Marine Biology 49, 197-202 (1978) MARINE BIOLOGY by Springer-Verlag 1978

Depth Analysis of Fatty Acids in Two Caribbean Reef Corals

P.A. Meyer=l, J.W. Porter2 and R.L Chad ]*

1Department of AtmoRdleric and Oceanic Science, The Univerd'ry of Michigan; Ann Arbor, Michigan, USA and 2Department of Zoology, Unlver=ity of Georgia; Athen=, Georgia, USA

Ab~

Total fatty acid compositions of colonies of two hermatypic, reef-building corals collected during the day-time over a depth range of 21 m were determined to assess the effect of depth-related environmental factors upon the lipid content of these organisms. No systematic changes were found, suggesting a steady-state balance be- tween algal and animal lipogenesis in these symbiotic partnerships. Stephanocoenia michelinii, a day and night feeder, contained lipids indicative of external dietary sources such as copepods, whereas Montastrea annularis, a night feeder, did not.

I nlaroduction labeled animal-tissue lipid was deacy- lated, 14C was detected only in the glyc- Hermatypic reef corals are characterized erol moiety suggesting animal synthesis by a mutualistic association between of these lipids from simpler components. host cnidarians and their dinoflagellate Recent evidence, however, suggests an symbionts. A feature of this relation- algal origin for some lipid synthesis ship is the translocation of photosyn- (Patton et al., 1977), and it has been thetic products from the algae to their speculated that coral fatty acid compo- animal parters. Carbon fixed by the al- sitions are to a large extent controlled gae can appear in the host in a variety by algal biosynthesis, and that much of of forms. To date, synthesis and move- the fatty acid content of the host ani- ment has been demonstrated for sugars, mal is derived unaltered from the zoo- primarily glucose and glycerol (Musca- xanthellae (Meyers, 1977; Patton et al., tine, 1967; Muscatine and Cernichiari, 1977). 1969; Lewis and Smith, 1971; Trench, Since fatty acids can be photosynthe- 1971a, b); amino acids, for instance tically derived, whether as direct prod- alanine or leucine (Muscatine and Cerni- ucts of translocation or indirect prod- chiari, 1969; Lewis and Smith, 1971); ucts of animal synthesis from algal com- proteins (Young et al., 1971); chitin ponents, their production might be a (Young et al., 1971) ; and lipids, primari- function of depth-related changes in ly triglycerides (Muscatine and Cerni- light intensity or spectral quality. chiari, 1969; Young et al., 1971; Patton Other parameters show such trends. For et al., 1977). Von Holt and Von Holt instance, with increasing depth, light- (1968) demonstrated with the Caribbean controlled, stable carbon isotopic compo- coral Scolymia lacera (Pallas) that roughly sitions of Caribbean reef corals become one-half of all CO 2 fixed during photo- progressively lighter in 13C in both tis- synthesis and translocated into water- sues (Land et al., 1975) and in the car- soluble or alcohol-soluble extracts is bonate skeletons (Weber et al., 1976). located in the lipid fraction. This Further, colonies of the Pacific coral amount fixed into animal lipid repre- Pavona praetorta (Dana) from depths of 10 sents almost 20% of all the CO 2 fixed and 25 m show distinctive oxygen produc- during photosynthesis. Muscatine and tion and consumption rates indicative of Cernichiari (1969) found that when this physiological adaptations to their indi- vidual light regimes (Wethey and Porter, *Present address: Department of Biology, Uni- 1976a, b). These photoadaptation pat- versity of South Florida, Tampa, Florida 33612, terns reoccur in the Caribbean coral Mon- USA. tastrea annularis (Ellis and Solander) from

0025-3162/78/0049/0197/S01.20 198 P.A. Meyers et al. : Depth Analysis of Coral Lipid

Jamaica (Davies, 1977; Wethey and Porter, plankton for lipid analysis for periods in preparation). This species also shows up to 9 months (Morris, 1972). a close correlation between ambient Water temperature was measured at light conditions and skeletal morphology each collection location and was found (Graus and Macintyre, 1976). to be isothermal at 26.O~ over the en- tire depth range. Salinity was deter- In view of these observed depth- mined by taking samples of water from related variations in coral composition, each depth back to the laboratory, for we were interested in determining if measurement with an American Optical Com- there existed changes in coral fatty pany Refractometer Salinometer. A small acid content with increasing depth. If salinity increase of 2~ was found in the such changes were found, they might in- top 14 m. At depths below this, salinity dicate variations in the dependence of did not vary from 35~. coral on an algal source of coral lipids, Total fatty acids in the combined cor- and hence an increasing or decreasing al and algal tissue were prepared for dependence on heterotrophically derived analysis by the procedure of Meyers et al. food over depth ranges of decreasing (1974), as modified by Meyers (1977). Ex- light supply. Differences in total fatty tracted fatty acids were converted to acid composition might also indicate their methyl esters as described by variations in the amount of translocated Meyers et al. (1974) using a procedure photosynthates with depth. adapted from that of Metcalfe et al. (1966). Analysis of these esters was by gas-liquid chromatography (Meyers, 1977). Materiels and Methods Individual components of the total fatty acid composition were identified by com- Coral samples from the seaward edge of parison of their retention times to the fringing-barrier reef at Discovery those of authentic standards. Replicate Bay, Jamaica, were hand-collected be- analyses yielded coefficients of varia- tween 10.OO and 13.OO hrs using SCUBA in tion of less than 3%. March, 1976. Small pieces of colonies of two hermatypic species, Montastrea annula- ris and Stephanocoenia michelinii (Milne- Results Edwards and Haime), were obtained at depths ranging from 3 to 24 m. S. ann~a- Fatty acid compositions for the 9 indi- ris was one of the first scleractinian vidual tissue samples of Montastrea annu- corals in which the movement of photosyn- laris from various depths are listed in thetically fixed carbon was experimental- Table I. Single samples from 4 entirely ly demonstrated (Goreau and Goreau, separate colonies of M. ann~aris were col- 1960), and, as cited above, has been the lected at 9 m in order to assess vari- object of a variety of physiological ability between colonies of this species studies, many of them being conducted at from one depth. The most abundant compo- Discovery Bay. It is a major frame build- nent in the composition of these 4 repli- er, with colonies at Discovery Bay oc- cate samples from 9 m is palmitic acid curring from sea level to 80 m (Goreau (16:O) with a mean weight percent of 61% and Wells, 1967). It is a capable plank- and a coefficient of variation of 11.3% ton feeder, expanding at night and con- of the mean. The second-most abundant tracting during the day (Porter, 1974). fatty acid is oleic (18:1). Its mean s. michelinii is similar in many respects contribution is 11.7% in these 4 samples, in size, overall shape, and depth range and its coefficient of variation is on the reef, but differs in position on 15.O% of the mean. The other major acids, the substrate and in activity periods. defined as contributing I% or more to S. mi~alinii generally grows closer to the to~al composition, show considerable the substrate, often in the understory variability. The data from these 4 sam- layer with numerous branching coral spe- ples support the findings of Meyers et al. cies overtopping it. It too is a capable (in preparation) from analysis of 8 planktivore, but most colonies are ex- replicate samples of Manicina areolata (Lin- panded fully both day and night instead naeus). Acids comprising 20% or more of of just at night. The result of this be- the total composition have considerably havior is that it is probably feeding less variability than lesser components, continuously on ambient plankton sup- and such acids may be useful in compara- plies. tive studies of the type reported here. The samples were frozen immediately No systematic change with depth of at -20~ and remained frozen until anal- collection was observed in the total fat- ysis was started in July 1976. Freezing ty acid composition of the samples of at -30~ has been shown to be a satis- Montastrea annularis analyzed in this study. factory method for preservation of zoo- Furthermore, there appeared to be no sig- P.A. Meyers et al.: Depth Analysis of Coral Lipid 199

Table i. Montastrea annularis. Fatty acid weight percent compositions of colonies from various depths

Major acid Depth (m) 3 6 9 9 9 9 12 18 24

Myristic (14:O) 2.5 3.8 1.0 0.8 2.4 1.6 2.5 0.9 1.8 Palmitic (16:O) 65.4 62.5 59.6 52.0 64.5 67.8 66.4 72.3 70.3 Palmitoleic (16:1) 4.2 5.1 4.3 0 0.9 2.8 4.8 2.6 0 Stearic (18:0) 12.2 8.2 2.7 18.5 5.5 6.6 9.4 12.1 18.8 Oleic (18:1) 8.8 12.4 11.6 12.5 9.3 13.4 10.3 7.8 7.0 Linoleic (18:2) i.i 1.6 2.5 0.3 3.1 1.9 i.i 0.6 0.6 Arachidic (20:0) 0.i 0.5 0 0.5 O 0 0.5 1.0 O Eicosenoic (20:1) 0.i O O 2.4 0 0 2.0 2.8 1.5 Docosahexaenoic (22:6) O 2.2 2.6 O 3.1 0 O O 0

Table 2. Stephanocoenia michelinii. Fatty acid weight percent compositions of colonies from various depths

Major acid Depth (m) 3 6 9 12 14 18 24

Myristic (14:O) 03 1.9 0.6 2.6 1.0 1.5 1.0 Palmitic (16:O) 6 9 43.2 54.9 68.3 29.3 50.7 52.6 Palmitoleic (16:1) 0 5 3.0 O 2.9 1.8 2.1 2.1 Stearic (18:O) 26 9.7 19.1 11.6 7.6 13.9 15.3 Oleic (18:1) 21 7.8 15.3 8.4 18.9 17.4 15.8 Linoleic (18:2) O4 2.5 i.i 2.1 1.5 3.0 1.9 Arachidic (20:0) 06 3.7 4.4 2.0 2.0 5.4 4.5 Eicosenoic (20:1) O.5 O 4.8 2.1 5.9 5.9 4.0 Docosahexaenoic (22:6) 82.8 16.4 0 0 15.0 0 0

nificant difference between the shal- The low level of polyunsaturated fatty lowest and the deepest samples. The mean acids in most of these samples agrees palmitic acid composition of samples with other reports of low levels or ab- from all depths is 64.5%, with a coeffi- sence of such acids in stoney corals cient of variation of 9.5%. This is es- (Pasby, 1965; Meyers et al., 1974; Patton sentially the same as the 4 replicate et al., 1977). However, some corals have samples from 9 m. Similarly, oleic acid been found to contain relatively large has a mean composition of 10.3% of the amounts of polyunsaturation (Meyers, mean. Other minor components are more 1977; Sassen, 1977). It is possible that variable, both within depths as well as some loss of polyunsaturated acids may between depths. have occurred during desiccation of the Fatty acid compositions of the 7 sam- coral tissues or during thin-layer chro- ples of Stephanocoenia michelinii in Table 2 matography (TLC) isolation of fatty acid show more variability than those of Mon- methyl esters. Nichaman et al. (1963) tastrea annularis, but also show only minor, have observed losses of polyunsaturated non-significant depth-related trends, acids during TLC visualization by iodine Palmitic acid is again the major fatty vapors, and Schultz and Quinn (1977) acid in most of the samples, and aver- have determined that 19% of the 22:6 fat- ages 44% of the total acids versus 64.5% ty acid can be lost by this method. Al- for M. annularis. However, the shallowest though iodine was the visualizing agent sample contains a low percentage of this in the present study, exposure to vapors acid. Its composition is dominated was kept brief to minimize loss of poly- (82.8%) instead by the marine-type, poly- unsaturated components. Furthermore, the unsaturated docosahexaenoic acid (22:6), overall procedure used here has been which also appears in sizeable amounts able to detect substantial amounts of in the samples of this species collected polyunsaturated acids and hydrocarbons from depths of 6 and 14 m but not from in a previous study (Meyers, 1977). any of the other depths. This acid is Therefore, while some losses may have present in some of the M. annularis sam- been encountered, it is not likely that ples only as a minor component (<3.1%). they would have been complete, and the 200 P.A. Meyers et al. : Depth Analysis of Coral Lipid low level of polyunsaturated acids found changes in translocated lipids could be in these samples is probably real. determined. It is known that lipid mate- Because docosahexaenoic acid has been rials such as glycerol (Muscatine, 1967; previously detected in substantial Muscatine and Cernichiari, 1969; Young amounts in only 8 out of 28 coral spe- et al., 1971) and fatty acids (Patton et cies studied, not including Stephanocoenia al., 1977) are transferred from zooxan- michelinii (Meyers, 197~), we were curi- thellae to their hosts. It is further ous to see the effect of omittingthis speculated (Patton et al., 1977) that fat- acid from our tabulated data. When the ty acids contributed by zooxanthellae compositions of s. michelinii in Table 2 are incorporated largely intact into the were recalculated without docosahexaeno- host-animal tissue. Because of distinc- ic acid, the coefficients of variation tive differences between animal and of palmitic, stearic (18:O), and oleic plant fatty acids (Gunstone, 1967), the acids were reduced by half. This implies total fatty composition of the algal- that these three acids may comprise the animal association should show depth- bulk of the coral fatty acid composition, related changes. These could be the re- whereas docosahexaenoic acid may be a sult of a decrease in the amount of al- dietary component still in the process gal biomass relative to animal material of being digested, or that the presence or due to a reduction in algal lipogene- of this acid is due to some other spe- sis. Both of these possibilities could cial circumstances, accompany the reduced light levels en- countered with increasing depth. Either possibility would cause a depth-related Di~n~, shift to decreasing amounts of algal- like saturated fatty acids and a rela- It is interesting that no systematic tive increase in amounts of animal-like depth trend was found in these determina- polyunsaturated acids. tions of total fatty acids in corals. However, fatty acid compositions of Weber et ~. (1976) found important depth- Montastreaannularis in Table I are not af- related variations in stable carbon iso- fected by depth between 3 and 24 m. tope ratios to occur in skeletal carbon- Therefore, it must be assumed either ate of Montastrea annularis samples from St. that coral metabolism is in fine balance Croix, U.S. Virgin Islands. These changes with algal photosynthetic rates and thus resulted in a steady decrease in 13C con- decreases with depth, or that the coral tent from surface samples to samples col- animal is altering translocated lipids lected at a depth of 18 m. Between 19 for its own use. Both these assumptions and 22 m, a discontinuity in the 13C:12C have interesting implications. The first ratio was found which was associated with implies that coral metabolic rates are morphological changes in M. annularis skel- related to depth. Indirect evidence ex- eton that also occur at this depth. In ists to support this possibility. Davies contrast, our fatty acid data from this (1977) found that oxygen consumption same species show no differences over this rates per unit area of tissue in samples critical depth range. This can be inter- of M. annularis from 40 m at Discovery preted to mean that skeletal morphology Bay, Jamaica, were half those in samples has no facile correlation with the total from 2 m. Therefore, it is possible that fatty acid composition of hermatypic metabolism and tissue growth rate also coral tissue. decrease with depth. The second assump- However, analysis of 13C:12C ratios tion implies that fatty acids synthe- in whole tissues of Montastrea annularis sized de novo or transformed from algal and of separated extracts of its endo- lipids by the coral animal are predomi- symbiotic algae by Land et al. (1975) nantly saturated, plant-like materials. showed that, while both total tissue and This means that coral animals have excep- algal isotope ratios were very similar, tional lipogenesis schemes because most no depth-related changes occurred above marine animals are characterized by poly- 40 m in samples from the same Jamaican unsaturated lipids (Youngken and Shimizu, site we sampled. The amount of 13C in 1975). tissues decreased in deeper samples, sim- Three of the Stephanocoenia michelinii ilar to skeletal 13C, but not until a samples in Table 2 contained substantial depth of 40 m was reached and exceeded. amounts of the marine-type polyunsatu- Therefore, it is possible that changes rated docosahexaenoic acid. This acid is in fatty acid composition should not be found in large percentages in many ma- expected until samples from greater rine animals, including copepods (Jef- depths than the 24 m reached in our col- fries, 1970; Morris, 1971). It is pos- lection are analyzed. sible that these samples may have compo- One of the objectives of this study sitions which give chemical evidence of was to determine if depth-related their last prey. As stated, the samples P.A. Meyers et al. : Depth Analysis of Coral Lipid 201

were collected between 10.00 and 13.00 biotic zooxanthellae. Limnol. Oceanogr. 20, hrs, a time period when Montastrea annula- 283-287 (1975) ris does not typically feed but s. miche- Lewis, D.H. and D.C. Smith: The autotrophic nu- linii does. Along this depth transect in trition of symbiotic marine coelenterates Discovery Bay, copepods constitute over with special reference to hermatypic corals. 90% of the planktonic individuals avail- I. Movement of photosynthetic products be- able for capture both day and night (Por- tween the symbionts. Proc. R. Soc. (Set. B) ter, in preparation). These prey are di- 178, 111-129 (1971) gested in less than 3 h (Porter, 1974), Metcalfe, L.D., A.A. Schmitz and J.R. Pelka: but assimilation rates for the products Rapid preparation of fatty acid esters from of digestion are not known. It seems un- lipids for gas chromatographic analysis. likely that recent prey items could ac- Analyt. Chem. 38, 514-515 (1966) count for the high percentage composi- Meyers, P.A.: Fatty acids and hydrocarbons of tion of the 22:6 fatty acid (nearly 83%), Caribbean corals. Proc. int. Symp. coral and it seems necessary to postulate some Reefs I, 529-539 (1977). (Third; Ed. by D.L. means of accumulation or manufacture to Taylor. Miami: University of Miami) account for so large an amount. It is -, J.G. Quinn and N. Marshall: A method for the possible that the presence of substan- analysis of fatty acids in coral. Limnol. tial amounts of this polyunsaturated Oceanogr. 19, 846-848 (1974) acid in the tissues of s. michelinii is Morris, R.J.: Comparison of the composition of due to partial alteration of dietary oceanic copepods from different depths, comp. lipids and accumulation of these prod- Biochem. Physiol. 40B, 275-281 (1971) ucts as well as recent evidence of diet. - The preservation of some oceanic animals for If this is true, then corals that active- lipid analysis. J. Fish. Res. Bd Can. 29, ly feed both night and day should have 1303-1307 (1972) lipid compositions which are more re- Muscatine, L.: Glycerol excretion by symbiotic lated to those of their prey than corals algae from corals and Tridacna and its con- which feed only at night. trol by the host. Science, N.Y. 156, 516-519 (1967) - and E. Cernichiari: Assimilation of photosyn- Acknowledgements. This research was supported thetic products of zooxanthellae by a reef by a grant from the Scott Turner Memorial Fund coral. Biol. Bull. mar. biol. Lab., Woods of The University of Michigan to P.A. Meyers, Hole 137, 506-523 (1969) and National Science Foundation Grant 0CE- Nichaman, M.Z., C.C. Sweeley, N.M. Oldham and R. 7726781 to J.W. Porter. Contribution No. 251 of E. Olson: Changes in the fatty acid composi- the Department of Atmospheric and Oceanic tion during preparative thin-layer chromatog- Science, and No. 168 of the Discovery Bay Marine raphy. J. Lipid Res. 4, 484-485 (1963) Laboratory, University of the West Indies. Pasby, B.F.: A characterization of the lipids of the organisms which make up the main bulk of a coral reef with main emphasis on the hydro- Literature Cited carbons, 149 pp. Ph.D. thesis, Texas A&M Uni- versity 1965 Davies, P.S.: Carbon budgets and vertical zona- Patton, J.S., S. Abraham and A.A. Benson: Lipo- tion of Atlantic reef corals. Proc. int. Symp. genesis in the intact coral Pocillopora capi- coral Reefs I, 391-396 (1977). (Third; Ed. by tata and its isolated zooxanthellae: evidence D.L. Taylor. Miami: University of Mia/ni) for a light-driven carbon cycle between sym- Goreau, T.F. and N.I. Goreau: Distribution of biont and host. Mar. Biol. 44, 235-247 (1977) labeled carbon in reef-building corals with Porter, J.W.: Zooplankton feeding by the Carib- and without zooxanthellae. Science, N.Y. 131, bean reef-building coral Montastrea caver- 668-669 (1960) nosa. Proc. int. Symp. coral Reefs 1, 111-125

- and J.W. Wells: The shallow-water Sclerac- (1974). (Second; Brisbane: Great Barrier Reef tinia and their vertical distribution range. Committee) Bull. mar. Sci. 17, 442-453 (1967) Sassen, R.: The fate of fatty acids from corals Graus, R.R. and I.G. Macintyre: Light control of and mangroves in Holocene sediments of St. growth form in colonial reef corals: computer Croix: significance with respect to petroleum simulation. Science, N.Y. 193, 895-897 (1976) genesis. Proc. int. Symp. coral Reefs 2, 135- Gunstone, F.D.: An introduction to the chemistry 141 (1977). (Third; Ed. by D.L. Taylor. Miami: and biochemistry of fatty acids and their University of Miami) triglycerides, 2nd ed. 209 pp. London: Chap- Schultz, D.M. and J.G. Quinn: Note on the chro- man & Hall Ltd. 1967 matographic analyses of marine polyunsatu- Jeffries, H.P.: Seasonal composition of temper- rated fatty acids. Mar. Biol. 40, 117-120 ate plankton communities: fatty acids. Limnol. (1977) Oceanogr. 15, 419-426 (1970) Trench, R.K.: The physiology and biochemistry of Land, L.S., J.C. Lang and B.N. Smith: Prelimi- zooxanthellae symbiotic with marine co- nary observations on the carbon isotopic com- elenterates. I. Assimilation of photosynthet- position of some reef coral tissues and sym- ic products of zooxanthellae by two marine 202 P.A. Meyers et al.: Depth Analysis of Coral Lipid

coelenterates. Proc. R. Soc. (Ser. B) 177, ratio of skeletal carbonate deposited by the 225-235 (1971a) Caribbean reef-frame building coral Monta- The physiology and biochemistry of zooxan- strea annularis: further implications of a thellae symbiotic with marine coelenterates. model for stable isotope fractionation by II. Liberation of fixed 14C by zooxanthellae sceleractinian corals. Geochim. cosmochim. in vitro. Proc. R. Soc. (Ser. B) 177, 236-250 Acta 40, 31-39 (1976) (1971b) Wethey, D.S. and J.W. Porter: Sun and shade dif- Von Holt, C. and M. Von Holt: Transfer of photo- ferences in productivity of reef corals. Na- synthetic products from zooxanthellae to co- ture, Lond. 262, 281-282 (1976a) elenterate hosts. Comp. Biochem. Physiol. 24, - - Habitat-relatedpatterns of productivity of 73-81 (1968) the foliaceous reef coral, Pavona praetorta Young, S.D., J.D. O'Conner, and L. Muscatine: Dana. In: Coelenterate ecology and behavior, Organic material from scleractinian coral pp 59-66. Ed. by G.O. Mackie. New York: Ple- skeletons. II. Incorporation of 14C into pro- num Publishing Corp. 1976b tein, chitin, and lipid. Comp. Biochem. Physiol. 40B, 945-958 (1971) Youngken, H.W. and Y. Shimizu: Marine drugs: chemical and pharmacological aspects. In: Dr. Philip A. Meyers Chemical oceanography, 2nd ed., Vol. 4, PP Department of Atmospheric and Oceanic Science 269-317. Ed. by J.P. Riley and G. Skirrow. The University of Michigan London: Academic Press 1975 2455 Hayward Avenue Weber, J.N., P. Deines, P.H. Weber and P.A. Ann Arbor, Michigan 48109 Baker: Depth related changes in the 13C/12C USA

Date of final manuscript acceptance: June 30, 1978. Communicated by M.R. Tripp, Newark