Comp. Biochem. Physiol. Vol. 68B, pp. 25 to 29 0305-0491/81/0101-0025502.00/0 © Pergamon Press Ltd 1981. Printed in Great Britain

THE ROLE OF CAROTENOIDS IN THE COLOR POLYMORPHISM OF THE SEA ANEMONE,

BUNODOSOMA GRAN ULIFERA (ANTHOZOA: ACTINIARIA)

ROBERT D. LEBOEUF 1, STEVEN A. McCOMMAS t, NATHAN R. HOWE 1 and J. DAVID TAUBER 2

~University of Houston Marine Science Program, 4700 Avenue U, Galveston, TX 77550, U.S.A. and 2Department of Chemistry, McNeese State University, Lake Charles, LA 70609, U.S.A.

(Received 6 May 1980)

Abstrac t - - l . The carotenoids responsible for the column color of Bunodosoma granulifera were exam- ined.

2. Three color morphs derive their colors from various proportions of the three major carotenoid diesters of astaxanthin (2), 2'-norastaxanthin (4) and 2,2'-bisnorastaxanthin (5).

3. Population samples from Curacao and Puerto Rico differ in the frequencies of the color morphs and, consequently, in the mean proportions of the major carotenoids.

4. This is interpreted as evidence of genetic differentiation between these populations at the enzyme loci involved.

INTRODUCTION

Coloration in sea anemones is due mainly to the pres- ence of carotenoproteins and/or carotenoids (Abe- looz-Parize & Abelooz-Parize, 1926; Lederer, 1933; de Nicola & Goodwin, 1954). While polymorphism in the column color within many actiniarian species is well documented (e.g. Stephenson, 1928; Hand, 1954; Cain, 1974; Dunn, 1977), studies of the underlying qualitative and/or quantitative differences in carote- noids between color morphs of sea anemones are few (Fox & Pantin, 1941; Fox et al., 1967; West, 1979). These earlier studies that address the question of the influence of specific carotenoids on animal coloration are hindered by the use of whole animal extracts. These extracts contain not only the carotenoids involved in coloration but also the carotenoids of ingested food and.internal tissues. A further drawback to previous studies was that pooling of individuals before carotenoid analysis obscured individual differ- ences in carotenoid composition.

The objectives of the present study were (1) to examine the carotenoid composition of tissues di- rectly responsible for external coloration and (2) to compare the qualitative and/or quantitative caro- tenoid relationships between individuals of a poly- morphic species so that the chemical basis for the observed color polymorphism may be elucidated.

Bunodosoma granulifera (Leseur) seems well suited for just such a study since (1) it has been reported to show variability in column color from brick-red to brown (Duerden, 1902) and in collections from Puerto Rico and Curacao we observed animals which were subjectively classified as "dark-red", "orange-red" and "orange" morphs and (2) possession of samples from Puerto Rico and Curacao allows an examination for inter-populational differences in color polymorphism. In addition, this species lacks zooxanthellae (endo- symbiotic dinoflagellates), whose carotenoids could contaminate the samples.

25

MATERIALS AND METHODS

Specimens of Bunodosoma granulifera were collected in Cabo Rojo, Puerto Rico, in July 1979 and Awa Blanku, Curacao, in March 1980. Animals were shipped live and frozen (-60°C) immediately upon arrival at the laboratory in Galveston, Texas.

Removal of the brightly colored epidermis was ac- complished by thawing animals at room temperature and re-freezing (-60°C). After thawing again, the epidermal layer could be easily scraped off with a spatula, revealing a dull gray underlying tissue. This method allowed for the assay of those tissues directly responsible for animal color- ation without contamination by carotenoids in reproduc- tive structures or ingested food. In addition, subsequent extractions revealed that this method provided carotenoid extracts which were relatively free of the interfering lipids typically associated with whole animal extracts of marine invertebrates. After tissues were removed, they were lyophi- lized and stored at -20°C until used. The epidermis from each animal was assayed for carotenoids individually.

Quantitation of carotenoid composition of individual animals Carotenoids from a portion of the lyophilized tissue

from each animal were extracted with acetone, concen- trated under a stream of N2, separated by TLC using silica gel 0.25 mm layer thickness, (E. Merck No. 5763) devel- oped in dichloromethane--diethyl ether (96:4) = System A. After development, the separated carotenoids were quanti- fied by scanning the chromatogram at 470 nm on a densit- ometer equipped with an internal integrator (Helena Quick Scan R & D). The contribution of each carotenoid fraction was calculated by dividing that fraction's total integrated absorption by the total absorption integral for all peaks and expressing as a percentage.

Characterization of carotenoids To obtain sufficient material for carotenoid identifi-

cation, remaining tissue samples were pooled and extracted with acetone. The acetone extracts were centrifuged for 10min, the supernatant decanted and filtered through Whatmann phase-separating paper and concentrated under a stream of N2. The extracted carotenoids were

26 ROBERT D. LEBOEUF et al.

separated by preparative TLC using System A. The caro- tenoid bands were removed from the plates and further purified by TLC using silica gel 0.25 mm layer thickness, (E. Merck No. 5763) developed in hexane-acetone (7:3) = System B. Characterization of individual caro- tenoids was accomplished by adsorptive properties, co- chromatography, electronic and mass spectra. Acetylation was performed by the method described by Hertzberg & Liaaen-Jensen (1968). All solvents used were of reagent or spectra grade. Partition coefficients were determined in hexane-90% methanol according to the procedure of Petracek & Zechmeister (1956).

Visible light absorption spectra were recorded on a Gil- ford 250 Scanning Spectrophotometer. Low resolution mass spectra were recorded on a Finnigan 4023 GC/MS Spectrometer using a direct insertion probe, ionizing volt- age of 70 eV, and an electron trap current of 50/~A.

Carotenoprotein extraction Equal volumes of acetone and hexane were added to

aqueous extracts of lyophilized tissues of samples from B. granulifera. The hexane layer was filtered thru Whatmann phase-separating paper and assayed for carotenoids chromatographically and spectrophotometrically.

RESULTS

Carotenoprotein analysis

Analysis of aqueous extracts from the lyophilized epidermis of B. granulifera did not indicate the pres- ence of carotenoproteins within these tissues.

Carotenoids of the epidermis orB. granulifera

The following carotenoids were isolated from ace- tone extracts of the epidermal tissue of B. granulifera. R: values were recorded in System A and ~max in hexane.

fl,fl-carotene (1 ) - -Rf=0 .84 ; 2~,x=(423), 447, 472 nm. 1 isolated in B. granulifera could not be separated from authentic samples of 1.

Astaxanthin diester (2)--Rf = 0.73; :'max = 473 nm; m/e 726, 634, 620, 596, 594, 592, 578 (726-92-56), 564 (726-106-56); partition coefficient = 100:0. The loss of 106 and 92 mass units is typical of carotenoid mass spectra. The loss of 56 mass units is typical of com- pounds with 4, 4' keto groups and is ascribed to the loss of a C(4)-C(5) fragment (Enzell et al., 1969). From this data it is concluded that 2 is a diester of astaxan- thin.

Unidentified fraction (3)--R: = 0.63; 2max = 452 nm. The unsymmetrical nature of the absorption spectra of 3 was similar to that of the monoketone, echinenone. Co-chromatography of 3 and authentic echinenone (R: = 0.51) in System A resolved these into two bands.

2'-norastaxanthin diester (4)---R: = 0.57; "~max = (458), 481, 512nm; m/e 718, 620, 604, 578, 550 (578-28), 522 (578-56), 458 (578-92-28), 444 (578-106-28); partition coefficient = 100:0. Francis et a/. (1972) have shown that the 2'-norastaxanthin dies- ter from Actinia equina forms roserythtrin (M = 578) upon oxidation. Some oxidation of 4 during purifi- cation could account for presence of the m/e 578, 550, 522, 458 and 444 peaks in the upper mass region. Further support for this conclusion was provided by the m/e 203 ion in the spectra of 4 which is character- istic of diosphenols such as the 6-membered ring in roserythrin (Baldas et al., 1969). From the experimen-

tal evidence, we conclude that 4 is a 2'-norastaxanthin diester with similar properties to the 2'-norastaxan- thin carotenoid isolated from A. equina (Francis et al., 1972) and A. tenebrosa (Tauber et al., 1980). The ester(s) moiety of 4 was not established.

Actinioerythrin (5)--Ry = 0.42; ~'max = (466), 492, 525nm; m/e 568, 564, 536, 444 (564-92-28), 430 (564-106-28); partition coefficient = I00:0. The mass peaks at m/e 568, and 536 have also been reported for 5 isolated from A. equina and synthetically prepared actinioerythrol diacetate. The occurrence of these two peaks is attributed to the similarity between these two compounds and similar mechanisms operating in the cleavage of the ester function (Hertzberg et al., 1969). The presence of a m/e 564 peak may indicate the formation of the tetraketone, violerythrin, during mass spectrometric analysis of 5. From the polarity on TLC, the mass and electronic spectra, and failure of 5 to undergo acetylation we conclude that 5 is a 2, 2'-bisnorastaxanthin diester.

Astaxanthin monoester (6)--R: = 0.28; 2 .... = 472; partition coefficient = 95:5 (hexane-90~o metha- nol). A carotenoid with absorptive and chromato- graphic properties corresponding to 6 has also been isolated from Actinia equina and A. tenebrosa (Tauber et al., 1980).

Unidentified fraction (7)--R: = 0.24. Due to insuf- ficient material further characterization was not poss- ible.

C4o diol (8)--R: = 0.04; "~'max ~- 460 nm. Chroma- tographic properties of 8 are similar to those recorded for astaxanthin (R: = 0.04). However, the 2max is not consistent with the Amax for astaxanthin (473 nm).

Actinioerythrol (9)--R: = 0.03; -~max = (422), 488, 522. The spectral and chromatographic properties of 9 correspond to those for actinioerythrol isolated from A. equina and A. tenebrosa (Tauber et al., 1980).

Unidentified fraction (10}--R: = 0.02. The quantity of this fraction in extracts of B. granulifera precluded further characterization.

Unidentified fraction ( l l )---R: = 0.00. A strongly absorbed carotenoid such as 11 has also been extracted from A. equina and A. tenebrosa and has been assigned as having a fl,fl-carotene-tetrol struc- ture (Tauber et al., 1980). The lack of sufficient ma- terial from B. granulifera prevents further comparison.

Inter-animal variation in epidermal carotenoids

Astaxanthin diester (2), 2'-norastaxanthin diester (4) and actinioerythrin (5) are the major carotenoids of the epidermis of B. granulifera as judged by their aver- age percentages (see Table 1).

These three pigments represent on an average 71-75% of the carotenoids in the epidermis and there- fore contribute maximally to the color of an indi- vidual. Further support for this conclusion is that the color of these three carotenoids corresponds to the column color of individuals from Puerto Rico and Curacao.

Individual analysis reveals that the percentages of all carotenoids in the epidermis of B. granulifera dis- play variation. However the percentages of astaxan- thin diester (2) and actinioerythrin (5) show the greatest variability. The observed percentages of these two carotenoids in individuals from Curacao and Puerto Rico are presented in Fig. 1. The plot reveals

Color polymorphism in Bunodosoma granul~era

Table 1. Mean percentage ~r each carotenoid ~action isolated ~om Curacao (n = 22) and Puerto Rico (n = 26) samples

Mean Percent ± SEM of Carotenoids in Epidermis

Fraction Curacao Puerto Rico

i

2

3

4

5

6

7

8&9

I0 & Ii

6±0.6

46±2 8

7±1.1

8±1 3

21±3.4

1±0.5

4±0.5

2±0.2

4±0.5

9±1

25±2

4±0

12±1

34±3

i±0

2±0

4±0

8±1

27

that the proportions of 2 and 5 display interanimal variation which ranges from individuals with 69~o of their epidermal carotenoids as 2 to individuals with 70~o 5. All individuals in both populations possess 2, but some do not contain 4 or 5. Individuals which lack 4 also lack 5, and vice versa. In no instance does an individual possess 5 but lack 2 or 4.

Inter-population variation in epidermal carotenoids

The mean percentages of each carotenoid present in samples from Curacao and Puerto Rico are shown in Table 1. These data show that individuals from Cura-

cao on an average have a greater proportion of 2 than the Puerto Rico population. Conversely individuals from Puerto Rico possess a greater proportion of 5 than the Curacao population. These trends are also evident in Fig. 1.

DISCUSSION

Astaxanthin diester (2) 2'-norastaxanthin diester (4) and the 2, 2'-bisnorastaxanthin diester (-= actinio- erythrin) (5), are the major carotenoids (71-75~o of total carotenoid) found in the epidermis of specimens

70

60

50

40 (

30 o

20

I0

0 0 • ° •

., °

• ° o o

o o o o

o o ~ o

o d o

o o

I I t I I I I

1o 2o 30 40 so 6o 7o

% Actinioerythln

0

0

Fig. 1. Percentages of astaxanthin diester and actinioerythrin for individuals from Puerto Rico (0) and Curacao (O).

28 ROBERT D. LEBOEUF et al.

of B. granulifera from Curacao and Puerto Rico. Column color polymorphism in B. granulifera is due to the proportions of these three pigments present in the epidermis. Individuals classified subjectively as "dark-red" possessed a relatively large proportion of 5, which is maroon or deep red in color. "Orange" individuals yield predominantly the orange carote- noid 2. Some orange individuals possessed only 2 as the major carotenoid, indicating that they lacked the ability to form the norcarotenoids 4 and 5. Between these two extremes in column color one could recog- nize specimens subjectively classified "orange-red', but within this heterogeneous group a range of hues was encountered. For example, some individuals pos- sessed 50~ of their major carotenoid as 2 and 30~o as 5 and would be called an "orange-red'. In other indi- viduals within this group the proportions of 2 and 5 are reversed (See Fig. 1) and the individual has a "red- orange" hue. In all specimens examined the column coloration of an individual reflected the ratio of epi- dermal carotenoids.

Qualitatively the carotenoids of B. granulifera are generally consistent with those reported for other actiniarians. Astaxanthin diester (2) has been isolated as the major carotenoid from whole animal extracts of Actinostola eallosa (Upadhyay & Liaaen-Jensen, 1970) and samples of A. equina from the North Sea and Atlantic (Czygan & Seefried, 1970). Actinoeryth- rin (5) is the major carotenoid and 2 and 4 are minor carotenoids in A. equina from Norwegian waters (Hertzberg & Liaaen-Jensen, 1968; Hertzberg et al., 1969; Francis et al., 1972), A. tenebrosa (Tauber et al., 1980), and Bolocera tuediae (Upadhyay & Liaaen-Jen- sen, 1970).

Quantitative comparison between B. granulifera and these actiniarians is difficult due to the use of whole animal extracts and the pooling of individuals. Comparing the ratio of 2 to 5 for individuals in Fig. 1 with the mean relative percentages of 2 and 5 in Table 1, one can see that the pooling of individuals conceals individual variation in carotenoid propor- tions. The use of whole animal extracts in previous studies may obscure the role of certain carotenoids in column coloration because carotenoids have also been extracted from the reproductive tissue in sea anemones (Fox et al., 1967, 1978). The contribution of carotenoids from reproductive tissues to column coloration is unlikely, especially if the conversion of carotenoids is tissue specific.

The methods employed in the present study reveal that the variation in column-color in B. granulifera is principally based upon variation in the ratios of the free carotenoid esters, 2, 4 and 5.

Several different explanations for the intraspecific differences observed in the ratios of these carotenoids among individuals could be advanced. The observed differences in column color cannot be explained by differential assimilation of carotenoids from the diet, since actinioerythrin has been reported only from sea anemones. Also, previous studies have shown that the column color of animals maintained on diets with varying carotenoid content is stable (Abeloos-Parize & Abeloos-Parize, 1926; Fox & Pantin, 1941; Fox et al., 1967). It is conceivable that the proportions of carotenoids are a response to some environmental factors such as salinity or sunlight. This seems un-

likely, however, since individuals representing the whole range of values can be found living side by side on the same small rock. It seems most likely, then, that the different proportions of carotenoids seen are genetically determined. Genetic control of coloration in anemones has also been suggested from natural history and ecological studies (Dalyell, 1848; Cain, 1974; Dunn, 1977). The correspondence in color between parents and asexually-produced offspring is documented in many species of sea anemones, includ- ing Metridium senile, Haliplanella luciae, and Anthop- leura elegantissima (e.g. Hoffman, 1976; Atoda, 1954; Francis, 1973), implying genetic control.

However, actiniarians whose carotenoids have been studied previously (Metridium senile, Actinia equina, A. tenebrosa, and Epiactis prolifera) present a problem to the investigator. Because of their breeding systems, individuals of these species tend to be surrounded by others of identical coloration (Torrey, 1902; Cain, 1974; Ottaway & Kirby, 1975; Dunn, 1977). The result can be that a study which seeks to understand the variation in carotenoid composition for the species as a whole may examine only one or a very small number of genotypes. Indeed, an investigator who obtains all his samples from one locality may not even realize that individual variation exists in the species. On the other hand, analysis of gene frequency data from an electrophoretic survey of B. granulifera populations from Puerto Rico and Panama (McCommas & Lester, 1980) demonstrates that this species is a sexually reproducing outcrosser. There- fore, individuals sampled from even a small area are the products of genetic recombination and are likely to give a good indication of the variation in the species.

Genetic control of coloration imparts a greater sig- nificance to quantitation studies of carotenoids. Vary- ing proportions of carotenoids are the result of modi- fications in the carotenoid biotransformation path- way. Hertzberg & Liaaen-Jensen (1968) proposed that actinoerythrin is formed in vivo from 2 with 4 as an intermediate. Although this pathway has not been established in B. granulifera by such methods as labeled substrates or in vitro conversion, it is strongly suggested by the existence of the 2'-norastaxanthin diester in any animal possessing 5. If this proposed pathway is correct, one would expect at least two enzymes to be involved; one to convert 2 to 4, and another to convert 4 to 5. These enzymes would probably be encoded by at least two loci. Therefore, variation in carotenoid proportions among indivi- duals reflects an underlying genetic variation at these enzyme loci. Individuals possessing 2 and lacking 4 and 5 are interpreted as being incapable of producing a functional enzyme for the conversion of 2 to 4.

Because differences in carotenoid proportions re- flect genetic variation, they can serve as markers of genetic differentiation among populations. Indeed, we observe differences in carotenoid proportions between Curacao and Puerto Rico populations. This indicates that the two populations differ in allele frequencies at the enzyme loci concerned, which agrees with findings of geographic differentiation between these popu- lations for other enzyme loci, assayed electrophoreti- cally (McCommas, unpublished data).

The variation in the proportions of the carotenoid

Color polymorphism in Bunodosoma granulifera 29

dlesters of astaxanthin, 2 ' -noras taxanthin and 2,2'-bis- noras taxanth in are responsible for the observed vari- at ion in column color in individuals of B. granulifera. These pigment propor t ions are mainta ined by genetic control of the b io t ransformat ion pathway of caro- tenoids in this sea anemone. The data presented emphasize the variability in carotenoid conversion among individuals and populat ions which provides a basis for further comparat ive studies.

Acknowledgements--The authors wish to gratefully ac- knowledge the help of Charles Cutress, Dr Ingvar Kristen- sen, Frits Vaandrager and Susan Brunenmeister for their help in obtaining samples. Mass spectrometric analysis was kindly performed by Dr James E. Hudson, The University of Texas, Austin. This research was supported by Moody Foundation Grant No. 78/141. We thank the NOAA laboratories in Galveston, Texas for the use of facilities and equipment. We would like to thank Dr Alfred R. Loeblich III, Mr William Quast and Ms Barbara Riddle for assist- ance in the preparation of this manuscript.

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