Tissues Of Marine Invertebrates Research Articles

Employing marine invertebrate cell culture media for isolation and cultivation of thraustochytrids

Abstract Thraustochytrids, a common group of marine eukaryotic protists, have drawn considerable scientific and industrial interest due to their ability to synthesize high levels of bioactive compounds, including polyunsaturated fatty acids, docosahexaenoic acid, squalene and carotenoids, and their new applications for biofuels. The pharmaceutical and industrial potential of thraustochytrids necessitate effective isolation of new strains and establishment of axenic cultures. To date, existing isolation protocols have used baiting and direct plating methods to generate axenic cultures with varied media compositions that contain peptone and yeast extracts as nitrogen sources, glucose as carbon source, seawater and antibiotics. Here we reveal a new approach for the isolation of thraustochytrids from tissues of marine invertebrates using (a) primary cell cultures in a liquid medium containing basal medium, 50% artificial seawater, vitamins, proteins and antibiotics, and (b) cultivation in the same cell culture medium. Using the colonial tunicate Botryllus schlosseri as a model system, thraustochytrid cells thrived in the medium from the day of extraction, grew and proliferated for the next five weeks (five-passages, up to 1.9 × 106 cells ml−1 in passage 5; 1.45-fold multiplication week−1). This new approach for isolation and cultivation of axenic thraustochytrid cultures enables the isolation of new species with promising bioactive compounds.

Kinetic Studies of Antioxidant Properties of Ovothiol A.

Ovothiol A (OSH) is one of the strongest natural antioxidants. So far, its presence was found in tissues of marine invertebrates, algae and fish. Due to very low pKa value of the SH group, under physiological conditions, this compound is almost entirely present in chemically active thiolate form and reacts with ROS and radicals significantly faster than other natural thiols. In biological systems, OSH acts in tandem with glutathione GSH, with OSH neutralizing oxidants and GSH maintaining ovothiol in the reduced state. In the present work, we report the rate constants of OSH oxidation by H2O2 and of reduction of oxidized ovothiol OSSO by GSH and we estimate the Arrhenius parameters for these rate constants. The absorption spectra of reaction intermediates, adduct OSSG and sulfenic acid OSOH, were obtained. We also found that OSH effectively quenches the triplet state of kynurenic acid with an almost diffusion-controlled rate constant. This finding indicates that OSH may serve as a good photoprotector to inhibit the deleterious effect of solar UV irradiation; this assumption explains the high concentrations of OSH in the fish lens. The unique antioxidant and photoprotecting properties of OSH open promising perspectives for its use in the treatment of human diseases.

Sea Cucumber Derived Type I Collagen: A Comprehensive Review.

Collagen is the major fibrillar protein in most living organisms. Among the different types of collagen, type I collagen is the most abundant one in tissues of marine invertebrates. Due to the health-related risk factors and religious constraints, use of mammalian derived collagen has been limited. This triggers the search for alternative sources of collagen for both food and non-food applications. In this regard, numerous studies have been conducted on maximizing the utilization of seafood processing by-products and address the need for collagen. However, less attention has been given to marine invertebrates and their by-products. The present review has focused on identifying sea cucumber as a potential source of collagen and discusses the general scope of collagen extraction, isolation, characterization, and physicochemical properties along with opportunities and challenges for utilizing marine-derived collagen.

Studying Properties of Prospective Biologically Active Extracts from Marine Hydrobionts

Biologically active substances (BAS) extracted from marine hydrobionts are characterized by high diversity and efficiency. They are represented by carotenoids, phospholipids, saponins and other compounds possessing high antioxidant (AOA), antitumor, immunomodulatory, radioprotective, and hypolipidemic activities. Because of extremely high marine BAS activity, the BAS dose necessary for treatment and prevention of many diseases is very small. The aim of present work was to assess biological properties of BAS complexes extracted from Black Sea marine inverterbrates for their following using in pharmacological preparations including liposomal drug delivery. BAS complexes were extracted from marine invertebrate tissues by two-phase extraction in combination with ultrasonication. Antioxidant activity of BAS extracts was determined with potassium permanganate discoloration method in an aqueous sulfuric acid medium. Antimicrobial activity was investigated by germination of BAS extracts. BAS influence on lymphocyte proliferative activity was studied with help of cytogenetic analysis. In this paper, the assessment of antioxidant, antimicrobial and mitogenic activities of biologically active complexes extracted from marine invertebrate tissues is presented. BAS properties in the oil and alcohol-water phases are compared. For the first time BAS complexes from Black Sea invertebrates are extracted by the resource-light method of two-phase extraction in combination with ultrasonication. Conditions for obtaining BAS extracts with optimal characteristics are proposed.

The differences of bacterial communities in the tissues between healthy and diseased Yesso scallop (Patinopecten yessoensis)

The tissues of marine invertebrates are colonized by species-rich microbial communities. The dysbiosis of host’s microbiota is tightly associated with the invertebrate diseases. Yesso scallop (Patinopecten yessoensis), one of the most important maricultured scallops in northern China, has recently suffered massive summer mortalities, which causes huge production losses. The knowledge about the interactions between the Yesso scallop and its microbiota is important to develop the strategy for the disease prevention and control. In the present study, the bacterial communities in hemolymph, intestine, mantle and adductor muscle were compared between the healthy and diseased Yesso scallop based on the high-throughput sequencing of 16S rRNA gene. The results indicated obvious difference of the composition rather than the diversity of the bacterial communities between the healthy and diseased Yesso scallop. Vibrio, Francisella and Photobacterium were found to overgrow and dominate in the mantle, adductor muscle and intestine of the diseased scallops, respectively. The prediction of bacterial community metagenomes and the variations of KEGG pathways revealed that the proportions of the pathways related with neurodegenerative diseases and carbohydrate metabolism both increased significantly in the mantle and hemolymph of the diseased scallops. The abundance of the metabolism pathways including carbohydrate metabolism, lipid metabolism and amino acid metabolism decreased significantly in the intestine of diseased scallops. The results suggested that the changes of bacterial communities might be closely associated with the Yesso scallop’s disease, which was helpful for further investigation of the pathogenesis as well as prevention and control of the disease in Yesso scallop.

Formation and mosaicity of coccolith segment calcite of the marine algae Emiliania huxleyi.

Coccolithophores belong to the most abundant calcium carbonate mineralizing organisms. Coccolithophore biomineralization is a complex and highly regulated process, resulting in a product that strongly differs in its intricate morphology from the abiogenically produced mineral equivalent. Moreover, unlike extracellularly formed biological carbonate hard tissues, coccolith calcite is neither a hybrid composite, nor is it distinguished by a hierarchical microstructure. This is remarkable as the key to optimizing crystalline biomaterials for mechanical strength and toughness lies in the composite nature of the biological hard tissue and the utilization of specific microstructures. To obtain insight into the pathway of biomineralization of Emiliania huxleyi coccoliths, we examine intracrystalline nanostructural features of the coccolith calcite in combination with cell ultrastructural observations related to the formation of the calcite in the coccolith vesicle within the cell. With TEM diffraction and annular dark-field imaging, we prove the presence of planar imperfections in the calcite crystals such as planar mosaic block boundaries. As only minor misorientations occur, we attribute them to dislocation networks creating small-angle boundaries. Intracrystalline occluded biopolymers are not observed. Hence, in E.huxleyi calcite mosaicity is not caused by occluded biopolymers, as it is the case in extracellularly formed hard tissues of marine invertebrates, but by planar defects and dislocations which are typical for crystals formed by classical ion-by-ion growth mechanisms. Using cryo-preparation techniques for SEM and TEM, we found that the membrane of the coccolith vesicle and the outer membrane of the nuclear envelope are in tight proximity, with a well-controlled constant gap of ~4nm between them. We describe this conspicuous connection as a not yet described interorganelle junction, the "nuclear envelope junction". The narrow gap of this junction likely facilitates transport of Ca2+ ions from the nuclear envelope tothe coccolith vesicle. On the basis of our observations, we propose that formation of the coccolith utilizes the nuclear envelope-endoplasmic reticulum Ca2+ -store of the cell for the transport of Ca2+ ions from the external medium to the coccolith vesicle and that E.huxleyi calcite forms by ion-by-ion growth rather than by a nanoparticle accretion mechanism.

Diverse profiles ofN-acyl-homoserine lactone molecules found in cnidarians

Many marine habitats, such as the surface and tissues of marine invertebrates, including corals, harbour diverse populations of microorganisms, which are thought to play a role in the health of their hosts and influence mutualistic and competitive interactions. Investigating the presence and stability of quorum sensing (QS) in these ecosystems may shed light on the roles and control of these bacterial communities. Samples of 13 cnidarian species were screened for the presence and diversity of N-acyl-homoserine lactones (AHLs; a prevalent type of QS molecule) using thin-layer chromatography and an Agrobacterium tumefaciens NTL4 biosensor. Ten of 13 were found to harbour species-specific, conserved AHL profiles. AHLs were confirmed in Anemonia viridis using liquid chromatography tandem mass spectrometry. To assess temporal role and stability, AHLs were investigated in A.viridis from intertidal pools over 16h. Patterns of AHLs showed conserved profiles except for two mid-chain length AHLs, which increased significantly over the day, peaking at 20:00, but had no correlation with pool chemistry. Denaturing gel electrophoresis of RT-PCR-amplified bacterial 16S rRNA showed the presence of an active bacterial community that changed in composition alongside AHL profiles and contained a number of bands that affiliate with known AHL-producing bacteria. Investigations into the quorum sensing-controlled, species-specific roles of these bacterial communities and how these regulatory circuits are influenced by the coral host and members of the bacterial community are imperative to expand our knowledge of these interactions with respect to the maintenance of coral health.

Identification of a cytochrome b5-fusion desaturase responsible for the synthesis of triunsaturated sphingolipid long chain bases in the marine diatom Thalassiosira pseudonana.

Triunsaturated sphingolipid long chain bases (LCBs) have previously been reported in some specialised tissues of marine invertebrates. We report the presence of similar LCBs in the marine diatom Thalassiosira pseudonana and identify the cytochrome b5-fusion desaturase responsible for the introduction of the third double bond at the Δ10 position in d18:3Δ4,8,10. This study extends the catalytic repertoire of the cytochrome b5 fusion desaturase family, also indicating the presence of orthologues in other marine invertebrates. The function of these polyunsaturated sphingolipid LCBs is currently unknown but it was previously suggested that they play an essential role in primitive animals. The identification of the desaturase responsible for their synthesis paves the way for further studies.

Stem cells of marine invertebrates: Regulation of proliferation and induction of differentiation in vitro

The review surveys own and the literature data on the plasticity of marine invertebrate stem cells. Stem and embryonic cell cultures of marine invertebrates are a novel model system characterized by a high level of physiological and synthetic processes. The production of biologically active substances in vitro may be an alternative to chemical synthesis and aquaculture. The factors involved in determination and maintenance of the pluripotency of marine invertebrate stem cells have been analyzed. The technology of the directed differentiation of marine invertebrate stem cells into certain functionally active cells in vitro embraces the use of different growth factors, various natural and artificial substrates, and unique bioactive compounds from marine invertebrate tissues. To increase the expression levels of regulatory genes, we applied genetically engineered constructions with foreign genes. The regulation of growth and differentiation of marine invertebrate stem cells opens new prospective uses for their application in marine biotechnology and is helpful for research in developmental biology.

Enzymological characteristic of monoamine oxidase from the visual ganglia of the Pacific squid Todarodes pacificus

The study of catalytic properties of mitochondrial monoamine oxidase (MAO; monoamine:О2 oxi� doreductase, deaminating; EC 1.4.3.4), which main� tains physiological level of mediators in the presynap� tic nerve terminal, is of general biological interest and a great importance for enzymology, in particular, for characterizing redox enzymes. The biological role of MAO is associated with the regulation of the level of mediators in nerve structures and, therefore, with dif� ferent functions of the nervous system, which is con� firmed, in particular, by experimental and clinical data on the use of MAO inhibitors [1]. The study of the sub� strate specificity and multiplicity of MAO forms in the central nervous system of different animals will proba� bly provide an opportunity to differentially determine the functional role of MAO in neural processes. The majority of available papers in this field are devoted to studying MAO of terrestrial vertebrates, whereas MAO of aquatic animals has been studied quite insuffi� ciently. It was established that neural tissues of marine invertebrates contain a relatively high MAO activity as compared to other organs [2, 3]. Monoamine oxidase activity was detected in the visual ganglia, which fulfill the function of the central nervous system, of some cephalopods, such as octopuses Octopus vulgaris [4], Eledone moschata [5], and Eledone cicchosa [6] and squids Loligo forbesi [7], Loligo pealei [8], and Berry� teuthis magister [9]. Although over 350 squid species are known in the World Ocean [10], the monoamine oxidase activity of visual ganglia has been studied so far only in a few of them [7–9], which is possible caused by difficulties in obtaining biological material. Almost all papers devoted to the MAO of visual ganglia of squids contain only qualitative results. The list of kinetically studied enzymes with enzymological parameters of substrate–inhibitor specificity is quite scanty [8]. The substrate and inhibitor analyses of monoamine oxidase activity of visual ganglia of the Pacific squid Todarodes pacificus were undoubtedly of interest. The kinetic parameters of enzymatic deamination of tyramine, serotonin, benzylamine, histamine, tryptamine, and β�phenylethylamine as well as bimo� lecular rate constants for the interaction of inhibitors deprenyl and chlorgilin with the enzyme were calcu� lated. It should be noted that this was the first work to study the catalytic properties of MAO from the visual ganglia of this commercially important squid species.

Quick DNA preparation for direct PCR in marine invertebrates and fishes

A modified protocol was developed for extracting DNA and direct PCR from tissues of marine invertebrates and finfishes. Protocol represented combination of classical lysis using a modified buffer system comprising KCl, Tris buffer and MgCl2. The DNA obtained was quantified and tested by mtDNA-PCR and RAPD. Test results indicated usefulness of this method in studies involving screening of large numbers of samples, such as evolutionary, forensic and population studies.

Mechanism of acute silver toxicity in marine invertebrates

In freshwater crustaceans and in both freshwater and marine fish, the key mechanism of acute silver toxicity involves ionoregulatory impairment. An inhibition of the Na +,K +-ATPase located at the basolateral membrane of the gill epithelium seems to be the key site for silver toxicity. However, studies to determine if the same mechanism of toxicity is occurring in marine invertebrates, which also are ionoregulators, had not been done. Thus, the present study was carried out to determine acute silver effects on hemolymph osmo- and ionoregulation in three marine invertebrates: the shrimp Penaeus duorarum, the sea hare Aplysia californica, and the sea urchin Diadema antillarum. Animals were exposed to silver (1 or 10 μg/L), as silver nitrate, in seawater for 48 h. Results show that acute silver exposure did not affect hemolymph osmolality or ion concentration (Na +, Cl −, K +, Ca 2+ and Mg 2+) in the three species studied. However, silver induced significant changes in the water content in shrimp gill and sea hare gill and hepatopancreas. Silver also caused significant changes in Na +,K +-ATPase activity and in both total and intracellular ion (Cl −, Na +, K +, Mg 2+, and Ca 2+) concentrations in different tissues of the three species studied. Overall, these results show that the key mechanism of acute silver toxicity in marine invertebrates is not associated with an osmotic or ionoregulatory impairment at the hemolymph level, as observed in freshwater fish and crustaceans and in seawater fish. However, they indicate that acute waterborne silver induces significant changes in Na +,K +-ATPase activity and probably affects other mechanisms involved in water and ion transport at the cell membrane level, inducing impairments in water and ion regulation at the cellular level in different tissues of marine invertebrates. These results indicate the need to consider other “toxic sites” than gills in any future extension of the biotic ligand model (BLM) for seawater.

Enzymatic hydrogen sulfide production in marine invertebrate tissues

At least some mammalian tissues produce H 2S in vitro from l-cysteine at rates sufficient to have physiological effects. To determine whether tissues of macrofaunal invertebrates have the same capacity, we measured H 2S production in tissue homogenates of the Manila clam Tapes philippinarum and the lugworm Arenicola marina. Tissue homogenates from both animals produced significant quantities of H 2S gas upon addition of l-cysteine and the enzyme cofactor pyridoxal -5′-phosphate (10 mmol l −1 and 2 mmol l −1, respectively), while only tissues from T. philippinarum produced measurable H 2S in the absence of added substrate or cofactor. In T. philippinarum tissues, H 2S production was completely inhibited by the cystathionine β-synthase (CBS) inhibitor aminooxyacetic acid (AOAA), suggesting that the majority of H 2S production was via CBS pathways, while in A. marina body wall, AOAA inhibited only half of the total H 2S production, indicating that the CBS pathway was not the only major source of H 2S production. H 2S production in tissues of T. philippinarum but not A. marina was doubled by the addition of a second thiol substrate (2.5 mmol l −1 2-mercaptoethanol), suggesting the presence of an ‘activated serine sulfhydrase pathway’, which had previously been demonstrated only in some microfauna.

Ecology of the marine protists, the Labyrinthulomycetes (Thraustochytrids and Labyrinthulids)

The Labyrinthulomycetes, comprising thraustochytrids and labyrinthulids, are marine osmoheterotrophic, straminipilan protists which have been isolated from a variety of habitats all over the world. The limited knowledge on their ecological role(s) is brought together in this review. Labyrinthulids are prevalent on or in living marine algae and sea grasses as parasites, commensals or mutualists. Thraustochytrids, on the contrary, are rarely found on these living plants and appear to be inhibited by plant antimicrobials. Members of both groups often abound on dead autochthonous, as well as allochthonous, plant material such as macroalgae and submerged mangrove leaves, and probably play an important role as saprobes by virtue of extracellular enzyme production and chemical alteration of detritus. Labyrinthulomycetes reportedly cause diseases in animals and there are indications that they live as commensals or mutualists within the guts and tissues of marine invertebrates, as well as being saprobic on such animal materials as faeces and mollusc shells. Thraustochytrids are common in the neritic and oceanic water column and sediments, including the deep sea. Seasonal studies indicate poor correlation with phytoplankton blooms, but often reveal thraustochytrid biomass equivalent to that of bacteria during times of phytoplankton decay. It is concluded that they may play an important role in mineralisation of phyto- and zooplankton detritus in the sea. Their high content of ω-3 polyunsaturated fatty acids suggests that they may form an important link in the food-web. More knowledge about their ability to degrade various forms and concentration levels of organic carbon in the sea, as well as possible phagotrophy will help to resolve their exact ecological niche vis-a-vis the bacteria in the marine ecosystem.

Genetics and marine pollution (review)

The development of cytogenetic methods applied to cells and tissues of marine invertebrates has been hampered by (1) a lack of in vitro cell lines, (2) inadequate karyotypic information (partly as a result of too few workers chasing too many organisms), and (3) the failure of their chromosomes to band satisfactorily. Compared to mammalian cytogenetics, our knowledge of marine invertebrates lags behind by several decades. With the current concern about mutagens in the marine environment, and the recognition that the cells of marine species have sensitivities to DNA-damaging agents similar to those of higher organisms, there is a need for methods which can be used (a) in environmental monitoring and (b) to screen potentially harmful substances in the laboratory. In the absence of in vitro cell lines, embryos and larvae have been used to provide a supply of dividing cells for mutation studies, although the advent of molecular methods has now brought with it the means to detect DNA damage without any need for the cells to be in a dividing state. Moreover, the use of FISH (Fluorescence In Situ Hybridisation) now makes it possible to study numerical and structural chromosomal aberrations with far greater accuracy than was previously possible. A new marine genotoxicity assay is described, based on the embryos and larvae of a tube-dwelling polychaete worm (Pomatoceros lamarkii), suitable for both laboratory studies and field monitoring. This new Pomatoceros assay provides, at the same time, a useful model for studying the consequences of adult exposure on the offspring. A novel application of marine cytogenetic research is the study of the evolutionary adaptations of invertebrates living in naturally polluted extreme environments viz. deep sea hydrothermal vents, which are typified by high levels of toxic heavy metals and radionuclides, substances known to inflict damage to DNA. Given these new methodological and conceptual advances, it is predicted that our understanding of the role played by mutation in the marine environment, both in an evolutionary and toxicological context, will increase dramatically over the next decade.

Production of H 2S in marine invertebrate tissues

Production of H 2S in marine invertebrate tissues

Marine invertebrate collagens: The prevalence of type V and XI like collagens in some marine crustacean and molluscan tissues

While the invertebrates constitute almost 95% of the animal kingdom, our knowledge on their extracellular matrices, particularly on collagen, is very scanty. Our group has been studying the collagen polymorphism in some marine invertebrate tissues with focus on structure-function relationships and molecular evolution. We have established methods to purify unique collagen molecules from some rare tissues of crustaceans and molluscs. Some of these include the intramuscular tissues of the crustaceans and the cartilage and cornea of molluscs. The biochemical parameters in these tissues relating to collagen content, solubility and carbohydrate composition have been determined. The chain composition of these collagens were deduced by SDS-PAGE. We have analyzed the amino acid compositions of these collagens and that of isolated single alpha chains. The physicochemical properties and ultrastructural characteristics of some of these collagens were also studied. The results indicate that the principal component of crustacean muscle is a type V like homotrimer and that of molluscan cartilage and cornea is a unique heteromeric collagen resembling vertebrate type V and XI collagens. These collagens were invariably highly crosslinked, stabilized largely by bound carbohydrates and had significantly high denaturation temperatures. While the crustacean type V collagen formed regularly banded fibrils, the V/XI like collagen of molluscan cartilage lacked periodicity in fibril structure. We correlate the significance of our key observations to the possible functional consequence as well as evolutionary significance, based on the available data on other similar collagens.

Ecotoxicology of arsenic in the marine environment

AbstractArsenic has a complex marine biogeochemistry that has important implications for its toxicity to marine organisms and their consumers, including humans. The average concentration of total arsenic in the ocean is about 1.7 μg/L, about two orders of magnitude higher than the U.S Environmental Protection Agency's human health criterion (fish consumption) value of 0.0175 μg/L. The dominant form of arsenic in oxygenated marine and brackish waters is arsenate (As V). The more toxic and potentially carcinogenic arsenite (As III) rarely accounts for more than 20% of total arsenic in seawater. Uncontaminated marine sediments contain from 5 to about 40 μg/g dry weight total arsenic. Arsenate dominates in oxidized sediments and is associated primarily with iron oxyhydroxides. In reducing marine sediments, arsenate is reduced to arsenite and is associated primarily with sulfide minerals. Marine algae accumulate arsenate from seawater, reduce it to arsenite, and then oxidize the arsenite to a large number of organoarsenic compounds. The algae release arsenite, methylarsonic acid, and dimethylarsinic acid to seawater. Dissolved arsenite and arsenate are more toxic to marine phytoplankton than to marine invertebrates and fish. This may be due to the fact that marine animals have a limited ability to bioconcentrate inorganic arsenic from seawater but can bioaccumulate organoarsenic compounds from their food. Tissues of marine invertebrates and fish contain high concentrations of arsenic, usually in the range of about 1 to 100 μg/g dry weight, most of it in the form of organoarsenic compounds, particularly arsenobetaine. Organoarsenic compounds are bioaccumulated by human consumers of seafood products, but the arsenic is excreted rapidly, mostly as organoarsenic compounds. Arsenobetaine, the most abundant organoarsenic compound in seafoods, is not toxic or carcinogenic to mammals. Little of the organoarsenic accumulated by humans from seafood is converted to toxic inorganic arsenite. Therefore, marine arsenic represents a low risk to human consumers of fishery products.

誘導結合プラズマ質量分析法による海洋生物中の｀２３８´Ｕの定量

Determination of 238U in fifty-five species of marine organisms was carried out by inductive-ly coupled plasma mass spectrometry which showed some advantages such as high sensitivity, wide dynamic range and small interferences from matrices for the analysis of high mass elements. The concentrations of 238U in soft tissues of marine animals ranged from 0.076 to 5000ng/g wet wt. Especially, the branchial heart of cephalopod molluscs showed the specific accumulation of 238U. The concentration factor of the branchial heart of Octopus vulgaris, which indicated the highest value, was calculated to be about 103 by comparing it with the concentration of 238U (3.2±0.2ng/ml) in coastal seawaters of Japan. The concentrations of 238U in hard tissues of marine invertebrates were similar to those in soft tissues. In contrast, hard tissues like bone, scale, fin, etc. of fishes showed much higher concentrations of 238U than soft tissues like muscle and liver. The concentrations of 238U of twenty species of algae ranged from 10 to 3700ng/g dry wt.

D-Alanine transport and metabolism by the coelomocytes of the bloodworm, Glycera dibranchiata (Polychaeta)

1. 1. A saturable transport system for d-alanine exists in Glycera RBCs which is Na dependent and which appears from competition experiments to be stereoselective for a broad range of d-amino acids. 2. 2. The kinetic constants for d-alanine for this system are: K T = 0.14 ± 0.01 mM and J max = 0.15 ± 0.02 mmol/min 1 cell per water. An apparent linear diffusion component was also present where K D = 0.059 ± 0.004/ min. 3. 3. Incubation of Glycera RBCs with 14C- d-alanine results in the conversion of some of the d-alanine to at least two other compounds. 4. 4. These data provide evidence that marine invertebrate tissues may possess specific d-amino acid transport systems which accumulate and maintain intracellular pools of free d-amino acids. These d-amino acids are available to the cellular metabolic machinery for utilization.