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Carotenoid fractionation in the plumose anemone Metridium—II. Search for dietary sources of ovarian astaxanthin
Comp. Biochem. PhysioL, 1978, VoL 59B, pp. 289 to 294. Peraaraon Press. Printed in Great Britain
CAROTENOID FRACTIONATION IN THE PLUMOSE ANEMONE METRIDIUM--II. SEARCH FOR DIETARY SOURCES OF OVARIAN ASTAXANTHIN* DENIS L. FOX, DONALD W. WILKIE and FRANCIS T. HAXO Division of Marine Biology, Scripps Institution of Oceanography, University of California, San Diego, La Jo|la, CA 92093, U.S.A.
(Received 15 June 1977) Abstract--1. The Pacific Coast planktotrophic, oviparous, piumose anemone Metridium senilefimbriaturn (representing a cosmopolitan genus) stores in its developing ova long-chained esters of astaxanthin as virtually the only carotenoid. 2. The animals stored ovarian astaxanthin if fed diets containing that carotenoid itself, or canthaxanthin, but failed to develop colored otcytes if furnished diets involving only //-carotene, zeaxanthin or echinenone, or such mixed algal xanthophyils as fucoxanthin, peridinin, diatoxanthin, dinoxanthin or diadinoxanthin. 3. Thus, while they could di-hydroxylate the diketone canthaxanthin, they failed to ketonize//-carotene, zeaxanthin or even echinenone to afford astaxanthin precursors; and they failed to reduce the acetylenic, allenic or epoxy groups in the typical algal xanthophylls. 4. This anemone presumably must rely chiefly upon certain zooplankton, notably microcrustaceans, and any leptopelic detrital matter containing astaxanthin or canthaxanthin, for developing its stores of ovarian astaxanthin. It thus lacks some broader biochemical faculties exhibited by crustaceans and some fishes and birds.
INTRODUCTION
Earlier studies in these laboratories had revealed astaxanthin as the greatly preponderant carotenoid in somatic and ovarian tissues of the sea anemone Metridium senile; also, that in the adult Pacific Coast subspecies, M.s. fimbriatum, the ovarian astaxanthin is entirely esterified with long-chained fatty acids, and usually is the only carotenoid present there (Fox et al., 1967). Aware that large, adult Metridium is a preferential ingester of finely particulate food including phytoand zooplanktonic organisms, various marine ova, larvae and organic leptopelic detritus, therefore doubtless swallowing incidental carotenoids of various kinds, we wished to discover which carotenoid species might serve as dietary precursors to the stored astaxanthin esters. MATERIALS AND METHODS We hoped to simplify our approach to the problem, and to minimize the expenditure of necessary time, holding the term insofar as possible within whatever might be the period of o/Sgenesis and of favorably low ambient seawater temperatures, although we later were able to cool incoming water as the season of slowly warming trends progressed. We chose ripe or ripening females of the dominant, whitebodied genotype, taking large (ca.200-300 g) adult specimens sub-tidally from Hopkins' Reef near Monterey, California, for our experimental specimens. The sexes manifestly cannot be discerned in other intact Metridium colorvariants, wherein the seapus or column is of red or brown coloration, but in the white-bodied type, the ova, develop* Contributions from the Scripps Institution of Oceanography. 289
ing along about the upper half or two-thirds of the free septal margins within the gastrovaseular cavity, can be recognized by the soft pinkish color seen through the body wall, notably when the relaxed animal is viewed by illumination from her opposite side with a bright source of white light. In contrast, males thus inspected remain matt. Separate lots of from 5 to 8 white female specimens were maintained in large (441-1. capacity) fiber re-enforced plastic tanks, each supplied at a controlled level with 342-1. of gently flowing seawater, at ambient temperatures, i.e. from about t3 ° to 15°C during the colder months from December, 1975 into March, 1976, until, on March 20, 1976, they began to receive water cooled to temperatures between 8° and 11°C, to ensure their continued health (see below). Those anemones receiving astaxanthin were fed a seawater suspension of finely chowdered red salmon flesh, which contained free astaxanthin as the only carotenoid. Canthaxanthin was afforded by brine shrimp, ArtemM salina, which characteristically contains ca.95% of its rich carotenoid supply in the form of that compound, the remaining 5% being echinenone (see below). Rich stores of echinenone were supplied by administering finely cornminuted, ripe gonad material from the sea urchin Strongylocentrotus franciscanus, wherein this carotenoid constituted some 87% of totals, the remaining 13% being fl-carotene (this investigation). The algal carotenoids were administered by supplying seawater suspensions rich in separetely cultured, unicellular algae of two marine species, along with some chowdered white tuna flesh (itself carotenoid-free) as an additional source of protein. Of the two algal species, the chrysophyte lsochrysis oalbana contains ca.67% of its total carotenoids as fucoxanthin, with about 11% each of fl-carotene, diatoxanthin and diadinoxanthin as the remaining portions. The dinoflagellate form Gymnodinium splendens carries some 71% of its carotenoid complement as peridinin, about 22% as diadinoxanthin, and 4% dinoxanthin, along with ca.l% fl-car-
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DENIS L. FOX, DONALDW. WILKIE and FRANCIST. HAXO
otene. (See respectively Jeffrey, 1961, and Johnsen et al., 1974.) Animals receiving either/~-carotene or zeaxanthin in test diets were administered a seawater chowder of the finely comminuted white tuna flesh, to which was added the particular crystalline carotenoid, dissolved in salad oil (Wesson) and, in this, absorbed upon dry, finely porous crumbs of toasted bread or upon fine, colloidally soluble starch, to give a kind of gruel. All such gruel materials were administered from a hard plastic syringe, through a soft rubber applicator, directly upon the exposed oral disc and expanded tentacles, or by insertion into the stomodaeum of each animal, depending respectively upon its expanded, receptive condition or alternatively its somewhat contracted state. At about four-weekly or monthly intervals, animals from each dietary regime were sampled for ova (and for digestive filaments or tubules), usually by withdrawing the material via hypodermic needle of sizeable bore (No. 12:ca.2.8 mm dia) passed through the body wall into the enteron; alternatively, and less frequently, the samples were taken through an incision cut throiJgh the side of the body wall. In either case the animals soon assumed their normal posture, repaired the lesion, and fed normally, provided care had been exercised to leave the pedal disc undamaged. We had hoped to recover maximally ripe, spawned ova during whatever might be the appropriate season for this species. But we observed no spawning at any time, either in our experimental animals or in those maintained in the display aquarium. Dr Fu-Shiang Chia, of the University of Alberta tells us, however (per litteras, 1976), that he has known this oviparous animal to spawn in June at Friday Harbor, Washington, and that he suspects spawning to occur annually in early summer. We have not solved our local puzzle over the animals' failure to spawn, but collected all eggs in the manner described. An account by Reimann-Ziimeck (1975) relates her finding of well-developed ova in specimens of Metridium senile subspecies tim. briatum taken in South Atlantic waters in both summer and winter; hence it was not possible for her to assign a peak time-point of reproductive activity. Her observations of apparent perennial sexual ripeness thus were confirmed by our own early findings in the same species. For occasional quantitative purposes, the ovarian and filamentous materials were segregated, insofar as feasible, on clean, white paper-cloth, whence they were removed separately with forceps after most of any adventitious seawater, mucus and allied fluids had been fairly well absorbed. Ova and digestive filaments were then wetweighed separately and analyzed for kinds and relative quantities of carotenoids. Each weighed sample was stored in ethanol under a nitrogen atmosphere and placed in a dark refrigerator, usually for only an overnight period, until analyses could be carried out. Each sample was finely comminuted in ethanol, whether in a blender or with pure, fine sand in a mortar. The ethanol-rinsed filter bed of celite on sintered glass retained all residual, pigment-free, particulate bits of extracted tissue, while the ethanolic filtrate, containing all carotenoid material, was now diluted with sodium chloride solution to preclude emulsification, and shaken with hexane, thus extracting all carotenoids. After complete transfer of the pigment thus into hexane, the resulting solution, rinsed free of alcohol, was adjusted to suitable volume, whether by adding fresh solvent or evaporating the excess hexane by passing a gentle stream of nitrogen across the surface of the liquid at warm-bath temperature~ A Carey Model 14, or a Bausch & Lomb 505 recording spectrophotometer provided characteristic absorption profiles and showed quantities of carotenoid present. Resolution of an aliquot sample by column or thin-layer chromatrography (on MgO and Celatom, 1:1, or on pure silica) was appl!ed when necessary to separate carotenoid frac-
tions, or to confirm the presence of but a single component. CONDITIONS, VARIABLE FACTORS AND RESULTS
That all, or very nearly all, of the carotenoid stored in the ripe or ripening ova proved to be astaxanthin was confirmed by (1) its single, rounded absorption maximum centering in hexane at ca.470 nm, and (2) its consistent emergence as the only carotenoid on a chromatogram; moreover, its complete esterification with long-chained fatty acids was established repeatedly by observing the raw pigment's consistent, totally epiphasic behavior when a hexane extract was shaken with an equal volume of 95% methanol; all pigment remained in the upper, hexane layer. (3) Finally, after an alcoholic aliquot of the raw carotenoid extract had been exposed to a little N a O H , under air, while warming the system in a water bath, the generated artifact, astacene, appeared as a red or orange interfacial Na soap when the diluted alkaline alcohol digest was shaken with added hexane, preferably in the presence of some NaC1 for precluding emulsions. The upper layer of hexane and the nether stratum of aqueous alkaline alcohol then were no longer colored, or might occasionally manifest at most minute traces, usually of colloidally suspended, contaminating lipid material. Despite the fact that we were rarely dealing with more than a single carotenoid type, i.e. esterified astaxanthin, and this moreover stored only in ova (of the white-bodied, female Metridium) we had difficulties to contend with. Firstly, our specimens, gathered together from the same natural habitat and manifesting the presence of colored ova, discernible by their pink appearance when viewed through the body wall (thus serving to discriminate between the sexes) were found, nevertheless, not necessarily to be in matching stages of ripeness inter se, either as to comparable size or numbers of ova or as to relative concentrations of pigment therein. Moreover differences in degree of pigmentation associated with orcytic developmental stages, even within a given individual female, precluded our collection of an assignable representative sampling from any one animal. Hence there was no ready way whereby we could estabhsh a basic reference concentration level of stored carotenoid at the outset of feeding experiments. In the second place, ripe females were found to retain at least some pigmentation in their eggs over relatively long periods, e.g. in some observed instances for as long as 6-8 months, even when disallowed carotenoids in the diet. Granted that there resulted progressive losses in ovarian astaxanthin with the passage of time, still the overall and inconsistent circumstances called for protracted periods of experimental feeding and sampling before complete confidence might be assignable to ultimate findings. These observations reflected the relatively low metabolic rate that characterizes anthozoans in general, and at low temperatures withal. As an example, the following data are recorded from a set of white female Metridium collected on 23 October, 1974 and placed on a protein-rich but
Carotenoid fractionation in plumose anemone Metridiunv-II strictly carotenoid-free regime on 8 January, 1975; pooled samples of ova collected on the tabulated dates afforded the indicated concentrations of astaxanthin in milligrams per 100grams fresh, wet ova. Date
Elapsed days
5-1-75 5-15.75 6-10-75 6-24-75 7-14-75
112 126 153 167 187
Concentration 1.86 0.87 0.56* 0.56* 0 (ovaries degenerated)
* In each of these two measurements, a whole specimen was opened, thus sacrificed, for gathering of all ova. Because our first lot of Metridum had been maintained is seawater at La Jollan ambient temperatures, which might climb gradually to as high as 21°C in late spring and summer, i.e. levels not considered to be favorable to the species' optimum habitat, we collected, for continuing the experiments, a new lot of white female specimens from Monterey waters. These were accommodated temporarily at Sea World in San Diego until placed in our tanks in the experimental aquarium, when average temperatures were recorded as follows: Date Oct. 29-31, 1975 Nov. 1-30, 1975 Dec. 1-31, 1975. Jan. 1-31, 1976 . Feb. 1-29, 1976 . Mar. 1-20, 1976
°C . . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
16.2 15.0 13.8 13.6 14.1 14.6
Beginning on March 20, 1976, the water serving our Metridum tanks was maintained at temperatures between about 8 ° and l l ° C throughout the year. It is to this lot that our observations and data below reported refer. Due to the discovered impossibility of knowing individual females' stages of egg development, and to the fact that even from any one individual, multiple sampling could yield ova in different stages of maturity and commensurate pigmentation, we were compelled to rely primarily upon careful microscopic confirmation as to the comparative status of overall, general ovarian ripeness and pigmentation between recorded dates of inspection. Thus, eggs seen at one inspection of an animal's condition resulting from the diet, contrasting in numbers, condition and pigmentation at a later date, or even found to have vanished, afforded reliable qualitative data as to the comparable dietary effects. Moreover, the genesis of ova observed to have become free of pigment afforded sound information as to the effect of the experimental diet being consumed. And when later, on a new diet, the same animals' new oOcytes had become pigmented, this was a positive sign of a successful trial. When feasible or desirable for orientative purposes, we gathered quantitative data to indicate stages or relative richness, but we were not able to apply such numerical data in the tabular manner which we should have preferred. We had to rely largely on fairly gross differences between rich vs pale ova, or ova vs none, or in some instances, the manifestation of colorless eggs.
291
"This mode of oblique research, when a more direct one is denied, we find to be the only one in our power" [De Quincy]. Suffice it to add that these sea anemones have been the most recalcitrant animals with which one of us at least (D.L.F.~ ever has had to contend on an attempted, even approximately quantitative approach. Thus, since individuals often seemed not to be in matching stages of o0genesis or ripeness, there were occasions when we were compelled to rely upon qualitative findings in one or two animals within our experimental group. If, for example a given diet had led to ovarian degeneration, and if then a change of food were to evoke the development of colorless, otherwise normal appearing ova, in one or two members of the group, the latter diet was considered to have supported oiSgenesis while not providing the precursor pigment for the elaboration of astaxanthin. If later a revised regime were to call forth the return of red or orange-colored ova in any of such specimens, we concluded that this sufficed as positive evidence in favor of the administered carotenoid's having served as a metabolic precursor to the ovarian astaxanthin. This despite possible failure of some animals in the group to have attained exactly the same status of o6cytic development. Experimentation was carried out over a period aggregating some 752 elapsed days, i.e. 208 days of preliminary exploration, during which we discovered the apparently perennial ovigerous character of these anemones, their protracted content of o ~ y t e s in various developmental stages and pigmentary content, the relative tenacity of the stored pigment, even when the animals were denied pigmentarily usable dietary carotenoid; and latterly and revealingly, their ability under the latter condition, to generate colorless ova. The ensuing 498 days covered the period of experimental feeding of a new batch of anemones, on the varying dietary carotenoids as here recorded. And a final 46-day period was invested in the concluding test of the experimental animals' sustained ability to store esterified astaxanthin when fed once more on salmon gruel or Artemia. Following are our recorded observations: 1. fl-carotene diet: first trial. Fine seawater gruel of canned, white tuna flesh with added//-carotene in vegetable oil, adsorbed to powdered toast-crumbs and blended. 10-30-75 to 1-8-76 (71 days). An earlier observation had shown that both female and male Metridium accumulated fl-carotene in their digestive filaments, rendering these quite yellow, at concentrations of from 0.30 (~) to 0.35 (3)nag carotene per I00 g wet tissue. In this experiment also the filaments were again yellow, yielding similar concentrations of//-carotene (0.27 rag/100 g), but ova of each of two females used were now but a pale tan color, and yielded only traces of residual astaxanthin esters. There was thus no evidence of Metridium's ability to utilize//-carotene for elaborating astaxanthin. 2. Astaxanthin diet. Fine seawater brei of canned red salmon containing free astaxanthin as the sole carotenoid. 1-13-76-2-10-76 (29 days)~ Digestive filaments now salmon-colored, containing only astaxanthin, about
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DENIS L. Fox, DONALD W. WILKIE and FRANCIS T. HAXO
three-quarters of it still unesterified. One of two earlier sterile animals now storing some large, ripe ova, yielding 1.79rag astaxanthin per 100g wetweight. 3-10-76 (58 days). Four of six animals now showed colored ova, one quite rich. Salmon-colored filaments yielded astaxanthin at 0.35 mg per 100 g wet weight, mostly in free state, some esterified. 4-14-76 (93 days). By now the four ripening females showed rich ovarian pigmentation, all astaxanthin esters, showing that Metridium assimilates, esterifies and stores this carotenoid in the ova. 3. E-carotene diet: Second trial. (Diet as in first trial using powdered starch in place of toast-crumbs.) 4-14-76-4-29-76 (16 days). No ova in the two previously sterile animals, but their digestive filaments again yellow with verified E-carotene. 5-18-76 (35 days). No ova in two still sterile animals, while others still retained decreasing ovarian pigment. 8-6-76 (115 days). One of two surviving sterile animals had been sacrificed earlier for total ova search (negative). Its survivor still without ova. Remaining four now had pale to very pale ova. Conclusion: Dietary E-carotene does not support generation of astaxanthin by Metridium. 4. Zeaxanthin diet. Herein, pure zeaxanthin was substituted for E-carotene in otherwise same diet. 8-9-76-9-8-76 (30 days). Ova scantier and paler than before in four animals; still absent in the sterile one. 10-11-76 (63 days). Ova still more scanty and pale in four, while the fifth, remaining animal showed numerous ova of normal size, but all colorless. 10-29-76 (81 days). Even originally richest animal now had but few recognizable ova, very pale pinkish in color, while the earlier sterile animal still yielded colorless oteytes of various sizes. Conclusion: Metridium cannot use zeaxanthin as a metabolic precursor for ovarian astaxanthin. 5. Phytoplanktonic algal xanthophylls. (a) Heavy suspensions of purely cultured algae in seawater, with some added white tuna-brei. (a) The marine chyrysophyte Isochrysis 9albana, containing, as the prominent carotenoid, fucoxanthin, 67.2% of totals, with ca.ll% each of: diatoxanthin, diadinoxanthin and E-carotene (see Jeffrey, 1961). 11-13-76 to 12-13-76 (31 days), following zeaxanthin tests. Four of the animals still showed but very pale pink ova, many of reduced size. Final "loner" specimen still yielded but colorless ova, now without discemible nuclei. Conclusion: Neither fucoxanthin, diatoxanthin, nor diadinoxanthin may serve as metabolic precursor to the elaboration of astaxanthin in Metridiu~ 6. Canthaxanthin diet. Frozen "brine shrimp" Artemia salina, thawed daily in seawater and administered directly to anemones. Carotenoids in Artemia are but two: canthaxanthin: 95% and echinenone: 5% of total present (Krinsky, 1955, also Davies et al., cited by Fox, 1976). Our own analyses of Artemia larvae likewise afforded respective values of 96.7 and 3.3%. 12-13-76--1-13-77 (31 days). All animals now had large eggs and some new, small o/~¢ytes, all well colored and nucleated. This observation included the erstwhile white-egged specimen.
1-17-77 (36 days, now including 5 days of fasting). Similar observations. Analyzed sample of richest ova at 0.94 mg astaxanthin (all esterified) per 100 g wet weight. Conclusion: Canthaxanthin is readily utilized Metridium (see below re: echinenone results) as a precursor to the formation of esterified ovarian astaxanthin. 7. Echinenone diet. Brei of ripe female sea urchin gonads, Strongylocentrotus franciscanus, in sea water. Rich in carotenoid proportions. Mixed male and female ripe gonads had shown 87.4% echinenone and 12.6% B-carotene (see also references by Fox, 1976). 1-26-77-3-10-77 (43 days). Two specimens contained softened, wrinkled and folded ova. Of two others, one with the previously rich ova on the Artemia diet, and the other the long sterile one that finally had colored some of its white ova on the same canthaxanthin food, neither now yielded any observable ova.
Conclusion: The monoketone echinenone, in contrast to its diketonic homologue canthaxanthin, did not serve as a metabolic precursor to astaxanthin in
M etridiu~ 8. Phytoplankton algal xanthophylls. (b) Seawater and tuna brei heavy suspension of cultured dinoflagellate Gymnodinium splendens, containing, as prominent carotenoids: peridinin, 71%; diadinoxanthin, 22%; dinoxanthin, 4%, and about 1% each of two other xanthophylls and of/~-carotene (see Johansen et al., 1974). 3-15-77-4-11-77 (26 days). One animal still carried a few ova, but of tan color, without red pigment. The other four showed no ova whatever. Conclusion: neither peridinin, dinoxanthin, nor diadinoxanthin (confirmed herein) may serve as metabolic precursors to astaxanthin production in Metri-
dium. At this time we resumed administering the earlier successful sources of ovarian pigmentation, namely red salmon brei, and later larval and thawed adult
Artemia. 4-11-77-4-29-77 (18 days on salmon). All animals developed pink-orange digestive tubules and acontia. One or two now yielded ova about three-quarters developed in size (ca.250 #m) and numbers of smaller o6cytes, all pale orange-colored. Pale orange ova 300/~m were observed by 5-13-77. 5-13-77-5-27-77 (14 days on larval and some thawed adult Artemia). One animal yielded wellformed, large ova (300 #m), nucleated, pale orange color, while another also had a few light-orange colored ova, nucleated, some unattached, and some young o6cytes, all pigmented. While, after 32 days of actual feeding, these observations did not reflect a dramatic response to the administered astaxanthin, the results were positive. Metridium of the same species, may of them indeed from the same original field collection as our experimental animals, had been maintained in the public display aquarium, where they received flowing water at the cool temperatures applying to our special lot, and received a diet including Artemia larvae. A number of these were also examined for any incidence of ripe gonads. Of five examined on one occasion, following the
Carotenoid fractionation in plumose anemone Metridium--II final inspection of our experimental animals, only one yielded ripe ova. A few days later, careful scrutiny was given to six more individuals, not :one of which was observed to posses ~ther ova or sperm. The observations would suggest that the animals were in a temporary phase of minimal gametogenesis, at least in the overall site that they occupied as captives. Hence the findings applying latterly to our experimental group of five remaining specimens, wherein we encountered but two instances of o6genesis, not involving the usual rapid pigmentation, may be accepted as of positive significance regarding their continuing ability to resume storage of ovarian astaxanthin, given the appropriate precursors. It was our consistent observation, incidentally, that our Metridium specimens clearly welcomed the administration of the brine shrimp (source of metabolizable canthaxanthin) or the red salmon brei (direct source of assimilable astaxanthin) far above the five other diets. That is, the consumers often indeed were relaxed, fully expanded with tentacles extended in the food-gathering attitude, even on the morning after the previous days' feeding on shrimp or salmon. On the other diets, the animals were likely to be found often closed, sometimes even "sulking" or firmly constricted; then it was necessary to introduce the food through the stomodaeum with the soft syringe-applicator. The animals clearly showed chemical sensitivity and selectivity of an olfactory or gustatory nature. After a tank-cleaning, entailing a complete water change, the anemones remained fully or partially closed for considerable periods, expanding once more when a favorite meal was offered.
293
groups. It is a general rule that animals, unless they split certain carotenoid molecules, e.g. into vitamin A or smaller fragments, either leave them unaltered or introduce oxygen functions. But chemical reduction of carotenoids by hydrogenation must be rare indeed if it occurs at all in strictly animal metabolism. There remains, however, the possibility of its implementation by microflora endemic to animals' alimentary tracts. Reference to our earlier publication (Fox et al., 1967) will afford reminders that Metridium senile timbriatum, while storing astaxanthin as the major carotenoid by far, and storing no carotenes, may manifest at times minor proportions of esterified zeaxanthin, notably in somatic tissues of pigmented phenotypes, but occasionally in minor quantities in ovarian material, e.g. in a large, red female, and one observation indicated a minor fraction of esterified zeaxanthin in gonadal material from a white female phenotype. This single sample, however, had included not only ova, but adventitious accessory tissues, i.e. before we had developed the refinement of isolating the ova proper. There was accordingly the possibility that some digestive diverticula might have been included in the dissected material. At any rate, esterified astaxanthin constituted the only recognizable carotenoid present in the ova of any specimens analyzed in the present work. While these anemones, being specialized for capturing finely particulate food materials, must ingest many forms of planktonic organisms, as well as much organic detritus, and must profit therefrom in a general nutritional sense, the results of this investigation would strongly suggest that the animals' source of ovarian astaxanthin must reside in zooplanktonic DISCUSSION AND SUMMARY food, notably among microcrustacean forms, whether It becomes manifest, from the recorded observa- copepods, euphasiids, or eggs and larvae of even tions, that Metridium senilefimbriatum is able to assi- larger species of the same or other phyla which store milate its ovarian astaxanthin, storing it in esterified astaxanthin, canthaxanthin or both. form, only from the consumption of this carotenoid Metridium thus appears to be less biochemically initself, or from the 4, 4'-diketo-fl-carotene derivative, novative in its carotenoid metabolism than are many canthaxanthin, which it is then able to di-hydroxylate other forms, indeed in more elevated phyla including and to subsequently esterify, in the 3,3'-positions. certain crustaceans, some species of fish, and even a The animals failed to place a ketone group on the number of birds, which are able to convert various 4' carbon of echinenone, which else would have ena- carotenes or neutral xanthophylls into canthaxanthin bled them to achieve the conversion, through the and astaxanthin (see Fox, 1976, for references). resulting canthaxanthin, into astaxanthin. A concluding tabular summary follows below in The species did not introduce ketonic or hydroxyl abbreviated form. groups into t-carotene for storage. Nor did they ketonize zeaxanthin on its 4,4'-carbons to produce astaxanthin. Stored as Although phoenicoxanthin (3-hydroxy-canthaxanovarian astaxanthin? thin) was not tried as a precursor, due to paucity Carotenoid fed in diet of available supplies, there should be very little doubt No that Metridium could add thereto the remaining //-Carotene (2 trials) Astaxanthin (tried near Yes 3'-hydroxyl, as it adds both such radicals to the beginning and at end) 3-positions of canthaxanthin. Zeaxanthin No It was less surprising to find Metridium failing to Canthaxanthin (tried early Yes metabolize for storage any of the several typical algal and at end) xanthophyils, fucoxanthin, peridinin, diatoxanthin, Echinenone No dinoxanthin, or diadinoxanthin, into astaxanthin, Algal xanthophylls (mixed): Fucoxanthin since such a biochemical innovation would have Diatoxanthin required chemical reduction, i.e. by adding hydrogen Diadinoxanthin None to triple (acetylenic) or anenicaUy bonded carbon-toPeridinin carbon linkages, and/or by substituting hydrogen for Dinoxanthin oxygen in various epoxy and linearly occurring keto
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DENIS L. FOX, DONALD W. WILKIE and FRANCIST. HAXO
Living matter guards more secrets than ever it yields to sustained research. The lowly coelenterates are conspicuous examples of this. They want further pursuit of possible roles played by A-vitamins, precursors thereof, and other carotenoids (abundantly present in planktonic and other larval and adult crustaceans according to Kon and his associates, cited by Fox, 1976), in the development and pigmentation of their ova.
tration of our prescribed diets to the experimental anemones; John Blevins for help in adjusting and monitoring the cooling of incoming ambient seawater; and, for a computerized literature reference search, Jean F. Munroe, Reference Librarian at S.I.O. We appreciate also the kindness of Sea World's staff in holding our Metridium specimens in their facilities until our own were prepared for accommodating them.
Acknowled#ements--We gladly record with many thanks our appreciation of help from a number of colleagues and others, including the following: for a sample of purified, crystalline echinenone as a reference compound, Drs U. Gloor and F. Weber, of Hoffmann-La Roche & Co., Basle, from which company we had earlier received also our supplies of crystalline fl-carotene; Nancy Withers and Barbara Prezlin for conducting some of the spectrophotometric measurements; James Lance for preparing mass cultures of pure marine algal organisms used; and, for providing gonadal material from local sea urchins: Elizabeth Baker, David Nishioka, George Perry and Nick Cross, for the Strongylocentrotus purpuratus material, and Dr Mia Tegner, Mark Aeder and Raul Walker for the S. franciscanus material used; Pat Kampmann, Mark Ferguson, Carol Slette, Charles Farwell and Scott Mahr, for various kinds of help related to sometime preparation or adminis-
Fox D. L. (1976) Animal Biochromes and Structural Colours. Second and Supplemented edn. University of California Press. Fox D. L., CROZIERG. F. & SMIm V. E. (1967) Carotenoid fractionation in the plumose anemone Metridium. Comp. Biochem. Physiol. 22, 177-188. KRINSKY N. I. (1965) The carotenoids of the brine shrimp Artemia salina. Comp. Biochem. Physiol. 16, 181-187. JEFFREY S. W. (1961) Paper chromatographic separation of chlorophylls and carotenoids from marine algae. BiDchem. J. 80, 336-342. JOHANSEN J. E., SVEC W. A., LIANN-JENSENS., & HAXO F. T. (1974) Carotenoids of the Dinophyceae. Phytochemistry 13, 2261-2271. REIMANN-Zt)RNECKK..(_1975) Actiniaria des Siidwestatlantik II. Segartiidae und Metridiidae. Helg. wiss. Meeresum. 27, 70-95.
REFERENCES
CAROTENOID FRACTIONATION IN THE PLUMOSE ANEMONE METRIDIUM--II. SEARCH FOR DIETARY SOURCES OF OVARIAN ASTAXANTHIN* DENIS L. FOX, DONALD W. WILKIE and FRANCIS T. HAXO Division of Marine Biology, Scripps Institution of Oceanography, University of California, San Diego, La Jo|la, CA 92093, U.S.A.
(Received 15 June 1977) Abstract--1. The Pacific Coast planktotrophic, oviparous, piumose anemone Metridium senilefimbriaturn (representing a cosmopolitan genus) stores in its developing ova long-chained esters of astaxanthin as virtually the only carotenoid. 2. The animals stored ovarian astaxanthin if fed diets containing that carotenoid itself, or canthaxanthin, but failed to develop colored otcytes if furnished diets involving only //-carotene, zeaxanthin or echinenone, or such mixed algal xanthophyils as fucoxanthin, peridinin, diatoxanthin, dinoxanthin or diadinoxanthin. 3. Thus, while they could di-hydroxylate the diketone canthaxanthin, they failed to ketonize//-carotene, zeaxanthin or even echinenone to afford astaxanthin precursors; and they failed to reduce the acetylenic, allenic or epoxy groups in the typical algal xanthophylls. 4. This anemone presumably must rely chiefly upon certain zooplankton, notably microcrustaceans, and any leptopelic detrital matter containing astaxanthin or canthaxanthin, for developing its stores of ovarian astaxanthin. It thus lacks some broader biochemical faculties exhibited by crustaceans and some fishes and birds.
INTRODUCTION
Earlier studies in these laboratories had revealed astaxanthin as the greatly preponderant carotenoid in somatic and ovarian tissues of the sea anemone Metridium senile; also, that in the adult Pacific Coast subspecies, M.s. fimbriatum, the ovarian astaxanthin is entirely esterified with long-chained fatty acids, and usually is the only carotenoid present there (Fox et al., 1967). Aware that large, adult Metridium is a preferential ingester of finely particulate food including phytoand zooplanktonic organisms, various marine ova, larvae and organic leptopelic detritus, therefore doubtless swallowing incidental carotenoids of various kinds, we wished to discover which carotenoid species might serve as dietary precursors to the stored astaxanthin esters. MATERIALS AND METHODS We hoped to simplify our approach to the problem, and to minimize the expenditure of necessary time, holding the term insofar as possible within whatever might be the period of o/Sgenesis and of favorably low ambient seawater temperatures, although we later were able to cool incoming water as the season of slowly warming trends progressed. We chose ripe or ripening females of the dominant, whitebodied genotype, taking large (ca.200-300 g) adult specimens sub-tidally from Hopkins' Reef near Monterey, California, for our experimental specimens. The sexes manifestly cannot be discerned in other intact Metridium colorvariants, wherein the seapus or column is of red or brown coloration, but in the white-bodied type, the ova, develop* Contributions from the Scripps Institution of Oceanography. 289
ing along about the upper half or two-thirds of the free septal margins within the gastrovaseular cavity, can be recognized by the soft pinkish color seen through the body wall, notably when the relaxed animal is viewed by illumination from her opposite side with a bright source of white light. In contrast, males thus inspected remain matt. Separate lots of from 5 to 8 white female specimens were maintained in large (441-1. capacity) fiber re-enforced plastic tanks, each supplied at a controlled level with 342-1. of gently flowing seawater, at ambient temperatures, i.e. from about t3 ° to 15°C during the colder months from December, 1975 into March, 1976, until, on March 20, 1976, they began to receive water cooled to temperatures between 8° and 11°C, to ensure their continued health (see below). Those anemones receiving astaxanthin were fed a seawater suspension of finely chowdered red salmon flesh, which contained free astaxanthin as the only carotenoid. Canthaxanthin was afforded by brine shrimp, ArtemM salina, which characteristically contains ca.95% of its rich carotenoid supply in the form of that compound, the remaining 5% being echinenone (see below). Rich stores of echinenone were supplied by administering finely cornminuted, ripe gonad material from the sea urchin Strongylocentrotus franciscanus, wherein this carotenoid constituted some 87% of totals, the remaining 13% being fl-carotene (this investigation). The algal carotenoids were administered by supplying seawater suspensions rich in separetely cultured, unicellular algae of two marine species, along with some chowdered white tuna flesh (itself carotenoid-free) as an additional source of protein. Of the two algal species, the chrysophyte lsochrysis oalbana contains ca.67% of its total carotenoids as fucoxanthin, with about 11% each of fl-carotene, diatoxanthin and diadinoxanthin as the remaining portions. The dinoflagellate form Gymnodinium splendens carries some 71% of its carotenoid complement as peridinin, about 22% as diadinoxanthin, and 4% dinoxanthin, along with ca.l% fl-car-
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DENIS L. FOX, DONALDW. WILKIE and FRANCIST. HAXO
otene. (See respectively Jeffrey, 1961, and Johnsen et al., 1974.) Animals receiving either/~-carotene or zeaxanthin in test diets were administered a seawater chowder of the finely comminuted white tuna flesh, to which was added the particular crystalline carotenoid, dissolved in salad oil (Wesson) and, in this, absorbed upon dry, finely porous crumbs of toasted bread or upon fine, colloidally soluble starch, to give a kind of gruel. All such gruel materials were administered from a hard plastic syringe, through a soft rubber applicator, directly upon the exposed oral disc and expanded tentacles, or by insertion into the stomodaeum of each animal, depending respectively upon its expanded, receptive condition or alternatively its somewhat contracted state. At about four-weekly or monthly intervals, animals from each dietary regime were sampled for ova (and for digestive filaments or tubules), usually by withdrawing the material via hypodermic needle of sizeable bore (No. 12:ca.2.8 mm dia) passed through the body wall into the enteron; alternatively, and less frequently, the samples were taken through an incision cut throiJgh the side of the body wall. In either case the animals soon assumed their normal posture, repaired the lesion, and fed normally, provided care had been exercised to leave the pedal disc undamaged. We had hoped to recover maximally ripe, spawned ova during whatever might be the appropriate season for this species. But we observed no spawning at any time, either in our experimental animals or in those maintained in the display aquarium. Dr Fu-Shiang Chia, of the University of Alberta tells us, however (per litteras, 1976), that he has known this oviparous animal to spawn in June at Friday Harbor, Washington, and that he suspects spawning to occur annually in early summer. We have not solved our local puzzle over the animals' failure to spawn, but collected all eggs in the manner described. An account by Reimann-Ziimeck (1975) relates her finding of well-developed ova in specimens of Metridium senile subspecies tim. briatum taken in South Atlantic waters in both summer and winter; hence it was not possible for her to assign a peak time-point of reproductive activity. Her observations of apparent perennial sexual ripeness thus were confirmed by our own early findings in the same species. For occasional quantitative purposes, the ovarian and filamentous materials were segregated, insofar as feasible, on clean, white paper-cloth, whence they were removed separately with forceps after most of any adventitious seawater, mucus and allied fluids had been fairly well absorbed. Ova and digestive filaments were then wetweighed separately and analyzed for kinds and relative quantities of carotenoids. Each weighed sample was stored in ethanol under a nitrogen atmosphere and placed in a dark refrigerator, usually for only an overnight period, until analyses could be carried out. Each sample was finely comminuted in ethanol, whether in a blender or with pure, fine sand in a mortar. The ethanol-rinsed filter bed of celite on sintered glass retained all residual, pigment-free, particulate bits of extracted tissue, while the ethanolic filtrate, containing all carotenoid material, was now diluted with sodium chloride solution to preclude emulsification, and shaken with hexane, thus extracting all carotenoids. After complete transfer of the pigment thus into hexane, the resulting solution, rinsed free of alcohol, was adjusted to suitable volume, whether by adding fresh solvent or evaporating the excess hexane by passing a gentle stream of nitrogen across the surface of the liquid at warm-bath temperature~ A Carey Model 14, or a Bausch & Lomb 505 recording spectrophotometer provided characteristic absorption profiles and showed quantities of carotenoid present. Resolution of an aliquot sample by column or thin-layer chromatrography (on MgO and Celatom, 1:1, or on pure silica) was appl!ed when necessary to separate carotenoid frac-
tions, or to confirm the presence of but a single component. CONDITIONS, VARIABLE FACTORS AND RESULTS
That all, or very nearly all, of the carotenoid stored in the ripe or ripening ova proved to be astaxanthin was confirmed by (1) its single, rounded absorption maximum centering in hexane at ca.470 nm, and (2) its consistent emergence as the only carotenoid on a chromatogram; moreover, its complete esterification with long-chained fatty acids was established repeatedly by observing the raw pigment's consistent, totally epiphasic behavior when a hexane extract was shaken with an equal volume of 95% methanol; all pigment remained in the upper, hexane layer. (3) Finally, after an alcoholic aliquot of the raw carotenoid extract had been exposed to a little N a O H , under air, while warming the system in a water bath, the generated artifact, astacene, appeared as a red or orange interfacial Na soap when the diluted alkaline alcohol digest was shaken with added hexane, preferably in the presence of some NaC1 for precluding emulsions. The upper layer of hexane and the nether stratum of aqueous alkaline alcohol then were no longer colored, or might occasionally manifest at most minute traces, usually of colloidally suspended, contaminating lipid material. Despite the fact that we were rarely dealing with more than a single carotenoid type, i.e. esterified astaxanthin, and this moreover stored only in ova (of the white-bodied, female Metridium) we had difficulties to contend with. Firstly, our specimens, gathered together from the same natural habitat and manifesting the presence of colored ova, discernible by their pink appearance when viewed through the body wall (thus serving to discriminate between the sexes) were found, nevertheless, not necessarily to be in matching stages of ripeness inter se, either as to comparable size or numbers of ova or as to relative concentrations of pigment therein. Moreover differences in degree of pigmentation associated with orcytic developmental stages, even within a given individual female, precluded our collection of an assignable representative sampling from any one animal. Hence there was no ready way whereby we could estabhsh a basic reference concentration level of stored carotenoid at the outset of feeding experiments. In the second place, ripe females were found to retain at least some pigmentation in their eggs over relatively long periods, e.g. in some observed instances for as long as 6-8 months, even when disallowed carotenoids in the diet. Granted that there resulted progressive losses in ovarian astaxanthin with the passage of time, still the overall and inconsistent circumstances called for protracted periods of experimental feeding and sampling before complete confidence might be assignable to ultimate findings. These observations reflected the relatively low metabolic rate that characterizes anthozoans in general, and at low temperatures withal. As an example, the following data are recorded from a set of white female Metridium collected on 23 October, 1974 and placed on a protein-rich but
Carotenoid fractionation in plumose anemone Metridiunv-II strictly carotenoid-free regime on 8 January, 1975; pooled samples of ova collected on the tabulated dates afforded the indicated concentrations of astaxanthin in milligrams per 100grams fresh, wet ova. Date
Elapsed days
5-1-75 5-15.75 6-10-75 6-24-75 7-14-75
112 126 153 167 187
Concentration 1.86 0.87 0.56* 0.56* 0 (ovaries degenerated)
* In each of these two measurements, a whole specimen was opened, thus sacrificed, for gathering of all ova. Because our first lot of Metridum had been maintained is seawater at La Jollan ambient temperatures, which might climb gradually to as high as 21°C in late spring and summer, i.e. levels not considered to be favorable to the species' optimum habitat, we collected, for continuing the experiments, a new lot of white female specimens from Monterey waters. These were accommodated temporarily at Sea World in San Diego until placed in our tanks in the experimental aquarium, when average temperatures were recorded as follows: Date Oct. 29-31, 1975 Nov. 1-30, 1975 Dec. 1-31, 1975. Jan. 1-31, 1976 . Feb. 1-29, 1976 . Mar. 1-20, 1976
°C . . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
16.2 15.0 13.8 13.6 14.1 14.6
Beginning on March 20, 1976, the water serving our Metridum tanks was maintained at temperatures between about 8 ° and l l ° C throughout the year. It is to this lot that our observations and data below reported refer. Due to the discovered impossibility of knowing individual females' stages of egg development, and to the fact that even from any one individual, multiple sampling could yield ova in different stages of maturity and commensurate pigmentation, we were compelled to rely primarily upon careful microscopic confirmation as to the comparative status of overall, general ovarian ripeness and pigmentation between recorded dates of inspection. Thus, eggs seen at one inspection of an animal's condition resulting from the diet, contrasting in numbers, condition and pigmentation at a later date, or even found to have vanished, afforded reliable qualitative data as to the comparable dietary effects. Moreover, the genesis of ova observed to have become free of pigment afforded sound information as to the effect of the experimental diet being consumed. And when later, on a new diet, the same animals' new oOcytes had become pigmented, this was a positive sign of a successful trial. When feasible or desirable for orientative purposes, we gathered quantitative data to indicate stages or relative richness, but we were not able to apply such numerical data in the tabular manner which we should have preferred. We had to rely largely on fairly gross differences between rich vs pale ova, or ova vs none, or in some instances, the manifestation of colorless eggs.
291
"This mode of oblique research, when a more direct one is denied, we find to be the only one in our power" [De Quincy]. Suffice it to add that these sea anemones have been the most recalcitrant animals with which one of us at least (D.L.F.~ ever has had to contend on an attempted, even approximately quantitative approach. Thus, since individuals often seemed not to be in matching stages of o0genesis or ripeness, there were occasions when we were compelled to rely upon qualitative findings in one or two animals within our experimental group. If, for example a given diet had led to ovarian degeneration, and if then a change of food were to evoke the development of colorless, otherwise normal appearing ova, in one or two members of the group, the latter diet was considered to have supported oiSgenesis while not providing the precursor pigment for the elaboration of astaxanthin. If later a revised regime were to call forth the return of red or orange-colored ova in any of such specimens, we concluded that this sufficed as positive evidence in favor of the administered carotenoid's having served as a metabolic precursor to the ovarian astaxanthin. This despite possible failure of some animals in the group to have attained exactly the same status of o6cytic development. Experimentation was carried out over a period aggregating some 752 elapsed days, i.e. 208 days of preliminary exploration, during which we discovered the apparently perennial ovigerous character of these anemones, their protracted content of o ~ y t e s in various developmental stages and pigmentary content, the relative tenacity of the stored pigment, even when the animals were denied pigmentarily usable dietary carotenoid; and latterly and revealingly, their ability under the latter condition, to generate colorless ova. The ensuing 498 days covered the period of experimental feeding of a new batch of anemones, on the varying dietary carotenoids as here recorded. And a final 46-day period was invested in the concluding test of the experimental animals' sustained ability to store esterified astaxanthin when fed once more on salmon gruel or Artemia. Following are our recorded observations: 1. fl-carotene diet: first trial. Fine seawater gruel of canned, white tuna flesh with added//-carotene in vegetable oil, adsorbed to powdered toast-crumbs and blended. 10-30-75 to 1-8-76 (71 days). An earlier observation had shown that both female and male Metridium accumulated fl-carotene in their digestive filaments, rendering these quite yellow, at concentrations of from 0.30 (~) to 0.35 (3)nag carotene per I00 g wet tissue. In this experiment also the filaments were again yellow, yielding similar concentrations of//-carotene (0.27 rag/100 g), but ova of each of two females used were now but a pale tan color, and yielded only traces of residual astaxanthin esters. There was thus no evidence of Metridium's ability to utilize//-carotene for elaborating astaxanthin. 2. Astaxanthin diet. Fine seawater brei of canned red salmon containing free astaxanthin as the sole carotenoid. 1-13-76-2-10-76 (29 days)~ Digestive filaments now salmon-colored, containing only astaxanthin, about
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DENIS L. Fox, DONALD W. WILKIE and FRANCIS T. HAXO
three-quarters of it still unesterified. One of two earlier sterile animals now storing some large, ripe ova, yielding 1.79rag astaxanthin per 100g wetweight. 3-10-76 (58 days). Four of six animals now showed colored ova, one quite rich. Salmon-colored filaments yielded astaxanthin at 0.35 mg per 100 g wet weight, mostly in free state, some esterified. 4-14-76 (93 days). By now the four ripening females showed rich ovarian pigmentation, all astaxanthin esters, showing that Metridium assimilates, esterifies and stores this carotenoid in the ova. 3. E-carotene diet: Second trial. (Diet as in first trial using powdered starch in place of toast-crumbs.) 4-14-76-4-29-76 (16 days). No ova in the two previously sterile animals, but their digestive filaments again yellow with verified E-carotene. 5-18-76 (35 days). No ova in two still sterile animals, while others still retained decreasing ovarian pigment. 8-6-76 (115 days). One of two surviving sterile animals had been sacrificed earlier for total ova search (negative). Its survivor still without ova. Remaining four now had pale to very pale ova. Conclusion: Dietary E-carotene does not support generation of astaxanthin by Metridium. 4. Zeaxanthin diet. Herein, pure zeaxanthin was substituted for E-carotene in otherwise same diet. 8-9-76-9-8-76 (30 days). Ova scantier and paler than before in four animals; still absent in the sterile one. 10-11-76 (63 days). Ova still more scanty and pale in four, while the fifth, remaining animal showed numerous ova of normal size, but all colorless. 10-29-76 (81 days). Even originally richest animal now had but few recognizable ova, very pale pinkish in color, while the earlier sterile animal still yielded colorless oteytes of various sizes. Conclusion: Metridium cannot use zeaxanthin as a metabolic precursor for ovarian astaxanthin. 5. Phytoplanktonic algal xanthophylls. (a) Heavy suspensions of purely cultured algae in seawater, with some added white tuna-brei. (a) The marine chyrysophyte Isochrysis 9albana, containing, as the prominent carotenoid, fucoxanthin, 67.2% of totals, with ca.ll% each of: diatoxanthin, diadinoxanthin and E-carotene (see Jeffrey, 1961). 11-13-76 to 12-13-76 (31 days), following zeaxanthin tests. Four of the animals still showed but very pale pink ova, many of reduced size. Final "loner" specimen still yielded but colorless ova, now without discemible nuclei. Conclusion: Neither fucoxanthin, diatoxanthin, nor diadinoxanthin may serve as metabolic precursor to the elaboration of astaxanthin in Metridiu~ 6. Canthaxanthin diet. Frozen "brine shrimp" Artemia salina, thawed daily in seawater and administered directly to anemones. Carotenoids in Artemia are but two: canthaxanthin: 95% and echinenone: 5% of total present (Krinsky, 1955, also Davies et al., cited by Fox, 1976). Our own analyses of Artemia larvae likewise afforded respective values of 96.7 and 3.3%. 12-13-76--1-13-77 (31 days). All animals now had large eggs and some new, small o/~¢ytes, all well colored and nucleated. This observation included the erstwhile white-egged specimen.
1-17-77 (36 days, now including 5 days of fasting). Similar observations. Analyzed sample of richest ova at 0.94 mg astaxanthin (all esterified) per 100 g wet weight. Conclusion: Canthaxanthin is readily utilized Metridium (see below re: echinenone results) as a precursor to the formation of esterified ovarian astaxanthin. 7. Echinenone diet. Brei of ripe female sea urchin gonads, Strongylocentrotus franciscanus, in sea water. Rich in carotenoid proportions. Mixed male and female ripe gonads had shown 87.4% echinenone and 12.6% B-carotene (see also references by Fox, 1976). 1-26-77-3-10-77 (43 days). Two specimens contained softened, wrinkled and folded ova. Of two others, one with the previously rich ova on the Artemia diet, and the other the long sterile one that finally had colored some of its white ova on the same canthaxanthin food, neither now yielded any observable ova.
Conclusion: The monoketone echinenone, in contrast to its diketonic homologue canthaxanthin, did not serve as a metabolic precursor to astaxanthin in
M etridiu~ 8. Phytoplankton algal xanthophylls. (b) Seawater and tuna brei heavy suspension of cultured dinoflagellate Gymnodinium splendens, containing, as prominent carotenoids: peridinin, 71%; diadinoxanthin, 22%; dinoxanthin, 4%, and about 1% each of two other xanthophylls and of/~-carotene (see Johansen et al., 1974). 3-15-77-4-11-77 (26 days). One animal still carried a few ova, but of tan color, without red pigment. The other four showed no ova whatever. Conclusion: neither peridinin, dinoxanthin, nor diadinoxanthin (confirmed herein) may serve as metabolic precursors to astaxanthin production in Metri-
dium. At this time we resumed administering the earlier successful sources of ovarian pigmentation, namely red salmon brei, and later larval and thawed adult
Artemia. 4-11-77-4-29-77 (18 days on salmon). All animals developed pink-orange digestive tubules and acontia. One or two now yielded ova about three-quarters developed in size (ca.250 #m) and numbers of smaller o6cytes, all pale orange-colored. Pale orange ova 300/~m were observed by 5-13-77. 5-13-77-5-27-77 (14 days on larval and some thawed adult Artemia). One animal yielded wellformed, large ova (300 #m), nucleated, pale orange color, while another also had a few light-orange colored ova, nucleated, some unattached, and some young o6cytes, all pigmented. While, after 32 days of actual feeding, these observations did not reflect a dramatic response to the administered astaxanthin, the results were positive. Metridium of the same species, may of them indeed from the same original field collection as our experimental animals, had been maintained in the public display aquarium, where they received flowing water at the cool temperatures applying to our special lot, and received a diet including Artemia larvae. A number of these were also examined for any incidence of ripe gonads. Of five examined on one occasion, following the
Carotenoid fractionation in plumose anemone Metridium--II final inspection of our experimental animals, only one yielded ripe ova. A few days later, careful scrutiny was given to six more individuals, not :one of which was observed to posses ~ther ova or sperm. The observations would suggest that the animals were in a temporary phase of minimal gametogenesis, at least in the overall site that they occupied as captives. Hence the findings applying latterly to our experimental group of five remaining specimens, wherein we encountered but two instances of o6genesis, not involving the usual rapid pigmentation, may be accepted as of positive significance regarding their continuing ability to resume storage of ovarian astaxanthin, given the appropriate precursors. It was our consistent observation, incidentally, that our Metridium specimens clearly welcomed the administration of the brine shrimp (source of metabolizable canthaxanthin) or the red salmon brei (direct source of assimilable astaxanthin) far above the five other diets. That is, the consumers often indeed were relaxed, fully expanded with tentacles extended in the food-gathering attitude, even on the morning after the previous days' feeding on shrimp or salmon. On the other diets, the animals were likely to be found often closed, sometimes even "sulking" or firmly constricted; then it was necessary to introduce the food through the stomodaeum with the soft syringe-applicator. The animals clearly showed chemical sensitivity and selectivity of an olfactory or gustatory nature. After a tank-cleaning, entailing a complete water change, the anemones remained fully or partially closed for considerable periods, expanding once more when a favorite meal was offered.
293
groups. It is a general rule that animals, unless they split certain carotenoid molecules, e.g. into vitamin A or smaller fragments, either leave them unaltered or introduce oxygen functions. But chemical reduction of carotenoids by hydrogenation must be rare indeed if it occurs at all in strictly animal metabolism. There remains, however, the possibility of its implementation by microflora endemic to animals' alimentary tracts. Reference to our earlier publication (Fox et al., 1967) will afford reminders that Metridium senile timbriatum, while storing astaxanthin as the major carotenoid by far, and storing no carotenes, may manifest at times minor proportions of esterified zeaxanthin, notably in somatic tissues of pigmented phenotypes, but occasionally in minor quantities in ovarian material, e.g. in a large, red female, and one observation indicated a minor fraction of esterified zeaxanthin in gonadal material from a white female phenotype. This single sample, however, had included not only ova, but adventitious accessory tissues, i.e. before we had developed the refinement of isolating the ova proper. There was accordingly the possibility that some digestive diverticula might have been included in the dissected material. At any rate, esterified astaxanthin constituted the only recognizable carotenoid present in the ova of any specimens analyzed in the present work. While these anemones, being specialized for capturing finely particulate food materials, must ingest many forms of planktonic organisms, as well as much organic detritus, and must profit therefrom in a general nutritional sense, the results of this investigation would strongly suggest that the animals' source of ovarian astaxanthin must reside in zooplanktonic DISCUSSION AND SUMMARY food, notably among microcrustacean forms, whether It becomes manifest, from the recorded observa- copepods, euphasiids, or eggs and larvae of even tions, that Metridium senilefimbriatum is able to assi- larger species of the same or other phyla which store milate its ovarian astaxanthin, storing it in esterified astaxanthin, canthaxanthin or both. form, only from the consumption of this carotenoid Metridium thus appears to be less biochemically initself, or from the 4, 4'-diketo-fl-carotene derivative, novative in its carotenoid metabolism than are many canthaxanthin, which it is then able to di-hydroxylate other forms, indeed in more elevated phyla including and to subsequently esterify, in the 3,3'-positions. certain crustaceans, some species of fish, and even a The animals failed to place a ketone group on the number of birds, which are able to convert various 4' carbon of echinenone, which else would have ena- carotenes or neutral xanthophylls into canthaxanthin bled them to achieve the conversion, through the and astaxanthin (see Fox, 1976, for references). resulting canthaxanthin, into astaxanthin. A concluding tabular summary follows below in The species did not introduce ketonic or hydroxyl abbreviated form. groups into t-carotene for storage. Nor did they ketonize zeaxanthin on its 4,4'-carbons to produce astaxanthin. Stored as Although phoenicoxanthin (3-hydroxy-canthaxanovarian astaxanthin? thin) was not tried as a precursor, due to paucity Carotenoid fed in diet of available supplies, there should be very little doubt No that Metridium could add thereto the remaining //-Carotene (2 trials) Astaxanthin (tried near Yes 3'-hydroxyl, as it adds both such radicals to the beginning and at end) 3-positions of canthaxanthin. Zeaxanthin No It was less surprising to find Metridium failing to Canthaxanthin (tried early Yes metabolize for storage any of the several typical algal and at end) xanthophyils, fucoxanthin, peridinin, diatoxanthin, Echinenone No dinoxanthin, or diadinoxanthin, into astaxanthin, Algal xanthophylls (mixed): Fucoxanthin since such a biochemical innovation would have Diatoxanthin required chemical reduction, i.e. by adding hydrogen Diadinoxanthin None to triple (acetylenic) or anenicaUy bonded carbon-toPeridinin carbon linkages, and/or by substituting hydrogen for Dinoxanthin oxygen in various epoxy and linearly occurring keto
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DENIS L. FOX, DONALD W. WILKIE and FRANCIST. HAXO
Living matter guards more secrets than ever it yields to sustained research. The lowly coelenterates are conspicuous examples of this. They want further pursuit of possible roles played by A-vitamins, precursors thereof, and other carotenoids (abundantly present in planktonic and other larval and adult crustaceans according to Kon and his associates, cited by Fox, 1976), in the development and pigmentation of their ova.
tration of our prescribed diets to the experimental anemones; John Blevins for help in adjusting and monitoring the cooling of incoming ambient seawater; and, for a computerized literature reference search, Jean F. Munroe, Reference Librarian at S.I.O. We appreciate also the kindness of Sea World's staff in holding our Metridium specimens in their facilities until our own were prepared for accommodating them.
Acknowled#ements--We gladly record with many thanks our appreciation of help from a number of colleagues and others, including the following: for a sample of purified, crystalline echinenone as a reference compound, Drs U. Gloor and F. Weber, of Hoffmann-La Roche & Co., Basle, from which company we had earlier received also our supplies of crystalline fl-carotene; Nancy Withers and Barbara Prezlin for conducting some of the spectrophotometric measurements; James Lance for preparing mass cultures of pure marine algal organisms used; and, for providing gonadal material from local sea urchins: Elizabeth Baker, David Nishioka, George Perry and Nick Cross, for the Strongylocentrotus purpuratus material, and Dr Mia Tegner, Mark Aeder and Raul Walker for the S. franciscanus material used; Pat Kampmann, Mark Ferguson, Carol Slette, Charles Farwell and Scott Mahr, for various kinds of help related to sometime preparation or adminis-
Fox D. L. (1976) Animal Biochromes and Structural Colours. Second and Supplemented edn. University of California Press. Fox D. L., CROZIERG. F. & SMIm V. E. (1967) Carotenoid fractionation in the plumose anemone Metridium. Comp. Biochem. Physiol. 22, 177-188. KRINSKY N. I. (1965) The carotenoids of the brine shrimp Artemia salina. Comp. Biochem. Physiol. 16, 181-187. JEFFREY S. W. (1961) Paper chromatographic separation of chlorophylls and carotenoids from marine algae. BiDchem. J. 80, 336-342. JOHANSEN J. E., SVEC W. A., LIANN-JENSENS., & HAXO F. T. (1974) Carotenoids of the Dinophyceae. Phytochemistry 13, 2261-2271. REIMANN-Zt)RNECKK..(_1975) Actiniaria des Siidwestatlantik II. Segartiidae und Metridiidae. Helg. wiss. Meeresum. 27, 70-95.
REFERENCES