Pigmentation of the marine isopod idothea granulosa (rathke)

Comp. Biochem.Physiol., 1966, Vol. 19, pp. 13 to 27. PergamonPressLtd. Printedin Great Britain

PIGMENTATION OF THE MARINE ISOPOD IDOTHEA G R A N U L O S A (RATHKE) WELTON

L. L E E *

Bedford College, University of London (Received 23 February 1966) A b s t r a c t - - 1 . Tissue and chromatophore pigments were isolated from the

three color varieties of the marine isopod Idothea granulosa. These included t-carotene, isocryptoxanthin, echinenone, 4-hydroxy-4"-keto-fl-carotene, canthaxanthin, isozeaxanthin and lutein. 2. The red, green and brown coloration of this species is primarily a result of the pigmentation of the epidermis although the animals' red chromatophores can effect a rapid change of color before epidermal pigments can be changed. 3. The chromatophore pigment is a reduced ommochrome. 4. The cuticles of all three color varieties contain two pigmented layers, the exocuticle and the endocuticle. The exocuticle is red and contains canthaxanthin and the endocuticle is yellow and contains lutein. 5. Red animals have canthaxanthin as their chief epidermal pigment. 6. Green animals have a green protein complex deposited in their epidermis. The complex appears to be a lipoprotein with strongly bound canthaxanthin and loosely bound or adsorbed lutein. 7. Brown animals have a mixture of the green complex and canthaxanthin in their epidermis. 8. A possible pathway has been suggested for the production of canthaxanthin from t-carotene. 9. A comparison has been made of the pigments and pigmentary systems in I. granulosa and I. montereyensis. INTRODUCTION RECENT work on pigmentation and color change of the marine isopod Idothea montereyensis (Lee, 1966; 1967) has indicated that similar investigations of related idotheids might prove especially valuable with regard to a better understanding of carotenoid pathways in invertebrates. T h e present report is a study of the pigments responsible for the various color varieties of the British species, Idothea granulosa. I. granulosa is abundant in the intertidal regions of almost all British shores (Naylor, 1955a, b) and it occurs in red, green and b r o w n color varieties as does its American counterpart, I. montereyensis. T h e British species resides and feeds on a wide range of intertidal algae including species of Fucus, Cladophora, Enteromorpha, Rhodymenia and Gigartina (Naylor, 1955a). T h e present investigation is utilized as the basis for a detailed comparison of the pigmentary systems in two closely related species, L montereyensis (cf. Lee, 1966) and I. granulosa. T h e ecological significance of color change in I. granulosa will be the subject of a later report. * Present address: Hopkins Marine Station of Stanford University, Pacific Grove, California. 13

14

WELTON L. LEE

METHODS

Animals All animals utilized for this investigation were collected at Black Rock, Brighton, Sussex. As soon as possible these were sorted into red, green and brown color varieties, frozen and stored until needed in a deep freezer at - 1 5 ° C . Approximately 100 animals were selected for each extraction and the digestive tracts of these were removed immediately before the animals were to be utilized. In addition, approximately 50 animals of each color variety were selected for the investigation of the gut diverticula pigments. T h e gut diverticula of these animals were isolated just before the extractions were made. Cuticles of each of the color varieties were separately prepared for extraction by cutting the animals in half down the medial sagittal plane. T h e dorsal and ventral sections were then separated and each piece of cuticle was thoroughly scraped so that nearly all the epidermis and underlying connective tissue was cleared away.

Extraction and pigment separation T h e material to be extracted (whole animals, cuticles or gut diverticula) was homogenized repeatedly in cold (5°C) acetone until no further pigment could be obtained. T h e separate acetone solutions were then combined and concentrated under a stream of nitrogen. Petroleum ether (b.p. 30-40°C) was added and the pigments transferred to the petroleum ether phase by the addition of water. T h e petroleum ether solution was repeatedly washed until free of acetone, dried over anhydrous sodium sulphate and again concentrated under nitrogen. After such treatment the residue of whole animal extracts was bright red due to the presence of chromatophores. T h e chromatophore pigments were easily extracted by the addition of methanol containing 5% conc. HC1. Carotenoids were initially separated chromatographically on columns of aluminum oxide (BDH, for chromatographic adsorption analysis). T h e aluminum oxide was activated by exposure to 100°C for 2 hr in an oven prior to packing the columns. T h e columns were of two sizes, measuring approximately 1 cm × 15 cm and 1'5 cm × 17 era. Pigments were placed on the columns in petroleum ether and developed with acetone-petroleum ether mixtures of different proportions. In all eases the resulting bands were eluted, transferred again to petroleum ether and rechromatographed for purification. Further purification was often carried out on columns containing a mixture of magnesium oxide (BDH, for chromatographic adsorption analysis) and "celite" (BDH, 30-80 mesh), 1 : 1 w/w. These columns were likewise developed with acetone-petroleum ether mixtures. Further separation was obtained by the use of thin-layer chromatography. Shandon equipment was utilized throughout and the plates were spread with aluminum oxide (Aluminumoxid-G-nach Stab.l, E. Merck AG., Darmstadt) to a depth of 200/z. T h e pigments were developed in one of four acetone-petroleum ether mixtures: 5%, 7% or 12% (v]v) acetone-petroleum ether mixtures were used to separate the carotenes, monohydroxy and mono- and di-keto derivatives, whereas a 25% mixture (v/v) was used for all others. It should be noted that the recorded R I values can only be considered as valid for individual plates, as for one and the same pigment these varied from plate to plate even when extreme precautions were taken to assure identical conditions.

Pigment identification T h e chromatographic and partition characteristics were recorded and the absorption maxima of the extracted pigments determined in petroleum ether, hexane and carbon disulfide. Furthermore, all of the extracted pigments were compared with known samples on thin-layer plates. Samples of echinenone and canthaxanthin were kindly supplied by the F. Hoffman-La Roche Company of Basle, Switzerland. Isocryptoxanthin and isozeaxanthin were produced by the borohydride reduction of echinenone and canthaxanthin, respectively. Lutein and fl-carotene were isolated and purified from dried nettle. Cryptoxanthin and zeaxanthin were isolated from maize meal.

PIGMENTATION OF IDOTHEA GRANULOSA

15

Additional procedures were utilized in identifying the isolated carotenoids. Among these was the determination of partition coefficients as expressed by the M-50 values and calculation of the corresponding relative polarities of each of the pigments according to the methods described b y Krinsky (1963). It was likewise often necessary to reduce ketocarotenoids to their corresponding hydroxy derivatives. This was done by adding a small amount of potassium borohydride to a solution of the pigment in 95 % ethanol and allowing this to stand under nitrogen overnight (cf. Krinsky & Goldsmith, 1960). Furthermore, the presence of allylic hydroxyl (or methoxyl) groups was determined by the acid--chloroform test (Karrer & Leumann, 1951). Finally, methyl ester derivatives were formed according to the method described by Petracek & Zechmeister (1956). Saponification was carried out by dissolving the pigment in question in a 15 %~ solution of K O H in methanol, and incubation in the dark, under nitrogen, at room temperature for 24 hr.

Quantitative determinations Quantitative determinations were made with a Unicam S.P. 500 spectrophotometer and the relative amount of each pigment given as a percentage of the total. This was based on the extinction at the wavelength of maximum absorbance. It should be emphasized that these values can only be taken as approximations as no allowance was made for differences in molar extinction. Estimations of the total amount of carotenoids present per unit weight of animal were based on the extinction at the wavelength of maximum absorbance of the entire extract in petroleum ether. In this case the extinction coefficient (E 1%/1 cm) was arbitrarily taken as 2500.

Protein complex Protein complexes were extracted with 0"01 M phosphate buffer p H 7 (KI-I2PO4 + NaaHPO4) from whole animals whose digestive tracts had been removed. T h e extracted protein complex was then precipitated with saturated ammonium sulfate, centrifuged and redissolved in 0"05 M phosphate buffer, p H 7. T h i s was diluted by the addition of about 2 vol. of distilled water, placed on columns of DEAE-cellulose (Whatman D E 50) and eluted by stepwise increase in the ionic concentration of the perfusing phosphate buffer. T h e DEAE-cellulose columns were prepared and run according to the methods described by Lee (1966). Horizontal micro-electrophoresis on starch gel (Connaught, lot 228-1) was carried out by utilizing methods similar to those developed for agar by Wieme (1959). 0-05 M phosphate buffer, p H 7"5, was used in the buffer compartment and 0.025 M phosphate buffer for the gel. Separation was carried out at 20 V/era for 8 hr. T h e gel was stained with a 1 ~o solution of Amidoschwartz 10 B in glycerol/water/acetic acid (50 : 50 : 20 v/v) and ultimately washed in the glycerol/water/acetic acid solution for several days. RESULTS

Whole animals--all color varieties W h o l e a n i m a l s of all t h r e e c o l o r v a r i e t i e s w e r e p r e p a r e d , e x t r a c t e d a n d t h e p i g m e n t s s e p a r a t e d c h r o m a t o g r a p h i c a l l y as o u t l i n e d above. A t o t a l o f e i g h t c a r o t e n o i d p i g m e n t s w e r e s e p a r a t e d ( F i g . 1). T h e s e w e r e : / 8 - c a r o t e n e , e c h i n e n o n e , isocryptoxanthin, canthaxanthin, 4-hydroxy-4'-keto-/%carotene, isozeaxanthin, l u t e i n a n d l u t e i n i s o m e r s . T h e c h a r a c t e r i s t i c s o f t h e s e p i g m e n t s are d e s c r i b e d b e l o w a n d t h e a b s o r p t i o n m a x i m a in p e t r o l e u m e t h e r , h e x a n e a n d c a r b o n d i s u l f i d e are r e c o r d e d in T a b l e 1.

16

WELTON L. LF~E Fraction

Piqment

Elufe

!.i.;i',:'.:.' Lutein isomer Lufein

I

I

or methanol

55% Acetone

I sozeoxonthin

5

100% Acetone

45% Acetone

4-Hydroxy, 4'- Kefo, B-Carotene

45% Acetone

Confhoxonthin

10%Acetone

4

~

3

l~ Isocrypfoxanthin 5-7%Acefone I I ~J Echinenone 2-5% Acetone I I JTIT~ ~- Carotene I-2% Acetone

2 I

\2 FIG. 1. Carotenoids from whole animal extracts of 1. granulosa as they appear on alumina when developed with petroleum ether-acetone mixtures. T h e percentage of acetone needed to elute each of the pigments is included.

T A B L E 1 - - A B s o R P T I O N MAXIMA I N PETROLEUM ETI-IER~ HEXANE AND CARBON DISULFIDE OF EACH OF THE CABOTENOIDS ISOLATED FROM WHOLE ANIMAL EXTRACTS OF I . granulosa

Absorption maxima (m/z) Fraction 1 2 3 4 5 6 7 8

Pigment fl-carotene Echinenone Isocryptoxanthin Canthaxanthin 4-hydroxy-4"-ketofl-carotene Isozeaxanthin Lutein Lutein isomer

Pet. ether

Hexane

Carbon disulfide

448-477 455 447-474 462

449-476 458 449-477 464

483-510 495 482-509 500

453 446-473 443"5-472 444-471

456 450-475 445-473 --

490 479-508 475-505 475-505

PIG1MiEN'TATIOI~I OF I D O T H E A G R A N U L O S A

17

Fraction 1, ~3-carotene. Fraction 1 elutes from alumina with 1-2% acetone in petroleum ether. It is epiphasic when partitioned between 90 and 95% methanol and petroleum ether both before and after saponification. The absorption spectrum in petroleum ether (448-477 mt~) is in full agreement with that reported for/3carotene (Krinsky & Goldsmith, 1960; Wolfe & Cornwell, 1965). Fraction 1 is inseparable from known samples of/3-carotene on mixed thinlayer chromatographs, and on such chromatographs (using 5 ~o acetone in petroleum ether as the solvent mixture) both show R! values of 0.97. Fraction 2, echinenone (4-keto-/3-carotene). Fraction 2 elutes from alumina with 2-5% acetone in petroleum ether. It is completely epiphasic to 90 and 95% methanol before and after saponification. This pigment exhibits a single absorption maximum at 455 rn~ in petroleum ether. The absorption maxima in petroleum ether, hexane and carbon disulfide are identical to those of known eehinenone in the same solvents. When purified, Fraction 2 has an M-50 value of 114.3 which corresponds to a relative polarity of 0.71. The relative polarity calculated for pure echinenone is 0.72. The pigment is inseparable from known echinenone on thin-layer plates and exhibits an Rt value of 0.91 (solvent = 12% acetone in petroleum ether). In addition, the borohydride reduction products of Fraction 2 and known echinenone (isocryptoxanthin = 4-hydroxy-/3-carotene) are inseparable on mixed thin-layer chromatograms (solvent = 7% acetone in petroleum ether). Fraction 3, isocryptoxanthin (4-hydroxy-/3-carotene). Fraction 3 elutes from alumina with 5-7% acetone in petroleum ether. It is epiphasic to 90% methanol Front

....

_(~,~-~

.....................

Rt =0.64 /~=0.64 Rf=0'64 ~)R! =0.42

Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (I)

(2)

(3)

(4)

(5)

FIG. 2. Thin-layer chromatograph on aluminum oxide showing the separation of (1) /3-carotene, (2) cryptoxanthin, (3) Fraction 3 (= isocryptoxanthin), (4) reduced echinenone (= isocryptoxanthin) and (5) a mixture of Fraction 3 and reduced echinenone. The solvent mixture was 7~o acetone in petroleum ether (v/v). The RI values are given for each of the spots.

18

WELTONL. LEE

but partially epiphasic and partially hypophasic to 95~o methanol before and after saponification. This pigment has absorption maxima at 447-474 m/~ in petroleum ether. It exhibits an M-50 value of 108.2 which corresponds to a relative polarity of about 0.91. The relative polarity of isocryptoxanthin is calculated to be 0-89. On mixed thin-layer chromatograms the pigment is inseparable from reduced echinenone but easily separable from cryptoxanthin and fl-carotene (solvent -----7 ~ acetone in petroleum ether). The R! values of these pigments are given in Fig. 2. The absorption maxima of reduced echinenone (isocryptoxanthin) and Fraction 3 were identical in petroleum ether, hexane and carbon disulfide, whereas those of cryptoxanthin were very slightly lower. Fraction 4, canthaxanthin (4, 4'-di-keto-fl-carotene). This pigment elutes with 10°/0 acetone in petroleum ether and is partially epiphasic and partially hypophasic to 90 and 95 ~o methanol both before and after saponification. It exhibits a single absorption maximum at 462 m/~ in petroleum ether. When purified the pigment has an M-50 value of 93.9 which corresponds to a relative polarity of 1.43. The relative polarity calculated for canthaxanthin is 1.44. Fraction 4 is inseparable from known canthaxanthin on thin-layer chromatograms. Both show an R! value of 0-85 (solvent =- 12% acetone in petroleum ether). The absorption spectra of the two are likewise identical. Fraction 4 can be reduced with borohydride to give a pigment which has absorption maxima at 451-476 m/z in 95% ethanol. This is identical to the reduction product of known canthaxanthin (----isozeaxanthin) and indeed the two reduction products cannot be separated on thin-layer chromatograms. Fraction 5, 4-hydroxy-4'-keto-fl-carotene. This pigment elutes with 45~o acetone in petroleum ether and is mostly epiphasic to 90~o methanol and mostly hypophasic to 9 5 ~ methanol both before and after saponification. The pigment exhibits a single absorption maximum which peaks at 453 m/~ in petroleum ether. The M-50 value was found to be 98.8 which corresponds to a relative polarity of 1.63. The relative polarity calculated for 4-hydroxy-4'-keto-fl-carotene is 1.61. If this pigment is the 4-hydroxy-4'-keto-fl-carotene derivative then it should be possible to reduce it with borohydride to isozeaxanthin (4-4'-di-hydroxy-flcarotene), the reduction product of canthaxanthin. Upon reduction with borohydride the pigment showed a fl-carotene-like absorption spectrum which was identical to that of isozeaxanthin formed by reducing canthaxanthin. The two reduction products were inseparable on mixed thin-layer chromatograms (R! ~ 0.79) but clearly separable from a non-reduced portion of Fraction 5 (R 1 ---- 0.89). A solvent mixture of 25% acetone in petroleum ether was used. The partitioning behavior, relative polarity, absorption maxima and similarity of the reduction products of this pigment with those of reduced canthaxanthin strongly suggest the presence of 4-hydroxy-4'-keto-fl-carotene. Fraction 6, isozeaxanthin (4, 4'-di-hydroxy-fl-carotene). This fraction elutes slowly with 45~o acetone in petroleum ether. It is mostly hypophasic to 90% methanol and completely hypophasic to 95% methanol before saponification. The

P I G M E N T A T I O N OF I D O T H E A G R A N U L O S A

19

pigment exhibits very unusual behaviour after saponification. Thin-layer chromatograms of the saponified material yield three, sometimes four, products. One is the original material (inseparable from the non-saponified material), one is similar to Fraction 5 (4-hydroxy-4'-keto-/3-carotene) and one is similar to echinenone (Fraction 2). This strange behavior is like that of isozeaxanthin when it is treated with chloroformic hydrogen chloride where one of the products is 3', 4'dehydro-echinenone (Weedon, 1965, Petracek & Zechmeister, 1956). By way of comparison, isozeaxanthin formed by the borohydride reduction of canthaxanthin was subjected to the same saponification procedure. The same process was seen to occur, although the yield of the various products and the number of them varied from sample to sample. Fraction 6 exhibits an absorption spectrum similar to that recorded for isozeaxanthin (Petracek & Zechmeister, 1956) with maxima at 446473 rn/z in petroleum ether. The absorption maxima in petroleum ether, hexane and carbon disulfide are identical to those of reduced canthaxanthin. The M-50 of this pigment is 80.2 which corresponds to a relative polarity of 1.97. Although this is much higher than that calculated for isozeaxanthin (1.78), it is not surprising in view of the apparent ease with which changes can occur in this pigment. When chromatographed on thin-layer plates with reduced canthaxanthin (isozeaxanthin) and zeaxanthin (3, 3'-di-hydroxy-fi-carotene), using a solvent mixture of 25°'0 acetone in petroleum ether, Fraction 6 was inseparable from the former (R 1 = 0-79) and clearly separable from the latter (R 1 = 0.37). Furthermore, it was possible to produce a methoxy derivative (4, 4'-dimethoxy-fl-carotene) by the treatment of methanolic solutions of the pigment with acid chloroform. The methoxy derivative of Fraction 6 was inseparable from the methoxy derivative formed in the same manner from reduced canthaxanthin. Finally, the presence of allylic hydroxyls was determined by the addition of acid chloroform to a chloroformic solution of the pigment. The result was similar to that already described. Numerous by-products were formed and many of these were similar to the derivatives isolated from the acid chloroform treatment of reduced canthaxanthin. Since isozeaxanthin has not yet been reported in nature it was imperative to compare Fraction 6 with reduced canthaxanthin as extensively as possible. Microanalysis and other more refined methods of analysis would have been desirable but there was insufficient pigment available. From the comparisons with reduced canthaxanthin described above it seems reasonable to believe that this pigment is the 4, 4'-di-hydroxy-fi-carotene, isozeaxanthin. Fraction 7, lutein (3, 3'-di-hydroxv-o~-carotene). This fraction elutes with 550/'0 acetone in petroleum ether. It is entirely hypophasic to 90 and 95% methanol both before and after saponification. The pigment has absorption maxima at 443.5 and 472 mt~ in petroleum ether. The M-50 value was 80"8 which corresponds to a relative polarity of 1-91, in close agreement with that calculated for lutein (1.89). The absorption maxima in petroleum ether, hexane and carbon disulfide are identical to those of known samples of lutein and the pigment is inseparable from lutein on mixed chromatograms (R l = 0.33).

20

WELTONL. LEE

Fraction 8, htein isomers. Fraction 8 ehtes with either 100% acetone or methanol and is strictly hypophasic to both 90 and 95% methanol before and after saponification. The absorption maxima are close to those reported for Fraction 7 (lutein) and differ only in the shape of the curve. The pigment is inseparable from one of the isomers produced by iodine catalysis of known htein. Cuticle pigments Cuticles of all three color varieties are generally of an orange or yellow-orange color and extracts of them produced only two major pigments, canthaxanthin and htein. These were always present in approximately equal proportions. Traces of mono-hydroxy-mono-keto-l~-carotene, echinenone and isozeaxanthin were sometimes encountered but these were presumably derived from residual epidermis which had not been completely removed from the cuticles before extraction.

Pigments of the gut diverticula The gut diverticula of adult animals, whatever color variety, are invariably light to deep yellow-orange in color. Gut diverticula from each of the color varieties were removed and the pigments were extracted and compared with known carotenoids on thin-layer chromatograms. The results are tabulated in Table 2. Detailed quantitative data could not be obtained because of the extremely small amounts of pigments involved and the information listed is only the result of visual comparisons of the intensity of pigment spots on the thin-layer plates. TABLE 2--THECAROTENOIDS ISOLATED FROM THE GUT DIVERTICULAOF THE RED, BROWN AND GREEN COLOR VARIETIES OF

J~. granulosa

Color variety

Major pigment Others

Red

Brown

Green

Isozeaxanthin Canthaxanthin Lutein

Isozeaxanthin fl-Carotene Canthaxanthin Lutein

Lutein Isozeaxanthin

Those pigments which were dominant are listed as major pigments whereas those present in only moderate amounts are listed as others. Pigments present only in traces have not been listed.

Quantitative determinations Determinations of the total carotenoid content in mg/100 g wet wt. of animal were made for each of the three color varieties. The results, although only rough approximations, indicate that the red and green color varieties have approximately the same total amount of carotenoid, namely 4.9 mg/100 g for the red color variety

PIGMENTATION OF

IDOTHEA GRANULOSA

21

and 4.7 mg/100 g for the green. The surprising feature is that nearly twice this amount (7.8 mg/100 g) can be found in the brown color variety. Table 3 illustrates the relative amounts of each of the individual carotenoids in the three color varieties as a percentage of the total carotenoid content. Although the eight carotenoids originally described are found in all three color varieties, the relative amounts of these differ greatly. Canthaxanthin and isozeaxanthin together account for nearly half of the pigment in red and brown animals, whereas lutein alone accounts for nearly half of the pigment in green animals although it is present in large amounts in all three color varieties. TABLE 3 - - T H E

RELATIVE PERCENTAGES OF THE PIGMENTS ISOLATED FROM THE RED, BROWN AND GREEN COLOR VARIETIES OF I. gTa?lulosa

Color variety (relative %) Pigment fl-carotene Isocryptoxanthin Echinenone 4-hydroxy-4'-keto-fl-carotene Canthaxanthin Isozeaxanthin Lutein Lutein isomers

Red

Brown

Green

1"88 1"41 3"77 15-11 25"50 22"90 28"33 1"06

5"42 1"86 3"20 14"57 21"68 27"20 20"97 5-06

4"60 1-11 0"87 16"91 12"56 16"66 46"26 0-99

Chromatophore pigment If whole animals are homogenized in phosphate buffer and the resulting suspension is lightly centrifuged for about 5 min the material forms a series of layers with a very significant brick-red layer in the middle. Examination of this material shows it to consist primarily of red chromatophores from which a single pigment was isolated whose characteristics were those of a reduced ommochrome (Lee, 1966). It should be noted that this pigment was identical to the pigment extracted from the reddish residue of acetone-extracted whole animals.

Protein complex Phosphate buffer extracts of whole green animals yield a green water-soluble pigment with absorption maxima at 683-(620)-280 m/z with very heavy absorption in the 400-500 m/z range. This green pigment can be precipitated with saturated ammonium sulfate and upon centrifugation a yellow lipid fraction separates from the blue-green precipitate. Thin-layer chromatograms of acetone extracts of this lipid fraction showed the presence of all of the eight reported carotenoids in approximately the same proportions as found in whole animal extracts. This lipid fraction was even more noticeable when the precipitated protein complex was redissolved in phosphate buffer and shaken with diethyl ether. After centrifugation the water

22

WELTONL. LrE

phase was bright blue, the diethyl ether yellow and a large amount of yellow lipid gathered at the interface. The green to blue-green redissolved protein complex separated into three main fractions when chromatographed on DEAE-ceUulose. The main fraction was blue in color and eluted with 0.0125 M phosphate buffer, pH 7. A second fraction, likewise blue in color, was eluted with 0.25 M phosphate buffer, pH 7. The last fraction was bright yellow and eluted with methanol. Fraction 1 was similar to the crude extract in that it had absorption maxima at 683-(620)-280 m# but differed in having a peak at 375 m# and little absorbance inthe 400-500 m/~ range. Fraction 2 had absorption maxima at 683-670-280 m~ and appeared to contain denatured material. The third fraction contained carotenoids in the same proportion as found in the earlier-mentioned lipid fraction. Acetone extracts were made of Fractions 1 and 2. This treatment freed the carotenoids and formed a white flocculent protein precipitate. The carotenoids were transferred to petroleum ether and the absorption spectrum determined. The more or less single-peak nature of this absorption spectrum suggested the presence of a keto-carotenoid and this was substantiated by thin-layer chromatography of the material. Canthaxanthin was by far the most abundant pigment, with only small traces of the remaining carotenoids. Repeated attempts were made on various samples to try to detect the presence of bile pigments but none were found. The precipitated protein complex gave no sign of a Gmelin reaction and no fluorescent zinc complexes could be found. The investigations reported above suggest that the green color of the green (and brown) variety of I. granulosa is a protein carotenoid complex involving a yellow lipo-protein containing all of the animals' carotenoids in approximately the same proportions as occur in whole animal extracts, and a blue canthaxanthinprotein complex. However, when subjected to micro-electrophoresis on starch gel the green protein complex does not separate into a yellow and a blue component as expected but remains as a single green band. Since only phosphate buffer of a single pH value was utilized it is not yet possible to know to what extent this result is influenced by pH and/or differences in buffer systems. However, this preliminary work does at least suggest that the protein complex is derived from a lipo-protein to which canthaxanthin is strongly bound and the remaining carotenoids only loosely bound or absorbed. The separation into yellow and blue components on DEAE-ceUulose might well be due to the strong affinities of this ion exchange medium for the more loosely bound or adsorbed carotenoids and/or its attraction for part or all of the lipid component of the complex. The absorption spectrum of the crude extract, then, appears to represent a better picture of the complex, with the blue canthaxanthin-linked portion showing maxima at 683-(620)-280 m# concurrently with the heavy absorption in the 400-500 m~ range which represents the composite spectrum of the loosely bound carotenoids. The information reported here is by no means considered complete and further investigations are now in progress so that the relationships between the various elements of this complex can be more thoroughly known.

P I G M E N T A T I O N OF 1 D O T H E A G R A N U L O S A

23

DISCUSSION The present study indicates that although the same carotenoids occur in each of the three color varieties of I. granulosa their relative abundance differs radically from one variety to the other. In addition, the animals differ in the relative abundance of a green carotenoid-protein complex. The red, green and brown color varieties seem to result from the presence of red canthaxanthin, a green protein complex or a mixture of these, respectively. These pigments appear to be largely restricted to the epidermis and it is epidermal pigmentation which is primarily responsible for color change in this species. Cuticles in all three color varieties are similar in that they are almost always of an orange-yellow color. This is due to the presence of red canthaxanthin in the exocuticle and yellow lutein in the endocuticle. However, the intensity of cuticle color may be greater in red animals. Since the algae on which these isopods feed have not been shown to contain canthaxanthin (Goodwin, 1954) it is suggested that they are metabolizing the 4, 4'-di-keto-fl-carotene from fl-carotene, fl-Carotene and lutein (the only ~x-carotene derivative isolated from these animals) are exceedingly abundant in the algae on which these idotheids cling and feed and would be readily available in their food. Four of the isolated pigments could be involved in the metabolism of fl-carotene to canthaxanthin. The proposed pathway is presented in Fig. 3. It would appear that the first step is the addition of an hydroxyl group to the 4 or 4' position of fl-carotene. The isocryptoxanthin which is thus formed would then be oxidized to the 4-keto derivative, echinenone. An identical procedure might occur on the other fl-ionone ring to first form 4-hydroxy-4'-keto-fl-carotene and finally the 4, 4'-di-keto derivative, canthaxanthin. It is difficult to ascertain, however, whether isozeaxanthin (4, 4'-di-hydroxy-fl-earotene) is directly involved in this pathway or not. As shown in part in Fig. 3, a number of alternative routes could exist. These include the double hydrogenation of fl-carotene or the addition of a second hydroxyl group to isocryptoxanthin (4-hydroxy-fl-carotene), both of which would form isozeaxanthin, the di-hydroxy derivative. Furthermore, isozeaxanthin and 4-hydroxy-4'-keto-fl-carotene could both form canthaxanthin independently, isozeaxanthin might well be a necessary intermediate between 4-hydroxy-4'-keto-fl-carotene and canthaxanthin, or indeed 4-hydroxy-4'-keto-fl-carotene the necessary intermediate between isozeaxanthin and canthaxanthin. The choice between these is a difficult one, but a glance at the relative abundance of each of the pigments in all three color varieties (Table 3) suggests an answer to the dilemma. Three rather definite facts emerge from a consideration of the data given in Table 3. First, fl-carotene, isocryptoxanthin and echinenone occur in rather low yield in all three color varieties, each amounting to less than about 5°/~ of the total pigment present. Furthermore, 4-hydroxy4'-keto-fl-carotene occurs in almost the same relative amount in all of the color varieties. Finally, isozeaxanthin and canthaxanthin always occur in roughly equal amounts in all three color varieties. The relatively small amounts of the

24

WI~LTONL. LEE

first three pigments in the series suggest the rapid metabolism of fl-carotene to 4-hydroxy-4'-keto-fl-carotene. The approximately equal amounts of isozeaxanthin and canthaxanthin suggest that these two pigments are being derived in equal amounts from 4-hydroxy-4'-keto-fl-carotene. This might be thought of in terms

,8-C~ rot'erie

OH I socryptoxonfhin

0

0

0

0

Confhoxonthin

Utilized in Color c honge

Echinenone

,8-Carotene OH

OH

OH

Isozeoxenfhin

?

FIG. 3. Possible pathway for the production of canthaxanthin from fl-carotene in 1. granulosa. Alternate routes are shown as dotted lines. of a disproportionation reaction in which two molecules of 4-hydroxy-4'-ketofl-carotene disproportionate to produce one molecule of canthaxanthin and one of isozeaxanthin. In such a case one molecule would act as a hydrogen acceptor for the oxidized member of the pair. This would explain the almost equal amounts of canthaxanthin and isozeaxanthin in each of the color varieties. The constancy of 4-hydroxy-4'-keto-fl-carotene might well be explained in the following manner.

PIGMENTATION OF I D O T H E A G R A N U L O S A

25

The series of reactions from E-carotene to 4-hydroxy-4'-keto-~-carotene can be thought of in terms of a single, partially reversible, enzymatic series. If this were so, then the direction of the reactions involved might be altered by relatively small changes in the abundance of either of the end-products, E-carotene or 4-hydroxy4'-keto-3-carotene. When the animal is producing canthaxanthin it presumably would be forming one molecule of canthaxanthin and one of isozeaxanthin from every two molecules of 4-hydroxy-4'-keto-fl-carotene. In so doing the relative amount of the latter would be reduced so that further formation of it from 3-carotene would be stimulated. Thus the amount of mono-hydroxy-mono-ketofl-carotene would remain at approximately the same level. If, however, canthaxanthin was not needed and as a consequence was not produced there might be a slight increase in the abundance of 4-hydroxy-4'-keto-fl-carotene, reversing the direction of the pathway: fl-carotene ~4-hydroxy-4'-keto-3-carotene. If this is indeed what happens we might expect only a slight increase in mono-hydroxymono-keto 3-carotene in green animals with a consequent decrease in echinenone, a slight decrease in isocryptoxanthin and an increase in E-carotene. Although the relative percentages presented in Table 3 are subject to inaccuracies, the changes in carotenoid content suggested above do appear to occur. Since all of the pigments in the series can be found in the gut diverticula and all of the pigments but E-carotene can be found in the epidermis it is suggested that carotenoid modification occurs here. The presence of nearly double the total carotenoid content in brown animals as against that found in both red and green ones might be explained in the following manner. Red animals are primarily producing canthaxanthin, thus only this pathway would be operative. In green animals the emphasis is on the green protein complex in which lutein appears to play an important role, thus lutein would be expected to be actively taken in (the canthaxanthin present before the animal changed color could suffice for the protein complex). In brown animals, however, both canthaxanthin and the protein complex are needed, hence both lutein and E-carotene would be taken in. This might easily cause a nearly twofold increase in total carotenoid content since both systems would be operative. The data shown in Table 2 are consistent with this contention. Figure 4 represents the proposed activities of the gut diverticula in the red, green and brown color varieties of I. granulosa. The pathways as well as the proposed mechanisms presented here can only be regarded as tentative. Further verification must come from attempts to isolate the enzyme systems involved and such investigations are being considered. The work recently carried out on a related idotheid, I. montereyensis (Lee, 1966), presents an opportunity to compare systems of carotenoid transformations in two closely related animals. I. granulosa differs from I. montereyensis in a number of ways. Perhaps the most significant difference is the emphasis on the epidermis rather than the cuticle as the vehicle for color change. Cuticles of all three color varieties of I. granulosa tend to be an orange color and color change is largely dependent on epidermal color alone (although in both species chromatophore changes are also involved). This is the prime factor responsible for the ability of

26

WELTON Lo LEE

1. granulosa to change its color without a molt, hence producing a much quicker response to its background color. This is achieved, however, only at the expense of limiting the intensity of the required color. I. montereyensis requires a molt, hence a longer time interval to change color, but since the red, green or brown color is a function of cuticle color the animal can achieve a far greater color intensity. This seems to be sound from the ecological standpoint since the British species is faced with an environment dominated by browns of varying intensity (i.e. redbrown to green-brown) while I. montereyensis occurs on distinctly differently colored plants; green Phyllospadix or red algae of various species, almost never on brown algae. Color Eplcufide Ex0cuflcle End0cuticle Epidermis

Red

variety Brown

II

Conthoxonthin Lufein [oJololololglolololo

f

Green

'

Confhoxonthin I Lufein Iololololol9191ojolo

Confhoxonthin Lutein ololololglololololo ~J

ololOlololofC>l-~lololc~lololoJ

Echrne ~one

Is0cry! ~x0nthin

/

Lutein7lipid (71 l

ololololcIololc~loloIololol

E

/~-Car0tene

ololo

/

0

rotein-Lutein

.

.

~1olololo

Lutein

loIQIololololololololololOlololololol FIG. 4. Diagrammatic representation of some of the chemical events taking place in the gut diverticula of I. granulosa and their relationship to epidermal pigmentation in the red, green and brown color varieties.

In addition to these differences there appears to be a rather significant difference in the pathways involved in the formation of canthaxanthin from fl-carotene. 1. montereyensis was shown to have a pathway which presumably involved the series: fl-carotene-->echinenone >4-hydroxy-4'-keto-fl-carotene >canthaxanthin. It was suggested in this earlier paper that isocryptoxanthin might have been present but overlooked due to its relative scarcity and the abundance of fl-carotene isomers. From the results reported in this paper the presence of this pigment in I. montereyensis now seems probable. The pathways in the two species, then, are similar with respect to the steps from fl-carotene to 4-hydroxy-4'-keto-fl-carotene. The chief difference occurs in the method of forming canthaxanthin from the latter B-carotene derivative.

PIGMENTATION OF I D O T H E A G R A N U L O N A

27

It appears on the evidence thus accumulated that I. granulosa produces isozeaxanthin as a direct result of canthaxanthin production. T h e isozeaxamhin produced in this m a n n e r seems to be of dubious function and merely reflects the result of alternate molecules of 4-hydroxy-4'-keto-~-carotene acting as hydrogen acceptors. I. montereyensis seems to have solved this p r o b l e m and is capable of forming canthaxanthin directly f r o m 4-hydroxy-4'-keto-~-carotene. T h i s too might in some way be related to the differences in intensity of color found in the two species. T h e present investigation has shown that the pigmentary systems in at least two related idotheid isopods, although not identical, are alike in a n u m b e r of respects. T h e general basis for color change likewise appears to be similar, although slight differences do occur. T h e s e differences appear to have ecological significance, a point which will be taken up in future work on the species. T h e protein complexes in b o t h species are also similar, and work is now in progress to try to elucidate more fully the nature of these complexes. F u r t h e r investigation on other invertebrates m a y show some important c o m m o n denominators in the production of canthaxanthin, perhaps even in the formation of astaxanthin, from E-carotene. Acknowledgements--This research was supported in part by a NATO Postdoctoral Fellowship. I would like to thank Professor N. Millott of Bedford College for so kindly providing the necessary facilities. My very special thanks go to Dr. Barbara Gilchrist whose enthusiasm and valuable help made this work possible. REFERENCES GOODWIN T. W. (1954) Carotenoids, their comparative Biochemistry. Chemical Publishing, New York. K~RER P. & LEUMANN E. (1951) Eschscholtzxanthin and anydro-eschscholtzxanthin. Helv. Chim. Acta 34, 445-453. KRI~qSKgN. I. (1963) A relationship between partition coefficients of carotenoids and their functional groups. Analyt. Biochem. 6, 293-302. KRINSKYN. I. & GOLDSMITHT. H. (1960) The carotenoids of the flagellated alga, Euglena gracilis. Archs Biochem. Biophys. 91, 271-279. LE~ W. L. (1966) Pigmentation of the marine isopod, Idothea montereyensis. Comp. Biochem. Physiol. 18, 17-36. LEE W. L. (1967) Color change and the ecology of the marine isopod Idothea (Pentidotea) montereyensis Maloney, 1933. Ecology (To be published). NAYLOR E. (1955a) The ecological distribution of British species of Idothea (Isopoda). J. Anim. Ecol. 24, 255-269. NAYLOrt E. (1955b) The comparative external morphology and revised taxonomy of the British species of Idothea. J. mar. Biol. Ass. U.K. 34, 467-493. PETRACEKF. J. & ZECHMEISTERL. (1956) Reaction of E-carotene with N-bromosuccinimide : the formation and conversions of some polyene ketones, ft. Am. Chem. Soc. 78, 14271434. WEEDON B. C. L. (1965) Chemistry of the carotenoids. In Chemistry and Biochemistry of Plant Pigments (Edited by GooDwIN T. W.). Academic Press, London & New York. WIEtCm R. J. (1959) Studies on Agar Gel Electrophoresis. Arscia Ultgavern, N.V., Brussels. WOLFE D. A. & COmqWELLD. G. (1965) Composition and tissue distribution of carotenoids in crayfish. Comp. Biochem. Physiol. 16, 205-213.