In order to better understand the role of the gills in the accumulation of contaminants, branchial uptake and bioaccumulation of inorganic (HgCl2) and monomethylmercury (CH3HgCl) were quantified in the shore crab Carcinus maenas, using living animals and an in vitro perfused gill preparation exposed to 50 mug l(-1) of either chemical forms in the external medium. In addition, localization of accumulated mercury was studied using the histochemical autometallographic technique by both light and electron microscopy. Gill tissue strongly accumulated either inorganic or methylated mercury at similar levels in vitro and in vivo. For both chemical forms of the metal, only about 1% of the total mercury input was recovered in the effluent fluid from in vitro perfused gills and could thus be considered available to distribute inside the animal via the circulatory system. Inorganic Hg was histochemically found to be accumulated at 2 markedly different locations in the gills: at the cuticular surface in direct contact with the contaminated medium and at high local levels in the central vacuole of gill nephrocytes. Although also present at these 2 locations, methylmercury was distributed more diffusely and more evenly in all cells. These results suggest that the high affinity of gill tissue for both forms of mercury may confer on this organ the role of an external barrier strongly limiting the invasion of the metal toward other compartments of the body.

Carbon dioxide contents and partial pressures were measured during the MAR 93 cruise on several water samples collected from black smoker chimneys and around mussels and shrimps at the Snake Pit (MARK) vent field (Mid-Atlantic Ridge). High end-member values for total CO2 (10.63 mmol l(-1)) and CO2 partial pressure (335 Ton directly measured at 33 degrees C; 1 Ton = 0.133 kPa) were recorded for black smokers samples. Water sampled in close proximity of organisms was found moderately but significantly enriched in carbon dioxide.

As in many aquatic organisms, the gill epithelium is the main site of the uptake of copper and toxicity in the shore crab. Long term pre-exposure to sublethal doses of water-borne copper has been shown to lead after some weeks to progressive recovery from the initial toxic effects. We report experimental results that shed light on some of the mechanisms underlying these acclimatory effects. The metal adaptation seems related to an important reduction of copper uptake from the ambient medium and to a better efficiency of metal transfer from hemolymph to the tissues. As a consequence, steady-state hemolymph levels of exogenous copper are strongly decreased during a further lethal exposure in pre-acclimated crabs compared to controls. A 17-day sublethal pre-acclimation period appears, however, to be too short to improve the resistance of the animals to a further challenge at usually lethal doses of copper.

Deep-sea hydrothermal vents, first discovered in 1977, are characterized by variable and extreme conditions, in particular by a low level of oxygen, a low pH, a high temperature, a high sulphide content, and a high level of carbon dioxide. Decapod crustaceans, which are found in all the hydrothermal vents yet known at the East Pacific Rise (e.g. Bythograea thermydron Williams, 1980, Cyanagraea praedator de Saint-Laurent, 1984, Alvinocaris lusca Williams & Chace Jr., 1982) and at the Mid-Atlantic Ridge (e.g. Rimicaris exoculata, Williams & Rona 1986, Chorocaris chacei (Williams & Rona, 1986), Segonzacia mesatlantica Williams, 1988), had to develop adaptation mechanisms, in particular respiratory adaptations to survive in this environment. The respiratory pigment of decapod crustaceans is haemocyanin that binds reversibly oxygen to a pair of copper atoms. It is a large extracellular protein, with a blue colour, which has a typical hexameric structure (Markl & Decker, 1992) and a large potential for functional plasticity (Truchot & Lallier, 1992).

In addition to metabolic CO2 production and gill ventilatory flow rate, expired water Pco2 is very dependent on water acid-base balance in a complex way. This is particularly true in carbonated waters at low ambient Pco2 and high pH, where CO2 excreted in the gill water may be buffered by carbonate ions, leading to an increased CO2 capacitance coefficient. The higher the carbonate alkalinity (CA) and the lower the inspired Pco2 (i.e., the higher the inspired water pH), the stronger the carbonate buffering and the smaller the increase of Pco2 in the gill water during respiratory CO2 exchanges. As a consequence, as shown by a number of reported data, increasing the CA leads to blood hypocapnia and respiratory alkalosis at constant low, but not at high, inspired Pco2. In the low range of inspired Pco2, internal Pco2 becomes very sensitive to even small changes of water Pco2, which may explain at least in part the large variability of reported blood Pco2 values in gill breathers. Water CA also influences the amplitude of respiratory acid-base disturbances caused by changes of the gill ventilatory flow rate. Carbonate buffering of excreted CO2 and thus dependence of blood Pco2 on water alkalinity requires catalysis of CO2 hydration by carbonic anhydrase, that must be available from the water side of the gill epithelium.

Tissue mercury concentrations were analysed in the whole organism and various organs of the euryhaline shore crab Carcinus maenas after 3 and 15 days of exposure to inorganic mercury (Hg(II)) and methylmercury (MeHg) at a nominal concentration of 1 μgHg l−1. Chemical speciation of the metals was varied by using four pH/salinity conditions (combinations between pH 7.0 and 8.5/salinity 560 and 140 mM NaCl). In all conditions, the results showed a particularly important Hg accumulation in gill tissue, smaller levels in the carapace and internal organs, and very low concentrations in circulating haemolymph. For Hg(II), accumulation was generally favoured at low salinity, but the data revealed complex pH/salinity interactions. Positive correlations were found between tissue metal levels and the abundance of some inorganic Hg species: HgCl2, HgCl−3, Hg2+, HgCl+, indicating that chemical speciation may be an important factor governing metal uptake. The balance of evidence favours HgCl2 and CH3HgCl as the most bioavailable species for uptake of Hg(II) and MeHg, respectively.

Rimicaris exoculata is a caridean shrimp from the family Alvinocarididae which forms the dominant species around deep-sea hydrothermal vents from the Mid-Atlantic Ridge (MAR). Seeking respiratory adaptations to the hydrothermal environment, we have analysed the oxygen-binding properties of Rimicaris hemocyanin (Hc) in relation with temperature, pH, and lactate variations. Rimicaris native Hc is mostly composed of hexamers. It showed a high oxygen affinity (P-50 approximately 3 Torr at pH 7.5, 15 degrees C), a large Bohr effect (Delta logP(50)/Delta pH = 1-1.87 +/- 0.25, n = 6), a moderate lactate effect (Delta logP(50)/Delta log[lac] = -0.12) and almost no temperature effect (Delta H = -1.23 kJ.mol(-1) 15-35 degrees C). Most surprisingly, dialysis of native hemolymph elicited a large increase of Hc-O-2 affinity, an effect opposite to the usual trend observed for crustacean Hcs. Moreover, this increase in affinity could be reversed by adding an ultrafiltrate of native hemolymph to a dialysed sample, thus unveiling the existence of a dialysable yet unknown cofactor which decreases Hc-oxygen affinity. (C) 1997 Wiley-Liss, Inc.

The combined effects of pH and salinity were investigated on the bioaccumulation of inorganic mercury in the Turkish crayfish Astacus leptodactylus, at the whole organism and organ levels. Effects on the ionic balance were also analysed during the acclimatization phase and after 3 and 15 days of exposure, via the direct route. The experimental protocol, based on a complete factorial design, took into account nine experimental conditions, resulting from the combination of three levels for pH (6, 7.5 and 9) and for salinity (1, 10 and 100 mM Cl-). The results showed an important Hg accumulation in the organs or tissues in direct contact with the surrounding medium, e.g. gills-and carapace. The effects of the nine pH and salinity conditions were complex and variable from one organ to another. Correlations were studied between chemical species concentrations in the water (HgCl2, HgOHCl, HgCl3-, HgCl42-, Hg(OH)(2)) and amounts of metal accumulated in the different organs. A positive correlation was found between the neutral species HgCl2 and metal accumulated in the gills and in the carapace. Perturbations of the haemolymph ionic concentrations were no significant, except for Na+ after 3 days of exposure.

Copper of exogenous origin is present in crab hemolymph mainly in a protein-bound form. Various approaches were used to assess the toxicity of this protein-bound exogenous metal. Mortality due to copper directly injected into the hemolymph space was apparent at circulating concentrations of non-hemocyanic copper much higher than that (about 150 μM) attained during exposure to a lethal level of waterborne copper (31.5 μM in water). No cytological damage to the gill epithelium was apparent in crabs injected with the metal to attain a hemolymph exogenous copper level of 150 μM, in contrast to those observed at similar hemolymph concentrations during waterborne metal exposure. The cytotoxicity of copper added to an in vitro hepatopancreatic cell preparation was greatly diminished in the presence of hemolymph proteins, probably in relation to a reduced copper uptake by these cells. Thus, binding of copper to hemolymph proteins and particularly hemocyanin plays a significant role, not only for transport and distribution, but also for detoxification of the metal within the organism.

Exogenous (i.e. non-haemocyanic) copper was quantified in the haemolymph of shore crabs Carcinus maenas and the fate of the metal was studied in animals contaminated either by exposure to waterborne copper or by injection into the haemolymph space. Direct contamination from the ambient water resulted in a relatively steady level of haemolymph exogenous copper by 2–3 days. Following a single injection, exogenous copper disappeared rapidly from haemolymph, following an exponential time course (half time 22.4 h). Insignificant amounts of the metal were excreted to the ambient water but dose-dependent tissue uptake could be demonstrated in vivo and by using an in vitro hepatopancreatic cell preparation. Almost all haemolymph exogenous copper was protein-bound and non-filterable. By ultracentrifugation and gel filtration experiments, the only protein binding exogenous copper was shown to be the respiratory pigment haemocyanin. These results emphasize the role of haemolymph and haemocyanin as an efficient copper transport system in the crustaceans.

Changes in circulatory, ventilatory and acid-base variables were studied in Siberian sturgeon (Acipenser baeri) exposed to acute and severe hypoxia (Pw(O2) = 10 torr), followed by a rapid return to normoxia. This treatment caused a significant stress, revealed by the high levels of plasma catecholamines and cortisol. The moderate circulatory changes firstly observed would represent the effects of increased plasma catecholamine levels together with an increased adrenergic nervous tone on the cardiovascular system. Then, these effects were masked by a possible vagal reflex resulting in bradycardia. Deep hypoxia induced a ventilatory alkalosis combined with a moderate metabolic acidosis. The latter amplified concomitantly with a massive flush of lactate into the blood stream. The initial hyperventilation was followed by a deep ventilatory depression. During return to normoxia, hyperventilation resumed consistent with the repayment of an oxygen debt. Thus, the sturgeon, although considered as an archaic fish, developped the same adaptative responses as teleosts submitted to comparable hypoxic conditions.

Keeping an appropriate acid-base state in the various body compartments of animals is of prime importance for many basic living processes. What is preserved is not a constant pH value but rather a constant relationship between pH and body temperature, which tends to stabilize the protein electrical charge and, more generally, conformation and function of macromolecules. Acid-base homeostasis requires a balance between metabolic production and controlled excretion of two classes of acids or bases : the volatile carbonic acid whose elimination depends on respiratory regulations ; and fixed acids and bases, usually excreted in association with ion exchanges. In aquatic animals, these functions are heavily challenged by large natural changes of respiratory gases, oxygen and carbon dioxide, as well as of total salinity or of particular ions in the environment. The effects of each of these factors in isolation have been well studied in laboratory conditions, but integrated responses to the changes of many factors as it occurs in the natural setting are less well known. Variations of ambient or internal CO2 are not a strong stimulus to breathing in aquatic crustaceans and fishes, and respiratory compensations are thus of little importance in acid-base homeostasis. On the contrary, aquatic organisms are usually able to quickly get rid of large fixed acid or alkaline loads by coupling their excretion with gill ionic exchanges. Such excretory processes also serve to compensate acid-base disturbances induced by changes of the respiratory qualities of the water. The well-known impact of various pollutants (heavy metals, ammonia, acid waters...) on gill structure and ionoregulatory mechanisms can also considerably disturb acid-base balance in aquatic animals. Such disturbances may serve as very sensitive tests of sublethal toxicity.

The time courses of extracellular ionic and acid-base adjustments were studied in juvenile turbot (Scophthalmus maximus) following a decrease of water salinity, either abruptly from 32 to 10%. or after a first step (4 weeks) in 19%. salinity followed by a direct transfer to 10%. brackish water (BW). Net exchanges of acid-base equivalents with the external water were also determined after transfer from 32%. SW to 10%. BW. Direct transfer from seawater (SW) to 10%. BW induced a transient decrease in plasma osmolarity, plasma sodium and chloride concentrations, associated with a marked and transient metabolic alkalosis in the blood. A significant net outflux of acidic equivalents was also measured only during the first day in BW. Four weeks preadaptation in 19%. BW reduced the intensity of the osmotic disturbances elicited by a subsequent abrupt transfer to 10%. BW. These ionic readjustments were also coupled with minimal acid-base changes, of lesser magnitude than those described after directly from SW to 10%. BW.

Changes in respiratory and acid-base variables were studied in siberian sturgeon, Acipenser baeri, during progressive deep hypoxia followed by recovery under normoxic conditions. During hypoxia, both ventilatory frequency and amplitude increased and this sturgeon was able to maintain standard oxygen consumption down to a low critical level of ambient PO2 (PWO2 < 40 mmHg). During the posthypoxic period, an O2 debt was repaid by an elevated oxygen consumption (nearly double control value at 1 h), indicating that a shift to anaerobic metabolism had occurred during exposure to severe hypoxia. Gradually increasing ambient hypoxia initially induced a respiratory alkalosis. Below the critical PWO2 level and during normoxic recovery, a sudden flush of lactate into the blood was associated with a typical metabolic acidosis which was almost totally compensated 3.5 h after return to normoxia. Thus, as for most other fish, respiratory responses of the sturgeon to progressive hypoxia reveal a typical O2 regulatory behavior.

The ultrastructure of gill lamellae was studied in shore crabs, Carcinus maenas, exposed to sublethal (0.5 mg∙L−1) and lethal (2 mg∙L−1) concentrations of waterborne copper for various durations. Oxygen tension, pH, and lactate concentration in arterial blood were determined in parallel studies. Extensive structural alterations involving cellular hyperplasia, vacuolization, and necrosis were found after 5–6 days of exposure to both sublethal and lethal copper levels. This led to considerable thickening of the gill epithelium and reduction of haemolymph spaces, resulting in restriction of respiratory gas exchange as shown by a marked hypoxemia. Ensuing lactacidemia suggests that tissue hypoxia was probably the major effect of the toxicant at lethal levels. In sublethal conditions, partial repair of gill tissue and recovery of normal blood oxygenation and pH were observed after 18 days of exposure.

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.