Mohanraju Raju
,
Narsinh Thakur
,
Laurent Dufossé

Marine tunicates are identified as a potential source of marine natural products (MNPs), demonstrating a wide range of biological properties, like antimicrobial and anticancer activities. The symbiotic relationship between tunicates and specific microbial groups has revealed the acquisition of microbial compounds by tunicates for defensive purpose. For instance, yellow pigmented compounds, “tambjamines”, produced by the tunicate, Sigillina signifera (Sluiter, 1909), primarily originated from their bacterial symbionts, which are involved in their chemical defense function, indicating the ecological role of symbiotic microbial association with tunicates.

  • tunicates
  • symbiotic microbes
  • pigments
  • bioactive compounds
  • alkaloids & peptides

1. Introduction

Tunicates and sea squirts are soft-bodied solitary or colonial (60%) sessile marine organisms belonging to the family Ascidiacea under the subphylum Urochordata, phylum Chordata [1][2]. These organisms are hermaphroditic, filter feeders, and appear in different body colors, such as translucent to blue, green, yellow, red, and brown, with a life span ranging from two months to one year [1][2][3][4]. Currently, tunicates are classified into four major clades such as (a) Appendicularia, (b) Thaliacea + Phlebobranchia + Aplousobranchia, (c) Molgulidae, and (d) Styelidae + Pyuridae, on the basis of the phylogenomic transcriptomic approach [5]. Globally, around 2815 tunicate species have been recorded from shallow coastal waters to deep waters [1]. Tunicate larvae resemble tadpole larvae of members of Chordata, but soon after the retrogressive metamorphosis, they lose the notochord and post-anal tail; thus, these organisms are often referred to as the “evolutionary connecting link” between invertebrates and chordates [6][7]. Therefore, tunicates are considered as important model organisms for several research aspects, such as evolution [6], development biology [8][9], invasion success [10], and bioactive compounds.
Tunicates are prolific producers of marine natural products (MNPs), and certain species are also known to release toxins, such as Bistramide A [11][12]. However, a few species, like Halocynthia roretzi and Pyura michaelseni, are eaten in southeast Asian countries like Korea [13][14]. The strong immune defensive system [15] and their associated symbiotic microbes with bioactive properties [16], makes tunicates highly preferential drug resources in the ocean [15][17]. Since the majority of the tunicate species are known to produce MNP’s, attempts are being undertaken in the culturing of these tunicates (e.g., mangrove tunicate Ecteinascidia turbinata) in large scale for various applications [18][19]. The process of accumulation of vanadium by vanadocytes of tunicates from seawater is well-known [20]. In contrast, investigations on the acquisition of MNPs by tunicates from their symbiotic bacteria are very limited, except for the antitumor products ecteinascidins [21][22], didemnin [23], and talaropeptides [24]. A recent review highlighted the association of bacteria, actinomycetes, fungi, and cyanobacteria with the tunicates and their bioactive nature [25]. It was also observed that actinomycetes, fungi, and bacteria are the predominant microbes associated with the tunicates, showing cytotoxic and antimicrobial activities [26], with the production of alkaloids as the major source of MNPs [27].

2. Ecological Importance of Tunicates

The tunicates population plays an important role in the marine food web through filter feeding [4]. Earlier studies have suggested that phytoplankton productivity in a shallow fjord is controlled by the tunicates population [28]. Tunicates are known to trap the sinking particulate organic matter and generate mucus rich organic matter and fecal pellets with carbohydrates and minerals [29][30], thereby triggering the downward biogeochemical flux (e.g., carbon flux) patterns from surface to deep waters [29][31][32]. Some obligate photosymbiotic tunicates have been suggested to act as environmental stress indicators [33]. The unknown ecological functions of a few tunicate MNPs [34] in understanding their ecological role is yet to be understood.

3. Profile of MNPs from Tunicates and Associated Microbes

Tunicates are known to produce a wide range of MNPs with various bioactive properties. These organisms are considered as a rich source of cellulose, which varies with different species [35]. Alkaloids and peptides are the major chemical constituents observed in tunicates [36]. Metabolites originated from tunicate hemocytes are also found to be cytotoxic to foreign particles [37] and various cell lines [38]. Microorganisms associated with the invertebrate hosts have also been identified as a source of bioactive metabolites [39]. In fact, bioactive metabolite-producing invertebrate-associated microorganisms have special implications in solving the “supply problem” in the initial steps of drug discovery [40]. Recently, Chen et al. reviewed the biological and chemical diversity of ascidian-associated microorganisms [41].
Microbes associated with tunicates have been found to produce potential metabolites showing antimicrobial and anticancer activities (Figure 1, Figure 2 and Figure 3). Tunicate-associated bacteria such as Bacillus, Pantoea, Pseudoalteromonas, Salinicola, Streptomyces, Vibrio and Virgibacillus have recently been identified with potential antimicrobial activities [16]. The introduced tunicate species are also reported to harbor diverse host-specific microbial populations [42] that produce species-specific metabolites [43]. In general, tunicate associated bacteria and fungi are known to produce a variety of MNPs with various biological properties [41]. The chemistry of yellow pigment-producing parasitic bacteria in the interstitial and blood-filled spaces of planktonic tunicates, Oikopleura vanhoeffeni and Oikopleura dioica, are yet to be characterized [44].
Figure 1. Important anticancer drugs of tunicates and their associated microbes in clinical trials.
Figure 2. Tunicate-associated epibiotic and endobiotic symbionts. (the small inserted empty box provides more details in Figure 3).
Figure 3. Illustration depicting various MNPs released from endobiotic and epibiotic microbes associated with tunicate’s endostyle and tunic.

4. Antimicrobial Applications

Tunicates [45], with their associated epi-symbionts [16][46] and endosymbionts [47], are prolific producers of antimicrobial and antifungal compounds inhibiting pathogens. The brominated alkaloids [48] and other compounds from tunicates have been reported to possess several biological activities [25][26]. Pseudoalteromonas tunicata produces alkaloid tambjamine (425 nm), an antifungal yellow pigment [49][50], and violacein (575 nm), a purple pigment with antiprotozoal activity [51][52], in addition to a range of bioactive compounds [51][53]. Methanol extraction of Lissoclinum fragile displayed antibacterial, antifungal, hemolytic, and cytotoxic activities [54]. The kuanoniamine A metabolite produced by Eusynstyela tincta inhibited pathogenic bacteria such as B. subtilis, E. coli, S. aureus, V. cholerae, and V. parahaemolyticus and fungi A. fumigatus and C. albicans [55]. A diffusible 190-kDa protein produced by tunicate Ciona intestinalis associated bacterium Pseudoalteromonas tunicata was found to show antibacterial activity against marine isolates [56]. The four α-helical peptides “clavanins A, B, C, and D” isolated from the hemocytes of tunicate Styela clava showed antibacterial activity against pathogenic Listeria monocytogenes strain EGD and antifungal activity against Candida albicans [57]. Halocidin, an antimicrobial peptide purified from tunicate Halocynthia aurantium showed antibacterial activity against methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa [58]. Similarly, halocyntin and papillosin peptides isolated from tunicate Halocynthia papillosa also displayed antibacterial activity against both Gram-positive and Gram-negative marine bacteria [59]. Halocyamine peptides synthesized by the hemocytes of Halocynthia roretzi showed antimicrobial activity against various bacteria and yeasts [60]. Similarly, Halocyamines produced by Styela clava also displayed antimicrobial properties [61]. A salt-tolerant peptide isolated from hemocytes of Ciona intestinalis showed both antibacterial and antifungal activity [62]. Vanadium chloride and vanadyl sulfate also displayed antibacterial activity against various pathogens [63].
An endobiont, Streptomyces sp., isolated from the tunicate, Styela canopus, produced antibacterial compounds such as granaticin, granatomycin D, and dihydrogranaticin B [64]. Similarly endosymbiotic fungi associated with the tunicates, Polycarpa aurata [65] and Rhopalaea crassa [66], showed antimicrobial activity. The fungi Talaromyces sp., isolated from an unidentified tunicate, produced talaropeptides A and B, two antibacterial metabolites that inhibited Gram-positive bacteria, Bacillus subtilis [24]. The endophytic fungus Penicillium sp. isolated from Didemnum sp. produced antifungal and cytotoxic compounds, terretrione C and D [67].
Some tunicates produced antiviral molecules, indicating their chemical defense function against environmental viruses. The Caribbean tunicate, Trididemnum sp., was found to produce depsipeptides, particularly didemnin A and B, exhibiting antiviral activity against DNA and RNA viruses in vitro [68][69]. Another species of Caribbean tunicate, Eudistoma olivaceum, produced prolific MNPs, such as eudistomins A, D, G, H, I, J, M, N, O, P, and Q, which possessed antiviral activity [70]. The ascidian Didemnum guttatum was found to produce the cyclodidemniserinol trisulfate compound that showed anti-retroviral activity by inhibiting HIV-1 integrase [71]. The tunicate, Didemnum molle, released lanthipeptide divamide A that promised to be a potential anti-HIV drug [72].

5. Anticancer and Antitumor Applications

Trabectedin (Ecteinascidin; ET-743; Yondelis®), an alkaloid extracted from the orange tunicate, Ecteinascidia turbinata, is approved as a first anticancer drug [73] to treat breast cancer [74][75], soft tissue sarcoma [76], and ovarian cancer [77][78][79]. This molecule is suggested to originate from E. turbinata symbiotic bacteria, Candidatus Endoecteinascidia frumentensis [80]. However, plitidepsin (Aplidin®), a depsipeptide isolated from the Mediterranean tunicate, Aplidium albicans, is in phase II clinical trials [73][81] as an anticancer drug against breast cancer [82], human kidney carcinoma cells [83], and multiple myeloma [84]. Didemnin B is also in phase II trials [85], showing anticancer activity against leukaemia P388 cells [68]. Significantly, 60% of the human cervical carcinoma cell lines (HeLa) were inhibited by Eudistomins H extracts (IC50 0.49 μg/mL) obtained from E. viride [86]. Clavepictine A and B alkaloids originated from Clavelina picta demonstrated potential cytotoxic activity (IC50 12 μg/mL) against murine leukemia and human solid tumor cell lines [87]. Lamellarin sulfates originated from Didemnum ternerratum [88] and polycarpine dihydrochloride, a disulfide alkaloid extracted from an ascidian Polycarpa clavata, were found to inhibit human colon tumor cell lines [89].
Cystodytins A, B, and C, three teracyclic alkaloids isolated from Okinawa tunicate Cystodytes dellechiajei, were reported to show antitumor activities [90]. Macrolides isolated from tunicates Lissoclinum patella (Patellazole C) [91] and Eudistoma cf. rigida (Lejimalides A, B, C, and D) [92][93] possessed anticancer activity [94]. Diplamine, an orange pigment alkaloid produced by Diplosoma sp., demonstrated cytotoxic activity against leukemia cells [95]. Halocyamine A and B peptides extracted from H. roretzi showed anticancer activity against various cell lines [60]. A depsipeptide, dehydrodidemnin B, produced by Aplidium albicans inhibited Ehrlich carcinoma cells in mice and reduced 80–90% tumor cells [96]. Bryostatins Ecteinascidins products, such as ET-729, 743, 745, 759 A, 759B, and 770, extracted from the Caribbean tunicate Ecteinascidia turbinata showed immunomodulator activity and antitumor activity against various leukemia cells [97] and breast, lung, ovary, and melanoma cells [98]. The Brazilian ascidian, Didemnum granulatum, produced G2 checkpoint-inhibiting aromatic alkaloids, granulatimide and isogranulatimide [99]. The ascidian Cystodytes dellechiajei produced topoisomerase II-inhibiting ascididemin, which has antitumor activity against various tumor cell lines [100]. This marine alkaloid exhibits marked cytotoxic activities against a range of tumor cells. The kuanoniamine A metabolite extracted from E. tincta displayed 100% inhibition of Dalton’s lymphoma and Ehrlich ascites tumor cell lines [55]. Cynthichlorine, an alkaloid isolated from the tunicate Cynthia savignyi, showed cytotoxicity against Artemia salina larva at an LD50 of 48.5 μg/mL [101]. Siladenoserinols A and B derivatives isolated from didemnid tunicates possessed antitumor activity by inhibiting the interaction of p53-Hdm2 [102].

6. Antifouling and Anti-Deterrent Activities

The colonial tunicate, Eudistoma olivaceum, was found to produce brominated alkaloids, Eudistomins G and H, which acted as antifouling substances and fish antifeedants; thus, the E. olivaceum surface was completely free from fouling epibionts [34]. A dark green pigmented bacteria, Pseudoalteromonas tunicata, isolated from the surface of Ciona intestinalis, collected originally from off the west coast of Sweden, showed antifouling activity against algal spores, invertebrate larvae, and diatoms [53][103][104]. The yellow pigmented Pseudoalteromonas tunicata mutants have demonstrated antifouling activity against algal spore germination, bacterial growth, fungal growth, and invertebrate larvae [51]. Diindol-3-ylmethane products isolated from an unidentified ascidian-associated bacteria, Pseudovibrio denitrificans, displayed nearly 50% antifouling activity against barnacle Balanus amphitrite and bryozoan Bugula neritina [105].
Deterring activity of vanadium acidic solutions, such as vanadyl sulfate and sodium vanadate, was observed against Thalassoma bifasciatum when incorporated into food pellets [63][106]. Didemnimides C and D from Didemnum conchyliatum [107], nordidemnin B [108] and didemnin B [109] from Trididemnum solidum, and granulatamides from Didemnum granulatum [110] displayed antifeedant effects on various fishes in laboratory experiments. The kuanoniamine A molecule from E. tincta displayed feeding-deterrent activities against carnivore gold fish, Carassius auratus [55]. MNPs isolated from Antarctic tunicates have demonstrated variability in anti-deterrent activities [111]. Both the yellow pigmented tambjamine metabolites and blue tetrapyrrole metabolite released from Sigillina sp. (i.e., Atapozoa sp.) showed feeding-deterrent activity against various carnivore fishes [112][113]. The blue tetrapyrrole pigment was suggested to originate from the associated bacteria Serratia marcescens [114]. Tambjamines and tetrapyrrole chemical constituents from both adult and larvae were reported to function as defensive chemicals against predators [108]. Lipophilic crude extracts from Antarctic tunicate, Distaplia cylindrica [115], and polyandrocarpidines from Polyandrocarpa sp. [108][116] demonstrated deterrent activity against certain sea-stars, hermit crabs, and snails.

7. Miscellaneous Applications

The chiton Mopalia sp. spawned when injected with 1.0 mg/L of gonadotropin releasing hormone (GnRH2) of a tunicate [117]. Lumichrome, a compound extracted from tunic, gonads, and eggs of ascidian, Halocynthia roretzi, was involved in the larval metamorphosis [118]. Similarly, sperm-activating and attracting factors (SAAF) were isolated from eggs of the ascidians Ciona intestinalis and Ascidia sydneiensis [119]. Lipids extracted from H. roretzi have demonstrated the antidiabetic and anti-obese properties in mice models [120]. Two novel alkaloids, mellpaladine and dopargimine, isolated from Palauan tunicate have demonstrated neuroactive behavior in mice [121]. Two new alkaloids, polyaurines A and B, isolated from the tunicate, Polycarpa aurata, inhibited blood-dwelling Schistosoma mansoni [122]. Lepadin and villatamine alakaloids isolated from Clavelina lepadiformis [123] and lepadins from Didemnum sp. [124] displayed potential antiparasitic and cytotoxic activities. The ascidian species, Didemnum psammathodes, collected from the central west coast of India was extracted in organic solvents. These extracts showed antimicrobial and antifouling properties [125].

This entry is adapted from the peer-reviewed paper 10.3390/md19060308

References

  1. Shenkar, N.; Swalla, B.J. Global diversity of Ascidiacea. PLoS ONE 2011, 6, e20657.
  2. Holland, L.Z. Tunicates. Curr. Biol. 2016, 26, R141–R156.
  3. Gasparini, F.; Ballarin, L. Reproduction in Tunicates. In Encyclopedia of Reproduction, 2nd ed.; Elsevier Inc.: Amsterdam, The Netherlands, 2018; Volume 6, pp. 546–553.
  4. Bone, Q.; Carré, C.; Chang, P. Tunicate feeding filters. J. Mar. Biol. Assoc. UK 2003, 83, 907–919.
  5. Delsuc, F.; Philippe, H.; Tsagkogeorga, G.; Simion, P.; Tilak, M.K.; Turon, X.; López-Legentil, S.; Piette, J.; Lemaire, P.; Douzery, E.J.P. A phylogenomic framework and timescale for comparative studies of tunicates. BMC Biol. 2018, 16, 39.
  6. Berna, L.; Alvarez-Valin, F. Evolutionary genomics of fast evolving tunicates. Genome Biol. Evol. 2014, 6, 1724–1738.
  7. Swalla, B.J. Building divergent body plans with similar genetic pathways. Heredity 2006, 97, 235–243.
  8. Jeffery, W.R. Tunicates: Models for Chordate Evolution and Development at Low Genomic Complexity. In Comparative Genomics; Clark, M.S., Ed.; Springer Science + Business Media: New York, NY, USA, 2000; pp. 43–69.
  9. Lemaire, P. Evolutionary crossroads in developmental biology: The tunicates. Development 2011, 138, 2143–2152.
  10. Zhan, A.; Briski, E.; Bock, D.G.; Ghabooli, S.; MacIsaac, H.J. Ascidians as models for studying invasion success. Mar. Biol. 2015, 162, 2449–2470.
  11. Watters, D.J. Ascidian toxins with potential for drug development. Mar. Drugs 2018, 16, 162.
  12. Gouiffes, D.; Juge, M.; Grimaud, N.; Welin, L.; Sauviat, M.P.; Barbin, Y.; Laurent, D.; Roussakis, C.; Henichart, J.P.; Verbist, J.F. Bistramide A, a new toxin from the urochordata Lissoclinum bistratum Sluiter: Isolation and preliminary characterization. Toxicon 1988, 26, 1129–1136.
  13. Oh, K.-S.; Kim, J.-S.; Heu, M.-S. Food Constituents of Edible Ascidians Halocynthia roretzi and Pyura michaelseni. Korean J. Food Sci. Technol. 1997, 29, 955–962.
  14. Ali, A.J.H.; Tamilselvi, M. Ascidians in Coastal Water: A Comprehensive Inventory of Ascidian Fauna from the Indian Coast; Springer Nature: Cham, Switzerland, 2016; ISBN 9783319291185.
  15. DeFilippo, J.; Beck, G. Tunicate Immunology. In Reference Module in Life Sciences; Elsevier: Amsterdam, The Netherlands, 2018; pp. 1–10.
  16. Ayuningrum, D.; Liu, Y.; Riyanti; Sibero, M.T.; Kristiana, R.; Asagabaldan, M.A.; Wuisan, Z.G.; Trianto, A.; Radjasa, O.K.; Sabdono, A.; et al. Tunicate-associated bacteria show a great potential for the discovery of antimicrobial compounds. PLoS ONE 2019, 14, e0213797.
  17. Franchi, N.; Ballarin, L. Immunity in protochordates: The tunicate perspective. Front. Immunol. 2017, 8, 674.
  18. Walters, T.L.; Gibson, D.M.; Frischer, M.E. Cultivation of the marine pelagic tunicate Dlioletta gegenbauri (Uljanin 1884) for experimental studies. J. Vis. Exp. 2019, 150, e59832.
  19. Fusetani, N. Drugs from the Sea; Krager: Basel, Switzerland, 2000.
  20. Michibara, H.; Uyama, T.; Ueki, T.; Kanamori, K. The mechanism of accumulation and reduction of vanadium by ascidians. In The Biology of Ascidians; Sawada, H., Yokosawa, H., Lambert, C.C., Eds.; Springer: Tokyo, Japan, 2001; pp. 363–373.
  21. Shen, G.Q.; Baker, B.J. Biosynthetic studies of the eudistomins in the tunicate Eudistoma olivaceum. Tetrahedron Lett. 1994, 35, 1141–1144.
  22. Le, V.H.; Inai, M.; Williams, R.M.; Kan, T. Ecteinascidins. A review of the chemistry, biology and clinical utility of potent tetrahydroisoquinoline antitumor antibiotics. Nat. Prod. Rep. 2015, 32, 328–347.
  23. Xu, Y.; Kersten, R.D.; Nam, S.J.; Lu, L.; Al-Suwailem, A.M.; Zheng, H.; Fenical, W.; Dorrestein, P.C.; Moore, B.S.; Qian, P.Y. Bacterial biosynthesis and maturation of the didemnin anti-cancer agents. J. Am. Chem. Soc. 2012, 134, 8625–8632.
  24. Dewapriya, P.; Khalil, Z.G.; Prasad, P.; Salim, A.A.; Cruz-Morales, P.; Marcellin, E.; Capon, R.J. Talaropeptides A-D: Structure and biosynthesis of extensively N-methylated linear peptides from an Australian marine tunicate-derived Talaromyces sp. Front. Chem. 2018, 6, 394.
  25. Dou, X.; Dong, B. Origins and bioactivities of natural compounds derived from marine ascidians and their symbionts. Mar. Drugs 2019, 17, 670.
  26. Casertano, M.; Menna, M.; Imperatore, C. The ascidian-derived metabolites with antimicrobial properties. Antibiotics 2020, 9, 510.
  27. Palanisamy, S.K.; Rajendran, N.M.; Marino, A. Natural products diversity of marine ascidians (Tunicates; Ascidiacea) and successful drugs in clinical development. Nat. Prod. Bioprospect. 2017, 7, 1–111.
  28. Petersen, J.K.; Riisgard, H.U. Filtration capacity of the ascidian Ciona intestinalis and its grazing impact in a shallow fjord. Mar. Ecol. Prog. Ser. 1992, 88, 9–17.
  29. Morris, R.J.; Bone, Q.; Head, R.; Braconnot, J.C.; Nival, P. Role of salps in the flux of organic matter to the bottom of the Ligurian Sea. Mar. Biol. 1988, 97, 237–241.
  30. Pomeroy, L.R.; Deibel, D. Aggregation of organic matter by pelagic tunicates. Limnol. Oceanogr. 1980, 25, 643–652.
  31. Gorsky, G.; Da Silva, N.L.; Dallot, S.; Laval, P.; Braconnot, J.C.; Prieur, L. Midwater tunicates: Are they related to the permanent front of the Ligurian Sea (NW Mediterranean)? Mar. Ecol. Prog. Ser. 1991, 74, 195–204.
  32. Sutherland, K.R.; Madin, L.P.; Stocker, R. Filtration of submicrometer particles by pelagic tunicates. Proc. Natl. Acad. Sci. USA 2010, 107, 15129–15134.
  33. Hirose, E.; Nozawa, Y. Latitudinal difference in the species richness of photosymbiotic ascidians along the east coast of Taiwan. Zool. Stud. 2020, 59, e19.
  34. Davis, A.R. Alkaloids and ascidian chemical defense: Evidence for the ecological role of natural products from Eudistoma olivaceum. Mar. Biol. 1991, 111, 375–379.
  35. Zhao, Y.; Li, J. Excellent chemical and material cellulose from tunicates: Diversity in cellulose production yield and chemical and morphological structures from different tunicate species. Cellulose 2014, 21, 3427–3441.
  36. Menna, M.; Aiello, A. The Chemistry of Marine Tunicates. In Handbook of Marine Natural Products; Fattorusso, E., Gerwick, W.H., Taglialatela-Scafati, O., Eds.; Springer Science + Business Media B.V.: Berlin/Heidelberg, Germany, 2012; pp. 295–385.
  37. Franchi, N.; Ballarin, L. Cytotoxic cells of compound Ascidians. In Lessons in Immunity: From Single-Cell Organisms to Mammals; Ballarin, L., Cammarata, M., Eds.; Elsevier Inc.: London, UK, 2016; pp. 193–199.
  38. Parrinello, N. Cytotoxic activity of tunicate hemocytes. In Invertebrate Immunology; Rinkevich, B., Müller, W.E.G., Eds.; Springer: Berlin/Heidelberg, Germany, 1996; pp. 190–217.
  39. Liu, L.; Zheng, Y.-Y.; Shao, C.-L.; Wang, C.-Y. Metabolites from marine invertebrates and their symbiotic microorganisms: Molecular diversity discovery, mining, and application. Mar. Life Sci. Technol. 2019, 1, 60–94.
  40. Leal, M.C.; Sheridan, C.; Osinga, R.; Dionísio, G.; Rocha, R.; Silva, B.; Rosa, R.; Calado, C. Marine Microorganism-Invertebrate Assemblages: Perspectives to Solve the “Supply Problem” in the Initial Steps of Drug Discovery. Mar. Drugs 2014, 12, 3929–3952.
  41. Chen, L.; Hu, J.S.; Xu, J.L.; Shao, C.L.; Wang, G.Y. Biological and chemical diversity of ascidian-associated microorganisms. Mar. Drugs 2018, 16, 362.
  42. Evans, J.S.; Erwin, P.M.; Shenkar, N.; López-Legentil, S. Introduced ascidians harbor highly diverse and host-specific symbiotic microbial assemblages. Sci. Rep. 2017, 7, 11033.
  43. Tianero, M.D.B.; Kwan, J.C.; Wyche, T.P.; Presson, A.P.; Koch, M.; Barrows, L.R.; Bugni, T.S.; Schmidt, E.W. Species specificity of symbiosis and secondary metabolism in ascidians. ISME J. 2015, 9, 615–628.
  44. Flood, P. Yellow-stained oikopleurid appendicularians are caused by bacterial parasitism. Mar. Ecol. Prog. Ser. 1991, 71, 291–295.
  45. Karthikeyan, M.M.; Ananthan, G.; Balasubramanian, T. Antimicrobial activity of crude extracts of some ascidians (Urochordata: Ascidiacea), from Palk Strait, (Southeast Coast of India). World J. Fish. Mar. Sci. 2009, 1, 262–267.
  46. Ayuningrum, D.; Kristiana, R.; Nisa, A.A.; Radjasa, S.K.; Muchlissin, S.I.; Radjasa, O.K.; Sabdono, A.; Trianto, A. Bacteria associated with tunicate, Polycarpa aurata, from Lease sea, Maluku, Indonesia exhibiting anti-multidrug resistant bacteria. Biodiversitas 2019, 20, 956–964.
  47. Litaay, M.; Christine, G.; Gobel, R.B.; Dwyana, Z. Bioactivity of endo-symbiont bacteria of tunicate Polycarpa aurata as antimicrobial. In Proceedings of the 23 National Seminar of Indonesia Biology Society, Jayapura, Indonesia, 18 September 2015.
  48. Menna, M.; Fattorusso, E.; Imperatore, C. Alkaloids from marine ascidians. Molecules 2011, 16, 8694–8732.
  49. Franks, A.; Haywood, P.; Holmström, C.; Egan, S.; Kjelleberg, S.; Kumar, N. Isolation and structure elucidation of a novel yellow pigment from the marine bacterium Pseudoalteromonas tunicata. Molecules 2005, 10, 1286–1291.
  50. Franks, A.; Egan, S.; Holmström, C.; James, S.; Lappin-Scott, H.; Kjelleberg, S. Inhibition of fungal colonization by Pseudoalteromonas tunicata provides a competitive advantage during surface colonization. Appl. Environ. Microbiol. 2006, 72, 6079–6087.
  51. Egan, S.; James, S.; Holmström, C.; Kjelleberg, S. Correlation between pigmentation and antifouling compounds produced by Pseudoalteromonas tunicata. Environ. Microbiol. 2002, 4, 433–442.
  52. Matz, C.; Webb, J.S.; Schupp, P.J.; Phang, S.Y.; Penesyan, A.; Egan, S.; Steinberg, P.; Kjelleberg, S. Marine biofilm bacteria evade eukaryotic predation by targeted chemical defense. PLoS ONE 2008, 3, e2744.
  53. Holmström, C.; James, S.; Neilan, B.A.; White, D.C.; Kjelleberg, S. Pseudoalteromonas tunicata sp. nov., a bacterium that produces antifouling agents. Int. J. Syst. Bacteriol. 1998, 48, 1205–1212.
  54. Kumaran, N.S.; Bragadeeswaran, S.; Meenakshi, V.K.; Balasubramanian, T. Bioactivity potential of extracts from ascidian Lissoclinum fragile. Afr. J. Pharm. Pharmacol. 2012, 6, 1854–1859.
  55. Devi, S.; Rajasekharan, K.; Padmakumar, K.; Tanaka, J.; Higa, T. Biological activity and chemistry of the compound ascidian Eusynstyela tineta. In The Biology of Ascidians; Sawada, H., Yokosawa, H., Lambert, C.C., Eds.; Springer: Tokyo, Japan, 2001; pp. 341–354.
  56. James, S.G.; Holmström, C.; Kjelleberg, S. Purification and characterization of a novel antibacterial protein from the marine bacterium D2. Appl. Environ. Microbiol. 1996, 62, 2783–2788.
  57. Lee, I.H.; Zhao, C.; Cho, Y.; Harwig, S.S.L.; Cooper, E.L.; Lehrer, R.I. Clavanins, α-helical antimicrobial peptides from tunicate hemocytes. FEBS Lett. 1997, 400, 158–162.
  58. Jang, W.S.; Kim, K.N.; Lee, Y.S.; Nam, M.H.; Lee, I.H. Halocidin: A new antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium. FEBS Lett. 2002, 521, 81–86.
  59. Galinier, R.; Roger, E.; Sautiere, P.E.; Aumelas, A.; Banaigs, B.; Mitta, G. Halocyntin and papillosin, two new antimicrobial peptides isolated from hemocytes of the solitary tunicate, Halocynthia papillosa. J. Pept. Sci. 2009, 15, 48–55.
  60. Azumi, K.; Yokosawa, H.; Ishii, S.I. Halocyamines: Novel antimicrobial tetrapeptide-like substances isolated from the hemocytes of the solitary ascidian Halocynthia roretzi. Biochemistry 1990, 29, 159–165.
  61. Menzel, L.P.; Lee, I.H.; Sjostrand, B.; Lehrer, R.I. Immunolocalization of clavanins in Styela clava hemocytes. Dev. Comp. Immunol. 2002, 26, 505–515.
  62. Fedders, H.; Michalek, M.; Grötzinger, J.; Leippe, M. An exceptional salt-tolerant antimicrobial peptide derived from a novel gene family of haemocytes of the marine invertebrate Ciona intestinalis. Biochem. J. 2008, 416, 65–75.
  63. Odate, S.; Pawlik, J.R. The role of vanadium in the chemical defense of the solitary tunicate, Phallusia nigra. J. Chem. Ecol. 2007, 33, 643–654.
  64. Sung, A.A.; Gromek, S.M.; Balunas, M.J. Upregulation and identification of antibiotic activity of a marine-derived Streptomyces sp. via co-cultures with human pathogens. Mar. Drugs 2017, 15, 250.
  65. Nurfadillah, A.; Litaay, M.; Gobel, R.B.; Haedar, N. Potency of tunicate Polycarpa aurata as inoculum source of sebagai sumber endosimbyotic fungi that produce antimicrobe. J. Alam Lingkung. 2015, 6, 10–16.
  66. Tahir, E.; Litaay, M.; Gobel, R.B.; Haedar, N.; Al, E. Potency of tunicate Rhopalaea crassa as inoculum source of endosymbiont fungi that produce antimicrobe. Spermonde 2016, 2, 33–37.
  67. Shaala, L.A.; Youssef, D.T.A. Identification and bioactivity of compounds from the fungus Penicillium sp. CYE-87 isolated from a marine tunicate. Mar. Drugs 2015, 13, 1698–1709.
  68. Rinehart, K.L.; Gloer, J.B.; Hughes, R.G.; Renis, H.E.; Patrick McGovren, J.; Swynenberg, E.B.; Stringfellow, D.A.; Kuentzel, S.L.; Li, L.H. Didemnins: Antiviral and antitumor depsipeptides from a Caribbean tunicate. Science 1981, 212, 933–935.
  69. Canonico, P.G.; Pannier, W.L.; Huggins, J.W.; Rienehart, K.L. Inhibition of RNA viruses in vitro and in Rift Valley fever-infected mice by didemnins A and B. Antimicrob. Agents Chemother. 1982, 22, 696–697.
  70. Kobayashi, J.; Harbour, G.C.; Gilmore, J.; Rinehart, K.L. Eudistomins A, D, G, H, I, J, M, N, O, P, and Q, Bromo-, Hydroxy-, Pyrrolyl-, and 1-Pyrrolinyl-β-carbolines from the antiviral Caribbean tunicate Eudistoma olivaceum. J. Am. Chem. Soc. 1984, 106, 1526–1528.
  71. Mitchell, S.S.; Rhodes, D.; Bushman, F.D.; Faulkner, D.J. Cyclodidemniserinol trisulfate, a sulfated serinolipid from the Palauan ascidian Didemnum guttatum that inhibits HIV-1 integrase. Org. Lett. 2000, 2, 1605–1607.
  72. Smith, T.E.; Pond, C.D.; Pierce, E.; Harmer, Z.P.; Kwan, J.; Zachariah, M.M.; Harper, M.K.; Wyche, T.P.; Matainaho, T.K.; Bugni, T.S.; et al. Accessing chemical diversity from the uncultivated symbionts of small marine animals. Nat. Chem. Biol. 2018, 14, 179–185.
  73. Mayer, A.M.S.; Glaser, K.B.; Cuevas, C.; Jacobs, R.S.; Kem, W.; Little, R.D.; McIntosh, J.M.; Newman, D.J.; Potts, B.C.; Shuster, D.E. The odyssey of marine pharmaceuticals: A current pipeline perspective. Trends Pharmacol. Sci. 2010, 31, 255–265.
  74. Zelek, L.; Yovine, A.; Brain, E.; Turpin, F.; Taamma, A.; Riofrio, M.; Spielmann, M.; Jimeno, J.; Misset, J.L. A phase II study of Yondelis® (trabectedin, ET-743) as a 24-h continuous intravenous infusion in pretreated advanced breast cancer. Br. J. Cancer 2006, 94, 1610–1614.
  75. Atmaca, H.; Bozkurt, E.; Uzunoglu, S.; Uslu, R.; Karaca, B. A diverse induction of apoptosis by trabectedin in MCF-7 (HER2−/ER+) and MDA-MB-453 (HER2+/ER−) breast cancer cells. Toxicol. Lett. 2013, 221, 128–136.
  76. Grosso, F.; Jones, R.L.; Demetri, G.D.; Judson, I.R.; Blay, J.-Y.; Cesne, A.L.; Lippo, R.S.; Casieri, P.; Collini, P.; Dileo, P.; et al. Effi cacy of trabectedin (ecteinascidin-743) in advanced pretreated myxoid liposarcomas: A retrospective study. Lancet Oncol. 2007, 8, 595–602.
  77. Sessa, C.; De Braud, F.; Perotti, A.; Bauer, J.; Curigliano, G.; Noberasco, C.; Zanaboni, F.; Gianni, L.; Marsoni, S.; Jimeno, J.; et al. Trabectedin for women with ovarian carcinoma after treatment with platinum and taxanes fails. J. Clin. Oncol. 2005, 23, 1867–1874.
  78. Krasner, C.N.; McMeekin, D.S.; Chan, S.; Braly, P.S.; Renshaw, F.G.; Kaye, S.; Provencher, D.M.; Campos, S.; Gore, M.E. A Phase II study of trabectedin single agent in patients with recurrent ovarian cancer previously treated with platinum-based regimens. Br. J. Cancer 2007, 97, 1618–1624.
  79. Monk, B. A randomized phase III study of trabectedin with pegylated liposomal doxorubicin (PLD) versus PLD in relapsed, recurrent ovarian cancer (OC). Eur. J. Cancer Suppl. 2008, 19, viii1–viii4.
  80. Rath, C.M.; Janto, B.; Earl, J.; Ahmed, A.; Hu, F.Z.; Hiller, L.; Dahlgren, M.; Kreft, R.; Yu, F.; Wolff, J.J.; et al. Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic natural product ET-743. ACS Chem. Biol. 2011, 6, 1244–1255.
  81. Tohme, R.; Darwiche, N.; Gali-Muhtasib, H. A journey under the sea: The quest for marine anti-cancer alkaloids. Molecules 2011, 16, 9665–9696.
  82. González-Santiago, L.; Suárez, Y.; Zarich, N.; Muñoz-Alonso, M.J.; Cuadrado, A.; Martínez, T.; Goya, L.; Iradi, A.; Sáez-Tormo, G.; Maier, J.V.; et al. Aplidin® induces JNK-dependent apoptosis in human breast cancer cells via alteration of glutathione homeostasis, Rac1 GTPase activation, and MKP-1 phosphatase downregulation. Cell Death Differ. 2006, 13, 1968–1981.
  83. Cuadrado, A.; García-Fernández, L.F.; González, L.; Suárez, Y.; Losada, A.; Alcaide, V.; Martínez, T.; Máa Fernández-Sousa, J.; Sánchez-Puelles, J.M.; Muñoz, A. AplidinTM induces apoptosis in human cancer cells via glutathione depletion and sustained activation of the epidermal growth factor receptor, Src, JNK, and p38 MAPK. J. Biol. Chem. 2003, 278, 241–250.
  84. Mitsiades, C.S.; Ocio, E.M.; Pandiella, A.; Maiso, P.; Gajate, C.; Garayoa, M.; Vilanova, D.; Montero, J.C.; Mitsiades, N.; McMullan, C.J.; et al. Aplidin, a marine organism-derived compound with potent antimyeloma activity in vitro and in vivo. Cancer Res. 2008.
  85. Rinehart, K.L. Antitumor compounds from tunicates. Med. Res. Rev. 2000, 20, 1–27.
  86. Rajesh, R.P.; Annappan, M. Anticancer effects of brominated indole alkaloid eudistomin H from marine ascidian Eudistoma viride against cervical cancer cells (HeLa). Anticancer Res. 2015, 35, 283–294.
  87. Raub, M.F.; Cardellina, J.H.; Choudhary, M.I.; Ni, C.Z.; Clardy, J.; Alley, M.C. Clavepictines A and B: Cytotoxic Quinolizidines from the Tunicate Clavelina picta. J. Am. Chem. Soc. 1991, 113, 3178–3180.
  88. Bracegirdle, J.; Robertson, L.P.; Hume, P.A.; Page, M.J.; Sharrock, A.V.; Ackerley, D.F.; Carroll, A.R.; Keyzers, R.A. Lamellarin Sulfates from the Pacific Tunicate Didemnum ternerratum. J. Nat. Prod. 2019, 82, 2000–2008.
  89. Kang, H.; Fenical, W. Polycarpine dihydrochloride: A cytotoxic dimeric disulfide alkaloid from the Indian ocean ascidian Polycarpa clavata. Tetrahedron Lett. 1996, 37, 2369–2372.
  90. Kobayashi, J.; Cheng, J.F.; Nakamura, H.; Ohizumi, Y.; Walchli, M.R.; Hirata, Y.; Sasaki, T. Cystodytins A, B, and C, novel tetracyclic aromatic alkaloids with potent antineoplastic activity from the Okinawan tunicate Cystodytes dellechiajei. J. Org. Chem. 1988, 53, 1800–1804.
  91. Zabriskie, T.M.; Mayne, C.L.; Ireland, C.M. Patellazole C: A novel cytotoxic macrolide from Lissoclinum patella. J. Am. Chem. Soc. 1988, 110, 7919–7920.
  92. Kobayashi, J.; Cheng, J.F.; Nakamura, H.; Ohta, T.; Nozoe, S.; Hirata, Y.; Sasaki, T. Lejimalides A and B, novel 24-membered macrolides with potent antileukemic activity from the Okinawan tunicate Eudistoma cf. rigida. J. Org. Chem. 1988, 53, 6147–6150.
  93. Kikuchi, Y.; Ishibashi, M.; Sasaki, T.; Kobayashi, J. Lejimalides C and D, new antineoplastic 24-membered macrolide sulfates from the okinawan marine tunicate Eudistoma cf. rigida. Tetrahedron Lett. 1991, 32, 789–797.
  94. Nguyen, M.H.; Imanishi, M.; Kurogi, T.; Wan, X.; Ishmael, J.E.; McPhail, K.L.; Smith, A.B. Synthetic access to the mandelalide family of macrolides: Development of an anion relay chemistry strategy. J. Org. Chem. 2018, 83, 4287–4306.
  95. Charyulu, G.A.; McKee, T.C.; Ireland, C.M. Diplamine, a cytotoxic polyaromatic alkaloid from the tunicate Diplosoma sp. Tetrahedron Lett. 1989, 30, 4201–4202.
  96. Urdiales, J.L.; Morata, P.; De Castro, I.N.; Sánchez-Jiménez, F. Antiproliferative effect of dehydrodidemnin B (DDB), a depsipeptide isolated from Mediterranean tunicates. Cancer Lett. 1996, 102, 31–37.
  97. Rinehart, K.L.; Holt, T.G.; Fregeau, N.L.; Stroh, J.G.; Keifer, P.A.; Sun, F.; Li, L.H.; Martin, D.G. Ecteinascidins 729, 743, 745, 759A, 759B, and 770: Potent antitumor agents from the Caribbean tunicate Ecteinascidia turbinata. J. Org. Chem. 1990, 55, 4512–4515.
  98. Izbicka, E.; Lawrence, R.; Raymond, E.; Eckhardt, G.; Faircloth, G.; Jimeno, J.; Clark, G.; Von Hoff, D.D. In vitro antitumor activity of the novel marine agent, Ecteinascidin-743 (ET-743, NSC-648766) against human tumors explanted from patients. Ann. Oncol. 1998, 9, 981–987.
  99. Berlmck, R.G.S.; Britton, R.; Piers, E.; Lim, L.; Roberge, M.; Moreira Da Rocha, R.; Andersen, R.J. Granulatimide and isogranulatimide, aromatic alkaloids with G2 checkpoint inhibition activity isolated from the Brazilian ascidian Didemnum granulatum: Structure elucidation and synthesis. J. Org. Chem. 1998, 63, 9850–9856.
  100. Dassonneville, L.; Wattez, N.; Baldeyrou, B.; Mahieu, C.; Lansiaux, A.; Banaigs, B.; Bonnard, I.; Bailly, C. Inhibition of topoisomerase II by the marine alkaloid ascididemin and induction of apoptosis in leukemia cells. Biochem. Pharmacol. 2000, 60, 527–537.
  101. Abourriche, A.; Abboud, Y.; Maoufoud, S.; Mohou, H.; Seffaj, T.; Charrouf, M.; Chaib, N.; Bennamara, A.; Bontemps, N.; Francisco, C. Cynthichlorine: A bioactive alkaloid from the tunicate Cynthia savignyi. Farmaco 2003, 58, 1351–1354.
  102. Torii, M.; Hitora, Y.; Kato, H.; Koyanagi, Y.; Kawahara, T.; Losung, F.; Mangindaan, R.E.P.; Tsukamoto, S. Siladenoserinols M-P, sulfonated serinol derivatives from a tunicate. Tetrahedron 2018, 74, 7516–7521.
  103. Holmstrom, C.; James, S.; Egan, S.; Kjelleberg, S. Inhibition of common fouling organisms by marine bacterial isolates with special reference to the role of pigmented bacteria. Biofouling 1996, 10, 251–259.
  104. Holmstrom, C.; Rittschof, D.; Kjelleberg, S. Inhibition of settlement by larvae of Balanus amphitrite and Ciona intestinalis by a surface-colonizing marine bacterium. Appl. Environ. Microbiol. 1992, 58, 2111–2115.
  105. Wang, K.L.; Xu, Y.; Lu, L.; Li, Y.; Han, Z.; Zhang, J.; Shao, C.L.; Wang, C.Y.; Qian, P.Y. Low-toxicity diindol-3-ylmethanes as potent antifouling compounds. Mar. Biotechnol. 2015, 17, 624–632.
  106. Stoecker, D. Resistance of a tunicate to fouling. Biol. Bull. 1978, 155, 615–626.
  107. Vervoort, H.C.; Pawlik, J.R.; Fenical, W. Chemical defense of the Caribbean ascidian Didemnum conchyliatum. Mar. Ecol. Prog. Ser. 1998, 164, 221–228.
  108. Lindquist, N.; Hay, M.E.; Fenical, W. Defense of ascidians and their conspicuous larvae: Adult vs. larval chemical defenses. Ecol. Monogr. 1992, 62, 547–568.
  109. Lindquist, N.; Hay, M.E. Can small rare prey be chemically defended? The case for marine larvae. Ecology 1995, 76, 1347–1358.
  110. Seleghim, M.H.R.; De Lira, S.P.; Campana, P.T.; Berlinck, R.G.S.; Custódio, M.R. Localization of granulatimide alkaloids in the tissues of the ascidian Didemnum granulatum. Mar. Biol. 2007, 150, 967–975.
  111. Núñez-Pons, L.; Carbone, M.; Vázquez, J.; Rodríguez, J.; Nieto, R.M.; Varela, M.M.; Gavagnin, M.; Avila, C. Natural products from antarctic colonial ascidians of the genera Aplidium and Synoicum: Variability and defensive role. Mar. Drugs 2012, 10, 1741–1764.
  112. Paul, V.J.; Lindquist, N.; Fenical, W. Chemical defenses of the tropical ascidian Atapozoa sp. and its nudibranch predators Nembrotha spp. Mar. Ecol. Prog. Ser. 1990, 59, 109–118.
  113. Lindquist, N.; Fenical, W. New tambjamine class alkaloids from the marine ascidian Atapozoa sp. and its nudibranch predators. Origin of the tambjamines in Atapozoa. Experientia 1991, 47, 504–506.
  114. Wasserman, H.H.; Friedland, D.J.; Morrison, D.A. A novel dipyrrolyldipyrromethene prodigiosin analog from Serratia marcescens. Tetrahedron Lett. 1968, 6, 641–644.
  115. McClintock, J.B.; Amsler, M.O.; Koplovitz, G.; Amsler, C.D.; Baker, B.J. Observations on an association between the dexaminid amphipod Polycheria antarctica f. acanthopoda and its ascidian host Distaplia cylindrica. J. Crustac. Biol. 2009, 29, 605–608.
  116. Cheng, M.T.; Rinehart, K.L. Polyandrocarpidines: Antimicrobial and Cytotoxic Agents from a Marine Tunicate (Polyandrocarpa sp.) from the Gulf of California. J. Am. Chem. Soc. 1978, 100, 7409–7411.
  117. Gorbman, A.; Whiteley, A.; Kavanaugh, S. Pheromonal stimulation of spawning release of gametes by gonadotropin releasing hormone in the chiton, Mopalia sp. Gen. Comp. Endocrinol. 2003, 131, 62–65.
  118. Tsukamotol, S.; Kato, H.; Hirota, H.; Fusetane, N. Lumichrome Is a putative intrinsic substance inducing larval metamorphosis in the ascidian Halocynthia roretzi. In The Biology of Ascidians; Sawada, H., Yokosawa, H., Lambert, C.C., Eds.; Springer: Tokyo, Japan, 2001; pp. 335–340.
  119. Watanabe, T.; Shibata, H.; Ebine, M.; Tsuchikawa, H.; Matsumori, N.; Murata, M.; Yoshida, M.; Morisawa, M.; Lin, S.; Yamauchi, K.; et al. Synthesis and complete structure determination of a sperm-activating and -attracting factor isolated from the ascidian ascidia sydneiensis. J. Nat. Prod. 2018, 81, 985–997.
  120. Mikami, N.; Hosokawa, M.; Miyashita, K. Effects of sea squirt (Halocynthia roretzi) lipids on white adipose tissue weight and blood glucose in diabetic/obese KK-Ay mice. Mol. Med. Rep. 2010, 3, 449–453.
  121. Uchimasu, H.; Matsumura, K.; Tsuda, M.; Kumagai, K.; Akakabe, M.; Fujita, M.J.; Sakai, R. Mellpaladines and dopargimine, novel neuroactive guanidine alkaloids from a Palauan Didemnidae tunicate. Tetrahedron 2016, 72, 7185–7193.
  122. Casertano, M.; Imperatore, C.; Luciano, P.; Aiello, A.; Putra, M.Y.; Gimmelli, R.; Ruberti, G.; Menna, M. Chemical investigation of the indonesian tunicate Polycarpa aurata and evaluation of the effects against Schistosoma mansoni of the novel alkaloids polyaurines A and B. Mar. Drugs 2019, 17, 278.
  123. Kubanek, J.; Williams, D.E.; de Silva, E.D.; Allen, T.; Andersen, R.J. Cytotoxic alkaloids from the flatworm Prostheceraeus villatus and its tunicate prey Clavelina lepadiformis. Tetrahedron Lett. 1995, 36, 6189–6192.
  124. Wright, A.D.; Goclik, E.; König, G.M.; Kaminsky, R. Lepadins D-F: Antiplasmodial and antitrypanosomal decahydroquinoline derivatives from the tropical marine tunicate Didemnum sp. J. Med. Chem. 2002, 45, 3067–3072.
  125. Thakur, N.L. Studies on Some Bioactive Aspects of Selected Marine Organisms; Goa University: Goa, India, 2001.
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