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Camp. Biochem. Phmiol. Vol. I I IA. No. 4. pp. 533-538. 1995 Elsevier Science Ltd

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Effect of different dietary carotenoids on the pigmented pattern of the hermit crab Clibanarius erythropus Latreille (Crustacea: Decapoda)

Renk Castillo and Genevikve NGgre-Sadargues

Laboratoire d’Ecophysiologie des Inverttbrts, Universite Montpellier II Sciences et Techniques du Languedoc, 34095 Montpellier, France

The purpose of this work is to investigate the pigmentary fate of dietary carotenoids in the hermit crab Cfibunarius erythrupus receiving synthetic pigments. The animals have been previously depigmented and fed on a carotenoid-free diet during three consecutive moulting cycles. Three experimental diets containing b-carotene from carrots, synthetic canthaxanthin, and astaxanthin were used. The abilities of the animal to metabolize carotenoids having different oxidation levels, have been investigated.

Key words: Carotenoid metabolism; Astaxanthin; Canthaxanthin; B-Carotene; Crustacea;

Decapoda; Anomoura; Pigmentation.

Camp. Biochem. Physiol. I I IA, 533-538, 1995.

Introduction

Pagurians, like many other crustaceans, have a coloured appearance depending essentially on the presence of carotenoid pigments. Neverthe- less, this genus has received little attention concerning this topic, probably due to the fact that these animals are not considered as edible species and therefore having no economical significance. The studies on the pig- mentation of the hermit crab Pagurus bern- hardus have permitted one to demonstrate the occurrence of different xanthophylls in the ani- mal (Lonnberg, 193 l), while the species Eupagu- rus prideauxi was found to contain mainly o-carotene (Lederer, 1938). The works on Pagurus pollicaris (McNamara et al., 1962). Eupagurus bernhardus (Cheesman and Prebble, 1966) and Clibanarius erythropus (Zagalsky et af., 1970) indicated the presence of

- Received 5 December 1994; revised 14 February 1995;

accepted 16 February 1995. Correspondence to: R. Castillor, Laboratoire

d’Ecophysiologie des Invertkbrks, Universitb Montpel- lier II Sciences et Techniques du Languedoc, 34095 Montpellier, France.

carotenoprotein complexes in different tissues of these pagurians.

Further investigations on the carotenoids of Clibanarius misanthropus from the Black Sea (Czeczuga and Czerpak, 1969) Dardanus arro- sor (Castillo and Lenel, 1971, 1973) Pagurus prideauxi (Castillo et al., 1980) and Clibanarius erythropus (Castillo and Lenel, 1978; Lenel et al., 1978; Castillo, 1980) have led to the determination of the series of metabolic trans- formations occurring in the animal.

More recently, the works on the effect of neurosecretory organs (Negre-Sadargues et ~1.. 1983; Castillo and Negre-Sadargues, 199 I ) have allowed investigation of the relationships be- tween carotenoids and hormonal effecters; cer- tain results corroborate those concerning the metabolism of carotenoids during the moulting cycle (Castillo et al., 1988, 1989).

The aim of this work is to obtain further information on the pigmentary fate of dietary carotenoids for crustacean aquaculture pur- poses. To this approach, the capabilities of the animal to metabolize molecules having different oxidation levels, have been investigated.

533

534 R. Castillo and G. Nigre-Sadargues

Materials and Methods

The species C. erythropus was found to be very suitable for this kind of work, since it shows a remarkable endurance in rearing con- ditions; added to this, the length of its moulting cycle enables nutritional experiments to be per- formed. The animals collected at Cap d’Agde (Mediterranean Languedoc shores) have been maintained in a rearing system consisting of four series of 20 translucent plastic boxes, each one divided into six individual compartments, under a continuous flow of filtered seawater. The salinity was adjusted to 1100 mOsm kg-’ (37.4%0), the temperature was constant (20°C) and a 12L-12D cycle was adopted.

Preliminary attempts have shown that the effects of any experimental dietary supply were masked by the rather sophisticated carotenoid pattern of natural pigmentation. For this reason, the animals have been previously depig- mented after being reared on carotenoid free diet (Cfd) during three consecutive moulting cycles. At the end of this period, the carotenoid content of the whole animal represents little more than 12% of that of standard individuals (Std) fed on mussels (Mytilus edulis). However, it must be noted that, at the final step of the experiment, the carotenoid content of standards corresponds to about 82% of that of freshly caught hermit crabs.

Three experimental diets containing, respect- ively, tram /?,/I-carotene (P-car), synthetic can- thaxanthin (P,/3-carotene-4,4’-dione) (Cantha), and astaxanthin (3, 3’-dihydroxy-/?$-carotene- 4,4’-dione) (Astax) (Hoffmann-La Roche), were prepared by adding 200 mg/kg of each pigment to a carotenoid-free basic meal (Castillo, 1980); the daily ration (0.5 g) was expected to contain 0.1 mg of pigment.

Male depigmented animals, divided into three groups, received daily a specific diet during two complete moulting cycles, prior to being sac- rificed by freezing during the C4 moulting stage. The dissected tissues: hepatopancreas, abdomi- nal epidermis and carapace (cephalothoracic dorsal shield) were extracted separately for quantitative evaluations; however, the extracts were pooled for the series of qualitative analy- ses, The material was extruded with acetone, and the carotenoid extract thus obtained was transferred into light petroleum (45-60”) by washing the mixture with fresh water (Castillo and Lenel, 1978).

The quantification was made by ponderal and spectrophotometrical methods (Arpin et al., 1969); the carotenoid relative amounts, ex- pressed as a percentage, were established by using a Vernon scan-recorder photointegrator. The qualitative determinations are a result of

the combined use of visible absorption spectra (380-550 nm), chemical reactions (Castillo and Lenel, 1978) and co-chromatography with auth- entic carotenoid samples.

Despite its undisputable efficiency, the HPLC technique has often appeared inoperative for chromatographic analysis of animal caroten- oids, in which esterified forms are preponderant. These are not always separable with the Cl8 reversed phase, commonly used for the study of plant carotenoids. For this reason, the TLC method using Silicagel G coated plates (Merck), appears much more adapted. Different eluents containing either 5% methanol or 20% acetone in petroleum ether have been used. The former enables the separation of carotenes and low polar esterified xanthophylls, while the latter has been found to be very useful for the separation of polar fractions. The mix- ture benzene, diethyl ether, methanol (17,2,1) is very suitable for hypophasic strongly adsorbed fractions.

Results

The pigmented pattern of animals receiving dietary p-carotene during two moulting cycles was not fundamentally different to that of indi- viduals lacking carotenoids. Besides this, the abdominal region of those of canthaxanthin and astaxanthin groups appeared ‘coloured’ at the end of the second moulting cycle under the experimental feeding conditions. Nevertheless, the quantitative data indicated, in all cases, a carotenoid content significantly lower than that recorded for standards fed on mussels.

The hepatopancreas of astaxanthin-fed ani- mals exhibited a light pink colouration, whereas that of p-carotene and canthaxanthin groups was visually colourless; however, all the extracts are found to contain detectable amounts of pigment (Table 1, Fig. 1). On the other hand, astaxanthin as well as canthaxanthin feeding tests were found to improve the pigmentation of the epidermis. In both cases the tissue appeared lightly bluish.

The epidermis of individuals receiving ,0- carotene exhibited a light violet colour, closely comparable with that of animals having a caro- tenoid-free diet; however, the former showed an increase of pigment concentration (Table 1, Fig. 2).

Only astaxanthin fed individuals were found to have a pigmented thoracic dorsal shield; nevertheiess, although there were marked quan- titative differences (Table 1, Fig. 3) the visual aspect of the dissected tissue was quite compar- able with those of the other feeding groups.

The comparisons of quantitative data ob- tained for exuvial cuticles I and II indicate an

Effect of different dietary carotenoids on pattern of hermit crab

Table 1. Effect of diet on the carotenoid content of the different tissues

535

Hepato- pancreas Epidermis Carapace

Ecdysial Ecdysial cuticle I cuticle II

Standard diet

Wd) Carot-free

diet

(Cfd) P-carotene

(p-car)

Canthaxanthin (Cantha)

Astaxanthin (Astax)

461.2 105 6

8 131.5 35.3 6 214.4 71.7

387.2 95.6 5

1427 971.9 72.1 131.0 5 5 155.9 208.4 81.9 38.4 6 5 419.4 186.3 77.3 49.0 7 6 680.8 241.0 299.3 162.7 9 5 1184.3 403.2 393 196.1 8 6

987.4 54. I 9 56. I 36.2

191.9 65.0 12 233.3 56.4 14 295.9 87.3 9

1001.8 m @g/g) 83.4 tsm 8 n 67.4

4 174.3 75.8 8 245.4 58.2 9 367.2 117.9 8

m: mean values expressed in pg per g of dry weight. tsm: confidence interval. n: number of data.

increase of carotenoid concentration in canthax- anthin and astaxanthin groups (Table 1, Fig. 4).

Photodensitometrical data, obtained after chromatography of hepatopancreas extracts, in- dicate different pigment proportions. The or- gan, regardless of the diet, was found to contain free astaxanthin; however, the preponderant fraction corresponds systematically to the in- gested pigment, while p-carotene represents the major carotenoid of the standard group (Table 2).

Although important differences in carotenoid concentration were observed in the carapace of the different groups, free astaxanthin relative amounts were very similar. Nevertheless, it is noteworthy that astacene proportions scored for the astaxanthin group appear significantly higher (Table 4).

Concerning the epidermis, one may observe that astaxanthin and canthaxanthin dietary supplies were both found to enhance the pig- mentation of the tissue; nevertheless, free astax- anthin relative amounts appeared higher in the /?-carotene group. On the other hand canthax- anthin represented a minor fraction in the astax- anthin one, in which, S-carotene has also been detected (Table 3). The percentage of astaxan- thin diester was closely comparable for all ex- perimental diets; however, a higher astaxanthin monoester value was also scored for animals receiving P-carotene.

The rather small amounts of pigment avail- able from ecdysial cuticles, added to the vari- ability of the data, have not permitted the determination of the incidence of specific diet- ary carotenoids on the pigmented pattern of the tissue.

Concerning standard individuals, it is useful to note that none of the major carotenoids of the mussel (Campbell, 1970; Hertzberg et al., 1988) has been detected in the animal (Castillo and Lenel, 1978).

Discussion

The digestive gland of C. erythropus, invari- ably poor in carotenoids, appears, however,

Std Cfd p-car Cantha Astax

Fig. 1. Hepatopancreas carotenoid content under different exnerimental dietarv conditions.

1600

1400

1200

1000

3 600

600

400

200

0

Std Cfd P-car Cantha AStaX

Fig. 2. Epidermis carotenoid content under different exper- tmental dietary conditions

536 R. Castillo and G. Nkgre-Sadargues

1000

900

600

700

600

3 500

400

300

200

100

0

Fig. 3. Carapace carotenoid content under different exper- imental dietary conditions.

formally pigmented in experimental animals fed either canthaxanthin or astaxanthin.

As stated for other crustaceans such as the lobster Panulirus japonicus (Katayama et al., 1973) most metabolic conversions of dietary carotenoids do not occur in the hepatopancreas. These are rapidly transferred into other tissues, epidermis in particular, following the intake of food; the essential of oxidative metabolic con- versions occurring in the pagurian takes place at this level. Therefore, the results reported above may be ascribed to the fact that the concen- tration of pigment in the experimental daily ration, is probably higher than that occurring in natural conditions, in which the pigmentation of the organ therefore reflects the availability of environmental carotenoids. The important storage thus observed may be also due to a certain metabolic inertia of the digestive gland concerning carotenoid bioconversions; this opinion could be supported by the fact that, in C. eryrhropus, the bulk of metabolic transform- ations occurs in the epidermis. According to this, one may assume that, in the experimental conditions thus defined, the surplus of ingested

I II I I, I II I II I II

std Cfd p-car Cantha &tax

Fig. 4. Ecdysial cuticle carotenoid content I and II under different experimental dietary conditions.

Table 2. Hepatopancreas relative carotenoid concentration. expressed as a percentage

Std Cfd p-car Canth Astax

Free astax 29 T(lOO%) II I4 8x b-car 56 84 T?

Canthax 77 Other 15 5 9 I?

T: traces < I %.

pigment is not readily transferred, being, thereof, retained in the absorbent cells (R) of the organ. In this case, p-carotene appears less retained than canthaxanthin or astaxanthin; so. theoretically, most of the ingested /?- carotene could be easily transferred, thereby inducing an increase of pigment storage at the epidermal level. Nevertheless, the low caroten- oid concentration scored in the epidermis of p-carotene-fed animals invalidates this hypoth- esis. In fact, the analyses of some faecal residues indicate that important amounts of unmetabo- lized pigment were excreted during the exper- imental period; unfortunately, it has not been possible to determine the proportion of ingested carotenoid.

The decreasing pigment content of individ- uals fed on mussels suggests the inability of the animal to metabolize the majority of the caro- tenoids of this mollusc.

The fact that the carapace of the p-carotene group appears less pigmented than that of indi- viduals lacking carotenoids, suggests a very low metabolic rate, hindering the transfer of pig- ment towards the calcified layer; on the other hand, the presence of astaxanthin in the hepato- pancreas of p-carotene group could be regarded as a recovering of this pigment from the epider- mis, after the series of metabolic conversions occurring in this tissue. The presence of free astaxanthin in the organ of individuals having a carotenoid free diet supports this opinion.

Astaxanthin as well as canthaxanthin diets were found to improve epidermis pigmentation; the light bluish colour of the epithelial layer corresponds very likely to the reconstitution of the carotenoprotein complex (Zagalsky et al., 1970).

The series of oxidative reactions leading to astaxanthin formation from p-carotene, prob-

Table 3. Epidermis relative carotenoid concentration, ex- pressed as a percentage

Std Cfd p-car Canth Astax

Astax diest 37 67 44 42 46 Astax II <2 20 10 I6

monoest Free astax I6 28 34 28 30 Canthax 2 6 5

Other 34 <3 12 14 3

Effect of different dietary carotenoids on pattern of hermit crab

Table 4. Carapace relative carotenoid concentration, ex- pressed as a percentage

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537

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Other 22 4 5 7 <3

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