Amino Acids in Sea Water

Free amino acids in the waters of the English Channel have been measured over a twelve month period using a newly developed field technique. Samples have been taken over a wide area both from the surface and at depth. The results show that the levels of amino acids found in these studies are greater than those previously generally accepted. These findings are of considerable importance in productivity studies since amino nitrogen as a source of nutrient for phytoplankton has been largely neglected. This paper shows that the levels of amino acids in the upper layers of the sea are not insignificant and should not be disregarded as an alternative nitrogen source for phytoplankton.     

Phagostimulatory properties of various chemical compounds to sea hares (Aplysia kurodai and A. dactylomela)

Phagostimulatory properties of amino acids, carbohydrates, vitamins, fatty acids, and cholesterol were assessed for sea hares (Aplysia kurodai and A. dactylomela). A. kurodai were collected at Shirahama, Japan, during March-May 1979, and A. dactylomela were collected at Barbados, West Indies, during August-November 1978. Greatest phagostimulatory responses were elicited by starch, glutamic and aspartic acids, maltose, oleic acid, and certain combinations of vitamins. Combinations of nutrients resulted in negative synergistic or masking effects of the phagostimulatory properties of the component nutrients. The widespread sensitivity and responsiveness to glutamic and aspartic acids by marine invertebrates, and especially by sea hares, are discussed from the standpoint of possible use by marine herbivores as feeding cues.     

Investigations of some trace elements in the Bay of Lim

Sea water was collected at several stations in the Bay of Lim at a depth of one meter below the surface, and before analysis, was filtered with 0.45-μm Millipore filters. Shallow water sediments were collected at the same locations. Humic acids used in this work were separated from near-shore sediments taken from various saline waters of the Bay of Lim. The prepared humic acids were then analysed for their elementary composition and also for positions of their hydrolytic products to obtain more data on how and to which characteristic part, the trace elements were related. The hydrolysis of humic acids was done by chemical methods in order to obtain four main components: amino acids, sugars, phenols and condensed benzene core. Neutron activation analysis was used for trace element analysis in evaporated portions of filtered sea water, sediments, soil, living organisms, humic acids and their hydrolytic products. This work was undertaken to obtain more data on the organic matter present in sediments and in seagrass flats, and also to collect more data on trace elements that are associated with typical and representative samples for the Bay of Lim. Analyses of trace elements associated with either humic acids or their hydrolytic products were performed with the purpose of gaining evidence as to which part of humic acids metals are bound. Humic acids or their decomposition products play an important role in the distribution and availability of a number of essential or nonessential trace elements. Results of this work indicate that humic acids influence the process of redistribution of trace elements in the investigated local coastal area.     

Untersuchungen über freie gelöste Aminosäuren in Meerwasser

In the literature concerning the determination of free dissolved amino acids in sea water, the evaluation of the various methods employed varies considerably. During our experiments, a two-stage preparatory method—evaporation of sea water to a small volume and desalination of the solution by ion exchange — which in principle was first used by Palmork (1963a), proved satisfactory. Recovery was about 80%, and reproduction of results sufficiently good. Following this preparatory method, and using the Beckman-Amino acid analyzer “Unichrom” for final analysis, a series of aged seawater samples was submitted to amino acid determination. The water samples always contained very small amounts of amino acids. A larger number of samples was collected in April and May, 1969, near Helgoland (54°11.3′ N, 7°54.0′ E). The samples were immediately filtered through a membrane filter (pore size 0.45 μ) and stored at-20°C until processed. Of the total amino acid content, glycine and serine were present regularly in the largest amounts, followed at some distance by lysine, glutamic acid, proline, and threonine. Quantitative determinations of aspartic acid were not usually possible, as it could not be separated sufficiently from another substance, probably methioninsulfoxide. Particular attention was paid to the danger of contamination of the samples by ninhydrinpositive compounds. The ion exchange resins used proved to be a considerable source of such foreign substances.     

Der Einfluß physikalischer und chemischer Faktoren auf die Aufnahme in Meerwasser gelöster Aminosäuren durch Aktinien

Changes in abiotic environmental factors do not adversely influence the capacity of Anemonia sulcata (Coelenterata, Anthozoa) to take up dissolved amino acids from sea water; e. g., A. sulcata resorbs also under anaerobic conditions. The influence of temperature on amino acid uptake (Q₁₀=2) indicates that the translocation of the organic molecules through membranes of the ectoderm depends on energy. Uptake of amino acids is possible against a gradient of up to 9×10⁶:1. It can be postulated that L-amino acids (also D-isomers) and glucose are resorbed by different “carrier systems”, since glucose does not influence the uptake of amino acids; however, amino acids can interfere with each other, e. g. phenylalanine/glycine. Blocking of anaerobic glycolysis results in uptake reduction; at the same concentrations “oxidative” blockers have a much smaller effect on amino acid uptake. The possibility of exploiting the surrounding water as an energy source by taking up dissolved organic material is discussed with reference to ecological and evolutionary aspects.     

Growth of Uronema marinum in chemically defined medium

A strain of the marine hymenostome ciliate Uronema marinum was isolated in axenic culture. This strain, designated PW2, was cultivated in a chemically defined medium containing 17 amino acids, 4 nucleotides, 5 fatty acids, stigmasterol, 8 growth factors and an artificial sea water. Increased populations were observed when either phosphatidylinositol or Tween 80 were included in the medium. The vitamins thiamine, riboflavin, pyridoxal, nicotinamide, folic acid and pantothenate were required by U. marinum PW2. No requirement could be demonstrated for exogenous thioctic acid or biotin, although growth was diminished in their absence. The amino acid and nucleotide requirements were found to be higher than the levels reported for other ciliates. This strain was found to be euryhaline, with maximum growth observed in media of specific gravity 1.007, corresponding to approximately 25% sea water. Although no requirement could be demonstrated for Fe, Zn, Mn or Cu, high levels of these metals were observed to be toxic. Growth was observed over a pH range of 6.6 to 8.3, with an optimum observed near 7.3.     

Isolation and sequence of cDNA encoding a 3-methylcholanthrene-inducible cytochrome P450 from wild red sea bream,Pagrus major

We have isolated a cDNA clone of mRNA for the cytochrome P450 from a 3-methylcholanthrene (MC)-treated red sea bream,Pagrus major, using a cDNA fragment for rat P4501A2 as a probe. The cloned cDNA is ca. 1.8 kb long and contains an open reading frame of 1545 nucleotides for polypeptides of 515 amino acids. The deduced N-terminal amino acid sequence of the cDNA is very similar to that for purified cytochrome P450 protein from the marine fish scup, which was reported previously (Klotz et al. 1983). A conserved amino acid sequence containing a putative heme-binding cysteine is present in the equivalent position proximate to the C-terminus of the molecules. The deduced amino acid sequence shows more than 50% positional identity with known members of the mammalian aromatic hydrocarbon-inducible P450 family. RNA blot analysis indicates that P450 mRNA (s) is expressed in the liver, kidney, gill and gut of the MC-treatedP. major.     

Essential amino acid synthesis and nitrogen recycling in an alga–invertebrate symbiosis

When aseptically-cultured sea anemones, Aiptasia pulchella, were incubated with ¹⁴C-labelled glucose, aspartate and glutamate, radioactivity was incorporated into animal protein. Radioactivity was recovered from all amino acids in the protein hydrolysates of A. pulchella bearing the symbiotic alga Symbiodinium sp., and from all but seven of the amino acids in A. pulchella experimentally deprived of their algae. These data suggest that these seven amino acids (histidine, isoleucine, leucine, lysine, phenylalanine, tyrosine and valine) may be synthesized by the symbiotic algae and translocated to the sea anemone's tissues; and that methionine and threonine, two amino acids traditionally considered as dietary essentials for animals, are synthesized by A. pulchella. Essential amino acid translocation from the symbiotic algae to the animal host is a core element in symbiotic nitrogen-recycling. Its nutritional value to the animal host is considered in the context of the amino acid biosynthetic capacity of the host. Received: 26 October 1998 / Accepted: 28 June 1999     

Kinetics of osmoregulation in the crab Carcinus maenas

When shore crabs Carcinus maenas are transferred from 11 to 38 S at 11°C, new constant levels of hemolymph freezing points and of concentrations of Na, K, Ca, and Mg in the hemolymph are accomplished within 24h. From a decrease in serum protein and in serum free amino acids and an increase in the relative amounts of individual essential free amino acids in the body fluids of whole crabs, a participation of proteolytic activities and a transport of amino acids from hemolymph to cell is deduced; the stationary concentration of total intracellular free amino acids increases up to a nearly two-fold value, compared with the concentration in crabs remaining in diluted sea water. Also, the low molecular neutral sugars increase in whole crabs after high salinity stress, reaching values of more than two-fold initial concentrations within a period of 10 days. This increase is fully accounted for by a 6.7-fold increase in trehalose levels. The processes of increasing the concentrations of low molecular organic material seem to be slower than those of establishing new osmotic pressures in the hemolymph. The oxygen consumption decreases by 30 to 45% to new constant values within 8 to 12 h after the salinity change from 11 to 38, reflecting similar kinetics as the establishment of new osmotic pressures in the hemolymph.     

Gelöste Aminosäuren in Oberflächenwasser der Nordsee bei Helgoland: Konzentrationsveränderungen im Sommer 1970

In order to obtain additional information on changes in dissolved amino-acid content of the surface water of the North Sea, more than 30 water samples taken near Helgoland (54/11.3′ N; 7/54′ E) between April and October, 1970, were investigated. The method was the same as reported by Bohling (1970). In addition to free amino acids in a number of cases, also that fraction of combined amino acids was analyzed which passed a cation exchange column in H⁺ form. The average concentration of free amino acids was higher in 1970 than in 1969, reaching a total maximum of 6 μM/l at the end of May. The amino-acid content fluctuated considerably even over short periods of time. The relative amounts of single compounds, however, remained rather constant. During Summer, 1970, the relationship between the concentrations of free and “combined” amino acids varied. As far as can be judged from the limited number of analyses, combined amino acids increased with the phytoplankton mass; such increase was not observed in the free amino acids. A possible explanation of the results, based on microbial activity, is discussed.     

Effect of salinity adaptation on nitrogen metabolism in the freshwater fish Tilapia mossambica. I. Tissue protein and amino acid levels

Water, proteins and total free amino acids were estimated in different tissues of the euryhaline fish Tilapia mossambica after adaptation to various strenghts of sea water. The water content did not vary significantly in any tissue on salinity adaptation. The soluble and insoluble proteins displayed a general and considerable decrease in muscle, liver and heart; the decrease in the soluble fraction in the heart and the proteins of the muscle in 75% sea water (100% sea water=32.5 S) were significant. The gill proteins did not alter with salinity; the kidney proteins tended to increase slightly in 100% sea water (SW). The total free amino-acid content decreased insignificantly in all tissues on adaptation to 25% SW; in higher salinities, however, the content increased significantly. This increase was sudden and steep in 50% SW, and gradual and less steep in 75 and 100% SW. It is suggested that constancy in water content may contribute to the great adaptability of T. mossambica to heterosmotic media, and that the total free amino acids may be involved in isosmotic intracellular regulation. The possibility of amino acid increase as a result of protein breakdown is also indicated.     

Effect of nitrogen deprivation on amino acid uptake by the chlorophyte Platymonas subcordiformis

Platymonas subcordiformis (UTEX 171) was cultured axenically for 4 d in constant light in a nitrate-containing medium and harvested in the log-phase of cell division. Cells were resuspended in artificial sea water without nutrients and either kept in constant light or placed in constant darkness. High-performance liquid chromatography was used to measure the free amino acid pools of the cells and to determine rates of net entry of each of a mixture of 18 amino acids at daily intervals for 5 d. Free amino acid pools decreased both in light and darkness in the absence of a nutrient sypply. The influx of amino acids in cells maintained in the light increased selectively. Comparison of the rate of entry of ¹⁴C-labeled glycine and net disappearance of glycine from the medium indicated extrusion of non-volatile labeled carbon that did not interact with reagents specific for amine groups. Light was required for synthesis of additional transporter protein which was apparently responsible for increased influx in cells maintained in the light. This response was blocked in the presence of cycloheximide. Cells maintained in the dark for prolonged periods retained the capacity to respond to light by synthesis of new transporter protein. Analysis of incorporation of amino acids into macromolecules indicated that both the overall rate and the pattern of amino acid incorporation were modified in the light. Analysis of the kinetics of glycine entry at a series of temperatures indicated that the concentration of glycine at which entry is half the maximum rate is approximately 2.7 M at the cell surface.     

Acides aminés libres chez les larves de Crangon septemspinosa (Caridea). Variation de leur taux de l'éclosion à la métamorphose. Leur rôle au cours du développement et leur importance dans la nutrition

Studies have been carried out on the variations in the pool of free amino-acids throughout the larval development of artificially reared Crangon septemspinosa, and their relationship with the nutrition of this species. Although quantitative variations are less apparent for essential than for non-essential amino acids, a certain uniformity appears. During larval development, the concentration of each amino acid reveals two optimal values: the first at the third larval stage; the second at the final larval stage, just before metamorphosis. Each of these peak values is followed by a more or less rapid drop in amino-acid concentration. An attempt is made to explain these variations in relation to their specific function in general metabolism and growth. By the addition of certain amino acids during phases of high metabolic activity, it may be possible to overcome difficulties encountered during critical stages of rearing experiments.     

Co-occurrence of copepods and dissolved free amino acids in shelf sea waters

The vertical distribution of chlorophylla, copepods, dissolved free amino acid concentration and the fixation of¹⁴C by phytoplankton were monitored in the springs of 1983, 1987 and 1988 in the Ushant front region, shelf edge of the Celtic Sea and central Irish Sea, respectively. In each area, two stations characterized by mixed and stratified water conditions were compared. Vertical distributions of amino acids coincided with the distribution of copepods. A positive and significant correlation was found between the abudance of copepods and the concentration of amino acids dissolved in seawater. A negative and significant correlation was found between chlorophylla and the concentration of amino acids. Enrichment of amino acids ( 20 to 500 nM l^–1 at specific depths) due to aspartic and glutamic acids, glutamine and ornithine, was assumed to reflect copepod feeding activity and faecal production. At these depths, the natural concentration and diversity of amino acids, including aspartic acid, glutamic acid, asparagine, serine, histidine, glutamine, arginine, threonine, glycine, alanine, tyrosine, valine, phenylalanine, ornithine and lysine, were high enough and in the correct proportions for triggering feeding and swimming and swarming behavior of copepods, as well as their remote detection of food at the micro- and meso-scales (1 to 10 m). This accumulation of amino acids also constitutes a potential additional source of organic nitrogen for bacteria and phytoplankton.     

Biodegradation of aromatic hydrocarbon in marine sediments of three North Sea oil fields

On a survey around three North Sea oil fields in 1980 the biodegradation of the hydrocarbons ¹⁴C-naphthalene and ¹⁴C-benzo[a]pyrene was measured. An amino acid mixture was taken as reference substrate for total heterotrophic activity. The rate of uptake of naphthalene was found to increase with decreasing distance from the oil platform at all fields with maximum values at stations 0.5 miles (ca 0.8 km) from the centre of oil activity. Data on naphthalene uptake parallel GC-MS data for aromatic hydrocarbons and allow an estimation of the actual uptake velocity of naphthalene in the sediment to be made. A significant mineralisation of benzo[a]pyrene could only occasionally be detected. The respiration of amino acids did not show any pattern related to the oil field activities.     

Regulation of in vitro and in vivo CaCO3 crystallization by fractions of oyster shell organic matrix

Correlations between structural properties of bivalve shell organic matrix and its proposed functions in the regulation of biomineralization were examined using proteinaceous fractions obtained from the shell of the oyster Crassostrea virginica (Gmelin) following dissolution of the mineral with ethylenediaminetetraacetate (EDTA). Matrix isolated in this way contains a continuous size distribution of proteins ranging from relatively small molecular weight (M_r) soluble matrix (SM) components to insoluble matrix (IM) components. Regulation of mineralization by these components was determined primarily by their reduction of crystal growth rate in an in vitro assay. For all fractions tested, there was an inverse correlation between M_r and regulatory activity. However, matrix properties other than molecular size may be important in regulation of crystal growth in vivo in that the larger but less acidic of two soluble matrix proteins was a more effective inhibitor of spicule formation by sea urchin embryos. Base treatment of IM and high molecular weight SM fractions that had little or no inhibitory activity when untreated, resulted in constituents that had a molecular weight distribution and in vitro inhibitory activity in the same range as whole SM. These results, combined with the finding that a major fraction of IM has an amino acid composition very similar to the highly charged SM fractions, suggest that much of the matrix is made up of similar molecules, and that their function in crystal growth regulation may change as they interact to form units of increasing size. A second class of IM was isolated which contained dihydroxyphenylalanine (dopa) and a preponderance of hydrophobic amino acids. This material may represent the basic structural framework of the matrix.     

Trophic and reproductive biochemistry of a deep-sea gelatinous octopus, Opisthoteuthis calypso

The present study provides a comprehensive analysis of the biochemical composition (proximate composition, amino acids and fatty acids in gonad, digestive gland and muscle) of the cirrate octopod, Opisthoteuthis calypso, collected off the coast of Portugal. Protein and energy contents of O. calypso were significantly lower (p < 0.05) than those observed for coastal octopods, suggesting that the gelatinous musculature of the cirrate octopus may not be a direct consequence of food limitation, but rather associated with reduced selective pressure for strong swimming ability in the deep sea. Moreover, principal component analyses of fatty acid (FA) data clearly separated coastal octopods from O. calypso. Saturated FA biomarkers (namely 16:0, 17:0, 18:0) and monounsaturated FA 22:1 suggest a higher phytodetrital influence on diet of O. calypso. A clear separation between groups was also observed with amino acid (AA) data. However, essential AA and non-essential AA designations (based on the flow of carbon through biochemical systems) do not necessarily provide an accurate picture of the origins of amino nitrogen in the deep-sea environment. Consequently, the interpretation of the present data is a challenging task but opens a new window of opportunity to unravel new trophic biomarkers in the deep sea.     

Energy metabolism during development of eggs and larvae of gilthead sea bream (Sparus aurata)

Developing eggs and larvae of laboratory-reared gilthead sea bream (Sparus aurata) maintained in filtered seawater (40 ppt) at 18°C, were measured for oxygen uptake, ammonia excretion, contents of free amino acids (FAA), protein, fatty acids (FA) accumulated ammonia, and volumes of yolk-sac and oil globule. Absorption of the yolk coincided with the consumption of FAA and was complete ca. 100 h post-fertilisation. Amino acids from protein were mobilised for energy in the last part of the yolk-sac stage. Absorption of the oil globule occurred primarily after hatching following yolk absorption, and correlated with catabolism of the FA neutral lipids. Overall, FAA appear to be a significant energy substrate during the egg stage (60 to 70%) while FA from neutral lipids derived from the oil globule are the main metabolic fuel after hatching (80 to 90%).     

Organic matter composition in sediments of the Oman Margin

The quantity and biochemical composition of organic matter (OM) in the sediments underlying and below oxygen minimum zone (OMZ) in the Arabian Sea were studied to provide information on the diagenetic processes of organic carbon under different environmental conditions. Concentrations of total organic carbon (TOC), total nitrogen (TN) and total hydrolysable amino acids (THAA) were significantly higher in sediments within rather than below OMZ, while those of total carbohydrates (TCHO) were slightly lower in the latter, suggesting the presence of a larger supply of labile compounds into the sea bottom at the shallower site (i.e. within OMZ). Hydrolysable amino acid and carbohydrate contribution to TOC were even lower (about 10% in surficial sediments) than the values obtained from the abyssal oligotrophic North Pacific, suggesting that OM food availability in the Oman Margin sediments within OMZ was lower than that observed at abyssal depths. The presence of the highest THAA and TCHO concentrations in the top 40 mm of the sediment core at both sites reflected the presence of bioturbation processes. In contrast with the general view of the deep sea as a stable and constant system, below OMZ in the Arabian Sea sediments, some differences were observed in the two investigated cores indicating the presence of a certain spatial variability in OM content and diagenesis.     

Organic matter composition in sediments of the Oman Margin

The quantity and biochemical composition of organic matter (OM) in the sediments underlying and below oxygen minimum zone (OMZ) in the Arabian Sea were studied to provide information on the diagenetic processes of organic carbon under different environmental conditions. Concentrations of total organic carbon (TOC), total nitrogen (TN) and total hydrolysable amino acids (THAA) were significantly higher in sediments within rather than below OMZ, while those of total carbohydrates (TCHO) were slightly lower in the latter, suggesting the presence of a larger supply of labile compounds into the sea bottom at the shallower site (i.e. within OMZ). Hydrolysable amino acid and carbohydrate contribution to TOC were even lower (about 10% in surficial sediments) than the values obtained from the abyssal oligotrophic North Pacific, suggesting that OM food availability in the Oman Margin sediments within OMZ was lower than that observed at abyssal depths. The presence of the highest THAA and TCHO concentrations in the top 40?mm of the sediment core at both sites reflected the presence of bioturbation processes. In contrast with the general view of the deep sea as a stable and constant system, below OMZ in the Arabian Sea sediments, some differences were observed in the two investigated cores indicating the presence of a certain spatial variability in OM content and diagenesis.