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Fatty acid profile of the digestive gland and mantle in the bivalve Macoma balthica
Camp. Biochera. Physiol.Vol. 97B,No. 2, pp. 269-273, 1990 Printed in Great Britain
0305-0491/90$3.00+ 0.00 © 1990PergamonPress pie
FATTY ACID PROFILE OF THE DIGESTIVE G L A N D A N D MANTLE IN THE BIVALVE MACOMA BALTHICA S. TURUNEN and M. PEKKARINEN Department of Zoology, University of Helsinki, 00100 Helsinki, Finland (Tel: 358 0 1911) (Recewed27February 1990) Abstract--l. Digestive gland and mantle fatty acids were studied in spring and summer in the bivalve Macoma balthica off the southern coast of Finland. The presence of lipids was also examined histochemically in various clam tissues. 2. The neutral lipid content of the digestivegland increased ca 4.5-fold during the annual growth period. 3. Neutral lipid fatty acids of the digestive gland, of which palmitoleic, eicosapentaenoic and palmitic acids were predominant, were clearly distinguished from phospho- and giycolipid fatty acids. 4. The degree of unsaturation of phospholipid fatty acids was higher in the cold season both in the digestive gland and mantle, mainly due to the titer of eicosapentaenoic acid.
INTRODUCTION Macoma balthica is a small bivalve widely distributed on soft bottoms in both marine and brackish water. The biology of this species has received increasing attention in recent years, in part owing to the use of this clam in long term monitoring of water pollution (ICES, 1974). Off the coast of southern Finland M. balthica reproduces from late April to June, when marked changes occur in the carbohydrate, protein, and lipid content of the clam (Pekkarinen, 1983, 1986; Bonsdorff and Wenne, 1989). Comparable changes have been observed in other sites in northern Europe (Beukema and de Bruin, 1977; Graf et al., 1982; Polak et al., 1987). Lipids account for a substantial proportion of the stored energy in M. balthica. Histological observations have suggested that the digestive gland (digestive diverticula, hepatopancreas) is a major organ of lipid storage in M. balthica (Pekkarinen, 1983). The purpose of the present study was to examine the fatty acid composition of digestive gland neutral-, glyco-, and phospholipids at the beginning and at the end of the annual growth season at one sampling site, for possible seasonal changes. Comparable data were obtained from mantle phospholipids. Histochemical studies were carried out to map the accumulation of neutral lipids in different clam tissues. MATERIALS AND METHODS
Lipid and fatty acid analyses Macoma balthica were collected from the southern coast of Finland (Tv/irminne Zoological Station) in April and July, 1989 (Table 1). Spring clams were maintained in the laboratory in darkness for 7 days at 5-6°C, and summer clams were maintained similarly for 4 days at 10°C before lipid analyses. For histological studies, clams were additionally collected in May at the same site. The digestive gland and the mantle were dissected under cold brackish seawater, carefully rinsed, and homogenized in chloroform: methanol (2 : 1, v: v), using a Potter-Elvehjem glass homogenizer. Lipids were purified (Folch et al., 1957), and
fractionated in a column of activated silicic acid (Unisil, column size 9 x 75 ram) into neutral lipids, glycolipids,and phospholipids (Christie, 1982). Lipid fractions were transesterified for gas-liquid chromatography (GLC) with a methanolic-base (0.5 N) reagent (Supelco). This method is suitable for preparing methyl esters of glycerides,sterol esters and phospholipids. Any free fatty acids present in the sample were converted into salts. Gas-liquid chromatography of fatty acid methyl esters was carried out with a Hewlett-Packard 5890 A instrument equipped with an automatic sampler (HP 7673 A) and an integrator (HP 3392 A). A 25 m capillary column (S.G.E., Australia; 0.22 mm id, FFAP as liquid phase, He as carrier) was used. Fatty acid methyl ester standards were from Nu Chek Prep and Sigma. The column was calibrated using a fatty acid methyl ester mixture of known composition. Histological studies For an examination of the stage of the reproductive cycle, the soft parts of clams were fixed in Bouin's fluid, routinely embedded in paraflin, and sectioned at 6-7 #m. The sections were stained with hematoxylin-chromotrope fast green (Gray, 1954). Comparison of digestive tubules in spring and summer clams was based on outer diameters of type II tubules (Morton, 1970; Langton, 1975). Neutral lipids were examined histochemicallyfrom clam bodies fixed with cold formal-Ca (4% formaldehyde solution made to 1% with CaC12, and saturated with CaCO~). Sagittal cryostate sections (10 #m) were stained with hematoxylin and Fettrot (Barka and Anderson, 1963). Material collected in previous years was also used in the assessment of tissue lipid distribution. The general condition of the clams was monitored by various indices used in previous studies of M. balthica (Pekkarinen, 1983). RESULTS Tissue lipids and fatty acids Lipid analyses were made from clams of an average shell length of 16.4 to 16.6 mm (Table 1). Data show that the clams deposited neutral lipids in the digestive gland during spring and early summer (ca 0.4 mg extracted/gland in April, vs ca 1.8 rag/gland in July), indicating an increase of ca 4.5-fold. The phospholipid content of this tissue, in contrast, did not change during the same period. In addition to neutral lipids
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S. TURUNENand M. PEKKARINEN
Table 1. Data on Macoma balthica collected for fatty acid analysis. Both samples were taken at the same site, from a depth of 7-8 m, salinity of ca 6--7%o, off Tv/irminne Zoological Station in southwestern Finland
Date collected Temperature of water (bottom sediment) Shell length (mm) Digestive gland NL (mg)* Digestive gland PL (mg)*
Spring clams
Summer clams
April 21, 1989 + 3°C
July 20, 1989 + 10.5°C
16.4 ± (n = 0.390 ± 0.538 ±
16.6 ± (n = 1.827 ± 0.550 ±
1.1 11) 0.129 0.053
1.0 12) 0.261 0.156
*Data per clam, mean ± SD (n = 3); a total of 12 clams were analyzed per sample. NL, neutral lipids; PL, phospholipids.
and phospholipids, the digestive gland contained a small proportion of glycolipids. Fatty acids were studied after fractionating total lipid extracts into neutral lipids (NL), glycolipids (GL), and phospholipids (PL). Characteristically, all fractions of the digestive gland contained a relatively high titer of palm±tic (16:0) and eicosapentaenoic (20:5n3) acids (Table 2). The overall fatty acid patterns of spring and summer clams showed few differences. Digestive gland NL contained a high proportion of palmitoleic acid (16:1), which accounted for c a 25% of all NL fatty acids in spring and summer clams. Digestive gland NL contained at least two isomers of 18:1 (oleic, palmitvaccenic), relatively small amounts of linoleic (18:2n6) and linolenic acid (18:3n3), two isomers of 20:1, and several highly unsaturated 20 and 22 carbon fatty acids. Neutral lipids typically had a relatively small titer of docosahexaenoic acid (22: 6n 3). Among digestive gland PL fatty acids, eicosapentaenoic acid (20:5n 3) was predominant, followed by 22:6n3 (Table 2). Phospholipids were distinguished from NL by a much lower titer of palmitoleic acid (7. ! % in the PL vs 25.4% in the NL of spring clams). Phospholipids contained less 18:4n3 than did NL, but more arachidonic (20:4n6) and eicosenoic (20: I n 9 and 20: I n 7) acids.
The digestive gland yielded a small amount of acetone-soluble lipids, which upon transesterification were found to have a fatty acid pattern distinct from both NL and PL (Table 2). Docosahexaenoic acid was the predominant fatty acid, followed by palm±tic acid. The fatty acids of digestive gland glycolipids showed more variability and more minor fatty acids under our analytical conditions than did the fatty acids of neutral lipids and phospholipids. Comparison of spring and summer clams showed a marked stability of the fatty acid profile of the digestive gland, despite the large increase in the overall NL content during the growth period. A consistent feature of NL fatty acids was a decline in the relative titer of 20:ln7 from spring to summer (6.5% vs 2.4%, Table 2). The proportion of NL monounsaturated fatty acids, in general, decreased during this period. Digestive gland PL did not change quantitatively from spring to summer, but the content of polyunsaturated fatty acids (PUFA) decreased in PL from 48.1% in spring clams to 42.3% in summer clams. Most of this difference was due to a declined titer of eicosapentaenoic acid. Summer clams tended to contain more unidentified and minor fatty acids especially in their digestive gland neutral lipids than did spring clams, a possible influence of the diet. Comparative data on fatty acids were also obtained from the mantle. Histological observations had indicated little or no neutral lipids present in the mantle, and column chromatography showed PL as the main lipid fraction. Phospholipid fatty acids of the digestive gland and the mantle differed only little, an exception being a distinctly higher content of arachidonic acid in the mantle (10.0% in the mantle vs 4.2% in the digestive gland of spring clams, Table 3). Another difference was the mantle's high titer of 20: ln9. PUFA accounted for c a 49% of the identified fatty acids of mantle PL in both spring and summer clams, i.e. no decline comparable to digestive gland PUFA was observed from spring to summer.
Table 2. Per cent fatty acid composition of the digestive gland of M a c o m a balthica collected off the coast of southern Finland Fatty acid 14:0 16:0 16:1 16:2n6 18:0 18:1n9 18:1n7 18:2n6 18:3n 3 18:4n3 20:In9 20:ln7 20:3n6 20:4n6 20:4n3 20:5n3 22:1n9 22:3n6 22:5n3 22:6n3 Other*
NL
Spring PL
GL
NL
Summer PL
GL
2.0 ± 0.28 11.8 ± 0.55 25.4 ± 2.41 0.7 ± 0.08 1.7 + 0.21 8.3 ± 0.22 1.8 ± 0.23 1.9 ± 0,05 0.9 ± 0.06 3.1 _+0.01 2.3 ± 0.15 6.5 ± 0.49~ tr 1.4 ± 0.17 0.7 ± 0.11 13.7 ± 1.00 tr tr 1.6 ± 0.06 5.5 ± 0.66 10.7
1.5 ± 0.14 10.9 ± 0.60 7.1 ± 1.60 tr 6.2 ± 0.46 5.4 ± 0.26 0.5 ± 0.01 1.0 ± 0.01 0.5 _+ 0.05 1.6±0.10 8.2 ± 0.68 4.1 ± 0.32 tr 4.2 ± 0.12 l.I ± 0.06 22.0 + 2.10"I" tr 2.8 + 1.10 1.6 ± 0.10 13.3 ± 0.40 9.0
3.6+ 1.27 14.4 ± 3.40 7.8 ± 1.60 4.0 ± 0.57 4.5 ± 1.76 4.2 ± 1.70 tr tr -3.1 ±0.71 5.5 ± 1.48 3.5 ± 0.98 -tr tr 12.8 ± 2.23 ---27.9 ± 3.96 8.7
3.5±0.44 11.3 ± 0.96 23.9 _4_-0.81 1.0 ± 0.06 0.5 ± 0.38 9.0 ± 0.70 1.3 ± 0.05 1.5 ± 0.25 1.2 ± 0.06 3.2 ± 0.49 1.0 ± 0.10 2.4 ± 0.47:~ tr 0.8 _ 0.05 0.8 ± 0.10 14.5 ± 1.00 tr 0.5 ± 0.06 tr 5.8 ± 0.87 17.8
1.8±0.17 11.5 ± 0.46 7.2 ± 0.25 tr 5.8 ± 0.26 7.4 ± 0.12 0.6 ± 0.01 1.1 ± 0.10 0.5 +_ 0.01 1.2_.+0.17 7.7 ± 0.76 3.3 ± 0.15 tr 4.0 + 0.10 1.0 ± 0.07 17.7 ± 0.40~" tr 0.9 ± 0.06 1.7 ± 0.10 14.2 ± 0.29 11.8
4.8±0.95 16.7 ± 2.83 13.5 ± 2.25 1.0 3.7 + 0.43 6.9 ± 0.81 0.8 1.8 + 0.25 -1.8 ± 0.07 3.0 ± 1.27 2.7 ± 0.38 -tr tr 12.7 ± 2.20 ---19.5 _+ 1.41 I 1.1
Twelve clams were analyzed for spring and summer in three determinations (mean ± SD). *Unidentified and minor peaks, including 20:2n6, 20:3n3 and 22:4n3. t P < 0.01 (Student's t-test); ~tP < 0.001 (Student's t-test).
Fatty acid profile in bivalve Among individual fatty acids, the relative proportions of 20:5n3 and 20:ln9 tended to decrease from spring to summer, whereas the proportion of 22:6n 3 increased during this period. The small amount of NL and GL present in mantle tissue allowed only an approximate determination of fatty acids. The major NL fatty acid in the mantle was palmitic acid. Mantle NL had only small amounts of 20:1n9 (ca 3.4% in spring clams), but appreciable amounts of 20:1n7 (ca 8.1% in spring clams). Mantle NL also had relatively small concentrations of 20: 4n 6 and 20: 5n 3 (ca 3.8% and 4.2%, respectively, in spring clams).
Clam condition and tissue histology At the time of the first sampling in April, about a quarter of the clams with a shell length above 12 mm had spawned, about 60% had not yet started spawning, and the rest were in the process of spawning. In late July, the gonads of all clams were developing, with large numbers of spermatozoa present in the testes of many males. The clams were lean and contained relatively much water, as judged by various condition indices (Pekkarinen, 1983). The average "condition factor" of 12-16 mm clams was 7.6, and that of > 16 mm clams was 6.6 in April. Corresponding values were 10.7 and 8.1 in July.
271
In the digestive glands of both spring and summer clams type II and Ill tubules predominated. Outer diameters of type II tubules were comparable in spring (106#m) and summer clams ( l l 8 # m ) . Cryostate sections stained with Fettrot contained only few neutral lipid droplets in the digestive tubule cells in April. Analyses of the clams in May showed an increase in the number of lipid droplets, and in July they were numerous or were fused into larger droplets in the basal region of digestive gland cells. In spring clams, when lipid was scarce in the digestive cells of the tubules, the duct cells occasionally contained numerous lipid droplets (Fig. 1). Neutral lipids were found throughout the epithelium of the digestive tract, sometimes also in the esophagus and rectum (Fig. 1). In addition to the digestive gland, neutral lipid was common in the dorsal epithelium of the stomach and the epithelium of the gastric appendage. In the intestine, epithelial cells of the anterior region bearing a typhlosole were occasionally found to contain lipids, especially in May. No lipid inclusions were observed in mantle tissue, and lipid was scarce in the foot and other clam tissues examined at the light microscopic level.
0
i * , * °----.,.,
MD
.i
':!
DG
-g-
ss
1'
A DG DH DT E GS H HG IG LC LP MD MG R RC S SD SS
Appendage Digestive gland Dorsal hood Digestive tubule Esophagus
Gastric shield Heart Hindgut Intestinal groove Left caecum Left pouch Main duct Midgut Rectum Right caecum Stomach
Secondary Style sac
Fig. 1. Outline of the digestive tract of Macoma balthica, showing areas where neutral lipid was found histochemicaUy in epithelial cells during an annual cycle. Data from several years are incorporated. Amount of lipid droplets is indicated by a scale from 0 to * *; 0 indicating infrequent occurrence of lipid droplets, and * * indicating maximum amount of lipid inclusions. Lipid droplets were most numerous in summer clams. In the midgut, lipids were observed especiallyin spring, and less frequently toward summer.
duct
S. TURUNENand M. PEKKARINEN
272
Table 3. Per cent fatty acid composition of mantle phospholipids of Macoma balthica collected on the coast of southern Finland Fatty acid Spring Summer 14:0 0.6 + 0.06 0.6 __.0.28 14:2 1.0 + 0.26 0.9 + 0.21 16:0 9.3 _+0.5t 9.1 _+0.64 16: 1 4.8 + 0.35 3.6 -+0.21 16:2 1.4-+0.25 0.9_+0.07 18:0 7.3 -+0.32 6.8 _+0.14 18:1n9 7.5 + 0.20 8.3 _+0.08 18:2n6 tr 0.6 + 0.06 18:3n3 tr tr 18:4n3 0.5 + 0.05 tr 20 : In 9 13.4 _+1.06 11.6 _+0.99 20:ln7 4.5 _+0.25 4.3 _+0.28 20:4n6 10.0 _+0.64 9.3 _+0.57 20 :4n 3 tr tr 20:5n3 16.6_+ 1.67" 14.1 _+0.35* 22:3n6 tr 0.7 +_0.01 22:3n3 tr 0.7 _+0.21 22:4n3 0.7 _+0.05 0.9 _+0A7 22:5n3 2.5 _+0.10 2.6 _+0.14 22:6n 3 14.8 _+0.76 17.6 _+0.42 Saturated 17.2 _+0.2 16.5 +_0.8 Monounsaturated 30.2 _+ 1.4 27.8 + 0.6 Polyunsaturated 48.9 _+3.7 49.0 _+1.6 Unidentified and minor peaks 5.1 6.7 Mean +_SD. A total of 12 clams were analyzed per sample (n = 3). *P < 0.05 (Student's t-test). DISCUSSION The data show that the digestive gland is an important neutral lipid storage site in M. balthica. Digestive gland neutral lipid fatty acids reflect the composition of fatty acids observed in unicellular algae, which are the primary food of M. balthica. The spring phytoplankton bloom at the sampling site reaches a peak in A p r i l - M a y , and consists mainly of diatoms (e.g. Achnanthes taeniata) and dinoflagellates (e.g. Gonyaulax catenata) (H/illfors et al., 1981). Diatoms contain substantial amounts of 16:1n7 and 20:5n 3, both of which are among the major N L fatty acids in the digestive gland of M. balthica (cf. K a y a m a et al., 1989). Another distinct feature of the fatty acid composition of diatoms is a virtual absence of 18:3n 3, and the presence of only small amounts of 18:2n6. These fatty acids are minor components in all lipid fractions of M. balthica. The digestive gland stores lipids in many species of bivalves (Reid, 1966; Giese et al., 1967; de Moreno et al., 1976). The storage cells may differ in different species: in Ostrea edulis and Crassostrea angulata, for example, lipid was stored especially in the connective tissue surrounding the digestive gland (Mathers, 1973). In several species, including C. angulata, C. virgata and Modiolus demissus (George, 1952; Mathers, 1973), as well as Macoma balthica, epithelial cells of digestive gland ducts also contained lipid droplets. Relatively small geographic variations, variations in depth, as well as seasonal changes may affect the fatty acid, total lipid or sterol profiles of M. balthica (Jarzebski et al., 1986; Polak et al., 1987; Bonsdorff and Wenne, 1989). In many studies of M. balthica, whole body soft parts have been used in fatty acid analyses, and such data are difficult to compare with the results of the present study. Our data showed a much lower content o f tissue 16:1 and a much higher content of 22: 6n 3 than was found in
a recent study in the southern Baltic (Polak et al., 1987). Phospholipid P U F A appear to change seasonally in M. balthica from the northern Baltic Sea. It may be significant that the relative titer of 20:5n 3 (instead of 22:6n3) was elevated in the cold season. The melting point of 20:5n 3 is lower than that of most other P U F A found in the tissues of M. balthica ( - 5 9 ° C , vs - 4 9 ° C for 20:4n6, and - 4 4 ° C for 22: 6n 3). One function of membrane 20: 5n 3 may be the maintenance of fluidity of the membranes during the cold season. Seasonal changes were observed in Tapes decussatus and T. philippinarum, but in these species PL fatty acids varied less during the annual cycle than did N L fatty acids (Beninger and Stephan, 1985).
Acknowledgements--This study was supported by a grant to M.P. from the Walter and Andrre de Nottbeck's Foundation. REFERENCES
Barka T. and Anderson P. J. (1963) Histochemistry. Harper & Row, New York. Beninger P. G. and Stephan G. (1985) Seasonal variations in the fatty acids of the triacylglycerols and phospholipids of two populations of adults clam (Tapes decussatus L. and T. philippinarum) reared in a common habitat. Comp. Biochem. Physiol. 81B, 591~i01. Beukema J. J. and de Bruin W. (1977) Seasonal changes in dry weight and chemical composition of the soft parts of the tellinid bivalve Macoma balthica in the Dutch Wadden Sea. Neth. J. Sea Res. 11, 42-55. Bonsdorff E. and Wenne R. (1989) A comparison of condition indices of Macoma balthica (L.) from the northern and southern Baltic Sea. Neth. J. Sea Res. 23, 45 55. Christie W. W. (1982) Lipid Analysis. Isolation, Separation, Identification, and Structural Analysis of Lipids, 207 pp. Pergamon Press, Oxford. Folch J., Lees M. and Sloan-Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497 509. George W. C. (1952) The digestion and absorption of fat in Lamellibranchs. Biol. Bull. 102, 119-127. Giese A. C., Hart M. A., Smith A. M. and Cheung M. A. (1967) Seasonal changes in body component indices and chemical composition in the pismo clam Tivela stultorum. Comp. Biochem. Physiol. 22, 549-561. Graf G., Bengtsson W., Diesner U., Schulz R. and Theede H. (1982) Benthic response to sedimentation of a spring phytoplankton bloom: process and budget. Mar. Biol. 67, 201-208. Gray P. (1954) The Microtomist's Formulatory and Guide. Blakiston, New York. H/illfors G., Niemi A., Ackefors H., Lassig J. and Lepp/ikoski E. (1981) Biological oceanography. In The Baltic Sea (Edited by Voipio A.), pp. 219-274. Elsevier Oceanography Ser. 30, Amsterdam. ICES (International Council for the Exploration of the Sea) (1974) Research programmes for investigations of the Baltic as a natural resource with special reference to pollution problems. Coop. Res. Rep. 42. Jarzebski A., Polak L., Wenne R. and Falkowski L. (1986) Microgeographic differentiation in the lipid composition of a bivalve Macoma balthica (L.) from the Gulf of Gdansk (Southern Baltic). Mar. Biol. 91, 27-31. Kayama M., Araki S. and Sato S. (1989) Lipids of marine plants. In Marine Biogenic Lipids, Fats, and Oils (Edited by Ackman R. G.), Vol. II, pp. 3-48. CRC Press, Boca Raton, FL.
Fatty acid profile in bivalve Langton R. W. (1975) Synehrony in the digestive diverticula of Mytilus edulis L. J. mar. biol. Ass. UK 55, 221-229. Mathers N. F. (1973) A comparative histochemical survey of enzymes associated with the process of digestion in Ostrea edulis and Crassostrea angulata (Mollusca: Bivalvia). J. Zool., Lond. 169, 169-179. Moreno J. E. A. de, Moreno V. J. and Brenner R. R. (1976) Lipid metabolism of the yellow clam, Mesodesma mactroides: I. Composition of the lipids. Lipids 11, 334-340. Morton B. (1970) A note on the cytological structure and function of the digestive diverticula of Macoma balthica correlated with the rhythm of the tide. Malacol. Rev. 3, 115-119.
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Pekkarinen M. (1983) Seasonal changes in condition and biochemical constituents in the soft part of Macoma balthica (Lamellibranchiata) in the Tv/irminne brackish water area (Baltic Sea). Ann. Zool. Fenn. 20, 311-322. Pekkarinen M. (1986) Notes on the spawning, egg cleavage and early development of the bivalve Macoma balthica. Ann. Zool. fenn. 23, 71-75. Polak L., Jarzebski A., Wenne R. and Falkowski L. (1987) Seasonal changes in condition and lipids composition of the bivalve Macoma balthica L. from the Gulf of Gdansk (Southern baltic). Comp. Biochem. Physiol. ggB, 881-885. Reid R. G. B. (1966) Digestive tract enzymes in the bivalves Lima hians G-melin and Mya arenaria L. Comp. Biochem. Physiol. 17, 417-433.
0305-0491/90$3.00+ 0.00 © 1990PergamonPress pie
FATTY ACID PROFILE OF THE DIGESTIVE G L A N D A N D MANTLE IN THE BIVALVE MACOMA BALTHICA S. TURUNEN and M. PEKKARINEN Department of Zoology, University of Helsinki, 00100 Helsinki, Finland (Tel: 358 0 1911) (Recewed27February 1990) Abstract--l. Digestive gland and mantle fatty acids were studied in spring and summer in the bivalve Macoma balthica off the southern coast of Finland. The presence of lipids was also examined histochemically in various clam tissues. 2. The neutral lipid content of the digestivegland increased ca 4.5-fold during the annual growth period. 3. Neutral lipid fatty acids of the digestive gland, of which palmitoleic, eicosapentaenoic and palmitic acids were predominant, were clearly distinguished from phospho- and giycolipid fatty acids. 4. The degree of unsaturation of phospholipid fatty acids was higher in the cold season both in the digestive gland and mantle, mainly due to the titer of eicosapentaenoic acid.
INTRODUCTION Macoma balthica is a small bivalve widely distributed on soft bottoms in both marine and brackish water. The biology of this species has received increasing attention in recent years, in part owing to the use of this clam in long term monitoring of water pollution (ICES, 1974). Off the coast of southern Finland M. balthica reproduces from late April to June, when marked changes occur in the carbohydrate, protein, and lipid content of the clam (Pekkarinen, 1983, 1986; Bonsdorff and Wenne, 1989). Comparable changes have been observed in other sites in northern Europe (Beukema and de Bruin, 1977; Graf et al., 1982; Polak et al., 1987). Lipids account for a substantial proportion of the stored energy in M. balthica. Histological observations have suggested that the digestive gland (digestive diverticula, hepatopancreas) is a major organ of lipid storage in M. balthica (Pekkarinen, 1983). The purpose of the present study was to examine the fatty acid composition of digestive gland neutral-, glyco-, and phospholipids at the beginning and at the end of the annual growth season at one sampling site, for possible seasonal changes. Comparable data were obtained from mantle phospholipids. Histochemical studies were carried out to map the accumulation of neutral lipids in different clam tissues. MATERIALS AND METHODS
Lipid and fatty acid analyses Macoma balthica were collected from the southern coast of Finland (Tv/irminne Zoological Station) in April and July, 1989 (Table 1). Spring clams were maintained in the laboratory in darkness for 7 days at 5-6°C, and summer clams were maintained similarly for 4 days at 10°C before lipid analyses. For histological studies, clams were additionally collected in May at the same site. The digestive gland and the mantle were dissected under cold brackish seawater, carefully rinsed, and homogenized in chloroform: methanol (2 : 1, v: v), using a Potter-Elvehjem glass homogenizer. Lipids were purified (Folch et al., 1957), and
fractionated in a column of activated silicic acid (Unisil, column size 9 x 75 ram) into neutral lipids, glycolipids,and phospholipids (Christie, 1982). Lipid fractions were transesterified for gas-liquid chromatography (GLC) with a methanolic-base (0.5 N) reagent (Supelco). This method is suitable for preparing methyl esters of glycerides,sterol esters and phospholipids. Any free fatty acids present in the sample were converted into salts. Gas-liquid chromatography of fatty acid methyl esters was carried out with a Hewlett-Packard 5890 A instrument equipped with an automatic sampler (HP 7673 A) and an integrator (HP 3392 A). A 25 m capillary column (S.G.E., Australia; 0.22 mm id, FFAP as liquid phase, He as carrier) was used. Fatty acid methyl ester standards were from Nu Chek Prep and Sigma. The column was calibrated using a fatty acid methyl ester mixture of known composition. Histological studies For an examination of the stage of the reproductive cycle, the soft parts of clams were fixed in Bouin's fluid, routinely embedded in paraflin, and sectioned at 6-7 #m. The sections were stained with hematoxylin-chromotrope fast green (Gray, 1954). Comparison of digestive tubules in spring and summer clams was based on outer diameters of type II tubules (Morton, 1970; Langton, 1975). Neutral lipids were examined histochemicallyfrom clam bodies fixed with cold formal-Ca (4% formaldehyde solution made to 1% with CaC12, and saturated with CaCO~). Sagittal cryostate sections (10 #m) were stained with hematoxylin and Fettrot (Barka and Anderson, 1963). Material collected in previous years was also used in the assessment of tissue lipid distribution. The general condition of the clams was monitored by various indices used in previous studies of M. balthica (Pekkarinen, 1983). RESULTS Tissue lipids and fatty acids Lipid analyses were made from clams of an average shell length of 16.4 to 16.6 mm (Table 1). Data show that the clams deposited neutral lipids in the digestive gland during spring and early summer (ca 0.4 mg extracted/gland in April, vs ca 1.8 rag/gland in July), indicating an increase of ca 4.5-fold. The phospholipid content of this tissue, in contrast, did not change during the same period. In addition to neutral lipids
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S. TURUNENand M. PEKKARINEN
Table 1. Data on Macoma balthica collected for fatty acid analysis. Both samples were taken at the same site, from a depth of 7-8 m, salinity of ca 6--7%o, off Tv/irminne Zoological Station in southwestern Finland
Date collected Temperature of water (bottom sediment) Shell length (mm) Digestive gland NL (mg)* Digestive gland PL (mg)*
Spring clams
Summer clams
April 21, 1989 + 3°C
July 20, 1989 + 10.5°C
16.4 ± (n = 0.390 ± 0.538 ±
16.6 ± (n = 1.827 ± 0.550 ±
1.1 11) 0.129 0.053
1.0 12) 0.261 0.156
*Data per clam, mean ± SD (n = 3); a total of 12 clams were analyzed per sample. NL, neutral lipids; PL, phospholipids.
and phospholipids, the digestive gland contained a small proportion of glycolipids. Fatty acids were studied after fractionating total lipid extracts into neutral lipids (NL), glycolipids (GL), and phospholipids (PL). Characteristically, all fractions of the digestive gland contained a relatively high titer of palm±tic (16:0) and eicosapentaenoic (20:5n3) acids (Table 2). The overall fatty acid patterns of spring and summer clams showed few differences. Digestive gland NL contained a high proportion of palmitoleic acid (16:1), which accounted for c a 25% of all NL fatty acids in spring and summer clams. Digestive gland NL contained at least two isomers of 18:1 (oleic, palmitvaccenic), relatively small amounts of linoleic (18:2n6) and linolenic acid (18:3n3), two isomers of 20:1, and several highly unsaturated 20 and 22 carbon fatty acids. Neutral lipids typically had a relatively small titer of docosahexaenoic acid (22: 6n 3). Among digestive gland PL fatty acids, eicosapentaenoic acid (20:5n 3) was predominant, followed by 22:6n3 (Table 2). Phospholipids were distinguished from NL by a much lower titer of palmitoleic acid (7. ! % in the PL vs 25.4% in the NL of spring clams). Phospholipids contained less 18:4n3 than did NL, but more arachidonic (20:4n6) and eicosenoic (20: I n 9 and 20: I n 7) acids.
The digestive gland yielded a small amount of acetone-soluble lipids, which upon transesterification were found to have a fatty acid pattern distinct from both NL and PL (Table 2). Docosahexaenoic acid was the predominant fatty acid, followed by palm±tic acid. The fatty acids of digestive gland glycolipids showed more variability and more minor fatty acids under our analytical conditions than did the fatty acids of neutral lipids and phospholipids. Comparison of spring and summer clams showed a marked stability of the fatty acid profile of the digestive gland, despite the large increase in the overall NL content during the growth period. A consistent feature of NL fatty acids was a decline in the relative titer of 20:ln7 from spring to summer (6.5% vs 2.4%, Table 2). The proportion of NL monounsaturated fatty acids, in general, decreased during this period. Digestive gland PL did not change quantitatively from spring to summer, but the content of polyunsaturated fatty acids (PUFA) decreased in PL from 48.1% in spring clams to 42.3% in summer clams. Most of this difference was due to a declined titer of eicosapentaenoic acid. Summer clams tended to contain more unidentified and minor fatty acids especially in their digestive gland neutral lipids than did spring clams, a possible influence of the diet. Comparative data on fatty acids were also obtained from the mantle. Histological observations had indicated little or no neutral lipids present in the mantle, and column chromatography showed PL as the main lipid fraction. Phospholipid fatty acids of the digestive gland and the mantle differed only little, an exception being a distinctly higher content of arachidonic acid in the mantle (10.0% in the mantle vs 4.2% in the digestive gland of spring clams, Table 3). Another difference was the mantle's high titer of 20: ln9. PUFA accounted for c a 49% of the identified fatty acids of mantle PL in both spring and summer clams, i.e. no decline comparable to digestive gland PUFA was observed from spring to summer.
Table 2. Per cent fatty acid composition of the digestive gland of M a c o m a balthica collected off the coast of southern Finland Fatty acid 14:0 16:0 16:1 16:2n6 18:0 18:1n9 18:1n7 18:2n6 18:3n 3 18:4n3 20:In9 20:ln7 20:3n6 20:4n6 20:4n3 20:5n3 22:1n9 22:3n6 22:5n3 22:6n3 Other*
NL
Spring PL
GL
NL
Summer PL
GL
2.0 ± 0.28 11.8 ± 0.55 25.4 ± 2.41 0.7 ± 0.08 1.7 + 0.21 8.3 ± 0.22 1.8 ± 0.23 1.9 ± 0,05 0.9 ± 0.06 3.1 _+0.01 2.3 ± 0.15 6.5 ± 0.49~ tr 1.4 ± 0.17 0.7 ± 0.11 13.7 ± 1.00 tr tr 1.6 ± 0.06 5.5 ± 0.66 10.7
1.5 ± 0.14 10.9 ± 0.60 7.1 ± 1.60 tr 6.2 ± 0.46 5.4 ± 0.26 0.5 ± 0.01 1.0 ± 0.01 0.5 _+ 0.05 1.6±0.10 8.2 ± 0.68 4.1 ± 0.32 tr 4.2 ± 0.12 l.I ± 0.06 22.0 + 2.10"I" tr 2.8 + 1.10 1.6 ± 0.10 13.3 ± 0.40 9.0
3.6+ 1.27 14.4 ± 3.40 7.8 ± 1.60 4.0 ± 0.57 4.5 ± 1.76 4.2 ± 1.70 tr tr -3.1 ±0.71 5.5 ± 1.48 3.5 ± 0.98 -tr tr 12.8 ± 2.23 ---27.9 ± 3.96 8.7
3.5±0.44 11.3 ± 0.96 23.9 _4_-0.81 1.0 ± 0.06 0.5 ± 0.38 9.0 ± 0.70 1.3 ± 0.05 1.5 ± 0.25 1.2 ± 0.06 3.2 ± 0.49 1.0 ± 0.10 2.4 ± 0.47:~ tr 0.8 _ 0.05 0.8 ± 0.10 14.5 ± 1.00 tr 0.5 ± 0.06 tr 5.8 ± 0.87 17.8
1.8±0.17 11.5 ± 0.46 7.2 ± 0.25 tr 5.8 ± 0.26 7.4 ± 0.12 0.6 ± 0.01 1.1 ± 0.10 0.5 +_ 0.01 1.2_.+0.17 7.7 ± 0.76 3.3 ± 0.15 tr 4.0 + 0.10 1.0 ± 0.07 17.7 ± 0.40~" tr 0.9 ± 0.06 1.7 ± 0.10 14.2 ± 0.29 11.8
4.8±0.95 16.7 ± 2.83 13.5 ± 2.25 1.0 3.7 + 0.43 6.9 ± 0.81 0.8 1.8 + 0.25 -1.8 ± 0.07 3.0 ± 1.27 2.7 ± 0.38 -tr tr 12.7 ± 2.20 ---19.5 _+ 1.41 I 1.1
Twelve clams were analyzed for spring and summer in three determinations (mean ± SD). *Unidentified and minor peaks, including 20:2n6, 20:3n3 and 22:4n3. t P < 0.01 (Student's t-test); ~tP < 0.001 (Student's t-test).
Fatty acid profile in bivalve Among individual fatty acids, the relative proportions of 20:5n3 and 20:ln9 tended to decrease from spring to summer, whereas the proportion of 22:6n 3 increased during this period. The small amount of NL and GL present in mantle tissue allowed only an approximate determination of fatty acids. The major NL fatty acid in the mantle was palmitic acid. Mantle NL had only small amounts of 20:1n9 (ca 3.4% in spring clams), but appreciable amounts of 20:1n7 (ca 8.1% in spring clams). Mantle NL also had relatively small concentrations of 20: 4n 6 and 20: 5n 3 (ca 3.8% and 4.2%, respectively, in spring clams).
Clam condition and tissue histology At the time of the first sampling in April, about a quarter of the clams with a shell length above 12 mm had spawned, about 60% had not yet started spawning, and the rest were in the process of spawning. In late July, the gonads of all clams were developing, with large numbers of spermatozoa present in the testes of many males. The clams were lean and contained relatively much water, as judged by various condition indices (Pekkarinen, 1983). The average "condition factor" of 12-16 mm clams was 7.6, and that of > 16 mm clams was 6.6 in April. Corresponding values were 10.7 and 8.1 in July.
271
In the digestive glands of both spring and summer clams type II and Ill tubules predominated. Outer diameters of type II tubules were comparable in spring (106#m) and summer clams ( l l 8 # m ) . Cryostate sections stained with Fettrot contained only few neutral lipid droplets in the digestive tubule cells in April. Analyses of the clams in May showed an increase in the number of lipid droplets, and in July they were numerous or were fused into larger droplets in the basal region of digestive gland cells. In spring clams, when lipid was scarce in the digestive cells of the tubules, the duct cells occasionally contained numerous lipid droplets (Fig. 1). Neutral lipids were found throughout the epithelium of the digestive tract, sometimes also in the esophagus and rectum (Fig. 1). In addition to the digestive gland, neutral lipid was common in the dorsal epithelium of the stomach and the epithelium of the gastric appendage. In the intestine, epithelial cells of the anterior region bearing a typhlosole were occasionally found to contain lipids, especially in May. No lipid inclusions were observed in mantle tissue, and lipid was scarce in the foot and other clam tissues examined at the light microscopic level.
0
i * , * °----.,.,
MD
.i
':!
DG
-g-
ss
1'
A DG DH DT E GS H HG IG LC LP MD MG R RC S SD SS
Appendage Digestive gland Dorsal hood Digestive tubule Esophagus
Gastric shield Heart Hindgut Intestinal groove Left caecum Left pouch Main duct Midgut Rectum Right caecum Stomach
Secondary Style sac
Fig. 1. Outline of the digestive tract of Macoma balthica, showing areas where neutral lipid was found histochemicaUy in epithelial cells during an annual cycle. Data from several years are incorporated. Amount of lipid droplets is indicated by a scale from 0 to * *; 0 indicating infrequent occurrence of lipid droplets, and * * indicating maximum amount of lipid inclusions. Lipid droplets were most numerous in summer clams. In the midgut, lipids were observed especiallyin spring, and less frequently toward summer.
duct
S. TURUNENand M. PEKKARINEN
272
Table 3. Per cent fatty acid composition of mantle phospholipids of Macoma balthica collected on the coast of southern Finland Fatty acid Spring Summer 14:0 0.6 + 0.06 0.6 __.0.28 14:2 1.0 + 0.26 0.9 + 0.21 16:0 9.3 _+0.5t 9.1 _+0.64 16: 1 4.8 + 0.35 3.6 -+0.21 16:2 1.4-+0.25 0.9_+0.07 18:0 7.3 -+0.32 6.8 _+0.14 18:1n9 7.5 + 0.20 8.3 _+0.08 18:2n6 tr 0.6 + 0.06 18:3n3 tr tr 18:4n3 0.5 + 0.05 tr 20 : In 9 13.4 _+1.06 11.6 _+0.99 20:ln7 4.5 _+0.25 4.3 _+0.28 20:4n6 10.0 _+0.64 9.3 _+0.57 20 :4n 3 tr tr 20:5n3 16.6_+ 1.67" 14.1 _+0.35* 22:3n6 tr 0.7 +_0.01 22:3n3 tr 0.7 _+0.21 22:4n3 0.7 _+0.05 0.9 _+0A7 22:5n3 2.5 _+0.10 2.6 _+0.14 22:6n 3 14.8 _+0.76 17.6 _+0.42 Saturated 17.2 _+0.2 16.5 +_0.8 Monounsaturated 30.2 _+ 1.4 27.8 + 0.6 Polyunsaturated 48.9 _+3.7 49.0 _+1.6 Unidentified and minor peaks 5.1 6.7 Mean +_SD. A total of 12 clams were analyzed per sample (n = 3). *P < 0.05 (Student's t-test). DISCUSSION The data show that the digestive gland is an important neutral lipid storage site in M. balthica. Digestive gland neutral lipid fatty acids reflect the composition of fatty acids observed in unicellular algae, which are the primary food of M. balthica. The spring phytoplankton bloom at the sampling site reaches a peak in A p r i l - M a y , and consists mainly of diatoms (e.g. Achnanthes taeniata) and dinoflagellates (e.g. Gonyaulax catenata) (H/illfors et al., 1981). Diatoms contain substantial amounts of 16:1n7 and 20:5n 3, both of which are among the major N L fatty acids in the digestive gland of M. balthica (cf. K a y a m a et al., 1989). Another distinct feature of the fatty acid composition of diatoms is a virtual absence of 18:3n 3, and the presence of only small amounts of 18:2n6. These fatty acids are minor components in all lipid fractions of M. balthica. The digestive gland stores lipids in many species of bivalves (Reid, 1966; Giese et al., 1967; de Moreno et al., 1976). The storage cells may differ in different species: in Ostrea edulis and Crassostrea angulata, for example, lipid was stored especially in the connective tissue surrounding the digestive gland (Mathers, 1973). In several species, including C. angulata, C. virgata and Modiolus demissus (George, 1952; Mathers, 1973), as well as Macoma balthica, epithelial cells of digestive gland ducts also contained lipid droplets. Relatively small geographic variations, variations in depth, as well as seasonal changes may affect the fatty acid, total lipid or sterol profiles of M. balthica (Jarzebski et al., 1986; Polak et al., 1987; Bonsdorff and Wenne, 1989). In many studies of M. balthica, whole body soft parts have been used in fatty acid analyses, and such data are difficult to compare with the results of the present study. Our data showed a much lower content o f tissue 16:1 and a much higher content of 22: 6n 3 than was found in
a recent study in the southern Baltic (Polak et al., 1987). Phospholipid P U F A appear to change seasonally in M. balthica from the northern Baltic Sea. It may be significant that the relative titer of 20:5n 3 (instead of 22:6n3) was elevated in the cold season. The melting point of 20:5n 3 is lower than that of most other P U F A found in the tissues of M. balthica ( - 5 9 ° C , vs - 4 9 ° C for 20:4n6, and - 4 4 ° C for 22: 6n 3). One function of membrane 20: 5n 3 may be the maintenance of fluidity of the membranes during the cold season. Seasonal changes were observed in Tapes decussatus and T. philippinarum, but in these species PL fatty acids varied less during the annual cycle than did N L fatty acids (Beninger and Stephan, 1985).
Acknowledgements--This study was supported by a grant to M.P. from the Walter and Andrre de Nottbeck's Foundation. REFERENCES
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