Lipid composition of marine and estuarine invertebrates. Part II: Mollusca

Pro O. Lipid Res. Vol. 21, pp. 109 153. 1982 Prinled in Great Britain. All rights reserved

0163 7827/82/020109 45522.50/0 Copyright • 1982 Persamon Press Lid

LIPID C O M P O S I T I O N OF MARINE AND ESTUARINE INVERTEBRATES. PART II: M O L L U S C A * JEANNE D. JOSEPH Department of Commerce, NOAA, NMFS, Southeast Fisheries Cemer, Charleston Laboratory, P.O. Box 12607, Charleston, South Carolina 29412, U,S.A. CONTENTS I. INTRODUCTION I1. NEUTRAL LIPlDS

A. Hydrocarbons 1. Polyplacaphora 2. Gastropoda

3. Bivalvia B. Wax esters C. Glyceryl ethers and plasmalogens 1. Polyplacaphora 2. Gastropoda

3. Bivalvia 4. Cephalopoda

D. Fatty acids 1. Polyplacaphora 2. Gastropoda (a) Order Archaeogastropoda (b) Order Mesogastropoda (c) Order Neogastropoda 3. Bivalvia (a) Order Mytiloidea (b) Order Pteroidea (c) Order Veneroida 4. Cephalopoda IlL POLAR LIPIOS A. Polar glyceryl ethers and plasmalogens 1. Polyplacaphora 2. Gastropoda 3. Bivalvia 4. Cephalopoda B. Phosphonolipids 1. Polyplacaphora 2. Gastropoda 3. Bivalvia C. Sphingolipids IV. SUMMARY ACKNOWLEDGEMENTS REFERENCES APPENDIX

109 i10 110 110 I!1 II1 I11 12 12 13 13 14 14 14 15 15 17 120 121 122 124 126 131 132 132 132 134 135 135 137 139 140 144 145 146 146 146 149

I. I N T R O D U C T I O N

The molluscs constitute one of the more important invertebrate groups in the animal kingdom and are second only to the insects in the number of living species. 45 Molluscs are divided, taxonomically, into seven classes, but published information on lipid composition or metabolism is available for only four: Polyplacaphora, Gastropoda, Bivalvia (also known as Pelecypoda) and Cephalopoda. The three latter classes comprise a major marine fishery resource and are commercially important, world-wide. In addition to their commercial value, current interest in the role of dietary polyunsaturated fatty acids in human health, particularly that of eicosapentaenoic acid (20:5(n-3)) in amelioration of certain cardiovascular diseases, 16"41-'43,1s6 also focuses attention on the molluscs, as well as other marine fishery products, which are excellent sources of polyunsaturates. Exler and Wcihrauch have compiled published fatty acid values and recalculated the data as g *Contribution number 81-37C, Southeast Fisheries Center, National Marine Fisheries Service, NOAA, Charleston, SC 29412. 109

110

Jeanne D. Joseph

fatty acid/100 g food for molluscs and crustaceans 4s and for finfish. 46'47 Because of the commercial importance and perceived nutritional value of molluscs, there is a great deal of published information on lipid composition and metabolism of representatives of this phylum. Therefore, to inform the reader of the availability of additional data not specifically discussed in this review and to clarify taxonomic relationships between the species, an appendix of species, taxonomic position and appropriate references is included. As in Part I of this review of marine and estuarine invertebrate lipids, 98 certain limits have been placed upon the literature selected for review. First, the decision was made to include only information published since the mid-1960's since increasingly available sophisticated instrumentation and technology have afforded far more accurate data than could be attained earlier. Second, sterols have been omitted from discussion because of their highly specialized structure, function and chemistry and because of the availability of recent excellent reviews. 57'5s'138 The fatty acid short-hand notation employed in this review has been suggested by the IUPAC-IUB Commission on Biochemical Nomenclature 9° as a replacement for the "o9" system which has been in wide use for many years, but there is no basic difference in the two systems. Both specify first, the number of carbon atoms, and second, the number of double bonds followed by the position of the terminal olefinic bond relative to the hydrocarbon end of the molecule, i.e. the end-carbon chain which is designated as ogx or (n--x), where the symbols "co" and "n-" are synonymous and x equals the end-carbon chain length. This perspective of the monoenoic or methylene interrupted polyunsaturated fatty acid molecule clarifies fatty acid family relationships in discussions of fatty acid biosynthesis by chain elongation and desaturation since, in this process, the end carbon chain is unaltered. These systems are also useful since they provide the basis for semilog plotting techniques and calculations utilized in identification of unsaturated fatty acids separated by gas-liquid chromatography (GLC). 2-4'91 However, if the polyunsaturates contain ethylenic bonds separated by more than one methylene group (i.e. nonmethylene interrupted fatty acids), these systems cannot be used and the position of each double bond in the molecule must be specified. In this review, fatty acids of this type are identified by the number of carbons in the chain followed by the number of double bonds and the symbol A which indicates that the positions of the double bonds in the chain are determined relative to the carboxyl group of the molecule. Structures of lipids other than fatty acids, for example, hydrocarbons, alcohols and long-chained bases, are abbreviated like the fatty acids, i.e. number of carbon atoms followed by the number of double bonds and (n--x) bond position, if necessary. II. N E U T R A L

LIPIDS

A. Hydrocarbons 1. Polyplacaphora Yasuda and Fukamiya have investigated hydrocarbons of the chiton, Liolophira japonica, a herbivorous grazer, collected from the shores of Hiroshima Bay. 195 Hydrocarbons of muscle tissue, 0.007~o of wet weight of the tissue, were separated by silver nitrate-thin layer chromatography (AgNO3-TLC) into saturated (30~), unsaturated (38~) and highly unsaturated (32~) fractions. Although the saturated fraction was dominated by n-17:0 (31~ of total alkanes), there was a slight even-carbon predominance in this fraction. Analysis of the unsaturated fraction as the O-isopropylidine derivatives by gas liquid chromatography-mass spectrometry (GLC-MS) showed that the major components were cis-15: l(n-12), cis-15: l(n-13), cis-15: l(n-14) and cis-17: l(n-12). The highly unsaturated fraction was identified as squalene. The presence of an unresolved peak envelope in the gas chromatogram of the n-alkanes suggested mild pollution by petroleum hydrocarbons but the authors concluded that the percentages of alkenes and squalene that they observed indicated that the major portion of the chiton hydrocarbons was of biogenic origin.

Lipid composition of marine and estuarine invertebrates

111

2. Gastropoda Hydrocarbons have been isolated from the unsaponifiable fraction of Haliotis discus hannai visceral lipids and identified by GLC. 7s Both saturates (28%) and unsaturates (71%), primarily monoenes, were dominated by odd-carbon components and there was no evidence of an unresolved peak envelope in the published chromatogram. Neither pristane (2,6,10,14-tetramethylpentadecane) nor squalane (2,6,10,15,19,23-hexamethyitetracosane) was observed but squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22tetracosahexaene) was present at 0.4% of the unsaponifiable fraction. An analysis of the carnivorous whelk, Buccinum undatum, collected from coastal waters of the United Kingdom showed low levels of hydrocarbons, averaging 2.8/zg/g wet weight (range, 0.2-15.6 #g/g).19o In an earlier investigation of hydrocarbons in the marine environment, this class of compounds was isolated from lipids of B. undatum, collected in the Firth of Clyde, and separated by GLC. ~26 Pristane was found to be the major component (25.3%), and although 23:0 was the major n-alkane present (13.3%), the sums of the odd-carbon and even-carbon n-alkanes were identical (31%). In this study, significant differences were noted in hydrocarbon composition of finfish muscle a n d liver tissues. Hydrocarbons of liver tissues were predominantly odd-carbon components as were those of sediment samples taken in the Firth, suggesting a food-web relationship, but there was no odd-carbon predominance in muscle tissues. The authors concluded that analysis of the entire body of the whelk perhaps obscured the odd-carbon predominance of its liver hydrocarbons. No conclusions could be drawn as to the origin of the whelk hydrocarbons.: 26,190 3. Bivalvia Biumer et al. compared the hydrocarbon content of scallops, Aequipectin irradians, from petroleum-polluted and non-polluted waters. 23 The major hydrocarbons identified in scallops from unpolluted waters were n-17:0, n-21:0 and n-heneicosahexaene (21:6), a polyenoic hydrocarbon previously reported to be synthesized by marine algae.95.~ 2t.122. ~97.19s In this report, Blumer et al. explicitly defined the standard whereby they assessed the origin of the scallop hydrocarbons, stating "the presence of olefinic hydrocarbons of st/'ongly predominant C17 and C21 n-alkanes, the absence of isoprenoids outside the C19-C20 range, and the lack of an unresolved peak envelope from cyclic saturates and aromatics is a clear indicator of biological origin and of the absence of pollution. ''2a Young mussels, Mytilus edulis, averaging 0.3 g (dry weight) have been found to contain about 1 mg naturally-occurring hydrocarbons per individual, the major components being n-20:l, pristane and 21:6.123 In contrast, however, biogenic hydrocarbons could not be detected in hard clams, Mercenaria mercenaria, collected from non-polluted waters.S B. Wax Esters Among the four major molluscan classes, wax esters have been found only in the cephalopods.63a,119.120.164 Since they are present in representatives of many phyla, it has been suggested that wax esters have no taxonomic significance, except for some copepods but are, rather, the result of environmental adaptation. 2°.164 In general, they are prominent lipids in many marine animals inhabiting deep or cold waters and which, as a result, have an intermittent food supply, lz°'~64 They are also found in many animals that undergo a large diurnal vertical migration, and in such animals, wax esters may have a hydrostatic function. 2°.12s.196 In a study designed to gather information on the origin of wax esters in lipids of the sperm whale, Physeter macrocephalus, Hansen and Cheah analyzed the lipids of a portion of undigested giant squid (species not stated) tentacle recovered from the stomach of a sperm whale. 63a Wax esters (48.6?/o of total lipid) were isolated by TLC, methylated and

112

Jeanne D. Joseph TABLE 1. Fatty Acids and Alcohols of the Giant Squid ~'3" Lipid class: Component: Chain length

Wax esters Triacylglycerols Alcohols Acids Acids Percent Composition

10:0

--

12:0 14:0 14: l(n-9) 14: l(n-5) 15:0 16: 0 16:1 16:2 17:0 17:1 17:3 18:0 18:1 18:2 19:0 19:1 20:0 20:1 20:2 20:4 20:5 22:1 22:5 22:6 24:1 24:4

2.2 --0.5 38.0 1.8 -0.9 0.9 -12.1 38.0 -0.3 0.4 0.3 4.3 0.1 tr tr -

--

0.3

tr*

3.0 5.0 7.6 tr 0.2 8.7 8.0 0.2 0.2 0.6 0.5 2.0 30.1 2.2 --0.3 0.4 17.3 0.5 3.8 0.7 1.9 1.5 3.8

1.4 5.8 1.2 -0.6 20.5 8.1 0.4 0.5 0.6 -4.6 36.8 1.3 --0.2 10.8 0.2 0.3 1.0 2.6 0.5 1.3

--

0.5

0.7

0.6

*Trace.

the product alcohols and methyl esters separated by TLC and analyzed by GLC. A comparison of the esters and alcohols of the squid (Table 1) with those of the whale head and blubber oils showed 14: l(n-9), rather than the more commonly occurring 14: l(n-5), to be the only 14 carbon monoenoic fatty acid in both squid and whale head waxesters. 63~, C. Glyceryl Ethers and Plasmalogens Although in common usage, the trivial terms "glyceryl ethers" and "plasmalogens'" are more properly designated "alkyldiacylglycerols" and "alk-l-enyldiacylglycerols," respectively. However, for the sake of brevity, the simpler terms will be used in this review. Structural formulae of a neutral alkyldiacylglycerol (I) and a neutral alk-l-enyldiacylglyceroi (II) are shown below. The polar forms differ, of course, in that they possess an esterified phosphate-nitrogenous base group on the third carbon of the glycerol molecule. H2C-- O - - R

o

H2C--OCH~CH--R

/

o

:i

R'--C--O--C

R'--C--O--C

o

I

i

H2C

0

C

R

(I~ Alkyldiacylglycerol

H2C

o i

0

C

R

(II) Alk-l-enyldiacylglycerol

1. Polyplacaphora Glyceryi ethers have been isolated from the lipids of two chitons, lsay et al. reported that total iipids of Ischnochiton hakodadensis, collected in the Sea of Japan, included 8.4°Jo

Lipid composition of marine and estuarine invertebrates

113

T^et£ 2. Principal n-Alkyl Constituents of Mollwwam Neutral (N), Polar (P) and Total Lipid (T) Glyceryl Ethers ( > 5Yo) Chain length: Lipid: CLASS Species (Ref.)

N

14:0 P T

N

15:0 P T

N

16:0 P T

16:1 N P T

18:0 N P T

N

18:1 P T

N

20:0 P T

N

20:1 P T

Percentage

Polyplacaphora

Katherina tunicata(177)

-8 --

72 75

8

6--

16 10

i

Gastropoda

Haliotis discus (78) Thaislamellosa(177)

11 1 3 - - -

26 37-

5----49

5---

34 - - - - 16 - - - - 17 - - - - - 21 12 13

Bivalvia

Chlamys nipponensis(79) Protothaca staminea(177) Mytilusedulis(61)

16 ---

5 9

2335-5 21 51 - - - -

38 - - - 6--36 216 - - 42 18 - -

6---9

21 - - - -

15 - - - - - -

7 11

5-

Cephaiopoda

Octopus dofleni (177) Tentacle Hepatopancreas

38

------134

- -

- -

30--i--

- -

---

7 - - 20 - - - 34 41 ~ " 9

7 6

--

glyceryl ethers, aa Similar values were found in Katherina tunicata lipids by Thompson and Lee. 177 In this species, neutral lipids contained 4% glyceryl ethers and the polar lipids, 7.8% (see below). Analyses of the isopropylidine derivatives by GLC showed that the principal alkyl groups of the neutral glyceryl ethers were 16:0 (72%), 18:1 (16%) and 18:0 (8%) (Table 2). 2. Gastropoda

In a report on visceral lipids of the abalone, Haliotis discus hannai, Hayashi and Yamada noted the presence of 26.7% glyceryl ethers in the unsaponifiable fraction of the acetone-soluble lipids.7s The major alkyl constituents detected by GLC were 18:0 (33.7%), 16:0 (25.7%), 20:0 (16.5%) and 18:1 (15.8%) (Table 2). These investigators also found small quantities of glyceryl ethers in the unsaponifiable fractions of Thais clavioera and Chiorostoma argyrostoma lischkei acetone-soluble visceral lipids, s° Other species of gastropods in which small amounts of glyceryl ethers have been observed in the total lipid fraction are: Collisella dorsuosa (4.9%),ss Collisella sp. (2.8%),ss Acmaea pallida (1.9%),ss Littorina brevicula (1.4%)" and Thais lamellosa (1.7%).177 In the latter species, the major alkyl chains were found to be 16:0 (49%), 18:0 (21%), 14:0 (11%) and branched 19:0 (10%) (Table 2). 3. Bivalvia

Isay et al. found that glyceryl ethers were present in the total lipids of a number of bivalves, ranging from a high of 9.9% in Macoma sp. to a low of 0.8?/0 in Mactra sulcataria, ss The average value reported was 2-4%. Using column chromatography, Hayashi and Yamada isolated 10.9% glyceryl ethers from the unsaponifiable fraction of acetone-soluble visceral lipids of the Japanese prickly scallop, Chlamys nipponensis akazawa. 79 The principal alkyl groups were reported to be 18:0 (37.9%), 18:1 (21.1%), 16:0 (15.37/o) and 20.0 (14.7%) (Table 2). Small quantities of both straight-chained and 2-methoxy alkyl glyceryl ethers have been identified in neutral lipids of the mussel, Mytilus edulis. ~ Compositions of the n-alkyl constituents are shown in Table 2. Hallgren et al. pointed out that as a phytanyl glyceryl ether has been identified in cod liver oil, the probable source of the phytanyl group being chlorophyll, the alkyl chain composition must be dependent upon dietary input, e~

114

Jeanne D. Joseph

Although only polar plasmalogens were identified in the clam, Protothaca staminea, ~77 Sampugna et al. reported that 28.3~o of the total plasmalogen was present in the neutral lipid fraction of the oyster, Crassostrea virginica. 162 Similarly, plasmalogens have been reported to be abundant (23.8~) in neutral lipids of the yellow clam, Mesodesma mactroides, a South American species, 38 but far less prominent in neutral lipids of the scallop, Chlamys tehuelcha, and the mussel, Mytilus platensis, both also from South American waters. Neutral plasmalogens of C. tehuelcha gonads and remaining soft tissue lipids were reported to be only 0.7~o and 3.8~, respectively, ~51 while that of gonad and total soft tissue lipids of M. platensis were stated to be 5.6~o and 6.3~°o, respectively. 4° 4. Cephalopoda

An analysis of tentacle and hepatopancreas lipids of Octopus dofleini revealed 2~ glyceryi ethers in hepatopancreas neutral lipids but only traces in tentacle neutral lipids. 177 The principal alkyl constituents were reported to be 16:0 and 18:0 (Table 2). Plasmalogens were also observed in O. dofleini lipids, but chromatographic separation and re-analysis showed that less than 5~o of the plasmalogen was present in the neutral lipid fraction. 177 D. Fatty Acids

Because of the relative simplicity of technology and general availability of gas-liquid chromatographic instrumentation, there is a wealth of information on fatty acid composition of molluscs, particularly for those of commercial importance. In recent years, improvements in instrument design, the development of wall-coated open-tubular (WCOT) glass and flexible fused silica columns and increased application of ancillary analytical techniques such as AgNO3-TLC, ozonolysis and hydrogenation/rechromatography of sample, for example, have led to far more detailed and accurate data. As a result, it has become increasingly clear that the fatty acid composition of molluscs and, indeed, of marine invertebrates generally, is influenced by a host of environmental and biological factors. Two important environmental factors are temperature and food availability which, together, may contribute to a seasonal variation in fatty acid composition. Among the biological factors are taxonomic relationships, differences in sex and influence of reproductive cycles, diet, and distribution of the different esterified lipid classes in specialized body tissues. The goal of this section is not only to detail fatty acid composition of representative molluscs but also to describe differences in composition as a function of these impacting variables. 1. Polyplacaphora

Unlike the commercially important gastropods, bivalves and cephalopods, little published information on lipids of chitons exists. One recent fatty acid analysis of a representative of this class, Ponerplax costata, has been published. 96 Although the data are somewhat limited, due to the use of a nonpolar GLC column (SE-30) which will not separate certain critical pairs of acids (20:4(n-6) from 20:5(n-3) and 22:4(n-6) from 22:5(n-3)), they are adequate to show that P. costata fatty acid composition (Table 3) is similar in several respects to that of the abalones and limpets, gastropod molluscs which, like the chitons, are algal grazers on rocky shorelines (see below, Tables 4 and 5). The principal polyunsaturates of P. costata tissues were found to be 20:4(n-6) plus 20:5(n-3), particularly in gills and viscera; 22:6(n-3) was virtually absent. These features also characterize red algae (Rhodophyceae) and brown algae (Phaeophyceae), 14'77"92 which were present at the collection site. 95"96 Modest amounts of 20 and 22 carbon nonmethylene interrupted dienes (NMID), first observed in the oyster, C. viroinica, 18v and the clam, Arctica islandica, ~42 were also recognized in P. costata lipids; their structures were assumed to be identical to those previously isolated from lipids of C. virginica and

Lipid composition of marine and estuarine invertebrates

115

TABLE3. Distribution of Major Fatty Acids in Tissues of the Chiton, Ponerplax costata96 Tissue: Collection date: Fatty acids 14:0 15:0 16:0 17:0 18:0 20:0 22:0 4,8,12 TMTD* TotaH" 14: l(n-7) 14:1(n-5) 16: l(n-13)trans 16: l(n-9) 16: l(n-7) 16: l(n-5) 18: l(n-9) 18: l(n-7) 18:1(n-5) 20: l(n-ll) "~ 20:1 (n-9) J 20: l(n-7) 22: l(n-7) Totali" 18:2(n-6) "~ 18: 3(n-3) J 18:3(n-6) "~ 18: 4(n-3) J 20:2(n-6) 20:2NMID ° 20:4(n-6) "~ 20: 5(n-3) J 22:2NMID "~ 22:4(n-6) "~ 22: 5(n-3) J 22: 6(n-3) Totalt '~

Whole Foot Gill Viscera 3/77 10/77 9/77 9/77 Weight percent composition 8.4 0.9 13.2 0.5 1.3 0.1 tr 1.0 26.8 tr 0.1 1.3 tr 2.1 0.1 13.1 4.8 tr 3.0

6.7 1.6 17.1 0.2 1.4 0.2 0.1 2.3 31.6 0.1 0.1 0.2 -3.2 0.1 24.0 2.2 tr 5.7

3.1 2.0 10.4 0.2 3.9 0.1 1.3 0.8 26.0 0.1 0.3 0.2 2.6 tr 0.2 5.8 6.0 0.1 4.3

3.1 1.1 12.8 0.4 1.8 tr # -3.0 27.0 0.1 0.1 0.2 0.4 2.1 0.1 12.4 5.0 0.1 4.1

tr 0.1 27.4 0.7

-0.1 35.6 1.1

--19.8 0.5

tr 0.1 24.4 3.3

0.3

1.3

0.7

0.9

0.9 1.9 22.7

1.7 3.2 14.3

0.8 1.9 26.6

1.6 2.5 30.9

1.9 6.0

3.0 6.2

6.6 9.7

2.4 9.3

0.9 35.9

-30.8

-44.5

0.4 50.5

*4,8,12-Trimethyltridecanoate. #Trace. °Nonmethylene interrupted dienes. tTotals as reported by authors. i"'~NMIDs not included in totals of polyunsaturates.

c h a r a c t e r i z e d as 2 0 : 2 A5,11; 2 0 : 2 A5,13; 2 2 : 2 A7,13 a n d 2 2 : 2 A7,15. ~4a N o details o f lipid c o n t e n t in the c h i t o n tissues were given, 96 so it is i m p o s s i b l e to d e t e r m i n e the relative c o n t r i b u t i o n o f each tissue lipid to w h o l e b o d y c o m p o s i t i o n .

2. Gastropoda G a s t r o p o d s are s u b d i v i d e d b y t a x o n o m i s t s into three orders, A r c h a e o g a s t r o p o d a , M e s o g a s t r o p o d a a n d N e o g a s t r o p o d a . A r c h a e o g a s t r o p o d s a r e the m o s t p r i m i t i v e o f the g a s t r o p o d s and, b e c a u s e t h e y a r e h e r b i v o r o u s , t h e y a r e restricted to r o c k y b o t t o m s o r s h o r e l i n e s w h e r e they feed o n e n c r u s t i n g algae. In contrast, m e s o g a s t r o p o d s i n c l u d e b o t h h e r b i v o r o u s a n d c a r n i v o r o u s species; the m o s t a d v a n c e d g a s t r o p o d s , the N e o g a s t r o p o d a , a r e all c a r n i v o r o u s . 45 (a) Order Archaeooastropoda. T h e a b a l o n e is a g a s t r o p o d o f c o n s i d e r a b l e c o m m e r c i a l i m p o r t a n c e a n d fatty acids o f several species h a v e been i n v e s t i g a t e d (Table 4).1 a.34,7 a.l os.~4o, 174.~ 91 G e n e r a l l y , t h e r e p o r t e d fatty a c i d c o m p o s i t i o n s o f all the species are similar. As in the chiton, P. costata, the p r o m i n e n c e of 14:0 (especially in the viscera),

116

Jeanne D. Joseph TABLE 4. Principal Fatty Acids of Abalones (Family Haliotidae, Genus Haliotisl

Tissue: Species:

Whole

Muscle

Viscera

H.

H.

H,

H.

H.

H.

japonicus

midae

iris

discus

japonicus

discus

Ref: Fatty acids

140

34 °

18

14:0 15:0 16:0 17:0 18:0 20:0 14:1 16:1 18: I 20:1 22:1 16:2 18:2 20:2 22:2 16:3 18:3 20:3 18:4 20:4 (n-61 (n-3) 22:4 20:5 22:5 22:6

2.3 n.r." 8.1 1.0 4.8 2.4 n.r, 1.2 7.6 4.8 n,r. n.r. 1.5 0.3 3.6 n,r. n.r, n.r. n.r. 7.6

167

108

167

108

191

78 •

6.3 1.1 23.1 1.4 3.1 0.8 0.3 0,9 10.2 4,7 0.4 0.2 2.1 1.6 4.2 tr 4.4 0.3 2.6

12.6 0.7 24.7 0.4 2.8 0.6 1.8 8.8 22.0 5.5 1.9 tr 3.3 tr n.r. tr 0.6 tr 3.8

14.0 0.7 24.1 0.5 2.9 0,3 0.8 5.6 19.3 7.4

7.6 0.8

2.2 0.7 n.r. 3.8 2.6 1.0

Weight percent composition 6 l 25 n.r. 8 n.r. n.r. 11 22 3 3 n.r. 3 1 n.r. n.r. n.r. n.r. n.r, 4

5.1 0.7 22.8 1.0 6.7 n.r, 0.3 1.8 15.7 3.7 5.3 0.2 0.5 n.r. n.r. n.r. 1.3 n.r. n.r. 13.4

3.9 2.9 20.9 n.r. 5.1 n.r. n.r. 3.3 16.4 4.8* n.r. 1.2 n.r. 5.5 n.r. n.r. n.r. 10.7t

3.0 1.0 21.9 1.3 4.5 n.r. tr 0.9 12.0 3.7 0.3 tr 1.3 0.7 4.9 tr 2,9 0.2 1.0

4.9 3.2 19.8 tr '~ 3.9 n.r. n.r. 4.4 17.1 5.9* n.r. 1.6 n.r, 3.9 n.r. n.r. 0.9 12.3t

8.7 1.3 2.4 2.8 7.8 1.4

n.r. 6 2 1

3.2 8.0 10.4 n.r.

8.8 7.3 n.r.

8.8 9.3 0.4

2.6 10.0 8.4 n.r.

9.3 5.0 tr

tr n.r. 2.8 n.r. n.r, n.r. 3.4 n.r. 3.6 5.1

n.r. 5.9 1.4

tr

°Neutral lipids. °Acetone soluble lipids. " -Not reported. '~Trace. *Includes 18:3. ~'lncludes 22: 1.

20:4(n-6), and 20:5(n-3) suggests a diet of red and brown algae, and 22:6(n-3) was found only in trivial amounts. Oleic acid (18:l, all isomers) appears to be the most variable of the reported fatty acids, varying from a low of 7.6~'o to a high of 22.0°; the range of 16:0 is similarly broad (8.1~o-25~o) but all the values except one 14° are clustered at the high end of the range (19.8~o-25~o). Using a pair of dissimilar GLC columns (DEGS and ApL liquid phases) and mathematical calculations to solve the problem of co-eluting esters on each of the columns. Noguchi et al. concluded that 24:3(n-6), rather than 22:6(n-3), was the principal component of the major, commonly observed, late-eluting GLC peak of marine oils. 14° However, they did not support their argument through the simple device of sample hydrogenation and rechromatography. The fatty acid percentages reported for Haliotis japonicus by these investigators are lower than the other values in Table 4, at least in part, because they reported the presence of 24:5(n-6) (11.4%), but again, this observation was not confirmed by any ancillary analysis. Dienoic 20 and 22 carbon fatty acids were observed in two species, H. japonicus 14°'167 and H. discus, 29"44 and although they were not identified as NMIDs, this would seem to be likely. An analysis of fatty acids of the limpet, Patella peroni (whole body), 96 showed that the composition was similar in many respects to that of the chiton and the abalones, the more prominent components being 14:0, 16:0, 18:1 and 20:4(n-6) plus 20:5(n-3) (Table 5). Specialized tissues dissected from animals collected six months later differed somewhat from whole bodies in fatty acid composition (note differences in total values for saturates, monoenes and polyunsaturates), but whether this reflects true differences in organ composition or seasonal variation cannot be determined from the data. These same investigators found significant differences in fatty acid composition of male and

Lipid composition of marine and estuarine invertebrates

117

TABLE5. Distribution of Major Fatty Acids in Tissues of the Gastropod, Patella peroni96 Tissue: Collection date: Fatty acids

Whole Foot Gonad 3/77 10/77 10/77 Weight percent composition

14:0 15:0 16:0 17:0 18:0 20:0 22:0 4,8,12 TMTD* Totalt 14: l(n-7) 14: l(n-5) 16:1(n-13)trans 16: l(n-9) 16: l(n-7) 16: l(n-5) 18: l(n-9)

7.4 1.0 25.4 0.3 2.0 0.1 0.1 3.1 41.2 0.1 0.3 0.2 0.6 2.7 0.3

18: l(n-7) 18: l(n-5)

20: l(n-I I) "~ 20: l(n-9) J 20: l(n-7) 22: l(n-7) Totalt 18: 2(n-6) 18:3(n-3) ) 18:3(n-6) ~ 18:4(n-3) f 20:2(n-6) 20:2NMID° 20:4{n-6) 20: 5(n-3) 22:2NMID° 22:4(n-6) 22: 5(n-3) 22:6(n-3) Totalt ° %

./

11.3 6.1 tr

2.9 0.6 14.4 0.3 4.7 0.2 tr # 0.5 25.2 tr 0.1 0.2 0.2 0.8 0.1 6.6

5.7 0.2 19.8 0.6 2.3 0.8 0.1 0.4 32.3 -0.4 1 0.6 2.4 0.3 12.0

5.3 --

9.2

6.2

9.5

tr 10.7

0.1 0.1 27.8

tr 0.2 23.1

tr 0.2 34.7

5.1

0.8

1.8

0.7

0.8

1.1

4.3 2.3 10.6 0.9 1.0 -25.1

3.7 2.3 28.8 3.4 5.8 -46.2

1.7 3.3 21.5 1.5 3.1 -33.0

~Trace. *4,8,12-Trimethyltridecanoate. tTotals as reported by authors. °Nonmethylene interrupted dienes. t°NMIDs are not included in totals of polyunsaturates.

female g o n a d s of a n o t h e r limpet, Subninella undulata 9~ (Fig. 1). While the female g o n a d a l lipid contained a p p r o x i m a t e l y equal percentages of saturates, m o n o e n e s and polyunsaturates, 37%, 29% and 34%, respectively, polyunsaturates of male g o n a d a l lipid totaled m o r e than half (57%) of the fatty acid content, and saturates (27%) and m o n o e n e s (15%) were, correspondingly, m u c h lower. O t h e r a r c h a e o g a s t r o p o d s for which published fatty acid data are available are: Patella coerulea L., 17s P. vuloata, 55 and Cellana tramoserica 9~ (family patellidae); Chlorostoma argyrostoma lischkei, s° Omphalius pfefferi carpenteri l°s (family Trochidae); Batillus cornutus s l n ° a and Turbo cornutus 167 (family Turbinidae). The reported fatty acid compositions of all these species are similar, in most respects, to those described above. (b) Order Mesogastropoda. Relatively, m u c h less research effort in fatty acid analysis has been devoted to the m e s o g a s t r o p o d s than to the a r c h a e o g a s t r o p o d s ; the most detailed work has been that carried out by A c k m a n and his colleagues. In one o f their earlier studies of molluscan lipids and fatty acids (a study of saturated and isoprenoid fatty acids in three species), they observed that, in c o m p a r i s o n with the filter-feeding bivalves, C. virginica and Venus mercenaria (now Mercenaria mercenaria), the m u d snail,

Jeanne D. Joseph

118

.=_o2 0 - El.

E "E

°o 15

~-~ 5 0

140

160

18:0

161 [n-7)

18:1 (n-9)

18:1 (n-7)

20:4 [n-6)

20:5 (n-3)

22:.5 (n-3)

Fatty acids FIG. 1. Sexual differences in gonadal fatty acid composition of the gastropod, Subninel/a undulata (male, black bar: female, white bar). 96

Littorina littorea, contained little pristanic acid (2,6,10,14-tetramethylpentadecanoic) or phytanic acid (3,7,11,15-tetramethylhexadecanoic) but very high levels of 4,8,12-trimethyltridecanoic acid (4,8,12 TMTD). 12 These three branched-chain acids arise from metabolism of dietary chlorophyll, and Ackman et al. postulated that the herbivorous mud snail does not accumulate phytol, pristanic and phytanic acids because of efficient degradation of these compounds to 4,8,12-TMTD (Scheme 1). Other fatty acids of interest in the mud snail total lipids were 16:0 (10.0To), 18:1 (11.2%), 18:2(n-6) (5.3To), 18:3(n-3) (4.0°,;), 20:1 (12.2%) and 22:1 (5.0To).12

C

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C

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0

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t

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\/\/\/\/\/\/\/\ c

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Phytanic Acid

C

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\

C

C

i

C

L

C

/

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C

c

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Y o

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~/\/\/\/\/\/ C

C

C

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C

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c

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C C

\ C

/ HO

4,8,12-Trimethyltridecanoic Acid SCHEME I. (Modified from Ackman 3)

In a later comparative study of fatty acids of the mud snail and the carnivorous moon snail, Lunatia triseriata (Table 6), the use of WCOT GLC columns demonstrated the presence of structurally unusual 20 and 22 carbon dienes, generally not separable from 20

c

Lipid composition of marine and estuarine invertebrates

119

Table 6. Fatty Acid Composition of a Herbivorous and a Carnivorous Mesogastropod Species: Lipid: Reference: Fatty acids 14:0 15:0 16:0 17:0 18:0 20:0 Other linear 4,8,12-TMTD* Other branched Total 16:1 (n-9) (n-7) 18:1 (n-13) (n-ll) + (n-9) (n-7) 20:1 (n-15) (n-11) (n-9) (n-7) 22:1 (n-ll) + (n-13) (n-9) (n-7) Other Total 16 PUFAt 18:2 (n-6) 20:2 Total 20:2 NMID # 22:2 Total 22:2 NMID # 18:3(n-3) 20:3(n-3) 18:4(n-3) 20:4(n-6) 22:4(n-6) 20:5(n-3) 22:5(n-3) 22:6(n-3) Other Total

Littorina littorea

Lunatia triseriata

Total 11,144

Total 144

Trig. 11

Phospho. 11

6.0 0.4 13.6 0.8 4.8 0.1 0.3 4.0 1.1 31.1

4.9 0.8 13.0 1.2 10.4 0.8 2.9 1.4 1.3 36.7 3.4 n.r. n.r. 7.5 n.r. n.r. n.r. 7.2 0.1 n.r. n.r. n.r. 0.5 n.r. n.r. n.r. 0.7 19.3 -1.0 2.4 0.4 4.4 4.0 0.2 0.2 0.2 5.1 1.0 13.4 3.2 7.7 5.2 44.0

5.8 0.8 12.2 1.1 6.8 0.5 3.8 + 2.0 33.0

4.0 0.8 12.2 1.2 14.0 1.1 2.8 1.9 1.5 39.5

1.2 4.3

0.1 0.8

0.3 8.2 2.7

0.2 3.7 1.0

0.2 4.0 2.8 4.3

3.3 0.8 1.2

0.6 0.4 0.2

----

30.5 -0.8 2.9 1.1 3.9 3.9 0.2 0.2 0.3 2.9 0.8 8.8 2.3 10.8 8.5 34.6

11.8 -1.3 2.9 0.1 5.0 5.0 0.3 0.1 0.1 7.1 1.2 16.1 4.0 4.3 0.5 41.3

0.3 3.0 0.3 2.2 2.0 0.1 4.3 0.5 1.6 0.2 0.2 0.1 2.1 17.1 2.6 2.6 2.2 0.4 1.7 1.7 6.5 2.5 3.5 7.9 -18.1 2.3 0.2 1.7 51.8

*4,8,12-Trimethyltridccanoate. tPolyunsaturatcd fatty acids. #Nonmethylene interrupted dienes.

a n d 22 c a r b o n m o n o e n e s b y p a c k e d c o l u m n G L C , in lipids o f b o t h species, 11 which p r o v i d e d a n e x p l a n a t i o n for the high p e r c e n t a g e s o f 20:1 a n d 22:1 p r e v i o u s l y f o u n d in the m u d snail lipids. 12 O t h e r details o f the fatty acid c o m p o s i t i o n o f these t w o snails, also n o t o b s e r v a b l e b y p a c k e d G L C , l e d A c k m a n a n d H o o p e r to suggest t h a t these dienes h a d n o n - m e t h y l e n e i n t e r r u p t e d e t h y l e n i c b o n d systems. 11 Specifically, they n o t e d the high p r o p o r t i o n o f 18:1(n-7) t o 18:1(n-9) a n d the p r e p o n d e r a n c e o f 20:I(n-7) o v e r 20: l(n-9) in lipids o f b o t h snails, b o t h u n u s u a l in m o s t a n a l y s e s o f m a r i n e lipids. T h e y p r o p o s e d t h a t the t w o m a j o r 20 c a r b o n N M I D s were f o r m e d f r o m the 20 c a r b o n m o n o e n e s b y d e s a t u r a t i o n o f the 5 - 6 c a r b o n b o n d ; 22 c a r b o n N M I D s c o u l d t h e n be b i o s y n t h e s i z e d b y t w o - c a r b o n c h a i n e l o n g a t i o n (Scheme II). T h e p o s s i b i l i t y o f a A5 d e s a t u r a s e was s u g g e s t e d b y the presence o f 18:1(n-13) a n d 20:1(n-15), p r o d u c t s o f 5 - 6

120

Jeanne D. Joseph 16:1 (n-7) A9 Chain elongation 18:1 (n-7) A11

18:1 (n-9) A9 Chain elongation

20:1 (n-7) A13 J, 20:2A5,13

A5 desaturation

20:1 (n-9) All ,~ 20:2A5,11

Chain elongation 22:2A7,15

22:2A7,13 SCHEME II. 11

carbon desaturation of the respective saturated fatty acids, in both molluscs. Subsequently, these proposed structures (Scheme II) were proven correct by a number of chemical, chromatographic and spectrometric techniques 143 (see below). One feature which distinguished the carnivorous moon snail fatty acid composition from that of the herbivorous mud snail and archaeogastropods (discussed above), was the presence of 22:6(n-3) in the triacylglycerol fraction of the moon snail lipids. The preferred tood of the moon snail has been reported to be the soft clam, Mya arenaria, ~1 which, like a number of other filter-feeding bivalves, contains 22: 6(n-3) in substantial amounts 24 (see below). (c) Order Neogastropoda. Hayashi and Yamada have examined the acetone-soluble lipids, essentially triacylglycerols, of several neogastropods, four species of carnivorous whelks (family Buccinidae) al and the oyster borer, Thais clavioera (family Muricidae) 8° (Table 7). The deep-water whelks were collected at depths of 1100--1200 meters while the intertidal oyster borer was collected from the shoreline at low tide. The animals were boiled, briefly, prior to analysis to facilitate removal from the shells, perhaps explaining the low percentages of 22:6(n-3) observed, although 20: 5(n-3), which should be almost as labile, was prominent in the flesh lipids of all species. The fatty acid composition of the oyster borer differed significantly in several respects from that of the whelks. In both flesh

TABLE 7. Acetone-Soluble Lipids of Some Carnivorous G a s t r o p o d s : Fatty Acid Composition Species:

Neptunea intersculpta

Reference: 81 Tissue: Flesh Viscera Total lipid 0.4 3.3 (~o wet wt.): Acetone-soluble 42.6 80.8 lipid: ( ~ of total lipid) Fatty acids 14:0 14:1 16:0 16:1 17:0 17:1 18:0 18:1 18:2 18:3 18:4 20:1 20:2 20:4 20:5 22:1 22:2 22: 5 22:6

5.8 0.4 15.9 4.8 1.3 0.6 3.8 12.2 0.8 1.4 1.5 5.8 2.9 14.8 19.1 0.8 0.4 2.4 1.4

5.0 1.7 14.1 10.9 1.3 0.7 1.3 28.2 1.1 0.4 2.4 8.5 1.0 3.3 8.3 0.9 0.2 0.9 --

Buccinum straitissimura

Buccinum bayani

Buccinum tsubai

Thais clavigera

Flesh 0.5

81 Viscera 3.8

Flesh 0.5

81 Viscera 6.7

Flesh 0.8

81 Viscera 5.0

Flesh 1.5

80 Viscera 5.9

34.9

84.7

42.6

90.8

43.7

89.7

30.5

72.5

5.3 0.7 15.1 7.5 1.1 0.6 3.4 20.3 1.3 0.7 2.3 4.9 1.8 5.1 19.1 0.7

7.2 3.5 13.0 9.7 1.4 0.6 2.2 20.1 1.2 0.3 3.1 14.5 1.1 3.6 8.7 0.4

1.3

0.8 1.1

2.9 tr 10.9 3.9 2.1 1.9 4.5 7.4 2.0 1.0 6.0 18.0 2.4 9.3 15.5 1.7 0.5 3.6 1.7

3.8 0.4 12.8 4.5 1.4 0.2 4.2 10.2 2.4 1.2 7.9 18.7 1.6 7.0 14.5 1.2 tr 2.3 2.1

Weight percent composition 6.5 1.6 17.6 5.8 1.0 0.3 4.4 16.0 1.3 0.1 2.3 8.1 3.9 5.5 14.2 0.7 . 1.6 1.4

4.0 0.6 17.2 8.0 1.0 0.4 1.9 27.7 1.1 0.2 2.9 10.5 1.3 3.1 9.2 0.6 .

. 0.4 . .

7.1 0.3 18.8 6.1 0.9 0.2 3.6 18.4 1.2 0.1 1.8 4.5 2.2 5.5 20.1 0.6 . 2.5 .

5.7 1.0 16.3 10.3 1.2 0.8 1.9 28.6 1.4 0.4 2.6 8.0 1.3 2.8 7.4 1.1 . . 0.6 . .

Lipid composition of marine and estuarine invertebrates

121

2o

15

o

"¢'r'r

I

"xm'r

~

I

"Jr 1968

"a,-r

I

"rrr

!

"rt

I

"m" 1969

"re"

"¢"

FIG. 2. Seasonal changes in selected fatty acids of the gastropod, Murex brandaris L. (16:0, white bar: 20: 4(n-3), black bar: 20: 5(n-3), hatched bar: 22: 6(n-3), cross-hatched bar).29

and visceral lipids of the oyster borer, 18:1 was present at one-half to one-third of that found in the whelks, but 20:1 was much more prominent in the oyster borer lipids. The question of whether this latter component also included 20 carbon NMIDs, which seems likely since this gastropod is an oyster predator, is an intriguing one, indeed. Calzolari et al. followed seasonal changes in fatty acid composition of a number of edible Adriatic molluscs, including one neogastropod. 29 Those changes observed in some of the more prominent or biochemically important fatty acids of Murex brandaris L, also family Muricidae, are shown in Fig. 2. Although 16:0 reached a minimum during the winter months, there was little compensatory increase in polyunsaturates during this period. In fact, 20:5(n-3) reached a maximum in the early spring and 22:6(n-3) remained relatively constant throughout the year. Eicosatetraenoic acid (20:4(n-3)) was unusually prominent in lipids of this species and, except during April and May, was present at about twice the percentage of 20:4(n-6) plus 22:1, which were inseparable in this analysis. Linolenic acid (18:3(n-3)) and 20:1, also inseparable, showed little seasonal variation (9.5-11.0%). Since 18:3(n-3) in significant amounts would not be a predictable fatty acid in a carnivorous animal, most of this component must have been 20:1 or, equally possible, 20 carbon NMIDs. Unlike the observations of Hayashi and Yamada in their study of the whelks, 22:6(n-3) was not an insignificant polyunsaturate in the lipids of M. brandaris, as it was present at 5% or slightly more throughout the year, a percentage comparable to that observed in the moon snail.t1 Shown in Table 8 is the distribution of fatty acids in the major esterified lipid classes of three species of gastropods, two herbivores (Patella vulgata and Crepidula fornicata) and one carnivore (Neptunea antiqua) of British coastal waters. 55 Due to a typesetting error, the published fatty acid composition of P. vulffata lipids was erroneous; the corrected data are listed in Table 8 (D. R. Gardner, personal communication). The moderate percentages of 18: 2(n-6), 18: 3(n-3), 20: 4(n-6) and 20: 5(n-3) in P. vuloata triacylglycerols suggest that this limpet may feed primarily on green and/or brown algae, but no similar conjecture can be made concerning the diet of C. fornicata. In fact, the most prominent triacylglycerol fatty acid.reported in this species, 16:2(n-4), has been found only as a minor component in green, red and brown algae, 14'92 Twenty carbon monoenes (20 carbon NMIDs?) were observed in all three species, comprising the major component in N. antiqua lipids but only C. fornicata lipids contained 22:1. In addition, 22:6(n-3) was present in the carnivorous N. antiqua lipids, less than that observed in the moon snail tt (Table 6), but more than that found in the whelks sl (Table 7). In contrast, the percentage of 20:5(n-3)in N. antiqua lipids was much less than that observed in the whelks and the moon snail.

3. Bivalvia Numerous publications over the last 15 years have dealt with the fatty acid composition of bivalves, valued seafoods of maritime countries, world-wide. Several different

122

Jeanne D. Joseph TABLE 8. Fatty Acid Distribution in Major Esterified Lipid Classes of Gastropods. 55

Order: Species: Lipid Class :t Fatty acids 14:0 15:0 16:0

17:0 18:0 16: l(n-7) 18:1(n-9) 20: l(n-9) 22: l(n-9) 22:1(n-11) 16: 2(n-7) 16:2(n-4)

18:2(n-6) 20:2(n-9) 22: 2? ° 16: 3(n-4) 18:3(n-6) 18:3(n-3) 20: 3(n-6) 22:3? ° 16:4(n-1) 18:4(n-3) 20:4(n-6) 20:4(n-3) 22:4? ° 20:5(n-3) 22:5(n-6) 22:6(n-3)

Archaeogastropoda Patella vulgata Trig.

Phos.

S.e.

2.3 0.8 10.7 0.7 2.9 4.8 17.8 3.5 1.7 --1.2 6.2 3.3 0.9 1.1

2.3 0.4 13.5 0.4 3.3 1.4 9.2 6.0 2.7 -0.3 0.8 1,0 1,1 0,7 1,4 0,5 0.7 -0.7 0.4 2.8 16.5 8.4 --20.1 -tr

1.9 1.1 11.5 0.9 1.9 3.8 18.8 5.9 2.0 --2.2 3.9 5.2 0.6 4.2 1.9 --tr 1.8 1.6 3.4 4.7 8.2 2.0 1.8 2.1

7.9 -1,3 0.7 2.6 5.9 3.0 0.9 9.7 0.8 1.7

Mesogastropoda Crepidula fornicata Trig. Phos. S.e. Weight percent composition 1.5 0.9 11.0

1.9 6.2 3.1 9.3 11,4 3.9 6.8 1.0 13.1 2.5 2.7 -2.2 1.3 0.3 0.6 0.7 1.1 3.7 2.6 1.1 , 1.6 5.7 1.6 1.3

2.2 0.7 11.7

1.4 tr* 10.8

Neogastropoda N eptunea antiqua Trig.

Phos.

S.c.

1.4 0.6 7.8

2.4 1.8 9.1

2.1 1.4 7.7 1.7 3.4 1.7 7.7 12.1 tr -1.9 2.4 1.1 1.6 1.1 1.7 tr -tr 1.8 2.3 2.9 4.0 0.9 3.0 4.0 3.7 2.1

1.4

1.0

1.6

1.0

6.3 8.3 9.2 12.2

5.7 8.9 7.4 7.1

4.4 2.9 8.0 12.1 3.5 -1.9 1,7 4.8 1,2 1,9 12 tr 0,3 tr 3.3 0.7 2.2 7.3 1.5 1.1 6.2 5.1 3.6

4.8 3.9 4.6 18.0 1.0 -0.6 1.7 3.3 2.3 0.8 tr -1.5 -4.1 0.6 2.3 3.7 1.0 0.3 3.1 3.5 1.9

tr 4.6 2.8 0.8 2.1 1.5 0.7 2.6 1.1 1.1 1.0 1.4 8.6 4.8 0.7 0.5 -5.4 1.3 1.3

tr 6.9 3.1 2.9 3.0 4.3 ~ 2.1 1.3 1.2 -• tr 4.9 1.0 1.5 ~ 7.8 5.8 # ~

tTriacylglycerols, phospholipids and steryl esters. °Authors' designation. ~Included in peak listed as 22:5(n-6). *Less than 1 ~ . '~Includes all 22 carbon polyunsaturates.

systems of taxonomic classification have been proposed for the class, based upon criteria such as gill structure or hinge morphology. The system adopted in this review is in use at the American Museum of Natural History in New York City and is based upon shell structure and the nature of the hinge teeth. 45 Within this system, fatty acid data are available for representatives of ten different families of bivalves; within one family alone, Pectinidae, at least 13 different species have been analyzed in recent years. Therefore, it seems advisable to limit the scope of this section, somewhat, to a consideration of the major bivalve families for which comparative published data are available. Not coincidentally, these include the bivalves of greatest commercial importance: mussels, scallQps, oysters and clams. Species not included in this discussion may be found, however, together with the pertinent references in the appendix to this section. (a) Order Mytiloidea. Included in the data of Table 9 are fatty acids of mussels from the northwest Atlantic Ocean (Mytilus edulist44), the Adriatic Sea (M. gailoprovincialis 29"31) and from South American waters (M. platensis4°). While it is not possible tO make an in-depth comparison of animals from such widely divergent locations, it is evident from the data in Table 9 that 16:0, 16:1, 18:1, 20:5(n-3) and 22:6(n-3) are prominent, to a greater or lesser extent, in all three species, deMoreno et al. followed the fatty acid composition of M. platensis over a two-year period, sampling monthly with few exceptions. 4° They observed, contrary to expectations, that lipid polyunsaturation was higher during the warmer summer months than during the winter months and attributed this to the masking effect of a greater availability of food (diatoms), resulting in high dietary intake of polyunsaturated fatty acids during the summer. The values which they observed for

Lipid composition of marine and estuarine invertebrates

123

TABLE 9. Major Fatty Acids of Mussels (Family Mytilidae).

M ytilus edulis

Species:

Reference: Fatty acids

144

14:0 16:0 18:0 20: 0 16:1 18:1 20:1 22:1 16:2 ) 16:3 ~ 16:4 18:2 18:3 18:4 20:2 20:3 20:4 (n-6) (n-3) 20:5 22:4 22:5 (n-6) (n-3) 22: 6 20:2 NMID 22:2 NMID

3.2 15.9 3.7 0.2 7.5 11.1 7.5 1.4

M ytilus galloprovincialis

M ytilus platensis

29,31 40 Weight percent composition 4.9 22.9 2.8 0.4 23.0 5.6 5.2* 1.01" tr" tr -2.3 6.2 0.2 0.6

4.2 21.8 4.2 -9.5 4.8 9.9 ° n.r. ~ n.r. n.r. n.r. 1.5 1.8 n.r. n.r. n.r.

3.8 n.r. 10.2 0.3

2.7 9.2 1.3

2.5 tr 17.9 1.I

n.r. 1.2 13.7 4.0 2.9

n.r. 0.7 6.4 n.r. n.r.

n.r. 0.7 11.5 n.r. n.r.

0.8 1.0 1.1 1.9 0.3 0.2

*Includes 18:3. °Includes 18:4. flncludes 20:4. CNot reported. "Trace.

16:0 and 22:6(n-3) are plotted in Fig. 3. Plots obtained by joining the monthly data points indicate the extent of variation of these two fatty acids in each monthly sample of 30 individuals. However, when a smooth curve, fitted by eye, is plotted to eliminate monthly variation, it becomes apparent that food availability must have varied not only 3 0 ~| ( o I

'°I, I

I

I

I

I

I

I

I

)

I

I

I

I

I

I

I

I

30~_ (b)

1976

Month

1977

I 197B

Yea r

FIG. 3. Seasonal changes in Mytilus platensis fatty acids: (A) 16:0; (B) 22:6(n-3). 4° ~.P.L.~.

21/2--c

124

Jeanne D. Joseph

16:0

L. m 16:1

18:0

18:1

20:5

22:6

Fa?ty acid FIG. 4. Selected fatty acids of

Mytilus californianus male (black bar) and female (white bar) gonads. ~~7

seasonally, but also annually during this period of time. In October 1976, 16:0 reached a minimum value over the period of this study but one year later, in October 1977, it reached its maximum value. An inverse change was observed in the percentage of 22:6(n-3). Annual fluctuations in phytoplankton populations due to differing climatic conditions are not uncommon, and these data show clearly the inherent danger in comparing fatty acid compositions of filter-feeders, even within a single species or population. Sexual differences in fatty acid composition of M. californianus were investigated by Rodegker and Nevenzel, 157 and some of the more biochemically important fatty acids of the mussel gonads are shown in Fig. 4. Unlike the differences observed in the limpet, Subninella undulata, in which the male gonad fatty acids were much more unsaturated than those of the female (Fig. 1), the female mussel gonad contained more unsaturated fatty acids than did that of the male. (b) Order Pteroidea. (1) Family Pectinidae. Reports of scallop fatty acid composition have all shown high levels of 20:5(n-3), ranging from 14.0°,o to 22.2%, and 22:6(n-3), ranging from 10.0% to 30.7% (Table 10), except for one notable exception, the purple hinge rock scallop, Hinnites multirugosus. Phleger et al. observed only traces of 22:6(n-3) in their study of this species in which they sampled two wild populations and a group of scallops that had been held in an aquarium for two months without f o o d . 149 The values shown for this species in Table 10 are average values observed in six individuals, three from each of the two wild populations. There was no significant difference in the composition of the aquarium-held scallops. What is remarkable about this fatty acid composition is the high percentages of 18:2(n-6) (14.8%, range 13-16~o) and 18:3(n-3) (13.2To, range 10-16%), which are generally present at far lower levels in marine animals. However, one or both of these particular fatty acids are found in substantial amounts in the Chlorophyceae (green algae) 14.t 5. t 9.32,92.97 and some photosynthetic flagellated algae s 5 as well as in the salt marsh grass, Spartina alterniflora, and its suspended detritus. 165 While the scallop's fatty acid composition suggests that green algae or plant detritus may constitute an important dietary component in this species, the similarity in the composition of the unfed scallops suggests the possibility of a species-specific composition such as that observed in the oyster by Watanabe and Ackman lsT'~ss (see below). Eicosenoic acid (20:1) was reported at levels (14.5~o, range 12-18%) which might indicate the inclusion of NMIDs in this component. An unknown 22 carbon fatty acid was also reported (3-5%) but the use of the highly nonpolar liquid phase, SP-2100, in the analysis makes identification difficult at best, since nonpolar columns do not permit adequate separation of unsaturated fatty acid methyl esters. 2"3 (2) Family Ostreidae. Watanabe and Ackman have investigated the influence of dietary algal fatty acids on the composition of two species of oysters, t87'~88 In this study, C.

Lipid composition of marine and estuarine invertebrates

125

TABLE10. Principal Fatty Acids of Scallops (Family Pectinidae) Species: Reference: Fatty acids 14:0 16:0 18:0 14:1 16:1 18:1 20:1 22:1 18:2 18:3 18:4 20:2 20:3 20:4 (n-6) (n-3) 20:5 22:4 22: 5 (n-6) (n-3) 22:6

Aquepectin irradians

Aquepectin Patinopectin Placopectin C h l a m y s Hinnites gibbus caurinus magellanicus nipponensis multiruyosus

112

112

112 112 Weight percent composition

2.6 14.3 5.6 1.0 5.5 6.1 4.7 n.r. 3.2 2.1. 2.8 1.8 0.9

3.3 14.5 6.5 0.5 4.5 5.5 2.6 n.r. 2.6 1.5 1.8 1.1 0.9

1.1 13.3 5.1 !.6 5.2 6.4 2.8 n.r. 1.2 1.7 2.5 0.9 0.5

1.6 14.4 5.9 0.9 3.6 6.4 3.0 n.r. 1.8 1.4 2.l 0.8 tr

2.6 1.2 15.8 3.7

3.5 0.9 14.0 4.1

2.2 0.7 16.1 1.1

1.7 0.7 16.1 1.7

1.4 2.5 18.1

0.5 3.6 19.7

0.8 1.2 30.7

1.6 1.6 26.8

79

149

3.7 23.4 5.3 2.3 8.7 9.9 1.8 n.r. 0.7 1.0 1.0 tr ° n.r. 1.7

1.7 1.3 2.3 n.r.* 14.7 6.2 14.5 4.5 14.8 13.2 n.r. n.r. n.r. n.r.

14.1 n.r. tr

22.2 n.r tr

10.0

tr

*Not reported. °Trace.

virginica and Ostrea edulis, imported one year previously from the U.K. and held in the same submerged tray with C. virginica, were each fed axenic cultures of lsochrysis galbana or Dicrateria inornata. After 6 hr exposure to the algal cultures, during which time the oysters actively fed for approximately 3 hr, the oysters were sacrificed and their iipids extracted for analysis. Calculations based upon the rate of disappearance of the algal cells from the cultures during the feeding period and the lipid content of the algae indicated that up to 2 1 ~ of the recovered oyster lipids originated in the algae, but distinctive algal fatty acids, 16: 3(n-3) in particular, were not enhanced in the oyster lipids. The absence of this algal fatty acid in O. edulis lipids indicated a rapid, species-oriented metabolism in this bivalve. Fatty acid compositions of the control oysters for this study are shown in Table 11. During this oyster feeding study, unsaturated fatty acids of unusual structure were observed within the 20 and 22 carbon chain lengths of C. virginica fatty acids, Further study, utilizing W C O T columns for analysis of these c o m p o u n d s isolated by A g N O a - T L C and preparative G L C , and coupled with oxidative and reductive ozonolysis and spectrometric techniques (MS, N M R and IR), led to the identification of these c o m p o u n d s as nonmethylene interrupted dienes, t43 The major isomers were found to be 20:2 A5,13 and 22:2 A7,15, accompanied by lesser amounts of 20:2 A5,11 and 22:2 A7,13. The biosynthetic pathways were illustrated in Scheme II. A search for N M I D fatty acids within the marine food web revealed their presence in additional molluscs, a coelenterate, an echinoderm, an arthropod, a teleost fish and the depot fat of a turtle, but not in mantle lipids of two species of squid. 144 Later studies have shown that N M I D s are prominent in some echinoderms, t°9-11t.t73 penaeid shrimps t°°'t4s and hepatopancreas fatty acids of a n u m b e r of other decapod crustaceans. 1°° Paradis and Ackman concluded that knowledge of the distribution of N M I D s a m o n g marine biota could be more useful, potentially, in elucidating food web relationships than the more c o m m o n l y observed polyunsaturates, t 58 and, indeed, in fatty acid analyses carried out in my laboratory, 22-carbon N M I D s have been observed in lipids of the teleost fish, Micropogonias undulatus, and were attributed to a diet which included polychaete worms, of which some species are good sources of N M I D s . 99"15°

126

Jeanne D. Joseph TABLE 11. Major Fatty Acids of Oysters (Family Ostreidae) Species: Lipid: Reference: Fatty acids 14:0 16:0 18:0 16:1 (n-9) (n-7) 18:1 (n-9) (n-7) 20:1 (n-11) (n-9) (n-7) 22:1 (n-ll + 13) (n-9) (n-7) 18:2 18:3 18:4 20:2 20:3 20:4 20:5 22:5 22:6 20:2 NMID 22:2 NMID

Crassostrea viroinica

Ostrea edulis

Total Total Polar Trig. Total Total Polar Trig. 144 188 144 188 Weight percent composition 2.1 19.1 4.3 2.4 5.4 6.6

0.2

1.7 2.8 2.6 0.2 0.3 4.2 12.0 2.0 18.6 1~7 5.8

3.5 2.0 4.4 3.0 8.9 6.0 8.1 28.9 26.7 34.8 20.8 33.8 31.5 42.0 3.6 6.2 2.2 4.0 10.1 12.7 7.5 3.3 1.0 0.6 1.3 1.7 1.5 1.7 3.2 1.8 4.0 4.3 2.5 5.9 Z3 3.9 3.7 4.8 4.1 2.7 5.4 4.3 3.1 5.6 2.7 1.3 5.9 4.1 1.5 1.9 0.8 0.5 1.0 0.6 0.6 1.5 0.5 0.9 0.8 0.4 2.9 4.9 1.5 1.9 2.2 2.6 1.0 NSA # NSA NSA 0.3 NSA 0.2 NSA NSA NSA 0.2 0.1 0.1 0.3 0.8 tr* 0.5 1.3 0.6 2.0 0.9 2.62.5 0.8 0.9 1.3 3.3 3.8 3.0 4.6 4.1 4.9 1.2 2.6 1.1 3.0 5.0 1.0 1.0 0.6 n.r. ° n.r. n.r. 0.3 n.r. n.r. n.r. n.r. n.r. n.r. 0.2 n.r. n.r. n.r. 2.3 4.6 1.1 3.2 0.7 2.6 0,5 11.2 8.1 9.1 19.9 3.4 6.9 2.2 n.r. n.r. n.r. 0.4 n.r. n.r. n.r. 9.7 10.3 4.7 9.8 1.3 2.3 0,9 n.r. n.r. n.r. 0.2 n.r. n.r. n,r. n.r. n.r. n.r. 1.6 n.r. n.r. n.r.

#No significant amount. *Trace. °Not reported.

(c) Order Veneroida. (1) Families Mactridae and Solenidae. A s e a s o n a l s t u d y of the yellow clam, Mesodesma mactroides, by d e M o r e n o et aL as s h o w e d q u i t e clearly t h a t s e a s o n a l v a r i a t i o n in fatty acid c o m p o s i t i o n , d u e to the t y p e a n d a m o u n t o f f o o d available, is to b e e x p e c t e d in filter-feeders. As was o b s e r v e d in lipids of the mussel, M. platensis, 4° the a v a i l a b i l i t y o f d i a t o m s , rich in p o l y u n s a t u r a t e s d u r i n g the s u m m e r m o n t h s , e n h a n c e d the p o l y u n s a t u r a t i o n o f the c l a m lipids to such an extent d u r i n g this s e a s o n t h a t a n y i n c r e a s e in p o l y u n s a t u r a t i o n d u r i n g the winter, in r e s p o n s e to low e n v i r o n m e n t a l t e m p e r a t u r e as o b s e r v e d p r e v i o u s l y in a q u a t i c o r g a n i s m s , 49'5 o.106.107.179 was n o t e v i d e n t in M. mactroides, d e M o r e n o et al. also d e t e r m i n e d fatty a c i d c o m p o sition of s p e c i a l i z e d y e l l o w c l a m o r g a n s in S e p t e m b e r , w h e n t h e g o n a d s h a d r e a c h e d m a t u r i t y (Fig. 5). T h e c o m p o s i t i o n o f t h e h e p a t o p a n c r e a s a n d the g o n a d s was q u i t e s i m i l a r in m o s t respects, suggesting t r a n s f e r e n c e o f s t o r e d lipids f r o m h e p a t o p a n c r e a s to the d e v e l o p i n g g o n a d ) 8 T a b l e 12 s h o w s fatty a c i d c o m p o s i t i o n s for two species of surf clams, Mactra sulcataria a n d Spisula solidissima (family M a c t r i d a e ) , a n d t w o species of r a z o r clams, Solen strictus a n d Siliqua patula (family Solenidae). L i k e the y e l l o w clam, M. mactroides, the r a z o r clam, S. patula o f the n o r t h w e s t Pacific coast, feeds o n d i a t o m s , b u t its fatty a c i d c o m p o s i t i o n differed significantly in a n u m b e r of respects f r o m t h a t o f the p r e v a l e n t d i a t o m , Chaetoceros armature, of the c o l l e c t i o n site. 124 T h e m o s t n o t a b l e differences o b s e r v e d were in the p e r c e n t a g e s o f 14:0, 16:1, 20:5(n-3) a n d 22:6(n-3) which, in the d i a t o m , were f o u n d to be 8.1%, 11.0%, 38.0% a n d 6.1%, respectively. Thus, it seems p r o b a b l e t h a t S. patula, like the oyster, c a t a b o l i z e s d i e t a r y fatty a c i d s w h i c h are n o t r e q u i r e d b y its m e t a b o l i s m . (2) Families Arcticidae, Veneridae and Myidae. A c k m a n et al. h a v e investigated, in detail, the fatty a c i d c o m p o s i t i o n of t h e o c e a n q u a h a u g , Arctica islandica (family Arctici-

Lipid composition of marine and estuarine invertebrates

•- 4 0 o

127

--

~3c ~, 2C

r~ -

4..

-- c

t-

.~ olN 8

14:0

16:0

16:1

IB:O

18:1

20:5

Fatty acids FIG. 5. Selected fatty acids of Mactra mactroides mantle and gill (white bar), gonad (black bar), hepatopancreas (hatched bar) and foot (cross-hatched bar). a8

dae), collected from two different sites in the Canadian maritime provinces, and a great deal of information is contained with this one publication, t° While fatty acid compositions of molluscs from the two sites were similar, generally, there were differences in the percentages of certain fatty acids, the most obvious being the absence of 22: 6(n-3) from TABLE 12. Principal Fatty Acids of Surf Clams (Family Mactridae) and Razor Clams (Family Solenidae) Family: Species: Reference: Fatty acids 14:0 16:0 18:0 20:0 16:1 18:1 20:1 22:1 16:2 16:3 16:4 18: 2

18:3 18:4 20:2 20:3 20:4 (n-6) (n-3) 20:5 22: 2 22:4 22:5 (n-6) (n-3) 22:6 20:2NMID 22:2NMID

Mactridae Solenidae M actra Spisula Solen Siliqua sulcataria solidissima strictus patula ° 167 24 108 124 Weight percent composition 4.0 29.8 9.7 n.r.* 10.5 8.2 6.7~" 1.7 # n.r. n.r. n.r. tr 2.0 tr n.r.

2.3 8.3 2.9 0.1 3.1 5.0 4.0 -n.r. n.r. n.r. 2.8 0.7 2.7 0.4 1.3

n.r. 9.5 1.9 5.8

4.2" 2.8 11.4 n.r. n.r.

4.0" 0.6 7.0 4.1 1.0

2.6 1.6 20.9 n.r. 0.6

"Includes 20:3.

2.5 8.4 9.4 n.r. 4.1 4.3 2.1 o n.r. 5.5 n.r. 2.3

3.3 6.9 11.5 n.r. n.r.

n.r. 2.1 14.0 1.1 n.r.

n.r. 2.3 15.4 n.r. n.r.

n.r.

3.7 2.4 0.3

--4.6 n.r. n.r.

eAverage of three analyses. *Not reported. tlncludes 18:3. °Includes 18:4. ~Includes 20:4. @Trace.

1.8 16.3 3.5 0.1 1.2 10.6 4.4 0.3 0.2 0.2 tr ° 3.2 1.4 2.5 1.5 0.3

128

Jeanne D. Joseph 3o F ( o l

!o 20

30 ~ Z~ 2o ~

io

16:0

[8:0

16;I 18:1 20:5 (n-7) (n-7) Fatty acid

22:6

Fro. 6. Selectedhepatopancreas fatty acids of Nova Scotia (black bar) and New Brunswick (white bar) quahaugs. Arctica is/andica: (A) triacylglycerols:(B) phospholipids.l°

hepatopancreas triacylglycerols of the Nova Scotia population (Fig. 6a) and the prominence of 20:5(n-3) in muscle triacylglycerols of the New Brunswick population as compared with that of the Nova Scotia molluscs (Fig. 7a). Both 16: l(n-7) and 18: l(n-7) were more prominent in hepatopancreas triacyglycerols of the New Brunswick quahaugs (Fig. 6a), but no differences were observed in these fatty acids in muscle triacylglycerols of the two populations (Fig. 7a). Shown in Table 13 are the percentages of fatty acids implicated in the biosynthesis of 20 carbon NMIDs in the two populations of bivalves. Also included are values (in parentheses) found in lipids of New Brunswick quahaugs held in aquaria without food 4o r ( a I /

30

20

o

~o

30

2°rz =

16:0

16:0

16:1 18:1 205 (n-7) in-7) Fatty acids

22:6

FtG. 7. Selected muscle fatty acids of Nova Scotia (black bar) and New Brunswick (white bar) quahaugs. Arctica is/am/lea : (A) triacylglycerols:(B) phospholipids.lo

Lipid composition of marine and estuarine invertebrates

129

TABLE13. Precursors and Products of NonmethyleneInterrupted Diene Biosynthesis in Lipids of Ocean Quahaug TissuesI° Lipid class: Tissue: Coll. site: # Fatty acids

Phospholipids Triacylglycerols Muscle Hepatopancreas M u s c l e Hepatopancreas N.S. N.B. N.S. N.B. N.S. N.B. N.S. N.B. Weight percent composition

16: l(n-7)

1.7

18: l(n-7)

2.9

20: l(n-7)

1.5

20:2A5,13

10.6

18: l(n-9)

1.9

20: l(n-9t

1.1

20:2A5,11

0.8

2.0 (2.5)* 2.0 (1.6) 4.2 (2.8) 7.2 (13.5) 1.9 (2.0) 0.9 (0.7) 2.7 (0.9)

1.2 5.2 2.6 7.3 3.2 1.6 1.2

3,1 (2.1) 3.4 (2.5) 1.5 (2.1) 7.6 (11.2) 1.7 (2.0) 1.1 (0.7) 1.4 (1.7)

12.6 8.3 5.3 0.9 3.7 1.7 1.8

12.8 (12.7) 6.8 (6.3) 3.1 (2.8) 0.3 (0.3) 3.6 (4.6) 0.9 (1.4) 1.5 (1.1)

14.4 10.5 3.4 0.6 2.7 1.6 1.1

7.8 (11.9) 7.3 (5.3) 3.6 (!.2) 0.6 (0.6) 3.3 (3.7) 1.1 (0.6) 1.0 (1.1)

~Nova Scotia (N.S.); New Brunswick(N.B.). *Values found in quahaugs unfed for ten weeks. for 10 weeks. 1° Palmitoleic acid (16: l(n-7)) was a major component in tissue triacylglycerols of both populations and was not diminished in the unfed quahaugs, demonstrating that this mollusc, like other animals, is able to biosynthesize 16: l(n-7) by A9 desaturation of 16:0. Successor (n-7) acids assumed less importance as chain length increased and only trivial amounts of 20:2 A5,13 were found in the triacylglycerol fraction of both groups. In contrast, however, this N M I D was a significant component in tissue phospholipids of both populations and was enhanced considerably in the quahaugs held without food, suggesting that 20:2 A5,13 must have some important metabolic/structural function or be a non-metabolizible fatty acid. It is interesting that the (n-9) pathway leading to 20:2 A5,11 was not equally active in this species during this study. Perhaps the percentages of the primary precursors in the lipids, i.e. the respective monoenes, determine the ultimate level of the two major NMIDs. The 20 carbon NMIDs of A. islandica were not chainelongated to any significant extent, as were those of the oysters, O. edulis and C. virginica (Table 11), which may indicate a species-specific biosynthetic pattern. The complete analysis of esterified lipid class fatty acids of the Nova Scotia quahaug is shown in Table 14, permitting comparison with other molluscs for which similar data have been published, particularly those of the gastropods (Table 8) and the oysters (Table 11). Among the molluscs of commercial importance in the United States, the hard clam, Mercenaria mercenaria, is second in value only to the oyster, C. virginica. Early American Indians of the Atlantic seacoast used the shells of this species as a currency called "wampum", and this usage probably inspired Linnaeus to select the name Mercenaria, derived from the Latin word, merces, meaning "hire, pay, reward". 4s The range of this species extends from Canada to Florida, while that of the soft, or steamer clam, Mya arenaria (family Myidae), is restricted to the colder waters of the northeast Atlantic coast of the United States and Canada. 45 Two analyses of M. mercenaria fatty acids, by different investigators, are shown in Table 15, along with analyses of an oriental species of Venus clam, Phacosoma japonica, and the soft clam, M. arenaria. With exception of the M. mercenaria analysis by Bonnet et al., 24 the reported fatty acid spectra were quite similar; 20:5(n-3) ranged from about 10%-18% and 22:6(n-3), from 10% to 17~o. Values for these fatty acids were low in the analysis of Bonnet et al., and the possibility of sample oxidation prior to G L C analysis must be considered; alternatively, these clams may have originated in more southerly, warmer waters. This publication is useful in that it lists the ranges of values observed in replicate fatty acid analyses, although the number of individual animals in each replicate was not indicated. However, some of the listed ranges for 20: 5(n-3) are suspect, i.e. ranges of 0.0-41.8% in S. solidissima and 0.0-36.9% in the bay

130

Jeanne D. Joseph TaBLe 14. Fatty Acids of Esterified Lipids of Nova Scotian Ocean Quahaug {Arctica islandica) Muscle and Hepatopancreas 1° Tissue: Lipid class: Fatty acids 14:0 4,8,12-TMTD o iso-15:0 anteiso- 15: 0 15:0 iso-16:0 Pristanic 16:0 7-MHD* iso-17:0 anteiso- 17: 0 17:0 Phytanic iso-18:0 18:0 19:0 20:0 14: l(n-7) 15:1 16:1(n-9) 16: l(n-7t 16: l(n-5)

17:1 18: l(n-I 3) 18: l(n-9) 18 : l(n-7) 18: l(n-5) 19:1 20: l(n-I I) 20: l{n-9) 20: l(n-7) 22:1{n-11 + 137 22: l(n-9) 22: l{n-7) 18 : 2(n-6) 20: 2(n-6) 20: 2"a" ~ 20:2"b" '~ 22:2-A"~ 22:2"B" '~ 18:3, n-6) 18:3 n-3) 20: 3 n-6) 20:3 n-3) 18:4 n-3) 20:4 n-6) 20:4 n-3) 22:4 1-6) 20:5 n-3) 21 : 5 n-2)f 22: 5 n-6) 22: 5 ,1-3) 22: 6 ,i-3)

Phos.

Muscle Hepatopancreas Trig. S. esters Phos. Trig. S. esters Weight percent composition

0.37 0.18 0.04 0.02 0.41 0.29 0.18 12.91 0.11 0.44 0.29 1.74 ND 0.36 6.26 0.21 ND ND ND 0.15 1.74 0.15 0.14 2.07 1.92 2.85 ND 0.14 0.28 1.06 1.47 ND ND ND 0.32 0.91 10.64 0.84 1.85 ND 0.44 1.32 ND ND ND 2.06 2.88 0.60 15.28 0.61 0.15 2.21 23.91

3.53 0.64 0.21 0.11 0.54 0.21 ND ° 32.35 0.23 0.84 0.84 1.69 ND 0.63 7.93 0.41 0.05 0.22 ND 0.84 12.64 ND ND 0.83 3.72 8.26 0.41 0.10 0,41 1,69 5,31 0,10 0.25 0.08 0.36 0.56 0.92 1.82 0.66 ND 0.03 1.27 ND ND 0.65 0.17 ND ND 5.48 0.14 ND 0.36 2.75

4.19 1,64 0.28 0.55 0.83 0.66 0.43 18.64 0.52 0.81 2.71 0.81 ND 1.88 7.79 0.53 ND ND 0.44 2.44 2.71 ND ND 1.06 6.92 5.32 ND ND 0,79 3.95 18.77 0.65 1.30 0.65 0.93 0.65 2.63 5.99 1.63 ND ND 0.27 ND ND 0.28 ND ND ND 0.83 ND ND ND ND

1.39 4,78 0.23 ND 0.88 0.38 0.41 22.46 0.08 0.45 0.15 2.25 0.15 0.15 11.60 ND 0.15 0.23 0.27 ND 1,20 ND ND 1.91 3,24 5,16 ND ND 0,29 1.60 2.60 ND ND ND 0,44 1.15 7,29 1,15 1,62 ND ND 2.07 ND ND 0.36 0.82 3.49 0.12 9.56 0.36 0.08 1.01 8.39

4.60 2.76 0.13 0.06 0.47 0.20 ND 20.04 0.10 0.25 0.12 1.61 ND 0.25 5.03 0.30 0.12 0.13 0.13 0.50 14.38 0.25 0,49 0,85 2,67 10,45 0,36 0.30 0.36 1.62 3,39 ND ND ND 1.03 0.36 0.60 1.07 0,42 ND 0.14 0.75 ND ND 2.82 0.27 0.22 ND 20.09 0.40 ND ND ND

7.92 2.70 0.68 1.36 1.71 1.18 1.64 20.27 ND 0.33 1.34 0.67 0.16 0.66 7.78 2.46 0.97 1.89 1.35 4.86 4.35 ND ND 0,33 9.84 3.93 ND 0.57 0,49 2.19 6.43 0.32 0.40 ND 0,08 0.40 0,81 1.61 0,80 ND ND 1.47 ND ND 0.86 0.27 1.36 ND 0.87 0.16 ND 1.02 0.75

°4,8,12-Trimethyltridecanoic, °Not detected. *7-Methylhexadecanoic ffrom hydrogenated sample). '~Nonmethylene interrupted diene. tRecent structural analysis indicates terminal olefinic bond position to be (n.3)13~

scallop are questionable. It should also be noted that, as originally published in the journal, a significant portion of the table of molluscan and crustacean polyunsaturated fatty acids was omitted, due to a type-setting error. Corrected reprints are available from the authors.

Lipid composition of marine and estuarine invertebrates

131

TABLE 15. Major Fatty Acids of Venus Clams (Family Veneridae) and Soft Clams (Family Myidae) Species: Tissue: Ref: Fatty acids 14:0 16:0 18:0 20:0 14:1 16:1 18:1 20:1 16:2

Phacosoma Mercenaria Mya japonica mercenaria arenaria Muscle Viscera Whole W h o l e Whole 108 144 24 24 Weight percent composition

16:4

0.7 18.2 0.3 0.6 0.2 0.5 8.2 3.1 trf tr 0.2

18: 2 18: 3

1.7 1.3

18:4 20:2 20:3 20:4 (n-6) (n-3) 20:5 22:2 22:4 22:5 (n-6) (n-3) 22:6 20:2 NMID 22:2 NMID

3.3 1.6 0.2

2.4 16.1 2.5 0.4 0.2 2.9 14.8 4.7 tr tr 0.1 4.3 2.2 2.2 1.2 0.2

3.7 0.5 10.7 4.2 1.0

2.7 0.7 9.8 4.3 0.6

3.6 n.r. 18.3 0.1 0.4

4.0 3.5 4.5 n.r. 5.6

4.4 1.2 11.2 n.r. 3.8

n.r. 2.3 15.1 0.8 n.r.

n.r. 1.5 9.8 1.1 n.r.

n.r. 2.0 15.0 3.6 0.3

-4.0 5.1 n.r. n.r.

0.5 3.9 13.4 n.r. n.r.

16: 3

2.0 12.3 5.5 0.4 n.r.* 4.8 8.4 8.6

2.2 8.8 4.1 2.7 1.1 3.1 5.3 1.2 n.r.

2.2 12.5 3.0 0.8 0.9 4.5 6.2 1.1 n.r.

0.9

n.r.

n.r.

n.r.

0.2 0.9 1.1 0.2 0.1

n.r. 3.5 2.6 4.6 2.3 1.0

1.9 1.4

1.8 1.2 0.8

*Not reported. fTrace.

Ueda has investigated the influence of environmental temperature on lipid content and fatty acid composition of muscle of the short-neck clam, Tapes philippinarium. 179 Analyses of samples taken approximately semimonthly from late August to mid-January showed statistically significant differences in total, neutral and polar lipid content as well as in fatty acid composition. Application of cubic, quadratic and linear regression equations of fatty acid compositions in relation to temperature showed strong dependence of composition upon temperature. Thus, Ueda's observations differ markedly from those of d e M o r e n o and co-workers who found food availability rather than temperature to be more important in regulating polyunsaturation in M. platensis 4° and M. mactroides 38 lipids. The decrease in mud temperature and changes in 16:0 and 22:6(n-3) which Ueda observed in the total lipid fraction of the clam are shown in Fig. 8. In addition to the species named and references cited, other fatty acid analyses of bivalves have been published. 5Z'55,63,82,94,151,189 4. Cephalopoda In comparison with the gastropods and bivalves, there is a paucity of data on fatty acids of the carnivorous squids and octopi, b u t the data which have been published show m a n y similarities. The fatty acid compositions of eight species of planktonic squids are shown in Table 16, and few, if any, significant differences between the species are evident. In all species, 16:0, 20:5(n-3) and 22:6(n-3) were predominant; modest amounts of 16:1, 18:1 and 20:1 were also present. This fatty acid profile is very similar to that of Loligo pealaii 144 and lllex illecebrosus mantle lipids 93 (Table 17). However, liver oils of squids

132

Jeanne D. Joseph 3O

20

ro

? a7

E

0 20

15

'

I-

i!I 26 vrrr

6 rE

4 ~r

20 Z

III

27 "r

5 "lrr

16 ~£I

26 ZI

I "TIT

8 "Err

15 :EII

6 I

17 I

Doy Mont h

FIG. 8. Declinein environmentalmud temperature and changes in 16:0 (white bar) and 22:6(n-3~ (black bar) of Tapes philippinarium total lipids with the onset of winter.~9 (Table 17) differed significantly from mantle lipids in that the liver oils have been found to contain substantially more monoenoic fatty acids and correspondingly less 20:5(n-3) and 22:6(n-3) than the mantle lipids, s9'93'191 M onoenoic fatty acids are prominent components of the lipids of a number of north Atlantic Ocean fishes, capelin (Mallotus rillosus), herring (Clupea harengus) and sand launce (Ammodytes americanus), 6-s'l 3.44.145 for example. They are also important fatty acids in the triacylglycerols of some coldwater pelagic crustaceans such as the euphausid, Meganyctiphanes norvegica (M. Sars), 5"9 and in the wax esters (as alcohols as well as acids) of many arctic upper-water copepods and amphipods, l16-118.t63`t64 All of these species are probably dietary components for the carnivorous squids. While the analyses of the squids, shown in Table 17, are in excellent agreement, they differ from that of the giant squid tentacle (Table I), in which wax ester and triacylglycerol acids were primarily 16:0, 18:1 and 20:1, which totaled 56°0 in the wax ester fraction and 68°'0 in the triacylglycerois: 20 and 22 carbon polyunsaturates were present only in trivial amounts. 63~ Culkin and Morris have reported the analysis of a single individual of a planktonic octopus, EIdonella pygmaea, in which the fatty acid composition was most unusual in a number of respects. 33 They observed a component (33.7°,) with a retention time of 0.87, relative to that of 18:0, in chromatograms of both nonhydrogenated and hydrogenated esters, which they tentatively identified as iso-18:0, while recognizing the inexplicably high percentage. In this analysis, only traces of 20 carbon polyunsaturates were observed, also confirmed on hydrogenated samples, and 22:5(n-3). rather than 22:6(n-3), was the most unsaturated 22-carbon fatty acid present. II1. POLAR LIPIDS

A. Polar Glycerrl Ethers and Plasmalogens 1. Polyplacaphora In a study of glyceryl ether lipids of molluscs, Thompson and Lee found that most of the glyceryl ethers of the chiton, Katherina tunicata, were concentrated in the polar lipid fraction and comprised 7.8% of the phospholipids. The alkyl chain composition was similar to that of the neutral glyceryl ethers (Table 2), but also included 15:0.17~

TABLE 16. P r i n c i p a l F a t t y Acids o f P l a n k t o n i c S q u i d s 33 Family: Sp.*:

Onychoteutidae

Onychot euthis banksi

Enoploteutidae

Pyroteuthis margaritifera

Pterygioteuthis giardi

Chiroteutidae -

Abraliopsis morrisii

Fatty acids

Chiroteuthis veranyi

Mastiooteuthis flammea

Cranchidae

Sepiolidae

Pyrogopsis

H eteroteuthis dispar

~ciflca

t"

W e i g h t percent c o m p o s i t i o n e~

14:0 16:0 18:0 16: l(n-9)t 18:1 (n-9)t 20: i(n-9)t 22: l(n-9)t 18:

2(n-6)

3(n-6) 3(n-3) 18:4(n-3) 20: 2(n-6) 20: 3(n-3) 20: 3(n-6) 20:4(n-3) 20:4(n-6) 20:5(n-3) 22:5(n-3) 22:6(n-3)

5.1 30.9 5.7 2.7 8.0 4.3 --

2.9 26.8 5.2 2.'/" 7.6 5.5 --

0.2

--

tr ----1 -13.8 1.6 25.3

tr -0.4 1.0 __ __ 0:2 15.7 2.1 26.7

18:

18:

*Species. t O t h e r isomers m a y a l s o be present. °Trace.

3.1 26.0 4.5 2.2 5.7 4.8 0.4 --

1.8 21.0 5.6 2.4 7.4 5.2 -tr °

2.3 23.1 4.7 3.9 13.9 4.0 ---

--

tr

1

tr

tr

0.5

--

tr

--

3.8 19.8 4.6 7.2 12.7 6.2 2.0 -0.8 --

6.4 23.6 4.9 4.6 9.1 4.2 --0.5 --

3.4 22.4 4.1 2.9 6.9 4.1 -1.0

tr

--

--

0.2 1.3

0.2 2.9

tr

0.2

--

--

o ~: = ~l r~. o= .~ e~

0.3 0.7 tr -1

15.3 8.4

26.0

0.2 2.4 .

0.2 1.5 .

0.5 -.

.

.

tr

tr

--

1.7 13.1 0.4 36.3

1.0 14.9 0.7 25.1

16.2 0.9 20.3

-

-

-

-

12.0 1.3 27.7

-

¢-

2. ~o

-

17.3 -31.9

-

~"

134

Jeanne D. Joseph TABLE 17. Principal Fatty Acids of Squid Tissues

Species:

Loligo pealaii

Tissue: Reference: Fatty acids

Mantle 144

lllex illecebrosus Mantle 93

Liver 93

Liver 89

191

Liver 191

4.2 15.1 3.4

2.2 15.5 10.2

6.3 15.9 9.1 5.8

3.4 15.0 11.2 2.6

Weight percent composition

14:0 16:0 18:0 20:0 16:1 18:1 20:1 22:1 16:2 16:3 16:4

2.0 28.2 4.5

2.2 27.6 4.4

4.4 13.7 1.8

3.9 15.4 2.3

0.5 4.1 4.2 0.3

0.4 4.9 4.9 0.5

5.9 16.4 11.8 7.7

0.1

0.3

18:2

0.2 0.1 0.1 0.1 0.5

0.3 0.1 0.1 0.3

9.0 16.3 12.4 8.2 0.5 0.1 n.r.* 0.8 0.4 0.8 0.2 0.1

0.8 n.r. 14.3 0.1

0.8 n.r. 15.8 0.1

0.4 0.4 13.9 0.3

1.3 0.6 12.1 --

n.r. 0.3 36.4

n.r. 0.3 37.1

0.1 1.3 16.9

-1.3 15.8

18:3 18:4 20:2 20:3 20:4 In-6) (n-3) 20:5 22:4 22:5 (n-6) 0~-3) 22:6

Thysanoteuthis rhombus

Ommastrephis solani pacificus

--

. n.r. 1.5 0.9 0.7

1.5

.

.

. n.r. 1.6 0.9 1.6 . .

n.r. 0.9 tr tr .

.

1.8 1.1 9.6 n.r. 1.1

3.3 tr 8.6 n.r. tr

13.8

15.1

*Not reported.

Polar plasmalogens were found to be prominent in phospholipids of Ischnochiton hakodadensis. By means of reaction micro-TLC chromatography, ~81 Dembitsky determined that 77.4% of the phosphatidylethanolamines, 45.3~o of the phosphatidylserines and 6.2% of the phosphatidyicholines were present in the plasmalogen form. 36

2. Gastropoda As in the chiton, K. tunicata, most of the glyceryl ethers (72%o of the total) of Thais lamellosa were found to be present in the phospholipids, 9a The major alkyl constituents are shown in Table 2. From their analysis of 22 species of marine invertebrates belonging to six phyla, Dembitsky and Vaskovsky concluded that the invertebrates can be grouped according to the distribution of plasmalogens in their phospholipids. 37 Plasmalogens of Group I invertebrates exist only as phosphatidylethanolamines (Coelenterata and Echinoidea); those of Group II as both phosphatidylethanolamines and phosphatidylcholines (Annelida, Arthopoda, Ascidia and most of the Echinodermata); and those of Group Ill as phosphatidylethanolamines, phosphatidylcholines and phosphatidylserines (Mollusca). Among all of the invertebrates studied by Dembitsky and Vaskovsky, only molluscs contained phosphatidylserine plasmalogens. The animals included in this study had been collected during the winter months and the authors acknowledged the limited nature of the data, due to the known seasonal variation in lipid composition of marine invertebrates. A later study of invertebrates collected during the summer months was conducted to remedy this deficiency; 36 and partial results of the two studies are shown in Table 18, Based upon the data of two (Collisella heroldi and Littorina kurila) of the three species collected in the two seasons, it appears that the percentage of phosphatidylserine plasmaIogen increases substantially during the summer months while the percentage of phosphatidylethanolamine plasmalogen increases less markedly. It seems possible that this

Lipid compositionof marine and estuarineinvertebrates

135

TABLE18. SeasonalVariation in Distribution of Plasmalogensin MarineGastropod Phospholipids36'37 Season: Phospholipid: Species Collisella heroldi Littorina kurila Tectonactica janthostoma Acmaea pallida Littorina brevicula Littorina squalida Nuccella heyseana Tequla rustica

Winter -Ethanolamine-Choline 53.4 68.7 52.3

29.6 6.9 13.5

Summer -Serine -Ethanolamine -Choline Percent of class 24.3 19.2 52.9

73.2 69.8 69.9 61.5 75.I 73.6 70.1 77.0

-Serine

20.9 10.5 7.8 11.5 11.6 15.0 3.0 --

55.2 66.7 57.6 39.1 75.8 71.8 45.2 65.3

seasonal change in composition is related to the reproductive cycle and is a result of gonadal development or loss of gametes after spawning. Dembitsky's data also indicate a striking similarity in plasmalogen composition of the three species of the genus Littorina. 36 3. Bivalvia

Glyceryl ethers were reported to comprise 3.2% of the polar lipids of the clam, Protothaca staminea.l 77 The primary alkyl constituents observed were 16: 0 (35%), 18: 0 (21%) and branched 17:0 (11%)(Table 2). Polar glyceryl ethers with both straight-chained and 2-methoxy alkyl groups were isolated from lipids of M . edulis. 61 In a study of oyster phospholipids, Sampugna et al. identified 8.3% glyceryl ether phospholipid and 2.4% glyceryl ether phosphonolipid in C. virginica, t62 This same detailed analysis of C. virginica phospholipids showed that 68% of the oyster plasmalogens was present in the polar lipid fraction, which included 21.8% plasmalogen phospholipid and 6.4% plasmalogen phosphonolipid (Table 19).162 Since 22.8% of the plasmalogens and 22.1% of the glyceryl ethers were present as phosphonates, the authors suggested a precursor-product relationship between these two classes, supporting Thompson's earlier proposal, that glyceryl ethers are precursors of plasmalogens, based upon the results of radioisotopic labeling studies with Arion ater, a terrestrial snail. 176 A TLC study of lipids of the clam, Tapes japonica, demonstrated the presence of phosphatidylethanolamine plasmalogen (9.2% of phospholipid), but no phosphatidylcholine or phosphatidylserine plasmalogens were detected. 19s In contrast, the research of Dembitsky and Vaskovsky indicated that phosphatidylserine plasmalogen was prominent in the phospholipids of many bivalves collected during the summer season but somewhat less so in those collected during the winter, with the exception of Crenomytilus orayanus (Table 20).36.37 As in the gastropods (Table 18), phosphatidylcholine plasmalogen was only a minor fraction of this polar lipid class in these bivalves. 4. Cephalopoda

A study of glyceryl ethers of representatives of the four major molluscan classes indicated that tentacle polar lipids of Octopus dofleini contained 9.1% glyceryl ethers and TABLE19. Distributionof Phosphorus in Polar Lipidsof the Oyster, Crassostrea virginicat62 Diacyl Phosphorus (Percent of polar lipid) Percent of polar lipid as: Phosphate Phosphonate Percent of class as phosphonate

Lipid class Plasmalogen Sphingolipid Glycerylether

40.7

28.2

20.4

10.6

38.1 2.6 6.5

21.8 6.4 22.8

6.9 13.5 66.2

8.3 2.4 22.1

136

Jeanne D. Joseph TABLE 20. Seasonal Variation in Distribution of Plasmalogens in Marine Bivalve Phospholipids 3~'37

Season: Phospholipid: Species

-Ethanolamine

Crenomytilus grayanus Chlamys swifti Crassostrea gigas Spisula sachalinensis Mercenaria stimpsoni Andara boughtoni Arca boucardi Callista brevisiphonata Chlamys farreri Glycemeris yessoensis Mactra sulcataria Modiolus difficilis Rapana thomasiana thomasiana Peronidia venulosa Patinopectin yessoensis Swiftopectin swifti

57.2 27.6 54.5 71.5 19.4

Winter -Choline

Summer

-Serine -Ethanolamine Percent of class

-3.4 12.9 I 1.2 10.0

72.1 33.3 37.7 21.4 20.1

81. l 72.9 68.9 72.2 77.4 85.7 77.3 85.7 67.0 80.7 74.2 63.5 66.7 66.0

-Choline

-Serine

6.7 10.0 3.2 9.0 20.0 2.1 2.7 0.0" 4.4 14.8 2.4 5.1 9.7

60.5 46.3 48.5 53.8 36.0 37.8 38.3 77.2 51.9 75.0 56.0 50.0 29.0 28.9

*Probably a misprint since authors have otherwise used a " -" to indicate absence.

hepatopancreas polar lipids, 6.5~/o. 177 Differences were noted in the alkyl side chain composition (Table 2). In the tentacle ethers, the two major alkyl chain lengths were 16:0 (38%) and 18:0 (20~o), while in the hepatopancreas ethers, 18:0 (41~o) predominated over 16:0 (30%). Most of the plasmalogen in O. dofleini tentacle iipids was present in the polar lipid fraction. 177 The polar lipids were reduced with LiAIH4 and fatty alcohols (from the reduction of fatty acids), unesterified glyceryl ethers and "cyclic acetals" were separated and recovered by silicic acid column chromatography. After aqueous HCI hydrolysis of the acetal hydrocarbon chain, the resulting aldehydes were analyzed by GLC. The major aldehydes of octopus tentacle plasmalogens were identified as 18:0 (41~o), branched 15:0 (18%) and 16:0 (14%). In a note added in proof, Thompson and Lee stated that further study indicated that the "cyclic acetals" were, more probably, the unesterified, ~t-fl unsaturated glyceryl ethers, but this observation does not, however, invalidate their results.17 v In one of a series of publications on phospholipids of marine animals, deKoning reported that plasmalogens comprise 14~ of the polar lipids of O. vuloaris and are concentrated in the phosphatidylethanolamine fraction (31%).35 In a histochemical study, using Feulgen stain, Boucaud-Camou observed the presence of plasmalogens in hepatopancreatic cell cytoplasm of the cuttlefish, Sepia officinalis. Lipid analysis by TLC showed that the major lipids present were phosphatidylethanolamine and phosphatidylcholine and the author speculated that they were present primarily in the plasmalogen form. 25 Dembitsky's study of summer-collected molluscs included two cephalopods, the squid, Todarodes pacificus, and Octopus sp., neither of which contained phosphatidyiserine; consequently, they also lacked phosphatidylserine phasmalogen. 36 The percentage of plasmalogens in phospholipids of T. pacificus and Octopus sp. is shown in Table 21. Based upon these data, it appears that, generally, plasmalogens comprise a significantly greater percentage of phosphatidylethanolamine and phosphatidylcholine in gastropods TABLE 21. Distribution of Plasmalogens in Marine Cephalopod

Phospholipids a6 Phospholipid: Species

Todarodes pacificus Octopus sp.

-Ethanolamine -Choline Percent of class 26.5

32.1

1.9 0.6

-Serine

---

Lipid compositionof marine and estuarine invertebrates

137

and bivalves than in cephalopods. Although Dembitsky's results 36 are in agreement with the evidence of deKoning 35 and Boucaud-Camou, 2s the data are too limited at this time to conclude that cephalopods are unique among molluscs in lacking phosphatidylserine and the related plasmalogen.

B. Phosphonolipids In the early 1960's, Kittredge and co-workers isolated an acid-stable, ninhydrin positive compound from lipids of the sea anemone, Anthropleura eleoantissima, which proved to be a glyceryl ester of 2-aminoethylphosphonic acid (2-AEP, III), an analog of ethanolamine containing a highly stable C - P bond. t°5 Further investigation by this group of investigators also established the biological occurrence of phosphonic acid analogs of choline (IV) and serine (V). 1°4 OH

OH

J

/

H2N--CH2--CH2--P=O

(H3C)3 - N ~

* ~--CH2--CH2--1~---O

OH

COOH

OH

(IID

OH

I I H2N--CH--CH2-P~O

(IV)

OH (v)

A short time later, Hayashi aand his colleagues, Matsubara and Matsuura, began an intensive study of molluscan phosphonolipids which has spanned more than a decade. This series of detailed, elegant studies, utilizing sophisticated chromatographic and spectrometric techniques, has demonstrated that phosphonolipids are widely distributed in the phylum Mollusca and, in some cases, comprise the major polar lipid class present. Although diacyl, plasmalogen and glyceryl ether ester phosphonates have been identified in the American oyster, C. virginica 162 (Table 19), published information and oral presentations in marine lipid symposia sponsored by the American Oil Chemists' Society6a'69'Ta'a6 indicate that in molluscs, phosphonates are present primarily as components of sphingolipids. The first sphingophosphonolipid isolated from a marine animal, the sea anemone, was structurally characterized as ceramide-2-aminoethylphosphonic acid (CAEP, VI), ~6°'16a an analog of sphingomyelin (VII). CH3--(C[-12)12

H

\C--C / O~- j / \ I H CH--CH--CH 20-- P--CH2--CHz--NH~*'

~H

I

It

NH

O

I

R--C---~O (VI) Ceramide-2-aminoethylphosphonicacid (CAEP) CH3--(CH2h2

H C=C

/ H

\

O I CH--CH --CH20--P --O--CH2--CHz--N ~+~--(CH3)a

J

J

OH NH

If

O

I

R--~O

(Vll) Sphingomyelin However, later investigations have revealed significant differences in CAEP and molluscan sphingophosphonolipids. Unlike the CAEP of the sea anemone, molluscan sphingophosphonolipids do not contain sphingosine as the sole long-chained base (LCB) component but, rather, a complex array of dihydroxy monoenoic and dienoic, and trihydroxy

138

Jeanne D. Joseph

saturated, monoenoic and dienoic bases of 16 to 22 odd and even carbon chain lengths. In addition, 2-N-methylaminoethylphosphonic acid (2-MAEP VIII) supplements or replaces 2-AEP in some gastropod sphingophosphonolipids (Table 22). 128 The structures of some representative molluscan LCBs (IX-XIV) are shown below. Trivial names used in this review are shown in parentheses: the number of hydroxyl groups is denoted by the letters "d" (dihydroxy) and "t" (trihydroxy). Excellent reviews on the chemistry, structure and occurrence of sphingolipid long chained bases are available. 1°1"102 CH 3

OH

HN]- - C H 2--CH2-- ~----O

I

OH

(VIII) 2-N-methylaminoethylphosphonic acid CH3--(CH2)I 2

\

/

H

C--~C

/

\

H

CH--CH--CH2OH

I

I

OH

NH2

(IX) Sphingosine

CH3--(CH2)10

\ /

H

/ ~C

\

H

CH--CH--CH2OH

I

I

OH NH2 (X) 1,3-Dihydroxy-2-aminohexadecane (Hexadecasphingosine: d 16 : l)

CH3--(CH2)s

\

/

/

~C

H

H

\ CH2--CH 2

\

H

/ ~C

H/

" ' C H - - C H - - C H 2OH

I

I

OH NH2 (XI) 1,3-Dihydroxy-2-amino-trans-4,trans-8-octadecadiene(Octadeca-4,8-sphingadienine: d 18 : 2)

CH3--(CH2)7

H

\c___c/ / \ H

(CH2) 5

\ /

H

/ ~C

H

\ CH--CH--CH2OH

L

I

OH NH2 (XII) 1,3-Dihydroxy-2-amino-4,1 l-eicosadiene (Eicosa-4,11-sphingadienine; d 20:2)

Lipid composition of marine and estuarine invertebrates CH3--(CH2)s

\ /

/ C-~C

139

H

\

H

(CH~)9

\ /

H

/ C-~C

\

H

CH--CH--CH2OH

I

I

OH

NH2

(XIII) 1,3-Dihydroxy-2-amino-4,15-docosadiene (Docosa-4,15-sphingadienine: d 22: 2)

CH3--(CH2) s

\ /

H

/ C-~C

H

\ (CH2)Io--CH--CH--CH--CH2OH OH

OH

NH2

(XIV) 1,3,4-Trihydroxy-2-amino-trans- 15-docosene (4-Hydroxy-docosa- 15-sphingadiene: t 22: l )

1.

Polyplacaphora

In a study of long chained bases of molluscan lipids, Matsubara isolated a fraction from lipids of the chiton, Liolophira japonica, which had an infrared spectrum with absorption bands characteristic of sphingophosphonolipids but no ester absorption bands. 12s This fraction comprised 37.4% of the adductor muscle total lipids (Table 22). Both 2-AEP and 2-MAEP were identified, with the latter acid predominating. Fatty acid and LBC compositions of the chiton sphingophosphonolipids are shown in Tables 23 and 24. The structure of the LCB, d 20:2, was determined to be eicosa-4,11-sphingadienine through mass spectrometry of the N-acetyl-O-TMS derivative, as well as of the product aldehydes (following sodium metaperiodate oxidation), alcohols (sodium borohydride reduction of aldehydes) and short-chained fatty acids (periodate-permanganate oxidation), t~s The geometry of the ethylenic bonds was unspecified. TABLE 22. Sphingophosphonolipid Content and Phosphonate Constituents

in Marine Molluscs ~2s Class Species Polyplacaphora Liolophira japonica Gastropoda Turbo cornutus Muscle Viscera Monodonta labio Tegula lischkei Conomurex luhanus Celluna eucosmia Bivalvia Crassostrea gioas'~ Adductor Gills Mantle Viscera Mytilus edulis

Spingophosphonolipid Content (%)*

AEP

MAEP

37.4

+ +

+

10.6 8.0 16.2 33.1 20.7 14.5

--+ + + + + + +

45.2 22.2 21.0 19.2 25.6

+ + + + +

*Percent of total lipid (see comment in text). ?Formerly Ostrea oigas. ~.P.t..R. 21/2--D

Phosphonate

+ + + + +

+ + + + +

+++ +++ ++ ++ +

m

140

J e a n n e D. J o s e p h TABLE 23. C o n s t i t u e n t F a t t y A c i d s o f M o l l u s c a n S p h i n g o p h o s p h o n o l i p i d s 12s

Class Species

Fatty acids (Percent composition) 17:0 18:0

16:0

br* 17:0

18:1(A2)

2-OH 16:0

52,1

--

3.8

7.7

--

36,4

86.7 68.7 78.7 71.3 80.1 73,0

-tr 6,5 11.4 tr tr

8.4 5,5 3,3 6.2 0.8 4.4

4.4 4,8 4,5 5.3 6.7 4.4

--5,9 ----

tr 21.1 1.2 5.8 12,4 18.2

89.7 77.2 76.2 77.2 76.7

----12.1

7,0 4.8 5.0 5.1 3.0

3,3 4.7 3.2 4.0 1.5

----, --

tr 13.2 15.1 13,7 5.7

Polyplacaphora

Liolophira japonica Gastropoda

Turbo cornutus muscle viscera

Monodonta labio Tegula lischkei Conomurex luhanus Celluna eucosmia Bivalvia

Crassostrea gigas't adductor gills mantle viscera

Mytilus edulis *Branched chain. ? F o r m e r l y Ostrea

gigas,

2. Gastropoda

Ceramide-2-aminoethylphosphonate has been reported to comprise 6% of the polar lipid of the abalone, Haliotis midae. 3a The principal fatty acids observed were 16:0 (53%), 18:0 (15%) and 20:5 (11%). This is the only known report of 20:5 as a constituent fatty acid in molluscan sphingophosphonolipids and its presence may indicate contamination of the CAEP fraction by esterified polar lipids. Hayashi and co-workers, in their study of molluscan sphingophosphonolipids, have focused their attention, in particular, on Turbo cornutus as a representative of the class, Gastropoda.6 7,7 0--7 2.7 4---76,12 8,13 2.13 4 I n 1 9 6 9 , they d e s c r i b e d a sphingophosphonolipid from visceral tissue of T. cornutus, which had a molar ratio of 2.0:1.1:1.0:1.0 for N, P, fatty acid residue and sphingosine, respectively. 7s GLC of the TMS derivatives of the constituent LCBs showed the presence of one major component which had a retention time of 0.4 and an equivalent chain length (ECL) of 15.55, both relative to those of the TMS derivative of an authentic dihydrosphingosine standard (I.0 and 18.00, respectively), TABLE 24. L o n g - C h a i n e d B a s e C o m p o s i t i o n o f M o l l u s c a n S p h i n g o p h o s p h o n o l i p i d s 12a

Class Species

Long-chained base (Percent composition ) d19:l d20:l t22:1

d18:2

d20:2

--

29,8

d16:l

d17:l

d18:l

18.9

17.1

18.8

10.6

4.9

5,4 10.2 15.0 4.4 13.0 13.9

11.0 8.2 7.2 5.3 12.1 6.9

37.2 41.5 37,6 39.4 32.6 10.2

2.7 3.8 19.1 18,4 10.9 21.9

-~----

75.9 18.4 20.4 10.8 17.1

-4.1 5.4 4.2 4.0

4,7 25.3 23.0 17.9 40.0

-1Z9 13.1 6.4 5.3

d22:2

Polyplacaphora

Liolophira japonica

--

Gastropoda

Turbo cornutus Muscle Viscera

Monodonta labio Tegula lischkei Conomurex luhanus Celluna eucosmia

-18,0 16.4 11.7 -13.5 -29.9 -30.1 . . . . . . .

----2.6 -47.1

19.3 30.7 29.0 52.3 33.2

7.6 8.5 8.1 --

-

-

Bivalvia

Crassostrea gigas't Adductor Gills Mantle Viscera

Mytilus edulis 5"Formerly

Ostrea gigas.

25.3 8.2 5.2 --

-

--

Lipid compositionof marine and estuarine invertebrates

141

TABLE 25.

DihydroxyLong-ChainBase and Fatty Acid Compositions of T. cornutus CMAEP i Ceramides13'* Peak No. Long-chainbase 1

2 3 4 5 6

16:1 17:1 18:1 br* 18:2 18:1 18:1 22:2

Fatty acid

Percent

16:0 16:0 16:0 16:0 } 16:0 16:0 16:0

26 I1 5 32 23

*Branched chain. suggesting that the major LCB had 16 rather than 18 carbon atoms in the molecule. Palmitic acid comprised 89.4% of the fatty acids and 2-MAEP was identified as the principal phosphonate present. For this lipid, the structure, N-palmitoylsphingosyl (C16)-2-N-methylaminoethylphosphonate was proposed. 75 Sphingophosphonolipids of T. cornutus viscera were later separated into two CMAEP fractions which differed only in fatty acid composition. 67 The principal fatty acid of one fraction was 16:0 (88.8%); the second contained 42.0% 16:0 and an unknown fatty acid (37.7%) with a G L C retention time between that of 21:0 and iso 22:0. The authors considered that the difference in fatty acid composition explained the observed difference in TLC retention time of the two fractions. The presence of sugar residues was also noted in these early studies. 67'134 In a more detailed analysis, Hayashi and co-workers separated T. cornutus visceral sphingophosphonolipids into three fractions, CMAEP I, C M A E P II and CMAEP III.t34 The first fraction, CMAEP I, was hydrolyzed with phospholipase C to eliminate the phosphonate group (2-MAEP), and the intact ceramides were derivatized to TMS ethers for G L C and G C - M S analysis. G L C of the derivatized ceramides showed the presence of at least six components and G C - M S indicated that 16:0 was the only constituent fatty acid (Table 25). Peak 4 (Table 25) consisted of two overlapping components and totaled 32% of the G L C response; however, the ratio of the the LCB 18:2/16:0 fatty acid component to the LCB 18:1/16:0 fatty acid component of the ceramides of this peak was calculated to be 7:25 from peak areas of a mass fragmentogram of ions at role 309 and role 311, respectively. The structure of the d 22:2 LCB was shown to be docosa-4,15sphingadienine by G C - M S of: (1) TMS ether derivatives of the LCBs; (2) aidehydic products of sodium metaperiodate oxidation; and (3) TMS ether derivatives of alcohols resulting from sodium borohydride reduction of the aldehydes. 7° Unlike C M A E P I, C M A E P II contained only 2-hydroxy (2-OH) fatty acids and trihydroxy LCBs. 72 After phospholipase C hydrolysis, liberating 2-MAEP, and derivatization of the product ceramides to TMS ethers, nine different ceramides were identified by G L C (Table 26). Peaks 3 and 4 both consisted of phytosphinogosine (18:0 LCB) and TABLE 26. TrihydroxyLong-ChainBase and

Compositions of Turbo cornutus Ceramides72 Peak No. Long-chainbase 1

16:0

2 3 4 5 6

17:0 18:0 18:0 19:0 f18:0 [19:0 ~19:0 1.20:0

7 8

X*

9

22:1

*Unknown structure.

Fatty Acid CMAEP II

Fatty acid

Percent

16:0 2-OH 16:0 2-OH 16:0 2-OH 16:0 2-OH 16:0 2-OH 16:0 2-OH 16:0 2-OH 16:0 } 2-OH 16:0 2-OH 16:0 2-OH 16:0

0.8 2.4 10.0 35.7 16.8

2-OH

2.9 2.9 1.5 27.0

142

Jeanne D. Joseph

2-OH 16:0 fatty acid and had identical mass spectra but different retention times; the structural difference between the two was not known. 72 The structure of the trihydroxy 22:1 LCB was established as 4-hydroxy-docosa-15-sphingadiene (XIV) by GC-MS analysis of: (1) the N-acetyl-O-TMS long chained base derivatives; (2) the aldehydes formed from the LCBs by sodium metaperiodate oxidation; and (3) the TMS ethers of the alcoholic products of sodium borohydride reduction of the aldehydes. 7° The authors noted that the two additional hydroxy groups in CMAEP II, as compared with CMAEP I, might confer upon this fraction an important role in the metabolism of sphingophosphonolipids in T. cornutus. 72 The third fraction isolated from T. cornutus visceral lipids (CMAEP III) was shown to consist of sphingosine (83.5% of total LCBs), 2-MAEP and fatty acids, primarily 16:0 (70.2%), 16:1 (9.7%) and 2-OH 16:0 (7.1%). In addition, sugars were extracted with pyridine and GLC analysis of TMS ether derivatives of this extract on two different columns indicated that the only sugar present was galactose. The molar ratio of P, N, fatty acid, sphingosine and sugar was 1.0:2.1:1.2:0.7:0.8. 71 Additional detailed degradative, chromatographic and spectrophotometric analyses indicated that CMAEP III was 1 - O ( 6 ' - O - ( N - m e t h y l a m i n o e t h y l p h o s p h a n y l ) - g a l a c t o p y r a n o s y l ) ceramide (XV), abbreviated by Matsuura as PnSGL (PhoslShonoSphingoGlycoLipid))s2 Thus, PnSGL is a cerebroside linked to an aminoalkylphosphonic acid.

O CH20--P--CH2--CH2--NH--CH 3 R'--CH--CH--CH2

0---~ OH IOH

(XV) l-O-(6'-O-(N-Methylaminoethylphosphanyl)-galactopyranosyl) ceramide The constituent fatty acids and LCBs of PnSGL are shown in Table 27. Unlike CMAEP I, which consisted solely of dihydroxy LCBs and normal acids, and CMAEP II, which consisted only of trihydroxy bases and 2-OH fatty acids, PnSGL contained, primarily, dihydroxy bases associated with both normal and 2-OH acids, which may have accounted for the two spots observed on TLC plates. 132 Turning their attention to muscle tissue of T. cornutus, Hayashi and Matsuura isolated and characterized two aminoalkylphosphonyl cerebrosides of muscle lipids. 74 In addition to the major component, PnSGL, identified previously in visceral lipids, 132 a new phosphonosphingoglycolipid which differed from visceral PnSGL only in the phosphonic acid component, was described. Employing the same degradative, chromatographic and spectrometric techniques used in their earlier studies of visceral sphingophosphonolipids, 2-AEP was identified as the phosphonic acid moiety of the minor muscle PnSGL. Fatty acid composition was similar to that observed for visceral PnSGL; 16:0 comprised 53.3~o and 2-OH 16:0, 14.6~ of the total fatty acids. Long chain base composition was also similar. The principal LCBs observed were the dihydroxy bases, 18:1 (14.6~), 18:2 (11.3~o) and 22:2 (36.6%) and the trihydroxy base 22:1 (6.3~). While the sphingophosphonolipids of T. cornutus have been most intensively investigated, other species of gastropods also contain these complex lipids. Hori et al. isolated a phosphonolipid from M o n o d o n t a labio which was analyzed by a number of chromatographic and spectrometric methods. Results of these analyses agreed well with calculated values for palmitoylsphingosyl-2-N-methylaminoethylphosphonate, i.e. CMAEP. s7 However, another study of sphingophosphonolipids of this species showed that, in ad-

Lipid composition of marine and estuarine invertebrates

143

TABLE27. Long-Chain Base and Fatty Acid Compositions of Turbo cornutus Phosphonosphingoglycolipid(PnSGL)132 Structure

15:0 16:0 17:0 br # 17:0 18:0 br 18:0 19:0 20:0br 20:0 21:0 22:0 23:0 24:0

Fatty acids Long-chain bases Percentage Structure Percentage Normal 2-Hydroxy +* 32.6 1.3 4.1 ++ 3.0 + ++ ++ ++ 1.8 1.0 +

++ 31.2 3.4 6.1 ++ 5.4 + -1.6 ++ 1.3 + --

Dihydroxy 16:1 17:1 18:1 br 18:1 18:2 19:1 br X° 19:1 20:1 22:1 Trihydroxy 16:0 17:0 18:0br 18:0 18:1 19:0br 19:0 20:0 22:1

2.7 5.7 7.0 18.0 14.7 3.1 4.6 1.3 1.9 23.5 + + ++ 2.5 ++ + ++ + 11.2

*+ (<0.5%), + + (0.5-1.0%),- (not present). *Branched chain. °Unknown structure. dition to sphingosine, a number of other dihydroxy bases (16:1, 15.0%; 19:1, 19.1%; 18:2, 13.5%) were also present in substantial amounts (Table 24). 12s Phosphonosphingoglycolipids (PnSGL) isolated from M. labio lipids were shown to be identical to those first observed in T. cornutus lipids, i.e. 2-AEP and 2-MAEP analogs of galactosylpyranosyl ceramides, the latter being the major analog present. 133 As in Z cornutus PnSGL, 16:0 (49.7%) and 2-OH 16:0 (12.2%) were the principal fatty acid residues, but there were differences in LCB.composition. In M. labio PnSGL, the major LCBs observed were dihydroxy 18:1 (26.0%), anteiso 19:1 (18.3%), 18:2 (12.4%), 22:2 (9.6%) and 16:1 (9.0%). 133 Other species of marine gastropods which have been investigated for total sphingophosphonolipids and sphingophosphonolipid composition are Tegula lischkei, Conomurex luhanus and Celluna eucosmia (Tables 22, 23 and 24). 12s Although the percentage of sphingophosphonolipids was stated to be the percent of total lipid (Table 22), it is probable that the values represent percentage of polar lipid since the tissues were first extracted exhaustively with acetone to eliminate neutral lipids and water. Another point to be made concerning the data of Table 22 is that, at the time of publication (1975), sphingophosphonolipids containing 2-AEP had not yet been observed in T. cornutus tissues. As noted earlier by Matsuura et al., in T. cornutus visceral sphingophosphonolipids, normal fatty acid residues were associated with dihydroxy bases and 2-OH fatty acids with trihydroxy bases 134 (Tables 25 and 26), except for the P n S G L fraction of both muscle and viscera, in which dihydroxy LCBs were associated with both normal and 2-OH acids (Table 27). 71'74 As shown in Tables 23 and 24, these relationships do not hold for LCBs and fatty acids of C. luhanus and C. eucosmia sphingophosphonolipids, in that both species contain considerable percentages of 2-OH 16:0 but no trihydroxy base. 12B As in T. cornutus visceral sphingophosphonolipid, the dihydroxy base of the muscle sphingophosphonolipid was established as docosa-4,15-sphingadienine by G C - M S of the N-acetyl-O-TMS deriva_tive:_ Similarly, the structure of the dihydroxy base, 20:2, of C. eucosmia sphingophosphonolipids was shown to be eicosa-4,11-sphingadienine. The 18:1 (trans A2) fatty acid, observed in M. labio sphingophosphonolipids, was

144

Jeanne D, Joseph TABLE 28. Distribution of Phosphorus in Tissue Lipids of Fresh, Starved and Postspawning Oyster, C r a s s o s t r e a t,iroinica ~72

Tissue Whole body

M antle/gills

Muscle

Viscera

Condition Fresh starved post-spawn Fresh starved post-spawn Fresh starved post-spawn Fresh starved post-spawn

Total lipid P (/~g/gwet weight) 237 313 212 267 281 209 228 289 156 242 404 297

Percent phosphorus Phosphate Phosphonate 76.8 66.1 63.7 76.0 61.7 71.2 59.6 61.2 73.7 87.2 72.8 71.0

23.2 33.9 36.3 24.0 38.3 28.7 40.4 38.8 26.3 12.8 27.2 29.0

identified by comparison of the mass spectrum of the methyl ester derivative with that of an authentic sample. 12a

3. Bivalvia Sampugna et al. carried out an intensive analysis of polar iipids of the American oyster, C. virginica, and found that a minimum of 13% of the phosphorus in the oyster lipids was present in the form of phosphonolipids. ~62 The distribution of phosphorus as phosphates and phosphonates in the four major classes of polar lipids is shown in Table 19. Although only small amounts of diacyl ester phosphonates were observed (6.5% of the polar diacyl esters), a significantly greater, and equal, percentage of the plasmalogen and glyceryl ether ester classes existed as phosphonates (22,8% and 22.1%, respectively), leading the authors to suggest a biosynthetic precursor-product relationship between the two classes. The major portion of the sphingolipid class (66.2%) existed as phosphonates. Research by Swift has demonstrated that during periods of physiological stress such as starvation or spawning, phosphonates are conserved in the tissues of C. virginica, in contrast with the esterified phospholipids which are metabolized, along with glycogen and other energy stores. 17~ As shown in Table 28, starved and post-spawning oysters contained more phosphonate than fresh oysters, a difference reflected in all tissues except the adductor muscle. These results lend support to the supposition that phosphonates may play a unique role in membrane structure or function.t72 An in-depth study of tissues of the Pacific oyster, Crassostrea oioas (= Ostrea oioas), showed that CAEP constituted 45% of total (polar) lipids in the adductor muscle, but only half as much in gills (22%), mantle (21%) and viscera (19%) (Table 22). 127'128 Fatty acid and LCB compositions of C. gioas sphingophosphonolipids are listed in Tables 23 and 24. By means of analytic techniques described in the previous section, ethylenic bonds in the 16:1 and 18:1 bases were located in the A4 position of the molecules, and the structure of the dihydroxy 18:2 base was shown to be octadeca.trans-4,trans-8sphingadienine. As shown in Table 23, 16:0 was the predominant fatty acid in all tissues of the oyster, although a modest amount of 2-OH 16:0 (13-15%) was also present in tissues other than adductor. In contrast, however, 16:1 LCB was concentrated only in adductor sphingophosphonolipids; in the other tissues, 18:2 LCB predominated, but 16:1 and 18:1 LCBs were also present in significant amounts (Table 24). 127A28 The structure of C. gioas adductor muscle CAEP was also investigated by GC-MS analysis of TMS ethers of the unhydrolyzed, intact CAEP molecule. ~29 Purified CAEP, which represented 47.8% (by weight) of the total phospholipid fraction, contained 16:0 as the major fatty acid residue (94.3%). The principal LCB present was d 16:1 (75.9%), but lesser amounts of d 18:2 (19.3%) and d 18:1 (4.7%) were also observed. The only phosphonic acid identified was 2-AEP. From these mass spectral fragments, the major cera-

Lipid compositionof marineand estuarineinvertebrates

145

mide of oyster adductor CAEP was identified as N-hexadecanoylhexadeca-4-sphinganine. Minor ceramides were N-heptadecanoylhexadeca-4-sphinganine, N-hexadecanoyloctadeca-4,8-sphingadienine and N-hexadecanoyloctadeca-4-sphinganine. As in the oyster, LCBs of mussel (Mytilus edulis) sphingophosphonolipids consisted principally of the dihydroxy bases, 16:1, 18:1 and 18:2 (Table 24) and AEP was the only phosphonic acid present. ~2s The two species differed, primarily, in fatty acid composition of the sphingophosphonolipids, in that branched 17:0 was the second most abundant fatty acid in the mussel but absent from oyster sphingophosphonolipids (Table 22). 12s Sphingophosphonolipids of other bivalves have also been investigated. Karlsson and Samuelsson reported that CAEP comprised 17% of muscle phospholipid of the scallop Hinnites gigantum. 1°3 The only phosphonic acid observed in Tapes japonica, 194 Barnea (Umitakea) dilatata japonica 137 and Pinctata martensii 72 was 2-AEP; no 2-MAEP was present. The principal constituent fatty acid in sphingophosphonolipids of these three species was 16:0 although P. martensii sphingophosphonolipids also contained 2-OH 16:0. 72 Only dihydroxy bases were found in these bivalves: 18:1 LCB in T. japonica ~94 and 18:2 LCB in B. japonica. ~37

C. Sphingolipids

For the purposes of this review, sphingolipids are defined as those lipids that contain sphingosine or other LCBs, with or without a sugar moiety (cerebrosides and ceramides, respectively) and which, unlike sphingophosphonolipids, lack a phosphonic acid component, although a phosphate ester group may be present, as in sphingomyelin (VII), Sug~ta et al. identified sphingomyelin, in addition to the sphingophosphonolipid, CAEP, in the bivalve, Pinctata martensii. 171 The results of chemical analyses agreed well with values that could be calculated for paimitoylsphingosylphosphorylcholine. Sphingosine constituted 85% of the LCB component and a branched chain LCB, O-methyl sphingosine (perhaps iso d-18:0 i°2) accounted for the balance. The fatty acids were identified as 16:0 (39.3%), 17:0 (34.9%) and 18:0 (25.8%). The 17:0 fatty acid was identified by comparison of a mass spectrum of the methyl ester with that of an authentic sample. Sphingoglycolipid extracted from oyster (C. oigas) mantle was shown by a variety of chromatographic methods (paper, column, GLC) to be a globoside-type mucolipid containing sphingosine, fatty acids and a number of sugars: glucose, galactose, fucose, O-alkyl fucose and the amino sugar, glucosamine.64 In later research by the same investigators, galactosamine was also identified in sphingoglycolipids of oyster gill tissue and the galactose moiety was recognized as having a branched structure; i.e. 4,6-di-O-methylgalactose. 13o More recently, 3-O-methylgalactosamine was identified as one of the components of oyster glycolipid. T M As Sugita et al. had noted in sphingoglycolipids of P. martensii, ~71 the fatty acids of oyster sphingoglycolipid were found to be, primarily, 16:0 (39.2%), 17:0 (17.4%) and 18:0 (23.4%).64 These percentages represent average values reported for two chromatographically separated fractions. 64 The major LCB had an equivalent chain length of 17.3, relative to that of sphingosine (18.0) and its structure was unknown. Hayashi and Matsubara later investigated the structure of the major LCB of oyster sphingogiycolipid (57.4%), which they determined to be octadeca-4,8-sphingadienine by GC-MS of TMS ether and N-acetyl TMS ether derivatives of the LCB mixture. 6s,66 Confrming evidence of structure was provided by GC-MS analysis of aldehydes produced from the LCBs by sodium metaperiodate oxidation, and of short chained fatty acid products of periodate-permanganate oxidation of the N-acetyl LCB derivatives. In addition, double bond positions were confirmed by GC-MS of TMS derivatives of the polyhydroxylated products after osmium tetroxide oxidation of the N-acetyl LCB derivatives. The authors noted strong infrared absorption at 980/cm, indicating that one, if not both of the ethylenic bonds, was trans in stereochemical configuration.73

146

Jeanne D. Joseph

Sphingoglycolipid of the bivalve, Barnea (Umitakea) dilatala japonica was found to be a galactosyl ceramide containing dihydroxy 18:2 as the primary LCB and 16:0 as the principal fatty acid component.137 IV. S U M M A R Y

Lipid and fatty acid compositions of molluscs differ in a number of respects from those of sponges and cnidarians, described previously. 9a While plasmalogens were observed in iipids of both of the more primitive phyla, this class of compounds, particularly the polar plasmalogens, has been found to be particularly prominent in the lipids of many molluscs. Ceramide-2-aminoethylphosphonic acid, first identified in an anthozoan cnidarian, the sea anemone, is only one of a large number of homologous sphingophosphonolipids that seem to characterize the phospholipids of the four major molluscan classes. The fatty acids of sponges were reported to include substantial percentages of longchained (26-28 C) NMIDs having 5,9 unsaturation ("demospongic acids") and cnidarian fatty acids have frequently been found to contain quantities of (n-6) polyunsaturates, particularly 20:4(n-6). The fatty acid composition of molluscs is more similar to that of phylogenetically more advanced marine animals than that of sponges and cnidarians and may reflect the influence of dietary habit, phylogenetic position or, more probably, a combination of the two factors. It is evident that 22:6(n-3), for example, is virtually absent in the primitive algal grazers, indicating that these molluscs are unable to desaturate and elongate 20:5(n-3) to 22:6(n-3), but prominent in the lipids of the more advanced carnivorous species. The presence of 20 and 22 carbon NMIDs, differing in double-bond position from those of sponges, have been demonstrated in several species and may b e suspected in others. These unusual fatty acids are probably biosynthesized by the herbivorous molluscs since they have been reported in no more than trace amounts in marine phytoplankton and algae. Acknowledgements--I thank Dr. Akira Hayashi (Kinki University, Higashi-Osaka, Japan) and Dr. R. B. Johns (University of Melborne, Parkville, Victoria, Australial for their assistance with taxonomy of molluscs from their geographic regions.

(Received 25 November 198I) REFERENCES 1. ABBOT, R. T. American Seashells, (2nd edn), 663 pp., Van Nostrand Reinhold Co., New York, 1974. 2. ACKMAN, R. G. In Methods in Enzymology, Vol. 14, pp. 329-381 (LowENSTEIN, J. M., ed.) Academic Press, New York, 1969. 3. ACKMAN,R. G. In Progress in the Chemistry of Fats and Other Lipids, Vol. 12, pp. 165-284 (HOLMAN, R. T.. ed.) Pergamon Press, London and New York, 1972. 4. ACKMAN, R. G. and BURGHER, R. D. J. Chromatogr. I1, 185-194 (1963). 5. ACKMAS, R. G. and EATON, C. A. Can. J. Biochem. 44, 1561-1566 (1966). 6. ACKMAN, R. G. and EATON, C. A. J. Fish. Res. Bd. Can. 27, 1669-1683 (1970). 7. ACKMAN, R. G. and EATON, C. A. J. Fish. Res. Bd, Can. 28, 601-606 (1971). 8. ACKMAN, R. G., EATON, C. A. and HINGLEY, J. d. Inst. Can. Sci. Technol. Aliment. 8, 155-159 (1975), 9. ACKMAN, R. G., EATON, C. A., SIPOS. J. C., HOOPER, S. N. and CASTELL J. D. J. Fish. Res. Bd. Can. 27, 513-533 (1970). 10. ACKMAN, R. G., EPSTEIN, S. and KELLEHER, M. J. Fish. Res. Bd. Can. 31, 1803-1811 (1974). 11. ACKMAN, R. G. and HOOPER, S. N. Comp. Biochem. Physiol. 46B, 153-165 (1973). 12. ACKMAN, R. G., I'tOOPER, S. N. and KE, P. J. Comp. Biochem. Physiol. 39B, 579-587 (1971). 13. ACKMAN, R. G., KE, P. J., MACCALLUM, W. A. and ADAMS, D. R. J. Fish. Res. Bd. Can. 26, 2037-2060 (1969). 14. ACKMAN, R. G. and McLACrlLAN, J. Proc. N.S. Inst. Sci. 28, 47-64 (1977). 15. ACKMAN, R. G., TOC,ER, C. S. and MCLACHLAN, J. J. Fish. Res. Bd. Can. 25, 1603-1620 (1968). 16. ANON. Prevention, 52-55, August (1980). 17. ANSELL, A. D. Mar. Biol. 27, 115-122 (1974). 18. BANNATYNE,W. R. and THOMAS, J. N.Z.J. Sci. 12, 207-212 (1969), 19. BEACH, D. H., HARRINt3TON, G. W. and Hot, z, G. G., JR. J. Protozool. 17, 501-510 0970). 20. BENSON,A. A., LI~E, R. F. and NEVENZEL J. C. In Current Trends in the Biochemistry of Lipids, pp. 175-187 (GANGULY, J. and SMELLIE, R. M. S., eds.) Academic Press, New York, 1972. 21. BLUMER, M., MtJLUN, M. M. and GUILLARD, R. R. L. Mar. Biol. 6, 226-235 (1970). 22. BLUMER, M., MULLn~, M. M. and THOMAS, D. W. Helgolander wiss, Meersunters. 10, 479~,88 (1972).

Lipid composition of marine and estuarine invertebrates 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

147

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160. 161. 162. 163.

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150

Jeanne D. Joseph H. japonicus 140.167 H. midae 34"48"153 H. discus 78"t°8'167"19~ N o r d o t i s discust 08,1 t t

Family Patellidae Patella coerulea L. 1~8 P. ~,ulgata 55 P. peroni 96 Cellana eucosmia 68" 128 C. traraoserica 96

Family Acmaeidae A c m a e a pallida 36"88"113.1s2.183 Collisella sp. 88'182'183 Collisella dorsuosa 26"88"113 C. heroldi 36"37 Patelloidea alticosta 96

Family Trochidae M o n o d o n t a labio 68"87A 11.133 C h l o r o s t o m a argyrostoma lischkei 8° Omphatius pfefferi carpenteri t oa Tegula lischkei 128 T. r u s t i c a 36'182"183 Gibbula ceneraria 186 Austrocochlea constricta 96

Family Turbinidae Batillus cornutus 81"1°8 T u r b o c o r n u t u s 6 7 ' 6 8 " 7 0 - 7 2 " 7 4 - 7 6 ' 128.132.134.1,67 Lunella coronata 62 Subninella undulata 9`6

Order Mesogastropoda Family Littorinidae Littorina brevicula 36.88.1 s2.1s3 L. littorea 11"12"27"8'1"18"4186 L. L. L. L.

manshurica i 83 neritoides 88 saxatilis 84"136 squalida 36"113"182"183

L. littoralis 84 L. kurila 3`6"37

Family Potamididae Batillaria multiformis 62

Family Crepidulidae Crepidula plana `6° C. f o r n i c a t a 55

Family Telescopiidae Telescopium telescopium 9`6 Terebralia palustris 96

Family Naticidae Lunatia triseriata 11.1~4 N a t i c a josephina 17 a Tectonatica j a n t h o s t oma 36"3 7.183

Order Neogastropoda Family Muricidae M u r e x brandaris L. 29"31 T h a i s clavigera s° T. lamellosa 177 Nucella lapillus 186 N . heyseana36"l 13.t 82

Family Buccinidae Buccinum bayani 81 B. stratissimum 81 B. undatum 126"19° B. tsubai 81 N e p t u n e a antiqua 5~ N . arthritica ~~5 N . constricta 182"183 N . intersculpta 81

Family Melongenidae Busycon canaliculatura 152,153.155

Family Nassariidae Tritia f e s t i v u s 62

Subclass Opistobranchia Order Thecosomata

Lipid composition of marine and estuarine invertebrates Family Limacinidae Limacina retroversa 22

Family Cuvieridae Diacria balantium I t 9 Clio pyramidata i t 9

Order Gymnostomata Family Clionidae Clione limacina t 17,118

Family Aplysiidae Aplysia depilans 1as A. fasciata 17 s A. punctata ~ °

Family Pleurobranchidae Pleurobranchaea meckeli Leue 178

Order Nudibranehia Family Glaucida¢ Glaucus $p.119 Order Basommatophora Family Siphonariidae Siphonaria diemensis 96

Class Bivalvia Subclass Palaetaxodonta Order Nueuioida Family Nuculidae Yoldia limatula 52

Subclass Pteriomorphia Order Arcoida Family Archidae Anadara brouyhtoni 36'113.1 s2 Arca noae L.29'31'48 A. boucardi 36'1 a 3

Family Limopsacea Glycymeris yessoensis s6'113,1s3

Order Mytiloida Family Mytilidae M ytilus edulis 36,4s'5 5.61.os'83,t t 3,123.12s'144"15 2,l s 3A sS'l ~9"t s6 M. aalloprovincialis Lemarek 29.3LIsa.Is9 M. canaliculus ls'4s M . californianus 4a'I s 7 Crenomytilus grayanus a 7 ,~ $z.~ s a Modiolus barbatus a t,4s M. difficilis 36.t13,t s2"l sa Lithophaga lithophaoa L. 31

Family Pinnidae Pinna pectinata~40

Order Pterioidae Family Pteriidae Pinctata martensii 171

Family Pectinidae Pectin irr adians ~s s P. j a c o b a e u s L. 2L4a P. m a x i m u s 55,190,i99

Patinopectin courinus ~12 P. yessoensis 36,s2.H3A74Aa2 Placopectin magellanicus 24,26,27,tlz.t2L144 Mizuhopectin yessoe_nsjs ls3 Aquepectin irradians 23,112 A. gibbus 24,112 Chlarays nipponensis akazara TM C. nipponensis 113,1 s2,tsa C. opercularis L.31'5s,19° C. olabra L. 29'31 C. ferrari 36 C. varia L. 31 C. herica Gould 1s4 C. swifti 37'1s3 C. tehuelcha 1~1 Swiftopectin swifti 36.1 i Hinnites raultirugosus 149 H. gigantum l °a

Family Limidae Lima hians 17

Family Ostreidae

151

152

JEANNE D. JOSEPH Crassostrea , q / ~ a s 3 6 " 3 7 ' 4 8 ' 5 9 ' 6 3 ~ 0 6 ' 6 8 " t 2 7 - l 31 ' 1 6 7 ' 1 8 2 ' 183 C. Dir~inica 12"24"28"30'48'60'143'144"162,172A87

189

Ostrea edulis as'la4"187 139 O, 9ryphea 53"54'139.16 t O. lutaria ta'4a

Subclass Heterodonta Order Veneroida Family Cardiidae Cardium edule Is6

Family Mactridae Mesodesma mactroides 3s'39 M a c t r a s u l c a t a r i a 36313,167,183 S pisula solidissima 24'14 °'t 4 7 S. sachalinensis 36"37"113"14°A82'183

Family Solenidae Solen strictus Gould 63'1°83 ~' Siliqua patula t 24 Family Tellinidae Tellina lutea t t3 M a c o m a sp. ~85 Perionidia venulosa 3°'t s3

Family Semelidae Semele (formerly Amphidesma) ventricosum Za Family Arcticidae Arctica islandica Jo. zo~

Family Veneridae Tapes decussatus L. 3~ T. philippinarium t 40.179.180 T. japonicus Deshayes °3"67"t92-194 Paphia indulata 63 Venl~S I;errucosa 3 !'48

1/. gallina 29.31,43 Mercenaria ( f o r m e r l y Venus} m e r c e n a r i a 12,24`51,94"la4"152A53"155A69 M. stimpsoni 36"37"1 t 3.183 M e r e t r i x meretrix luseria ~o7 Callista brevisiphonata 36"t t 3"182.t 83 Protothaca staminea 59"z ~7 Phacosoma japonica L08.1 ~

Family Myidae M y a sp. 6° M. arenaria 24"155"z58"259 M, japonica t s 5

Family Pholadidae Barnea (Umitakea) dilatata japonica ~37

Class Cephalopoda Order Sepioidea Family Sepiolidae Rossia pacifica t s 2

Family Sepiidae Sepia officinalis 25"tat Sepia sp. zs4 Heteroteuthis dispar s3

Order Teuthoidea Family Loliginidae Loligo vulgaris 3~'48"178 Loligo sp. 154 L. pealeii zaa'~55

Family Enoploteuthidae Abraliopsis morrisii 33 Pyroteuthis margaritifera 33 Pterygioteuthis giardi 3~

Family Onychoteuthidae Onychoteuthis banksi 33

Family Bathyteuthidae l llex itlec ebrosus 2 7"93"144

Family Ommastrephidae Todarodes pacificus T M Ommastrephes sloanie pacificas 89"166"t 9 t

Family Thysanoteuthidae Thysanoteuthis rhombus 191

Family Chiroteuthidae Chiroteuthis veranyi 33

Family Mastigoteuthidae

Lipid composition of marine and estuarine invertebrates Mastigoteuthis flammea33

Family Cranchidae Pyrooopsis pacifica 33

Order Octopoda Family Octopodidae Octopus sp. 36.1 s 2 O. dofleinP ~ O. vulgaris 35. I ~s

Family Bolitaenidae Eledonella pygmaea 33

153