Skeletal fatty acids in fish from different depths off Jamaica

Comp. Biochem, Physiol.Vol. 94B, No. 2, pp. 329-334, 1989 Printed in Great Britain

0305-0491/89 $3.00 + 0.00 © 1989 Pergamon Press plc

SKELETAL FATTY ACIDS IN FISH FROM DIFFERENT DEPTHS OFF JAMAICA C. F. PHL~GER* and R. J. LAUBt *Department of Natural Science and fDepartment of Chemistry, San Diego State University, San Diego, CA 92182, USA (Tel: 619 594 5157)

(Recewed 22February 1989) The cardinal snapper, Pristopomoides macropthalmus, collected from 500m depth off Jamaica, had 4.0-4.4% lipid in its neurocranium and vertebral centra, comprised of 85-86% triacylglycerol. 2. The rare arrowtail, Melanonous zugmayeri, from 1000 m depth, had 1.3-1.8% lipid in these same tissues, comprised of 47.6-76.7% triacylglycerol. 3. Fatty acids of these deep fish when compared with two shallow reef herbivores, Acanthurus bahianus and A. chirurgus, were enriched in polyunsaturated fatty acids, particularly 22:6o3, 20:5093, and 16:4o93/m 1. 4. Monounsaturated fatty acids in Melanonous zugmayeri were 31.7-46.3% of the total fatty acids and were dominated by 18:lo99 + 11 and 20:1. 5. Monounsaturated fatty acids in the Acanthurus species were 27.0-36.4% and 23.3-30.7% in the bigeye, Priacanthus arenatus, a nocturnal carnivore from 25 m depth. 6. The principal monounsaturated fatty acid in Acanthurus spp. was 16:1oJ7 (8.7-24.6%) whereas P. arenatus was similar to the two deep fish with respect to monounsaturated fatty acids and polyunsaturated fatty acids. 7. Fatty acid composition of P. arenatus is probably controlled by its diet consisting of larval forms from deeper colder water.

Abstract--1.

INTRODUCTION Forty-four species of fish from 28 families have been analysed for bone lipid; 10 of these families have representatives with more than 24% lipid as percent dry weight (Phleger et al., 1989; Phleger, 1988a). Lee et al. (1975) demonstrated that triacylglycerol was the principal lipid class present, with minor amounts of cholesterol and phospholipid in the bone. Lipid may comprise up to 93% of the total body lipid in the sheepshead wrasse, Semicossyphus pulcher (Phleger et al., 1976) and the utilization of triacylglycerol from neurocranium and vertebral centra during starvation stress has been demonstrated in the ocean surgeon, Acanthurus bahianus (Phleger, 1988b). Bones of the deep ocean orange roughy (Hoplostethus atlanticus), appear to contain wax ester rather than triacylglycerol (Grigor et al., 1983). The purpose of the present study is to compare skeletal lipids in fish from different depths (5-1000 m) in the ocean with principal emphasis on the triacylglycerol and phospholipid fatty acids. An opportunity to perform this study occurred when a species of the rare arrowtail, Melanonous zugmayeri, was obtained from 1000m depth off the Discovery Bay Marine Laboratory on Jamaica's north shore, which has a steep continental slope resulting in deep water very close to shore (Land, 1979). It was then possible to compare lipids of this fish with a snapper (Pristopomoides macropthalmus) from 500 m depth, a nocturnal carnivorous bigeye (Priacanthus arenatus) from 25 m depth, and two diurnal herbivorous surgeonfish, Acanthurus bahianus and A. chirurgus, from 5 m depth. Such a study allowed careful examination

of tropical fish skeletal fatty acids for the first time and consideration of such factors as temperature, diet, and depth on their distribution. MATERIALS AND METHODS

The ocean surgeon, Acanthurus bahianus, and the doctorfish, A. chirurgus, were collected by spear near Discovery Bay Marine Laboratory on the north coast of Jamaica, West Indies (Phleger, 1988a). Both species of Acanthurus were collected at a depth of 5 m in coral reef areas. The bigeye, Priacanthus arenatus, was collected by spear at 25 m depth beneath overhanging coral near the Rio Bueno west wall located approximately 7 miles west of Discovery Bay Marine Laboratory. The cardinal snapper, Pristopomoides macropthalmus, and the arrowtail, Melanonous zugmayeri, were collected by free vehicle vertical set line (Phleger and Soutar, 1971) from depths of 500 and 1000 m, respectively, located 1-2 miles north of Discovery Bay Marine Laboratory. Species identifications of Acanthurus spp. and P. arenatus were made according to Randall (1968) and confirmed by K. Aiken, Department of Zoology, University of West Indies. The cardinal snapper was identified according to Fischer (1978) and also confirmed by Aiken. Melanonous zugrnayeri (Norman, 1930) was identified by P. S. Lobel who transported it frozen to Woods Hole Oceanographic Institution [see Hubbs et al. (1979) for a brief description of this species]. Each fish was frozen immediately after collection and thawed only just prior to dissection of bones and other organs. The neurocranium and the vertebral centra were dissected out of all fishes, rigorously cleaned to remove flesh and brain or nerve cord tissue, chopped into fine pieces with a razor, and extracted with chloroform-methanol (2 : 1, v/v) (Lee et al., 1975; Phleger, 1987). Liver, heart, gut and flesh were dissected out of all fish and the lipids extracted with chloroform-methanol (2 : 1 v/v). Flesh refers to all parts of 329

C. F. PHLEGERand R. J. LAUB

330

Table I. Bone lipids of two species of deep water Jamaican fish Tissue

Lipid (mg}

Lipid (percent weight)

Triacylglycerot (percent of lipid)

Cholesterol (percent of lipid)

Pbospholipid (percent of lipid)

Vertebral centra Neurocranium

106 76

Lipids of Pristopomoides macropthalrnus (Lutjanidae) 4.4 85.1 1.7 4.0 86.2 2.5

13.2 11.3

Vertebral centra Neurocranium

14 15

Lipids of Melanonous zugmayeri (Melanonidae) 1.8 76.7 ND 1.3 47.6 ND

23.3 52.4

ND, not determined.

the fish excluding the bones, liver, heart, and gut. Lipids were dried under N: or on a roto-evaporator and weighed. The lipids were applied to silica gel (6(~80 mesh) columns and the neutral lipids were eluted with chloroform followed by elution of the polar lipids by methanol. Neutral lipids were assayed for cholesterol by a Liebermann-Burchard reaction (Kabara, 1957). The bone lipid showed only three lipid classes by thin layer chromatography (petroleum ether: diethyl ether: glacial acetic acid, 80:20:1): phospholipid, cholesterol, and triacylglycerol. The triacylglycerol percentage was calculated by subtracting the value for cholesterol from the neutral lipids: the polar lipids were phospholipid. The neutral and polar lipid fractions from the silica gel columns were methylated without further purification. Methyl esters were prepared by base-catalysed transmethylation with 14% sodium methoxide in methanol added to lipid aliquots in screw cap vials with teflon lined caps. The gas chromatography (Carlo Erba Model 4160 GC: flame ionization detector, hydrogen carrier; 100:1 split injection) column was a model WCOT fused silica (50 m, 0.25 mm I.D.) CP-Si188 capillary column obtained from Chrompack (Raritan, New Jersey) that contained a phenylmethylsilicone stationary phase. The oven temperature was programmed from 100 to 210C at 4/min. Component fatty acid methyl esters were identified by comparison with authentic FAME standard mixtures obtained from Chrompack, Nu-Chek, and the National Marine Fisheries Service. Quantitation was carried out with a Hewlett-Packard Integrator (3380A) and checked manually by triangulation. Chromatograms of over 100 samples were taken; replicate analyses of those arising from the same tissue of a given fish differed by no more than 2 3%. RESULTS

Skeletal lipid ranged between 4.0 and 4.4% (percent dry weight) for the neurocranium and vertebral centra of P. macropthalmus and between 1.3 and 1.8% for these same tissues in M. zugmayeri (Table 1). Most of this lipid is triacylglycerol (85.1 86.2%) in P. macropthalmus with phospholipid (11.3-13.2%) and cholesterol (1.7-2.5%) present as minor components. In the vertebral centra of M. zugmayeri triacylglycerol is the major lipid class (76.7%), whereas in the neurocranium it is less than one-half of the lipid (47.6%). Phospholipid (52.4%) is the principal lipid class in the neurocranium of M. zugmayeri, but this simply reflects lower triacylglycerol and lower percentages of lipid (Table 1). Skeletal lipid (for neurocranium plus vertebral centra) as a percentage of the total body lipid in these two species of deep water Jamaican fish was 6.4-16.2% (Table 2). The viscera is similar to the bones containing 6.8-13.6% lipid is percent total body lipid. Most of the body lipid in these species is found in the flesh; 86.6% for P. macropthalmus and 70.1% for M. zugmayeri (Table 2).

There are less total polyunsaturated fatty acids (PUFA) in the two species of Acanthurus from shallow water (0.6-8.6%) than in the three deeper species (4.1-28.1%) (Table 3), Priacanthus arenatus total P U F A range from 4.1 to 19.9%, Pristopomoides macropthalmus P U F A range from 10.6 to 12.4%, and M. zugmayeri P U F A range from 2.3 to 28.1%. There are more P U F A in neutral lipid (triacylglycerol) than polar lipid in all cases except the vertebral centra of A. chirurgus. The principal P U F A in most deep fish is 22:6093 with 20:5{03 and 16:4o93/o9 1 also important. The Acanthurid fishes lack 22:6093 except in trace amounts (Table 3), whereas the deep fish all have significant percentages of 22:6093 (up to 9.2%). The neurocranium of M. zugmayeri is an exception to this with only trace amounts of 22:093 detected and rather low amounts of P U F A (2.3-4.5%). The vertebral centra neutral lipid fraction of M. zugmayeri has 28.1% P U F A including 22:6093 (9.2%), 20: 5093 (9.0%), 20 : 420: 3 (5.3 %) and 22 : 509 3 (2.8%) (Table 3). Monounsaturated fatty acids ( M U F A ) were generally present in greater quantities in Melanonous zugmayeri (31.7-46.3), particularly in the neurocranium neutral lipid (46.3%) (Table 3). The M U F A ranged between 28.8 and 31.4% in Pristopomoides macropthalmus, from 23.3 to 30.7% in Priacanthus arenatus, and from 27.0 to 36.4% in Acanthurus spp. The d o m i n a n t M U F A in Acanthurus spp. 16:1097 (8.7-24.6%) whereas 18:1099 + 11 was dominant in all other fish species (9.2 23.6%). Also important was 18:1o97 (1.4-9.2% in all fish species), whereas 20:1 was more important in the P. arenatus, P. Macropthalmus, and M. zugmayeri (tr to 4.6%) (Table 3). The neutral lipid fraction was enriched in 16:1097 relative to the polar lipid fraction, particularly in the Acanthurids where it comprised 18.6-24.6% of the NL and 8.7 12.0% of the PL (Table 3). This was also true to a lesser extent in P. arenatus and P. macropthalmus and the neurocranial lipid of M. zugmayeri. The vertebral centra lipid of M. zugmayeri was an exception with almost twice as much 16:lo97 in the PL as the NL. The opposite was true of 18:1099 + 11 where more was present in the polar lipid (10.6-23.6%) than in the neutral lipids (5.1-13.3%) except in the neurcranium of M. zugrnayeri where 18 : 1099 + 11 was approximately equal in both neutral and polar fractions. Table 2. Tissue lipids as percent total body lipid for two species of

deep water Jamaican fish species

Pristopomoidesmacropthalmus Melanonous zugmayeri

Bone

Viscera

Flesh

6.4 16.2

6.8 13.6

86.6 70.1

331

Skeletal fatty acids in fish from different depths off Jamaica The saturated fatty acids were relatively more important in the Acanthurids, ranging from 32.9 to 63.4% in A. bahianus and 47.3 to 64.7% in A. chirurgus (Table 3). These included predominantly 16:0 (10.2-44.7%), 14:0 (3.9-18.6%) and 18:0 (3.8-15.9%). Saturated fatty acids were less important in the other fish species, particularly P. macropthalmus (37.7-50.0%) and M. zugmayeri (26.1--41.9%). In these two deep fish the saturated fatty acids mostly included 16:0 (10.4-28.2%), 14:0 (2.5-9.3%) and 18:0 (3.6-10.8%). The fatty acids 15: 0 and 17: 0 were of intermediate importance (tr-4.3% for 15:0 and 1.1-4.5% for 17:0) in these deep fish. DISCUSSION

These fatty acid analyses are the most complete available for tropical fish skeletal tissue to date. Lee et al. (1975) analysed 12 fatty acids from bone triacylglycerol and phospholipid of the Atlantic ocean Stromateoid Peprilus simillinus and the Pacific Ocean Anoplopomatid Anoplopoma fimbria. Phleger et al. (1989) analysed 27 fatty acids in spawning Pacific Ocean pink salmon, Oncorhynchus gorbuscha. The analyses reported here include 25-27 identified fatty acids generally comprising 80-90% of the total (Table 3). Some of our chromatograms had up to 50 unknown peaks; most, however, were minor compo-

nents. Recent GC analyses of sardine (Sardinops melanosticta) and pollack (Theragra chalcogramma) include 73 peaks for fatty acids, 66 of which have been identified (Itabashi and Takagi, 1980). In contrast, exoskeleton triacylglycerols from the Colorado beetle Leptinotarsa decemlineata contain only seven fatty acids (Dubis et al., 1986). Although there is not much lipid in the skeleton of M. zugmayeri and P. macropthalmus (as a percentage of total body weight, 1.3--4.4% lipid; Table 1), the percentage of total body lipid is substantial. M. zugmayeri had 16.2% bone lipid as percent total body lipid and P. macropthalmus had 6.4% bone lipid as percent total body lipid (Table 2). Therefore, one might expect the skeletal triacylglycerol, which is the major skeletal lipid class (Table 1), to be important as an energy storage lipid. The shallow reef fishes in this study have substantially more oil in their bones than the deep ocean M. zugmayeri and P. macropthalmus. Vertebral centra lipid (percent weight) ranges from 4.4 to 10.1% and neurocranium lipid ranges from 14.7 to 29.7% in Acanthurus bahianus, A. chirurgus and Priacanthus arenatus (Phleger, 1988a). Some individuals of A. bahianus have 53-57% of their total body lipid in the neurocranium, and this triacylglycerol-rich lipid is rapidly utilized during starvation stress (Phleger, 1988b), which occurs during violent storms in the Caribbean. The seasonal change in bone-marrow lipid of African

T a b l e 3. Triglyceride and phospholipid fatty acids of skeletal lipids in fish from different depths off Jamaica 5m*

5m

Acanthurus bahianus

Acanthurus chirurgus

NCi"

VCt

F a t t y acid

NL:~

PL:~

12 : 0 13:0 14:0 15:0 16:0 17:0 18:0 20:0 22:0 24:0

tr§

Total 14:1o97 16:1o97 18:1o99 + 11 18:1o97 20:1 22 : 1 24:1 Total 16:4o93/16:4o9 1 18: 2 18:3o93 20: 2 20:3 20: 4/20: 3 20: 5o9 s 22: 4? 22: 5o9 3? 22: 6o9 3 Total Total, these c o m p o n e n t s

NC

VC

NL

PL

NL

PL

NL

12.4 1.1 44.7 0.9 3.8 tr tr 0.5

tr tr 6.8 1.2 16.4 1.5 15.9 4.8 0.7 1.5

7.1 0.9 31.1 0.9 7.4 0.7 0.8 tr

tr tr 5.0 0.9 10.2 0.9 9.7 4.9 0.8 0.5

tr tr 18.6 1.3 20.9 1.2 5.6 1.2 2.0 tr

tr 10.0 0.9 43.5 0.6 9.7 -

63.4

48.8

48.9

32.9

50.8

64.7

54.7

47.3

tr 21.1 5.1 2.9 tr 0.7

tr 9.4 14.1 2.5 tr tr 1.3

24.6 8.6 4.9 tr tr tr

tr 8.7 10.6 8.3 0.5 9.6 1.5

18.6 7.8 4.9 1.0 tr 0.5

12.0 13.0 2.0 tr -

20.0 9.0 4.9 tr tr

0.7 9.5 19.2 5.3 tr tr 1.7

29.8

27.3

38.1

30.2

32.8

27.0

33.9

36.4

0.5 1.2 0.8 tr

0.6 1.4 0.6 tr tr

-

tr tr 0.9 tr tr tr tr tr

0.6 tr -

tr 2.0 tr tr tr tr

0.8 1.9 1.2 1.4 0.7 2.6 tr tr tr

tr tr tr 2.7 -

tr tr 0.6 tr tr tr tr tr tr

0.5 0.7 tr tr 1.5 0.6 tr tr tr

2.5

0.9

3.2

2.0

8.6

2.7

0.6

3.3

95.7

77.0

90.2

65.1

92.2

94.4

89.2

87.0

17.2 1.3 22.5 1.4 11.3 1.0 tr

PL 3.9 0.5 19.3 15.3 tr 1.9 6.5

continued overleaf

332

C . F. PHLEGER a n d R . J. LAUB Table 3. Cont. 25m

500 m

Priacanthus arenatus

Pristopomoides macropthalmus

NC

VC

NC

VC

Fatty acid

NL

PL

NL

PL

NL

PL

NL

PL

12: 0

tr tr 4.9 1.4 25.1 2.3 9.2 0.8 0.5 tr 44.2

tr

tr

tr tr 9.3 4.3 10.4 4.5 9.2 1.1 0.6 tr 39.4

tr

tr tr 8.6 2.9 14.2 3.2 7.3 0.9 0.6 tr 37.7

0.6

1.9 0.9 27.0 2.8 11.9 tr tr 1.6 46.1

tr tr 8.3 4.0 12.4 5.3 11.0 IA 0.7 tr 42.8

3.4 1.3 28.2 2.4 9.5 tr 3.0 1.6 50.0

4.4

6.7 11.8 2.6 2.8 3.4

6.0 21.8 2.1 tr tr

13 : 0

14:0 15:0 16:0 17:0 18:0 20:0 22:0 24:0 Total 14:1o97 1 8 : t o 9 9 + 11 18:1097 20 : 1 22 : 1 24 : 1 Total

3.9 13.3 2.8 1.3 2.0 tr 23.3

2.3 23.6 1.4 tr 3.4 tr 30.7

tr 8.2 11.0 5.9 2.5 1.9 tr 29.5

16:4co3/16:409 1 18:2 18 : 3093 20:2 20 : 3 20 : 4/20:3 20 : 509 3 22 : 4? 22 : 5~o 3? 22:6~93 Total

0.6 2.1 tr 0.5 1.8 1.6 3.5 0.9 1.5 7.4 19.9

1.4 0.8 tr tr tr

1.6 2.0 tr 1.1 tr

1.9 1.4 1.2 6.8 13.5

tr 0.7 1.0 3.0 9.4

Total, these components

87.4

90.3

81.7

16 : t09 7

4.1 1.2 38.0 16.6 0.6 0.6 61.1

tr 9.3 9.2 5.4 3.4 2.6

2.9 22.0 2.1 tr

1.0

1.0

28.8

29.3

30.9

1.0

1.6 1.8 1.4 0.6 0.6

1.4 0.9 tr

1.0 1.7 0.8 0.5 tr

1.3 0.9 tr tr 0.6

1.9

0.6 0.8 4.1

1.4 0.7 0.8 3.5 12.4

0.7 7.0 11.9

3.3 1.4 1.3 1.9 I 1.9

1.9 0.6 0.7 4.6 10.6

92.2

83.2

79.9

78.9

91.5

1.7

VC

NL

PL

NL

PL

12:0

tr

tr 1.1 2.6 tr 21.3 I.I 10.8 2.6 1.2 1.2 41.8

tr

tr

5.2 tr 11.1 2.0 3.6 2.2 1.5 0.5 26.1

11.0 2t.5 9.2 4.6 tr 46.3

4.9 21.3 5.2 1.3 0.7 4.8 38.2

tr 4.4 12.8 4.0 2.6 7.9 tr 31.7

36.1

1.7 tr tr

1.3 1.0 tr

1.9 tr tr 1.2 0.8 2,8 tr 1.1 7.8 85.8

14:1097 16:1097 18:1099+11 18:1~o7 20:1 22:1 24:1 Total 16:4~o3/16:4o91 18:2 18 : 309 3 20:2 20 : 3 20 : 4/20 : 3 20:5093 22:4? 22 : 509 3? 22:6093 Total

1.2

tr

tr 1.6 tr 4.5

tr tr tr 2.3

tr 0.9 tr tr tr 5.3 9,0 0.9 2.8 9.2 28.1

Total, these components

91.3

82.3

85.9

4.2 18.2 2.0

31.4

Fatty acid

6.8 0.6 19.3 1.1 8.2 0.6 2.3 1.6 40.5

39.2

27.0

Melanonous zugmayeri

13:0 14:0 15:0 16:0 17:0 18:0 20:0 22:0 24:0 Total

tr

1.5

1000 m NC

2.5 1.4 25.2 2.7 7.4

tr 7.3 1.1 17.3 2.5 6.3 3.7 3.7 41.9 tr 7.6 18.8 8.5 1.2 tr

* D e p t h o f collection. J'NC, neurocranium; VC, vertebral centra. :~NL, neutral lipid (triacylglycerol); PL, polar lipid (phospholipid). §tr, trace a m o u n t ( < 0 . 5 % ) .

Skeletal fatty acids in fish from different depths off Jamaica wildebeast is highly correlated with changes in dietary crude protein (Sinclair, 1977). Bone-marrow lipid is the last fat deposit in wildebeast to be utilized during starvation and the first to be replaced (Sinclair and Duncan, 1972). Cholesterol and phospholipids were relatively minor lipid components in the bone ofP. macropthalmus (1.7-13.2%). There was more phospholipid in the bone of M. zugmayeri (23.3-52.4%). Triacylglycerol was the principal bone lipid in these fishes (47.6-86.2%). Chebomareva et al. (1987) have shown that total lipid and triacylglycerol of the vertebral column of rabbits increase while cholesterol and phospholipid decrease as these animals age from 4.5 to 12 months. No such studies have been performed on skeletal lipid changes in fish of different ages. M. zugmayeri and P. macropthalmus were probably adults because they had well-developed gonads. The specimen of M. zugmayeri is over twice as large as any Atlantic ocean specimen heretofore collected (P. Lobel, private communication). The increase in PUFA in the deeper fish species (Table 3) undoubtedly reflects lower temperatures at greater depths in the ocean. There is a gradual decrease in temperature to depths greater than 1000 m in the tropical north Atlantic ocean. The two species of Acanthurus live in water of 25-30°C, whereas P. macropthalmus at 500 m depth lives in water of 10°C, and M. zugmayeri at 1000m depth probably experiences water of 3-4°C or less (Tchernia, 1980). It is noteworthy that PUFA in P. arenathus (collected from 25 m depth) comprise a significantly greater percentage of the FAME than in the Acanthurus species from 5 m depth, whereas the temperature at 25 m off Jamaica does not differ significantly from that at 5 m. P. arenatus is a nocturnal carnivore, whereas the Acanthurids are diurnal herbivores which can be observed actively grazing on algae. According to Randall (1967), P. arenatus is a zooplankton feeder, consuming primarily fish and fish larvae (atherinids, Dactylopterus volitans, and Lactophrys sp.), shrimp, polychaetes, and crabs and crab larvae (Cronius tumidulus, portunids). Some of these larval forms are derived from adults found in colder, deeper water, and whose lipids are enriched in PUFA. The more highly unsaturated fatty acids in P. arenatus may therefore be a consequence of diet rather than temperature. Both P. macropthalmus and M. zugmayeri are active carnivores that probably roam the sea floor widely in search of food. A red unidentified shrimp-like organism was observed in the gut of M. zugmayeri; diet may influence the fatty acid composition in these fish also. The increase in PUFA of the phospholipids in the deep living fish (Table 3) causes a decrease in the melting points of these structural membrane lipids (Lewis, 1962). As the number of double bonds increases in the fatty acid molecule, the number of possible molecular conformations increases (Brenner, 1984). This decreases the membrane packing of phospholipids, making the membrane more fluid, and increasing its surface and reducing its thickness. Interestingly, increasing the number of double bonds over 3 does not significantly increase membrane fluidity in pig and rat liver microsomes. However, since PUFA such as 20:5co3 and 22:6093 are so

333

abundant (Table 3), this may not be the case in deep sea fish such as M. zugmayeri. The increase in P U F A and M U F A in the triacylglycerols of the deeper fish (Table 3) may also help maintain skeletal flexibility. Certain deep ocean fish from 500 to 1000 m, such as Anoplopoma fimbria, have 50-80% of the total body lipid in the bones (Phleger et al., 1976). In this fish and others like it, solid lipid in bone could hinder swimming movement.

Acknowledgements--We thank J. Woodley, K. Aiken, and D. Steele in Jamaica for their advice and support. We also thank N. Phleger, R. Gates, and G. Bruno for their help in the field and as diving partners. J. Nevenzel generously performed preliminary fatty acid analyses which prepared us for this study. The field work was completed at the Discovery Bay Marine Laboratory in Jamaica where CFP was a 1985-1986 Fulbright Lecturer in the Department of Zoology, University of West Indies. The analytical work was supported by grants in part from the Department of Energy Office of Basic Energy Sciences and from the National Science Foundation to R. J. Laub.

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C.F. PHLEGERand R.J. LAUB

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