)r Biology 24, 279--285 (1974) 9 by Springer-Verlag 1974

Wax Ester Biosynthesis by Midwater Marine Animals M. K a y a m a 1 and J. C. Nevenzel 2. 1 Department of Fisheries, Hiroshima University; Fukuyama, Japan and 2 Laboratory of Nuclear Medicine and Radiation Biology, University of California; Los Angeles, California, USA

Abstract

Lipid is a major energy reserve in many aquatic animals, and wax esters are the principal type of lipid present in most pelagic marine invertebrates and teleost fishes from deep water or near-surface cold waters. It has been suggested that these wax esters are biosynthesized by only a few organisms, and are then transferred along the food web to the fishes and marine mammals. We found that accessible mesopelagic myctophid (Lampanyctus ritteri, Stenobrachius leucopsarus and Triphoturus mexieanus) and gonostomatid (Cyelothone atraria and Gonostoma gracile) fishes and crustaceans (Gaussia princeps, Calanus helgolandicus, Acanthephyra quadrispinosa and Sergestes prehensilis) biosynthesize wax esters from acetate, longchain alcohol or fatty acid precursors, in vivo or in vitro. In the latter experiments, organ tissues (hepatopancreas and gut) are more active than muscle, although, overall, fish muscle is probably a major site of wax ester biosynthesis in the species studied. Therefore, wax esters are not persistent dietary survivors in the food web of the oceanic midwaters; rather, most invertebrates and fishes in this environment make wax esters, modifying ingested fats to their own characteristic patterns. Introduction The midwater environment (the meso- and bathypelagic zones of the ecologists (Hedgpeth, 1957) is one of relatively stable conditions: very low to undetectable light intensity, a narrow range of temperatures (approximately 4 ~ to 10 ~ with negligible diel or seasonal variation, but continuous vertical distribution), high hydrostatic pressure increasing regularly with depth (Bruun, t957), isolation from the turbulence of the surface waters, and a supply of food of very low concentration widely scattered in space and temporally highly variable. This environment is below the photie zone, but sufficient light penetrates into its upper regions to permit the larger animals to follow isolumes (Boden and Kampa, 1967) in diel migrations up to, or into, the epipelagic zone in search of food. The biochemical studies presented in this paper were performed with such vertical migrators because they are comparatively easily obtained alive. * All enquiries regarding reprints, etc. should be addressed to Dr. J. C. Nevenzel, Laboratory of Nuclear Medicine and l~adiation Biology, University of California, 900 Veteran Avenue, Los Angeles, California 90024, USA. 37

Marine Biology, Vol. 24

However , the importance of wax esters as an energy reserve lipid has been established for mcso- and bathypelagic animals generally (Nevenzel et al., 1969; Nevenzel, 1970; Lee et al., i971a), and the meager evidence available (e.g. for Gnathophausia gigas) points to its relevance in abyssal pelagic animals as well. I n the simplified food chain, phytoplankton - , herbivorous copepod--* myctophid fish, wax esters are known to be biosynthesized by the copepods (Lee et al., 1970, i971b), and therefore the wax esters found in many myctophids and other mesopelagic animals could be of dietary origin [cf. the postulated direct origin of sperm-whale wax esters from those of the dietary giant squid (Hansen and Cheah, t969)]. On the other hand, some epipelagie fishes (herring, anchovy, sardine, young salmon) which feed on waxester-containing copepods do not retain any wax esters in their lipids. Several explanations for this striking difference between herring and myctophids ingesting very similar fats can be offered, the most appealing of which assumes that midwater animMs biosynthesize wax esters. I t is the latter hypothesis with which this study is concerned. The approach chosen was the use of isotopically labelled precursors to show that: (1) the expected products of digestion of wax esters, namely fatty acids plus long-chain alcohols, could be incorporated into wax esters by living lantern fishes; (2) wax esters are synthesized de hove from acetate by these animals; and (3) even tissue slices or homogenates could biosynthesize wax esters from appropriate precursors. Preliminary results have been reported (Nevenzel and Kayama, 1968; Nevenzel, t970; Kayama, t971). Materials and Methods

Animals Three species of Myctophidae (Lampanyctus ritteri, Ste~obrachius leucopsaru8 and Triphoturus mexicanus, collected off Santa Barbara, California, USA) and two Gonostomatidae (Cyclothone atraria and Gonostoma gracile, collected in Suruga and Sagami Bays, Japan)

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were used as experimental fish. Their wax ester contents varied from 20% of the total lipid ofG. gracile and 70% for C. atraria (Kayama et al., 1972) to nearly 90% for the myctophids (Nevenzel et al., t969). The standard body lengths and body wet weights of specimens used for our experiments were in the ranges of 3 to 8 cm and I to 6 g for the myctophid fishes, and 2 to 5 cm and 0.5 to t.5 g for the gonostomatids. Fish reaching the surface in good condition lived for up to 6 h in cold (10~ seawater. The midwater eopepod Gaussia princeps, the epipelagic copepod Calanus helgolandicus, and the deeapods Acanthephyra quadrispinosa and Sergestes prehensilis (Kayama et al., 1972) were also used in homogenate experiments.

Labelled Compounds Sodium (l-14C)acetate, (t-14C)palmitie acid, (9,t0-aH)oleic acid and some of the (1-14C) hexadecanol (cetyl alcohol) used were commercial products. Except for the acetate, they were purified by silicie acid column chromatography, where necessary, to a final purity of better than 99% (radiochemical) as determined with the radioehromatogram scanner (Packard Model 7200 or Aloka TLC-I, Nippon Musen Co.). Some (1-14C) hexadeeanol was also synthesized from (l-14C)palmitic acid by LiA1H4 reduction (Brown, t95t) and chromatographic purification. For the in vivo experiments, the (14C)aeetate was dissolved in isotonic saline, and the labelled hexadeeanol was emulsified in the same medium with the aid of sodium taurocholate and monopalmitin (Hansen and Mead, t965). The most satisfactory technique for solubilizing the f a t t y acids was as their bovineserum albumin complexes (Fillerup et al., 1958) in isotonic saline. In some early experiments, the fatty acids and hexadecanol were dissolved in reagentgrade dimethylsulfoxide (J. T. Baker Chemical Co.) in concentrations (29--200 raM, 8--48 ~zg/~xl)sufficient to contain 2/xCi/tzl. For the homogenate experiments, the labelled compounds were dissolved or dispersed in 0.1 M phosphate buffer of p K 7.0 or 7.8, using Triton X-t00 (Sigma Chemical Co.) to emulsify both the Meohol and fatty acid substrates. Details are given in the footnote to Table 2.

Incubations Thirty to 100 9Ci of the labelled substrates in an appropriate aqueous medium (i0 to 100~zl) were administered to each fish by either intraperitoneal or, more satisfactorily, intramuscular injection, best given dorsally in two equal portions on either side of the spine. The fish were kept alive in cold seawater (10 ~ for up to 3 h, and then killed by freezing on dry ice. They were transported in the frozen state to a shore laboratory for analyses.

Mar. Biol.

The homogenates of the fish hepatopanereas (including the pyloric caeca), digestive organ ( = stomach plus intestinal epithelium) and muscle tissues and of whole copepods and decapods were prepared in a Potter tissue homogenizer in 5 or t0 volumes of 0.1 M phosphate buffer of pI-I 7.0 or 7.8 (see Table 2 footnote for details). By low-speed centrifugation (approximately 500 x g for 2 min) unbroken cells and tissue debris were eliminated. The resulting supernatant (0.8 to 2.0 ml) was incubated with the appropriate substrate (i EzM containing 5~zCi) with the addition, in some runs, of nonlabelled aeceptor long-chain alcohols or acids. These in vitro incubations were carried out either at the ship's ambient temperature (approximately i9 ~ for 6 h, or in a motor-driven shaker ("Personal Shaker", Taiyo Kagaku Kogyo Co.) at 30 ~ for 4 h. Reaction was terminated by the addition of chloroform-methanol (t :2, v/v). In all cases the lipids were extracted with chloroform-methanol by the method of Bligh and Dyer (i959). Subsequent manipulations of the lipids were carried out under nitrogen, to minimize losses due to autoxidation.

Chromatography For thin-layer chromatography (TLC) 5 • 20 cm plates were coated with silica gel G (E. Merck) of about 250 ~z thickness. The plates were washed before use with chloroform-methanol (4:t, v/v - - S k i p s k i et al., 1965), and a channel in the form of a trapezoid was defined on the plate (Kayama and Nevenzel, i972). The sample was spotted in a i-cm-wide line 3 cm from the edge at the narrow end of the trapezoid, and the plate was developed with petroleum ether (b.p. 60 ~ to 70 ~ - - isopropyl ether - - acetic acid (87.5:12.5 : i, v/v/v). The distribution of radioactivity on the developed plate was determined with the radioehromatogram scanner before the plates were charred. Lipids from the homogenate experiments were separated by TLC, the radioactive bands located, and each band scraped off and suspended in scintillation solution containing Cab-O-Sil ("Thixotropie gel powder", Packard) for radioactivity assay in the liquid scintillation spectrometer (Packard Tri-earb Model 3003). The laC-labelled fatty acid and alcohol moieties were analyzed (as their methyl esters and trifluoroacetates, respectively) by gas-liquid chromatography (GLC), with simultaneous measurement of mass and radioactivity in the Cary-Loenco Model 70 Hi-Flex instrument. Silicic acid columns were used to separate the various lipid classes; the wax esters were elated with 2% diethyl ether in petroleum ether (Kayama and Tsuchiya, 1964). A Florisil column was used to separate the alcohols and acids derived from the wax esters by hydrolysis (Nevenzel et al., 1965). Quantitative analyses were derived from the weights of the chromatographic fractions.

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Table 1. In vivo incorporation o/ precursors into wax esters by lantern fish (R, Lampanyctus ritteri; L, Stenobrachius leueopsarus," M, Triphoturus mexicanus). Substrates administered in saline solution or emulsion by intramuscular injection, unless otherwise noted Labelled compound (activity injected/fish)

% of injected radioactivity Specific activity recovered in (disintegrations per sec/mg) Total Wax ester Phospholipidb Wax ester Phospholipidlipid~

Distribution Of wax ester radioactivity (%) Alcohol Fatty acid

Sodium (l-14C)acetate (100 ~Ci), 1~ (l-~dC)palmitic acid (40 ~Ci)o, L (1-1~C)palmitic acid (30 ~Ci), L (9,i0-3H)oleic acid (60 ~Ci), M (t-14C)hexadecanol (30 ~Ci), R + M (1-14C)hexadecanol (30 y,Ci)% L (~t-14C)hexadecanol (40 ~zCi)o, 1%

2.7 6.2 10.6 13.2 2.7 2.7 8.5

89 -49 5 88 92 96

1.1 0.27 3.8 5.2 t.5 1.2 5.2

0.50 5.9 5.4 5.0 1.2 1.5 2.6

i05 6.0 112 623 40.4 20.7 t42

621 2,980 2,460 6,800 560 696 t,200

li -51 95 12 8 4

Excluding unmetabolized starting material. b Crude total phospholipids eluted from silicic acid by methanol. Dissolved in dimethyl sulfoxide. Intraperitoneal injection.

Results

I n Vivo E x p e r i m e n t ~ Acetate Incorporation Results were highly variable, b u t in the best e x p e r i m e n t (highest incorporation) 3.5% of the radioa c t i v i t y injected as acetate was recovered i n t o t a l lipids. The average i n c o r p o r a t i o n i n 5 experiments using i n t r a m u s c u l a r injection was 1.6 +_ i.5 (standard deviation); i n t r a p e r i t o n e a l i n j e c t i o n was m u c h less

Fig. 1. Lampanyctus ritteri. In vivo incorporation of (1-14C) acetate into various lipid classes. The silica gel G thin-layer plate was developed in petroleum ether - - isopropyl ether - acetic acid (87.5:12.5:1, v/v/v). After scanning on the radiochromatogram scanner (upper trace) the plate was sprayed with sulfuric acid and charred on a hot plate. Operating conditions for the scan: counting rate, linear range, t000 counts per min; time constant, 30 see; scan speed, l cm/min; collimator width, 2.5 mm. 0: origin; F: solvent front; PL: phospholipid; AL: free long-chain alcohol; AC: free fatty acid; TG: triglyceride; WE: wax ester 37*

satisfactory, as only 0.14% incorporation was found in two trials. From one of the experiments using intramuscular injection, the wax ester fraction, separated by silicic-acid column chromatography, was found to contain 41% of the total lipid radioactivity (i.1% of that administered; cf. Table I). The long-chain alcohols and fatty acids resulting from saponification of these wax esters had specific activities of 2i6 and 18.6 disintegrations per see/rag, respectively

Fig. 2. Stenobrachius leucopsarus. Distribution of radioactivity between fatty acid and alcohol moieties of wax esters, after injection of (l-14C)palmitic acid as the albumin complex. Thin-layer chromatography (TLC), radioactivity scan and abbreviations as for Fig. t

The r a d i o c h r o m a t o g r a m scan of the TLC separa t i o n of the t o t a l lipids is shown in Fig. l ; other radioactive lipid classes detected are phospholipid

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2liar. Biol.

Table 2. I n vitro incorporation el precursors into wax esters, n.d. : no data; tr : trace - less than 0.2 % incorporation

Tissue

Gonostomatid fish~ Hepatopancreas Digestive organ Viscera Decapodsb

Labelled substrate

% of initial radioactivity found in wax esters when unlabelled substrata was Present Absent

(1 14C)acetic acida, f

n.d. n.d. n.d. n.d.

17.7 7.8 3.2 2.9

n.d.

2.9

Calanus helgolandicus

Gonostomatid fish~ Hepatopancreas Digestive organ Muscle Gonostomatid fish~ Hepatopanereas Digestive organ Muscle Decapodsb

(oleyl alcohol) 3.5 1.0 tr (hexadecanol) 6.4 2.5 2.2 2.t

1.6 0.9 0.6 n.d.

(palmitic acid) 7.3 n.d. 2.5

4.0 5.9 ~/.6

Gaussia princeps

1.8

1.5

Gonostomatid fish~ ttepatopancreas Digestive organ Muscle Decapodsb

(oleic acid) 4.1 6.0 0.6 i .0

Myc~phid fish~ Kepatopanoreas Digestive organ Muscle

(~l ltC)palmitie acida

(1 l~C)oleic acid~

(~ tdC)hexadecar~ol~

tr tr tr

3.1 3.9 0.7 n.d.

Cyclothone atraria and Gonostoma gracile were used interchangeably. b Acanthephyra quadrispinosa or Sergestes prehensilis. o Lampanyctus ritteri or Triphoturus mexicanus.

One ml of a 5-volumes homogenate supernatant in 0A ~ phosphate buffer (pH 7.0) was incubated with 5 mg Triton X-100 plus 3 IzM of each substrata at 30 ~ for 4 h; total radioactivity used was 2 tzCi per incubation vial. e As bovine serum albumin complex in isotonic saline. f Used 50 ~xCitotal radioactivity. g Two ml of a 10-volumes homogenate in 0.1 M phosphate buffer (pH 7.8) were incubated with 5 mg Triton X-100 plus 1 ~zM of each substrate at 19~ to 2t ~ for 6 h; total radioactivity used was 5 ~Ci per incubation vial.

(at t h e origin), free alcohol, free f a t t y acid a n d triglyceride. G L C - r a d i o c h r o m a t o g r a p h i c analyses of t h e w a x ester alcohols a n d f a t t y acids showed t h a t a c e t a t e h a d been i n c o r p o r a t e d p r e d o m i n a n t l y into h e x a d e e a n o l a n d p a l m i t i c a ~ d oleic acids. P a l m i t i c a n d Oleic Acids M e t a b o l i s m I n t r a m u s c u l a r injection of free (l-14C)pMmitic acid dissolved in d i m e t h y l sulfoxide r e s u l t e d in v e r y slight i n c o r p o r a t i o n into t h e w a x esters, a n d t h e m a i n r a d i o a c t i v i t y a p p e a r e d in t h e free f a t t y a c i d (und o u b t e d l y r e c o v e r e d s t a r t i n g substrata) a n d t h e p h o s p h o l i p i d fractions. On t h e other hand, 4 to 5%

of t h e (t-14C)palmitic a n d (9,t0-aH)oleic acids administered i n t r a m u s c u l a r l y in t h e form of t h e i r a l b u m i n complexes were i n c o r p o r a t e d into w a x esters; triglycerides a n d p h o s p h o l i p i d s were also labelled. T h e d i s t r i b u t i o n o f r a d i o a c t i v i t y ~n t h e alcohol a n d a c i d moieties of t h e w a x esters was d e t e r m i n e d ; from p a l m i t i c acid 51% of t h e t o t a l w a x ester radioa c t i v i t y was found in t h e acids a n d 49 % in t h e alcohols (cf. Fig. 2). Clearly t h e i n j e c t e d p a l m i t i e a c i d was c o n v e r t e d into alcohols. G L C - r a d i o c h r o m a t o graphic a n a l y s e s of t h e w a x ester alcohols a n d acids showed t h a t m o s t of t h e r a d i o a c t i v i t y was in p a l m i t i c a c i d a n d hexadecanol. R a d i o a c t i v i t y from t h e t r i t i a t e d oleic acid was i n c o r p o r a t e d into w a x esters to a slightly

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greater extent t h a n was palmitic acid, but most of the activity remained in the acid portion (95%), and very little appeared in the alcohol moiety (5 %). Hexadeeanol ?r When (t-ltC)hexadecanol (cetyl alcohol) was given intramuscularly, t.5% of the radioactivity injected appeared in the wax esters, distributed between the acid and alcohol moieties in the ratio t2:88. The palmitic acid recovered from these wax esters was degraded by the Schmidt reaction (Brady et al., t960) to ascertain the proportion of label located in carbon a t o m - t of the acid. An average of 92.5% of the total activity recovered was in carbon-t, confirming that the palmitic acid was formed predominantly by direct oxidation of the (t-14C)hexadceanol substrate, rather t h a n via de novo synthesis from (l-14C)aectate formed b y catabolism of the labelled alcohol. I n one experiment, intramuscular injection of the labelled hexadecanol dissolved in dimethyl sulfoxide gave better incorporation into all lipid classes tha~_ use of the standard saline emulsion. For the wax esters this is in marked contrast to the results obtained with palmitie acid (Table t) and is largely due to enhanced incorporation into the alcohol moiety. Homogenate Experiments Representative results are given in Table 2. With all tissues studied, 14C-hexadecanol was incorporated into wax esters more efficiently t h a n was ~dC-palmitic acid. The oleic acid data are less clear, but presenting the acid as the albumin complex improved its incorporation into wax esters compared to use of the free acid. I n this crude system, activation of the acid by use of the CoA ester did not improve the incorporation of label into the wax esters unless the Triton X-100 was omitted, when the amount of 14C found in wax esters doubled; no other potential cofactors were investigated. The homogenates incorporated earbon-i4 from acetate into wax esters more efficiently than did intact fish in vivo b y a factor of more than two; the distribution of label among the lipid classes was similar in both cases. When living copepods (Calanus helgolandicus) were kept in seawater containing sodium (t-14C)acetate (about 20 specimens in t5 ml of seawater containing a total of 50~zCi of carbon-t4; 0.i mM in sodium acetate), some carbon-t4 was found in the copepod ]ipids. I n this case, the phospholipids and free f a t t y acids were the most intensively labelled lipid classes, but some radioactivity was present in the wax esters also. A typical radiochromatogram of lipid synthesized in vitro from labelled hexadecanol by a homogenate of lantern-fish hepatopancreas is shown in Fig. 3; most of the radioactivity was recovered in nnreacted hexadecanol (the large peak at the left).

Fig. 3. Pooled Lampanyctus ritteri and Triphoturus mexicanus. In vitro synthesis of wax ester in homogenates of lantern fish hepatopanoreas from (l-14C)hexadeeanol and nonradioactive palmitie acid. TLC, radioactivity scan and abbreviations as for Fig. t

Discussion

The most important conclusion from our data is that all midwater animals studied do biosynthesize wax esters from the universally available precursors, acetate and f a t t y acids, as well as from long-chain alcohols. W a x esters, then, are not persistent survivors in the midwater food web originating from one or a limited number of organisms; rather, each species in this environment makes wax esters, modifying dietary fats to its own characteristic pattern. T h a t these patterns are very similar is a consequence of the uniformity of the environment and the distance of these animals (number of transfers) along the food chain from the source of essential f a t t y acids, the cpipelagic phytoplankton. There is no reason to believe t h a t f a t t y acid metabolism in midwater fishes is different from t h a t of shallower species (cf. Mead and K a y a m a , 1967), so we can safely assume that, while able to introduce the first double bond into the chain (usually between carbon atoms 9 and l0 from the carboxyl end), they cannot introduce a second double bond further away from the carboxyl end and so are unable to synthesize de hove linoleic and linolenic acids (9,12--i8:2 and 9,12,15--18 : 3, respectively, shorthand designations for acids each with t8 carbon atoms, the first having 2 double bonds at carbons 9 and i2 and the second with 3 double bonds at carbons 9, i2 and i5). 0 n l y photosynthetic organisms such as the epipelagic phytoplankton are known to be able to biosynthesize these essential f a t t y acids. Midwater fishes and crustaceans can, however, by chain-elongation and further desaturation, convert exogenous 18:2 and i8 : 3 f a t t y acids into

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the 20 : 5, 22 : 5 and 22 : 6 acids characteristic of marine oils. Consequently, exogenous polyunsaturated fatty acids are conselved for incorporation into phospholipide, which are essential for proper functioning of membranes. The carbon chains of reserve lipide (triglycerides and wax esters), on the other hand, are predominantly saturated and monounsaturated, refleeting both the available exogenous acids and those the animal biosynthesizes de nero from acetate. Control of chain length and degree of unsaturation is exercised at several points, as the fatty acids are metabolized via various pathways (catabolism for energy - - particularly of exogenous acids; chainelongation and desaturation; reduction to the alcohol), each at a rate characteristic for the individual acid. We found (Table 2) that in both myctophid and gonostomatid fishes the hepatopancreas and gut were most active in wax synthesis. Recognizing the uncertainties involved in extrapolating from our crude homogenate experiments - - no added co-factorsi, optimal p H and substrate concentration not defined, the difficulties in presenting the water-insoluble substrates to the enzyme system (which was assumed to be water soluble ~) - - still, from the relative weights of white muscle (a few grams) compared to organ tissues (no more than t50 mg per fish) and the relative wax syntheses per assay (musele/(hepatopancreas plus gut) = 0.2 to 0.5) the muscle seems capable of producing at least as much wax as the combined hepatopancreas and gut. Certainly most of the wax esters are stored in the muscle, but the relative contributions of the various potential sites of their in rive synthesis or those of their key precursor, the long-chain alcohols, remain undetermined. An interesting sidelight to the in vivo experiments of Table I is the effect of dimethyl sulfoxide (DMSO), which apparently inhibits the incorporation of exogenous fatty acid into wax esters but stimulates the incorporation of hexadecanol into this lipid type. Biosynthesis of phospholipids is perhaps slightly stimulated by this solvent in both cases. These facts, taken together with its known oxidant properties (Fieser and Fieser, 1967), strongly suggest that DMSO blocks the NADH-mediated reduction of fatty acid to long-chain alcohol (Sargent et al., 1972b). As discussed above (p. 281), intraperitoneal injection of labelled acetate gave poor incorporation into lipide. However, note in Table i that, when hexadeeanol was administered by this route, the results were comparable to those achieved with intramuscular injection. Possibly the water-soluble acetate diffused into the lumen of the intestine and was lost via the 1 Other than alcohol or acid. Sargent et al. (1972 a, b) and Holtz et al. (t973) found that ATP was required for maximum wax ester synthesis in copepod homogenates. 2 Holtz et al. (1973) have shown that in eopepods (Calanus helgolandicus and C. pacificus) the wax ester synthetase is bound to membrar~es,

Mar. Biol.

anus 3 more rapidly than it was absorbed into the vascular system; the insoluble hexadecanol would diffuse only slowly. Summary i. Several species of myctophid (Lampanyctws ritteri, Stenobrachius leucopsarus and Triphoturus mexieanus) and gonostomatid ( Cyclothone atraria and Gonostoma gracile) fishes incorporated radioactive label from appropriate lipid precursors into wax esters in rive. 2. Similarly, homogenates of muscle, hepatopancreas and gut tissues from the same fishes and of whole calanoid copepods and decapods (Gaussia

princeps, Calanus helgolandicus, Acanthephyra quadrispinosa and Sergestes prehensilis) also synthesized radioactive wax esters from sodium (l-l~C)acetate, (t-idC)hexadecanol, (l-14C)palmitic acid and (l-idC)oleic acid. 3. De novo synthesis from acetate of both the alcohol and acid moieties of the wax esters occurred in vivo with the myctophid fishes. About eight times as much activity was incorporated into the alcohol as into the acid moiety, probably as a consequence of the smaller pool size and more rapid turnover of the former. The results are consistent with a ready interconversion of fatty acid and long-chain alcohol - - at least of saturated C~s chains. 4. Since some of the most abundant inhabitants of the oceanic midwaters, both invertebrates and fishes, can biosynthesize wax esters from any ingested fat, we believe that this lipid type is in general not a persistent dietary survivor. However, until we know more about the digestion and absorption of dietary wax esters and the turnover of tissue wax esters in individual midwater animals, we cannot decide what portion, if any, of their carbon chains m a y have passed along the food web unchanged.

Acknowledgements. We thank Dr. A. W. Ebeling, Department of Biological Sciences, University of California, Santa Barbara, USA, for our participation in the t966--1967 cruises of the R. V. "Swan" (ship time financed by NSF grants GB2867 and GB-4698 to UCSB) and for identification of the lantern fish used; Dr. T. Kasuka, Ocean Research Institute, Tokyo University, Japan, and Dr. Y. Komaki, Department of Fisheries, Tokyo University (present address: Nihonkai Regional Fisheries Research Laboratory) for the joint cruise of R. V. "Tansei Maru"; Drs. M. Omori and H. Kawaguchi, Ocean Research Institute, Tokyo University, for identification of respectively, the crustaceans and the gonostomatid fishes used. We are grateful for the encouragement of Professor J. F. Mead, Laboratory of Nuclear Medicine and ]~adiation Biology, UCLA, and Professor Y. Hashimoto, Department of Fisheries, Tokyo University; and for many helpful discussions with Dr. J. Hirota, Scripps Institution of Oceanography, during the preparation of this manuscript. The work was partly supported by a grant from the Ministry of Education of Japan to MK (Title No. 786036) and by contract AT(04d) GEN-12 between the U. S. Atomic Energy Commission and the University of California. 3 Injection was made via the anus and out through the wall of the lower intestine.

Vol. 24, No. 4, 1974

M. Kayama and J. C. Nevenzel : Lipids of Midwater Animals

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Date of final manuscript acceptance: November 23, i973. Communicated by J. Bunt, Miami