Comp. Biochem. PhysioL, 1973, Vol. 44B, pp. 953 to 962. Pergamon Press. Printed in Great Britain

CHITIN-BOUND KETO-CAROTENOIDS IN A CRUSTACEAN CARAPACE

DENIS L. FOX

Division of Marine Biology, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92037

(Received 24 ffuly 1972)

Abstract- -1 . Chitinous parts from the carapace of the red kelp crab, Taliepus nuttallii, yielded no or negligible pigment to common organic solvents (save to pyridine) and/or protein denaturants unless first decalcified.

2. Hot aqueous acetic acid readily decalcified the shell and extracted all pigment, which was resolved into but four fractions, in the following order of increasing proportion and decreasing polarity; astaxanthin, phoenicoxanthin, canthaxanthin and echinenone, all of which had been firmly bonded to chitin.

INTRODUCTION

IT IS WELL recognized that many animal species are able to convert ingested, yellow plant carotenoids into oxygenated, thus more polar, orange or red keto derivatives, and in some instances to then conjugate these to give variously colored chromo- proteins (e.g. in blood, eggs, skin or feathers), or in certain hydrocoral skeletons, calcareous esters (Fox, 1953, 1972a, b; Fox & Hopkins, 1966; Cheesman et al., 1967; Thommen, 1971).

The recent finding, in this laboratory, of carotenoids chemically bonded to calcium carbonate, e.g. in the orange-yellow spicules of the gorgonian coral Eugorgia ampla (Fox et al., 1969), and in purple, red or orange calcareous skeletons of several hydrocoral species (Fox & Wilkie, 1970; Fox, 1972b) have evoked questions as to the status of chromogens involved in the hard parts of other animals, such as crustacean carapaces. Granted that there are numerous reports of chromoproteins recovered from exoskeletal parts of crustaceans (Cheesman et al., 1967; Thommen, 1971), the fact remains that not all pigment (indeed in some instances relatively little) is recoverable from some chitinous crustacean material merely by exposure to the usual carotenoid solvents and/or protein denaturants, unless the shell has first been decalcified, e.g. with citric acid (Cheesman et al., 1967).

The red kelp crab, Taliepus nuttallii, inhabiting coastal waters off southern California, possesses a very hard, brittle exoskeleton, and was suspected of perhaps storing calcareous carotenoid esters in the hard parts, notably since it retained the shell pigment even in the presence of hot alkali. It was of interest to discover, however, that the carotenoids therein are instead firmly bonded in some fashion to the chitin itself.

953

954 DENIS L. Fox

MATERIALS AND METHODS Specimens were collected freshly from intertidal kelp beds off Point Loma, San Diego.

One which had sustained an accidental open fracture on the dorsal surface of its carapace had just died, and was placed in a freezer at once, while the others were placed on display and for experimental feeding purposes. The expired specimen was a mature-sized female whose carapace measured 5"3 cm wide by 6"5 cm long from rostral tips to posterior, omitting the ventrally folded telson.

Living TaIiepus nuttallii consume algal blades, e.g. Macrocystis pyrifera, readily, voiding copious fecal rods of the comminuted plant material, and hence enjoy ample supplies of this kind of carotenoid-rich food. The active feeding and fecal accumulation were demon- strated and photographed in the aquarium.

Pure chemicals, spectrophotometric instrumentation and thin-layer chromatographic equipment, as described earlier (e.g. Fox & Hopkins, 1966), were used in this investigation. Silica gel-H was the adsorbent on the plates, and acetone in hexane (30/70) the chromato- graphic eluant; 5% methanol in hexane (v/v) was employed for eluting individual fractions from the developed plate. Evaporation of hexane for adjusting to suitable volume or for treatment with added ethanolic alkali, was conducted in a stream of nitrogen while the containing vessel stood in a warm water-bath at ca. 65°C.

RESULTS

It was desirable to investigate first whether the red pigment were perchance associated, as in some hydrocorals, with the calcareous component of the exo- skeleton of this crab, since 54 per cent by weight of the cleaned and dried carapace parts had been determined by ashing procedures, to comprise such inorganic material. Aqueous citric acid (pH = ca. 2), aqueous Na2EDTA (pH = ca. 5) or dilute HC1 dissolved the calcium carbonate, leaving all pigment firmly associated with the tough, now pliable chitinous pieces. This animal, accordingly, does not store calcareously bound carotenoids, as do some of the hydrocorals (Fox & Wilkie, 1970; Fox, 1972b).

I t was then necessary to learn whether or not the pigments were bound to scleroprotein as occurs, e.g. in some feathers. However, boiling chips of red carapace in strong alkali, either without or after previous decalcification, appeared to digest none of the material at all; nor did the treatment release pigment, whereas exposure of red feathers to alkali digests the keratin and releases the carotenoids. Moreover, pieces of decalcified exoskeleton failed to give evidence for the presence of protein, e.g. when tested for reduced sulphur through the addition of lead acetate after treatment with boiling alkali, or when examined for phenyl groups through treatment with hot, concentrated nitric acid. Th e acid rendered the chitinous chips transparent even in the cold; on heating it dissolved them with very little mani- festation of any yellow color, and presented a colorless solution on minor dilution; moreover, alkalization with ammonia contributed only a pale yellow color, not the orange to red typical of the xanthoproteic reaction. I t should be added also that, during the ashing operation on the alkali-cleaned and dried material, there was detectable only an initial slight, passing scent reminiscent of burning protein (perhaps from remaining traces of impurity), and that the rest of the smoking phase of incineration lacked that odour. Repetition of this test, using a large fragment,

C H I T I N - B O U N D K E T O - C A R O T E N O I D S I N A CRUSTACEAN CARAPACE 955

pre-extracted with acetic acid, gave no trace of that characteristic odor during incineration.

Typical of the behavior of chitin, but not of keratin, was the ready and complete dissolution of carapace chips in hot, concentrated HCI. Following the evolution of COs, as the calcareous matrix was digested without the release of pigment, the chips suddenly dissolved, conferring upon the acid solution a bright red color. Clearly the pigment in situ is bonded in some manner to the chitin itself. Chips of the exoskeletal material could be cleaned of adventitious tissue shreds by exposure to strong alkali which, even if boiled, appeared in no way to modify the subsequent behavior of the pigmentary material or its substrate. Such chips yielded unaltered astaxanthin, for example, even after the boiling alkali treatment. Moreover, the pigment was not extracted or chemically altered by hot alkali even after the chitin had been decalcified.

However, decalcified chips of cleaned carapace yielded their pigment readily to ethanol or acetone, while pyridine at ca. 85°C also gradually leached the pigment even from calcareous pieces of exoskeleton. Warmed acetic acid, diluted with an equivalent volume of water, extracted the carotenoids more readily since it first attacked the carbonate material.

Aqueous KBH4 solution, which slowly turned a red flamingo feather orange in color through chemical reduction of its keto-carotenoids, left red, decalcified Taliepus chitin unchanged in appearance over the same period of days. The chitin- bound carotenoids seemingly are afforded considerable protection against chemical attack and, in the intact exoskeleton, against leaching by common organic solvents.

In a quantitative approach, pieces of carapace from the dorsal surface, scraped free of most adhering tissue fragments, were exposed to ca. 5 N NaOH solution with magnetic stirring overnight at ambient temperatures to assure removal of all adventitious matter. The bright red color of the outer surface appeared to remain unchanged, save through cleaning, by such treatment, and no true skeletal pigment could now be extracted from the brittle fragments even with boiling alkali.

Two g of the (moist) carapace chips were exposed to ca. 50 per cent acetic acid while stirring at ambient temperatures. This cold process evolved the COs, but leached away only an insignificant fraction of pale yellow-orange pigment which exhibited no spectrally familiar properties.

Stirring was resumed in freshly added aqueous acetic acid, now warmed to ca. 80°C. This gradually leached away orange-red pigment, leaving the chitinous flakes snow-white and flexible.

The system was cooled, the acetic acid decanted and refrigerated awaiting analysis. An aliquot, transferred to an equal volume of hexane, then rinsed free of residual acid, was adjusted to volume for spectrophotometric measurement, then was concentrated in a warm water-bath, using a stream of N 2 preparatory to chromatography.

The chromatographic procedure was conducted upon a thin-layer plate of silica gel-H, enclosed within a special chamber holding a shallow solution of 30 per cent acetone in hexane (v/v) as a developing eluant. At the completion of the

956 Dm~Is L. Fox

operation, the plate, removed and blown briefly to dryness, exhibited but four separate, distinct zones. These were scraped away individually, each then eluted in a separate receiver containing 5% methanol in hexane (v/v), finally adjusted to a small volume in a N~ stream, and measured in a Cary recording spectrophotometer.

The four fractions recognized were echinenone, canthaxanthin, phoenico- xanthin and astaxanthin, in that order of increasing relative proportion and relative polarity, as outlined in Table 1. No evidence of isomers was detected, and no carotenes or mere hydroxy-carotenes were found. The aggregated concentration of carotenoids present amounted to 16mg/100g (non-dried) shell (astaxanthin equivalents). When an account is accorded the fact that the carotenoids are associ- ated only with the chitin, and none with the 54 per cent calcareous portion of the skeletal material, the resulting concentration of carotenoids becomes nearly 35 mg/ 100 g chitin, a relatively high value among natural products.

DISCUSSION There are logical reasons for entertaining a prevalent view that carotenoids

bearing keto groups in one or both of the 4-positions may likely owe their capacity to conjugate with terminal amino groups of proteins by formation of a carbonyl- amino structure or a protonated Schiff's base (Cheesman et al., 1967).

II ~ II

I CH2 I

CH, I

R And while the relative lability of such a carotenoid moiety's disposition toward

water-miscible solvents may introduce a note of doubt into this concept, there remain certain factors tending to support it in some instances. Thus a carbonyl- amino compound or a Schiff's base should obey principles governing chemical equilibria in aqueous media wherein the reactants are soluble or hydrated and colloidally dispersed; therefore, a reagent capable of depressing the solubility of the protein moiety, e.g. by actual denaturation, while also serving as a solvent for the involved polyene, should thus shift such equilibria far or completely toward the dissociated state (e.g. as in water-dispersible carotenoid proteins treated with alcohol or acetone). But, in solid, insoluble carotenoid-protein complexes, such as are present in red flamingo feathers, the association no longer is in an equilibrial state. The bonds are very firm and not affected by the presence of alcohol, acetone or ether, although they can be unchained by the application of certain polar, water-miscible reagents such as pyridine or acetic acid, especially in warmed systems.

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958 DENTS L. Fox

The same basic principles should be applicable to conditions wherein relatively polar keto-carotenoids are firmly bonded to chitin, a widely occurring acetyl- glucosaminopolysaccharide, endowed with terminal basic N-groups, as are kera- tinous proteins, thus offering sites for the establishment of carbonylamino or Schiff's base linkages.

In the Taliepus material examined, we have one 4-keto and three 4,4'-diketo- E-carotene derivatives firmly bonded to the chitin of the exoskeleton. A calcareous shield seems to play an effective role in precluding the dissociation of the carotenoid- complex on exposure to such neutral, water-miscible solvents as ethanol or acetone, although not against extraction by hot pyridine, while acetic acid gradually dissolves the carbonate, then extracts the pigments.

No potential function suggests itself as a role of chitinocarotenoids in the metabolic economy of the crab, nor of keratinocarotenoids or calcareous carotenoid esters in hard structures of other animals. Some comparative observations con- cerning these complexes, however, are listed in Table 2, which is limited to con- sideration of conjugated carotenoid systems of solid, relatively refractory character.

It was revealed that the carotenoids are readily liberated from their conjugated state with chitin by exposure to ethanol or acetone following decalcification of the exoskeleton with aqueous solutions of Na~EDTA or citric acid. This dissociability is in contrast with the status of carotenoids conjugated with feather-keratin, and suggests the possible linkage to be that of a pre-existing carbonylamino structure rather than a Schiff's base, unless perhaps the decalcification process might have involved also the hydration of Schiff's base linkages in situ, thus converting this into a carbonylamino configuration.

Other contrasts, however, suggest the likelihood of a firmer bond in chitino- carotenoids than might apply to keratin-carotenoid complexes. Chemical reduction by mercaptoethanol and by KBH v for example, applied to the latter association, but not to the chitin-bound carotenoids. Moreover, treatment with cold or hot alkali, which solubilizes keratin, releasing the carotenoids thereby and catalyzing rapidly the atmospheric oxidation of astaxanthin to astacene (and of phoenico- xanthin to phoeniconone), exercised no detectable effect upon these tautomeric keto bodies in the chitin-bonded state. Still, it is too early to conclude the exact status of the chemical linkage between such carotenoids and keratin or chitin.

Account has to be taken of the general structure of crustacean exoskeleton, and of reports mentioning the suspected presence of water-soluble protein and of chitin-bound or other insoluble protein in the carapace of some species. Thus Dennell (1960) depicts four main strata in the structure of a decapod cuticle: (1) a very thin, calcified, non-chitinous lipoproteinoid epicuticle, and a so-called endo- cuticle composed of (2) a calcified, chitinous, pigmented layer, (3) a succeeding colorless, calcified, chitinous stratum and at the bottom (4) a chitinous, non- calcified bed. Dennell mentions this relatively minor, uncalcified layer at the bottom as a possible chitinoprotein complex, but writes nothing of any pigmenta- tion therein.

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C H I T I N - B O U N D K E T O - C A R O T E N O I D S I N A CRUSTACEAN CARAPACE 961

The fairly vulnerable, dark red-purplish epicuticle of some Taliepus specimens apparently is readily stripped away by alkali treatment employed for preliminary cleaning of carapace pieces, which then are of bright red color (see above).

Lafon (1948) stated that calcareous exoskeletons of marine crustaceans are relatively rich in chitin, but poor in protein content, e.g. amounting to some 88 vs. 12 per cent respectively, in the organic portion of Carcinas maenas carapace; while in insects, arachnids and other arthropod dwellers on land, the relative proportions of hardened protein to chitin are of reversed order.

It was for such reasons that special attention was given, in this study, to the possible presence of substantial protein in (cleaned) Taliepus exoskeleton, since such a contingency might have provided an alternative and more familiar basis for the bonding of rich deposits of ketocarotenoids. The complete insolubility of decalcified carapace pieces in strong, boiling alkali, their ready dissolution in hot concentrated mineral acids, and the negativity of other tests for scleroproteins, such as that for reduced sulphur (no blackening on addition of lead acetate to a decalcified chip boiled in alkali) and for phenyl groups with the familiar xantho- proteic test (no distinct yellowing on treatment with hot, concentrated nitric acid; colorless on minor dilution and not becoming orange in color on ammoniflcation) all substantiated the conclusion that, in Taliepus at least, we have an instance where- in the ketocarotenoids, greatly preponderating in the commonest member, astaxan- thin, are chemically bonded, not to a scleroprotein but to chitin.

The recognition of the four carotenoids recovered from the carapace of the red kelp crab as 4-keto derivatives of fl-carotene leads to the supposition that the most logical precursor is that hydrocarbon, as has been demonstrated in another crus- tacean, Artemia salina (Davies et al., 1965) and in the American flamingo Phoeni- copterus tuber (Fox et al., 1969).

The ready and continuous consumption of brown seaweeds (Phaeophyceae) by T. nuttallii assures to the crab ample sources of carotene, which constitutes as much as 10 per cent of total carotenoids in a dozen species reported by Goodwin (1965).

Acknowledgements--My thanks are due to Dr. F. T. Haxo and Mr. Donald W. Wilkie for their friendly interest, and respectively for the loan of instrumentation and procurement of specimens; to Mr. Robert Kiwala for collecting and refrigerating the specimens used; and to Miss Yolanda Montejanos for technical assistance.

REFERENCES CrrS~MAN D. F., LEE W. L. & ZAGALSKY P. F. (1967) Carotenoproteins in invertebrates.

Biol. Rev. 42, 132-160. DAVIES B. H., Hsu W. J. & CHICHESTER C. O. (1965) The metabolism of carotenoids in the

brine shrimp Artemia salina. Biochem. J. 94, 26P. (Abstract.) DENNELL R. (1960) Integument and exoskeleton. In The Physiology of Crustacea (Edited by

WATERMAN T. H.), Chapt. 14, pp. 449-472. Academic Press, New York. Fox D. L. (1953) Animal Biochromes and Structural Colours. Cambridge University Press. Fox D. L. (1972a) Chromatology of animal skeletons. Am. Sc/. 60, 436-447.

962 DENIS L. Fox

Fox D. L. (1972b) Pigmented calcareous skeletons of some corals. Comp. Biochem. Physiol. 43B, 919-927.

Fox D. L. • HOPKINS T. S. (1966) Comparative metabolic fractionation of carotenoids in three flamingo species. Comp. Biochem. Physiol. 17, 841-856.

Fox D. L., SMITH V. E., GRIGG R. W. & MACLEOD W. D. (1969) Some structural and chemi- cal studies of the microspicules of the fan-coral Eugorgia ampla VerriU. Comp. Biochem. Physiol. 28, 1103-1114.

Fox D. L. & WILKIE D. W. (1970) Somatic and chemically fixed carotenoids of the purple hydrocoral Allopora californica. Comp. Biochem. Physiol. 36, 49-60.

Fox D. L., WOLFSON A. A. & McB~TH J. W. (1969) Metabolism of/~-carotene in the American flamingo Phoenicopterus tuber. Comp. Biochem. Physiol. 29, 1223-1239.

GOODWIN T. W. (1965) Algal carotenoids. In Aspects of Terpenoid Chemistry and Bio- chemistry (Edited by GooDwlN T. W.), Chapt. 11. Academic Press, New York.

LAFON M. (1948) NouveUes recherches biochimiques et physiologiques sur le squelette t6gumentaire des Crustaceae. Bull. Inst. Oceanogr. 45, 1-28.

THOMMEN H. (1971) Metabolism. In Carotenoids (Edited by ISL~ O., GUTMANN H. & SOLES U.), Chapt. 8, pp. 637-668. Birkhauser, Basle.

Key Word Index---Taliepus nuttallii ; astaxanthin; phoenicoxanthin; canthaxanthin; echinenone; carotenoids.