Marine Natural Products from New Caledonia—A Review

Marine micro- and macroorganisms are well known to produce metabolites with high biotechnological potential. Nearly 40 years of systematic prospecting all around the New Caledonia archipelago and several successive research programs have uncovered new chemical leads from benthic and planktonic organisms. After species identification, biological and/or pharmaceutical analyses are performed on marine organisms to assess their bioactivities. A total of 3582 genera, 1107 families and 9372 species have been surveyed and more than 350 novel molecular structures have been identified. Along with their bioactivities that hold promise for therapeutic applications, most of these molecules are also potentially useful for cosmetics and food biotechnology. This review highlights the tremendous marine diversity in New Caledonia, and offers an outline of the vast possibilities for natural products, especially in the interest of pursuing collaborative fundamental research programs and developing local biotechnology programs.


Introduction
Located approximately at 165˝E and 21˝30 1 S, the New Caledonia archipelago enjoys a privileged position east of the city of Rockhampton, central province of the Great Barrier Reef, 1500 km away from the Australian Queensland coast, with surface seawater temperatures mild enough to avoid recurrent coral bleaching episodes.
This tropical subregion of Melanesia is a relic of a continental landmass that drifted away from the super-continent Gondwana at the end of the Cretaceous period (see Pelletier 2007) [1]. Successive geological events have resulted in an archipelago that comprises a main island called Grande Terre, Ile des Pins in the south, the Entrecasteaux reefs in the north, the Loyalty Islands in the east and, finally, the Chesterfield-Bellona plateau in the west, located at mid-distance to Australian coast ( Figure 1).
New Caledonian waters represent an exclusive economic zone (EEZ) spanning more than 1,700,000 km 2 . The EEZ includes a vast and complex reef system of 4537 km 2

of reef area and
Only since the 19th century has attention been paid to diversity of the New Caledonian reef systems. Many expeditions have been conducted to study the oceanic life of these hitherto unexplored oceanic zones. The first oceanographic cruise, the Challenger expedition (1872-1876), led to the collection of thousands of previously unknown marine species [6]. Since the 19th century, scientific interest has shifted from the study of "exotic" specimens stored in museum collections and classified according to their morphological features, to the study and understanding of marine ecosystems. Charles Darwin's landmark expeditions in the Eastern Pacific islands and his remarkable observations on the formation of atolls from subsiding volcanic islands sparked continued interest in ecosystems biology in oceans across the world. Research vessels are now equipped with sophisticated equipment to carry out on-site sample analysis (e.g., molecular biology) and data transmission via satellite systems. For example, the schooner Tara, specially fit out with on-board facilities, recently sailed around the world to study the impact of global warming on plankton and coral reef systems across the Pacific [7].
Pioneering scientific investigations in New Caledonian waters were carried out locally after World War II. The naturalist René Catala initiated ecological surveys and species census around Ile aux Canards [8], an island off Nouméa now directly exposed to human activities, with recent 2009 [2], with kind permission from the author).
Only since the 19th century has attention been paid to diversity of the New Caledonian reef systems. Many expeditions have been conducted to study the oceanic life of these hitherto unexplored oceanic zones. The first oceanographic cruise, the Challenger expedition (1872-1876), led to the collection of thousands of previously unknown marine species [6]. Since the 19th century, scientific interest has shifted from the study of "exotic" specimens stored in museum collections and classified according to their morphological features, to the study and understanding of marine ecosystems. Charles Darwin's landmark expeditions in the Eastern Pacific islands and his remarkable observations on the formation of atolls from subsiding volcanic islands sparked continued interest in ecosystems biology in oceans across the world. Research vessels are now equipped with sophisticated equipment to carry out on-site sample analysis (e.g., molecular biology) and data transmission via satellite systems. For example, the schooner Tara, specially fit out with on-board facilities, recently sailed around the world to study the impact of global warming on plankton and coral reef systems across the Pacific [7].
Pioneering scientific investigations in New Caledonian waters were carried out locally after World War II. The naturalist René Catala initiated ecological surveys and species census around Ile aux Canards [8], an island off Nouméa now directly exposed to human activities, with recent changes in reef zonation and species composition. In addition, in 1956, Catala founded the Aquarium of Nouméa (now the Aquarium des Lagons), a first-of-its-kind structure specifically designed to observe rare and fragile marine species in their environment, including homegrown corals. Catala incidentally discovered the

Sample Collection Sites
Collection sites were selected to cover the large diversity of habitats ranging from shallow lagoons to deep parts of the outer slopes of the barrier reefs. They include hard and sandy bottoms, sheltered and exposed areas of the mainland (Grande Terre), Loyalty Islands, as well as remote reefs and atolls (Entrecasteaux, Chesterfield) and seamounts ( Figure 1).
Each site was georeferenced and described using geomorphological and biological descriptors. Historical data have been sorted, standardized and stored along with recent information in the dedicated database LagPlon [24].

Biological Material Sampling
For all the groups collected for chemical purposes, specimens from each taxon were sampled for taxonomical identification and preserved in ethanol as vouchers after labelling. Collections were duplicated, one sample was kept for the IRD reference collection to facilitate new sampling efforts and the duplicate was sent to specialists for taxonomy work. In situ macrophotographs were taken as well as additional ex situ laboratory photographs when necessary. Specimens used to describe new species (holotypes) have been deposited in various museum collections in Australia (Northern Territory Museum, Darwin and Queensland Museum of South Brisbane), New Zealand, mainland France (National Museum of Natural History, Paris), Belgium (Université Libre de Bruxelles). Paratypes were systematically filed in the IRD reference collection in Nouméa, along with photographs and field records.

Biological Material
Chemical characterization and biological assays were initially carried on samples with wet weights ranging from 300 to 3000 g. However, recent progress in analytical techniques allows molecular inventory and pharmacological tests from smaller amounts of material in compliance with international regulations on the use of biological material for research purposes.
Below, we describe the basic protocols used locally at the IRD laboratories in Nouméa. Each of the molecules that have been investigated locally or with national and international partners have been treated separately ( Figure 3). It is beyond the scope of this review to detail each protocol individually; they can be found in the original research papers, or in a natural products database.

Conditioning Samples for Chemistry
Freshly collected material is sorted by taxon and frozen on board at´20˝C, or at least placed in 70% ethanol in distilled water (slightly acidified to avoid oxidation of polar compounds) until it can be deep frozen and freeze-dried for subsequent use. Material used for enzymology or genome studies is snap-frozen on board (liquid nitrogen or dry ice in pure alcohol). For short collecting sessions (less than one full day), the material may be kept alive if each item is appropriately handled.
Back at the laboratory, the material must be ground/chopped/crushed then freeze-dried or ethanol-preserved and stowed away for later use, or sent abroad to partner laboratories.

Routine Procedure
Unless otherwise specified, a standard protocol is used for extracting compounds and separating them into crude non-polar (organic) and polar (aqueous) fractions ( Figure 4).

Conditioning Samples for Chemistry
Freshly collected material is sorted by taxon and frozen on board at −20 °C, or at least placed in 70% ethanol in distilled water (slightly acidified to avoid oxidation of polar compounds) until it can be deep frozen and freeze-dried for subsequent use. Material used for enzymology or genome studies is snap-frozen on board (liquid nitrogen or dry ice in pure alcohol). For short collecting sessions (less than one full day), the material may be kept alive if each item is appropriately handled.
Back at the laboratory, the material must be ground/chopped/crushed then freeze-dried or ethanol-preserved and stowed away for later use, or sent abroad to partner laboratories.

Routine Procedure
Unless otherwise specified, a standard protocol is used for extracting compounds and separating them into crude non-polar (organic) and polar (aqueous) fractions ( Figure 4). Organic fractions are generally evaporated under vacuum (Rotovap) and kept in the dark at −20 °C. Water solubles may need to be desalted using size-exclusion gel chromatography (SEC) or by membrane filtration under nitrogen (Amicon ® , Alsace, France) prior to separation, but for long-term storage, desalting is not necessary. Aliquoting is necessary for multiple chemical characterizations and for bioassays.

Peptide Protease Inhibitors
Live tissues are processed (minced or fragmented) on board and placed in an acidic solution (methanol/2 N acetic acid) to optimize the extraction of polar substances and block any potential hydrolysis by proteolytic enzymes. The cold (−20 °C) slurry is filtered twice and filtrates are reduced by evaporation and neutralized to pH 5.5 prior to freeze-drying. The total protein content of the crude extract is assayed by measuring optical densities with a UV spectrophotometer. Organic fractions are generally evaporated under vacuum (Rotovap) and kept in the dark at 20˝C. Water solubles may need to be desalted using size-exclusion gel chromatography (SEC) or by membrane filtration under nitrogen (Amicon ® , Alsace, France) prior to separation, but for long-term storage, desalting is not necessary. Aliquoting is necessary for multiple chemical characterizations and for bioassays.

Peptide Protease Inhibitors
Live tissues are processed (minced or fragmented) on board and placed in an acidic solution (methanol/2 N acetic acid) to optimize the extraction of polar substances and block any potential hydrolysis by proteolytic enzymes. The cold (´20˝C) slurry is filtered twice and filtrates are reduced by evaporation and neutralized to pH 5.5 prior to freeze-drying. The total protein content of the crude extract is assayed by measuring optical densities with a UV spectrophotometer.

Separation, Purification
Classical separation and purification techniques (TLC, HPLC, SEC) were not routinely used unless requested for products presenting an interesting bioactivity profile. This data is mentioned in the original publications for each novel structure.

Structural Analysis
Basic spectroscopic (NMR, UV, IR) and spectrometric (MS) methods are used when available on site, mostly to avoid replication.

Chemical Databases
The IRD proprietary information system Cantharella [25] compiles pharmacochemical data of all organisms collected in New Caledonia and cross-Pacific oceanographic cruises for the study of their natural substances, with restricted access via Internet. The information system provides access to biological data, accounts for all chemical processes from extraction to purification, and presents the bioactivity profile detected using the complete range of tests.

Preliminary Testing
Vacuum-dried crude extracts or non-purified fractions thereof can be used for describing general characteristics using toxicity assays on various invertebrate, vertebrate and plant models, and cultures of microbial reference strains (Table 1).
Preliminary tests performed on site are useful for making appropriate decisions as to whether a given sample from a newly collected organism is worth investigating further. Along with taxonomic criteria and dereplication issues, such decisions are now taken after consulting the scientific literature and specialized databases, especially the IRD databases LagPlon [24] and Cantharella [25], which, in addition to the more traditional MarinLit and other databases, considerably aid local researchers. The brine shrimp (Artemia salina) toxicity bioassay is one of the most basic and widely used tests to detect cytotoxicity. Newly hatched brine shrimp nauplii are exposed to various concentrations of soluble or solubilized test substances and mortality is recorded, leading to a median lethal dose (LD 50 ) estimate. A typical protocol is detailed in [27].

Mosquito Fish Toxicity Assay
The mosquito fish Gambusia affinis is often used to evaluate the toxicity of substances to be screened for antimitotic activities, and it has been adapted from [28], a study that used this species for comparative ecotoxicological studies of soft corals. The mosquito fish toxicity test has been extended to include multiparametric behavioral observations and can provide valuable neurophysiological information as well as ecotoxicological data on crude extracts that are water soluble or solubilized [29].

Fertilization of Sea Urchin Eggs
The aim of this test is to observe the division pattern of eggs of the test sea urchin Echinometra mathaei fertilized in the laboratory, by adapting the method Kobayashi [30] originally designed for testing the toxicity of heavy metals on early embryonic stages. This test is highly sensitive and works at extremely low concentrations of the test substances. Cell division arrest at specific early and embryonic stages provides a preliminary indication of specific enzymatic inhibition at determined steps of the cell cycle.

Anti-Serpin Activity
Serine protease inhibition assays are conducted locally using bovine trypsin (protocol inspired from Green et al., 1953) [36] and porcine elastase [37], with respectively, benzoyl-arginine ethyl ester (BAEE) and succinyl (Alanyl) 3 para-nitro anilide (Suc [Ala] 3 pNa) as substrates. End-point titration using pH-stat benchtop equipment is used to evaluate the inhibition potential of the crude extracts on bovine trypsin. The colorimetric method is used to evaluate the anti-elastase activity of the crude extracts.

Further Biological Testing
For the identification of antiproliferative natural products, cancerous and non-cancerous mammalian cell lines are tested. The comprehensive list of cell lines that are used to characterize active molecules from sponges is given in the footnote of Table 2.

General Comments
Sponges are multicellular, filter-feeding diploblastic invertebrates, i.e., without organized tissues or organs, but with a special inner layer of ciliated cells called choanocytes that generate constant water flow through numerous feeding/excretory channels. There are two major subphyla of Porifera based on their biomineralization patterns: the Calcarea or calcareous sponges and the Silicispongia or siliceous sponges, the latter group being further subdivided into Hexactinellida (glass sponges), Demospongiae, which contain a proteinaceous matrix (spongin), and Homoscleromorpha, now recognized as distinct from the latter class. Calcareous sponges are primarily found in shallow waters, and particularly abundant and diverse in tropical coral reefs. Siliceous sponges, which represent most of the living sponge species today, are found at all bathymetric levels and also in freshwater, with large populations in some rivers and lakes. Most glass sponges live in deep waters, and are difficult to access [16]. Since the 1980s, deep-water dredging on the outer reef slope of the barrier reef down to 800 m has led to the discovery of a number of non-described species. As of 2007, 149 species of Porifera have been recorded in New Caledonian waters [5].
Most shallow-water sponge species live in symbiosis with Archaea, eubacteria and cyanobacteria, forming holobiont systems that represent the main source of bioactive compounds [38,39] primarily isolated from host tissues, but occasionally isolated from cultures of associated microbiota. Sponges contain a wide range of so-called secondary metabolites, some of which afford interesting biological activities [40] (Table 2).

Porifera Success Stories
New Caledonian sponges have revealed more exciting chemicals than any other studied taxon, in terms of carbon skeleton, degree and patterns of unsaturation, halogenation, functional group originality, presence of unusual heteroatoms etc. Some of these features are responsible for the rather exceptional bioactivity profiles encountered. Some outstanding examples are described below:
Chemical analyses led to the isolation of sterols that contain a 3β-hydroxymethyl-A-norcholestane skeleton, a typical feature of the Axinellidae (1-3, Figure 5B) [100], along with alkaloids such as odiline (4, Figure 5B), dibromocantharelline (5, Figure 5B) and dibromophakellin (6, Figure 5B) [53]. Studies performed on the crude ethanol extracts of C. cantharella led to the isolation of girolline (7, Figure 5B). Its absolute configuration has been established by X-ray diffraction [56] and total synthesis was achieved few years later [53,57,58,63,100]. Girolline (7, Figure 5B) (or girodazole) has demonstrated potent antiproliferative activity in vitro on several cell lines such as P388 murine leukemia and P388/dox, a sub-line resistant to doxorubicin, an anthracycline, or KB naso-pharyngeal and T24 bladder carcinoma human cell lines. The half maximal inhibitory concentration (IC 50 ) values are 0.06 µM, 0.08 µM, 0.21 µM and 0.19 µM, respectively [55]. Subsequent in vivo assays have been performed on several grafted murine tumors used for preclinical evaluation including P388 and L1210 leukemia, and solid tumors such as M5076 histiocytosarcoma and MA/16C mammary adenocarcinoma cells [54,59,63]. Moreover, girodazole (7, Figure 5B) also has antitumor activity in vivo on the P388/dox cell line. This compound may inhibit the termination step of protein synthesis in vivo [60,61]. Although toxicological studies in mice and dogs do not show any major toxic effects, girodazole (7, Figure 5B) clinical development was interrupted in 1991 in phase II due to severe side effects, in particular cardiovascular toxicity [59]. Girolline (7, Figure 5B) has also shown promising results for the treatment of malaria. This compound has demonstrated potent antiplasmodial activities against four Plasmodium falciparum strains with IC 50 values ranging from 77 to 215 nM, and may act synergistically with chloroquine in vitro by affecting protein synthesis [62].

Echinochalina Bargibanti
The demosponge Echinochalina bargibanti (voucher #R1858, IRD Nouméa, Figure 6A) was collected along the northeastern coast of Grande Terre between 18 and 25 m depth during the SMIB program. A bioassay-guided fractionation of its organic extracts led to the isolation of arsenicin A (14, Figure 6B), the first polyarsenic compound ever found in nature.
Arsenicin A (14, Figure 6B) demonstrates potent bactericidal and fungicidal activities against human pathogenic strains such as Staphylococcus aureus, Escherichia coli and Candida albicans with inhibition circles of 24, 28 and 26 mm respectively at 10 µg per disk. In contrast, gentamicin, which is the reference antibiotic, induces inhibition circles of 22, 30 and 22 mm [68]. Arsenicin A (14, Figure 6B) has been synthesized and its crystal structure determined [101]. An improvement in this synthesis has led to (˘)-arsenicin A which shows potent antiproliferative activity on acute promyelocytic leukemia cell lines. This compound is more potent than arsenic trioxide (Trisenox) which is used for treating acute promyelocytic leukemia. (˘)-Arsenicin A has also demonstrated antiproliferative activity against pancreatic adenocarcinomas and glioblastomas [102].

Echinochalina Bargibanti
The demosponge Echinochalina bargibanti (voucher #R1858, IRD Nouméa, Figure 6A) was collected along the northeastern coast of Grande Terre between 18 and 25 m depth during the SMIB program. A bioassay-guided fractionation of its organic extracts led to the isolation of arsenicin A (14, Figure 6B), the first polyarsenic compound ever found in nature.
Arsenicin A (14, Figure 6B) demonstrates potent bactericidal and fungicidal activities against human pathogenic strains such as Staphylococcus aureus, Escherichia coli and Candida albicans with inhibition circles of 24, 28 and 26 mm respectively at 10 μg per disk. In contrast, gentamicin, which is the reference antibiotic, induces inhibition circles of 22, 30 and 22 mm [68]. Arsenicin A (14, Figure 6B) has been synthesized and its crystal structure determined [101]. An improvement in this synthesis has led to (±)-arsenicin A which shows potent antiproliferative activity on acute promyelocytic leukemia cell lines. This compound is more potent than arsenic trioxide (Trisenox) which is used for treating acute promyelocytic leukemia. (±)-Arsenicin A has also demonstrated antiproliferative activity against pancreatic adenocarcinomas and glioblastomas [102]. Dendrilla sp.
Dendrilla sp. (voucher #R171, IRD Nouméa) is a common shallow-water (lagoon, at approximately 20 m depth) dendroceratid sponge belonging to the Demospongiae. It features an unusual "scouring pad" appearance with its spongin fibers protruding from an elastic tissue mass that can vary in color from dark green to reddish. R171 is cytotoxic in brine shrimp larvae bioassays [103], and its crude aqueous extract is toxic to the mosquito fish Gambusia affinis, causing erratic swimming patterns followed by 100% mortality within 12 h [29]. Reported biological activities of the crude organic extracts of Dendrilla nigra ( Figure  7) include antitumor, anti-inflammatory and antimicrobial [103] properties with potential interest for shrimp aquaculture for treating Vibrio pathogens [104].
Chemically, dendrillid sponges are known to contain lamellarins A-B (15-18, Figure 8), aromatic alkaloids of probable symbiotic origin. These pyrrole derivatives are antitumoral (HIF-1 inhibitors) [105]. Dendrilla cactos contains cyclic bastadins, which are bromotyrosine-derived peptides endowed with Gram+ antibacterial and anti-inflammatory properties; in addition they inhibit topoisomerase II, dehydrofolate reductase and the endothelin A receptor [106]. Cold-water Dendrilla membranosa contains membranolides used as natural antifeedants and other microbe-derived bioactive compounds [107]. The suspicion that indwelling bacteria may be responsible for much of the reported bioactivities in sponges in the Dendrilla genus has prompted the taxonomic characterization of culturable strains in Dendrilla nigra [108]. Dendrilla sp.
Dendrilla sp. (voucher #R171, IRD Nouméa) is a common shallow-water (lagoon, at approximately 20 m depth) dendroceratid sponge belonging to the Demospongiae. It features an unusual "scouring pad" appearance with its spongin fibers protruding from an elastic tissue mass that can vary in color from dark green to reddish. R171 is cytotoxic in brine shrimp larvae bioassays [103], and its crude aqueous extract is toxic to the mosquito fish Gambusia affinis, causing erratic swimming patterns followed by 100% mortality within 12 h [29]. Reported biological activities of the crude organic extracts of Dendrilla nigra ( Figure 7) include antitumor, anti-inflammatory and antimicrobial [103] properties with potential interest for shrimp aquaculture for treating Vibrio pathogens [104].
Chemically, dendrillid sponges are known to contain lamellarins A-B (15-18, Figure 8), aromatic alkaloids of probable symbiotic origin. These pyrrole derivatives are antitumoral (HIF-1 inhibitors) [105]. Dendrilla cactos contains cyclic bastadins, which are bromotyrosine-derived peptides endowed with Gram+ antibacterial and anti-inflammatory properties; in addition they inhibit topoisomerase II, dehydrofolate reductase and the endothelin A receptor [106]. Cold-water Dendrilla membranosa contains membranolides used as natural antifeedants and other microbe-derived bioactive compounds [107]. The suspicion that indwelling bacteria may be responsible for much of the reported bioactivities in sponges in the Dendrilla genus has prompted the taxonomic characterization of culturable strains in Dendrilla nigra [108].  Another interest in Dendrilla R171 stems from bioassays as serine protease inhibitors ( § 4.1.4) with remarkable anti-trypsin and anti-elastase activities in preliminary benchtop assays on crude extracts. With up to 100% inhibition on both bovine trypsin and porcine elastase activities, this sponge displays the most potent anti-serpin activity of the 133 invertebrate species tested in total. In 1988, collaborators in Montpellier isolated the active compound (a peptide) from the water-ethanol extract, bioguided by an anti-elastase assay (porcine and human) and they sequenced a 43-residue amino acid, but the structure was never published. This was long before aeruginosins, peptide anti-serine proteases first isolated from cyanobacteria in 1994. A whole family of more than 20 aeruginosins has been described since then, some from sponges of the genus Dysidea [109]. The presence of such compounds may explain the anti-trypsin activity if aeruginosins are also present in Dendrilla R171 (which has not been verified), but anti-elastase activity has never been associated with these molecules.
Thus, the identity of the trypsin and elastase inhibitor(s) from Dendrilla R171 remains unknown (or at least unpublished) to date. The fact that another tested Dendrilla (voucher #1225) does not display any inhibitory activity suggests that microbial symbionts-possibly cyanobacteria-are involved in the reported R171 activities.
A new C47 polyoxygenated acetylenic acid, nepheliosyne B (20, Figure 9B), along with the previously described nepheliosyne A (19, Figure 9B   Another interest in Dendrilla R171 stems from bioassays as serine protease inhibitors ( § 4.1.4) with remarkable anti-trypsin and anti-elastase activities in preliminary benchtop assays on crude extracts. With up to 100% inhibition on both bovine trypsin and porcine elastase activities, this sponge displays the most potent anti-serpin activity of the 133 invertebrate species tested in total. In 1988, collaborators in Montpellier isolated the active compound (a peptide) from the water-ethanol extract, bioguided by an anti-elastase assay (porcine and human) and they sequenced a 43-residue amino acid, but the structure was never published. This was long before aeruginosins, peptide anti-serine proteases first isolated from cyanobacteria in 1994. A whole family of more than 20 aeruginosins has been described since then, some from sponges of the genus Dysidea [109]. The presence of such compounds may explain the anti-trypsin activity if aeruginosins are also present in Dendrilla R171 (which has not been verified), but anti-elastase activity has never been associated with these molecules.
Thus, the identity of the trypsin and elastase inhibitor(s) from Dendrilla R171 remains unknown (or at least unpublished) to date. The fact that another tested Dendrilla (voucher #1225) does not display any inhibitory activity suggests that microbial symbionts-possibly cyanobacteria-are involved in the reported R171 activities.
A new C47 polyoxygenated acetylenic acid, nepheliosyne B (20, Figure 9B), along with the previously described nepheliosyne A (19, Figure 9B  Another interest in Dendrilla R171 stems from bioassays as serine protease inhibitors ( § 4.1.4) with remarkable anti-trypsin and anti-elastase activities in preliminary benchtop assays on crude extracts. With up to 100% inhibition on both bovine trypsin and porcine elastase activities, this sponge displays the most potent anti-serpin activity of the 133 invertebrate species tested in total. In 1988, collaborators in Montpellier isolated the active compound (a peptide) from the water-ethanol extract, bioguided by an anti-elastase assay (porcine and human) and they sequenced a 43-residue amino acid, but the structure was never published. This was long before aeruginosins, peptide anti-serine proteases first isolated from cyanobacteria in 1994. A whole family of more than 20 aeruginosins has been described since then, some from sponges of the genus Dysidea [109]. The presence of such compounds may explain the anti-trypsin activity if aeruginosins are also present in Dendrilla R171 (which has not been verified), but anti-elastase activity has never been associated with these molecules.
Thus, the identity of the trypsin and elastase inhibitor(s) from Dendrilla R171 remains unknown (or at least unpublished) to date. The fact that another tested Dendrilla (voucher #1225) does not display any inhibitory activity suggests that microbial symbionts-possibly cyanobacteria-are involved in the reported R171 activities.
This deep-sea sponge was collected by beam trawl at a depth of 350 m in the Coral Sea. The crude dichloromethanol extract shows cytotoxic activities against the KB naso-pharyngeal carcinoma cell line with an IC50 of 10 μg/mL. Subsequent chemical analyses led to the isolation of a free porphyrin called corallistin A (38, Figure 10) [50]. Its total synthesis followed a few years later [110].
This deep-sea sponge was collected by beam trawl at a depth of 350 m in the Coral Sea. The crude dichloromethanol extract shows cytotoxic activities against the KB naso-pharyngeal carcinoma cell line with an IC 50 of 10 µg/mL. Subsequent chemical analyses led to the isolation of a free porphyrin called corallistin A (38, Figure 10) [50]. Its total synthesis followed a few years later [110].

General Comments
Ascidians are invertebrate filter feeders that belong to phylum Chordata. They represent the most evolved invertebrates with a heart and a respiratory system. Living in both solitary and colonial sessile modes, ascidians can be found in all the world's seas and at all depths. The external tunic of sea-squirts (tunicates) is made of tunicin, a cellulosic substance that is extremely rare in the animal kingdom [19]. Didemnids are soft-bodied, bag-like organisms. Like sponges, didemnids can harbor cyanobacterial photosymbionts, e.g., the photosynthetic genus Prochloron which lives in symbiosis with its host [111]. This class has a special "defense" feature: they can concentrate some toxic elements including heavy metals (notably vanadium), hydrocarbons and elementary sulfur, which may be found in Polycarpa aurata, or even sulfuric acid concentrated inside tiny vesicles of the outer tunic. Studies have also revealed high amounts of nitrogenous compounds (cyclic peptides and alkaloids) with powerful biological activities [112] (Table 3). As of 2007, 290 tunicates had been identified in New Caledonian waters [5].

General Comments
Ascidians are invertebrate filter feeders that belong to phylum Chordata. They represent the most evolved invertebrates with a heart and a respiratory system. Living in both solitary and colonial sessile modes, ascidians can be found in all the world's seas and at all depths. The external tunic of sea-squirts (tunicates) is made of tunicin, a cellulosic substance that is extremely rare in the animal kingdom [19]. Didemnids are soft-bodied, bag-like organisms. Like sponges, didemnids can harbor cyanobacterial photosymbionts, e.g., the photosynthetic genus Prochloron which lives in symbiosis with its host [111]. This class has a special "defense" feature: they can concentrate some toxic elements including heavy metals (notably vanadium), hydrocarbons and elementary sulfur, which may be found in Polycarpa aurata, or even sulfuric acid concentrated inside tiny vesicles of the outer tunic. Studies have also revealed high amounts of nitrogenous compounds (cyclic peptides and alkaloids) with powerful biological activities [112] (Table 3). As of 2007, 290 tunicates had been identified in New Caledonian waters [5].

Lissoclinum bistratum
The didemnid Lissoclinum bistratum (voucher # UA264, IRD Nouméa, Figure 11A) was collected near the Ua islet in the southwest lagoon. Chemical studies followed when two cases of human intoxication were observed during manipulation of the lyophilized powder. The crude dichloromethanol extract of L. bistratum demonstrates acute toxicity on rats, mice and rabbits. Moreover, it has revealed cytotoxic activities with IC 50 values of 45 nM, 20 nM and 22 nM on KB, P388 and normal human endothelial cells, respectively [115].
Separation and purification led to the isolation of a tetrahydropyran derivative called bistramide A (43, Figure 11B) (or bistratene). Its partial structure has been characterized using two dimensional NMR techniques [125].
Bistramide A (43, Figure 11B) appears to block the division of NSCLC-N6 (L16) cells at the G1 phase after a 24 h incubation period at a concentration of 1.42 µM. The IC 50 value is 0.49 µM. Bistramide A (43, Figure 11B) may also induce polyploidy, causing unsuccessful cytokinesis [116]. It has revealed immunomodulator activities (suppressor/stimulator) on the proliferation of T and B cells [120]. Moreover, demonstrated using the voltage-clamp technique, bistramide A (43, Figure 11B) has proven to be responsible for a resting block by inhibiting sodium channels [118] and can bind to contractile proteins in competition with calcium in frog skeletal muscle fibers [119].

Lissoclinum Bistratum
The didemnid Lissoclinum bistratum (voucher # UA264, IRD Nouméa, Figure 11A) was collected near the Ua islet in the southwest lagoon. Chemical studies followed when two cases of human intoxication were observed during manipulation of the lyophilized powder. The crude dichloromethanol extract of L. bistratum demonstrates acute toxicity on rats, mice and rabbits. Moreover, it has revealed cytotoxic activities with IC50 values of 45 nM, 20 nM and 22 nM on KB, P388 and normal human endothelial cells, respectively [115].
Separation and purification led to the isolation of a tetrahydropyran derivative called bistramide A (43, Figure 11B) (or bistratene). Its partial structure has been characterized using two dimensional NMR techniques [125].
Bistramide A (43, Figure 11B) appears to block the division of NSCLC-N6 (L16) cells at the G1 phase after a 24 h incubation period at a concentration of 1.42 μM. The IC50 value is 0.49 μM. Bistramide A (43, Figure 11B) may also induce polyploidy, causing unsuccessful cytokinesis [116]. It has revealed immunomodulator activities (suppressor/stimulator) on the proliferation of T and B cells [120]. Moreover, demonstrated using the voltage-clamp technique, bistramide A (43, Figure 11B) has proven to be responsible for a resting block by inhibiting sodium channels [118] and can bind to contractile proteins in competition with calcium in frog skeletal muscle fibers [119].

Cnidaria
This phylum is divided into two subclasses, Octocorallia, which includes alcyonarians, gorgonians and pennatularians, and Hexacorallia, which includes scleratinarians. The chemical and biological properties of Cnidaria collected in New Caledonia are reported in Table 4.

Cnidaria
This phylum is divided into two subclasses, Octocorallia, which includes alcyonarians, gorgonians and pennatularians, and Hexacorallia, which includes scleratinarians. The chemical and biological properties of Cnidaria collected in New Caledonia are reported in Table 4.

General Comments
Alcyonarians also called "soft corals" because they lack a calcium carbonate skeleton. This carbonate skeleton is replaced in most species by aragonitic sclerites, which are of major taxonomic importance for identification. They are benthic, sessile, colonial organisms often composed of many individual polyps stemming from a common sterile trunk, which is attached to the substratum by a glycoprotein "glue". The main families are the Alcyoniidae, Nephtheidae and Xeniidae.
Alcyonarians often possess endodermal zooxanthellae which provide them with oxygen and photosynthates due to their photosynthetic activity. A total of 173 species have been reported in New Caledonian waters [5].
"Cocktails" of toxic secondary metabolites, especially diterpenes, occur in their tissues, and serve anti-predator (toxic and emetic effects) [130] and anti-competitor defense functions (contact necrosis and allelopathic growth inhibition [131], but also as anti-fouling substances and as egg protectants [132]).

Xenia Garciae
The "soft coral" Xenia garciae (voucher #3526, Northern Territory Museum, Darwin, Australia) was collected in shallow-water fringing reefs at a depth less than five meters. Chemical analyses isolated an original xenicane diterpene (48, Figure 12) that inhibits the growth of Ceramium codii, which is a common benthic Rhodophyta contributing to the process known as "fouling" [33]. Around the xenicane skeleton, as around the cembrane skeleton of alcyonids, are found dozens of bioactive diterpenes. The xenicane diterpenes, also found in some brown algae (Dictyotales), are particularly interesting as synthetic templates for the design of antitumor drugs [134].

General Comments
Alcyonarians also called "soft corals" because they lack a calcium carbonate skeleton. This carbonate skeleton is replaced in most species by aragonitic sclerites, which are of major taxonomic importance for identification. They are benthic, sessile, colonial organisms often composed of many individual polyps stemming from a common sterile trunk, which is attached to the substratum by a glycoprotein "glue". The main families are the Alcyoniidae, Nephtheidae and Xeniidae.
Alcyonarians often possess endodermal zooxanthellae which provide them with oxygen and photosynthates due to their photosynthetic activity. A total of 173 species have been reported in New Caledonian waters [5].
"Cocktails" of toxic secondary metabolites, especially diterpenes, occur in their tissues, and serve anti-predator (toxic and emetic effects) [130] and anti-competitor defense functions (contact necrosis and allelopathic growth inhibition [131], but also as anti-fouling substances and as egg protectants [132]).

Xenia Garciae
The "soft coral" Xenia garciae (voucher #3526, Northern Territory Museum, Darwin, Australia) was collected in shallow-water fringing reefs at a depth less than five meters. Chemical analyses isolated an original xenicane diterpene (48, Figure 12) that inhibits the growth of Ceramium codii, which is a common benthic Rhodophyta contributing to the process known as "fouling" [33]. Around the xenicane skeleton, as around the cembrane skeleton of alcyonids, are found dozens of bioactive diterpenes. The xenicane diterpenes, also found in some brown algae (Dictyotales), are particularly interesting as synthetic templates for the design of antitumor drugs [134].

General Comments
Also known as sea whips or sea fans, gorgonians are "animal-flowers" that are supported either by a calcareous skeleton (suborder Scleraxonia) or by a flexible horny skeleton (suborder Holaxonia) made of a fibrous protein, gorgonin. Exclusively marine, these sessile invertebrates can live as solitary animals or in colonies. They are found in many places in all oceans and can occur in different growth forms such as bushes, whip-like branches and sometimes as blunt lobes or even flat crusts [18]. As of 2007, 93 species of gorgonians have been described in New Caledonian waters [5].

General Comments
Also known as sea whips or sea fans, gorgonians are "animal-flowers" that are supported either by a calcareous skeleton (suborder Scleraxonia) or by a flexible horny skeleton (suborder Holaxonia) made of a fibrous protein, gorgonin. Exclusively marine, these sessile invertebrates can live as solitary animals or in colonies. They are found in many places in all oceans and can occur in different growth forms such as bushes, whip-like branches and sometimes as blunt lobes or even flat crusts [18]. As of 2007, 93 species of gorgonians have been described in New Caledonian waters [5].

Melithea cf. Stormii
The sea fan Melithea cf. stormii ( Figure 13A) is a large scleraxonian gorgonian commonly found on outer reef slopes. The voucher sample and the biological material used for sequencing and bioassays were taken from only one medium-sized specimen (voucher #HG163, IRD Nouméa) collected in Uitoe Pass, Southern Province at 20 m depth. Below, we summarize the main highlights of this specimen HG163; all details can be found in [37]. The sea fan Melithea cf. stormii ( Figure 13A) is a large scleraxonian gorgonian commonly found on outer reef slopes. The voucher sample and the biological material used for sequencing and bioassays were taken from only one medium-sized specimen (voucher #HG163, IRD Nouméa) collected in Uitoe Pass, Southern Province at 20 m depth. Below, we summarize the main highlights of this specimen HG163; all details can be found in [37].  (49). Half cysteine residues are noted in red, and correspond to half-cysteines of the non-classical Kazal-type inhibitor. The blue pair indicates P1-P1' residues around the putative active site. One-letter codes for amino acids: A, alanine; C, cysteine; D, aspartate; E, glutamate; G, glycine; I, isoleucine; K, lysine; L, leucine; M, methionine; P, proline; S, serine; T, threonine; V, valine; X, unknown; Y, tyrosine.
From HG163, a 95% pure novel peptide elastase inhibitor was isolated using SEC and reversed-phase chromatography. This peptide of 39 residues in length was hereafter referenced as iela melst (49, Figure 13B) according to nomenclature. The amino acid sequence of the first 20 N-terminal residues of iela melst (49, Figure 13B) is homologous to that of iela anesu from the sea anemone Anemonia sulcata, a non-typical Kazal-type elastase inhibitor [135]. The Cha procedure [136] was used to monitor the inhibition kinetics of the amidolysis of the synthetic substrate [Suc(Ala)3pNA] by porcine pancreatic elastase (PPE) at different concentrations using iela melst (49, Figure 13B). This inhibitor works as a slow, tight-binding inhibitor of PPE. The equilibrium dissociation constant Ki (1.5 nM) of the complex is quite similar to that of the PPE-elafin complex, the latter being an antileuko-proteinase isolated from patients with psoriasis. The anti-elastase activity of iela melst (49, Figure 13B) is very comparable to those of most natural or synthetic PPE inhibitors. The presence of such a strong anti-elastase substance in gorgonian cortical tissues may hinder various necrotic processes caused by epibiotic fouling or extracoelenteric digestion by other coelenterates [37].
From HG163, a 95% pure novel peptide elastase inhibitor was isolated using SEC and reversed-phase chromatography. This peptide of 39 residues in length was hereafter referenced as iela melst (49, Figure 13B) according to nomenclature. The amino acid sequence of the first 20 N-terminal residues of iela melst (49, Figure 13B) is homologous to that of iela anesu from the sea anemone Anemonia sulcata, a non-typical Kazal-type elastase inhibitor [135]. The Cha procedure [136] was used to monitor the inhibition kinetics of the amidolysis of the synthetic substrate [Suc(Ala) 3 pNA] by porcine pancreatic elastase (PPE) at different concentrations using iela melst (49, Figure 13B). This inhibitor works as a slow, tight-binding inhibitor of PPE. The equilibrium dissociation constant K i (1.5 nM) of the complex is quite similar to that of the PPE-elafin complex, the latter being an antileuko-proteinase isolated from patients with psoriasis. The anti-elastase activity of iela melst (49, Figure 13B) is very comparable to those of most natural or synthetic PPE inhibitors. The presence of such a strong anti-elastase substance in gorgonian cortical tissues may hinder various necrotic processes caused by epibiotic fouling or extracoelenteric digestion by other coelenterates [37].
Villogorgin A (50, Figure 14) inhibits (i) the contraction of the guinea pig ileum induced by acetylcholine; (ii) the aggregation of human platelets induced by thrombin and calcium ionophore A23187; this inhibition may be due to modulation of calcium/calmodulin-dependent enzymes. Villogorgin A (50, Figure 14) and B (51, Figure 14) are structurally related to the marine tunicate β-carboline alkaloid eudistomidin-A (56, Figure 14), a strong calmodulin antagonist [127].

Pennatularians General Comments
Sea pens (sea feathers)-also called pennatularians-are a specialized and morphologically distinct group of octocorallian cnidarians.
Pennatularians are made of a thin tissue called the coenenchyme. These sessile animals live in colonies that are built from a single large primary polyp, the oozoid. They have been encountered in all oceans down to 6100 m depth [137].

Pennatularians General Comments
Sea pens (sea feathers)-also called pennatularians-are a specialized and morphologically distinct group of octocorallian cnidarians.
Pennatularians are made of a thin tissue called the coenenchyme. These sessile animals live in colonies that are built from a single large primary polyp, the oozoid. They have been encountered in all oceans down to 6100 m depth [137].

Scleractinians
Scleractinians are also called "stony corals" or "hard corals". Scleractinian colonies act as biological photosystems during sunlit hours and feed on plankton at night. Closely related to sea anemones, they are also armed with cnidocytes that can be used for predation as well as for territorial defense [138].
"Stony corals" have a large range of colonial growth forms, from crustose to massive, but mostly branching, corymbose, tabular or lamellate, with a few solitary and free-living forms, e.g., mushroom corals [14]. All corals originate from a minute swimming planula that settles and metamorphoses into a single solitary polyp, which itself undergoes successive budding to form a colony that encases itself in an aragonitic (calcium/magnesium carbonate) shell. Growth rates can vary from a few centimeters per year for fast growing branching acroporids that are shorter-lived and rarely very large, to a few millimeters per year at most for large poritid corals that can grow up to 10 m in diameter and live for many centuries.
Shallow-water corals contain Symbiodinium (symbiotic unicellular microalgae known as zooxanthellae) within their endodermal cells. These microalgae give the coral its color, which thus can vary in hue depending on the symbiont species. Scleractinians are usually photosymbiotes.
Echinoderms are exclusively marine invertebrates that have an endoskeleton consisting of magnesium calcite. In all, 257 species of echinoderms have been reported in New Caledonian waters as of 2007 [5].

Scleractinians
Scleractinians are also called "stony corals" or "hard corals". Scleractinian colonies act as biological photosystems during sunlit hours and feed on plankton at night. Closely related to sea anemones, they are also armed with cnidocytes that can be used for predation as well as for territorial defense [138].
"Stony corals" have a large range of colonial growth forms, from crustose to massive, but mostly branching, corymbose, tabular or lamellate, with a few solitary and free-living forms, e.g., mushroom corals [14]. All corals originate from a minute swimming planula that settles and metamorphoses into a single solitary polyp, which itself undergoes successive budding to form a colony that encases itself in an aragonitic (calcium/magnesium carbonate) shell. Growth rates can vary from a few centimeters per year for fast growing branching acroporids that are shorter-lived and rarely very large, to a few millimeters per year at most for large poritid corals that can grow up to 10 m in diameter and live for many centuries.
Shallow-water corals contain Symbiodinium (symbiotic unicellular microalgae known as zooxanthellae) within their endodermal cells. These microalgae give the coral its color, which thus can vary in hue depending on the symbiont species. Scleractinians are usually photosymbiotes.
Echinoderms are exclusively marine invertebrates that have an endoskeleton consisting of magnesium calcite. In all, 257 species of echinoderms have been reported in New Caledonian waters as of 2007 [5].

Actinopyga Flammea
The sea cucumber Actinopyga flammea (voucher #EH 025, IRD Nouméa) was collected at depths ranging from 35 to 50 m on the outer reef slope on the New Caledonian west coast. Highly ichthyotoxic, this species is used by fishermen in the Philippines to kill fish in coral holes. A specimen similar to that shown in Figure 16A reportedly caused the death of all fish overnight after a caretaker accidentally placed it in a fish tank at the Aquarium of Nouméa.

Actinopyga Flammea
The sea cucumber Actinopyga flammea (voucher #EH 025, IRD Nouméa) was collected at depths ranging from 35 to 50 m on the outer reef slope on the New Caledonian west coast. Highly ichthyotoxic, this species is used by fishermen in the Philippines to kill fish in coral holes. A specimen similar to that shown in Figure 16A reportedly caused the death of all fish overnight after a caretaker accidentally placed it in a fish tank at the Aquarium of Nouméa.
Further studies have revealed that photoexcitation of gymnochrome A (65, Figure 17B) may induce electrophysiological effects such as the blockage of background K + current in the atrial region of the frog heart muscle [147]. Moreover, gymnochrome B (66, Figure 17B) has demonstrated powerful virucidal and antiviral photoactivity with median effective dose (ED 50 ) of 0.042 nM/mL and 0.029 nM/mL, respectively. This photoactivity may be partly due to its brominated hypericin core [148].
Further studies have revealed that photoexcitation of gymnochrome A (65, Figure 17B) may induce electrophysiological effects such as the blockage of background K + current in the atrial region of the frog heart muscle [147]. Moreover, gymnochrome B (66, Figure 17B) has demonstrated powerful virucidal and antiviral photoactivity with median effective dose (ED50) of 0.042 nM/mL and 0.029 nM/mL, respectively. This photoactivity may be partly due to its brominated hypericin core [148].

Thromidia Catalai
Thromidia catalai (voucher #EA065, IRD Nouméa, Figure 18A) is one of the largest known starfish species, discovered by René Catala in the inner lagoon close to Dumbea Pass in the barrier reef of the Southern Province, around 25 m in depth. This massive five-armed species reaches up to 60 cm in diameter and weighs several kilograms.

Thromidia Catalai
Thromidia catalai (voucher #EA065, IRD Nouméa, Figure 18A) is one of the largest known starfish species, discovered by René Catala in the inner lagoon close to Dumbea Pass in the barrier reef of the Southern Province, around 25 m in depth. This massive five-armed species reaches up to 60 cm in diameter and weighs several kilograms.

Thromidia Catalai
Thromidia catalai (voucher #EA065, IRD Nouméa, Figure 18A) is one of the largest known starfish species, discovered by René Catala in the inner lagoon close to Dumbea Pass in the barrier reef of the Southern Province, around 25 m in depth. This massive five-armed species reaches up to 60 cm in diameter and weighs several kilograms.

Macroalgae
Macroalgae belong to three different divisions: red algae (Rhodophyta), brown algae (Heterokontophyta, also known as the Ochrophyta, class Phaeophyceae) and green algae (Chlorophyta). All three evolutionary lineages diverged early on according to their respective plastid configurations, reflecting their endosymbiotic history [150].
Algae have a relatively unspecialized vegetative apparatus called a thallus. Important differences are noted in many ultrastructural and biochemical features including photosynthetic pigments, storage compounds, composition of cell walls, presence/absence of flagella, ultrastructure of mitosis, connections between adjacent cells, and the fine structure of chloroplasts. They vary from small, single-celled forms to complex multicellular forms, such as the giant kelp Macrocystis pyrifera that can grow up to 49 m in length in New Zealand and holds the record of the fastest growing organism (0.6 m per day).
Marine macroalgae generally live attached to rocks or other hard substrata in coastal areas but can be also found in sandy areas such as the Udoteaceae in tropical areas. They occur in all of the world's oceans where they occupy the seabed (phytobenthos), although a few species float freely on the surface (e.g., the pelagic algae of the Sargasso Sea). In New Caledonia [151], the large brown algae are mainly Fucales (Sargassum, Turbinaria) and Dictyotales and form large stands on rocky lagoon bottoms, while several Chlorophyta species from the genera Halimeda and Caulerpa colonize sandy bottoms [152][153][154][155]. The large fleshy Rhodophyta are mainly restricted to outer slopes or the cooler waters of the southern lagoon. As of 2007, 454 species of algae and marine angiosperms had been reported in New Caledonian waters [5].
Despite their capacity to produce large amounts of natural products, macroalgae from New Caledonia have been little documented in this respect. Caulerpa species were investigated for caulerpicin (76-80, Figure 19) activity in the 1980s for cosmetic purposes, but the highly publicized invasion of Caulerpa taxifolia in Mediterranean Sea brought the project to an immediate halt [156]. The genus Lobophora (Phaeophyta) has been investigated in biological interaction studies between macroalgae and several corals as part of a PhD thesis. Three new compounds have been isolated and described [34], and their bioactivity experimentally tested on corals results in severe coral bleaching.

Macroalgae
Macroalgae belong to three different divisions: red algae (Rhodophyta), brown algae (Heterokontophyta, also known as the Ochrophyta, class Phaeophyceae) and green algae (Chlorophyta). All three evolutionary lineages diverged early on according to their respective plastid configurations, reflecting their endosymbiotic history [150].
Algae have a relatively unspecialized vegetative apparatus called a thallus. Important differences are noted in many ultrastructural and biochemical features including photosynthetic pigments, storage compounds, composition of cell walls, presence/absence of flagella, ultrastructure of mitosis, connections between adjacent cells, and the fine structure of chloroplasts. They vary from small, single-celled forms to complex multicellular forms, such as the giant kelp Macrocystis pyrifera that can grow up to 49 m in length in New Zealand and holds the record of the fastest growing organism (0.6 m per day).
Marine macroalgae generally live attached to rocks or other hard substrata in coastal areas but can be also found in sandy areas such as the Udoteaceae in tropical areas. They occur in all of the world's oceans where they occupy the seabed (phytobenthos), although a few species float freely on the surface (e.g., the pelagic algae of the Sargasso Sea). In New Caledonia [151], the large brown algae are mainly Fucales (Sargassum, Turbinaria) and Dictyotales and form large stands on rocky lagoon bottoms, while several Chlorophyta species from the genera Halimeda and Caulerpa colonize sandy bottoms [152][153][154][155]. The large fleshy Rhodophyta are mainly restricted to outer slopes or the cooler waters of the southern lagoon. As of 2007, 454 species of algae and marine angiosperms had been reported in New Caledonian waters [5].
Despite their capacity to produce large amounts of natural products, macroalgae from New Caledonia have been little documented in this respect. Caulerpa species were investigated for caulerpicin (76-80, Figure 19) activity in the 1980s for cosmetic purposes, but the highly publicized invasion of Caulerpa taxifolia in Mediterranean Sea brought the project to an immediate halt [156]. The genus Lobophora (Phaeophyta) has been investigated in biological interaction studies between macroalgae and several corals as part of a PhD thesis. Three new compounds have been isolated and described [34], and their bioactivity experimentally tested on corals results in severe coral bleaching.

Microalgae and Cyanobacteria
Less than 1% of described marine phytoplanktonic species are known to produce potent toxins [157]. Microalgal benthic dinoflagellates species are involved, including Gambierdiscus toxicus that produces maitotoxin (81, Figure 20). In vivo toxicity in mice indicates that intraperitoneal injection of 0.13 μg/kg is lethal [158]. A culture of ca. 4000 L of dinoflagellate cells is needed for the purification of 20 mg of MTX, enough to kill about four million mice. MTX activates both voltage-sensitive and receptor-operated calcium channels in the plasma membrane, thus inducing calpain protease that rapidly leads to cell death. MTX has since been considered as a useful tool for investigating the physiological and pathophysiological roles of calpain in neuronal cells [159].

Microalgae and Cyanobacteria
Less than 1% of described marine phytoplanktonic species are known to produce potent toxins [157]. Microalgal benthic dinoflagellates species are involved, including Gambierdiscus toxicus that produces maitotoxin (81, Figure 20). In vivo toxicity in mice indicates that intraperitoneal injection of 0.13 µg/kg is lethal [158]. A culture of ca. 4000 L of dinoflagellate cells is needed for the purification of 20 mg of MTX, enough to kill about four million mice. MTX activates both voltage-sensitive and receptor-operated calcium channels in the plasma membrane, thus inducing calpain protease that rapidly leads to cell death. MTX has since been considered as a useful tool for investigating the physiological and pathophysiological roles of calpain in neuronal cells [159].

Microalgae and Cyanobacteria
Less than 1% of described marine phytoplanktonic species are known to produce potent toxins [157]. Microalgal benthic dinoflagellates species are involved, including Gambierdiscus toxicus that produces maitotoxin (81, Figure 20). In vivo toxicity in mice indicates that intraperitoneal injection of 0.13 μg/kg is lethal [158]. A culture of ca. 4000 L of dinoflagellate cells is needed for the purification of 20 mg of MTX, enough to kill about four million mice. MTX activates both voltage-sensitive and receptor-operated calcium channels in the plasma membrane, thus inducing calpain protease that rapidly leads to cell death. MTX has since been considered as a useful tool for investigating the physiological and pathophysiological roles of calpain in neuronal cells [159].  Species belonging to the genus Gambierdiscus have also been shown to produce ciguatoxin analogues (CTX analogs) causing ciguatera fish poisoning (CFP). New Caledonia is a CFP-endemic area. CFP constitutes a global health problem with more than 50,000 people worldwide affected by Mar. Drugs 2016, 14, 58 37 of 60 this disease annually. CTXs arise from biotransformation/metabolization of CTX analogs through the food chain: from herbivorous fish that accumulate dinoflagellate toxins by eating dead coral and marine algae to carnivorous fish [157]. In Pacific areas, the principal and most potent CTX is Pacific ciguatoxin-1 (82, Figure 21). CTXs are potent activators of voltage-sensitive sodium channels and cause an increase in neuronal excitability and neurotransmitter release [160]. In New Caledonia, traditional remedies are commonly employed in the treatment of CFP: 90 plant species have been catalogued as useful CFP remedies, including the leaves of Heliotropium foertherianum used to prepare the most popular herbal remedy [161].
A study conducted by D. Laurent et al. following the observation of many cases of seafood poisoning on Lifou Island between 2001 and 2005 (Loyalty Islands, New Caledonia, Figure 1), indicates that bloom-forming cyanobacteria, such as the filamentous Trichodesmium erythraeum, can also produce CTX-like compounds [160]. Species belonging to the genus Gambierdiscus have also been shown to produce ciguatoxin analogues (CTX analogs) causing ciguatera fish poisoning (CFP). New Caledonia is a CFP-endemic area. CFP constitutes a global health problem with more than 50,000 people worldwide affected by this disease annually. CTXs arise from biotransformation/metabolization of CTX analogs through the food chain: from herbivorous fish that accumulate dinoflagellate toxins by eating dead coral and marine algae to carnivorous fish [157]. In Pacific areas, the principal and most potent CTX is Pacific ciguatoxin-1 (82, Figure 21). CTXs are potent activators of voltage-sensitive sodium channels and cause an increase in neuronal excitability and neurotransmitter release [160]. In New Caledonia, traditional remedies are commonly employed in the treatment of CFP: 90 plant species have been catalogued as useful CFP remedies, including the leaves of Heliotropium foertherianum used to prepare the most popular herbal remedy [161].
A study conducted by D. Laurent et al. following the observation of many cases of seafood poisoning on Lifou Island between 2001 and 2005 (Loyalty Islands, New Caledonia, Figure 1), indicates that bloom-forming cyanobacteria, such as the filamentous Trichodesmium erythraeum, can also produce CTX-like compounds [160]. In line with programs exploring the biodiversity of planktonic species such as OCEANOMICs (see [162] for details), the project AMICAL (Aquaculture of MIcroalgae in New CALedonia, [163]) instigated by IFREMER-New Caledonia, the Physiology and Biotechnology of Algae (PBA) laboratory (Nantes, mainland France) and ADECAL (Caledonian Economic Development Agency-Nouméa, [164]), aims to develop industrial microalgal production based on indigenous microalgae species isolated along the mainland coasts ( Figure 1). Partners of this program will transfer the cultivation and extraction techniques to selected algal species for industrial programs in New Caledonia targeting the animal nutrition market and high added-value compounds (cosmetics, health food, etc.).

Other Biological Sources
Statistically "minor" phyla in terms of the number of studies on local species include prokaryotes, several invertebrate phyla, and snakes as the sole vertebrate representatives.

Micrococcus Luteus
Micrococcus luteus is a Gram+ bacterium that was isolated from Xestospongia exigua sponges collected off Nouméa in the Southwest Pacific. Chemical studies reveal that this bacterium can produce lutoside (83, Figure 22), an acyl-1-(acyl-6′-mannobiosyl)-3-glycerol, and also triclosan (84, Figure 22) (2,4,4′-trichloro-2′-hydroxydiphenylether), with both molecules presenting antibacterial activities [165,166]. In line with programs exploring the biodiversity of planktonic species such as OCEANOMICs (see [162] for details), the project AMICAL (Aquaculture of MIcroalgae in New CALedonia, [163]) instigated by IFREMER-New Caledonia, the Physiology and Biotechnology of Algae (PBA) laboratory (Nantes, mainland France) and ADECAL (Caledonian Economic Development Agency-Nouméa, [164]), aims to develop industrial microalgal production based on indigenous microalgae species isolated along the mainland coasts ( Figure 1). Partners of this program will transfer the cultivation and extraction techniques to selected algal species for industrial programs in New Caledonia targeting the animal nutrition market and high added-value compounds (cosmetics, health food, etc.).

Other Biological Sources
Statistically "minor" phyla in terms of the number of studies on local species include prokaryotes, several invertebrate phyla, and snakes as the sole vertebrate representatives.

Bacteria from Pseudoalteromonas and Vibrio Genus
Along the west coast of New Caledonia, 205 environmental samples were collected on a variety of surfaces (sediments, intertidal rocks, invertebrates, plants, fish and biofilms found on organic substrates). This sampling led to the isolation of 493 marine bacteria [167,168]. Studies were at first performed to assess their ability to produce exopolysaccharides (EPSs). Among them, the new strain Vibrio neocaledonicus sp. nov. (NC470), isolated from a biofilm found on Holothuroidea in St. Vincent Bay, has demonstrated the production of EPSs that have a high N-acetyl-hexosamine and uronic acid content with a low amount of neutral sugars. Preliminary experiments conducted on these EPSs have shown high metal-binding capacity [167]. Further studies have shown that four other strains (NC282, NC412, NC272 and NC120), which belong to the genus Pseudoalteromonas, have antibacterial potential against reference and multidrug-resistant pathogen strains such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis [168].

Bacteria from Pseudoalteromonas and Vibrio Genus
Along the west coast of New Caledonia, 205 environmental samples were collected on a variety of surfaces (sediments, intertidal rocks, invertebrates, plants, fish and biofilms found on organic substrates). This sampling led to the isolation of 493 marine bacteria [167,168]. Studies were at first performed to assess their ability to produce exopolysaccharides (EPSs). Among them, the new strain Vibrio neocaledonicus sp. nov. (NC470), isolated from a biofilm found on Holothuroidea in St. Vincent Bay, has demonstrated the production of EPSs that have a high N-acetyl-hexosamine and uronic acid content with a low amount of neutral sugars. Preliminary experiments conducted on these EPSs have shown high metal-binding capacity [167]. Further studies have shown that four other strains (NC282, NC412, NC272 and NC120), which belong to the genus Pseudoalteromonas, have antibacterial potential against reference and multidrug-resistant pathogen strains such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis [168].

Venomous Cone Snails
Venomous marine cone snails (Conoidea) are neogastropod mollusks that predate on marine worms and small benthic invertebrates (thick-shelled snails, e.g., Conus textile) or that actively "hunt" fish (thin-shelled snails, e.g., Conus geographus) primarily by inflicting cocktails of peptide neurotoxins produced by venom glands via their modified harpoon-like radula (see review in Kaas

Venomous Cone Snails
Venomous marine cone snails (Conoidea) are neogastropod mollusks that predate on marine worms and small benthic invertebrates (thick-shelled snails, e.g., Conus textile) or that actively "hunt" fish (thin-shelled snails, e.g., Conus geographus) primarily by inflicting cocktails of peptide neurotoxins produced by venom glands via their modified harpoon-like radula (see review in Kaas and Craik, 2014 [171]). Fish-hunting cone snails may switch from prey-stimulated to defensive envenomation strategies as shown by the use of different and more potent "high threat" neurotoxins if threatened, the latter being regarded as a specialization in response to predation pressure from fish and cephalopods [172]. In addition, C. geographus (common in New Caledonia) diffuses a "cloud" of fish-like insulin that literally "tetanizes" the fish by eliciting hypoglycemic shock prior to envenomation [173], a possible metabolic cost-saving feature. Pharmacological applications of cone snail venoms have stemmed from the work of Olivera's group on Conus magus, which inspired the biosynthesis of the most potent pain-killer on the market (ziconotide, Prialt ® , Dublin, Ireland), claimed to be 1000 times more potent than morphine for the sedation of terminally ill cancer patients. The CONCO cone snail genome project for health funded by the European Commission was launched in 2005 to study the toxins composing the venom of the cone snail Conus consors (Figure 24) from the Chesterfield Islands using genomic, transcriptomic and proteomic approaches. The mitochondrial genome of C. consors has now been sequenced and gene annotated. The authors report the presence of a novel 700 bp control region absent from the hitherto known mitochondrial genomes of cone shells [174]. The species richness of venomous cone mollusks in New Caledonia is exceptional and prompts further investigations in the molecular biology, pharmacology and ecology of this fascinating group.

Venomous Cone Snails
Venomous marine cone snails (Conoidea) are neogastropod mollusks that predate on marine worms and small benthic invertebrates (thick-shelled snails, e.g., Conus textile) or that actively "hunt" fish (thin-shelled snails, e.g., Conus geographus) primarily by inflicting cocktails of peptide neurotoxins produced by venom glands via their modified harpoon-like radula (see review in Kaas and Craik, 2014 [171]). Fish-hunting cone snails may switch from prey-stimulated to defensive envenomation strategies as shown by the use of different and more potent "high threat" neurotoxins if threatened, the latter being regarded as a specialization in response to predation pressure from fish and cephalopods [172]. In addition, C. geographus (common in New Caledonia) diffuses a "cloud" of fish-like insulin that literally ʺtetanizesʺ the fish by eliciting hypoglycemic shock prior to envenomation [173], a possible metabolic cost-saving feature. Pharmacological applications of cone snail venoms have stemmed from the work of Olivera's group on Conus magus, which inspired the biosynthesis of the most potent pain-killer on the market (ziconotide, Prialt ® , Dublin, Ireland), claimed to be 1000 times more potent than morphine for the sedation of terminally ill cancer patients. The CONCO cone snail genome project for health funded by the European Commission was launched in 2005 to study the toxins composing the venom of the cone snail Conus consors (Figure 24) from the Chesterfield Islands using genomic, transcriptomic and proteomic approaches. The mitochondrial genome of C. consors has now been sequenced and gene annotated. The authors report the presence of a novel 700 bp control region absent from the hitherto known mitochondrial genomes of cone shells [174]. The species richness of venomous cone mollusks in New Caledonia is exceptional and prompts further investigations in the molecular biology, pharmacology and ecology of this fascinating group.

Vertebrates: Venomous Marine Snakes
Globally, snake venom studies are of primordial importance for the design of novel antivenoms, for research tools in neurophysiology, and for a better understanding of the adaptive history of reptiles.
Like Australia, New Caledonia has no terrestrial venomous snakes, and Gail and Rageau established the first census of venomous marine snakes from the Elapidae family in 1955 [176]. Of the marine snakes that have been investigated by toxicologists, the most common ones are Laticauda laticaudata and its congener Laticauda colubrina, both highly venomous snakes that essentially hunt small fish underwater at night and rest on land during the day ( Figure 25A).
L. laticaudata and L. colubrina rank, respectively, 9th and 46th among the 163 most venomous snakes (terrestrial and marine species of all continents). Being related to well-known Asian cobras and African mambas (Elapidae), but being much easier to handle (the mouth is very small and the hooks are adapted to bite prey during the swallowing process), laticaudas are an excellent model for toxicologists. A major component, erabutoxin b (91, Figure 25B) from Laticauda semifasciata binds with high affinity to muscular nicotinic acetylcholine receptors (nAChRs), but with low affinity to neuronal alpha-7 nAChRs and inhibits acetylcholine from binding to the receptor, thereby impairing neuromuscular transmission [177]. As a result, it produces peripheral paralysis by blocking neuromuscular transmission at the postsynaptic site. The first cDNA studies undertaken as part of a collaboration between the Centre d'Etudes Nucléaires de Saclay and Tokyo University [178] later included New Caledonian snakes in a review of the evolution of Elapid snake venoms [179].
Aipisurus laevis is a fully pelagic species, collected in surface waters of the lagoon in the Southern Province. A. laevis ranks 10 out of the 163 known most "globally dangerous" snakes, and 30 out of 163 in terms of toxicity (LD 50 by injection in mice). Two cDNAs of short-chain neurotoxins were cloned and sequenced by Ducancel et al., (1990) [180], and compared with similar work on erabutoxin b (91, Figure 25B) from Laticauda snakes.
L. laticaudata and L. colubrina rank, respectively, 9th and 46th among the 163 most venomous snakes (terrestrial and marine species of all continents). Being related to well-known Asian cobras and African mambas (Elapidae), but being much easier to handle (the mouth is very small and the hooks are adapted to bite prey during the swallowing process), laticaudas are an excellent model for toxicologists. A major component, erabutoxin b (91, Figure 25B) from Laticauda semifasciata binds with high affinity to muscular nicotinic acetylcholine receptors (nAChRs), but with low affinity to neuronal alpha-7 nAChRs and inhibits acetylcholine from binding to the receptor, thereby impairing neuromuscular transmission [177]. As a result, it produces peripheral paralysis by blocking neuromuscular transmission at the postsynaptic site. The first cDNA studies undertaken as part of a collaboration between the Centre dʹEtudes Nucléaires de Saclay and Tokyo University [178] later included New Caledonian snakes in a review of the evolution of Elapid snake venoms [179].
Aipisurus laevis is a fully pelagic species, collected in surface waters of the lagoon in the Southern Province. A. laevis ranks 10 out of the 163 known most "globally dangerous" snakes, and 30 out of 163 in terms of toxicity (LD50 by injection in mice). Two cDNAs of short-chain neurotoxins were cloned and sequenced by Ducancel et al., (1990) [180], and compared with similar work on erabutoxin b (91, Figure 25B) from Laticauda snakes.

Recent Advances on Selected New Caledonian Marine Natural Products
Following their discovery in the course of ongoing New Caledonian scientific programs, and after promising results were revealed by preliminary biological screening and spectral characterization, further biological and chemical investigations on several molecules have been pursued to carry out (i) total synthesis; (ii) synthesis of chemical derivatives selected on the basis of structure-activity relationship (SAR) studies; (iii) determination of the mechanism of action; (iv) determination of additional bioactivities including in vivo and receptor studies. These studies and relevant updates are listed in Table 6.

Recent Advances on Selected New Caledonian Marine Natural Products
Following their discovery in the course of ongoing New Caledonian scientific programs, and after promising results were revealed by preliminary biological screening and spectral characterization, further biological and chemical investigations on several molecules have been pursued to carry out (i) total synthesis; (ii) synthesis of chemical derivatives selected on the basis of structure-activity relationship (SAR) studies; (iii) determination of the mechanism of action; (iv) determination of additional bioactivities including in vivo and receptor studies. These studies and relevant updates are listed in Table 6. Table 6. Subsequent and ongoing development of selected natural products isolated from New Caledonian marine organisms. -Importance of an electronegative functional group at position C-13 for CLL activity. [182] suvanine -Anti-inflammatory activity, Hsp 60 inhibitor.

Natural Product and/or Analogs Chemical Synthesis and Biological Activity Reference
Analogs of bistramide A -Design and synthesis of analog of bistramide A targeting cytoskeletal organization of cancer cells in vivo (combination of reversible G-actin binding and effective F-actin severing). -Potent and reversible binding of monomeric actin (K d 9.0 nM), in vitro depolymerization of actin; in vitro and in vivo inhibition of A549 cells. [206] -Computational analyses of non-covalent actin-inhibitor interactions (AutoDock and DrugScore scoring function).
-Design of a novel, highly modular class of hybrid analogs with a rationale to address both the bistramide and rhizopodin binding sites. -Antiproliferative activity is conserved in analogs resembling the original side chain of rhizopodin. [207] luzonicoside A -Potent immunomodulatory agent: more effective in stimulating lysosomal activity, intracellular ROS level elevation, and NO synthesis up-regulation in RAW 264.7 murine macrophages cells (0.01-0.1 µM).
[208] Despite recent advances in developing therapeutic strategies, cancer is still one of the leading causes of death worldwide. Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. Drug-induced programmed cell death holds promise for cancer therapeutic approaches. Apoptosis is one of the well-known pathways used by cells to die. It is a morphological event characterized by chromosomal DNA fragmentation, nuclear disintegration, cell shrinkage, translocation of phosphatidyl serine moieties to the outer membrane leaflet, and membrane blebbing [209]. Numerous anticancer drugs, both on the market and in development, have apoptosis-modulating properties (e.g., Yondelis ® , Madrid, Spain, has been recently approved by the American Food and Drug Administration (FDA) to treat patients with advanced soft tissue sarcoma [210]). Apoptogens are agents that can induce rapid death by modulating the apoptosis pathway. Numerous marine natural products reported in Table 6 are apoptogenic compounds associated with antitumor activity.
Agelastatins, members of the chemically diverse pyrrole-imidazole alkaloids (PIA), are among the best examples of this class of molecules. Since the discovery of agelastatin A (92, Figure 26) in 1993 by Pietra et al., in the New Caledonian coral sea sponge Agelas dendromorpha, more than ten different research groups have reported innovative solutions to synthesize it. As reported in Han et al., 2013, recent development of a concise, stereo-controlled, and biosynthetically inspired strategy to synthesize agelastatin alkaloids and a new synthetic methodology for azaheterocycle synthesis has given rise to many agelastatin derivatives. These developments also facilitate the first side-by-side testing of all known agelastatin alkaloids for their ability to induce cell death in various cancer cells [181]: U-937 (lymphoma), HeLa (cervical carcinoma), A549 (non-small-cell lung carcinoma), BT549 (breast carcinoma), and IMR90 (immortalized lung fibroblasts) human cell lines. Agelastatins A (92, Figure 26) and D (95, Figure 26) both induce dose-dependent apoptosis (in particular cell membrane permeabilization, activation of procaspase-3 to active caspase-3 and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP)) and exhibit dose-dependent G2/M cell cycle arrest in synchronized U-937 cells without affecting tubulin dynamics within cells [181]. In addition, the potency of all agelastatins (92-97, Figure 26) has been evaluated in five human blood cancer cell lines. Interestingly, agelastatin C (94, Figure 26) is only weakly active and agelastatin F (97, Figure 26) is inactive against the tested cell lines. Agelastatin A (92, Figure 26) is remarkably active, particularly against CEM from acute lymphoblastic leukemia and against Daudi from Burkitt's lymphoma (values reported in Table 6) and specifically target these white blood cell lines over normal red blood cells: it is 16,650 times more active on cancer cells than on normal cells! These results are in line with a previous study showing the inhibition of osteopontin-mediated neoplastic transformation (notably by β-catenin inhibition) and metastasis by agelastatin A (92, Figure 26) in MDA-MB-231 and MDA-MB-435 human breast cancer cell lines [211]. Therefore, agelastatins exhibit considerable potential as antitumor drugs that can simultaneously inhibit cancer cell growth as well as act as a potent anti-metastatic drug [211]. However, the mechanism through which agelastatin A (92, Figure 26) causes this cellular effect, particularly cycle arrest, still needs to be elucidated. Identification of potential intracellular targets of agelastatins can be performed by affinity chromatography on immobilized agelastatin. This approach has previously provided valuable insights into the cellular targets of various protein kinases inhibitors including hymenialdisine (12, Figure 5) [212].
Mar. Drugs 2016, 14, x Despite recent advances in developing therapeutic strategies, cancer is still one of the leading causes of death worldwide. Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. Drug-induced programmed cell death holds promise for cancer therapeutic approaches. Apoptosis is one of the well-known pathways used by cells to die. It is a morphological event characterized by chromosomal DNA fragmentation, nuclear disintegration, cell shrinkage, translocation of phosphatidyl serine moieties to the outer membrane leaflet, and membrane blebbing [209]. Numerous anticancer drugs, both on the market and in development, have apoptosis-modulating properties (e.g., Yondelis ® , Madrid, Spain, has been recently approved by the American Food and Drug Administration (FDA) to treat patients with advanced soft tissue sarcoma [210]). Apoptogens are agents that can induce rapid death by modulating the apoptosis pathway. Numerous marine natural products reported in Table 6 are apoptogenic compounds associated with antitumor activity.
Agelastatins, members of the chemically diverse pyrrole-imidazole alkaloids (PIA), are among the best examples of this class of molecules. Since the discovery of agelastatin A (92, Figure 26) in 1993 by Pietra et al., in the New Caledonian coral sea sponge Agelas dendromorpha, more than ten different research groups have reported innovative solutions to synthesize it. As reported in Han et al., 2013, recent development of a concise, stereo-controlled, and biosynthetically inspired strategy to synthesize agelastatin alkaloids and a new synthetic methodology for azaheterocycle synthesis has given rise to many agelastatin derivatives. These developments also facilitate the first side-by-side testing of all known agelastatin alkaloids for their ability to induce cell death in various cancer cells [181]: U-937 (lymphoma), HeLa (cervical carcinoma), A549 (non-small-cell lung carcinoma), BT549 (breast carcinoma), and IMR90 (immortalized lung fibroblasts) human cell lines. Agelastatins A (92, Figure 26) and D (95, Figure 26) both induce dose-dependent apoptosis (in particular cell membrane permeabilization, activation of procaspase-3 to active caspase-3 and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP)) and exhibit dose-dependent G2/M cell cycle arrest in synchronized U-937 cells without affecting tubulin dynamics within cells [181]. In addition, the potency of all agelastatins (92-97, Figure 26) has been evaluated in five human blood cancer cell lines. Interestingly, agelastatin C (94, Figure 26) is only weakly active and agelastatin F (97, Figure 26) is inactive against the tested cell lines. Agelastatin A (92, Figure 26) is remarkably active, particularly against CEM from acute lymphoblastic leukemia and against Daudi from Burkitt's lymphoma (values reported in Table 6) and specifically target these white blood cell lines over normal red blood cells: it is 16,650 times more active on cancer cells than on normal cells! These results are in line with a previous study showing the inhibition of osteopontin-mediated neoplastic transformation (notably by β-catenin inhibition) and metastasis by agelastatin A (92, Figure 26) in MDA-MB-231 and MDA-MB-435 human breast cancer cell lines [211]. Therefore, agelastatins exhibit considerable potential as antitumor drugs that can simultaneously inhibit cancer cell growth as well as act as a potent anti-metastatic drug [211]. However, the mechanism through which agelastatin A (92, Figure 26) causes this cellular effect, particularly cycle arrest, still needs to be elucidated. Identification of potential intracellular targets of agelastatins can be performed by affinity chromatography on immobilized agelastatin. This approach has previously provided valuable insights into the cellular targets of various protein kinases inhibitors including hymenialdisine (12, Figure 5) [212]. Among the molecules reported in Table 6, heteronemin (102, Figure 26), a marine sesterterpene, has been shown to induce apoptosis in A-498 human renal carcinoma cells by downregulating apoptosis inhibitors Bcl-2 and Bcl-xL and upregulating the death agonist Bax, leading to the disruption of the mitochondrial membrane potential and the release of cytochrome c from mitochondria [192]. As also reported for agelastatins, these effects are associated with the activation of caspases (3/8 and 9), followed by PARP cleavage. Interestingly, the same study showed that heteronemin (102, Figure 26) also induces autophagy in A-498 cells, but the inhibition of autophagy enhances the anticancer effect of heteronemin (102, Figure 26) in A-498 cells. This result suggests that the combination of heteronemin (102, Figure 26) with autophagy inhibitors further enhances its therapeutic effects for cancer treatment [192]. Therefore, marine molecules can represent chemical tools for the exploration of novel combined therapeutic strategies. Petrosaspongiolide M (109, Figure 26) may also represent an equally valuable chemical tool for its capacity to modulate intracellular proteolysis through dual inhibition of the immunoproteasome and autophagy [202].
Synergistic effects (e.g., treatment with heteronemin (102, Figure 26) and inhibitors of autophagy such as chloroquine) of marine products and therapeutic drugs on disease-related phenotypes highlight the concept of combination therapy. The cytotoxicity of new marine molecules should be tested alone or in combination with known anticancer compounds to overcome drug resistance, as reported in Elmallah and Micheau (2015) for the resistance of cancer cells to TNF-related apoptosis inducing ligand (TRAIL)-induced cell death [209]. TRAIL is a molecule that selectively kills-via apoptosis-transformed and cancer cells, but not most normal cells [213]. Manzamine A, an alkaloid originally isolated from an Okinawan sponge Haliclona sp., may restore TRAIL-induced apoptotic cell death in the TRAIL-resistance pancreatic AsPC-1 cell line via inhibition of glycogen synthase kinase-3β (GSK-3β) and subsequent inhibition of the survival factor NF-κB [209]. It will thus be very interesting to evaluate the TRAIL-sensitizing activity of agelastatin A (92, Figure 26); which is known to inhibit GSK-3β with an IC50 of 12 μM [44] (manzamine A has an IC50 for GSK-3β inhibition of 10 μM [214]). All new marine natural products, including those from biodiversity hotspots such as the New Caledonia archipelago, represent putative new hope for cancer treatments, in particular for overcoming defects in apoptosis signaling.
As highlighted above, targeting apoptotic pathways may have a direct role in inducing tumor cell death, as confirmed by recent advances on marine molecules found in New Caledonia. In addition to agelastatins and heteronemin (102, Figure 26), naamidine A (107, Figure 26), microsclerodermin A (108, Figure 26), aeroplysinin-1 (127, Figure 26), fistularin-3 (128, Figure 26) have been shown to induce apoptosis (Table 6). Cancer is the principal target of the marine molecules released on the market today. Of the seven marine compounds currently on the market, only three (Prialt ® , Dublin, Ireland; Yondelis ® , Madrid, Spain and Lovaza ® , London, UK) were not chemically modified to become drugs [215,216]. The four other molecules underwent lead Among the molecules reported in Table 6, heteronemin (102, Figure 26), a marine sesterterpene, has been shown to induce apoptosis in A-498 human renal carcinoma cells by downregulating apoptosis inhibitors Bcl-2 and Bcl-xL and upregulating the death agonist Bax, leading to the disruption of the mitochondrial membrane potential and the release of cytochrome c from mitochondria [192]. As also reported for agelastatins, these effects are associated with the activation of caspases (3/8 and 9), followed by PARP cleavage. Interestingly, the same study showed that heteronemin (102, Figure 26) also induces autophagy in A-498 cells, but the inhibition of autophagy enhances the anticancer effect of heteronemin (102, Figure 26) in A-498 cells. This result suggests that the combination of heteronemin (102, Figure 26) with autophagy inhibitors further enhances its therapeutic effects for cancer treatment [192]. Therefore, marine molecules can represent chemical tools for the exploration of novel combined therapeutic strategies. Petrosaspongiolide M (109, Figure 26) may also represent an equally valuable chemical tool for its capacity to modulate intracellular proteolysis through dual inhibition of the immunoproteasome and autophagy [202].
Synergistic effects (e.g., treatment with heteronemin (102, Figure 26) and inhibitors of autophagy such as chloroquine) of marine products and therapeutic drugs on disease-related phenotypes highlight the concept of combination therapy. The cytotoxicity of new marine molecules should be tested alone or in combination with known anticancer compounds to overcome drug resistance, as reported in Elmallah and Micheau (2015) for the resistance of cancer cells to TNF-related apoptosis inducing ligand (TRAIL)-induced cell death [209]. TRAIL is a molecule that selectively kills-via apoptosis-transformed and cancer cells, but not most normal cells [213]. Manzamine A, an alkaloid originally isolated from an Okinawan sponge Haliclona sp., may restore TRAIL-induced apoptotic cell death in the TRAIL-resistance pancreatic AsPC-1 cell line via inhibition of glycogen synthase kinase-3β (GSK-3β) and subsequent inhibition of the survival factor NF-κB [209]. It will thus be very interesting to evaluate the TRAIL-sensitizing activity of agelastatin A (92, Figure 26); which is known to inhibit GSK-3β with an IC 50 of 12 µM [44] (manzamine A has an IC 50 for GSK-3β inhibition of 10 µM [214]). All new marine natural products, including those from biodiversity hotspots such as the New Caledonia archipelago, represent putative new hope for cancer treatments, in particular for overcoming defects in apoptosis signaling.
As highlighted above, targeting apoptotic pathways may have a direct role in inducing tumor cell death, as confirmed by recent advances on marine molecules found in New Caledonia. In addition to agelastatins and heteronemin (102, Figure 26), naamidine A (107, Figure 26), microsclerodermin A (108, Figure 26), aeroplysinin-1 (127, Figure 26), fistularin-3 (128, Figure 26) have been shown to induce apoptosis (Table 6). Cancer is the principal target of the marine molecules released on the market today. Of the seven marine compounds currently on the market, only three (Prialt ® , Dublin, Ireland; Yondelis ® , Madrid, Spain and Lovaza ® , London, UK) were not chemically modified to become drugs [215,216]. The four other molecules underwent lead optimization during the various different stages of their development. Developments in structural chemistry must be followed by the characterization of biological targets to optimize therapeutic applications.

Conclusions
Though far from being exhaustive, this review outlines 40 years of exciting research on the chemodiversity of marine organisms, ranging from microbes and invertebrates to vertebrates, from microalgae to macroalgae and halophytes, belonging to very different biota in association with the complex coral reef systems of New Caledonia.
Traditionally "interesting" lead groups such as sponges, cnidarians and ascidians have been intensely investigated because they not only provide the most interesting array of original chemical structures, but they also show the most potent anticancer, anti-inflammatory and antibiotic properties. Other groups have occasionally led to original and stimulating research: echinoderms, mollusks, etc. This review focused on novel products although several studies have been performed on New Caledonian marine species leading to the rediscovery of some secondary metabolites (e.g., Acremonium neocaledoniae, p. 41).
The fact that "minor" groups have been largely left aside does not reflect their lack of intrinsic interest for chemical exploration, but rather the technical difficulties to collect or cultivate them in New Caledonia. Only about 2% of marine bacteria can be cultivated using classical growth media. Some small invertebrates require time-consuming field and laboratory work to get enough molecular material to work on (e.g., bryozoans), are haphazardly encountered (e.g., many "naked" nudibranch and opistobranch mollusks) or are on an endangered species list, etc. Noteworthy are independent investigations on different models: e.g., biomaterials from scleractinian corals for maxillofacial replacement of bone tissue [217] or from oyster nacre as an osteoinductor in odontology [218]. Another example is that of the endemic Nautilus macromphallus cephalopod excretory metabolism. This metabolism relies on a unique and seemingly very stable symbiosis with specific bacterial symbionts to produce molecular nitrogen in the chambered shell and regulate the buoyancy [219]. Figure 2 indicates that the "golden age" of traditional screening for novel molecules and bioactivity has ceased after several periods of intense investigation. This reflects the successive explorations of new "territories", e.g., sea mounts, outer reef slopes and deep benthos in general. Based on what has been achieved thus far, taking into account the exceptional richness of the local biota and the growing need to develop local economy in a balanced and sustainable way, it appears necessary to develop a permanent, reliable and collaborative research and development pipeline linking field exploration to: -drug development, including (i) isolation of active principles; (ii) high-throughput bioactivity screening; (iii) structure-activity investigations and structural elucidation; (iv) cultivation technologies or bioinspired synthesis; (v) clinical trials and beyond; -aquaculture focusing on the treatment of locally grown species of prawns and oyster varieties that are sensitive to seasonal blooms of toxigenic bacteria and microalgae.
Molecular approaches have now come of age, making new biological models more attractive and promising. New Caledonia represents a living laboratory with its unique source of marine organisms that have not been investigated to date. There is now a need to better understand the relationships between host organisms and their microbial flora. This requires on-site operations and specialized equipment (aquaria, cultivation facilities for microorganisms).
Finally, in the general context of "blue growth", New Caledonia is ideally located for investigating the production of third-generation biofuels and of high value-added products (e.g., cosmetics, nutraceuticals) via the biomass of microalgal primary producers.
New Caledonia is a treasure chest for scientists to explore, but is also fragile. With the perspective of climate change due to global warming and emerging anthropogenic forcings, the sustainability of the sources of molecules, including resident bacteria, must take precedence. New Caledonian coral reefs-of which some portions were added to UNESCO's World Heritage list in 2008-must be protected for the livelihood of resident populations, as well as for biological inspiration for the sciences and the arts. Extensive studies of their chemodiversity should contribute to their protection.