Metabolic responses of the limpet Patella caerulea (L.) to anoxia and dehydration.

This study examined the metabolic responses of the limpet Patella caerulea (L.) to anoxia and dehydration, attempting to tease apart the effect of these two stressful conditions, which are often not clearly distinguished in experiments. Specimens were exposed to: (a) oxygen-free sea water; (b) oxygen-saturated water (controls); (c) low-humidity air (55% RH); and (d) high-humidity air (100% RH). For each of the treatments, we took samples of five specimens after 6 and 18 h of exposure to the experimental conditions and determined the concentrations in the foot muscle of succinate, acetate, propionate, aspartate and alanine. Exposure to anoxia caused an increase in the levels of succinate (6 and 18 h) and acetate and propionate (18 h) with respect to control specimens. Anoxia also induced a decrease of aspartate and an increase of alanine after both 6 and 18 h. Exposure to both moist and dry air generally had negligible effects on the organic acid levels. Aspartate content increased after 18 h of exposure to moist air. Alanine levels also increased with respect to control values after exposure to air, with dry air having the more pronounced effect. In conclusion, the results of this study suggest that one should be cautious when inferring anaerobic conditions from the simple exposure of intertidal species to air, without strict control of the experimental conditions and actual respiration rates.     

Microbial responses to salt-induced osmotic stress

Summary The construction and assembly of a model root region is described. The model was used to manipulate the soil matrix, soil microorganisms, and to simulate release of root exudates. The design of the apparatus facilitated long-term, direct microscopic observations of microbial activity in soil and on artificial roots. Preliminary studies indicate that microbial responses to osmotic stress and to changes in components of exudate solutions are easily monitored.     

Microbial responses to salt-induced osmotic stress

Summary Soil columns were exposed to balanced (low Na⁺) or unbalanced (high Na⁺) high-salt solutions for a period of 7 days followed by 7 days of stress reflief. Total numbers of bacteria released into the perfusates rose under both types of stress, but the proportion of displaced bacteria that were viable fell significantly. Relief from both types of stress stimulated rapid increases in the number of viable micro-organisms released from soil. Examination of the soils at the end of the relief periods revealed that soils exposed to stress contained more viable bacteria than the non-stressed controls. However, high levels of balanced stress led to a significant decrease in species diversity within the microbial population, but a similar effect was not observed in soils exposed to unbalanced, high Na⁺ stress. These results suggest that, while salt stress may cause a significant reduction in the number of microorganisms in a soil, a large portion of the microbial population can rapidly adapt to marked changes in salinity.     

Seasonal variation in glycoconjugates of the pedal glandular system of the rayed Mediterranean limpet,Patella caerulea (Gastropoda: Patellidae)

Glycoconjugates secreted by the pedal system of the rayed limpet, Patella caerulea, were characterised in situ by histochemical and lectin-histochemical methods in individuals collected around the annual cycle, in November, March, and June. Stainings with periodic acid–Schiff (PAS), Alcian blue pH 2.5 (AB pH 2.5), Alcian blue pH 1.0 (AB pH 1.0), high-iron diamine–Alcian blue pH 2.5 and lectin binding assays with 9 lectins (Con A, WGA, succinylated-WGA, PNA, DBA, SBA, AAA, UEA-I, LTA) were performed. Four secreting cell types were observed in the sole, one in the peripheric region, and two in the sidewall. Glycoconjugate composition varied among cell types and also in one and the same cell type throughout the year. β-Elimination followed by PAS and AB pH 2.5 stainings indicated that most saccharidic chains were O-linked to the protein backbone. Secretion by sole and peripheric region was acidic, carboxylated and/or sulfated, whereas that of the sidewall was neutral. Glucosaminylated and 1,4-fucosylated residuals were predominant in the cell types along the year, 1,2-fucosylated residuals being observed only in the sidewall cells in June. Mannosylated and/or glycosylated residuals were observed in all cells mostly in November. Galactosylated/galactosaminylated residuals were present mostly in the sidewall cells and in the sole subepidermal mucocytes in June. Mannosylated and/or glycosylated residuals in November are probably linked to gonad maturation or to higher locomotion and foraging activity, whereas galactosaminylation in the sole cells and 1,2-fucosylation and glucosaminylation in the sidewall cells in June are linked to a prolonged stationary state, increasing water adsorption to counteract dehydration and/or to modulate microbial interactions.     

Physiological responses of Zygosaccharomyces rouxii to osmotic stress

When cell suspensions of Zygosaccharomyces rouxii were subjected to osmotic shock with NaCl, the cell volume decreased sharply and plasmolysis was observed. The cell subsequently recovered and volumes similar to those of cells growing at the respective water activity (a_w) values were found. Cycloheximide prevented cell recovery, indicating the involvement of protein synthesis in the recovery process. The intracellular glycerol concentration of Z. rouxii incubated in the presence of [¹⁴C]glycerol increased from 13 to 96 mmol/l during the initial 20 min after an upshock from 0.998 a_w to 0.96 a_w. All the intracellular glycerol was labelled and therefore derived from the medium. Labelled glycerol was subsequently utilized and replaced by unlabelled glycerol produced by the cell within 90 min. The initial increase in glycerol concentration following the upshock was confirmed by ¹³C-nuclear magnetic resonance (NMR) spectroscopic studies of cell extracts. The combined dihydroxyacetone and dihydroxyacetone phosphate concentrations fluctuated during this period, whereas glycerol-3-phosphate initially increased and then remained constant. This indicates that the production of glycerol is regulated. Decreases in ATP and polyphosphate levels were observed following osmotic upshock and may reflect a greater demand for ATP during the period of adjustment to decreased a_w. The changes in cell volume and in ATP concentration following osmotic upshock may serve as osmoregulatory signals in Z. rouxii, as suggested previously for other microorganisms.Correspondence to: S. G. Kilian     

Time-course of covalent modification of pyruvate kinase during anaerobiosis in the mantle muscle and the hepatopancreas of the limpet Patella caerulea (L)

Summary The changes in some kinetic properties of pyruvate kinase from the mantle muscle and the hepatopancreas of Patella caerulea, over a time-course of 16 or 8 h of acute hypoxia were studied. Up to the second hour of anaerobiosis PK exhibited changes of the kinetic properties in the direction of activation, whereas in the late stages of anaerobiosis in both tissues examined these changes occurred in the direction of inactivation. Although PKs from these tissues seemed to be regulated in a similar pattern during the early stages of anaerobiosis, they exhibited different kinetic properties under both normoxic and hypoxic conditions. Two forms of PK were found to be present in both the mantle muscle and the hepatopancreas of P. caerulea with different kinetic properties. The ratio of the enzyme units distributed between the two forms of PK (designated peak I and peak II) was varied during anaerobiosis indicating that the two forms of PK are interconvertible.Abbreviations ADP adenosine diphosphate - ATP adenosine triphosphate - EGTA ethyleneglycol-bis--aminoethylether N,N,N,N-tetraacetic acid - PK pyruvate kinase - PEP phosphoenolpyruvate - PMSF phenylmethysulfonyl fluoride     

New polymorphic microsatellite markers for the limpet Patella rustica and cross-priming testing in four Patella species

Eighteen microsatellite markers have been characterized from the Patella rustica genome. An average of 10 alleles per locus and an expected heterozygosity ranging from 0.054 to 0.937 were observed in a sample of 32 wild individuals from Viana do Castelo, Portugal. Distinct cross-priming amplification rates were recovered on four additional Patella species. Three monomorphic P. rustica loci were polymorphic in other Patella species. The microsatellites developed herein could be a useful intraspecific genetic tool to undertake fine population studies in the genus Patella.     

Cardiolipin and the osmotic stress responses of bacteria

Cells control their own hydration by accumulating solutes when they are exposed to high osmolality media and releasing solutes in response to osmotic down-shocks. Osmosensory transporters mediate solute accumulation and mechanosensitive channels mediate solute release. Escherichia coli serves as a paradigm for studies of cellular osmoregulation. Growth in media of high salinity alters the phospholipid headgroup and fatty acid compositions of bacterial cytoplasmic membranes, in many cases increasing the ratio of anionic to zwitterionic lipid. In E. coli, the proportion of cardiolipin (CL) increases as the proportion of phosphatidylethanolamine (PE) decreases when osmotic stress is imposed with an electrolyte or a non-electrolyte. Osmotic induction of the gene encoding CL synthase (cls) contributes to these changes. The proportion of phosphatidylglycerol (PG) increases at the expense of PE in cls⁻ bacteria and, in Bacillus subtilis, the genes encoding CL and PG synthases (clsA and pgsA) are both osmotically regulated. CL is concentrated at the poles of diverse bacterial cells. A FlAsH-tagged variant of osmosensory transporter ProP is also concentrated at E. coli cell poles. Polar concentration of ProP is CL-dependent whereas polar concentration of its paralogue LacY, a H⁺-lactose symporter, is not. The proportion of anionic lipids (CL and PG) modulates the function of ProP in vivo and in vitro. These effects suggest that the osmotic induction of CL synthesis and co-localization of ProP with CL at the cell poles adjust the osmolality range over which ProP activity is controlled by placing it in a CL-rich membrane environment. In contrast, a GFP-tagged variant of mechanosensitive channel MscL is not concentrated at the cell poles but anionic lipids bind to a specific site on each subunit of MscL and influence its function in vitro. The sub-cellular locations and lipid dependencies of other osmosensory systems are not known. Varying CL content is a key element of osmotic adaptation by bacteria but much remains to be learned about its roles in the localization and function of osmoregulatory proteins.     

Escherichia coli and Lactobacillus plantarum responses to osmotic stress

Escherichia coli and Lactobacillus plantarum were subjected to final water potentials of −5.6 MPa and −11.5 MPa with three solutes: glycerol, sorbitol and NaCl. The water potential decrease was realized either rapidly (osmotic shock) or slowly (20 min) and a difference in cell viability between these conditions was only observed when the solute was NaCl. The cell mortality during osmotic shocks induced by NaCl cannot be explained by a critical volume decrease or by the intensity of the water flow across the cell membrane. When the osmotic stress is realized with NaCl as the solute, in a medium in which osmoregulation cannot take place, the application of a slow decrease in water potential resulted in the significant maintenance of cell viability (about 70–90%) with regard to the corresponding viability observed after a sudden step change to same final water potential (14–40%). This viability difference can be explained by the existence of a critical internal free Na⁺ concentration. Received: 20 May 1998 / Received revision: 31 July 1998 / Accepted: 31 July 1998     

Molecular and genetic aspects of plant responses to osmotic stress

Drought, high salinity and freezing impose osmotic stress on plants. Plants respond to the stress in part by modulating gene expression, which eventually leads to the restoration of cellular homeostasis, detoxification of toxins and recovery of growth. The signal transduction pathways mediating these adaptations can be dissected by combining forward and reverse genetic approaches with molecular, biochemical and physiological studies. Arabidopsis is a useful genetic model system for this purpose and its relatives including the halophyte Thellungiella halophila, can serve as valuable complementary genetic model systems.     

Physiological and genetic responses of bacteria to osmotic stress.

The capacity of organisms to respond to fluctuations in their osmotic environments is an important physiological process that determines their abilities to thrive in a variety of habitats. The primary response of bacteria to exposure to a high osmotic environment is the accumulation of certain solutes, K+, glutamate, trehalose, proline, and glycinebetaine, at concentrations that are proportional to the osmolarity of the medium. The supposed function of these solutes is to maintain the osmolarity of the cytoplasm at a value greater than the osmolarity of the medium and thus provide turgor pressure within the cells. Accumulation of these metabolites is accomplished by de novo synthesis or by uptake from the medium. Production of proteins that mediate accumulation or uptake of these metabolites is under osmotic control. This review is an account of the processes that mediate adaptation of bacteria to changes in their osmotic environment.     

Development of the musculature in the limpet Patella (Mollusca, Patellogastropoda)

 Whole-mount technique using fluorescent-labelled phalloidin for actin staining and confocal laser scanning microscopy as well as semi-thin serial sectioning, scanning and transmission electron microscopy were applied to investigate the ontogeny of the various muscular systems during larval development in the limpets Patella vulgata L. and P. caerulea L. In contrast to earlier studies, which described a single or two larval shell muscles, the pretorsional trochophore-like larva shows no less than four different muscle systems, namely the asymmetrical main head/foot larval retractor muscle, an accessory larval retractor with distinct insertion area, a circular prototroch/velar system, and a plexus-like pedal muscle system. In both Patella species only posttorsional larvae are able to retract into the shell and to close the aperture by means of the operculum. Shortly after torsion the two adult shell muscles originate independently in lateral positions, starting with two fine muscle fibres which insert at the operculum and laterally at the shell. During late larval development the main larval retractor and the accessory larval retractor become reduced and the velar muscle system is shed. In contrast, the paired adult shell muscles and the pedal muscle plexus increase in volume, and a new mantle musculature, the tentacular muscle system, and the buccal musculature arise. Because the adult shell muscles are entirely independent from the various larval muscular systems, several current hypotheses on the ontogeny and phylogeny of the early gastropod muscle system have to be reconsidered. Received: 23 June 1998 / Accepted: 25 November 1998     

Modelling past and present geographical distribution of the marine gastropod Patella rustica as a tool for exploring responses to environmental change

A climate envelope approach was used to model the distributions of the intertidal gastropod Patella rustica , to test the robustness of forecast responses to climate change. The model incorporated variables that were likely to determine the abundance and the northern range limit of this species in the NE Atlantic. The model was built using classification and regression tree analysis (CART) trained with historical distribution data from the mid 1950s and a set of corresponding climatic and oceanographic variables. Results indicated air and sea temperature, in particular during the reproductive and settlement periods, as the main determinants of the Atlantic distribution of P. rustica . The model was subsequently fed with contemporary climatic data and its output was compared with the current distribution and abundance of P. rustica , assessed during a 2002–2003 survey. The model correctly hindcasted the recent collapse of a distributional gap in northern Portugal, as well as an increase in abundance at locations within its range. The predicted northward expansion of the northern range limit did not occur because the absence of the species was confirmed in a survey encompassing the whole Atlantic French coast up to Brest. Stretches of unsuitable habitat too long to be overcome by dispersal are the likely mechanism controlling the northern limit of the distribution of this intertidal species.     

Changes in membrane properties during in-vitro meiotic maturation of the limpet Patella vulgata

Changes in the membrane properties of the oocyte of the mollusk, Patella vulgata, were analyzed following the induction of meiosis reinitiation by paleopedial ganglia extract or by the weak base ammonia. During maturation it was possible to distinguish between an early phase characterized by an initial hyperpolarization and a late phase consisting of a depolarization which triggers an action potential with a long-term overshoot (20 minutes) of the membrane potential. Major changes in individual ionic permeabilities were studied using both current and voltage clamp conditions. The depolarizing phase appears to depend on decreases in K+ membrane permeability. Finally we observed that the overshoot did not appear to be directly related to germinal vesicle breakdown (GVBD) since it was absent in Na-deprived artificial sea water and could be elicited in the presence of TEA bromide, which did not induce maturation. This last observation suggests that it may result from a change in specific K+ ion permeability due to the possible activation of stretch channels.