Purification and partial characterisation of phenoloxidase from the colonial ascidian Botryllus schlosseri

Phenoloxidase (PO) from the colonial ascidian Botryllus schlosseri was purified using two different chromatographic strategies. A three-step purification was developed in order to maintain enzyme activity, whereas an easier purification procedure was adopted to obtain enough PO for the production of specific polyclonal antibodies. The enzyme showed optimal pH and temperature values of 7.0 to 7.5 and 35 °C, respectively, and a K _m value of 4.62 ± 0.76 mM was estimated using l-DOPA as substrate. A molecular weight of 160 kDa was determined after SDS-PAGE under non-reducing conditions. The addition of the reducing agent β-mercaptoethanol caused the disappearance of the 160 kDa band and the appearance of a new band at 80 kDa, suggesting that active PO is a dimer and the two subunits are linked by disulphide bridges. Received: 14 December 1998 / Accepted: 24 August 1999     

NADP+-dependent glutamate dehydrogenase from Acropora formosa: purification and properties

As an initial step in our study of nitrogen metabolism in the coral/algal symbiosis we have purified glutamate dehydrogenase (EC 1.4.1.4) to homogeneity from polyp tissue of the staghorn coral Acropora formosa collected from Magnetic Island (North Queensland) in 1985–1986. The purified enzyme had a specific activity of 78 U mg^-1. The native enzyme had a relative molecular weight, M _r, of 360 000 (±20 000), and appears to be a hexamer with subunits of M _r=56000 (±3 000). Like the enzyme from other coelenterates, the coral glutamate dehydrogenase (GDH) was absolutely specific with respect to the coenzyme substrate (NADP⁺/NADPH), and was insensitive to allosteric regulation by nucleotides; unlike other coelenterate GDHs, the coral enzyme was absorlutely specific for ammonium as amino group donor in the reductive amination reaction, and major differences in kinetic properties were apparent. Linear Michaelis-Menten kinetics were observed for the substrates a-ketoglutarate, NADPH and NADP⁺, the K _m values being 0.93, 0.11 and 0.03 mM, respectively. However glutamate dehydrogenase displayed biphasic kinetics with respect to l-glutamate and ammonium, indicating two apparent K _m values (18 and 81 mM for l-glutamate and 9.2 and 416 mM for ammonium). The enzyme also exhibits Scatchard plots, Hill coefficients and cooperativity indices characteristic of enzymes displaying negative cooperativity.     

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.     

Temporal and spatial variation in mountain birch foliar enzyme activities during the larval period of <Emphasis Type="Italic">Epirrita autumnata</Emphasis>

Summary. Mountain birches are deciduous trees consisting of several clonal and partly autonomous ramets. Autumnal moth, Epirrita autumnata, is an extremely harmful pest of mountain birches that during outbreak years may cause the death of trees over large areas. During the larval development of E. autumnata, leaf characters and nutritional suitability vary considerably both temporally and spatially among and within mountain birches. Compared to variation between trees, however, the extent of within-tree variation is known for only a limited number of potentially defensive traits. Plant oxidases, polyphenoloxidases (PPOs) and peroxidases (PODs) may play an important part in dictating the suitability of a tree for plant-eating animals, including E. autumnata. In this study, we observed changes in the activities of oxidative and antioxidative enzymes along leaf development during the larval period of E. autumnata. We also estimated the relative amounts of spatial variation among and within trees. Our results show that POD activities were remarkably high during the rapid elongation growth phase of leaves but decreased with leaf maturation. Thus PODs are suggested to take part in leaf elongation growth. Time and within tree variation accounted for the most of variation in POD activities whereas there was no among tree variation. In contrast, the activities of antioxidative CATs, which act as antagonists to PODs, were low in young leaves and increased towards leaf maturation, reflecting an increase in the oxidative status of source leaves. Within tree variation accounted for the most of the variation in CAT activities. The absolute PPO activities decreased along leaf growth due to the dilution effect whereas the specific activity of PPOs, which has been shown to be defensive against E. autumnata larvae, did not vary temporally that might reflect the importance of these enzymes in the defence of birches. Acidic PPOs showed marked within- and among-tree variation, which may impact herbivores performance on certain genotypes and increase larval mobility within the tree canopy.     

Light quality in relation to growth,photosynthetic rates and carbon metabolism in two species of marine plankton algae

The effect of light quality on growth, photosynthesis and carbon metabolism in two species of marine algae,Cyclotella nana (Hustedt) andDunaliella tertiolecta (Butcher), was examined. Relative growth constants forC. nana were 0.37, 0.29 and 0.25 in blue, white and green light, respectively. Corresponding constants were 0.41, 0.31 and 0.29 forD. tertiolecta. Photosynthetic rates in both species were higher in blue light and lower in green light compared with white light of the same intensity. More than 60% of¹⁴C assimilated byC. nana orD. tertiolecta grown in blue or green light was incorporated into the ethanol-insoluble fraction, compared with 10 to 30% in this fraction in white light. The relative importance of the various components within this fraction was independent of light quality. Although less¹⁴C was assimilated into the ethanol-soluble fraction in blue or green light, there was a relative increase in some amino acids and organic acids in this fraction and a decrease in sugars and sugar phosphates relative to white light of the same intensity. These differences were independent of light intensity, photosynthetic rate and cell density in the cultures.     

Heterotrophic nitrogen fixation (acetylene reduction) during leaf-litter decomposition of two mangrove species from South Florida, USA

Heterotrophic nitrogen-fixation (acetylene reduction) was measured during decomposition (under dark conditions) of Rhizophora mangle L. and Avicennia germinans (L.) Stearn leaf litter. Nitrogen-fixation rates in leaf litter increased following 24 d incubation, then decreased after ≃44 d for both species. Maximum rates of 66.2 and 64.6 nmol C₂H₄ g⁻¹ dry wt h⁻¹ were reached by R. mangle and A. germinans leaf litter, respectively. Higher fixation rates of leaf litter were associated with an increase in water content and sediment particles on leaf surfaces of both species. Rates of nitrogen fixation by diazotrophs attached to sediment particles were not significantly different from zero. With additions of d-glucose, ethylene production rates increased by factors of 625-, 34- and 7-fold for sediment, R. mangle and A.␣germinans leaf litter, respectively, compared to rates prior to enrichment. These organically enhanced rates of nitrogen fixation on leaves could be accounted for by increased activity associated with attached sediment particles and not the leaf material. Total phenolics [reported as tannic acid equivalent (TAE) units] decreased nitrogen-fixation rates when added to d-glucose-enriched sediment at >20 mg TAE l⁻¹. Phenolic compounds could explain the initial lag in rates of nitrogen fixation during leaf-litter decomposition of R. mangle (initial content of 110.8 mg TAE g⁻¹ dry wt), but not of A. germinans (initial content of 23.4 mg TAE g⁻¹ dry wt). The higher phenolic content and reportedly lower carbon substrate of R. mangle did not result in species-specific differences in either the magnitude or temporal pattern of nitrogen fixation compared to A. germinans leaf litter. We conclude that the availability of organic substrates leached from the leaf litter along with colonization by the heterotrophic diazotrophs (as indicated by sediment accumulation) controls nitrogen-fixation rates in a similar manner in the leaf litter of both species. Received: 8 August 1997 / Accepted: 4 December 1997     

N-acyl homoserine lactone production by bacteria within the sponge Suberites domuncula (Olivi, 1792) (Porifera, Demospongiae)

Many bacteria live in close association with sponges. Within these consortia, molecules of communication such as quorum-sensing and hormone-like molecules may occur in order to regulate the partnership. Of particular interest, bacterial N-acyl-l-homoserine lactones (AHLs) were screened in supernatants from Suberites domuncula-associated bacteria using an E. coli bioluminescent reporter system. These sponge-associated bacteria were beforehand isolated on several media supplemented or not with a sponge extract to attempt to isolate sponge-specific bacteria. Out of 81 AHL-producing bacteria, three strains requiring sponge extract to grow were selected for AHL characterization. The in vitro produced AHLs, that is, in bacterial culture supernatants, were identified as N-(3-butanoyl)-l-homoserine lactone and N-(3-oxododecanoyl)-l-homoserine lactone and quantified using LC–ESI–MS/MS. The in vivo production of AHLs by sponge-associated bacteria has also been demonstrated in a healthy host for the first time: N-(3-oxododecanoyl)-l-homoserine lactone, N-(3-hexanoyl)-l-homoserine lactone, and N-(3-heptanoyl)-l-homoserine lactone. This AHL production in sponges may suggest a potential role of these molecules between sponge-associated bacteria and/or between sponge-associated bacteria and the sponge.     

Sodium-dependent amino acid transport in the chlorophyte Platymonas subcordiformis

Sodium-dependent transport of alanine and serine by Platymonas subcordiformis was demonstrated by evaluating the kinetics of entry of these substrates over a range of concentrations of ambient sodium. The reciprocal of the concentration at which entry rate was half maximal (K _t ) was linearly related to ambient sodium concentration; maximum entry (V _max) was not affected. Entry of labeled amino acids as measured by determining radioactivity in the medium was close to but slightly less than rates of net entry as determined by high-performance liquid chromatography. This difference presumably reflects extrusion of labeled carbon by the cells in a form not detectable by the chromatographic techniques employed. The coupling coefficient (sodium ions per amino acid molecule) for entry of alanine and serine was evaluated and determined to be 2. Methylaminoisobutyric acid was not taken up by P. subcordiformis and had no inhibitory effect on uptake of alanine or serine. B-2-aminobicycloheptane-2-carboxylic acid competitively inhibited uptake of both substrates. The internal sodium concentration of P. subcordiformis was measured, and the maximum gradient energetically favorable for amino acid transport was calculated from data in the present work and drawn from the literature. Assuming that uptake of amino acids is strictly sodium-dependent, an amino acid concentration gradient of the order of 10⁶ (cell:medium) can be achieved. This concentration differential permits net uptake of amino acids by P. subcordiformis from ambient amino acid levels in the nanomolar range.     

镉对单环刺螠非特异性免疫及组织蓄积的影响

为探究镉(Cd)对海洋动物的毒性效应,考察了不同质量浓度(0.005、0.05和0.5 mg·L~(-1)) Cd处理对单环刺螠相关免疫生理指标及主要组织中Cd蓄积量的影响。结果表明:Cd胁迫下,血细胞密度(density of hemocyte, DHC)先升后降再升,酚氧化酶(phenoloxidase, PO)活力先升后降,溶菌酶(lysozyme, LSZ)活力先降后升再降,至处理96 h,处理组DHC均显著高于对照组(P0.05),总蛋白含量变化不显著;除低浓度胁迫组(0.005 mg·L~(-1))PO活力与对照组相近外,其他处理组的PO和LSZ活力均显著低于对照组(P0.05)。超氧化物歧化酶(superoxide dismutase, SOD)活力和丙二醛(malondialdehyde, MDA)含量总体呈上升趋势,过氧化氢(H_2O_2)含量先升后降再升,至胁迫96 h,各处理组SOD活力、MDA和H_2O_2含量均显著高于对照组(P0.05)。总之,Cd胁迫对单环刺螠非特异性免疫影响显著,Cd的短期胁迫可刺激DHC、PO和LSZ升高,免疫力增强,而高浓度Cd的胁迫时间较长会造成机体解毒能力下降,持续刺激机体产生过氧化反应,MDA含量升高,可作为单环刺螠在Cd长期胁迫下的免疫检测指标。不同胁迫浓度组单环刺螠肌肉和消化道中的Cd含量随胁迫时间均呈上升趋势,且存在显著的时间-剂量-效应关系(P0.05),其蓄积量水平为:消化道肌肉,具有组织特异性。     

The relation between spontaneous activity cycles and diurnal rhythms of metabolism in the polychaetous annelid Glycera dibranchiata

The oxygen consumption rate of the bloodworm Glycera dibranchiata Ehlers was measured 4 times each day. Aerobic metabolism during daylight hours is significantly greater than at night, regardless of the phase of the spontaneous activity cycle. There is no reason to suppose that increased metabolism reflects an overall increase in motor activity.Supported by National Science Foundation research grant GB-6884.     

Effect of amino acids on the swimming activity of newly hatched turbot larvae (Scophthalmus maximus)

The swimming behaviour of newly hatched turbot (Scophthalmus maximus L.) larvae was observed in artificial seawater (ASW) and in solutions of 21 l-amino acids at a concentration of 10⁻⁵  M. The behaviour of 20 larvae was analysed in each solution. Each larva was observed for 1 min. Individual movements were recorded on video and analysed using a computer-assisted program. The larvae swam in convoluted, randomised three-dimensional paths, rested and started swimming again. There were large variations in the swimming behaviour of turbot larvae during ontogeny. In ASW the mean frequency of trajectories longer than a body length of 4 mm larva⁻¹ min⁻¹ increased from 1.2 at Day 1, to 10 at Day 4. Analysing the data (Dunnett's method) revealed that the frequency of swimming trajectories increased in the presence of glycine, histidine and glutamine, and decreased in the presence of proline. The total distance swum increased for glycine but decreased for proline. The threshold concentration for glycine detected by turbot larvae was 10⁻⁵  M. The straightness index did not change in the presence of the amino acids. The possible role of these changes in behaviour is discussed. Received: 12 June 1997 / Accepted: 13 January 1998     

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.     

Molecular mechanisms of adaptation to low temperature in marine poikilotherms. Some regulatory properties of dehydrogenases from two arctic species

The properties of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from gill tissue of the tanner crab Chionocetes bairdi, and lactate dehydrogenase (LDH) and glyceraldehyde dehydrogenase from skeletal muscle of C. bairdi and the yellowfin sole Limanda aspera were examined over the physiological temperature range of the animals. Both animals were obtained in the Bering Sea in winter, and their enzymes appear to be remarkably cold-adapted. Affinity of sole LDH for substrate appears to increase with decreasing temperature, thus keeping reaction rate essentially independent of temperature at physiological concentrations of the substrate. Calculated values of activation energy are low, in keeping with the argument that organisms from cold environments have enzymes with a reduced energy of activation. In addition, Hill plots of the substrate saturation curves for lactate dehydrogenase from muscle of sole indicate that there is a facilitation of allosteric behaviour at low temperatures. Maximum affinity of sole LDH for substrate in the absence of univalent cations occurs at 3°C, while in the presence of 150 mN K⁺, it occurs between 0° to-2°C. The effects of Mg²⁺ on enzyme activity appear to be determined by concentration of substrate and temperature. Thus, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are stimulated more effectively by Mg²⁺ at low temperature and at low substrate levels whereas, at high concentrations of substrate, they are relatively independent of the bivalent cation. All four dehydrogenases are affected by the univalent cations Na⁺, K⁺ and NH₄ ⁺ in a manner which appears to be determined, in part at least, by concentration of substrate and by temperature. These findings suggest mechanisms for the maintenance and regulation of enzyme activity in poikilothermic tissues at low and changing temperatures.     

Glycohistochemistry of a marine sponge, Chondrilla nucula (Porifera, Desmospongiae), with remarks on a possibly related antimicrobial defense strategy and a note on exopinacoderm function

Based on carbohydrate histochemistry, including the use of lectins, and TEM, the study describes the distribution of terminal sugars in different structures of the demosponge Chondrilla nucula. The results of the general and specific carbohydrate histochemical approaches confirmed the presence of acidic and neutral glycoconjugates in the cells, and, with declining amounts from the ectosome to the mesohyl, in the extracellular matrix (ECM). AB-PAS staining indicated acidic complex carbohydrates particularly in the exopinacoderm, and more neutral ones in the cells and the ECM of the mesohyl. The PO-lectins applied demonstrated a general spectrum of free saccharide residues (α-d-mannose, α-/β-d-N-acetylglucosamine, α-d-N-acetylgalactosamine, α-d-galactose, β-d-galactose) in both sponge parts; α-l-fucose was only distinct in the ectosome. Sialic acids [siaα(2,3)-galactose, siaα(2,6)-N-acetylgalactosamine] were dominant in the very thin exopinacoderm, indicating O-linked high molecular weight glycoproteins. In this way a glycophysiologically ‘rigid’ outer mucus cover is developed as protection against mechanical hazards. Some of the free sugars (α-d-mannose, N-acetylglucosamine, N-acetylgalactosamine β-d-galactose, α-l-fucose) are known to prevent the adherence of different bacteria and fungi to cellular surfaces. Thus a high concentration of such sugars, may impede massive attacks of micro-inhabitants on mobile sponge cells, pinacocytes, and the exopinacoderm layer.     

Larval growth and settlement of the European oyster (Ostrea edulis) as a function of food quality measured as fatty acid composition

Larval growth rate and settlement of the European flat oyster Ostrea edulis were experimentally studied as a function of the composition of dietary fatty acids. Diets differing in fatty acid composition were composed by mixtures of the microalgae Isochrysis galbana, Pavlova lutheri and Chaetoceros calcitrans. Fatty acid content in the tissue of the feeding larvae, analyzed by gas chromatography and mass spectrometry, reflected the composition in the diet. Larval growth rate was significantly correlated to the three omega-3 polyunsaturated fatty acids (PUFA) C18:3, C18:4 and C22:6, with minor differences for neutral and polar lipids. No relation between growth rate and the omega-3 PUFA C20:5 was detected, a PUFA often implied as essential for bivalves. It is suggested that naturally occurring variability in fatty acid composition may constrain larval growth. In settlement experiments in both still water and flume flow little substrate selectivity was found for some contrasting substrates. It is concluded that differences in dietary fatty acids may explain as much of settlement success as the variability of substrates. Received: 12 October 1998 / Accepted: 6 April 1999     

Isolation of novel metal-binding proteins distinct from metallothionein from spotted seatrout (Cynoscion nebulosus) and Atlantic croaker (Micropogonias undulatus) ovaries

Metal-binding proteins were isolated from ovaries of the spotted seatroutCynoscion nebulosus and the Atlantic croakerMicropogonias undulatus collected in 1988 near Port Aransas, Texas, USA. Gel-filtration analysis of spotted seatrout trout ovarian cytosolic fraction on Sephadex G-75 revealed the presence of three zincbinding protein fractions. A major zinc/calcium-binding protein fraction had a low molecular weight (M _r)(6 000 to 10 000), similar to mammalian hepatic metallothionein (MT). All the metals were displaced from this fraction following saturation with exogenous cadmium. After exposure of Atlantic croaker to 2 mg cadmium l^–1 seawater for 2 mo, the majority of the cadmium in the ovarian cytosolic fraction was associated with a similar low molecular weight protein fraction. These proteins were further purified by heat treatment and sequential acetone precipitation. Three isoforms were isolated by reversephase high-performance liquid chromatography. All the isoforms were found to be distinct from mammalian MT, based on amino acid composition. The major isoform contained low amounts of cysteine (approximately 5 residues per molecule) and aromatic amino acids, compared to high amounts of cysteine (typically 17 to 20 residues/molecule) and a lack of aromatic amino acids for mammalian MT. All the ovarian protein isoforms contained more glutamate than mammalian MT. The spotted seatrout and Atlantic croaker ovarian isoforms showed a high degree of homology with metal-binding proteins isolated from mammalian gonadal tissues. The results suggest a physiological role for these metal-binding proteins in developing vertebrate ovaries as well as an involvement in the sequestration of cadmium following environmental exposure.     

Heterotrophic nutrition of the marine pennate diatom Nitzschia angularis var. affinis

The nutritional pattern for heterotrophic growth of Nitzschia angularis var. affinis (Grun.) Perag. is more complex than for other diatom species studied previously. This species grew slowly in the dark in the presence of single amino acids, either glutamate or alanine; other amino acids when supplied singly were not used as substrates. Carbon from glutamate was converted to cell carbon with an efficiency of 43%. Glutamine was inhibitory both in the light and in the dark, and aspartate inhibited heterotrophic growth on glutamate. Glucose and tryptone supplied singly did not support heterotrophic growth, but when combined, together they allowed for rapid growth of N. angularis (generation time of 16 h). Glucose in combination with glutamate, alanine, aspartate, or asparagine (but not with any other amino acids) also supported growth in the dark, at a rate considerably more rapid than with glutamate alone. In the presence of excess glucose and limiting concentrations of glutamate, approximately 50% of the cell carbon for heterotrophic growth came from glucose, while in combination with tryptone about 25% of the cell carbon came from glucose. Amino acids were taken up by cells grown either photoautrophically or in the dark in the presence or absence of organic substrates; uptake rates were some-what higher for dark-grown than for light-grown cells. Glucose was taken up only by dark-grown cells; induction of a glucose uptake system in the dark required the presence of glutamate but not of glucose. The rates of uptake of glutamate and glucose by cells incubated in the dark with glutamate were sufficiently high to account for the observed rates of growth on these substrates in the dark. The uptake systems of N. angularis have relatively high affinities for glucose (K _s =0.03 mM) and glutamate (K _s =0.02 mM).Contribution No. 890 from the Department of Oceanography, University of Washington, Seattle, Washington 98195, USA.     

Ammonia production and kinetic properties of glutamate dehydrogenase in the sipinculid Phascolosoma arcuatum exposed to anoxia

The amounts of total NH ₄ ⁺ detected in the external media in which Phascolosoma arcuatum had been exposed to various periods of anoxia were significantly greater than those in which the worms were exposed to normoxia for a similar period. The increased NH ₄ ⁺ production by P. arcuatum during anoxic exposure was unlikely to be due to an increased catabolism of adenine nucleotides or urea. In contrast, there were significant decreases in the concentrations of several free amino acids in the coelomic plasma and body tissues of individuals during the 48 h of anoxic exposure. The amount of NH ₄ ⁺ produced by the anoxic P. arcuatum could be accounted for by the decreases in the concentrations of aspartate or glycine. Increases in the catabolism of free amino acids (FAA), leading to the increased production of NH ₄ ⁺ , in P. arcuatum during anoxia were supported by the detection of significant changes in the kinetic properties of glutamate dehydrogenase (GDH), in the deaminating direction, from worms exposed to anoxia for 48 h. The apparent increase in the affinity of GDH from the anoxic worm to glutamate would bring about a greater deaminating activity at physiological concentrations of ths substrate. P. arcuatum used in these experiments were collected from the mangrove swamp at Mandai, Singapore between 1990 and 1993.     

Abundance and diversity of the phyllosphere bacterial communities of Mediterranean perennial plants that differ in leaf chemistry

Summary.  We studied the epiphytic bacterial communities of the summer leaves of eight perennial species naturally occurring in a Mediterranean ecosystem. The species differ in essential-oil content (from rich in essential oil to non-producers) and composition, and also in life form (from herbaceous species to tall shrubs). We compared the epiphytic bacterial communities on the basis of (i) their abundance, (ii) their metabolic profile (derived by use of the BIOLOG Ecoplate system) and (iii) richness and diversity of substrates that they use, as a measure of functional diversity. Among all species, the aromatic Melissa officinalis was the most abundantly colonized. The bacterial communities on the leaves of the aromatic Myrtus communis, Calamintha nepeta and Melissa officinalis, and also of Cistus incanus catabolized all 31 substrates offered; those on the evergreen-sclerophyllous species, Arbutus unedo and Quercus coccifera, catabolized only 14 and 17 substrates, respectively. Carbohydrates were consistently used abundantly by all communities, whereas carboxylic acids were most variably used. On average, the group of aromatic plants scored higher regarding bacterial abundance, and richness and diversity of substrates used by the bacterial communities on their leaves; the lowest values for both substrate-use indices were recorded in A. unedo. Bacterial abundance or richness or diversity of substrates used did not vary with leaf oil content. Abundance was positively correlated with both substrate-use indices. Results support claims that the antimicrobial effects of essential oils are not exerted so much under natural conditions as reports based on biassays with pathogens usually show. Although essential oils play a part in the microbial colonization of the phyllosphere, it is not likely that inhibition of phyllosphere bacteria is essential oils’ primary role, at least in the Mediterranean environment.     

Hepatopancreas mitochondria from Mytilus edulis:

A method for the isolation of intact coupled mitochondria from the hepatopancreas of Mytilus edulis has been developed. These mitochondria prefer lipid substrates and accept a range of fatty acid chain lengths. Acetyl carnitine is also oxidized at high rates. Evidence for the presence of the tricarboxylate and the dicarboxylate transporters is presented. The absence of the pyruvate transporter or inhibition of pyruvate dehydrogenase is indicated by the absence of pyruvate oxidation. Glutamate is oxidized at high rates with a pH optimum of 7.5 based on the RCR. Cysteine oxidation may indicate the presence of the enzymes involved in the synthesis of taurine. Ornithine transport is also indicated, implying the existence of the urea cycle. Alanine, glycine, proline, asparagine, arginine, aspartate and citrulline were not oxidized at detectable levels. The osmotic optimum for the oxidation of glutamate extends from 600 to 1000 mOsm l^-1. The state 3 rate is more profoundly affected by the osmolarity than the state 4 rate. The role of osmolarity in the control of oxidation of amino acids during volume regulation of osmotically stressed M. edulis is suggested. Mitochondrial volume changes correlate with the optimal range of osmolarity for glutamate oxidation based on the RCR.