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Carotenoids in Eudiaptomus amblyodon marenz (crustacea) during ontogenetic development
Camp. Biochem.Physiol., 1975, Vol. 50A, pp. 665 to 668. PergamonPress. Printed in Great Britain
CAROTENOIDS (CRUSTACEA)
IN EUDIAPTOMUS AMBL YODON MARENZ DURING ONTOGENETIC DEVELOPMENT B. CZECZUGA
Department
of General Biology, Medical Academy, Bialystok, Poland (Received 4 January 1974)
Ah&a&-l. By means of columnar and thin-layer chromatography, the presence of carotenoids in Eudiaptomusamblyodonwas studied. 2. The following carotenoids were found: (a) in eggs: p-carotene (2.73 per cent), isozeaxanthm (5.16 per cent) and astacene (20.16 per cent); (b) in pink copepoidal form: isozeaxan thin (504 per cent), 3,4dihydroxy-g-carotene (1.20 per cent), 1’,2’-dihydro-l’-hydroxy Cketo-r-carotene (1.25 per cent) and astaxanthin (8644 per cent); (c) in bluish copepoidal form: canthaxanthin (1.72 per cent), 4-hydroxy4-keto-flcarotene (5.29 per cent), isoxeaxan thin (8.78 per cent) and astaxanthin (77.15 per cent); (d) in the mature female: isocryptoxanthin (1.48 per cent), 4_hydroxy4keto-g-carotene (2.66 per cent), lutein (7.29 per cent) and astaxanthin (76.56 per cent).
INTRODUCTION THE BIOLOGYof the species Eudiaptomus amblyodon is for many reasons extremely interesting. In small
ponds the winter eggs of this crustacean hatch just after the ice melts and pink forms of nauplii appear. The nauplii then change into a pink copepoidal form which, before their transformation into the adult form, become bluish in colour. The adults are dark blue in colour. The females of these crustaceans lay eggs which hibernate in the bed of the pond until the next spring and thus the cycle begins again. It is, therefore, a typically monocyclic species which appears only in the early spring (Rylow, 1920). If we take the average seasonal changes in the northeastern part of Poland, the first form of nauplii appears more or less in the middle of April and by the end of May a few mature specimens of the species can occasionally be seen. The preliminary investigations carried out some time ago (Czeczuga & Czerpak, 1966) on this species revealed the presence of a number of carotenoids which give its members their coloration. In view of the changes in its colour during its ontogenetic development, we became interested in the question of what if any qualitative and quantitative changes in carotenoids accompany these changes in colour. MATERIALS AND METHODS The E. amblyoabn specimens were collected in AprilMay from the ponds of Dojlidy near Bialystok. Pigments The carotenoid pigments were extracted by means of 95% acetone in a dark room. Saponification was carried
out by means of 6% KOH in methanol at a temperature of about 40°C for several minutes in the dark in a nitrogen
atmosphere. Chromatography Columnar and thin-layer chromatography, described in detail in our previous papers (Czecxuga, 1971a, b), were used for the separation of the various carotenoids. A glass column approximately 1 cm dia and 15-u) cm in length, filled with AlsOa, was used in column chromatography. The E. ambfyodonextract was passed through the column after which the different fractions were eluted with the solvent systems given in the tables. Silica gel was used for thin-layer chromatography, with the appropriate solvent systems, the R, values being determined for each spot. For identification of &carotene and astaxanthin identical co-chromatography was applied using carotenoids.
The pigments were identified by the following methods: (a) behaviour on column chromatography; (b) absorption spectra of the pigments in various solvents were recorded by a Beckman spectrophotometer Model 2400DU; (c) the partition characteristics of the carotenoid between hexane and 95% methanol using the method of Petracek and Zechmeister (1956); (d) comparison of R, on thin-layer chromatography (with authentic B-carotene and astaxanthin); (e) the presence of allylic hydroxyl groups was determined by the acid chloroform test (Karrer & Leumalm, 1951); and (f) the epoxide test (Krinsky & Goldsmith, 1960a; Curl & Bailey, 1961). Quantitativedeterminations Quantitative determinations of the concentrations of carotenoid solutions were made from the quantitative absorption spectra. These determinations were based on 665
666
B. CZECZUGA
the extinction coefficient E$$ at the wavelengths of maximal absorbance in petroleum ether or hexane (Davies, 1965; Foppen, 1971). RESULTS The results of the columnar chromatographic analysis of the acetone extract of E. ambfyodon eggs are given in Table 1. The eggs of this crustacean contain p-carotene, isozeaxanthin and astacene. Two fractions were not identified. The last fraction, which was not identified (maximum absorption in benzene 416,442 and 468), comprised almost 70 per cent of the total carotenoid content of the eggs. Of Table 1. Column chromatogram No. of fraction I II III IV V
Eluent
Table 2. Column chromatogram
No. of fraction
from E. amblyodon eggs (3.943 pg/g live weight)
Solvent
2% Acetone in petroleum ether 20% Acetone in petroleum ether 30% Acetone in petroleum ether Methanol (100%) Ethyl ether, cold acetic acid (20 : 1) 15% KOH in 90% methanol
VI
of carotenoids
the carotenoids identified, astacene occurred in the largest amount, 20.2 per cent of the total carotenoid content. The pink copepoidal form of this species contained 11.555 pg/g of carotenoids per live weight. Four fractions were identified: isozeaxanthin, 3,4-dihydroxy-P-carotene, and y-carotene derivative and astaxanthin (Table 2). The predominant carotenoid was found to be astaxanthin which comprised 86.4 per cent of the carotenoid content of the pink forms of copepoidal E. amblyodorz. The results of the chromatographic separation of the acetone extract of the blue copepoidal forms of
Eluent
Maximum absorption (nm)
Petroleum ether 423,451,478 452 Hexane 451,478 Ethanol 451,478 Ethanol 495 Benzene Benzene
416,442,468
Partition ratio 100 0 25 20 10
0
100 75 80 90
7 93
of carotenoids from the pink copepoidal (11.555 pg/g live weight)
Solvent
Partition ratio
2% Acetone in petroleum ether 10% Acetone in petroleum ether 20% Acetone in petroleum ether
Petroleum ether Petroleum ether Hexane
418,435,465 446,475 424,452,481
80 : 20 25 : 75 40 : 60
IV V VI VII
30% Acetone in petroleum ether 50% Acetone in petroleum ether Methanol (100%) Ethyl ether, cold acetic acid (20 : 1)
Hexane Ethanol Ethanol Ethanol
416,438,470 451,478 455 464
100:o 17 : 83 34 : 66 20 : 80
VIII
15% KOH in 90% methanol
Benzene
485
Table 3. Column chromatogram
No. of fraction
Eluent
Solvent ether ether ether ether
Maximum absorption (nm)
Petroleum ether 465 Petroleum ether 466 468 Hexane 455 Hexane
I II III IV
2% Acetone in petroleum 10% Acetone in petroleum 20% Acetone in petroleum 30% Acetone in petroleum
V VI VII
Methanol (100%) Ethanol Ethyl ether, cold acetic acid (20 : l)Benzene 15% KOH in 90% methanol Benzene
451,478 464 485
20 : 80 34 : 66 8 : 92
2.73 2.02 2.53 2.63 20.16
Unknown
69.89
Identification
Amount (%) 1.47 1.15 1.20 2.85 3.89 l-71 1.25
8644
form of E. amblyodon
Partition ratio 50 : 50 25 : 75 25 : 75 27 : 73
p-carotene Unknown Isozeaxanthin Isozeaxanthin Astacene
Unknown Isozeaxanthin 3,CDihydroxyp-carotene Unknown Isozeaxanthin Unknown l’J-Dihydro-l’hydroxy-l-ketoy-carotene Astaxanthin
17 : 83
of carotenoids from the blue copepoidal (3.416 pg/g live weight)
Amount (%)
form of E. amblyodon
Maximum absorption (nm)
I II III
Identification
Identification Canthaxanthin Astaxanthin Astaxanthin 4-Hydroxy+ketop-carotene Isozeaxanthin Unknown Astaxanthin
Amount (%) 1.72 2.62 2.70 5.29 8.78 7.02 71.83
Carotenoids in Eudiaptomusamblyodon
667
Table 4. Column chromatogram of carotenoids from the mature female of E. ambfyodon (6.127 pg/g live weight) No. of
fraction
Eluent
Solvent
I II III IV
2% Acetone in petroleum ether 10% Acetone in petroleum ether 20% Acetone in petroleum ether 30% Acetone in petroleum ether
V VI VII
50% Acetone in petroleum ether Ethanol Ethyl ether, cold acetic acid (20 : 1) Ethanol Benzene 15% KOH in 90% methanol
Petroleum ether 447,478 Petroleum ether 420,442 Hexane 467 Hexane 457
E. amblyodon are shown in Table 3. The presence of the following carotenoids was determined: canthaxanthin, astaxanthin, 4-hydroxy-4-keto-j3-carotene, isozeaxanthin and astaxanthin. As in the pink copepoidal forms, the predominant carotenoid in the blue forms was found to be astaxanthin, 77.2 per cent of the total carotenoid content, i.e. 3.416 pg/g of the live weight of the crustaceans. The results of the investigations on the mature female specimens are presented in Table 4. Isocryptoxanthin, canthaxanthin, 4-hydroxy-4-keto-pcarotene, lutein and astaxanthin were identified. In this case, too, astaxanthin was predominant, 76.6 per cent of the total amount of carotenoids found in the adult female specimens of E. amblyodon (6.127 pg/g live weight). DISCUSSION The
Maximum absorption (nm)
carotenoids found in E. amblyodon eggs have been reported in other Crustacea both in fresh water and marine species (Czeczuga, 1971c, 1973). In the shrimp, Leander serratus, however, the carotenoids echinenon, isocryptoxanthin and lutein were also found. These carotenoids were not found in E. amblyodon eggs. In Nephrops norvegicus eggs, however, only astaxanthin was present. A difference in the carotenoid content of the pink and blue copepoidal forms was noted. In the pink forms 3,4-dihydroxy-p-carotene and a y-carotene derivative were identified whereas the blue forms did not contain these carotenoids. Canthaxanthin and 4-hydroxy-4-keto-@-carotene were present in the blue forms but not in the pink forms. In the blue mature forms two carotenoids, not found in either the pink or the blue copepoidal forms, were found, namely, isocryptoxanthin and lutein. Like the blue copepoidal forms, the adult E. amblyodon specimens contained canthaxanthin and 4hydroxy-4-keto-@arotene. It would seem that the most probable explanation of the changes in colour of E. amblyodon during its ontogenetic development is as follows. The pink
420,445,475 472 485
Partition ratio 89: 11 71 :29 50 : 50 33 : 67 14 : 86 25 : 75 8 : 92
Identification
Amount (%)
Isocryptoxanthin 8.16 Unknown 3.81 Canthaxanthin l-48 4-Hydroxy-4-keto- 2.66 p-carotene Lutein 7.29 Astaxanthin l-25 Astaxanthin 75.31
colour of the copepoidal forms is due to the caroten-
oids present, with the predominance of astaxanthin which comprises 84.6 per cent of the total carotenoid content of pink copepoidal E. amblyodon. The blue colour of some of the copepoidal forms and of mature E. amblyodon is the result of the formation of protein-carotenoid complexes as was shown by the studies of Zagalsky et al. (1967, 1970) and Zagalsky & Herring (1972). These complexes are quite common in different organs of crustacea. These complexes are also peculiar to the eggs of fresh water and marine crustaceans (Ball, 1944; Green, 1965; Cheesman & Prebble, 1966; Zagalsky et al., 1967). As Cheesman et al. (1967) state, the prosthetic group of these protein-carotenoid complexes usually consists of canthaxanthin, astaxanthin or astaxanthin ester. Canthaxanthin and astaxanthin were identified in the blue copepoidal forms and in the mature specimens of E. amblyodon. In all the investigated forms of this crustacean, astaxanthin was found to be predominate. The blue coloration of the body is, as the data given by Herring (1965, 1967) show, peculiar to many species of marine organisms belonging to various systematic groups which live in the upper layers of the water. According to that author, too, the blue colour is due to the protein-carotenoid complexes formed in the prosthetic groups of which astaxanthin comprises the prosthetic group. The author also considers that the blue colour of marine organisms is due to the processes of adaption to their surroundings. How then can we explain the blue colour of the E. amblyodon species which live in small, shallow ponds which dry up during the summer months? Future studies of this problem will without doubt give the answer to this question. REFERENCES
BALL E. G. (1944) A blue chromoprotein found in the ews of the goose barnacle. J. biol. Chem. 152.627-634. C&&AN D:F., LEE W. L. & ZAGALSKY P.-F. (1967) Carotenoproteins in invertebrates. Biol. Rev. 42, 131160.
668 CHEESMAN D.
B.
CZECZUGA
F. & PREBBLEJ. (1966) Astaxanthin ester as a prosthetic group: a carotenoprotein from the hermit crab. Comp. Biochem. Physiol. 17,929-935. CURL A. L. & BAILEYG. F. (1961) An improved test for carotenoid epoxide. Agric. Fd Chem. 9,403-405. CZECZUGAB. (1971a) Composition and tissue distribution of carotenoids and vitamin A in the craytish Astacus leptodactylus (Esch.) (Crustacea, Decapoda). Comp. Biochem. Phvsiol. 39B. 945-953. CZECZUGAB. i1971b) Assimilation of carotenoids with food by the beetle Leptinotarsa decemlineata. J. Insect Physiol. 17, 2017-2025. CZECZUGA B. (1971c) Studies on the carotenoids in Artemia salina L. eggs. Comp. Biochem. Physiol. 4OB, 47-52. CZECZUGAB. (1973) Investigations of arotenoids in some fauna of the Adriatic Sea-III. Leander (Palaemon) serratus and Nephrops norvegicus (Crustacea: Decapoda). Mar. Biol. 21, 139-143. CZECZUGA B. & CZERPAK R. (1966) Carotenoids in certain Diaptomidae (Crustacea). Comp. Biochem. Physiol. 17, 523-534. DAVIESB. H. (1965) Chemistry and Biochemistry ofPlant Pigments, pp. 489-532. Academic Press, New York. FOPPEN F. H. (1971) Tables for the identification of carotenoid pigments. Chrom. Rev. 14, 133-298. GREEN J. (1965) Chemical embryology of the Crustacea. Biol. Rev. 40,580-600. HERRINGP. J. (1965) A blue pigment of a surface living oceanic copepod. Nature, Land. 205, 103-104.
HERRINGP. J. (1967) The pigments of plankton at the sea surface. Symp. zoo/. Sot. Lond. 19,215-235. KARRERP. & LEUMANNE. (1951) Eschscholtzxanthin and anhydro-eschscholtzxanthin. Helv. chim. Acta 34, 445453. KRINSKY N. J. & GOLDSMITHT. H. (1960a) The carotenoids of the flagellated alga, Euglena gracilis. Archs Biochem. Biophys. 91, 2711279. _ PETRACEKF. J. & ZECHMEISTER L. (1956) \ , Determination of partition coefficients of carotenoids as a tool in pigments analysis. Analyt. Chem. 26, 1484-1485. RYLOW W. M. (1920) K biologii Diaptomus amblyodon Marenz. Tr. Petroar. Obshch. Estestwoisv. 51. 29-36. ZAGAL~KYP. F., CE&ALDI H. J. & DA&AS R: (1970) Comparative studies on some Decapod crustacea carotenoproteins. Comp. Biochem. Physiol. 34, 579607. ZAGALSKYP. F., CHEE~MAND. F. & CECCALDIH. J. (1967) Studies on carotenoid-containing lipoproteins isolated from the eggs and ovaries of certain marine invertebrates. Comp. Biochem. Physiol. 22, 851-871. ZAGAL~KYP. F. & HERRING P. J. (1972) Studies on a carotenoprotein isolated from the copepod Labidocera acutifrons and its relationship to the Decapod carotenoproteins and other polyene-binding proteins. Comp. Biochem. Physiol. 41B, 397-415. Key Word Index-Carotenoids; mus amblyodon Marenz.
Crustacea;
Eudiapto-
CAROTENOIDS (CRUSTACEA)
IN EUDIAPTOMUS AMBL YODON MARENZ DURING ONTOGENETIC DEVELOPMENT B. CZECZUGA
Department
of General Biology, Medical Academy, Bialystok, Poland (Received 4 January 1974)
Ah&a&-l. By means of columnar and thin-layer chromatography, the presence of carotenoids in Eudiaptomusamblyodonwas studied. 2. The following carotenoids were found: (a) in eggs: p-carotene (2.73 per cent), isozeaxanthm (5.16 per cent) and astacene (20.16 per cent); (b) in pink copepoidal form: isozeaxan thin (504 per cent), 3,4dihydroxy-g-carotene (1.20 per cent), 1’,2’-dihydro-l’-hydroxy Cketo-r-carotene (1.25 per cent) and astaxanthin (8644 per cent); (c) in bluish copepoidal form: canthaxanthin (1.72 per cent), 4-hydroxy4-keto-flcarotene (5.29 per cent), isoxeaxan thin (8.78 per cent) and astaxanthin (77.15 per cent); (d) in the mature female: isocryptoxanthin (1.48 per cent), 4_hydroxy4keto-g-carotene (2.66 per cent), lutein (7.29 per cent) and astaxanthin (76.56 per cent).
INTRODUCTION THE BIOLOGYof the species Eudiaptomus amblyodon is for many reasons extremely interesting. In small
ponds the winter eggs of this crustacean hatch just after the ice melts and pink forms of nauplii appear. The nauplii then change into a pink copepoidal form which, before their transformation into the adult form, become bluish in colour. The adults are dark blue in colour. The females of these crustaceans lay eggs which hibernate in the bed of the pond until the next spring and thus the cycle begins again. It is, therefore, a typically monocyclic species which appears only in the early spring (Rylow, 1920). If we take the average seasonal changes in the northeastern part of Poland, the first form of nauplii appears more or less in the middle of April and by the end of May a few mature specimens of the species can occasionally be seen. The preliminary investigations carried out some time ago (Czeczuga & Czerpak, 1966) on this species revealed the presence of a number of carotenoids which give its members their coloration. In view of the changes in its colour during its ontogenetic development, we became interested in the question of what if any qualitative and quantitative changes in carotenoids accompany these changes in colour. MATERIALS AND METHODS The E. amblyoabn specimens were collected in AprilMay from the ponds of Dojlidy near Bialystok. Pigments The carotenoid pigments were extracted by means of 95% acetone in a dark room. Saponification was carried
out by means of 6% KOH in methanol at a temperature of about 40°C for several minutes in the dark in a nitrogen
atmosphere. Chromatography Columnar and thin-layer chromatography, described in detail in our previous papers (Czecxuga, 1971a, b), were used for the separation of the various carotenoids. A glass column approximately 1 cm dia and 15-u) cm in length, filled with AlsOa, was used in column chromatography. The E. ambfyodonextract was passed through the column after which the different fractions were eluted with the solvent systems given in the tables. Silica gel was used for thin-layer chromatography, with the appropriate solvent systems, the R, values being determined for each spot. For identification of &carotene and astaxanthin identical co-chromatography was applied using carotenoids.
The pigments were identified by the following methods: (a) behaviour on column chromatography; (b) absorption spectra of the pigments in various solvents were recorded by a Beckman spectrophotometer Model 2400DU; (c) the partition characteristics of the carotenoid between hexane and 95% methanol using the method of Petracek and Zechmeister (1956); (d) comparison of R, on thin-layer chromatography (with authentic B-carotene and astaxanthin); (e) the presence of allylic hydroxyl groups was determined by the acid chloroform test (Karrer & Leumalm, 1951); and (f) the epoxide test (Krinsky & Goldsmith, 1960a; Curl & Bailey, 1961). Quantitativedeterminations Quantitative determinations of the concentrations of carotenoid solutions were made from the quantitative absorption spectra. These determinations were based on 665
666
B. CZECZUGA
the extinction coefficient E$$ at the wavelengths of maximal absorbance in petroleum ether or hexane (Davies, 1965; Foppen, 1971). RESULTS The results of the columnar chromatographic analysis of the acetone extract of E. ambfyodon eggs are given in Table 1. The eggs of this crustacean contain p-carotene, isozeaxanthin and astacene. Two fractions were not identified. The last fraction, which was not identified (maximum absorption in benzene 416,442 and 468), comprised almost 70 per cent of the total carotenoid content of the eggs. Of Table 1. Column chromatogram No. of fraction I II III IV V
Eluent
Table 2. Column chromatogram
No. of fraction
from E. amblyodon eggs (3.943 pg/g live weight)
Solvent
2% Acetone in petroleum ether 20% Acetone in petroleum ether 30% Acetone in petroleum ether Methanol (100%) Ethyl ether, cold acetic acid (20 : 1) 15% KOH in 90% methanol
VI
of carotenoids
the carotenoids identified, astacene occurred in the largest amount, 20.2 per cent of the total carotenoid content. The pink copepoidal form of this species contained 11.555 pg/g of carotenoids per live weight. Four fractions were identified: isozeaxanthin, 3,4-dihydroxy-P-carotene, and y-carotene derivative and astaxanthin (Table 2). The predominant carotenoid was found to be astaxanthin which comprised 86.4 per cent of the carotenoid content of the pink forms of copepoidal E. amblyodorz. The results of the chromatographic separation of the acetone extract of the blue copepoidal forms of
Eluent
Maximum absorption (nm)
Petroleum ether 423,451,478 452 Hexane 451,478 Ethanol 451,478 Ethanol 495 Benzene Benzene
416,442,468
Partition ratio 100 0 25 20 10
0
100 75 80 90
7 93
of carotenoids from the pink copepoidal (11.555 pg/g live weight)
Solvent
Partition ratio
2% Acetone in petroleum ether 10% Acetone in petroleum ether 20% Acetone in petroleum ether
Petroleum ether Petroleum ether Hexane
418,435,465 446,475 424,452,481
80 : 20 25 : 75 40 : 60
IV V VI VII
30% Acetone in petroleum ether 50% Acetone in petroleum ether Methanol (100%) Ethyl ether, cold acetic acid (20 : 1)
Hexane Ethanol Ethanol Ethanol
416,438,470 451,478 455 464
100:o 17 : 83 34 : 66 20 : 80
VIII
15% KOH in 90% methanol
Benzene
485
Table 3. Column chromatogram
No. of fraction
Eluent
Solvent ether ether ether ether
Maximum absorption (nm)
Petroleum ether 465 Petroleum ether 466 468 Hexane 455 Hexane
I II III IV
2% Acetone in petroleum 10% Acetone in petroleum 20% Acetone in petroleum 30% Acetone in petroleum
V VI VII
Methanol (100%) Ethanol Ethyl ether, cold acetic acid (20 : l)Benzene 15% KOH in 90% methanol Benzene
451,478 464 485
20 : 80 34 : 66 8 : 92
2.73 2.02 2.53 2.63 20.16
Unknown
69.89
Identification
Amount (%) 1.47 1.15 1.20 2.85 3.89 l-71 1.25
8644
form of E. amblyodon
Partition ratio 50 : 50 25 : 75 25 : 75 27 : 73
p-carotene Unknown Isozeaxanthin Isozeaxanthin Astacene
Unknown Isozeaxanthin 3,CDihydroxyp-carotene Unknown Isozeaxanthin Unknown l’J-Dihydro-l’hydroxy-l-ketoy-carotene Astaxanthin
17 : 83
of carotenoids from the blue copepoidal (3.416 pg/g live weight)
Amount (%)
form of E. amblyodon
Maximum absorption (nm)
I II III
Identification
Identification Canthaxanthin Astaxanthin Astaxanthin 4-Hydroxy+ketop-carotene Isozeaxanthin Unknown Astaxanthin
Amount (%) 1.72 2.62 2.70 5.29 8.78 7.02 71.83
Carotenoids in Eudiaptomusamblyodon
667
Table 4. Column chromatogram of carotenoids from the mature female of E. ambfyodon (6.127 pg/g live weight) No. of
fraction
Eluent
Solvent
I II III IV
2% Acetone in petroleum ether 10% Acetone in petroleum ether 20% Acetone in petroleum ether 30% Acetone in petroleum ether
V VI VII
50% Acetone in petroleum ether Ethanol Ethyl ether, cold acetic acid (20 : 1) Ethanol Benzene 15% KOH in 90% methanol
Petroleum ether 447,478 Petroleum ether 420,442 Hexane 467 Hexane 457
E. amblyodon are shown in Table 3. The presence of the following carotenoids was determined: canthaxanthin, astaxanthin, 4-hydroxy-4-keto-j3-carotene, isozeaxanthin and astaxanthin. As in the pink copepoidal forms, the predominant carotenoid in the blue forms was found to be astaxanthin, 77.2 per cent of the total carotenoid content, i.e. 3.416 pg/g of the live weight of the crustaceans. The results of the investigations on the mature female specimens are presented in Table 4. Isocryptoxanthin, canthaxanthin, 4-hydroxy-4-keto-pcarotene, lutein and astaxanthin were identified. In this case, too, astaxanthin was predominant, 76.6 per cent of the total amount of carotenoids found in the adult female specimens of E. amblyodon (6.127 pg/g live weight). DISCUSSION The
Maximum absorption (nm)
carotenoids found in E. amblyodon eggs have been reported in other Crustacea both in fresh water and marine species (Czeczuga, 1971c, 1973). In the shrimp, Leander serratus, however, the carotenoids echinenon, isocryptoxanthin and lutein were also found. These carotenoids were not found in E. amblyodon eggs. In Nephrops norvegicus eggs, however, only astaxanthin was present. A difference in the carotenoid content of the pink and blue copepoidal forms was noted. In the pink forms 3,4-dihydroxy-p-carotene and a y-carotene derivative were identified whereas the blue forms did not contain these carotenoids. Canthaxanthin and 4-hydroxy-4-keto-@-carotene were present in the blue forms but not in the pink forms. In the blue mature forms two carotenoids, not found in either the pink or the blue copepoidal forms, were found, namely, isocryptoxanthin and lutein. Like the blue copepoidal forms, the adult E. amblyodon specimens contained canthaxanthin and 4hydroxy-4-keto-@arotene. It would seem that the most probable explanation of the changes in colour of E. amblyodon during its ontogenetic development is as follows. The pink
420,445,475 472 485
Partition ratio 89: 11 71 :29 50 : 50 33 : 67 14 : 86 25 : 75 8 : 92
Identification
Amount (%)
Isocryptoxanthin 8.16 Unknown 3.81 Canthaxanthin l-48 4-Hydroxy-4-keto- 2.66 p-carotene Lutein 7.29 Astaxanthin l-25 Astaxanthin 75.31
colour of the copepoidal forms is due to the caroten-
oids present, with the predominance of astaxanthin which comprises 84.6 per cent of the total carotenoid content of pink copepoidal E. amblyodon. The blue colour of some of the copepoidal forms and of mature E. amblyodon is the result of the formation of protein-carotenoid complexes as was shown by the studies of Zagalsky et al. (1967, 1970) and Zagalsky & Herring (1972). These complexes are quite common in different organs of crustacea. These complexes are also peculiar to the eggs of fresh water and marine crustaceans (Ball, 1944; Green, 1965; Cheesman & Prebble, 1966; Zagalsky et al., 1967). As Cheesman et al. (1967) state, the prosthetic group of these protein-carotenoid complexes usually consists of canthaxanthin, astaxanthin or astaxanthin ester. Canthaxanthin and astaxanthin were identified in the blue copepoidal forms and in the mature specimens of E. amblyodon. In all the investigated forms of this crustacean, astaxanthin was found to be predominate. The blue coloration of the body is, as the data given by Herring (1965, 1967) show, peculiar to many species of marine organisms belonging to various systematic groups which live in the upper layers of the water. According to that author, too, the blue colour is due to the protein-carotenoid complexes formed in the prosthetic groups of which astaxanthin comprises the prosthetic group. The author also considers that the blue colour of marine organisms is due to the processes of adaption to their surroundings. How then can we explain the blue colour of the E. amblyodon species which live in small, shallow ponds which dry up during the summer months? Future studies of this problem will without doubt give the answer to this question. REFERENCES
BALL E. G. (1944) A blue chromoprotein found in the ews of the goose barnacle. J. biol. Chem. 152.627-634. C&&AN D:F., LEE W. L. & ZAGALSKY P.-F. (1967) Carotenoproteins in invertebrates. Biol. Rev. 42, 131160.
668 CHEESMAN D.
B.
CZECZUGA
F. & PREBBLEJ. (1966) Astaxanthin ester as a prosthetic group: a carotenoprotein from the hermit crab. Comp. Biochem. Physiol. 17,929-935. CURL A. L. & BAILEYG. F. (1961) An improved test for carotenoid epoxide. Agric. Fd Chem. 9,403-405. CZECZUGAB. (1971a) Composition and tissue distribution of carotenoids and vitamin A in the craytish Astacus leptodactylus (Esch.) (Crustacea, Decapoda). Comp. Biochem. Phvsiol. 39B. 945-953. CZECZUGAB. i1971b) Assimilation of carotenoids with food by the beetle Leptinotarsa decemlineata. J. Insect Physiol. 17, 2017-2025. CZECZUGA B. (1971c) Studies on the carotenoids in Artemia salina L. eggs. Comp. Biochem. Physiol. 4OB, 47-52. CZECZUGAB. (1973) Investigations of arotenoids in some fauna of the Adriatic Sea-III. Leander (Palaemon) serratus and Nephrops norvegicus (Crustacea: Decapoda). Mar. Biol. 21, 139-143. CZECZUGA B. & CZERPAK R. (1966) Carotenoids in certain Diaptomidae (Crustacea). Comp. Biochem. Physiol. 17, 523-534. DAVIESB. H. (1965) Chemistry and Biochemistry ofPlant Pigments, pp. 489-532. Academic Press, New York. FOPPEN F. H. (1971) Tables for the identification of carotenoid pigments. Chrom. Rev. 14, 133-298. GREEN J. (1965) Chemical embryology of the Crustacea. Biol. Rev. 40,580-600. HERRINGP. J. (1965) A blue pigment of a surface living oceanic copepod. Nature, Land. 205, 103-104.
HERRINGP. J. (1967) The pigments of plankton at the sea surface. Symp. zoo/. Sot. Lond. 19,215-235. KARRERP. & LEUMANNE. (1951) Eschscholtzxanthin and anhydro-eschscholtzxanthin. Helv. chim. Acta 34, 445453. KRINSKY N. J. & GOLDSMITHT. H. (1960a) The carotenoids of the flagellated alga, Euglena gracilis. Archs Biochem. Biophys. 91, 2711279. _ PETRACEKF. J. & ZECHMEISTER L. (1956) \ , Determination of partition coefficients of carotenoids as a tool in pigments analysis. Analyt. Chem. 26, 1484-1485. RYLOW W. M. (1920) K biologii Diaptomus amblyodon Marenz. Tr. Petroar. Obshch. Estestwoisv. 51. 29-36. ZAGAL~KYP. F., CE&ALDI H. J. & DA&AS R: (1970) Comparative studies on some Decapod crustacea carotenoproteins. Comp. Biochem. Physiol. 34, 579607. ZAGALSKYP. F., CHEE~MAND. F. & CECCALDIH. J. (1967) Studies on carotenoid-containing lipoproteins isolated from the eggs and ovaries of certain marine invertebrates. Comp. Biochem. Physiol. 22, 851-871. ZAGAL~KYP. F. & HERRING P. J. (1972) Studies on a carotenoprotein isolated from the copepod Labidocera acutifrons and its relationship to the Decapod carotenoproteins and other polyene-binding proteins. Comp. Biochem. Physiol. 41B, 397-415. Key Word Index-Carotenoids; mus amblyodon Marenz.
Crustacea;
Eudiapto-