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ATP content and mortality in mytilus edulis from different habitats in relation to anaerobiosis
.Netherlands Journal of Sea Research 10 (1) : 140-148 (1976)
ATP
CONTENT AND MORTALITY IN MYTILUS EDULIS FROM DIFFERENT HABITATS IN RELATION TO ANAEROBIOSIS by T. C. M. W I J S M A N (Laboratory of Chemical Animal Physiolog),, Utrecht, The Netherlm~ds) CONTENTS
I. Introduction . . . . . . . . . . . . . . . II. Materials and Methods . . . . . . . . . . . . 1. Animals . . . . . . . . . . . . . . . . 2. Preparation of tissue extract . . . . . . . . . 3. Enzymatic assay of ATP . . . . . . . . . . III. Results . . . . . . . . . . . . . . . . . IV. Discussion . . . . . . . . . . . . . . . . V. Summary . . . . . . . . . . . . . . . . VI. References . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . .
140 142 142 142 143 143 145 147 147
I. I N T R O D U C T I O N All investigations in our l a b o r a t o r y a b o u t the energy metabolism in Mytilus edulis in relation to oxygen availability were p e r f o r m e d on c u l t u r e d mussels (DE ZWAAN • ZANDEE, 1972; DE ZWAAN & VAN MARREWIJK, 1973; D E ZWAAN, DE BONT & KLUYTMANS, 1975; KLUYTMANS, VEENHOF & DE ZWAAN, 1975). These cultured mussels live in the W a d d e n Sea at a d e p t h of 4 to 7 m and are never lying dry at all. Nevertheless they are a d a p t a t e d to anoxia and can survive anaerobic periods of several days (THAMDRUP, 1935). Even in sea water spontaneous shell closure is observed for u p to 5 hours (WIJSMAN, 1975). T h e W a d d e n Sea p o p u l a t i o n is not restricted to the maritime zone, favoured by continuous oxygen and food supply, but is in exchange with animals in the intertidal zone (wild mussels) by means of the free swimming larvae. It is not likely that wild and cultured mussels will differ in their basic pathways of anaerobic energy production. M a n y schemes have been proposed to account for the f e r m e n t a t i o n of carbohydrates, the m a i n fuel substrate (CItEN, 1969; GILLES, 1972; HOCHACHKA & MUSTAFA, 1972; HOCHACHKA, FIELDS & MUSTAFA, 1973; DE ZWAAN, VAN ,?VIARREWlJK & HOLWERDA, 1973; DE ZWAAN, KLtJYTMANS & ZANDEE, 1976). In any concept, however, the total A T P - y i e l d is strongly diminished d u r i n g anoxia, because oxydative phosphorylation via the c y t o c h r o m e
ATP
IN
MYTILUS
141
system is lacking whereas a clear Pasteur effect cannot be demonstrated (DE ZWAA~r & WUSMAN, 1975). By reducing various energy demanding processes the sea mussel will try to maintain balance between A T P consumption and regeneration. Imbalance between offer and demand of ATP, however, will influence its steady state concentration. It might be possible that this A T P level in wild mussels, which are exposed to air during low tide twice a day, is less affected in comparison with the cultured animals. This might be due to a more extensive use of the energy delivering processes or a more sufficient reduction of the overall metabolism. Therefore, it is interesting to follow the course of the ATP levels in both groups during anaerobiosis. Most of the direct available energy is stored in the high energetic "r-phosphate-bound of ATP, which, together with A M P and ADP*, form the adenylate pool as the main system for energy transport. Equilibrium between the three components is maintained by the enzyme adenylate kinase, which is probably ubiquitous in living material (MARKLAND& WADKINS,1966). The charge of this system is given by the mean number of anhydride bound phosphates per adenosine moiety and varies between 0 (only A M P present) and 2 (only A T P present). Dividing by 2 gives the quotient (ATP + ½ A D P ) / ( A T P + ADP + AMP), the energy charge of the adenylate system as introduced by ATKINSON & WALTON (1967), varying between 0 and 1. This parameter is a better estimation of the energy state than the ATP level or the A T P / A D P ratio. From a previous investigation (WIjSM~'% 1976), however--in which the concentration of AMP, ADP and ATP were determined in M. edulis, kept in well aerated sea water and exposed to air up to 7 days--the relation between ATP, expressed as percentage of the control, and the energy charge can be determined (Fig. 1). For this reason only ATP was determined in this investigation. ATP+I'~ADP ATP+ADP +AMP
1.(~ 09
09
0.~
~o ~o /o ~o ~o ~o A'I'P (% of control )
Fig. 1. Relationship between ATP content, as percentage of the control, and the energy charge in Mytilus edulis. * Abbreviations used: AMP, ADP, ATP for adenosine~5'-mono-,di-, triphosphate; NADH for [~-nicotinamide-adeninedinucleotide (reduced).
142
T.C.M. WIJSMAN
Acknowledgements.--The author is much indebted to Dr J. H. F. M. Kluytmans, Prof. Dr D. I. Zandee and Dr A. de Zwaan for their guidance and valuable criticism, to Mr D. Smit for drawing the figures and to Miss J. C. Lobato for her help in preparing the manuscript. II. MATERIALS
AND METHODS
1. ANIMALS Mussels (Mytilus edulis L.) were supplied by the Institute of Mussel Research, Texel, The Netherlands. Cultured animals, living in deeper parts of the Wadden Sea never exposed to air, were sampled with a trawl at the end of May 1975; wild animals, exposed to air for 5 to 6 hrs a day, were collected near the south point of the island of Texel. After transport to Utrecht cultured and wild mussels were placed in perforated polyethylene racks in recirculating sea water (13 ° C) without food supply. They were exposed to air by placing the racks just above the sea water level. At fixed times during the experiments 2 samples of 10 animals each were taken from both groups and the A T P concentration of the total soft tissues was determined. Dead animals were removed and the cumulative mortality was calculated, taking into account the decreased number of mussels due to the ATP determinations. Experiments started at J u n e 5th, 1975 and were finished within 2 weeks. Cultured and wild mussels, kept in sea water continuously, were used to obtain control values of ATP and mortality at the end of the experiments. The mean wet weight of the soft tissue, shell length, and weight of the shells were 3.83 g, 5.10 cm and 8.84 g for wild mussels and 5.83 g, 6.19 cm and 7.39 g for cultured ones respectively (determined for 50 mussels). 2. P R E P A R A T I O N
OF T I S S U E E X T R A C T
Extracts were made from samples of" 10 mussels. The shells were opened by cutting the posterior adductor muscle. The tissue was excised, blotted on a soft paper tissue and dropped in liquid nitrogen. After evaporation of the nitrogen the wet weight was determined. The material was homogenized during 5 min with 3 volumes ice-cold 7% (w/v) trichloroacetic acid in a Sorvall Omnimix at maximum speed. The homogenate was centrifuged at 35,000 g for 15 min at 0 ° C (Sorvall RC-2B) and 5 ml of the supernatant was neutralized with 5 M K~CO 3 and phenolred as indicator.
ATP
143
IN MYTILUS
3. E N Z Y M A T I C ASSAY OF ATP
A T P was determined according to JAWOREK, GRUBER & BERGMEYER (1970), with tris (hydroxymethyl)aminomethane-HC1 as buffering system, while the final volume of the assay mixture was 1 ml. Determinations were performed in fourfold with 100 ~1 tissue extract. The absorbance at 340 nm was followed with a Zeiss P M - Q I I spectrophotometer coupled with a Vitratron UR-400 recorder. All reactions were finished within 10 min. The A T P concentration was calculated from the change in absorbance (extrapolated to the starting point of the reaction) and the extinction coefficient of N A D H (6.22 X 103 1 mo1-1 cm-a). Standard samples in a range of 4 to 80 ~M gave a reproducible linear response with a quantitative recovery. Tissue samples were within this range, but showed an inhibition of 7 ~o. Corrections could be made by use of internal standards. Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), 3-phosphoglycerate kinase (EC 2.7.2.3), N A D H and A T P were obtained from Boehringer, Mannheim. All other chemicals were "Baker Analyzed" from J. T. Baker Chemicals. III. RESULTS
Cultured and wild mussels were exposed to air for 7 days, and then reimmersed in sea water (Fig. 2). The A T P levels, with normal values of about 2 mM, decrease during 3 days of anoxia to minima of 1.18 and 0.94 m M in cultured and wild animals, respectively. After reimmersion in sea water, the A T P concentrations reach levels equal k\\-,~
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Fig. 2. ATP concentration ( © , e ) and mortality (z%&) in cultured ( © , A ) and wild (O,A) mussels (Mytilus edulis) during exposure to air (blank part of bar) and subsequent immersion in sea water (hatched part). Control values were obtained for animals, covered by sea water during the whole experiment.
144
T.C.M. WIJSMAN
to those of the control groups within 2 to 3 days. Although statistical evidence is lacking it seems that the ATP concentration in wild mussels, compared to that in cultured ones, shows a stronger decrease under anoxia while it takes more time to restore the normal situation. The general course of both curves, however, is identical. There is no conformity, however, with respect to mortality. The increase in mortality of wild mussels, which starts at the 7th day of anoxia, immediately ends when the animals are placed in sea water again. Mortality in cultured mussels shows a remarkable increase during exposure to air, starting from the 4th day, reaches a value of 24% after 7 days of anoxia, and is not stopped by reimmersion in sea water, with the consequence that mortality is over 50~/o after 4 subsequent days of aerobiosis. To study the effect of a periodical aerobic-anaerobic transition, cultured and wild mussels were alternately exposed to air and reimmersed in sea water for 24 11 over a period of 10 days. Each day mortality was calculated while ATP was determined after each anaerobic period (Fig. 3). ATP concentrations, obtained after the first anaerobic kk\\'x\\\\\\\'~
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Fig. 3. ATP concentration (2),O) and mortality (/~.,,A) in cuhured (, ,,1~: and wild (O,A) mussels (Mytilus edulis), alternately immersed in sea water (hatched part of bar) and exposed to air (blank part). Control values were obtained for animals, covered by sea water during the whole experiment.
period of 24 h (1.43 m M for cultured and 1.23 m M tbr wild mussels) are in agreement with the corresponding data of Fig. 2 (1.48 and 1.31 m M for cultured and wild mussels, respectively). Mean ATP levels (with standard deviation), calculated from these data, are 1.46 4:0.09 m M for cultured and 1.27 i 0.07 m M for wild animals, corresponding to 729/o and 61% of the "aerobic" values. During subsequent periods of exposure to air ATP concentrations in both groups hardly drop further,
ATP
IN M Y T I L U S
145
even when exposure was extended to 5 days beginning at the 9th day o f this experiment (Fig. 3). In the last case, A T P levels of 1.38 m M (cultured) and 1.14 m M (wild) were determined after 3 days of continuous anoxia, which is both 0.2 m M higher than the corresponding data of Fig. 2. U p to the 10th day mortality is nearly equal for both groups and does not exceed the control values. After that time the course of mortality is identical to the one in Fig. 2. IV. DISCUSSION The two groups of animals used in the experiments live under quite different conditions in their natural habitat. The cultured mussels are continually covered by sea water, whereas the wild animals are exposed to air for a quarter of their life during which period no food and oxygen are available. These differences are reflected in their phenotype: tissue wet weight of cultured mussels was 50% higher (5.83 versus 3.83 g) and shell length was 20% longer (6.19 versus 5.10 cm) than of wild animals, whereas the weight of the shells was 18% lower (7.39 versus 8.84 g). Prolonged exposure to air showed a remarkable difference in mortality between the two groups (Fig. 2), indicating that wild mussels are better adapted to these circumstances than cultured animals. The continued mortality of cultured mussels after reimmersion in sea water indicates that processes are started, leading to death, which cannot be stopped under aerobic conditions. No significant difference was obtained between the "aerobic" A T P levels of cultured (2.02 4- 0.14 raM) and wild mussels (2.08 4- 0.09 mM). But A T P levels, determined after anaerobic periods of 24 hrs and during a subsequent anoxic period of 5 days (Fig. 3), were almost 0.2 m M higher in cultured than in wild mussels. Even after 7 days of continuous anaerobiosis, when mortality was more than two times higher in cultured mussels than in wild ones, and during reimmersion in sea water, when mortality in cultured mussels increased to the fivefold of that in wild animals, the ATP concentration was equal or even higher compared to wild mussels (Fig. 2). These higher values cannot be attributed to a greater desiccation of the cultured mussels, because the quotient of dry weight and wet weight was equal for both groups, being unchanged during exposure to air: 0.203 4- 0.010 g dry weight/g wet weight (n ---- 24) for cultured mussels versus 0.202 ± 0.09 g dry weight/g wet weight (n = 26) for wild animals. As discussed in the Introduction, anoxia causes a pronounced fall in ATP production which will result in decreased A T P levels when
146
T.C.M. WI~SMAN
ATP-consuming processes are not reduced to the same extent. So it might be possible that the steady state concentration of ATP is less affected when mussels are better adapted to anaerobiosis. According to this supposition, the ATP data suggest that cultured mussels are better adapted to sustain anaerobiosis than wild animals. But the opposite is true, as is evident from the data on mortality. Probably the ATP concentration (and the energy charge) is not indicative for the physiological state of Mytilus edulis, as can be deduced also from the high concentration of A T P in dead animals (WIJSMAN, 1976). Apparently survival depends not only on maintenance of an adequate A T P level. While glycogen stores in M. edulis are sufficient to survive anoxia for months, the derived end products, as succinate and free volatile fatty acids, are accumulated to amounts which may disorder the whole organism. Maximum survival times under experimental anaerobic conditions are strongly different for various species of molluscs (YONBRAND, 1946). These differences might be related to the extent in which the animal can reduce its energy demand. At room temperature, for example, Mytilus edulis can withstand anoxia for several days, whereas the pond snail Limnaea stagnalis dies after a few days. In both species carbohydrates are catabolized to the same end products during anaerobiosis, but in spite of its relative low tolerance for anoxia only in L. stagnalis a clear Pasteur effect is observed (DE ZWAAN& ZANDEE, 1972 ; DE ZWAAN, MOHAMED & GERAERTS, 1976). This investigation illustrates that A T P levels and changes in the steady state concentration during anoxia reveal nothing about survival under anaerobic conditions. A better approach to this seems the study of the actual energy demand during anaerobiosis. This has been started in our team (DE ZWAAN & WUsMAN, 1975). It takes several days to restore the A T P concentration and probably the energy charge (Fig. 1) in Mytilus edulis after prolonged exposure to air. In this situation energy yielding processes are stimulated while energy demanding processes are inhibited according to the theory of ATrdNSON (1968). Inhibition of pyruvate kinase by the anaerobic drop in p H will be releaved within an hour after reimmersion of M. edulis in sea water (WusMAN, 1975), whereas activation of this enzyme by the decreased energy charge (EBBERIN~, DE ZWAAN & WUSMAN, 1976) is probably maintained over a longer period. In this way the enhanced catalytic capacity of pyruvate kinase can serve to increase the flux of acetyl-CoA units into the tricarboxylic acid cycle, facilitated by the accumulation of some intermediates of this cycle during anaerobiosis. This is in agreement with the observed oxygen debt in M. edulis after anoxia (ScHLIEPER& KOWALSKI,1959).
ATP
IN MYTILUS
147
V. S U M M A R Y
Mortality and A T P content during experimental exposure to air were determined in two groups of Mytilus edulis L., one lying dry during low tide (wild), the other continuously covered by sea water in its habitat (cultured). When kept in sea water continuously, ATP levels were 2.02 4- 0.14 m M in cultured and 2.08 -4- 0.09 m M in wild mussels. Mortality was negligible and almost equal for both groups (Fig. 2). In mussels, exposed to air for 7 days, a m a x i m u m decrease in ATP content to 1.18 m M (cultured) and 0.94 m M (wild) was determined after 3 days. Mortality in wild mussels reached 9% and stopped after reimmersion in sea water, whereas mortality in cultured animals was 24~/o at the end of exposure and continued under aerobic conditions, being 50% after 4 days of aerobiosis. In both groups normal A T P levels were restored within 2 to 3 days of aerobiosis (Fig. 2). When mussels were exposed to air and immersed in sea water alternately for periods of 24 h, ATP levels were maintained at about 1.4 m M for cultured and 1.2 m M for wild animals. Mortality was equal in both groups and did not exceed the control values. During subsequent exposure of 5 days, no further decrease in ATP concentration was observed in both groups (Fig. 3). VI. REFERENCES ATKINSON, D. E., 1968. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers.---Biochemistry, N.Y. 7t 4030-4034. ATKmSON, D. E. & G. M. WALTON, 1967. Adenosine triphosphate conservation in metabolic regulation.--J, biol. Chem. 242: 3239-3241. BRAND, T. YON, 1946. Anaerobiosis in invertebrates. In: B. J. LOYET. Biodynamiea Monographs.~Biodynamica 4: 90-91. CHEN, C., 1969. A variation of the Embden-Meyerhof-Parnas scheme in molluscs. Ph.D. Thesis, Rice University. E~BERINK, R. H. M., A. DE ZWAAN & T. C. M. WIJSMAN, 1976. Regulation at the phosphoenolpyruvate branchpoint by the adenylate energy charge in Mytilus edulis.---Biochem. Soc. Trans. 4: 444-447. GmLES, R., 1972. Biochemical ecology of Mollusca. In: M. FLORI~N & B. T. SCHEER. Chemical Zoology. Academic Press, New York, London 7: 467-495. HOCHACHKA, P. W., J. FIELDS ~; T. MUSTAFA, 1973. Animal life without oxygen: basic biochemical mechanisms.--Am. Zoologist 13- 543-555. HOCHACHKA, P. W. • T. MUSTAFA, 1972. Invertebrate facultative anaerobiosis.-Science, N.Y. 178: 1056-1060. JAWOREK, D., W. GRUBER & H. U. BERGMEYER, 1970. Adenosin-5"-triphosphat. In: H. U. BERGMEYER. Methoden der enzymatischen Analyse. Weinheim Vetlag Chemic 2" 2020-2024. KLUYTMANS,J. H., P. R. VEENHOF & A. DE ZWAAN, 1975. Anaerobic production of
148
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volatile fatty acids in the sea mussel Mytilus edulis L.--J. cell. comp. Physiol. 104: 71-78. MARKLAND, F. S. & C. L. WADKINS, 1966. Adenosine triphosphate-adenosine 5'monophosphate phosphotransferase of bovine liver mitochondria. I. Isolation and chemical properties.--J, biol. Chem. 241: 4124-4135. SCHLmI'ZR, C. & R. KOWALSKb 1959. Ein zelluliirer Regulationsmechanismus ftir erh6hte Kiemenventilation nach anoxybiose bei Mytilus edulis L.--Kieler Meeresforsch. 14: 42-47. THAmDRUP,H. M., 1935. Beitriige zur Okologie der Wattenfauna.--Meddr Kommn Danm. Fisk.-og Havunders. Ser. Fisk. 10 (2): 1-130. WIJSMAN, T. C. M., 1975. pH fluctuations in Mytilus edulis L. in relation to shell movements under aerobic and anaerobic conditions. In: H. BARNES. Proc. 9th Europ. mar. biol. Syrup. Aberdeen University Press: 139--149. , 1976. Adenosine phosphates and energy charge in different tissues of Mytilus edulis L. under aerobic and anaerobic conditions.--J, cell. comp. Physiol. 107: 129-140. ZWAAN, A. DE, A. M. T. DE BONT & J. H. F. M. KLUYTMANS, 1975. Metabolic adaptations on the aerobic-anaerobic transition in the sea mussel, Mytilus edulis L. In: H. BARNES.Proc. 9th Europ. mar. biol. Syrup. Aberdeen University Press: 121-138. ZWAAN, A. DE, J. H. F. M. KLUYTMANS& D. I. ZANDEE, 1976. Facultative anaerobiosis in molluscs.---Biochem. Soc. Symp. l l : 133-167. ZWAAN, A. DE & W. J. A. VAN MARREWlJK, 1973. Anaerobic glucose degradation in the sea mussel Mytilus edulis L.--Comp. Biochem. Physiol. 4413: 429439. ZWAAN, A. DE, W. J. A. VAN MARREWIJK & D. A. HOLWERDA, 1973. Anaerobic carbohydrate metabolism in the sea mussel Mytilus edulis L.--Neth. J. Zool. 23: 225-228. ZWAAN, A. DE, A. M. MOHA~ED & W. P. M. GERAERTS, 1976. Glycogen degradation and the accumulation of compounds during anaerobiosls in the fresh water snail Lymnaea stagnalis .--Neth. J. Zool. (in press.). ZWAAN, A. DE & T. C. M. WUSMAN, 1975. Anaerobic metabolism in Bivalvia (Mollusca). Characteristics of anaerobic metabolism.--Comp. Biochem. Physiol. 54B: 313-324. ZWAAN,A. DE & D. I. ZANDEE, 1972. The utilization of glycogen and accumulation of some intermediates during anaerobiosis in Mytilus edulis L.--Comp. Biochem. Physiol. 43B: 47-54.
ATP
CONTENT AND MORTALITY IN MYTILUS EDULIS FROM DIFFERENT HABITATS IN RELATION TO ANAEROBIOSIS by T. C. M. W I J S M A N (Laboratory of Chemical Animal Physiolog),, Utrecht, The Netherlm~ds) CONTENTS
I. Introduction . . . . . . . . . . . . . . . II. Materials and Methods . . . . . . . . . . . . 1. Animals . . . . . . . . . . . . . . . . 2. Preparation of tissue extract . . . . . . . . . 3. Enzymatic assay of ATP . . . . . . . . . . III. Results . . . . . . . . . . . . . . . . . IV. Discussion . . . . . . . . . . . . . . . . V. Summary . . . . . . . . . . . . . . . . VI. References . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . .
140 142 142 142 143 143 145 147 147
I. I N T R O D U C T I O N All investigations in our l a b o r a t o r y a b o u t the energy metabolism in Mytilus edulis in relation to oxygen availability were p e r f o r m e d on c u l t u r e d mussels (DE ZWAAN • ZANDEE, 1972; DE ZWAAN & VAN MARREWIJK, 1973; D E ZWAAN, DE BONT & KLUYTMANS, 1975; KLUYTMANS, VEENHOF & DE ZWAAN, 1975). These cultured mussels live in the W a d d e n Sea at a d e p t h of 4 to 7 m and are never lying dry at all. Nevertheless they are a d a p t a t e d to anoxia and can survive anaerobic periods of several days (THAMDRUP, 1935). Even in sea water spontaneous shell closure is observed for u p to 5 hours (WIJSMAN, 1975). T h e W a d d e n Sea p o p u l a t i o n is not restricted to the maritime zone, favoured by continuous oxygen and food supply, but is in exchange with animals in the intertidal zone (wild mussels) by means of the free swimming larvae. It is not likely that wild and cultured mussels will differ in their basic pathways of anaerobic energy production. M a n y schemes have been proposed to account for the f e r m e n t a t i o n of carbohydrates, the m a i n fuel substrate (CItEN, 1969; GILLES, 1972; HOCHACHKA & MUSTAFA, 1972; HOCHACHKA, FIELDS & MUSTAFA, 1973; DE ZWAAN, VAN ,?VIARREWlJK & HOLWERDA, 1973; DE ZWAAN, KLtJYTMANS & ZANDEE, 1976). In any concept, however, the total A T P - y i e l d is strongly diminished d u r i n g anoxia, because oxydative phosphorylation via the c y t o c h r o m e
ATP
IN
MYTILUS
141
system is lacking whereas a clear Pasteur effect cannot be demonstrated (DE ZWAA~r & WUSMAN, 1975). By reducing various energy demanding processes the sea mussel will try to maintain balance between A T P consumption and regeneration. Imbalance between offer and demand of ATP, however, will influence its steady state concentration. It might be possible that this A T P level in wild mussels, which are exposed to air during low tide twice a day, is less affected in comparison with the cultured animals. This might be due to a more extensive use of the energy delivering processes or a more sufficient reduction of the overall metabolism. Therefore, it is interesting to follow the course of the ATP levels in both groups during anaerobiosis. Most of the direct available energy is stored in the high energetic "r-phosphate-bound of ATP, which, together with A M P and ADP*, form the adenylate pool as the main system for energy transport. Equilibrium between the three components is maintained by the enzyme adenylate kinase, which is probably ubiquitous in living material (MARKLAND& WADKINS,1966). The charge of this system is given by the mean number of anhydride bound phosphates per adenosine moiety and varies between 0 (only A M P present) and 2 (only A T P present). Dividing by 2 gives the quotient (ATP + ½ A D P ) / ( A T P + ADP + AMP), the energy charge of the adenylate system as introduced by ATKINSON & WALTON (1967), varying between 0 and 1. This parameter is a better estimation of the energy state than the ATP level or the A T P / A D P ratio. From a previous investigation (WIjSM~'% 1976), however--in which the concentration of AMP, ADP and ATP were determined in M. edulis, kept in well aerated sea water and exposed to air up to 7 days--the relation between ATP, expressed as percentage of the control, and the energy charge can be determined (Fig. 1). For this reason only ATP was determined in this investigation. ATP+I'~ADP ATP+ADP +AMP
1.(~ 09
09
0.~
~o ~o /o ~o ~o ~o A'I'P (% of control )
Fig. 1. Relationship between ATP content, as percentage of the control, and the energy charge in Mytilus edulis. * Abbreviations used: AMP, ADP, ATP for adenosine~5'-mono-,di-, triphosphate; NADH for [~-nicotinamide-adeninedinucleotide (reduced).
142
T.C.M. WIJSMAN
Acknowledgements.--The author is much indebted to Dr J. H. F. M. Kluytmans, Prof. Dr D. I. Zandee and Dr A. de Zwaan for their guidance and valuable criticism, to Mr D. Smit for drawing the figures and to Miss J. C. Lobato for her help in preparing the manuscript. II. MATERIALS
AND METHODS
1. ANIMALS Mussels (Mytilus edulis L.) were supplied by the Institute of Mussel Research, Texel, The Netherlands. Cultured animals, living in deeper parts of the Wadden Sea never exposed to air, were sampled with a trawl at the end of May 1975; wild animals, exposed to air for 5 to 6 hrs a day, were collected near the south point of the island of Texel. After transport to Utrecht cultured and wild mussels were placed in perforated polyethylene racks in recirculating sea water (13 ° C) without food supply. They were exposed to air by placing the racks just above the sea water level. At fixed times during the experiments 2 samples of 10 animals each were taken from both groups and the A T P concentration of the total soft tissues was determined. Dead animals were removed and the cumulative mortality was calculated, taking into account the decreased number of mussels due to the ATP determinations. Experiments started at J u n e 5th, 1975 and were finished within 2 weeks. Cultured and wild mussels, kept in sea water continuously, were used to obtain control values of ATP and mortality at the end of the experiments. The mean wet weight of the soft tissue, shell length, and weight of the shells were 3.83 g, 5.10 cm and 8.84 g for wild mussels and 5.83 g, 6.19 cm and 7.39 g for cultured ones respectively (determined for 50 mussels). 2. P R E P A R A T I O N
OF T I S S U E E X T R A C T
Extracts were made from samples of" 10 mussels. The shells were opened by cutting the posterior adductor muscle. The tissue was excised, blotted on a soft paper tissue and dropped in liquid nitrogen. After evaporation of the nitrogen the wet weight was determined. The material was homogenized during 5 min with 3 volumes ice-cold 7% (w/v) trichloroacetic acid in a Sorvall Omnimix at maximum speed. The homogenate was centrifuged at 35,000 g for 15 min at 0 ° C (Sorvall RC-2B) and 5 ml of the supernatant was neutralized with 5 M K~CO 3 and phenolred as indicator.
ATP
143
IN MYTILUS
3. E N Z Y M A T I C ASSAY OF ATP
A T P was determined according to JAWOREK, GRUBER & BERGMEYER (1970), with tris (hydroxymethyl)aminomethane-HC1 as buffering system, while the final volume of the assay mixture was 1 ml. Determinations were performed in fourfold with 100 ~1 tissue extract. The absorbance at 340 nm was followed with a Zeiss P M - Q I I spectrophotometer coupled with a Vitratron UR-400 recorder. All reactions were finished within 10 min. The A T P concentration was calculated from the change in absorbance (extrapolated to the starting point of the reaction) and the extinction coefficient of N A D H (6.22 X 103 1 mo1-1 cm-a). Standard samples in a range of 4 to 80 ~M gave a reproducible linear response with a quantitative recovery. Tissue samples were within this range, but showed an inhibition of 7 ~o. Corrections could be made by use of internal standards. Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), 3-phosphoglycerate kinase (EC 2.7.2.3), N A D H and A T P were obtained from Boehringer, Mannheim. All other chemicals were "Baker Analyzed" from J. T. Baker Chemicals. III. RESULTS
Cultured and wild mussels were exposed to air for 7 days, and then reimmersed in sea water (Fig. 2). The A T P levels, with normal values of about 2 mM, decrease during 3 days of anoxia to minima of 1.18 and 0.94 m M in cultured and wild animals, respectively. After reimmersion in sea water, the A T P concentrations reach levels equal k\\-,~
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Fig. 2. ATP concentration ( © , e ) and mortality (z%&) in cultured ( © , A ) and wild (O,A) mussels (Mytilus edulis) during exposure to air (blank part of bar) and subsequent immersion in sea water (hatched part). Control values were obtained for animals, covered by sea water during the whole experiment.
144
T.C.M. WIJSMAN
to those of the control groups within 2 to 3 days. Although statistical evidence is lacking it seems that the ATP concentration in wild mussels, compared to that in cultured ones, shows a stronger decrease under anoxia while it takes more time to restore the normal situation. The general course of both curves, however, is identical. There is no conformity, however, with respect to mortality. The increase in mortality of wild mussels, which starts at the 7th day of anoxia, immediately ends when the animals are placed in sea water again. Mortality in cultured mussels shows a remarkable increase during exposure to air, starting from the 4th day, reaches a value of 24% after 7 days of anoxia, and is not stopped by reimmersion in sea water, with the consequence that mortality is over 50~/o after 4 subsequent days of aerobiosis. To study the effect of a periodical aerobic-anaerobic transition, cultured and wild mussels were alternately exposed to air and reimmersed in sea water for 24 11 over a period of 10 days. Each day mortality was calculated while ATP was determined after each anaerobic period (Fig. 3). ATP concentrations, obtained after the first anaerobic kk\\'x\\\\\\\'~
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Fig. 3. ATP concentration (2),O) and mortality (/~.,,A) in cuhured (, ,,1~: and wild (O,A) mussels (Mytilus edulis), alternately immersed in sea water (hatched part of bar) and exposed to air (blank part). Control values were obtained for animals, covered by sea water during the whole experiment.
period of 24 h (1.43 m M for cultured and 1.23 m M tbr wild mussels) are in agreement with the corresponding data of Fig. 2 (1.48 and 1.31 m M for cultured and wild mussels, respectively). Mean ATP levels (with standard deviation), calculated from these data, are 1.46 4:0.09 m M for cultured and 1.27 i 0.07 m M for wild animals, corresponding to 729/o and 61% of the "aerobic" values. During subsequent periods of exposure to air ATP concentrations in both groups hardly drop further,
ATP
IN M Y T I L U S
145
even when exposure was extended to 5 days beginning at the 9th day o f this experiment (Fig. 3). In the last case, A T P levels of 1.38 m M (cultured) and 1.14 m M (wild) were determined after 3 days of continuous anoxia, which is both 0.2 m M higher than the corresponding data of Fig. 2. U p to the 10th day mortality is nearly equal for both groups and does not exceed the control values. After that time the course of mortality is identical to the one in Fig. 2. IV. DISCUSSION The two groups of animals used in the experiments live under quite different conditions in their natural habitat. The cultured mussels are continually covered by sea water, whereas the wild animals are exposed to air for a quarter of their life during which period no food and oxygen are available. These differences are reflected in their phenotype: tissue wet weight of cultured mussels was 50% higher (5.83 versus 3.83 g) and shell length was 20% longer (6.19 versus 5.10 cm) than of wild animals, whereas the weight of the shells was 18% lower (7.39 versus 8.84 g). Prolonged exposure to air showed a remarkable difference in mortality between the two groups (Fig. 2), indicating that wild mussels are better adapted to these circumstances than cultured animals. The continued mortality of cultured mussels after reimmersion in sea water indicates that processes are started, leading to death, which cannot be stopped under aerobic conditions. No significant difference was obtained between the "aerobic" A T P levels of cultured (2.02 4- 0.14 raM) and wild mussels (2.08 4- 0.09 mM). But A T P levels, determined after anaerobic periods of 24 hrs and during a subsequent anoxic period of 5 days (Fig. 3), were almost 0.2 m M higher in cultured than in wild mussels. Even after 7 days of continuous anaerobiosis, when mortality was more than two times higher in cultured mussels than in wild ones, and during reimmersion in sea water, when mortality in cultured mussels increased to the fivefold of that in wild animals, the ATP concentration was equal or even higher compared to wild mussels (Fig. 2). These higher values cannot be attributed to a greater desiccation of the cultured mussels, because the quotient of dry weight and wet weight was equal for both groups, being unchanged during exposure to air: 0.203 4- 0.010 g dry weight/g wet weight (n ---- 24) for cultured mussels versus 0.202 ± 0.09 g dry weight/g wet weight (n = 26) for wild animals. As discussed in the Introduction, anoxia causes a pronounced fall in ATP production which will result in decreased A T P levels when
146
T.C.M. WI~SMAN
ATP-consuming processes are not reduced to the same extent. So it might be possible that the steady state concentration of ATP is less affected when mussels are better adapted to anaerobiosis. According to this supposition, the ATP data suggest that cultured mussels are better adapted to sustain anaerobiosis than wild animals. But the opposite is true, as is evident from the data on mortality. Probably the ATP concentration (and the energy charge) is not indicative for the physiological state of Mytilus edulis, as can be deduced also from the high concentration of A T P in dead animals (WIJSMAN, 1976). Apparently survival depends not only on maintenance of an adequate A T P level. While glycogen stores in M. edulis are sufficient to survive anoxia for months, the derived end products, as succinate and free volatile fatty acids, are accumulated to amounts which may disorder the whole organism. Maximum survival times under experimental anaerobic conditions are strongly different for various species of molluscs (YONBRAND, 1946). These differences might be related to the extent in which the animal can reduce its energy demand. At room temperature, for example, Mytilus edulis can withstand anoxia for several days, whereas the pond snail Limnaea stagnalis dies after a few days. In both species carbohydrates are catabolized to the same end products during anaerobiosis, but in spite of its relative low tolerance for anoxia only in L. stagnalis a clear Pasteur effect is observed (DE ZWAAN& ZANDEE, 1972 ; DE ZWAAN, MOHAMED & GERAERTS, 1976). This investigation illustrates that A T P levels and changes in the steady state concentration during anoxia reveal nothing about survival under anaerobic conditions. A better approach to this seems the study of the actual energy demand during anaerobiosis. This has been started in our team (DE ZWAAN & WUsMAN, 1975). It takes several days to restore the A T P concentration and probably the energy charge (Fig. 1) in Mytilus edulis after prolonged exposure to air. In this situation energy yielding processes are stimulated while energy demanding processes are inhibited according to the theory of ATrdNSON (1968). Inhibition of pyruvate kinase by the anaerobic drop in p H will be releaved within an hour after reimmersion of M. edulis in sea water (WusMAN, 1975), whereas activation of this enzyme by the decreased energy charge (EBBERIN~, DE ZWAAN & WUSMAN, 1976) is probably maintained over a longer period. In this way the enhanced catalytic capacity of pyruvate kinase can serve to increase the flux of acetyl-CoA units into the tricarboxylic acid cycle, facilitated by the accumulation of some intermediates of this cycle during anaerobiosis. This is in agreement with the observed oxygen debt in M. edulis after anoxia (ScHLIEPER& KOWALSKI,1959).
ATP
IN MYTILUS
147
V. S U M M A R Y
Mortality and A T P content during experimental exposure to air were determined in two groups of Mytilus edulis L., one lying dry during low tide (wild), the other continuously covered by sea water in its habitat (cultured). When kept in sea water continuously, ATP levels were 2.02 4- 0.14 m M in cultured and 2.08 -4- 0.09 m M in wild mussels. Mortality was negligible and almost equal for both groups (Fig. 2). In mussels, exposed to air for 7 days, a m a x i m u m decrease in ATP content to 1.18 m M (cultured) and 0.94 m M (wild) was determined after 3 days. Mortality in wild mussels reached 9% and stopped after reimmersion in sea water, whereas mortality in cultured animals was 24~/o at the end of exposure and continued under aerobic conditions, being 50% after 4 days of aerobiosis. In both groups normal A T P levels were restored within 2 to 3 days of aerobiosis (Fig. 2). When mussels were exposed to air and immersed in sea water alternately for periods of 24 h, ATP levels were maintained at about 1.4 m M for cultured and 1.2 m M for wild animals. Mortality was equal in both groups and did not exceed the control values. During subsequent exposure of 5 days, no further decrease in ATP concentration was observed in both groups (Fig. 3). VI. REFERENCES ATKINSON, D. E., 1968. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers.---Biochemistry, N.Y. 7t 4030-4034. ATKmSON, D. E. & G. M. WALTON, 1967. Adenosine triphosphate conservation in metabolic regulation.--J, biol. Chem. 242: 3239-3241. BRAND, T. YON, 1946. Anaerobiosis in invertebrates. In: B. J. LOYET. Biodynamiea Monographs.~Biodynamica 4: 90-91. CHEN, C., 1969. A variation of the Embden-Meyerhof-Parnas scheme in molluscs. Ph.D. Thesis, Rice University. E~BERINK, R. H. M., A. DE ZWAAN & T. C. M. WIJSMAN, 1976. Regulation at the phosphoenolpyruvate branchpoint by the adenylate energy charge in Mytilus edulis.---Biochem. Soc. Trans. 4: 444-447. GmLES, R., 1972. Biochemical ecology of Mollusca. In: M. FLORI~N & B. T. SCHEER. Chemical Zoology. Academic Press, New York, London 7: 467-495. HOCHACHKA, P. W., J. FIELDS ~; T. MUSTAFA, 1973. Animal life without oxygen: basic biochemical mechanisms.--Am. Zoologist 13- 543-555. HOCHACHKA, P. W. • T. MUSTAFA, 1972. Invertebrate facultative anaerobiosis.-Science, N.Y. 178: 1056-1060. JAWOREK, D., W. GRUBER & H. U. BERGMEYER, 1970. Adenosin-5"-triphosphat. In: H. U. BERGMEYER. Methoden der enzymatischen Analyse. Weinheim Vetlag Chemic 2" 2020-2024. KLUYTMANS,J. H., P. R. VEENHOF & A. DE ZWAAN, 1975. Anaerobic production of
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volatile fatty acids in the sea mussel Mytilus edulis L.--J. cell. comp. Physiol. 104: 71-78. MARKLAND, F. S. & C. L. WADKINS, 1966. Adenosine triphosphate-adenosine 5'monophosphate phosphotransferase of bovine liver mitochondria. I. Isolation and chemical properties.--J, biol. Chem. 241: 4124-4135. SCHLmI'ZR, C. & R. KOWALSKb 1959. Ein zelluliirer Regulationsmechanismus ftir erh6hte Kiemenventilation nach anoxybiose bei Mytilus edulis L.--Kieler Meeresforsch. 14: 42-47. THAmDRUP,H. M., 1935. Beitriige zur Okologie der Wattenfauna.--Meddr Kommn Danm. Fisk.-og Havunders. Ser. Fisk. 10 (2): 1-130. WIJSMAN, T. C. M., 1975. pH fluctuations in Mytilus edulis L. in relation to shell movements under aerobic and anaerobic conditions. In: H. BARNES. Proc. 9th Europ. mar. biol. Syrup. Aberdeen University Press: 139--149. , 1976. Adenosine phosphates and energy charge in different tissues of Mytilus edulis L. under aerobic and anaerobic conditions.--J, cell. comp. Physiol. 107: 129-140. ZWAAN, A. DE, A. M. T. DE BONT & J. H. F. M. KLUYTMANS, 1975. Metabolic adaptations on the aerobic-anaerobic transition in the sea mussel, Mytilus edulis L. In: H. BARNES.Proc. 9th Europ. mar. biol. Syrup. Aberdeen University Press: 121-138. ZWAAN, A. DE, J. H. F. M. KLUYTMANS& D. I. ZANDEE, 1976. Facultative anaerobiosis in molluscs.---Biochem. Soc. Symp. l l : 133-167. ZWAAN, A. DE & W. J. A. VAN MARREWlJK, 1973. Anaerobic glucose degradation in the sea mussel Mytilus edulis L.--Comp. Biochem. Physiol. 4413: 429439. ZWAAN, A. DE, W. J. A. VAN MARREWIJK & D. A. HOLWERDA, 1973. Anaerobic carbohydrate metabolism in the sea mussel Mytilus edulis L.--Neth. J. Zool. 23: 225-228. ZWAAN, A. DE, A. M. MOHA~ED & W. P. M. GERAERTS, 1976. Glycogen degradation and the accumulation of compounds during anaerobiosls in the fresh water snail Lymnaea stagnalis .--Neth. J. Zool. (in press.). ZWAAN, A. DE & T. C. M. WUSMAN, 1975. Anaerobic metabolism in Bivalvia (Mollusca). Characteristics of anaerobic metabolism.--Comp. Biochem. Physiol. 54B: 313-324. ZWAAN,A. DE & D. I. ZANDEE, 1972. The utilization of glycogen and accumulation of some intermediates during anaerobiosis in Mytilus edulis L.--Comp. Biochem. Physiol. 43B: 47-54.