Terpenoids in Marine Heterobranch Molluscs

Heterobranch molluscs are rich in natural products. As other marine organisms, these gastropods are still quite unexplored, but they provide a stunning arsenal of compounds with interesting activities. Among their natural products, terpenoids are particularly abundant and diverse, including monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, tetraterpenoids, and steroids. This review evaluates the different kinds of terpenoids found in heterobranchs and reports on their bioactivity. It includes more than 330 metabolites isolated from ca. 70 species of heterobranchs. The monoterpenoids reported may be linear or monocyclic, while sesquiterpenoids may include linear, monocyclic, bicyclic, or tricyclic molecules. Diterpenoids in heterobranchs may include linear, monocyclic, bicyclic, tricyclic, or tetracyclic compounds. Sesterterpenoids, instead, are linear, bicyclic, or tetracyclic. Triterpenoids, tetraterpenoids, and steroids are not as abundant as the previously mentioned types. Within heterobranch molluscs, no terpenoids have been described in this period in tylodinoideans, cephalaspideans, or pteropods, and most terpenoids have been found in nudibranchs, anaspideans, and sacoglossans, with very few compounds in pleurobranchoideans and pulmonates. Monoterpenoids are present mostly in anaspidea, and less abundant in sacoglossa. Nudibranchs are especially rich in sesquiterpenes, which are also present in anaspidea, and in less numbers in sacoglossa and pulmonata. Diterpenoids are also very abundant in nudibranchs, present also in anaspidea, and scarce in pleurobranchoidea, sacoglossa, and pulmonata. Sesterterpenoids are only found in nudibranchia, while triterpenoids, carotenoids, and steroids are only reported for nudibranchia, pleurobranchoidea, and anaspidea. Many of these compounds are obtained from their diet, while others are biotransformed, or de novo biosynthesized by the molluscs. Overall, a huge variety of structures is found, indicating that chemodiversity correlates to the amazing biodiversity of this fascinating group of molluscs.


Background
Marine organisms produce a wide variety of natural products, often unique and critical for their survival and ecological performance [1][2][3][4]. Among these molecules, terpenes are remarkably abundant, with about 60% of all known natural products being terpenoids [1,2]. Terpenoids (or isoprenoids) are a large and diverse class of naturally occurring organic chemicals derived from terpenes; they are the largest class of natural products, with estimates of > 70 000 distinct compounds providing a vast pool of complexity that can interact with biological targets in a huge variety of ways [5]. They usually are multicyclic structures with oxygen-containing functional groups. Biochemical modifications of terpenes produce the related terpenoids. Terpenes are biosynthetically derived from units of isopentenyl pyrophosphate (IPP). Animals produce terpenes through the HMG-CoA reductase pathway, the mevalonate pathway, which also produces cholesterol. These reactions take place in the cytoplasm of the cells, where IPP and dimethylallyl pyrophosphate (DMAPP) condense  [3,19,20]. In brackets: number of terpenoids reviewed here.
Overall, heterobranchs display a huge diversity of biological and ecological strategies based on their chemical defenses. The presence of symbiotic microorganisms, which could in fact be producing some of these natural compounds, is unknown for this group [3]. The natural products of heterobranchs, however, are pivotal for their ecological specialization, as said above, although many aspects of their chemical ecology, as in other marine organisms, remain currently unknown [3,23,28,29]. As already mentioned, heterobranchs may feed upon a wide range of other organisms, such as algae (Chlorophyta, Ochrophyta, Rhodophyta (green, red, and brown algae, respectively)), sea grasses, Porifera, Cnidaria, Annelida, Bryozoa, Chordata (tunicates), other Mollusca, and others. Therefore, they are relevant in marine ecosystems because they occupy many different ecological niches and display a wide array of trophic relationships with organisms from many different taxonomic groups, comprising macroalgal or plant herbivory, carnivore prey-predator relationships, and occasionally cannibalism. They are also able to use cleptochemistry, incorporating chemicals from their diet (cleptochemicals), often called cleptochemodefenses when used for their own defensive means [15,[30][31][32].
Marine organisms are a still underexplored source of unique natural compounds produced or accumulated both by micro-and macroorganisms with pharmacologically interesting properties to be used as drugs [1,2,23,[33][34][35], displaying specific biological activities and unique skeletons, rarely found in non-marine organisms [36][37][38][39][40]. The pharmacological potential of terpenoids from marine organisms has been reviewed in the past [12]. For heterobranchs, their pharmacological potential in drug discovery is remarkable. Promising heterobranch compounds include several aplyronines and dolastatins from the anaspidean Dolabella auricularia and bursatellanins from Bursatella leachii, kahalalides from the sacoglossan Elysia rufescens, jorumycin from the doridacean Jorunna funebris,

Monoterpenoids
Monoterpenes are formed by two isoprene units and have a molecular formula C 10 H 16 . They may be linear (acyclic) or contain rings, and are derived biosynthetically from units of IPP, which is formed from acetyl-CoA via mevalonic acid in the HMG-CoA reductase pathway. IPP is further isomerized to DMAPP by the enzyme IPP isomerase. GPP is the precursor to monoterpenes, and their biosynthesis is mediated by terpene synthases [6][7][8]. Within heterobranchs, in this time period, only anaspideans and sacoglossans are found to possess monoterpenoids, which may be linear or cyclic (Table 1). When cyclic monoterpenoids are present (in sea hares and one sacoglossan), these are always monocyclic. Table 1. Number of monoterpenoids in the different Heterobranch groups in the reviewed period. In brackets: number of species with monoterpenoids.

Linear Monoterpenoids
Anaspideans and sacoglossans possess several kinds of linear monoterpenoids, either from their diet or transformed from dietary compounds.
specifically, these are four unusual acetates of linear polyhalogenated monoterpenes and four cyclic derivatives.
Some shelled sacoglossans are also rich in monoterpenoids, which they can obtain from their algal food and further transform into more bioactive compounds. The Mediterranean species Oxynoe olivacea, Lobiger serradifalci, and Ascobulla (= Cylindrobulla) fragilis feed on different parts of the green algae Caulerpa prolifera, which contains the sesquiterpenoid caulerpenyne [67]. O. olivacea and A. fragilis are able to modify caulerpenyne, which can be found in their digestive gland, into the potent ichthyotoxic aldehydes, oxytoxin-1 (3) and -2 (4) (Figure 2), and transport them to be secreted to the mucus and mantle. L. serradifalci instead contains only oxytoxin-1 in the parapodial lobes and in its defensive mucus [68]. Similarly, in the Caribbean species Ascobulla ulla (feeding on Caulerpa fastigiata), Oxynoe antillarum (feeding on Caulerpa sp.), and Lobiger souberveii (feeding on Caulerpa racemosa), the same chemistry is found, accumulating caulerpenyne (only this molecule is found in L. souberveii) and further transforming it into oxytoxins [69]. In the Indian species Volvatella sp. caulerpenyne has also been reported [70]. Caulerpenyne is a highly bioactive molecule, presenting anticancer activity, cell grown inhibition, neurotoxic activity, and inhibiting lipoxygenases, among others [71][72][73][74].  Some shelled sacoglossans are also rich in monoterpenoids, which they can obtain from their algal food and further transform into more bioactive compounds. The Mediterranean species Oxynoe olivacea, Lobiger serradifalci, and Ascobulla (= Cylindrobulla) fragilis feed on different parts of the green algae Caulerpa prolifera, which contains the sesquiterpenoid caulerpenyne [67]. O. olivacea and A. fragilis are able to modify caulerpenyne, which can be found in their digestive gland, into the potent ichthyotoxic aldehydes, oxytoxin-1 (3) and -2 (4) (Figure 2), and transport them to be secreted to the mucus and mantle. L. serradifalci instead contains only oxytoxin-1 in the parapodial lobes and in its defensive mucus [68]. Similarly, in the Caribbean species Ascobulla ulla (feeding on Caulerpa fastigiata), Oxynoe antillarum (feeding on Caulerpa sp.), and Lobiger souberveii (feeding on Caulerpa racemosa), the same chemistry is found, accumulating caulerpenyne (only this molecule is found in L. souberveii) and further transforming it into oxytoxins [69]. In the Indian species Volvatella sp. caulerpenyne has also been reported [70]. Caulerpenyne is a highly bioactive molecule, presenting anticancer activity, cell grown inhibition, neurotoxic activity, and inhibiting lipoxygenases, among others [71][72][73][74].
The shell-less sacoglossans of the genus Elysia usually also feed on Caulerpa species and related green algae [75], from which they obtain and transform their defensive compounds. Some Caribbean

Sesquiterpenoids
Sesquiterpenoids are formed by three isoprene units and have molecular formula C 15 H 24 . They may be acyclic or contain rings, displaying many unique combinations. The reaction of GPP with IPP results in 15-C FPP, an intermediate in their biosynthesis [6]. Cyclic sesquiterpenes are more common than cyclic monoterpenes because of the increased chain length and the additional double bond in sesquiterpene precursors. The FPP backbone can be rearranged in several different ways and further complemented with different functional groups, thus producing a wide variety of sesquiterpenoids. Within heterobranchs, in this time frame, nudibranchs, anaspideans, sacoglossans, and pulmonates, have been found to possess sesquiterpenes, which are mostly cyclic, while no sesquiterpenoids have been found in Pleurobranchoidea or other heterobranchs (Table 2).

Bicyclic Sesquiterpenoids
Bicyclic sesquiterpenoids are mainly present in sea slugs, with a couple of examples in sea hares, and none in the remaining heterobranch groups. Within nudibranchs, the Antarctic doridacean Bathydoris hodgsoni contains the drimane sesquiterpene hodgsonal (20) (Figure 4) [104,105]. Hodgsonal is found in the most exposed body parts (mantle and dorsal papillae) and is probably de novo biosynthesized by the nudibranch. Hodgsonal is used as a feeding deterrent against sympatric predators, such as the sea star Odontaster validus and the anemone Epiactis sp. [105]. Furthermore, austrodorins A and B, and the two nor-sesquiterpenes austrodoral (21) ( Figure 4) and austrodoric acid, were found in the Antarctic slug Doris kerguelenensis, but their ecological role is still unknown [106][107][108][109][110].
The widely studied nudibranch Dendrodoris possesses drimane sesquiterpenes distributed in different body parts [3,15]. In this slugs, drimane esters are generally associated to the reproductive organs and eggs, while drimane sesquiterpenes are detected in the mantle. D. arborescens possess the known sesquiterpene 7-deacetoxy-olepupuane (22) (Figure 4) [111], while D. carbunculosa presents several cytotoxic drimane sesquiterpenes, the dendrocarbins A-N [112]. Moreover, D. krebsi from Mexico also possess drimane sesquiterpenes and esters [113], allocated in the body in a way similar to that previously described for other Dendrodoris species [114]. D. denisoni from New Zealand presents cinnamolide, olepupuane, and polygodial in its mantle [78]. The phylogenetically related genus Doriopsilla also presents compounds related to those of Dendrodoris. Pelseneeriols-1 (23) and -2, two furanosesquiterpene alcohols, are present in the mantle of Doriopsilla pelseneeri from the Atlantic [115]. De novo biosynthesis of drimane esters, sesquiterpenes, and 15-acetoxy-ent-pallescensin was demonstrated for D. areolata and Doriopsilla sp. through the mevalonic pathway [116][117][118][119]. Two more diastereomeric acetates of pelseneeriol-1 and -2 ( Figure 4) were further described in that study. Moreover, both D. albopunctata from the Pacific and D. areolata from the Atlantic also contain drimane sesquiterpenes and ent-pallescensin A [113], located in their bodies in a way similar to the Dendrodoris species [114]. Doriopsilla pharpa contains polygodial, and their extracts are deterrent to two fish, the blenny Chasmodes bosquianus, and the mummichog Fundulus heteroclitus, which learned to avoid food items that contained extracts from D. pharpa [120], while extracts of the slug were also rejected by the crabs Callinectes similus and Panopeus herbstii in the field. Similarly, the Arminacean Leminda millecra from South Africa presents several sesquiterpenes, probably from its octocoral prey (Alcyonium foliatum, A. valdiviae, A. fauri, and Capnella thyrsoidea) [157,158]. These are millecrones A (32) and B and millecrols A and B (Figure 4). Among them, millecrone A inhibited the growth of Candida albicans at 50 μg/disk, while millecrone B was active against Staphylococcus aureus and Bacillus subtilis at 50 μg/disk; millecrol B was active only against B. Another well-studied nudibranch group is that of the Phyllidids, which are often colorful animals containing isocyanate compounds [15]. These isocyanates display a wide array of activities, such as antifouling, antibiotic, antifungal, and antitumor properties, and have been studied in depth during recent years [121][122][123][124][125]. Several studies on dietary sesquiterpene isocyanides suggest that the nudibranchs sequester them from different demosponges, showing a much broader feeding variability than previously reported [46]. One of the most studied species is Phyllidiella pustulosa, where compounds are obtained from the demosponge Acanthella cavernosa [126]. Specimens from China and Vietnam also contain sesquiterpene isocyanides and related compounds, some of them found also in Acanthella sponges [126][127][128][129]. A recent chemical analyses of the South China Sea nudibranchs Phyllidiella pustulosa and Phyllidia coelestis, as well as their possible sponge prey Acanthella cavernosa, led to the isolation of a nitrogenous cadinane-type sesquiterpenoid, xidaoisocyanate A (24) (Figure 4), among other sesquiterpenoids and diterpenoids [130]. Moreover, P. pustulosa from Fiji presents an isothiocyanate, axisonitrile-3 (25) (Figure 4), and several minor related sesquiterpenes [131]. The isothiocyanate displays a moderated antiplasmodial activity, being weakly cytotoxic (IC 50 > 20 µg/ml) but strong growth inhibitor of Mycobacterium tuberculosis (MIC 2 µg/ml) [132]. Furthermore, a sesquiterpene isonitrile was also isolated as an antifouling agent from the Japanese P. pustulosa [123], and some other studies on the antifouling potential of Phyllidia ocelata, P. varicosa, Phyllidiella pustulosa, and Phillidiopsis krempfi, found three more sesquiterpene isonitriles, the 10-epi-axisonitrile-3, 10-isocyano-4-cadinene, and 2-isocyanotrachyopsane, as well as a peroxide, 1,7-epidioxy-5-cadinene, and some more sesquiterpene isonitriles [122,133]. P. pustulosa and Phyllidia ocellata from Australia, also present some stereoisomers of 4-isocyano-9-amorphene and of 10-isocyano-4-amorphene, respectively, while Phyllidia picta from Bali contained the axane sesquiterpenoids pictaisonitrile-1 and pictaisonitrile-2 [134]. Phyllidia sp. from Sri Lanka also contains 3-isocyano-theonellin, closely related to a cyanide from the demosponge Axinyssa [135]. Some nitrogenous bisabolene sesquiterpenes from these species also possess a potent antifouling activity against barnacle larvae in in vitro assays [136,137]. P. varicosa presents two 9-thiocyanatopupukeanane sesquiterpenes, isolated as an epimeric mixture, found also in its demosponge prey Axinyssa aculeata [138]. One of these compounds is present in the mantle, suggesting an implication in chemical defense, while both are present in the digestive gland, supporting a dietary origin. Both compounds display mild toxicity against brine shrimp and some antimicrobial activity against Candida albicans and Bacillus subtilis. On the other hand, P. coelestis from Thailand also presents two cytotoxic pupukeanane sesquiterpenoids [139]. Several studies suggest all these compounds may play an important role in the chemical defense of phyllidids against fish predators, and that this is also related to visual defenses [3,140]. Contrastingly, Reticulidia fungia from Okinawa is different from the rest of the family members in presenting two sesquiterpenes of a rare class of sponge compounds, the cytotoxic carbonimidic dichlorides, reticulidins A (26) and B ( Figure 4) [141]. Furthermore, the dorid Hexabranchus sanguineus from South China presents a couple of sesquiterpenes, as well as other compounds, suggested to originate from its sponge diet [142].
In Dendronotids, the species Tritonia hamnerorum from Florida sequesters julieannafuran (31) (Figure 4), a furanogermacrane from its food, the sea fan Gorgonia ventalina, which is deterrent against reef fish in field assays [155]. Furthermore, some byciclic sesquiterpenes are found in the mantle of Tochuina tetraquetra and its food, the soft coral Gersemia rubiformis from British Columbia [156]. These are tochuinyl acetate and dihydrotochuinyl acetate, two cuparane sesquiterpenoids, found together with some diterpenes (see below). Their ecological activity has not been studied so far.
Similarly, the Arminacean Leminda millecra from South Africa presents several sesquiterpenes, probably from its octocoral prey (Alcyonium foliatum, A. valdiviae, A. fauri, and Capnella thyrsoidea) [157,158]. These are millecrones A (32) and B and millecrols A and B ( Figure 4). Among them, millecrone A inhibited the growth of Candida albicans at 50 µg/disk, while millecrone B was active against Staphylococcus aureus and Bacillus subtilis at 50 µg/disk; millecrol B was active only against B. subtilis at 50 µg/disk [45]. In other sampling areas, L. millecra presents some quinones, and some of its chemicals are described to be obtained from its diet, the gorgonian Leptogorgia palma [158]. Again, their ecological activity has not been studied yet. In Japan, the arminid Dermatobranchus otome possesses germacrane sesquiterpenoids DO1, DO2, and DO3, with some antibacterial activity against B. subtilis [159].

Tricyclic Sesquiterpenoids
Only nudibranchs and sea hares present tricyclic sesquiterpenoids. Pallescensin A and several other terpenes are found in the Patagonian doridacean Tyrinna nobilis [89]. Hypselodoris infucata from Bali yielded the known (−)-furodysinin (34) (Figure 5), being active against the HeLa cell line with an IC 50 at 102.7 µg/mL, while its crude extract is repellent to the sympatric shrimp Penaeus vannamei at natural concentration [169]. Similarly, the species H. kanga from India possesses furodysinin, suggesting a trophic relationship, since furodysinin is also found in the associated demosponge Dysidea sp. [170]. H. lajensis from Brazil contains furodysinin lactone (35) (Figure 5), also found in Dysidea sponge species [171]. Goniobranchus reticulatus from Australia presents a dialdehyde sesquiterpene, together with the ring-closed acetal, both bioactive against P388 mouse leukemia cells, as well as some diterpenes [90]. These two compounds are also found in Goniobranchus sinensis (previously Chromodoris sinensis) from China, where they are reported to be deterrent [90].

Tricyclic Sesquiterpenoids
Only nudibranchs and sea hares present tricyclic sesquiterpenoids. Pallescensin A and several other terpenes are found in the Patagonian doridacean Tyrinna nobilis [89]. Hypselodoris infucata from Bali yielded the known (−)-furodysinin (34) (Figure 5), being active against the HeLa cell line with an IC50 at 102.7 μg/mL, while its crude extract is repellent to the sympatric shrimp Penaeus vannamei at natural concentration [169]. Similarly, the species H. kanga from India possesses furodysinin, suggesting a trophic relationship, since furodysinin is also found in the associated demosponge Dysidea sp. [170]. H. lajensis from Brazil contains furodysinin lactone (35) (Figure 5), also found in Dysidea sponge species [171]. Goniobranchus reticulatus from Australia presents a dialdehyde sesquiterpene, together with the ring-closed acetal, both bioactive against P388 mouse leukemia cells, as well as some diterpenes [90]. These two compounds are also found in Goniobranchus sinensis (previously Chromodoris sinensis) from China, where they are reported to be deterrent [90].  Two brominated compounds, aplysin (36) and aplysinol (37) (Figure 5), are found in Aplysia kurodai from Japan [172]. This species is also a source of several alkaloids and other compounds [3]. On the other hand, dactylomelatriol (38) (Figure 5) is found in Aplysia dactylomela from the Atlantic, derived from an omphalane skeleton and previously described only in terrestrial fungi [173]. Dactylomelatriol is suggested to originate by a modification of a precursor obtained from Laurencia red algae. In fact, the sea hare A. dactylomela is one of the most prolific sources of natural products, containing mixtures of compounds, including many sesquiterpenes, along with polyketides, diterpenes, and triterpenes, which are usually biotransformed from red algal compounds. This species is now considered to be in fact two species: A. dactylomela and A. argus, from the Atlantic and Indo-Pacific, respectively [174]. The dietary compounds found in these sea hares display very diverse structures and characteristics and have been recently reviewed [3].

Diterpenoids
Diterpenoids are composed of four isoprene units (two terpene units) and usually have the molecular formula C 20 H 32 . They are biosynthesized via the HMG-CoA reductase pathway, with geranylgeranyl pyrophosphate (GGPP) being a primary intermediate; structural diversity is achieved mainly by diterpene synthases [6]. Diterpenoids in heterobranchs often appear within mixtures of compounds, including other terpenes or even structurally different compounds, and are mostly found in nudibranchs (Table 3). In addition, many halogenated and brominated monoterpenes and diterpenes are allocated into different body parts in sea hares, for example in Aplysia kurodai (see below).

Bicyclic Diterpenoids
Nudibranchs, pleurobranchoids, sea hares, and pulmonates possess bicyclic diterpenoids. In sea slugs, Phyllidiella pustulosa from China represents the first finding of isocyanide diterpenoids in the family, some of them previously identified from Acanthella sponges, thus confirming the prey-
In pleurobranchoids, Pleurobranchaea meckelii from the Mediterranean possesses two labdane aldehyde diterpenes in the mantle [189], which are similar to those of the pulmonate Trimusculus reticulatus (see below).
Some natural products of heterobranchs, particularly in sea slugs and sea hares, include different combinations of terpenoids with glycerides, guanidines, and others. These include the diterpene glycerides found in several doridacean slugs, such as the Mediterranean Doris verrucosa, possessing the verrucosins 1-9 (68) (Figure 8) [15,216] and a further series of diterpenoid glycerides. Among these compounds, verrucosin A (69) (Figure 8) is de novo biosynthesized, as demonstrated in experiments with 13 C-and 14 C-labelled precursors [217][218][219]. Biosynthesis is commonly found in this group [3]. Another example is the doridacean Archidoris pseudoargus, which possesses several ichtyotoxic diterpene glycerides in mantle and egg masses in UK specimens [220]. Similarly, Doris (Austrodoris) kerguelenensis from several Antarctic locations, presents a series of diterpene diacylglycerides in the mantle, which effectively protect them from predation by sympatric sea stars and anemones, along with the corresponding monoacylglycerides, and monoacylglycerides of regular fatty acids [15,106,221,222]. Additional diterpene glycerides and clerodane diterpenes, such as palmadorins, were described in specimens of D. kerguelenensis from diverse Antarctic populations [223,224]. The existence of different chemotypes in D. kerguelenensis indicates that different terpene synthases may be regulating the biosynthesis of this wide arsenal of terpene glycerides [225], and this may be related to their genetic variability and/or cryptic speciation [224][225][226][227]. Palmadorin A, B, D, M, N, and O inhibit human erythroleukemia cells (HEL) at low micromolar IC 50 , while palmadorin M inhibits Jak2, STAT5, and Erk1/2 activation in HEL cells and produces apoptosis at 5mM [224].
The aeolids Phyllodesmium briarieum, P. longicirrum and P. magnum possess several diterpenes along with some sesquiterpenes, possibly obtained from their octocoral prey [182,228]. Further studies report four polycyclic chatancin diterpenes in Australian specimens of P. longicirrum, along with other deterrent compounds against fish [229,230]. Tritoniopsins A-D (70) (Figure 8) are found in the dendronotid Tritoniopsis elegans in China, from its coral prey Cladiella krempfi [86,231]. The arminid Dermatobranchus ornatus from China possesses a calicophirin diterpenoid in the mantle, suggested to be obtained from its potential gorgonian prey Muricella sp. in South China [232]. D. ornatus also presents four eunicellin diterpenes, where two of them are suggested to be obtained from its prey, the gorgonian Muricella sinensis [46], and another one was isolated earlier from an unidentified Pacific soft coral [233]. These compounds display moderate cytotoxicity and inhibition of cell division in fertilized starfish eggs, but their ecological role is not described [46]. Similar briarane diterpenoids are known in the Mediterranean Armina maculata and its prey, the pennatulacean octocoral Veretillum cynomorium [234][235][236].

Sesterterpenoids
Sesterterpenoids possess 25 carbons, five isoprene units, and they originate from GFPP. Biogenetically, sesterterpenoids are derived from five DMAPP monomers that form the C25 GFPP backbone (10). Cyclisation of GFPP is catalysed by sesterterpene synthases. They are rare in nature relative to other types of terpenes. In Heterobranch molluscs, only linear, bicyclic, and tetracyclic sesterterpenes have been reported, and in all cases, they are found in nudibranchs (Table 4). Table 4. Number of sesterterpenoids in the different Heterobranch groups in the reviewed period. In brackets: number of species with sesterterpenoids.

Linear Sesterterpenoids
Only one nudibranch species has been described to possess linear sesterterpenoids. Interestingly enough, the Antarctic cladobranch slug Charcotia granulosa presents the unique linear homosesterterpene, granuloside (77) (Figure 10) [249], suggested to be de novo biosynthesized and stored in MDF-like structures, and probably released as a deterrent [250].

Tetracyclic Sesterterpenoids
This type of sesterterpenoids is only found in chromodorid nudibranchs. Cadlina luteomarginata from Canada and its prey, the demosponge Phorbas sp., both possess the sesterterpenoid ansellone A (78) (Figure 10), with unknown ecological role, but activating the cAMP signaling pathway [251]. The

Tetracyclic Sesterterpenoids
This type of sesterterpenoids is only found in chromodorid nudibranchs. Cadlina luteomarginata from Canada and its prey, the demosponge Phorbas sp., both possess the sesterterpenoid ansellone A (78) (Figure 10), with unknown ecological role, but activating the cAMP signaling pathway [251]. The South African Chromodoris hamiltoni, a part of the compounds already mentioned above, presents the sesterterpene hamiltonin E (79) (Figure 10), together with some macrolides (latrunculins A and B) [208]. The Indian Glossodoris atromarginata and its prey, the demosponge Spongia sp. present two scalaranes in their mantle dermal formation-like structures [242]. Moreover, these sea slugs were found on two other potential prey, the sponge Hyattella cribriformis presenting pentacyclic scalaranes, and an unidentified sponge, possibly Spongia sp., possessing heteronemin (80) (Figure 10) and other scalaranes [46,170,242]. Heteronemin was further reported to display antimycobacterial activity towards Mycobacterium tuberculosis H 37 Rv with an MIC of 6.25 µg/ml [252]. Deoxoscalarin (81) (Figure 10), previously reported in a Mediterranean sponge, was found in G. atromarginata and the sponge H. cribriformis, supporting its dietary origin. The specimens found on Spongia instead, presented heteronemin, known from the sponge Heteronema erecta, as well as two scalaranes, one of them also reported from an unidentified sponge. Furthermore, G. rufomarginata from China presents scalarane compounds derived from feeding on an unidentified sponge, which are biotransformed into scalaradial derivatives. In particular, scalaradial, a potent anti-inflammatory metabolite [253], and its 12-deacetyl derivative are present in the sponge, while the absence of scalaradial in the slug supports their ability to transform this toxic compound into related scalaranes [254]. Similarly, G. pallida specimens from China, as well as G. vespa and G. averni from Australia, contain 12-deacetoxy-12-oxoscalaradial [255], while G. pallida from Guam presents different sesquiterpenes including scalaradial, deacetylscalaradial, and deoxoscalarin; these compounds are used as feeding deterrents against sympatric crabs and reef fish and are located in the mantle border of the slugs [256,257]. Scalaradial and deacetylscalaradial are also present in the demosponge Cacospongia sp., which is preyed upon by G. pallida. As reported above, Glossodoris species probably biotransforms their dietary scalaranes into related molecules in a detoxification process. Supporting this, the injection of scalaradial in the viscera of G. pallida was reported to be not toxic for the slug but resulted instead in rapid transformation of the metabolite in less than 24 h [3]. All these data support the dietary acquisition and further biotransformation of the scalarane compounds in chromodorid nudibranchs [46,170]. On the other hand, Glossodoris sedna from Costa Rica possess several scalarane sesterterpenes, 12-deacetyl-23-acetoxy-20-methyl-12-epi-scalaradial, 12-deacetyl-23-acetoxy-20-methyl-12-epi-deoxoscalarin, and 12-deacetyl-20-methyl-12-epi-deoxoscalarin, the first of which moderately inhibits mammalian phospholipase A2 (IC 50 18 µM) and is ichthyotoxic at 0.1 ppm against the allopatric fish Gambusia affinis [258]. Contrastingly, specimens of G. dalli from the same place contain the scalarane sesterterpenes 20-deoxoscalarin and 12-epi-20-deoxoscalarin [258]. Additionally, in Japan, Chromodoris inornata contains three cytotoxic sesterterpenes, inorolides A (82), B, and C (Figure 10), and five scalaranes [259]. Recently, Glossodoris hikuerensis and G. vespa from Australia have been reported to contain heteronemin in the viscera, while scalaradial, 12-deacetoxy-12-oxoscalaradial, and 12-deacetoxy-12-oxo-deoxoscalarin are present in the mantle [154].

Triterpenoids
Triterpenoids consist of six isoprene units (three terpene units) with the molecular formula C 30 H 48 . The linear triterpene squalene is derived from the reductive coupling of two molecules of FPP and is the precursor to all steroids [6], as mentioned above. Triterpenes exist in a great variety of structures.
Nearly 200 different skeletons have been identified in nature [6]. These skeletons may be broadly divided according to the number of rings present. In general, pentacyclic structures tend to dominate in the organisms. Triterpenes are biosynthesized through the head-to-head condensation of two FPP units to form a squalene. In turn, the squalene serves as a precursor for the formation of triterpenoids, including eukaryotic sterols. The squalene synthase is a prenyltransferase that catalyzes a complex series of cationic rearrangements to join the C-1 carbons of two farnesyl residues. Differently from other marine invertebrates, such as echinoderms, heterobranch molluscs do not seem to present saponins (triterpenoid glycosides). As far as we know, no linear triterpenoids are described in heterobranchs, while cyclic triterpenoids are present only in nudibranchs, pleurobranchoids, and anaspideans (Table 5).

Group (Species)/Compounds Nudibranchia (1) Pleurobranchoidea (2) Anaspidea (2)
Cyclic Triterpenoids 1 2 3 The doridacean nudibranch from the North Sea, Adalaria loveni, contains lovenone (83) (Figure 11), a cytotoxic degraded triterpenoid suggested to come from an unidentified bryozoan prey [260]. Testudinariols A and B (84) (Figure 11), from the Mediterranean pleurobranchoid Pleurobranchus testudinarius are present in the mantle and mucus and are similar to some sponge compounds [261] although they are thought to be biosynthesized. Testudinariols are chemically related to limatulone of the limpet Lottia limatula [262]. Similarly, an unidentified species of Pleurobranchus from the South China Sea also possesses testudinariol B [263]. The doridacean nudibranch from the North Sea, Adalaria loveni, contains lovenone (83) ( Figure  11), a cytotoxic degraded triterpenoid suggested to come from an unidentified bryozoan prey [260]. Testudinariols A and B (84) (Figure 11), from the Mediterranean pleurobranchoid Pleurobranchus testudinarius are present in the mantle and mucus and are similar to some sponge compounds [261] although they are thought to be biosynthesized. Testudinariols are chemically related to limatulone of the limpet Lottia limatula [262]. Similarly, an unidentified species of Pleurobranchus from the South China Sea also possesses testudinariol B [263]. In sea hares, aplysiols A and B are tetracyclic triterpene polyethers found in the mantle of Aplysia argus from China, similar to compounds from the red algae Laurencia [264]. On the other hand, Dolabella auricularia modifies its molecules from dietary brown and red algae, while de novo synthesizing polypropionates and peptides [265]. Among the metabolites derived from red algae, the polyether bromotriterpene aurilol (85) (Figure 11) is cytotoxic to the HeLa tumor cell line [266].

Carotenoids (Tetraterpenoids)
Carotenoids are tetraterpenoids produced by plants and algae, as well as several bacteria and fungi. All carotenoids are derivatives of tetraterpenes, and are thus produced from 8 isoprene In sea hares, aplysiols A and B are tetracyclic triterpene polyethers found in the mantle of Aplysia argus from China, similar to compounds from the red algae Laurencia [264]. On the other hand, Dolabella auricularia modifies its molecules from dietary brown and red algae, while de novo synthesizing polypropionates and peptides [265]. Among the metabolites derived from red algae, the polyether bromotriterpene aurilol (85) (Figure 11) is cytotoxic to the HeLa tumor cell line [266].

Carotenoids (Tetraterpenoids)
Carotenoids are tetraterpenoids produced by plants and algae, as well as several bacteria and fungi. All carotenoids are derivatives of tetraterpenes, and are thus produced from 8 isoprene molecules (four terpene units); they contain 40 carbon atoms. Tetraterpenes are produced by the head-to-head union of two molecules of GGPP, similar to the formation of a squalene [267]. In general, carotenoids absorb wavelengths from 400 to 550 nm (violet to green light). This causes these compounds to be characteristically colored in yellow, orange, or red. The general structure of the carotenoid is a polyene hydrocarbon chain consisting of 9-11 double bonds and sometimes terminated by rings, with or without additional oxygen atoms attached. This structure of conjugated double bonds leads to a high reducing potential [268]. When carotenoids are present in heterobranchs, they are always diet derived ( Table 6). Although many heterobranchs probably possess carotenoids, very few studies have properly described them. Eight carotenoids and 22 polyunsaturated fatty acids, all of them with anti-inflammatory potential, have been found in the digestive gland of the sea hare Aplysia depilans [269]. These carotenoids are obtained from the algae they feed upon.

Steroids
Steroids consist of four rings arranged in a specific molecular configuration. They are important components of cell membranes altering membrane fluidity, and may also act as signaling molecules. Hundreds of steroids are found in plants, animals, and fungi. In animals, all steroids are manufactured in cells from lanosterol, which is derived by cyclization of the triterpene squalene [9]. They are typically composed of seventeen carbon atoms, bonded in four fused rings: three six-member cyclohexane rings and one five-member cyclopentane ring. Steroids vary by the functional groups attached to this four-ring core and by the oxidation state of the rings. Sterols are steroids with a hydroxy group at position 3 and a skeleton derived from cholestane [9].
Only nudibranchs and anaspideans are reported to present steroids (Table 6); many other heterobranchs probably do so as well, albeit they have not been investigated. Among sea slugs, the doridacean Aldisa smaragdina from Spain presents a progesterone homologue in their external tissues [270]. The related species Aldisa sanguinea was previously reported to have similar steroids and to use them against predators [15]. The dorid Doris aff. verrucosa from Brazil presents a variety of common sterols in the mantle [271], and another Doris (Archidoris) sp. also possess similar glycerids, some of them de novo biosynthesized [15,272]. On the other hand, among the caryophyllidid doridoidea, diaulusterol A (86) (Figure 12) is partially biosynthesized by Diaulula sandieguensis [273]. Phyllidiella pustulosa from Vietnam also contained some sterols [129]. Moreover, the aeolidacean species Cratena peregrina, Flabellina affinis, and Flabellina (Coryphella) lineata present several hydroxy and acetoxysterols from their Eudendrium hydroids diet in the Mediterranean [274]. manufactured in cells from lanosterol, which is derived by cyclization of the triterpene squalene [9]. They are typically composed of seventeen carbon atoms, bonded in four fused rings: three sixmember cyclohexane rings and one five-member cyclopentane ring. Steroids vary by the functional groups attached to this four-ring core and by the oxidation state of the rings. Sterols are steroids with a hydroxy group at position 3 and a skeleton derived from cholestane [9]. Only nudibranchs and anaspideans are reported to present steroids (Table 6); many other heterobranchs probably do so as well, albeit they have not been investigated. Among sea slugs, the doridacean Aldisa smaragdina from Spain presents a progesterone homologue in their external tissues [270]. The related species Aldisa sanguinea was previously reported to have similar steroids and to use them against predators [15]. The dorid Doris aff. verrucosa from Brazil presents a variety of common sterols in the mantle [271], and another Doris (Archidoris) sp. also possess similar glycerids, some of them de novo biosynthesized [15,272]. On the other hand, among the caryophyllidid doridoidea, diaulusterol A (86) (Figure 12) is partially biosynthesized by Diaulula sandieguensis [273]. Phyllidiella In sea hares, Aplysia fasciata from the Mediterranean possesses certain ichthyotoxic degraded sterols, like 4-acetylaplykurodin-B, aplykurodinone B, and 3-epi-aplykurodinone B (87) (Figure 12) [275], in their external tissues, similar to the steroids found in the Atlantic specimens of the same species [276]. These steroids are closely related to aplykurodin B found in the Pacific species A. kurodai [200]. In addition, two secosterols have been found in the sea hare A. kurodai [277]. Mucus secretions in both Atlantic and Mediterranean specimens of A. punctata present epidioxy sterols, as those of A. depilans found in its digestive gland [195].

Concluding Remarks
Heterobranch molluscs are rich in natural products; however, only a small proportion has been studied so far. As reported here, their terpenoids are particularly abundant and diverse; they include monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, tetraterpenoids, and steroids. This review discusses more than 330 metabolites isolated from ca. 70 species of heterobranch molluscs. The monoterpenoids reported here may be linear or monocyclic, while sesquiterpenoids may include linear, monocyclic, bicyclic, or tricyclic molecules. Diterpenoids may include linear, monocyclic, bicyclic, tricyclic, or tetracyclic compounds. Sesterterpenoids, instead, are linear, bicyclic, or tetracyclic. Triterpenoids, tetraterpenoids, and steroids are not as abundant as the previously mentioned types. Remarkably, no terpenoids have been described in this period in tylodinoideans, cephalaspideans, or pteropods; and most terpenoids have been found in nudibranchs, anaspideans, and sacoglossans, with very few compounds in pleurobranchoideans and pulmonates. Many of these compounds found in heterobranchs are obtained from their diet, while others are biotransformed, or de novo biosynthesized by the molluscs. Bacterial origin has been suggested for several types of compounds in heterobranchs [280]. However, very few studies rigorously demonstrate this. The structural similarity and dietary origin of some natural products from microorganisms (particularly cyanobacteria) indicate that this is possible, but further studies are required to shed light on this very interesting topic.
Monoterpenoids are present mostly in anaspidea and are less abundant in sacoglossa. Nudibranchs are especially rich in sesquiterpenes, which are also present in anaspidea, and in less numbers in sacoglossa and pulmonata. Diterpenoids are also abundant in nudibranchs, present also in anaspidea, and scarce in pleurobranchoidea, sacoglossa, and pulmonata. Sesterterpenoids are only found in nudibranchia, while triterpenoids, carotenoids, and steroids are only reported for a few nudibranchia, pleurobranchoidea, and anaspidea. Anaspidea include many halogenated and non-halogenated terpenoids, mostly from diet. Nudibranchs possess many sesquiterpenoids and diterpenoids, mostly from diet, but also de novo biosynthesized. Some compounds are present as glyceryl esters, such as verrucosins. De novo biosynthesis has been demonstrated in a few cases, as well as the putative symbiotic origin of some metabolites [3,15], probably related to the intrinsic difficulties of this kind of research. Dietary origin is much better established, with compounds originating from many diverse organisms, from different kinds of algae and seagrasses to metazoans, such as Porifera, Cnidaria, Mollusca, Bryozoa, Tunicata, and others. In some cases, however, it remains a hypothesis that should be further demonstrated.
The ecological role of these compounds includes many examples of feeding deterrence against sympatric predators, mostly fish and crabs, as well as toxicity. However, many assays are still conducted with species that do not live in the same habitat as the heterobranchs, and thus the ecological significance of many compounds continues to be further examined. The low numbers of reliable field experimental data available is evident. The pharmacological potential of heterobranch terpenoids is clear from the examples reported above-from cytotoxicity to antiplasmodial activity, antituberculosis, antifouling, antifungal, antibacterial, antitumoral, and apoptosis inducers.
Overall, a huge variety of terpenoid structures is found in heterobranchs, indicating that chemodiversity correlates to the biodiversity of this amazing group of molluscs. The potential to vastly increase the number and diversity of known natural products in heterobranchs, as well as their bioactivity, is open to further research. The search for effective and efficient methods of integral and/or selective extraction of terpenoids from heterobranchs, in particular, deserves more attention, since environmentally friendly practices are needed. How global change will affect biodiversity, and thus chemodiversity, in heterobranch molluscs remains to be further investigated, hopefully before both species and molecules disappear.
Funding: Support for this work was provided by a BLUEBIO grant to C.A. (CTM2016-78901/ANT).