The carotenoids composition of the corals and the tridacnid clam were similar to each other (Table 1). β,β-Carotene, peridinin (including the 9′Z isomer), pyrrhoxanthin, diatoxanthin, and diadinoxanthin were found in these animals. These carotenoid patterns resembled those of symbiotic zooxanthellae [5,6]. The results indicate that corals and the tridacnid clam directly absorb carotenoids from symbiotic zooxanthellae and accumulate them without metabolic modification. In the eggs of corals, peridinin and pyrroxanthin were present as major carotenoids. It was assumed that peridinin and pyrroxanthin play important roles in reproduction in corals, as with astaxanthin in salmonid fishes [7].

Recently, Daigo et al. studied carotenoids of more than 20 species of coral inhabiting reefs in Okinawa [8]. They reported that carotenoids found in these corals were not only peridinin and diadinoxanthin, that originated from symbiotic zooxanthellae, but also zeaxanthin, lutein, and, fucoxanthin, that originated from cyanobacteria, green algae, and diatoms. Cyanobacteria, green algae, and diatoms were epizoic and/or endolithic algae that grew in association with the corals. Corals accumulated carotenoids from these epizoic and/or endolithic algae [8]. However, the present study found that carotenoids in Acropora corals, inhabiting the Kuroshio current coast of Kochi, only consisted of those that originated from zooxanthellae. These differences might reflect the constitution of associating algae with corals.

Peridinin and pyrrhoxanthin were found to be major carotenoids in the tridacnid clam. In general, major carotenoids found in clams are fucoxanthin and its metabolites originating from diatoms [9–11]. On the other hand, neither fucoxanthin nor its metabolites were found in the tridacnid clam. This indicates that the tridacnid clam only ingested carotenoids from dinoflagellate algae. Similar results were reported in carotenoids of the bivalves, Modiolus modiolus and Pecten maximus [12].

The crown-of-thorns starfish, A. planci, is a large, nocturnal sea star that mainly preys upon coral polyps. Like other starfish [13], 7,8-didehydroastaxanthin and astaxanthin were found to be major carotenoids along with pectenolone, 7,8,7′,8′-tetradehydroastaxanthin, diatoxanthin, and alloxanthin (Table 2). In general, the starfish can introduce a hydroxy group at C-3 and carbonyl group at C-4 in the β-end group of carotenoids [6]. So, 7,8-didehydroastaxanthin and astaxanthin were oxidative metabolites of diatoxanthin and β-carotene, respectively, ingested from dietary corals. Echinenone and canthaxanthin were metabolic intermediates from β,β-carotene to astaxanthin. The acetylenic carotenoids, pectenolone, pectenol A, and pectenol B, were also metabolic intermediates from diatoxanthin to 7,8-didehydroastaxanthin. Peridinol, one of the major carotenoids in the crown-of-thorns starfish, was converted from peridinin, which originated from corals, by hydrolysis. Furthermore, four new carotenoids; 4-ketodeepoxyneoxanthin, 4-keto-4′-hydroxydiatoxanthin, 3′-epigobiusxanthin, and 7,8-dihydrodiadinoxanthin, were isolated [14]. Details of the structural elucidation of those compounds were described previously [14]. In the present paper, the biosynthetic origins of these compounds are discussed (Figure 2). 4-Keto-4′-hydroxydiatoxanthin was one of the metabolic intermediates from diatoxanthin to 7,8-didehydroastaxanthin. 4-Ketodeepoxyneoxanthin might be an oxidative metabolite of deepoxyneoxanthin derived from neoxanthin by deepoxydation. 3′-Epigobiusxanthin might be derived from diadinoxanthin. 7,8-Dihydrodiadinoxanthin, which has a unique single bond in the 7,8-saturated polyene chain, may be a reduction metabolite of diadinoxanthin. Therefore, it was concluded that carotenoids ingested from corals were oxidatively metabolized and accumulated in the crown-of-thorns starfish.

Like the crown-of-thorns starfish, the small sea snail D. fragum also feeds on corals. The carotenoid composition of this snail resembled that of the dietary corals (Table 3). This indicated that D. fragum also accumulated carotenoids from dietary corals without metabolic modification, except for the esterification of peridinin. In the present study, peridinin 3-acyl esters were fully characterized based on ¹H-NMR and FAB MS spectral data. The ¹H-NMR signal of H-3 (δ 4.95), which showed 1.04 ppm downfield shift relative to the corresponding signal in peridinin [15,16], indicated that the hydroxy group at C-3 was acylated. Fatty acids esterified with peridinin were assigned as palmitic acid, palmitoleic acid, and myristic acid based on FAB-MS data. Previously, peridinol fatty acid ester was characterized by Moaka et al. [10] and Sugawara et al. [17]. However, peridinin 3-acyl esters have not yet been reported. The origin of zeaxanthin in this snail was unclear. It might have originated from associated algae such as cyanobacteria [8].

The UV-Visible (UV-VIS) spectra were recorded with a Hitachi U-2001 in diethyl ether (Et₂O). The positive ion FAB-MS spectra were recorded using a JEOL JMS-700 110A mass spectrometer with m-nitrobenzyl alcohol as a matrix. The ¹H-NMR (500 MHz) spectra were measured with a Varian UNITY INOVA 500 spectrometer in CDCl₃ with TMS as an internal standard. HPLC was performed on a Shimadzu LC-6AD with a Shimadzu SPD-6AV spectrophotometer set at 470 nm. The column used was a 250 × 10 mm i.d., 10 μm Cosmosil 5C18-II (Nacalai Tesque, Kyoto, Japan) with acetone:hexane (3:7, v/v) at a flow rate of 1.0 mL/min. The optical purity of astaxanthin was analyzed by chiral HPLC using a 300 × 8 mm i.d., 5 μm Sumichiral OA-2000 (Sumitomo Chemicals, Osaka, Japan) with n-hexane/CHCl₃-ethanol (48:16:0.8, v/v) at a flow rate of 1.0 mL/min [18].

The corals A. japonica, A. secale, and A. hyacinthus, the tridacnid clam T. squamosa, the crown-of-thorns starfish A. planc, and the sea snail D. fragum were collected along the Ootsuki coast, Kochi Prefecture, Japan from July to August 2009 and 2010.

The extraction and identification of carotenoids were carried out according to our routine methods [19]. Carotenoids were extracted from living or fresh animal specimens with acetone. The acetone extract was transferred to ether-hexane (1:1) layer after the addition of water. The total carotenoid contents were calculated employing an extinction coefficient of E cm 1 % = 2100 (astaxanthin) [20] for the starfish A. planci and E cm 1 % = 1350 (peridinin) [20] for A. japonica, T. squamosa, and D. fragum at λ max. The ether-hexane solution was evaporated. The residue was subjected to HPLC on silica gel. Carotenoid compositions were estimated by the peak area of the HPLC on silica gel with acetone-hexane (3:7) monitored at 470 nm.

Individual carotenoids were identified by UV-VIS (ether), FAB MS, and partial ¹H NMR (500 MHz, CDCl₃).

Identification of individual carotenoids were carried out on UV-VIS and FAB MS spectral data and compared with chromatographic property with authentic samples [19]. Optical isomer of astaxanthin in the crown-of-thorns starfish Acanthaster planci was analyzed by Chiral HPLC [18]. Astaxanthin fraction in Acanthaster planci was consisted of three optical isomers (3R,3′R):(3R,3′S):(3S,3′S) with a ratio of 32:14:54. Furthermore, peridiniol, peridinin and 9′Z-Peridinin were characterized by ¹H NMR [15,16]. Structures of 7,8-ihydrodiadinoxanthin, 3′-epigobiusxanthin, 4-keto-4′-hydroxydiatoxanthin, 4-ketodeepoxyneoxanthin, and deepoxyneoxanthin were fully characterized by NMR [14].

Peridinin-3-acyl esters. FAB-MS: m/z 868.5860 [M]⁺ (calcd. for C₅₅H₈₀O₈, 868.5856) peridinin 3-palmitate, m/z 866.5698 [M]⁺ (calcd. for C₅₅H₇₉O₈, 866.5703) peridinin 3-palmitolate, m/z 840.5550 [M]⁺ (calcd. for C₅₃H₇₆O₈, 840.5547) peridinin 3-myristate; UV-VIS 455, 475 nm; ¹H NMR (CDCl₃), δ 0.88 (3H, t, J = 7.5 Hz, CH₃ in fatty acid moiety), 0.99 (3H, s, H-16), 1.07 (3H, s, H-17′), 1.20 (3H, s H-17), 1.21 (3H, s H-18), 1.25 (about 24H, s, -CH₂- in fatty acid moiety), 1.35 (3H, s, H-18′), 1.39 (3H, s, H-16′), 1.41 (1H, dd, J = 13, 7 Hz,H-2′β), 1.51 (1H, dd, J = 13, 12.5 Hz, H-4′β), 1.64 (1H, dd, J = 12.5, 12 Hz, H-2α eq), 1.79 (1H, dd, J = 12, 7 Hz, H-4β ax), 1.81 (3H, s, H-19′), 2.00 (H, ddd, J = 13, 4, 2 Hz, H-2′α), 2.04 (3H, s, CH₃CO-), 2.23 (3H, s, H-20), 2.29 (1H, overlapped, H-α), 2.28 (2H, t, J = 7.5 Hz, -CH₂-COOH in fatty acid moiety), 2.41 (1H, ddd, J = 14, 5, 1.5 Hz, H-4α), 4.95 (1H, m, H-3), 5.37 (1H, m, H-3′), 5.74 (1H, s, H-12), 6.06 (1H, s, H-8′), 6.11 (1H, d, J = 11 Hz, H-10′), 6.38 (1H, dg, J = 14, 11 Hz, H-11′), 6.40 (1H, d, J = 16 Hz, H-8), 6.46 (1H, d, J = 11 Hz, H-14′), 6.51 (1H, dd, J = 14, 11 Hz, H-15′), 6.61 (2H, dd, J = 14, 11 Hz, H-11′, 15′).