Pigments of the Sea-Anemone Isactinia olivacea (Hutton, 1878) (Coelenterata, Actinozoa)
Thin layer chromatography followed by spectrophotometry shows thirteen pigments in both the green and brown varieties of Isactinia olivacea (Hutton, 1878) collected from well illuminated, mid-tidal zone rock pools in the Island Bay area, Cook Strait. Phaeophytin, anthocyans, β-carotene and carotenoid pigments are found in both colour varieties. Colour variation is due to marked differences in density of carotenoid compounds. Two new pigments, an anthocyan and a flavone are described and named. The pigments have three functions; protective colouration, shielding against harmful effects of ultraviolet light, and in the case of flavin, contributing to basic metabolism.
Research during recent years has revealed the importance to plants and animals of biochromes. Functions such as oxygen transport, the transfer of substances between the vascular system and the tissues, as well as animal colouration have long been recognized.
The pigments described in the present paper were extracted from two colour varieties of Isactinia olivacea collected at Island Bay, Wellington, from well illuminated and exposed rock pools. The rock pools receive both morning and afternoon sun and are situated between the low and high tide marks. The anemones are always covered with water and are sheltered from strong wave action by surrounding calcareous algae. Moreover, I. olivacea is well concealed in its natural habitat, as it is usually covered with stand and small stones, except for the oral disc. Tentacles surrounding the oral disc are of similar colour and appearance to adjacent sea weed, i.e. anemones living in rock pools in which olive-green algae predominate have olive-green tentacles, while those amongst brown sea weeds have brown tentacles. The young of the green variety do not change colour to brown when transferred to a rock pool where the brown animals are found, and vice versa. Many animals have been transferred for experimentation and after almost three years there was no change in coloration.
A. Collection of material
Specimens were collected when the water level just covered the animals. If they were not tightly adhered to the rock the anemones were gently removed by separating the base from the substratum with a blunt scalpel. Otherwise it was better to chip off a piece of rock wih the animal attached.
For transferring anemones from sea-shore to laboratory, 20 to 30 animals were placed in a glass screw-topped jar about ¾ full of sea water. The anemones were transferred to an aquarium within two hours of being brought back to the laboratory, and left overnight so that all undigested food could be ejected, leaving the coelenteron empty.
B. Extraction of pigments
Ten to twenty animals were removed from the aquarium, placed on a clean towel for about 5 minutes and gently squeezed a few times to dry out sea water from page 2coelenteron and body wall. They were then ground in a mortar with acid washed sand. Anhydrous sodium sulphate was then added until the mixture became fairly dry, and lastly the solvent of one part methanol to three parts petroleum ether (40-60°C m.p.) was added. The mixture is best left to soak overnight, but 30 minutes in the dark will give fairly good results. For the extraction of actiniochrome however, glycerine was used (with freshly ground material) as the pigment extraction medium.
The solvent extract was separated from the solid debris by suction filtration through two thicknesses of Whatman No. 1 on a Buchner funnel. The residue was re-extracted by adding more solvent. The filtered extract was collected in a separating funnel and allowed to stand for 15 minutes. Two distinct layers were obtained. The upper hydrocarbon layer was of an intense brown (epiphasic pigments) and the bottom layer was green (hypophasic pigments). The two layers were separated, placed in a dessicator in the dark and allowed to evaporate to dryness at room temperature. A few drops of petroleum ether or acetone were added to the dry residues to redissolve the pigments for thin layer chromatographic separation.
C. Separation of pigments
The condensed extract of each layer was spotted separately as a line, 2.5 cm from the edge of a 20 × 10 cm glass plate coated with an 0.50 mm thick layer of Merck Silica gel.
The plate was developed with a solvent containing one part methanol to five parts petroleum ether. The standard method of ascending thin-layer chromatography was used. The plates were air dried and the coloured bands then removed and eluted with either petroleum ether, methanol, ethanol, water or acetone, etc...., depending on the solubility of the pigments. The pigments so obtained were stored in the dark because the coloured bands on the plates decolourize very rapidly in the light. The eluted pigments were chromatographed on other plates using different solvents in case they were not homogenous.
D. Identification methods
(i) Absorption spectra
Absorption curves were obtained using a Perkin Elmer Model 137 ultra-violet and visible spectrophotometer. The ultraviolet and visible regions are covered on separate quarto size charts, 190 to 390 and 350 to 750 millimicrons. Each range represents one drum revolution, and turning from one range to the other automatically switches the hydrogen and tungsten lamp sources.
(ii) Rf values
Rf. values for all the pigments were calculated and compared with those of known pigments.
The main criterion for identification was the value obtained for the absorption curves, as it is difficult to reproduce Rf. values exactly from one run to another in chromatography. No great reliance therefore was praced on the Rf. values obtained.
A. Epiphasic pigments
The upper brown hydrocarbon layer with thin-layer chromatography using five parts petroleum ether to one part methanol as solvent gave ten bands of pigments. Starting from the uppermost, these are referred to as follows: A yellow band (A) close to the front line; an orange band (A'); a yellow-pink band (A"); a light pink band (B); an intense violet-red band (B'); an orange band (C); a green band (D); page 3a pink band (E); a yellow band (E') and a greeny-yellow band (F).
Table 1 details the characteristics of these bands.
Identification of the Epiphasic Pigments
Comparison of the absorption peaks obtained for A (450 and 440 millimicrons) as mentioned above and those given by Fox (1953) suggests that band A (fig. 2) is a flavin or lyochrome. The wavelength of the absorption peak of band A' (fig. 7) suggests that this pigment is β-carotene. The nature of A" (fig. 3) is still not known. However, the peaks between 450 and 500 millimicrons and the colour of the pigment suggest that it might be a carotenoid compound. The values given by Fox (1953) for cyanidin were 510 and 269 millimicrons. The characteristics of B' (fig. 2) and its absorption peaks identify the pigment as cyanidin which is an anthocyan. Band B (fig. 1) only differs from B' by the last peak. Band B is probably also an anthocyan compound. Further work is needed, however, to fully identify this pigment. The yellow-orange colour of pigment C (fig. 4) and the absorption peaks noted earlier when dissolved in ethanol suggest that band C contains a group of two or three flavones, but this needs to be verified by further separation, purification and chemical analysis.
The absorption curve of D (fig. 6) in petroleum ether corresponds to that of phaeophytin, (660 millimicrons) as illustrated by Goodwin (1965). The green colour and the typical absorption curve show that this band is a chlorophyll (phaeophytin). By comparing the values obtained for E (fig. 4) and those of the known pigments of the two groups, anthocyans and flavonoid compounds, not all the absorption peaks correspond exactly. The value 267 millimicrons corresponds to one peak of pelargonidin. Apart from this peak pelargonidin also has peaks at 504.5; 450; 400.5 and 311 millimicrons. The range of maximum wavelength of the second peak of E is between 250-270 millimicrons. This corresponds to that of either flavones or flavonols. However the colour of the pigment, its solubility in water or cold methanol, its insolubility in fat solvents and its change of colour to green in the presence of ammonia shows that the pigment is more an anthocyan than a flavone. I have called this pigment "Isactinin" in the meantime. It could however be an incidental breakdown product of anthocyans accumulated as a result of the phytophagous nutrition of the animals.
Peaks of band E' (fig. 2) suggest that this pigment is probably a flavone compound. The peak for chrysin is 266 millimicrons, and that of quercitin 258 millimicrons. Pigment F (fig. 1) has peaks at 267 and 255 which are very near to those of the above-mentioned pigments. However, the peaks of F do not correspond exactly to any of the known pigments. This unidentified pigment which has most of the properties of flavone and is similar to quercitin and chrysin is tentatively called "olivacitin" because of its faint greenish-yellow colour.
In all previous investigations of animal pigments, most workers had to identify the pigments tentatively on the basis of absorption spectra together with any available chemical and physical characteristics. The two new pigments of bands E and F do not have all the characteristics of known pigments. However some of their properties indicate the group of compounds to which they may belong.
B. Hypophasic Pigments
The bottom layer of the extract when developed on a thin layer chromatogram showed three faint bands which decolorised very rapidly. (The solvent used was five parts petroleum ether to one part methanol). Nevertheless, one can distinguish three layers which did not move very far from the initial spot. This is to be expected page 4 page 5 because the mixture of pigments obtained was extracted in the aqueous-alcoholic layer. The three bands in ascending order are:
- Band G — greenish-yellow
- Band H — also greenish-yellow
- Band I — yellow
Identification of the Hypophasic Pigments
The individual pigments of the hypophasic layer have yet to be identified conclusively. However, some of their constituents and physical properties suggest the presence of xanthophylls. In a crude extract, xanthophylls are preferentially soluble in 90% methanol. In the presence of two immiscible solvents, petroleum ether and 90% methanol shaken in a separating funnel, xanthophylls are expected to become concentrated in the lower aqueous-alcoholic layer. The colours obtained from this lower layer also agree with those obtained by other workers for xanthophylls. If the lower layer pigments are xanthophyll, the coloration of Isactinia olivacea agrees with Goodwin's (1962) statement that in coelenterates the predominant carotenoid pigments are xanthophyll and acidic carotenoids.
From the results of MacMunn (1885) and the present experiments it seems that in some actinians the respiratory requirements of the animal are provided by pigments found mainly in its symbiotic algae.
The phaeophytin of band D in I. olivacea is a chlorophyll pigment, and symbiotic algal cells are present in large numbers in epithelial cells.
Green glycerol extracts of fresh material of Isactinia have absorption curves with peaks at 211 millimicrons in the U.V. region and 397 millimicrons in the visible range. These values however do not correspond with the characteristic peaks of the two respiratory pigments actiniochrome and actiniohaematin known to be specific to sea-anemones. In glycerol, actiniochrome has peaks at 563 and 595 millimicrons (centre at 579) and 458 and 477 millimicrons. Actiniohaematin has the characteristic absorption of a, b1, c, of cytochrome. These cannot be recognized as present in Isactinia olivacea. The absence of actiniohaematin is compensated for by other respiratory pigments.
The results also show the presence of flavins or lyochromes. These compounds are important in biological oxidation-reduction systems. Flavins are universal in plants and animals (especially riboflavin), and promote cell respiration, growth and fat absorption. In animals, flavins are derived either from symbiotic algae or from food. As Fox (1953) suggested, this class of biochrome plays an indispensable role in the basal metabolism of animals, thus explaining their presence in I. olivacea.
Band A' and A" were mentioned above as possible carotenoid compounds. Carotenoids have been found in various coelenterates by several previous workers. Therefore their presence in I. olivacea is not surprising. Extensive work on the pigments of Actinia equina by Abeloos-Parize (1926) and Fabre and Lederer (1934) demonstrated the presence of actinioerythrin, a- and b-carotenes and violerythrin. Anemonia sulcata was found to have carotenes (Elmhirst and Sharpe, 1919) mainly sulcatoxanthin. Several carotenoids were found in Metridium senile by Fox and Pantin (1941). They found ectodermal cells contained red fat droplets evenly distributed throughout the cell or nearer the cell's free surface. In I. olivacea sections do not show conspicuous fat droplets similarly placed. The presence of carotenoids in I. olivacea is here attributed to the presence of symbiotic algae. Goodwin (1962) emphasized that all carotenoids in animals are of dietary origin. He stated further that "animals", especially invertebrates, do not possess the ability to oxidize tertiary carotenoids to produce pigments which are often characteristic of the species".page 6 page 7 page 8
It was suggested by Cheesman, Lee and Zagalsky (1967) that carotenoids function either in the stabilization of protein and carotenoid, or as protective coloration; they also suggested that carotenoids play an active role in electron transport and enzymatic activity.
In the case of J. olivacea, there is no evidence of carotenoids playing an important part in the stabilization of protein and carotenoid compounds. However, further research may isolate and identify carotenoid compounds similar to that of "metridene" or "astaxanthin". This is not impossible because band A' which is not yet finally identified, could be one of these carotenoid compounds.
There is the further possibility that in I. olivacea carotenoids are used for protection against harmful effects of radiation. The anemones are found in well-illuminated rock pools, and in the laboratory they tend to move towards the light. They therefore need a layer of pigments which could form a shield and filter out harmful irradiations. With regard to the survival value of colour, I. olivacea may be classified as a predator. The close similarity of its colour to that of surrounding sea-weeds makes its presence less conspicuous to the crustaceans, worms and other small animals constituting its food. The density of the carotenoid pigments is important in this respect, because green and brown varieties of this species differ by the concentration of the two bands A' and A". For example, the green variety found in rock pools where olive-green sea-weeds are predominant, show only very faint traces of these pigments on the chromatogram. The brown variety however, has very concentrated bands of these pigments and they confer on the animal the intense brown colour that makes it virtually indistinguishable from its immediate environment.
Bands E, E' and F, although not positively identified, do give some indication of their importance in being light sensitive because their absorption curves show peaks in the ultra-violet region and no peaks in the visible range. They absorb in the wave length from 210 to 267 millimicrons. These pigments could be of vital importance to I. olivacea because the animal lives in well illuminated rock pools and it therefore needs some device for filtering the ultra-violet wavelength. As Herring (1965) stated, "the ultra-violet wavelengths are generally considered as the most biologically harmful. ..." It seems likely therefore that these pigments either protect the animal itself from excessive sunlight, or provide a filter for the protection of the symbiotic algal cells within the tissues of the animal.
The results obtained in this paper are very similar to those obtained by Strain, Manning and Hardin (1944). They made a thorough analysis of pigments of algal zooxanthellae which inhabit the tissues of the sea anemone Cibrina xanthogammica. An extract of pigments yielded, on chromatographic separation, no fewer than a dozen pigments, including chlorophyll a, a', and c, phaeophytin a-carotene and several newly described xanthophylls.
I would like to express my sincere thanks to Dr. P. M. Ralph, Zoology Department, V.U.W., for her kind encouragement and helpful discussions. I am also very grateful to Mr. M. Loper, Senior Technical Officer, and Mr. A. Hoverd for reproduction of text figures, to the Biochemistry Department for the use of the spectrophotometer, and to Mr. W. H. Johnston, Botany Department, V.U.W., for the use of the thin layer chromatography equipment. Mr. M. J. Williams, N.Z. Wildlife Service and Professor J. A. F. Garrick, V.U.W., made helpful criticisms of the manuscript and I thank them for their considerable effort.
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