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AMER. ZOOL., 31:522-534 (1991) Origins and Evolution of Pathways of Anaerobic Metabolism in the Animal Kingdom1 DAVID ROBERT LIVINGSTONE Plymouth Marine Laboratory, Prospect Place, West Hoe, Plymouth PL1 3DH, United Kingdom SYNOPSIS. Energetic characteristics and functional roles define two main types of anaerobic pathways in the animal kingdom: high efficiency/low rates of energy production pathways geared to anoxia survival (aspartate-succinate and glucose-succinate pathways), and low efficiency/high rates of energy production pathways geared to maintaining or increasing metabolic activity (multiple opine pathways and lactate pathway). The aspartate-succinate and opine pathways require both amino acids and carbohydrate as substrates, whereas the glucose-succinate and lactate pathways are dependent on carbohydrate only. Phylogenetic, functional and chemical considerations indicate an evolutionary progression from amino acid-linked to carbohydrate-based anaerobic pathways. The tauropine and strombine pathways are possibly the most ancient opine pathways so far discovered, and the octopine pathway the most advanced. The roles of the aspartate-succinate and opine pathways may originally have been not too dissimilar. A hierarchy of "rates of energy production pathways" of phosphagen > lactate > octopine > other opine pathways is proposed, which defines much of their phylogenetic selection and how they are used. The different properties of phosphocreatine compared to other phosphagens is indicated to have been a key factor in the emergence of vertebrates. INTRODUCTION In a previous paper (Livingstone, 1983), the evolution of anaerobic metabolism in animals was considered from the viewpoint of the functional nature of the pathways and the interaction of this with the physical and biological aspects of the environment. A review of anaerobic metabolism in the invertebrate and vertebrate phyla identified four pathway-types of interest: the lactate and opine pathways used for maintaining or increasing metabolic activity, and the glucose-succinate and aspartate-succinate pathways used for anoxia survival. Based on the phylogenetic distribution of these pathways, and other considerations, a hypothetical scheme of the evolution of anaerobic pathways was proposed which indicated early presences for amino acid-based, opine and aspartate-succinate pathways. A consideration of chemical, fossil and functional approaches to the reconstruction of the beginning of life suggested an early role for opine pathways in providing energy for the burrowing of infaunal worms of the Precambrian era. In this paper, I examine these and other proposals in the light of recent theories of biochemical evolution, and the substantial amount of information since published on the quantitative aspects and energetic characteristics of anaerobic metabolism, including the discovery of new pathways. Also considered are phosphagens, metabolic acidosis and constraints that operate on biochemical evolution. ANAEROBIC PATHWAYS AND PHOSPHAGENS Pathway types and characteristics The structural, functional and energetic characteristics of anaerobic pathways in animals have been extensively analyzed (see references below for details of pathways). Various stoichiometric/redox-balance combinations of oxidation (carbonyl to hydroxyl, aldehyde to carboxyl), reduction (carbonyl to hydroxyl, double bond saturation, reductive condensation) and energy providing (substrate level and electron transfer level phosphorylations) reactions result in the many different anaerobic pathways seen across the animal kingdom (Fields, 1988). Carbohydrate with its multiple hydroxyl groups for coupled oxidoreduction reactions is an ideal storage substrate, whereas the use of proteins and amino acids as sub1 From the Symposium on The Origin and Evolution strates is limited by the lack of hydroxyl and of Metabolic Pathways in Animals presented at the presence of carboxyl groups: fatty acids repAnnual Meeting of the American Society of Zoologists, resent the limit of carbohydrate fermenta27-30 December 1989, at Boston, Massachusetts. 522 EVOLUTION OF ANAEROBIC METABOLISM tion (i.e., maximum reduced state) and as an end-product have the advantage of passing most easily through lipid membranes (Fields, 1988). Branched pathways with multiple end-products (glucose-succinate and aspartate-succinate pathways) produce low rates of energy output at relatively high efficiencies (5 to 7 ATP molecules per glucose unit) for environmental anaerobiosis, e.g., aerial exposure, parasitism, whereas linear (lactate pathway) and semi-linear (opine pathways) pathways with single endproducts produce high rates of energy outputs at low efficiences (3 ATP molecules per glucose unit) for functional anaerobiosis, e.g., exercise, recovery from anoxia (Livingstone, 1982; Ellington, 1983; Gade, 1983a; De Zwaan and Putzer, 1985; De Zwaan and Van den Thillart, 1985). The aspartate-succinate and opine pathways have in common a requirement for both carbohydrate and amino acids as substrates, whereas the glucose-succinate and lactate pathways are dependent on carbohydrate only. The rate of substrate consumption is much greater for the low efficiency/high rates of energy production than for the high efficiency/low rates of energy production pathways. Anoxia survival is generally characterized by a marked decrease in total rates of energy production, whereas anaerobic muscular activity involves a marked increase. Switching from aerobic metabolism to low or high output modes of anaerobic energy production generally requires, respectively, a decrease and marked increase in glycolytic flux (De Zwaan and Van den Thillart, 1985). Thermodynamic considerations indicate that the different anaerobic pathways can be designed for economy (energetic efficiency) or power (high rates of energy production), but not for both (Gnaiger, 1983). The aspartate-succinate and glucose-succinate pathways are closely related pathways, often found coupled together in the same tissues (De Zwaan and Putzer, 1985). Although the functions of the pathways have been considered similar in both providing low rates of energy production during environmental anaerobiosis (Livingstone, 1983), differences are evident, indicative of the aspartate-succinate pathway being used in 523 situations, or tissues, where relatively higher rates of energy production are required. Where the two pathways occur together, the aspartate-succinate pathway functions during the early stages of environmental anaerobiosis, whereas the glucose-succinate pathway does not become operative until later. A regulatory role for aspartate, via its activation of pyruvate kinase (E.C. 2.7.1.40), has been proposed in anoxia tolerant molluscs (Storey, 1986): a depletion of aspartate, and inhibition of pyruvate kinase, would presumably favour carbon flow through the glucose-succinate pathway. Phosphagen is also used during the early stages and the total rates of energy production, although reduced from aerobic rates, are greater than in the later stages, e.g., the total rates in fimol ATP min"1 g~' wet weight for four species of marine bivalve (Mytilus edulis, Cardium edule, Geukensia demissa granosissima and Lima hians) were 0.021 to 0.121 in the first four hours of anaerobiosis compared to 0.005 to 0.021 after ten hours (De Zwaan and Putzer, 1985). The glucose-succinate pathway is found in most, if not all, tissue types, whereas the aspartatesuccinate pathway is typical of active tissues, such as adductor muscle and heart tissue of bivalves, which are characterized by high aspartate pools (De Zwaan and Putzer, 1985). The aspartate-succinate pathway in the posterior adductor muscle of the common mussel M. edulis is used during hypoxia when the response of the whole animal is to try and maintain metabolic rates (Bayne and Livingstone, 1977; Livingstone and Bayne, 1977). During the early stages of environmental anaerobiosis, in various marine invertebrates, the formation of opines and lactate is minimal and the aspartate-succinate pathway is argued to outcompete the glycolytic end-product pathways by virtue of its greater affinity for NADH (De Zwaan and Putzer, 1985). The utilization of aspartate usually exceeds the production of succinate through its oxidative decarboxylation to alanine via malic enzyme (E.C. 1.1.1.40) and transaminase reactions, and this pathway has been proposed to functionally replace the well-known aspartatemalate shuttle in rapidly transferring reducing equivalents into the mitochondria 524 DAVID ROBERT LIVINGSTONE during environmental anaerobiosis (De Zwaan and Putzer, 1985; De Zwaan and Van den Thillart, 1985). All of these features are indicative of a pathway suited for shorter rather than longer term anaerobiosis, when reduction of metabolic rate is less than later, and the intrinsic scope for ATP power output of the aspartate-succinate pathway has been proposed to be greater than that of the glucose-succinate pathway (De Zwaan and Putzer, 1985). The use of the aspartate-succinate pathway is presumably limited by the size of the free aspartate pool, i.e., equals low capacity relative to glycogen pool (De Zwaan and Putzer, 1985). A functionally equivalent pathway to the aspartate-succinate pathway in freshwater and terrestrial invertebrates, lacking substantial free amino acid pools, is thought to be the malate-succinate pathway, e.g., malate levels are high in the midge Chaoborus crystallinus and earthworm Lumbriculus variegatus and are converted to succinate during environmental anaerobiosis (De Zwaan and Putzer, 1985; De Zwaan and Van den Thillart, 1985). The lactate pathway is primarily a high rate of energy production pathway for functional anaerobiosis whose usefulness for anoxia tolerance is very limited. Some longterm survival is seen with various vertebrate species which possess a lactate pathway only, but this is usually associated with hypoxic rather than anoxic conditions, and with a significant depression of metabolic rate (De Zwaan and Van den Thillart, 1985). Lactate is formed as a major end-product during environmental anaerobiosis in nonmarine invertebrates such as gastropods and parasitic platyhelminths and nematodes, but often, although not always, in conjunction with the simultaneous operation of the glucose-succinate pathway (Livingstone, 1982, 1983). Some lactate is also formed during recovery from anoxic conditions, e.g., in various crustaceans (De Zwaan and Putzer, 1985). L-lactate is formed by vertebrates and higher invertebrates such as crustaceans and echinoderms, e.g., the crab Potamon warreni (Van Aardt and Wolmarans, 1987) and the sea urchin Echinus esculentus (Spicer et al., 1988), whereas D-lactate is typical of molluscs, e.g., the ormer Haliotis lamel- losa (Gade, 1988) and certain other groups such as arachnids, e.g., the spider Filistata hibernalis (Prestwich, 1988a, b). The importance of the lactate pathway in providing high rates of energy production for muscular activity and locomotion, and for other forms of maintained or increased metabolic activity, is reflected in the direct scaling of lactate dehydrogenase (LDH; E.C. 1.1.1.27 and 1.1.1.28) activities with body mass in fish (Somero and Childress, 1980) and other vertebrates (Hochachka et al., 1988), and possibly with its apparent selection in the active and evolutionarily advanced nereid polychaetes and the group of bivalves (Cardium sp. and Anodonta cygnea) whose metabolic rate is not drastically reduced during aerial exposure (Livingstone, 1982; Livingstone et al., 1983). The opine pathways are functionally analogous to the lactate pathway, at least in energetic terms, but the maximal rates of energy production that might be realised from these pathways are argued to be less than the lactate pathway (Livingstone, 1983). The terminal reaction of the opine pathway involves the reductive condensation of pyruvate with an amino acid to form an imino acid derivative (opine), viz.: pyruvate + amino acid + NADH + H + = opine + H2O + NAD + The major opine pathways so far characterized involve reductive condensation of pyruvate with arginine, alanine or glycine to produce, respectively, octopine, alanopine and strombine. The substrate specificities of the enzymes catalyzing the terminal reactions vary, but they are generally known, respectively, as octopine dehydrogenase (ODH; E.C. 1.5.1.11), alanopine and strombine dehydrogenases (ADH and SDH). The characteristics of the opine pathways have been extensively studied and the enzyme activities are highest in muscular tissues (Livingstone, 1982; Ellington, 1983; Gade, 1983a; De Zwaan and Dando, 1984; De Zwaan and Putzer, 1985; Gade and Grieshaber, 1986). More recently, additional opine pathways have been discovered, viz. tauropine and /3-alanopine formed, respectively, from taurine and /3-alanine through the actions of tauropine and /3-alanopine EVOLUTION OF ANAEROBIC METABOLISM dehydrogenases (TDH and BDH) (Gade, 1986, 1988; Sato et al, 1987; Doumen and Ellington, 1987), and there is the possibility that others may exist, utilizing other free amino acids. Opine pathways are predominantly employed during exercise, e.g., tauropine in the shell adductor muscle of H. lamellosa (Gade, 1988), and recovery from exercise or anoxia, e.g., octopine in the phasic adductor muscle of the scallop Placopecten magellanicus (Livingstone, 1982). The formation of strombine in the posterior adductor muscle of M. edulis is related to valve opening and closing (De Zwaan and Dando, 1984; Shick et al, 1986) and was significantly enhanced when movement of the shells was prevented (De Zwaan et al, 1983). Multiple opines can be formed and different opines can be formed under different conditions, e.g., alanopine and strombine were formed in the body wall musculature of the lugworm Arenicola marina, the former predominating during exercise (ratio of 6:1) and the latter during anoxia (ratio of 1:3.5) (Siegmund et al, 1985). Minor amounts of opines are formed during environmental anaerobiosis, e.g., octopine and alanopine/strombine in the posterior adductor muscle of the bivalve Scapharca inaequivalvis (Isani et al, 1989), and (in decreasing proportions) /3-alanopine, strombine, tauropine and alanopine in adductor muscle of the blood shell Scapharca broughtonii (Sato et al, 1988). Various energetic functions have been proposed for opine formation during recovery from environmental or functional anaerobiosis, including physical (valve) movements, maintenance of metabolic rates (Livingstone, 1982), and supplementing normal (or elevated) aerobic ATP yielding processes involved in recharging of phosphagen and ATP pools, resynthesis of aspartate and glycogen, clearance of acidic end-products and restoration of normal physiological activities such as feeding (De Zwaan and Putzer, 1985; Shick et al, 1988). Based on enzyme kinetics and other observations, it has been argued that the octopine pathway is likely to realise higher rates of energy production than either the strombine or alanopine pathways (Livingstone et al, 1983). 525 Other anaerobic pathways exist in addition to these four major types. Ethanol formation is used as a means to limit metabolic acidosis in certain anoxia tolerant fish species, e.g., goldfish Carassius auratus (De Zwaan and Van den Thillart, 1985). Discrepancies between theoretical anoxic energy output (calculated from accumulated fermentation products) and that measured directly by calorimetry has suggested the existence of unidentified anaerobic pathways, e.g., accumulated end-products in whole M. edulis (Shick et al, 1988) and isolated ovaries of the sea urchin Strongylocentrotus droebachiensis (Bookbinder and Shick, 1986) accounted for only, respectively, 50% and 37% of total anoxic heat dissipation. However, similar "exothermic" gaps are also seen for vertebrate species (see Shick et al, 1988) and it may be they reflect phenomena other than endproduct accumulation. Phosphagens and a hierarchy of rates of energy production pathways A second important source of anaerobic energy are the various phosphoguanidine compounds, or phosphagens, which are transphosphorylated by specific phosphokinases to yield ATP according to the reaction: phosphoguanidine + MgADP + H + = guanidine + MgATP Phosphagens are used during both muscular activity and anoxia survival, e.g., for a number of species of mollusc, total rates of anaerobic energy production (mean ± SEM in jtmol min~' g~' wet wt.; n = 5 to 12) during exercise and short-term anaerobiosis were, respectively, 6.4 ± 1.7 and 0.044 ± 0.007, of which, respectively, 55.4 ± 5.2% and 50.0 ± 10.6% were provided by the phosphagen (calculated from Table 1 of De Zwaan and Van den Thillart, 1985). Maximum rates of energy production from phosphagens generally exceed those of the lactate or opine pathways (Table 1), and, possibly for this reason, phosphagen utilization during exercise often precedes, to varying degrees, the accumulation of glycolytic end-products. This tendency is observed across the animal kingdom, e.g., 526 DAVID ROBERT LIVINGSTONE TABLE 1. Maximum observed rates of energy production (in y.mol min~' g-' wet weight) during exercise (or recovery from anaerobiosis*) from phosphagen and lactate or opine anaerobic pathways in vertebrates and invertebrates. Organism Phosphagen breakdown Man Other mammals Reptile Amphibian Fish Arachnid Crustacean Annelid Mollusc 96-360 16.9 — 37.1 12.0 41.3 ± 10 (3) 20.0 ± 2.5 (3) 0.55 ± 0.5 (2) 3.6 ± 0 . 8 (21) Anaerobic pathway Lactate Lactate Lactate Lactate Lactate Lactate Lactate Lactate Octopine Octopine* Strombine/alanopine Strombine/alanopine* Strombine* Alanopine* Tauropine Anaerobic energy References 60-210 5.1 ± 3 (3) 16.1 ± 4 (3) 6.9 ±2.1 (4) 8.4 ± 3.4(8) 9.9 ± 3.1 (3) 2.9 ± 1.6(3) 0.8 ± 0.3 (5) 1.4 ±0.5 (8) 0.39 ± 0.2 (3) 0.28 ±0.1 (4) 0.12 0.03 ± 0 (3) 0.03 0.46 1,2 3 3,4 3,5 3,6 7,8 9 9, 10 3, 9, 11, 12 12 11 11 13, 14 13 15 Data presented are comprehensive for marine invertebrates, but illustrative for other animal groups. Maximum rates were calculated for each species and results presented as single values, means ± range (n = 2) or ±SEM (number of species given in parentheses). Rates are generally for muscular tissues although some whole animal data were used. For these and other details see references, viz., 1: Hochachka (1985); 2: Livingstone (1982); 3: De Zwaan and Van den Thillart (1985); 4: Gleeson and Dalessio (1989); 5: Miller and Sabol (1989); 6: Dalla Via et al. (1989); 7: Prestwich (1988a); 8: Prestwich (19886); 9: De Zwaan and Putzer (1985); 10: Siegmund et al. (1985); 11: Baldwin and England (1982); 12: Livingstone (1982); 13: Eberlee et al. (1983); 14: De Zwaan et al. (1983); 15: Gade (1988). in spiders (Prestwich, 19886), molluscs, crustaceans and fish (De Zwaan and Van den Thillart, 1985). Phosphagen concentrations are highest in muscular tissues and many observations testify to their importance in high energy demanding activities, e.g., exhaustion in scallops, decreased sprinting speeds in spiders and reduced escape responses in gastropods coincide with phosphagen depletion (De Zwaan and Van den Thillart, 1985; Prestwich, 19886). Phosphagen is the main energy source for contraction of isolated anterior byssus retractor muscle of M. edulis, with octopine formation being invoked with increasing energy demand (Zange et al., 1989). A hierarchy of rates of energy production pathways of lactate > octopine > alanopine and strombine has been proposed based on observed maximum rates of energy production, pathway design and enzyme kinetics (Livingstone, 1982, 1983; Livingstone # al., 1983). To the top of this list, with the greatest intrinsic potential for high rates of energy production, can be added the phosphagens (Table 1). Although many factors will influence rates of energy production, the comparisons between the pathways hold within the different animal groups, and, to some extent, it could be argued between them, e.g., the lactate pathway in crustaceans and arachnids compared to the octopine pathway in molluscs. Differences are also seen between the phosphagens, the relative equilibrium constants for phosphagen formation being lower for phosphoarginine, phosphoglycocyamine, phosphotaurocyamine and phospholombricine than for phosphocreatine, i.e., the latter is thermodynamically less stable (Ellington, 1989). Information on the newly discovered ^-alanopine and tauropine pathways is limited, but observed rates of energy production (Table 1) and the high apparent K,,, values for amino acid substrate of the dehydrogenases (Gade, 1986; Doumen and Ellington, 1987) indicate their energetic potential is similar to that of the strombine and alanopine pathways. At the bottom of the rates of energy production pathways list can be placed the glucose-succinate pathway designed for energetic efficiency and anoxia survival, e.g., 0.008 to 0.13 ^mol min"1 g~' wet weight for muscular tissue of various EVOLUTION OF ANAEROBIC METABOLISM bivalve and gastropod species (Livingstone, 1982). 527 genetic distribution of the lactate and glucose-succinate pathways accords with the anoxia tolerances of the organisms. The Phylogenetic distribution marked presence of the lactate pathway and The distributions of phosphagens and the nature of the phosphagens in the Choranaerobic pathways across the animal king- data and Echinodermata are consistent with dom are well characterized, including to a their phylogenetic relationship in the Deureasonable degree the opine pathways (Liv- terostomia. A consistency is also seen in the ingstone, 1982; Gade and Grieshaber, 1986; phylogenetic relationships between the De Zwaan and Putzer, 1985; Ellington, Annelida and the Arthropoda, and the 1989). Phosphocreatine and phosphoargi- Lophophorata (Brachiopoda) and the Deunine occur predominantly in, respectively, terostomia, in that the former group of each vertebrates and invertebrates. The excep- pair contain both opine and lactate pathtions are the Echinodermata, which contain ways, whereas the latter groups have lost both (the primitive crinoids contain argi- the opine pathways (Livingstone et ai, nine kinase [E.C. 2.7.3.3] only), and the 1983). Annelida which contain four other major The distribution of individual opine phosphagens in addition to these two, i.e., pathways has been studied in terms of both phosphoglycocyamine, phosphotaurocy- in vivo end-product formation and, to a amine, phosphohypotaurocyamine and much greater extent, the presence of specific phospholombricine. The flagellated, highly dehydrogenase activities. The correlation motile sperm of echinoderms and poly- between the two is by no means absolute chaetes contain creatine kinase (E.C. 2.7.3.2) but nevertheless reasonable, e.g., tauropine only, whereas the eggs of echinoderms and formation in H. lamellosa (Gade, 1988) and tissues of polychaetes contain arginine /8-alanopine formation in S. broughtonii kinase or other phosphokinases. The sub- (Sato et ai, 1987,1988) corresponded with, units of the different kinases often hybridize respectively, high TDH and BDH activities. together, e.g., sea cucumber Caudina are- The octopine pathway is absent from the nicola arginine kinase with rabbit brain cre- Polychaeta, characteristic of the Mollusca, atine kinase (Seals and Grossman, 1988), and present in other marine invertebrate and the different kinases are thought to have phyla. Octopine formation has been originated from an ancestral arginine kinase observed in some 15 species of bivalve, gasgene. tropod and cephalopod mollusc, Sipunculus The lactate pathway is present to some nudus (Sipuncula) and Cerebratulus lacteus degree in all phyla, but is the sole major (Nemertina) (Gade, 1983&; De Zwaan and anaerobic pathway of the higher or evolu- Putzer, 1985; Isani et ai, 1989). Alanopine tionarily most advanced ones (Chordata, or strombine formation has been observed Echinodermata, Arthropoda). In contrast, in S. nudus, A. marina (Annelida) and 15 the opine pathways are essentially found in species of bivalves and gastropods, but not marine species of the lower (Porifera, Cni- in cephalopods (Korycan and Storey, 1983; daria, Nemertina) and middle (Mollusca, De Zwaan and Putzer, 1985; Isani et ai, Annelida, Brachiopoda, Sipuncula) phyla. 1989); tauropine and /3-alanopine formaThe glucose-succinate pathway is charac- tion occurs in S. broughtonii and tauropine teristic of organisms regularly experiencing in the archaeogastropod H. lamellosa (see anaerobiosis (most lower and middle phyla, before). Consistencies in the phylogenetic including the parasitic Nematoda and distribution of opine dehydrogenase activPlatyhelminthes), and is also found in insect ities are observed down to the level of phyla, larvae. The aspartate-succinate pathway class, order and even family (Table 2). SDH occurs in molluscs and polychaetes, and is characteristic of the Porifera, and ODH minor presences of the succinate pathways and ADH are high in the Cnidaria. ODH is are found in crustaceans and vertebrates (De the sole major dehydrogenase activity of Zwaan and Van den Thillart, 1985; Van some of the more active molluscs, i.e., Aardt and Wolmarans, 1987). The phylo- Cephalopoda, Scaphopoda and swimming, 528 DAVID ROBERT LIVINGSTONE TABLE 2. Relative pyruvate oxidoreductase activities in muscular or whole tissues of various marine invertebrate groups. Phyla, class, order or family Porifera Cnidaria Nemertina Brachiopoda Annelida Polychaeta Mollusca Polyplacophora Gastropoda Archaeogastropoda Mesogastropoda Neogastropoda Bivalvia Myidae Ostreidae Veneridae Mytilidae Tellinidae Cardiidae Pectinidae Scaphopoda Cephalopoda Crustacea Echinodermata Dehydrogenases Number of species Lactate Octopine Alanopine Strombine 6 10 3 3 0.41 (0.20) 0.23(0.10) 0.44 (0.29) 0.02 (0.02) C). 17 (0.17) C).70(0.15) C).67 (0.33) 0.03 (0.03) 0 (0) 0.52(0.12) 0.25(0.10) 0.83(0.13) 0.57 (0.20) 0.29(0.11) 0.09 (0.06) 0.71 (0.29) 15 0.31(0.11) 0.07(0.07) 0.54(0.11) 0.38(0.12) 4 29 9 10 10 50 2 5 10 5 3 2 3 1 10 11 8 1.0 (0) 0.40 (0.08) 0.47(0.12) 0.53(0.14) 0.07 (0.02) 0.21(0.05) 1.0 (0) 0.03 (0.02) 0.08 (0.02) 0.18(0.13) 0.07 (0.03) 0.34 (0.33) 0 (0) 0.28 0.05 (0.02) 1.0 (0) 1.0 (0) 0.04(0.02) ().33 (0.08) 0.11(0.07) (). 15 (0.10) ().67(0.13) 0.59(0.06) () (0) C) (0) 0.44 (0.09) C).76(0.17) .0 (0) 0.01 (0.01) 0.54 (0.08) 0.46(0.16) 0.53(0.11) 0.63(0.12) 0.43 (0.06) 0 (0) 0.90 (0.03) 0.86 (0.06) 0.42(0.15) 0.14(0.03) 0.12(0.06) 0.03 (0) 0.01 0 (0) 0 (0) 0 (0) 0.02 (0.02) 0.15(0.04) 0.12(0.08) 0.24 (0.08) 0.15(0.04) 0.46 (0.06) 0 (0) 0.97 (0.02) 0.96 (0.02) 0.40(0.11) 0.12(0.03) 0.19(0.05) 0.03(0.01) 0 0 (0) 0.06 (0.05) 0.03 (0.02) •0 (0) .0 (0) .0 .0 (0) () (0) ().07 (0.06) For each species the highest dehydrogenase specific activity (in activity g~' wet weight) was given a value of 1 and the others calculated relative to this. Means and SEM were than calculated for each dehydrogenase activity for each particular group of animals. SEM given in parentheses. Original data taken from Baldwin (1982), Baldwin and England (1982), Eberlee et al. (1983), Kluytmans et al. (1983), Korycan and Storey (1983), Livingstone et al. (1983), Bowen (1987), Sato et al. (1987) and D. R. Livingstone, W. B. Stickle, M. Kapper, S. Wang and W. Zuburg (unpublished). Activities were mainly for muscular tissues, although in some cases whole animals were used. burrowing and jumping bivalves (Pectinidae, Solenidae, Cardiidae and Tellinidae). ODH is prominent in advanced Neogastropoda, whereas ADH features in this gastropod order, the Mesogastropoda and the more primitive Archaeogastropoda. Specific activities of ADH (not shown) are much lower in Archaeogastropoda than in the other two gastropod orders (Livingstone et al., 1983). Much less is known of the distribution of the recently discovered TDH and BDH activities: BDH is prominent in S. broughtonii, whereas TDH appears characteristic of Archaeogastropoda and the ancient Brachiopoda (TDH activity in pedicle of Glottidea pyramidata was about x 2 that of ADH) (Doumen and Ellington, 1987; Sato et al., 1987; Gade, 1988; Hammen, 1989). TDH or BDH could possibly be pres- ent in certain marine groups lacking ADH, SDH and ODH, accounting for the apparent absence of opine dehydrogenase activities, viz., in the primitive Polyplacophora and possibly in the Myidae and Nudibranchia (Table 2). EVOLUTION OF ANAEROBIC PATHWAYS AND PHOSPHAGENS The earliest environmental conditions and organisms were anaerobic, the transition to various degrees of oxygen-based existence occurring between some 2,700 and 1,600 million years ago with the appearance of, respectively, the photosynthetic blue-green algae and the first aerobes: key biochemical pathways are proposed to have arisen in this order—rudiments of glycolysis, electron transport chain, polyphosphate phospha- EVOLUTION OF ANAEROBIC METABOLISM gens, arginine phosphate and creatine phosphate (Fox, 1988). The first discernible invertebrates (Porifera) occurred some 700 million years ago, followed by the appearance of most major invertebrate phyla around 700 to 570 million years ago (Precambrian/Cambrian border). Oxygen was a key factor in the development of metazoan life through its requirement for hydroxyproline, hydroxylysine and resultant collagen synthesis (Towe, 1981). Anaerobic pathways An hypothetical scheme for the evolution of anaerobic pathways is given in Figure 1. An early existence for rudiments of the glycolytic pathway is indicated (Fox, 1988), and it is interesting to speculate whether the glucose-succinate pathway is a modification of the later evolved Krebs cycle, or whether the reverse occurred and the rudimentary reactions of the anaerobic pathways that gave rise to the glucose-succinate pathway also gave rise to the Krebs cycle. A number of observations support the proposal for the early appearance of amino acid based pathways. Amino acids were prominent components of the primaeval soup, and the relative abundance of the four major ones present in various cosmic environments (moon, meteorites, hot terrestrial lava) and formed in laboratory simulations (reducing atmosphere and electric discharge, heating) is exactly the same, viz., in order of decreasing abundance, glycine, alanine, glutamic acid and aspartic acid (Fox, 1988). The next two to five most abundant amino acids vary for the different sources and include proline (required for hydroxyproline synthesis), but not arginine (required for phosphoarginine and octopine synthesis). Glycine, alanine and aspartate are utilized, respectively, by the strombine, alanopine and aspartate-succinate pathways: /3-alanine is an end-product of pyrimidine metabolism and can also be derived from the decarboxylation of aspartate. Protein, not carbohydrate, is indicated to be the major substrate for anoxic energy metabolism in lower organisms, such as coelenterates (Shick et ah, 1988), and in the juvenile forms of others, such as larvae of the oyster Crassostrea virginica (Widdows et al, 1989). The major anaerobic end- 529 products of coelenterates are alanine and glutamate, e.g., in the sea anemone Actinia equina (Navarro and Ortega, 1984). The use of the aspartate-succinate pathway is thought to have a sparing effect on what carbohydrate stores there may be (De Zwaan and Putzer, 1985), and ammonia production during anaerobiosis can be used for acidbase balance (Shick et al, 1988). A phylogenetic and functional line of consistency therefore appears to exist for an early prominence for amino acids in anaerobic metabolism. The divergence of the amino acid pathways into the aspartate-succinate and opine pathways resulted in the appearance and subsequent evolution of the two main types of anaerobic pathway geared to, respectively, anoxia survival and maintaining or increasing metabolic activity. The functional distinction between the two types may initially not have been great, given the energetic features of aspartate-succinate pathway and the fact that both types of pathways would be similarly limited by the size of their free amino acid pools (also see below). However, with selection for particular features such as ATP yield and decreased or increased glycolytic flux, their roles would have become better denned. Correlations between the size of free amino acid pools and the presence of opine pathways are seen in modern day species, e.g., /3-alanine in S. broughtonii (Sato et al., 1987), and presumably this must have been a factor in their selection. Multiple opine pathways increase the capacity of the system, i.e., the size of the amino acid pool is effectively the sum of components, and have been argued to guarantee continuous glycolytic flux during conditions of exercise or anoxia/hypoxia followed by recovery (Gade and Grieshaber, 1986). Strombine and tauropine may be the oldest opine pathways, a major presence being indicated in, respectively, the primitive Porifera and the ancient Brachiopoda (the status of the tauropine pathway in the Porifera and the other lower phyla is unknown). Glycine and taurine are often the largest free amino acid pools found in species, and this could have given the opine pathways an early advantage over the aspartate-succinate pathway in functional an- 530 DAVID ROBERT LIVINGSTONE aerobiosis, or even allowed them to have a role in anoxia survival, i.e., generating a low rate of energy production over a long period of time. The selection of the octopine pathway in active molluscs, including the evolutionarily advanced cephalopods and neogastropods, may have been linked to its capability for higher rates of energy production and/or its relationship with the molluscan phosphagen, phosphoarginine. Phosphoargine breakdown provides arginine for octopine formation, so freeing it from the constraints of any interaction with other aspects of amino acid metabolism, although integration between the two processes in modern day molluscs is generally minimal (De Zwaan and Putzer, 1985). The octopine pathway is usually found associated with high phosphoarginine pools, and is the only opine pathway found in freshwater invertebrates (bivalves) (De Zwaan and Putzer, 1985). A novel origin for the octopine pathway in molluscs from bacterial transfection has been suggested (Hochachka, 1988), but if this is so, then, given the wide phylogenetic distribution of the pathway (Nemertina, Sipuncula) and/or ODH activity (Table 2), it must have been a general phenomenon and occurred early in the evolution of the Animalia. The advantages of carbohydrate over protein and amino acids as an anaerobic substrate would have been a major factor in the selection of the glucose-succinate and lactate pathways. Movement to freshwater and terrestrial existences would also have contributed to the loss of the amino acidlinked pathways from the lower and middle phyla. The possible relationships between the aspartate-succinate and glucose-succinate pathways have been discussed before (Livingstone, 1983). The lactate pathway could have originated independently of the opine pathways, or possibly from them. An evolutionary relationship (progression) between monomeric opine dehydrogenases, dimeric D-LDH and tetrameric L-LDH has been speculated at (Livingstone et ai, 1983) (N.B., a monomeric LDH, is present in primitive fish but a tetrameric LDH is found in ascidians—Baldwin, 1988). The functions of the pathways would have become HIGH EFFICIENCY / LOW RATES LOW EFFICIENCY / HIGH RATES OF ENERGY PRODUCTION OF ENERGY PRODUCTION PATHWAYS PATHWAYS FIG. 1. Hypothetical scheme of the evolution of the pathways of anaerobic metabolism (modified from Livingstone, 1983). increasingly distinct by selection for their key features, viz. fuel stores, ATP yield or rates on energy production, levels and regulation of glycolytic enzymes, and mechanisms for dealing with metabolic acidosis, such as excretable (volatile) end-products and others (see below) (De Zwaan and Putzer, 1985; Hochachka, 1985; Hochachka et ai, 1988). For example, the magnitude of the Pasteur effect (increased glycolytic flux during functional anaerobiosis) increases through the Mollusca, Crustacea and Vertebrata (De Zwaan and Putzer, 1985; De Zwaan and Van den Thillart, 1985), e.g., x 50 for swimming bivalves compared to x 2,300 for man (Livingstone, 1983). Similarly, total rates of ATP turnover during anoxia are reduced about x 75 in molluscs compared to xlO in crustaceans, so contributing to the prolonged survival of the former (De Zwaan and Putzer, 1985). In addition, selection for other features would have occurred, such as enzyme polymorphism for organ specialization, e.g., see Gade and Grieshaber (1986) and metabolic efficiency, e.g., allozyme heterozygosity in P. magellanicus is correlated with increased octopine formation and may be related to the scallop's scope for movement (Volckaert and Zouros, 1989). Phosphagens The appearance of phosphagens allowed the storage of energy for subsequent use during periods of high energy need. The change EVOLUTION OF ANAEROBIC METABOLISM 531 from phosphoarginine to phosphocreatine for the prolonged burrowing of infaunal is argued to be a central factor in the appear- worms of the Precambrian era (Livingstone, ance of chordates, providing energy not only 1983). A flagellate ancestor is generally for increased motility during, for example, assumed for the metazoans, but the status embryological development, but also driv- of the proposed ancestoral acoelomate (see ing the transition from rudimentary to higher Livingstone, 1983) {i.e., primitive or seclevel nervous activity (Fox, 1988). The spe- ondarily derived from a coelomate condicific presence of phosphocreatine in highly tion) is a matter of debate (Barnes, 1983). motile polychaete and echinoderm sperm is Phosphagens would have been present for presumably testimony to its energetic nervous activity and no doubt also been advantages. A mechanistic basis for this important in burrowing. The aspartate-sucadvantage is provided by the lower ther- cinate pathway could have made a contrimodynamic stability of phosphocreatine, bution to functional anaerobiosis, but in the allowing the maintenance of higher ATP/ longer term the opine pathways would have ADP ratios, necessary for flagellar protein predominated. Movement would have movement of sperm and rapid contraction/ intensified animal-environmental and anirelaxation of vertebrate muscle, i.e., the mal-animal interactions (Fox, 1988), and cycles of contraction are promoted by the allowed the invasion of new aerobic and rapid dissociation of ADP away from spe- anaerobic habitats. Different anaerobic cific proteins (Ellington, 1989). A selective strategies could have been employed at difadvantage is also conveyed on the other ferent stages of an animal's lifecycle, e.g., phosphagens by virtue of their greater ther- anaerobic energy output decreases and modynamic stability, making them better anoxia tolerance increases of Crassostrea buffers of ATP levels under the conditions virginica larvae with developmental stage of reduced pH that occur during long-term and size, indicating a switch from functional anoxia survival (Ellington, 1989). The phe- to environmental-type anaerobic pathways nomenon of pluriphosphagens (more than (Widdows et ai, 1989). The combined, or one phosphagen in one tissue) could be due complementary, use of aerobic and anaerto functional compartmentalization, or obic pathways to create new niches would involve their sequential use during energy be accompanied by an increase in their integrated control (Simon et ai, 1978). Other deficit (Ellington, 1989). aspects of the evolutionary interaction and The phosphagens and anaerobic path- biochemistry and biology have been disways are used together, or sequentially, to cussed elsewhere (Livingstone, 1983; Livmeet the particular energy demands of the ingstone et ai, 1983). organism. The scope for energy output is generally increased up the phylogenetic tree, e.g., total energy output during functional Constraints on the evolution of anaerobiosis in various species of mollusc anaerobic pathways and crustacean was raised, respectively, The speed and direction of evolutionary about x20 and x60 (De Zwaan and Van change are continually influenced by selecden Thillart, 1985). Phosphagens can pro- tive constraints operating at various levels vide the energy crucial for nerve and other of biological organization: the past history excitable tissue function, whereas anaerobic of evolutionary change may act to constrain pathways are effective in short- and longer- further adaptive modifications in a manner term muscular activity. that may differ between taxonomic groups simply because of the different evolutionary Biological considerations trajectories followed (see Pogson, 1988). Advances in energy metabolism, regula- Such "local" constraints have been invoked tion and storage have been hypothesized to to explain the presence of the octopine pathhave been the impetus to phylogenetic way (as opposed to the lactate pathway) in metamorphosis (Fox, 1988). A prominent the fast-moving Cephalopoda, and the lacrole was assigned to the opine pathways in tate pathway in the slow-moving Echinothe rise of metazoans in providing energy dermata (Livingstone, 1983), although in the 532 DAVID ROBERT LIVINGSTONE latter case other considerations are possible (Livingstone et al, 1983). The absence of the glucose-succinate pathway in the higher phyla is of interest (Fields, 1988), and could be due to such constraints, or to the fact that evolution works to produce energetically efficient but also different animals, i.e., with physiologies capable of filling/creating unique niches. Acid production is a potential threat to most cellular activities, and the evolution of anaerobic pathways was probably closely linked to the development of mechanisms/ strategies for combating this (Livingstone, 1983). 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