Proteome changes in muscles, ganglia, and gills in Corbicula fluminea clams exposed to crude oil: Relationship with behavioural disturbances.

The use of online remote control for 24/7 behavioural monitoring can play a key role in estimating the environmental status of aquatic ecosystems. Recording the valve activity of bivalve molluscs is a relevant approach in this context. However, a clear understanding of the underlying disturbances associated with behaviour is a key step. In this work, we studied freshwater Asian clams after exposure to crude oil (measured concentration, 167 ± 28 µg·L-1) for three days in a semi-natural environment using outdoor artificial streams. Three complementary approaches to assess and explore disturbances were used: behaviour by high frequency non-invasive (HFNI) valvometry, tissue contamination with polycyclic aromatic hydrocarbons (PAH), and proteomic analysis. Two tissues were targeted: the pool adductor muscles - retractor pedal muscle - cerebral and visceral ganglia, which is the effector of any valve movement and the gills, which are on the frontline during contamination. The behavioural response was marked by an increase in valve closure-duration, a decrease in valve opening-amplitude and an increase in valve agitation index during opening periods. There was no significant PAH accumulation in the muscle plus nervous ganglia pool, contrary to the situation in the gills, although the latter remained in the low range of data available in literature. Major proteomic changes included (i) a slowdown in metabolic and/or cellular processes in muscles plus ganglia pool associated with minor toxicological effect and (ii) an increase of metabolic and/or cellular processes in gills associated with a greater toxicological effect. The nature of the proteomic changes is discussed in terms of unequal PAH distribution and allows to propose a set of explanatory mechanisms to associate behaviour to underlying physiological changes following oil exposure. First, the first tissues facing contaminated water are the inhalant siphon, the mantle edge and the gills. The routine nervous activity in the visceral ganglia should be modified by nervous information originating from these tissues. Second, the nervous activity in the visceral ganglia could be modified by its own specific contamination. Third, a decrease in nervous activity of the cerebral ganglia close to the mouth, including some kind of narcosis, could contribute to a decrease in visceral ganglia activity via a decrease or blockage of the downward neuromodulation by the cerebro-visceral connective. This whole set of events can explain the decrease of metabolic activity in the adductor muscles, contribute to initiate the catch mechanism and then deeply modify the valve behaviour.

Asiatic clam Corbicula fluminea exhibits distinguishable behavioural responses to crude oil under semi-natural multiple stress conditions.

Aquatic ecosystems are subject to many anthropogenic disturbances, and understanding their possible impacts is a real challenge. Developing approaches based on the behaviour of bivalve mollusks, an integrating marker of the state of the organisms, and therefore of their environment, is relevant, whether within a natural ecosystem or an ecosystem subject to industrial activities. The main objective of this study was to identify by HFNI Valvometry a reliable and reproducible clam behavioural response in the presence of crude oil in a multistress context. To closely replicate actual field conditions, Corbicula fluminea was exposed in outdoor artificial streams that were subject to natural variations and were continuously fed by fresh water from the Gave de Pau (S.W. France). After a period of 26 days in these artificial streams, the clams (n = 14-16 per condition) were separately exposed for 10 days to crude oil alone, crude oil and barium, crude oil and noise pollution, crude oil and turbidity pulses, barium alone, noise pollution alone, turbidity pulses alone or natural changes alone. The secondary objective was to characterize the accumulation of polycyclic aromatic hydrocarbons (PAH) in 3 tissues (gills, adductor muscles and foot) in clams exposed for 10 days to crude oil alone or under multistress conditions (n = 5 clams per condition) and then to compare the accumulation and behaviour of clams under these conditions. The response of clams to crude oil alone or under multistress conditions was visually and statistically significant and not confounded by the other disturbances tested, despite large variations in water temperature. In the presence of crude oil, the behaviour of clams was characterized by an increase in valve-closure duration, a decrease in valve-opening amplitude and an increase in valve agitation index. In the presence of crude oil, the clam behaviour showed no direct relationship with PAH accumulation in the gills, adductor muscles or foot, although hypothetical mechanisms are discussed. This work supports the growing interest in studying the behaviour of bivalve mollusks in the context of biomonitoring of the aquatic environment surrounding oil facilities.

Comparative effects of dietary methylmercury on gene expression in liver, skeletal muscle, and brain of the zebrafish (Danio rerio).

Effects of dietary methylmercury (MeHg) on gene expression were examined in three organs (liver, skeletal muscle, and brain) of the zebrafish (Danio rerio). Adult male fish were fed over 7, 21, and 63 days on three different diets: one control diet (C0: 0.08 microg of Hg g(-1), dry wt) and two diets (C1 and C2) contaminated by MeHg at 5 and 13.5 microg of Hg g(-1), dry wt. Total Hg and MeHg concentrations were determined in the three organs after each exposure duration, and a demethylation process was evidenced only in the liver. Thirteen genes known to be involved in antioxidant defenses, metal chelation, active efflux of organic compounds, mitochondrial metabolism, DNA repair, and apoptosis were investigated by quantitative real-time RT-PCR and normalized according to actin gene expression. Surprisingly, no change in the expression levels of these genes was observed in contaminated brain samples, although this organ accumulated the highest mercury concentration (63.5 +/- 4.4 microg g(-1), dry wt after 63 days). This lack of genetic response could explain the high neurotoxicity of MeHg. coxI and cytoplasmic and mitochondrial sod gene expressions were induced early in skeletal muscle and later in liver, indicating an impact on the mitochondrial metabolism and production of reactive oxygen species. Results demonstrated that skeletal muscle was not only an important storage reservoir but was also affected by MeHg contamination. The expression of the metallothionein mt2 and the DNA repair rad51 genes was up-regulated in liver between 21 and 63 days, whereas in skeletal muscle, mt2 remained uninduced, and gadd and rad51 were found to be repressed.

Primitive, and protective, our cellular oxygenation status?

The primitive atmosphere where aerobic life started on earth was hypoxic and hypercapnic. Remarkably, an adaptation strategy whereby O2 partial pressure, PO2, in the arterial blood is maintained within a low and narrow range of 1-3 kPa, largely independent of inspired PO2, has also been reported in modern water-breathers. In mammalian tissues, including brain, the most frequently measured PO2 is also in the same low range. Based on the postulate that basic cellular machinery has been established since the early stages of evolution, we propose that this similarity in oxygenation status is the consequence of an early adaptation strategy which, subsequently, throughout the course of evolution, maintained cellular oxygenation in the same low and primitive range independent of environmental changes. Specialized enzymes aimed at protecting cells against O2 toxicity are thought to have appeared very early in evolution but we suggest that preventing high PO2's is also the simplest and most efficient tool for limiting reactive oxygen species (ROS) production. It could be a cue mechanism to widen our understanding of the ageing process.

From low arterial- to low tissue-oxygenation strategy. An evolutionary theory.

The primitive atmosphere where aerobic life started on earth was hypoxic and hypercapnic. Remarkably, an adaptation strategy whereby O(2) partial pressure, P(O(2)), in the arterial blood is maintained within a low and narrow range of 1-3 kPa, largely independent of inspired P(O(2)), has also been reported in modern water-breathers. In mammalian tissues, including brain, the most frequently measured P(O(2)) is in the same low range. Based on the postulate that basic cellular machinery has been established since the early stages of evolution, we propose that this similarity in oxygenation status is the consequence of an early adaptation strategy which, subsequently throughout the course of evolution, maintained cellular oxygenation in the same low and primitive range independent of environmental changes. The rational for such an evolutionary theory is discussed in terms of an equilibrium between physiological and pathological reactions associated with O(2) excess vs O(2) lack and emerging concepts about the importance of cellular O(2)-dependent mechanisms in the low but physiological P(O(2)) range.

A modulatory role for oxygen in shaping rhythmic motor output patterns of neuronal networks.

It is becoming increasingly evident that O(2)-uptake in animal tissue is not only devoted to energy production. Here we review recent findings on a novel role of tissue oxygenation, notably in controlling the operation of neuronal networks in the central nervous system. Electrophysiological recordings in vivo and in vitro from rhythmically-active motor pattern generating networks in the lobster stomatogastric ganglion (STG) have revealed that oxygen is able to act in a manner equivalent to a classical neuromodulator. Local P(O(2)) variations within the low, but physiological range of 1-6 kPa are able to shape ongoing activity of these networks and therefore the motor behaviours in which they are involved. Oxygen's contribution to two of these, feeding and moulting, have been investigated. Importantly, the P(O(2)) effects are not related to hypoxic depression but are highly specific in terms of the network, neuron and even the synapse targeted. Our results are discussed in terms of functional significance and new research directions for mammalian physiology.

How water oxygenation level influences cadmium accumulation pattern in the Asiatic clam Corbicula fluminea: a laboratory and field study.

The level of O2 in water is highly variable in the aquatic environment and is a major ventilatory drive in all animals breathing water. Low O2 partial pressure (PO2) strongly stimulates ventilatory activity compared to air-equilibrated or O2-enriched water. We studied the influence of ventilatory activity on the bioaccumulation rate of Cd in the freshwater Asiatic clam Corbicula fluminea for PO2 ranging from 4 to 40 kPa (2-20 mg/L at 15 degrees C) during steady-state exposure to controlled concentrations of Cd of approximately 2 or 0.5 microg/L under both laboratory and field conditions. The concentration of Cd in the expired water and its apparent extraction coefficient (EwCd) from the ventilated water were calculated. Results show that a low PO2 strongly enhanced Cd bioaccumulation rate in the whole soft body and modified the distribution pattern and the relative burden in the organs. Whatever the water PO2, values for the concentration of Cd in the expired water remained close to the Cd concentration in the inspired water and EwCd varied from 2 to 12%. Because the field results conformed to the laboratory analysis, the suggestion is made that the influence of O2 on bioaccumulation patterns of metals in water-breathers should be classified as of primary importance.

Selective trapping of organochlorine compounds in mountain lakes of temperate areas.

The study of fish concentrations and sediment inventories in 19 European high mountain lakes (40-67 degrees N) shows that a fraction of organochlorine compounds (OCs), the less volatile compounds (LVC; subcooled liquid vapor pressure < or = 10(-2.5) Pa), are trapped in the higher locations. This general trend is not significantly influenced by possible local sources. Compound distribution is related to average air temperatures. The phase-change pseudoenthalpies calculated from the sediment inventories closely match the summed theoretical volatilization and dissolution enthalpies. This fractionation effect is responsible for the accumulation of high concentrations of the LVC, the more persistent and toxic according to literature data, in organisms inhabiting sites far from the locations of synthesis or use.

Is the resting rate of oxygen consumption of locomotor muscles in crustaceans limited by the low blood oxygenation strategy?

Numerous water-breathers exhibit a gas-exchange regulation strategy that maintains O(2) partial pressure, P(O2), in the arterial blood within the range 1-3 kPa at rest during the daytime. In a night-active crustacean, we examined whether this could limit the rate of O(2 )consumption (M(O2)) of locomotor muscles and/or the whole body as part of a coordinated response to energy conservation. In the crayfish Astacus leptodactylus, we compared the in vitro relationship between the M(O2) of locomotor muscles as a function of the extracellular P(O2) and P(CO2) and in vivo circadian changes in blood gas tensions at various values of water P(O2). In vitro, the M(O2) of locomotor muscle, either at rest or when stimulated with CCCP, was O(2)-dependent up to an extracellular P(O2) of 8-10 kPa. In vivo, the existence of a night-time increase in arterial P(O2) of up to 4 kPa at water P(O2) values of 20 and 40 kPa was demonstrated, but an experimental increase in arterial P(O2) during the day did not lead to any rise in whole-body M(O2). This suggested that the low blood P(O2) in normoxia has no global limiting effect on daytime whole-body M(O2). The participation of blood O(2) status in shaping the circadian behaviour of crayfish is discussed.

The ability to feed in hypoxia follows a seasonally dependent pattern in shore crab Carcinus maenas.

The ability of the adult shore crab Carcinus maenas, native to the Bay of Arcachon (SW France), to feed in hypoxia was determined at various seasons. Crabs previously kept at field temperature were fed after a 5-day fasting period at 15 degrees C. Their blood oxygenation and pH regulation strategy and also their gill anatomy were analysed. From May to October, C. maenas feed at levels of O(2) partial pressure (p(O(2))) in the water, pwO(2)=2 kPa (1 mg l(-1)), without switching to their anaerobic metabolism. In March-April, before the main moulting period, the same food intake at pwO(2)=4 kPa induced a systematic blood lactate increase associated with some mortality. An analysis performed at pwO(2)=4 kPa at that time showed that in intermoult crabs the development of a coating of foreign material over the gill cuticle interfered with O(2)-supply, preventing the small arterial p(O(2)) increases (from 0.7 to 1 kPa) which occurred at other seasons. This led to a cellular hypoxia despite a systematic postprandial blood-pH alkalinisation which favoured O(2)-loading at gill level and increased arterial O(2) concentration. In March-April, alkalinisation appeared at pwO(2) values >/=6 kPa and from May to at least July at pwO(2)>/=2 kPa. Results are discussed in terms of season-related physiological performance, as hypoxic events mainly occur during the hot season.

In vivo modulation of interacting central pattern generators in lobster stomatogastric ganglion: influence of feeding and partial pressure of oxygen.

The stomatogastric ganglion (STG) of the European lobster Homarus gammarus contains two rhythm-generating networks (the gastric and pyloric circuits) that in resting, unfed animals produce two distinct, yet strongly interacting, motor patterns. By using simultaneous EMG recordings from the gastric and pyloric muscles in vivo, we found that after feeding, the gastropyloric interaction disappears as the two networks express accelerated motor rhythms. The return to control levels of network activity occurs progressively over the following 1-2 d and is associated with a gradual reappearance of the gastropyloric interaction. In parallel with this change in network activity is an alteration of oxygen levels in the blood. In resting, unfed animals, arterial partial pressure of oxygen (PO2) is most often between 1 and 2 kPa and then doubles within 1 hr after feeding, before returning to control values some 24 hr later. In vivo, experimental prevention of the arterial PO2 increase after feeding leads to a slowing of pyloric rhythmicity toward control values and a reappearance of the gastropyloric interaction, without apparent effect on gastric network operation. Using in vitro preparations of the stomatogastric nervous system and by changing oxygen levels uniquely at the level of the STG within the range observed in the intact animal, we were able to mimic most of the effects observed in vivo. Our data indicate that the gastropyloric interaction appears only during a "free run" mode of foregut activity and that the coordinated operation of multiple neural networks may be modulated by local changes in oxygenation.

Modulation of a neural network by physiological levels of oxygen in lobster stomatogastric ganglion.

Although a large body of literature has been devoted to the role of O2 in the CNS, how neural networks function during long-term exposures to low but physiological O2 partial pressure (PO2) has never been studied. We addressed this issue in crustaceans, where arterial blood PO2 is set in the 1-3 kPa range, a level that is similar to the most frequently measured tissue PO2 in the vertebrate CNS. We demonstrate that over its physiological range, O2 can reversibly modify the activity of the pyloric network in the lobster Homarus gammarus. This network is composed of 12 identified neurons that spontaneously generate a triphasic rhythmic motor output in vitro as well as in vivo. When PO2 decreased from 20 to 1 kPa, the pyloric cycle period increased by 30-40%, and the neuronal pattern was modified. These effects were all dose- and state-dependent. Specifically, we found that the single lateral pyloric (LP) neuron was responsible for the O2-mediated changes. At low PO2, the LP burst duration increased without change in its intraburst firing frequency. Because LP inhibits the pyloric pacemaker neurons, the increased LP burst duration delayed the onset of each rhythmic pacemaker burst, thereby reducing significantly the cycling frequency. When we deleted LP, the network was no longer O2-sensitive. In conclusion, we propose that (1) O2 has specific neuromodulator-like actions in the CNS and that (2) the physiological role of this reduction of activity and energy expenditure could be a key adaptation for tolerating low but physiological PO2 in sensitive neural networks.

When are resting water-breathers lacking O2? Arterial PO2 at the anaerobic threshold in crab.

The minimum arterial O2 partial pressure (PaO2) at which, in resting conditions, O2 consumption (MO2) can be maintained and below which anaerobic metabolism is initiated was studied in the crabs Eriocheir sinensis and Carcinus maenas at 15 degrees C. Arterial PO2, MO2 (in E. sinensis), blood lactate concentration ([lact]b) and blood copper concentration ([Cu]b, an index of the blood O2 carrying capacity) were determined after 24 h exposure to inspired PO2 (PIO2) ranging from 2.7-2.1 kPa. They were compared to normoxic controls. In normoxia, the most frequently measured PaO2 ranged between 1 and 3 kPa in both species. In hypoxia, the threshold for blood lactate appearance was PaO2 = 2.1 kPa in E. sinensis and 1.3 kPa in C. maenas, but in many individuals anaerobic metabolism was initiated at lower PaO2's. The lowest PaO2 with [lact]b approx. 0 was 0.7 kPa in both species. MO2 was maintained in 4 E. sinensis out of 6 with PaO2 ranging from 0.7-1.2 kPa (PIO2 = 2.1 kPa). The arterial PO2 at which anaerobic metabolism occurred was not related to blood O2 carrying capacity.

How is O2 consumption maintained independent of ambient oxygen in mussel Anodonta cygnea?

The mechanisms of adaptation allowing resting freshwater mussels Anodonta cygnea to maintain their oxygen consumption (MO2) constant when the O2 partial pressure in the inspired water (PIO2) varied were studied at 13 degrees C. Steady-state values of oxygen consumption and/or shell valve activity were determined at prefixed PIO2 for periods ranging from 2 to 15 days. Values of PO2, O2 concentration and acid-base status of arterial blood in the heart were determined after two days. MO2 was maintained constant over PIO2 ranging from 35 to less than 1 kPa. At 0.3 kPa it decreased by 50%. Valves remained open (and MO2 constant) most of the time even during a 4.5 day period at PIO2 approximately 1.5 kPa. Between 35 and 1 kPa, blood PO2 at the heart level can remain low and within a narrow range independent of PIO2. Blood PCO2 increased at high PIO2 and decreased at low PIO2. Data are compared to previous results in crayfish and in wels (sheat-fish). On the basis of data similarity it is proposed that these three animals exhibit the same basic strategy for maintaining resting MO2 when PIO2 varies. This common feature relies mainly on the ability to maintain PO2 in the arterial blood at a value which is low and independent of PIO2. Hence, in terms of O2, homeostasis of the milieu intérieur is accomplished.

Oxygen-sensitive chemoreceptors in the branchio-cardiac veins of the crayfish, Astacus leptodactylus.

Oxygen-sensitive activity was recorded from the branchial nerve of the crayfish Astacus leptodactylus in vitro. After the podobranchial and arthrobranchial nerves branch off to the gill, the branchial nerve terminates in the branchio-cardiac vein wall and its surroundings. When the former 2 branches were cut, irregular spontaneous activity could be recorded from a few fiber preparations innervating the branchio-cardiac vein. The branchio-cardiac vein was superfused or perfused with hypoxic or hyperoxic Ringer solution. Impulse frequency increased in response to hypoxia and decreased in hyperoxia. NaCN and almitrine strongly stimulated nerve activity. Baroreceptor activity was also observed. These response characteristics demonstrate that these receptors are Heymans-type chemoreceptors.

Circadian rhythm of extracellular pH in crayfish at different levels of oxygenation.

The extracellular pH regulation was studied in the crayfish Astacus leptodactylus (a night animal) as a function of circadian rhythm. The venous acid-base balance (ABB) was determined in the morning (10 a.m.-12 a.m.) and in the evening (10 p.m.-12 p.m.) at PO2 ranging from 29 to 6 kPa and constant ABB in the water at 13 degrees C. In the morning the venous pH (pHv) was maintained constant by metabolic means independently of PO2 from 29 to 10 kPa. In the evening pHv again was constant and independent of PO2 but it was more alkaline by 0.1 unit corresponding to a shift along the in vitro buffer line. At that time, the ventilation required for providing a unit quantity of O2 (i.e. the ventilatory requirement) increased more than for simply providing O2. The related circadian changes of sensitivity of the ventilatory control system were assessed by comparing morning and evening ventilatory responses to 1-h periods of hypoxia and then hypercapnia. In the evening, the amplitude of the responses to both O2 and CO2 increased but the increase in CO2 sensitivity was proportionally more important. This is consistent with the increase of ventilatory requirement and the related decrease of hemolymph PCO2 during this period. It is concluded that in this animal there exists a circadian rhythm of extracellular pH that is achieved by controlling the CO2 partial pressure in the hemolymph. Results are discussed in terms of O2 transport processes and metabolic modulation through pH adjustments.

Ventilatory regulation of extracellular pH in crayfish exposed to changes in water titration alkalinity and NaCl concentration.

The mechanisms of extracellular pH regulation were studied in normoxic crayfish Astacus leptodactylus during changes in water ionic composition at 13 degrees C. In artificial waters all ambient physico-chemical properties were controlled. Ventilatory changes and the time course of hemolymph acid-base balance, ABB, were followed after a decrease of water titration alkalinity, TAw, from 4 to 2 meq X L-1 simultaneously associated with either an increase of NaCl concentration, [NaCl]w, from 0.5 to 5 mmol X L-1, or a decrease of [NaCl]w, from 0.5 to 0.15 mmol X L-1. The ABB changes were characterized by a hypercapnic acidosis attributable to the decrease of TAw. Depending on the simultaneous change of [NaCl]w, two different mechanisms of compensation were observed. When [NaCl]w increased, the compensation was metabolic: the ventilatory requirement, VW X MO2-1, did not vary. When [NaCl]w decreased, the compensation was ventilatory: VW X MO2-1 doubled. It is concluded that in water-breathers ventilation, contrary to what is generally accepted, can play a role in extracellular ABB regulation.