Carotenoids in Marine Invertebrates Living along the Kuroshio Current Coast

Carotenoids of the corals Acropora japonica, A. secale, and A. hyacinthus, the tridacnid clam Tridacna squamosa, the crown-of-thorns starfish Acanthaster planci, and the small sea snail Drupella fragum were investigated. The corals and the tridacnid clam are filter feeders and are associated with symbiotic zooxanthellae. Peridinin and pyrrhoxanthin, which originated from symbiotic zooxanthellae, were found to be major carotenoids in corals and the tridacnid clam. The crown-of-thorns starfish and the sea snail D. fragum are carnivorous and mainly feed on corals. Peridinin-3-acyl esters were major carotenoids in the sea snail D. fragum. On the other hand, ketocarotenoids such as 7,8-didehydroastaxanthin and astaxanthin were major carotenoids in the crown-of-thorns starfish. Carotenoids found in these marine animals closely reflected not only their metabolism but also their food chains.

starfish. Carotenoids found in these marine animals closely reflected not only their metabolism but also their food chains.

Introduction
Marine animals, especially marine invertebrates, contain various carotenoids, with structural diversity [1][2][3][4]. Interesting structural carotenoids are still being found in marine animals [4]. In general, animals do not synthesize carotenoids de novo, and so those found in animals are either directly accumulated from food or partly modified through metabolic reactions [2]. The major metabolic conversions of carotenoids found in marine animals are oxidation, reduction, transformation of double bonds, oxidative cleavage of double bonds, and cleavage of epoxy bonds [2,3]. Therefore, various structural varieties are found in carotenoids of marine animals [4].
We have studied carotenoids in several marine invertebrates from chemical and comparative biochemical points of view [4]. In the present study, we focused on carotenoids of the corals Acropora japonica, A. secale, and A. hyacinthus, the tridacnid clam (elongate giant clam) Tridacna squamosa, crown-of-thorns starfish Acanthaster planci, and small sea snail Drupella fragum, inhabiting the Kuroshio current coast. These animals are closely associated within the food chain. Corals and the tridacnid clam are filter feeders and are associated with symbiotic zooxanthellae (dinoflagellate algae). On the other hand, the crown-of-thorns starfish and small sea snail D. fragum are carnivorous and mainly prey upon corals. Therefore, carotenoids that originated from zooxanthellae are passed to starfish and small sea snails through this food chain. In the present paper, we describe the carotenoids of these marine invertebrates.

Results and Discussion
Structural formulae of carotenoids identified from Acropora corals, the tridacnid clam T. squamosa, starfish A. planci, and sea snail D. fragum are shown in Figure 1.

Carotenoids of Corals and the Tridacnid Clam
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][10][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].

Carotenoids of the Sea Snail D. fragum
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 1 H-NMR and FAB MS spectral data. The 1 H-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].

Animal Material
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.

Analysis of Carotenoids
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 1% cm = 2100 (astaxanthin) [20] for the starfish A. planci and E 1% cm = 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.

Conclusions
In conclusion, carotenoids found in the coral A. japonica, clam T. squamosa, starfish A. planci, and sea snail D. fragum well reflected not only their metabolism but also the food chain. The accumulation and metabolism of carotenoids that originate from zooxanthellae to the starfish through the food chain are summarized in Figure 3.