Astaxanthin and Lutein Compete for Accumulation Into the Middle Silk Gland Via Yellow Cocoon Gene (C)-Dependent Control and Produce a Red Cocoon of Bombyx Mori

Journal of Insect Biotechnology and Sericology 83, 1-11 (2014)

Astaxanthin and lutein compete for accumulation into the middle silk gland via Yellow cocoon gene (C)-dependent control and produce a red cocoon of Bombyx mori

Masashi Yuasa1＊, Akitoshi Kitamura2＊, Takashi Maoka3, Takashi Sakudoh4, Toru Shimada1 and Kozo Tsuchida4＊＊

1 Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo, Tokyo 113-8657, Japan 2 Life Science Division, Fuji Chemical Industry Co., Ltd, Nakaniikawa, Toyama 930-0397, Japan 3 Research Institute for Production and Development, Sakyo, Kyoto 606-0805, Japan 4 Division of Radiological Protection and Biology, National Institute of Infectious Diseases, Shinjuku, Tokyo 162-8640, Japan (Received February 20, 2014; Accepted June 19, 2014)

Newly ecdysed fifth instar larvae of the Bombyx mori strain N4 were divided into three groups and fed one of three different diets until they began spinning a cocoon. One group was fed the basal diet, in which carotenoids consisted mainly of lutein (53.3 μg/g wet) and β-carotene (14.4 μg/g wet), while the two other groups were fed the basal diet supplemented with astaxanthin from AstaREAL® or capsanthin from paprika-red®. High perfor- mance liquid chromatography was performed to identify the carotenoids in lipophorin, the middle silk glands, and the cocoons. Lutein, as well as astaxanthin and capsanthin, was accumulated in the cocoons after feeding with the astaxanthin- or capsanthin-supplemented diet. Quantitative analysis of the strain N4 and c10 demonstrated competitive accumulation in the cocoon between lutein and astaxanthin. In fifth instar larvae of the white cocoon producer, the strain c05 fed a diet containing lutein and astaxanthin, however, they produced only white cocoon. These results indicated that lutein and astaxanthin transport share common pathways, and that the delivery sys- tem in the silkworm can mediate the transfer and accumulation of carotenoids which are not derived from mul- berry leaves, such as astaxanthin and capsanthin. Key words: Silkworm, Red cocoon, Carotenoids, Astaxanthin, Capsanthin

the accumulation of specific carotenoids into the middle INTRODUCTION silk gland. Carotenoids are lipid-soluble pigments comprising two The strain c10 (c10) larva that has the dominant C al- diterpenoid units joined tail to tail. The xanthophylls lutein, lele of the C gene accumulates lutein in the middle silk astaxanthin, zeaxanthin, and capsanthin are the oxidized gland and produce yellow cocoon, while the strain c05 derivatives of β-carotene. Due to their hydrophobicity, (c05) larva that has homozygous for the recessive +C al- they are located in lipophilic sites of cells, such as mem- lele of C gene does not accumulate lutein and produces branes bilayers, and seem to play roles in determining the white cocoon. Both strains do not accumulate β-carotene strength and fluidity of membranes (Britton, 1995). in the middle silk gland, because they have homozygous The genetics of the silkworm Bombyx mori clearly in- for the recessive +F allele of F gene which controls dicates the presence of carotenoid molecule-specific deliv- β-carotene uptake in the middle silk gland. As c05 larva ery. As the silkworm has been cultured for over 5000 has homozygous for both the +C allele and the +F allele, years, numerous mutants have been noted. Among these the larva produces white cocoon. are mutants in cocoon color, such as Y (Yellow blood), C We examined the hypothesis that the molecular speci- (Yellow cocoon), and F (Flesh cocoon) genes (Tazima, ficity of carotenoid delivery requires at least two compo- 1964). The yellow, orange, and flesh cocoons are due to nents: a tissue-specific carotenoid-binding protein in the carotenoids, and mutants with white cocoons are defective cytosol and a non-internalizing lipophorin receptor/trans- in one of the steps involved in transporting carotenoids porter on the surface of the cells of the middle silk gland from the midgut lumen to the middle silk gland where (Sakudoh and Tsuchida, 2010). We have identified carot- they are incorporated into the cocoon (Nakajima, 1963). enoid-binding protein (CBP), which is involved in the The middle silk gland accumulates specific carotenoids transport of carotenoids across the enterocyte and the epi- from the hemolymph lipophorin that binds several differ- thelium of the middle silk gland (Tabunoki et al., 2002; ent carotenoids with lipids (Chino et al., 1969; Tsuchida Tsuchida et al., 2004; Sakudoh et al., 2007). CBP has et al., 1998). The different colored cocoons are caused by broad specificity for carotenoids and binds both lutein and  β-carotene. This is the first study to report that the cellu- * These authors contributed equally to this work. lar carotenoid transport is facilitated by the participation **To whom correspondence should be addressed. of a specific cellular transporter. The C locus associated Email: [email protected] membrane protein homologous to a mammalian high den- 2 Yuasa et al. sity lipoprotein receptor 2 (Cameo2) and Scavenger recep- 300 mL of boiling water and mixed well. The mixed diet tor class B member 15 (SCRB15), may function as a non- was steamed for 15 min, again mixed and allowed to cool. internalizing lipophorin receptor and control the cellular AstaREAL® 50F (AstaREAL) (Fuji Chemical Industry uptake of carotenoids in the middle silk glands (Sakudoh Co., Toyama, Japan), which was used as the source of et al., 2010, 2013). Cameo2 and SCRB15 are single-chain astaxanthin, is the oil fraction extracted from the unicellu- transmembrane glycoproteins that belong to the scavenger lar microalga, Haematococcus pluvialis and contains 5% receptor class B type I (SR-BI) family. astaxanthin (w/w) (Tominaga et al., 2012). Paprika-red Using transgenic expression of three genes (Y, C, and F N15A (Paprika-red) (Yaegaki Bio-Industry, Inc., Himeji, genes) based on the binary GAL4/upstream-activating se- Japan) was used as the source of capsanthin. Carotenoid quence (UAS) system (Imamura et al., 2003), we showed supplementation was performed by mixing the steamed that Y, C, and F genes encode CBP, Cameo2, and SilkMate PM diet (the basal diet) with the corresponding SCRB15, respectively (Sakudoh et al., 2007, 2010, 2013). amount of carotenoids to reach a final concentration of Although these data provide the first evidence of discrimi- 100 μg/g astaxanthin (the A diet) or 75 μg/g capsanthin nating carotenoid molecules in lipophorin, how certain ca- (the C diet). All diets were kept at 4°C. rotenoids were preferentially accumulated over other carotenoids in the middle silk gland from the circulating Experimental design of astaxanthin and capsan- hemolymph lipophorin remained to be elucidated. thin feeding In this study, to better understand the mechanism of se- All feeding experiments with the supplemented diet lective carotenoid uptake, we examined whether the deliv- were performed on fifth instar larvae. N4 was fed on the ery system in the silkworm can mediate the transfer and basal diet from newly hatched larval stage to the fourth accumulation of astaxanthin and capsanthin, which are not molt stage. The larvae of c10 and c05 were fed on mul- derived from mulberry leaves. Accumulation of astaxan- berry leaves from newly hatched larval stage to the third thin and capsanthin into the cocoon would suggest that li- molt stage and switched to the basal diet during the fourth pophorin and CBP are also bound with affinities against instar larval stage. Larvae and pupae were reared at 25°C these carotenoids. These findings provide useful informa- under 12 h light /12 h dark cycle. tion for clarifying the discrimination of carotenoid mole- Three groups of 30 fifth instar larvae of N4 each were cules on the cell surface of the middle silk gland. fed the diets in disposable lunch boxes (200 × 150 × Furthermore, the cocoon with accumulation of astaxanthin 25 mm). One group was fed the basal diet, while the oth- or capsanthin may change color from yellow to red, thus er groups were fed the AstaREAL- or Paprika-red-supple- facilitating the production of various colored silks. To our mented diet. At the beginning of the fifth larval instar, all knowledge, no previous studies have examined the distri- diets were sliced into small pieces and supplied to larvae, bution of astaxanthin or capsanthin in various tissues after with the diet changed each day. When using c10 or c05, feeding. In addition, this is the first report of high-perfor- larvae were divided into two groups. One group was fed mance liquid chromatography (HPLC) analyses of astax- the basal diet, and the other was fed the A diet. anthin or capsanthin in the cocoon. Carotenoid extraction from the diets A small amount of ethanol was added to 2 g of mulber- MATERIALS AND METHODS ry leaves or the basal diet and ground in a glass mortar. Silkworms Samples were transferred into glass centrifuge tubes and The N4 strain of B. mori (N4) has been preserved at vortexed for 30 s with 5 g of glass beads. After centrifu- the National Institute of Infectious Diseases, Tokyo, Ja- gation at 1000 ×g for 5 min, the supernatant was collected pan. The larvae of c10 and c05 strains (c10, c05) were into a 100-mL measuring flask. Extraction with 5 mL of kind gifts from the silkworm stock center of Kyushu Uni- ethanol containing 10 μg/mL of butylhydroxytoluene (BHT) versity, Fukuoka, Japan. N4 has the genotype [Y, C, F] was repeated three times and extractions with 5 mL of ac- (Y/Y, C/C, F/F), c10 has the genotype [Y, C, +F] (Y/Y, etone were repeated until the residue became colorless. C/+C, +F/+F), and c05 has the genotype [Y, +C, +F] (Y/Y, All of the collected extracts were pooled and made up to +C/+C, +F/+F). 100 mL with acetone in the measuring flask. Five millili- ter samples of the extract were removed into glass tubes Diets and evaporated. The residue was dissolved with 5 mL of The silkworm diets were purchased from a commercial diethyl ether, after which 5 mL of 5% potassium hydrox- source and supplied as powder (SilkMate PMTM and ide in methanol was added and vortexed well. After keep- SilkMate 2MTM, Nihon Nosan Kogyo Co., Yokohama, Ja- ing the tubes at room temperature for 3 h, 1 mL of pan). One hundred grams of diet powder was added to deionized water was added and centrifuged at 1000 ×g for Red cocoon with astaxanthin 3

5 min. The upper phase was transferred to a new glass repeated until the residue became colorless. All of the col- tube, and the lower phase was extracted twice with 5 mL lected solutions were pooled, and 1 g of sodium sulfate of diethyl ether:hexane (1:1). After the extract was evapo- and 5 mL of petroleum ether were added, then vortexed. rated, the residue was dissolved in 5 mL of acetone, fil- After centrifugation at 1000 ×g for 10 min, the upper tered through 0.45-μm PTFE membrane (Sigma-Aldrich, phase was collected. The extraction with petroleum ether St. Louis, MO), and subjected to HPLC. was repeated three times and all extracts were pooled. The extract was dried over anhydrous sodium sulfate and High-density lipophorin (HDLp) preparation and evaporated. The residue was dissolved in acetone and carotenoid extraction from HDLp used for carotenoid analysis by HPLC. The larval hemolymph was isolated from the fifth instar larvae on day 4 according to the method described by Carotenoid extraction from the cocoon Yokoyama et al. (2013). The hemolymph was centrifuged Then 100-mg samples of minced cocoon were added to at 800 ×g for 5 min to remove hemocytes. Then 8.9 g of 3 mL of distilled water and 2 g of glass beads in glass potassium bromide (KBr) was added to the supernatant, tubes, then heated at 90°C for 10 min. The water was col- and the volume was adjusted to 20 mL with a bleeding lected in new tubes and 2 mL of 100% ethanol and 0.5 g solution (20 mM sodium phosphate, pH 6.8, 150 mM of sodium sulfate decahydrate were added. The solution NaCl, 5 mM ethylenediaminetetra acetic acid, 1 mM glu- was then extracted with 3 mL of petroleum ether. The pe- tathione and 1 mM 4-2-aminoethyl benzenesulfonylfluo- troleum ether fraction was collected. The cocoon residue ride). The solution was transferred to 36.2-mL OptiSealTM was added to 2 mL of 80% ethanol containing 10 μg/mL centrifuge tubes (Beckman Coulter, Brea, CA) and over- of BHT and kept at 80°C for 10 min. The solution was laid with the bleeding solution. The tubes were centri- collected and the ethanol was evaporated off by purging fuged at 4°C and 50,000 rpm for 4 h in a VTi 50 rotor under nitrogen gas. Next, 2 mL of 100% ethanol and 0.5 g (Beckman Coulter). After centrifugation, HDLp and lipid of sodium sulfate decahydrate were added to the residue transfer particle (LTP) formed two yellow bands in the ul- and extracted with 3 mL of petroleum ether. After centrif- tracentrifuge tubes. The fractions from the middle yellow ugation at 1000 ×g for 5 min, the petroleum ether phase band, which contained mainly HDLp, were pooled. Pro- was collected. The extraction with petroleum ether was tein concentration of HDLp was determined using the repeated three times. All of the collected extracts were BCA kit (Thermo Scientific Inc., Rockford, IL). pooled and evaporated. The residue was dissolved in 3 mL Next, 500-μg aliquots of HDLp in bleeding solution of acetone and filtered through 0.45-μm PTFE membrane. were used for extraction. The sample was added to 3 mL The solution was subjected to HPLC. of distilled water and 2 mL of ethanol containing 10 μg/ mL BHT and vortexed for 30 s, after which 0.2 g of sodi- Carotenoid analysis by HPLC um sulfide decahydrate was added to the sample. Then, The carotenoids were analyzed by comparing their re- 3 mL of petroleum ether was added to the sample and the tention times and absorption spectra with the following supernatant was removed after centrifugation at 1000 ×g reference compounds: lutein, α-carotene, β-carotene and for 5 min. This extraction with petroleum ether was re- zeaxanthin (Wako Pure Chemicals Industries Ltd., Tokyo, peated three times. The supernatant was pooled and evap- Japan), and astaxanthin (Sigma-Aldrich). orated, and the residue was dissolved in 5 mL of acetone. Twenty-μL aliquots of samples were subjected to HPLC This sample was subjected to HPLC. (Waters 2695 Separation Module: Waters Co., Milford, MA). A reverse-phase column [YMC carotenoid 5 μm (4.6 Carotenoid extraction from the middle silk gland × 250 mm); Waters Co.] was used under the following Then 500-mg samples of middle silk glands were ho- conditions: temperature, 25°C; flow rate, 1 mL/min; mo- mogenized in a mortar with distilled water and a small bile phase, A: methanol, B: t-butylmethylether, C: 1% (v/v) amount of sandstone. A small amount of ethanol contain- aqueous phosphoric acid; a 15-min linear gradient from ing 10 μg/mL BHT was added to the mortar and the solu- 81% A, 15% B, 4% C to 66% A, 30% B, 4% C; an 8-min tion was collected. The residue was transferred into a linear gradient to 16% A, 80% B, 4% C; a 4-min hold at glass centrifuge tube and 3 mL of distilled water was add- 16% A, 80% B, 4% C, then back to 81% A, 15% B, 4% ed. The tube was incubated at 90°C for 10 min with vor- C; and an 8-min hold at 81% A, 15% B, 4% C. The ab- texing at constant intervals. The solution was collected, sorbance was recorded at 450 nm. and 3 mL of 100% ethanol was added to the residue and incubated at 90°C for 10 min with vortexing at constant HPLC analysis of carotenoids in Paprika-red and intervals. The solution was collected. These extractions cocoon from the silkworm fed the C diet with distilled water, 80% ethanol, and 100% ethanol were Carotenoids in Paprika-red were extracted with metha- 4 Yuasa et al. nol. The extract was saponified with 5% KOH/methanol signment was validated by comparison of the retention at room temperature. Then unsaponifiable matter was ex- times with those of authentic standard carotenoids (lutein, tracted with ether/hexane (1:1, v/v) by addition of water. α-carotene, β-carotene and zeaxanthin, and astaxanthin). The organic phase was evaporated to dryness and was As shown in Fig. 1, several minor peaks were observed. submitted to HPLC analyses. Carotenoids of the cocoon Peaks from 1 to 8 showed the expected retention times for from silkworm fed the C diet were extracted as the same 3’-dehydrolutein, lutein isomer/derivative-1, lutein isomer/ method described above. derivative-2, 1,3-cis lutein, 9-cis lutein, unknown, 1,3-cis- Carotenoids in Paprika-red and the cocoon from silk- β-carotene, and 9-cis-β-carotene, respectively. However, worm fed the C diet were analyzed by normal phase HPLC they were not verified carotenoids identification, because system. HPLC was performed with a Hitachi L-6000 in- we did not use their authentic standard of carotenoids. telligent pump and an L-4250 UV-VIS detector set at And these eight carotenoids were mostly not detectable in 450 nm. The column used was a 250 × 4.6 mm i.d., 5 μm the hemolymph, the middle silk gland, and the cocoon. Cosmosil 5SL-II (Nacalai Tesque, Kyoto, Japan) with Table 1 shows the compositions of carotenoids in fresh acetone:hexane (3:7) as a solvent at a flow rate of 1.0 ml/ mulberry leaves, SilkMate PM (the basal diet), and min. The reference compounds, capsanthin, capsorubin, SilkMate 2M. Carotenoid analysis indicated that lutein and cryptocapsin, and β-cryptoxanthin, were prepared from β-carotene were enriched in fresh mulberry leaves. Lutein Paprika-red oleoresin. Structure and purity of these com- was present at 110.1 μg/g, and the level of β-carotene was pounds were checked by 1H-NMR and MS spectral data. 70.3 μg/g. Zeaxanthin and α-carotene were present as mi- nor components at concentrations of 1.2 μg/g and 0.8 μg/g, Transfer efficiency (% of a given dose) respectively. The carotenoids compositions of the com- Transfer efficiencies of carotenoids from the diet to the mercially available artificial diets [SilkMate PM (the basal cocoon were expressed as the amount of the carotenoid diet), SilkMate 2M] were also analyzed. Carotenoids were found in the cocoons (μg/g) divided by that in the diet extracted from SilkMate PM (the basal diet) which had (μg/g). already been steamed and allowed to cool. Lutein and β-carotene were found, but the levels of both carotenoids were lower by as much as half of the respective concentra- RESULTS tion in fresh mulberry leaves, and a decrease in β-carotene Compositions of carotenoids in fresh mulberry was remarkable rather than that in lutein. The level of lu- leaves, SilkMate PM (the basal diet), and SilkMate tein was 53.3 μg/g and that of β-carotene was 14.4 μg/g in 2M the wet basal diet. α-Carotene was found at a much high- Fig. 1 shows a representative HPLC profile of mulberry er concentration in the basal diet (5.5 μg/g wet basal diet) leaf extract. Two large peaks correspond to the standards than in fresh mulberry leaves (0.8 μg/g). Small amounts translutein and β-carotene; one of the other minor peaks of carotenoids were found in SilkMate 2M. was α-carotene, which eluted at around 24 min. Peak as-

Fig. 1. HPLC-profile of mulberry leaf extract. The carotenoids were analyzed by comparing their retention times and absorption spectra with the following reference compounds: lutein, α-carotene, β-carotene and zeaxanthin. Peaks from 1 to 8 showed the expected retention times for 3’-dehydrolutein, lutein isomer/ derivative-1, lutein isomer/derivative-2, 1,3-cis lutein, 9-cis lutein, unknown, 1,3-cis-β-carotene, and 9-cis-β-carotene, respectively. Red cocoon with astaxanthin 5

Table 1. Carotenoid concentrations of three diets, mulberry leaves, SilkMate PM (Basal diet), and SilkMate 2M (μg/g wet) Mulberry SilkMate PM Carotenoids SilkMate 2M leaves (Basal diet) Lutein 110.1 53.3 8.7 Zeaxanthin 1.2 1.7 0.2 α-Carotene 0.8 5.5 0.7 β-Carotene 70.3 14.4 6.2 Data are values of single analysis

N4 fed the basal diet and the A diet As shown in Fig. 2, the reddish color of the middle silk gland and the cocoon increased intensity with increases in the amount of AstaREAL in the basal diet. The optimum Fig. 2. Color change of the middle silk glands and the co- content of AstaREAL in the basal diet was determined us- coons of N4 by astaxanthin concentration in the basal diet. ing N4. Fifth instar larvae were fed the basal diet supple- Scale bar: 1 cm. mented with 0.1, 0.25, 0.5, 1, or 2 mg/g wet of AstaREAL; the amounts of astaxanthin in 1 g wet diet were equiva- lent to 5, 12.5, 25, 50, or 100 μg, respectively. All diets supplemented with different amounts of AstaREAL also supported good and synchronized growth. On day-4 of fifth instar larvae after 5 days of feeding, no difference in body weight was noted between the larvae fed the basal diet and those fed the A diet containing 100 μg of astax- anthin derived from AstaREAL. The results indicated that supplementation with 2 mg of AstaREAL in 1 g of the basal diet (the A diet) had no inhibitory effect on feeding or growth. We examined the hemolymph color through the skin of N4 larvae. As shown in Fig. 3A, fifth instar N4 larvae were fed the basal diet, the skin was light yellow in color. While fifth instar N4 fed the A diet became redder skin andthe body became reddish in color after 4 h of feeding. The hemolymph of day-4 fifth instar larvae was collected after 5 days of feeding. HDLp was fractionated after KBr den- sity gradient ultracentrifugation. The coloration of HDLp Fig. 3. Color change of the larvae, lipophorin, and eggs af- from larvae fed the A diet turned a deep orange color, but ter feeding with the basal diet and the A diet. (A) Fifth instar larvae of N4, which have genotype [Y, C, F], after 5 days that fed the basal diet was still yellow (Fig. 3B). Further- feeding of the basal diet (left) or the A diet (right). (B) KBr more, the egg color shifted from yellow to a more intense density gradient ultracentrifugation of hemolymph from day-4 red color with AstaREAL supplementation (Fig. 3C). fifth larvae fed the basal diet (left) or the A diet (right). Arrows The carotenoids were extracted from the HDLp and the show the positions of HDLp and LTP in the ultracentrifugation tubes. (C) Eggs of N4 adults laid after fifth instar larvae were cocoon to determine carotenoid compositions by HPLC fed the basal diet (left) or the A diet (right) for 5 days. Scale (Table 2). Ninety % of total carotenoids in the hemolymph bars: 1 cm. were found in HDLp. In the HDLp of larvae fed the basal diet, lutein was the major carotenoid, and zeaxanthin, present at 219.70 μg/g cocoon, and the level of β-carotene α-carotene, and β-carotene were also detected as minor was 4.75 μg/g. Zeaxanthin and α-carotene were present at components. One milligram of HDLp protein contained levels of 4.33 μg/g and 0.50 μg/g, respectively (Table 2). 7.95 μg of lutein, 0.19 μg of zeaxanthin, 0.05 μg of In the HDLp and the cocoon of larvae fed the A diets, α-carotene, and 0.11 μg of β-carotene. Although lutein and astaxanthin and lutein were found as major carotenoids in β-carotene were present as major carotenoids in the basal the HDLp and the cocoon, and the astaxanthin level was diet, the concentration of β-carotene in HDLp was mark- more than double that of lutein in both the HDLp and the edly lower than that of lutein. The concentrations of ca- cocoon. Amounts of astaxanthin and lutein in the HDLp rotenoids in the cocoons were determined. Lutein was were 11.86 μg/mg or 5.60 μg/mg respectively, and that in 6 Yuasa et al.

Table 2. Carotenoid concentrations in HDLps and cocoons of N4 after 5 days of feeding with the basal diet or the A diet. HDLp Cocoon Carotenoids (μg/mg protein) (μg/g) Basal diet A diet Basal diet A diet Astaxanthin – 11.86 ± 0.70 – 347.11 ± 0.00 Lutein 7.95 ± 0.98 5.60 ± 0.36 219.70 ± 1.21 158.41 ± 0.87 Zeaxanthin 0.19 ± 0.04 0.12 ± 0.01 4.33 ± 0.03 3.38 ± 0.02 α-Carotene 0.05 ± 0.00 0.03 ± 0.01 0.50 ± 0.06 0.37 ± 0.04 β-Carotene 0.11 ± 0.04 0.07 ± 0.01 4.75 ± 0.30 4.04 ± 0.37 Bars in table: not analyzed. Data are mean ± SD of three independent experiments. the cocoon were 347.11 μg/g and 158.41 μg/g, respective- Table 3. Carotenoid concentrations of the C diet and the co- ly (Table 2). As the AstaREAL contained 5% astaxanthin, coons of N4 after feeding with the C diet C diet Cocoon the diet supplemented with 2 mg/g AstaREAL contained Carotenoids 100 μg/g astaxanthin (the A diet). This corresponds to (μg/g of wet diet) (μg/g) around double the concentration of lutein in the A diet. Capsanthin* 75.2 50.7 Lutein 53.3 240.6 N4 fed the C diet Zeaxanthin 25.0 77.7 Cryptocapsin* 15.5 ND As the source of capsanthin, 1 mg of Paprika-red was Capsorubin* 9.9 ND added to 1 g of wet basal diet (the C diet) and fifth instar β-Cryptoxanthin* 7.5 5.2 N4 larvae were fed the C diet. The carotenoid composi- α-Carotene 5.5 ND tions of the C diet and the cocoon from silkworm fed the β-Carotene 24.2 7.9 C diet are shown in Table 3. Paprika-red contains a wide range of different carotenoids, and capsanthin and zeaxan- ND: not detectable. Date are values of single analysis. thin were enriched. In the C diet, capsanthin was present *: Four carotenoids were supplemented from paprika-red 15 and at 75.2 μg/g, while lutein, zeaxanthin and β-carotene were were not detected in the basal diet. 53.3 μg/g, 25.0 μg/g, and 24.2 μg/g, respectively. Crypto- capsin, capsorubin, and β-cryptoxanthin were present at black striped skin. When larvae of both strains were 15.5 μg/g, 9.9 μg/g, and 7.5 μg/g, respectively. raised on the diet A, the larvae of c10 clearly accumulat- The carotenoids were extracted from the cocoon to de- ed astaxanthin with lutein in the middle silk gland and the termine carotenoid compositions by HPLC. Lutein, zea- cocoons. While the middle silk gland and the cocoon of xanthin, and capsanthin were found as major carotenoids c05 showed no such color change; the middle silk gland in the cocoons. β-Carotene was also detected as minor of this strain was colorless and the cocoon was white, al- components. In accordance with their minor occurrence in though the body color and the hemolymph of both strains Paprika-red, the level of β-cryptoxanthin in the cocoons changed to a reddish color. As controls, c10 silkworms was lower. Although the C diet contained approximately made yellow cocoons and c05 made white cocoons when the same amount of β-carotene (24.2 μg/g) as zeaxanthin the larvae of both strains were fed the basal diet. (25.0 μg/g), the amount of β-carotene (7.9 μg/g) in the co- The carotenoid compositions in HDLps and the co- coons was lower than that of zeaxanthin (77.7 μg/g) (Ta- coons of c10 and c05 were analyzed by HPLC and the re- ble 3). Cryptocapsin, capsorubin, and α-carotene were not sults were shown in Table 4. The levels of HDLp lutein in detected in the cocoons. The cocoon color of N4 fed the c10 or c05 larvae fed the basal diet contained 4.18 μg/mg C diet was yellow to light orange (Fig. 4). protein or 19.36 μg/mg protein, respectively. The lutein levels of HDLp in c10 were lower than those of c05, pre- C10 and c05 fed the basal diet or the A diet sumably because lutein is taken up from the hemolymph The c10 and c05 silkworms were reared on the basal to the middle silk gland more actively in c10 than in c05. diet or the A diet. In this experiment, supplementation Due to the low uptake of carotenoids in c05, lutein and with AstaREAL supported good growth of both strains. To zeaxanthin remained in the hemolymph. Indeed, in c10, determine whether astaxanthin can be transported from large amounts of lutein were accumulated in the middle the midgut lumen to the cocoon via the hemolymph, we silk gland (84.41 μg/g) and cocoon (278.83 μg/g), while examined the changes in color of the larval body, hemo- lutein amounts in the middle silk gland and cocoon of c05 lymph, silk gland, and cocoon (Fig. 5). In the pictures of were 3.30 μg/g and 2.02 μg/g, respectively. The levels of larvae of Fig. 5, c10 larvae have white skin and c05 has HDLp β-carotene in c10 and c05 were 4.27 μg/mg and Red cocoon with astaxanthin 7

3.23 μg/mg, respectively. β-Carotene was transferred from tained around double of the concentration of lutein. the diet to HDLp in the hemplymph of both strains, but did rarely accumulate in the cocoons of either strain. Transfer efficiency The cocoons of the c10 silkworms raised on the A diet The transfer efficiency (TE) was calculated as the value was used for extraction of carotenoids. The astaxanthin of the carotenoid concentration of cocoon devided by that level in the cocoon (376.37 μg/g) was more than double of the diet (Table 5). The TEs of carotenoids were mea- of lutein (161.09 μg/g), and the amount of lutein was de- sured when the larvae of different strains were fed the dif- creased by supplementation with astaxanthin in the basal ferent diets. When N4 and c10 larvae were fed the basal diet. As mention before, astaxanthin in the A diet con- diet, TEs of lutein were 4.12 in N4 and 5.23 in c10. After feeding with the capsanthin-supplemented diet (the C diet), TE of lutein was 4.51 in N4. However, when N4 and c10 larve were fed the astaxanthin-supplemented diet (the A diet), TEs of lutein decreased to 2.97 in N4 and 3.02 in c10, suggesting that competition between lutein and astaxanthin could occur at the step(s) for transport in both strains. TEs of astaxanthin were determined when larvae were fed the A diet that contained 100 μg/g wet diet of astaxan- thin. TEs of astaxanthin were 3.47 in N4 and 3.76 in c10. The TEs of zeaxanthin were 2.54 and 3.00 in N4 and c10, respectively, when larvae of both strains were fed the bas- al diet. The TEs of other xanthophyll carotenoids, such as Fig. 4. Cocoons from N4 larvae fed the basal diet (left) or β-cryptoxanthin and capsanthin, were determined; when the C diet (right) for 5 days. Scale bar: 1 cm. the larvae were fed the C diet, their TEs were low (0.69

Fig. 5. Pictures of the larvae, the hemolymph, and the silk glands from fifth instar on day 4, and cocoons of c10 (left) and c05 (right), which have genotypes [Y, C, +F] and [Y, +C, +F], respectively, fed the basal diet or the A diet. In the picture showing larvae, c10 has white skin, and c05 has black striped skin. Scale bars: 1 cm. 8 Yuasa et al.

Table 4. Carotenoid concentrations of the HDLps, the middle silk glands, and the cocoons of the strain c10 or c05 after feeding with the basal diet, and carotenoid concentrations of the cocoons from c10 after feeding with the A diet. HDLp Middle silk gland Cocoon (μg/mg protein) (μg/g ) (μg/g ) Strain c10 c05 c10 c05 c10 c05 c10 Diet Basal diet Basal diet Basal diet A diet Astaxanthin – – – – – – 376.37 ± 1.84 Lutein 4.18 ± 0.56 19.36 ± 1.95 84.41 ± 32.6 3.30 ± 1.06 278.83 ± 5.43 2.02 ± 0.11 161.09 ± 1.94 Zeaxanthin 0.10 ± 0.03 0.44 ± 0.13 2.00 ± 0.01 0.07 ± 0.00 5.11 ± 0.09 ND 5.59 ± 0.36 α-Carotene 0.05 ± 0.01 0.05 ± 0.01 ND ND ND ND 0.93 ± 0.05 β-Carotene 4.27 ± 0.10 3.23 ± 0.63 0.40 ± 0.11 0.49 ± 0.18 1.00 ± 0.06 0.06 ± 0.02 ND ND: not detectable. Bars in table: not analyzed. Data are mean ± SD of three independent experiments. and 0.67 for β-cryptoxanthin and capsanthin, respective- transport can occur at many steps of digestion, including ly). The TEs of two carotenes, α-carotene and β-carotene, carotenoid uptake into micelles (Tyssandier et al., 2003), were very low in both N4 and c10. These strains showed uptake via the apical membrane of enterocytes, incorpora- significantly impaired carotene transport. These results were tion into CBP, and efflux from the basolateral membrane in agreement with those of previous studies (Nakajima, of enterocytes. In addition, absorbed β-carotene may be 1963). The strain c05 showed little accumulation of any converted to vitamin A in the midgut, but the site of con- carotenoids, so the TEs for all carotenoids were signifi- version has not been confirmed (von Lintig et al., 2001). cantly low. However, no differences were observed between the amounts of lutein (4.18 μg/g) and β-carotene (4.27 μg/g) in the he- molymph of c10 (Table 4). The simplest explanation for DISCUSSION carotenoid transport is that one or more proteins/transport- The transport of dietary carotenoids is a multistep pro- ers are involved in β-carotene transport and that N4 may cess initiated by absorption in the midgut. No data are have impairments of the protein(s) for β-carotene absorp- available regarding carotenoid absorption in insects. In tion or efflux of β-carotene into the hemolymph. The pro- some plants, xanthophylls find in the form of esters or teins involved in β-carotene absorption on the brush border free. Xanthophylls esterified with fatty acids, lutein in of the midgut and secretion on the basolateral membrane marigold flower (Tagetes erecta L.) is more than 95% as are currently unknown. diester (Breithaupt et al., 2002), astaxanthin from H. plu- After 5 days of feeding, a greater accumulation of vialis occurs mainly monoesters (Miao et al., 2006) and astaxanthin was observed in the cocoons of N4 fed the capsanthin in paprika is also esterified (Biacs et al., 1989, diet supplemented with higher concentrations of astaxan- Breithaupt and Schwack, 2000). Nothing is available in thin, while the accumulation of lutein, which was already the information concerning the characterstic xanthophylls present in the basal diet, was diminished by supplementa- in mulberry leaves and artificial diets. Our results detected tion with astaxanthin (100 μg/g wet diet, A diet) (27.9% by HPLC indicate that carotenoids in mulberry leaf and inhibition). Also, in c10, compared with the control co- the basal diet are free form. In general it is thought that coons (feeding the basal diet), feeding the A diet resulted the esterified fatty acids are hydrolysed before absorption in significant decrease of the accumulation of lutein in the of the resulting free xanthophylls in the gut lumen cocoon by 42.3%. The results indicated not only that (Tyssandier et al., 2002). The present study indicates that astaxanthin from the unicellular microalga, H. pluvialis in the hemolymph and the middle silk gland, carotenoids could be absorbed and transported to the cocoon as easily are transported in their free form, and xanthophylls are as that from natural sources, such as lutein from mulberry not esterified by local enzymes even when they enter the leaves, but also that competition occurred between lutein cocoon as depository site. and astaxanthin transport from absorption to uptake in the In B. mori N4, β-carotene concentration in larval HDLp middle silk gland. These results suggest that lutein and was considerably lower than that of lutein. Although the astaxanthin transport share common pathways. amount of β-carotene in the basal diet was about a quarter Capsanthin in the diet was also accumulated into the that of lutein, the amount of β-carotene in larval HDLp cocoons of N4, but the transfer efficiency was very low. was 1/70 that of lutein. Our data suggested that β-carotene Even if the amount of capsanthin in the C diet was great- transfer from the midgut lumen to the hemolymph was er than that of lutein, the levels of capsanthin accumulat- significantly impaired in N4. The decrease of β-carotene ed into the cocoons were less than a quarter of that of Red cocoon with astaxanthin 9

Table 5. Effects of diet, strain, and carotenoid structure on carotenoid transfer efficiencies (TEs). TE of Strains Carotenoids Diets N4 c10 c05 Basal – – – A 3.47 3.76 – C – – – Basal 4.12 5.23 0.04 A 2.97 3.02 – C 4.51 – – Basal 2.54 3.00 0 A 1.99 3.28 – C 3.11 – – Basal – – – A – – – C 0.69 – –

Basal – – –

A – – –

C 0.67 – –

Basal – – –

A – – –

C 0 – –

Basal – – –

A – – –

C 0 – – Basal 0.09 0 0 A 0.07 0.17 – C 0 – – Basal 0.28 0.07 0.004 A 0.33 0 – C 0.33 – – Bars in table: not analyzed. lutein. Lutein transport was not reduced by capsanthin parable, independent of strain, diet, and concentrations of supplementation (75.2 μg/g wet), which may reflect poor carotenoids in the diet. As mentioned above, in c10, both capsanthin absorption compared to lutein or astaxanthin, lutein and β-carotene were present at the same levels in but further investigations are required because the distri- larval HDLp, but the β-carotene level of c10 cocoons was bution in the hemolymph was not analyzed in this study. diminished compared to that of lutein. Thus, the discrimi- Monohydroxycarotenoids, β-cryptoxanthin was also de- nation for β-carotene occurred at the cellular uptake of the tected in the cocoons of N4 fed the C diet, but the con- apical membrane of the middle silk gland of c10. centrations were extremely low. The accumulation of Two carotenoids, lutein and β-carotene, which exhibit other monohydroxycarotenoids, crypocapsin in the cocoon different structural characteristics, use different pathways is negligible. during transport except that in the hemolymph with lipo- The transfer efficiency is affected by numerous factors, phorin and cellular transport with CBP. Lutein and zea- but the results of this study indicated that the transfer effi- xanthin are dihydroxycarotenoids, and astaxanthin is ciencies of lutein, zeaxanthin, and astaxanthin were com- additionally with two carbonyl groups, diketodihyroxyca- 10 Yuasa et al. rotenoid with β-ionone rings on two ends of the polyene Imamura, M., Nakai, J., Inoue, S., Quan, G.X., Kanda, T. and chain, while α-carotene and β-carotene are hydrocarbon Tamura,T. (2003) Targeted gene expression using the GAL4/ carotenoids with unsubstituted rings. In lipophorin, lipids UAS system in the silkworm Bombyx mori. Genetics, 165, 1329-1340. organize their positions on the basis of their structural Katagiri, C., Sato, M. and Tanaka, N. (1987) Small-angle x- properties; outer shell with phospholipids and a core with ray scattering study of insect lipophorin. J. Biol. Chem., hydrocarbon (Katagiri et al., 1987). Carotenoids may oc- 262, 15857-15861. cupy their positions in lipophorin: the dihydroxycarot- Miao, F., Lu, D., Li, Y. and Zeng, M. (2006) Characterization enoids, which are more polar, may be near the surface, of astaxanthin esters in Haematococcus pluvialis by lipid chromatography-atmoapheric pressure chemical ionization and the hydrocarbon carotenoids may lie in the core of li- mass spectrometry. Anal. Biochem., 352, 176-181. pophorin. The different types of receptors may need to Nakajima, M. (1963) Physiological studies on the function of uptake the carotenoids which distribute the different posi- genes concerning carotenoid permeability in the silkworm. tions in the lipophorin. Thus, Cameo2 and SCRB15 may Bull. Fac. Agric. Tokyo Univ. Agric. Tech., 8, 1-80. pick carotenoids up from the surface and from the core of Sakudoh, T. and Tsuchida, K. (2010) Transport of carotenoids by a carotenoid-binding protein in the silkworm. In Carot- hemolymph lipophorin, respectively. However, Cameo2 enoids (ed. by Landrum, J.T.), pp.511-523. CRC Press, New shows specificity not only for lutein, but also for other di- York. hydroxycarotenoids, such as zeaxanthin and astaxanthin, Sakudoh, T., Sezutsu, H., Nakashima, T., Kobayashi, I., so it seems to have broad substrate specificity for dihy- Fujimoto, H., Uchino, K., Banno, Y., Iwano, H., Maekawa, droxycarotenoids rather than single molecular substrate H., Tamura, T., Kataoka, H. and Tsuchida, K. (2007) Carot- enoid silk coloration is controlled by a carotenoid-binding specificity. protein, a product of the yellow blood gene. Proc. Natl. The in vivo observation of carotenoid transfer by sup- Acad. Sci. USA, 104, 8941-8946. plementation of the diet with astaxanthin and capsanthin Sakudoh, T., Iizuka, T., Narukawa, J., Sezutsu, H., Kobayashi, has yielded interesting data on the competition between I., Kuwazaki, S., Banno, Y., Kitamura, A., Sugiyama, H., different carotenoids, the silkworm can transfer and accu- Takada, N., Fujimoto, H., Kadono-Okuda, K., Mita, K., Tamura, T., Yamamoto, K. and Tsuchida, K. (2010) A mulate carotenoids, which are not derived from mulberry CD36-related transmembrane protein is coordinated with an leaves, in the cocoon. Our findings have not only opened intracellular lipid-binding protein in selective carotenoid new avenues of research on this topic, but will also facili- transport for cocoon coloration. J. Biol. Chem., 285, 7739- tate the production of yellow, orange, and red silk using 7751. certain carotenoids. Sakudoh, T., Kuwazaki, S., Iizuka, T., Narukawa, J., Yamamoto, K., Uchino, K., Sezutsu, H., Banno, Y. and Tsuchida, K. 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