Carotenoids in thirteen species of gammaridae from Lake Bajka Ł

Comp. Biochem. Physiol., 1975, Vol. 5013,pp. 259 to 268. PergamonPress. Printed in Great Britain

CAROTENOIDS IN THIRTEEN SPECIES OF GAMMARIDAE FROM LAKE BAJKAL B. CZECZUGA Department of General Biology, Medical Academy Bialystok, Poland

(Received 31 January 1974) Abstract--1. By means of columnar and thin-layer chromatography, the presence of carotenoids in the Gammaridae from Lake Baikal was studied. 2. The following carotenoids were found: in Crypturopus pachytus: /8-carotene, eryptoxanthin, cantaxanthin, 4-hydroxy-4-keto-/8-carotene, isozeaxanthin, astaxanthin free and astaxanthin ester; in Micruropus wahli: E-carotene, ~,-¢arotene, ~,-carotene derivative, lutein ester, lutein free, zeaxanthin, astaxanthin and astacene; in Mieruropus vortex: y-carotene, lutein, lutein epoxy, isozesxanthin, astaxanthin and astaxanthin ester; in Gmelinoides faseiatus: ~,-carotene, canthaxanthin, astaxanthin, lutein epoxy, zeaxanthin and astacene; in Brandtia iata:/8-carotene, isocryptoxanthin, caathaxanthin, lutein ester, lutein epoxy, zcaxanthin and astaxanthin; in Acanthogammarus albus: canthaxanthin, zeaxanthin, astaxanthin, astaxanthin ester and astacene; in Pallasea eaneellus:/8-carotene, isocryptoxanthin, lutein, tunaxanthin, zeaxanthin, astaxanthin and astaxanthin-like carotenoid; in Pallasea cancelloides: 4-hydroxy-3,4-dehydro-/8-carotene,canthaxanthin, lutein ester, lutein epoxy, zeaxanthin, astaxanthin and astacene; in Pallasea viridis: canthaxanthin, zeaxanthin, astaxanthin and astaxanthin ester; in Eulimnogammarus cruentus: y-carotene, canthaxanthin, lutein ester, lutein, ~,-carotene derivative, zeaxanthin, and astaxanthin; in Eulimnogammarus maacki: /8-carotene, y-carotene derivative, isocaryptoxanthin, lutein ester, lutein and astaxanthin; in Eulimnogammarus grandimanus: ~ n i a x a n . thin-like, keto-carotenoid?, lutein ester, lutein, zeaxanthin, astaxanthin and astacene; and in Macrohectopus branickii: isozeaxanthin, astaxanthin ester, astaxanthin and astacene.

INTRODUCTION LAKE BAJKAL, one of a few hydrobiologieal rarities of the world, has, to a great extent, endemic fauna and flora making it unique among other such lakes. Seals characteristic of this lake are to be found there and a number of typical fish species but, above all, an abundance of invertebrates inhabit this lake. Among these invertebrates the Gammaridae occupy an important position. These species live in different ecological niches in the lake. So far about 200 species have been described; such a variety of species has not been observed in any other lake in the world. The variety consists not only in their occupying different niches but perhaps to an even greater extent in the morphology and even physiology of the members of this group of crustaceans. There are species which are phytophagous, others which are typical planktophages, e.g. Macrohectopus branickii, and yet others which feed on fish usually in nets, e.g. species of the Acanthogammarus genus. The body coloration also varies greatly. Some species are of a blood-red colour (e.g. species of the Eulimnogamraarus genus) or green species of the Pallasea genus), some species of the Brandtia genus are violet, and some species of the Crypturopus and Micruropus genera, which burrow in the sand, are light grey in colour. The species which live in the depths, e.g.

species of the Acanthogammarus genus, are armed with spikes and their carapace is a yellowish-pink colour. The pelagic species, M. branickii, which is adapted to a plankton way of life has an almost transparent carapace. The colour of the eye pigment also varies according to the depths of water inhabited by the various species. In the littoral species the eyes are black, those of species in the deeper water, red or pink, whereas those of species living in the deepest parts of the lake are practically colourless. There have been comparatively few publications on carotenoids in the Gammaridae. In 1949, Beatty discussed the carotenoids in several Amphipoda species without, however, giving the names of the earotenoids. The presence of carotenoids in two species of the Gammarus genus were mentioned in that paper. Later, Grangaud (1951) reported the presence of carotenoids in specimens of Gammarus pulex. Andres(1956a, b) also studied the carotenoids in that species. The presence of protein--carotenoid complexes in some representatives of the Amphipoda was reported by Wieser (1965). Czeczuga (1970) investigated the occurrence of various carotenoids in marine species of the Amphipoda (Hyale perieri) and later in two species of Niphargus living in spring water (Czeczuga & Skalski, 1973). 259

260

B. CZECZUG,, MATERIALS AND METHODS

The material, consisting of thirteen species of Gammaridae, was collected in August and September 1971 during my stay on the southern shore of Lake Bajkat at the Biological Station at Bolshe Koty. It was gathered from a boat by means of a drag net from the area near the Station with the exception of the Acanthogammarus albus specimens which were collected on the beach at Piesczany Biereg. (a) Pigments The carotenoid pigments were extracted by means of 95~o acetone in a dark room. Saponification was carried out by means of 6 ~ K O H in ethanol at a temperature of about 40°C for several minutes in the dark in a nitrogen atmosphere. (b) Chromatography Columnar and thin-layer chromatography, described in detail in our previous paper (Czeczuga, 1971a), were used for the separation of the various carotenoids. A glass column approximately 1 cm dia and 15-20 cm in length, filled with AI~Os, was used in column chromatography. The extracts were passed through the column in 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 I values being determined for each spot. For identification of E-carotene and astaxanthin, cochromatography was applied using identical carotenoids. (c) Identification of pigments 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~o methanol using the method of Petracek & Zechmeister (1956); (d) comparison of R I on thin-layer

chromatography (with authentic fl-carotene and astaxanthin, (e) the presence of allylic hydroxyl groups was determined by the acid chloroform test {Karrer & Leumann, 1951); and (f) the epoxide test (Krinsky & Goldsmith, 1960; Curl & Bailey, 1961). (d) Quantitative determination Quantitative determinations of the concentrations of carotenoid solutions were made from the quantitative absorption spectra. These determinations were based on the extinction coefficient Ec~ at the wavelengths of maximal absorbance in petroleum ether or hexane (Da;,,,~ 2965; Foppen, 1971). RESULTS In discussing the results of the chromatographic analysis o f the carotenoids, I have given the body colour of each species together with the ecological data. The data comes f r o m the m o n o g r a p h of Bazika~owa (1945) in which the G a m m a r i d a e of Lake Bajka~ are described. 1. Crypturopus pachytus (Dyb.) (Tablel) The body length of this species reaches 18 m m and its colour is greyish-white. It is usually found at a depth of 15-30 m in the sandy bed all over the lake. The total a m o u n t of carotenoids was 52.337/zg/g raw weight. The predominant carotenoid was astaxanthin comprising 39-17~ of the total a m o u n t of carotenoids. A b o u t 15~ consisted of fl-carotene and canthaxanthin and about 1 0 ~ cryptoxanthin and 4-hydroxy-4-keto-fl-carotene, 5 . 5 ~ consisted of isozeaxanthin. 2. Micruropus wahli (Dyb.) (Table 2) The body of this species is whitish in colour and its length is 8-9 mm. It lives in sand and when the

Table 1. Column chromatogram of carotenoids from C. pachytus (Dyb.) Maximum absorption (nm)

Partition ratio

Petroleum ether Petroleum ether

423,448,478 424, 451,478

100 : 0 70 : 30

/3-Carotene Cryptoxanthin

14.28 9'91

Petroleum ether

462

53 : 47

Canthaxanthin

14.78

Hexane

457

30 : 70

9.03

Hexane

466

20 : 80

4-hydroxy-4-keto/3-carotene Astaxanthin

Ethanol

464

Astaxanthin ether

5.91

Ethanol Benzene

424,451,478 455,475

Isozeaxanthin Unknown

5.50 7.31

Benzene

485

No.

of fraction I II

Eluent

Petroleum ether 2~o Acetone in petroleum ether III 10~ Acetone in petroleum ether IV 20~ Acetone in petroleum ether V 30~ acetone in petroleum ether VI 50~ Acetone in petroleum ether VII Methanol (100~o) VIII Ethyl ether, cold acetic acid (20 : 1) IX 15~ K O H in 90~ methanol

Solvent

100 : 0 20 : 80 20 : 80 7 : 93

Identification

Astaxanthin

Amount (~)

5.54

27-72

Carotenoids in Gammaridae

261

Table 2. Column chromatogram of carotenoids from M. wahli (Dyb.) No. of fraction

Eluent

I Petroleum ether II 2% acetone in petroleum ether III 10% Acetone in petroleum ether IV 30~oAcetone in petroleum ether V 50~ Acetone in petroleum ether VI Methanol (100%) VII Ethyl ether, cold acetic acid (20 : I) VIII 15~oKOH in 90% methanol

Solvent

Maximum absorption (rim)

Partition ratio Identification

Amount (%)

Petroleum ether 426,448, 478 100 : 0 Petroleum ether 435,462, 495 100 : 0

//-Carotene y-Carotene

3.06 10.16

Hexane

437, 464, 497

29 : 71

7.86

Hexane

424, 442, 468

34 : 66

y-Carotene derivative Lutein ester

Ethanol

424, 446, 476

12 : 88

Lutein

26.50

Ethanol Benzene

451,483 485

10 : 90 10 : 90

Zeaxanthin Astaxanthin

5.03 20.52

Benzene

495

Astacene

23.28

water of the lake is calm it even swims under the surface of the water. It is found at a depth of 3-4 m. Of the total amount of carotenoids, 21.143/,g/g weight, over 40% consisted of astaxanthin and 26.5% lutein. About 10% was y-carotene and 7"86~o a derivative of y-carotene, fl-Carotene comprised 3~o of the total amount of carotenoids found. Lutein ester was more or less of the same order whereas 5-03~o consisted of zeaxanthin. 3. Micruropus vortex (Dyb.) (Table 3) The body length was approximately 5 mm. It is usually found on stones covered with algae at a depth of 0.5-200 m in the southern part of Lake Bajkat. The total amount of carotenoids was 67.294/,g/g of raw weight. The following carotenoids were identified: ),-carotene (4.99%), lutein (53"26~o), lutein epoxy (11.35%), isozeaxanthin (3.22%), pure astaxanthin (17.12~o) and astaxanthin ester (8-44%).

5 : 95

3.47

4. Gmelinoides fasciatus (Stebb) (Table 4) The members of this species are found at a depth of 0-5 m on stones overgrown with algae or on the sandy bed and are frequently found to burrow into the sand. During windless weather they swim under the lake surface. The total amount of carotenoids was 67.045/~g/g raw weight. As can be seen from Table 4 these consisted of y-carotene (6.25%), canthaxanthin (15.47%), astaxanthin (12.20~o), together with astacene (11.34%), lutein epoxy (20.05%) and zeaxanthin (10.87.%o). 5. Brandtia lata (Dyb.) (Table 5) This species is found in the southern part of Lake Baikal at a depth of 0.5--65 m, usually on stones covered with sponges and rarely on the sandy bed. Its body length varies between 15 and 18 m. The results of the chromatographic analysis show that the total content of carotenoids in the material

Table 3. Column chromatogram of carotenoids from M. vortex (Dyb.) No. of fraction

Eluent

I Petroleum ether II 2~o Acetone in petroleum ether III 10% Acetone in petroleum ether IV 20% Acetone in petroleum ether V 30°/0Acetone in petroleum ether VI 50°/0Acetone in petroleum ether VII 15% KOH in 90°/0 methanol

Solvent

Maximum absorption (nm)

Partition ratio Identification

Amount (%)

Petroleum ether 466 0 : 100 Astaxanthin Petroleum ether 435, 462, 495 100 : 0 y-Carotene

4.57 4.99

Petroleum ether 466

77 : 23

Astaxanthin ester

8.44

Hexane

424, 446, 476

12 : 88

Lutein

53"26

Hexane

424, 446, 476

20 : 80

Lutein epoxy

11.35

Hexane

425, 451,478

23 : 77

Isozeaxanthin

3.22

Benzene

485

17 : 83

Astaxanthin

12.66

262

B. CZECZUGA Table 4. Column chromatogram of carotenoids from G. fasciatus (Stebb.) No. of fraction

Eluent

I

2 ~ Acetone in petroleum ether 1I 10~ Acetone in petroleum ether Ili 20~ Acetone in petroleum ether IV 30~o Acetone in petroleum ether V 50~ Acetone in petroleum ether VI 10~on. Propanol in acetone VII Methanol (100~o) VIII IX

Ethyl ether, cold acetic acid (20 : 1) 15~o K O H in 9 0 ~ o methanol

Solvent

Maximum absorption (nm)

Partition ratio Identification

Petroleum ether

437, 462, 495

100 : 0

Petroleum ether

464

Hexane

Amount (o/)

y-Carotene

6.25

55 : 45

Canthaxanthin

5-08

466

13 : 87

Astaxanthin

Hexane

464

50 : 50

Canthaxanthin

Ethanol

425,442, 470

23 : 77

Lutein epoxy

15.04

Ethanol

425,445,470

17 : 83

Lutein epoxy

5.01

Ethanol 451,483 Carbondisulphide 450, 481, 518 Benzene 468

12 : 88

Zeaxanthin

10.87

14 : 86

Unknown

24.68

5 : 95

Astacene

11.34

Benzene

495

12.20 9-49

Table 5. Column chromatogram of carotenoids from B. lata (Dyb.) No. of fraction

Eluent

I

2~o Acetone in petroleum ether II 10~ Acetone in petroleum ether III 20~ Acetone in petroleum ether IV 30~ Acetone in petroleum ether V 50~o Acetone in petroleum ether VI Acetone (100~) VII 10~on, Propanol in acetone VIII Methanol (100~) IX X

Ethyl ether, cold acetic acid (20 : 1) 15~ K O H in 90~ methanol

Solvent

Maximum absorption (nm)

Partition ratio Identification

Petroleum ether

425,448,478

100 : 0

Petroleum ether

425,451,478

Hexane

Amount (~o)

/3-Carotene

0"96

86 : 14

Isocryptoxanthin

6.27

462

50 : 50

Canthaxanthin

4'77

Hexane

425,442, 470

30 : 70

Lutein ester

Ethanol

425,442, 470

20 : 80

Lutein expoxy

7.52

Hexane Ethanol

425, 445, 470 425,445, 470

40 : 60 20 : 80

Lutein ester Lutein epoxy

13.45 0.48

Ethanol 451,481 Carbondisulphide 448, 483, 514 Benzene 485

11 : 89

Zeaxanthin

12.76

4 : 96

Astaxanthin

14.84

2 : 98

Unknown

28.73

Benzene

470

10.20

investigated was 82.263/~g/g raw weight. The following carotenoids were identified: /3-carotene (0"96~), isocryptoxanthin (6.27~), canthaxanthin (4.77~), an ester f o r m of lutein (23"65~) and lutein cpoxy (8-00~). In addition, zeaxanthin (12.76~) and astaxanthin (14-84~o) were found. The last fraction was not identified.

usually at a depth of 5 0 - 3 0 0 m on a loamy or loamy-sandy bed. It is usually yellowish in colour with darker diagonal stripes. The total carotenoid content was 3.225 tzg/g raw mass. Astaxanthin ester (13.56~), pure astaxanthin (5.02~), astacen (43"34~o), canthaxanthin (1'95~o) and zeaxanthin (36"08~o) were found.

6. A c a n t h o g a m m a r u s albus (Graj.) (Table 6)

7. Pallasea cancellus (Pall.) (Table 7~

This is one of the more numerous representatives o f the G a m m a r i d a e and its body length can be up to 43-54 turn. It is found in all parts of Lake Bajkat

This species is to be found on a sandy-stony bed in all parts of the lake at a depth of 1-52 m. The body is 63-65 m m in length and is green in colour.

Carotenoids in Gammaridae As a result of the chromatographic analysis, a carotenoid content of 196.154/~g/g raw mass was found comprising fl-carotene (2.76%), isocryptoxanthin (2.71%), pure lutein (15.38%) and its ester (17.80~o), astaxanthin (2.71~o), and astaxanthin-like carotenoid (24.62%) and also zeaxanthin (3.84%) and tunaxanthin (17.2%). 8. Pallasea cancelloides (Gerstf.) (Table 8) This species inhabits sandy beds and sometimes stony beds in all parts o f the lake at a depth of 1-178m. It is of a greenish colour. The total carotenoid content was found to be 181.974/~g/g raw weight. Astaxanthin (37.21~o) and lutein epoxy (25.39%) were found in the largest amounts. Canthaxanthin comprised 10-92% of the total amount ofcarotenoids. The carotenoids 4-hydroxy-3'-4'-dehydro-~-carotene, zeaxanthin and astacene were also found. These comprised only a small percentage of the total content.

263

9. Pallasea viridis (Graj.) (Table 9) This species is found on a loamy-sandy bed in all parts of Lake Baikal usually at a depth of 0-63 m. It is greenish in colour. The total carotenoid content was 76.760 #g/g raw weight. Astaxanthin ester (3.94%), canthaxanthin (25.26~o), zeaxanthin (25.55~o) and pure astaxanthin (15.79%) were identified. It was not possible to identify two fractions comprising over 28% of the total carotenoid content. 10. Eulimnogamrnarus cruentus (Dot.) (Table 10) The body length of this species is 20-23 mm and the colour is blood-red. The specimens inhabit stony beds overgrown with algae and sponges generally at a depth of 1-35 m. The analysis showed that the total carotenoid content was 33.754/~g/g raw mass. The following carotenoids were identified: y-carotene (3.38%) and

Table 6. Column chromatogram of carotenoids from ,4. albus (Graj.) No. of fraction

Eluent

I 2% Acetone in petroleum ether II 10°/0Acetone in petroleum ether III 20~ Acetone in petroleum ether IV 30~oAcetone in petroleum ether V Methanol (100°/0) VI 15~oKOH in 90~ methanol

Solvent

Maximum absorption (nm)

Partition ratio Identification

Amount (~)

Petroleum ether 466

83 : 17

Astaxanthin ester

8.27

Hexane

462

66 : 34

Astaxanthin ester

5-29

Ethanol

472

17 : 83

Astaxanthin

5.02

Ethanol

462

50 : 50

Canthaxanthin

1.95

10 : 90

Zeaxanthin

36.08

I0 : 90

Astacene

43.34

Ethanol 451,478 Carbondisulphide 450, 485, 518 Benzene 495

Table 7. Column chromatogram of carotenoids from P. eaneellus (Pall.) No. of fraction

Eluent

I Petroleum ether II 2% Acetone in petroleum ether III 20% Acetone in petroleum ether IV 30°/0Acetone in petroleum ether V 50°/0Acetone in petroleum ether VI 10~on. Propanol in acetone VII Methanol (100%) VIII Ethyl ether, cold acetic acid (20 : 1) IX 15~oKOH in 9 0 ~ o methanol

Solvent

Maximum absorption (tun)

Petroleum ether 448, 478 Petroleum ether 448, 480

Partition ratio Identification 100 : 0 80 : 20

//-Carotene Isocryptoxanthin

Amount C%) 2.76 2.71

Hexane

425, 442, 470

29 : 71

Lutein ester

17.80

Hexane

425, 444, 472

16 : 84

Lutein

15.38

Ethanol

415, 435, 466

20 : 80

Tunaxanthin

17.20

Ethanol

466

10 : 90

Astaxanthin

2-71

Ethanol Benzene

425, 451,481 470

10 : 90 5 : 95

Zenxanthin Unknown

3.48 13-29

Benzene

480

Astaxanthin-like

24-62

2 : 98

264

B. CZECZUGA Table 8. Column chromatogram of carotenoids from P. cancelloides (Gerstf.) No. of fraction I II III IV V VI VII VIII IX X XI

Solvent

Maximum absorption (nm)

Petroleum ether 2% Acetone in petroleum ether

Petroleum ether Petroleum ether

472 460

20 : 80 80 : 20

10% Acetone in petroleum ether 20% Acetone in petroleum ether 30% Acetone in petroleum ether 50% Acetone in petroleum ether Acetone (100%) 10% n. Propanol in acetone Methanol (100%) Ethyl ether, cold acetic acid (20 : 1) 15~ K O H in 9 0 ~ methanol

Petroleum ether

472

52 : 48

Astaxanthin 4-hydroxy-3',4'dehydro-/3carotene Canthaxanthin

Hexane

425,442, 470

33 : 67

Lutein ester

3.65

Hexane

466

10 : 90

Astaxanthin

3.21

Ethanol

420, 440, 470

25 : 75

Lutein epoxy

25"39

Hexane Ethanol

420, 450, 480 466

11 : 89 23 : 77

Zeaxanthin Astaxanthin

1.87 27.54

Ethanol Benzene

448,481 485

24 : 76 8 : 92

Unknown Astaxanthin

5"90 4-01

Benzene

495

10 : 90

Astacene

6.50

Eluent

Partition ratio Identification

Amount o/ (/o) 2-45 7"95 10.92

Table 9. Column chromatogram of carotenoids from P. viridis (Garj.) No. of fraction I II III IV V VI

Eluent 10~ Acetone in petroleum ether 20--30% Acetone in petroleum ether 50% Acetone in petroleum ether Methanol (100%) Ethyl ether, cold acetic acid (20 : 1) 15% K O H in 90% methanol

Solvent

Maximum absorption (nm)

Petroleum ether

466

70 : 30

Astaxanthin ester

Hexane

464

50 : 50

Canthaxanthin

Ethanol

415, 463, 495

17 : 83

Unknown

Carbondisulphide 450, 483, 516 Benzene 470

10 : 90 11 : 89

Zeaxanthin Unknown

25"55 23.89

Benzene

10 : 90

Astaxanthin

15.79

485

Partition ratio Identification

Amount (%) 3.94 25'26 4.51

Table 10. Column chromatogram of carotenoids from Eulimnogammarus cruentus (Dor.) No. of fraction I II III IV V VI VII VIII IX

Eluent 2% Acetone in petroleum ether 10% Acetone in petroleum ether 2 0 ~ Acetone in petroleum ether 30% Acetone in petroleum ether 50% Acetone in petroleum ether 10% n. Propanol in acetone Methanol (100~) Ethyl ether, cold acetic acid (20 : 1) 15~ K O H in 9 0 ~ methanol

Solvent

Maximum absorption (nm)

Partition ratio Identification

Petroleum ether

440, 465, 492

100 : 0

Petroleum ether

466

Hexane

Amount (~)

y-Carotene

3"38

51 : 49

Canthaxanthin

5.32

464

10 : 90

Astaxanthin

1'23

Hexane

424, 442, 468

30 : 70

Lutein ester

8.62

Ethanol

424, 448,472

12 : 88

Lutein

Ethanol

433, 465, 492

14 : 86

Ethanol Benzene

466 440, 462, 492

12 : 88 10 : 90

~,-Carotene derivative Astaxanthin Zeaxanthin

1.32 31.47

Benzene

485

1 : 99

Astaxanthin

34.27

11'66 2.67

Carotenoids in Garnmaridae its derivatives which were not more precisely identified (2"67~o), canthaxanthin (5-32%), astaxanthin (36"82~o), pure lutein (11"66~o) and its ester form (8.62%) and zeaxanthin which comprised 31-47% of the total amount of carotenoids.

265

(19.38%) and its ester (11.64%) and astaxanthin (51.27%) were found. In these specimens the predominant carotenoids were found to be astaxanthin and lutein.

11. Eulirrmogammarus maacki (Gerstf.) (Table 11)

12. Eulin'mogammarus grandimanus (Bazik.) (Table 12)

The body sometimes is as long as 27 mm and its colour is yellowish-green with dark stripes on the segments of its bright-red flippers. It is found in all parts of the lake at a depth of from 1 to 15 m. The total amount of carotenoids comprised 37.571/~g/g of the raw mass of the material investigated. E-Carotene (4,10%), a y-carotene derivative (6.51~o), isocryptoxanthin (3.26%), pure lutein

The body of this species is 6-8 mm in length and dark red in colour. It occurs in all parts o f Lake Bajkal at depths of 0.5-15 m on a stony bed. The chromatographic analysis showed that the total carotenoid content was 83.240/zg/g of raw mass. The following carotenoids were identified: pure lutein (12.05%) and an ester form (12.36%), zeaxanthin (3.81%), astaxanthin (13.31%) and astacene

Table 11. Column chromatogram of carotenoids from E. maacki (Gerstf.) No. of fraction

Eluent

I 2% Acetone in petroleum ether H 10,%,Acetone in petroleum ether HI 20.%oAcetone in petroleum ether IV 30% Acetone in petroleum ether V 50% Acetone in petroleum ether VI Acetone (100°/0) VII 10% n. Propanol in acetone VIII Ethyl ether, cold acetic acid (20 : 1) IX 15% KOH in 90% methanol

Solvent

Maximum absorption (rim)

Petroleum ether 425, 448,478

Partition ratio Identification 100 : 0

Amount (~.)

//-Carotene

4.10 6.51

Petroleum ether 435, 462, 490

64 : 46

Hexane

425, 448, 478

80 : 20

y-Carotenederivative Isocryptoxanthin

Hexane

425, 442, 470

46 : 54

Lutein ester

11"64

Ethanol

425, 448, 478

10 : 90

Lutein

19"38

Hexane Ethanol

425, 454, 475 445, 465, 485

61 : 39 40 : 60

Unknown Unknown

Benzene

485

9 : 91

Astaxanthin

15.29

Benzene

485

4 : 96

Astaxanthin

35.98

3.26

1.05 2.75

Table 12. Column chromatogram of carotenoids from E. grandimanus (Bazik.) No. of fraction

Eluent

I Petroleum ether II 2~o Acetone in petroleum ether III 10~oAcetone in petroleum ether IV 20% Acetone in petroleum ether V 30% Acetone in petroleum ether VI 50~oAcetone in petroleum ether VII Methanol (100~o) VIII Ethyl ether, cold acetic acid (20 : 1) IX 15~oKOH in 90OA methanol

Solvent

Maximum absorption (nm)

Partition ratio Identification

Amount (~o)

Petroleum ether 435, 462, 493 Petroleum ether 456

40 : 60 70 : 30

Gazaniaxanthin-like 2.64 Keto-carotenoid ? 6.75

Petroleum ether 466

32 : 68

Astaxanthin

Hexane

435, 462, 493

30 : 70

Gazaniaxanthin-like 1"26

Hexane

423, 442, 470

30 : 70

Lutein ester

12.36

Ethanol

424, 446, 472

11 : 89

Lutein

12.05

Ethanol Benzene

426, 451,483 485

10 : 90 4 : 96

Zeaxanthin Astaxanthin

Benzene

495

10 : 90

Astacene

5.64

3.81 7.67 47.80

266

B. CZECZUGA

(47.80~). In all probability, as shown by the chromatographic analysis, a carotenoid similar to gazanixanthin (3.90~) is peculiar to this species. In addition, a carotenoid of the fraction which appeared to be some kind of keto-carotenoid (6.75~) was found. 13. Macrohectopus branickii ( Dyb.) (Table 13) The length of this species varies between 25 and 30 ram. Unlike the above-mentioned species, M. branickii lives as a necton organism in water at the surface to a depth of 1410 m and migrates daily. Its body is almost transparent. It is the main food of the Bajkat mussel (Ko~ow, 1962). The carotenoid content was comparatively small, namely 4.545/xg/g raw mass of the material investigated. Pure astaxanthin (27.167/o) and an ester form (3.54~), isozeaxanthin (2.00~o) and astacene were identified, the last carotenoid being found to be the predominant carotenoid (51.50~). The third fraction was not identified. DISCUSSION On comparing the results obtained from all the species of Gammaride investigated, it is seen that astaxanthin occurs in all the species in amounts from 2.71 to 51.27~. In the specimens from six species, astacene in addition to astaxanthin was found to be present. This carotenoid comprised from 6.50 to 51.50~ of the total amount of carotenoids. In addition, an astaxanthin ester was found in the specimens of five species in amounts from 3.54 to 13.56~. As regards the other carotenoids, the commonest carotenoids in the material investigated were zeaxanthin, lutein and canthaxanthin. In only one species, E. maacki, was zeaxanthin or its derivative, isozeaxanthin not found. In the A. albus the

zeaxanthin content comprised 36"08~o of the total carotenoid content. The next commonest carotenoid, lutein, was found in its pure form and in the ester form. In three species it occurred in the form of lutein epoxy. Only in the species C. pachytus, Acanthogammarus, P. viridis and M. branickii was this carotenoid not found. In some cases lutein comprised 53.26~ of the total carotenoid content (M. vortex). It should also be emphasized that Lee & Gilchrist (1972) found large amounts of lutein (several per cent) in the species Idotea resecata. In six species of the Gammaridae investigated were considerable amounts of canthaxanthin (25.16~o) in P. viridis found. The presence of ycarotene or its derivatives found in certain species of the Bajkat Gammaride is worthy of note. y-Carotene was identified in M. wahli, M. vortex, G. fasciatus and E. cruentus. The derivatives of this carotenoid were found in M. wahli and in all the three species of the Eulimnogammarus genus. In E. granidimanus the y-carotene derivatives were very similar to gazanioxanthin. It should be noted that y-carotene, or its derivatives, was found in the species of the Eulimnogammarus genus whose body is yellowish-red or dark red in colour and in two species which burrow into the sand (M. wahli and G. fasciatus). In the two species of Niphargus which burrow into the sand around springs or in wells y-carotene derivatives have been found (Czeczuga & Skalski, 1973). y-Carotene has also been found in fresh-water crustaceans and in some marine species (Czeczuga & Czerpak, 1969, 1970a, b; Czeczuga, 1971, 1974). j3-Carotene was found in the specimens of only five species. In C. pachytus fl-carotene comprised 14"28~o of the total amount of carotenoids found. In the remaining four species, however, the /3carotene content varied between 0-96~o (B. lata) and 4"10~o (E. maacki). Cryptoxanthin was found only in the C. pachytus specimens (9"91~o), whereas

Table 13. Column chromatogram of carotenoids from M. branickii (Dyb.) No.

of fraction

Eluent

I Petroleum ether II 2~ Acetone in petroleum ether III 10~ Acetone in petroleum ether IV 20°,/0Acetone in petroleum ether V 30~ Acetone in petroleum ether VI 50~oacetone in petroleum ether VII Methanol (100~) VIII Ethyl ether, cold acetic acid (20 : 1) IX 15~oKOH in 90~o methanol

Solvent

Maximum absorption (nm)

Partition ratio Identification

Amount (%)

Petroleum ether 452, 475, 515 Petroleum ether 452, 485, 505

80 : 20 53 : 47

Unknown Unknown

Petroleum ether 464

26 : 74

Astaxanthin

Hexane

445,475, 495

40 : 60

Unknown

6.22

Hexane

466

Astaxanthin ester

3-54

Ethanol

468

20 : 80

Astaxanthin

11.94

Ethanol Benzene

425, 451,483 495

25 : 75 7 : 93

Isozeaxanthin Astacene

2.00 15.84

Benzene

495

Astacene

35.66

100 : 0

6 : 94

5"72 3.82 15.22

Carotenoids in Gammaridae isocryptoxanthin was found in B. lata, P. cancellus and E. maacki in comparatively small amounts (2.71-6.27%). In addition, the carotenoid 4hydroxy-4-keto-fl-carotene was found only in C. pachytus (9.03Yo), and 4-hydroxy-3-4-dehydro-flcarotene (7.95~o) in P. cancelloides. It should be noted that carotenoid 4-hydroxy-4-keto-fl-carotene is quite frequently to be found in aquatic animals. This carotenoid has been found in other crustaceans (Lee, 1966), echinoderms (de Nicola, 1959) and in several fish species (Hata & Hata, 1971; Czeczuga, 1974). As we know, the specimens of Pallasea investigated here are of a greenish shade and P. viridis is marked by its green colour. The Eulimnogammarus species taken for examination are, on the other hand, of various shades of red. It is known that such carotenoids as astaxanthin, canthaxanthin, among others, form complexes with proteins which give the crustaceans a different coloration (Cheesman et al., 1967). The carotenoids which form part of the protein carotenoid complex in the crustaceans investigated have been identified. This probably explains the colour of the Pallasea and Eulimnogammarus species, though without a doubt ycarotene and its derivatives are responsible for the reddish coloration of the Eulimnogammarus species. The carotenoids found in M. branickii specimens are also worthy of note. It is known that this species leads a plankton way of life (Bazikatowa, 1945; Ko~ow, 1962), as a result of which this crustacean has undergone a series.of adaptations. The body is extremely elongated and, above all, its transparency makes it practically unnoticeable in the water to predators. The chromatographic analysis showed only astaxanthin, astacene and isozeaxanthin to be present in this species, Isozeaxanthin comprised only 2.0% of the carotenoid content, whereas about 82% consisted of astaxanthin and astacene which we know gives crustaceans their pink colour. In the case of M. branickii astaxanthin probably forms colourless complexes with protein which makes the members of this species practically invisible in their environment. Altogether very small amounts of carotenoids were noted in the M. branickii specimens being only a few/zg/g of raw mass. Like this species, A. albus also contained comparatively small amounts of carotenoids (3.225/zg/g of raw mass), whereas in certain species of the Pallasea genus the carotenoid content reached 200/zg/g of raw mass. In the remaining species the total carotenoid content varied between 21.143 and 83.240/xg/g of raw mass. This is more or less in the range of the quantities of carotenoids found in the other species of Crustacea investigated to date. Acknowledgements--The author wishes to express his thanks to Dr. O. M. Ko~owa and Dr. G. S. Kaplina from the Biological Station of Bolshie Koty for their valuable assistance in identifying the species under investigation.

267 REFERENCES

ANDRES A. (1956a) Untersuchungen fiber die Pigments einiger Augenfarbmutanten yon Gammarus pulex L. ZooL Anz. 19, 286-291. ANDRESA. (1956b) LPoer Ausbildung und Vererbung der K6rperfarbe bei Gammarus pulex sp. subterraneus (Schneider), einer normalerweise pigmentlosen H6hlenform des gemeinen Bachitohkrebses. Z. indukt. Abstamm. Vereblerhre 87, 567-579. BAZlKALOWAA. (1945) Les Amphipodes du Bajkat. Tray. de la Station de Limnologie du Lac Baical 11, 1--440. BEATrY R. A. (1949) The pigmentation of cavemicolous animals--III. The carotenoid pigments of some amphipod Crustacea. J, expl. BioL 26, 125-130. CHEESMAND. F., LEE W. L. & ZAGALSKYP. T. (1967) Carotenoproteins in invertebrates. Biol. Rev. 42, 131160. CURL A. L. & BAILEYG. F. (1961) An improved test for carotenoid epoxides. Agric. Fd Chem. 9, 403-405. CZECZU6A B. (1970) Presence of B-carotene and a number of xanthophylls in Hyale perieri (Crustacea: Amphipoda) from the Black Sea. Mar. Biol. 6, 117120. CZECZUGA B. (1971a) Composition and tissue distribution of carotenoids and vitamin A in the crayfish Astacus leptodactylus (Esch.) (Crustacea, Decapoda). Comp. Biochem. Physiol. 39B, 945-953. CZECZUGAB. (1971b) Carotenoids in the eggs of Artemia salina. Comp. Biochem. Physiol. 40B, 47-52. CZECZUGAB. (1974) Carotenoids in fish--IV. Salmonidae and Thymallidae from Polish waters. Hydrobiologia. (In press.) CZECZUGA B. & CZERI°AK R. (1969) Pigments of the Limnadia lenticularis L. (Crustacea: Branchipoda). Comp. Biochem. Physiol. 28, 221-226. CZECZUGA B. & CZEPa'AKR. (1970a) Investigations on the pigments of Apus cancriformis (Bosc.) (Crustacea: Branchipoda). Hydrobiologia 35, 163-173. CZECZU~A B. & CZEP.rAK R. (1970b) Pigments in the crab Rhithropanopeus harrisi (Gould) subsp, tridentatus (Macland) (Crustacea: Decapoda). Int. Rev. ges. Hydrobiol. 55, 213-220. CZECZUGAB. & SKALSrdA. (1973) Presence of B-carotene and xanthophylls in two species of Niphargus (Crustacea: Amphipoda). Hydrobiologia 42, 355-362. DAVIESB. H. (1965) Chemistry and Biochemistry of Plant Pigments, pp. 489-532. Academic Press, New York. FOl~PEN F. H. (1971) Tables for the identification of carotenoid pigments. Chrom. Rev. 14, 133-298. GP,ANCAUD R. (1951) L'astaxanthine, nouveau facteur vitaminique. A. Actul. bio.-chim. 15, 336--338. HATA M. & HATA M. (1971) Carotenoid pigments in goldfish ( Carassius auratus). Int. J. Biochem. 2, 11-19. KARgERP. & LEUMANNE. (1951) Eschscholtzxanthin and anhydro-eschscholtzxanthin. Heir. chim. Acta 34, 445453. Ko~ow, M. M. (1962) Biotogia oziera Baikal. Moscow. KRINSKYN. J. & GOLDSMITHT. H. (1960) The carotenoids of the flagellated alga, Euglenagracilis. Archs Biochem. Biophys. 91, 271-279. LEE W. L. (1966) Pigmentation of the marine isopod Idothea granulosa (Rathke). Comp. Biochem. Physiol. 19, 13-27.

268

B. CZECZUGA

LEE W. L. & GILCHRISTB. M. (1972) Pigmentation, color change and the ecology of the marine isopod Idotea resecata (Stimpson). J. exp. mar. Biol. EcoL 10, 1-27. DE I'qICOLA M. G. (1959) I carotenoidi negli Astroidi: Marthasterias glacialis. La Rie, Sci. 29, 78-84. PETRACEKF. J. & ZECHMEISTERL. (1956) Determination of partition coefficients of carotenoids as a tool in pigment analysis. Analyt. Chem. 26, 1448-1485.

WIESER W. (1965) Electrophoretic studies on blood proteins in an ecological series of isopod and amphipod species. J. mar. biol. Ass. U.K. 45, 50%523. Key Word Index--Carotenoids; astaxanthin; Gammarids; Lake Bajkat; lutein; carotene; astacene; zeaxanthin; canthaxanthin.