**3. Discussion**

It has been reported that 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 [6–8]. *L. helicina* is a herbivorous animal that feeds on micro algae such as diatoms and dinoflagellates [2]. Sea angels, *C. limacina* and *P.*  *doliiformis* are carnivorous animals that exclusively feed on the small mollusk *L. helicina* [1]. Therefore, carotenoids produced by micro algae are made available to sea angels through *L. helicina* in the food chain. As shown in Table 1, Ά-carotene, zeaxanthin, diatoxanthin, and Ά-cryptoxanthin were found to be major carotenoids along with alloxanthin, fucoxanthin, and diadinoxanthin in *L. helicina*. They are characteristic carotenoids in diatoms and microalgae belonging to Cyanophyceae, Rhodophyceae, *etc.* [5,6]. The results indicate that *L. helicina* directly absorbs carotenoids from dietary algae and accumulates them without metabolic modification. On the other hand, keto-carotenoids such as pectenolone, 

7,8-didehydroastaxanthin, 4-ketoalloxanthin, and echinenone were found to be major components in sea angels. The results clearly indicate that sea angels oxidatively metabolize ingested carotenoids from *L. helicina*. So, Ά-carotene was oxidatively converted to astaxanthin via echinenone and canthaxanthin. Ά-Cryptoxanthin was also metabolized to astaxanthin via asteroidenone and adonirubin, as shown in Figure 3. There are three optical isomers of astaxanthin in nature. However, sea angels contain only one (3*S*,3*<sup>ȝ</sup>S*) isomer. This shows that hydroxylation at C-3 and/or C-3ȝ of 4-keto and/or 

4ȝ-keto Ά-end group of carotenoid in sea angels is stereo-selective to form (3*S*,3*<sup>ȝ</sup>S*)- astaxanthin. This stereo-selective hydroxylation has also been reported in other snails: *Fushinus perplexus*, *F. perplexus ferrugineus*, *F. forceps* [11,12], *Cipangopaludina chinensis laeta*, *Semisulcospia libertina* [13], and *Pomacea canaliculata* [14]. 

**Figure 3.** Accumulation and metabolic pathways of carotenoids that originated from phytoplankton in the sea angels *C. limacina* and *P. doliiformis*.

Sea angels also introduced a carbonyl group at C-4 and/or C-4ȝ in the 3-hydroxyand/or 3ȝ-hydroxy-Ά-end group. Namely, zeaxanthin was metabolized to astaxanthin via adonixanthin and idoxanthin. Similarly, an acetylenic carotenoid, diatoxanthin, was metabolized to 7,8-didehydroastaxanthin via pectenol, pectenolone, and 4ȝ-hydroxypectenolone. Alloxanthin was also oxidatively metabolized to 7,8,7<sup>ȝ</sup>,8<sup>ȝ</sup>-tetradehydroastaxanthin via 4ȝ-hydroxy-4-ketoalloxanthin, and 4-ketoalloxanthin, as shown in Figure 3. By introducing a carbonyl group at C-4 and/or C-4ȝ in the 3-hydroxy- and/or 3ȝ-hydroxy-Ά-end group, carotenoids changed their color from yellow to red. Therefore, the red color of the gonads of sea angels is due to the presence of keto-carotenoids such as pectenolone, 7,8- didehydroastaxanthin, and 7,8,7<sup>ȝ</sup>,8<sup>ȝ</sup>-tetradehydroastaxanthin. Epoxy carotenoids, diadinoxanthin and fucoxanthin, which are present in *L. helicina*, were not found in sea angels*.* It is suggested that sea angels cannot absorb epoxy carotenoids. 

Chum salmon, *O. keta*, feeds not only on micro crustaceans but also on sea angels [3–5]. Astaxanthin, which consists of three optical isomers, was found to be a major carotenoid, along with the acetylenic carotenoids pectenolone and 7,8- didehydroastaxanthin, in *O. keta*. It is well-known that astaxanthin in crustaceans such as krill also consists of three optical isomers [6–8,15]. Therefore it is clear that astaxanthin in salmon originates from crustaceans. On the other hand, the acetylenic carotenoids pectenolone and 7,8-didehydroastaxanthin were not found in these crustaceans [6–8,15]. So, they are suggested to originate from sea angels. 

It has been reported that marine animals accumulate carotenoids in their gonads, such as astaxanthin in salmon, pectenolone in scallops, and echinenone in sea urchins and that carotenoids are essential for reproduction in marine animals [8]. For example, astaxanthin supplementation in cultured salmon and red sea bream increased ovary development, fertilization, hatching, and larval growth [16]. In the case of sea urchins, supplementation with Ά-carotene, which was metabolized to echinenone, also increased reproduction and the survival of larvae [17]. 

As described above, sea angels converted dietary carotenoids to corresponding keto-carotenoids by introducing a carobonyl group and accumulated these ketocarotenoids in their gonads. Several investigators have reported that introducing a carobonyl group at C-4 and/or C-4ȝ of the Ά-end group of carotenoids enhanced their antioxidant effects, such as the quenching of singlet oxygen (1O2), inhibiting lipid peroxidation, and protection from photo-oxidation [18–21]. As well as astaxanthin, pectenolone, an oxidative metabolite of diatoxanthin, showed excellent antioxidative activity by inhibiting lipid peroxidation [22] and quenching singlet oxygen (1O2). Therefore, keto-carotenoids such as pectenolone may contribute to protection against oxidative stress and promote the reproduction of sea angels through antioxidative activity. 
