The Regulation of Respiratory Metabolism in Parasitic Helminths

The Regulation of Respiratory Metabolism in Parasitic Helminths C. BRYANT

Department of Zoology, Australian National University, Canberra, Australia I. Introduction ................................................................................. 11. Cestodes ....................................................................................... A. Hymenolepis diminuta-The Biochemistry of the Host-Parasite Relationship .............................................................................. B. Moniezia expansa and Other Cestodes-Studies of Metabolic Regulation 111. Nematodes .................................................................................... A. Ascaris: Aerobic or Anaerobic? ................................................ B. Other Nematodes ..................................................................... IV. Trematodes ................................................................................. A. Fasciola hepatica-Metabolic Regulation in a Non-intestinal Parasite B. Other Trematodes and the PK/PEPCK Branch-point ..................... V. Conclusions ................................................................................. Acknowledgements ........................................................................ References ....................................................................................

311 312 312 315 318 318 321 322 322 325 326 321 321

I. INTRODUCTION The literature on the uptake of carbon dioxide by parasitic helminths and the mechanisms which control carbon flow along respiratory metabolic pathways has recently been reviewed (Bryant, 1975). Unfortunately, an unusually long time elapsed between the completion of the review (early 1973) and its publication (mid 1975). In the interim, a number of important papers have been published which go far in providing answers to questions raised therein. The present work is intended to update the earlier one, and to repair a number of omissions. Abbreviations used in the text are summarized in Table I. Readers unfamiliar with the field may find it helpful to refer to the earlier review. There, the answer was sought for a number of important questions in biochemical parasitology; why the path leading to succinate and its derivatives is so important in parasite respiration; what role the carbon dioxide incorporation plays in the pathway; how metabolism is regulated at the crucial PEP branch-point in glycolysis. Inevitably, definitive answers were not available and so the questions remain central to the study of respiratory metabolism in helminths. To them should now be added the problems of whether one or more types of mitochondrion, with different functions, are involved in 311

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respiration in a single parasite; whether the TCA cycle has an important role in parasite metabolism ; what are the mechanisms controlling mitochondria1 permeability and oxidative phosphorylation; and how, during respiration, the redox potentials of the various compartments within parasite cells are maintained. The problems are compounded by the nature of the environment of the helminth, which may present all degrees of anoxia. This brief account does not promise to answer the questions; it assembles and, I hope, critically evaluates some of the data which may be used as a foundation upon which, in due course, the answers will be built. TABLE1 Abbreviations used in the text

ATP, ADP, AMP : adenosine tri-, di- and monophosphates FDPase: fructose-1; 6-diphosphatase (EC 3.1.3.11) FDP: fructose-1; 6-diphosphate

F6P: fructose-6-phosphate HK : hexokinase (EC 2.7.1.1) LDH: lactate dehydrogenase (EC 1.1.1.27) MDH: malate dehydrogenase (EC 1.1.1.37) ME: “malic enzyme” (EC 1.1.1.39 and 1.1.1.30) NAD(P), NAD(P)H : oxidized and reduced nicotinamide adenine dinucleotide (phosphate) PEP : phosphoenolpyruvate PEPCK: phosphoenolpyruvate carboxykinase (EC 4.1.32) PK: pyruvate kinase (EC 2.7.1.40) PFK: phosphofructokinase (EC 2.7.1.1 1) TCA: tricarboxylic acid

A number of reviews on parasite metabolism have been published recently. They include one on bioenergetics in helminths, by Barrett (1976a); two on trematodes by Coles (1973, 1975); a more general one by Kurelec (1975a); and a very complete account, by Pappas and Read (1975), of membrane transport in helminth parasites. I shall endeavour here not to duplicate these works. 11. CESTODES A. H YMENOLEPZS DZMZNUTA-THE BIOCHEMISTRY OF THE HOST-PARASITE RELATIONSHIP

Hymenolepis diminuta possesses a mechanism for the mediated uptake of glucose (McCracken and Lumsden, 1975a, b). The mechanisms by which glucose is subsequently oxidized under aerobic and anaerobic conditions have been discussed in the earlier review (Bryant, 1975). The major end products of respiration are succinic, acetic and lactic acids. They are derived not only from glucose absorbed from the medium in which the parasite is bathed, but also from endogenous carbohydrate reserves which are readily utilized or replaced under conditions of starvation or plenty.

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The relative proportions of excretory products change as the parasite ages (Watts and Fairbairn, 1974). More succinate is produced, during incubation in vitro, by 10 and 14 day old organisms than by 6-7 day old ones. In the younger animals, isozymic forms of LDH are found (Walkey and Fairbairn, 1973),together with isozymic forms of PK (Carter and Fairbairn, unpublished ; quoted in Watts and Fairbairn, 1974). Isozymes of these enzymes are less apparent in older specimens of H. diminuta, which may be symptomatic of the increasing dependence on the succinate producing pathway. Watts and Fairbairn (1974) note, from measurements of fresh weights of their samples, that the youngest parasites have considerably greater surface : volume ratios than the oldest ones. Following a suggestion of Fairbairn (1970)-that the excretion of lactic acid by helminths is related to the ease with which strong acids can be excreted when surface :volume ratios are high -they correlate the relative decrease in lactic acid production with increase in this ratio. They point out that the excretion of acetic acid (a weaker acid than lactic) remains constant. The same authors detected high activities of the pyruvate dehydrogenase complex in H. diminuta (77 nmoles acetyl phosphate formed min-lmg-l protein). Excreted acetic acid is therefore most probably produced by the action of this enzyme system, which supports the anaerobic reaction scheme summarized by Bryant (1975). When levels of adenine nucleotides in the tissue of H. diminuta were measured, after freeze-clamping the parasite immediately after its removal from the host, Barrett and Beis (1973a) found that, in spite of the essentially anaerobic metabolism of the parasite, its adenylate energy charge-given by [ATPI $[ADPI -is high (0.71). the expression [ATPI [ADPI [AMPI They also found that about 75 % of the NAD in the tissues is present in the oxidized form, whereas about 60 % of the NADP occurs in the reduced form. Although Barrett and Beis were unable to determine the proportions of bound and unbound cofactor, nor were they able to distinguish between the contents of different sub-cellular compartments, their results are consistent with the view that NAD is utilized primarily in respiratory metabolism, whereas NADPH is required for synthetic purposes. If this be so, then clearly, mechanisms must exist for reoxidizing NADH under the relatively anaerobic conditions in which H. diminuta lives. LDH and MDH undoubtedly assist in this function. Although it is widely accepted that the intestinal environment is “relatively anaerobic”, until recently there has been surprisingly little information about the physical characteristics of the environments occupied by intestinal helminths. During the last three years an important series of papers has been published which provides insight into the physical, chemical and biological relationships which exist between H. diminuta and the rat intestine. The foundation for this work was detailed comparison of the physiologies of normal rat gut and gut carrying an infection of H. diminuta (Mettrick, 1971a, b, c, 1972, 1973). Factors investigated included microbial fauna, pH, parasite migration and the competition between parasite and host for

+

+

+

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ingested nutrients. The studies established that these factors fluctuate in the normal rat intestine and that, in parasitized animals, the characteristics are very different. In turn, these observations led to a close re-examination of a large number of the other assumptions about the nature of the environment occupied by H. diminuta (Mettrick, 1975; Podesta and Mettrick, 1974a, b; 1975; 1976). Included in this work are accounts of the effect of the parasite on pH, gastrointestinal function and, most importantly, on oxygen and carbon dioxide tensions within the rat gut. The concept that the lumenal contents of the mammalian intestine are anoxic, while the region “close to the mucosa” contains measurable amounts of oxygen is shown to be, to say the least, an oversimplification. Podesta and Mettrick (1974a) found that, in the rat intestine, the aqueous phase of the lumenal contents has an oxygen tension of 40-50 mm Hg. In uninfected intestines, they observed that anoxic conditions may occur in the distal ileum and colon, but that H. diminuta, by reducing fluid absorption by the gut, helps to maintain the level of oxygen. Other factors contribute to this phenomenon. The parasitized mucosa decreases in weight, thus reducing the barrier to the diffusion of oxygen across the gut wall. As the parasitized intestine also displays reduced substrate transport and glucose metabolism, less energy is expended within epithelial cells, less oxygen is utilized and PO, is effectively raised. pH in the infected intestine is also lower than normal. Since increase in hydrogen ion concentration is accompanied by electron uptake, and since decrease in pH elevates redox potential, then, in the more acidic parasitized intestine, less oxygen is consumed by oxidizing agents in the lumenal contents. Another important effect of depressed pH is to elevate pC0,. Mettrick (1975) quotes values for the oxidation-reduction potential (Eh) in uninfected (-195 to -28 mV) and infected (-75 to +76 mV) rats. Generally, the greater the worm biomass, the greater is the increase in Eh. This shift, from strong reducing tendencies to relatively less reduced conditions may provide an explanation for the occurrence of high levels of cytochromes of the b type in intestinal helminths. Such cytochromes have Eh values of about +50mV (Lehninger, 1970) and are well fitted to act as terminal oxidases under the conditions described by Mettrick (1975). It is possible that here we have the reconciliation of the “aerobic” versus “anaerobic” schools of respiratory metabolism championed most notably by Smith (1969) and Cheah (for example, l972,1976a, b) on the one hand and by Saz (for example, 1971, 1972) on the other. This, when taken into account with increased pC0, and the capacity of intestinal helminths to utilize CO, may mean that, in the words of Podesta and Mettrick (1974a) “the worms, by combining the versatility of CO, fixation and aerobic energy metabolism, would appear to be ideally suited for a lumenal existence”. Podesta and Mettrick (1974b, 1975, 1976a) have explored the relationships between transport across the parasite integument and changes in pH. It is beyond the scope of this review to comment on these results in detail, except in so far as they illuminate the process of HC0,- absorption in H. diminuta. They showed that the parasite absorbs bicarbonate (it is the main anion accompanying Na+ absorption) and that a net influx of ions occurs in the

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absence of concomitant water influxes. The parasite also secretes hydrogen ions into the gut lumen. Organic acid secretion cannot, of itself, account for the latter observation. The high pC0, (250-500 mm Hg) in the parasitized environment, as a consequence of depressed pH, results in the diffusion of CO, down its concentration gradient into worm tissue. Within the tissue the equilibrium of the CO,/HCO,- system favours the production of H+ and HC0,-, thus: HC0,-

+ H+ r' CO, + H,O

The presence of HCO,- within the worm leads to buffering problems in which calcareous corpuscles may play a part, and which favours its use in metabolism. It follows from this that the characteristic metabolic patterns so frequently encountered in helminths-not only parasitic ones-living in so-called anaerobic environments may not be a response to anoxia but to the high, and otherwise toxic levels of CO, encountered. Further support for this hypothesis derives from Mettrick et al. (1976) who examined the kinetic behaviour of PEPCK and PK from H . diminuta. PEPCK has an acid pH optimum, and is activated by HCO,-; PK, however, is inhibited by HC0,- and has a low affinity for PEP at acid pH. Accumulation of CO, in the environment would thus favour the PEPCK reaction. PEPCK from M . expansa has a similarly acidic pH optimum (Behm and Bryant, 1975c) and the suggestion was made that here, too, a C0,-rich environment might drive the PEPCK reaction. The hypothesis put forward by Mettrick, Podesta and co-workers may well have a very wide application. The work quoted in this section has shown how, in the establishment of the rat/H. diminuta host-parasite relationship, the intestinal environment alters to allow the parasite to compete more favourably with the host. This can be further illustrated. Lesser et al. (1975) have shown that maximal rates of glucose transport by H. diminuta are maintained o"er a wider range of pH (5.8-8-5) than is the case for host intestine (6.5-7.5). This may confer an advantage upon the parasite, although the authors remain cautious, both about this and about extrapolating to other parasites. They point out that much higher pH's are encountered in other parasite-intestine systems, Even with this caveat, there is little doubt that what has been done with H. diminuta is a paradigm of what must be done for other parasite-host systems. One can only await eagerly the extension of this type of work to, say, the ruminant gut, as the insight gained by such an approach is of the greatest importance. B. MONZEZIA EXPANSA A N D OTHER CESTODES-STUDIES OF METABOLIC REGULATION

The recent work on H. diminuta has been directed primarily at an analysis of the interactions between the gut and across the body wall of the parasite. Recent studies on Moniezia expansa, however, describe in some detail metabolic events consequent on the uptake of glucose from an artificial medium. Behm and Bryant (1975a) measured the activities of a number of enzymes in

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the scolex and anterior portion of this sheep tapeworm. The activities of HK, PFK, FDPase, aldolase, PK and LDH are high in the cytosol fraction only. PEPCK and MDH activities are present in this and the mitochondrial fraction. Special attention was devoted to the assay of ME, as in H . diminuta it occupies a key role in the oxidative pathway. Only very low ME activity was found in the cytosol fraction from M . expunsa and it was primarily in the carboxylation direction. Traces were found in the mitochondria. On the basis of these results it is not possible to draw any conclusions save that ME plays a very minor role in the oxidative pathway in M . expansa. Determinations of intermediate and end-product concentrations were also made. It was found that considerably more (51 %) lactate is produced by M . expansa under anaerobic conditions than under aerobic conditions. Succinate production is virtually unaffected. The internal pool size of malate increases during aerobiosis. Mass action ratios were calculated from intermediate concentrations and compared with apparent equilibrium constants. The reactions catalysed by HK, PFK and PK were found to be rate limiting, and thus may regulate the pathway of energy metabolism. The status of the reaction catalysed by PEPCK remains equivocal, as a number of approximations were made in the calculation of the mass action ratio. However, the analysis of PEPCK activity in H . diminuta by Mettrick et ul. (1976) renders it likely that, in M . expansa too, the enzyme is regulatory. In M . expansa it was also found that ATP/ADP ratios remain high (1.571.67), as do adenylate energy charges (0*70-0.76),irrespective of whether the parasite is maintained under aerobic or anaerobic conditions. Further studies of three of the potentially regulatory enzymes were made. Earlier work on PK (Bryant, 1972) was extended to include a consideration of the effect of ATP on the enzyme (Behm and Bryant, 1975b). ATP exerts an inhibition (competitivewith ADP) on the unactivated enzyme. In the presence of FDP, activated PK is still inhibited by ATP, but the results suggest a mixed type of inhibition. A partially purified preparation of PFK from M . expansa was found to have a pH optimum in the range 7.4-8.0, to be activated by Mg2+or Mn2+, and to exhibit sigmoid kinetics with F6P. ATP decreases the affinity of the enzyme for F6P, an effect partially relieved by F6P, AMP and NH4+. The degree of sigmoidicity at pH 8 is markedly different from that observed in enzymes from mammalian sources. Generaily, however, PK and PFK can be considered to regulate the pathway in a manner similar to that observed in other organisms. PEPCK has been partially purified from supernatant and mitochondrial fractions from M . expansa (Behm and Bryant, 1975~).The enzymes from the two sources differ. Mitochondria1 PEPCK is specifically activated by Mn2+; the cytosolic enzyme is also activated by Mg2+.The forward and back reactions proceed at similar rates, suggesting that the reaction may be readily reversed in vivo. Of a number of possible activators and inhibitors, only nucleotide triphosphates were found to have an inhibitory effect. Guanosine diphosphate as substrate permits greater maximal activity than ADP.

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A report, by Rasero et al. (1968), that there exists in M. expansa a pathway from glutamate or a-oxoglutarate to succinate, mediated by succinic semialdehyde dehydrogenase and y-aminobutyrate :a-oxoglutarate transaminase, was investigated by Cornish and Bryant (1975). They found no evidence to support this contention and concluded that glutamate could be metabolized via a preliminary transamination to a a-oxoglutarate followed by oxidation to succinate. These studies have been combined in a model which gives a postulated sequence of events which may occur in M. expansa scoleces during transition from aerobiosis to anaerobiosis in vitro (Bryant and Behm, 1976). Briefly, it suggests that there are two sorts of mitochondrial function. In the first, succinate or NADH may be oxidized aerobically by an electron transfer system using oxygen as the terminal electron acceptor. Malate accumulates within the mitochondria, whence it travels to the cytosol. The second function is the reduction of fumarate to succinate with the concomitant oxidation of NADH by the fumarate reductase system. The source of fumarate is cytosolic malate. The different functions may be located in the same or different mitochondria, cells or tissues. If little oxygen is available, the activity of the aerobic system is suppressed and less mitochondrial malate is produced. As the malate pool is also depleted by the production of fumarate for anaerobic respiration, the activity of PK, hitherto inhibited by high concentrations of malate, is enhanced. Lactate production increases. Reducing equivalents to drive the fumarate reductase reaction must originate outside the pathway of energy metabolism (possibly from glutamate oxidation) as the combined LDH and MDH reactions account for all NADH produced earlier in the pathway. The whole system is finely controlled by relative concentrations of adenine nucleotides. During the transition from aerobiosis to anaerobiosis, aerobic production of ATP falls, derepressing PFK and permitting a greater flow of carbon, thus allowing increased anaerobic production of ATP. Although it is necessary to invoke “transient” changes in concentrations of adenine nucleotides, it is unlikely that they can be measured, as the regulatory mechanisms adjust carbon flow to maintain optimum levels. The adjustments may be seen indirectly as increases in lactate output and decreases in the internal concentration of malate. There are relatively few studies on the biochemistry of respiration in cestodes other than H. diminuta and M . expansa. Salminen (1974) investigated electron transfer in adults and plerocercoids of Diphyllobothrium latum. Succinate dehydrogenase, as measured by the phenazine methosulphate technique, was found in both stages in similar activities, and K, values for succinate fall within the range reported for other organisms. Further experiments showed that only adults possess an electron transfer system involving both cytochrome c and cytochrome oxidase. The evidence therefore suggests that adult D. latum is capable of complete aerobic oxidation of substrates, whereas the plerocercoid conforms to the widely accepted anaerobic pattern. Studies on other plerocercoids tend to confirm this. McManus (1975) has shown that PK from the plerocercoid of Ligula intestinalis is similar to that

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described for other parasites. It shows optimal activity at pH 7.0, is activated by FDP, while ATP and malate inhibit co-operatively. An active PEPCK is also present. The author speculates that, as in M . expansa, co-ordinated changes in intracellular concentrations of ATP, malate, FDP and PEP combine to control the activity of PK and thus the major end-products of glucose degradation in vivo. Plerocercoids of Triaenophorus crassus and Schistocephalus solidus also conform to the general anaerobic pattern (Korting, 1976). They, the adults of these species, and the adults of Bothriocephalus gowkongensis and Khawia sinensis are parasites of fresh water fish and are similar in general metabolism to intestinal parasite of homeotherms. Kohler and Hanselmann (1974) have carried out a more intensive study on the tetrathyridia larvae of Mesocestoides corti. They found that glucose is consumed under aerobic or anaerobic conditions, that lactate and succinate are major end-products, with traces of acetate. Succinate production increases anaerobically, which suggests that it might be a substrate under aerobic conditions. The larva probably has an energy metabolism similar in some respects to that described here for H. diminuta, as it has a PK of low activity, regulated by FDP, an active PEPCK, the capacity to decarboxylate malate, and a fumarate reductase. It also possesses all the enzymes necessary for TCA cycle activity, with the probable exception of an NAD-dependent isocitrate dehydrogenase. The authors therefore conclude that the larva is capable of both aerobic and anaerobic metabolism.

111. NEMATODES A. ASCARZS-AEROBIC

OR ANAEROBIC?

Ascaris has been fortunate enough to receive the attentions of biochemists since the 1920s, when Keilin included the parasite in the list of organisms in which he had detected cytochrome. Studies on Ascaris have contributed hugely to the understanding of the biochemical basis of parasitism, and it is no accident that some most significant advances in the study of respiration in the last three years have been made with Ascaris mitochondria. Cheah (1974) has refined his methods for isolating Ascaris mitochondria, and reports that they contain outer and inner membranes, outer compartments and inner cristal spaces similar to those observed in mammalian mitochondria. Direct estimations of ATP synthesis by the preparations were not made. However, the occurrence of oxidative phosphorylation within them was inferred from the observations that ADP stimulated oxygen uptake and that oligomycin (an inhibitor of electron transport-mediated ATP synthesis) depressed it. The preparations proved to be only loosely coupled, as oxygen uptake due to succinate or a-glycerophosphate oxidation was not affected by ADP. There are two points in this study that remain to be clarified. The first is that no transitions between state 3 and state 4 respiration rates were observable, implying either that the technique for isolating mitochondria needs further improvement, or that the analogy between Ascaris and mammalian

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mitochondria is incomplete. The second is that Barrett and Beis (1973b) were not able to observe a-glycerophosphate oxidation in their studies of glycolysis in Ascaris. Cheah (1974) also assayed cytochrome oxidase in Ascaris mitochondrial preparations. He found that ADP stimulates oxygen uptake during ascorbate oxidation, and this is prevented by oligomycin. Once again, the transition from state 3 to state 4 respiration was not clearly observed. Two further papers by Cheah (1976a, b) suggest the presence of a branched respiratory chain with terminal oxidase activity (cytochromes a3, a, and 0).He postulates that the terminal oxidases are controlled by variations in oxygen tension in the small intestine of the host, and that the parasite makes use of

Glucose--+

To fumarole reductase

Molafe

Fumarase Fumorate Outer membrane

U ‘ cyfosol

space

membrane

Matrix

1

J

Mitochondrion

FIG.1. The oxidation of malate by Ascaris, after Rew and Saz (1974).

this cytochrome system in vivo, since its components are found to be reduced by the endogenous substrates of the mitochondrial preparations. While accepting the broad hypothesis, it is not yet unequivocal that branched, rather than parallel, cytochrome chains occur. Barrett (1976a) has also expressed the view that branched electron transport systems must be less efficient than straight chain systems, unless they possess a more elaborate regulation mechanism for which there is, as yet, no evidence. Further support for the anaerobic hypothesis is to be found in Rew and Saz (1974). They used a fractionation procedure, developed for mammalian mitochondria by Sottacasa et al. (1967), which enabled them to separate Ascaris mitochondria into inner membrane, outer membrane, matrix and intermembrane space fractions. The distribution of enzymes in each fraction conforms to the mammalian pattern except that fumarase and NAD-linked “malic” enzyme are found to be associated with the intermembrane space, not with the matrix as in mammals. They thus postulate the metabolic respiratory scheme outlined in Fig. 1, which poses the following problem. If, as in mammals, the inner membrane is impermeable to NADH, and as succinic dehydrogenase is associated with the inner membrane, how then does the energy-yieldingreduction of fumarate take place? The solution appears to reside in the active NADH/NAD transhydrogenase

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associated with the inner mitochrondrial membrane of Ascaris (Fioravanti and Saz, quoted in Rew and Saz, 1974). The authors suggest that it transports protons from NADH across the inner membrane, resulting in NADH accumulation within the matrix (Fig. 1). Kohler (1976) has achieved a partial purification of the transhydrogenase, has shown that NAD is an acceptor, and that NAD and NADH compete for binding sites on the enzyme. Experiments with inhibitors of NADH:quinone reductase and NADH oxidase support the participation of a quinone system in electron transport, but leave the role of the cytochromes obscure. There have been two important studies on the mechanisms of oxidative phosphorylation and adenine nucleotide transport in Ascaris muscle mitochondria. Van den Bossche (1974) examined the membrane-bound ATP-ase activity (which is generally considered to be a reversal of oxidative phosphoryIation) of intact and fragmented mitochondria. He found that intact mitochondria possess little ATP-ase activity, as compared with mammals, and that, although the activity is increased in submitochondrial particles, it still remains relatively low. In addition, the P:2-ATP exchange reaction is also slow. In contrast, Beis and Barrett (1974) found that adenine nucleotide exchange in Ascaris muscle mitochondria is similar to that found in mammals, as it is specific for ATP and ADP, activated by K+ and Rag2+,temperature dependent and inhibited by atractyloside. Barrett (1 976b) has also investigated the intermediary metabolism of developing Ascaris eggs. The profiles of the enzymes of the respiratory pathways show only quantitative changes during embryonation and the acquisition of infectivity. None of the changes is sufficient to alter the carbon flux through the pathways ; dormancy is therefore not associated with these phenomena. Onset and termination of dormancy appear, however, to be associated with changes in the concentrations of some metabolites. Dormant eggs have high ATP/ADP and low cytoplasmic NAD/NADH ratios. Calculations of mass action ratios thus indicate that phosphorylase, HF, PFK and PK are inhibited. The metabolism of the infective egg is apparently intermediate between that of the aerobic developing egg and that of the adult as it possesses a complete TCA cycle, as well as the capacity to fix CO, and reduce fumarate. It is difficult to summarize this section. One may remark that Ascaris mitochondria have some notable similarities to mammalian mitochondria (ultrastructure, possession of “mammalian” cytochromes, ATP-ADP exchange) and some notable dissimilarities (ultrastructure, enzyme distribution, ATP-ase activity, possession of “helminthine” cytochromes). The nature of the parasite (aerobic or anaerobic) is still unresolved and will remain so until a detailed study of its environment has been carried out. On the one hand, Bueding, Saz and co-workers (e.g. Kmetec and Bueding, 1961; Saz, 1971, 1972) suggest that respiration is mediated by a fumarate reductase system involving flavoproteins, and is thus fully anaerobic. On the other hand, a second group (e.g. Kikuchi et al. 1959; Kikuchi and Ban, 1961; Smith, 1969; Cheah, 1976a) claim that Ascaris is aerobic, and that its muscle contains substrate-reducible cytochromes of types a, b and c, and a variety of

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terminal oxidases. Cheah (1974) asserts that these observations collectively do not support the anaerobic hypothesis. Nonetheless, they must be considered in conjunction with the observations of the opposing school which show that the parasite does have an anaerobic capacity. It seems likely that both capacities co-exist within the organism, which is thus adapted to possible fluctuations of oxygen tension within its environment and also to high CO, concentrations, if they occur. Ascaris, it must be remembered, is a whale among nematodes; it is large, probably atypical, and its deeper body tissues may experience different redox conditions from the more superficial tissues. It is unfortunate that the smaller and perhaps more typical nematodes have not been investigated, for obvious reasons, in the same detail. B. OTHER NEMATODES

A nematode parasite which has received considerable recent attention is the rabbit stomach worm, Obeliscoides cuniculi (Hutchinson and Fernando, 1974, 1975). The presence of all the enzymes of glycolysis has been demonstrated, and although variations were observed in developmental stages (early fifth-stage larvae, young and mature adults) they were variations in activities rather than in presence or absence of enzymes. Gluconeogenic capacity is indicated by an active glucose-6-phosphatase and, possibly, pentose phosphate pathway capacity by glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. All the enzymes of the TCA cycle except a-oxoglutarate dehydrogenase were found, although with commendable caution the authors point out the logical problem involved in establishing the absence of a component of a pathway. Hutchinson and Fernando (1975) therefore suggest that substrate enters the TCA cycle as oxaloacetate produced by PEPCK, the activity of which is much greater than that of PK. The oxaloacetate is rapidly converted to malate by MDH, thus regenerating NAD. A high level of cytosolic fumarase activity and an unusually low level of mitochondria1 fumarate reductase activity in the adults lead the authors to suggest that succinate may not be an end-product of metabolism. They note, however, that it is possible that their techniques fail to disrupt the mitochondria, which would result in erroneously low fumarate reductase estimations because the reagents would not have access to the enzyme. Unlike Ascaris, an NADP-dependent malate specific ME was found in the supernatant fraction. The fate of its product, pyruvate, is not clear. With these few reservations, it seems likely that respiratory metabolism in 0. cuniculi follows the general pattern encountered in other helminths, with, of course, the differences in detail that one has become accustomed to in each genus. A similarly truncated TCA cycle also seems to be present in Hyostrongylus rubidus (Stockdale and Fernando, 1974) and in Trichinella spiralis larvae (Boczon, 1974, 1976). Both lack isocitrate dehydrogenase and possess a high MDH activity. In T. spiralis the presence of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase implicates the presence of the

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pentose phosphate pathway. T. spiralis also excretes relatively large amounts of short chain aliphatic amines (Castro et al. 1973). However, they are probably derived from the decarboxylation of endogenous amino acids, and the authors suggest that the CO, so formed may be utilized in respiratory metabolism at the PEPCK reaction. Thus, if CO, levels were to drop during parasitically-induced gut inflammation, the parasite would be able to sustain respiration. One of the by-products of research on anthelmintics is the infoimation which accrues about parasites which might not otherwise be studied. In two recent papers, Saz and co-workers (Wang and Saz, 1974; Saz and Dunbar, 1975) reported experiments on the respiratory metabolism of three filarial nematodes, Litomosoides carinii, Dipetalonema viteae and Brugia pahangi. Only L. carinii is incapable of sustained motility under anaerobic conditions. Studies of l4CO, and 14C- acetate production from differentially labelled glucose suggest that this nematode has little TCA cycle activity and that, probably, the aerobic requirement of the parasite derives from the single, oxidative conversion of pyruvate to acetate. D . viteae and B . pahangi do not yield acetate; most of the carbon is recovered in glycogen and lactale. There is little TCA cycle activity; Wang and Saz (1974) concluded that the parasites were most probably “homolactate” fermenter s. Saz and Dunbar (1975), in an extension of the above work, examined some of the properties of the P F K s and aldolases from three filariids. They found that the P F K s are similar to those of other helminths ( H . diminuta, Ascaris and Schistosoma mansoni) but differ from mammalian PFK in heightened sensitivity to antimonial drugs. If this result is taken in conjunction with the observation of Behm and Bryant (1975b) that PFK from M . expansa displays allosteric properties different from those observed in mammals, it seems possible that in this important regulatory enzyme lies an adaptation common to organisms with low TCA cycle activity. Ward (1974) studied, in vitro, the metabolism of glucose by Haemonchus contortus. Chief among his findings is the observation that propan-1-01 is a major excretory product; the author indicates that this need not be considered a radical departure from orthodoxy as the alcohol may be formed by the simple reduction of propionate. Adult H. contortus rapidly consumes oxygen ; for what purpose it is not yet clear. However, much of the time the parasite was maintained under anaerobic conditions, and like T. spiralis, seems capable of utilizing endogenously produced CO, in the pathway to propionate.

IV. TREMATODES A. FASCZOLA HEPATICA-METABOLIC

REGULATION IN A NON-INTESTINAL PARASITE

The evidence which supports the view that there is a general similarity between the pathways of energy metabolism in Fasciola and intestinal helminths has already been summarized (Bryant, 1975). Recently, Cheah and Prichard (1975) have studied the electron transport system of F. hepatica and,

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as in Ascaris and M . expansa, have found the classical cytochromes of the mammalian type as well as a b-type of cytochrome component containing a CO-reactive “0’’cytochrome. Once again, this suggests a dual system which may have evolved in response to an environment of low or fluctuating oxygen tensions and/or high pC0,. In F. hepatica, the oxidation of carbohydrate leads, in vitro, to the production of propionate, acetate, lactate and a little succinate. Wright and Isseroff (1973) have indicated a possible flaw in these observations. They found that the kinetics of acetate absorption in F. hepatica indicated that it is an active process, enhanced by pH and inhibited by propionate, butyrate and valerate. They speculate on the role of such a mechanism in an organism which ostensibly produces acetate, and offer the hypothesis that, in vivo, acetate production may be low; instead, it is incorporated into higher fatty acids and lipids. This illustrates yet again the potential gulf between in-vivo and in-vitro experimentation. It is therefore most desirable that extrapolations from experiments in vitro to the situation in vivo should be made with experimental systems whose validity has in some way been tested. Even then, such extrapolations should be made with caution. Two attempts have been made to establish and evaluate in-vitro approaches to the study of aspects of metabolism in F. hepatica. Hanna and Threadgold (1975) described a method for the preparation of fluke slices and their subsequent maintenance in a completely defined tissue culture medium (M199). Morphological criteria which formed the basis of evaluation included electron microscopical examination of mitochondria, vacuoles and membranous bodies, which retained their structural integrity for up to ten hours. Threadgold and Hanna (1975) extended the criteria of integrity to include oxygen consumption and glucose uptake, the rates for which were rather higher than in intact flukes, although corrections for dry weight tended to abolish the difference. Both processes were inhibited by iodoacetate and accelerated by 2,4-dinitrophenol. The authors concluded that the criteria they had adopted were valid, and that the slices remain in good condition for experimentation for up to 12 hours. The above-mentioned aspects of morphology and physiology may well be valid criteria of viability but they are not necessarily sufficient to establish metabolic validity. Other criteria could include stability of concentrations of metabolic intermediates, constant rates of excretion of end-products and maintenance of energy status and carbohydrate reserves. Some of these are difficult (if not impossible) to observe in vivo but measurements of energy status, as given by adenine nucleotide and glycogen concentrations, and measurements of intermediate levels can be obtained in at least a rough approximation of reality by the modern techniques of freeze-clamping and spectrophotometric assay. Barret and Beis (1973) give a value of 0.61 for the adenylate energy charge in freshly removed F. hepatica. This value is only slightly lower than those encountered in mammalian systems and shows that, in spite of the lower efficiency entailed in the excretion of highly reduced end-products, the parasite

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maintains a relatively high energy status. Assays of the nicotinamide nucleotides show that most NAD is in the oxidized state, whereas most NADP is reduced, consistent with the role played by the former in energy metabolism and by the latter in synthetic reactions. Cornish and Bryant (1976) determined how some of these criteria varied during in-vitro maintenance of the whole animal in a simple balanced salt solution with glucose as a carbon source. The adenylate energy change is 0.62 immediately after removal from the host (which agrees well with the value obtained by Barrett and Beis, 1973) and rises in culture to 0.68 and 0.65 after

b2{

Carbon balance

+21

3'12 Glucose

I

6 NADH

f

--

-''IL-

- -+ I NADH

I

7 NADH NAD

-

L t :

7 Phosphoenolpyruvate

I Pyruvale

4

I LoCtate

- 3 + 6

6 Oxolaacetate

6 NADH 6 NAD

6 Molole

2 Acetate

4 NAD

+

4 NADH 4 N A D

4 Fumarole-

4C02 ( v i a p y r u v a t e )

-

8

4 NADH

4 Succinate

4COz

4 Proplonote

- I6 0

FIG. 2. The oxidation of glucose and the yield of respiratory end-products in Fasciola hepatica.

24 and 48 hours, respectively. Glycogen levels remain constant after an initial drop, probably due to expulsion of eggs, and levels of respiratory intermediates are maintained within fairly narrow limits. Output of excretory products approaches linearity after ten hours, and occurs in the remarkably exact proportions of lactate (1.0), acetate (2.02) and propionate (4.05). This ratio supports the contention of Prichard (1976) and Van Vugt et al. (1976) that the acetate producing pathway provides the reducing equivalents to drive the energy yielding fumarate reductase reaction. The relative proportions result from the distribution of carbon, as dictated by the NAD/NADH ratio, at the PEP and malate branch-points in the pathway (Fig. 2). The pathway illustrated in Fig. 2 is entirely self sufficient in that all NADH produced to the level of PEP is reoxidized by MDH and LDH. Van Vugt et al. (1976) have investigated the fate of NADH produced in the acetate branch of the pathway. They observed that, in mitochondria from F. hepatica, malate utilization is tightly coupled to phosphorylation as it is ADP dependent, and can be stimulated by the addition of an uncoupler. Determination of phosphorylation efficiencies under anaerobic and aerobic conditions led them to the view that reducing equivalents formed during the oxidation of

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malate can be transported either to fumarate or oxygen. In the latter case more ATP is produced, which supports the views of Cheah and Prichard (1975) about electron transport in F. hepatica. Under aerobic conditions succinate can act as substrate for the production of ATP. One of the characteristics of F. hepatica is that it produces large quantities of proline, a process which has generated much interest (Ertel and Isseroff, 1974e; Isseroff and Ertel, 1976; Kurelec, 1975a, b). It has been suggested that proline production may be related to the maintenance of redox potential within the cell. Prichard (quoted by Kurelec, 1975a) has criticized this on the basis that the pathway as outlined by Kurelec (1975b) would not result in the oxidation of any more NADH than that produced by the fluke in providing the precursors of the pathway. In any event, the pathway of energy metabolism is, as stated earlier, self-sufficient with respect to the oxidation and reduction of NAD. Kurelec (1975a) argues that only exogenous arginine is necessary to maintain the pathway, the other necessary precursor, pyruvate, being recycled. He also points out that the same pathway occurs in Schistosoma mansoni, a trematode which is a so-called “homolactate” fermenter, and which therefore reoxidizes NADH at the LDH level. The significance of the pathway therefore remains to be resolved. There are few studies on liver fluke developmental stages other than adults. Prichard” (1974) found that the 6-week migratory juveniles were generally similar to adults, but suggested that there might be greater dependence on the TCA cycle. Metzger and Duwel* (1974) followed changes in the activities of a large number of enzymes from 5 weeks to 41 weeks after infection; it appears that, during maturation, the activities of the enzymes of energy metabolism are lowered whereas those of synthetic pathways are increased. This phenomenon seems to be related to the vast egg production of the adult. B. OTHER TREMATODES A N D THE PK/PEPCK BRANCH-POINT

The PEP branch-point in the energy metabolism of parasites has been the object of attention of many biochemical parasitologists. PKjPEPCK ratios are frequently assumed to give a measure of the distribution of carbon along the branches to lactate and succinate. This assumption has been criticized (Bryant, 1975) on the grounds that PK and PEPCK assays are usually conducted at sub-optimal conditions in vitro that take no account of the regulatory properties of the enzymes. Further support for this criticism derives from the recent studies of allosterism in PEPCK and PK from M . expansa (Behm and Bryant, 197.5~)~ H. diminuta (Mettrick et al. 1976) and F. hepatica (Prichard, 1976). In the daughter sporocysts of Microphallus aerobic oxidation proceeds via glycolysis and the TCA cycle (Marshal et al. 1974; McManus and James, 1975a). Anaerobically, succinate, alanine and lactate are produced in the ratio of 2:l:l (McManus and James, 1975b). The authors suggest that PEPCK and ME are involved and point out the striking similarity between the pattern of excretory products in the parasite and its molluscan host, suggesting that it

* Quoted by Kurelec, 1975a.

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is indicative of a long association between the Digenea and the Mollusca. There is circumstantial evidence that PEPCK operates in the direction of CO, fixation even under aerobic conditions (McManus and James, 1975~). PK from the daughter sporocysts of Microphallus similis (McManus and James 1975d) is strongly activated by FDP and inhibited by ATP; it is apparently not affected by malate. Kohler and Hanselmann (1973) have investigated the enzyme activities of the pathways of respiratory metabolism in Dicrocoelium dendriticum and have concluded that they bear a close similarity to those described in F. hepatica. Although all enzymes of the TCA cycle are present, the low activity of a-oxoglutarate dehydrogenase suggests that there is little cycle activity. Pentose phosphate pathway activity is indicated by the detection of glucose-6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and the appearance of radiocarbon in CO, when the flukes were incubated with l-l4C glucose. CO, fixation occurs via PEPCK. A detailed analysis of the kinetics of PK from D. dendriticum shows slight co-operativity between PEP and the enzyme, and that it is strongly activated by low concentrations of FDP (Kohler, 1974). It requires either Mg2+ or Mn2+ but at the concentrations of ADP prevailing in the tissues, greater reaction velocities are achieved with Mg2+.ATP is inhibitory, especially to the FDP-activated reaction; malate inhibited the reaction only in the absence of FDP. Kohler (1974) therefore suggests a regulatory scheme similar to that postulated for M . expansa (Bryant, 1972; Bryant and Behm, 1976), in which, under aerobic conditions, increased concentrations of malate and ATP depress PK and the lactate producing branch. In F. hepatica, Prichard (1976) has shown that PK required Mn2+and K+ for maximum activity but that in the presence of FDP, Mg2+can substitute for Mn2+.FDP increases the activity of the Mn2+activated enzyme, decreasing the apparent K, values for PEP and ADP. FDP also relieved the inhibition caused by ATP. In the same paper, Prichard (1976) reported that NADH stimulated the activity of PEPCK and that, when Mn2+was present, citrate appeared to act synergistically with NADH. Estimates of the concentrations of metabolites suggest that PK would be saturated by in-vivo concentrations of PEP and ADP, whereas PEPCK would not be saturated by PEP. The net effect would therefore be that the enzymes would act to ensure the steady production of pyruvate in accordance with the demands for NADH to generate energy, whilst allowing the respiratory pathways to accommodate carbon flow to the degree of anaerobiosis. Schistosomes do not appear to have the same problem. They are “homolactate” fermenters; levels of PEPCK are very low, and it is possible that they possess a functional TCA cycle (Coles, 1973a, b). Immature forms are apparently similar to adults.

V. CONCLUSIONS The last three years have seen a number of significant advances in the understanding of respiratory metabolism in helminths. The application of

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more sophisticated experimental techniques and of methods of analysis of results is largely responsible. It now appears that the major groups of parasites have more in common than was previously thought; metabolic pathways show generic similarities suggesting that the environments occupied by parasites have a few predominating features in common which dictate the respiratory strategies of the organisms occupying them. Whether the critical factor is oxygen tension or the concentration of carbon dioxide remains to be proved. The next major advance will no doubt be consequent upon two approaches. The first is the analysis of the distribution of metabolites and enzymes within the cells and tissues of parasites, and also of the transport of molecules across cellular and subcellular barriers. Rew and Saz (1974) have pointed the way with their study on Ascaris mitochondria. The second is the analysis of the physical/chemical interactions between environment and parasite, in the manner of Mettrick et al. (1976). The extension of all these studies to other parasites and to other life stages is now essential. ACKNOWLEDGEMENTS

I thank my colleagues, Carolyn Behm, Roslyn Cornish and Saidur Rahman for their helpful comments on the manuscript. Grants from the Australian Research Grants Committee, the Commonwealth Scientific and Industrial Research Organisation, the Australian Wool Board and Merck, Sharp and Dohme financed that of my own work which appears in this review. REFERENCES Barrett, J. (1976a). Bionergetics in helminths. In “The Biochemistry of Parasites and Host-Parasite Relationships” (H. Van den Bossche, ed.). Elsevier NorthHolland, Amsterdam. Barrett, J. (1976b). Intermediary metabolism in Ascaris eggs. In “The Biochemistry of Parasites and Host-parasite Relationships” (H. Van den Bossche, ed.). Elsevier North-Holland, Amsterdam. Barrett, J. and Beis, I. (1973a). Nicotinamide and adenosine nucleotide levels in Ascaris lumbricoides, Hymenolepis diminuta and Fasciola hepatica. International Journal for Parasitology 3, 271-273. Barrett, J. and Beis, I. (1973b). Studies on glycolysis in the muscle tissues of Ascaris lumbricoides (Nematoda). Comparative Biochemistry and Physiology 44B,751-761. Behm, C. A. and Bryant, C. (1975a). Studies of regulatory metabolism in Moniezia expansa : general considerations. International Journal for Parasitology 5 , 209-217. Behm, C . A. and Bryant, C. (1975b). Studies of regulatory metabolism in Moniezia expansa: the role of phosphofructokinase (with a note on pyruvate kinase). International Journal for Parasitology 5 , 339-346. Behm, C. A. and Bryant, C. (197%). Studies of regulatory metabolism in Moniezia expansa: the role of phosphoenolpyruvate carboxykinase. International Journal for Parasitology 5 , 347-354.

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Beis, I. and Barrett, J. (1974). Studies on adenine nucleotide exchange in mitochondria from the muscle tissue of Ascaris lumbricoides (Nematoda). Znternational Journal for Parasitology 4, 663-666. Boczon, K. (1974). The tricarboxylic acid cycle and pentose pathway enzymes in T. spiralis larvae. Wiadomosci Parazytologiczne 20, 29-39. Boczon, K. (1976). Effects of some anthelmintics on succinate dehydrogenasecomplex in Trichinella spiralis larvae. In “The Biochemistry of Parasites and Host-Parasite Relationships” (H. Van den Bossche ed.). Elsevier NorthHolland, Amsterdam. Bryant, C. (1972). Metabolic regulation in Moniezia expansa (Cestoda): the role pyruvate kinase. International Journal for Parasitology 2, 333-340. Bryant, C. (1975). Carbon dioxide utilisation, and the regulation of respiratory metabolic pathways in parasitic helminths. In “Advances in Parasitology” (Ben Dawes, ed.), Vol. 13, pp. 35-69. Academic Press, New York and London. Bryant, C. and Behm, C. A. (1976). Regulation of respiratory metabolism in Moniezia expansa under aerobic and anaerobic conditions. In “The Biochemistry of Parasites and Host-Parasite Relationships” (H. Van den Bossche, ed.). Elsevier North-Holland, Amsterdam. Castro, G . A., Ferguson, J. D. and Jorden, C. W. (1973). Amine excretion in excysted larvae and adults of Trichinella spiralis. Comparative Biochemistry and Physiology 45A, 819-828. Cheah, K. S. (1972). Cytochromes in Ascaris and Moniezia. In “Comparative Biochemistry of Parasites” (H. Van den Bossche, ed.), pp. 417-432. Academic Press, London and New York. Cheah, K. S . (1974). Oxidative phosphorylation in Ascaris muscle mitochondria. Comparative Biochemistry and Physiology 47B,237-242. Cheah, K. S . (1976a). Electron transport system of Ascaris muscle mitochondria. In “The Biochemistry of Parasites and Host-Parasite Relationships” (H. Van den Bossche, ed.). Elsevier North-Holland, Amsterdam. Cheah, K. S . (1976b). Ascaris lumbricoides cytochrome b-560. In “The Biochemistry of Parasites and Host-Parasite Relationships” (H. Van den Bossche, ed.). Elsevier North-Holland, Amsterdam. Cheah, K. S. and Prichard, R. K. (1975). The electron transport systems of Fasciola hepatica mitochondria. International Journal for Parasitology 5, 183186. Coles, G . C. (1973a). The metabolism of schistosomes: a review. International Journal of Biochemistry 4, 319-337. Coles, G . C. (1973b). Enzyme levels in cercariae and adult Schistosoma mansoni. International Journal of Parasitology 3, 505-510. Coles, G . C. (1975). Fluke biochemistry-Fasciola and Schistosoma. Helminthological Abstracts 44, 147-162. Cornish, R. A. and Bryant, C. (1975). Studies of regulatory metabolism in Moniezia expansa: glutamate, and the absence of the y-aminobutyrate pathway. International Journal for Parasitology 5, 355-362. Cornish, R. A. and Bryant, C. (1976). The metabolic integrity of Fasciola hepatica during in vitro maintenance. International Journal for Parasitology 6, 387-392. Ertel, J. and Isseroff, H. (1974). Proline in fascioliasis: 1. Comparative activities of ornithine-6-transaminase and proline oxidase in Fasciola and in mammalian livers. Journal for Parasitology 60,574-577. Fairbairn, D. (1970). Biochemical adaptation and loss of genetic capacity in helminth parasites. Biological Reviews 45, 29-72. Hanna, R. B. and Threadgold, L. T. (1975). Development of an in vitro technique

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for cytological investigations of slices of Fasciola hepatica : evaluation by morphological criteria. International Journal for Parasitology 5, 321-331. Hutchinson, G. W. and Fernando, M. A. (1974). Enzymes of gylcolysis and the pentose phosphate pathway during development of the rabbit stomach worm Obeliscoides cuniculi. International Journal for Parasitology 4, 389-395. Hutchinson, G. W. and Fernando, M. A. (1975). Enzymes of the tricarboxylic acid cycle in Obeliscoides cuniculi (Nematoda; Trichostrongylidae) during parasitic development. International Journal for Parasitology 5, 77-82. Isseroff, H. and Ertel, J. C. (1976). Proline in fascioliasis: 111. Activities of pyrroline-5-carboxylicacid reducase and pyrroline-5-carboxylicacid dehydrogenase in Fasciola. International Journal of Parasitology 6, 183-188. Kikuchi, G. and Ban, S. (1961). Cytochrome in the particulate preparation of the Ascaris lumbricoides muscle. Biochimica Biophysica Acta 51, 387-389. Kikuchi, G., Ramirez, J. and Barron, E. S. G. (1959). Electron transport system in Ascaris hmbricoides. Biochemica Biophysica Acta 36, 335-342. Kmetec, E. and Bueding, E. (1961). Succinic and reduced diphosphopyridine nucleotide oxidase system of Ascaris muscle. Journal of Biological Chemistry 236,584-591. Kohler, P. (1974). Metabolic role of pyruvate kinase in the trematode Dicrocoelium dendriticum. Comparative Biochemistry and Physiology 49B, 335-344. Kohler, P. (1976). Hydrogen transport in the muscle mitochondria of Ascaris suum. In “The Biochemistry of Parasites and Host-Parasite Relationships” (H. Van den Bossche, ed.) Elsevier North-Holland, Amsterdam. Kohler, P. and Hanselmann, K. (1973). Intermediary metabolism in Dicrocoelium dendriticum (Trematoda). Comparative Biochemistry and Physiology 45B, 825845. Kohler, P. and Hanselmann, K. (1974). Anaerobic and aerobic energy metabolism in the larvae (Tetrathyridia) of Mesocestoides corti. Experimental Parasitology 36, 178-188. Korting, W. (1976). Metabolism in parasitic helminths of freshwater fish. In “The Biochemistry of Parasites and Host-Parasite Relationships” (H. Van den Bossche ed.). Elsevier North-Holland, Amsterdam. Kurelec, B. (1975a). Molecular biology of helminth parasites. International Journal of Biochemistry 6, 375-386. Kurelec, B. (1975b). Catabolic path of arginine and NAD regeneration in the parasite Fasciola hepatica. Comparative Biochemistry and Physiology 51B, 151156. Lehninger, A. L. (1970). “Biochemistry.” 1st edition p. 368. Worth Publishers Inc., New York. Lesser, R. D., McCracken, R. 0. and Lumsden, R. D. (1975). The effect of ambient pH changes on glucose absorption by the tapeworm Hymenolepis diminuta. Comparative Biochemistry and Physiology 52A, 97-100. Marshal, I., McManus, D. P. and James, B. L. (1974). Glycolysis in the digestive gland of healthy and parasitised Littorina saxatilis rudis (Maton) and in the daughter sporocysts of Microphallus similis (Jag.) Comparative Biochemistry and Physiology 49B, 291-299. McCracken, R. 0. and Lumsden, R. D. (1975a). Structure and function of parasite surface membranes - I. Mechanism of phlorizin inhibition of hexose transport by the cestode Hymenolepis diminuta. Comparative Biochemistry and Physiology 50B, 153-157. McCracken, R. 0. and Lumsden, R. D. (1975b). Structure and function of parasite surface membranes - 11. Concanavalin A absorption by the cestode

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Podesta, R. B. and Mettrick, D. F. (1976). The inter-relationships between the in situ fluxes of water, electrolytes and glucose by Hymenolepis diminuta. Znternational Journal for Parasitology 6, 163-172. Prichard, R. K. (1976). Regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase activity in adult Fasciola hepatica (Trematoda). International Journal for Parasitology 6, 227-233. Rasero, F. S., Monteoliva, M. and Mayor, F. (1968). Enzymes related to 4-aminobutyrate metabolism in intestinal parasites. Comparative Biochemistry and Physiology 25, 693-701. Rew, R. S. and Saz, H. J. (1974). Enzyme localization in the anaerobic mitochondria of Ascaris lumbricoides. Journal of Cellular Biology 63, 125-135. Salminen, K. (1974). Succinate dehydrogenase and cytochrome oxidase in adult and plerocercoid Diphyllobothrium latum. Comparative Biochemistry and Physiology 49B, 87-92. Saz, H. J. (1971). Facultative anaerobiosis in the invertebrates: pathways and control systems. American Zoologist 11, 125-135. Saz, H. J. (1972). Comparative Biochemistry of carbohydrates in nematodes and cestodes. In “The Comparative Biochemistry of Parasites”. (H. Van den Bossche, ed.), pp. 33-47. Academic Press, New York and London. Saz, H. J. and Dunbar, C. A. (1975). The effects of stibophen on phosphofructokinases and aldolases of adult filariids. Journal of Parasitology 61, 794-801. Smith, M. H. (1969). Do intestinal parasites require oxygen? Nature London 223, 1129-1 132. Sottocasa, G . L., Kuylenstierna, B., Ernster, L. and Bergstrand, A. (1967). An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. Journal of Cellular Biology 32, 415-438. Stockdale, P. H. G . and Fernando, M. A. (1974). Some enzymes associated with the metabolism of phosphoenolpyruvate in Hyostrongylus rubidus. Journal of Parasitology 60, 178. Threadgold, L. T. and Hanna, B. (1975). Development of an in vitro technique for cytological investigations of slices of Fasciola hepatica: evaluation by physiological criteria. International Journal of Parasitology 5, 333-337. Van den Bossche, H. (1974). Adenosine triphosphatase activity in Ascaris muscle mitochondria. Comparative Biochemistry and Physiology 49B, 71-78. Van Vugt, F., Kalaycioglu, L. and Van den Bergh, S. G. (1976). ATP production in Fasciola hepatica mitochondria. In “The Biochemistry of Parasites and HostParasite Relationships” (H. Van den Bossche, ed.). Elsevier North-Holland, Amsterdam. Walkey, M. and Fairbairn, D. (1973). L-( +)-lactate dehydrogenase from Hymenolepis diminuta (Cestoda). Journal of Experimental Zoology 183,365-374. Wang, E. J. and Saz, H. J. (1974). Comparative biochemical studies of Litomosoides carinii, Dipetalonema viteae, and Brugia pahangi adults. Journal of Parasitology 60, 3 16-321. Ward, P. F. V. (1974). The metabolism of glucose by Haemonchus contortus, in vitro. Parasitology 69, 175-190. Watts, S. D. M. and Fairbairn, D. (1974). Anaerobic excretion of fermentation acids by Hymenolepis diminuta during development in the definitive host. Journal of Parasitology 60, 621-625. Wright, R. W. and Isseroff, H. (1973). Further studies on the absorption of acetate by Fasciola hepatica. Comparative Biochemistry and Physiology 45B, 95-99.