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][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][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][6,7]. Therefore, tunicates are considered as important model organisms for several research aspects, such as evolution [6], development biology ^[8][9][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][11,12]. However, a few species, like Halocynthia roretzi and Pyura michaelseni, are eaten in southeast Asian countries like Korea ^[13][14][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][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][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][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][29,30], thereby triggering the downward biogeochemical flux (e.g., carbon flux) patterns from surface to deep waters ^[29][31][32][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][49] that produce species-specific metabolites ^[43][50]. 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][51].

4. Antimicrobial Applications

Tunicates ^[45][123], with their associated epi-symbionts ^[16][46][16,124] and endosymbionts ^[47][125], are prolific producers of antimicrobial and antifungal compounds inhibiting pathogens. The brominated alkaloids ^[48][126] and other compounds from tunicates have been reported to possess several biological activities ^[25][26][25,26]. Pseudoalteromonas tunicata produces alkaloid tambjamine (425 nm), an antifungal yellow pigment ^[49][50][127,128], and violacein (575 nm), a purple pigment with antiprotozoal activity ^[51][52][129,130], in addition to a range of bioactive compounds ^[51][53][129,131]. Methanol extraction of Lissoclinum fragile displayed antibacterial, antifungal, hemolytic, and cytotoxic activities ^[54][92]. 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][88]. A diffusible 190-kDa protein produced by tunicate Ciona intestinalis associated bacterium Pseudoalteromonas tunicata was found to show antibacterial activity against marine isolates ^[56][132]. 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][44]. Halocidin, an antimicrobial peptide purified from tunicate Halocynthia aurantium showed antibacterial activity against methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa ^[58][47]. Similarly, halocyntin and papillosin peptides isolated from tunicate Halocynthia papillosa also displayed antibacterial activity against both Gram-positive and Gram-negative marine bacteria ^[59][46]. Halocyamine peptides synthesized by the hemocytes of Halocynthia roretzi showed antimicrobial activity against various bacteria and yeasts ^[60][90]. Similarly, Halocyamines produced by Styela clava also displayed antimicrobial properties ^[61][108]. A salt-tolerant peptide isolated from hemocytes of Ciona intestinalis showed both antibacterial and antifungal activity ^[62][133]. Vanadium chloride and vanadyl sulfate also displayed antibacterial activity against various pathogens ^[63][95]. An endobiont, Streptomyces sp., isolated from the tunicate, Styela canopus, produced antibacterial compounds such as granaticin, granatomycin D, and dihydrogranaticin B ^[64][121]. Similarly endosymbiotic fungi associated with the tunicates, Polycarpa aurata ^[65][134] and Rhopalaea crassa ^[66][135], 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][136]. 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][111,137]. 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][83]. The ascidian Didemnum guttatum was found to produce the cyclodidemniserinol trisulfate compound that showed anti-retroviral activity by inhibiting HIV-1 integrase ^[71][72]. The tunicate, Didemnum molle, released lanthipeptide divamide A that promised to be a potential anti-HIV drug ^[72][74].

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][138] to treat breast cancer ^[74][75][139,140], soft tissue sarcoma ^[76][141], and ovarian cancer ^[77][78][79][142,143,144]. This molecule is suggested to originate from E. turbinata symbiotic bacteria, Candidatus Endoecteinascidia frumentensis ^[80][145]. However, plitidepsin (Aplidin^®), a depsipeptide isolated from the Mediterranean tunicate, Aplidium albicans, is in phase II clinical trials ^[73][81][138,146] as an anticancer drug against breast cancer ^[82][147], human kidney carcinoma cells ^[83][52], and multiple myeloma ^[84][53]. Didemnin B is also in phase II trials ^[85][148], showing anticancer activity against leukaemia P388 cells ^[68][111]. 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][62]. Lamellarin sulfates originated from Didemnum ternerratum ^[88][78] and polycarpine dihydrochloride, a disulfide alkaloid extracted from an ascidian Polycarpa clavata, were found to inhibit human colon tumor cell lines ^[89][97]. Cystodytins A, B, and C, three teracyclic alkaloids isolated from Okinawa tunicate Cystodytes dellechiajei, were reported to show antitumor activities ^[90][64]. Macrolides isolated from tunicates Lissoclinum patella (Patellazole C) ^[91][94] and Eudistoma cf. rigida (Lejimalides A, B, C, and D) ^[92][93][149,150] possessed anticancer activity ^[94][151]. Diplamine, an orange pigment alkaloid produced by Diplosoma sp., demonstrated cytotoxic activity against leukemia cells ^[95][79]. Halocyamine A and B peptides extracted from H. roretzi showed anticancer activity against various cell lines ^[60][90]. A depsipeptide, dehydrodidemnin B, produced by Aplidium albicans inhibited Ehrlich carcinoma cells in mice and reduced 80–90% tumor cells ^[96][54]. 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][152] and breast, lung, ovary, and melanoma cells ^[98][153]. The Brazilian ascidian, Didemnum granulatum, produced G2 checkpoint-inhibiting aromatic alkaloids, granulatimide and isogranulatimide ^[99][154]. The ascidian Cystodytes dellechiajei produced topoisomerase II-inhibiting ascididemin, which has antitumor activity against various tumor cell lines ^[100][66]. 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][88]. Cynthichlorine, an alkaloid isolated from the tunicate Cynthia savignyi, showed cytotoxicity against Artemia salina larva at an LD₅₀ of 48.5 μg/mL ^[101][63]. Siladenoserinols A and B derivatives isolated from didemnid tunicates possessed antitumor activity by inhibiting the interaction of p53-Hdm2 ^[102][69].

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]}[131,155,156]. The yellow pigmented Pseudoalteromonas tunicata mutants have demonstrated antifouling activity against algal spore germination, bacterial growth, fungal growth, and invertebrate larvae ^[51][129]. 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][118]. 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][95,157]. Didemnimides C and D from Didemnum conchyliatum ^[107][158], nordidemnin B ^[108][102] and didemnin B ^[109][159] from Trididemnum solidum, and granulatamides from Didemnum granulatum ^[110][73] 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][88]. MNPs isolated from Antarctic tunicates have demonstrated variability in anti-deterrent activities ^[111][58]. 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][59,160]. The blue tetrapyrrole pigment was suggested to originate from the associated bacteria Serratia marcescens ^[114][120]. Tambjamines and tetrapyrrole chemical constituents from both adult and larvae were reported to function as defensive chemicals against predators ^[108][102]. Lipophilic crude extracts from Antarctic tunicate, Distaplia cylindrica ^[115][161], and polyandrocarpidines from Polyandrocarpa sp. ^[108][116][101,102] 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][48]. Lumichrome, a compound extracted from tunic, gonads, and eggs of ascidian, Halocynthia roretzi, was involved in the larval metamorphosis ^[118][89]. Similarly, sperm-activating and attracting factors (SAAF) were isolated from eggs of the ascidians Ciona intestinalis and Ascidia sydneiensis ^[119][162]. Lipids extracted from H. roretzi have demonstrated the antidiabetic and anti-obese properties in mice models ^[120][163]. Two novel alkaloids, mellpaladine and dopargimine, isolated from Palauan tunicate have demonstrated neuroactive behavior in mice ^[121][68]. Two new alkaloids, polyaurines A and B, isolated from the tunicate, Polycarpa aurata, inhibited blood-dwelling Schistosoma mansoni ^[122][96]. Lepadin and villatamine alakaloids isolated from Clavelina lepadiformis ^[123][61] and lepadins from Didemnum sp. ^[124][71] 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][164].