Seasonal Variations of Fatty Acid Contents of Saccostrea cucullata
at Intertidal Zone of Chabahar Bay
Seasonal variations of fatty acids were studied in the
lipid fractions of the bivalve mollusk, Saccostrea cucullata, at
the intertidal zone of Chabahar bay in the northern part of Oman Sea (Iran).
Samples were collected in rocky shores between two stations. The analysis
were carried by GC/MS chromatography. Thirteen fatty acids were identified,
of which, the most important saturated fatty acids (SFA) were 14:0, 4,
8, 12 tri Me- 13:0, 16:0 and 18:0, the mono unsaturated fatty acids (MUFA)
included 16:1n-9, 18:1n-9 and 20:1n-11, the polyunsaturated fatty acids
(PUFA) were linoleic acid 9,12 18:2 , eicosapentaenoic acid EPA 20:5n-3
and arachidonic acid 20:4n-6.Variability of the fatty acid components
were studied in four seasons. Maximum percentage level in Saccostrea
cucullata for 14:0, 4, 8, 12 tri Me 13:0, 16:0 and 15:0 as saturated
fatty acids was observed in summer, while for 18:1n-9, 20:1n-11 and 20:5n-3
(as unsaturated fatty acids) maximum concentration was observed in winter.
The environmental factors were monitored monthly and their effects on
seasonal variations of the fatty acids were studied by applying pearson
coefficient correlation. The results showed the significant dependency
of 20:1n-11 fatty acid concentration to ambient temperature and 9,12 18:2
fatty acid to silicate as environmental factors. Also, principal component
analysis was done to establish the fatty acid groups. After Varimax rotation,
three factors were extracted, of which first and second factors contributed
to 86% of the data matrix. These were mainly dependent on the seasonal
variations of the fatty acids.
Chabahar bay with northern 60E 37′ 45" longitude and 27E 15′ 45"
latitude is located along the coast of Sistan Province in the South east of
Iran, northern part of Oman Sea in Indian Ocean. This area posses one of the
highest rates of primary productivity in the world due to seasonal upwelling
(Passow et al., 1993; Barlow
et al., 1999). This phenomenon provides a diverse and unique dietary
selection for mollusks which in turn might result in different compositions
of fatty acids. However, according to available data there is no earlier report
about fatty acids in this region.
Investigations of fatty acids in marine mollusks have been carried out by Ackman
et al. (1980), Ackman (2000), Joseph
(1989), Dembitsky et al. (1993), Misra
et al. (2002) and Abad et al. (1995)
in many habitats. Among the mollusks, bivalves are more important because of
their commercial value for use in human foodstuffs, the biological and pharmacological
role of their polyunsaturated fatty acids and especially the fatty acid 20:5n3,
which is useful in the treatment of some cardiovascular diseases (Joseph,
1982). Seasonal variations in lipid and fatty acid compositions of bivalves
have been reported for several species including Argopecten irradians,
Tapes decusssatus, T. Philippinrum, Chlamys tehelcha,
C. Opercularis, Venus pallina, Seapharca inequivalvis and
Crassostrea virginica (Barber and Blake, 1981;
Beninger and Stephan, 1985; Kluytmans
et al., 1985; Piretti et al., 1987,
1988; Chu et al., 1990).
The composition of the lipid fraction of mollusks may be affected by external
factors such as the changes of environmental conditions or internal factors
such as sexual maturation (Piretti et al., 1988).
The purpose of this study was to provide information on fatty acids compositions
of Saccostrea cucullata, class Bivalvia; order Pterioida; family
Ostreidae and their seasonal variations with the aim of understanding
their relationship with certain physico-chemical parameters of the environment.
MATERIALS AND METHODS
Oysters, with the same size (S. cucullata; 6-7 cm in length) to
minimize compositional variations, were collected from Chabahar bay, in
the rocky shores between two stations at a distance of about 7 km in four
seasons (April, July, October 2007 and February 2008). The environmental
conditions were monitored monthly from June 2007 until March 2008.
The whole body tissue of fifteen oysters were dissected from the shells in
every sampling, frozen and stored in pre-weighted containers at -80°C for
later analysis of the fatty acids. Wet fresh weight of the standard animal was
7 g. Five gram of each sample was taken, extracted using a homogenizer (Wagtech
T1813) with a solvent mixture of chloroform/methanol 2:1 (v/v) and volume to
weight ratio of 20:1. About 50 ppm of BHT (Butylated Hydroxy Toluene) was added
as an antioxidant into the mixture . The total extract was filtered under vacuum
using glass fiber filter (Whatman, S and S, GF6) and 0.5% NaCl (0.2 vol. of
the extract ) was added. The aqueous layer was re-extracted with chloroform.
The combined organic layers were evaporated to about 3-5 cm3 and
then hydrolyzed with 5% aqueous KOH (20 cm3) and methanol (100 cm3)
for 2-3 h at reflux temperature. After cooling, water (50 cm3) was
added and the basic solution was extracted twice with n-heptane/ diethyl ether
1:1 (v/v). The aqueous methanolic layer was acidified to pH = 2 and the fatty
acids were extracted with n-heptane/diethyl ether 1:1(v/v, 3x100 cm3).
Then they were dried over anhydrous MgSO4 and filtered. The filtrate
was concentrated to 2-3 cm3 (Johns et al.,
1980). The fatty acids were esterified with BF3-methanol (Morrison
and Smith, 1964) and heated in boiling water for 5 min. After cooling, 1
mL water and 2 mL pentane were added, vortexed (Stuart SA8) for 1 min and centrifuged
(Heraeus Biofuge). Then the upper phase was collected. Pentane was evaporated
and the residue dissolved immediately in 50-100 Fl of n-hexane for injection
to the gas chromatograph. Each sample was treated and analyzed for three times.
The samples were injected into GC/Mass and the separation of the fatty acids
methyl esters was performed using a Gas Chromatograph (Agilent Technologies,
6890) with a mass selective detector (6973N). GC/Mass analysis was done with
an Electron Impact (EI) mode of 70 eV as ionization source and quadrupole mass
filter with Chemstation data analysis system. The capillary column used was
HP-5 (5% diphenyl 95% dimethyl siloxane copolymer) with 30 m length, 320 μm
internal decimeter and 1 μm film thickness. The carrier gas was helium
(purity , 99.999%). 0.5 μL of the extract containing the fatty acids methyl
esters was injected into the injector using split mode with 50:1 split ratio.
The injector temperature was 200°C, the detector temperature was 280°C
and the oven temperature was programmed from 75°C min-1 to 270°C
at 30°C min-1, while the final temperature was held for 7 min
(Casado et al., 1998).
To insure that all the components were detected, the final temperature
was held for 20 min in the replicated test runs.
The fatty acids methyl esters were separated and identified in composition
with the chromatograms of commercial fatty acids standards and in order
to eliminate quantitative differences between the samples, the fatty acids
composition was expressed in the relative terms, as a percentage of the
total fatty acids. Peaks < 0.2% of the total area were not included
in the profiles.
The pearson correlation coefficients and regression analysis were used
in order to determine if there are relationships among the fatty acids
variations and environmental parameters.
Principal Component Analysis (PCA) was performed to distinguish similarities
between the fatty acids and categorizing them, on a personal computer
using the statistical software package SPSS version 11.5. The data matrix
for analysis consisted of the concentrations of identified fatty acids
at four seasons. Factors were extracted by PCA. The factors which determined
were extracted only in cases where the eigenvalues were over one. If the
eigenvalue of the extracted factor was less than one, it was not considered
in the data set. Then by rotation of axis (Varimax rotation), the factors
treated mathematically for maximizing the load of factors and each factor
was assumed to be independent.
The sea water conditions from June 2007 until March 2008 (the sampling
period) are presented in Fig. 1. As shown in Fig.
1A with the advance of winter a decrease in water temperature was
observed until reaching a minimum (21°C) in February and an increase
of temperature during spring to summer until its maximum (33.07°C
) in July. Chlorophyll-a showed relatively low values during the winter
(Fig. 1B), with minimum concentrations (0.1 ppb) and
increases in May, then shortly decrease and reach its maximum at fall
(1.28 ppb). The trend of silicate and phosphate is slightly similar to
each other, have their maximum levels in summer (Fig.
1C, E), as 0.6 and 0.504 ppm, respectively. Nitrate
(Fig. 1D) has its minimum (0.050 ppm) in April and
reach to its maximum (2.817 ppm) in May.
Increased differences in the salinity (Fig. 1F),
were observed from May till September and in October has minimum of 36.43
PSU and then followed by a sharp increase in November, thereafter decreasing
The analysis of fatty acid contents of Saccostrea cucullata during
four seasons are reported in Table 1. Thirteen fatty
acids were identified including seven saturated. The proportion of the
saturated fatty acids varied from 64.135 to 86.211% of total lipids in
Saccostrea cucullata (Table 2).
The major saturated fatty acids were 4,8,12-tri Me-13:0 , 16:0, 14:0
and 18:0. The monoenoics were 16:1n-9, 18:1n-9 and 20:1n-11. Linoleic
acid 9,12- 18:2 was only identified in the dienoics group of the fatty
acids. 20:4n-6 and 20:5n-3 were identified as tetra and penta enoic acids.
PCA, a multivariate technique with the aim of reduce the number of variables
(measured fatty acids content in four seasons) to a smaller set of factors
was applied to the fatty acids data set. The number of factors extracted
from the variables was determined according to Kaiser′s rule. This
criterion retains only factors with eigenvalues that exceed one. The analysis
of the fatty acid data established three factors. Varimax rotation for
the correlations was applied. Concentrations of thirteen fatty acids as
active variables in four seasons were selected. The cumulative variance
among these three factors was 100%. The first factor was the most effective
of the three factors and accounted for 60.134% of the variance (Table
3). Fatty acids with high factor loading values in the first factor
were 14:0,4,8,12-tri Me-13:0, 15:0, 16:0, 17:0, 9,12- 18:2 , 18:1n-9,
18:0, 20:5n-3 and 20:1n-11. The second (PC2) and the third (PC3) explained
25.721 and 14.145% of the total variances, respectively. PC2 dominated
by Me17:0 (0.929) and 20:4n-6 (0.95) and PC3 dominated by 16:1n-9 (0.945).
Seasonal variations of fatty acids related to these three factors are
represented in Fig. 2.
||Variations of environmental conditions in four seasons
at Chabahar bay
||Fatty acid contents in Saccostrea cucullata during
||Total saturated fatty acid contents in Saccostrea
cucullata during four seasons
The use of pearson correlation matrix among the environmental factors
including salinity, temperature, chlorophyll-A, silicate, phosphate and
nitrate (as nutrients), with fatty acids showed a strong negative correlation
between the temperature and 20:1n-11 fatty acid (r = -0.97) and also for
silicate and 9,12 18:2 fatty acid (r = -0.95).
||Seasonal variations in the three groups of fatty acids
of Saccostrea cucullata
||Factor analysis for three principal components (Varimax
with Kaiser Normalization) for fatty acids in four seasons in Chabahar
According to the results of pearson correlation, regression method applied
for temperature and 20:1n-11 fatty acid; silicate and 9,12 18:2 fatty
acid, which resulted r2 = 0.96 and 0.91, respectively and indicate
the important role of the above ecological parameters in the mentioned
fatty acids variations.
From the above context, total saturated fatty acids varied between 64.135
and 86.211% in Sacostrea cucullata. Thirteen fatty acids
including seven saturated, three monoenoics, one dienoic, one tetraenoic
and one pentaenoic fatty acids were determined.
By applying PCA meth od fatty acids categorised in three groups, due to their
similar trends. Based on PCA results, PC1 consist of the saturated fatty acids
(14:0, tri Me-13:0, 15:0 and 16:0) which had their maximum level in summer and
the unsaturated fatty acids (20:5n-3, 18:1n-9 and 20:1n-11) which had their
maximum level in winter. This result confirm the earlier reports which have
shown an inverse relationship between temperature and the amount of polyunsaturated
fatty acids in tissue lipids of invertebrates and diminution of the saturated
fatty acids during winter (Chu and Greaves, 1991).
Accumulation of polyunsaturated fatty acids (especially 20:5n-3) and minimum
levels of saturated fatty acids during winter and reverse change in summer may
be due to the adaptive regulation of melting point of cellular lipids (Pazos
et al., 1996). Also, tri Me 13:0 (4,8,12 tri Methyl Thridecanoic
acid) could be originated from the bivalves diet (Wood, 1974;
Johns et al., 1979) and related to composition
of the food sources which is abundant in summer in the sampling area. Considering
the fact that 20:5n-3 could not be synthesized de novo (De
Moreno et al., 1980) and is also related to bivalves diet, maximum
percentage level of tri Me 13:0 in summer and 20:5n-3 fatty acids in winter
could be originated from different diet sources.
Also 18:0 fatty acid in PC1 has a slightly constant trend throughout the year,
which is in line with other similar researches regarding bivalves (De
Moreno et al., 1980). In addition to other parameters in PC1, 9,12
18:2 fatty acid reached to its maximum level in spring which in this time the
available food source from phytoplankton origin gives an increase in polyunsaturated
fatty acids (Langdon and Waldock, 1981).
PC2 consists of Me17:0 and 20:4n-6 fatty acids which have their maximum in
fall. These levels probably arise from selective retention of the 20:4n-6 fatty
acid for use in reproductive processes and it has been shown that the 20:4n-6
fatty acid in the oyster can be implicated in the synthesis of various neurotransmitters
related to the reproductive processes, such as the prostaglandins 2 (Freites
et al., 2002). Besides 20:4n-6 might be used as an energy reserve
during periods of food scarcity (Gardner and Riley, 1972).
16:1n-9 fatty acid had been categorized in an individual group (PC3) which reached
its maximum level in spring, decreased toward summer; thereafter increased in
fall slightly similar to spring and decreased in winter again similar to summer.
Therefore, it had a unique trend among the others which categorised in a separate
group. Also its maximum in spring confirms again the research of Langdon
and Waldock (1981), about increasing such polyunsaturated fatty acids in
this time in relation to available food source from phytoplankton origin.
According to pearson correlation, temperature among ecological factors
showed a significant correlation with the 20:1n-11 fatty acid, i.e., with
the increasing of temperature, the amount of fatty acid will decrease
with a strong correlation and a good regression which is mentioned before
that unsaturated fatty acids must be increased in low temperatures because
of their higher melting point compared to the saturated fatty acids and
reflect that these fatty acids could be used in metabolism of cells.
A significant negative correlation was observed between silicate and 9,12 18:2
fatty acid. This can be explained by the fact that in high temperatures a decrease
occurs in silicate availability and highly saturated fatty acids, which will
increase unsaturated ones (Mortensen et al., 1988).
Similar results had been observed that within a silicate deprivation diet, 18:2n-6
fatty acid increased in fatty acid composition of sea Scallop Placopecten
magellanicus larvae (Pernet and Tremblay et al.,
This research was a part of first authors Ph.D Thesis which was supported
by Iranian National Centre for Oceanography (INCO) grant number 385-0111.
Authors dedicate their thanks to Prof. Isa Yavari for his precious recommendations/guidelines.
Abad, M., C. Ruiz, D. Martinez, G. Mosquera and J.L. Sanchez, 1995.
Seasonal variations of lipid class and fatty acids in flat oyster, Ostrea edulis
, from San Cibran, (Galicia, Spain). Comp. Biochem. Physiol. Part C: Pharmacol. Toxicol. Endocrinol. 108: 109-118.Direct Link |
Ackman, R.G., 2000.
Fatty Acids in Fish and Shellfish. In: Fatty Acids in Foods and their Health Implications, Chow, C.K. (Ed.). Marcel Dekker Inc., New York, pp: 153-172
Ackman, R.G., J.L. Sebedio and M.I.P. Kovacs, 1980.
Role of eicosenoinc and docosenoic fatty acids in fresh water and marine lipids. Mar. Chem., 9: 157-167.Direct Link |
Barber, B.J. and N.J. Blake, 1981.
Energy storage and utilization in relation to gametogenesis in Argopecten irradians concentricus
(say). J. Exp. Mar. Biol. Ecol., 52: 121-134.CrossRef | Direct Link |
Barlow, R.G., R.F.C. Mantoura and D.G. Cummings, 1999.
Monsoonal influence on the distribution of phytoplankton pigments in the Arabian Sea. Deep Sea Res. Part II: Top. Stud. Oceanogr., 46: 677-699.Direct Link |
Beninger, P.G. and G. Stephan, 1985.
Seasonal variations in the fatty acids of the triacylglycerols and phospholipids of two populations of adult clam (Tapes decussatus
L. and T. philippinarum
) reared in a common habitat. Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol., 81: 591-601.Direct Link |
Casado, A.G., E.J.A. Hernandez , P.E. Espinoza and J.L. Vilchez, 1998.
Determination of total fatty acids (C8
) in sludge by gas chromatography-mass spectrometry. J. Chromatogr. A, 826: 49-56.Direct Link |
Chu, F.L.E., K.L. Webb and J. Chen, 1990.
Seasonal changes of lipids and fatty acids in oyster tissues (Crassostrea virginica
) and estuarine particulate matter. Comp. Biochem. Physiol. Part A: Physiol., 95: 385-391.Direct Link |
Chu, F.L.E. and J. Greaves, 1991.
Metabolism of palmitic, linoleic and linolenic acids in adult oysters. Crassostrea virginica
. Mar. Biol., 110: 229-236.Direct Link |
Dembitsky, V.M., T. Rezanka and A.G. Kashin, 1993.
Comparative study of the endemic freshwater fauna of Lake Baikal-l. Phospholipid and fatty acid compositions of two mollusc species Baicalia oviformis
and Benedictia baicalensis
. Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol., 106: 819-823.Direct Link |
De Moreno, J.E.A., R.J. Pollero, V.J. Moreno and R.R. Brenner, 1980.
Lipids and fatty acids of the mussel (Mytilus Platensis
d’Orbigny) from South Atlantic waters. J. Exp. Mar. Biol. Ecol., 48: 263-276.Direct Link |
Freites, L., U. Labata and M.J. Fernandez-Reiriz, 2002.
Evolution of fatty acid profiles of subtidal and rocky shore mussel seed (Mytilius galloprovincialis
LmK.). Influence of environmental parameters. J. Exp. Mar. Biol. Ecol., 268: 185-204.Direct Link |
Gardner, D. and J.P. Riley, 1972.
The component fatty acids of the lipids of some species of marine and freshwater molluscs. J. Mar. Biol. Assoc. UK., 52: 827-838.CrossRef | Direct Link |
Johns, R.B., P.D. Nichols and G.J. Perry, 1979.
Fatty acid composition of ten marine algae from Australian waters. Phytochemistry, 18: 799-802.Direct Link |
Johns, R.B., P.D. Nichols and G.J. Perry, 1980.
Fatty acid components of nine species of molluscs of the littoral zone from Australian waters. Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol., 65: 207-214.Direct Link |
Jones, I.D., L.S. Buttler, E. Gibbs and R.C. White, 1972.
An evaluation of reversed phase partition for thin-layer chromatographic identification of chlorophylls and derivatives. J. Chromatogr. A, 70: 87-98.Direct Link |
Joseph, J.D., 1982.
Lipid composition of marine and estuarine invertebrates. Prog. Lipid Res., 21: 109-153.Direct Link |
Joseph, J.D., 1989.
Distribution and Composition of Lipids in Marine Invertebrates. In: Marine Biogenic Lipids, Fats and Oils, Ackman, R.G. (Ed.). CRC Press, Boca Raton, FL., USA., pp: 49-144
Kluytmans, J.H., J.H. Boot and R.C.H.M. Oudejans, 1985.
Fatty acid synthesis in relation to gametogenesis in the mussel Mytilus edulis
L. Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol., 81: 959-963.Direct Link |
Langdon, C.J. and M.J. Waldock, 1981.
The effect of algal and artificial diets on the growth and fatty acid composition of Crassostrea gigas
spat. J. Mar. Biol. Assoc. UK., 61: 431-448.CrossRef | Direct Link |
Misra, K.K., I. Shkrob, S. Rakshit and V.M. Dembitsky, 2002.
Variability in fatty acids and fatty aldehydes in different organs of two prosobranch gastropod mollusks. Biochem. Syst. Ecol., 30: 749-761.CrossRef | Direct Link |
Morrison, W.R. and L.M. Smith, 1964.
Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J. Lipid Res., 5: 600-608.PubMed | Direct Link |
Mortensen, S.H., K.Y. Borsheim, J.R. Rainuzzo and G. Knutsen, 1988.
Fatty acid and elemental composition of the marine diatom chaetoceros gracilis
Schütt. Effects of silicate deprivation, temperature and light intensity. J. Exp. Mar. Biol. Ecol., 122: 173-185.Direct Link |
Passow, U., R. Peinert and B. Zeitzsche, 1993.
Distribution and sedimentation of organic matter during the inter-monsoon period off Oman (West Arabian Sea). Deep Sea Res. Part II: Top. Stud. Oceanogr., 40: 833-849.CrossRef | Direct Link |
Pazos, A.J., C. Ruiz, O. Garcia-Martin, M. Abad and J.L. Sanchez, 1996.
Seasonal variations of the lipid content and fatty acid composition of Crassostrea gigas cultured in E1 Grove, Galicia, N.W. Spain. Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol., 114: 171-179.CrossRef | Direct Link |
Pernet, F. and R. Tremblay, 2004.
Effect of varying levels of dietary essential fatty acid during early ontogeny of the sea scallop Placopecten magellanicus
. J. Exp. Mar. Biol. Ecol., 310: 73-86.Direct Link |
Piretti, M.V., F. Zuppa and G. Pagliuca, 1987.
Investigation of the seasonal variations of sterol and fatty acid constituents in the bivalves mollusk Venus gallina
and Scapharca inaequivalvis
(Bruguiere). Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol., 88: 1201-1208.Direct Link |
Piretti, M.V., F. Zuppa, G. Pagliuca and F. Taioli, 1988.
Investigation of the seasonal variations of fatty acid constituents in selected tissue of the bivalve mollusc, Scapharca inaequivalvis
(Bruguiere). Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol., 89: 183-187.Direct Link |
Wood, B.J.B., 1974.
Fatty Acids and Saponifiable Lipids. In: Algal Physiology and Biochemistry, Stewart, W.D.P. (Ed.). Oxford University Press, Oxford, pp: 236-265