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The distribution of saturated and isoprenoid fatty acids in the lipids of three species of molluscs, Littorina littorea, Crassostrea virginica and Venus mercenaria
Comp. Biochem. Physiol., 1971, Vol. 39B, pp. 579 to 587. Pergamon Press. Printed in Great Britain
T H E D I S T R I B U T I O N OF SATURATED AND ISOPRENOID FATTY ACIDS IN T H E LIPIDS OF THREE SPECIES OF MOLLUSCS, L I T T O R I N A LITTOREA, C R A S S O S T R E A VIRGINICA AND VENUS MERCEN.4RIA R. G. ACKMAN, S. N. H O O P E R and P. J. KE* Fisheries Research Board of Canada, Halifax Laboratory, Halifax, Nova Scotia (Received 17 November 1970)
Abstract--1. Marine molluscs (periwinkle, oyster, quahaug) were found to contain isoprenoid, iso, anteiso and normal odd-numbered fatty acids qualitatively similar to those in higher aquatic life forms. 2. In all three species the branched-chain and normal odd-numbered fatty acids tended to be quantitatively associated with trends in the neighbouring normal even-numbered fatty acids, specifically with myristic, palmitic and stearic acids. 3. Periwinkle lipids had very little phytanic or pristanic acids, suggesting an adaptation to rapidly degrade ingested phytol. The obvious degradation product was 4,8,12-trimethyltridecanoic acid. INTRODUCTION THE MINOR saturated fatty acids occurring in lipids of aquatic animals have been studied (Ackman & Sipos, 1955 ; Ackman & Hooper, 1958, 1970). Fish and marine mammal oils showed only minor differences in proportions of iso and anteiso fatty acids, suggesting formation by more primitive organisms in the food chain. The proportions of the three common isoprenoid fatty acids 4,8,12-TMTD (4,8,12trimethyltridecanoic), pristanic (2,6,10,14-tetramethylpentadecanoic) and phytanic (3,7,11,15-tetramethylhexadecanoic), were determined for marine mammal samples and for fish fairly high in the aquatic food web (Ackman & Hooper, 1958). Fresh-water fish oils did not show any sharp distinction from the marine fish results (Ackman & Hooper, 1970). At a lower trophic level the larger planktonic crustacea (krill) may ingest unicellular plants or be partially or wholly carnivorous, depending on species, on the stage in the life of the animal, or on food availability (Mauchline & Fisher, 1969). Recent results from three krill species (Ackman et al., 1970; Hansen & Meiklen, 1970) were also not strikingly dissimilar in phytanic indext or phytanic acid diastereoisomer ratio 3L, 7D, 10D/3D, 7D, 10D in comparison with higher life forms. * Present address: Chemistry Department, University of Windsor, Windsor, Ontario, Canada. I" Phytanic index = 100 x wt (%) 4,8,12-TMTD (or pristanate) wt (%) phytanate 579
580
R. G.
ACKMAN, S. N. HOOPER AND P. J. KE
We have now extended these investigations to molluscs. Starved filter-feeding oysters and quahaugs both conformed to the previously known pattern for the proportions of the three isoprenoid acids, but a freshly collected grazing herbivore, the c o m m o n periwinkle, showed an exceptionally high proportion of 4 , 8 , 1 2 - T M T D . Other fatty acid data are presented to amplify knowledge of the fatty acid pattern in marine invertebrates, especially for the periwinkle. MATERIALS AND METHODS Animals examined were of the following origins :
Periwinkle (Littorina littorea): A. Collected 18 December 1967 at Terrence Bay, N.S., and sacrificed 19 December 1967. B. Collected 25 May 1967 at Black Rock, Halifax Harbour, N.S., and sacrificed 27 May 1967. Oyster (Crassostrea virginica): Collected in the fall of 1966 near Ellerslie, P.E.I., and sacrificed on 2 October 1967. Bay quahaug (Venus mercenaria): Collected in May of 1966 near Ellerslie, P.E.I., and sacrificed 10 October 1967. All animals were held in filtered sea water in dimly lit tanks. Periwinkles were confined in nylon net bags. Random samples were taken to give approximately 100 g of meat for the analysis. Extractions of animals were carried out on total wet organic tissue (excluding the operculum in the periwinkles) with chloroform-methanol (Bligh & Dyer, 1959). Polar lipids and triglycerides were separated on and recovered from a divinylbenzene copolymer bead gel column (Sipos and Ackman, 1968). The balance of the lipid (sterols, sterol esters, hydrocarbons, etc.) was not examined. Removal of unsaponifiable materials from total lipids, preparation of methyl esters (by MeOH-BF3 esterification of acids, or by transesterification of lipids), all GLC operations (packed or open-tubular columns), and isolation of saturated fatty acids by Florisil-AgNO3 followed procedures described previously (Sipos & Ackman, 1968 ; Ackman& Hooper, 1970). Presentation of GLC data to two decimal places is solely to show small proportions of minor components. Accuracy is estimated at + 5 per cent for large (> 10 per cent components), but may not be better than + 30 per cent for small (< 0"1 per cent) components. Interfering compounds appeared to be associated principally with phospholipids and may be derived from plasmalogens. Some were apparently artifacts promoted or concentrated by the AgNO3-Florisil separation of saturated fatty acids. There were distinguished from methyl esters of fatty acids by GLC analysis on different polarity columns, and were usually removed by the urea complex treatment for concentration of isoprenoids. Where apparent they did not affect the proportionation of the other two isoprenoid fatty acids based on the easily recognizable 4,8,12-TMTD. RESULTS AND D I S C U S S I O N T h e low recovery of lipid for the oysters and quahaugs (Table 1) reflects the extended holding period for these animals (compare Ackman & Cormier, 1967). H o w e v e r the substantial residue of triglyceride indicates that the depletion of lipids is not especially abnormal in view of seasonal limitations on food supply and winter lowering of water t e m p e r a t u r e in the original habitat in Prince Edward Island (compare Ansell et al., 1964, for additional quahaug data). T h e gravimetric isolation of saturated methyl esters by c h r o m a t o g r a p h y of total methyl esters f r o m lipid fractions on Florisil impregnated with silver nitrate shows that in oysters and quahaugs the triglycerides contained only one-third of the saturated fatty acids found in the polar lipids (presumably mostly phospholipids)
581
FATTY ACIDS IN THE LIPIDS OF MOLLUSCS
T A B L E 1 - - L I P I D RECOVERIES (PER CENT OF WET W E I G H T OF ORGANIC TISSUE)~ AND PROPORTION OF TRIGLYCERIDES AND POLAR LIPIDS IN TOTAL L I P I D FOR THE THREE SAMPLES OF MOLLUSCS STUDIED
Lipid composition T o t a l lipid
Oysters Quahaugs D e c e m b e r periwinkles May periwinkles
Non-saponifiable Triglycerides
Polar lipids
wt (%)
wt (%)
wt (%)
wt (%)
0"37 0-47 2-35 1-62
--11"0 12"6
28 12 -76
50 68 -6*
* S u s p e c t value, see text.
(Table 2). In retrospect it appears possible that in these lipid-depleted samples the "triglyceride" may in fact contain a proportion of glyceryl ethers in which the glycerol l-position, which frequently contains a saturated acid (Brockerhoff et al., 1963), is occupied by the ether linkage. For these two molluscs there is a gradual differential proportionation of fatty acids from emphasis on 14:0 in the triglyceride to 18:0 in the polar lipids, but in most other respects triglycerides and polar lipids show little difference in the respective species. In an interspecies comparison the proportion of iso and anteiso 17:0, but not of the corresponding 15:0 acids, is higher in the quahaug. This is probably part of a trend towards additional normal 17:0 and 18:0 in the quahaug. These effects are balanced by the presence of smaller amounts of 14:0 and 15:0 in the quahaugs, but also partly by the approximately 40 per cent 16:0 in the quahaug in contrast to the approximately 64 per cent of the oyster lipids. Some suspicion attended the presence in the quahaug of the iso 16:0 and iso 18:0 components which would coincide on butanediol-succinate polyester with the degradation product of the dimethyl acetals of 16:0 and 18:0 aldehydes, these originating in plasmalogens (Ackman, unpublished studies; Viswanathan, 1968). However quantitation on Apiezon-L verified these as fatty acid methyl esters. The isoprenoid fatty acids in oysters and quahaugs (Table 3) show no unusual features when compared with other available marine lipid data (Ackman & Hooper, 1968; Ackman et al., 1970). The higher proportion of 4,8,12-TMTD in the triglycerides relative to polar lipids reflects the behaviour of this acid in lipid metabolism in association with 14:0 (compare Table 2). If the basic biochemistry by which vertebrates preferentially form the LDD diastereoisomer of phytanic acid from phytol (Baxter & Milne, 1969) can be extended to invertebrates (Ackman et al., 1970; compare capelin discussion in Ackman & Hooper, 1968), then the low ratio of LDD to DDD phytanate in the quahaug may reflect the lipid depletion. Seasonal variation studies currently in progress show that oysters taken from the natural summer environment show phytanic indices as high as 2000 for 4,8,12T M T D , but usually accompanied by pristanate at approximately the phytanate level.
582
R.G.
ACKMAN, S. N. HOOPER AND P. J. KE
T A B L E 2 - - C O M P O S I T I O N OF SATURATED FATTY ACIDS [IN w t (°."o)] ISOLATED FROM LIPIDS OF OYSTERS, QUAHAUGS AND PERIV¢INKLES~ AND CHAIN LENGTH DISTRIBUTION OF TOTAL FATTY ACIDS FOR TWO LOTS OF P E R I W I N K L E S
Oysters Triglycerides isolated saturates = 6'1% of esters
Polar lipids isolated saturates = 20"8% of esters
Fatty acid 12:0 Iso 13:0 13:0 Iso 14:0 14:0 4,8,12-TMTD Iso 15:0 Anteiso 15:0 15:0 Iso 16:0 Pristanic 16:0 Iso 17:0 Anteiso 17:0 17:0 P hytanic Iso 18:0 18:0 19:0 20:0 21:0 22:0 iso (°'o) anteiso (%) Ratio--iso: anteiso linear odd numbered
(%)
. . . . 12-10 1.26 0'63 0-12 2-85 0'67 0'05 67.68 1.24 0.93 3.88 0- 31 0.38 * 7-23 0-18 0.49 . . 2-92 1.05 2.78 6-91
Quahaugs
. . . .
. .
Triglycerides isolated saturates = 4'8°0 of esters
. . . . 6"12 1'30 0"47 0"05 3'20 0'61 0'71 61 "49 1'90 0"89 5"46 1"51 0"99 * 15'17 0'12 -. . 3"97 0.94 4-22 8'78
Polar lipids isolated saturates -- 13"3% of esters
. . . . 6-02 3"63 0"72 0"36 2'03 4'06* 0'87 44"38 10"22 6'60 3"84 1"30 2"17 * 13'78 ---
1.46 1"09 0"56 0"07 1"82 4'48* 0"30 40'57 10'53 8'19 7'09 1-01 1-96 20"12 0'15 0'21
17"17 6'96 2-47 5'87
17"53 8-26 2-12 9"06
. .
* Verified on both polar and non-polar liquid phases. T h e p e r i w i n k l e l i p i d y i e l d s ( T a b l e 1) c o r r e s p o n d closely to values r e c o r d e d for t h e s a m e m o n t h s ( W i l l i a m s , 1970) if d r y w e i g h t of m e a t is t a k e n (as an a p p r o x i m a tion) as 20 p e r c e n t of w e t w e i g h t . T h e s e are t h e r e f o r e n o r m a l values for t h i s species, a l t h o u g h l i p i d levels are v a r i a b l e a n d sensitive to food i n t a k e ( c o m p a r e A c k m a n & C o r m i e r , 1967). T h e f a t t y acid c o m p o s i t i o n in t e r m s o f total c h a i n l e n g t h for t h e t w o p e r i w i n k l e l i p i d e x t r a c t s are c o m p a r e d in T a b l e 2. T h e r e are m i n o r s y s t e m a t i c differences, as in t h e h i g h e r levels of 14:0, 15:0 a n d 16:0 in t h e D e c e m b e r s a m p l e , b u t it is likely t h a t this is s i m p l y d u e to a h i g h e r p r o p o r t i o n of t r i g l y c e r i d e
FATTY ACIDS IN THE LIPIDS OF MOLLUSCS TABLE
583
2--(Cont.)
12:0 Iso 13:0 13:0 Iso 14:0 14:0 4,8,12-TMTD Iso 15:0 Anteiso 15:0 15:0 Iso 16:0 Pristanic 16:0 Iso 17:0 Anteiso 17:0 17:0 Phytanie Iso 1 8 : 0 18:0 19:0 20:0 21:0 22:0 Unidentified iso (%) anteiso (%) Ratio---iso : anteiso linear odd numbered (%)
December periwinkles
May periwinkles
Total lipids hydrogenated
Total lipids hydrogenated
Triglycerides isolated saturates= 10% of esters
Polar lipids isolated saturates= 17°./oof esters
0.16 -0.10 -6.72 2.20 0.32 0.08 0,69 0.25 0"15 17"79 1"31 0.49 1"01 0.11 0.06 25.98 0.52 32.55
---
0-52 0.22
0"02 --
--
--
-2-77 2.06 0"17 0"08 0"36 0"56 0'06 15"29 0"75 0"25 0"70 0.02 Trace 24.94 0"39 39.07 0.18
0"21
0"18 13"67 11"19 0"38 0"10 1.35 0"40 0"25 50"38 3"38 1'54 1"52 0.08 0-36 12.31 0.68 0.29 --
--
0"03 7-88 7.24 0"21 0-12 1.00 0"28 0"13 37"14 1"57 0"79 2"53 0.04 0-25 33.60 0"08 0'18 --
9-51 --
10.49 --
-1.55
-7.32
2"54 0"57 4.46 2"32
1.48 0.33 4.48 1-63
4.92 1-64 3-00 3"76
2"34 0"91 2"57 3"43
rich in these acids. T h e non-saponifiable materials in the two samples suggest a December triglyceride-rich lipid (by the method Ca-6b-53 of the American Oil Chemists' Society). T h e lipid material balance, based on the data of Table 1, suggests that the May value for polar lipid (presumably mostly phospholipid) may be too low, and should be 10-12 per cent which would correspond more closely to phospholipid levels anticipated for cellular lipid ( ~ 0.2 per cent) as distinct from depot lipid. T h e value of 6 per cent could arise from carry-over of phospholipid into the triglyceride during lipid fractionation, or to lipid hydrolysis before fractionation.
584
R . G . ACKMAN, S. N. HOOPERAND P. J. KE TABLE 3--DETAILS
OF ISOPRENOID FATTY ACID PROPORTIONS AND PHYTANIC ACID DIASTEREOISOMERISM
Isoprenoids as °o of total %of saturated acids acids
Phytanic indices 4,8,12TMTD
Pristanic
Phytanic acid LDD/DDD ratio
Oysters Triglycerides Polar lipids
0"10 0'71
1"6 3'5
400 86
16 47
2"7 8"5
Quahaugs Triglycerides Polar lipids
0"27 0'31
5"8 2"3
280 107
67 29
0-9 0"6
December periwinkles Total lipid
2"15
--*
11,000
370
4'3
May periwinkles Triglycerides Polar lipids
1' 13 1"26
11' 5 7'4
14,000 18,000
310 320
4.4 2"3
* Sample hydrogenated, data not available. The common normal, iso, and anteiso saturated fatty acids isolated from periwinkle triglycerides and polar lipids (Table 2) follow the same trends as the oyster and quahaug acids in terms of proportion of lipid and in the trends for fatty acids with chain length. The isoprenoid fatty acids were dramatically unbalanced because of the accumulation of 4,8,12-TMTD both in absolute terms (Table 2) and relative to pristanic and phytanic acids (Table 3). It can only be assumed that the periwinkle has a very high intake of phytol because of its purely herbivorous diet. The oyster and quahaug ingest, as a proportion of their diet, particulate matter with possibly no phytol at one extreme or whole algae at the other extreme (Galtsoff, 1964). Probably the periwinkle prevents accumulation of phytol or phytanie and pristanic acids by degrading phytol via trans-2-phytenic acid, or an intermediate pristanic acid, to 4,8,12-TMTD, which then participates in the saturated fatty acid pool with 14:0. The observation that the L D D / D D D ratio for the periwinkle phytanate is not especially high may have an as yet unproven significance. The crustacean Euphausia superba has an L D D / D D D ratio of 18, and it is thought to be purely herbivorous, whereas two crustacea with more diverse diets have ratios of 2.2-3.4 (Ackman et al., 1970). Molluscs therefore apparently do not form L D D phytanic acid specifically from phytol via trans-2-phytenic acid (Baxter and Milne, 1969). To further explore the dietary intake of the periwinkle the fatty acid composition of the May sample lipids was determined by packed-column G L C (Table 4). The total lipid analysis includes fatty acids from sterol esters and other lipids not
FATTY ACIDS IN THE LIPIDS OF MOLLUSCS
585
T A B L E 4 - - - F A T T Y ACID COMPOSITIONS OF LIPIDS FROM MAY SAMPLE OF PERIWINKLES I N WT. °,o
Fatty acid
Total Polar lipid Triglyceride lipids
12:0 13:0 14:0" 14:1 t 15:0 Pristanic, etc. 16:0 16:1 16:2oJ7 (?) 16:2co4~ 16:3oJ4§ 16:4col 17:0
17:1 18:0 18:1 18:2oJ6 18:3t06 18:3oJ3 18:4oJ3 19:0 19:1
0"2 0"1 4"6 0"4 0"3
0.1 Trace 5.1 0"5 0"3
0.2 0.1 4"2 0-3 0"4
Trace 10.0 5.6 0"2 0"9 0"8 1"0 0"4 0"1 4"0 11"2 5"3 0'4 4"0 2"2 ---
0.1 10.2 8-6 0"2 0"9 0"8 0"9 0"4 0"1 3"8 15"0 6"1 0-4 4"6 2"5 Trace --
Trace 9.2 2"7 0"2 0"9 0"3 1"6 0"4 0"2 7"3 4"8 4"0 0-4 2"9 1"2
Fatty acid
Total lipid Triglyceride
20:0 20:1 20:2oJ6 20:3oJ6 20:4m6
-12.2 3.7 Trace 5"7
Trace 13.6 2"3 -3"3
20:4o~3 20:5o~3 21:4~o2 ? 22:0 22:1 22:2oJ6 22:4oJ6 22:5~o6 22:5¢o3 22:6w3 24:1
0"6 16.8 0.1 -5"0 --0"2 0"7 1"4 0"4
0"5 13"7 0.2 -2"9 --0"2 1"2 2"6 0"3
Polar lipids -9.5 5.2 10.0 0.4 22.7 --8'5 ----
1.4 0"8 0"3
--
* Includes 4,8,12-TMTD. t Includes iso and anteiso 15:0. § Includes anteiso 17:0.
++Includes iso
17:0
included in the two isolated lipids. Basically the total lipid fatty acid composition resembles that of the triglyceride modified by that of the polar lipids in some cases. A m o n g details of interest are the moderately high proportions, for a marine lipid, of 18:2oJ6 and 18:3oJ3 (compare, for example, crustacean respective value of ~ 2 and ~ 1 per cent respectively reported by Ackman et al., 1970). T h e two co6 successor series 20:2~o6 and 20:4w6 are presumably associated with a high 18:2xo6 intake. T h e high proportion of 20:5oJ3 in lieu of 22:&o3, especially in the polar lipids, probably represents direct dietary deposition of ingested fatty acids as 20:5co3 is m o r e characteristic of marine algal polyunsaturated fatty acids than is 22:6o~3 (Ackman et al., 1970; see also Brockerhoff et al., 1963). T h e occurrence of 20:1 and 22:1 in the triglyceride and especially in the polar lipid was unexpected on the basis of our work with other species. H o w e v e r there is relatively little 18:1 for a marine lipid and it is not impossible that periwinkles elongate 16:1 or 18:1 precursors to 20:1 and 22:1. As metabolism in molluscs is in a n u m b e r of cases "lipid oriented", considerable activity in lipid biosynthesis could be expected if this were applicable (compare other species discussed by 20
586
R. G. ACKMAN, S. N. HOOPER AND P. J. KE
Stickle and Duerr, 1970). T h e drop in lipid from 4"80 to 1"21 per cent in two weeks of periwinkle starvation could indicate this to be the case for L. littorea (Ackman & Cormier, 1967). T h e matter has been tested experimentally in two terrestrial snails, and in addition to active incorporation of 14C-labelled acetate into fatty acids, the isolation and identification of iso and anteiso fatty acids in chain lengths up to Cz~, C2a or C24 suggests a tendency for these animals to elongate fatty acids of middle chain lengths (van der Horst & Voogt, 1969a, b). A large n u m b e r of species of marine molluscs are reported as having appreciable (2-10 per cent) 20:1 and/or 22:1 fatty acids in total, neutral, and/or polar lipids (Gruger et al., 1964; Shimma & Taguchi, 1964; de Koning, 1966; Tibaldi, 1966; Jeffries, 1967, 1968; Hamada & Ueno, 1968, 1969; Hayashi et al., 1969; Takama et al., 1969). These particular fatty acids are the object of further study in our laboratory. T h e ratio of iso to anteiso acids for all three species of molluscs (Table 2) was not especially different from previously published results for fish lipids (Ackman & Sipos, 1965 : Ackman & Hooper, 1970) or krill (Ackman et al., 1970). This does not help distinguish an exogenous origin in the form of bacterial lipids from an endogenous origin involving fatty acid biosynthesis from amino acid skeletons as starting points. T h e proportions of saturated linear o d d - n u m b e r e d fatty acids in the oysters and quahaugs are higher than previous results for other marine animals but further work is also required to elaborate on the significance of this finding. Acknowledgement--This work was supported in part by the Industrial Development Service of the Department of Fisheries of Canada.
REFERENCES ACKMAN R. G. & CORMIERM. G. (1967) ~-Tocopherol in some Atlantic fish and shellfish with particular reference to live-holding without food. ft. Fish. Res. Bd Can. 24, 357-373. ACK/vIANR. G., EATONC. A., SIPOSJ. C., HOOPERS. N. & CASTELLJ. D. (1970) Lipids and fatty acids of two species of North Atlantic krill (Meganyctiphanes norvegica and Thysano~ssa inermis) and their role in the aquatic food web. ft. Fish. Res. Bd Can. 27, 513-533. ACKMANR. G. & HOOVERS. N. (1968) Examination of isoprenoid fatty acids as distinguishing characteristics of specific marine oils with particular reference to whale oils. Cornp. Biochem. Physiol. 24, 549-565. ACKMANR. G. & HOOPERS. N. (1970) Branched-chain fatty acids of four fresh-water fish oils. Comp. Biochem. Physiol. 32, 117-125. ACKMAN, R. G. & SIPOS J. C. (1965) Isolation of the saturated fatty acids of some marine lipids with particular reference to normal odd-numbered fatty acids and branched-chain fatty acids. Comp. Biochem. Physiol. 15, 445-456. ANSELL A. D., LOOSMOREF. A. & LANDER K. F. (1964) Studies on the hardshell clam, Venus mercenaria, in British water--II. Seasonal cycle in condition and biochemical composition, ft. appl. Ecol. 1, 83-95. BAXTERJ. H. & lklILNEG. W. A. (1969) Phytenic acid : Identification of five isomers in chemical and biological products of phytol. Biochim. biophys. Acta 176, 265-277. BLmH E. G. & DYeR W. J. (1959) A rapid method of total lipid extraction and purification. Can. ft. Biochem. Physiol. 37, 911-917. BROCKERHOFFH., ACKMANR. G. & HOYLER. J. (1963) Specific distribution of fatty acids in marine lipids. Archs Biochem. Biophys. 100, 9-12.
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DE KONINO A. J. (1966) Phospholipids of marine origin IV. The abalone (Haliotis midae). J. Sci. Fd Agric. 17, 460-464. GALTSOFF P. S. (1964) T h e American oyster Crassstrea virginica Gmelin. Fish. Bull. U.S. Dept. Interior No. 64. GaU6Ea E. H., JR., NELSON R. W. & STANSBYM. E. (1964) Fatty acid composition of oils from 21 species of marine fish, freshwater fish and shellfish. J. Am. Oil Chem. Soc. 41, 662-667. HAMADA S. & UENO S. (1968) On the lipids of shells--I. Yukagaku 17, 39-42. HAMADA S. & UENO S. (1969) On the lipids of shells--II. Yukagaku 18, 478-480. HANSEN R. P. & MEIKLEN S. M. (1970). Isoprenoid fatty acids in Antarctic krill (Euphausia superba). J. Sci. Fd Agric. 21, 204-206. HAYASHI A., MATSUBARAT. & MATSUURA F. (1969) Biochemical studies on the lipids of Turbo cornutus--I. Yukagaku 18, 118-123. JEFERIES H. P. (1967) Chemical responses by marine organisms to stress. Phase 1. In
Technical Report No. 1, Graduate School of Oceanography, Narragansett Marine Laboratory, University of Rhode Island, Kingston, R.L, U.S.A. JEEFRIES H. P. (1968) Chemical responses by marine organisms to the quality of the environment. In Proc. Ann. North Eastern Reg. Antipollution Conf., 22-24 July, University of Rhode Island, R.I., U.S.A., pp. 84-93. MAUCHLINE J. & FISHER L. R. (1969) The biology of Euphausiids. In Advances in Marine Biology (Edited by RUSSELL F. S. and YONGE M.), Vol. 7, pp. 174-199. Academic Press, New York and London. SHIMMA Y. ~l; TAGUCHI H. (1964) A comparative study on fatty acid composition of shellfish. Bull. Jap. Soe. Sci. Fish. 30, 153-160. SIPoS J. C. & ACKMANR. G. (1968) Jellyfish (Cyanea capillata) lipids: fatty acid composition. J. Fish. Res. Bd Can. 25, 1561-1569. STICKLE W. B. & DUERR F. (1970) T h e effects of starvation on the respiration and major nutrient stores of Thais lamellosa. Comp. Biochem. Physiol. 33, 689-695. TAKAMA K., ZAMA K . & IGARASHI H . (1969) Lipids in whelk, Neptunea arthritica. Bull.Jap. Soc. Sci. Fish. 35, 1184-1188. TmALDI E. (1966) Richerche preliminari sugli acidi grassi di alcune specia di mollusehi marini. Rend. Accad. Naz. Lined (Sc. Fis.) 40, 921-925. VAN DER HORST D. J. & VOOGT P. A. (1969a) Investigation of the fatty acid composition of the snail Arianta arbustorum. Archs int. Physiol. Biochem. 77, 507-514. VAN DER HORST D. J. & VOOGT P. A. (1969b) Investigation of fatty acid composition of the snail Succinea putris L. Comp. Biochem. Physiol. 31, 763-769. VISWANATHAN C. V. (1968) Chromatographic analysis of plasmalogens. Chromatographic Rev. 10, 18-36. WILLIAMS E. E. (1970) Seasonal variations in the biochemical composition of the edible winkle Littorina littorea (L). Comp. Biochem. Physiol. 33, 655-661.
Key Word Index--Molluscs; fatty acids ; isoprenoid fatty acids ; oysters; quahaugs ; periwinkles; Cr assostrea virginica ; Venus mercenaria ; Littorina litorrea.
T H E D I S T R I B U T I O N OF SATURATED AND ISOPRENOID FATTY ACIDS IN T H E LIPIDS OF THREE SPECIES OF MOLLUSCS, L I T T O R I N A LITTOREA, C R A S S O S T R E A VIRGINICA AND VENUS MERCEN.4RIA R. G. ACKMAN, S. N. H O O P E R and P. J. KE* Fisheries Research Board of Canada, Halifax Laboratory, Halifax, Nova Scotia (Received 17 November 1970)
Abstract--1. Marine molluscs (periwinkle, oyster, quahaug) were found to contain isoprenoid, iso, anteiso and normal odd-numbered fatty acids qualitatively similar to those in higher aquatic life forms. 2. In all three species the branched-chain and normal odd-numbered fatty acids tended to be quantitatively associated with trends in the neighbouring normal even-numbered fatty acids, specifically with myristic, palmitic and stearic acids. 3. Periwinkle lipids had very little phytanic or pristanic acids, suggesting an adaptation to rapidly degrade ingested phytol. The obvious degradation product was 4,8,12-trimethyltridecanoic acid. INTRODUCTION THE MINOR saturated fatty acids occurring in lipids of aquatic animals have been studied (Ackman & Sipos, 1955 ; Ackman & Hooper, 1958, 1970). Fish and marine mammal oils showed only minor differences in proportions of iso and anteiso fatty acids, suggesting formation by more primitive organisms in the food chain. The proportions of the three common isoprenoid fatty acids 4,8,12-TMTD (4,8,12trimethyltridecanoic), pristanic (2,6,10,14-tetramethylpentadecanoic) and phytanic (3,7,11,15-tetramethylhexadecanoic), were determined for marine mammal samples and for fish fairly high in the aquatic food web (Ackman & Hooper, 1958). Fresh-water fish oils did not show any sharp distinction from the marine fish results (Ackman & Hooper, 1970). At a lower trophic level the larger planktonic crustacea (krill) may ingest unicellular plants or be partially or wholly carnivorous, depending on species, on the stage in the life of the animal, or on food availability (Mauchline & Fisher, 1969). Recent results from three krill species (Ackman et al., 1970; Hansen & Meiklen, 1970) were also not strikingly dissimilar in phytanic indext or phytanic acid diastereoisomer ratio 3L, 7D, 10D/3D, 7D, 10D in comparison with higher life forms. * Present address: Chemistry Department, University of Windsor, Windsor, Ontario, Canada. I" Phytanic index = 100 x wt (%) 4,8,12-TMTD (or pristanate) wt (%) phytanate 579
580
R. G.
ACKMAN, S. N. HOOPER AND P. J. KE
We have now extended these investigations to molluscs. Starved filter-feeding oysters and quahaugs both conformed to the previously known pattern for the proportions of the three isoprenoid acids, but a freshly collected grazing herbivore, the c o m m o n periwinkle, showed an exceptionally high proportion of 4 , 8 , 1 2 - T M T D . Other fatty acid data are presented to amplify knowledge of the fatty acid pattern in marine invertebrates, especially for the periwinkle. MATERIALS AND METHODS Animals examined were of the following origins :
Periwinkle (Littorina littorea): A. Collected 18 December 1967 at Terrence Bay, N.S., and sacrificed 19 December 1967. B. Collected 25 May 1967 at Black Rock, Halifax Harbour, N.S., and sacrificed 27 May 1967. Oyster (Crassostrea virginica): Collected in the fall of 1966 near Ellerslie, P.E.I., and sacrificed on 2 October 1967. Bay quahaug (Venus mercenaria): Collected in May of 1966 near Ellerslie, P.E.I., and sacrificed 10 October 1967. All animals were held in filtered sea water in dimly lit tanks. Periwinkles were confined in nylon net bags. Random samples were taken to give approximately 100 g of meat for the analysis. Extractions of animals were carried out on total wet organic tissue (excluding the operculum in the periwinkles) with chloroform-methanol (Bligh & Dyer, 1959). Polar lipids and triglycerides were separated on and recovered from a divinylbenzene copolymer bead gel column (Sipos and Ackman, 1968). The balance of the lipid (sterols, sterol esters, hydrocarbons, etc.) was not examined. Removal of unsaponifiable materials from total lipids, preparation of methyl esters (by MeOH-BF3 esterification of acids, or by transesterification of lipids), all GLC operations (packed or open-tubular columns), and isolation of saturated fatty acids by Florisil-AgNO3 followed procedures described previously (Sipos & Ackman, 1968 ; Ackman& Hooper, 1970). Presentation of GLC data to two decimal places is solely to show small proportions of minor components. Accuracy is estimated at + 5 per cent for large (> 10 per cent components), but may not be better than + 30 per cent for small (< 0"1 per cent) components. Interfering compounds appeared to be associated principally with phospholipids and may be derived from plasmalogens. Some were apparently artifacts promoted or concentrated by the AgNO3-Florisil separation of saturated fatty acids. There were distinguished from methyl esters of fatty acids by GLC analysis on different polarity columns, and were usually removed by the urea complex treatment for concentration of isoprenoids. Where apparent they did not affect the proportionation of the other two isoprenoid fatty acids based on the easily recognizable 4,8,12-TMTD. RESULTS AND D I S C U S S I O N T h e low recovery of lipid for the oysters and quahaugs (Table 1) reflects the extended holding period for these animals (compare Ackman & Cormier, 1967). H o w e v e r the substantial residue of triglyceride indicates that the depletion of lipids is not especially abnormal in view of seasonal limitations on food supply and winter lowering of water t e m p e r a t u r e in the original habitat in Prince Edward Island (compare Ansell et al., 1964, for additional quahaug data). T h e gravimetric isolation of saturated methyl esters by c h r o m a t o g r a p h y of total methyl esters f r o m lipid fractions on Florisil impregnated with silver nitrate shows that in oysters and quahaugs the triglycerides contained only one-third of the saturated fatty acids found in the polar lipids (presumably mostly phospholipids)
581
FATTY ACIDS IN THE LIPIDS OF MOLLUSCS
T A B L E 1 - - L I P I D RECOVERIES (PER CENT OF WET W E I G H T OF ORGANIC TISSUE)~ AND PROPORTION OF TRIGLYCERIDES AND POLAR LIPIDS IN TOTAL L I P I D FOR THE THREE SAMPLES OF MOLLUSCS STUDIED
Lipid composition T o t a l lipid
Oysters Quahaugs D e c e m b e r periwinkles May periwinkles
Non-saponifiable Triglycerides
Polar lipids
wt (%)
wt (%)
wt (%)
wt (%)
0"37 0-47 2-35 1-62
--11"0 12"6
28 12 -76
50 68 -6*
* S u s p e c t value, see text.
(Table 2). In retrospect it appears possible that in these lipid-depleted samples the "triglyceride" may in fact contain a proportion of glyceryl ethers in which the glycerol l-position, which frequently contains a saturated acid (Brockerhoff et al., 1963), is occupied by the ether linkage. For these two molluscs there is a gradual differential proportionation of fatty acids from emphasis on 14:0 in the triglyceride to 18:0 in the polar lipids, but in most other respects triglycerides and polar lipids show little difference in the respective species. In an interspecies comparison the proportion of iso and anteiso 17:0, but not of the corresponding 15:0 acids, is higher in the quahaug. This is probably part of a trend towards additional normal 17:0 and 18:0 in the quahaug. These effects are balanced by the presence of smaller amounts of 14:0 and 15:0 in the quahaugs, but also partly by the approximately 40 per cent 16:0 in the quahaug in contrast to the approximately 64 per cent of the oyster lipids. Some suspicion attended the presence in the quahaug of the iso 16:0 and iso 18:0 components which would coincide on butanediol-succinate polyester with the degradation product of the dimethyl acetals of 16:0 and 18:0 aldehydes, these originating in plasmalogens (Ackman, unpublished studies; Viswanathan, 1968). However quantitation on Apiezon-L verified these as fatty acid methyl esters. The isoprenoid fatty acids in oysters and quahaugs (Table 3) show no unusual features when compared with other available marine lipid data (Ackman & Hooper, 1968; Ackman et al., 1970). The higher proportion of 4,8,12-TMTD in the triglycerides relative to polar lipids reflects the behaviour of this acid in lipid metabolism in association with 14:0 (compare Table 2). If the basic biochemistry by which vertebrates preferentially form the LDD diastereoisomer of phytanic acid from phytol (Baxter & Milne, 1969) can be extended to invertebrates (Ackman et al., 1970; compare capelin discussion in Ackman & Hooper, 1968), then the low ratio of LDD to DDD phytanate in the quahaug may reflect the lipid depletion. Seasonal variation studies currently in progress show that oysters taken from the natural summer environment show phytanic indices as high as 2000 for 4,8,12T M T D , but usually accompanied by pristanate at approximately the phytanate level.
582
R.G.
ACKMAN, S. N. HOOPER AND P. J. KE
T A B L E 2 - - C O M P O S I T I O N OF SATURATED FATTY ACIDS [IN w t (°."o)] ISOLATED FROM LIPIDS OF OYSTERS, QUAHAUGS AND PERIV¢INKLES~ AND CHAIN LENGTH DISTRIBUTION OF TOTAL FATTY ACIDS FOR TWO LOTS OF P E R I W I N K L E S
Oysters Triglycerides isolated saturates = 6'1% of esters
Polar lipids isolated saturates = 20"8% of esters
Fatty acid 12:0 Iso 13:0 13:0 Iso 14:0 14:0 4,8,12-TMTD Iso 15:0 Anteiso 15:0 15:0 Iso 16:0 Pristanic 16:0 Iso 17:0 Anteiso 17:0 17:0 P hytanic Iso 18:0 18:0 19:0 20:0 21:0 22:0 iso (°'o) anteiso (%) Ratio--iso: anteiso linear odd numbered
(%)
. . . . 12-10 1.26 0'63 0-12 2-85 0'67 0'05 67.68 1.24 0.93 3.88 0- 31 0.38 * 7-23 0-18 0.49 . . 2-92 1.05 2.78 6-91
Quahaugs
. . . .
. .
Triglycerides isolated saturates = 4'8°0 of esters
. . . . 6"12 1'30 0"47 0"05 3'20 0'61 0'71 61 "49 1'90 0"89 5"46 1"51 0"99 * 15'17 0'12 -. . 3"97 0.94 4-22 8'78
Polar lipids isolated saturates -- 13"3% of esters
. . . . 6-02 3"63 0"72 0"36 2'03 4'06* 0'87 44"38 10"22 6'60 3"84 1"30 2"17 * 13'78 ---
1.46 1"09 0"56 0"07 1"82 4'48* 0"30 40'57 10'53 8'19 7'09 1-01 1-96 20"12 0'15 0'21
17"17 6'96 2-47 5'87
17"53 8-26 2-12 9"06
. .
* Verified on both polar and non-polar liquid phases. T h e p e r i w i n k l e l i p i d y i e l d s ( T a b l e 1) c o r r e s p o n d closely to values r e c o r d e d for t h e s a m e m o n t h s ( W i l l i a m s , 1970) if d r y w e i g h t of m e a t is t a k e n (as an a p p r o x i m a tion) as 20 p e r c e n t of w e t w e i g h t . T h e s e are t h e r e f o r e n o r m a l values for t h i s species, a l t h o u g h l i p i d levels are v a r i a b l e a n d sensitive to food i n t a k e ( c o m p a r e A c k m a n & C o r m i e r , 1967). T h e f a t t y acid c o m p o s i t i o n in t e r m s o f total c h a i n l e n g t h for t h e t w o p e r i w i n k l e l i p i d e x t r a c t s are c o m p a r e d in T a b l e 2. T h e r e are m i n o r s y s t e m a t i c differences, as in t h e h i g h e r levels of 14:0, 15:0 a n d 16:0 in t h e D e c e m b e r s a m p l e , b u t it is likely t h a t this is s i m p l y d u e to a h i g h e r p r o p o r t i o n of t r i g l y c e r i d e
FATTY ACIDS IN THE LIPIDS OF MOLLUSCS TABLE
583
2--(Cont.)
12:0 Iso 13:0 13:0 Iso 14:0 14:0 4,8,12-TMTD Iso 15:0 Anteiso 15:0 15:0 Iso 16:0 Pristanic 16:0 Iso 17:0 Anteiso 17:0 17:0 Phytanie Iso 1 8 : 0 18:0 19:0 20:0 21:0 22:0 Unidentified iso (%) anteiso (%) Ratio---iso : anteiso linear odd numbered (%)
December periwinkles
May periwinkles
Total lipids hydrogenated
Total lipids hydrogenated
Triglycerides isolated saturates= 10% of esters
Polar lipids isolated saturates= 17°./oof esters
0.16 -0.10 -6.72 2.20 0.32 0.08 0,69 0.25 0"15 17"79 1"31 0.49 1"01 0.11 0.06 25.98 0.52 32.55
---
0-52 0.22
0"02 --
--
--
-2-77 2.06 0"17 0"08 0"36 0"56 0'06 15"29 0"75 0"25 0"70 0.02 Trace 24.94 0"39 39.07 0.18
0"21
0"18 13"67 11"19 0"38 0"10 1.35 0"40 0"25 50"38 3"38 1'54 1"52 0.08 0-36 12.31 0.68 0.29 --
--
0"03 7-88 7.24 0"21 0-12 1.00 0"28 0"13 37"14 1"57 0"79 2"53 0.04 0-25 33.60 0"08 0'18 --
9-51 --
10.49 --
-1.55
-7.32
2"54 0"57 4.46 2"32
1.48 0.33 4.48 1-63
4.92 1-64 3-00 3"76
2"34 0"91 2"57 3"43
rich in these acids. T h e non-saponifiable materials in the two samples suggest a December triglyceride-rich lipid (by the method Ca-6b-53 of the American Oil Chemists' Society). T h e lipid material balance, based on the data of Table 1, suggests that the May value for polar lipid (presumably mostly phospholipid) may be too low, and should be 10-12 per cent which would correspond more closely to phospholipid levels anticipated for cellular lipid ( ~ 0.2 per cent) as distinct from depot lipid. T h e value of 6 per cent could arise from carry-over of phospholipid into the triglyceride during lipid fractionation, or to lipid hydrolysis before fractionation.
584
R . G . ACKMAN, S. N. HOOPERAND P. J. KE TABLE 3--DETAILS
OF ISOPRENOID FATTY ACID PROPORTIONS AND PHYTANIC ACID DIASTEREOISOMERISM
Isoprenoids as °o of total %of saturated acids acids
Phytanic indices 4,8,12TMTD
Pristanic
Phytanic acid LDD/DDD ratio
Oysters Triglycerides Polar lipids
0"10 0'71
1"6 3'5
400 86
16 47
2"7 8"5
Quahaugs Triglycerides Polar lipids
0"27 0'31
5"8 2"3
280 107
67 29
0-9 0"6
December periwinkles Total lipid
2"15
--*
11,000
370
4'3
May periwinkles Triglycerides Polar lipids
1' 13 1"26
11' 5 7'4
14,000 18,000
310 320
4.4 2"3
* Sample hydrogenated, data not available. The common normal, iso, and anteiso saturated fatty acids isolated from periwinkle triglycerides and polar lipids (Table 2) follow the same trends as the oyster and quahaug acids in terms of proportion of lipid and in the trends for fatty acids with chain length. The isoprenoid fatty acids were dramatically unbalanced because of the accumulation of 4,8,12-TMTD both in absolute terms (Table 2) and relative to pristanic and phytanic acids (Table 3). It can only be assumed that the periwinkle has a very high intake of phytol because of its purely herbivorous diet. The oyster and quahaug ingest, as a proportion of their diet, particulate matter with possibly no phytol at one extreme or whole algae at the other extreme (Galtsoff, 1964). Probably the periwinkle prevents accumulation of phytol or phytanie and pristanic acids by degrading phytol via trans-2-phytenic acid, or an intermediate pristanic acid, to 4,8,12-TMTD, which then participates in the saturated fatty acid pool with 14:0. The observation that the L D D / D D D ratio for the periwinkle phytanate is not especially high may have an as yet unproven significance. The crustacean Euphausia superba has an L D D / D D D ratio of 18, and it is thought to be purely herbivorous, whereas two crustacea with more diverse diets have ratios of 2.2-3.4 (Ackman et al., 1970). Molluscs therefore apparently do not form L D D phytanic acid specifically from phytol via trans-2-phytenic acid (Baxter and Milne, 1969). To further explore the dietary intake of the periwinkle the fatty acid composition of the May sample lipids was determined by packed-column G L C (Table 4). The total lipid analysis includes fatty acids from sterol esters and other lipids not
FATTY ACIDS IN THE LIPIDS OF MOLLUSCS
585
T A B L E 4 - - - F A T T Y ACID COMPOSITIONS OF LIPIDS FROM MAY SAMPLE OF PERIWINKLES I N WT. °,o
Fatty acid
Total Polar lipid Triglyceride lipids
12:0 13:0 14:0" 14:1 t 15:0 Pristanic, etc. 16:0 16:1 16:2oJ7 (?) 16:2co4~ 16:3oJ4§ 16:4col 17:0
17:1 18:0 18:1 18:2oJ6 18:3t06 18:3oJ3 18:4oJ3 19:0 19:1
0"2 0"1 4"6 0"4 0"3
0.1 Trace 5.1 0"5 0"3
0.2 0.1 4"2 0-3 0"4
Trace 10.0 5.6 0"2 0"9 0"8 1"0 0"4 0"1 4"0 11"2 5"3 0'4 4"0 2"2 ---
0.1 10.2 8-6 0"2 0"9 0"8 0"9 0"4 0"1 3"8 15"0 6"1 0-4 4"6 2"5 Trace --
Trace 9.2 2"7 0"2 0"9 0"3 1"6 0"4 0"2 7"3 4"8 4"0 0-4 2"9 1"2
Fatty acid
Total lipid Triglyceride
20:0 20:1 20:2oJ6 20:3oJ6 20:4m6
-12.2 3.7 Trace 5"7
Trace 13.6 2"3 -3"3
20:4o~3 20:5o~3 21:4~o2 ? 22:0 22:1 22:2oJ6 22:4oJ6 22:5~o6 22:5¢o3 22:6w3 24:1
0"6 16.8 0.1 -5"0 --0"2 0"7 1"4 0"4
0"5 13"7 0.2 -2"9 --0"2 1"2 2"6 0"3
Polar lipids -9.5 5.2 10.0 0.4 22.7 --8'5 ----
1.4 0"8 0"3
--
* Includes 4,8,12-TMTD. t Includes iso and anteiso 15:0. § Includes anteiso 17:0.
++Includes iso
17:0
included in the two isolated lipids. Basically the total lipid fatty acid composition resembles that of the triglyceride modified by that of the polar lipids in some cases. A m o n g details of interest are the moderately high proportions, for a marine lipid, of 18:2oJ6 and 18:3oJ3 (compare, for example, crustacean respective value of ~ 2 and ~ 1 per cent respectively reported by Ackman et al., 1970). T h e two co6 successor series 20:2~o6 and 20:4w6 are presumably associated with a high 18:2xo6 intake. T h e high proportion of 20:5oJ3 in lieu of 22:&o3, especially in the polar lipids, probably represents direct dietary deposition of ingested fatty acids as 20:5co3 is m o r e characteristic of marine algal polyunsaturated fatty acids than is 22:6o~3 (Ackman et al., 1970; see also Brockerhoff et al., 1963). T h e occurrence of 20:1 and 22:1 in the triglyceride and especially in the polar lipid was unexpected on the basis of our work with other species. H o w e v e r there is relatively little 18:1 for a marine lipid and it is not impossible that periwinkles elongate 16:1 or 18:1 precursors to 20:1 and 22:1. As metabolism in molluscs is in a n u m b e r of cases "lipid oriented", considerable activity in lipid biosynthesis could be expected if this were applicable (compare other species discussed by 20
586
R. G. ACKMAN, S. N. HOOPER AND P. J. KE
Stickle and Duerr, 1970). T h e drop in lipid from 4"80 to 1"21 per cent in two weeks of periwinkle starvation could indicate this to be the case for L. littorea (Ackman & Cormier, 1967). T h e matter has been tested experimentally in two terrestrial snails, and in addition to active incorporation of 14C-labelled acetate into fatty acids, the isolation and identification of iso and anteiso fatty acids in chain lengths up to Cz~, C2a or C24 suggests a tendency for these animals to elongate fatty acids of middle chain lengths (van der Horst & Voogt, 1969a, b). A large n u m b e r of species of marine molluscs are reported as having appreciable (2-10 per cent) 20:1 and/or 22:1 fatty acids in total, neutral, and/or polar lipids (Gruger et al., 1964; Shimma & Taguchi, 1964; de Koning, 1966; Tibaldi, 1966; Jeffries, 1967, 1968; Hamada & Ueno, 1968, 1969; Hayashi et al., 1969; Takama et al., 1969). These particular fatty acids are the object of further study in our laboratory. T h e ratio of iso to anteiso acids for all three species of molluscs (Table 2) was not especially different from previously published results for fish lipids (Ackman & Sipos, 1965 : Ackman & Hooper, 1970) or krill (Ackman et al., 1970). This does not help distinguish an exogenous origin in the form of bacterial lipids from an endogenous origin involving fatty acid biosynthesis from amino acid skeletons as starting points. T h e proportions of saturated linear o d d - n u m b e r e d fatty acids in the oysters and quahaugs are higher than previous results for other marine animals but further work is also required to elaborate on the significance of this finding. Acknowledgement--This work was supported in part by the Industrial Development Service of the Department of Fisheries of Canada.
REFERENCES ACKMAN R. G. & CORMIERM. G. (1967) ~-Tocopherol in some Atlantic fish and shellfish with particular reference to live-holding without food. ft. Fish. Res. Bd Can. 24, 357-373. ACK/vIANR. G., EATONC. A., SIPOSJ. C., HOOPERS. N. & CASTELLJ. D. (1970) Lipids and fatty acids of two species of North Atlantic krill (Meganyctiphanes norvegica and Thysano~ssa inermis) and their role in the aquatic food web. ft. Fish. Res. Bd Can. 27, 513-533. ACKMANR. G. & HOOVERS. N. (1968) Examination of isoprenoid fatty acids as distinguishing characteristics of specific marine oils with particular reference to whale oils. Cornp. Biochem. Physiol. 24, 549-565. ACKMANR. G. & HOOPERS. N. (1970) Branched-chain fatty acids of four fresh-water fish oils. Comp. Biochem. Physiol. 32, 117-125. ACKMAN, R. G. & SIPOS J. C. (1965) Isolation of the saturated fatty acids of some marine lipids with particular reference to normal odd-numbered fatty acids and branched-chain fatty acids. Comp. Biochem. Physiol. 15, 445-456. ANSELL A. D., LOOSMOREF. A. & LANDER K. F. (1964) Studies on the hardshell clam, Venus mercenaria, in British water--II. Seasonal cycle in condition and biochemical composition, ft. appl. Ecol. 1, 83-95. BAXTERJ. H. & lklILNEG. W. A. (1969) Phytenic acid : Identification of five isomers in chemical and biological products of phytol. Biochim. biophys. Acta 176, 265-277. BLmH E. G. & DYeR W. J. (1959) A rapid method of total lipid extraction and purification. Can. ft. Biochem. Physiol. 37, 911-917. BROCKERHOFFH., ACKMANR. G. & HOYLER. J. (1963) Specific distribution of fatty acids in marine lipids. Archs Biochem. Biophys. 100, 9-12.
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587
DE KONINO A. J. (1966) Phospholipids of marine origin IV. The abalone (Haliotis midae). J. Sci. Fd Agric. 17, 460-464. GALTSOFF P. S. (1964) T h e American oyster Crassstrea virginica Gmelin. Fish. Bull. U.S. Dept. Interior No. 64. GaU6Ea E. H., JR., NELSON R. W. & STANSBYM. E. (1964) Fatty acid composition of oils from 21 species of marine fish, freshwater fish and shellfish. J. Am. Oil Chem. Soc. 41, 662-667. HAMADA S. & UENO S. (1968) On the lipids of shells--I. Yukagaku 17, 39-42. HAMADA S. & UENO S. (1969) On the lipids of shells--II. Yukagaku 18, 478-480. HANSEN R. P. & MEIKLEN S. M. (1970). Isoprenoid fatty acids in Antarctic krill (Euphausia superba). J. Sci. Fd Agric. 21, 204-206. HAYASHI A., MATSUBARAT. & MATSUURA F. (1969) Biochemical studies on the lipids of Turbo cornutus--I. Yukagaku 18, 118-123. JEFERIES H. P. (1967) Chemical responses by marine organisms to stress. Phase 1. In
Technical Report No. 1, Graduate School of Oceanography, Narragansett Marine Laboratory, University of Rhode Island, Kingston, R.L, U.S.A. JEEFRIES H. P. (1968) Chemical responses by marine organisms to the quality of the environment. In Proc. Ann. North Eastern Reg. Antipollution Conf., 22-24 July, University of Rhode Island, R.I., U.S.A., pp. 84-93. MAUCHLINE J. & FISHER L. R. (1969) The biology of Euphausiids. In Advances in Marine Biology (Edited by RUSSELL F. S. and YONGE M.), Vol. 7, pp. 174-199. Academic Press, New York and London. SHIMMA Y. ~l; TAGUCHI H. (1964) A comparative study on fatty acid composition of shellfish. Bull. Jap. Soe. Sci. Fish. 30, 153-160. SIPoS J. C. & ACKMANR. G. (1968) Jellyfish (Cyanea capillata) lipids: fatty acid composition. J. Fish. Res. Bd Can. 25, 1561-1569. STICKLE W. B. & DUERR F. (1970) T h e effects of starvation on the respiration and major nutrient stores of Thais lamellosa. Comp. Biochem. Physiol. 33, 689-695. TAKAMA K., ZAMA K . & IGARASHI H . (1969) Lipids in whelk, Neptunea arthritica. Bull.Jap. Soc. Sci. Fish. 35, 1184-1188. TmALDI E. (1966) Richerche preliminari sugli acidi grassi di alcune specia di mollusehi marini. Rend. Accad. Naz. Lined (Sc. Fis.) 40, 921-925. VAN DER HORST D. J. & VOOGT P. A. (1969a) Investigation of the fatty acid composition of the snail Arianta arbustorum. Archs int. Physiol. Biochem. 77, 507-514. VAN DER HORST D. J. & VOOGT P. A. (1969b) Investigation of fatty acid composition of the snail Succinea putris L. Comp. Biochem. Physiol. 31, 763-769. VISWANATHAN C. V. (1968) Chromatographic analysis of plasmalogens. Chromatographic Rev. 10, 18-36. WILLIAMS E. E. (1970) Seasonal variations in the biochemical composition of the edible winkle Littorina littorea (L). Comp. Biochem. Physiol. 33, 655-661.
Key Word Index--Molluscs; fatty acids ; isoprenoid fatty acids ; oysters; quahaugs ; periwinkles; Cr assostrea virginica ; Venus mercenaria ; Littorina litorrea.