Aspartic acid

ABSTRACT Effects of salinity and turbidity stresses on free amino acid (FAA) composition in Crassostrea gigas was investigated using high performance liquid chromatography. Oysters were exposed for 2 to 5 days to low salinity (LS, 7 [per thousand]), normal (control, 30 [per thousand]), high salinity (HS, 39 [per thousand]), and high turbidity in normal salinity (HT, 30 [per thousand], 0.326 g kaolin clay/l). Levels of aspartic acid, glutamic acid, serine, glycine, [beta]-alanine, taurine, and L-alanine in the gill tissues were then monitored using a HPLC. Taurine and glycine were the two major contributors to the total FAA in gill tissues of oysters collected from fields, ranging from 200.7-324.7 and 42.9-69.0 [micro]mol/mg dry tissue. On day 2, total FAA in HS and HT was significantly higher than the levels in the control and LS: taurine and glycine levels in HS and HT were significantly higher than the levels of the controls and LS (P < 0.05). In contrast, most FAA levels in LS had dropped except for glutamic acid, glycine, and [beta]-alanine; [beta]-alanine level of oysters in LS on day 2 was about 10-fold higher than the control. Aspartic acid and Glutamic acid levels in HT on day 2 were significantly higher than the level observed in HS. LS and control (P < 0.0.5). From day 2 to 5, total FAA in the control increased 11.4% whereas total FAA in HS, LS, and HT decreased 17.3%, 21.6%. and 19.3% respectively. Aspartic acid and glutamic acid levels in the control also increased 29.1% and 50.6% between day 2 and 5 whereas the levels in HS, LS, and HT dropped dramatically, from 47.1 (HT) to 72.0% (LS) in case of aspartic acid and 53.5 (HS) to 68.5% (LS) in glutamic acid. Response of FAA composition in the gill tissues to salinity and turbidity stress was unique in this study, suggesting that FAA analysis is a useful tool to diagnose environmental stress to C. gigas, as was observed in C. virginica.

KEY WORDS: free amino acids. Crassostrea gigas, salinity, high turbidity, stress, HPLC, oyster

INTRODUCTION

Marine invertebrates contain very high levels of tree amino acids (FAA) in their tissues and fluids, being 100 to 1000 times higher than those found in the extra-cellular fluid (Gilles 1972; 1979, Bayne et al. 1979). The FAAs are involved in a wide variety of biochemical and physiologic processes, and they are often monitored as a stress indicator in marine invertebrates under natural or man-made disturbances (Lynch & Wood 1966; Briggs 1979; Powell el al. 1984; Burton 1992). Epidermal tissues of marine bivalves in particular contain high level of FAAs, and the level of FAAs in the tissues often indicates their physiologic and nutritional status of an animal (Feng et al. 1970; Biondi & Noviello 1978: Sakaguchi & Murata 1989; Paynter et al. 1995). It is also well known that intracellular FAA in bivalve tissues are used in cellular volume regulation. An increase or decrease in salinity often results in an increase or decrease of FAA level in the tissues (Lynch & Wood 1966; Baginski & Pierce 1977: Shumway & Gabbott, 1977; Powell et al. 1982; Ellis et al. 1985).

C. gigas is an estuarine bivalve inhabiting intertidal to shallow subtidal areas in Korea, Japan, and China where they are commercially raised. The oysters are particularly abundant in coastal estuarine areas where fluctuations of temperature and salinity, as well as man-made pollution, often exert significant physiologic impacts (Feng et al. 1970; Livingston 1985; Tirard et al. 1997). C. gigas tissues are enriched with amino acids, and the levels of free amino acids in the tissues often reflect environmental changes as well as seasonal variations (Sakaguchi & Murata 1989). Although there are many reports on the effects of environmental changes on C. gigas physiology as measured with various biochemical assays, studies on the effects of environmental changes on the FAA pool are rare despite the commercial importance of this species. This study attempts to evaluate the effects of salinity and turbidity on the FAA pool of Pacific oysters.

MATERIALS AND METHODS

Oysters used in the experiment were collected from Kamakman Bay on the south coast of Korea, where the oysters are commercially cultured using the hanging culture system. Salinity in this bay fluctuates between 28 [per thousand] and 33 [per thousand], and the visibility of the water varies from 1.5-3 m annually (Hyun et al. 2001). Oysters of 8-12 cm long and 10-15 g wet tissue weight were collected from oyster grow-out hanging lines placed at a depth between 1 and 4 m below the surface. The gill tissues of 13 oysters were excised in situ and frozen immediately using dry ice and subjected to FAA analysis. The amino acid composition of the control oysters collected from Kamakman Bay were compared with that of other oysters collected from an intertidal area at Daechon on the west coast of Korea where salinity varies 25 [per thousand] to 35 [per thousand] annually.

For the experiment, oysters were placed in static 50-L glass tanks filled with normal salinity seawater (30 [per thousand]). After a 2-day acclimation period, the following experimental conditions were applied; (1) control at 30 [per thousand]; (2) high salinity stress at 39 [per thousand] (HS); (3) low salinity stress at 7 [per thousand] (LS); and (4) high turbidity (HT) stress created with kaolin clay with a constant concentration of 0.323 gm/l in 30 [per thousand] seawater according to Powell et al. (1982). A set of 20 oysters was placed in each 50-L glass tank, and the water was continuously stirred and aerated during the experimental period. Seawater in the tanks underwent a 100% water change twice daily. To maintain constant high turbidity in the tank, the kaolin concentration was checked four times a day and a proportional amount of clay was added if necessary.

On days 2 and 5, seven oysters were collected from each experimental tank to measure FAA in their gill tissues. Oysters were opened and gill tissues removed, weighed, and stored at -70[degrees]C until analyzed. Dry weight of the gill tissue was estimated from the empirical relation; dry weight = wet weight of gill tissue x 0.196 (Choi et al. 1993). The oyster gill tissue was homogenized in phosphate buffer (pH 7.3) using a mortar and ultrasonifier at 4 []. The homogenate was centrifuged and the supernatant collected for analysis. The supernatant was then treated with trichloric acid (TCA), from 10%-50%, to precipitate proteins and nucleic acids (Powell et al. 1982). TCA treated samples were centrifuged to remove protein and the supernatant containing FAA was harvested. TCA in the supernatant was then removed using six applications of ether. Types of FAA in the samples were determined with HPLC using methanol-acetate as a solvent. Orthophthaldialdehyde (OPA) was used as the detecting compound for identification and quantification of FAA. Aspartic acid, glutamic acid, serine, [beta]-alanine, L-alanine, glycine, and taurine were analyzed from the FAA pool in the oyster gill tissues and finally expressed as [micro]mol FAA/g dry tissue weight.

RESULTS

FAA Composition of Oysters Collected in Situ

Amino acid composition in gill tissues of control oysters is summarized in Table 1. Among the seven amino acids analyzed, taurine and glycine were the most abundant amino acids in the gill tissues sampled from the Daechon and Kamakman Bay. These two amino acids accounted for more than 70%-80% of the free amino acids measured in the oysters.

Changes in FAA Composition in Oysters in Control Tank

Table 2 summarizes changes in the FAA composition of oysters held in the control tank (30 [per thousand]) for 2 and 5 days. As observed in wild oysters, taurine and glycine were the most abundant compounds that accounted for more than 82% of total FAAs in oysters in the control tank for 2 days. In addition to taurine and glycine, L-alanine, aspartic acid, and glutamic acid were present in the oyster tissues at levels of 20.14, 16.72, and 14.23 [micro]mol/g dry tissue weight respectively. In contrast, serine and [beta]-alanine levels in the control oysters were <10 [micro]mol/g dry tissue. After 5 days in the control tank, glutamic acid levels increased by 50.6% relative to the level at day 2. In contrast, 13-alanine and L-alanine levels at day 5 dropped by 31.1% and 32.5%.

FAA Changes in HS Tank

All FAA levels in oysters in the increased salinity at day 5 were lower than the FAA levels in oysters at day 2, except serine. Aspartic acid, glutamic acid, glycine, and [beta]-alanine levels dropped by -54.1%, -53.5%, -55.8% and -36.1% respectively. Contrary to other FAAs, taurine levels between day 2 and day 5 remained largely unchanged (see Table 2). Due to the taurine, total FAAs had only dropped -17.3% between day 2 and day 5 in the high salinity tank.

Compared with the control oysters, total FAA levels in oysters held in HS tank for two days were found to be 1.61 fold elevated (Table 3). Particularly, the glycine level in the oysters at Day 2 was much higher, by a factor of 2.90, than the level in oysters held in the control tank (30 [per thousand]) at day 2. It was also noticed that the [beta]-alanine level in HS at day 2 was 3.05-fold higher than the level in oysters held in the control. The taurine level of oysters in the tank was also 1.47-fold higher than the level of control oysters. In contrast, the serine level remained largely unchanged at day 2. By day 5, aspartic acid and glutamic acid levels had dropped by a factor of 0.60 relative to the control oysters. However, the [beta]-alanine level of the oysters in the LS tank was much higher, a factor of 2.83, than the level in the control salinity tank. Serine, glycine, and L-alanine levels were also higher than the control oysters at day 5, by a factor of 1.70, 1.53, and 1.63 (see Table 3).

FAA Changes in LS Tank

Between day 2 and day 5 in the LS tank, all FAA levels in the oysters had dropped. Aspartic acid, glutamic acid, glycine, and [beta]-alanine levels fell by 72.0%, 68.6%, 34.6%, and 31.9%. In contrast, taurine, that accounts for more than 70% of the total FAA in the oysters, decreased by only 8.8% (see Table 2).

After 2 days in LS (7 [per thousand]), the taurine level dropped by a factor of 0.68 compared with the level of the control oysters (see Table 3). Aspartic acid and L-alanine levels in the oysters also decreased by factors of 0.59 and 0.46 relative to the control oysters at day 2. However, the glutamic acid and [beta]-alanine levels were elevated by a factor of 1.85 and 10.36 respectively. At day 5, the total FAA level dropped by a factor of 43% compared with the control oysters at the same time period. Aspartic acid and glutamic acid levels dropped by a factor of 0.13 and 0.39 relative to the control oysters. In contrast, the [beta]-alanine level in oysters at day 5 remained elevated as observed at day 2, 10.23-fold higher than the [beta]-alanine level in the contrail oysters (see Table 3).

FAA Changes in HT Tank

Between day 2 and day 5, most FAA levels in the oysters dropped more than 30%. Aspartic acid, glutamic acid, glycine, and [beta]-alanine levels dropped by 47.1%, 64.1%, 36.6%, and 30.1% respectively. However, the serine level had risen by 55.9% after 3 days in HT, between days 2 and 5 (see Table 2).

Oysters incubated in the HT tank for 2 days showed a very high level of taurine compared with the control oysters. Taurine level of the oysters was 1.85-fold higher than the level of the control oysters. Other FAA levels were also elevated by more than 30% for 2 days (see Table 3). Aspartic acid and glutamic acid levels also increased by a factor of 2.40 and 2.78 relative to the control oysters. Total FAAs of the oysters were also 1.81-fold higher than the total FAAs of the control oysters at day 2. At day 5, total FAA level in the high turbidity tank remained elevated by a factor of 1.31. The taurine level of the oysters at day 5 was 1.38-fold higher than the taurine level of the control oysters. In contrast, glutamic acid levels in the oysters at day 5 were lower than those of the control oysters by a factor of 0.66 for the same length of time.

DISCUSSION

FAA Composition in Field Oysters

Table 1 indicates that taurine is the most abundant FAA in the control oysters collected from Kamakman Bay, followed by glycine. Taurine and glycine account for 52% and 18% of total FAAs respectively in oysters collected from Daechon. Sakaguchi and Murata (1989) also reported that taurine and glycine are the most abundant FAAs in cultured C. gigas. High levels of taurine in the FAA pool also have been reported from various marine bivalves; it is believed that most marine bivalves use taurine in osmotic volume regulation (Sansone et al. 1978; Powell et al. 1982; Lynch & Wood 1966; Feng et al. 1970). Taurine levels in oysters collected from Kamakman Bay were much higher than the levels observed in Daechon oysters, whereas the glycine level of Kamakman Bay oysters was lower than those from Daechon. Taurine comprises 70% of the total FAA, whereas glycine accounts for only 9% in Kamakman oysters.

Different FAA levels observed between Kamakman Bay and Daechon oysters could be associated with different levels of tolerance to stress and anoxia during transportation to the laboratory. Daechon oysters were collected from the intertidal zone where the oysters are exposed to the atmosphere twice a day during low tide. In contrast, Kamakman Bay oysters were collected from an oyster farm where oysters were hanged on suspended rope and submerged from 2-5 m below the surface. It is likely that the intertidal oysters from Daechon are better adapted to anoxic condition formed during transportation by closing their valves. Several studies have reported that marine bivalves often exhibit elevated levels of taurine due to an intracellular response to change in extracellular fluid osmolarity (Pierce 1971; Powell et al. 1982; Paynter et al. 1995).

Effects of Changes in Environmental Factors on FAA Composition of Oysters

Man-made disturbances such as high concentrations of heavy metals, hydrocarbons, and drilling effluents, as well as natural stresses, such as starvation, decrease or increase in salinity, and parasitism, often alter the composition of FAAs in marine animals (Jeffries 1972; Pecon & Powell 1981; Roesijadi & Anderson 1979: Widows et al. 1982; Bayne et al. 1976; Kendall el al. 1985: Paynter et al. 1995) as well as the protein synthesis rate (Choi et al. 1994: Tirard et al. 1997). However, the response of the animals in terms of internal FAA change varies with different environmental or natural stresses. Hyper-osmotic, hypo-osmotic, and high turbidity stresses were applied to C. gigas in the present study. Oysters exposed to high salinity and high turbidity stress showed a remarkable increase in total FAA for the first 2 days (see Tables 2 and 3).

Other studies have also reported elevated FAA levels in marine bivalves exposed to hyper-osmotic conditions for a short period (Powell et al. 1982; Lynch & Wood 1966). C. virginica exposed to high salinity (38 [per thousand]) for 2 days showed elevated total FAAs. Glycine, alanine, and [beta]-alanine levels were elevated compared with the control oysters (Powell et al. 1982). C. gigas in this study also showed a remarkable increase in glycine, L-alanine, and [beta]-alanine levels when treated with high salinity stress. Baginski and Pierce (1977) also observed elevated alanine and glycine levels in Modiolus demissus (= Geukensia demissa) 1 to 3 days after holding the mussels in a hyper-osmotic environment. Accumulation of alanine and glycine during hyper-osmotic stress also has been reported for the mussels, Mytilus edulis and Geukensia demissa (Deaton et al. 1985, Deaton 2001). The increased FAA levels observed in the oysters held in the high salinity tank for the first 2 days are due to volume regulation by the cells of the oysters as reported in other marine bivalves (Deaton et al. 1985).

Oysters exposed to high turbidity water for 2 days also showed a remarkable increase in the FAA levels. Compared with the control oysters, all levels of FAAs in the gill tissue of those oysters were elevated for the 2 days of immersion in highly turbid water. However, the pattern of elevation in FAA is somewhat different between oysters treated with high salinity and high turbidity stress. Taurine, aspartic acid, and glutamic acid levels in the oysters in the high turbidity tank for 2 days were remarkably elevated by a factor of 1.85-2.78 relative to the FAA level in control oysters for the same length of time (see Table 3). In contrast, glycine and [beta]-alanine levels in oysters held in the high turbidity tank for 2 days increased by only a factor of 1,30 and 1.61 respectively. As shown in Tables 2 and 3, the taurine level in oysters held in the high salinity tank for 2 days increased only a factor of 1.47, whereas glycine and [beta]-alanine levels in oysters held in this tank for the same length of time were 2.90- and 3.05-fold higher than the levels in control oysters.

Powell et al. (1982) also observed elevated FAA levels in C. virginica held for 2 days in a high turbidity tank containing kaolin clay as turbid particles. Particularly, the oysters held in the kaolin-clay tank showed a remarkable increase in taurine and cystic acid levels. One possible explanation for the observed increase in taurine level in the oysters in this study would be the elevated mucus secretion activities. The oysters in the high turbidity tank would secrete more mucus to remove the kaolin clay particles captured in the gills. According to Powell et al. (1982), mucus of marine invertebrates, including oysters, contains high level of sulfate anion. An increase in mucus secretion in oysters may result in cystic acid levels in the FAA pool because an increase in sulfate anion level in the mucus also elevates a precursor compound for cystic acid. Powell et al. (1982) also observed that an increase in cystic acid levels accompanies an increase of taurine levels in their study. Thus, we postulate that, in this study, the observed increased taurine levels in the oysters in the high turbidity tank on day 2 was influenced by elevated cystic acid levels in the mucus, although cystic acid was not analyzed in this study.

Oysters in hypo-osmotic conditions for 2 days exhibited slightly decreased total FAA levels in the gill tissues. As shown in Table 2, aspartic acid, taurine, and L-alanine levels in the oyster gill tissues dropped more than 30% compared with the level of control oysters. In contrast, glutamic acid and [beta]-alanine levels in oysters in low salinity conditions were much higher than the levels observed among control oysters. Especially, the [beta]-alanine level in the oysters was 10 times higher than the level in the control oysters. and 3-5 times higher than the level in oysters held in the high salinity or high turbidity tanks. Although the [beta]-alanine levels showed dramatic changes after 2 days in the tank, the role of [beta]-alanine in the FAA pool's response to osmotic stress seems to be minor; [beta]-alanine accounts only for 8% of the total FAA. Elevation of [beta]-alanine levels was also observed in C. virginica held in increased salinity, anoxia, drilling effluent front oil drilling operations, and high turbidity treatments for 5 days (Powell et al. 1982). An increase in [beta]-alanine levels was also reported from sea anemones raised in a laboratory and in the field (Low et al. 1980). It is likely that the elevation of [beta]-alanine observed in C. gigas is a unique physiologic response to stress, although the mechanism of [beta]-alanine elevation in the FAA pool of either C. virginica or C. gigas is not fully understood.

FAA levels in oysters in the experimental tanks at day 5 were considerably decreased compared with either the day 5 controls or the day 2 treatment groups (see Table 2). Decreases in FAA between days 2 and 5 were very conspicuous in the oysters held in the low salinity tank. All amino acids analyzed in the oysters exhibited an 8.8%-72% decrease in FAA between days 2 and 5. Specifically the levels of glutamic acid declined dramatically between those two days. Those levels dropped 53.5%, 68.5%, and 64.1% respectively in the high salinity, low salinity, and high turbidity tanks. However, it is unlikely that the dramatic changes in glutamic acid observed in this study exert a great influence on osmotic balance of the oysters since glutamic acid is a minor constituent of the FAA pool. Glutamic acid accounts for only 2.8%-5.5% of the total FAA at day 5. In contrast, taurine, which comprises from 62.6%-82.0% of the total FAA pool in the oysters at day 5, remained less changed compared with other amino acids.

A considerable decrease in FAA was also observed in C. virginica held in drilling effluent, anoxia, and high turbidity tanks between days 2 and 5; the oysters exhibited a remarkable decrease in FAA, including taurine, glycine, and alanine (Powell et al. 1982). Particularly, the taurine level in the oysters dropped as much as 58% in the anoxia tank between days 2 and 5. Powell et al. (1982) postulated that such a considerable drop in amino acid levels in C. virginica could be attributed to a general loss of FAA front the gills. This was because of some changes in the tissue's ability to prevent FAA loss across the outer membrane of the cells in the gill tissues, as indicated by another study (Bishop et al. 1994). Although the extent of decrease in the taurine level in C. gigas was not as great in as C. virginica, the oysters held in the kaolin turbidity tank, in this study, also showed a remarkable decrease in the taurine level between days 2 and 5. It is believed that C. gigas, in the kaolin tank, also loses a large amount of taurine through the gills, as observed in C. virginica.

ACKNOWLEDGMENTS

We are grateful to E. N. Powell of Haskin Shellfish Laboratory of Rutgers University and Charles Bai of Pukyung National University for reviewing and commenting on the manuscript. This work was supported at the regional research center for coastal environmental of Yellow Sea at Inha University designated by Ministry of Science and Technology (MOST) of Korea and Korea Science and Engineering Foundation (KOSEF).

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NAM-HYUN LEE, (1) KYUNG-NAM HAN (1) AND KWANG-SIK CHOI (2), * (1) Department of Oceanography, College of Natural Science, In-Ha University 253 Yonghyun-Dong, Nam-Gu, Inchon 402-751, KOREA; (2) School of Applied Marine Science, College of Ocean Science, Cheju National University 1 Ara 1-Dong, Jeju, 690-756 Korea

* Corresponding author. E mail: skchoi@cheju.ac.kr

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