Comp. Biochem. Physiol. Vol. 99B, No. 1, pp. 187-192, 1991 0305-0491/91 $3.00 + 0.00 Printed in Great Britain © 1991 Pergamon Press pie

EFFECT OF EYESTALK ABLATION ON THE METABOLISM OF CAROTENOID PIGMENTS OF THE

HERMIT CRAB CLIBANARIUS ER YTHROPUS LATREILLE (1818) (CRUSTACEA DECAPODA ANOMOURA)

RENl~ CASTILLO and GENEVII~VE NEGRE-SADARGUES

Laboratoire d'Ecophysiologle des Invert6br6s Universit6 des Sciences et Techniques du Languedoc, Place E. Bataillon, 34095 Montpellier Cedex 2, France (Tel: 33 67 54 47 42)

(Received 24 September 1990)

Abstract--1. Histological data demonstrate the occurrence of the sinus gland and other neurosecretory groups of ceils in the eyestalks of Clibanarius erythropus.

2. Bilateral eyestalks removal induces flagrant modifications of the nature and the amounts of carotenoids in the different tissues.

3. The concentration of fl-carotene of ablated animals decreases substantially suggesting accelerated oxidative reactions leading to the formation of astaxanthin.

INTRODUCTION

The protocerebrum of Malacostraca contains neuro- secretory structures such as the sinus gland and the organ of Hanstrom which may be located entirely in the brain or pulled into the eyestalks as is the case for all studied pagurians (Hanstrom, 1937; Gabe, 1952; Nagabhushanam and Sarojini, 1969; Farges, 1973). The ablation of eyestalks leads to the suppression of various neurohormonal secretions, Moulting inhibit- ing hormone (MIH) in particular. Although eyestalks removal is not systematically reported to accelerate the moult of certain crustaceans (Sochasky, 1973) it has, however, been established that in most species, ablated animals undergo a shortening of their moult- ing cycle as a consequence of the activation of the Y-organ producing the moulting hormone (Adiyodi and Adiyodi, 1970). Therefore it is possible that such a treatment could consequently affect carotenoid transfer as well as metabolic interconversions within the animal.

The influence of eyestalk hormonal factors on carotenoid metabolism in crustacea has been the subject of few papers. Controversial if not contradic- tory data have not provided a clear idea of the relationships between neurohormones and caroten- oids. The reddish appearance of the shore crab Carcinus maenas after eyestalk ablation could result from the denaturation of the carotenoprotein com- pound occurring in the carapace (Lenel and Veillet, 1951; Lenel, 1957, 1959, 1961; Ceccaldi, 1968). The experiments made on the eyestalk ablated shrimp Palaemon serratus (Chassard-Bouchaud and Noel, 1971) enable the authors to infer that neurosecretory organs are not directly implicated in carotenoid metabolism, suggesting therefore that assimilation or destruction of these substances in the tissues depend merely on the "quantity of light passing through optical pathway" (Fox, 1953). Further studies have,

however, shown the effect of the sinus gland on the formation of a blue carotenoprotein compound in the ablated shrimp (Genofre, 1973), as well as a "disorganisation of carotenoid metabolism" (Chassard-Bouchaud et al., 1973).

The results related to the metabolism of caro- tenoids in the hermit crab Dardanus arrosor after eyestalk ablation indicate a flagrant modification of lipid metabolism involving hydrolysis of astaxanthin esterified forms (Castillo and Lenel, 1973). Analogous results have been obtained for the crab Pachygrapsus marmoratus (Negre-Sadargues et al., 1983) as well as for the freshwater prawn (Macro- brachium rosembergii (Maugle et al., 1980) which is found to undergo an increase of dietary carotenoid storage subsequent to the ablation.

The works on the carotenoids of the hermit crab Clibanarius erythropus (Castillo and Lenel, 1978; Castillo, 1980), as well as those related to the modifi- cations during the moulting cycle (Castillo et al., 1988, 1989), have incited us to study the effects of eyestalk neurosecretory organs on the metabolism of these substances.

MATERIALS AND METHODS

About 150 collected male individuals have been main- tained in laboratory rearing conditions (Castillo et aL, 1988) during two complete intermoult cycles. Bilateral eyestalk ablation has been accomplished at stage C4. in these conditions the survival rate was about 50% during the first intermoult period, reaching 85% at the second period; loss has been reported to occur mainly during the eedysis, some individuals being unable to withdraw from their old exoskeleton. Food consisting of frozen Artemia and mussels was delivered 3 days a week. The animals were killed by freezing (-18°C) at stage C4 following the third ecdysis. Carotenoid extraction as well as the essential analytical procedure were made according to the methods outlined previously (Castillo and Lenel, 1978; Castillo et al., 1988).

187

188 R. CASTILLO and G. NEGRE-SADARGUES

A

G

N:

B Fig. 1. (A) Eyestalk sagittal section. (B) Detail of sinus gland (SG). OM, ommatidia; RC, retinian ceils; RN, retinian nerve; LG, lamina ganglionaris; ME, medulla externa; MI, medulla interna; SG, sinus gland;

MT, medulla terminalis; NSC, neuro-secretory cells; ON, optic nerve.

For histological purposes, the eyestalks were fixed and decalcified with solution of Halmi. After dehydration in a series of ethanols (50, 70, 95 and 100%) the tissue was

embedded in paraffin in order to obtain sagittal thin sections (6 #m) and stained according to the technique of Gomori (Martoja and Martoja, 1967).

Metabol i sm o f carotenoid pigments 189

Table I. Effect of eyestalk ablation on the carotenoid content of different tissues. Data expressed in gg/g of dry wt (/~g/ml)* and in percentage (relative amounts)

Free Astax Astax Free ~t-Dorad Lutein Total Astax m.est diester ~t-dorad diester zeax fl-Carot cartenoids

M A 60.85 61.76 116.84 31.65 27.80 53.33 10.5 397.27 _ S E 5.54 9.57 21.14 14.01 7.65 14.08 5.71 41.36

n 9 9 9 9 8 9 9 9 % 15 15 30 8 7 10 2.6

Whole animal M C 66.76 117.39 186.61 32.80 48.02 51.19 25.3 554.17 _ SE 6.36 11.25 16.50 12.65 8.6 11.00 9.36 29.08

n 16 16 16 15 16 16 14 12 % 12 22 33 6 9 9 4.5 P 9.41 16.59 26.76 9.32 13.31 7.81 12.52 59.92

A M 5.91 55.90 69.77 0.63 20.22 2.14 14.80 156.96

M A 337.65 175.21 300.12 105.58 68.72 103.92 75.57 1169.71 _+ SE 15.66 29.88 19.8 12.65 12.40 9.92 9.21 152.48

n 21 21 21 21 21 21 19 13 Epidermis % 29 15 24 9 6 9 6

M C 346.69 214.64 275.33 80.60 132.60 134.21 87.45 1358.45 _+ SE 22.50 12.28 18.50 9.14 24.00 12.18 14.60 102.13

n 13 13 13 12 10 12 I I 10 % 25 16 20 6 10 10 6 P 33.06 39.33 15.54 18.12 24.34 15.89 16.14 199.84

AM 9.04 39.43 24.79 24.98 63.88 30.25 11.88 194.74

M A 33.87 - - T T - - T 19.64 92.80 + SE 7.8 8.91 19.68

n 7 7 9 % 36 21

Hepatopancreas M C 94.49 3.50 19.12 23.92 - - 20.16 140.46 389.71 _+ SE 16.41 - - 4.34 9.71 - - 11.09 8.29 28.56

n 7 2 7 5 5 7 8 % 25 1 5 7 5 36 P 17.46 . . . . 5.36 32.88

AM 60.62 120.78 296.91

M A 622.17 - - - - 86.62 - - 22.53 - - 987.58 + SE 71.77 9.54 - - 118.23

n 7 7 3 10 % 63 9 2

Carapace M C 510.01 57.86 29.55 1133.36 + SE 72.32 14.24 9.57 121.67

n 7 6 6 8 % 45 6 3 P 97.91 - - - - 15.86 - - - - 167.20

AM 112.16 29.56 145.78

M A 749.27 104.38 80.14 1248.79 _+ SE 52.60 22.57 21.09 114.01

n 16 16 16 12 % 60 9 7

Ecdysial M C 788.72 125.47 77.20 1792.56 layer _+ SE 59.43 25.00 19.46 266.02

n 11 11 9 8 % 44 8 4 P 79.48 33.82 31.56 246. 78

AM 39.45 21.09 2.94 543.77

Haemolymph* M A 71.09 T T T 104.50 M C 92.70 T T T 144.85

Fecal M A 116.66 - - T - - 166.66 pellets M C 33.50 - - - - T 52.17

A = ablated; C = control; P = Sdx; AM = M I - M 2 ; T = traces; astax = astaxanthin; m.est = monoester; ct-dorad = ct-doradexanthin; zeax = zeaxanthin; ~-carot = fl-carotene.

Italic P values indicate significant differences between means.

R E S U L T S

The eyestalks of C. erythropus are about one-tenth of the length of the animal. The distal portion contains the photoreceptive unit, represented by the ommatidia, the crystalline cones, the retinian cells packed together, and the rhabdoms. In the middle part one may identify the lamina gangiionaris, the medulla externa and the medulla interna (Fig. 1A). Located on the median dorsal surface of the latter

there is a group of swollen axon terminals and glial cells forming the sinus gland which abuts a large blood sinus (Fig. 1B). The proximal portion encloses the Medulla terminalis and the optic nerve.

In normal conditions, the length of the moulting cycle is about 56 + 10 days. The surgical suppression of the eyestalks leads to a drastic shortening corre- sponding to about 39 + 9 days, inducing a series of well-defined modifications of carotenoid metabolism in the pagurian. Comparative studies carried out on

190 R. CASTILLO and G. NEGRE--SADARGUES

o t °r

FA AME ADE FA AME ADE

60

50

40 % 30

20

10

40-

30-

%

20-

10-

FA

Car

60-

50-

40- % 30-

20-

10-

80-

HP (~) 6o-

%

40-

20-

FA

FA AMI: ADE FA

EL ©

Haem (~)

mA

Fig. 2. Astaxanthin contents in different tissues of ablated and control individuals (data expressed in percentage). (a) Whole animal (WA). (b) Carapace (Car). (c) Ecdysial layer (EL). (d) Epidermis (Ep). (e) Hepatopancreas (HP). (f) Haemolymph (Haem). A, ablated animals; C, control; FA, free astaxanthin;

AME, astaxanthin monoester; ADE, astaxanthin diester.

different tissues of ablated and normal individuals have permitted the determination of the nature and extent of such alterations.

Although both major and minor pigments appear to be affected, less attention has been paid to the latter; the weak amounts available have often hin- dered their evaluation, and they have therefore only been reported as occurring or not in a given tissue.

Whole animal earotenoids

Eyestalk ablation is accompanied by a noticeable decrease of the bulk of carotenoids (Table 1); fl- carotene decreases from 25.3 to 10,5#g/g. On the other hand, one may observe slight differences affect- ing the concentration as well as the relative amounts of astaxanthin; the diester form shows a slight in- crease while the monoester form has been found to decrease. Free astaxanthin appears unchanged; how- ever, the relative amounts of this carotenoid represent 15% of total pigment against 12% scored for stan- dard animals (Fig. 2a).

Carotenoids o f the carapace

The quantitative evaluation of the carotenoid content of the carapace leads to the following remarks:

The concentration of carotenoids of the dorsal shield of animals without eyestalks that have accom- plished two complete moulting cycles represents 987.58#g/g while that of standard animals is 1133.36 #g/g (Table 1).

The carotenoid pattern in ablated individuals is closely comparable to that observed for the standard group. The relative amounts (percentage) of free astaxanthin in ablated animals represent 63% of carotenoids (622.17#g/g) against 45% (510.01 #g) scored for non-ablated ones (Fig. 2b).

Statistical data indicate non-significant differences related to the concentration of free ~t-doradexanthin of ablated (9%) and normal individuals (6%); the low amounts of free lutein-zeaxanthin fraction have not permitted incidental variations to be recorded.

Metabolism of carotenoid pigments 191

Carotenoids of the ecdysial layer Two successive moults occurred within the interval

between eyestalk ablation and tissue sampling; the data refer to the second ecdysis at which some differences could be recorded. Basically, the caro- tenoid content of the ecdysial layer decreases in the ablated group (Table 1) although relative amounts of free astaxanthin undergo a slight increase---near 60% (749.27#g/g) against 44% (788.72#g/g) for non- ablated individuals (Fig. 2c). Small amounts of free ~-doradexanthin are also reported; however the differences are not significant.

Carotenoids of the epidermis The epidermis is found to contain most of the

carotenoids of the animal, free and esterified forms of astaxanthin in particular. The quantitative analysis indicates 1163.71 #g/g for ablated animals, while a value of 1358.45 #g/g has been scored for standard animals (Table 1). Monoester and free astaxanthin represent, respectively, 15% (175.21 #g/g) and 29% (337.65#g/g) against 16% (214.64#g/g) and 25% (346.69 #g/g) for the standard group (Fig. 2d). In the same manner, the relative amount of astaxanthin diester established for the standard group (20%) has been found to be slightly lower than that scored for the ablated group (24%). Relative amounts of ~- doradexanthin diester fall from little more than 10% to about 6%, but the percentage of the free form is found to increase. The exiguous amounts of other carotenoids, such as echinenone and phoenic- oxanthin, detected in the different extracts have not permitted their eventual variations to be established.

Carotenoids of the hepatopancreas The quantification of whole carotenoids of the

hepatopancreas indicates a marked difference be- tween standard (389.71 #g/g) and ablated individuals (92.8 #g/g), whose colourless digestive gland shows a rather low carotenoid content that represents a decrease of about 78% of pigment in the organ. Free astaxanthin and fl-carotene shift, respectively, from 94.49 and 140.46 #g/g for the control group to 33.87 and 19.64 #g/g in animals without eyestalks (Table 1). However, this "fall" of free astaxanthin corresponds to an increase of its relative amount since it represents 36% of total pigment in the organ against 25% scored for control (Fig. 2e). On the other hand, the tissue has not been found to contain astaxanthin diester, while small amounts of free ~-doradexanthin and lutein-zeaxanthin have been detected; ~-doradexanthin diester is not reported.

Carotenoids of the haemolymph The low availability of blood of either ablated or

normal individuals has only permitted the evaluation of the relative amounts of free astaxanthin. The poor number of data restricts the statistical approach; nevertheless the photodensitometrical analyses have shown that free astaxanthin yields about 72% of blood carotenoids against 64% in the standard group (Table 1 and Fig. 2f).

Carotenoid excretion

The analysis of fecal pellets from the ablated group has revealed the occurrence of variable amounts of

pigment, free astaxanthin in particular (Table 1), whereas few traces have been observed for non- ablated individuals.

DISCUSSION

A series of visual comparisons of ablated and normal animals, made a few minutes before death, have not permitted us to report conspicuous changes of their pigmented appearance other than a slight modification consisting of a reddish coloration of the abdominal region, attenuating the normal green one. This part of the animal does not possess a hard calcified exoskeleton, showing only a thin membrane whose pigmentation is the result of the occurrence of a blue caroteno-lipoprotein having astaxanthin as prosthetic group (Cheesman et al., 1966). The cara- pace is not found to contain esterified carotenoids which are located in the chromatophores at the epidermal level. The red colour induced by the abla- tion is very likely to be a result of the dissociation of the anhydride bonds between protein and carotenoid.

The fact that calcified parts such as the cephalotho- rax and limbs of ablated pagurians do not exhibit a noticeable colour change before ecdysis and no differ- ences affect the relative amounts of pigment, support the view of a metabolic inertness of carotenoids in this layer, beyond post-moult stages. The average carotenoid content of the carapace from the ablated group appears rather lower than that of the standard group, due very likely to an increased loss of pigment at moulting, as a consequence of the weakening of carotenoid-recovering capabilities in late premoult.

Compared with the noticeable carotenoid de- pletion undergone by the hepatopancreas after eye- stalk removal (Hariharan, 1966), the evaluation of the carotenoid content of the epidermis has not shown fundamental modifications. This suggests a strong retention of pigment following its transfer from the digestive gland via the haemolymph and supports the view that the epidermis may be con- sidered as the carotenoid storage site within the animal.

One must bear in mind that eyestalk removal has been made during stage C4, at which the digestive gland has been found to contain mainly fl-carotene and free astaxanthin. The important loss of pigment is very likely due to several simultaneous different effects; the lowering of fl-carotene might have re- sulted as a lack of this carotenoid in the diet, but the results obtained for the standard group invalidate this hypothesis, strengthening the idea of accelerated oxi- dative metabolic transformations. The fall of asta- xanthin diester in the hepatopancreas suggests a de-esterification reaction leading to the increase of unesterified form relative amounts (Table 1), explain- ing to some extent the upward tendency of free astaxanthin in blood and epidermis. The enhanced excretion of pigment, stated above for the ablated group, could also be involved in the depletion of carotenoids of the hepatopancreas.

Although a well-defined phenomenon could not be outlined from these experiments, it appears clear however that the removal of eyestalks induces a tremendous physiological upheaval affect- ing not only the moulting, but also most of the

192 R. CASTILLO and G. NEGRE-SADARGUES

biochemical reactions involved in the metabolism of carotenoids.

These observations must be regarded as prelimi- nary results in this species. Further investigations will permit us to improve our knowledge on the relation- ships between eyestalk neurosecretory cells and caro- tenoid metabolism in crustaceans.

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