Metabolites from marine invertebrates and their symbiotic microorganisms: molecular diversity discovery, mining, and application

Metabolites from marine organisms have proven to be a rich source for the discovery of multiple potent bioactive molecules with diverse structures. In recent years, we initiated a program to investigate the diversity of the secondary metabolites from marine invertebrates and their symbiotic microorganisms collected from the South China Sea. In this review, representative cases are summarized focusing on molecular diversity, mining, and application of natural products from these marine organisms. To provide a comprehensive introduction to the field of marine natural products, we highlight typical molecules including their structures, chemical synthesis, bioactivities and mechanisms, structure–activity relationships as well as biogenesis. The mining of marine-derived microorganisms to produce novel secondary metabolites is also discussed through the OSMAC strategy and via partial chemical epigenetic modification. A broad prospectus has revealed a plethora of bioactive natural products with novel structures from marine organisms, especially from soft corals, gorgonians, sponges, and their symbiotic fungi and bacteria.


Introduction
The marine environment has been proven to be a rich source of new bioactive natural products for drug discovery. Coral reefs are among the most productive ecosystems and are a source of a large group of structurally unique biosynthetic products. To date, more than 40,000 marine natural products (MNPs) have been identified from various marine organisms, such as sponges, cnidarians, tunicates, molluscs, echinoderms, bryozoans, red algae, brown algae, green algae, and microorganisms (Carroll et al. 2019;Deshmukh et al. 2017;Jiménez 2018;Leal et al. 2016;Newman and Cragg 2016b). The upward trend in the discovery of new MNPs sourced from marine microorganisms continues unabated and now represents 57% of the total new MNPs reported in 2017 (Carroll et al. 2019). Based upon the putative biogenetic origins, these MNPs can be classified as polyketides, terpenoids, alkaloids, steroids, lactones, peptides, phenols, and lipids. Also, a large proportion of MNPs display interesting pharmaceutical activities, such as cytotoxic, antimicrobial, hypolipidemic, anti-inflammatory, antimalarial, analgesic, and antiasthmatic activities (According to the following Edited by Chengchao Chen. * Chang-Lun Shao shaochanglun@ouc.edu.cn * Chang-Yun Wang changyun@ouc.edu.cn websites: http://marin ephar macol ogy.midwe stern .edu; Jiménez 2018). Hence, MNPs are considered as an excellent and potentially valuable source for new chemical entities with novel structures and distinct mechanisms of action. To date, there have been 13 approved therapeutic agents that could be considered derivatives of MNPs (Altmann 2017;Jiménez 2018;Newman 2019;Pereira et al. 2019). Moreover, more than 30 MNP derivatives constitute the global marine pharmaceutical clinical pipeline in Phases III, II or I of drug development (According to the following websites: http:// marin ephar macol ogy.midwe stern .edu; http://pharm a.id.infor ma.com (accessed on August 6, 2019); Jiménez 2018; Newman 2019; Pereira et al. 2019). The significant potential for new drug development based on MNPs in all disease areas has been previously discussed (Newman and Cragg 2016a). Marine invertebrates have proven to be a primary source of bioactive MNPs, as many serve as chemical defense tools against predators, competitors and other ecological pressures. It has been demonstrated that the true origin of most MNPs appears to be the microorganisms living in concert with invertebrates. Most invertebrates are sessile, soft-bodied and move slowly, and are thus subject to potential parasite predation and detrimental microbial colonization. Therefore, they require a complex arsenal of secondary metabolites produced by their symbiotic microorganisms to facilitate a form of chemical defense (Jiménez 2018;Wang et al. 2008). This is likely the reason why MNPs from marine invertebrates and their symbiotic microorganisms are a rich sources of diverse and bioactive secondary metabolites (Martins et al. 2014;Mayer et al. 2010;Newman and Cragg 2016b). The chemical ecology underlying invertebrate-microorganism interactions provides a great opportunity for natural product chemists to mine for novel drug discovery. Therefore, the invertebrates and the abundant microorganisms in their ecosystems have attracted widespread attention for producing novel structural metabolites with potential bioactive applications (Blunt et al. 2018).
In the recent decade, the China Sea, especially the South China Sea, has become a hot spot in searching for novel bioactive MNPs. The invertebrates including sponges, soft corals, gorgonians and tunicates are prolific in the coral reefs in the South China Sea, and the microorganisms associated with these invertebrates have been demonstrated as a distinctive source for new bioactive MNPs.
In recent years, we have initiated a research program to find biological active MNPs based on marine chemical ecology (Figs. 1, 2) (Hou et al. 2015(Hou et al. , 2019aWang et al. 2008). A total of 709 MNPs including 307 new compounds have been obtained from marine invertebrates and their symbiotic microorganisms collected from the South China Sea. In this review, we summarize the representative 287 MNPs (Table 1) obtained by our group, highlighting multiple structural types of compounds and demonstrating discovery, diversity, compound mining, and bioactive application. The goal is to provide further inspiration for the discovery of bioactive MNPs and subsequent drugs development.

Macrocyclic lactones
Macrocyclic lactones derived from marine organisms have attracted much attention on account of their multiple potent biological activities including antitumor (Hirata and Uemura 1986;Isaka et al. 2002Isaka et al. , 2009Qi and Ma 2011;Suo et al. 2018), antifungal (Shier et al. 2001), and antimalarial activities (Isaka et al. 2009). For instance, caniferolides A and B, two glycosylated 36-membered polyol macrolides from marine-derived Streptomyces sp., showed pronounced antifungal activity (Perez-Victoria et al. 2019). Peloruside E, a macrolide from the New Zealand marine sponge Mycale hentscheli, displayed potent antiproliferative activity (Hong et al. 2018). As part of our current research, 88 macrolides with unique structures and significant biological properties have been obtained from marine invertebrates and their symbiotic microorganisms Shao et al. 2011Shao et al. , 2015aShao et al. , 2018. To obtain more valuable undiscovered natural compounds, multiple strategies and methods, including OSMAC and chemical epigenetic manipulation, were applied to microorganisms. Structural modification and chemical synthesis were performed to provide more derivatives, and the structure-activity relationships were discussed . The molecular mechanisms of compounds with strong activities were also investigated .
Three new 14-membered resorcylic acid lactones (RALs), including compounds 1 and 2 (cochliomycins A and B) with a rare natural acetonide group and 3 (cochliomycin C) with a 5-chloro substituted lactone (Fig. 3), were isolated from the culture broth of Cochliobolus lunatus (M351) isolated from the gorgonian Dichotella gemmacea collected from the South China Sea . In an antifouling assay, compound 1 (cochliomycin A) showed significant antifouling activity against the barnacle Amphibalanus (= Balanus) amphitrite at nontoxic concentrations (EC 50 = 1.2 µg/mL). Thus, compound 1 merits further investigation as a molecular model for the discovery of new antifouling molecules .
The molecular mechanism underlying antifouling activity of 1 against the cyprids of barnacle A. amphitrite has been investigated using the isobaric tags for the relative or absolute quantification (iTRAQ) labeling proteomic method . Differentially expressed proteins were examined by analyzing the changes in the proteome of A. amphitrite cyprids in response to 1 treatment. The results suggested that compound 1 affected the cytochrome P450, glutathione S-transferase (GST) and NO/cGMP pathways. The results of real-time PCR further demonstrated that the NO/cGMP pathway was activated in response to 1. Larval settlement assays suggested that S-methylisothiourea sulfate (SMIS) and 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) rescued cyprids from 1 (cochliomycin A)-induced inhibition of larval settlement. These findings supported the hypothesis that 1 inhibited barnacle larval settlement through stimulating the NO/cGMP pathway .
It should be noted that many gene clusters in marinederived fungi for the production of undiscovered secondary metabolites generally remain unexpressed under common laboratory culture conditions. In order to obtain uncharacterized metabolites, the chemical epigenetic perturbation method was applied to C. lunatus (TA26-46) to manipulate the silent fungal genes. Two new 14-membered RALs characterized with bromine substitution, 30 (5-bromozeaenol) and 31 (3,5-dibromozeaenol) (Fig. 6), were obtained from the culture treated with histone deacetylase inhibitor, sodium butyrate . Compounds 30 and 31 represent the first examples of naturally occurring brominated RALs.
Facing the challenges of discovery repetition and silent biosynthetic pathways, only limited mining approaches were applied in our work to date. As well known, microorganisms are easily manipulated on the genetic level. With gene sequencing technology blooming, genetic techniques and bioinformatics algorithms will continue to provide more opportunities for disclosing the novel pathways within marine microorganisms to then enable exploration of the untapped valuable bioactive molecules encoded within.
Furthermore, co-culturing as an effective method to induce production of novel secondary metabolites from two interacting microbial strains was applied to mine macrocyclic lactones. In single strain cultivation, the sponge-derived actinomycete Streptomyces rochei MB037 could produce two 18-membered macrolides, 32 (borrelidin) and 33 (borrelidin F) (Yu et al. 2019). Interestingly, in the co-culture of S. rochei MB037 and the gorgonian-derived fungus Rhinocladiella similis 35, besides macrolides 32 and 33, S. rochei MB037 was induced to produce two new fatty acids with a rare nitrile group, 34 (borrelidin J) and 35 (borrelidin K) ( Fig. 7) (Yu et al. 2019), which are structurally related to borrelidins. Compounds 34 and 35 were derived from the actinomycete S. rochei MB037, suggesting that the silent hydrolytic enzyme genes for hydrolyzing lactone in the borrelidin macrolides could be activated by the co-culture approach. Notably, both 34 and 35, obtained only in coculture, exhibited stronger antibacterial activities against methicillin-resistant S. aureus than 32 and 33.
In addition to marine invertebrates and their symbiotic microorganisms, macrolides were also obtained from marine-derived bacteria. Compound 36 (bastimolide A) (Fig. 8), a polyhydroxy macrolide with a 40-membered ring, was obtained from a new genus of the tropical marine cyanobacterium Okeania hirsute ). Its complete structure and absolute configuration were unambiguously identified by X-ray diffraction analysis of the nona-p-nitrobenzoate derivative 36a (Fig. 8). Compound 36 is a complex 40-membered macrolactone with repeating 1,5-diol and 1,3-diol groups as well as a t-butyl group moiety which is quite rare among natural products. Compound 36 showed strong antimalarial activity against four resistant strains of P. falciparum (IC 50 = 80 to 270 nmol/L), including TM90-C2A (chloroquine, mefloquine, and pyrimethamine resistant), TM90-C2B (chloroquine, mefloquine, pyrimethamine, and atovaquone resistant), W2 (chloroquine and pyrimethamine resistant), and TM91-C235 (chloroquine, mefloquine, and pyrimethamine resistant). Although this compound displayed toxicity to the host Vero cells (IC 50 = 2.1 μmol/L), it still represents a potentially promising lead for antimalarial drug discovery .
After discovery of the potent antimalarial 40-membered macrolide, continued investigation of the biologicallyactive and structurally-complex polyketides from O. hirsuta led to the isolation of a novel analogue 37 (bastimolide B) (Fig. 9), a 24-membered macrolide characterized by a long aliphatic chain with polyhydroxy groups and bearing a unique terminal tert-butyl group . A methanolysis mechanism for 36 is proposed and one unexpected isomerization product of the C 2 -C 3 double bond, 38 (2-(E)-bastimolide A) ( Fig. 9), was also obtained. The natural product 37 showed pronounced antimalarial activity against chloroquine-sensitive P. falciparum strain HB3 (EC 50 = 5.72 ± 0.65 μmol/L). Compound 38 showed even stronger antimalarial potency with an EC 50 value of 1.41 ± 0.47 μmol/L. Thus, the bastimolides show considerable antimalarial activity and represent an intriguing new class of antimalarial agents .

Anthraquinones
The diverse activities of anthraquinones isolated from marine-derived fungi with cytotoxic (Chen et al. 2013a;Xie et al. 2010;Zhu et al. 2012), antiviral , antioxidant ) and other activities have attracted many interests for drug discovery. For example, SZ-685C, a hydroanthraquinone isolated from the marine-derived Halorosellinia fungus, exhibited potent cytotoxic activity (Chen et al. 2013a;Xie et al. 2010;Zhu et al. 2012). In our investigation of bioactive anthraquinone derivatives from marinederived fungi Zheng et al. 2012), a total of 54 anthraquinone monomers and dimers have been isolated from Nigrospora sp., Alternaria sp. and other fungal genera. Analysis of anthraquinones with various chemical structures possessing interesting biological activities revealed the key structural features required for their activities.
Structure-activity relationship analysis revealed that the introduction of the 4-OH/9-OH/10-OH groups had little effect on antibacterial activity (Fig. 11). Interestingly, transforming from a hydroxy group at C-3 to an acetoxy  The cycloaliphatic ring has a positive contribution to the antibacterial activity. Additionally, an aromatic B ring may be necessary for the antibacterial activity ).

Azaphilones
The azaphilone molecules are a structurally variable family of fungal polyketide metabolites with a highly oxygenated pyranoquinone bicyclic core and exhibiting multiple bioactivities such as cytotoxic (Yamada et al. 2008), antimicrobial (Che et al. 2002), antiviral (Wang et al. 2011a), and anti-inflammatory (Yasukawa et al. 2008) activities. For instance, sclerketide C, an azaphilone analogous isolated from gorgonian-derived fungus Penicillium sclerotiorin CHNSCLM-0013, exhibited significant anti-inflammatory activity . Pleosporalone B from the culture of marine-derived fungus Pleosporales sp. CF09-1 and penicilazaphilone C from Penicillium sclerotiorum M-22 displayed potent antimicrobial activities Zhou et al. 2016). Azaphilones have attracted much attention due to their fascinating structural features and distinguished bioactivities (Wei et al. 2017). Our previous work on metabolites produced by symbiotic microorganisms of marine invertebrates found 48 azaphilones, including 37 new compounds, many of which were reported to exhibit antifouling and antiviral activities Wei et al. 2017;Zhao et al. 2015a). Desiring promising antifouling molecules, a one-step semisynthetic method was applied to discover more new azaphilonoids derivatives.
The bisindole derivatives manifest significant biological activities, while the synthesis method for unsymmetrical bisindolylmethanes or triarylmethanes still remains a great challenge (Abe et al. 2013;Fu et al. 2013;Ma and Yu 2005;Yu and Yu 2009;Zhu et al. 2002). In our study, an efficient S N 1-type reaction was developed for 3-indolylmethanols with miscellaneous nucleophiles, featuring catalyst-free, low cost, wide substrate scope and mild reaction conditions (Figs. 21, 22). This approach provides an efficient and environmental friendly method for synthesis of diverse 3-substituted indolyl derivatives as well as unsymmetrical bisindolylmethanes and triarylmethanes (Wen et al. 2015).

Terpenoids
Terpenoids constitute a class of broadly active natural products isolated from a diverse range of marine organisms. For example, 11R-methoxy-5,9,13-proharzitrien3-ol, obtained from an endophytic fungus Trichoderma harzianum X-5 derived from the marine brown alga Laminaria japonica, displayed growth inhibition of some marine phytoplankton species (Song et al. 2018). Nakijinol G, a meroterpenoid obtained from a sponge Hyrtios sp. collected from the South China Sea, showed protein tyrosine phosphatase (PTP1B) inhibitory activity ). Trichodermanin C, a diterpenes obtained from a fungal strain Trichoderma harzianum OUPS-111D-4 derived from sponge Halichondria okadai, exhibited significant cytotoxic activity (Yamada et al. 2017). In our ongoing research on the marine invertebrates and their symboitic microorgnisms, 101 terpenoids including 43 new compounds with novel structures were isolated, which exhibited antibacterial, cytotoxic and antifouling activities (Cao et al. , 2017Li et al. 2012a).

Steroids
Steroid derivatives from marine organisms are noted for diverse unusual structures with multiple potent biological properties. For instance, petasitosterone B, a steroid isolated from a Formosan marine soft coral Umbellulifera petasites exhibited promising anti-inflammatory activity . Two 9,11-secosteroidal glycosides, sinularosides A and B, isolated from the South China Sea soft coral Sinularia humilis, exhibited potent antimicrobial activity (Sun et al. 2012). In our previous reports, 86 steroidal compounds were obtained from marine invertebrates and their symbiotic fungi, which exhibited cytotoxic, antiviral and antibacterial activities (Cao et al. 2014;Sun et al. 2015;Zhao et al. 2013).

Phenylpropanoids
Phenylpropanoids contain many chemical structure types, such as chromone, coumarin, flavone and lignin, which possess various biological activities including antioxidant, cytotoxic, antimicrobial, and enzyme inhibitory activities. For example, arthone C, a chromone derivative isolated from a deep-sea-derived fungus Arthrinium sp., exhibited potent antioxidant activity (Bao et al. 2018). Two tetracyclic coumarin derivatives and two coumarin dimers isolated from the marine-derived fungus Eurotium rubrum showed significant tyrosinase inhibitory activity (Kamauchi et al. 2018). In our previous studies, 80 phenylpropanoid compounds with 54 new compounds were obtained, which showed antifouling, antibacterial and cytotoxic activities (Qi et al. 2013;Zhao et al. 2015b). These findings provide further insight into the chemical diversity and biological activities of this class of MNPs.
Twelve new chromone derivatives, 211-222 (corynechromones A-L) (Fig. 38), were obtained from the spongederived fungus Corynespora cassiicola XS-200900I7 (Zhao et al. 2015b). These are the first chromone derivatives reported from the genus Corynespora. The absolute configurations of 211-220 were determined by the modified Mosher's method and TDDFT ECD calculations along with comparison of their CD spectra. Interestingly, 211/212, 213/214, 215/216, and 217/218 were pairs of epimers at C-2. A biogenetic pathway of the isolated chromone derivatives was proposed (Fig. 39). The different cyclization pathways from an uncyclized α,β-unsaturated ketone precursor may lead to production of the octalactones (Ebrahim et al. 2012(Ebrahim et al. , 2013. Alternatively, the decalactones may be produced by ester formation with the distal hydroxy group. A possible non-enzymatic cyclization route may be involved to form the epimeric pairs. A Michael reaction can occur with the nucleophile attacking from either face, leading to the formation of the epimeric pairs (Zhao et al. 2015b).

Peptides
Peptides isolated from many marine species present various biological activities, such as antimicrobial, antiviral, antitumor, antioxidative and cardioprotective activities. For instance, mirabamide A, a cyclic depsipeptide obtained from the sponge Siliquariaspongia mirabilis, showed potent inhibitory activity on HIV-1 fusion (Plaza et al. 2007).
Reniochalistatin E, an octapeptide isolated and characterized from the marine sponge Reniochalina stalagmitis, displayed significant cytotoxic activity (Zhan et al. 2014). In our studies, a total of 34 peptides were identified, of which more than half are cyclopeptides involved in 18 new peptides (Chen et al. 2014c;Hou et al. 2019bHou et al. , 2019c. Particularly, LC-MS/MS-dependent molecular networking and 1 H NMR techniques were applied in order to identify new peptides. The strategy of molecular networking based on MS/ MS cheminformatics could shed light on structural relationships and accelerate the dereplication of molecules in crude extracts to mine new molecules. Based on integrating LC-MS/MS-dependent molecular networking and 1 H NMR techniques, the targeted identification of ten cyclohexadepsipeptides were achieved from the gorgonian coral-derived fungus Penicillium chrysogenum (TA01-16) (Fig. 41), including the new compounds 235-237 (chrysogeamides A-C) and 240-244 (hrysogeamides D-H), as well as the known compounds 238 and 239 (scopularides A and B) ( Fig. 42) (Hou et al. 2019b). These cyclohexadepsipeptides contain the same D-Leu fragment, and 235 and 242 are reported for the first time featuring a 3-hydroxy-4-methylhexanoic acid (HMHA) moiety of cyclodepsipeptides. Interestingly, isotope labelling feeding experiments demonstrated that 13 C 1 -L-Leu was transformed into 13 C 1 -D-Leu moiety in 238 and 239 indicating that D-Leu in these molecules may be epimerized from L-Leu by a leucine racemase. Compounds 235-239 displayed selective activity in promoting angiogenesis toward the Tg (Flk1: EGFP) transgenic zebra fish D. rerio embryo (Hou et al. 2019b). Applying molecular networking techniques, three new cycloheptapeptides, 245-247 (asperversiamides A-C) (Fig. 43), were isolated from the coral-derived fungus Aspergillus versicolor (CHNSCLM-0063) (Hou et al. 2019c). Their complete structures including configuration were confirmed by total synthesis. These compounds showed strong inhibitory activity against Mycobacterium marinum (MICs = 23.4,81.2,87.5 μmol/L, respectively), demonstrating valuable application potential in treating M. marinum infection (Hou et al. 2019c).
Until now, the molecular networking approach has only been applied to mine the undiscovered cycloheptapeptides from marine-derived fungus in our research on MNPs. Undoubtedly, the molecular networking strategy based on LC-MS/MS and relevant data libraries should be considered an effectively targeted isolation technique to speed the discovery of new cryptic secondary metabolites. It is worth applying the molecular networking approach to exploit novel molecules from the pools of MNPs in extracts from marine organisms.

Phenyl ether derivatives
An increasing number of marine-derived phenyl ether derivatives with novel structures have been reported with diverse biological activities, including antimicrobial, antitumor, cardiotonic, antidiabetic, antiinflammatory and antiviral activities. For instance, 4-carbglyceryl-3,30-dihydroxy-5,50-dimethyldiphenyl ether, a diphenyl ether isolated from the deep-sea-derived fungus Aspergillus versicolor SCSIO 41502 showed potent antifouling activity . A series of antibacterial polybrominated diphenyl ethers were isolated from the Indonesian sponge Lamellodysidea herbacea (Hanif et al. 2007). In our studies, 35 phenyl ether derivatives were obtained in all, among them 9 compounds were first reported, showing antibacterial, antifouling and cytotoxic activities (Chen et al. 2013b;Shi et al. 2017). To clarify the structure-activity relationships, chemical synthesis was performed (Chen et al. 2013b). epidermidis (MIC = 2.71 μmol/L). A series of derivatives (256a, 258a-258g) were designed and synthesized for the study of structure-activity relationships, suggesting that one free hydroxy plays a critical role for antibacterial activity. In particular, 258g exhibited the strongest antibacterial activity toward S. epidermidis (MIC = 0.556 μmol/L). These results revealed that the ester functionality or bromination could increase antibacterial activity, but the simultaneous presence of both the ester and brominated benzene ring caused a loss of activity (Chen et al. 2013b).
In summary, our research on the discovery of MNPs resulted in the discovery of diverse structures from soft corals, gorgonians, sponges, tunicates, anemones and their symbiotic microorganisms collected from the South China Sea. Based on our findings, the statistical analysis of 709 MNPs including 307 new compounds demonstrated that the proportions of terpenoids, alkaloids, steroids, and macrocyclic lactones are larger than that of other structures (Fig. 48). It is also revealed that macrocyclic lactones, azaphilones and phenylpropanoids are the most potential classes for the discovery of novel MNPs (Fig. 48). In terms of biological activities, more than half of the obtained MNPs displayed tested biological activities, with the most frequent findings as antibacterial, antifouling, and cytotoxic activities (Fig. 49). Interestingly, many of the active MNPs exhibited multiple bioactivities, for example, MNPs with antiviral activities usually displayed cytotoxic activities, while MNPs with antibacterial activities commonly showed cytotoxic and antifouling activities. It is worth noting that more than 40 compounds were found to exhibit potent activities even stronger than the positive controls. Particularly, the macrocyclic  lactone cochliomycin A (1), the azaphilone sclerotioramine derivative (101), and the alkaloid aniduquinolone A (104) may be candidates for efficient antifouling agents, and the macrocyclic lactone bastimolide A (36), the anthraquinones nigrosporin B (41), and the steroid echrebsteroid C (165) might be promising lead compounds for further development of antimalarial, antibiotic, and antiviral drugs.
The South China Sea possesses rich and unique species resources, with more than 95% of the invertebrates mainly existing in the coral reefs in this sea area in China (Zhang et al. 2006). Not surprisingly, many investigations on marine invertebrates and their symbiotic microorganisms target this "biodiversity hotspot" as the center of sample collection. It should be mentioned that the diverse abundance of MNPs derived from marine invertebrates and their symbiotic microorganisms from the South China Sea were discovered and studied. In recent decades, research on the diversity of MNPs discovered from the South China Sea by other Chinese researchers has been well underway. These groundbreaking research efforts contributed to the discovery of a great number of novel and promising bioactive molecules usually from such sources as sponges Jiao et al. 2019;Wang et al. 2015b), corals Wu et al. 2019), bryozoans (Yu et al. 2015), and associated microorganisms (Cheng et al. 2016;Nong et al. 2016). Regarding the abundance of MNPs with diverse structures and a wealth of biological activities, we believe that only the proverbial "tip of the iceberg" has been explored from the South China Sea, and the resulting novel active metabolites with potential pharmacology applications are worthy of further exploration.

Conclusion
In this review, we exemplified nine types of structurally unique MNPs including macrocyclic lactones, anthraquinones, azaphilones, alkaloids, terpenoids, steroids, phenylpropanoids, peptides, and phenyl ether derivatives obtained from marine invertebrates and their symbiotic microorganisms. Our research provides several typical representative MNPs from marine invertebrates, especially sponges, soft corals, gorgonian corals, and their symbiotic microorganisms (mainly fungi). These MNPs display various potent bioactivities involved in not only chemoecological effects such as antifouling, ichthyootoxic, and brine shrimp lethal activities but also pharmaceutical activities including antibacterial, antiviral, fungicidal, cytotoxic, and antimalarial activities. Our studies demonstrate that MNPs derived from marine invertebrates and their symbiotic microorganisms in the South China Sea are a prolific resource for the discovery of bioactive MNPs. It could be expected that the symbiotic microorganisms associated with marine invertebrates have great potential as a significant source of structurally interesting molecules. It should be noted that the application of multiple discovery strategies and methods could effectively promote the exploitation of novel MNPs with diverse structures. In our study, different methodological approaches, including single culture, OSMAC, chemical epigenetic manipulation, co-culture, structural modification and chemical synthesis, have been applied in the search for new MNPs. Among them, co-culture, structural modification and chemical synthesis were found to be effective approaches to obtain potential bioactive MNPs. Particularly, LC-MS/ MS-dependent molecular networking has been applied as a promising approach to dereplicate complex natural product mixtures, contributing to the targeted isolation of a series of new compounds. It is anticipated that future genetic techniques and bioinformatics tools, especially metagenomic approaches, genome mining, and heterologue biosynthesis, could accelerate the exploration and accessibility of remaining undiscovered MNPs with novel structures and promising bioactivities from marine microorganisms.