Astaxanthin is bioconverted from carotenoids (beta-carotene, lutein, canthaxanthin, zeaxanthin) through the diet.
Astaxanthin is the predominant carotenoid pigment found in the marine crustacean Tigriopus californicus (Baker), giving these animals their orange-red color. Like all metazoans, T. californicus must convert carotene or hydroxy-carotenoids found in their algal diet into astaxanthin. Other astaxanthin-pigmented crustaceans have been shown to use precursor-specific bioconversion pathways to astaxanthin. The purpose of this study was to demonstrate that californicus bioconverts dietary carotenoids to astaxanthin. Before the experiments, copepods were maintained on a carotenoid-free diet, so they lost all carotenoid coloration. Copepods were then fed one of four dietary carotenoids (β-carotene, lutein, zeaxanthin or canthaxathin) that are precursors to the specific astaxanthin bioconversion pathway. We found that crustaceans from each precursor pigment group produce astaxanthin and that the amount produced depends on the type of carotenoid added. We also characterized the distribution of astaxanthin in the developing egg sac and showed that the red color of larval eyespots is not due to astaxanthin.
INTRODUCE
With some exceptions, animals cannot synthesize carotenoids de novo from basic biological precursors ( Britton and Goodwin, 1982 ). To use carotenoids as external colorants, animals as diverse as flamingos and lobsters must absorb carotenoids from their diet ( Cianci et al., 2002 ; Fox and Hopkins, 1966 ). Red animals can produce red carotenoids through two distinct pathways: they can ingest yellow pigments and oxidize them to produce red keto-carotenoids ( Figure 1 ), or they can ingest Direct red pigment. For most multicellular animals, yellow carotenoids are much more common components of the diet and thus most multicellular animals are red through the conversion of yellow pigments in diet ( Brush, 1990 ; Goodwin, 1984 ). Despite a long history of research on the evolution and distribution of carotenoid color across diverse taxa such as birds, fish, and crustaceans, an ideal model system for studying Research the genetic and physiological mechanisms related to carotenoid metabolism in animals.
Figure 1. Proposed biotransformation pathways for astaxanthin production in transformed animals from Rhodes (2007) . Pathway I is used by several fish species, including goldfish, which use lutein as a substrate. ?? represents the putative conversion of α to β-doradexanthin. Pathway II uses β-carotene or zeaxanthin as a substrate for astaxanthin. Pathway III begins with β-carotene and includes canthaxanthin as an intermediate of astaxanthin. The enzyme responsible for each transformation is italicized.
We recently began investigating the potential of the red marine crustacean Tigriopus californicus (Baker) to serve as a model system for the study of carotenoid physiology. Over the past four decades, the crustacean Tigriopus has become a model organism for studies in ecotoxicology ( Raisuddin et al., 2007 ), phytogeography ( Edmands, 2001 ), and local adaptation. mitosis ( Pereira et al., 2016 ) and nuclear-mitochondrial interactions ( Ellison and Burton, 2008 ). As a result, there is a wealth of physiological and genetic data that will facilitate detailed studies of the molecular mechanisms involved in carotenoid color. However, basic aspects of their pigmentation physiology remain unexplored.
In nature, Tigriopus californicus and other species in the genus Tigriopus typically have a bright orange-red color ( Figure 2 a,c) produced through the deposition of the red keto-carotenoid astaxanthin ( Davenport et al., 2004 ; Goodwin et al., 2004 ; Goodwin and Srisukh, 1949 ; Weaver et al., 2016 ). Like other carotenoid-pigmented animals, the crustacean Tigriopus must ingest carotenoids in its diet to use them as coloring agents. This basic claim is supported by the observation of Davenport et al. ( Davenport et al., 1997 ) that when Tigriopus crustaceans are fed a carotenoid-deficient diet, they lose their characteristic red color and become white. However, to date, the dietary requirements and bioconversion of dietary carotenoids to astaxanthin by Tigriopus californicus have not been the subject of well-controlled studies.
Figure 2. Characteristic color of Tigriopus californicus fed with food (A,C) and nutritional yeast (B). The red larval eyespot present in crustaceans fed both carotenoid-enriched and carotenoid-deficient diets suggests that the production of that eye pigment is independent of diet. (C) The female carries an egg sac that turns from dark gray to red as the embryo develops.
In this study, we investigated the molecular source of pigmentation in T. californicus. We first compared the body color and determined the carotenoid content of animals fed a live microalgae diet and a carotenoid-free yeast diet. We then used carefully controlled precursor carotenoid supplementation feeding experiments to study the bioconversion of dietary carotenoid precursors to astaxanthin by T. californicus . Finally, we tested whether the red color of larval eyespots ( Fig. 2 ) and the color of egg sacs of pregnant females ( Fig. 2c ) were due to the presence of astaxanthin.
METHOD
(a) Crustacean culture
Tigriopus californicus crustaceans collected from the wild in the vicinity of San Diego, California have been reared in our laboratory since 2014 in 10L tanks in filtered artificial seawater (ASW, 32 psu) at 24C for 12 hours of light: 12-hour light-dark cycle and feeding on live microalgae Tetraselmis chuai and Synechococcus spp. This diet contained the algal-derived carotenoids α- and β-carotene, lutein, and zeaxanthin ( Brown and Jeffrey, 1992 ; Guillard et al., 1985 ), which are hypothesized to be substrates for biological conversion to astaxanthin ( Figure 1 ). We call the crustaceans raised under these conditions our ‘reserve population’.
(b) Examination of the dietary origin of red coloration in T. californicus
To test the hypothesis that the characteristic orange-red color of T. californicus depends on the presence of carotenoids in its diet, in 2015 we transferred a sample of approximately 1,000 populations of copepods to carotenoid-free diet with nutritional yeast (Bragg, Santa Barbara, CA). Nutritional yeast diets contain inactive dry yeast and a B complex vitamin blend but lack carotenoids. We grow these crustaceans in 15L containers with opaque walls and lids to prevent algae growth under gently aerated conditions; Change the water and mix the crustaceans from separate containers about every three months. We call crustaceans grown under these conditions ‘yeast-fed copepods’. We sampled 10 adult crustaceans from the population three times and 10 yeast-feeding crustaceans three times, then assessed color by eye and tested for the presence of carotenoids in their bodies as described. in part (f) below.
(c) Qualitative carotenoid analysis of red eye spots
To determine whether the red color of larval eyespots is carotenoid-based, we dissected the cephalosome containing the eyespots from 10 yeast-fed adult crustaceans. All 10 dissected eyespots were pooled in one tube and the 10 corresponding bodies pooled in a separate tube were then processed and analyzed for carotenoids as described below.
(d) Maternal astaxanthin deposition for embryonic development
In a separate experiment, we sought to determine whether the red color of mature egg sacs associated with pregnant females in a colony is carotenoid based. We carefully removed the red egg sacs from three pregnant females using a fine needle under the dissecting scope and processed each clutch individually for carotenoid analysis as described below.
(e) Bioconversion to astaxanthin from carotenoid supplementation of yeast-fed crustaceans
To robustly test the bioconversion of dietary carotenoids to astaxanthin by T. californicus, yeast-fed crustaceans were supplemented with β-carotene, lutein, zeaxanthin, and canthaxanthin. We chose these four carotenoid precursors for the feeding experiments because they occupy various positions in the putative pathway used by crustaceans to produce astaxanthin ( Fig. 1 ). Stock solutions of β-carotene, lutein, zeaxanthin, and canthaxanthin were made from water-soluble carotenoid granules (DSM, Basel, Switzerland) in ASW and diluted to an active carotenoid concentration of 2 μg/mL. Each carotenoid supplement contains only the carotenoids listed, except lutein supplements. Of the total carotenoid content, 90.6% is pure lutein, but zeaxanthin accounts for 7.8%. Therefore, the “lutein” supplement contains 1.984 μg/mL lutein and 0.016 μg/mL zeaxanthin. For each carotenoid supplementation group, 10 adult crustaceans were placed in 5 mL of carotenoid solution in each well of a six-well plate (n = 6 for each supplementation group) with 0.75 mg nutritional yeast as food for 48 hours, then processed for carotenoid analysis (see below).
(f) Carotenoid analysis
After each experiment, crustaceans were placed into fresh ASW to clear the gut contents, then rinsed with deionized water, dried and – for bioconversion experiments – weighed to the nearest zero. 01 mg. The mass from one sample from the β-carotene supplemented group was not recorded, and one sample from the zeaxanthin supplemented group was destroyed before carotenoid analysis.
Carotenoids were extracted from copepods by sonication in 500 μL HPLC-grade acetone in a 1.7 mL microcentrifuge tube for 10 s at 10W; We then capped the tubes with nitrogen gas and incubated them overnight at 4o C in the dark. Samples were centrifuged at 3,000 g for 5 min, the supernatant was removed to a new tube and evaporated to dryness at 40C under vacuum, then resuspended in 50 μL of acetone. Carotenoids were separated using a Shimadzu HPLC system from a 40 µL pump onto a Sonoma C18 column (10 µm, 250 x 4.6 mm, ES Technologies) equipped with a C18 protective cartridge. We used mobile phases A) 80:20, methanol:0.5 M ammonium acetate, B) 90:10, acetonitrile:H 2 O and C) ethyl acetate in a cubic linear gradient as follows: 100% A to 100% B over 4 min, then to 80% C: 20% B for 14 min, back to 100% B over 3 min, and back to 100% A over 5 min and hold for 6 min ( Wright et al., 1991 ). Total run time is 32 minutes at a flow rate of 1 mL/min. Absorbance was measured at 450 nm using a UV/VIS detector. Carotenoids are identified and quantified by comparison with authentic (company) standards. Astaxanthin concentrations were normalized to crustacean dry weight.
(g) Statistical analysis
We tested for differences in the amount of astaxanthin produced by crustaceans from each group using ANOVA and evaluated pairwise comparisons between groups using the Tukey HSD post hoc test.
Figure 3. Astaxanthin content of T. californicus Copepoda after addition of upstream carotenoid precursor astaxanthin for 48 hours. Least squares means and standard error bars are shown, and significantly different means are indicated by separate letters.
RESULT
Origin of the red diet of T. californicus
The main carotenoid found in copepod populations was free astaxanthin (mean ± se; 49.38 ng copepod -1 ± 2.19). Small amounts of mono- and di-esterified astaxanthin were detected accounting for 3.22% and 8.83% of the total carotenoid content, respectively. We found that when crustacean populations were switched to a yeast-free diet without carotenoids, they lost their characteristic orange-red color and appeared transparent ( Figure 2b ). Biochemical analysis showed that small measurable amounts of astaxanthin were detected in yeast-eating crustaceans (mean ± s.e.; 0.54 ng copepods -1 ± 0.016).
Astaxanthin analysis of red eye spots
We detected no carotenoids in the red eyespot-enriched fraction of yeast-fed Tigriopus californicus. Analysis of the corresponding body part revealed astaxanthin concentrations similar to those from yeast-fed crustaceans in the previous experiment (0.3 ng copepod -1).
Astaxanthin analysis of red egg sacs
We found that the red egg sacs of the pregnant female population contained astaxanthin (mean ± se; 10.53 ng egg sac -1 ± 3.31).
Biotransformation of carotenoids added to the diet
We found that T. californicus copepods from each carotenoid supplementation group accumulated astaxanthin after 48 h when none was present in their diet ( Fig. 3 ). The amount of astaxanthin metabolized depends on the specific carotenoid supplemented (mean astaxanthin in μg/mg crustacean dry mass ± s.e.); zeaxanthin (0.99 ± 0.11) > canthaxanthin (0.90 ± 0.12) > β-carotene (0.38 ± 0.06) > lutein (0.21 ± 0.02). However, the amount of astaxanthin produced was not significantly different between the zeaxanthin and canthaxanthin groups (difference, 95% confidence interval: −0.09 μg/mg, −0.41 to 0.23, p= 0 ,91) or between the β-carotene and lutein groups (difference, 95 % CI: −0.17 μg/mg, −0.5 to 0.14, p= 0.49). Post hoc pairwise comparisons showed that crustaceans supplemented with zeaxanthin and canthaxanthin produced significantly more astaxanthin than crustaceans fed β-carotene or lutein ( p < 0.001). In each supplement group, only free astaxanthin and additional carotenoids were detected. It is possible that large amounts of intermediate carotenoids were present in the samples but were not detected in our system.
Discussions
In this study, we conducted carefully controlled precursor/product assays to document the bioconversion of dietary carotenoids to the red ketocarotenoid astaxanthin in the marine crustacean T. californicus . When they are maintained on a yeast diet that does not provide carotenoids, the crustaceans become transparent with no hint of red or yellow. Biochemical analysis confirmed that these animals were essentially deficient in carotenoids in their tissues. When we supplemented yeast-fed carotenoid-free crustaceans with β-carotene, lutein, zeaxanthin, or canthaxanthin for 48 h, we observed significant astaxanthin yields ( Figure 3 ). These experiments confirmed that T. californicus requires carotenoid-rich foods to obtain their characteristic orange-red color and that they bioconvert carotenoid precursors from their diet into astaxanthin.
The amount of astaxanthin produced depends on which carotenoid is the precursor for biotransformation ( Figure 3 ). Copepods fed zeaxanthin and canthaxanthin produced more astaxanthin in 48 h than crustaceans fed β-carotene or lutein. This model suggests that the number of oxidation reactions required to convert the added carotenoid precursor to astaxanthin may mediate the rate of astaxanthin production in this species. Canthaxanthin and zeaxanthin require two hydroxylation reactions or two ketolation reactions, respectively, to form astaxanthin; Meanwhile, β-carotene requires 4–7 reactions, and lutein requires 3 reactions and converts α-doradexanthin to β-doradexanthin ( Figure 1 ). Interestingly, similar effects of zeaxanthin supplementation compared to lutein were observed in a study of American Goldfinches, which convert dietary carotenoids to Canary Xanthophyll A and B, and Northern Cardinals. converts dietary pigments to astaxanthin. Both goldfinches and cardinals produced more oxidized pigments and more colorful skin structures when they were fed zeaxanthin than when they were fed lutein ( Mcraw et al., 2014 ).
In nature, crustaceans that feed on micro- and macroalgae ingest relatively large amounts of β-carotene, zeaxanthin and lutein ( Brown and Jeffrey, 1992 ; Buffan-dubau et al., 1996 ; Sigaud-Kutner et al., 2005 ; Takaichi, 2011 ; Wang et al., 2015 ). Bioconversion of dietary carotenoids to astaxanthin has been documented in other crustaceans ( Caramujo et al., 2012 ; Rhodes, 2007 ), crustaceans ( Hsu et al., 1970 ; Tanaka et al., 1976 ) and fish ( Hsu et al., 1972 ) and these authors concluded that this pathway begins with β-carotene. However, in addition to being used as a pigment, β-carotene is also a major precursor for vitamin A synthesis in animals ( Parker, 1996 ), which may cause an allostatic balance between vitamin A production and coloration. identity ( Hill and Johnson, 2012 ). Additionally, our results suggest that T. californicus can utilize multiple carotenoids as substrates for bioconversion to astaxanthin depending on which carotenoids are available in their diet and/or vitamin requirements A of the body. Zeaxanthin is unlikely to provide vitamin A, and we found that copepods fed this precursor produced significantly more astaxanthin than copepods supplemented with β-carotene. Although we did not detect any intermediates along the proposed biotransformation pathway, our results demonstrate that T. californicus uses zeaxanthin as a substrate for astaxanthin production. It is possible that zeaxanthin is the beginning of a more efficient bioconversion pathway for astaxanthin production in T. californicus. Future experiments analyzing large numbers of crustaceans over shorter sampling periods may identify intermediate carotenoids and help resolve which astaxanthin biotransformation pathway(s) is used by T. californicus .
It is unclear from this study whether T. californicus uses lutein as a substrate to produce astaxanthin because lutein supplements also contain small amounts of zeaxanthin. Lutein is also common in marine phytoplankton, and some marine animals are thought to preferentially use lutein as a substrate for biological conversion to astaxanthin and other ketocarotenoids. Hsu ( Hsu et al., 1972 ) and Katayama ( Katayama et al., 1973 ) have shown that goldfish ( Carassius auratus ) are capable of using lutein as a precursor to astaxanthin. However, this biotransformation pathway requires the isomerization of α-doradexanthin to β-doradexanthin, another transformation process shown to be unlikely in fungi, plants, and other marine animals ( Matsuno et al., 1999 ; Ohkubo et al., 1999 ). .
The red color of the eyespot does not depend on diet; Tigriopus crustaceans grown with algae or yeast all have red eyes ( Figure 2 ). We found that the bright red eyespot color of T. californicus did not come from astaxanthin or any other carotenoid that we could detect, and that the trace astaxanthin content of yeast-fed crustaceans was not within eye. These results clarify that the red eye color is not due to astaxanthin but may be due to the visual pigment rhodopsin which can use 3-hydroxyretinal as a chromophore (Vogt, 1983, 1984; Cronin, 1986).
We found that females deposit astaxanthin for developing embryos, supporting previous reports of this ketocarotenoid occurring in the egg sacs of other Tigriopus species ( Goodwin and Srisukh, 1949 ). It has been proposed that deposition of astaxanthin into the developing egg provides photoprotection of the embryo from solar ultraviolet radiation ( Dethier, 1980 ). Thang et al. (2014) have shown that experimental UV exposure reduces the likelihood of successful hatching of Paracyclopina nana larvae, although the specific role of carotenoids may ensure survival after exposure to ultraviolet rays are still unclear.
Studies on the genetic structure and physiological mechanisms involved in carotenoid metabolism in animals have only recently begun. The gene responsible for the ketolation of dietary yellow carotenoids in birds – known as the red gene – was discovered independently by Lopes et al (2016) and Mundy et al (2016) . This gene encodes the cytochrome P450 oxidoreductase enzyme, CYP2J, whose sequence motifs imply subcellular localization to the mitochondria. The red gene in birds is highly likely to be conserved in all animals that produce red carotenoids from yellow carotenoids ( Lopes et al., 2016 ). Determination of the physiological mechanisms and cellular locations involved in the hydroxylation and ketolation of carotenoids in crustaceans awaits future research.
View ORCID ProfileRyan J. Weaver, Paul A. Cobine, Geoffrey E. Hill