Marine Natural Products from Tunicates and Their Associated Microbes

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. This review has garnered comprehensive literature on MNPs produced by tunicates and their symbiotic microbionts. Various sections covered in this review include tunicates’ ecological functions, biological activities, such as antimicrobial, antitumor, and anticancer activities, metabolic origins, utilization of invasive tunicates, and research gaps. Apart from the literature content, 20 different chemical databases were explored to identify tunicates-derived MNPs. In addition, the management and exploitation of tunicate resources in the global oceans are detailed for their ecological and biotechnological implications.


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.  Tables 2 and 3 where details are from the literature, and therefore, references are cited.
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 [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 [83]. The ascidian Didemnum guttatum was found to produce the cyclodidemniserinol trisulfate compound that showed anti-retroviral activity by inhibiting HIV-1 integrase [72]. The tunicate, Didemnum molle, released lanthipeptide divamide A that promised to be a potential anti-HIV drug [74] (Table 4).

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 [131,155,156]. The yellow pigmented Pseudoalteromonas tunicata mutants have demonstrated antifouling activity against algal spore germination, bacterial growth, fungal growth, and invertebrate larvae [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 [118].

Miscellaneous Applications
The chiton Mopalia sp. spawned when injected with 1.0 mg/L of gonadotropin releasing hormone (GnRH2) of a tunicate [48]. Lumichrome, a compound extracted from tunic, gonads, and eggs of ascidian, Halocynthia roretzi, was involved in the larval metamorphosis [89]. Similarly, sperm-activating and attracting factors (SAAF) were isolated from eggs of the ascidians Ciona intestinalis and Ascidia sydneiensis [162]. Lipids extracted from H. roretzi have demonstrated the antidiabetic and anti-obese properties in mice models [163]. Two novel alkaloids, mellpaladine and dopargimine, isolated from Palauan tunicate have demonstrated neuroactive behavior in mice [68]. Two new alkaloids, polyaurines A and B, isolated from the tunicate, Polycarpa aurata, inhibited blood-dwelling Schistosoma mansoni [96]. Lepadin and villatamine alakaloids isolated from Clavelina lepadiformis [61] and lepadins from Didemnum sp. [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 [164].

Issues in Extraction & Identification of Tunicate MNPs
Marine organisms have developed diverse secondary metabolic pathways, which produce a vast number of unusual chemical moieties. These compounds belong to a wide variety of chemical classes, including terpenes, shikimates, polyketides, peptides, alkaloids, and many unidentified and uncharacterized structures (Houssen and Jaspars, 2012). There are several technologies in place to isolate and characterize the natural products from even a very small quantity of marine organisms. However, there are still hurdles in the isolation and characterization of bioactive molecules from ascidians. These include 1. taxonomic uncertainty: worldwide, there are very few taxonomists available for proper taxonomic assignments of tunicates. Sometimes the identification using molecular tools has been complicated by the difficulty in getting pure gDNA from the target species due to complex biotic associations (Houssen amd Jaspars, 2012). 2. Quantity of isolated molecules: most of the time, a small quantity of metabolites is available in the organisms, which is not even sufficient for spectroscopic analysis. 3. Instability of molecules: there are extremely labile compounds in the extracts, which decompose during the purification process, and we get artefacts. Of course, these problems are common in other marine invertebrates as well. Research funding has also become a hurdle for many young researchers; thus, many researchers are publishing their works with crude extracts instead of analyzing complete structural elucidation. If we could address these issues, we will be able to isolate and characterize novel bioactive molecules from this unique group of marine invertebrates. The quantity of molecules can be increased if we collect the target tunicate species at the right time (season) from the correct geographic location. This can be achieved by understanding the chemical ecology of the producing species. For this purpose, there should be joint efforts from marine biologists, ecologists, and natural product chemists.

Metabolic Origin of Some Tunicates and Their Predators
Several bioactive MNPs extracted from tunicates were believed to be originated from tunicates themselves. However, few studies have investigated the original origin of tunicate MNPs from their symbiotic microbes. Tambjamine pigments have been reported to be originated from tunicate-associated symbiotic bacteria like S. marcescens [160] and Pseudoalteromonas tunicata [116,131]. An identical dark blue pigmented tetrapyrrole compound isolated from an ascidian was observed from a bacterium [165]. The blue tetrapyrrole pigment was reported to have originated from the associated bacteria, Serratia marcescens [120]. Didemnins extracted from the tunicate, T. solidum [111], are found to be released by associated bacteria, Tistrella mobilis and Tistrella bauzanensis [23,122]. Similarly, the trabectedin compound identified from the Caribbean tunicate, E. turbinata [152,166], has now been observed to be produced by its symbiotic bacteria, Candidatus Endoecteinascidia frumentensis [145]. Meridianins isolated from Antarctic tunicates, Aplidium, Synoicum, and some sponges, are thought to have originated from their symbiotic microbes [58]. Similarly, tetrahydroisoquinoline constituents identified from the tunicate, Ecteinascidia turbinata, appeared to be released by the unculturable endosymbiotic bacterium, Candidatus Endoecteinascidia frumentensis [113]. Some of the bioactive MNPs identified from Didemnid tunicates also originated from their symbiotic cyanobacterial species, such as Synechocystis and Prochloron [167,168]. Namenamicin produced by the orange color ascidian, Polysyncraton lithostrotum, was suggested to originate from its symbiotic bacterium, Micromonospora species [100]. The anti-HIV lanthipeptide, divamide A, isolated from the tunicate, Didemnum molle, was found to be produced by uncultivable symbiotic bacteria [74].
Tunicates are known to produce more than 300 alkaloid compounds [126]. The tunicate predatory flatworm Prostheceraeus villatus was reported to obtain alkaloids, lepadins, and villatamines by preying (dietary origin) on the tunicate, Clavelina lepadiformis [61]. Likewise, tambjamine alkaloids observed in the ascidian Atapozoa sp. [160] and associated bacteria [131] were found to be acquired by the predatory nudibranchs, like Nembrotha sp., for defense functions [59,169]. Pyridoacridine metabolites observed in ascidians and some sponges indicate a possible microbial origin or convergent evolution of these molecules [170].

Utilization of Invasive Tunicates Resources
Tunicates usually occur in relatively low abundance in coastal waters. However, some tunicates are reported as invasive species in some coastal waters [171] and are known to cause space competition [172], damage to aquaculture [173,174] by harboring pathogenic viruses and bacteria [175], and ecosystem alteration within the spread area [176]. Few non-invasive tunicate species of the coral reef environment have also been reported to overgrow on massive corals and caused minimal [112] or partial inhibition or delayed development of coral polyps [177]. A study reported the outbreak of the invasive tunicate, Diplosoma similis, that overgrew on corals and macrophytes and resulted in 50% mortality of corals [178] (Table 5). Didemnum psammathodes India Indigenous Space competition [182] Didemnum vexillum USA Exotic Threat to eelgrass [183] Didemnum vexillum Wales Exotic Space competition [184] Diplosoma similis American Sāmoa Indigenous Kill corals [178] Therefore, such overwhelming invasive species may be utilized to investigate their biological properties, biotechnological implications, and drug development. The exploitation of antiviral and cytotoxic didemnins from the invasive tunicate, T. solidum, has already been investigated [111,112]. Antimicrobial activity of α-helical peptides "Clavanins" was identified from the hemocytes of the tunicate, Styela clava [44]. Thus, other invasive species need to be investigated for their bioactive properties. Seasonal studies on the spread of various invasive tunicates and their biomass estimations are an important research aspect for resource management and coastal conservation. A study suggested that ocean warming is triggering the rise of invasive species in coastal waters [185]. Therefore, identifying the key ocean-warming factors and their mitigation strategies is essential for a sustainable management of the global ocean bioresources.

Research Gaps and Future Perspective
Tunicates have been an important marine drug reservoir to treat a variety of diseases, including cancer. These resources from the ocean, particularly from the deep-sea, remain untapped for drug discovery. Therefore, exploration and exploitation of tunicate resources from coastal waters to the deep-sea and tropical to polar regions would open new insights in the drug discovery and evolutionary lineages. However, these efforts should be driven by chemical ecology of these organisms. The study of chemical ecology will help in bioprospecting and the efficient production of marine drugs from this unique group of organisms. On the other hand, the mode of colonization and pigment biosynthesis by associated microbes and the acquisition mechanism of pigments (e.g., tambjamines) by tunicates from their associated microbes are yet to be unveiled. Since tunicates have been reported to be colonized by pathogenic bacteria during filter feeding, the pathological implications of tunicates needs to be investigated to understand the possible transfer ways of pathogenic bacteria from tunicates to other biota and aquaculture setups. Therefore, regular biodiversity monitoring and population dynamics of tunicate resources should be performed to understand their distribution patterns and impact on the coastal resources.