Fisheries Science 64(4), 606-611 (1998)

Distribution of Free D-Amino Acids in Bivalve Mollusks and the Effects of Physiological Conditions on the Levels of D and L-Alanine inthe Tissues of the Hard Clam, Meretrix lusoria

miko Okuma, *1 Katsuko Watanabe, *2 and Hiroki Abe*2,t

*1 Department of Hematology, Research Institute, International Medical Center of Japan, Toyama,Shinjuku, Tokyo 162-8655, Japan *2Laboratory of Marine Biochemistry , Graduate School of Agricultural Life Science, The niversity of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan Received December 26, 1997)

The distribution of D-amino acids was examined on the tissues of seven species of marine bivalve mollusks belonging to two different subclasses, Pterimorphia and Heterodonta. High concentration of D-alanine was found in all tissues of only Heterodonta and not in the tissues of Pterimorphia except in gills and midgut gland. Several other D-amino acids including D-arginine also occurred in the several tis sues of both groups of bivalves. Along with the salinity stress on the hard clamMeretrix lusoria in 150% seawater, only D and L alanine increased in adductor muscle, gills, and midgut gland. The incorporation of D-alanine from ex ternal seawater was found onlyin gills, hemolymph, and midgut gland of hard clam, and the levels of D and L-alanine inadductor and foot muscles decreased in the seawater containing 50 HIMD-alanine and returned to the control level after recovery innormal seawater. During the starvation of hard clam for 22 days,D and L-alanine as well as other free amino acids decreased considerably in most tissues. The anoxia of hard clam for a week duration gave no large effect on the levels of free amino acids but resulted in the slight increases of D and L-alanine levels after sevendays. Key words: D-amino acid, D-alanine, free amino acid, distribution, bivalves, mollusk, Meretrix lusoria, osmoregulation

Several bivalve mollusks have been reported to contain in the muscle of crayfish ƒÏrocambarus clarkii during sea free D-alanine or D-aspartate in their tissues.1-8) Even the water acclimation 17) must be produced in muscle by the ac distribution of these D-amino acids, however, is still con tion of alanine racemase.15) In turn, we sought to learn troversial on various molluscan species. It is not clear whether this mechanism is also working in the other whether the distribution of D-amino acids depends on the animals. phylogenetic positions or ecological environments. We re In the present study, we examined the distribution of D cently established a sensitive HPLC method to determine amino acids in the tissues of bivalve mollusks belonging to all free D and L-amino acids simultaneously using (+)-1 two different subclasses. We also examined the changes of (9-fluorenyl)ethyl chloroformate (FLEC) as a pre-derivatiz D and L-alanine levels as well as other free amino acids ing reagent) and could detect several nano moles ofD-ami along with the changes of physiological conditions of hard no acids in the tissues. By using this method, we found a clam Meretrix lusoria, including hyperosmotic stress, star large amount of D-alanine aswell as small amounts of vation, anoxic stress, and rearing in the D-alanine added several other D-amino acids in the crustacean nervous tis seawater. sues,9) muscles, and hepatopancreas.10) The origin of free D-amino acids in mollusks has also Materials and Methods been controversial. Several researchers have found alanine racemase [EC 5.1.1.1] activity in molluscan tissues.8,12-14 Animals The exogenous D-amino acids are, however, also postulat Live specimens of marine bivalve mollusks were pur ed to be incorporated from external media8,12-14) or from chased at a local fish market in Tokyo. Seven species be symbiotic bacteria.',') In this respect, we found strong ac longing to two different phylogenetic subclasses used in tivity of alanine racemase in the muscle and hepatopan this experimentwere as follows: subclass Pterimorphia, creas of several crustaceans") and partially purified the en order Arcoida; ark shell Scapharca broughtonii, orde zyme from the muscle of black tiger prawnƒÏenaeus Pterioida; scallop Patinopecten yessoensis, order Ostrei monodon.16) Thus, D-alanine accumulated in large amount da; oysterCrassostrea gigas, and subclass Heterodonta,

•õ To whom correspondence should be addressed D-Amino Acids in Bivalve Mollusks 607 order Veneroida; hard clam Meretrix lusoria, short-neck ously9) using FLEC as a pre-derivatizing reagent. Briefly, ed clam Ruditapes philippinarum, Sakhalin surf-clam after derivatization of D andL-amino acids in the tissue ex Pseudocardium sachalinensis,and otter shell Tresus kee tract with FLEC in borate buffer and acetonitril, 19 amin nae. acids were separated into their D and L-enantiomers and from other 14 physiological amino compounds by Rearing of Hard Clam and the Changes of Physiological reversed-phase ion-pair HPLC, and monitored fluorimetri Conditions cally. Hard clam was reared in a laboratory aquarium sup plied with aerated circulating seawater prepared with an ar Results tificial seawater salt mixture. Water temperature was con trolled at 14-16•Ž in all experiments. Five individuals Distribution of D-Amino Acids in the Tissues ofBivalve were used for each experimental group written below. Mollusks When the hard clam received hyperosmoticstress, salinity Results are shown in Table 1 for Pterimorphia and of the rearing water was increased gradually to 150% sea Table 2 for Heterodonta. Several D-amino acids were water level with the salt mixture taking one week. Starva found in all tissues of mollusks examined. D-Alanine, D-ar tion of the hard clam was performed for 22 days in the ginine, and D-aspartate were the most widely distributed D aquarium with full-seawater. amino acids. Only two other D-amino acids, D-proline and Incorporation of D-alanine from seawater was D-asparagine, were detected in the several tissues of Heter confirmed as follows. The hard clam reared in seawater odonta and Pterimorphia, respectively. A small amount of for three days were transferred to an aquarium filled with D-arginine occurred in all tissues examined, irrespective of 101 of seawater containing50 MM D-alanine. After three species. The amount, however, ranged only from 0.05 to days, the animals were transferred again to the aquarium 2.3 ƒÊmol/g wet weightand the ratio of D-arginine to total filled only with the normal seawater and kept for three arginine was below 10% in most tissues. This ratio of D-ar days. In the case of giving anoxic stress, the shell of each ginine was rather high in midgut gland and gills of almost clam was tightly tied with a thread and left to stand inthe all species. The distribution of the other D-amino acids ex air at 5•Ž for a week. The shell was covered with moist cept for D-alanine was very limited in amount and inthe tis paper towels to avoid drying. sues of species, though the ratios of D-enantiomer to total D+L enantiomers were extremely high in some tissues as Preparation of Tissue Extracts seen in D-proline of hard clam tissues. Each tissue was dissected from three to 38 individuals of The most striking feature was seen in the distribution of each species or five individualsof hard clam and mixed D-alanine. D-Alanine was found in large amount in all tis well. From one gram portion of the tissues, perchloric acid sues of mollusks belonging to subclass Heterodonta, but extract was prepared according to the previous method.) almost not at all in those of Pterimorphia, though a small No extraction was performed on each individual in order amount was detected in the gills and midgut gland. In con to conserve costly pre-labeling reagent. trast, Heterodonta contained a much higher amount of D alanine in the muscle tissues than in the gills and midgut Analytical Method gland. The percentage ratio of D-alanine to total alanine Precolumn derivatization and HPLC determinationof was extremely high in these muscle tissues, ranging from D and L-amino acids were performed as described previ 35 to 84%.

Table 1. Distribution of free D-amino acids in the tissues of bivalve mollusks belonging to the subclass Pterimorphia

Values are expressed in ƒÊmol/g wet weight. Percentage of o/(o+L) are shown in parentheses.-; not detected. 608 Okuma et al.

Table 2. Distribution of free D-amino acids in the tissues of bivalve mollusks belonging to the subclass Heterodonta

Values are expressed in ƒÊmol/g wet weight. Percentage of D/(D+L) are shown in parentheses.-; not detected.

Effects of Hyperosmotic Stress tepwise up to five-fold of the control level. After acclimation of hard clam to high salinityseawater The highest increase of D-alanine was seen in gills which of 150%, total free amino acids increased slightly in adduc incorporated a large amount of D-alanine from seawater. tor muscle as seen in Fig. 1. In the gills and midgut gland, The amount reached 17-fold of the original level and the however, total free amino acids declined after hyperosmot D-alanineratio to total alanine also reached 85%. This lev ic stress. In adductor muscle, the increased amino acids in el declined considerably after recovery in the normal sea high salinity seawaterwere almost confined to D and L-ala water. Hemolymph originally contained only small nineand L-arginine. The concentration of the other vari amounts of freeamino acids incorporated a large amount ous amino acids were not changed or slightly reduced in of D-alanine from external water and released it after recov 150% seawater. In the other tissues, D and L-alanine also ery. There was almost no change in the amount of the increased largely but taurine and the other 15 amino acids other free amino acids in alltissues of hard clam. decreased along with the hyperosmotic stress. Effects of Starvation Incorporation of D-Alanine from External Seawater Figure 3 shows the effects of starvation for 22days on Hard clam was reared in seawater containing 50 MM D the concentrations of free amino acids in the several tis alanine for three days and returned to normal seawater sues of hard clam. The body weight of the clam decreased (Fig. 2). In adductor and foot muscles, D and L-alanine lar by only 1 % after starvation. As seen in the figure, total gely decreased in D-alanine added seawater and restored free amino acids declined in all tissues after starvation. the control level after recovery in the normal seawater. Almost all free amino acids decreased during starvation The ratio of D-alanine to total alanine was not changed but the most largest decrease was seen in taurine, irrespec throughout the experimental period, ranging from 61.5 to tive of the tissues. The concentrationsof D and L-alanine 65.5%. This decrease of D and L-alanine was responsible also decreasedduring starvation in many tissues except for for the decrease of total free amino acids in D-alanine midgut gland and gonad in which bothD and L-alanine in added seawater. In mantle and siphon, on the other hand, creased slightly. Other than taurine and alanine, the only D-alanine was elevated in D-alanine added seawater decrease of glycine and L-glutamate was rather large com and even after recovery in the normal seawater. This in pared with the other amino acids existing insmall quanti crease was larger in siphon inwhich D-alanine was almost ties in the clam tissues. doubled in D-alanine added seawater. This was also the case for midgut gland in which case D-alanine increased D-Amino Acids in Bivalve Mollusks 609

Fig. 1. Effects of high salinity stress on the levels of v and L-amino acids and other free amino acids in the tissues of hard clam. Salinity was gradually increased from 100% seawater (SW) to 150% seawater level taking six days. After three days acclimation in the high salin ity seawater, the animals were used for the experiment.

Fig. 2. Incorporation of D-alanine from external seawater into the tissues of hard clam. The animals were transferred from seawater (SW) to 50 mM n-alanine added seawater (+n) and kept for three days. They were then returned to seawater tank.

Effects of Anoxic Stress During anoxia of hard clam for seven days duration Discussion (Fig. 4), no large change of free amino acids was found in all tissues, though total amino acids were reduced at the We could find several D-amino acids in the various tis third day of anoxia. The levels of D and L-alanine, sues of bivalves except for oyster which contained only a however, almost unchanged at the third day but increased small amount of D-arginine. High concentration of D-ala at the seventh day of anoxia in all tissues examined. This nine wasfound in all tissues of Heterodonta as in those of increase was larger for L-alanine than D-form and there crustaceans.10) In addition to the concentration difference, fore the ratio of D-alanine to total alanine decreased from there exists very different features between D-alanine and 50.8% for the zero and three-day's specimen to 45.5% for the other D-amino acids. Bivalves belonging to Pterimor seven-day's specimen. phia did not contain any substantial amount of D-alanine in the tissues except for gills and midgut gland, indicating 610 Okuma et a!.

Fig. 3. Changes in the levels of D and L-alanine as well as other free amino acids in the tissues of hard clam during starvation. The well-fed animals were starved for 22 days in seawater tank without any feed.

Fig. 4. Effects of anoxic stress on the levels of n and L-alanine as well as other free amino acids in the tissues of hard clam. The shell of the animal was tied with a thread and left to stand in the air at 5•Ž for a week.

that the occurrence of D-alanine depends on the to total alanine was 30-60% in crustacean muscle.10)This phylogenetic situation and D-alanine in Heterodonta may ratio is rather higher in some muscle tissues of bivalves as take an important role in muscle tissues. No D-alanine has seen in the mantle and siphon of otter shell but in these been found in the several other species belonging to animals the equilibrium constant of D and L interconver Pterimorphia.2,7,8) Yamada and Matsushima') described sion reaction catalyzed by alanine racemase may be favora that they could not find any phylogenetic relationship for ble for L to D direction,if the enzyme works in the clam tis the occurrence ofD-alanine in 18 species of mollusks be sues. longingto 12 families. Our data show that thecomparison It is also an interesting feature that, except for D-ala should be done among much larger groups of phylogeny in nine, other D-amino acids occurred in high percentage of mollusks. Therefore, it is necessary to determine the occur D/(D + L) in gills and midgut gland. This suggests that D rence of D-alanine in a wide spectrum of molluscan spe amino acids other than D-alanine in the muscle tissues are cies. incorporated exogenously. In fact, D-alanine was incorpo A large amount of D-alanine in the muscle tissues of Het rated into gills, hemolymph, and midgut gland of hard erodonta suggests that it is synthesized in muscle probably clam (Fig. 2) but not intothe muscle tissues. It means that by alanine racemase. In this respect, the ratio of D-alanine D-alanineincorporated from gills is not transported to the D-Amino Acids in Bivalve Mollusks 611 adductor and foot muscles. Thus, these data also verify 4) O. Matsushima, H. Katayama, K. Yamada, and Y. Kado: Occur that D-alanine in the muscle tissues is produced in situ. rence of free D-alanine and alanine racemase activity in bivalve mol During the acclimation of hard clam to high salinity sea luscs with special reference to intracellular osmoregulation. Mar. water (Fig. 1), only D and L-alanine out of various physio Biol. Let., 5, 217-225 (1984). 5) O. Matsushima and Y. S. Hayashi: Metabolism of D and L-alanine logical amino acids increased considerably in the tissues and regulation of intracellular free amino acid levels during salinity including gills and midgut gland. Thus, D-alanine is synthe stress in a brackish-water bivalve Corbicula japonica. Comp. sizedeven in gills and midgut gland and is a major osmo Biochem. Physiol., 102A, 465-471 (1992). effector together with L-alanine for the tissue iso-osmotic 6) O. Matsushima and A. Yamada: Uptake of L and D-alanine by a regulation under hyperosmotic stress. brackish-water bivalve, Corbicula japonica, with special reference From the starvation experiments of hard clam (Fig. 3), to their transport pathways and the salinity effect. J. Exp. Zool., D-alanineas well as L-alanine is confirmed to be served as 263, 8-17 (1992). 7) R. L. Preston: Occurrence of D-amino acids in higher organisms: A an energy source as described earlier by Preston.7) Thus, D survey of the distribution of D-amino acids in marine invertebrates. alanine incorporated into gills, hemolymph, and midgut Comp. Biochem. Physiol., 87B, 55-62 (1987). gland of hard clam from external water (Fig. 2)may be uti 8) A. Yamada and O. Matsushima: The relation ofD-alanine and ala lized as a nutritional source. nine racemase activity in molluscs. Comp. Biochem. Physiol., Anoxic stress within seven days produced no large 103B, 617-621 (1992). change of the levels of free amino acids and n and L-ala 9) E. Okuma and H. Abe: Simultaneous determination of D and L amino acids in the nervous tissues of crustaceans using precolumn nine as seen in Fig. 4. At the seventh day of anoxia, derivatization with (+)-l-(9-fluorenyl)ethyl chloroformate and however, D and L-alaninewere elevated in all tissues. reversed-phase ion-pair high-performance liquid chromatography. Anaerobic end products in bivalve mollusks are well J. Chromatogr. B, 660, 243-250 (1994). known to be L-alanine, alanopine, and succinate or 10) E. Okuma, E. Fujita, H. Amano, H. Noda, andH. Abe: Distribu propionate.18) D-Alanine may be another good candidate tion of free D-amino acids in the tissues of crustaceans. Fisheries of the end products in bivalve mollusks during anaerobio Sci.,61, 157-160 (1995). sis. It may also be an interesting problem to be examined 11) W. P. Low, W. T. Ong, and Y. K. Ip: Different physiological func tions of free D and L-alanine in three body parts of the intertidal in the future. sipunculid Phascolosoma arcuatum. J. Comp. Physiol. B, 165, Our preliminary examination revealed that alanine 558-564,(1996). racemase activity was found in the footmuscle of otter 12) J. L. Bada: Racemization of amino acids in nature. Interdisciplina shell and hard clam, thoughthe activity was below 1 / 10 of ry Sci. Rev., 7, 30-46 (1982). that in crustacean muscle and hepatopancreas.15) Alanine 13) J. L. Bada and C. Lee: Decomposition and alteration of organic racemase has also been found recently in sipunculid Phas compounds dissolved in sea water. Marine Chem., 5, 527-534 colosoma arcuatum11) other than mollusks and crustace (1977). ans, suggesting the wide distribution of the enzyme in in 14) C. Lee and J. L. Bada: Dissolved amino acids in the equatorial Pacific, the Sargasso Sea and Byscayne Bay. Limnol. Oceanogr., vertebrates. 22, 502-510 (1977). 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