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A M . ZOOLOGIST, 11:125-135 (1971) Facultative Anaerobiosis in the Invertebrates: Pathways and Control Systems HOWARD J. SAZ Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556 SYNOPSIS. An increasing number of the invertebrates studied have been found to rely on an anaerobic energy metabolism during at least one stage in their development. Some of the bivalve mollusks, and particularly a relatively large number o£ parasitic nematodes, cestodes, trematodes and acanthocephala may be classified in this category. Regardless of the aerobic or anaerobic requirements of the parasitic helminths, two features appear to be common to all members of this group which have been examined. First, they are all capable of taking up oxygen. Second, none can oxidize substrates completely to CO2 and H2O; end-products of fermentation invariably accumulate, indicating either the complete absence or the presence of only a limited terminal respiratory pathway. The intestinal nematode, Ascaris lumbricoides, has served as a model system for the elucidation of an anaerobic energy-yielding pathway of carbohydrate dissimilation which appears to be operative in the anaerobic stages of a number of other invertebrates. This pathway differs in several major respects from those previously described for mammalian and other aerobic tissues. The Ascaris system is discussed in detail and is compared with other invertebrate and vertebrate metabolisms. For many years it was thought that essentially all multicellular tissues obtained their energy by employing similar aerobic terminal respiratory pathways. With the advent of biochemical investigations of some of the invertebrate groups, however, it became apparent that not all tissues had an aerobic requirement to fulfill their energy needs. It was suggested by von Brand (1950), Bueding (1949) and others since, (Saz, 1969), that the adult forms of the larger intestinal helminths, for example, possess a predominantly anaerobic energy metabolism. The early suggestions were based primarily on the fact that these worms reside in the intestine, an environment where the oxygen tension is very low. Further experimentation has substantiated these predictions for an increasing number of helminth parasites. It has been suggested also, by the reports of Galtsoff (1964) as well as those of Hammen and collaborators (See Wegener et al., 1969), that some of the bivalve mollusks, particularly the American oyster, tolerate long periods of anaerobiosis by utilizing energy-producing pathways simiThe author's current investigations are supported in part by the NIH, U.S. Public Health Service (Grant AI-09483). lar to those which occur in the intestinal nematode Ascaris lumbricoides. It appears likely that other invertebrates which depend upon intermittent or continuous periods of anaerobiosis may employ similar metabolic pathways. It would seem a simple matter to determine whether or not an organism requires oxygen for survival. In the case of many helminths, however, this basic question has remained unanswered for a number of reasons. All nematodes, as well as cestodes and trematodes which have been examined thus far, are capable of taking up oxygen when incubated in vitro in air. This includes Ascaris. On the other hand, the physiological significance of this oxygen uptake has been seriously questioned in the case of those worms which normally dwell in essentially anaerobic environments, such as the lumen of the intestinal tract. In addition, some of the intestinal helminths have been shown by a number of investigators to survive for extended periods of time in anaerobic environments. Oxygen may even be detrimental to survival in some instances. More recently, Berntzen (1961) and subsequently Schiller (1965) have cultured successfully the rat tapeworm, Hymenolepis diminuta. In 125 126 HOWARD J. SAZ agreement with earlier biochemical findings, the worms were cultured under anaerobic conditions, oxygen being detrimental to development. It would appear, therefore, that at least some of the helminths are anaerobes in the adult stage. It is particularly intriguing that none of the helminths studied to date, regardless of their oxygen requirements, be they aerobic or anaerobic, are capable of the complete oxidation of substrates to CO2 and water. All of those examined accumulate organic end-products. Since substrates are not completely metabolized, it would appear that terminal respiration is either absent or rate-limiting in this entire group of organisms. Although fermentation products always accumulate, they differ both qualitatively and quantitatively with each parasite. It is of interest, however, that a surprising number of parasitic helminths accumulate one product in common. That is succinate. Representative nematodes which form succinate or products presumably derived from succinate include Ascaris lurnbricoides, Heterakis gallinae, Trichuris vulpis, and the larval form of Trichinclla spirails. Cestodes which also show this property include the tapeworms Hymenolepis diminuta and Moniezia expansa, and cysts of Echinococcus granulosus and Taeiiia taeniaformis. The liver fluke, Fasciola hepatica and Moniliformis dubins represent trematodes and acanthocephalans, respectively, which fall into the category of succinate accumulating helminths (For references see Sa/. and Bueding, 1966). The invertebrate which has served as a model system for biochemical studies of this type has been the intestinal parasite, Ascaris lumbricoides. To a large extent, the anaerobic pathway for carbohydrate dissimilation has been elucidated in this nematode, and may be related to pathways occurring in other helminths and possibly in other groups of facultative invertebrates. In spite of the fact that Ascaris adults consume oxygen in vitro (Laser, 1944), as do all other helminths examined, early studies indicated that this parasite derived its energy from an anaerobic sequence of reactions. Weinland (1901) reported that Ascaris survives equally well under aerobic or anaerobic conditions, indicating that the adult stage obtains its energy via anaerobic pathways of metabolism. In accord with this, the organism is not sensitive to cyanide, and neither cytochrome c nor cytochrome oxidase is demonstrable in Ascaris tissues (Bueding and Charms, 1952). It would appear that a flavin system for terminal oxidation has replaced the cytochrome system, since, in the presence of air, hydrogen peroxide is formed (Laser, 1944). Hydrogen peroxide is toxic to the worm due to a deficiency of catalase (Laser, 1944; Magath, 1918). A further indication of a flavin system is the finding that the rate of oxygen utilization is dependent upon the oxygen tension, being almost negligible at the oxygen tensions found in the physiological environment of the adult ascarids (Bueding and Charms, 1952; Harnisch, 1933; Laser, 1944; Rathbone, 1955). One additional indication that the parasite is an anaerobe is that, qualitatively at least, the same products are formed in either air or N2-CO2 environments (Harpur and Waters, I960). These observations are consistent with the conclusion that this parasite is predominantly anaerobic and, unlike mammalian tissues, possesses a terminal flavin, rather than a cytochrome oxidase system. Succinate and a mixture of volatile fatty acids comprise the major end-products of Ascaris fermentations. With the aid of C u labeled substrates, Saz and Vidrine (1959) studied the anaerobic pathway for succinate formation in Ascaris and suggested the pathway outlined in Figure 1. Their findings were in accord with the dissimilation of glucose via glycolysis to a three carbon moiety, presumed to be pyruvate. Carbon dioxide was then fixed into the three carbon compound with subsequent reduction of the product to succinate. Succinate and pyruvate were then found to be precursors of the volatile fatty acid end-products. Of particular interest was the suggestion that succinate was formed by a reversal of the reactions which ANAEROBIOSIS IN INVERTEBRATES FIG. 1. Proposed pathway for the formation of succinate and volatile acids in Ascaris muscle. occur in aerobic tissues, that is, by the backward or reductive reactions of the tricarboxylic acid cycle. Succinate dehydrogenase in the parasite muscle acted physiologically in a manner opposite to that of mammalian tissues. Rather than a succinate dehydrogenase, Ascaris appeared to possess a fumarate reductase which would serve to reoxidize the reduced DPN formed during glycolysis. Kmetec and Bueding (1961) partially purified Ascaris succinate dehydrogenase and demonstrated that it did indeed behave as a fumarate reductase system. Studies of this nature, together with the report of Seidman and Entner (1961), made it clear that Ascaris muscle relied on this backward pathway for much of its energy supply, since ATP generation was found to be associated with the electron transport system coupled to the fumarate reductase reaction. Fumarate acts as the ultimate electron acceptor, reoxidizing reduced DPN with the concomitant formation of succinate. The transfer of electrons from DPNH to an intermediate flavin presumably results in the liberation of energy in the form of ATP. In Ascaris, therefore, fumarate takes the place of oxygen as the terminal electron acceptor. What, then, are the controlling factors which allow this parasite to maintain its metabolic flow in a direction opposite to that found in most other tissues? A hint at an answer to this question came with the demonstration of the presence of an active phosphoenolpyruvate carboxykinase in Ascaris muscle (Saz and Lescure, 1967). It 127 can be noticed that the accumulation of succinate by Ascaris requires the fixation of one molecule of COL. for each succinate formed. Phosphoenolpyruvate carboxykinase is an enzyme which has CO2 fixing capabilities and which was first described in chicken and, subsequently, mammalian liver by Utter and Kurahashi (1954). In contrast to Ascaris muscle, the enzyme could not be detected in mammalian muscle. Prescott and Campbell (1965) were the first to demonstrate the presence of this enzyme in a helminth, Hymenolepis diminuta. Subsequently, it has been shown to be present in a number of other helminths. The reaction catalyzed by phosphoenolpyruvate (PEP) carboxykinase is shown in Figure 2. The enzyme catalyzes OAA + ITP ?± PEP + CO, + IDP [GTP] [GDP] FIG. 2. Reaction catalyzed by phosphoenolpyruvate carboxykinase. the reversible decarboxylation of oxalacetate (OAA) in the presence of the nucleotide inosine triphosphate (ITP) to form phosphoenolpyruvate (PEP), CO2 and inosine diphosphate (IDP). Guanosine triphosphate can substitute partially for the inosine nucleotide. The reverse reaction, the fixation of CO2 into PEP to form oxalacetate, can also be catalyzed by this system, but the enzyme in mammalian tissues is thought to catalyze primarily the decarboxylation of oxalacetate (Utter et al, 1964). Therefore, once again, Ascaris appears to catalyze this reaction in a direction opposite to that found in mammalian tissues. Saz and Lescure (1967) incubated dialyzed Ascaris muscle homogenates in the presence of C14 labeled sodium bicarbonate plus unlabeled oxalacetate. After five minutes of incubation, the incorporation of radioactivity into the OAA was determined. Results are shown in Table 1. This assay procedure for PEP carboxykinase indicated a high activity in Ascaris muscle in the presence of ITP. Guanosine triphosphate (GTP) can partially replace ITP, but activity with ATP is quite low. Of major importance is the fact that the reaction is dependent upon the 128 HOWARD J. SAZ TABLE 1. Incorporation of NaHC^O, into OAA by Asearis Muscle Homogenate (PEP CarboxyTcinase Assay) System Dpm fixed/5 min. /iMoles fixed/5 min. Complete, with ITP Complete, with GTP Complete, with ATP No nueleotide added Without Oxalacetate 180,200 125,240 47,000 4,400 7.45 5.18 1.94 0.18 30 addition of a nueleotide. This dependency for a nueleotide distinguishes the PEP carboxykinase from some of the other known CO2 fixing enzymes. These, plus subsequent findings, indicated strongly that PEP carboxykinase is indeed the enzyme responsible for catalyzing the fixation of CO2 into PEP in Asearis muscle. Presumably the oxalacetate formed by the reaction ultimately would be reduced to succinate, thereby making phosphoenolpyruvate rather than pyruvate the point at which the metabolism of Asearis muscle branches off from that of the host tissues. A new question now presented itself. Why does the PEP carboxykinase of Asearis act primarily in a direction opposite to that of the equivalent mammalian enzyme? The helminth system, on the one hand, catalyzes the fixation of CO2; the mammalian system, on the other hand, catalyzes primarily the decarboxylation of oxalacetate, thereby forming PEP, which, in turn, could be transformed into glycogen by a reversal of the reactions of glycolysis. In an effort to determine whether or not the Ascnris PEP carboxykinase was similar to the mammalian enzyme, assay procedures were worked out and optimal conditions determined for assaying the reaction in both directions (Saz and Lescure, 1969). The worm enzyme, like the one from the host, was almost twice as TABLE 2. OpHmal Activities and Apparent Values of Asearis PEP Carboxykinase Km Reaction Assayed Optimal Activityz Apparent Km for Substrate M P E P + CO.. -> OAA OAA -> P E P + CO2 403 794 2.ti X 10-'3 1.8 X 10- " Activity expressed former! /min/mf* protein. m^mole^ oH product 0 active in the direction of oxalacetate decarboxylation (Table 2). It should be emphasized that these activities are based upon optimal conditions of substrate concentration. If now the effect of substrate concentration on enzymatic activity is studied and the Michaelis constant (Km) determined, a different picture is obtained. Knl values designate the concentration of substrate which results in half maximal activity. In this respect, it is an approximation of the affinity of the enzyme for the substrate under investigation. It is of particular interest that with the Asearis enzyme, half maximal activity in the direction of CO2 fixation was reached at approximately one-seventh the substrate concentration of the reverse decarboxylation reaction (Table 2). These findings indicate that under physiological conditions the reaction may be catalyzed primarily in the direction of CO2 fixation. This possiblity becomes particularly impressive in view of the fact that Asearis tissues contain surprisingly high concentrations of PEP which would tend to push the CO2 fixing reaction. In addition, a very high malate dehydrogenase activity is also present which should serve to pull off oxalacetate rapidly, thereby possibly pulling the enzyme again in the direction of CO2 fixation. Figure 3 presents a summary of some of the reactions of Asearis and mammalian tissues discussed so far. In both parasite and host tissues, carbohydrate is dissimilated to phosphoenolpyruvate. At this point, the two types of tissues diverge; Ascaris fixing CO2 via reaction (2), PEP carbox\kinase, to form oxalacetate which is ultimately reduced to succinate. Mammalian tissues, on the other hand, generally employ reaction (2) in the opposite ANAEROBIOSIS IN INVERTEBRATES FIG. 3. Comparison of metabolic reactions of mammalian and Ascnri.s tissues. (1) = Pyruvate kinase. (2) = Phosphoenolypyruvate carboxykinase. direction. That is, in mammalian tissues, oxalacetate formed either from succinate or pyruvate is decarboxylated to PEP which can then serve as a precursor of glycogen, the overall process being referred to as glyconeogenesis. Most significant is the fact that mammalian tissues utilize PEP rapidly in the presence of ADP to form pyruvate and ATP (reaction 1). This reaction is catalyzed by pyruvate kinase. Once pyruvate is formed it may be converted anaerobically (as in rapidly contracting muscle) to lactate, or aerobically to CO2 and water via the tricarboxylic acid cycle. The next question which arises is, why is the enzyme pyruvate kinase so successful in its competition for substrate PEP in mammalian tissues, but so unsuccessful in Ascaris where CO2 fixation appears to be favored? In an attempt to answer this question, various enzymatic activities of Ascaris muscle were determined under optimal conditions (Bueding and Saz, 1968). PEP carboxykinase was assayed spectrophotometrically in the direction of COo fixation. Results are presented in Table 3. The figures in parentheses indicate the TABLE 3. Enzymatic Activities of Ascaris Muscle Enzyme PEP Carboxykinase Lactate Dehydrogenase Malate Dehydrogennse Pyruvate Kinase Substrate Utilized (m^moles/min/mg protein) 168 (126-209) 143 (71-163) 5,278 (4,221-6,807) 7 (4.6-9.1) 129 range of activities observed. Lactate dehydiogenase and PEP carboxykinase were found to be present at comparable activities. This is of interest, in view of the fact that intact ascarids do not accumulate lactate. As mentioned previously, the malate dehydrogenase activity is very high, the highest of all Ascaris enzymes assayed. Most significant is the barely detectable activity of pyruvate kinase. This enzyme is essentially absent from Ascaris muscle. The very low level of pyruvate kinase activity, then, explains why pyruvate is not formed directly from PEP by this parasite. In addition, it would explain why the fixation of CO2 into PEP occurs as a major metabolic reaction in this tissue. Since phosphoenolpyruvate is not removed appreciably by pyruvate kinase in the parasite muscle, the endogenous levels of PEP and pyruvate were compared in Ascaris muscle. In accord with predictions, the existing concentrations of PEP were, on the average, approximately twice those of pyruvate (0.26 ^moles/gm versus 0.12 ^moles/gm). As pointed out earlier, this relatively high tissue level of PEP would favor the PEP carboxykinase reaction in the direction of CO2 fixation. Two major questions remained to be answered by the proposed scheme for succinate formation in Ascaris. First, fermentations by this nematode result in the accumulation of relatively large quantities of acetate. In addition, acetate serves as a precursor for alpha-methylbutyrate which is a major fermentation product of the parasite. If pyruvate can not be formed from PEP via the pyruvate kinase reaction, then from where do it and acetate originate in the Ascaris fermentation? Second, if pyruvate is formed by some pathway in the muscle, then why does lactate not accumulate in Ascaris fermentations, since the presence of an active lactate dehydrogenase has been demonstrated? Recent findings have helped elucidate answers to both questions. Saz and Hubbard (1957) described an active DPN linked malic enzyme from Ascaris muscle. This enzyme catalyzes the reaction shown in Figure 4. Malate is ox- 130 HOWARD J. SAZ 1-Malate + DPN ^ Pyruvate + CO2 + DPNH + H+ FIG. 4. Reaction catalyzed by the malic enzyme. idatively decarboxylated in the presence of DPN to pyruvate, CO2 and DPNH. Although this reaction is reversible, the Ascaris system is approximately 300 times faster in the direction of pyruvate formation from malate. This reaction then could serve as a means of pyruvate formation in Ascaris muscle, which would be independent of pyruvate kinase. Despite the fact that the malic enzyme was found in Ascaris muscle in 1957, its physiological function could not be understood until quite recently. Although the malic enzyme could provide a means for forming pyruvate, the question concerning the lack of lactate formation remained to be answered. One possible answer was compartmentalization within the cell such that pyruvate and lactate dehydrogenase would not appear within the same compartment. Ascaris has adapted itself remarkably well to its environment. Mitochondria are present in Ascaris tissues, but they function in a manner quite different from the corresponding organelles of the host tissues. That is, they function anaerobically. The distribution of several enzyme systems from Ascaris muscle between the soluble and mitochondrial fractions was determined by Saz and Lescure (1969). The enzymes examined were malic enzyme, fumarase, PEP carboxykinase and lactate dehydrogenase (Table 4). Ascaris muscle was homogenized and large particles removed by slow speed centrifugation. The 128g supernatant obtained contains both the mitochondria and the soluble fraction. This fraction contains essentially all of the activities being examined. If now we sediment the mitochondria by centrifugation at 9,000g the supernatant contains the so-called soluble fraction. It should be noted that all of the lactate dehydrogenase is recovered in this soluble fraction; none is retained by the mitochondria. Similarly, essentially all of the PEP carboxykinase is recovered in this fraction, plus a small amount in the wash of the mitochrondria. In the case of fumarase, approximately one-half was recovered in the soluble fraction; the remainder stayed with the mitochondrial pellet. In the case of the malic enzyme, approximately one-sixth of the activity remained in the supernatant; the remainder appeared in the mitochondrial fraction. YVhen the mitochondria were disrupted, most of the remaining activities of both malic enzyme and fumarase were liberated. Of interest now, if the disrupted mitochondrial fraction was spun at 96,000g essentially all of the malic enzyme activity was solubilized, whereas three-fourths of the fumarase activity remained in the insoluble (membrane-bound) fraction. The finding, therefore, of small quantities of malic enzyme activity in the 128g supernatant fraction may reflect a partial disruption of the mitochondria during preparation followed by leakage of the enzyme TABLE 4. Cellular Distribution of Ascaris Muscle Enzt/mrs TJmtsVgm of Muscle Fraction 128<7 Supernatant 9,0000 Supernatant Mitochondrial Wash Disrupted Mitochondria 9f>,000<7 Supernatant of Disrupted Mitochondria 96,0000 Residue of Disrupted Mitochondria Malic Enzyme Fumarase 6.13 1.37 0.70 3.90 3.6S 11.32 6.47 0.03 4.52 1.35 — 3.24 • Units express ^moleh of substrate utilized per minute. PEP Carboxykinase Lactate Dehydrogenase 7.60 7.06 0.38 2.05 2.02 0 II — 0 0 n — ANAEROBIOSIS IN INVERTEBRATES T. FIG. 5. Pathway of carbohydrate dissimilation in Ascaris muscle. into the soluble fraction. Dissimilatory pathways in mammalian tissues may now be compared with our current concepts of the comparable situation in Ascaris. The overall pathway by which mammalian tissues dissimilate carbohydrate is briefly described as follows. In the soluble, or cytoplasmic, portion of the cell, glucose arising either externally or from glycogen breakdown is phosphorylated and transformed by the glycolytic enzymes into two moieties of three carbons each, which are converted ultimately to phosphoenolpyruvate. Phosphoenolpyruvate, in turn, donates its high energy phosphate bond to ADP to form ATP and pyruvate, the reaction being catalyzed by pyruvate kinase. In mammalian tissues, pyruvate may then be converted to lactate, or it may enter the mitochondrion where it undergoes complete oxidation to CO2 and water via the aerobic reactions of the tricarboxylic acid cycle. These reactions are coupled through the cytochrome electron transport system to oxygen. In view of the more recent findings with Ascaris, the situation in the parasite muscle may be summarized as illustrated in Figure 5. According to the proposed pathway, the glycolytic enzymes of Ascaris, which are present in the soluble portion of the cell, function similarly to those of the host tissues up to the point of PEP accumulation. Since phosphoenolpyruvate can not be dephosphorylated directly to pyruvate, the Ascaris PEP carboxykinase, which is found in the soluble portion of the cell, fixes COS into this compound, 131 resulting in the formation of oxalacetate. This dicarboxylic keto acid is rapidly reduced by glycolytically formed DPNH, the reaction being catalyzed by the very active cytoplasmic malate dehydrogenase. It is this reaction which serves to regenerate cytoplasmic DPN, a function carried out by lactate dehydrogenase in mammalian tissues. Malate thus formed in the cytoplasm now crosses over into the mitochondrion and becomes the mitochondrial substrate. Since direct oxidative systems are absent in the Ascaris mitochondrion, malate must be utilized further by a dismutation system. Intramitochondrial reducing power, in the form of DPNH, is generated by the oxidative decarboxylation of malate to pyruvate and CO2, thereby giving rise to pyruvate in the absence of pyruvate kinase. This reaction is, of course, catalyzed by the mitochondrial malic enzyme. DPNH formed from this reaction then serves to reduce a corresponding amount of malate to succinate via fumarate and the fumarate reductase reaction with the concomitant formation of ATP. Pyruvate may then serve as a precursor for acetate, but again within the mitochondrion. This reaction would presumably result in additional reducing power inside the mitochondrion which might enter into the reductive formation of the volatile fatty acids which accumulate during Ascaris fermentations. The site of fatty acid formation is, however, still not known. It should be recalled that pyruvate, according to this scheme, is formed within the mitochondrion, while lactate dehydrogenase is a cytoplasmic enzyme. Therefore, lactate would not be an end-product of Ascaris fermentations by intact worms. It might be expected, however, that homogenates of Ascaris muscle in which the mitochondria are disrupted and the lactate dehydrogenase released would accumulate lactate. This is indeed the case. Homogenates of Ascaris muscle do form lactate. According to this proposed scheme, malate should be the mitochondrial substrate which would dismutate to a mixture of pyruvate, succinate and possibly other 132 HOWARD J. SAZ TABLE 5. The Anaerobic Utilization of Malate by Intact Asearis Muscle Mitochondria Compound Recovered (jiMoles) Acetate Propionate Pyruvate Succinate 2.05 0.97 5.25 5.29 Malate Disappearance = 13.29 ^Moles. volatile acids. In addition, since evidence has been presented indicating an electrontransport associated phosphorylation resulting from the fumaric reductase reaction, an uptake of inorganic P32 into organic phosphate would be expected. Incubation of L-malate with intact Asearis mitochondria under anaerobic conditions (Saz and Lescure, 1969) results in the accumulation of acetate, propionate, pyruvate and succinate (Table 5). These are the products which might be predicted, and essentially one pyruvate accumulates for each succinate formed. Presumably the acetate and propionate arise from pyruvate and succinate respectively. Asearis mitochondria also catalyze a rapid anaerobic malate dependent phosphorylation as determined by the incorporation of inorganic P32 into organic phosphates Mitochondria were again incubated under a nitrogen atmosphere. In the absence of malate, less than 0.1 //.moles of P32 are incorporated. In the presence of malate and P32, 11.6 ^moles of malate disappeared and 5.4 ^moles of P32 were esterifled in 20 minutes. In place of a P/O ratio, we may calculate a P32/malate ratio which, in this experiment, was 0.47. Since, for each mole of malate used, only one-half goes to succinate, the theoretical ratio would be 0.5. Therefore, the biochemical behavior of Asearis mitochondria is in direct accord with the proposed pathway. At this point, it might be wise to consider whether the anaerobic pathways discussed are peculiar or unique to Asearis. Apparently this is not the case. Although information is still incomplete, evidence is accumulating that more and more of the anaerobic or facultative anaerobic inverte- brates may obtain energy by similar mechanisms. Fairbairn et al., (1961) have shown that the rat tapeworm, Hymenolepis diminuta, forms succinate and requires CO2 for glycolysis to proceed. More recently, evidence has been presented indicating a pathway in H. diminuta similar to that of Asearis (Scheibel and Saz, 1966). A CO2 requirement has been demonstrated for a number of other helminths. Ward et al., (1968rt,fr) have demonstrated an almost complete lack of pyruvate kinase in larvae of the nematode Haemonchus contortus as well as the presence of PEP carboxykinase and a malic enzyme. Similarly, Prichard and Schofield (1968a,b) reported low pyruvate kinase and high PEP carboxykinase activities in the adult liver fluke, Fasciola hepatica. The findings of Wegener et al., (1969) that the American oyster during anaerobic incubation accumulates succinate, but no lactate, and possesses an active fumaric reductase are particularly intriguing. Considerable progress has been made in the area of the biochemistry of invertebrates over the past ten years. However, with the possible exception of the insects, comparatively little is known of this vast group of organisms. There is still much room for more progress in the years to come, particularly if one can envision the large gaps in our knowledge of invertebrate biochemistry, and can discard the concept that these organisms are necessarily biochemical miniatures of all other tissues. REFERENCES Berntzen, A. K. 1961. The in vitro cultivation of tapeworms. I. Growth of Hymenolepis diminuta (Cestoda:Cyclophyllidea). J. Parasitol. 47:351-355. Bueding, E. 1949. Metabolism of parasitic helminths. Physiol. Rev. 29:195-218. Bueding, E., and B. Charms. 1952. 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