SUMMARY The ubiquitin-proteasome system controls a variety of essential intracellular processes through directed protein turnover. The invertebrate proteasome has recently gained increasing interest with respect to central physiological processes and pathways in different taxa. A pitfall in proteasome activity assays, represented by the trypsin-like, chymotrypsin-like or caspase-like site, lies in the fact that most commonly used experimental substrates are susceptible to degradation by non-proteasomal proteolytic enzymes, which can lead to erroneous interpretation of activity data obtained. Through the use of a proteasome-specific inhibitor, epoxomicin, we showed that the shares of proteasomal and non-proteasomal activities in the degradation of a model polypeptide substrate for chymotrypsin-like activity vary considerably between invertebrate taxa. Crustacean muscle tissue and hemocytes showed almost exclusively proteasomal activity. In yeast, approximately 90% of total proteolytic activity can be attributed to the proteasome. In contrast, proteasomal activity comprises only 20–60% of the total proteolytic activity in bivalve tissues. These results reveal that, without verification of the shares of proteasomal and non-proteasomal activities in crude extracts through the use of highly specific inhibitors, common proteasomal enzyme assays should be used and interpreted with caution.

SUMMARY Arctica islandica is the longest-lived non-colonial animal found so far, and reaches individual ages of 150 years in the German Bight (GB) and more than 350 years around Iceland (IC). Frequent burrowing and physiological adjustments to low tissue oxygenation in the burrowed state are proposed to lower mitochondrial reactive oxygen species (ROS) formation. We investigated burrowing patterns and shell water partial pressure of oxygen ( P O 2 ) in experiments with live A. islandica . Furthermore, succinate accumulation and antioxidant defences were recorded in tissues of bivalves in the normoxic or metabolically downregulated state, as well as ROS formation in isolated gills exposed to normoxia, hypoxia and hypoxia/reoxygenation. IC bivalves burrowed more frequently and deeper in winter than in summer under in situ conditions, and both IC and GB bivalves remained burrowed for between 1 and 6 days in laboratory experiments. Shell water P O 2 was <5 kPa when bivalves were maintained in fully oxygenated seawater, and ventilation increased before animals entered the state of metabolic depression. Succinate did not accumulate upon spontaneous shell closure, although shell water P O 2 was 0 kPa for over 24 h. A ROS burst was absent in isolated gills during hypoxia/reoxygenation, and antioxidant enzyme activities were not enhanced in metabolically depressed clams compared with normally respiring clams. Postponing the onset of anaerobiosis in the burrowed state and under hypoxic exposure presumably limits the need for elevated recovery respiration upon surfacing and oxidative stress during reoxygenation.

SUMMARY The decline of cellular and especially mitochondrial functions with age is,among other causes, held responsible for a decrease in physiological fitness and exercise capacity during lifetime. We investigated size- and age-related changes in the physiology of exercising specimens of the short lived swimming scallop Aequipecten opercularis (maximum life span 8 to 10 years)from the Isle of Man, UK. A. opercularis swim mainly to avoid predators, and a decrease in swimming abilities would increase the risk of capture and lower the rates of survival. Bigger (older) individuals were found to have lower mitochondrial volume density and aerobic capacities (citrate synthase activity and adenylates) as well as less anaerobic capacity deduced from the amount of glycogen stored in muscle tissue. Changes in redox potential, tissue pH and the loss of glutathione in the swimming muscle during the exercise were more pronounced in young compared to older individuals. This indicates that older individuals can more effectively stabilize cellular homeostasis during repeated exercise than younger animals but with a possible fitness cost as the change in physiology with age and size might result in a changed escape response behaviour towards predators.

SUMMARY The interplay between antioxidants, heat shock proteins and hypoxic signaling is supposed to be important for passive survival of critical temperature stress, e.g. during unfavorable conditions in hot summers. We investigated the effect of mild (18°C), critical (22°C) and severe(26°C) experimental heat stress, assumed to induce different degrees of functional hypoxia, as well as the effect of recovery following heat stress on these parameters in liver samples of the common eelpout Zoarces viviparus . Upon heat exposure to critical and higher temperatures we found an increase in oxidative damage markers such as TBARS (thiobarbituric reactive substances)and a more oxidized cellular redox potential, combined with reduced activities of the antioxidant enzyme superoxide dismutase at 26°C. Together, these point to higher oxidative stress levels during hyperthermia. In a recovery-time series, heat-induced hypoxia and subsequent reoxygenation upon return of the fishes to 12°C led to increased protein oxidation and chemiluminescence rates within the first 12 h of recovery, therein resembling ischemia/reperfusion injury in mammals. HSP70 levels were found to be only slightly elevated after recovery from sub-lethal heat stress, indicating minor importance of the heat shock response in this species. The DNA binding activity of the hypoxia-inducible transcription factor (HIF-1) was elevated only during mild heat exposure(18°C), but appeared impaired at more severe heat stress. We suppose that the more oxidized redox state during extreme heat may interfere with the hypoxic signaling response.

SUMMARY The influence of seasonal and acute temperature changes on mitochondrial functions were studied in isolated mitochondria of the eurythermal lugworm Arenicola marina (Polychaeta), with special emphasis on the interdependence of membrane potential and radical production. Acclimatisation of lugworms to pre-spawning/summer conditions is associated with rising mitochondrial substrate oxidation rates, higher proton leakage rates, elevated membrane potentials, and increased production of reactive oxygen species (ROS)in isolated mitochondria, compared with mitochondria from winter animals. However, a high ROS production was compensated for by higher activities of the antioxidant enzymes catalase and superoxide dismutase, as well as lower mitochondrial densities in summer compared with winter animals. In summer animals, a higher sensitivity of the proton leakage rate to changes of membrane potential will confer better flexibility for metabolic regulation(mild uncoupling) in response to temperature change. These seasonal alterations in mitochondrial functions suggest modifications of energy metabolism in eurythermal and euryoxic organisms on intertidal mudflats during summer. In winter, low and less changeable temperatures in intertidal sedimentary environments permit higher respiratory efficiency at low aerobic metabolic rates and lower membrane potentials in A. marina mitochondria.