Some Responses of the Electric Ray (Torpedo Marmorata) to Low Ambient Oxygen Tensions

There is considerable variation in the extent to which different vertebrate species are adapted to survive prolonged hypoxic exposure. This ability is particularly well developed in diving species, such as turtles, seals and dolphins. For example, whales may remain submerged for up to 2 h during which time they are capable of diving to depths in excess of 1500 m (Andersen, 1966).

Among fish even more impressive anaerobic capacities are found not only among those which come out of water and show some analogous responses to behaviour of diving mammals but also aquatic species in their normal habitat. Blazka (1958) was probably the first to report that European carp survived for several months at near zero oxygen tensions in ice-locked lakes during the winter months. Coultner (1967) obtained evidence that several benthic species in Lake Tanganyika lived below the thermocline under totally anoxic conditions. There are also records of very low oxygen tensions in deep marine waters which are known to contain fish (Sverdrup, Johnson & Fleming, 1942; Macdonald, 1972).

Metabolic studies of adaptation to hypoxia in vertebrates are only just beginning to appear in the literature. For example, recent studies involving diving mammals (Hochachka, 1975; Hochachka et al. 1975; Owen & Hochachka, 1974) have provided evidence that anaerobic glycolysis is not the only energy-yielding pathway available to these species during anaerobic periods. In addition to lactate and pyruvate, succinate and alanine were observed to accumulate as anaerobic end products in the blood of these species following diving (Hochachka et al. 1975).

In contrast to vertebrates, the occurrence of multiple anaerobic end products including pyruvate, lactate, octopine, alanine, succinate, propionate, acetate and carbon dioxide in a variety of invertebrates with impressive anaerobic capabilities is well established (Mangum & van Winkle 1973; Hochachka, 1975; De Zwaan & Wijsman 1976). In these animals the Krebs cycle remains totally or partially functional under anaerobic conditions since fumarate or other specific electron acceptors are able to replace the function of molecular oxygen. These anaerobic pathways, although energetically less efficient than aerobiosis, nevertheless allow the cell to maintain redox balance under anoxic conditions and no doubt have contributed to the ecological success of these organisms in exploiting anaerobic environments.

The present study was undertaken following a series of investigations on the effect of hypoxia on the respiratory and cardiovascular mechanisms of the electric ray (Torpedo marmorata). It had been found that this species is able to withstand prolonged periods of very low oxygen and recover. At certain levels of hypoxia the ventilatory movements completely cease and during the initial investigation (Hughes, 1973) indications were found of relatively little bradycardia during the lowering of environmental oxygen. Cannulation techniques enable samples of post-branchial blood to be taken and were ideal for investigating changes in blood chemistry during hypoxia. The use of these indwelling cannulae allows the simultaneous monitoring of blood oxygen, pH, and anaerobic end products continuously for periods of up to 24 h. The small sample size and large blood volume make such fish suitable for such studies. A preliminary report of this work has been given (Hughes & Johnston, 1977).

Specimens of Torpedo marmorata were obtained from the Gulf of Gascony and kept in the sea water circulation of the Institute de Biologie Marine, Arcachon, France. The methods of cannulation and preparation for the experiment are described elsewhere (Hughes, 1978). The experimental set-up allowed the maintenance of a continuous flow of water over the fish, the oxygen content of which could be lowered by bubbling air and nitrogen at known flow rates through a gas exchange column. Fish were contained in a respirometer especially constructed for the study of rays. During experiments on Torpedo particular care was taken to avoid over-exciting the animal because of the danger of it producing electric discharges. The fish was usually placed in the respirometer circulation for at least 12 h before an experiment began and it was found that they could be maintained in the circulation for several days without any apparent upset. The fish remain surprisingly quiescent even during periods of extreme hypoxia and for this reason these fish are especially suitable for such experiments. Blood samples were taken from the cannulae into heparinized syringes and transferred to a Radiometer pH and electrode assembly.

Approximately 0·6 ml samples of fresh blood were transferred to polyethylene tubes and immersed in liquid nitrogen (– 170°C). Batches of samples were deproteinized by addition of 2 vols of 0·6 N-HCIO₄. Following centrifugation, aliquots of the clear supernatant were neutralized with KOH and stored in 15 mm Tris-HCl pH 7·1 at – 40°C until subsequent analysis.

A comparison was made between the steady-state concentrations of metabolites in freeze-clamped red muscle from normoxic fish and from fish subjected to an ambient oxygen level of less than 5 mm Hg for 2·5 h. Fish were killed by a blow to the head and muscle samples were immediately excised from the tail and dropped into liquid nitrogen (−170°C). Samples were stored in liquid nitrogen until subsequent processing. All muscle metabolites were assayed from a single 1−2 g sample of red muscle. Weighed samples of muscle were pulverized to a fine powder in a stainless steel pestle and mortar also cooled in liquid nitrogen. Metabolites were extracted in 0·6 N-HCIO4 for 3 min at 4°C with continuous stirring. Tissue debris was removed by centrifugation and an aliquot of the supernatant was exactly neutralized with KOH and Tris-HCl buffer was added to give a final concentration of 15 mm at pH 7·1. Samples were stored at − 40°C until analysis.

Metabolite concentrations were compared using the method of analysis of variance for unequal sample numbers.

Resting levels of postbranchial blood metabolite concentrations are given in Table 1. The time course of changes in the blood chemistry during hypoxia and recovery are illustrated in Figs. 1 (a)-(c) and 2(a)-(c). The initial phase of hypoxia during which the inspired oxygen tension fell to around 40 mmHg was characterized by a small increase in lactate level, indicative of a switch to anaerobic glycolysis. A further decrease of below 35 mmHg was associated with a dramatic increase in lactate and lactate/pyruvate ratio. The increase in blood lactate/pyruvate ratio paralleled the fall in blood pH. An increase in succinate concentration provides evidence for the accumulation of multiple anaerobic end products within the tissues. The blood oxygen rapidly approached zero following the cessation of ventilation, which occurred within 1 h of the inspired water falling to 10–15 mmHg. Hypoxia was maintained at this level for up to 5 h. All fish survived such periods of hypoxia and were able to make complete recovery. After such 5 h periods of anaerobiosis, lactate concentrations in the blood were often in excess of 7 μmol/ml, a rise of 300%, compared to 3·0 μmol for succinate. Blood pH fell from 7·9 to around 7·45 under extreme hypoxia. In some individual fish (Fig. 2) anaerobic end products accumulated at a slower rate once the hypoxia was established, possibly due to a slowing of the metabolic rate caused by cessation of ventilation and reduction in heart rate (Hughes, 1976).

When the oxygen tension of the inspired water was raised towards normoxic levels, there was a lag of 20–40 min before the of the arterial blood was restored to the prehypoxic level. This was probably because of a delay between the time at which the oxygen level was raised and the time when ventilation was re-established. In some individuals blood P_Os continued to rise above the prehypoxic level during recovery (Fig. 2). This is especially true of fish which had been subjected to slow and more prolonged hypoxia. Immediately after blood oxygen began to increase there was a rapid fall in succinate and, to a lesser extent, in pyruvate concentrations, presumably due to their in situ oxidation by the tricarboxylic acid cycle. In contrast lactate and lactate/pyruvate ratio continued to increase following the rise in blood oxygen and thus provided indirect evidence for vascular shunts whereby muscle and other peripheral tissues might be isolated from the general circulation during hypoxia. Re-establishment of their blood supply makes possible the release of their accumulated anaerobic end products following the transition to normoxia. Lactate levels fell slowly following a transition to normoxia and remained 200–300% elevated following 3–5 h recovery. Lactate only returned to prehypoxic values following 18 h recovery. Once again, blood pH mirrored the changes in blood lactate, there being a slow rise in pH during recovery.

The steady state concentrations of the following muscle metabolites from normoxic fish are summarized in Table 2: succinate, lactate, pyruvate, alanine, α-ketoglutarate and aspartate. The muscle lactate/pyruvate ratio increased from 239 to 350 following hypoxia, indicating an increase in anaerobic glycolysis (P < 0·05). Evidence was also obtained for the accumulation of multiple anaerobic end products in the red muscle during hypoxia, with increases in the concentrations of alanine and succinate in addition to pyruvate and lactate. The concentrations of free α-ketogutarate and aspartate were also found to fall significantly during anaerobiosis (P < 0 ·05).

The respiratory responses to hypoxia in fish have recently been reviewed (Hughes, 1973) and an account given of some responses of the respiratory and circulatory systems in Torpedo (Hughes, 1978). In the present study since the arterial blood oxygen levels approached zero following cessation of ventilation the tissues of the fish were probably totally anoxic for the remainder of the experiment. Under these conditions Torpedo must rely exclusively on anaerobic metabolism to meet its energy requirements. Torpedo obviously has a well-developed ability to withstand hypoxia since all fish survived and were able to recover from up to 5 h exposure at these very low ambient oxygen tensions. In common with most vertebrate tissues Torpedo muscle responds to periods of anoxia by an increase in anaerobic glycogenolysis. Numerous previous studies have revealed that large quantities of lactate accumulate following brief periods of anaerobic work by fish muscles (Black, Robertson & Parker, 1961; Peres et al 1972) and following short dives by turtles and other marine vertebrates (Jackson, 1968; Hochachka & Storey, 1975). It is interesting that although the hypoxic exposure was presumably far more severe in the Torpedo experiments than would be experienced by turtles following short dives, there was far less accumulation of lactate in the blood.

In view of the relatively low lactate accumulation we have explored the possibility that alternative pathways to glycolysis are operative as occurs in numerous facultative anaerobes and in diving mammals (Mangum & van Winkle, 1973; Hochachka, 1975; De Zwaan & Wijsman, 1976). It was decided to investigate anaerobic metabolism in the red muscle since evidence for extra-glycolytic energy production during hypoxia has already been reported for this tissue in another species (Johnston, 1975). Also fish red muscle, in contrast to the white muscle, is in almost continuous use in providing the motive power for slow-speed swimming (Bilinski, 1975). The red muscle of fish is a highly aerobic slow twitch muscle abundant in mitochondria with high concentrations of myoglobin and cytochromes, and high activities of TCA cycle and respiratory chain enzymes (Bilinski, 1975).

The accumulation of succinate and alanine and the depletion of free amino acids during hypoxia observed in the present study provides evidence that anaerobic glycolysis is not the only anaerobic strategy open to Torpedo under these conditions. It would seem probable that Torpedo can couple glycolysis to mitochondrial energyyielding reactions during periods of anoxia in a manner analogous to the mechanisms proposed for diving vertebrates such as the turtle and porpoise (Owen & Hochachka, 1974; Hochachka et al. 1975). The depletion of free pool sizes of aspartate and α-ketoglutarate have also been reported in the muscles of diving mammals following peripheral vasoconstriction (Hochachka, 1975). Succinate also accumulates in various mammalian tissues following hypoxia (Hoberman & Prosky, 1967; Kandrashova & Chahovets, 1971).

Succinate production during anaerobiosis has been extensively studied in molluscs. However, the nature and stoichiometry of the pathways involved remain a matter of controversy (see Hochachka (1975), and De Zwaan & Wijsman (1976) for differing viewpoints), particularly with respect to the extent to which free amino acids can serve as an energy pool during anaerobiosis. These discrepancies may be due to species differences or be related to other factors such as temperature, salinity and duration of anoxia which have been shown to affect the proportions of end-products accumulated during anaerobiosis (De Zwaan & Wijsman, 1976). A common feature of all the reaction schemes proposed are a series of substrate level phosphorylations within the mitochondria utilizing fumarate or other specific electron acceptors in place of oxygen. This allows both an extra yield of high-energy phosphates compared to glycolysis and the maintenance of redox balance during anoxia (Hochachka, 1975; De Zwann, Kluytmans & Zandee, 1976; De Zwaan & Wijsman, 1976).

Although the mechanisms of succinate production by fish tissues during hypoxia is unknown it is likely that this also represents an energetic advantage over classical glycolysis for species which might frequently experience sustained periods of anaerobiosis in their environment. Estimates of the energy yield in anoxic invertebrate muscles producing succinate vary from 5 to 7 mol ATP/mol glucose 6-phosphate (G6P) compared to 3 mol ATP/mol (G6P) for anaerobic glycolysis (Hochachka & Somero, 1976). Furthermore, it is probable that succinate is less toxic to cellular metabolism than lactate. Indeed, lactate accumulation has been implicated as a cause of death in certain fish species following severe exercise (Black et al. 1961). However, the accumulation of lactate was 3·5 times greater than that of succinate in the red muscle of Torpedo. The importance of the accumulation of succinate as an anaerobic end-product is clearly dependent on the abundance of mitochondria in the tissue. Fish white muscle fibres, which usually constitute around 90–95 % of the total muscle bulk, are characterized by their very low mitochondrial density. For example, the ratio of mitochondria in the red and white muscles of the crucian carp expressed as percentage volume occupied is 23:1 respectively (Patterson & Goldspink, 1973). Indeed, no evidence for succinate accumulation in the white muscle has been obtained for either crucian carp (Johnston, 1975) or mirror carp (Driedzic & Hochachka, 1975) following severe hypoxia. Both of these species, like Torpedo, have well-developed capabilities for surviving extended exposure to low ambient oxygen levels. It would therefore appear that anaerobic mitochondrial energy-yielding pathways are unlikely to provide a very large contribution to the total swimming musculature’s ability to do anaerobic work, simply because red fibres constitute such a small percentage of the muscle bulk. However, since mitochondria-rich fibres are utilized almost continually for slow-speed swimming and in maintaining the contractile activity of the heart, the operation of these pathways during periods of restricted oxygen availability may have significant survival value particularly in relation to the maintenance of redox balance.

After the fish breathes air-saturated water once more, there was a lag of about 40 min before blood was restored to prehypoxic levels. This is probably because ventilation did not begin immediately. As soon as the blood oxygen levels began to rise there was a rapid drop in circulating succinate and to a lesser extent pyruvate. This is undoubtedly because these substances are metabolic intermediates and underwent in situ oxidation as aerobic respiration was ‘switched on’ to provide the energy required to fuel the increased demand from the respiratory and cardiac muscles as ventilation and heart rate increased following hypoxia. This illustrates a further advantage of the formation of succinate as an anaerobic end-product, namely the ease with which it can be metabolized following a period of anoxia. Oxidation of succinate can be expected to yield 11 mol of ATP per mol (Hochachka, 1975). In the present study, succinate was reduced below prehypoxic levels within 15 min of an increase in the oxygen availability (Figs, 1, 2). Lactate on the other hand can only be oxidized or excreted, both of which processes occur only very slowly in fish. Lactate levels in the present experiments were 40 % of peak concentrations 5 h following transition to aerobiosis and had not returned to resting levels until 18 h later.

Similar time relationships have been reported for the metabolism of blood and white muscle lactate following exercise in salmonids, cod (Beamish, 1968; Black et al. 1961) and carp (Johnston & Goldspink, 1973). In contrast, blood lactate concentrations return to normal within 30 min of return to rest following maximal effort by man on a bicycle ergometer (Karlsson, 1971).

During recovery of some fish (Fig. 2), the arterial increased dramatically to about twice its previous resting normoxic value. This is adaptive in that it will increase the driving force for oxygen transfer to the cells, facilitating aerobic energy production and the restoration of redox balance. The increase in blood may be associated with a shift in the oxygen dissociation curve to the right caused by the more acid conditions and the Bohr effect which thus allow a higher partial pressure of oxygen for the same oxygen content.

The significance of anaerobic mitochondrial energy production in fish capable of surviving prolonged hypoxia is obviously an important area for future research as is the nature and regulation of these pathways. Differences in the particular anaerobic pathways predominating can be expected to be correlated with the ecology of the particular fish species. For those which normally only encounter short periods of hypoxia it may be energetically advantageous to rely on classical anaerobic glycolysis alone, because the lactate formed can be re-oxidized to pyruvate by the liver, red muscle and gills at a later time and then metabolized aerobically via the TCA cycle (Bilinski & Jonas, 1972). This would provide five times the energy available for the conversion of pyruvate to succinate under anoxic conditions (Hochachka, 1975). On the other hand such a strategy is not favourable in situations where prolonged periods of oxygen lack are obtained because of the vast build-up of lactate which would enhance the difficulties of maintaining intracellular pH.

In the case of Torpedo, its normal mode of life is often in deep water and perhaps buried in the seabed for certain periods. At Arcachon it is known that specimens often enter the Bassin in the autumn for reproductive purposes. At these times they are not infrequently found buried in the mud and even cut off in small pools on the oyster beds, where they will clearly suffer very low oxygen levels at low tide. The adaptations discussed here may provide ways in which these organisms are adapted to such a mode of life.

We wish to thank the director and staff of the Institut de Biologie Marine, Arcachon, for their help and hospitality. This work was possible because of grants from the Browne fund of the Royal Society and from NERC. The assistance of Richard Adeney is gratefully acknowledged. I.A.J. wishes to acknowledge, with thanks, a postdoctoral fellowship award from NERC.