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.