Variations in the Rate of Anaerobic Succinate Accumulation within the Central and Marginal Regions of an Euryoxic Bivalve Mantle

P.S.Z.N. I: Marine Ecology, 2 (4): 353362 (1981) Q 1981 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0173-9565/InterCode: MAECDR

Accepted: 12.2.1982

Variations in the Rate of Anaerobic Succinate Accumulation within the Central and Marginal Regions of an Euryoxic Bivalve Mantle ED GREENFIELD & MILES A. CRENSHAW

Curriculum in Marine Sciences and Dental Research Center, University of North Carolina, Chapel Hill, N.C., 275 14, U.S.A.

With 5 figures

Key words: Metabolic regulation, anaerobiosis, succinate, bivalve mantle, metabolite translocation, PEPCK, shell dissolution

Abstract. Anaerobic metabolism in the central and marginal portions of the mantle of Mercenaria mercenaria was compared. Anaerobic succinate accumulation was more rapid in the central region. This difference may be due to higher phosphoenolpyruvate carboxykinase activity in the central region. Thus, the central region is more specialized for anaerobic metabolism and the marginal region more for net shell growth. The original rate of succinate accumulation in the mantle is similar in isolated mantles and intact clams, suggesting that mantle succinate production does not require translocation of precursors from other tissues. However, in intact clams, the rate of succinate accumulation in the central region of the mantle slows after four hours. The reduced rate is probably caused by reducing the metabolic rate. Succinate accumulation and shell dissolution are slower in freshly collected clams than in clams that had been stored anaerobically. The difference may be due to induction of PEPCK synthesis during storage. Shell derived calcium did not accumulate in the mantle and, therefore did not aher the intracellular calcium concentration in the mantle.

Problem

Intertidal organisms are often exposed to the air during low tide. Many intertidal molluscs close their shells tightly during subaerial exposure to avoid dessication and terrestrial predators. However, shell closure restricts the organisms’ oxygen supply. In the clam, Mercenaria rnercenaria, the internal oxygen store is depleted within thirty minutes of shell closure (CRENSHAW & NEFF, 1969). Accordingly, many immobile molluscs must obtain oxygen from the atmosphere by shell “gaping” (BOYDEN, 1972) or rely on anaerobic metabolism for extended periods (THEEDE, 1973; HAMMEN, 1976). The ability to withstand extended shell closure can also protect the organism from a variety of other adverse environmental conditions including high ambient sulfide levels (THEEDE, 1973), low salinity (GILLES, 1972; BURRELL, 1977), predation (NIELSEN, 1975; VERMEIJ & VEIL, 1978), heavy metal pollution (AKERBALI & BLACK, 1980), loss of freshwa-

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354 GREENFIELD & CRENSHAW

ter habitats during droughts (HISCOCK, 1953) and flooding of terrestrial habitats (WIESER, 1981).

Anaerobic metabolism produces organic acids. In the conventional vertebrate pathway, lactate is the end product. However, this pathway is very inefficient in terms of energy (ATP) yield from a given amount of consumed substrate and, therefore, can only be used for short periods (BENNETT & RUBEN, 1979). During anaerobiosis, many euryoxic organisms reduce their metabolic rate greatly and utilize a modified pathway that converts stored glycogen to a variety of end products. Alanine is produced during the aerobic-anaerobic transition. Produc- tion of succinate begins at the onset of anaerobiosis and propionate begins accumulating after a lag period (DE ZWANN & WIJSMAN, 1976; DE ZWAAN et al., 1976; DE ZWANN, 1977; EBBERINK & DE ZWANN, 1980). This pathway is controlled at the phosphoenolpyruvate (PEP) branchpoint. The fate of PEP is determined by the relative, allosterically regulated activities of PEP carboxykinase (PEPCK) and pyruvate kinase. Because this pathway is coupled to an electron transport chain, the ATP yield is higher than from the lactate pathway (HOLWERDA & DE ZWAAN, 1979, 1980; ZANDEE et al., 1980).

Many intertidal molluscs are well adapted for anaerobic succinate production. The intertidal mussel, Myrilus edulis, produces more succinate during anaerobiosis and contains a higher PEPCK:pyruvate kinase ratio than the subtidal M. galloprovincialis (DE VOOYS, 1979, 1980). Similarly, intertidal gastropods and annelids produce more succinate during anaerobiosis than related subtidal species (SCHOITLER, 1979; WIESER, 1980). Different tissues within the 'same animal also have different rates of anaerobic metabolism and succinate accumulation (CHAPLIN & LOXTON, 1976; KLUYTMANS et al., 1977; DE ZWAAN, 1977; ZURBURG & KLUYTMANS, 1980). The metabolism in these tissues must be well integrated (HOCHACHKA, 1980). For example, HOLWERDA and DE ZWAAN (1980) have suggested from measurements of tissue enzyme levels that malate is produced primarily by the adductor muscle and then translocated to the mantle and digestive gland where it is converted to succinate. However, the mechanisms that coordinate the various tissues are not understood.

Since succinic is a weaker acid than lactic, succinic acid accumulation creates a less severe pH decrease than would lactate accumulation. This acidification must be controlled to maintain normal physiological conditions. Bivalves dissol- ve calcium carbonate from the internal surface of the shell to neutralize the acid (COLLIP, 1920; DUCAL, 1939; CRENSHAW & NEFF, 1969). This dissolution makes rapid shell repair and extension necessary during aerobic intervals to continue net shell growth (RHOADS & MORSE, 1971). Bivalves appear to control shell dissolution and calcification by separating the processes spatially, as well as temporally. Dissolution only occurs inside the pallial line, the site of attachment between the shell and the overlying mantle (Fig. 1) (DUGAL, 1939; WILKES & CRENSHAW, 1979). In comparison, net shell accretion and thickening occur primarily in the marginal portion of the shell. The rate of 45-calcium deposition outside the pallial line is two-to ten-fold faster than the rate inside (WILBUR & JODREY, 1952; ZISCHKE et al., 1970).

The two portions of the mantle differ both morphologically and physiologically. In the marginal region, the epithelial cells facing the shell are polar, columnar, secretory-like cells with numerous mitochondria and extensive basal

Variations in anaerobic metabolism within the bivalve mantle 355

Mantle Cells Inner Shell Surfaces

Fig. 1. Differences between the marginal and central portions in the ultrastructure of the outer mantle epithelium and in scanning electron micrographs of the inner shell surface. Reprinted from CRENSHAW (1980) in RHOADS & LUTZ (Eds.), Skeletal growth of aquatic organisms, Plenum Press: 115-132.

infolding, endoplasmic reticulum (ER) and golgi structures (Fig. 1). In the central region, the epithelial cells are cuboidal and virtually devoid of mitochondria, ER and golgi (TSUJII, 1976; CRENSHAW, 1980). During experimentally induced repair of the central portion of the shell, the central cells differentiate to resemble cells from the marginal region and, after the repair is complete, revert to their normal ultrastructure (BEEDHAM, 1965; TIMMERMANS, 1973; TSUJII, 1976). Scanning electron micrographs of the cell surfaces on the shell side also reveal differences between the two portions of the mantle (PETIT et al., 1978; WALLER, 1980).

Physiologically, the marginal region consumes oxygen faster (JODREY & WILBUR, 1955; TSUJII & ISONO, 1963), contains more of calcium binding glyco- protein component of the organic matrix (DOGTEROM, 1980), and takes up more 45-calcium and 32-phosphorous (OKADA et al., 1959). The central region permits passive intercellular diffusion of the calcium between the extrapallial and the


body fluids (MACHIN, 1977; CRENSHAW, 1980) and is the primary site of sodium pumping ATPase activity in the euryhalince clam, Rangia (SAINTSING & TOWLE, 1978). Thus, the marginal region of the mantle-shell complex appears morphologically specialized for shell growth and the central region for controlling ion concentration during shell dissolution and osmoregulation. These differences should be associated with a greater rate of anaerobic metabolism in the central region.

Most recent biochemical studies have treated the mantle as a single tissue. In this study, the metabolic differences between the two regions were studied in the intertidal bivalve, Mercenaria mercenaria. This organism is particularly suited for this work as the pallial line is distinct, and the mantle zones are easily separated.

Material and Methods

Clams were obtained commercially either near Morehead City, N. C. (hereafter referred to as fresh clams) or after being transported anaerobically 120 km inland to Chapel Hill, N. C. (referred to as transported clams). Fresh clams were maintained overnight in a flow-through seawater table at the Institute of Marine Science. Transported clams were maintained overnight in a recirculating seawater aquarium at 18 "C and fed the diatom, Acnanthes brevipes. Clams were incubated anaerobically by exposing intact clams to air or by incubating isolated central or marginal mantle portions in three volumes of nitrogen-saturated, 10 mM Tris-HCI (pH 8) buffered, artificial seawater (Instant Ocean). The culture media was Millipore@ filtered and the excised tissue rinsed in media before the incubations in an attempt to remove bacteria. Lactate. Tissue samples from three clams were pooled, homogenized in three volumes of 0.1 M HC1 and the protein precipitated with 0.9 M perchloric acid. The lactate concentration was measured by the standard lactic dehydrogenase spectrophotometric method (BERGMEYER, 1974). Succinate. A gas chromatographic procedure based on the methods of SMITH & HEATH (1980) and of SANSONE & MARTENS (1981) was developed (GREENFIELD, 1981). Portions of the homogenates used to measure lactate were mixed with one-half volume of acetone to precipitate proteins. The supernatant was dried and the residue incubated overnight with methanolic 3N sulfuric acid. The resulting methylated succinate was extracted into chloroform and chromatographed on a Lac-l-R- 296 Chromosorb W-AW column (Alltech Associates). The initial 80 "C column temperature (ten minutes) was followed by an increase of 32 Wminute to 130 "C. Unfortunately, this method could not be adapted to measure the amount of succinate released into the medium during the in virro incubations. However, isolated Myrifus adductor muscles do not release succinate into the medium (DE ZWAAN et a!., 1982). Calcium. Shell dissolution was monitored, in conjunction with end product accumulation, by measuring the increase in calcium levels in the mantle portions and in the remaining tissue and fluids. Samples were dried, ashed at 600 "C, dissolved in 1 M HCI, brought to 1 % lanthanum and the calcium measured by atomic absorption spectrophotometry. PEPCK. Enzyme activity was measured after incubating intact clams anaerobically for two days. Mantles were divided into central and marginal portions and homogenized in ten volumes of ice-cold 2 mM EDTA, 0.1 M imidazole-HCI (pH 7) and centrifuged at 40,000 g for 15 minutes. PEPCK was measured in the supernatant by the standard spectrophotometric malic dehydrogenase linked assay at 35 "C (DE ZWAAN & DE Born, 1974). However, omitting the bicarbonate from control assays did not exclude all bicarbonate from the solutions and resulted in unacceptably high blanks. Therefore, PEP or inosine diphosphate were omitted in control vessels. Protein. All assays were performed in at least duplicate and the results expressed per unit of total protein as measured by the bromosulphathelein method (NAWAR & GLICK, 1954). Student's t test was used to determine statistical significance.


Results

All of the results had a great deal of variability (Figs. 2, 3, 4, 5) . However, the variability between replicate succinate and lactate assays on the same homoge- nate averaged 4.6% (+ 4.0%) of the means. Therefore, the scatter is due to differences between individual clams and not to imprecision in the assays.

Lactate did not accumulate in either portion of the mantle during anaerobiosis (Fig. 2). In fact, the lactate level may have decreased slightly in the marginal portion. This confirms the idea that vertebrate type metabolism is not important in the mantle during anaerobiosis.

Intact clams that had been transported to Chapel Hill accumulated succinate rapidly in the central region of the mantle and more slowly in the marginal region (Fig. 3 a, b). After four hours of anaerobiosis, the central region contained 49% more succinate than the marginal region (p < 0.005). However, after 24 hours, the succinate ievels in the two regions were not significantly different.

In the initial studies with isolated mantles, succinate accumulated in prepara- tions saturated with nitrogen or air but not in cultures continuously bubbled with 95 % oxygen, 5 % carbon dioxide. Apparently, an extremely high oxygen level is needed to support aerobic metabolism in isolated tissue.

The rate of succinate accumulation in isolated mantles was similar to the rate found in intact clams (Fig. 3 c). After four hours of anaerobiosis, the levels of succinate in the central and marginal regions were not significantly different.

Fig. 2 . Lactate levels in the central (open circles) and marginal (closed circles) portions of intact clams during anaerobiosis. Fresh clams were exposed to air for the indicated time periods and lactate determined. Each point represents the combined tissue of three clams.

= 100 Fig. 3. Anaerobic succinate accumulation in the central (shaded bars) and 80

60 marginal (white bars) portions of transported clams. Groups of three clams were incubated for the indicated time pe- 40 riods and succinate concentration determined. Each bar represents the mean .i 20 (f S.D.) of 9-12 clams. A = Aerobic, B = Intact incubation, C = Isolated 0 mantle incubation.

3 z 5 -

I I

0

r 0 B 16 ' 24

Anaarobiasis (hrs)

T A B C

Anaerobiosis (hrs.)

358 GREEYFIELD & CRENSHAW

Fig. 4. Anaerobic succinate accumulation in the central (shaded bars) and marginal (white bars) portions of fresh clams. Each bar represents the mean (+ S.D. ) of 9-12 clams. A = Aerobic, B = Intact incubation.

Fig. 5 . Calcium accumulation during anaerobiosis. The same central (open circles) and marginal (closed circles) homogenqtes used for succinate analysis were used to determine calcium. In addition, the remaining tissue and fluids from transported (squares) and fresh (triangles) clams were assayed. Each point represents tissue from at least six clams.

Anaarobiosis (hrs)

0 4 24 Anaerobiosir (hrs)

However, after 24 hours, the central region contained twice as much succinate as the marginal region (p < 0.01).

Fresh clams accumlated less succinate than transported clams, particularly in the first four hours (Fig. 4). Preliminary studies on the time course of succinate accumulation in fresh clams revealed very little increase during the first twelve hours of shell closure (GREENFIELD, 1981). After 24 hours, the central region contained 85 % more succinate than the marginal region (p < 0.025).

Total calcium increased in the bulk tissue and fluids, but not in either portion of the mantle (Fig. 5) . Similarly to succinate, the calcium increase in fresh clams was considerably less than the increase in transported clams.

The central region of the mantle contained 155 units (pmoles substrate converted/mg protein X minute) of PEPCK in the 40,000 g supernatant. This was 29 % more than the 120 units found in the marginal region (p < 0.005).

Discussion . Succinate accumulated faster in the central region than in the marginal region during all types of anaerobic incubations studies. This difference may be caused by the relatively high activity of PEPCK in the central region. However, allosteric regulation may also be a factor in the more rapid central succinate accumulation. Thus, the central region is specialized for anaerobic succinate production and ATP synthesis, allowing the shell to function as an alkali reserve without reducing net shell formation at the growing margin.

In intact clams that had been transported, the succinate accumulation in the central region slowed after four hours. This might be due to succinate translocation out of the central region. However, ZURBURG & KLUYTMANS (1980) demonstrated that the succinate concentration in the hemolymph remains low in


bivalves during anaerobiosis and therefore, argued that succinate is not translocated. This does not exclude direct translocation to the marginal region. However, the increase in the marginal region succinate level between four and 24 hours can account for, at most, 15 % of the difference between the expected and the actual succinate increase in the central region during this period. Therefore, the reduced rate of succinate accumulation is probably due to either slower succinate synthesis or increased conversion of succinate to propionate.

In mantles isolated from transported clams, succinate accumulated in the central region throughout the 24 hour duration of the anaerobic incubations. Thus, isolated tissue i s similar to intact bivalves incubated in oxygen-free seawater, which accumulate end products faster than organisms exposed to the air (ZURBURG, 1981). Incubation in relatively large volumes of oxygen-free seawater isolates the tissue or the organism from the end products, protons and calcium that accumulate in the various body fluids during shell closure (CREN- SHAW & NEFF, 1969; BOOTH & MANGUM, 1978; ZURBURG & KLUYTMANS, 1980; WIESER, 1981). Reduced fluid acidification should coincide with slower tissue acidification (DE ZWAAN, 1977; BARROW et al., 1980). The intracellular pH drop during shell closure may slow succinate production by inhibiting a regulatory enzyme such as phosphofructokinase (SOLING et al., 1977; EBBERINK & DE ZWAAN, 1980). This would resemble the acidotic torpor of hibernating mammals (MALAN, 1980). Onset of propionate synthesis probably does not account for the decrease in the rate of central succinate accumulation during shell closure since propionate accumulation is also greater in oxygen-free seawater than in air (ZURBURG, 1981).

The initial rate of succinate production in isolated mantles approximated the rate in intact clams. This similarity indicates that the mantle does not require exogenous substrate for maximal succinate production and does not support the hypothesis that malate is translocated to the mantle from the adductor muscle prior to conversion to succinate (HOLWERDA & DE ZWAAN, 1980).

Fresh clams only accumulated succinate and calcium after twelve hours of anaerobiosis. The results in fresh clams should more closely reflect succinate accumulation in the natural habitat than should the results in transported clams. Thus, succinate synthesis and shell dissolution only occur during unusually long low tides. Most low tides may only induce a reduced metabolic rate and alanine production. However, if alanine is produced before succinate, alanine synthesis cannot be linked to succinate production from aspartate (EBBERINK et al., 1979). The rapid onset of succinate accumulation in transported clams may have been caused by allosteric activation or by inducing synthesis of a regulatory enzyme. However, the overnight aerobic incubation before the experiments should have eliminated any allosteric effects. Also, PEPCK is induced in Mytilus by anaerobic incubation (ROSS & REISH, 1976) and hormonally in mammals (TILGHMAN et al., 1976; IYNEDJIAN & JACOT, 1980). In other organisms, anaerobiosis induces the enzymes that regulate the anaerobic pathways (BINET- TE et al., 1977; SACHS et al., 1980). Therefore, transporting the clams anaerobically probably induced PEPCK synthesis resulting in rapid succinate production during the subsequent experimental incubations. In the natural habitat, this enzyme induction may allow accelerated succinate production and ATP synthesis during extended periods of anaerobiosis.


Calcium did not accumulate in the mantle. Most cells are able to precisely regulate their internal calcium concentration. Accordingly, the calcium that dissolves from the shell is stored in t.he body fluids rather than in the mantle. Thus, this calcium cannot regulate PEPCK and pyruvate kinase, as has been suggested from the in vitro kinetic properties of the enzymes (DE ZWAAN & DE BONT, 1975; DE ZWAAN et al., 1975).

Summary Anaerobic metabolism was more rapid in the central portion than in the marginal portion of the mantle of M . mercenaria. Thus, the central region is more specialized for anaerobic energy production and the marginal region more for net shell growth. The central region contained significantly more PEPCK activity and can, therefore, produce succinate rapidly. This tissue specialization allows the shell to function as an alkali reserve without directly reducing the rate of net shell accretion at the growing margin or damaging the previously deposited marginal portion of the shell. An intact shell margin may be important to withstand dessication and predation.

The primary response of Mercenaria to a wide variety of environmental stresses appears to be shell closure. The length of the resulting anoxia deter- mines which of the three types of metabolic response occurs. Short periods of anoxia probably cause a reduction in metabolic rate and alanine production. This response seems to be sufficient for most low tides. Longer periods without oxygen induce PEPCK synthesis, succinate accumulation and shell dissolution. If the anoxic interval extends for an extremely long period, the metabolic rate is reduced further, possibly by inactivating phosphofructokinase, and succinate accumulation stops. Evolution of both the metabolic potential for a variety of responses to anoxia and the ability to localize shell dissolution allows molluscs to endure a wide range of environmental stresses and to occupy seemingly inhos- pitable habitats.

Acknowledgements The authors would like to thank F. SANSONE and D. WILSON for discussing their procedures, C. MARTENS and R. RIECER for reviewing the manuscript, D. THOMPSON for preparing the figures and the Institute of Marine Science, Morehead City, N. C., for providing lab space. This work was supported, in part, by NIH DE 02668.

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Documents

Variations in the Rate of Anaerobic Succinate Accumulation within the Central and Marginal Regions of an Euryoxic Bivalve Mantle