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Adv. Mar. Biol. Vol. 16, 1979, pp. 309-381 I. 11. 111. Iv. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. xv. XVI. XVII. XVIII. XIX. xx. XXI. PIGMENTS OF MARINE INVERTEBRATES G. Y. KENNEDY The University of Shefield, Shefield, England Introduction . . .. .. .. .. .. .. .. Protozoa .. .. .. .. .. .. .. .. Porifera. . . . .. .. .. .. .. .. .. Coelenterata . . .. .. .. .. .. .. .. Ctenophora . . .. .. .. .. .. .. .. Platyhelmiiithes . . .. .. .. .. .. .. B. Acanthocephala .. .. .. .. .. .. Nemathelminthes . . .. .. .. .. .. .. A. Nematoda . . .. .. .. .. .. .. Rotifera .. .. .. .. .. .. .. .. Nemertini . . .. .. .. .. .. .. .. Annelida, Echiuroidea, Sipunculoidea, Priapuloidea and Phoronidea A. Crustacea .. .. .. .. .. .. .. B. Arachnida . . .. .. .. .. .. .. Mollusca .. .. .. .. .. .. .. .. Arthropoda .. .. .. .. .. .. .. .. C. Myriapoda . . .. .. .. .. .. .. Chaetognatha . . .. .. .. .. .. .. .. Brachiopoda . . .. .. .. .. .. .. .. Polyzoa .. .. .. .. .. .. .. .. Echinodermata .. .. .. .. .. .. .. Pogonophora . . .. .. .. .. .. .. .. Tunicata . . .. .. .. .. .. .. .. Comment . . .. .. .. .. .. .. .. Acknowledgements . . .. *. .. .. .. .. References . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 309 312 314 319 326 326 329 329 331 332 332 333 336 336 342 343 343 366 356 366 367 364 364 364 366 366 " 0 Sea! Old Sea! who yet knows half Of thy wonders or thy pride? " Gosse: Aquarium 226-227. I. INTRODUCTION Marine plants and animals are often very brilliantly coloured, especially those from tropical waters. Even in temperate climes many animals of the sea-shore, when viewed in quantity as in a rock pool association, present a fine sight, but in warmer waters, corals and their attendant fauna and flora provide a pageant of great beauty. Marine organisms also display many examples of pattern, an aspect discussed 309 A.P.B.-16 11

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Page 1: [Advances in Marine Biology] Advances in Marine Biology Volume 16 Volume 16 || Pigments of Marine Invertebrates

Adv. Mar. Biol. Vol. 16, 1979, pp. 309-381

I. 11.

111. Iv. V.

VI. VII.

VIII. IX. X.

XI.

XII. XIII. XIV. xv.

XVI. XVII.

XVIII. XIX. xx.

XXI.

PIGMENTS OF MARINE INVERTEBRATES

G. Y. KENNEDY

The University of Shefield, Shefield, England

Introduction . . .. . . . . .. .. .. .. Protozoa .. .. .. . . . . .. .. .. Porifera. . . . .. .. .. . . .. .. .. Coelenterata . . . . .. . . . . . . .. .. Ctenophora . . .. .. . . . . .. .. .. Platyhelmiiithes . . .. . . . . .. .. . .

B. Acanthocephala .. .. . . . . . . ..

Nemathelminthes . . . . .. . . . . . . .. A. Nematoda . . . . .. . . . . .. ..

Rotifera .. . . . . . . . . . . . . . . Nemertini . . . . . . . . . . . . . . . . Annelida, Echiuroidea, Sipunculoidea, Priapuloidea and Phoronidea

A. Crustacea .. .. .. . . . . .. .. B. Arachnida . . . . . . . . . . .. ..

Mollusca .. .. . . . . .. .. .. . .

Arthropoda .. . . . . . . . . . . . . . .

C. Myriapoda . . . . . . . . . . . . ..

Chaetognatha . . . . .. . . . . .. .. . . Brachiopoda . . .. .. . . . . .. .. .. Polyzoa .. . . . . . . . . .. .. .. Echinodermata .. .. . . .. .. .. .. Pogonophora . . .. .. . . . . .. .. .. Tunicata . . . . .. . . . . . . . . .. Comment . . .. .. . . . . . . .. . . Acknowledgements . . .. * . .. . . . . . . References . . .. .. .. .. . . .. ..

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309 312 314 319 326 326 329 329 331 332 332 333 336 336 342 343 343 366 356 366 367 364 364 364 366 366

" 0 Sea! Old Sea! who yet knows half Of thy wonders or thy pride? "

Gosse: Aquarium 226-227.

I. INTRODUCTION Marine plants and animals are often very brilliantly coloured,

especially those from tropical waters. Even in temperate climes many animals of the sea-shore, when viewed in quantity as in a rock pool association, present a fine sight, but in warmer waters, corals and their attendant fauna and flora provide a pageant of great beauty. Marine organisms also display many examples of pattern, an aspect discussed

309 A.P.B.-16 11

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The phylogenetic tree FIG. 1. The phylogenetio tree (from Scheuer, 1973).

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PIGMENTS OF MARINE INVERTEBRATES 311

in most fascinating style by the late T. A. Stephenson (1944) in his beautiful little book “Sea Shore Life and Pattern.”

In his stimulating article on Marine Natural Products, Thomson (1978) writes : “ There is no clear explanation for chemists’ neglect of marine products, although several contributory factors come to mind. Chief among these is the relative difficulty of collecting material which may be compounded by the problem of identification. While collecting intertidal species is easy enough, in deeper waters diving, trawling or dredging is necessary, and in some parts of the world the local marine fauna and flora have not been studied, and there is no taxonomic literature available. Sometimes there is no local expertise to hand where it might reasonably have been expected: e.g. there is no algologist in Aberdeen and only one in the whole of South Africa. Hence it is not unusual to find in the current literature interesting compounds reported from unidentified sources.” Later on : “ Numerous marine animals are brightly coloured but little is known about the pigments.”

It is true that at times the study of natural pigments has been desultory and empirical but, as we hope to show in this chapter, a good deal is known about many of them, and we owe our knowledge of many quinone pigments to Professor Thomson himself, and his school in Aberdeen.

Marion Newbigin (1898) gave three reasons why the biologist should be interested in the colours of organisms :

1. Conspicuousness of colour phenomena in an objective survey of

2. Relation of these colours to current theories of evolution ; 3. Their importance in comparative physiology.

animals and plants ;

The colours of living things are the visual result of three different processes :

1. Chemical: the metabolic formation of natural pigments, or the storage of ingested pigments, both consisting of coloured molecules which reflect and transmit parts of visible light : i.e. chemical pigments.

2. Physical: colourless structures which include laminations, striations, ridges, air bubbles, crystals, particles etc. which split light into its con- stituent colours by reflection, scattering and interference : i.e. structural colours.

3. A combination of 1 and 2. In this review, we shall consider only the chemical pigments of

marine invertebrates by a discussion of their occurrence, phylum by

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312 Q. Y. KENNEDY

phylum, with some mention of their metabolism and speculation on their functions. The order of classification followed is that of the Ply- mouth Marine Fauna List of the Marine Biological Association of the United Kingdom, 1957. There is a most useful table (Table 4.1) in the book by Needham (1974) embodying “ The major taxa of animals in relation to their chromatology.”

“ Their colours and their forms were then to me an appetite . . . . . . . 1 ,

Wordsworth: ‘‘ Lines composed a few mil@ from Tintern Abbey ”.

11. PROTOZOA Very little has been done on the pigments of marine Protozoa,

possibly because in many instances it is very difficult to obtain quan- tities sufficient to make a thorough chemical examination.

The amoeba Janickina (Paramoeba) pigmentifera is parasitic in the coelom of the chaetognaths Sagitta and Spadella, and contains pigments which have not yet been identified (Hyman, 1959). Several blue-green, brown, yellow and purple pigments are found in some heterotrich ciliates ; some of these are fluorescent and photodynamic. Carotenoids have also been reported.

Stentor coeruleus has been extensively studied by Tartar (1961) in a comprehensive monograph which describes some marine species :

8. multiformis is reported from salt or brackish water, and is blue- green, with pigment stripes ; S. pygmaeus with a chitinoid case is found attached to some gamma- rids in the depths of the Sea of Baikal. This has a dark pigment; S. auriculata Kent and S. auriculatus Kahl were shown by Faur6- Fremiet (1936a) to belong to the genus Condylostoma; S. acrobaticus said to be found on a branch of Fucus-reported unpigmented.

Some chemistry has been done on the pigments of Stentor and its species and relatives. The blue-green pigment of S. coeruleus, ‘‘ stentorin”, is probably also found in S. multiformis, S. amethystinus and S. introversus. Stentorin is very similar to hypericin (the pigment from some species of Hypericum, notably St John’s Wort, H . perforatum) in fluorescence and U.V. visible absorption spectra. The pigment has the structure of a tetra-cr-hydroxynaphthodianthrone. Another pigment, “ stentorol”, was extracted from Stentor niger by Lankester (1873), and this yellow pigment was studied by Barbier, FaurB-Fremiet and Lederer (1956) who

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PIGMENTS O F MARINE INVERTEBRATES 313

isolated it as a black powder giving a red solution in chloroform and red fluorescence in U.V. light. They suggested that it was a polyhydroxy- quinone.

Zoopurpurin extracted from Blepharisma undulans by Arcichovskij (1 905) and examined fairly recently by Sevenants (1 965) is a mixture of two compounds both very similar to stentorin and hypericin, and has been the subject of further study by Giese and Grainger (1970).

It may be seen from the discussion of these pigments that investiga- tion of the ma,rine species of Stentor and its relatives might be well worth while. The function of these pigments is still unkown. They may render the animals sensitive to light or, since they are toxic to other Protozoa, they may have some protective value. It is interesting that if Stentor engulfs another Stentor, the pigment of the victim is not assimi- lated but is ejected as a green excretion vacuole.

In the Folliculinidae, which often attach themselves to mollusc shells and tunicates, Faur6-Fremiet (1936b) found green, blue and red- dish-violet pigments which may be polycyclic quinones. In the blue Folliculina ampulla, closely related to the stentors, he found many blue granules close to the macronucleus. Another heterotrich Fabera salina, found in salt works in France and saline pools in Russia, Rumania and California, contains a dark pigment which, when extracted, is purple-red with a fine red fluorescence in U.V. (Fontaine, 1934). The yellow-green solution in pyridine gives absorption bands at 612 and 566 nm, and it has been suggested that this is also a polycyclic quinone.

Although the hypotrich ciliate Holosticha rubra (previously known as Kernopsis rubra) found in aquarium tanks at the Plymouth Labora- tory obviously invites investigation, nothing is known about its pig- ment ; there is also a yellowish variety H . rubra var. Jlava.

Some Protozoa require pterins, principally biopterin and neopterin, as co-enzymes for some redox systems (Kidder, 1967) and others need pteroylglutamic acid (Kaufman, 1967).

The hermit crab Eupagurus prideauxii is parasitized by ciliates Polyspira spp. and Gymnodioides spp., which take up blue caroteno- proteins from the host. The carotenoid is split off from the protein by digestion in the food vacuole and is attached to another protein, pro- ducing a new carotenoprotein which imparts a violet-red colour, or even blue or green, to the ciliate. This pigment is passed on to the daughter cells in fission. In similar vein, the copepod Idyafurcata contains a blue carotenoprotein in epidermis and retina, and a deep orange carotenolipo- protein in the blood and eggs. The parasitic ciliate Spirophrya takes up these pigments and reconstitutes them after digestion, to become pig- mented itself.

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314 Q. Y. EBNNEDY

There is an unidentified ethanol-soluble red pigment in cytoplasmic granules in the foraminiferan Myxotheca arenilega-probably a caro- tenoid derived from the crustaceans on which it feeds. Newbigin (1898) drew attention to this pigment, and also to some species of the rhizopod Globigerina, which were described by Agassiz (1888) in his account of the cruise of the Blake, as " floating scarlet masses on the surface of the sea."

Radiolarians often have a pigmented body, the phaeodium, which suggests a brown colour.

It is well known that the protozoans Noctiluca miliaris and Pyro- cystis noctiluca have great powers of phosphorescence, produced from minute granules of the system :

luciferase Luciferin + oxygen - oxyluciferin + water + light

Less well known is the pink colour of Noctiluca which " may be thrown upon the shore in such numbers as to form a coloured layer along the beach, the shore water resembling thick tomato soup " (Russell and Yonge, 1975). The identity of this pigment is unkown, but it is likely to be a form of oxyluciferin. The luciferins of known structure are heterocyclic chain-linked molecules whose colours, in their oxidized forms, may range from yellow through red to purple.

The protozoan Opalina ranarum, parasitic in the intestine of frogs, does not fall within the marine category, but is mentioned because of its unique green pigmentation by biliverdin from the bile of the host. Another protozoan, Nassula, has a blue pigment which is probably derived from the Oscillatoria of the food.

Nusslin (1884) described a beautiful violet pigment in Zoonomyxa violacea from the Herrenwieser Lake. The protoplasm is filled with many small violet vacuoles which impart a violet colour to the whole animal. Amphizonella violacea contains a granular pigment which is similar in many ways to that of Zoonomyxa.

111. PORIFERA The sponges provide many fine examples of vivid pigmentation as

well as the more sombre browns, black and grey with off-white. The lipid-soluble nature of some of the yellow and red pigments of sponges was described by Krukenberg in 1880-1882 (see Krukenberg, 1882). MacMunn (1883, 1890) examined Halichondria albescens, Halma buck- lanai and Leuconia gossei and found " lipochromes '' giving one strong absorption band in his simple spectroscope. The pigments producing the most vivid coloration of sponges are predominantly carotenoids

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PIGMENTS OF MARINE INVERTEBRATES 316

(Figs. 2, 3, 4), with a preponderance of carotenes over xanthophylls, but there are instances of the occurrence of other types of pigment.

Lonnberg (1931, 1932, 1933) examined many sponges (among other marine animals) for carotenoids, but his studies were not sufficiently

FIG. 2. Structures of some cmotenoids.

complete to enable the pigments in his extracts to be characterized; this is a great misfortune, considering the amount of work done. None of the absorption spectra given by Lonnberg are near enough to the accepted maxima of authentic carotenoids to identify his pigments. However, because of the striking coloration of many sponges, their

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316 0. Y. KENNEDY

FIG. 3. Structures of carotenoids.

availability in quantity and the continuing active interest in carotenoids coupled with well-developed analytical methods, much of the work of Lonnberg has been repeated and extended.

Astacene (Fig. 3) was isolated and crystallized from the red " cocks- comb " Axinella crista-gulli by Karrer and Solmssen (1935) (but see later discussion on astacene). Lederer (1938), working with Suberites domuncula and Ficulina $GUS, reported that all the carotenoids in his extracts were epiphasic, even after saponification, and maintained that the pigments present were torulene (as in the red yeast Torula rubra), lycopene (Fig. 2) (as in the epidermis of the fruits of the tomato) and

CH CH, v CH, CH,

/ \

Zeaxanthin \/

CH, I

CH, I

CHa I

CH, '\ / /"\ I CH, C ~ C I I - C H ~ C - C H C H - C H ~ C - C H C H - C f i C H ~ C ~ C H - C H C H - C ~ C H - C 1 I ~ C CH,

HOCH I 11 C.CH, I1,C.k AHOF

CH, /

CH,

I I1 BOCH C.Ct1,

Xant hophyll H&.d (!HOE

FIG, 4. Structures of carotenoids.

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PIGMENTS OF MARINE INVERTEBRATES 317

a-, /3- and y-carotenes (Fig. 2) with a small fraction of xanthophyll (Fig. 4) in Ficulina only. A propos this report of torulene by Lederer, Fox, Updegraff and Novelli (1 944) often encountered this carotenoid in deep marine muds, and they suggested that the sponges which Lederer had examined may have ingested numbers of red Torula species known to occur in the sea (ZoBell, 1946.) If this is true, it seems odd that torulene has not been found in other detritus feeders, although of course there are isolated cases of specific retention of carotenoids and other pigments by marine animals.

Drumm and O’Connor (1940) and Drumm, O’Connor and Renouf (1 945) isolated and crystallized echinenone (4-keto-fi-carotene) from Hymeniacidon perleve ( = Hymeniacidon sanguinea) and also detected a-carotene in traces. Lederer (1938) also reported a brown-orange carotenoprotein in Ficulina Jicus.

There are many papers describing carotenoids of sponges, and reference should be made to the books of Karrer and Jucker (1950), Goodwin (1952)) Fox (1953, 1976) and the short review by Goodwin ( 1 96th).

CH3

FIU. 5. Struoture of renieratene.

In recent times, the work of Yamaguchi (1957) who worked with the sponge Reniera japonica brought to light two new carotenoid hydro- carbons, together with ,%carotene. These were named renieratene, iso-renieratene and renierapurpurin. The two first-named are unique carotenoids in that they have aromatic rings (Fig. 5). Their absorption spectra are very near to those of y-carotene and /I-carotene, respectively. The sponge is able to aromatize the cyclic end-groups. Leprotene, present in some mycobacteria (Goodwin and Jamikorn, 1956) has been shown by Liaaen-Jensen and Weedon (1964) to be identical with iso- renieratene. Yamaguchi (1957) also found a pigment spectroscopically close to torulene in R. japonica, but it differs from torulene in having a keto-group. Goodwin (1968a) raises the question of the reported occurrence of torulene and y-carotene in sponges and wonders whether this will be supported by further work.

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318 0. Y. KENNEDY

Smith (1968) working with two red siliceous sponges Cyamon neon and Trikentrion helium, common on the sea floor off La Jolla, California, extracted a mixture of carotenoids containing unusual proportions of hydrocarbons from both. These included ,%carotene, 3, 4-dehydro- 8-carotene and some resembling 8-iso-renieratene. There was also a carotenoid tentatively identified as monohydroxy-fl-iso-renieratene and some 40% of the total carotenoid was a partially esterified dihydroxy- carotene not previously encountered, suggested by Smith to be a dihydroxy-bis-dehydro-8-carotene.

Tyrosinase has been found in the tissue fluids of the black Suberitee domuncub, the orange Tethya aurantia and Cydomium gigas by Cotte (1903), so that some of the dark colours and greys and blacks of sponges may be due to melanins. Some of the Aplysinidae contain pigments of the type described by Krukenberg (1882) as ‘‘ uranidines ”. These are yellow pigments, soluble in water and organic solvents with a green fluorescence (cf. Holothuria). The yellow pigment of Aplys im aerophoba blackens in situ after death, or when extracted ; it is blackened by boil- ing, alkaline pH or when shaken with air or oxygen. Acid pH prevents this change to some extent. The black material becomes insoluble and is precipitated or flocculated ; it is probably melanin. I have noticed that when the encrusting sponges Halichodria, Hymeniacidon and Hicrociona are collected, the torn edges readily darken during the period from the shore to the laboratory, and any material left overnight out of water is very dark the next day. Dark pigments also occur in Chondrosia.

Ray Lankester sent specimens of the Australian sponge Suberites wilsoni to MacMunn in 1890. Lankester had already named the striking purple pigment “ spongioporphyrin ”, and MacMunn found that it had a two-banded absorption spectrum with peaks at 571 and 627 nm. How- ever, he could not obtain a porphyrin after treating the pigment with concentrated sulphuric acid, and concluded that the name spongiopor- phyrin, while descriptively apt, was misleading. The pigment is still unidentified, but considering the absorption spectrum, colour and behaviour in various solvents with acid and alkali, one might speculate that it could be a trihydroxyanthraquinone.

MacMunn (1890) also examined another purple sponge, the hexa- cinellid Polypogon gigas, and found that the pigment was quite different from that of Suberites iuilsoni and very unstable. Kennedy and Vevers (1954) reported a small amount of a red-fluorescent pigment in Tethya aurantia. This had an acid number of 5 (non-esterified) so that it could have been free protoporphyrin but further investigation was prevented by scarcity of material. There were no chlorophyll derivatives.

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PIQMENTS OF MARIXE INVERTEBRATES 319

MacMunn (1890) extracted red-fluorescent “ chlorophyll-like ” pigments from a number of sponges, including Hymeniacidon sanguinea, Grantia seriata and Hircinia variabilis. These had a phaeophytin-like absorp- tion spectrum and almost certainly were derived from the chlorophyll of algal symbionts or epiphytes.

The green colour of the fresh-water sponge Spongilb viridis is due to symbiotic algae but that of the marine Halichondria panicea is not. This sponge contains two pigments-a yellow carotenoid resembling p-carotenone, and a blue one soluble in water which is pH and redox sensitive. This pigment is also soluble in ethanol but not in ether or acetone and is destroyed by boiling (Abeloos and Abeloos, 1932). It is interesting that the blue pigment is accumulated in the liver of the nudibranch Archidoris pseudoargus ( = Doris tuberculata) which feeds on the sponge. This may be a biliprotein like phycocyanin.

Hircinia variabilis and some of the sponges which Krukenberg examined (cited by Newbigin, 1898) were said by him to contain a red pigment “ similar to the pigment of red algae and which is readily decolourized by reducing agents.” This could be a biliprotein like phy coerythrin .

IV.COELENTERATA The Coelenterata have some of the most beautiful and spectacular

coloration, with the molluscs and echinoderms as runners-up. Members of the phylum, which includes the anemones, jellyfishes and corals, are found in greatest abundance in warm seas but, even in temperate climates, the colours to be seen in shallow water or in rock pools are striking.

There have been several reviews of coelenterate pigments, many of them extensive, the most recent being Fox (1974, 1976), Goodwin (1968b), and Fox and Pantin (1944), but the older accounts of Newbigin (1898) and of Verne (1926, 1930) still make fascinating reading and often provide useful and interesting facts, some of them long forgotten, from a time more tranquil and leisured than ours today. The reviews also provide many references to the original work so that the account to be given here will be confined to the most interesting work and the more recent discoveries.

There is good evidence that carotenoids are distributed through every branch of the coelenterates, as may be seen by referring to the useful table (Table 28) by Goodwin (1952).

The red pigment “ zoonerythrin ” first described by Bogdanow (1858) and later renamed tetronerythrin was found by Merejkowski (1881,1883) in Actinia ~ e ~ e ~ b ~ ~ a ~ ~ ~ e ~ u ~ ( =A. eguina), Aiptasia spp.,

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320 0. Y. KENNEDY

Cereactis spp and some hydrozoans. M. and R. Abeloos-Parize (1926) working with the red, brown and green forms of A. equina found an orange carotenoid in the red and brown variants and another red one in the red form. Further work by Lederer (1933) and Fabre and Lederer (1934) led to the isolation of a- and /3- carotenes, a xanthophyll and a red esterified carotenoid, actinioerythrin as carotenoprotein. A beautiful blue acidic carotenoid, violerythrin, was isolated by Heilbron, Jackson and Jones (1935) from hydrolysis of actinioerythrin.

Actinioerythrin is now known (Weedon, 1971) to be a mixture of fatty-acid esters of the parent carotenoid actinioerythrol, which has 2-nor end groups. Actinioerythrol has not been reported in nature, and neither has violerythrin, the exact structure of which is still unknown, but which certainly has cyclopentenedione end-groups.

The green variety of Actinia equina yielded a xanthophyll ester probably of taraxanthin or dinaxanthin, the green colour being due to the conjugation of the carotenoid with protein (see Cheesman et al., 1967).

Sulcatoxanthin, isolated by Heilbron et al. (1935) from Anemonia sulcata, has been identified with peridinin, the pigment of the alga Peridinium, but this may not be so (Weedon, 1971).

MacMunn (1890) investigated the bright red pigment from the red form of Actinoloba dianthus ( = Metridium senile) and much later, Heilbron et al. (1935) found this to be an ester of low melting-point which upon hydrolysis with sodium hydroxide gave a red sodium salt. Fox and Pantin (1 941) reported the pigment as distinct from astacene and named it " metridene " or more correctly, metridioxanthin. It is inter- esting that within the colour varieties of M . senile, the white ones contain some astaxanthin esters with free astaxanthin ; the brown have the least carotenoid, with esters of astaxanthin or metridioxanthin, carotenes and xanthophyll esters plus melanin ; the yellow, orange and red forms have large amounts of carotenoid with metridioxanthin or astaxanthin esters and some free pigments as well. This suggests that even very white animals (in any phylum) are worth investigating for pigments.

Fox et al. (1967) examined M . senile jimbriatum from the Pacific Coast and found rich quantities of red carotenoids in the eggs of the white genotype which rendered very little pigment while unripe. The carotenoid was reported as mainly astaxanthin accompanied by some unfamiliar ketones and " zeaxanthin-like " esters. The same was true of the ovarian tissue of the red and brown variants.

MacMunn (1885a) extracted a purple-brown pigment from Actinia mesembryanthemum ( = A . equina) with glycerine and named it actinio- haematin, a haemoprotein. Reduction produced a haemochromogen

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PIGMENTS O F MARINE INVERTEBRATES 321

spectrum, concentrated sulphuric acid formed haematoporphyrin. The same pigment was detected in Tealia felina and the white form of Metridium senile. Roche (1936) found actiniohaematin to be a mixture of cytochromes a,, b, and c with b, predominating. The whole spectrum of reduced cytochrome with b very intense may be seen in the muscula- ture of Hormathia coronata and Cereus pedunculatus. Band d of the cytochrome absorption spectrum can be seen in muscles of Actinia equina, Anemonia sulcata and Adamsia palliata. MacMunn also found biliverdin in the green base of Actinia equina.

The first reported observation of an invertebrate porphyrin was that of Moseley (1877)-the same year as the discovery of haematoporphyrin by Hoppe-Seyler. Moseley extracted a red pigment which he named “ polyperythrin ” from the anemones Discosoma and Actinia, the scyphozoans Cassiopeia, Rhizostoma and Cyanea capillata; he also obtained it from the corals Ceratotrochus diadema, Flabellurn variabile, Fungia symmetrica and Xtephanophyllia formosissima.

MacMunn (1886) examined polyperythrin and considered it to be identical with haematoporphyrin.

This work has never been repeated, so far as I know, certainly because of the dificulty in obtaining material, but quoting MacMunn’s original paper :

“ As the colouring matters in Uraster [ = Asterias], Limax, Arion and Lumbricus described above are identical with haematoporphy- rin, and as polyperythrin is identical with them, polyperythrin must also be identical with haematoporphyrin ”

and later :

“ In the eggshell of the Cochin-China hen, the bands almost exactly coincide with those of polyperythrin ”.

By the same reasoning, we now know that Asterias porphyrin is pro- toporphyrin (Kennedy and Vevers, 1953a and b), Lumbricus porphyrin is protoporphyrin (Hausmann, 1916 ; DhBrB, 1932) and the main porphy- rin of avian eggshells is protoporphyrin (Kennedy and Vevers 1973, 1976), axiomatically polyperythrin must be protoporphyrin too. It is therefore almost certain that the coelenterates which Moseley and Mac- Munn examined contained protoporphyrin (Fig. 6). However, it is now known that Arion and Limax contain uroporphyrin I (Kennedy, 1959), so that there must have been some spectroscopic error here.

Herring (1972) found free protoporphyrin in the bathypelagic scyphozoans Atolb wyvillei and Periphylla periphylla, but not in either of the shallow-living medusae Pelagia and Aurelia. He suggested that

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322 Q. Y. KENNEDY

the distribution of the pigment pointed to its possible function as a light absorber for the bioluminescence of prey in the gut, prevention of reflection of ambient day-light or bioluminescence from some of the tissues of the animals. The porphyrin fluorescence in vivo is quenched, yet it cannot be extracted as a protein complex by using buffers : it may be a complex with polysaccharide. Bonnett, Head and Herring (personal communication) have confirmed that the pigment is proto- porphyrin present in the free state in crystalline cell-inclusions. The purple-brown colour in many deep-sea medusae may be due partially or even entirely to porphyrin.

CH? CH2 I

LOOH COOH

FIQ. 6. Protoporphyrin IX.

Some medusae are conspicuous by the presence of striking blue pig- ments in the integument, and these are usually caroteno proteins. Merejkowski (1883) described such a blue pigment in the oceanic siphonophores Velella spirans ( ‘ I by-the-wind-sailor ”) and Porpita, and called the pigment “ velelline ”. He converted the blue pigment to “ zoonerythrin ” by adding alcohol. S. C. Crane (cited by Fox, 1976) demonstrated that the blue chromoprotein of Velella lata has astaxan- thin as chromophore, and Fox and Haxo (1959) confirmed this, suggest- ing also that the coloration protects the symbiotic zooanthellae against excessive sunlight.

Herring (1971a) found that the blue carotenoproteins from Velella and Porpita show reversibility in colour changes due to rising tempera- ture or depression of salinity, when normal conditions return.

Herring (1971b) reported a biliprotein in the venomous jellyfish Physalia physalia, the Portuguese Man-0’-War. He found the chromo-

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PIGMENTS OF MARINE INVERTEBRATES 323

phore to be a bilatriene, similar to but yet different from biliverdin. He suggested also that the lavender colour of the float, the pink crest and the green or purple of the gonodendra and gastrozoids might be due to other biliproteins. The reddish, purple and brown stripes of the large venomous jellyfish Pelagia colorata Russell (as P. noctiluca) were found to be due to melanins by Fox and Millott (1954). The violet chromo- proteins described by them are not carotenoproteins.

The sea-fan Eugorgia ampla is found at depths of up to 50 metres off the Baja California Coast. It is yellow-orange and has a pale yellow carotenoid with the cumbersome name of eugorgiaenoic acid firmly bound to the calcareous microspicules which are embedded in the soft parts. There are other yellow carotenoids and long-chain fatty acids bound to calcium carbonate. The eugorgiaenoic acid is a non-fluores- cent, non-aromatic unstable polyene resembling dihydrobixin (Fox, 1976).

Goldman (1 953) cultured isolated perisarc-enclosed segments of the hydroid Tubularia, without any source of exogenous pigment and found that the segments reconstituted with normal red pigmentation. The pigment is reported as astaxanthin (Goodwin, 1952) so that is the fist authenticated report of any animal being able to synthesize carotenoid de novo.

Astaxanthin was the only carotenoid extracted from the vermilion skeleton of the hydrocoral Distichopora violacea from Eniwetok Island in the Marshall group. From the purple aragonitic skeleton of the hydrocoral Allopora californica from depths of 50 metres near Catalina Island off Southern California came the same pigment (Fox and Wilkie, 1970). Other species of Distichopora, D. coccinea and D. nitida also had astaxanthin as the only carotenoid but the coenenchyme of Allopora californica yielded astaxanthin ester, free phoenicoxanthin, free astaxan- thin, a free polyhydroxy-/3-carotene and an unfamiliar hydroxydike- tonic carotenoid.

Fox (1972) investigated three species of Stylaster and reported that 8. roseus had astaxanthin as the only carotenoid in the purple skeleton, while the pink and orange skeleton of S. elegans and that of S. san- guineum revealed the enolically acidogenic astaxanthin and low con- centrations of a neutral dihydroxyxanthophyll which Fox considered to be '' bonded through formation of their respective calcium acid carbonate ester."

Not all coloured coral skeltons contain carotenoids. The brilliant blue Heliopora coerulea, the alcyonarian coral of Australian and West- Indo-Pacific waters has in its calcareous skeleton a bilichrome which was named helioporobilin by Tixier (1945). Riidiger et al. (1968)

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321 G . Y. KENNEDY

isolated the bilin, identified it as biliverdin IXa and suggested that helioporobilin was a mixture of biliverdin and the partially oxidized pigment. The free carboxylic radicals probably render the pigment sufficiently acid to form a calcium salt with the bicarbonate and carbonate of the skeleton.

The deep red skeletons of the Organ-pipe Coral Tubipora musica and the Precious Coral Corallium rubrum are not coloured by carotenoid. Ranson and Durivault (1937) found that these corals contain iron, confirmed in some experiments in the laboratory of the writer, which further suggest that the red pigmentation is due to ferric hydroxide bound in the form of a complex salt with aluminium and calcium to a mucopolysaccharide. This work is still under way. Alcyonium palmatum also contains iron in the spicules (Durivault, 1937).

Read andco-workers (1 968) examined some coelenterates in blue light, and reported that the madreporarian corals Montastrea cavernosa and Mussa angulosa were red-fluorescent. They were red or blue-grey a t a depth of 40 metres but brown at the surface : the zooantharianCorynactis californicus was also red-fluorescent. The pigments were not identified but, recalling the work of MacMunn, it is tempting to expect them to be porphyrins. The zooantharian may also be fluorescent because of symbiotic algae although if the chlorophyll (or derivatives) is linked to a protein, fluorescence would be quenched.

The pigment actiniochrome, occurring in the violet tentacle tips and stomodaeum of some anemones, e.g. Tealia felina, Anthopleura ballii and Anemonia sulcata, is still of unkown composition. It was first described by Moseley (1873) who found it to be a red pigment with an absorption band in the yellow-green. MacMunn (1885a) confirmed this and obtained the pigment from Anemonia sulcata. Fox and Vevers (1960) give the absorption band as 572 nm, and that of the green pig- ment which colours the tentacles of Anemonia sulcata as 512 mn. Both pigments are destroyed by boiling water (H. M. Fox, unpublished), the green one turning grey and losing its fluorescence, suggesting that the pigments may be linked to protein or, more likely, to a polysaccharide. Actiniochrome may be extracted from fresh tissue by glycerol as a red solution and this is changed to violet by alkali and darkened by ammonium sulphide, possibly on account of the presence of iron. That is all that is known about it so far.

Christomanos (1953) described a purple pigment from the acontia of Adamsia rondeleti, which had a blue fluorescence in u.v., was sensitive to pH and gave an absorption spectrum of 555, 465, 450 and 435 nm. He took matters no farther.

A violet pigment in the anemone Xagartia parasitica ( = Calliactis

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PIGMENTS OF MAXINE INVERTEBRATES 325

eSfoeta) was described by Abeloos and Teissier (1926) and then isolated in crystalline form by Lederer et al. (1940), who named the pigment calliactin, reporting its close chemical relationship to the bile pigments. Calliactin can be oxidized and reduced and is yellow with acid and blue, violet and red with increasing pH. Riidiger (1970) maintains that this is not a pyrrol pigment.

The beautiful anthozoan Cerianthus membranaceus from the Mediter- ranean may be violet, purple or even red. Krukenberg (1882) extracted a violet pigment from it, which he called “ purpuridin ” ; there was no definite absorption spectrum, and the colour was not discharged or changed by boiling. There seems to be a small amount of this pigment in C. lloydii. The chemical nature of this substance is unknown.

Bullock (1 970), working with sea-pens, reported free protoporphyrin in the soft parts of Pennatula borealis and Bolticina jinmarchia. The concentration was particularly high in the tentacles and was such that the animal could be photosensitive. Pennatula aculeata had only minute amounts of porphyrin, too small to characterize. Bullock suggested that the pigment must have a function of some selective value to the animals : they live a t moderate depths and it is possible that the photo- sensitivity may be an advantage.

V.CTEHOPHORA There do not seem to be any reports of pigments in this phylum of

beautiful, transparent and infinitely fragile animals. Fox and Vevers (1960) mention the fine colours produced by diffraction in ctenophores seen in the aquarium of the Stazione Zoologica at Naples. The diffraction grating is supplied by the moving ciliary combs. These combs, which may be reddish in Pleurobrachia pileus ( = Cydippe pileus), also give out waves of a greenish luminescence, which flashes for a few seconds, is extinguished and then returns. This is probably based on the luciferin- luciferase system.

The food of ctenophores consists of plankton, which may be worms, crustaceans, larvae, little fish, etc.-all pigmented-and in the words of Dr Strethill Wright, a naturalist of the last century, observing Cydippe pomiformia: “ The bright colouring of the prey so swallowed contrasts most conspicuously with the crystalline transparency of the body in which they are enclosed.” The ctenophores do not seem to store pigment from their food so must get rid of it by ejecting it or by metabolizing it into colourless compounds.

VI. PLATYHELMINTHES The somewhat grim associations which cluster about the platy-

helminths do not lead us to expect bright colours among them, yet in

< c

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326 G? Y. KENNEDY

FIQ. 7. Uroporphyrin I.

point of fact, the free-living forms often exhibit great brilliancy” (Newbigin, 1898).

Krugelis-Macrae (1 956) examined the planarian Dugesia doroto- cephalo and reported the presence of coproporphyrin and uroporphyrin (isomers unspecified). She suggested that these pigments may be the

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PIGMENTS OF MARINE INVERTEBRATES 327

L l l z I

Ln.2

I I ‘iH2 CH,

COOH COOH FIG. 9. Coproporphyrin 111.

cause of the well-known photosensitivity of planarians. In 1963, Krugelis-Macrae found porphyrins in the epidermally-derived rhabdites of some other Turbellaria, Dugesia tigrina, D . gonocephala and Cura foremnii. All had uroporphyrin (isomer unspecified) (Figures 7 , 8). Phagocata gracilis, Ph. iwanai, Ph. virida and 3iel lo~e~ha~a ~ r ~ n n e a all had coproporphyrin (isomer unspecified) (Fig. 9).

In discussing her results, Dr Krugelis-Macrae made these sugges- tions :

1. The occurrence of coproporphyrin in one genus and uroporphyrin in another may be reflecting a phylogenetic development ;

2. These porphyrins may be biochemical phenotypes which represent selected random mutations among the species ;

3. The porphyrins may be biochemically characteristic of species or genera, and might be added to the morphological characteristics by which they are generally classified.

Many turbellarians have melanins, and these may be soluble in acid- methanol (Needham, 1965)-they may be oligomeric melanins. Some of the violet pigments of some planarians may be intermediates in the indolic melanin biosynthetic pathway.

MacMunn (1890) found carotenoids with two absorption bands in

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328 0. Y. KENNEDY

Turbellaria spp. in tentacles and integument. In the Turbellaria, pig- ment is often found in the cells of the epidermis and in the interstices between these cells or in the epidermis of the body. Many, such as Convoluta paradoxa, have symbiotic green or brown algae, whose cells are not bounded by a cellulose wall, and have pyrenoids.

Moseley (1877) made some observations on two species of Rhyncho- demus found in Australia: of these, one was blue and the other red, living in similar environments. The blue pigment was insoluble in alcohol but when the solvent was acidified, the pigment turned red and dissolved immediately ; alkali restored the blue colour but did not affect the red pigment of the other species. Moseley concluded that the pig- ments were different. The epidermal pigment of Turbellaria is located in fluid vacuoles (Hyman, 1951), and Franchotte (1898) stated that the colours of some Turbellaria are due to carotenoids derived from the food (ascidians) on which they live.

Eggshells of platyhelminths are coloured and hardened by quinone- tanned proteins.

Tapeworms, being endoparasites, are not, as a rule, pigmented but may absorb pigment from the host. Trematodes feeding on the red blood of their hosts split the haemoglobin on digestion, absorb the globin break- down products and retain the haematin in faeces in their guts (Llewellyn, 1954).

Vernberg (1968) gives a useful table (his Table 111) of the distribu- tion of haemoglobin and cytochromes in the platyhelminths. Haemo- globin occurs round the anterior horns and vitellaria of trematodes (Lee and Smith, 1965) and, where separate vitellaria are formed, they are usually coloured.

Carotenoids are most frequently involved in the metabolism of parasitized animals, being abundant and easily transported across membranes. Marshall et al. (1934) reported that Galanus Jinlnarchicus when parasitized by trematodes and cestodes assumes a brilliant scarlet hue.

Melanins are found in the capsule of connective tissue which often surrounds metacercariae in the cod (Hsiao, 1941) ; the pigment may be incidental to the more essential capsule, but could also have strengthen- ing or toughening qualities.

Derrien (1927) claimed to have found protoporphyrin in the cestode Tetrathyridium (parasitic in the hedgehog) and in the cysticercus stage of Taenia solium but the report is vague. This is mentioned here a s a hint that porphyrins might be present in other platyhelminth parasites, and porphyrins and bilins might well be found in parasitic worms which feed on blood containing haemoglobin or chlorocruorin.

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PIGMENTS OF MARINE INVERTEBRATES 329

FIQ. 10. Haem (ferrous protoporphyrin).

VII. NEMATHELMINTHES The review of the pigments of this phylum, which includes the

Nematoda and Acanthocephala, by the late Malcolm Smith (1969) makes the point that the subject appears to have been given scant attention and gives some information about the occurrence of haemo- globins, bilins and some other pigments. However, the examples cited are mostly from parasites of terrestrial animals whose external appear- ance is usually white. There are some which are obviously coloured, even microscopic forms, but there do not appear to be any pigments characteristic of the phylum.

A. Nematoda Lee and Smith (1965) listed 18 species of nematode in which haemo-

globin is definitely known, and a further 13 related species in which its presence was judged possible from a study of the literature. Only three species without haemoglobin have been reported-Cruzia testudinis, Falcaustra oficinis and Oxyuris equi, all members of the order Oxyuroidea.

The free-living marine nematode Enoplus brevis has haemoglobin in the pharynx of both sexes and copulatory muscles of the male. There was less haemoglobin in E. communis (Ellenby and Smith, 1966). Croll (1966) suggested that the reddish-brown eye-spots of E . communis may

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330 0. Y. KENNEDY

contain melanic granules and similar pigmentation seen in other species may be due to melanin.

Although not a marine species, it should be noted that Faur6- Fremiet (1913) found bile pigments in Ascaris lumbricoides gut. The washed guts of these nematodes vary from light brown to dark green and the colour relates to the haemoglobin content of the perienteric fluid-redder worms having greener guts. Smith (1969) carried out some experiments on the pigmentation and came to the conclusion that " some form of tetrapyrrole " was present. Cain and Bassow (1976) found that only the enteric fluid of A . lumbricoides was red-fluorescent in U.V. light, due to the presence ofprotoporphyrin and coproporphyrin I11 (Fig. 9) in the proportions of 95.4% and 4.6% of the total porphyrin. Uroporphyrin was not detected and no porphyrins were recovered from other tissues of the worm. Proto- and copro-porphyrins in the fluid of worms with dark guts were nearly seven times those in worms with light guts, but the molar ratio of the two porphyrins remained relatively constant in both groups. The molar ratio of haem (Fig. 10) to protoporphyrin was almost identical in the fluid of worms with light guts or dark guts.

Cain and Bassow suggest three ways in which A . lumbricoides could obtain the porphyrins:

1. They could originate as breakdown products of endogenous haem proteins (unlikely if not impossible indicated by the presence of coproporphyrin) ;

2. Host gut contents-but then higher concentrations of coproporphy- rin would be expected ;

3. Accumulation of biosynthetic intermediates.

The constancy of porphyrin-haem ratios suggests (3), but even better support is given by the finding of coproporphyrinogen oxidase (Cain, in press). Cain and Basson feel, however, that the absence of uroporphy- rin is against (3)-but this is not necessarily a stumbling block, in view of the predominance of coproporphyrin I11 in those polychaetes which have free porphyrins (Kennedy and Dales, 1958 ; Mangum and Dales, 1965). Uroporphyrin has not been detected in Lumbricus, Allolobophra or Eisenia but was present in homogenates of some polychaete heart- body tissue incubated with ALA or PBG (Kennedy and Dales, 1968.).

The porphyrins reported by Cain and Bassow could be the usual " fall-out '' pigments in an inefficient haem biosynthetic pathway like that in many marine invertebrates. In this connection the reference to the report by Aduco (1888) of porphyrins in the red substance causing the coloration of the dog parasite Dioctophyme renale ( = Eustrongylus

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PIQBXENTS OF MARINE INVERTEBRATES 331

g i g a s ) cited by von Furth (1903) and by Lederer (1940) as ‘‘ semble avoir constate la, presence de porphyrines ” is of considerable interest.

There may be flavins in the ascarids since Smith (1969) found a,

yellow pigment with a green fluorescence, separable from haemoglobin by passage through CM-cellulose.

Folic acid (pteroylglutamic acid) is an essential vitamin for nema- todes, hydrolysis giving rise to Ascaris-blue (Torri, 1956). Sato and Ozawa (1969) found rhodoquinone-9 in the muscle of Ascaris lumbri- coides var suis and Metastrongylus elongatus. Frank and Fetzer (1968) detected carotenoids in a species of Filaria.

Weinstein (1966) found that the absorption spectrum of cyanocobala- min (vitamin B12) wm given by the coelomocytes of Nippostrongylus braziliensis. Viglierchio (1974) studied the pigments of the ocelli of some Antarctic and Pacific marine nematodes.

These references to non-marine nematodes are given here to indicate some points for the investigation of marine forms, especially the parasi- tic forms, e.g. in the Ascaroidea. In this connection, it would be interest- ing to enquire into the pigments of Contracaecum clavatum, an ascarid found in the stomach or intestine of several fishes-among them Cottzcs bubalis, Myoxocephalus scorpius and Conger conger, a11 of which have B

distinctive bile pigment metabolism.

B. Acanthocephala The knowledge of pigments in this class is even more limited than in

the Nematoda. Hyman (1951) mentions that the Acanthocephala have no intrinsic coloration, but sometimes are red, orange, yellow or brown, the colours coming from food absorbed from the host; This is not accepted by everyone, there being instances of red Pomphorhymus associated with colourless Acanthocephalus or Echinorhyncus, while the juvenile of Polymorphus minutus forms orange-red lipids (carotenoids?) in the colourless body of the host Clammarus.

Arhythmrhynus comptus is reported to have ,%carotene (Van Cleave and Rausch, 1950) so that other red or orange acanthocephalans, e.g. Polymorphus just mentioned, may have carotenoids. Acanthocephalan larvae increase the amount of ommochrome in the operculum of the isopod Asellus, so that the gill-covers become dark and conspicuous- an increase in the synthesis of the isopod’s ommochrome pigmentation (see Crustacea) and an extension of the normal chromatophore system.

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332 a. Y. KENNEDY

VIII. ROTIFERA The rotifers or “ wheel animalcules ” are mostly colourless and

transparent, but some are brightly coloured. Some rotifers have com- mensal algae which must contribute to their coloration, and extraction of these rotifers by the usual methods will yield chlorophylls or their derivatives, carotenoids and possibly bilins (de Beauchamp, 1909).

Rotifers living in high mountain lakes have an intense red colour due to carotenoids; the effect, if any, of altitude is unexplained (Brehni, 1938).

IX. NEMERTINI The Nemertini provide many examples of very bright coloration,

and M’Intosh (1910) in his great Monograph of British Annelids was moved to eloquence :

‘‘ The colours of many species of the group are of such beauty as to attract even the casual observer, while in this respect also they widely deviate from their supposed allies, the parasitic worms. The richest purples appear on velvety skins of deep brown or black, each of the soft and mobile folds giving shades that vary in intensity and lustre. Bright yellow contrasts with dark brown, white with ver- milion, brown and dull pink, while individual uniformity is charac- terized by such hues as rose-pink, white, green yellow and olive, the gradation of colour in the various parts of a single specimen being so subtle that enthusiasm as well as skill is necessary in the artist who sets himself to the task of faithful delineation.”

This description is based upon the British species only-the tropical nemertines are said even to surpass these in splendour. The writer has “ set himself to the task ” of identification of the nemertine pigments, and trusts that his skill may match his enthusiasm in the investigation of their subtleties! With the splendour of colour are found markings, such as longitudinal and transverse stripes, iridescence of cilia and a silver sheen. The colour seems to vary according to the amount of ex- posure to light, and it is interesting that Amphiporus lacti$oreus, which is white with an orange spot at the head, develops more pigment in strongly lit conditions so that the integument becomes deeper in colour.

Some nemertines are transparent so that the food in the gut can be seen and this contributes to the external coloration. Sex makes no dis- tinction in colour but in some cases, as in A . pulcher, the egg masses are bright red and can be seen through the body wall producing a striking effect.

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PIQMENTS O F MARINE INVERTEBRATES 333

The fine monograph by Burger (1895) which contains thirty-one beautiful colour-plates should be consulted. He describes the epithelium as consisting of three kinds of cell, thread-like cells, interstitial cells and gland cells, with the pigment appearing in any one of them. In Lineus and the related forms, the pigment is in the gland cells ; in Nemertopsis peronea, on the other hand, the pigment is restricted to the interstitial cells which form two long red-brown dorsal stripes.

The nemertines have haemoglobin in blood corpuscles and in the central nervous system ; they may have carotenoids in the blood cells also, since they appear more varied in colour than is accounted for by haemoglobin alone.

Carotenoids have been recorded in a number of species-Amphiporus pulcher, Carinella annulata, Cerebratulus fuscus, C. marginatus and Malacobdella grossa by Loiinberg (1931) and by Lonnberg and Hellstrom (1931), but their results were not such that the pigments could be identified, although Malacobdella was said to have a xanthophyll.

At the suggestion of the late H. Munro Fox, Kennedy (1962) ex- amined the boot-lace worm Lineus longissimus and found protoporphy- rin in the integument. Vernet (1966) reported an ommochrome in the integument of L. ruber and, in 1968, the presence of uroporphyrin I11 also. This heteronemertine secretes some of its ommochrome in its mucus and Vernet showed that it is able to transform 6-aminolaevulinic acid (ALA) into porphobilinogen (PBG) in tissue homogenates, so that the uroporphyrin of the integument is endogenous.

The writer has already done a considerable amount of work on some British nemertines at the Plymouth Laboratory-pigments revealed so far include carotenoids, ommochromes, porphyrins and bile pigments.

X. ANNELIDA, ECHIUROIDEA, SIPUNCULOIDEA, PRIAPULOIDEA AND PHORONIDEA

Kennedy (1969) published an extensive review of the pigments and coloration of these phyla, which includes a discussion of the origin, metabolism and function of the pigments, so far as these are known. It will only be necessary here to describe some of the more recent work.

Pigments found in these phyla include carotenoids, flavins, quinones, melanins, pterins, haematins, porphyrins, bilins and chlorophyll deri- vatives-a rich field for the chromatologist. It is interesting that the porphyrin occurring most frequently, in polychaetes at any rate, is coproporphyrin I11 with its haematin, usually in the body wall, coelomocytes or heart-body (if present) or a combination of all three (Mangum and Dales, 1965).

The red pigment in the integument of Halla parthenopeia, a eunicid

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334 Q. Y. KENNEDY

polychaete of the family Lysa.retidae found in the Bay of Naples is well known. Prota et al. (1971) re-examined the pigment and have shown it to be an hydroxy-methoxy-methyl-1, 2-anthraquinone. This is a most unusual anthraquinone, unsubstituted at positions 9 and 10 and, as yet, the only anthraquinone known in the Annelida. The same pigment has been found in another eunicid Lumbriconereis impatiens by Prota et al. (1971).

Arenicochrome from the lugworm Arenicola marina is a benzpyrene- quinone, but this is an artifact (Morimoto et al., 1970) and the structure

- CH,

CH2 HC-C=O I I

FH2 Coo.CH3 COO-C,,H3,

FIQ. 11. Chlorophyll a.

of the actual pigment in the integument is still unknown. During puri- fication of the extracted pigment, it oxidizes to a purple derivative- arenicochromine.

Sorby (1875) gave the name " bonelline " to the bright green pigment from the echiuroid Bonellia viridis. The pigment was reported by Lederer (1939) to be dioxymesopyrrochlorin, corresponding with the isocyclic nucleus of chlorophyll a (Fig. 11) . Pelter et al. (1976) have re-examined the pigment and found that it is indeed a chlorin, but an unique one. They reported that Lederer's suggestion of oxygen atoms placed at C-13 and (I-15 (because of its assumed relationship to chloro- phyll a ) was unacceptable on the grounds that the absorption spectrum would no longer be that of a simple chlorin.

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PIGMENTS OF MARINE INVERTEBRATES 335

Pelter et al. find bonelline to be a dihydroporphyrin (chlorin) with a gem-dimethyl group on the reduced ring and no C-15 substituent " im- plying an origin other than chlorophyll " (Fig. 12). The pigment is physiologically active and the similarity in structure to that postulated for sirohydrochlorins is noteworthy. Carotenoids are rare or even absent in Priapuloidea, Echiuroidea and Sipunculoidea.

Fuscin granules have a high endogenous oxygen uptake and Schreiber (1930) considered that fuscin of the nerve cells of Sipunculus

H

had a respiratory function. Sipunculus has two haemerythrins-one in the general cavity and one in the tentacular coelom-incidentally con- firming that the two systems are not connected.

The integumentary pigments of sipunculids are granular and as yet (1978) unidentified. The nephridia have a brown pigment. The purple- brown pigment of Priapulue may be haemerythrin or a derivative (Pange, 1969).

The polychaete Sabella penicillis has iso-cryptoxanthin. Czeczuga (1971) described the coloration of some Nereis xonata from the Black Sea, and reported the presence of p-carotene, cryptoxanthin, lutein, zeaxanthin, neoxanthin and astacene. Weedon (1971) believes that reports of the natural occurrence of astacene should be interpreted with caution.

XI. ABTHROPODA

A. Crustacea The crustacea show many beautiful colours-blue, green, purple,

red and orange-together with the more sombre black and brown. In spite of the whole spectrum of colour, there is considerable uniformity

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of pigments, the carotenoids being the group most widely represented, both in the free state and as carotenoproteins. Recent reviews on crustacean pigments include those of Fox (1953, 1974, 1976), Goodwin (1952, 1971), Cheesman et al. (1967) (carotenoproteins) and Gilchrist (1968) with Fox and Vevers (1960) for general discussion. Once again, our purpose will be to consider the most interesting of the older papers and include some recent work.

The investigation of the crustacean pigments has been active fairly continuously since 1873, when Pouchet separated two pigments, yellow and red, from a lobster. He crystallized the red pigment which was almost certainly astaxanthin. From that time onwards, it is fair to say that the carotenoids (originally called lipochromes) have been the most extensively investigated pigments, particularly since their relationship to vitamin A was discovered.

Merejkowski (1881, 1883) named the red pigment of Pouchet " zoonerythrin " and described its occurrence in some crustaceans. He is thought to be the first to notice the water solubility of many blue, green and grey pigments in the sub-phylum and in other invertebrates, and Newbigin (1897, 1898) extracted the blue pigment from the lobster Homarus vulgaris with ammonium chloride and concluded that it was " a compound of a red lipochrome with an unstable, complex organic base ". Verne (1921, 1923) established that the base was a protein. Lwoff (1925, 1927) examined the carotenoids of copepods, and especially the blue carotenoprotein in the eyes of the harpactfcoid Idya furcata. He considered thah the copepod was able to synthesize small amounts of carotenoid, even though much of the pigment in the animal came from the diet. Twenty years later, Lwoff confided to D. L. Fox that the synthesis of carotenoids by animals de novo had not been proven.

Heim (1892) had shown that female decapods can mobilize caro- tenoids from the hepatopancreas via the blood system to the ripening ovary, and when oviposition takes place, the blood shows no carotenoid pigment. This important work was taken up and extended by Abeloos and Fischer (1926) to the crab Carcinus maenas, and they reported that alimentary carotenoids were assimilated directly by the hepatopancreas and transferred by the blood to the ovary.

The monograph by Parker (1948) on the regulation of colour change by hormones includes much information on adaptation to environment by crustaceans.

The most prominent carotenoid in the Crustacea is astaxanthin and it was obtained by Kuhn and Lederer (1933) from the dark-brown chromoproteins of the carapace, the red hypodermis and the blue-green egg mass of the crayfish Astacus gammarus. It was also made from the

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eggs of the spider crab Maia squinado by the same team and called astacene. Subsequently, this pigment was described in Palinurus vulgaris, Portunm puber, Leander serratus, Potomobius astacus, Cancer pagurus and species of Nephrops.

The papers of D. L. Fox and his associates have done much to elucidate some of the problems of carotenoid metabolism and function and references to these may be found in Fox’s book (1953, 1976) and Goodwin’s (1971) review of the pigments of Arthropoda. The latter contains most useful tables which summarize the distribution of pig- ments among the sub-phyla. Fox was led to the conclusion that caro- tenoids in animals may have an important r61e in the maintenance of integumentary surfaces and in the secretion of calcareous or mucous substances. Porphyrins (see later) have a similar function.

The green pigment isolated from the eggs of the lobster Homarus americanus by Stern and Salomon (1938) was named by them “ ovover- din ”, and had been observed in earlier times when the eggs turned red on preservation in alcohol. They found that thermal dissociation of the polar carotenoid (referred to as astacene) and the protein conjugant could be reversed at temperatures not exceeding 7OOC.

It is now thought very doubtful that astacene ever occurs in nature, and the pigment is regarded as an artifact derived from astaxanthin,

The review by Cheesman et al. (1967) of invertebrate carotenopro- teins contains a useful table (their Table I) of the occurrence of these pigments, with some discussion of their chemistry and functions. The authors say that it is notable that within each of the phyla listed, the carotenoproteins appear primarily to be found in two general ways in the body-the exoskeleton and the epidermis, and the eggs and the ovaries. This, they suggest, is indicative of the participation of these pigments in development and possibly in protective coloration or storage of some kind.

Cheesman et al. also point out that the carotenoproteins listed in their table fall into two categories :

1. The majority, which are blue to green with their main absorption

2. The remainder, which are red to purple with maxima between 532

maxima between 680 and 560 nm ;

and 490 nm.

A few are of other colours.

There is another useful table (Table 3.2) listing the colours and composition of some carotenoproteins in Needham (1974). Some of the

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338 Q. Y. KENNEDY

green complexes which have been studied appear to be the result of blue stoichometric combination of protein with either astaxanthin or canth- axanthin and absorbed or dissolved (in lipid) free yellow carotenoid.

Liinnberg’s work on the occurrence of carotenoids is cited by Karrer and Jucker (1950) with many references and a list of 47 species of crustaceans (in which astaxanthin should be read for astacene) ; there is another list in Goodwin (1952) and one more up-to-date in Goodwin (1971) which covers ommochromes and insect pigments as well (See also Vuillaume, 1969).

The work of Kon (1 949) and co-workers gave weight to the impression that /I-carotene is only a minor component of crustacean carotenoids. These workers found only traces of this pigment in Meganyctiphanes norvegica, Thysanoessa raschii, Pandalus bonnieri, Xpirontocarus spinus, Crangon allmanni and C. vulgaris, while astaxanthin was present in large amounts.

Kuhn and Ssrensen (1938a, b) made the important discovery that astacene is an oxidative artifact of astaxanthin, made during the chemistry of the extraction and purification processes. Due to keto-enol tautomerism, astaxanthin and astacene both show acid properties and will dissolve in dilute aqueous alkali. It is interesting that Goodwin and Srisukh (1949) found that in Nephrops norvegicus (the Norway lobster famous as “ scampi ”) the hypodermal pigment is esterified astaxanthin, while that of the carapace is not esterified.

The case of the brine shrimp Artemia salina is curious. This little branchiopod is able to convert dietary p-carotene into its monoxy- and dioxy- derivatives echinenone and canthaxanthin (Davies, Hsu, Wan- Jean andchichester, 1965). These two ketones are the only carotenoids in the eggs and newly hatched nauplii of Artemia. The formation of canth- axanthin from ,&carotene is apparently stepwise through echinenone since thelatter, if fed to Artemia, is converted into canthaxanthin (Hsu et al., 1970). These workers also reported that zeaxanthin and iso-zeaxan- thin were absorbed by the gut but not converted to ketocarotenoids; iso-cryptoxanthin (4-hydroxy-p-carotene) was oxidized by the animal to 4-hydroxy-4’-keto-p-carotene but not echinenone or canthaxanthin.

The blue goose-barnacle Lepas fascicularis owes its bright colours in the outer and inner somatic tissue and its ripe eggs to an astaxanthin carotenoprotein. Several shades of blue are displayed, mainly in the cirri, the carapace and ripe ovary. The eggs have the richest content of carotenoid, of which some 93% is astaxanthin-protein (Fox, Smith and Wofson, 1967). Fox (1973) found an instance of keto-carotenoids firmly bound to chitin in the red kelp-crab Taliepus nuttallii, common in kelp beds in Southern California. The four carotenoids extracted from the

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carapaoe were echinenone, canthaxanthin, phoenicoxanthin and astaxanthin.

Lenel (1 961) working with the degenerate crustacean Sacculina carcina, parasitic on the crab Carcinus maenas, examined the carotenoids acquired from the host. He found that Sacculina was very selective, taking only @-carotene from the many carotenoids available. The larvae of the parasite contained @-carotene only.

Lee (1966a) studying the pigmentation of the isopods Idothea granu- losa and I . montereyensis showed that the red, green and brown colours of these crustacea resulted mainly from the combined colours of the epidermis, although the chromatophore pigment was a red reduced ommochrome. I . granulosa has a red exocuticle containing canthaxan- thin, while the endocuticle is yellow with lutein. The green specimens have a blue astaxanthin carotenoprotein, which, when associated with the yellow lutein (in lipid) produces the green colour. The brown colours are produced by mixtures of the green carotenoprotein with cantha- xanthin in the epidermis. I. granulosa could change colour without ecdysis ; I. montereyensis could not. Both these isopods stored cantha- xanthin, free or as a complex with protein, and lutein with its epoxide, @-carotene, echinenone and possibly monoketomonohydroxy-,%carotene (Lee, 1966b). Gilchrist and Lee (1972) studied the carotenoids of the Pacific shore mole crab Emerita analoga. They identified a-carotene, ,%carotene, echinenone, canthaxanthin, astaxanthin, zeaxanthin, dia- toxanthin and alloxanthin ; there was an orange carotenoprotein in the ovaries, eggs and blood.

There is increasing evidence for the genesis of ketocarotenoids in crustaceans, and these workers demonstrated that Emerita can use /?-carotene for the generation of astaxanthin through echinenone and canthaxanthin. It will be remembered that Artemia converts /3-carotene to canthaxanthin with echinenone as intermediate ; it obviously employs the same chemistry, but does not want astaxanthin.

Castillo and Lenel (1978), working with the hermit crab Clibanarius erythropus, isolated the following carotenoids :

/3-carotene, echinenone, (4-keto-/l-carotene) canthaxanthin, (4, 4’-diketo-fl-carotene) phoenicoxanthin, (3-hydroxy-4, 4’-diketo-/3-carotene) astaxanthin, (3, 3’-dihydroxy-4, 4’diketo-fl-carotene) astaxanthin esters, lutein, (3, 3’-dihydroxy-a-carotene) a-doradexanthin (3, 3’-dihydroxy-4’-keto-a-carotene) a-doradexanthin esters.

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These authors point out that a comparison of the structures of these carotenoids suggests a metabolic pathway :

/3-carotene + echinenone + canthaxanthin t phoenicoxanthin + astaxanthin

The a-doradexant hin may represent an intermediate form during the biosynthesis of astaxanthin from lutein. This has been considered improbable hitherto (Herring, 1968).

These crabs inhabit the shells of gastropods Trochococlea turbinata and Nassa reticulata ; their bodies are green with red lines on both sides of the dactylus but become red after death. This change of colour could be due to breakage of the carotenoprotein link by enzymes post-mortem.

Gilchrist and Lee suggest that carotenoid, and specifically astax- anthin, may be used by adult Emerita and in the eggs as a heat or light shield. Carotenoproteins may also function in the developing young as stabilizers and protectors of food reserves. Some indications were found of some relationship between carotenoids and carotenoproteins and reproduction, but without conclusive proof.

Crustacyanin, a blue-grey carotenoprotein from the carapace of the lobsters Homarus vulgaris and H . americanus, is an astaxanthin con- jugate with a simple protein. It also appears in carapace, mandibles and stomach wall of Aristeus antennatus and other decapods. Ovoverdin, the green carotenoprotein in the eggs of these lobsters, is astaxanthin conjugated with a glycolipoprotein rich in phospholipids (Weedon, 1971).

It is agreed that for true chemical conjugation between a carotenoid and a protein, the carotenoid must include one or more ketone radicals as components of either or both of the terminal cyclohexenyl rings. This is why astaxanthin is the chromophore by far the most frequently found in carotenoproteins. Even esterified astaxanthin, still with its two ketone groups, has been found in a carotenoprotein in the hermit crab Eupagurus bernharduv (Cheesman and Prebble, 1966).

Crustaxanthin, accompanying astaxanthin in Arctodiu~tomus sali- nus, is tetrahydroxy-/3-carotene and may be an intermediate in the biosynthesis of 3-hydroxy-4-ketocarotenoids (Weedon, 1971). The bright red colours of deep-sea crustaceans are very striking, but there are no reports of investigations so far.

There have been no reports of free porphyrins in the Crustacee. Protohaem was found in the eggshells of Artemia (Needham and Needham, 1930) and of Triops (H. M. Fox, 1957). Haemoglobin is widespread in the Entomostraca, in all main divisions of this group (H. M. Fox, 1957), and the pigment occurs in solution in the blood of all

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Notostraca, Anostraca and Conchostraca, Daphnia and most other Cladocera ; in some rhizocephalan cirripedes; but not in Malacostraca (which includes the lobsters and crabs).

In a survey of 48 species of marine invertebrates, Kennedy and Vevers (1964) did not find free porphyrins in any of the crustacea included in this number.

Artemia develops pink blood if kept for two or three weeks in water at temperatures of 18-20°C, with air saturation of only 18-20%, and synthesizes haemoglobin to meet the oxygen deficiency. Coupled with the enormous amount of haematin in the eggshells, this points to an active haem biosynthesis. Haemocyanin, which does not have the por- phyrin macrocyclic structure, but is a protein-copper complex, is the respiratory blood pigment in decapods and stomatopods but not in others. Busselen (1971) found haemocyanin in the eggs of Carcinus maenas, Eriocheir sinensis and Portunus holsatus, the first record of intracellular haemocyanin.

Bloch-Raphael (1948) found bile pigment in the parasitic cirripede Septosaccus cuenoti ; a biliverdin-like pigment in the sucking organs of the adults and a small amount in the eggs and larvae; it was assumed that this arose from haemoglobin ingested by the (‘ mother ”. The amount of bile pigment varied with the sexual cycle. H. M. Fox (1953) examined two other parasitic cirripedes, Rhizocephala peltogaster and Rh. partenopea. The (( roots ” of the parasite, anchored in the host, contained a Gmelin-positive green pigment which was shown to be biliverdin. Bradley (1908) found that the fresh-water crab Cambarus secreted green bilins, although the blood and muscles were free of haemoglobin.

Biliverdin is present in the eye of the cladoceran Polyphemus (Green, 1961), and in some ostracods, e.g. Heterocypris incongruens, Eucypris virens, biladienes of type a and b derived from the Cyano- phyceae of the food are found. Eucypris transfers some pigment to the valves of the carapace (Green, 1962). Green also reported a blue- green violinoid biladiene in the gut of Eucypris virens, also present on the valves of the carapace. Heterocypris has no bile pigment outside the gut. The colour of the carapace valves of the ostracod Cypridopsis aculeata may be due to bile pigments.

Ommochromes are likely to be distributed throughout the Artkro- poda in eyes and integument (Linzen, 1967). The isopod Asellus aquaticus has xanthommatin in the integument ; Ligia oceanica and the decapod Leander serratus have xanthommatinin the eyes, and L. serratus has ommins as well. Crangon vulgaris has ommins in the eyes and integument while Portunus holsatus, Carcinus maenas, Palinurus vul-

A.M.B.-16 12

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342 Q. Y. KENNEDY

garis and Penaeus mentbranaceus have these pigments in the eyes only. Xanthopterin and other pterins are found in the hepatopancreas and

hypodermal tissues of some crustaceans, Crangon, Hornarus, Palinurus, Eupagurus, Leander and Galathea. There is a pale yellow pterin with a strong blue fluorescence revealed by fluorescence microscopy in the epidermis of Crustacea. This appears in melanophores and is not visible in the living tissue but only when it is treated with acetic acid or am- monia. There are chromatophores in decapod crustaceans, e.g. prawns, which contain a yellow (by reflected light) pigment which may also be a pterin. The reflecting pigment in the eyes of lobsters is a mixture of xanthopterin with xanthine, hypoxanthine and uric acid. Guanine is absent (Kleinholz, 1959).

Riboflavin in the dorsal integument of crustaceans may have a photosensitizing effect (Beerstecher, 1950). Fontaine, Raffy and Col- lange (1943) found that some species of marine cirripedes and isopods Living in good conditions in well-oxygenated water contained 1-2 pg of flavin per gram of wet tissue, whereas others living amphibiously or in less favorable respiratory conditions had 3-14 pg flavin per gram.

According to Verne and Busnel (1943) amino-acidophore cells are precursors of melanoblasts in the integument of some crabs ; both con- tain flavin which may have a r61e in melanogenesis.

B. Arachnida The sea spider Nymphon rubrum is an intertidal example of a group

of animals which are very abundant in great depths of the ocean. As the specific name suggests, N . rubrum contains a red pigment which may well be carotenoid, since these pycnogonids feed on anemones and

Packard (1875) was the first to call attention to a bright-red organ in Limulus polyphemus, and Lankester (1884) referred to it as the " brick-red gland ". The tissue had structural resemblances to the colourless coxal glands of Scorpio, and Packard held the view that the gland is " renal in nature '' ; this idea has received support. The red colour of the gland has been shown by Ball (1977) to be carotenoid, averaging 111.0 pg per gram wet weight. The carotenoid consists of about eight components, including /3-carotene, two ketocarotenoids resembling echinenone and a santhophyll. The remaining pigments were not classified. The three main pigments were also found in the eggs and amoebocytes, but the hepatopancreas contained different carotenoids.

LimuZus has haemocyanin in the blood and guanine in the eyes as a white reflecting medium.

POlYPS.

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C . Myriapoda Needham (1960) examined some young individuals of the littoral

chilopod Scolioplanes maritima and found a mauve-pink pigment in a band on each side of the heart, above the heart, at the base of the legs and round the nerve-cord corresponding with the distribution of the chilopod fat-body. The exoskeleton, amber-coloured, is transparent enough to allow the pigment to contribute to the external appearance of the animal, which varies from orange to deep red.

The mauve-pink pigment is almost certainly related to the violet pigment isolated from the centipede Lithobius forJicatus, by Bannister and Needham (1971) and named by them lithobiliviolin. They con- sidered it to be an hydroxyquinone, concerned with the formation of the exoskeleton, and Needham (1974) has further suggested, in view of its distribution round the tracheae and under the epidermis and the coinci- dence of high concentrations of riboflavin at these loci, that litho- biliviolin is an oxygen carrier.

XII. MOLLUSCA This is a large and diverse phylum and includes many examples of

terrestrial, fresh-water and marine forms, the last named in particular exhibiting great brilliancy of colour. Once again, the carotenoids provide most of the orange, red, yellow, blue and green colours, but there are many examples of other pigment classes as well.

Marion Newbigin (1898) gave an account of the colours and pigments of molluscs so far as they were known at that time, but in spite of a fair volume of research over almost three-quarters of a century, there are still many mysteries. Reference may be made to several reviews of molluscan pigmentation, including those of Goodwin (1972), Fox (1966, 1974) and Comfort (1951), with good coverage in the books of Goodwin (1952), H. M. Fox and Vevers (1960), Fox (1953, 1976) and Needham (1974). The review of Goodwin (1972) is, once again, valuable for the separate tables of the distribution of carotenoids in the Gastropoda, Lamellibranchia and Cephalopoda. Our purpose here will be to outline the more important or interesting work and add some of the new material.

Lonnberg (1931-1935) cited by Fox (1953) examined more than eighty species of molluscs, and his conclusions may be found listed- with those of other workers-in the books by Karrer and Jucker (1950) and Goodwin (1 952). In many cases, the inclusion of the viscera clouded the results, but the main pigments are seen to be carotenoids.

Fabre and Lederer (1934) extracted and crystallized a xanthophyll

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344 a. Y. KENNEDY

from a bivalve Pectunculus glycymeris, and named the pigment “ glycy- merin ”. According to Weedon (1971), this may be pectenolone, an acetylenic ketone with the structure 3, 3‘-dihydroxy-7, 8-didehydro-4’- keto-/?-carotene. The pigment is not invariably present in this scallop.

Lederer (1938) extracted the gonads of Pecten maximus and named the new red-orange xanthophyll “ pectenoxanthin ”. There were other epiphasic pigments consisting of esterified xanthophylls with /?-carotene. Pectenoxanthin is now referred to as alloxanthin, and has a diacety- lenic structure (Weedon, 1971). The other xanthophylls are now known to be astaxanthin and pectenolone. P. jacobaeus and P. yessoensis also have alloxanthin.

Scheer (1940) working with the Californian mussel Mytilus califor- nianus, extracted Rome xanthopylls and a new acidic carotenoid which he called mytiloxanthin, but there were no carotenes. Mytiloxanthin is now (Weedon, 1971) believed to have an acetylenic end-group. Scheer observed that mytiloxanthin gradually disappeared during starvation and was replaced by what he took to be zeaxanthin ; Weedon states that the replacement pigment is alloxanthin-which also occurs in M . edulis. The mussel can apparently synthesize mytiloxanthin from a xantho- phyll precursor.

There is some new information on the Pacific Coast nudibranch Hopkinsia rosacea. This animal was shown by Strain (1949) to be col- oured by a xanthophyll which he took to be a ketone. No carotenes or esterified xanthophylls were found. Hopkinsiaxanthin is an orange apocarotenoid with one hydroxyl and one keto group on the single cyclohexenyl ring, an acetylenic linkage between C7 and C8 of the polyene chain and a keto group on the terminal carbon atom. The pink polyzoan Eurystomella bilabiata, which is the nudibranch’s main source of food, also contains hopkinsiaxanthin as its principal carotenoid (McBeth, 1970).

The very beautiful Flabellinopsis iodinea, which has a purple integu- ment and orange gills is also a native of the Californian foreshore. On extraction, this nudibranch gave astaxanthin as its only carotenoid, but in various forms and loci. The red rhinophores gave one free and two esterified fractions of astaxanthin ; the free pigment (up to 80% of total) was accompanied by two esters in the orange cerata ; the ripe egg-masses had only astaxanthin (free) and the purple integument contained an astaxanthin carotenoprotein.

The food of Hopkinsia is mainly the hydroid Eudendrium ramosum which is bright orange and has free astaxanthin and five esterified fractions of astaxanthin in the gastrozooids. It would seem, therefore, that the nudibranch derives its astaxanthin directly without having to

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spend energy on conversion. The nudibranchs Anisodoris nobilis (orange to light yellow), Bendrodoris fulva (yellow to orange-yellow) and Doriopdla albopunctata (brown) were found to have unusually high proportions of a- and 8-carotenes in their integuments, and they were also found to have a store of iso-renieratene (the aromatic carotenoid).

McBeth (1972a), whose work we are describing, found a new caro- tenoid resembling hopkinsiaxanthin in the nudibranch Triopha carpen- teri, and dubbed it triophaxanthin. It is fascinating that the polyzoans which form the food of this nudibranch were shown by McBeth to contain a mixture of carotenoids, seven of which matched those of Triophus and, above all, the predominant pigment from the polyzoan extract was triophaxanthin!

Cheesman et al. (1967) list some carotenoproteins of molluscs which include the blue-green of the mantle of Cerithidia californica (possibly containing a carotenoid acid ester), olive green ovary of Patella vulgata (a mixture), the red albumin gland and eggs of Pomacea canaliculata (a glycoprotein conjugate of astaxanthin) and the brilliant orange-red ovary of Pecten maximus (glycolipoprotein with alloxanthin, astaxan- thin and pectenolone.

The pigments of most importance in the Mollusca, after the caro- tenoids are the tetrapyrroles, which include the haemoglobins, haema- tins, porphyrins and bilins. Porphyrins in molluscan shells were reviewed by Comfort (1951), Rimington and Kennedy (1962), Kennedy (1975) and bilins by With (1968), Riidiger (1970) and Yamaguchi (1971). There is an interesting discussion on the haemoglobins of invertebrates by Wittenberg et al. (1965) and a stimulating discussion by Needham (1974) which has tables of distribution and physical constants. Although the respiratory pigments are outside the desiderata of this review, it is important, when discussing the occurrence of haematins, porphyrins and bilins, to know something of the distribution of haemoglobins.

In the Mollusca, haemoglobin is occasionally found in Amphineura, Gastropoda and Lamellibranchia, but not in the Cephalopoda. The pigment may occur in pharyngeal musculature, as in many gastropods and the amphineuran Chiton ; in Littorina the haemoglobin is in the buccal mass and the radula. Aplysia has haemoglobin in the central nervous system in the giant nerve cells, and in Limnaea it is in the connective-tissue sheath. Planorbis is the only gastropod with haemo- globin dissolved in the blood, and it is odd that the closely-linked Limnaea, living in the same (fresh-water) environment does not have the pigment in the blood (cited by Fox and Vevers, 1960). Some lamelli- branchs, e.g. Arca spp. and Solen legumen have haemoglobin in blood corpuscles .

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The Pacific pismo clam Tivela stultorum has haemoglobin in the gills, central nervous system and adductor muscles, and the wood-boring bivalve Bankia cetacea (a shipworm) has the pigment in the posterior adductor muscle and heart. Phear (1955) in her review of the gut haems in invertebrates, included her own observations on the molluscs, from which she found gut haemochromogens to be widely distributed. In Table I of her paper, Phear lists the species in which she found gut haemochromogens and those in which it was not detected. It is interest- ing, in view of the distribution of haemoglobin, to correlate this pigment with the incidence of gut haems (see Table I).

TABLE 1:. CORRELATION OF HAEMOGLOBIN DISTRIBUTION WITH INCIDENCE OF

GUT HAEMS (Data from Phear, 1955)

Molluac Hmmoglobin aut haem

Planorbis in blood - ve Limnaea connective tissue tract?

sheath Aplysia C.N.S. - ve Helix muscle of pharynx ++ Patella ,, ,, ++ Chiton ,, 9 s + I -

The occurrence of gut haems has apparently no connection with the presence of haemocyanin nor with absence of haemoglobin, since Planorbis guadaloupensis was found to have traces of gut haemochro- mogen and, incidentally, many Crustacea and Polychaeta with haemo- globin in the blood, have a gut haemochromogen. It appears possible that the gut haemochromogens are made by the animals in which they are found, from tissue rather than blood haem compounds.

MacMunn (1886) extracted a porphyrin from the integument of Limax jlavus, L. variegatus and Arion ater ( = A. empiricorum) (all terrestrial animals, of course) and identified this pigment with haemato- porphyrin, the only porphyrin known at that time. In 1887, MacMunn detected the same pigment in the shell of the bivalve Solecurtus ~tr~giZZ~tus, which is deep pink. Dh6r6 and Baumeler (1 928) confirmed the presence of a porphyrin in the integument of Arion ater but did not identify it ; Kennedy (1959) found it to be uroporphyrin I in concentra- tion so high that isolation and crystallization could be achieved from the integument of one large individual of the black form. The concentration

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PIGMENTS O F MARTNE INVERTEBRATES 347

of integumental porphyrin decreases in parallel with the amount of melanin until in the pallid forms there is no porphyrin at all.

Comfort’s (1951) review of the pigments of mollusc shells contains much of his own work on their chemistry and distribution. In it, he wrote : “ The distribution of porphyrins closely follows the accepted anatomical classification of molluscs. They occur widely in the Arch- aeogastropoda, though not in Patella, Pleurotomaria or most species of Haliotis, being replaced in the Turbinidae by linear pyrroles. They also occur in several families of Lamellariacea, in several Cypraea, in Mar- ginella ornata, in several tectibranchs and in Umbraculum, in several loricates, scaphopods and among bivalves in the Anomiidae (Placuna, Enigmonia), Pinctada, Malleus, Pinna and a few isolated Veneridae. They are absent from all land and freshwater shells which have been studied except for a few species of the Neritinidae ”. Comfort then gives a list of the main genera in which porphyrins have been shown to occur, and mentions that the distribution pattern in an individual species may or may not coincide with the visible pigment, in some forms generalized and in others confined to a single locus. Shell porphyrins have great stability, shown by their detection in post-Pleistocene and Upper Eocene fossil shells.

Free uroporphyrin I was isolated from the shells of Pteria, Pinctada and Trochus by Hans Fischer and his associates (1930, 1931, 1937). They reported accompanying traces of coproporphyrin and “ conchopor phyrin ” which was said to be pentacarboxylic. Nicholas and Comfort (1 949) re-examined Fischer’s material and extracted many different species of Pteria, but could not find any evidence for a 5-COOH por- phyrin. These workers reported that their investigation of eight species of mollusc shells revealed that uroporphyrin (unspecified) was the only porphyrin present, with the exception that in Pinctada vulgaris traces of coproporphyrin (unspecified) were found. In the light of considerable experience of pigment variation, it is possible that the 5-COOH porphy- rin may have been present in some individuals but not in others ; Kennedy and Vevers (1954) found such a porphyrin in Chaetopterus variopedatus, but have not encountered it since!

The reddish stippling on the siphon of Bankia setacea (the shipworm or “ pile worm ”) contains protoporphyrin as solid intracelluler granules and Lockwood, in a personal communication, has said that he found protoporphyrin in “ quite large amounts ” in the foot of a species of Haliotis. These are the only two instances known to me of a porphyrin other than uroporphyrin I occurring in the Mollusca (except for the squid Histioteuthis, described later).

It would be interesting to examine some other genera of wood-

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348 a. Y. KENNEDY

borers, e.g. Teredo, Nototeredo and Psiloteredo (which are British) and the massive tropical Kuphus polythalamia. Then there are two other genera of wood-boring molluscs-Xylophaga and Martesia. There may be some connection between the ability to bore through wood and the possession of protoporphyrin.

Kennedy and Vevers (1954) found uroporphyrin I in the upper integument of the nudibranch Duvaucelia ( = Tritonia) plebeia and the opisthobranch Aplysia punctata, but not in Archidoris britannica or Jorunna tomentom, which are usually of an off-white colour. Kennedy and Vevers (1956) isolated uroporphyrin I from the integument of the tectibranch Akera bullata, which made a sudden appearance in static

CH2

COO-C~,H~,

FIG. 13. Chlorophyll b.

water-tanks in H. M. Dockyard, Devonport, and Kennedy (1976, un- published) also found this porphyrin in the integument of the Califor- nian species Aplysia californica in the laboratory of the marine station of the University of Southern California at Catalina Island.

While working with Akera bullata, Kennedy and Vevers also found the copper chelate of phaeophorbide a in extracts of the viscera. Morton and Kennedy (unpublished) detected the same pigment in the viscera of Aplysia punctata.

The selective retention of a derivative of chlorophyll a in these two molluscs is most interesting and is but one more example of the selec- tivity of some animals for certain molecules. One is reminded at once of the polychaete Owenia fusiformis which was found by Dales (1957) to have granules of phaeophorbide b in the epithelial cells, and of the

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PIGMENTS O F MARINE INVERTEBRATES 349

polychaete Flabelligera afinis which has the copper chelate of phaeo- phorbide a in the body wall and gut. There are also animals which are able to discriminate between carotenes and xanthophylls, as w0 have seen. These chlorophyll derivatives must clearly come from the food and either the animal discards the unwanted pigment or may feed specifi- cally on material containing one chlorophyll only, such as the Xantho- phyceae which contain chlorophyll a only. The chelation of the copper may be a detoxication device.

The very striking defensive secretion (" ink ") of Aplysia species has been studied at various times. Ziegler (1866) asserted that the deep-violet-red coloiir was due to " natural aniline dyes '' ; Moseley (1877) made some experiments on solubility of the pigment and Mac-

COOH C O O H I I

C H 3 C H 2 CH, C H 2 I II CH, CH

N H H H H

FIO. 14. Structure of phycoerythrobilin.

Munn (1899) precipitated the pigment with saturated ammonium sul- phate and named it " aplysiopurpurin ". The aplysiopurpurin comes from the glands of Bohatsch in the operculum. These are pear-shaped vesicles, among which Mazzarelli (1891) distinguished three types : " cellules odorifhres, cellules cromatogbnes and cellules mucoses gigantes- ques." These produce, respectively, a white secretion strongly smelling of musk; a violet secretion and a slimy mucous secretion. Similar glands appear in all the Aplysidae, e.g. Dolabella, Aplysiella, Notarchus and, of course, Aplysia.

The first suggestion that aplysiopurpurin might be a bilin came from Derrien and Turchini (1925), and Fontaine and Raffy (1936) compared its properties with those of mesobiliviolin, suggesting that it was derived from the red algae upon which the animal browses. Lederer and Huttrer (1 942) reported that the pigment was a mixture of two biliproteins, aplysioviolin and aplysiorhodin. Riidiger (1967) working with A . limacina placed the identity of the pigment beyond doubt in an elegant degradation study, showing that the pigment is indeed a new type of

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350 0. Y. KENNEDY

bilin, the monomethyl ester of a biladiene dicarboxylic acid, phycoery- throbilin (Fig. 14). The same pigment occurs in A. punctata and A . californica.

A. depilans, by mechanical or electrical stimulation, emits a white secretion which is toxic to lower animals, causing paralysis. This is also produced by A . punctata accompanied by the violet aplysiopurpurin, but A . limacina secretes the purple “ ink ” alone which appears to be non-toxic.

The red-yellow accompanying pigment (Lederer’s aplysiorhodin) is more strongly polar than aplysioviolin, and has a single absorption band at 495 nm, so that it must be of the urobilin type and has, in fact, been named aplysiourobilin. Riidiger also (1967) found that the blue com- ponent gave an absorption spectrum with peaks at 688 and 366 nm (in acid-methanol) indicative of a bilatriene, and this was named aplysio- verdin. (Winkler in 1959 had found this pigment in A . californica and labelled it aplysioazurin.) Aplysioverdin appeared to be a mono-ester (Riidiger, unpublished).

Aplysia spp. are strict herbivores, and A. califronica obtains its pig- ment from the phycoerythrobilin (Fig. 14) of the larger Rhodophyceae, but the European species usually graze on Ulva (Chlorophyceae) so that the immediate source of their pigment is still unknown. Chapman and Fox (1969) exhausted the purple glands of A. californica, maintained the animals on a diet exclusively of Phaeophyceae, and found that the purple secretion did not develop until the normal diet of Rhodophyceae was restored. If a phycocyanin diet was provided in this experiment, the animals secreted small amounts of phycocyanobilin monomethyl ester and, in contrast t o phycoerytlwin, a diet of phycocyanin leads to the deposition of blue chromopeptides in the inner skin. Chapman and Fox do not consider the ink to be defensive.

The beautiful blue pigmentation of the shells and ink of the pelagic gastropod Janthina janthina has attracted attention for many years without stimulating any extended study. Moseley (1877) carried out a few experiments with the secretion and the shell, finding that it dis- solved easily in alcohoI to form a violet sohtion which turned light blue with acid ; the solution had an absorption spectrum showing two bands.

Comfort (1961) examined the secretion and shells of Janthina which had been stranded on beaches at Ballineden, north of Sligo Bay, and at Trawalua, on the west side of Mullaghmore. Working under difficulties, he had little to add to Moseley’s account, but found that the pigment was intensely red-fluorescent in U.V. light (like aplysiopurpurin) and that the violet component of the latter, chelated with zinc, was the nearest to janthinine in absorption spectrum :

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Violet component of aplysiopurpurin, Zn complex (cf. Riidiger, 1967) :

600 535 505 nm.

Violet pigment from Janthina: 605 (530) 505 nm.

Since Comfort found that the addition of zinc acetate did not alter the absorption spectrum of janthinine, and from the similarity to zinc- aplysioviolin, it is possible that janthinine is a naturally-occurring zinc complex. There is precedent for this in the study by Kennedy and Vevers (1973, 1976) on avian eggshells and the occurrence of copper complexes in the molluscs already mentioned.

Mollusca is the only phylum of marine invertebrates in which bili- proteins have been detected so far. (It is not yet known whether the biliverdin of the blue coral Heliopora (see p. 323) is associated with protein or not.)

The presence of bile pigments in the shells of molluscs was first reported by Krukenberg in 1883 in the genera Haliotis, Turbo and Trochus. His results were confirmed by Schulz (1904) which led eventu- ally to some new work. The shells of Haliotis rufescens are deep red and those of H . gigantea and H . californiensis are deep blue. Turbo olivaceus, T. radiatus, T. petholatus and T . regenfussi are greenish, T . sarmaticus and T . rugosus are red-brown.

Dhh.5 and Baumeler (1930) took up Haliotis rufescens, extracted the pigment, dubbed " rufescine ", and determined its absorption spectrum and reactions. They found that the pigment resembled bilirubin. Following further studies by Tixier (1945), Riidiger and Klose (in pre- paration) found, in H . rufescens, two green pigments (haliotisverdin) with rufescine and unconjugated haliotisviolin. Chapman and Riidiger (cited by Fox, 1974) examined a dozen Haliotis species and in every one found haliotisrubin ( = rufescine) which has keto-groups in place of the terminal hydroxyl radicals present in bilirubin. These workers suggest that this pigment is derived from the red algae on which the molluscs are voracious feeders.

Rudiger (1970) described pigments from H . Zamellosa, which in- cluded, in addition to the main component haliotisviolin, a bright red one, haliotisrhodin. From H . gigantea (Japan), haliotisviolin and halio- tisrhodin were obtained which were indistinguishable in absorption spectrum and chromatographic Rf values from the pigments of H . lamellosa. There were also two green pigments which were different in absorption spectrum from known bile pigments.

From H . cracherodii, the Black Abalone, described by Tixier (1952) and Riidiger (1970), only haliotisverdin is like that from H . gigantea;

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352 a. Y. KENNEDY

the blue-violet pigment is more strongly polar than that from other species and can be separated into two components, A and B, whose spectra are similar to that of haliotisviolin. They may be present in the form of conjugates. Using his chromic acid degradation method, Riidiger suggested that haliotisviolin may have a structure related to that of protoporphyrin.

Krukenberg (1883), as already mentioned, examined shells of Turbo spp. and obtained green solutions which did not give a clear absorption spectrum but gave a positive Gmelin reaction. However, the green pig- ment differed from biliverdin in that it was not extracted by chloroform from acid solutions. Krukenberg also extracted shells of Turbo sarma- ticus and T. rugosus, obtaining a brown pigment " turbobrunine " which became purple-red in acid and under certain conditions turned green. Tixier (1952) presented a study of the pigments of T . regenfussi from Nha Trang in what is now known as Vietnam. A calcareous crust covered and protected the pigmented surface of the shells, but Tixier isolated a green-blue pigment to which he gave the name " turboglauco- bilin ", and worked out its physical and chemical properties.

Turboglaucobilin had all the properties of a bilatriene, and an absorption spectrum midway between those of mesobiliverdin (glauco- bilin) and coproglaucobilin on the one hand, and biliverdin on the other. Tixier obtained the same pigment from T . marmoratus and T. elegans. Considering the data at his disposal, the empirical formula was very close to that for the methyl ester of coproglaucobilin, except for the presence of 12 or 13 oxygen atoms against the 10 for that pigment. Turboglaucobilin has 4 carboxyl groups, like coproglaucobilin and, if all is in order, must be the first occurrence in the animal kingdom of a bile pigment derived from coproporphyrin and not protoporphyrin. It would also be the first appearance of a derivative of coproporphyrin in the Mollusca, whose usual porphyrin is uroporphyrin I. Presumably, therefore, turboglaucobilin would then relate to coproporphyrin I , the macrocyclic ring having broken at the a-position (cf. Kennedy, 1969). This would also be the first bile pigment in nature with a series Ia configuration, so obviously further corroborative evidence is desirable.

The pink and green colours of pearls-apart from interference effects-are said to be due to traces of porphyrins and metalloporphy- rins (Kosaki, 1947 ; Takagi and Tanaka, 1955; Takagi, 1956) : the pink colours are due to free porphyrins and the green to metalloporphyrins. Confirmation should prove expensive! This could be another example of porphyrins being concerned in the formation of calcareous tissues, as in mollusc shells, avian eggshells and mammalian ossification.

Other pigments in Mollusca include the well known and ancient

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Tyrian Purple, formed from the crushed tissues of Murex, Mitra and Nucella ( = Purpura) species, when exposed to the sun. The tissue mash turns first green, then blue, red, and finally purple. RBaumur (1711) who described these colour-changes came to the conclusion that they took place under the influence of air-but we now know that the reaction is independent of air or oxygen. It is possible that the colours through which the crushed tissues pass are due to the sequence Indigo green, Indirubin and Indigotin.

Lacaze-Duthiers (1859) found that the secretion responsible for the formation of the Tyrian Purple comes from the hypobranchial (or adrectal) gland. The pigment was isolated and crystallized by Fried- lander (1909) and shown by him to be 6, 6'-dibromindigotin. Fischer (1925) reported it in the fluid surrounding the eggs in their capsule, observed long ago by RBaumur in 1711. Dubois (1902) had separated two fractions from the glands of Murex brandaris, which only formed the purple when mixed. He suggested the presence of an enzyme which he called " purpurase " and thought that there was a difference between the chromogenic systems of M . brandaris and M . trunculus. As we may see, Dubois was ahead of his time, for two prochromogens were isolated from the glands of M . trunculus and one from M . brandaris by Bouchil- loux and Roche (1955). All prochromogens released sulphate with a sulphatase (a fairly common enzyme in molluscs). Prochromogen I ( M . trunculus) seems to be indoxyl, oxidizable to indigotin. Prochro- mogen I1 ( M . trunculus and M . brandaris) is 6-bromoindigotin.

The use of the fine purple dye formed from Murex and Purpura (the old name) is as old as the hills and has considerable antiquarian interest. The Phoenicians were the first to create an industry for the extraction of purple-hence Tyrian Purple-in their sea-ports, and vast mounds of empty shells have been found at Tyre and Sidon. New towns arose in the western Mediterranean by Phoenicians looking for new grounds of Murex. Dedekind (1896a) discovered, from a study of hieroglyphic inscriptions, that the ancient Egyptians knew about purple. The Museum of Art in Vienna holds a shroud dyed with purple which had covered a mummy, obviously of someone of noble birth, and it is possible that the red shroud which covered the third mummiform sheath of Tutankhamen was dyed with the pigment. The dye was much used by the Babylonians, as we read in the Bible (Jeremiah X, 9) and the hangings of the Temples of the Israelites were also of that colour (Exodus XXV, 4).

The Assyrians knew purple, and Dedekind (1896b), with the help of the Sanskrit scholar Friedrich Muller, discovered that the etymology of the word purpur goes back to the indo-germanic root bharbhur. This

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364 a. Y. KENNEDY

word denotes something which rapidly moves or changes and might have a connection with the striking way in which the purple forms quickly under the influence of sunlight before the very eyes of the beholder.

In Asia, the value of Tyrian Purple surpassed that of silver. The Greeks and Romans used Murex although, according to Pliny, the genus Nucella ( = Purpura) was then known as Buccinum, and Murex was Purpura. The dignitaries and Senators of Rome were recognized as the Purpurati, and only those and the Emperors were allowed to wear the purple, hence the description “ porphyrogenite ”, one “ born to the purple ”. In Rome, the discarded shells accumulated, forming a hill known as Monte Testaccio.

The mollusca affording the best dye came from the rocks off the coast of Tyre, but they were also to be found at Meninge on the shores of Africa and on the coast of Laconia in Europe. The colour of the dye produced varied according to the district from which it was brought. From Pontus, in Galatia, the dye was very dark, almost black, while the warmer the region the more violet the dye until a t Rhodes the colour was a rich red. In the manufacture of the dye, animals from different districts were mixed together to produce the best effect. Pliny records that one hundred of one sort were mixed with one hundred-and-eleven of another to produce the finest purple.

The great esteem in which the dye was held stems from the fact that neither vegetable dyes nor cochineal (also known to Pliny) could resist the burning sun of Italy, Greece or the Orient, whereas purple was stable and fast. Fox (1974) quoting from Lucretius (99-55 BC) : “. . . . and the purple tint of the shell-fish is united . . . . with the body of the wool, yet it cannot be separated . . . . not . . . . if the whole sea should strive to wash it out with all its waves.” Light-resisting and fastness Considered, it is extraordinary that the discovery and use of kermes (Kennedy, 1969) should lead to a diminution of the demand for Tyrian Purple and its eventual eclipse.

Purple was the colour of the robes of cardinals until 1464 when Pope Paul I1 decreed that they should wear scarlet. Fabius Columna in 1616 and Major in 1675 made studies of Tyrian Purple and William Cole (1 683) a Bristol Master-dyer found a way to dye linen and silk bright crimson starting with the shellfish, exposing the fabric to sunlight and finally washing and boiling in soapy water (Sace, 1854).

The name of Ford’s famous murexide reaction for acidic pyrimidines comes from the similarity of the colour to that produced from Murex. According to Newbigin (1898), Clathrus ( = Scalaria) species (Gastro- poda : Epitoniidae) also has a purple secretion but nothing seems to have been written about it.

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Price and Hunt ( 1974) report fluorescent chromophore components from the egg-capsules of Buccinum undatum. These contain a yellow fluorophore with aldehyde functional groups and a strong blue-white iluorescence in U.V. light.

Melanins are found in molluscs in various guises and colours. In Littorina spp. the shells may be black, red, orange, yellow or off-white, most likely due to melanins ; Comfort (1951) suggests that they may be melanoproteins analogous to those found in feathers. Octopus bima- culatus the two-spotted octopus of the North-American Pacific coast stores dark brown melanin (or melanoprotein) in the glandular ink sac attached to the pancreas. The ink is squirted from the duct opening beside the anus. Melanin occurs in the ink of the cuttlefish Sepia oficinalis and the dried sacs used to be sold by artists’ colourmen as the brown pigment sepia. A similar, though much darker pigment, is found in the ink of the squid Loligo spp.

It is noteworthy that carotenoids are comparatively sparse in cephalopods. Lonnberg (1936) found lutein in the eyes of three species, but traces only elsewhere, save for the liver of Eledone. Carotenoids are also absent from the eggs of cephalopods. Fox and Crane (1942) reported carotenoids in Octopus ( = Paroctopus) bimaculatus in liver and ink: 8-carotene and xanthophylls in the liver and xanthophylls only in the ink.

Siuda (1 974) has reported the presence of 8-hydroxy-4-quinalone from the ink of the giant octopus 0. dofleini, together with tryptophan metabolites-the latter from the melanin biosynthetic pathway, of course. Tyrosinase was detected in the cephalopod ink sac by Gessard (1902) who demonstrated it in the dried Sepia sacs already men- tioned.

Nardi and Steinberg (1 974) have isolated adenochrome from the branchial hearts of Octopus vulgaris and studied its distribution and properties. This pigment was previously isolated by Fox and Updegraff (1944) from the branchial hearts of Octopus ( = Paroctopus) bimaculatus and dubbed adenochrome, but it was contaminated by a yellow pigment. The new method of Nardi and Steinberg is based upon the initial separation of pigment granules from whole tissue, followed by chromato- graphy on Bio-Gel P10, which allowed separation of the adenochrome from the yellow contaminant. The yellow pigment is stable at all pH values ; ferric chloride turns it red, and it has been suggested that it is a natural complexing agent with a high affinity for iron, the metal being essential for the purple-red colour of adenochrome.

Herring (unpublished) has found that the photophores in the squid Histioteuthis meleagroteuthis are red-fluorescent in U.V. light (Plate I)

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and holds the view that this is due to a porphyrin which acts as a filter pigment. His early studies point to the pigment being protoporphyrin and, if this is true, this would be another instance of the occurrence of this porphyrin in the molluscs, as mentioned earlier.

XIII. CHAETOGNATHA The chaetognaths do not appear to have haemoglobin in any species.

They are usually transparent with a silvery sheen, e.g. Sagitta (the arrow- worm), but those which live at great depths may be red or orange. Nothing seems known about these pigments although the colour and the fact that they feed largely upon small crustacea suggests that the pigment might be carotenoid.

XIV. BRACHIOPODA Carotenoids are frequent in the brachiopods, particularly in the

gonads (Hyman, 1959). Carotene and xanthophyll (both unspecified) were found by Ltmnberg (1931) in Crania anomala and Terebratulina caput-serpentis.

In some lingulids, e.g. Lingula unguis, the respiratory pigment is haemerythrin in coelomic corpuscles (Kawaguti, 1941), red in the oxidized state and colourless when de-oxygenated. (It should be remem- bered that this pigment has no porphyrin prosthetic group.)

Terbratella rubicunda from New Zealand (H. M. Fox, unpublished) displays a fine red fluorescence in U.V. light, which may be due to a porphyrin. The lophophores of some brachiopods are pink which may be due to porphyrin, and it is interesting that the shells of the Devonian fossil brachiopod Cranaena have wide radial maroon bands on the inner part which may be the preservation of the original pigment (Cloud, 1941). This might be porphyrin since these pigments have tremendous stability but, on the other hand, it may be that ferric oxide deposition has replaced the original organic pigment ; U.V. examination would decide this in a moment.

Ohuye (1936) found ;L naphthoquinone in Terbratalia corcanica which must be the first report of this class of pigment in marine invertebrates outside the echinoderms.

XV. POLYZOA MacMunn (1 890) reported the presence of astacene in Flustra

foliacea and Pentapora (Lepralia) foliacea : this was almost certainly astaxanthin. Lbnnberg and Hellstrom (1931) found a carotene and a

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0. Y. KENNEDY

1

2 PLATE I. Photophores of Histioteuthis meleagroteuthis:

1. Whole animal in white light; 2. Surrounding eye in white light.

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3

4 PLATE I (continued):.

3. Body and part of head in U.V. light; 4. Surrounding the eye, U.V. light. (Herring, P. J. Unpublished.)

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PIGMENTS O F MARINE INVERTEBRATES 357

xanthophyll (both unspecified) in Alcyonidium gelatinosum and in Flustra securifrons.

Krukenberg (1 882) described a carotenoid in Bugula neritina, together with a diffuse reddish pigment in branched cells in the living polyps. Immersion of the brown colonies in fresh water brought out a purple pigment into solution which was precipitated by the addition of alcohol and named Bugula-purpur. The pigment would not dissolve in ether, chloroform or carbon disulphide, and was very unstable to light ; it was also decolorized by hydrogen sulphide, chlorine and hydrogen peroxide. Ammonia and hydrochloric acid each produced a bluish- violet colour. The absorption spectrum had two bands in the green and the blue regions.

Bugula-purpur remained without further investigation until 1948 when Villela (1948a, b) examined B. neritina and B. Jlabellata in Brazil, both from deep water in Guanabara Bay. He reported the purple pig- ment described by Krukenberg and found that it occurred in granules at the distal portion of the zooecium and round the “ corpo castanho ” or brown body, considered to be concerned in excretion. Bugula-purpur has some resemblances to the adenochrome of Fox and Updegraff (1944) but the main point of difference is that of absorption spectrum : Bugula- purpur’s sharp peak lies at 525 nm, whereas the broad peak of adeno- chrome has a centre at 505 nm.

Needham (1974) quoting Villela (1948b) thinks that it may be significant that astaxanthin accumulates distally in the zooecia of Bugula. Newbigin (1898) mentions that the purple pigment disappears for a certain period during the development of the young polyps, and reappears later.

The colours of polyzoans, even in temperate waters are often very beautiful. The calcareous Pentapora (Lepralia) foliacea has a red pig- ment in addition to the carotenoid found by MacMunn (1890). Some polyzoans have commensal algae, so that care must be taken in inter- preting results.

Schizoporella unicornis forms bright red colonies and Villela (1948b) found two carotenes and a small amount of xanthophylls on extraction. Steginoporella magnilabris is also red, and here Villela found only caro- tenes (@-carotene predominating) while Trigonospora had carotenes, xanthophyll esters and an unfamiliar water-soluble yellow pigment, which might be a flavin.

XVI. ECHINODERMATA This phylum contains many beautiful and brightly-coloured

animals and among these red and orange seem to be the most frequent

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368 a. Y. KENNEDY

colours, but Alcock (1893), in his Indian Marine Survey, lists the colora- tion of twenty-five forms of asteroids of which 18 were pink or red, 1 black, 1 grey, 1 brown, 1 orange, 2 red or yellow and 1 yellow with brown. Many of these were from deep water but among the shallow water starfishes are Linckia lwvigata from the Great Barrier Reef in which the upper integument is bright blue and the tube-feet yellow. L. miliaris from the coral reefs of the Malay Archipelago is also blue, and Kiikenthal (1896) mentions that this species has a parasite (or com- mensal?) mollusc Capulus crystallinus of exactly the same blue colour. Pigments of the echinoderms include carotenoids, quinones, porphyrins, melanins and a few flavins and, of these, the most spectacular are the first two.

There are several reviews of echinoderm pigmentation, which include Fox (1953, 1976), Vevers (1966), Fox and Hopkins (1966), Cheesman et al. (1967) for carotenoproteins and Goodwin (1969). The last two are especially notable for the tables they contain showing the distribution of pigments with references, so that our task here is to give account of interesting and more recent work. Early observations of echinoderm pigments were by Krukenberg (1882) and MacMunn (1890) with Mere- jkowski (1881) and Heim (1891). Later came the studies of Lonnberg (1931-1934), Lederer (1935) and Fox and Scheer (1941).

The principal carotenoids are 8-carotene, cryptoxanthin, echinenone, astaxanthin, ketocarotenoids and small amounts of other xanthophylls. There may be carotenoproteins, as in Asterias rubens (Vevers, 1949) which may be yellow, brown, red or violet. It is interesting that in some colour variants of Henricia leviuscula, astaxanthin is absent (Fox and Hopkins, 1966). Hopkins (1967) also found an unusual apo-caro- tenoid neurosporoxanthin (4-apo-8-carotenoic acid).

A number of pigments have been found which may be intermediates between 8-carotene and astaxanthin, e.g. asteroidenone (3-hydroxy-4- keto-/?-carotene) and hydroxyasteroidenone (3, 3'-hydroxy-4-keto-8- carotene) (Giudice de Nicola, 1959).

Two of the most important pigments of the echinoderms are echine- none and echinochrome, the latter with its related spinochromes. Lederer (1938) obtained a mixture of carotenoids from the gonads of Xtrongylocentrotus lividus and, from the epiphasic fraction isolated echinenone, a monoketone (4-keto-/?-carotene)-incidentally, the first carotenoid of animal origin to exhibit vitamin A activity when fed to rats.

To MacMunn (1883) goes the discovery of the purple and brown pigments of the perivisceral fluid, ectodermal and endodermal tissues, spines and shell (test) of Echinus esculentus. He called these echino-

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PIQMENTS OF MARINE INVERTEBRATES 369

chromes. There was (I great deal of confusion in the naming and identi- fication of the “ echinochromes ” which Thomson (1971) has done much to clarify. Thomson gives valuable tables of distribution of echino- chromes and spinochromes in his book (1971). Echinochrome was crystallized by McClendon (1912) and a structure worked out by Kuhn and Wallenfels (1939), showing it to be a polyhydroxynaphthoquinone. Thomson points out that echinochrome A is a common spine pigment belonging to the spinochrome group (found in test and spines) so named by Lederer and Glaser (1938) to distinguish them from the pigments occurring in the perivisceral fluid, eggs and internal organs.

Gough and Sutherland (1964) have shown that the quinones pre- viously designated as spinochromes B, B1, M2, N and P are all identical, and Chang et al. (1964) have shown that spinochromes A and M are the same, as are spinochromes C and F. Spinochromes A to E and echino- chrome A are those most frequently found. The number of authentic spinochromes now exceeds twenty, so that the suggestion is made that these pigments should be named as substituted naphthazarins or juglones.

The distribution of spinochromes is well reviewed by Anderson et al. (1969). These pigments occur in spines and test as calcium and magne- sium salts, and are extracted with hydrochloric acid and ether. Most species of echinoid give up to six separate pigments, but Hawaiian echinoids Echinothrix diademu and E . calamaris were shown by Moore et al. (1966) to yield thirty pigments-half are spinochromes (some possibly as methyl esters), one is a benzoquinone and the remainder may not be quinones at all. Some ophiuroids have given eight spinochromes.

Two dihydroxy-dimethoxynaphthazarins are found in the “ Crown of Thorns ’’ starfish Acanthaster planci. Two novel bi-naphthoquinones have been isolated from Spatungus purpureus by Mathieson and Thom- son (1971) ; one was ethylidene-3,3’-bis(2, 6, 7-trihydroxynaphthazarin)

The first reported methoxylated spinochrome namakochrome-(2, 3, 6,-trihydroxy-7-methoxynaphthazarin), was found as a protein complex in the holothurian Polyckeira rufescens by Mukai (1958-1960). This animal is dark purple and the pigment occurs in the body wall.

Fucoxanthinol and paracentrone were isolated from the coelomic epithelium of Paracentrotus lividus by Galasko et al. (1969). Structures are given by Straub (1971). The presence of traces of fucoxanthin suggests that these two new allenes are formed by metabolism of fuco- xanthin in the diet. Pentaxanthin, the pigment reported by Lederer (1938) to be present in P . lividus, may have been iso-fucoxanthinol.

Asterinic acid, a xanthophyll occurring in the dorsal inetgument of Asterim rubens as a blue, violet or brown carotenoprotein (Vevers,

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360 a. Y. KENNEDY

1949) and thought to be astaxanthin (Liaaen-Jensen and Jensen, 1965), is now known to be a mixture of its mono- and di-acetylenic analogues (Liaaen-Jensen, 1969). Melanins are widely distributed in the Ophiu- roidea, Echinoidea and Holothuroidea ; they have not been clearly established in the Asteroidea.

Fontaine (1 962) examined the melanins in the ophiuroid Ophiocomina nigra and found the melanocytes well-developed but that there were no melanophores. He suggested that the varying colours of the integument were due to differences in the oxidation of the melanin but Vevers (1966) considers it possible that they may result from different degrees of polymerization.

In the echinoids, the melanin is found mainly in the skin and amoebocytes and has been seen in the axialorgan of Diadema antillarum, Paracentrotus lividus and Arbacia lixula (Vevers, 1967). Accompanying pigments were an hydroxynaphthaquinone, an unidentified pigment containing iron and a lipofuscin thought to be derived from phospholipid which was found in Diadema only.

Thyone briareus has large epidermal melanophores which contain melanin (Millott, 1950) and the body-wall of Holothuria forskali also. The amoebocytes of H . forskali have a phenolic system like that in Diadema (Millott, 1953).

1. Porphyrins MacMunn (1886) reported the presence of haematoporphyrin in the

integument of the starfish Uraster ( = Asterias) rubens. Kennedy and Vevers (1953a, b) confirmed the porphyrin but identified it as protopor- phyrin IX. They also examined Porania pulvillus, Palmipes mem- branaceus, Solaster papposus, Henricia sanguinolenta, Marthasterias glacialis, Ophiothrix fragilis, Ophiocomina nigra, Psammechinus miliaris and Antedon bi$da without finding a porphyrin in any of them.

Surprisingly, Luidia ciliaris and Astropecten irregularis had both protoporphyrin and chlorocruoroporphyrin in their integuments (Ken- nedy and Vevers, 1954). Warburg held that the presence of a carbonyl group in a side-chain (the formyl group in chlorocruoroporphyrin) is a primitive characteristic, and so the finding of this porphyrin in Luidia and Astropecten, both phanerozonian asteroids, may be regarded as additional evidence for the classification of the Phanerozonia as less specialized than the Forcipulata, e.g. Asterias.

Echinoids have not been shown to contain porphyrins with the exception of Arbacia lixula from Madeira, in which Kennedy and Vevers (1972) reported the occurrence of chlorin e6 in the test, with traces of coproporphyrin I. The tests of Arbacia lixula when viewed in U.V. light

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PIQMENTS OF MARINE INVERTEBRATES 361

show red fluorescence in the interambulacra, and the intensity of this fluorescence could be correlated with the visible appearance of these areas ; those tests which had no reddish-orange bands gave no fluore- scence. This is believed to be the only occurrence so far of a derivative of chlorophyll incorporated in a calcified animal tissue.

The naphthaquinones are present as calcium salts in the spines and test, and it is likely that the chlorin is also in this form, in view of its

TABLE 11. CORRELATION BETWEEN VISIBLE COLORATION AND RED FLUORES- CENCE (u.v.) OF THE TESTS OF Arbacia Zixula (Kennedy and Vevers, 1972)

Examined dry Examined wet

Animal viaible red visible colour red jlmreaceme naphthapinone jlmrescence oolour in U.V.

1

6 7 8 9

10 11

strong

nil nil

marked

nil

slight diffuse nil indefinite nil slight marked

strong

nil slight

very marked nil

diffuse nil very dull negligible slight strong

diffuse on either side of interambulacra ; ve'y strong especially in interambulacra

slight in interambulacra absent slight sIight ; acetone brings

up fluorescence strong strong ; no change with

acetone general slight slight; no change after

acetone slight general slight, diffuse nil nil nil slight strong slight strong intense strong intense but uneven

three carboxyl groups. It is reasonable that Arbacia, needing to rid it- self of photosensitizing pigment, should incorporate it into the calcified test, where it can do no harm, much in the same way that porphyrins are found in quantity in a few asteroids, some mollusc shells, avian eggshells and some bones and teeth in a few mammals. It is unlikely that the chlorin plays a part in calcification of the test, since no tetrapyrrole pigment has yet been found in the tests of any other echinoids. The naphthaquinones at the same locus may serve to prevent photosensiti- zation of the adjacent integumentary epithelium.

Harvey (1956) found that light-coloured Arbacia ZixuZa at Naples

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361 a. Y. KENNEDY

darkened in visible light and dark-coloured specimens paled in the dark over a period of a month or so. She suggested that visible light must play a part in the distribution of melanins or naphthaquinones or both. Certainly this observation points to the need for the formation of more pigment in light conditions, possibly to mask the photosensitivity caused by the chlorin e6, and this would lessen in the dark. Supporting this are the observations of Kennedy and Vevers (1972), see Table 11. It may be seen from this table that strongly-coloured tests have an intense red fluorescence in the interambulacra, while those tests which have very little colour have no fluorescence.

Holothurians do not seem to have porphyrins, although four species, Caudina, Cucumaria miniata and two species of Thyonella have special haemoglobins (Prosser and Brown, 196 1). Unidentified naphtho- quinones have been detected in Stichopus japonicus (Yamaguchi, 1961).

Carotenoids in holothurians have been known for some time. Goodwin (1969) discusses the occurrence of carotenoids in some holo- thurians, notably the report of red-violet and green carotenoproteins in Holothuria ( 3 species) and Cucumaria ( 3 species) by Toumanoff (1926).

The well-known yellow, green-fluorescent pigment (the “ uranidine ” of Krukenberg) seen when Holothuria forskali or H . nigra are preserved in alcohol may be a flavin together with melanogenic material (Riming- ton and Kennedy, 1962). Crude preparations were able to replace riboflavin when fed to riboflavin-deficient rats. Villela (1961) observed a similar pigment in H . grisea and H . lubrica. There may be pterins in holothurians too.

Fontaine (1962) reported a flavin from Ophiocomina nigra, but the site is uncertain since whole animals were used. Mattisson (1961) described a flavin with a blue fluorescence in Parastichopus tremulus muscle.

2. CThOid8 The crinoids have some interesting pigments which are usually

anthraquinones; the occurrence of carotenoids in many of them is doubtful.

Moseley (1877) is credited with the initiation of research into the pigments of crinoids, and characterized three different pigments ; purple pentacrinin and red pentacrinin from stalked crinoids and antedonin from the comatulid Antedon. Moseley’s specimens have now been identified as Hypalocrinus naresianus, Endoxocrinus alternicirris and several species of Metacrinus. The animal referred to as Antedon is now thought to have been Validia rotolaria.

Krukenberg (1882) extracted Antedon rosaceus (now A . adriaticus)

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PIGMENTS OF MARINE INVERTEBRATES 363

with alcohol to obtain the red comatulin contaminated with chlorophyll, and MacMunn (1890) examined extracts of Antedon rosacea (now known to have been A . bi@a) and only found " enterochlorophyll " from the stomach contents. He also worked with what is now Ptilometra australis and obtained antedonin ; there may have been a carotenoid present aa well.

Abeloos and Teissier (1926) reported two pigments in Antedon b i p a , and Lsnnberg (1931) observed that Antedon pentasus gave a brown red solution in methanol which had indicator properties. Karrer and Solmssen (1935) could not find much evidence of carotenoids in Antedon.

Dimelow (1958) described p-carotene, astaxanthin in free and esteri- fied form and xanthophyll in extracts of the arms and pinnules of Antedon bijida ; also present was an indicator pigment giving a green colour with ferric chloride. The behaviour and absorption peaks of this latter pigment were thought to be comparable with the hydroxynaph- thaquinone, probably echinochrome A, from the echinoid Diadema antillarum.

The bulk of the modern work on the crinoid pigments seems to have been done in Australia. The Australian crinoids Comatula pectinata and C . cratera were shown by Sutherland and Wells (1959, 1967) to contain three principal constituents ; the 6-methyl and the 6, 8-dimethyl ethers of rhodocomatulin, and a monomethyl ether of rubrocomatulin. The structure of the rhodocomatulin skeleton was shown by them to be 4-butyryl- 1, 3, 6, 8-tetrahydroxyanthraquinone.

Powell et al. (1967) gave details of the paper chromatography of hydroxyanthraquinones and the utility of various adsorbents for column chromatography. Powell and Sutherland ( 1967) examined Ptilometra australis and described a complex mixture of pigments, of which the principal components were :

1. 1 6,8-trihydroxy- 3-( 1 -hydroxypropyl)-anthraquinone 2. lJ6,8-trihydroxy-3-( 2-hydroxypropyl)-anthraquinone 3. 1,6,8-trihydroxy-3-propylanthraquinone carboxylic acid.

These authors state that the pigments of Comatula and Ptilometra con- form to the acetate rule and are probably endogenous in origin. Both black and yellow variants of Tropiometra afra contain the anthraquinone carboxylic acid (3).

The red pigment already described in Antedon bijida, the feather star, occurs in protein granules in superficial connective tissue and in the eggs (Dimelow, 1958). Miss Dimelow also found a-carotene and a lipofuscin in addition to the carotenoids already mentioned.

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364 0. Y. KENNEDY

XVII. POGONOPHORA Haemoglobin is the major pigment in the Pogonophora, and is

present in solution, not in corpuscles, in the blood (Southward and Southward, 1963). The pigment is in high concentration, approximately 2% of the whole animal, indicating the presence of the hnem biosyn- thetic system, but so far porphyrins have not been detected in any pogonophores (Kennedy, unpublished). The brown pigment of the epidermis is thought not to be a haemoglobin breakdown product.

XVIII. TUNICATA There are some very bright colours to be seen in tunicates, especially

among the sedentary species. There have been reports of a vanadium porphyrin in the vanadocytes of some ascidians, but these are certainly wrong. The coloured cells contain oxides of vanadium which may or may not be bound to proteins. The so-called " haemovanadin " is dealt with a t length by Needham (1974) where several important- references may be found. The striking thing about this pigment is that it is present in some ascidians in special intracellular bodies of cells which also contain 0.9 M sulphuric acid ; in others it is free in the plasma. Haemovanadin has a molecular weight of 900, so that it seems to be a fairly complex molecule.

The dark red Halocynthia papillosa contains, in the tunic, astaxan- thin, a- and p-carotenes and alloxanthin. The earlier papers gave cynthiaxanthin and pectenolone, but these diacetylenic carotenoids are known to be identical withalloxanthin (Campbell et al., 1967). The violet- red Dendrodoa grossularia has astaxanthin and the compound ascidian Botryllus schlosseri has alloxanthin.

There is an account of the pigments of some ascidians in Fox ( 1 953). Kennedy and Vevers (1954) examined Ascidiella aspersa, Ciona intesti- nalis and Botryllus schlosseri for porphyrins but found none.

XIX. COMMENT There is a full discussion of the origin, metabolism and function of

invertebrate pigments by Kennedy (1969, 1975) and in some chapters in Fox (1953, 1976) but one or two points may be added here by way of conclusion.

Ommochromes and melanins are concerned in the mechanism of colour change or intensity in response to hormones, and here the Macrura differ from the Brachyura in the responses of the black chro- matophores. The pigment in the Macrura is ommochrome and in the Brachyura it is melanin. The subject is well discussed by Needham

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PIGMENTS OF MARINE INVERTEBRATES 365

(1974) in his chapter dealing with the functions of integumental pig- ments in crypsis and semasis. Needham makes the interesting point that protection against light and heat is afforded by white reflecting surfaces and " this may be the biological reason for the prevalent white colora- tion of the Sargasso shrimps Latreutes, Leander and Hippolyte. Maximal protection through the whole solar spectrum could be given by a com- bination of white and black screens, and the fiddler crab Uca does display both black and white chromatophores in bright light ."

Haem enzymes and cytochromes are only present in trace amounts in the asteroid integument, and very likely in the tissues of echinoids too, although there seems to be no mention of this in the literature. It is very improbable that any animal is without cytochrome or haemo- protein of some kind. This is mentioned in view of the finding of traces of coproporphyrin I in Arbacia lixula test (Kennedy and Vevers, 1972). Coproporphyrin I11 is an intermediate in the haem biosynthetic system (in the form of coproporphyrinogen 111), but coproporphyrinogen I is not, and it may be that some echinoids, a long way back in evolution, having no use for haemoglobin, took the haem path as far as porphobi- linogen (PBG) and the uroporphyrinogen III-cosynthetase being absent, uroporphyrinogen I was formed, and then coproporphyrinogen I, which, having no part in the formation of haemoglobin, oxidized to the free porphyrin. The traces of coproporphyrin I in Arbacia may be the last remnants of this. The chlorin has a completely exogenous origin.

It is quite striking that throughout the invertebrates, free porphy- rins are scattered in a,n apparently random way but, since there must be a reason for everything, if one looks a t the list of porphyrins detected, items of evidence can be pieced together for the evolution of haemo- proteins and haemoglobin itself. Some animals have stopped a t uropor- phyrin I , as in the molluscs, through an evolutionary enzyme defect; others, like the polychaetes, go on as far as coproporphyrin I11 with small amounts of coproporphyrin I . Lumbricus has haemoglobin but protoporphyrin in the dark purple anterior dorsal integument, while Allolobophora has protoporphyrin plus coproporphyrin I11 and a tri- carboxylic porphyrin (Kennedy, 1969), demonstrating an active, though slightly inefficient, haem biosynthesis ; the green form has a bile pigment as well. Hexa- and penta-carboxylic porphyrins have been found in some annelids and formyl-porphyrin in two asteroids.

Lemberg (1949) suggested that the reason for sporadic and quantita- tively scanty occurrences of free porphyrins in nature may be the fact that protohaem production is so efficient. Comfort (1951) mentions the little known mollusc Enigmonia which has a great deal of free porphyrin in its thin shell and asks if the porphyrin is dietary in origin, is the

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366 Q. Y . KEKNEDY

animal unable to destroy it? Much more reasonable is his idea that in molluscs which have free porphyrin in their shells or integument (in the shell-less forms), there is a retention in phylogeny of the power of porphyrin synthesis by forms which can no longer dispose of it metaboli- cally-analogous to the formation of uric acid in man.

Readers of this review must be struck by the very frequent use of the words “ may ”, “ suggests ”, “ possibly ”, “ probably ” and so on, and if the impression gained is that there is still plenty of work to be done and many enigmas to be solved in this most satisfying and rewarding field, then our purpose will have been served.

XX. ACKNOWLEDGEMENTS I am most grateful to Dr Peter Herring of the Institute of Oceano-

graphic Sciences, Wormley, for permission to publish his beautiful pic- tures of Histioteuthis meteagroteuthis. Thanks are also due to Mr Roy Wilson of the Photographic Department in the University of Sheffield Library for much careful work. My own work on marine pigments has been supported for many years by grants from the Browne Fund of the Royal Society, and I offer my sincere thanks to their Committee.

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Abeloos, M. and Fischer, E. (1926). Sur I’origine et les migrations des pigments carotenoides chez les CrustacBs. Compte Rendu des Skances de la Socidtk de Biologie, 95, 383-384.

Abeloos, M. and Fischer, E. (1927). Les pigments carotenoides chez les Crustaces : sur l’origine des pigments de la carapace. Cornpte Rendu des Skances de la Sociktk de Biologie, 96, 374-375.

Abeloos, M. and Teissier, G. (1926). Notes sup les pigments animaux. Bulletirr de la Sociktc? de Zoologie de Prance, 51, 145-151.

Abeloos-Parize, M. and Abeloos-Parize, R. (1926). Sur l’origine alimentaire du pigment carotenoide de 1’Actinia equina L. Compte Rendu des Skances de la Socidtk de Biologie, 94, 560-562.

Aduco, V. (1888). La sostanza colorante rossa dell ‘Eustrongylus gigas I e 11. Rendiconti della R . Accademia dei Lincei, Roma, 4, 2, 187 e 213.

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Alcock, H. (1893). The Asteroidea of the Indian Marine Survey. Annals and Magazine of Natural History, 11, 73-121.

Anderson, H. A., Mathieson, J. W. and Thomson, R. H. (1969). Distribution of spinochrome pigments in echinoids. Comparative Biochemistry and Physio- logy, 28, 333-345.

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Arcichovskij, V. (1905). tfber das Zoopurpurin, ein neues Pigment der Protozoan Blepharkma lateritium. Archiv fiir Protistenhn.de, 6, 227-229.

Ball, E. G . (1977). Carotenoid pigments in the coxal gland of Limulus. Biological Bulletin, Woods Hole, 153, 10G112.

Ball, E. G. and Cooper, 0. (1949). Echinochrome: its absorption spectra, pK’ value and concentration in the eggs, amoebocytes and test of Asbacia punctulata. Biological Bulletin, Woods Hole, 97, 231-232.

Bannister, W. H. and Needham, A. E. (1971). Connective tissue pigment of the centipede Lithobius forficatus (L.). Naturzoissenschaften, 58, 58-69.

Barbier, M., Faur6-Fremiet, E. and Lederer, E. (1966). Sur les pigments du cili8 Stentor niger. Compte Rendu hebdomadaire des Sdances de I’Academie des Sciences, 242, 2182-2184.

Recherches sur les Rotifbres: les formations tbgumentaires et l’appareil digestif. These de Science Naturel, Paris. Archives de Zoologie ExpBrimentale et GBn6rale.

Beerstecher, E. (1950). The nutrition of the Crustacea. Vitamins and Hormonee,

Bloch-Raphael, C. (1948). Evolution de l’h6moglobine e t ses d6riv6s au cours du d6veloppment de Septosaccus cuenoti (Duboseq). Compte Rendu des Sdances de la SociBtd de Biologie, 142, 67-71.

Bogdanow, A. (1858). fitudes sur les causes de la coloration des oiseaux. Compte R e d u des Shnces de la Socidtk de Biologie, 46, 780-781.

Bonnett, R., Head, E. and Herring, P. H. (1978). Pigments of deep-sea fauna: porphyrin pigmentation in medusae. Personal communication.

Bouchilloux, S. and Roche, J. (1955). Contribution ti 1’8tude de la pourpre des Murex. Bulletin de l’lnstitut Ockanographique, Monaco, 52, 1-23.

Bradley, H. C. (1908). A green bilin in the digestive fluid of Cambarus. Journal of Biological Chemistry, 4, 36-37.

Brehm, V. ( 1938). Rotfiirbung von Hoohgebirgseeorganismen. Biological Reviews of the Cambridge Philosophical Society, 13, 307-31 8.

Bullock, E. (1970). Occurrence of free porphyrins in certain coelenterates. Com- parative Biochemistry and Physiology, 33, 71 1-713.

Burger, 0. (1895). “ Die Nemertinen des Golfes von Neapel ” Fauna und Flora Neapel, Monograph 22, 743 pp. Friedkinder, Berlin.

Busselen, P. (1971). The presence of haemocyanin and of serum proteins in the eggs of Carcinus moenas, Eriocheir sinensis and Portunua holsatus. Com- parative Biochemistry and Physiology, 38A, 317-328.

Cain, G. D. and Bassow, F. (1976). Porphyrins in the enteric fluid of Ascaris lumbricoidea. International Journal for Parasitology, 6, 79-82.

Campbell, S. A., Mallams, A. K., Waight, E. S., Weedon, B. C. L., Barbier, M., Lederer, E., and Salaque, A. (1967). Pectenoxanthin, Cynthiaxanthin and a new acetylenic carotenoid, Pectenolone. Chemical Communications of the Chemical Society, 941-942.

Castillo, R. and Lenel, R. (1978). Determination and metabolism of carotenoid pigments in the hermit crab Clibanariw erythropus Latreille ( 18 18). Com- parative Biochemistry and Physiology, SSB, 67-73.

Chang, C. W.-J., Moore, R. E. and Scheuer, P. J. (1964). Structure of Spino- chrome M . Journal of the American Chemical Society, 86, 2959-2961.

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Chatton, E., Lwoff, A. and Parat, M. (1926). L’origine, la nature et 1’6volution du pigment des Spirophyra, des Polyspira et des Gymnodinioides : pr6sence de carotinalbumins dans la mue des Crustac6s DBcapodes. Compte Rendu des sdances de la Sociitd de Biologie, 94, 567-570.

Cheesman, D. F., Lee, W. L. and Zagalsky, P. F. (1967). Carotenoproteins in Invertebrates. Biological Reviews of the Cambridge Philosophical Society, 42, 132-160.

Cheesman, D. F. and I’rebble, J. (1966). Astaxanthin ester as a prosthetic group : a carotenoproteiri from the hermit crab. Comparative Biochemistry and

Christomanos, A. (1953). Purple pigment and protein in the threads of the sea- anemone Adamsia rondeleti. Nature, London, 171, 886-887.

Cloud, P. E. (1941). Colour patterns in terebratulids. American Journal of Science, 239, 905-907.

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