Food sources of three bivalves living in two habitats of Jiaozhou Bay (Qingdao, China): Indicated by lipid biomarkers and stable isotope analysis.

ABSTRACT Food sources of three filter-feeding bivalves from two habitats (intertidal oyster Crassostreagigas, mussel Mytilus galloprovincialis, and subtidal cultured scallop Chlamys farreri) of Jiaozhou Bay (Qingdao, China) were determined by fatty acid and stable isotope analysis. Cultured scallop was characterized by significant diatom markers such as 16:1 / 16:0 close to 1 and high ratio of 20:5(n - 3)/22:6(n - 3), hence we assume that the scallop mainly feeds on diatoms. Fatty acid biomarkers specific to bacteria and terrestrial materials were also found in considerable amounts in scallop tissue, which suggested that there were substantial bacterial and terrestrial input into the food of the species. Intertidal oyster and mussel, however, exhibited significant flagellate marker, 22:6(n - 3), and lower level of diatom markers, which indicated that flagellates are also part of intertidal bivalves' planktonic food sources; meanwhile, high level of Chlorophyta fatty acid marker, [GAMMA]18:2(n - 6) + 18:3(n - 3), suggested that Ulva pertusa (Chlorophyta) seaweed bed supplied important food sources to intertidal bivalves. Additionally, result of stable isotope analysis showed that phytoplankton contributed 86.2 to 89.0% to intertidal bivalves' carbon budget; macroalga U. pertusa origin source had a contribution of 8.7% to 11.0%, which indicated its role as an important supplemental food source to intertidal bivalves. From this study, it is concluded that the dietary difference of three bivalves probably relates to the different potential food sources in the scallop farm and intertidal zone in Jiaozhou Bay.

KEY WORDS: bivalve, food sources, lipid biomarker, stable isotope

INTRODUCTION

Suspension-feeding bivalves, primarily scallops, oysters and mussels, are a significant element of near-shore communities, which profoundly impact pelagic and benthic processes. Through filter-feeding activity they can cycle large amounts of particulate matter within the environment, converting some of it into flesh and gametes, depositing varying amounts to the benthos and cycling complex molecules into inorganic forms. As herbivores, bivalves can exert top-down control on phytoplankton production, reducing the amount of suspended organic carbon available to stimulate anoxia. Grazing of particulates also reduces turbidity, thereby increasing light availability to the bottom and enhancing the growth of benthic macrophyte (Ward & Shumway 2004). Moreover, many species of bivalve are commercially important, playing crucial roles in the local economies of many states, provinces and countries. In China, for example, the production of bivalves was close to 10.7 x [10.sup.6] tons in 2005, which was about 77% of total mariculture production of China.

Suspension-feeding bivalves are confronted with a wide range of living and nonliving material. The seston consists of plankton of a wide range of sizes and palatability, material resuspended from the benthos, as well as detritus, fecal pellets and microorganisms (e.g., Alldredge & Gotschalk 1989, Crocker & Passow 1995, Passow et al. 1994). Particulate matter in the benthic environment is dominated by mineral grains of various sizes, mixed with a variety of organic matter including material derived from the plankton, benthic mieroalgae, feces, detritus, protozoa, and bacteria (Newell 1965, Lopez & Levinton 1987). The complex of organic particles around the bivalves resulted in the variation of their food source compositions. It is reported that Patagonian scallop Chlamys tehuelcha has a seasonal dietary change, and gut content analysis showed that benthic algae such as Synedra investens, Melosira sulcata, planktonic diatoms such as Chaetoceros and Thalassiosira species, and even scallop eggs during the spawning season, were all available to the scallop in different part of the year (Bricelj & Shumway 1991). Bachok et al. (2003) concluded that mangrove detritus and bacteria attached on decomposed leaf detritus were major food sources of the mud clam Geloina coaxans living in the mangrove forest during cold and warm seasons. The oyster Crassostrea virginica and the mussel Geukensia demissa were also proved capable to use bacteria with an efficiency as high as 15.8% (Langdon & Newell 1990).

Determination of the relative proportion of various food sources assimilated by suspension-feeding bivalves is an important question in understanding their feeding physiology. Gut content analysis, however, provide only rough estimates of the percentages of various materials ingested by bivalves and may reflect material that is not assimilated. Meanwhile, a large proportion of amorphous organic particles were frequently detected in bivalve's gut content. They probably have complex origins, yet it's often difficult to make a clear determination only depending on their morphology.

Recently, lipid and stable isotope biomarkers have been widely used in the food web analysis and dietary determination around the world. The concept of using lipids as biomarkers in marine ecosystems has been paid considerable attention in the past few decades (Volkman et al. 1989, Falk-Petersen et al. 2002). This trophic biomarker concept is based on observations that specific dietary lipid components, particularly fatty acids, are incorporated into the consumers' lipids largely unmodified (Graeve et al. 1994). This approach can provide information where the classical gut content analysis fails. Instead of a snapshot impression, lipid biomarkers integrate the trophic information over a longer time scale of several weeks.

The natural abundance of stable isotopes has emerged as another powerful means to estimate the dependence of organisms on specific diets and habitats. For carbon, there appears to be a slight enrichment of [sup.13]C in the consumer's tissue relative to its diet (0.3 [per thousand] to 1 [per thousand]), whereas that of 15N is greater (3 [per thousand] to 4 [per thousand]) (Michener & Schell 1994). Using carbon isotope solely can distinguish two food sources with clear differences in [[delta].sup.13]C values, whereas a dual-isotope integrating carbon and nitrogen isotopes together can give an evaluation of three food sources using a single mixing model (Peterson & Fry 1987, Rundel et al. 1989). Lately, a powerful software has been developed by Phillips and Gregg (2003) to cope with more than three food sources using one or two stable isotopes. This actually broke the limit of stable isotope analysis and enlarged its applying field.

Jiaozhou Bay, situated in southern part of Shandong Peninsula (China), is a typical temperate semienclosed bay. The intertidal and subtidal zone (0 ~ 5-m deep) covers over 80% of its total area, in which abundant invertebrates are widely distributed (Liu 1992). Oyster Crassostrea gigas and mussel Mytilus galloprovincialis are predominant bivalve species living in the rocky intertidal area, whereas scallop Chlamys farreri is the key species cultured in the subtidal zone with a production of about 2.1 x [10.sup.4] tons in 2004. However, previous researches on bivalves in Jiaozhou Bay most focused on the biomass, population dynamics and community ecology (Li et al. 2006, Yu et al. 2006, Liu 1992), and little work is done on the determination of bivalves' food sources.

In the present study, we selected three representative bivalve species living in intertidal area and subtidal raft-culturing farm in Jiaozhou Bay and determined their food sources using lipid biomarker and stable carbon isotope analysis. The relationship between bivalve's diet composition and the potential food sources in the habitat was also discussed.

METHODS

Study Area

Jiaozhou Bay locates in North China (35[degrees]38'-36[degrees]18'N, 120[degrees]04' - 120[degrees]23'E) with an area of about 390 [km.sup.2] and an average water depth of about 7 m. The bay connects to the Yellow Sea with a narrow mouth of only 2.5 km wide. There are more than 10 rivers entering Jiaozhou Bay such as the Dagu River with an annual average runoff of 6.61 x [10.sup.8] [m.sup.3]. Other rivers pass through the urban area, such as the Lincun River, Haibo River, and Loushan River (Liu 1992). The intertidal zone at the mouth of the bay is mainly rocky coast, whereas at the center of the bay situates bivalve's raft-culture farm with an area of over 1300 ha.

Sample Collection

Samples from aquaculture and intertidal zone were collected in the spring of 2005. Raft-cultured scallops C. farreri (n = 30, 1-year-old) were sampled in the aquaculture area (site a) at April 21 (Fig. 1); intertidal oyster C. gigas and mussel M. galloprovincialis (n = 30, both 1-year-old) were caught on the rocks at May 24. Two species of subtidal macroalgae, Ulva pertusa (which formed a flourishing seaweed bed) and Undaria pinnatifida, were sampled by hand at site b (Fig. 1). Net-towed phytoplankton samples (76-[micro]m mesh) at two sites were obtained by hauling horizontally in the subsurface water.

[FIGURE 1 OMITTED]

All samples were taken back to the laboratory in 1h. Nettowed phytoplankton sample was prefiltered with a 200-[micro]m mesh net to exclude large zooplanktons and particles. Then they were collected on Whatman GF/C filters (n = 2, precombusted under 450[degrees]C for 4 h). The filters were rinsed with Milli-Q water and stored under -20[degrees]C for further analysis. Intertidal macroalgae samples were cleaned by filtered seawater and Milli-Q water respectively and then freeze-dried. Mollusks were kept in filtered seawater for 24 h to empty their guts. Then they were dissected followed by rinsing the tissues carefully with Milli-Q water. Tissues were either stored in 5 mL dichloromethane under -20[degrees]C for lipid extraction, or freeze-dried for stable isotope analysis. Lipid biomarker analysis was used on samples from both habitats, whereas stable isotope method was used only on intertidal samples, because there were no other primary producers except phytoplankton in the mariculture area. For lipid extraction, three to four individuals were pooled to acquire enough tissue, and three replications were set.

Lipid Extraction, Separation and Fatty Acid Analysis

All of the organic solvents used for fatty acid analysis were HPLC grade. Bivalve tissues or phytoplankton samples were transferred to a glass homogenizer and homogenized in a mixture of dichloromethane-methanol (2:1, v/v). Lipids were extracted using a modified Folch et al. (1957) extraction procedure (Parrish et al. 1999).

In bivalves, digestive gland has proved to be the major storage organ for lipid reserves and can response significantly to different diets (Napolitano & Ackman 1992, Caers et al. 2003). Moreover, dietary influence on the fatty acid composition of total lipid extracts can be masked by the presence of structural lipids such as phospholipids, which are supposed to have a relatively stable fatty acid profile (Sargent et al. 1987). Instead, the dietary influence can be mirrored very well in storage lipid, mainly triglyceride (TG) for bivalves (Pazos et al. 2003). Therefore, we chose TG in the digestive gland of scallop for FA biomarker analysis so as to sensitively detect the possible expression of diet lipid marker in its tissue.

For bivalves, TG was separated from the total lipid extract by thin-layer chromatography (TLC) with a solvent mixture of hexane/diethyl ether/acetic acid (90:10:1, by vol.). Then TG was scrapped off the plate after spraying with 4% [I.sub.2] in methanol, extracted with dichloromethane and concentrated to approximately 1 mL under gentle [N.sub.2] airflow. To transform fatty acids to fatty acid methyl esters (FAMEs), an aliquot of TG extract was evaporated to near dryness and treated consecutively with 1 mL 0.5 M sodium hydroxide in methanol and 1 mL 14% boron trifluoride ([BF.sub.3]) methanol reagent and heated in a 80[degrees]C water bath for 2 h under a nitrogen atmosphere. FAMEs were purified by TLC in hexane/diethyl ether/acetic acid (90:10:1, by vol.), extracted into hexane and concentrated to appropriate volume. A GC-9A gas chromatograph (Shimadzu, Tokyo, Japan) was used for analysis. Separation of FAMEs was performed on a DB-FFAP capillary column (30 m x 0.32 mm i.d., Agilent Co., USA). The injector temperature was 250[degrees]C. Hydrogen was used as a carrier gas with a flow rate of 1.2 mL/min. Individual peaks of FAME were identified by comparing retention times with those of the authentic Cod Liver Oil FAME standard (Sigma, USA). The relative abundance of each FAME was quantified by peak area normalization of total chromatography peaks.

Isotopic Analysis

For isotopic analysis, intertidal phytoplankton filters were dried under 60[degrees]C overnight. To remove any possible carbonates, filters were exposed to concentrated HCl fume in a desiccator for 4 h. Then they were placed in a fume hood (1h) and then in an oven (overnight, 60[degrees]C). Freeze-dried bivalve and algal tissues were grounded to fine powder with a mortar and pestle and stored in a desiccator.

Stable carbon isotope ratio was measured on an Italian EuroEA3000 element analyzer plus Iso-Prime stable isotope mass spectrometer (GV Corporation). Data were expressed in the standard [delta] unit notation:

[delta]].sup.13]C([per thousand]) = [([R.sub.sample]/[R.sub.standard]) - 1] X [10.sup.3],

where R = [sup.13]C/[sup.12]C. Results were reported relative to the Pee Dee Belemnite standard.

Data Processing 

Significant differences (P < 0.05) of fatty acid biomarker and stable isotopic ratios among species were tested using one-way ANOVA and t-test. All tests were performed using SPSS 13.0 statistical software.

We applied the software IsoSource developed by Phillips and Koch (2002) to assess the contributions of different food sources to intertidal oyster and mussel. The increment and tolerance parameter in the procedure were set to 1% and 0.01 [per thousand]

respectively. The trophic shift for [sup.13]C between bivalve tissue and the diet was set as +0.3 [per thousand] (McCutchan et al. 2003). RESULTS

Fatty Acid Compositions of Phytoplankton and Bivalves

Fatty acid composition of net-towed phytoplankton is tabulated in Table 1. The fatty acids 16:0 and 14:0 were

predominant components in saturated fatty acids. 16:l(n - 7) and 20:5(n - 3) (EPA) were two most abundant components in monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA), which were 21.32% and 7.07% of the total fatty acids, respectively.

Fatty acid compositions of three bivalves were also presented in Table 1. In general, three bivalves contained between 24.75 ~ 29.96% SFAs, 27.43 ~ 31.48% MUFAs and 37.58 39.01% PUFAs. High percent of 16:l(n - 7) and 20:5(n - 3) (EPA) were found in MUFAs and PUFAs of cultured scallop C. farreri. In intertidal species C. gigas and M. galloprovincialis, 18:l(n - 9) was predominant in MUFAs, meanwhile, EPA and 22:6(n - 3) (DHA) were two abundant fatty acids in PUFAs.

Fatty Acid Biomarkers

Fatty acid biomarkers used in the determination of organic sources were listed in Table 2 (referred to Budge & Parrish 1998, Budge et al. 2001, Dalsgaard et al. 2003). There were five potential food sources differentiated in present study: diatoms, flagellates, bacteria, Chlorophyta, and terrestrial organic source. Biomarker values of three bivalves and net-towed phytoplankton sample were tabulated in Table 3.

In the scallop farming area, net-towed phytoplankton sample showed a high ratio of 16:1/16:0 (above 1) and EPA/ DHA (5.64), and these showed that diatom was predominant component in the sample (Budge & Parrish 1998, Budge et al. 2001, Reuss & Poulsen 2002). Cultured scallop, C. farreri, also showed remarkable diatom biomarkers in its tissue, compared with the other two bivalve species. Meanwhile, scallop tissue also had a higher bacterial marker level than intertidal species, such as higher percent of odd and branched fatty acids (4.08%, P < 0.05) and 18:l(n - 7)/18:1(n - 9) above 1 (P < 0.001).

Intertidal bivalves, C. gigas and M. galloprovincialis showed significantly higher level of flagellate marker (DHA) than cultured C. farreri, (5.26% to 6.35%, P < 0.001). Additionally, the percent of [SIGMA] 18:2(n - 6) + 18:3(n - 3) was over 2-folded that of C. farreri (10.03% to 11.67%, P < 0.001), meanwhile, there was no terrestrial plants distributing nearby, so the biomarker should be derived of the U. pertusa seaweed bed. Diatom biomarkers, however, stayed at a lower level such as 16:1/16:0 below 0.5 and EPA/DHA below 2.0.

Proportion of Different Food Sources of Intertidal Bivalves

Isotopic compositions of intertidal primary producers and bivalves were given in Table 4. Three primary producers had distinct [[delta].sup.13]C values ranging from -13.13 [per thousand] to -22.07 [per thousand], whereas the [[delta].sup.13]C values of oyster and mussel were -21.52 [per thousand] and -21.38 [per thousand], respectively, and no significance was found between them (t-test, P = 0.565). It suggested that the two bivalve species may have similar food compositions.

The food compositions of oyster and mussel, calculated by IsoSource software, were tabulated in Table 5. Phytoplankton was the predominant part of intertidal bivalves' diet, whereas the U. pertusa seaweed bed contributed to 8.7% to 11.0% of total food source. Another subtidal brown algae U. pinnatifida also had a small contribution of about 2.3% to 2.8% to bivalves' diet, although it had postbloomed.

DISCUSSION

Various studies have shown that suspension-feeding bivalves feed on whatever available in the habitat, and through particle selection, they separate the grain from the chaff and acquire the optimum energetic budget (reviewed by Ward & Shumway 2004). Results of present study figured out the correlation between bivalve's diet and the potential food sources in the habitat.

Diet of Cultured Scallop C. farreri

The potential food sources of coastal cultured scallop in Jiaozhou Bay include diatoms, bacteria, and terrestrial materials.

In present study, diatoms proved to be predominant in the phytoplankton community in the scallop farming area, and this is supported by previous phytoplankton investigation of Jiaozhou Bay, in which diatoms, such as Chaetocero spp. and Skeletone macostatum, accounted for 89.1% of total phytoplankton biomass in the spring bloom (Li et al. 2005). Large biomass of diatoms subsequently supplied abundant food source to cultured scallops, indicated by the remarkable diatom biomarkers in scallop's storage lipid.

The remarkable level of bacterial biomarker in the net-towed phytoplankton sample suggested the existence of bacteria in the sample. Bacteria, else than phytoplankton, represent another important organic source widely distributed in Jiaozhou Bay. It was reported that a peak bacteria concentration of 1.2 x [10.sup.6] cells/mL could be reached in April (Zhao et al. 2005). Meanwhile, a 4.08% of bacterial marker found in scallop's tissue indicated that the scallop ingested some amount of bacteria as its diet. On the other hand, free bacterioplankton, typically ranging in size from 0.3-1 [micro]m is usually not available as a food source for scallops (Bricelj & Shumway 1991); however, bacteria may colonize on large organic particles and ingested by scallop and other filter-feeding bivalves. In Langdon and Newell's research (1990), the attached bacteria associated with the breakdown of cellulosic material could mediate the flow of dissolved inorganic nitrogen from seawater to the oyster Crassostrea virginica. Cheng and Lopez (1991) also found that the bivalve Nucula proxima absorbed bacteria attached on sediment particles.

A similar characteristic of fatty acid composition in terrestrial plants and macroalgae Chlorophyta is the high concentration of [SIGMA] 18:2(n - 6) + 18:3(n - 3). This similarity is partially correlated to the evolutional homologue of terrestrial plant and marine Chlorophyta. Therefore, when [SIGMA]18:2(n - 6) + 18:3(n - 3) is used as a marker, it can indicate both organic sources. So the result must be discussed carefully, and when necessary, other markers (such as long-chain fatty acids) together with in situ investigation should be applied simultaneously to make precise estimation. When [SIGMA] 18:2(n - 6) + 18:3(n - 3) is used as a marker of terrestrial organic matter, a threshold of 2.5% can be set to indicate significant terrestrial organic source (Budge & Parrish 1998). In present study, 4.51% of [SIGMA] 18:2(n - 6) + 18:3(n - 3) was found in scallop's tissue; meanwhile, there was no species of Chlorophyta growing nearby, so the biomarker probably revealed that the scallop ingested some amount of terrestrial organic materials as its diet. The scallop farm is adjacent to the east coast of Jiaozhou Bay, and there locates two rivers, Haibo River and Licun River. Enormous trees and bushes grew on the downstream bank of Licun River, and a large area of estuarine wet land dominated by common reed also located at its entrance to the bay. Terrestrial organic materials, mainly derived from decomposed leaves and branches, were brought into the bay by water flow continuously. It probably formed an important input to the marine suspended particulate organic materials and became available for cultured scallops.

Diet of Intertidal Oyster and Mussel

The potential food sources of intertidal oyster and mussel include mainly diatoms, flagellates and macroalgae Chlorophyta.

Compared with cultured scallop, intertidal oyster and mussel exhibited a low level of diatom biomarkers, such as 16:1/16:0 < 0.5 and a low ratio of EPA/DHA (1.8). It indicated that diatoms may have a smaller contribution to intertidal bivalves' diet. Moreover, remarkable level of flagellate biomarker in both species suggested the importance of flagellates (mainly including species in Chrysophyta and Pyrrophyt) in the diet of two intertidal bivalves. Previous phytoplankton investigation of Jiaozhou Bay revealed the difference of biomass and community composition at the two sampling areas in this paper. Results showed that the total phytoplankton biomass at the scallop farm was 10-folded that of intertidal zone: in the scallop farming area, diatoms were predominant (99.6%) with rare species of Pyrrophyta (0.4%), whereas at the intertidal area, the proportion of diatoms fell to 70.1%, and species of Pyrrophyta and Chrysophyta occupied 25.4% and 4.5% of total biomass, respectively. These differences of phytoplankton composition at two habitats were mirrored very well into the fatty acid profiles of the bivalves.

Intertidal bivalves also exhibited high level of Chlorophyta biomarker, and this probably relates to the bloom of macroalgae U. pertusa (Chlorophyta), which formed a flourishing seaweed bed at the sampling site in May. The evaluation result of IsoSource software showed that U. pertusa had a contribution ranging from 8.7% to 11.0%, to the carbon budget of intertidal bivalves, and it proved that the seaweed bed served as a supplemental organic source for oysters and mussels. Macroalgal origin organic source in seaweed beds, mainly derived of the release of detritus and dissolved organic matter, have been proved to be available for bivalves living inside (Bustamante & Branch 1996, Dunton & Schell 1987, Fielding & Davis 1989). For example, Bustamante and Branch (1996) reported that, as high as 50% of carbon and 65% nitrogen assimilated by two filter-feeding bivalves in a kelp bed could be conservatively explained by the contribution of kelp-derived organic materials. In present research, the contribution of U. pertusa was not as high as kelp, yet it still supplied part of oyster and mussel's food source. Additionally, the evaluation result showed that brown algae U. pinnatifida, which bloomed in April, also had a small contribution to intertidal bivalves' carbon origin.

In summary, results of present study figured out the food sources of three bivalves in Jiaozhou Bay in spring: subtidal cultured scallop feeds mainly on diatoms, and it also ingests a small proportion of bacterial and terrestrial organic sources; flagellates are part of the planktonic diet of intertidal oyster and mussel besides diatoms, meanwhile, green alga U. pertusa seaweed bed supplied 8.7% to 11% of intertidal bivalves' carbon budget. The dietary difference of three bivalves probably relates to the different potential food sources in the scallop farm and intertidal zone. Results agreed well with the fact that suspension feeding bivalves' diet depend largely on their habitats.

ACKNOWLEDGMENTS

The authors thank Kui You, Xinling Xu, and Fei Gao for their help in field sampling. The research was funded by the National S & T Supporting Projects (No. 2006BAD09A02) and Hi-tech Research and Development Program of China (No. 2006AA 100304).

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QIANG XU (1,2) AND HONGSHENG YANG (1) *

(1) Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China; (2) Graduate School, Chinese Academy of Sciences, Beijing, 100039, China

* Corresponding author. E-mail address: [e-mail]

TABLE 1.

Fatty acid compositions of net-towed phytoplankton sample and scallop
(in the aquaculture farm) and intertidal oyster and mussel of Jiaozhou
Bay (Qingdao, China). % total fatty acids (SD); n = 3. "--" means not
detected; "tr" means concentration below 0.1%. "*": sample was
collected in the scallop farming area; "**": isomers.

Fatty Acid      Phytoplakton *    Chlamys farreri

SFA
 14:0            14.60 (1.27)      3.52 (0.46)
 15:0             0.75 (0.10)      0.44 (0.04)
 16:0            17.44 (1.12)     15.63 (1.10)
 17:0             0.70 (0.01)      1.01 (0.07)
 18:0             2.66 (0.45)      3.56 (0.19)
 20:0             1.01 (0.06)      0.36 (0.07)
 22:0             0.21 (0.00)      0.23 (0.06)
MUFA
 14:1(n - 5)      0.12 (0.01)      1.09 (0.02)
 16:1(n - 9)      4.99 (0.53)              --
 16:1(n - 7)     21.32 (0.57)     13.72 (0.84)
 16:1(n - 5)      0.50 (0.01)      0.32 (0.02)
 17:1(n - 9)              --       0.59 (0.07)
 18:1(n - 9)      2.14 (0.02)      5.04 (0.34)
 18:1(n - 7)      1.39 (0.08)      7.28 (0.29)
 18:1(n - 5)      0.21 (0.01)      0.27 (0.02)
 20:1(n - 9)      0.40 (0.15)      0.94 (0.03)
 20:1(n - 7)      0.24 (0.05)      1.72 (0.15)
 22:1(n - 11)              --      0.34 (0.12)
 22:1(n - 9)               --      0.19 (0.06)
PUFA
 16:2(n - 4)      3.77 (0.02)      1.00 (0.02)
 18:2(n - 6)      2.40 (0.37)      2.26 (0.10)
 18:2(n - 4)      0.20 (0.12)      0.87 (0.04)
 20:2(n - 9)      0.30 (0.08)      0.21 (0.09)
 20:2(n - 6)      0.47 (0.03)      0.41 (0.01)
 16:3(n - 3)      1.59 (0.03)      0.00 (0.03)
 18:3(n - 6)      0.37 (0.17)      0.24 (0.01)
 18:3 **          0.20 (0.08)      0.31 (0.00)
 18:3(n - 3)      0.33 (0.16)      2.26 (0.34)
 20:3(n - 6)      0.18 (0.05)      0.27 (0.01)
 20:3(n - 3)               --      0.26 (0.00)
 22:3(n - 9)               --      1.26 (0.04)
 22:3(n - 6)               --      0.36 (0.05)
 16:4(n - 3)      2.47 (0.05)      1.54 (0.12)
 18:4(n - 3)      0.96 (0.18)      5.22 (0.54)
 18:4 **          0.29 (0.02)      0.43 (0.01)
 20:4(n - 6)      0.24 (0.01)      0.65 (0.06)
 20:4(n - 3)      0.32 (0.01)      1.23 (0.03)
 22:4(n - 6)               --      0.43 (0.31)
 20:5(n - 3)      7.07 (1.66)     14.39 (0.88)
 22:5(n - 6)               --      0.46 (0.01)
 22:5(n - 3)      0.17 (0.00)      0.71 (0.17)
 22:6(n - 3)      2.06 (0.11)      2.57 (0.21)
BFA
 14-iso           0.57 (0.10)      0.15 (0.02)
 14-antiiso       0.17 (0.03)      0.58 (0.66)
 15-iso           0.19 (0.06)      0.14 (0.02)
 16-iso           2.50 (0.36)      0.58 (0.02)
 16-antiiso       0.59 (0.04)      0.23 (0.01)
 17-iso            --              0.37 (0.02)
 Total           95.72 (0.27)     96.57 (2.33)
 [SIGMA] SFA     37.36 (2.88)     24.75 (1.36)
 [SIGMA] MUFA    31.20 (0.02)     31.48 (1.27)
 [SIGMA] PUFA    23.13 (3.05)     38.30 (0.42)

Fatty Acid       Crassostrea      Mytilus
                 gigas            galloprovincialis
SFA
 14:0             3.10 (0.49)      2.50 (0.09)
 15:0             0.44 (0.02)      0.45 (0.02)
 16:0            19.97 (1.24)     16.80 (0.56)
 17:0             1.13 (0.06)      0.55 (0.03)
 18:0             4.32 (0.02)      4.44 (0.19)
 20:0             0.70 (0.08)      3.48 (0.41)
 22:0             0.29 (0.04)              Tr
MUFA
 14:1(n - 5)               --               --
 16:1(n - 9)               --               --
 16:1(n - 7)      4.37 (0.72)      7.99 (0.34)
 16:1(n - 5)      0.19 (0.03)      0.15 (0.01)
 17:1(n - 9)      0.11 (0.00)      0.10 (0.01)
 18:1(n - 9)     11.55 (0.59)     12.95 (0.38)
 18:1(n - 7)      5.34 (0.11)      4.01 (0.13)
 18:1(n - 5)      0.35 (0.02)      0.23 (0.06)
 20:1(n - 9)      2.37 (0.18)      0.68 (0.10)
 20:1(n - 7)      1.65 (0.04)      0.75 (0.68)
 22:1(n - 11)     0.41 (0.03)      0.39 (0.06)
 22:1(n - 9)      1.10 (0.11)      0.44 (0.11)
PUFA
 16:2(n - 4)      0.41 (0.06)      0.75 (0.09)
 18:2(n - 6)      7.82 (0.59)      9.29 (0.16)
 18:2(n - 4)      0.19 (0.02)      0.24 (0.03)
 20:2(n - 9)      0.49 (0.01)      1.06 (0.04)
 20:2(n - 6)      0.10 (0.02)      0.34 (0.03)
 16:3(n - 3)      0.36 (0.02)      0.43 (0.10)
 18:3(n - 6)      0.32 (0.01)      0.25 (0.01)
 18:3 **          0.19 (0.01)      0.17 (0.05)
 18:3(n - 3)      2.20 (0.28)      2.38 (0.18)
 20:3(n - 6)      0.23 (0.01)      0.14 (0.01)
 20:3(n - 3)      0.12 (0.03)      0.19 (0.00)
 22:3(n - 9)      0.84 (0.02)      0.65 (0.05)
 22:3(n - 6)              tr                --
 16:4(n - 3)      0.13 (0.06)      0.46 (0.12)
 18:4(n - 3)      5.29 (0.26)      4.07 (0.20)
 18:4 **          0.19 (0.02)      0.30 (0.08)
 20:4(n - 6)      1.00 (0.05)      1.15 (0.12)
 20:4(n - 3)      0.57 (0.03)      0.52 (0.08)
 22:4(n - 6)      0.15 (0.01)      0.11 (0.00)
 20:5(n - 3)     11.52 (1.19)      9.43 (0.33)
 22:5(n - 6)               --               --
 22:5(n - 3)      0.54 (0.00)      0.48 (0.15)
 22:6(n - 3)      6.35 (0.38)      5.26 (0.21)
BFA
 14-iso           0.15 (0.01)      0.12 (0.01)
 14-antiiso                --               --
 15-iso           0.17 (0.01)      0.16 (0.01)
 16-iso           0.45 (0.03)      0.46 (0.05)
 16-antiiso       0.27 (0.01)      0.38 (0.08)
 17-iso           0.38 (0.01)      0.24 (0.00)
 Total           97.81 (0.11)     94.94 (0.58)
 [SIGMA] SFA     29.96 (1.60)     28.23 (0.61)
 [SIGMA] MUFA    27.43 (0.06)     27.69 (0.19)
 [SIGMA] PUFA    39.01 (1.73)     37.58 (0.06)

TABLE 2.

Fatty acid biomarkers used in the food sources determination
of bivalves from Jiaozhou Bay (Qingdao, China).

Biomarker                           Main Source

16:1(n - 7)/16:0>1                  Diatoms
EPA/DHA                             Diatoms
DHA                                 Flagellates
Odd & br FAs                        Bacteria
18:1 (n - 7)/18:1 (n - 9)           Bacteria
[SIGMA] 18:2(n - 6) + 18:3 (n - 3)  Chlorophyta/terrestrial plants

References: Budge & Parrish, 1998; Budge et al.,
2001; Dalsgaard et al., 2003.

TABLE 3.

Values of fatty acid biomarkers in three bivalves and net-towed sample
of Jiaozhou Bay (Qingdao, China). Different letters mean significant
difference was observed between two values (n = 3, P < 0.05).

Species
                            16:1/16:0        EPA/DHA

Net-towed phytoplankton *   1.23     (0.11)  3.42     (0.62)
Chlamys farreri             0.88 (a) (0.01)   5.64 (a)(0.79)
Crassostrea gigas           0.22 (b) (0.02)   1.81 (b)(0.08)
Mytilus galloprovincialis   0.48 (c) (0.03)   1.80 (b)(0.11)

Species
                            DHA              Odd & br FAs

Net-towed phytoplankton *   2.06     (0.11)   4.73     (0.60)
Chlamys farreri             2.57 (a) (0.21)   4.08 (a) (0.54)
Crassostrea gigas           6.35 (b) (0.38)   3.09 (b) (0.03)
Mytilus galloprovincialis   5.26 (c) (0.21)   2.45 (b) (0.15)

Species                     18:1 (n - 7)/    18:2 (n - 6) +
                            18:1(n - 9)      18:3 (n - 3)

Net-towed phytoplankton *   0.65     (0.05)   2.73      (0.54)
Chlamys farreri             l.44 (a) (0.04)   4.51  (a) (0.44)
Crassostrea gigas           0.46 (b) (0.01)   10.03 (b) (0.30)
Mytilus galloprovincialis   0.31 (c) (0.01)   11.67 (c) (0.33)

"*": Sample was collected in the scallop farming area.
It was not included in the one-way ANOVA with bivalves
for its different trophic level.

TABLE 4.

Values of stable carbon isotope ratio for intertidal bivalves
and potential food sources in Jiaozhou Bay, (Qingdao, China.).
Mean (SD), n = 3.

Sample                       [[delta].sup.13]C

Net-towed phytoplankton      -22.07
Ova pertusa                  -18.07 (0.34)
Undaria pinnatifida          -13.13 (0.22)
Crassostrea gigas            -21.52 (0.09)
Mytilus galloprovincialis    -21.38 (0.25)

TABLE 5.

Contributions of primary producers to bivalves' diets
in intertidal area of Jiaozhou Bay (Qingdao, China)
calculated by IsoSource[TM] software: % (range).

Source                 Crassostrea         Mytilus
                          gigas        galloprovincialis

Phytoplankton          89.0 (86-91)      86.2 (83-90)
Ulva pertasa            8.7 (5-14)        11.0 (4-17)
Undaria pinnatifida     2.3 (0-4)          2.8 (0-6)