Carotenoid fractionation in the plumose anemone Metridium

Comp. Biochera. Physiol., 1967, Vol. 22, pp. 177 to 188. PergamonPress. Printed in Great Britain

CAROTENOID F R A C T I O N A T I O N IN THE PLUMOSE ANEMONE M E T R I D I U M * DENIS

L. FOX,

GEORGE

F. CROZIERt

a n d V. E L L I O T T

SMITH

Department of Marine Biology, Scripps Institution of Oceanography, University of California, La Jolla, California, U.S.A. (Received 31 January 1967)

A b s t r a c t - - 1 . Several colour-variants of the Pacific Coast plumose anemone, MetHdium senile fimbriatum, and the red form of the British species, M. senile sen//e, yielded astaxanthin esters as the preponderant carotenoid fraction stored in somatic or in gonadal tissues. 2. White males assimilated no carotenoid from a protracted diet of finely divided salmon flesh, rich in astaxanthin. Nor did the whole body of a large, mature white male yield more than traces of astaxanthin or of any other carotenoid. 3. Pacific Coast Metridium with white, red or various shades of brown somatic tissues stored in their gonads various concentrations of carotenoids, predominately asterified astaxanthin. Somatic tissues of coloured types, including red variants from Britain, likewise yield astaxanthin esters, mostly epiphasic, but with some ambiphasie, in systems of hexane+95~o methanol. 4. Esters of zeaxanthin (or certainly of a neutral xanthophyll exhibiting zeaxanthin's spectral and partitional properties) were usually present, while minor amounts of unfamiliar ketones or hydroxyketones were of occasional incidence. 5. T h e red specimens from Britain likewise yielded zeaxanthin and its esters, as well as occasional minor quantities of acidogenic carotenoid esters (or the free compound) different from astaxanthin. No carotenes were detected in either species. 6. Metridium senile fimbriatum exhibits some similarities to, and certain apparent differences from, its British relative, M. s. senile, with respect to the metabolic fractionation of dietary carotenoids. One of us (D. L. F.) would take this occasion to honor the late Professor Carl F. A. Pantin, F.R.S., to whose memory this extension of earlier collaborative researches is an affectionate tribute. It was Carl Pantin who, shortly after m y arrival in Cambridge in 1938 for a sabbatical year, first drew m y attention to the striking polychromy among Metridium senile individuals, and who encouraged the joint researches which led to our 1941 paper on the subject. M y own responsibility in that study lay in the biochemical area, i.e. the only portion to which certain revisions and extensions apply in the present work. It is personally gratifying to know that Carl Pantin had received and had been able to appreciate a preliminary report, by letter, of these present researches and current interpretations, less than a fortnight before his death. * Contribution from the Scripps Institution of Oceanography. This research was aided by Grant GB-2312 to the senior author from the National Science Foundation. t Present address: Department of Biology, University of Southern Mississippi, Hattiesburg, Miss. 177

178

MORE

DENIS L. Fox, GEORGE F. CROZIER AND V. ELLIOTT SMITH INTRODUCTION, MATERIALS AND METHODS than a quarter-century ago, researches at Cambridge University (Fox &

Pantin, 1941) revealed some qualitative biochemical features relating to several colour-variants within the species then called Metridium senile (now M. s. senile), the plumose anemone occurring commonly along the coasts of Britain and continental Europe (Stephenson, 1935). In that work it was reported that the white form, bearing little or no melanin, confined its carotenoids probably to the ripening gonads, wherein there occurred limited quantities of astaxanthin, both esterified and free (or more probably ambiphasic, short-chain esters thereof, accompanying the long-chain esters, in the light of what we now know). Individuals exhibiting various shades of brown (without any obvious red) coloration, due to corresponding amounts of melanin, had yielded but very minor fractions of carotenoids, including esters of astaxanthin if ripening ovaries were present and possibly of other acidogenic carotenoids, sometimes traces of fl-carotene, and several free or esterified, neutral xanthophylls. Yellow-orange animals with little or no melanin had been found to store several kinds of carotenoids in considerable concentrations, e.g. esters of acidogenic and of some conventional, neutral xanthophylls, as well as fl-carotene. Red-and-brown specimens (i.e. with red tentacles, oral disc, mouth and endoderm, but with darkbrown ectoderm over the scapus), showing various degrees of melanization, had yielded mainly esters of astaxanthin, or of this with other acidogenic carotenoid esters. Finally, non-melanistic, red animals were reported to possess the highest concentrations of carotenoids in the series, these being principally esters of acidogenic compounds, including astaxanthin, accompanied by neutral xanthophylls and infrequently by small amounts of carotene. (Note: the older, incorrect term, "astacene", which actually is the name of the acidic, oxidized artifact, has long since been replaced by the name of the naturally occurring pigment, astaxanthin, here and elsewhere, e.g. Fox, 1953. The provisional terms "metridine" or "metridioxanthin", used in the earlier work, also have been discontinued, for reasons to be explained below.) Intending at the outset of the present investigation to seek for some quantitative data applying to carotenoid fractionation in representatives of different colour-types, we had wondered whether our Metridium from colder waters along the Pacific Coast of North America (i.e. subtidally in the north and at greater depths in the south) might be conspecific with Metridium senile of the British waters, as had been assumed by some writers (cf. Pratt, 1935; Stephenson, 1935; Light et al., 1964). Professor Cadet Hand tells us, however, that minor morphological differences have distinguished the British form, Metridium senile senile, from the Pacific Coast one, known as M. s. fimbriatum. And, indeed, certain minor biochemical differences, not evident in comparisons between the white variants of the British and Pacific forms, emerged when we compared come of the other colour-types from the two localities. These differences will be detailed below. Source materials, reagents and instrumentation. The Pacific anemones were taken from submerged pier piling surfaces at Monterey, California, and by divers

C A R O T E N O I D F R A C T I O N A T I O B I I N T H E P L U M O S E A N E M O N E METPdDIUM

179

from rock-surfaces under cold, deeper waters off La JoUa, and were analyzed while fresh. The British specimens of the red variety, preserved with MgSO4 or with NaC1, were air-mailed from Plymouth, Swansea, Menai Bridge and Millport (Cumbrae Islands). Spectrophotometric and chromatographic instrumentation and reagents used in this work were the same as have been described earlier (Fox et aL, 1965 ; Fox & Hopkins, 1966). For extraction of the pigments, the tough somatic tissues were comminuted into chowder, under ethanol, with the fast-revolving blades of a Sorvall Omni-mixer. The same procedure was used for breaking up egg masses. Losses were precluded by containing the material, during the operation, within a gasket-sealed metal cup. Since earlier analyses by Fox & Pantin (1941) had revealed the presence of total mixed lipids at an average concentration of 1-65% (by wet weight), therefore when, in the present work, it was anticipated that such compounds, perhaps including yellow chromolipids, might interfere with good chromatographic resolution of separate fractions, the carotenoids were first adsorbed from the crude, ethanol-free hexane solution of extracted material by adding powdered MgO in small, successive "knife-point" amounts while the system was kept stirred by a rotating magnetic bar. The powder bearing the adsorbed carotenoids, after being rinsed several times with fresh hexane, was treated with hexane containing minor amounts (2.5-5% by volume) of methanol, following the method described for separating astaxanthin esters from much of the adventitious lipid in flamingo shank-skin (Fox & Hopkins, 1966). Following elution and recovery by filtration, the system was washed free of methanol with a few successive lots of distilled water. If necessary, the resulting hexane solution was then dried briefly with anhydrous Na2SO 4 before chromatography. For the Pacific Coast Metridium and for the Plymouth specimen, the first batch of four from Swansea and No. 2 from Menai Bridge, columnar MgO-celite chromatography was applied in the usual way. In the rest of the analyses, i.e. the other three from Menai Bridge, all five from the Cumbrae Islands and the second lot of six from Swansea, we substituted thin-layer chromatography, using SiO~ Grade G or H, with 25% acetone in hexane (v/v) as the eluant, within the concentration chamber described and illustrated earlier (Fox & Hopkins, 1966). RESULTS

A. Pacific Coast Metridium Sexual differences. Among the white variants, ripe females are readily distinguished from ripe males by inspection of the gonads through the lower part of the scapus, against strong light transmitted through the whole body, or more critically with the use of an electrically lighted clinical proctoscope inserted through the mouth down into the enteron. The creamy white spermaries are thus readily contrasted with pink to red or even purplish ovaries. Moreover, mature males may be squeezed manually (preferably in a towel because of the slime) to induce

0.50

0.57

0.22

0.20

1 '5

0"27

466 my. 424; 451; 477 mr*

Fraction 1 : 2:

Fraction 1 : 2:

Fraction 1 : ~ 2: 3: 4:

465 m p

466 m/x 466 m/*

467 mtz 468 m # 465 mtz

424; 450; 475 m/* 467 m/z 466 mtz 450 m/z 466 mtz

Fraction 1 : 466 m/* 2 : 466 m/z Fraction 1 : ~ 4 0 0 ; 425; 445; 472-4 m/z 2: 467 m/z 3 : ~ 458 m/*

4: ~ 4 5 0 ; 481 mtz

3 : ~ 450; 471 mt~

Fraction 1 : 2:

~ 454 mt~

Absorption maxima of chromatographic fractions in hexane

100/0

100/0 23/77

100/0 17.5/82.5 100/0

100/0 100/0 21/79 36/64 100/0

100/0 30/70 100/0 100/0 --

100j0 100/0 (13/87 on hydrolysis 58/42 (9/91 on hydrolysis) 46/54

--

Partition ratio in hexane/ 95% M e O H

(e) (a) (?) (e)

Astaxanthin esters (e)

Astaxanthin esters (e) Astaxanthin esters (a)

Astaxanthin esters (e) Astaxanthin esters (a) Astaxanthin esters (e)

Zeaxanthin esters Astaxanthin esters Astaxanthin esters Unfamiliar ketone Astaxanthin esters

Astaxanthin esters (e) Astaxanthin esters (a) Zeaxanthin esters (e) Astaxanthin esters (e) Unrecognized ketone

~ 100

80.7 19-2

80.7 19.2 ~ 100

0-8 74"0 21.6 3'5 ~ 100

85.6 14.4 1.8 95'6 2-6

10.5

7.1

Zeaxanthin esters (e) Unfamiliar hydroxy- "1 ketone esters (a)* .~ Unfamiliar ketone

82'4

Proportion o f total carotenoids (%)

Astaxanthin esters (e)*

Traces of contaminated astaxanthin

Identity

Metridium senilefimbriatum

* T h e ~0arenthetical letters (e) and (a) signify respectively epiphasic esters, selectively soluble in hexane, and ambiphasic esters, distributed between hexane and 95 ~o methanol in the partition test. Eplphaslc esters of astaxanthm are regarded as revolving long-chained fatty acids, while ambiphasic distribution has been found to characterize esters of the same c o m p o u n d conjugated with shorter-chained acids of the series.

Orange-red gonadal tissue analyzed, 14 g

Red-brown (immature or spent ~), 280 g Soma analyzed, 153 g 0.28

Ovaries, 4 g

Brown, ~, 45 g Soma analyzed, 29 g

Ovaries, 12 g

Tan, ~, 169 g Soma analyzed, 68 g

Ovaries, 13 g

1'0

0"70 (3'5)

White, ~, 259 g Ripe ovaries, 128 (26) g

Red, ~, 250 g Soma analyzed, 147 g

0'007

White, C2, 146 g

Colour variant (sex; wet weight, g)

Concentration of total carotenoids (mg/100 g wet)

TABLE I ~ M E T A B O L I C FRACTIONATION OF CAROTENOIDS AMONG COLOUR-VARIANTS OF

re

©

t"

r~

<

© S

(2

©

O

Z

CAROTENOID FRACTIONATION I N THE PLUMOSE ANEMONE M E T R I D I U M

181

maximal contraction, resulting finally in the extrusion of creamy spermary material through the lateral pores of the column. Microscopic inspection of this exudate revealed active spermatozoa. Eggs were not thus extruded by females, but were lost only if the integument, e.g. of the pedal disc, were accidentally torn. Adults with pigmented columns, even if sexually ripe, could not be diagnosed by translucence, and therefore had usually to be dissected for this purpose. A ripe, white female Metridium, when laid open, presented an aspect reminiscent of raspberry jam spread upon white ice-cream as the magenta-red to purplish ovaries lay over and against the completely white somatic tissue. The ovaries yielded relatively rich supplies of carotenoids (Table 1). A ripe, white male, on the other hand, revealed spermaries bearing only a faint cream colour, and yielded only traces of carotenoid material. Comparative findings. Table 1 reveals several conspicuous results of this brief survey. Total concentrations of carotenoids, in mg/100 g of wet tissue, are of a fairly low order, but are highest in the red genotype (exclusive of the ovarian tissues and eggs of the ripe white female). The greatly preponderant fraction in all types was esterified astaxanthin (i.e. from 82 to ca. 100 per cent). This was epiphasic for the most part, suggesting long-chain fatty acid conjugants, but in some instances minor complements of ambiphasic esters were found as well, indicating conjugation with short-chain fatty acids (as in the shank-akin of the American flamingo, Fox & Hopkins, 1966). There were, in addition, occasional minor fractions of esterified zeaxanthin and of some unfamiliar, suspectedly ketonic carotenoids. No carotenes were ever recognized. Astaxanthin was confirmed by its characteristic broad, rounded and symmetrical absorption curve, exhibiting a single maximum at from 465 to 468 m/z in hexane and 490 m/z in pyridine, while its acidic derivative, astacene, produced by alkaline oxidation, manifested somewhat elevated maximal absorption, e.g. at 472474 mt~ in hexane and about 492494 m/~ in pyridine. For the analysis, the ripe ovaries of the white female had been scooped away from the somatic tissues, along with much mucus and other adventitious material. The ovarian material itself amounted, in all probability, to no more than about one-fifth of the total mass taken. Hence the estimated corrected values shown in parentheses under that tabular entry. In subsequent analyses, the ovarian strands were merely lifted away with forceps, thus separating them essentially free from contaminating materials. From their microscopic appearance as relatively large and deeply pigmented, discrete ovate bodies, and in consideration of the relatively rich yield of carotenoids, the ova from the white Metridium were judged to have attained a more advanced stage of maturity than had those of other females analyzed. It may hardly be doubted that depth of pigmentation is a function of size and of ripeness. Hence the lower values for overall ovarian carotenoids in other pigmented variants examined in this work may signify merely less mature stages of sexual development. The large white male Metridium, bearing pale cream-coloured spermaries, yielded no more than about 7/zg of astaxanthin per 100 g wet tissues. Nor were

182

DENIS L.

Fox, GEORGE F. CROZIER AND V. ELLIOTT SMITH

five large, mature white males induced to store any obvious carotenoid material in their tissues after receiving daily for 6 weeks small bits or a fine brei of fresh, pink salmon flesh, well known to be rich in astaxanthin. The food was accepted by the animals, and wastes were discharged, but the carotenoids, whether assimilated or rejected, seemed to have been completely bleached or otherwise degraded. Clearly, an oxidase must be sought, presumably in the mesenterial digestive tissues, which must be destructive of all dietary carotenoids in the white males. One is led to reflect also upon possible correlative proportions of saturated to unsaturated lipids, e.g. fatty acids in glyceryl esters and ethers (cf. Lewis, 1962, 1965) stored by Metridium varieties suspected of possessing different degrees or levels of carotenoid oxidases. B. British Metridium Our finding no acidogenic carotenoids other than astaxanthin itself in the Pacific Coast Metridium led to a searching reinvestigation of the red variety of British Metridium, wherein an astaxanthin-like compound "metridene" (the acidic derivative) or "metridioxanthin" (the naturally occurring compound) had been referred to in the earlier survey (Fox & Pantin, 1941 ; Fox, 1953). In our current reexamination, however, we encountered only in occasional specimens minor fractions of an acidogenic congener of astaxanthin. Specimens 1, 3 and 4 from Menai Bridge and 2, 3 and 5 from the Cumbrae Islands yielded such fractions, which commonly showed a broad, rounded maximum centering at around 455460 m/~. In most instances the fraction occurred as a pinkish (sometimes a yelloworange) ester in the second, third or fourth zone of a chromatogram, but in one instance (Menai Bridge, No. 3) it appeared as a pink band nearest the origin, where it was unesterified, hence most firmly adsorbed. On hydrolysis, the unidentified esters yielded one or another acidogenic carotenoid exhibiting maximal absorption at 465 m/~ in hexane, 480 m/~ in pyridine, and from 495 to 500 m/~ in carbon disulphide. The material clearly was not astaxanthin, but gave a maximal absorption value in carbon disulphide recalling that of "metridene" (see Table 3). It must be remembered, however, that the analytical procedures and spectrometric instruments were far less reliable or accurate in 1939 than they are today. Reference is directed, for instance, to the relatively irregular absorption profiles published in the 1941 paper for astaxanthin, astacene and the other supposed fraction of acidogenic carotenoid; the earlier curves are in contrast with the smooth, rounded and symmetrical maxima determined now, and in the present work for such compounds. Because the profiles for astacene, astaxanthin and like compounds are now so well known (cf. Fox, 1962), they have not been reproduced in this paper (Tables 1 and 3). Column chromatography serves less critically than does the thin-layer technique, used in this work on most of the British forms, for the resolution of closely similar compounds. Moreover, the older visual spectrophotometer methods did not yield the smooth, reliable data obtainable today with the use of photoelectric equipment.

CAROTENOID FRACTIONATION IN THE PLUMOSE ANEMONE M E T R I D I U M

183

Hence the earlier analyses, based upon column chromatography, involving less critical eluants for fractional resolution, and providing somewhat less consistent or regular spectral profiles for the acidogenic fractions "metridene" (or "metridioxanthin", as the neutral compound was later called) than for astacene, most likely reflected the contamination of astaxanthin or of astacene by minor aberrant fractions such as were found in the present work, accompanying astaxanthin esters but now separable therefrom on thin-layer chromatograms. These minor fractions, involving from less than l per cent to as much as 9 per cent of total recovered pigment, exhibited in some instances (e.g. Cumbrae Islands specimens 2 and 3) spectral maxima in carbon disulphide reminiscent of those reported for the "metridene" type (see Table 3). From the analysis of twenty British specimens, fourteen of these individually, we are led to conclude that the red form of M . s. senile, containing astaxanthin esters as the greatly preponderant carotenoid fraction, in numerous instances stored also astaxanthin esters contaminated with unresolved fractions of the similar congener, and thus conferred upon the composite fraction spectral maxima somewhat lower than apply to the relatively pure material. Thus the issue of "metridioxanthin", as originally and only approximately described, now remains in considerable doubt. By reference to Table 2 it is manifest that, as in much of the American Metridium material, epiphasic and ambiphasic esters of astaxanthin together aggregate the preponderant carotenoid type present in the British sub-species. It amounts to 90 per cent or more in all save three analyses, wherein we find aggregate values of 67, 78.74 and 86.24 per cent. Uncombined astaxanthin was encountered or suspected in only a few instances. Zeaxanthin esters, usually but not always confined to the epiphasic class, represent the second most common carotenoid species; they were not invariably found, but showed a range of from 0-98 to as high as 17.8 per cent, while ambiphasic zeaxanthin esters and the free xanthophyll usually occurred in minor concentrations. The unidentified acidogenic carotenoids and/or their esters found in occasional specimens already have been discussed and are tabulated in Table 3. It has been a source of some surprise to find in the present analyses no evidence for carotenes, whereas fl-carotene had turned up in minor proportions in extracts of some red and of various brown varieties of M . s. senile during the earlier survey at Cambridge. Moreover, in that work, certain lutein-like or other xanthophylls had been encountered in various specimens, whereas in the present investigation the only conventional neutral xanthophyll in both Pacific and British forms was indistinguishable from zeaxanthin, mostly in the form of esters. Finally, we are not able to explain to ourselves the diversity between current quantitative data and the earlier comparative finding, e.g. for the white versus the red Metridium, wherein the respective figures of 1.76 and 14.96 rag/100 g had been reported. From the present studies, values more llke one-tenth of those earlier reported would have been consistent. True, the earlier work had been done with less precise methods, but only a gross error in the purity of the astaxanthin then

W h o l e : 13-92 (2"57)

5

(3"3) (1"8) (2"57) (2"14)

Whole: Whole: Whole: Whole:

1 2 3 4

43"86 40"67 29'19 12"67

W h o l e : 31.3 (0.81) W h o l e : 27.0 (1"55)

3 4

13 (21/79") 95 (63/37)

Ambiphasic

91"55

87"75 81"62 78'89 86"10

59.5 85"6

85"1 63"2

70"55

14"23 (e)

8"2 (37/63)

11 (e) 6 (e)

---

Zeaxanthin esterified

1'46

2"47 10"40 7"35 7"04

5'91 (e)

6"52 (e) -3"72 (e) 0"96 (e)

1"2 (e) 17.8 (e) +7"7 (43/57) 19.24 (12/88) 16.35 (e) 7"4 (10/90; 4"8 (e) free)

9'3 3'8 (free)

10'40

-89 (77/23) -94 (89/11) -ca. 100 (82/18) 59'5 (92/8) 32"3 (70/30)

77 --

Epiphasic

Astaxanthin esters

--

1"0 (acidogenic) 7"95 (Table 3) 9"05 (Table 3) 1-26 (unknown keto-ester) 4"72 (unrecognized) 0"73 (Table 3)

2"50 (Table 3) 0'9 (Table 3)

7'1 2"37 1'2 2"4 -1"03 --

3'8 (Table 3)

2"7

--__ --

---

Other

0"6

2"12

-----

10 5

Free

* Parenthesized fractions signify the distribution of a fraction between hexane and 95% methanol; the letter (e) denotes complete epiphasic behavior as observed visually. t T h e s e were animals of fairly uniform weight: 19"2, 23.6, 18.6, 23.2, 19"8 and 17"0 (average 20'2 g).

C u m b r a e Islands

W h o l e : 28"6 (1'40) W h o l e : 20'8 (1"45)

W h o l e : 8 (1"88) W h o l e : 14.2 (1.20) W h o l e : 36"0 (0'69) W h o l e : 38"0 (0'40) Six whole: 121"4 (1"07)

S o m a : 46 (0"21) G o n a d : 9 (0"95)

W e t weight (g) (total carotenoid conc. mg/100 g)

1 2

1 2 3 4 5

Swansea

Menai Bridge

1

Specimen no.

Plymouth

Locality of collection

Metridium senile senile

Carotenoid fractions per cent of total extracted

T A B L E 2 - - R E L A T I V E PROPORTIONS OF CAROTENOIDS IN THE RED FORM OF B R I T I S H

fa0

t" O ,q

.<

O b~

(3

O

O

4~

~o

CAROTENOID F R A C T I O N A T I O N I N THE P L U M O S E A N E M O N E M E T R I D 1 U M TABLE

3--SPECTRAL Metridium,

185

M A X I M A OF U N R E C O G N I Z E D A C I D O O E N I C CAROTENOIDS FROM B R I T I S H

COMPARED W I T H SPECTRAL M A X I M A OF A S T A X A N T H I N AND ASTACENE

Max. (mtz) Compound or source of fraction Astaxanthin B & L (1966) HR (1938-39) Astacene B & L (1966) HR (1938-39) "Metridioxanthin" HS (1938-39) HR (1938-39) "Metridene" (free acid) HS (1938-39) HR (1938-39) Menai Bridge No. 1 (Fraction 3; pink on column; epiphasic ester, 3"8%) Menai Bridge No. 3 (Fraction 6, pink on column; uncombined; 2"5%) Menai Bridge No. 4 (Fraction 4, pink on column; epiphasic ester; c a 1%) Cumbrae Islands No. 2 (Fraction 2; pink on column; epiphasic ester; 7"95%) Cumbrae Islands No. 3 (Fraction 4, yellow on column; epiphasic ester; 9-05%) Cumbrae Islands No. 5 (Fraction 4; yellow on column ; epiphasic ester; 0"73%)

Hexane

Pyridine

Chloroform

467 --

490 501

483

492 497

488

472-474 --

ca.

ca.

502 498-505

ca.

---

495 --

m

---

495 --

m

455 (465 after hydrolysis)

Carbon disulphide

m

508 511 495 500-505 495 495

480 (after hydrolysis)

456 460 (464 after hydrolysis) 456 461

495 480 (after hydrolysis)

500 (after hydrolysis)

456

B & L refers to the Bausch & Lomb 505 spectrometer used in this research; HR denotes the Hartridge Reversion spectroscope, and HS a Hilger spectrometer, both employed at Cambridge, 1938-39. used, or in the instrumentation or operation thereof, could have been responsible for the degree of departure. T h e value of 1-76 mg/100 g is, in any event, too high even for a female carrying fully ripe eggs, as shown b y the analysis of such a specimen in the present work (i.e. only some 0.7 rag/100 g of whole animal).

186

DENIS L.

Fox, GEORGE F. CROZIER AND V. ELLIOTT SMITH

In this respect, it is interesting to observe that the white female Metridium, while mobilizing no residual, intact carotenoids to her somatic tissues, manages, as do the other female colourotypes, to assimilate astaxanthin, perhaps as an end product of her carotenoid metabolism, in her ripening eggs. Moreover, the coloured varieties carry the same pigment in their somatic tissues as well. Such observations suggest the likely presence of carotenoid oxidases, capable of different levels of performance. In the soma of whites for example, such an oxidase system would appear to carry the oxidation of dietary carotenoids to completion (cf. the white males fed on astaxanthin-rich food). In the soma or ovaries of white and of other female varieties, and in the somatic tissues of red forms, an oxidase may be capable of carrying the process as far as the dihydroxy-diketonic stage prevailing in astaxanthin. Alternatively, the astaxanthin found may represent merely selective retention, e.g. from a microcrustacean source in the diet. It should be remembered, however, that (1) astaxanthin is rather less stable chemically than several other carotenoids, and that (2) it is known to be elaborated from other dietary sources by other animal species, such as flamingos (Fox, 1962; Fox & Hopkins, 1966). Moreover, many invertebrates are able to store modified carotenoids not commonly found in vertebrates. The fate of specific dietary carotenoids in Metridium calls for further investigation (e.g. what happens to ingested carotenes, not always found in the animal ?). This plumose anemone, possessing very numerous short, ciliated tentacles, is said to subsist in nature chiefly upon plankton (Stephenson, 1935). Hence assorted carotenoids of marine eggs, small animals, larvae, algae, bacteria or detritus from various sources should be available in the food. It was pointed out earlier (Fox & Pantin, 1941) that the various colour varieties of this anemone are found side by side in nature, and that the pigmentation does not arise from the intake of different foods, but that the capacity for selective fractionation of carotenoids is inherited. It was further suggested that pigmentary variations do not reflect environmental adaptation, such as warning coloration, camouflage or the like, also that there was no evidence to suggest that the pigments might serve directly any physiological needs or the general metabolic economy of the animals. The view then taken, and still endorsed, is rather that the fact of pigmentation in Metridium appears to illustrate an example of Poulton's category of "nonsignificant colours", wherein the pigment residues may represent by-products of metabolic processes serving other needs. The white variety, often found to be predominant among natural populations of this species, is regarded as being comparable to an instance either of dominant albinism or perhaps of a universal recessive (Fox & Pantin, 1941). The genetically controlled capacity to metabolize coloured molecules in a number of ways and combinations is regarded as indicating relative immunity from environmental restraints, rather than representing any direct functional adaptation. In summary: since wide individual variations occur even within colour-classes, as discovered in the Cambridge work, and in view of the small number of Pacific

CAROTENOID FRACTIONATION IN THE PLUMOSE ANEMONE M E T R I D I U M

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Metridium analyzed in the present study, only a few generalized comparisons may be drawn. First, as to similarities, astaxanthin esters constituted the greatly predominant fraction in ovaries of white and of all specimens analyzed, as well as in somatic tissues of reds in both sites, and in red-brown animals. Esters of a neutral xanthophyll, zeaxanthin, were found in secondary amounts in most of the animals. As to differences, these were relatively minor, and not always identifiable. T h e Pacific sub-species yielded astaxanthin as the only recognized hydroxyketonic carotenoid, while there were occasional and minor instances of unrecognized, neutral ketonic carotenoids as well. A number of the British specimens, however, yielded minor proportions of ketonic, acidogenic carotenoids, or esters thereof, in addition to the esters of astaxanthin. Astaxanthin esters were recovered from the somatic tissues as well as from the ovaries of brown Pacific Metridium, whereas there are recorded instances wherein brown forms of the British sub-species (immature ?) had yielded no acidogenic carotenoids, but instead a considerable variety of neutral xanthophylls in minor concentrations, along with/%carotene. Carotenes appeared in small amounts rather frequently in animals of the brown and the yellow-orange varieties, but infrequently in the red ones, studied at Cambridge. In the present work, no carotenes whatever were detected in Metridium from either locality. On the basis of these limited studies, it would appear to us that the minor morphological differences reported to exist between the two sub-species (Professor Hand) are accompanied by only trivial differences in their carotenoid biochemistry. It is, in fact, conceivable that the less common and relatively small carotenoid fractions sometimes encountered may represent, not a static condition of storage, but chemical steps in the progressive oxidation of dietary carotenoids toward the relatively longer-lasting end product, astaxanthin. Acknowledgements--We are grateful and indebted to several friends and colleagues for supplying us with anemones for our analyses, namely: Mr. Donald W. Wilkie, Director of the Aquarium and Museum at the Scripps Institution, for the various colour-variants of Metridium senile fimbriatum from Pacific Coast waters; Dr. J. E. Smith, Director of the Marine Biological Laboratory of the United Kingdom at Plymouth; Professor E. W. KnightJones of the Department of Zoology, University College of South Wales at Swansea; Dr. D. J. Crisp, Director of the Marine Laboratory at Menai Bridge, University College of North Wales; Dr. S. M. Marshal and Dr. Ronald Currie, Director, Marine Station at Millport, Cumbrae Islands, for numerous specimens of the pink to red variant of Metridium senile senile gathered at the respective sites and air-mailed to us preserved as requested. We gladly take this opportunity to thank also the National Science Foundation, Division of Metabolic Biology, for a research grant which has afforded extensive support for these investigations. REFERENCES

Fox D. L. (1953) Animal Biochromes and Structural Colours. Cambridge University Press. Fox D. L. (1962) Metabolic fractionation, storage and display of carotenoid pigments by flamingos. Comp. Biochem. Physiol. 6, 1--40.

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