Factors involved in the (near) anoxic survival time of Cerastoderma edule: associated bacteria vs. endogenous fuel.

The effect of several antibiotics, molybdate and hydrogen sulfide was tested on anoxic tolerance of the cockle Cerastoderma edule, as well as utilisation of glycogen. The aim was to evaluate the role of fuel depletion and growth of bacteria as a cause of mortality. The exponential increase of sulfide and ammonium occurred in anoxic natural seawater incubations and to a lesser extend in artificial, sulfate free, seawater. This could be strongly decreased by antibacterial agents, which led to improved survival time by approximately two-fold. Molybdate suppressed sulfide formation also, but did not affect survival time. Exogenous sulfide showed a negative effect on survival time at pH 6.8 and induced stronger accumulation of free glucose, D-lactate and L-alanine. This was not the case at pH 8.2. Fifty percent (LT50) of cockles in anoxic seawater died after 3.5 days still with half the initial glycogen concentration present. However, in the presence of chloramphenicol (LT50 7.9 days), the cockles utilised their endogenous fuel almost completely. In both incubations there was initially a strong increase of D-lactate and L-alanine. The D-lactate levels subsequently decreased again, probably due to bacterial consumption. This study strongly indicates that in anoxic closed systems, infection by pathogenic bacteria is the first cause of death and not exhaustion of endogenous fuel depots.

Anoxic survival of Macoma balthica: the effect of antibiotics, molybdate and sulphide.

In anoxic semi-closed systems, the survival time of the clam Macoma balthica was compared to clams which were incubated in the presence of several antibiotics (chloramphenicol, 5-oxytetracycline hydrochloride, penicillin, streptomycin, a mix of penicillin and streptomycin and a mix of chloramphenicol, polymyxin, neomycin and penicillin), sulphide and chloramphenicol at pH 6.8 and 8.2 and molybdate (specific inhibitor of the process of sulphate reduction). The aim was to detect maximum survival times of this clam and indications for the cause of mortality under the conditions tested. Median survival time (LT(50)) of the clam was 4.8 days (at 19 degrees C) in incubations without any addition. Added sulphide (200 µM) decreased survival time. At pH 8.2, LT(50) decreased by 20.8% and at pH 6.8 by 35.2%. However, added molybdate, which suppressed biotic sulphide formation, did not improve survival time (LT(50)=4.4 days). Biotic sulphide probably did not speed up mortality rate, but indicated excessive growth of sulphate reducing bacteria once mortality started. The presence of different antibiotics increased significantly survival time (LT(50)) from 8.9 to 14.9 days. Qualitative estimations were made of the numbers of bacteria present in the systems. Compared to a seawater control, highest numbers were observed in the incubation of clams without additions and in the presence of molybdate. Nevertheless, due to the presence of molybdate, bacteria numbers were significantly lower. However, very low numbers of bacteria were observed in the incubations of clams in the presence of chloramphenicol. These data demonstrated that the presence and proliferation of bacteria was probably the cause of death of the clams.

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

The 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] less than 1.5 ppm (4 kPa) 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Health care institutions in a regional network: a longitudinal case study of inter-organizing.

This is a second article reporting on the functioning of a regional federation in the Dutch health care field. It is a heterogeneous federation of health care delivery institutions (clinical, ambulatory, and primary care) within a geographical area of about 250,000 inhabitants. The first article on this subject, published in this journal in 1981, dealt with the facilities and obstacles during the founding process of the federation and with the problems concerning the making of a policy program (Soc. Sci. Med. 15A, 41-48, 1981). That was about five years after the inception of the federation and it seemed both possible and fruitful to carry out further evaluation of this particular case. A content analysis of all the 41 meetings of the federation could be performed on the agendas, minutes and other relevant documents. The results of this analysis, taken together with a substantial amount of information gathered by participant observation during these sessions, gave rise to certain conclusions and interpretations. They concerned the numbers and kinds of subjects discussed, the changes in their composition over the years, the varying amount of energy put into the development of the federation's organization, the evolution of the federation's task and its effectiveness in preparing regional plans for health care provision. The results of the analysis seemed to encourage this approach to regionalization. Developments during the most recent period, since 1982, will be described in a third article. Although in a legislative sense this year was important, the related enactment processes went on very slowly.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic adaptations of intertidal invertebrates to environmental hypoxia (a comparison of environmental anoxia to exercise anoxia).

By comparing environmental anaerobiosis with exercise anaerobiosis it appears that animals with high anoxia tolerance use (partly) different types of metabolic reactions to sustain energy metabolism, whereas low tolerance animals (Arthropoda, Echinodermata, Vertebrata) use the same pathway under both conditions. During exercise anaerobiosis the classical glycolysis (lactate pathway) is a main pathway among all multicellular organisms, although in marine invertebrates--except the Arthropoda and Echinodermata--it mostly does not terminate in lactate. During environmental anaerobiosis Cnidaria, Mollusca, Annelida and Sipunculida first couple additional pathways for energy extraction to the glycolytic pathway (the aspartate--succinate pathway) and later deviate the main carbon flow of glycogen at the level of phosphoenolpyruvate towards succinate, propionate and acetate production. Metabolic adaptations to anoxic cellular conditions in these groups are high fuel stores, increased ATP yield by anaerobic sources, formation of easily excretable (volatile) end products, an aspartate-dependent system for transport of hydrogen through the inner membrane of the mitochondrion and a rapid recovery from anaerobic metabolism. During anaerobic conditions three sources can contribute to the anaerobic power output, endogenous stores of both ATP and phosphagen and catabolism. Anaerobic power output rates have been calculated for a number of Mollusca, Annelida and Crustacea. Extreme anoxia resistance is coupled to a strongly reduced metabolic rate. In animals with high aspartate stores, the aspartate--succinate pathway and phosphagen hydrolysis can provide sufficient ATP during environmental anaerobiosis; however, with exercise anaerobiosis when ATP turnover rates may be increased by a factor of 20, pyruvate derivatives simultaneously accumulate in high amounts relative to succinate.

Facultative anaerobiosis in molluscs.

The glycolytic fermentation of molluscs is rather complex. Multiple end products accumulate (lactate, alanine, octopine, succinate, propionate, acetate and CO2), which are partly formed in the cytoplasm and partly in the mitochondrion. Various schemes have been presented to account for these end products as well as for the maintenance of the redox balance. With respect to the role of alanine there are two opinions: (1) alanine accumulation is continuous and is essential for the generation of the mitochondrial NADH required in the reduction of fumarate and (2) succinate and alanine (initial end products) accumulate in different compartments and their accumulation occurs independently. Both statements are evaluated in the light of the latest experimental observations including the regulatory properties at the phosphoenolpyruvate branchpoint and the effect of pH and 'energy charge'. For nervous tissue the function of oxygen can be replaced by the lipochrome pigment, which enables carbohydrates to be totally oxidized to CO2 and water. The simultaneous mobilization of carbohydrates and amino acids is not supported by the experimental data. Various advantages of the glycolytic fermentation in molluscs as compared with classical glycolysis in skeletal muscle are discussed.