Biotic and abiotic stress effects on nitrogen chemistry in the salt marsh cordgrassSpartina alterniflora (Poaceae)

Summary Planthopper (Insecta: Homoptera) feeding stress induces a senesence-like response in the leaves ofSpartina alterniflora characterized by decreased soluble protein, an increased total amino acid pool, and elevated levels of 10 individual amino acids. Increased proline and tryptophan in response to planthopper feeding could not be fully explained by protein degradation. Low degrees of soil salinity stress resulted in an increased total free amino acid pool and elevated levels of 7 amino acids. Anaerobic soil stress resulted in decreased glutamic acid and increased asparagine. Low salinity and anaerobic stress had no effect on soluble protein levels. Glycinebetaine was not affected by the stresses examined in this study.     

Water balance systems of Rangia cuneata: ionic and amino acid regulation in changing salinities

In order to study the interaction of the extracellular and intracellular osmoregulatory systems of the bivalve Rangia cuneata, we have measured blood osmotic and ionic concentrations together with intracellular free amino acid concentrations and total tissue water under identical salinity conditions. Like freshwater bivalves, the blood of R. cuneata is maintained hyperosmotic (50 mOsm) to the environment in salinities below 110 mosm by the regulation of Na⁺, Cl^-, K⁺ and Ca²⁺ concentrations. On the other hand in company with marine bivalves, R. cuneata also regulates intracellular free amino acids (FAA) as a mechanism to control cellular volume during osmotic stress over the entire non-lethal salinity range (3 to 620 mOsm). Alanine is the predominant intracellular osmotic effector. Thus, by utilizing the osmoregulatory mechanisms of both marine and freshwater bivalves, R. cuneata is able to tolerate salinities ranging from freshwater to 25 ppt and to traverse the faunal salinity boundary, known as the horohalinicum (5 to 8 ppt), controlling cell volume throughout.Please address requests for reprints to Dr. S. K. Pierce     

Aspects of nitrogen metabolism of the common mussel Mytilus edulis: Adaptation to abrupt and fluctuating changes in salinity

Mytilus edulis L. were exposed to abrupt (3015 and 1530) and fluctuating (sinusoidal 12 h cycles of 301530) changes in salinity, and the changes in the total osmoconcentration of the haemolymph were recorded. The response of nitrogen metabolism to the altered extracellular osmotic concentrations was investigated in terms of the concentrations of the total NPS (ninhydrin-positive substances) pool and the individual amino acids of the tissues, the concentration of the amino acids of the haemolymph, and the rates of excretion of ammonia and amino acids by whole individuals. The haemolymph became isosmotic with the seawater with abrupt changes in salinity, but with fluctuating salinity was slightly hyperosmotic as the salinity decreased and then slightly hypo-osmotic as the salinity increased. This resulted in a reduction in the extent of the extracellular osmotic change compared to the change in fluctuating salinity to which it was exposed. Total NPS of the tissues decreased with an abrupt decrease in salinity and increased with an abrupt increase in salinity, but a seasonal dependence of the response was indicated. The short-term response of tissue NPS to fluctuating salinity was equivocal, but with long-term exposure the concentration declined. Ammonia and amino acid excretion increased with both an abrupt decrease in salinity and fluctuating salinity and decreased with an abrupt increase in salinity. Haemolymph amino acids increased with an abrupt decrease in salinity. The increased rates of nitrogen excretion accounted for the reductions in the NPS concentrations of the tissues except in the early stages of fluctuating salinity. Taurine, aspartate, threonine, serine, glycine and arginine declined with an abrupt decrease in salinity while alanine and glutamate increased slightly. With an abrupt increase in salinity, alanine and ammonia accumulated in the tissues and then declined while the other amino acids increased slowly over a longer time-course. Similar individual amino acid responses were seen with long-term exposure to fluctuating salinity, except for taurine which did not decrease in concentration. On the basis of the changes in tissue amino acids and ammonia, it is suggested that the alanine dehydrogenase reaction is the primary nitrogen-fixing reaction in marine bivalves such as M. edulis.     

The effects of exposure to zinc and cadmium separately and jointly on the free amino acid pool of Gammarus pulex (L.)

Effects of the heavy metals zinc and cadmium on the free amino acid (FAA) pool of Gammarus pulex were studied at different metal concentrations and combinations as well as different exposure times. The dominant effect of these two metals was the reduction of most free amino acids and the whole FAA pool, except in the 10‐day low zinc and cadmium concentration exposures which resulted in a rise of free amino acid pool.

Among the free amino acids, the most sensitive to zinc exposure, were alanine, glutamic acid, arginine, and taurine; valine, leucine, asparagine, and isoleucine were among the most sensitive to cadmium. No predictable changes of individual free amino acids were shown in the mixed metals exposures. Elevation of taurine concentration was constant in seven of the eight treatments, it is suggested that this elevation may be related to the hepatopancreatic damage observed and induced synthesis of metallothioneins.     

Osmoregulation of the grass shrimp Palaemonetes pugio Exposed to polychlorinated biphenyls (PCBs).

Glycine was the most abundant free amino acid (FAA) in abdominal muscle of grass shrimp Palaemonetes pugio, accounting for over 50% of the total pool. Arginine, alanine, proline, taurine and serine were also present at relatively high concentrations. Following transfer from 17%. to 2 and 32%. S, new steady-state levels of total FAA were observed at 72 h. Total FAA and the sum of glycine, alanine and proline exhibited a positive correlation with salinity. Exposure to Aroclor 1254 did not have appreciable effects on total FAA levels, indicating that disruption of intracellular osmoregulation was not a major consequence of PCB toxicity. However, changes in individual amino acid concentrations in exposed shrimp were reflective of an altered metabolic state. Glycine, which did not show changes immediately following exposure, underwent large decreases after transfer to PCB-free water and indicated a delayed effect of PCB exposure. A correlation between salinity and the sum of glycine, alanine and proline did not exist in exposed shrimp.     

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.     

Modulation of the free amino acid pool and protein content in populations of the brine shrimp Artemia spp.

 Free amino acid (FAA) and protein content were measured in various developmental stages of Artemia franciscana, from cysts to Instar III metanauplii. In addition, decapsulated cysts of 15 Artemia populations from different localities were compared with respect to their FAA and protein content. Furthermore, the content and composition of the FAA pool were modulated by hatching the cysts at various salinities, and by enriching the nauplii with algae or a lipid-enrichment emulsion. The FAA content increased threefold from cysts to nauplii, and Instar III metanauplii contained nearly 50% taurine of total FAA. Cysts of A. franciscana were found to contain one-third the amount of FAA compared to the other Artemia species investigated. The content and pool composition of FAA was successfully modulated in 11 of 13 populations, where by the content of FAA was significantly increased when hatched at high salinity. Finally, enrichment elevated the content of FAA and changed the pool composition, thereby showing a dietary effect. Algal enrichment also increased the protein content. Received: 27 September 1999 / Accepted: 17 July 2000     

Amino acid metabolism and protein turnover in larval turbot (Scophthalmus maximus) fed natural zooplankton or Artemia

The present paper studied the influence of different food regimes on the free amino acid (FAA) pool, the rate of protein turnover, the flux of amino acids, and their relation to growth of larval turbot (Scophthalmus maximus L.) from first feeding until metamorphosis. The amino acid profile of protein was stable during the larval period although some small, but significant, differences were found. Turbot larvae had proteins which were rich in leucine and aspartate, and poor in glutamate, suggesting a high leucine requirement. The profile of the FAA pool was highly variable and quite different from the amino acid profile in protein. The proportion of essential FAA decreased with development. High contents of free tyrosine and phenylalanine were found on Day 3, while free taurine was present at high levels throughout the experimental period. Larval growth rates were positively correlated with taurine levels, suggesting a dietary dependency for taurine and/or sulphur amino acids. Reduced growth rates in Artemia-fed larvae were associated with lower levels of free methionine, indicating that this diet is deficient in methionine for turbot larvae. Leucine might also be limiting turbot growth as the different diet organisms had lower levels of this amino acid in the free pool than was found in the larval protein. A previously presented model was used to describe the flux of amino acids in growing turbot larvae. The FAA pool was found to be small and variable. It was estimated that the daily dietary amino acid intake might be up to ten times the larval FAA pool. In addition, protein synthesis and protein degradation might daily remove and return, respectively, the equivalent of up to 20 and 10 times the size of the FAA pool. In an early phase (Day 11) high growth rates were associated with a relatively low protein turnover, while at a later stage (Day 17), a much higher turnover was observed. Received: 19 March 1997 / Accepted: 14 April 1997     

Effects of starvation,season, and diet on the free amino acid and protein content of<Emphasis Type="Italic"> Calanus finmarchicus</Emphasis> females

Effects of food availability and season on the free amino acid (FAA) and total protein content of the copepod Calanus finmarchicus females were investigated in two mesocosm experiments on the Norwegian west coast in spring and autumn. Starved C. finmarchicus females showed no change in total FAA content, but the FAA pool composition changed drastically. During the first 10 days of starvation the protein content showed a moderate decline (<2 µg ind ^-1); however, during the following 21 days the total content was drastically reduced, from 63 to 9 µg ind ^-1. This supports the notion of a sequential catabolism of endogenous nutrients during starvation. In females at high food concentrations, the body protein content increased during spring, but decreased during autumn. The FAA pool composition of females differed between spring and autumn in 14 of the 18 FAA investigated. Reduced body protein content and increased proportion of essential free amino acid were observed during starvation. Similar changes were observed in females sampled at the end of the mesocosm experiments in the autumn. The results suggest that mature C. finmarchicus females are in a negative protein balance during autumn, despite high food concentrations, contributing to a lower fitness than in females maturing during the spring.     

Osmoregulation in the euryhaline flagellate Brachiomonas submarina (Chlorophyceae)

Brachiomonas submarina Bohlin (Chlorophyceae), a euryhaline marine flagellate, can osmoregulate over a wide range of external salinity. The alga exhibits maximum water content at 100% artificial seawater (ASW), and shows only a small water loss (<15%) when salinity is increased to 300% ASW. The non-aqueous volume of the cells is increased at salinities higher than 100% ASW. This is partially attributable to the accumulation of glycerol. Glycerol is the major osmoregulatory organic solute in this flagellate. The alga also shows an accumulation of amino acids in response to increased salinity. The contribution of glycerol and amino acids to intracellular osmolarity is only 9% at 10% ASW, but accounts for 49% at 300% ASW. The remainder of the osmotic balance is due to uptake and accumulation of inorganic ions, particularly sodium, potassium and chloride.     

Implications of salinity fluctuation for growth and nitrogen metabolism of the marine diatom Ditylum brightwellii in comparison with Skeletonema costatum

In 1987 effects of salinity fluctuations on growth of Ditylum brightwellii (West) Grunow, isolated from the Eastern Scheldt estuary (SW Netherlands) in 1981, were studied. D. brightwellii was grown in a 12 h light: dark cycle at constant salinity in brackish media. Ammonium-limited cultures were subjected to a salinity fluctuation. By decreasing the salinity to 4.8 photosynthesis and cell division were inhibited; cells were deformed. Protein and carbohydrate contents increased slightly, dark respiration was stimulated and cellular levels of glucose decreased at low salinity; this indicated a possible role of sugars in osmoregulation. Ammonium was accumulated in cultures, amino acids may have been stored; the role of the vacuole as a storage compartment was discussed. Both the ammonium uptake capacity and the affinity for ammonium decreased. Nitrogen limitation was relieved in the transient state. [With the activity of the nitrogen assimilation enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) being uninhibited by lower salinity.] Recovery from hypo-osmotic stress during a salinity increase was initiated by stimulated photosynthesis; chlorophyll a increased, but persistant contractions of cytoplasm (with chloroplasts) may have delayed cell growth. The glutamate dehydrogenase (GDH) activity decreased further whereas the cellular level of alanine increased in the presence of large ammonium pools; this may indicate a temporary activity of ADH (alanine dehydrogenase). Skeletonema costatum (Greville) Cleve, recovered faster from hypoosmotic stress than did D. brightwellii. Due to an osmotic shock from 13.6 to 7.1 S both species excreted amino acids and glucose; S. costatum accumulated more glucose, D. brightwellii accumulated more amino acids. S. costatum may with the competition for nitrogen in waters with an unstable salinity; it will replace D. brightwellii.Contribution no. 427 Delta Institute for Hydrobiological Research, Yerseke, The Netherlands     

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.     

Influence of anoxia on adaptation of euryhaline polychaetes to hyposmotic conditions

The metabolic reactions ofArenicola marina andNereis diversicolor (both euryoxic and euryhaline polychaetes from the intertidal) to hyposmotic shock under anaerobic conditions were investigated in 1987–1988 using specimens from the East Frisian Wadden Sea. Although reductions in salinity were within the limits of tolerance under normoxic conditions, during anoxia they resulted in increased stress and a significant reduction in survival time. Both species were unable to sufficiently intensify anaerobic energy production to match the increased requirement for energy during adaptation to hyposmotic conditions. Moreover, as an adaptation to anoxic conditions, worms reduced their metabolic activity after the first 6 h. Thus, specimens remained more swollen than at the same reduction in salinity under normoxic conditions. InA. marina only the extracellular volume was reduced all be it to a limited extent.N. diversicolor probably reacted in the same or similar way. In both species, cells remained swollen. The concentrations of cellular free amino acids were not reduced; that means the main mechanism of cellular volume regulation was not activated under these conditions.     

甜椒穴盘苗对不同程度水分胁迫-复水的生理生化响应

对温室甜椒穴盘苗进行水分处理,探讨了不同程度水分胁迫-复水过程中叶片生理生化特性的变化.叶片相对含水量(RWC)、渗透势随基质水分减少而降低,渗调能力逐步增强.POD、SOD、CAT酶活性随水分减少而上升,SOD对水分胁迫最敏感,复水后主要由POD、CAT负责清除H2O2和过氧化物.游离氨基酸、脯氨酸随基质水分减少急剧上升,复水后大幅下降,可能为水分胁迫下主要渗透调节物质.水分胁迫下,甜椒叶片可溶性蛋白主要降解为氨基酸的形式参与渗透调节.RWC降至45%(停水后d3)时,可溶性糖显著积累,但明显晚于氨基酸,但它基础含量高,主要在较为严重的水分胁迫时发挥作用.尽管水分胁迫使保护酶活性和渗透调节能力均提高,但任何程度的胁迫均不可避免伤害幼苗.穴盘苗生产中,建议“水分胁迫锻炼”时间以不超过3d逐步达到RWC≥45%为宜.图4表2参12     

Tolerance of Mediterranean seagrasses (<Emphasis Type="Italic">Posidonia oceanica</Emphasis> and <Emphasis Type="Italic">Cymodocea nodosa</Emphasis>) to hypersaline stress: water relations and osmolyte concentrations

The present study examines for the first time the effects of increased salinity on water relations and osmolyte (carbohydrates and amino acids) concentrations in two Mediterranean seagrass species, Posidonia oceanica and Cymodocea nodosa, which are adapted to growth in environments with contrasting salinity and have a known differential sensitivity to alterations in ambient salinity. The specific aim was to obtain insights into their respective capacities to cope with natural or anthropogenically induced (e.g. desalination plants) hypersaline stress and its ecological implications. To this end, large plant fragments of both seagrass species were maintained for 47 days in a laboratory mesocosm system under ambient salinity (37 psu; control) and three chronic hypersaline conditions (39, 41 and 43 psu). Analyses of leaf-tissue osmolality indicated that both species followed a dehydration avoidance strategy, decreasing their leaf water potential (Ψ_w) as the external salinity increased, but using different physiological mechanisms: whereas P. oceanica leaves exhibited a reduction in osmotic potential (Ψ_π), C. nodosa leaves maintained osmotic stability through a decrease in turgor pressure (Ψ_p) probably mediated through cell-hardening processes. Accordingly, the concentrations of soluble sugars and some amino acids (mainly Pro and Gly) suggested the activation of osmoregulatory processes in P. oceanica leaves, but not in C. nodosa leaves. Osmotic adjustments probably interfered with leaf growth and shoot survival of P. oceanica under hypersaline stress, whereas C. nodosa showed a more efficient physiological capacity to maintain plant performance under the same experimental conditions. These results are consistent with the more euryhaline ecological behaviour of C. nodosa and contribute to understanding the high vulnerability shown by P. oceanica to even mild increments in seawater salinity.     

Seasonal changes in nucleic acids,amino acids and protein content in juvenile Norway lobster (<Emphasis Type="Italic">Nephrops norvegicus</Emphasis>)

The objective of this study was to describe the seasonal variations in nucleic acid contents and amino acid profiles in the muscle of juvenile Nephrops norvegicus. RNA and protein contents, and RNA:protein and RNA:DNA ratios varied significantly between seasons, being highest in spring and lowest in autumn/winter ( P<0.05). Though DNA content increased significantly from autumn to summer ( P<0.05), protein:DNA ratio did not show significant seasonal variations ( P=0.05). In respect to protein-bound amino acid content (BAA), a significant increase was observed from winter to summer ( P<0.05). Both essential (EAA) and non-essential amino acids (NEAA) increased significantly (27.6% and 27.8%, respectively; P<0.05), mainly due to the considerable increase in arginine and proline (59.1% and 225.2%, respectively; P<0.05). A significant decrease was observed in the free amino acid content (FAA) from winter to summer ( P<0.05); and a higher percentage decrease occurred in free non-essential (FNEAA; 27.9%) in comparison to free essential amino acids (FEAA; 21.8%). The significant increase in RNA and BAA contents from winter to spring may be related to protein synthesis. On the other hand, the lowest values obtained in winter may be due to a reduction in feeding activity; in this period the muscle protein must be progressively hydrolysed, which is evident with the higher FAA content. The liberated amino acids enter the FAA pool and become available for energy production.     

Ultrastructural changes in the lysosomal-vacuolar system in digestive cells of Mytilus edulis as a response to increased salinity

The effects of increasing salinity on the ultrastructural morphology of the lysosomal-vacuolar system in digestive cells of the common mussel Mytilus edulis were investigated in order to relate structural changes to previous biochemical and cytochemical observations. After 3 h of increased salinity, from 21 to 35%., the digestive cells showed an increase in numbers of heterolysosomes. There was some evidence of digestive cell breakdown, the contents forming membrane-bound bodies and being released into the tubule lumen. After 12 h of increased salinity, heterolysosomes were prevalent in the digestive cells. There was increased evidence for digestive-cell breakdown, many of the tubule lumina being packed with membrane-bound bodies. It is concluded that increasing salinity from 21 to 35%. stimulates the lysosomal-vacuolar system, as a result of autophagocytosis or apoptosis; this is consistent with the hypothesis that intracellular, lysosomally-mediated, catabolism of proteins is a source for free amino acids during the adaptation of mussels to increased salinity.     

Inorganic nitrogen metabolism in marine bacteria: The intracellular free amino acid pools of a marine pseudomonad

The marine pseudomonad bacterium PL₁ contains an intracellular pool of free amino acids which consist mainly of glutamate with small amounts of glutamine and aspartate when grown in a nutrient medium containing 0.2 M NaCl. When the NaCl concentration of the growth medium is increased to 0.8 M, proline becomes a major component of the intracellular pool together with glutamate—at this molarity and under suitable nutrient conditions these amino acids comprise 20% of total bacterial amino acid nitrogen. When grown in a nutrient growth medium containing a constant level of NaCl, the intracellular pool size can vary by a factor of 4 depending on the concentration of carbon and nitrogen in the medium. Experiments show that the amino acid pool can act as a nitrogen reserve but has little function as a carbon reserve. At high NaCl concentrations there is a marked dependence for growth on the presence of sufficient potassium in the medium. However, no correlation between K⁺ and glutamate concentration in either nitrogen or K⁺-limited cultures has been found. None of the enzymes associated with glutamate biosynthesis was influenced by NaCl levels between 0.2 and 0.5 M. Neither Na⁺ or K⁺ stimulated the activity of these enzymes when tested in vitro.     

Physiological and behavioural responses of the intertidal scavenging gastropod Nassarius festivus to salinity changes

Physiology (oxygen consumption and ammonia excretion) and behaviour (feeding and activity) of the intertidal gastropod Nassarius festivus (Powys) at five different salinities [15, 20, 25 (control), 30 and 35‰] were studied for 4 weeks. Oxygen consumption and ammonia excretion rates were reduced immediately after salinity was either elevated or reduced. Subsequently, both rates were increased while the O:N ratio was decreased at all salinities, including the control, in the first 2 weeks and then levelled off. Such changes were probably attributed to osmotic adjustment and reproductive activity. Activity and feeding were reduced at low salinity, particularly in the first week. Reproductive output, in terms of the total number of egg capsules and the mean number of eggs per capsule, was also lowered at reduced salinities. Nevertheless, individuals at all salinities are able to maintain a positive energy balance. Results are discussed with respect to the distribution of N. festivus in Hong Kong waters. Received: 14 April 1997 / Accepted: 9 May 1997     

Regulation of body dimethylsulfoniopropionate (DMSP) content by the copepod Temora longicornis: a test of four mechanisms

Copepods contain dimethylsulfoniopropionate (DMSP) in their tissues in addition to the DMSP in their gut contents and therefore constitute an additional pool of DMSP in the ocean. In the estuarine copepod Temora longicornis Müller, this dynamic pool of DMSP is reduced when external salinity decreases. In the present study the mechanism(s) used by T. longicornis to adjust its DMSP content were examined. Four possible mechanisms were tested in experiments conducted between July 1997 and May 1999: (1) DMSP cleavage, (2) demethylation, (3) conversion to methionine, and (4) release from the body. Tissue extracts of T. longicornis did not exhibit the ability to cleave or demethylate DMSP. In incubation experiments, when external salinity decreased, T. longicornis individuals reduced their DMSP content without increasing their methionine content. The total amount of DMSP in the incubation vials was conserved regardless of salinity. The copepods retained most of the DMSP in their tissues in 30‰ water, but when salinity was reduced to 20‰, the copepods released 41 to 55% of their body DMSP to the surrounding medium; 89 to 91% of the total DMSP was recovered. This suggests that estuarine copepods represent a reservoir of DMSP, which is released as dissolved DMSP upon salinity decline. Therefore, while osmoregulation by these copepods may have little impact on the chemical properties of DMSP, it will affect the partitioning of DMSP between particulate and dissolved phases in the water column. Received: 20 August 1999 / Accepted: 28 March 2000