P. Cortesi

Differential sensitivities to hypoxia by two anoxia-tolerant marine molluscs: A biochemical analysis

The metabolic responses to a series of low oxygen tensions were compared for two species of Mediterranaean bivalves,Mytilus galloprovincialis andScapharca inaequivalvis. Whereas both species have well-developed and similar tolerances of anoxia, the metabolic responses ofS. inaequivalvis to low oxygen tensions indicate a substantially greater tolerance of hypoxia. Compared withM. galloprovincialis, the responses ofS. inaequivalvis included the ability to maintain a constant oxygen consumption down to a much lower pO2 value (ca. 1.7 vs 3.4 ppm), and a lower critical pO2 for the recruitment of fermentative pathways of ATP production (ca. 1 vs 3 ppm). Furthermore, a graded increase in the output of anaerobic products (succinate, alanine) occured at oxygen tensions below 3 ppm inM. galloprovincialis and reached a maximum at 1.6 ppm whereas inS. inaequivalvis the net accumulation of anaerobic products at the lowest oxygen tension tested (0.5 ppm) was still substantially less than the level of production output in complete anoxia. This suggests that fermentative pathways are maximally activated at all oxygen tensions below 1.6 ppm inM. galloprovincialis whereas rates of anaerobic pathways are still less than maximum at 0.5 ppm inS. inaequivalvis. These results indicate that in situations of declining oxygen tensions, such as occur due to eutrophication,M. galloprovincialis would not only begin to experience metabolic stress at higher oxygen tensions thanS. inaequivalvis but would experience greater stress at any given pO2. Such differences in hypoxia tolerances may explain the success of the recently introducedS. inaequivalvis in out-competing the nativeM. galloprovincialis in the Adriatic Sea.

Correlation between metallothionein and energy metabolism in sea bass, Dicentrarchus labrax, exposed to cadmium☆

Specimens of sea bass (Dicentrarchus labrax) were exposed to two different cadmium concentrations (0.5 and 5 μg Cd2+/ml seawater) for a period of 7 days. Cadmium accumulated in the tissues of D. labrax in the following order: kidney > liver > gills at both concentrations. Accumulation patterns in fish exposed to 0.5 μg Cd2+/ml seawater were different with respect to 5.0 μg Cd2+/ml seawater. At both Cd concentrations a similar stress situation occurred during the first 4 hr as noted by the depletion of glycogen stores and the increase in free glucose in the muscle; metallothionein was induced in the liver, but failed to bind all the cytosolic Cd, which was in part bound to high-molecular-weight ligands. Fish recovered from this initial stress situation within 24 hr as indicated by the increase in glycogen and the decrease of glucose. Long-term effects were clearly dependent upon metal concentration: at lower Cd exposure, metallothionein induction increased linearly with time and counteracted the toxic effect of the metal; on the other hand, when fish were exposed to 5.0 μg Cd2+/ml seawater a clear stress occurred at the end of the exposure, as indicated by the notable decrease of glycogen stores, the increase of free glucose, the decrease of AEC in the muscle and the increase of Cd bound to high-molecular-weight ligands in the liver.

Energy metabolism of the mussel, Mytilus galloprovincialis, during long-term anoxia

Abstract In the mussel, Mytilus galloprovincialis , the pattern of metabolic adaptations to long-term anoxia was clearly biphasic. The first phase (transition stage) lasted 24 hr and was characterized by the decrease of adenylate energy charge, the consumption of aspartate and the accumulation of succinate and alanine. The second phase (stationary stage) was characterized by the depletion of glycogen stores, the production of propionate, which was subsequently excreted into incubation water, and the metabolic depression. The ATP turnover rate was reduced by a factor of 3.9 from short-term anoxia to long-term anoxia.

Differential survival of Venus gallina and Scapharca inaequivalvis during anoxic stress: Covalent modification of phosphofructokinase and glycogen phosphorylase during anoxia

SummaryBiochemical mechanisms underlying anaerobiosis were assessed in two Mediterranean bivalve species, Scapharca inaequivalvis and Venus gallina, with widely differing tolerances for oxygen lack. These species displayed LT50 values for anoxic survival at 17–18°C of 17 and 4 d, respectively. Succinate and alanine were the major products of 24 h anaerobic metabolism in both species but only S. inaequivalvis further metabolized succinate to propionate. Both species reduced metabolic rate while anoxic but metabolic arrest was more pronounced in S. inaequivalvis. Calculated ATP turnover rate (MATP) during exposure to N2-bubbled seawater was only 4.51% of the aerobic rate in S. inaequivalvis but was 12.68% in V. gallina. To counteract a greater load of acid end products, V. gallina foot showed a significantly greater buffering capacity, 23.38±0.20 slykes, compared to 19.6±0.79 slykes in S. inaequivalvis. The two species also differed distinctly in the enzymatic regulation of anaerobiosis. In V. gallina anoxia exposure caused only a small change in PFK kinetic parameters (a decrease in Ka AMP) and had no effect on glycogen phosphorylase. By contrast, S. inaequivalvis foot showed a strong modification of enzyme properties in anoxia. The percentage of glycogen phosphorylase in the a form dropped significantly only in S. inaequivalvis. Other changes included alterations in the properties of PFK leading to a less active enzyme form in anoxia. Compared to the aerobic enzyme form, PFK from anoxic foot showed a reduced affinity for fructose-6-P (Km increased 2.4-fold), greater inhibition by ATP (I50 decreased 6.8-fold), and an increase in sensitivity to AMP activation (Ka decreased by 50%). These enzyme changes appear to be key to a glycolytic rate depression during anaerobiosis in S. inaequivalvis foot muscle.

Resistance of bivalves to anoxia as a response to pollution-induced environmental stress

The effect of environmental pollution stress factors on anoxic survival time was established in three bivalve molluscs (Mytilus edulis, Scapharca inaequivalvis and Venus gallina). In order to study relationships with energy status of the cell and ATP turnover rates the accumulation of fermentative end-products and changes in the glycogen, the adenylate phosphate and the phosphoarginine pool were also estimated. The response among the different species was not consistent. In contrast to Scapharca inaequivalvis, in Mytilus edulis and Venus gallina, accumulated cadmium reduced the tolerance to anoxic conditions. In Mytilus edulis this was accompanied with enhanced metabolic activity indicating a higher anaerobic ATP utilization rate. Additional evidence from literature shows that the anoxic survival response of mussels is a simple method to judge whether environmental variables might have perturbing effects on cellular processes which lead to reduced fitness.

Energy metabolism during anaerobiosis and recovery in the posterior adductor muscle of the bivalve Scapharca inaequivalvis (Bruguiere)

Abstract 1. 1. In the posterior adductor muscle of Scapharca inaequivalvis both aspartate and glycogen were substrates for anaerobic metabolism. ATP and arginine phosphate only contributed to a small extent. Alanine and succinate were major end products, whereas opines were minor end products. Lactate production was not observed. 2. 2. The ATP turnover rate was reduced by a factor of 6 in the later anoxic period. Glycolysis and aspartate contributed about 90% either during the initial step or during the prolonged anoxia. 3. 3. Recovery of 24 hr was not enough for a complete restoration of energy reserves, whereas anaerobic end products were eliminated over this period. There was no strombine or lactate accumulation.

Influence of long-term hypoxia on the energy metabolism of the haemoglobin- containing bivalve Scapharca inaequivalvis: critical O2 levels for metabolic depression

SummaryThe oxygen consumption rate of Scapharca inaequivalvis measured under normoxic conditions over 48 h showed a significant daily cycle with lowest values occurring shortly after the dark period; all hypoxia exposure experiments were carried out during the declining part of the cycle. Animals were exposed to a constant level of hypoxia for a 12-h period in a series of 14 experiments, each at a different oxygen tension. The oxygen consumption was measured continuously, and the extent of accumulation of end-products (succinate and propionate), and the inhibitory effect of adenosine triphosphate on phosphofructokinase were determined at the end of exposures. All three parameters (oxygen consumption, end-product accumulation, phosphofructokinase inhibition) showed a remarkable correlation with major changes occurring between 2.5 and 1.5 ppm (7 and 4 kPa) O2. The oxygen consumption rates showed a drop to 6% of the normoxic rate, but a consistent low consumption remained below 2 ppm (5.5 kPa) which partly recovered over the 12-h exposure period by about three-fold. Succinate and propionate accumulated progressively between 2.5 and 1.5 ppm (7 and 4 kPa); at [O2]<1.5 ppm (4kPa) the concentration did not increase further, indicating that anaerobic metabolism had reached a maximum. Over the same range, phosphofructokinase showed an increased sensitivity for adenosine triphosphate, the lower inhibitor concentration at 50% Vmax value pointing to depression of glycolytic rate. Despite the activation of anaerobic metabolism and the evident depression of aerobic metabolism, simple calculation demonstrates that Scapharca inaequivalvis relies mainly on aerobic metabolism even during severe hypoxia. It is assumed that the occurrence of haemoglobin in this species is essential for its capacity to survive long periods of hypoxia.

Energy metabolism of bivalves at reduced oxygen tensions

This paper describes physiological and biochemical responses of three bivalved molluscs of the Northern Adriatic Sea to reduced ambient oxygen tension. Since the regular occurrence of exceptional algal blooms, the accidentally-imported «Indo-Pacific» blood clam Scapharca inaequivalvis is out-competing the native Venus gallina and Mytilus galloprovincialis. One reason for their differential ability to survive eutrophication with concomitant oxygen depletion is probably the exclusive presence of haemoglobin containing erythrocytes in the blood clam

Effects of cadmium on anoxic survival, haematology, erythrocytic volume regulation and haemoglobin-oxygen affinity in the marine bivalve Scapharca inaequivalvis

Exposure to 50 ppb Cd for 6 wk reduced survival of Scapharca inaequivalvis in anoxic water (LT50 decreasing from ≈ 15 to 12 days). The treatment decreased packed erythrocyte volume (haematocrit) and erythrocyte count but increased mean cellular haemoglobin concentration. In vitro Cd administration had no significant effect on O2 affinity or co-operativity in O2 binding of the stripped haemolysate. Erythrocyte suspensions exhibited a slight volume regulatory decrease, following swelling in hypotonie media, and no significant capacity for volume regulatory increase under hypertonic stress. Neither pattern was markedly affected by in vivo or in vitro Cd administration, which however, tended to reduce the primary volume changes. The results are discussed as regards Cd toxicity and the cellular and molecular mechanisms implicated in volume regulation and tissue-O2 supply.

Long-term anaerobic metabolism of erythrocytes of the arcid clam Scapharca inaequivalvis

Abstract Metabolic events up to 5 days in vivo anoxia incubation are described in the red blood cells of the blood clam Scapharca inaequivalvis . In the first 3 h, respiration is sustained by endogenous oxygen stores. In the subsequent 9 h, the drop in aspartate equals the sum of increase in malate and succinate, and the utilization of aspartate also appears to be linked to the conversion of glucose into alanine. After 12 h anoxia there is no further utilization of aspartate, but alanine, malate and succinate continue to accumulate, whereas propionate accumulation in the whole tissue cells is initiated. This points to a gradual shift away from pyruvate towards oxaloacetate in the phosphoenolpyruvate conversion which seems to be completed after about 48 h. There is a gradual increase of glucose during the first 4 days (hyperglycemia). The transmembrane gradient for glucose is near unity. Only shortly after the accumulation of the volatile fatty acids propionate and acetate in the whole tissues has started, these compounds also appear in the incubation water. This indicates a very easy transport of these compounds. In vitro incubations of red blood cells show, however, that anaerobic propionate accumulation is a feature which have these cells in common with other tissue cells. From day 2 onwards anaerobic metabolism has reached a steady state with propionate and acetate as the sole fermentative products of glucose which are completely excreted.