Lipids of midwater marine fish: family gonostomatidae

Comp. Biochem. Physiol., Vol. 6511, pp. 351 to 355 Pergamon Press Ltd 1980. Printed in Great Britain

LIPIDS OF MIDWATER MARINE FISH: FAMILY GONOSTOMATIDAE JUDD C. NEVENZEL Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093, U.S.A. and NIRMALA K. MENON Lab. Nuclear Medicine and Radiation Biology, University of California, Los Angeles, Los Angeles, CA 90024, U.S.A.

(Received 1 May 1979) Abstract--1. Six species of marine teleost fishes of the family Gonostomatidae contained 3,1-30% dry wt lipid. 2. Wax esters were major neutral lipids in four species; a bathypelagic species, Cyclothone acclinidens, contained the highest percentage (44%), while a species caught at the surface, Vinciguerria lucetia, contained only traces. 3. The main wax esters were even numbered C3o-C42 components, most with one double bond. 4. The structures of the monoenoic alcohols and acids of C. acclinidens wax esters were consistent with Ag-desaturation of the saturated Ct6, Cls and C2o homologs, followed by chain elongation by one or two steps.

INTRODUCTION In terms of numbers of individuals, the lightfishes or bristlemouth fishes, family Gonostomatidae, of the middle depths of the open oceans are the most abundant vertebrates on earth (Fitch & Lavenberg, 1968; Mukhacheva, 1954; Beebe & Vanderpyl, 1944). However, because of their small size (to 11 cm in length, maximum fresh wt about 3 g) their biomass is less impressive. They have so far not been the objects of a major fishery. Pursuing our interest in the occurrence of wax esters in marine animals (Nevenzel et al., 1965, 1966, 1969, 1970) we found that this lipid type is an important component in the lipids of four species of bristlemouths from off the California coast. Two additional species investigated contained less than 4.5% of this lipid type. The variability is consistent with the literature values and is discussed below. MATERIAL AND METHODS The specimens were collected as follows: Cyclothone pallidae and C. pseudopallidae from midwater trawls in the Santa Cruz Basin (33°50'N, 119°30'W) in 1966, identified by R. J. Lavenberg. Most of the C. acclinidens and C. signata individuals used were collected in trawls to 300 m off Guadalupe Island, Baja California, Mexico (29°N, 118°20'W) in February/March, 1970, identified by R. Wisner. Danaphos occulatus and Vinciguerria lucetia were also collected off Guadalupe Island in daylight trawls to 300 m; the latter could also be dipnetted at the surface at night. Both species were identified by M. S. Gordon and B. B. Gordon. The lipids were extracted from the various specimens and fractionated by adsorption column chromatography on silicic acid (J. T. Baker Silica Gel, Powder) and the fatty acids and long-chain alcohols were analyzed by gas liquid 351

chromatography (GLC) using methods previously described (Nevenzel etal., 1965). For the GLC analyses of native wax esters a 1.02 m × 3 mm i.d. column of JXR (a methyl silicone polymer; 1.97% by weight on 60/80 mesh Gas-Chrom Q, both products of Applied Science Laboratories, Inc., State College, PA) was operated isothermally at 260°C and 1.05 kg/cm 2 argon carrier gas in a BarberColman Model 10 instrument fitted with a flame ionization detector. Separations on the JXR column were similar to those described previously for OV-1 (Ohio Valley Specialty Chemical, also a methyl silicone polymer; cf. Lee etal., 1971), so that wax esters of the same number of carbons emerged together, with little or no separation according to the number of double bonds. Where only a few milligrams of total lipid were available, quantitative estimation of some of the component lipid types present was made by densitometry, after TLC separation and precisely controlled charring (Fewster et al., 1969). When the total lipid extracts of C, pallidae, C. pseudopallidae and V. lucetia were injected into the gas chromatographic apparatus under conditions suitable for wax ester analyses the prodominant component had a retention time very near that for the C3o wax ester. In order to eliminate interference from cholesterol (which is not resolved from the C3o wax ester in this GLC system; cf. Lee et al., 1971) samples of a milligram or less of crude lipids from the first two species were fractionated on a micro column of silicic acid (0.27 x 20 cm, made from a l ml disposable glass pipette; one column vol about 0.9 ml), collecting separately the wax esters and cholesterol. We also determined the structures of the major longchain alcohol and fatty acids moieties of the wax esters of Cyclothone acclinidens. An attempt to first separate the alcohols according to the degree of unsaturation, using the mercuric acetate adducts of the trifluoroacetates in a modification of the preparative TLC method developed by Pohl (1969) for methyl esters, was unsatisfactory because the recoveries were low, By direct preparative GLC of the trifluoroacetates in the Varian Autoprep Model 700 (Varian

352

JUDD C. NEVENZELand NIRMALAK. MENON Table 1. Lipid compositions of gonostomatid fishes

Species

Cyclothone acclinidens

Cyclothone Cyclothone Cyclothone Danaphos signata pallidae pseudopallidae occulatus

7ineiguerria lucetia

Lipid as % of: wet wt

4.2

4.4

1.7

4.9

4.8+1.3

1.6+0.2

dry wt

30 + 2

25.2

3.1

6.5

18.2+2.2

8.3+0.4

Component lipid types : Hydrocarbons

.

.

.

.

.

.

.

.

.

.

2.3

0.8

Wax esters a

44.1

37,7

Triacylglycerols

.

.

.

% of total lipid . . . . . . . . . . . . . . .

.

19 -+ 3b

1.4

5

17 + 2b

4.2

5

38 + 3b

37.3

33.3

62 b

55.0

17

Polar lipids

9.8

6.8

-

19.3

37

Phospholipids

6.5

21.4

-

20. i

36

"Contain some sterol (cholesteryl)esters. b By quantitative TLC. Aerograph, Walnut Creek, CA) using a 1.52 x 0.95cm column of diethylene glycol succinate polymer (DEGS; 10~o by weight on 80/100 mesh Chromosorb W) at 175°C and 1.76kg/cm2 He, it was possible to collect a minimum of 2 mg of each of the major monoenes. The positions of the double bonds were determined by ozonolysis, followed by reductive cleavage of the ozonides with triphenyl phosphine and identification (and semiquantitation) by GLC of the aldehydes--from the methyl end of the original chain--or the aldehyde-esters--from the hydroxyl end (Stein & Nicolaides, 1962): O CH 3(CH2)m--CH~CH---(CH 2).--CH 2--O--C--CF3

species living above 250 m (although vertical migrators into this zone are included); known benthic species such as the flatfishes (halibut, soles etc.), macrurids, Moridae etc. or non-teleosts such as sharks. Note that C. pallidae and C. pseudopallidae were also analyzed by Kayama & Ikeda (1975) and that agreement between the two laboratories is poor for wax ester contents: 19 vs 40~o and 17 vs 50~o for the respective species. Several factors could account for these differences: (1) size, therefore age, of the animals

(a) O~ (b) ¢3 P

0 CHs---(CH2)m--CH~O aldehyde

RESULTS

The lipid contents and compositions of six species are given in Table 1. The amounts of total lipid found (1.6-4.9~ wet wt, 3.1-30~ dry wt) are consistent with the values reported by others of 1.1-6.0~o wet wt (Culkin & Morris, 1970; Kayama & Ikeda, 1975) or 9-29.4~ dry wt (Lee, Hirota & Barnett, 1971; Childress & Nygaard, 1973; Lee & Hirota, 1973). Polimanti's 1915 values for Cyclothone microdon of 0.035~o wet wt and 0.27~ dry wt seem too low. For meso- and bathypelagic fish of other families regularly captured in midwater trawls the range of fat contents is equally wide: 0.9-27~ wet wt, 1.5~4~o dry wt, for myctophids (lantern fishes), Sternoptychidae (hatchet fishes) and some 18 additional families (Ackman et al., 1972; Butler & Pearcy, 1972; Childress & Nygaard, 1973; Culkin & Morris, 1970; Kayama & Ikeda, 1975; Lee & Hirota, 1973; and Nevenzel et al., 1969). Not considered are values for large predators such as tunas, the castor oil fish, the coelacanth, the oreostomatid Allocytus verrucosus or the opah; epipelagic

\

+ ~CH--(CH2)n--CH2--O--C--CF3. aldehyde-ester

analyzed; (2) seasonal variations; (3) general nutritional state; (4) stomach contents, since Lee et al. (1971) and DeWitt & Cailliet (1972) reported that copepods were major food items found in gonostomatid stomachs, and the deep-living calanoid copepods are rich in wax esters (Lee et al., 1971) and (5) the possibility that two sub-populations are involved, in view of the 4500 nautical miles between the collecting sites. Analyses of the wax esters of five species are given in Table 2. The GLC pattern for Vinciguerria lucetia total crude lipid showed one main peak of "C30" (92~o), several small peaks corresponding nearly to C24 to C2a and no Ca2 or higher components. We conclude that at best there were only traces of wax esters in this species. The 5~o "wax esters" reported in Table 1 was largely eholesteryl esters (not detectable by the GLC technique we used). The "C3o wax ester" observed was free cholesterol. In the other species C32 to C42 were main constituents, again in the normal range for marine teleosts

Lipids of gonostomatid fish

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JUDD C. NEVENZEL and NIRMALAK. MENON

354

Table 4. Alcohols of gonostomatid wax esters

Cyclothone Cyclothone Danaphos acclinidens signata occulatus

Species

Component

Weight %

14:0

a

b

c

5.9

10.7

7.4

15:0

1.5

1.0

2.1

16:0

39.1

49.7

47.0

16:1

2.1

17:0-br 17:0

-

4.3

1.2

-

-

i.i

1.6

17:1

1.2

1.2

18:0

3.4

6.0

6.3

18:1

7.7

3.9

4.8

18:2

0.4

-

4.5

20:0

0.7

i.i

3.0

20:1

9.1

2.8

4.1

20:4

tr

-

6.4

22:1

18.1

8.3

6.6

22:6

0.5

-

24:1

4.3

11.2

1.2 ND

Additional components included 15:0-br, 0.9~o, 16:0-br 0.6~o, 18:0-br, 0.6~o and 23:1 1.1~o. b Additional components: 15:1 1.2~oand 22:0 0.8~o. c Additional component: 22:2 0.8~o. (Sargent et al., 1976). These wax ester chain lengths were confirmed by the component alcohol and fatty acid compositions, Tables 3 and 4. Taken together, however, the three Tables point up some deficiencies Table 5. Structures of the monoenes of Cyclothone acclinidens wax esters Moiety Position of double bond

ALCOHOL

FATTY ACID

Relative amount

Relative amount

100%

100%

Carbon number 16:1

A

18:1

A

9 9

Al l 9

20:1

A

22:1

A ll 9 A

64.1

80.4

35.9

19.6

81.1

17.5

18.9

82.5

Al l A 13 24:1

A 13

-

21.5

+

62.0

+ i00

- =not detected, + =present.

16.4 (24:1 fatty acid not detected)

of the data. Our GLC method lacked the sensitivity and instrumental stability to detect the low, wide peaks of small amounts of the long retention time C44 wax esters; and in the case of C. signata we inadvertently stopped the run before 22:6 methyl ester emerged. Assuming random combination of alcohols and acids, at least 1.2~o of C44 would be expected in both C. acclinidens and C. signata. The positions of the double bonds in the monounsaturated alcohols and acids of C. acclinidens wax esters are summarized in Table 5. Also, for C. acclinidens only, the phospholipids were separated by two-dimensional TLC (Parsons & Patton, 1967); phosphatidyl ethanolamine was the major phospholipid, with lesser amounts of phosphatidyl choline, phosphatidyl inositol, phosphatidyl serine and phosphatidic acid. DISCUSSION

The major point we make is that wax esters are the principal energy reserve of those gonostomatid fish living below 250 m (Cyclothone acclinidens and C. signata), whereas epipelagic species have mostly triacylglycerols with lesser amounts of wax esters. Indeed, in this work the one species present at the surface, Vinciguerria lucetia, contained only traces of wax esters, while the deepest living species, C. acclinidens (DeWitt, 1972) contained the highest percentage of this lipid type. Although using different terminologies, the literature (Kayama & Ikeda, 1975; Lee et al., 1971) is essentially in agreement that shallow-living gonostomatids (epipelagic species) contain little or no wax esters; deeper living species which migrate into this epipelagic zone to feed (usually at night) contain at least 10-29~o wax esters; and the deepest-living species so far analyzed, which do not undertake diel vertical migrations and remain below the epipelagic zone, contain the highest proportion of wax esters, 45-70~o of their total lipids. These relationships are further support for the theory first clearly stated by Lee et al. (1971) that wax esters provide an energy reserve for marine animals living in environments such as the deep oceans or boreal waters where food may be widely scattered temporally and spatially. Laboratory work with marine invertebrates has made it clear that wax esters are mobilized much more slowly than are triacylglycerols (Sargent et al., 1977; Lee & Barnes, 1975), in part because all known animal lipases hydrolyze wax esters at no more than a quarter of the rate at which triacylglycerols are hydrolyzed (Patton et al., 1975). Finally, the possibility that dietary wax esters could be the direct source of this lipid type in gonostomatids is unlikely, since (a) Kayama & Nevenzel (1974) showed that at least some gonostomatids (Cyclothone atraria and Gonostoma gracile) biosynthesized wax esters from fatty acid precursors and (b) normal digestion and absorption of this lipid involves an initial hydrolysis to fatty acids (which pass across the gut wall) and alcohols, which appear as the corresponding fatty acids in the blood. That is, the alcohol moieties of the original dietary wax esters are oxidized during absorption and hence are not available to the predator for wax ester biosynthesis (Patton & Benson, 1975).

Lipids of gonostomatid fish The structures found for the m o n o e n e s (Table 5) are similar to those found in other marine lipids (Calanus plumchrus alcohols, Lee & Nevenzel, 1979; Anthopleura elegantissima fatty acids, Blanquet et al., 1979; capelin alcohols a n d fatty acids, Pascal & Ackman, 1976; etc.), differing only in the relative proportions of the various isomers. Apparently the saturated acids t h r o u g h C2o serve as substrates for a Ag-desa turase, b u t chain elongation of the monoenes from 16:1 through 22:1 is also observed. Also from ozonolysis, b o t h 20:4 (n-3) and 20:4 (n-6) were present, the first being the principal isomer.

Acknowledgements--Laboratory work was completed in 1970-72, while both authors were employees of the Laboratory of Nuclear Medicine and Radiation Biology, University of California, Los Angeles under Contract AT(04-1)GEN-12 between the (then) U.S. Atomic Energy Commission and the University of California. We are grateful to R. J. Lavenberg, Los Angeles County Museum of Natural History, for animals from the Jay M. Savage and John S. Garth mid-water ecology project, financed by NSF Grants G-10691 and G-23647 to the University of Southern California and to M. S. Gordon, Dept. of Biology, UCLA for the opportunity to participate in the 1970 R/V Alpha Helix Guadalupe Cruise, supported by NSF Grants GB-5661 and GB-15180 and by the Zoology-Fisheries Program, UCLA. R. L. Wisner, Scripps Institution of Oceanography, identified some species.

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355

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