Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia

Comparative Biochemistry and Physiology Part A 131 Ž2002. 313᎐321

Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia Matthew H. Routley a , Goran ¨ E. Nilssonb, Gillian M.C. Renshaw c,U a

Faculty of Medicine, Uni¨ ersity of Sydney, Sydney, New South Wales 2006, Australia Di¨ ision of General Physiology, Department of Biology, Uni¨ ersity of Oslo, N-0316 Oslo, Norway c Hypoxia and Ischemia Research Unit, School of Physiotherapy and Exercise Science, Griffith Uni¨ ersity, Gold Coast Campus, PMB 50 Gold Coast Mail Centre, Queensland, 9726 Australia b

Received 11 December 2000; received in revised form 23 April 2001; accepted 8 October 2001

Abstract The majority of vertebrates are not tolerant to hypoxia but epaulette sharks Ž Hemiscyllium ocellatum) living on shallow reef platforms appear to tolerate hypoxic periods during tidal fluctuations. The effects of progressive hypoxia on the metabolic and ventilatory responses of these elasmobranchs were examined in a closed respirometer. In order to determine whether repeated exposure to hypoxia primes these sharks to alter their metabolism, one group of sharks was exposed to repeated sub-lethal hypoxia, at 5% of air saturation, prior to respirometry. In response to falling oxygen concentration wO 2 x, the epaulette shark increased its ventilatory rate and maintained its O 2 consumption rate Ž V O 2 . down to 2.2 mg O 2 ly1 at 25 ⬚C. This is the lowest critical wO 2 x ŽwO 2 x crit . ever measured for any elasmobranch. After reaching the wO 2 x crit , the shark remained in the respirometer for a further 4᎐5 h of progressive hypoxia. Only after the wO 2 x fell to 1.0 mg ly1 was there a decrease in the ventilatory rate followed by a rise in blood lactate levels, indicating that the epaulette shark responds to severe hypoxia by entering a phase of metabolic and ventilatory depression. Interestingly, hypoxia tolerance was dynamic because hypoxic pre-conditioning lowered the V O 2 of the epaulette shark by 29%, which resulted in a significantly reduced wO 2 x crit Ž1.7 mg O 2 ly1 ., revealing that hypoxic pre-conditioning elicits an enhanced physiological response to hypoxia. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Hypoxia tolerance; Ventilatory depression; Metabolic depression; Hypoxic pre-conditioning; Respirometry; Elasmobranch; Adaptive physiology

U

Corresponding author. Tel.: q61-7-5594-8392; fax: q61-7-5552-8674. E-mail address: [email protected] ŽG.M. Renshaw..

1095-6433r02r$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 1 . 0 0 4 8 4 - 6

314

M.H. Routley et al. r Comparati¨ e Biochemistry and Physiology Part A 131 (2002) 313᎐321

1. Introduction

In general most vertebrates are unable to tolerate more than a few minutes of oxygen deprivation, but a few species can survive extreme hypoxia and even anoxia. The best-studied examples include North American freshwater turtles Žgenera Chrysemys and Trachemys. and two closely related species of teleost fishes, the crucian carp Ž Carassius carassius L.. and the goldfish Ž Carassius auratus L.. ŽLutz and Nilsson, 1997; Ultsch, 1989.. Although these Temperate Zone vertebrates can survive up to 2 days in anoxia at room temperature, they have evolved their hypoxia tolerance in response to over-wintering in freshwater at temperatures close to 0 ⬚C. Some tropical freshwater fishes, such as the Oscar cichlid Ž Astronotus ocellatus Cuvier. of the Amazon River, have been reported to survive hours of anoxia ŽVal and de Almeida-Val, 1995.. The elephant-nose fish Ž Gnathonemus petersii Gunther . from Central ¨ Africa can tolerate severe hypoxia Ž10% air saturation. but is unable to survive anoxia ŽNilsson, 1996.. Hypoxia and anoxia tolerance in a tropical marine environment is less common than in a freshwater environment. However, there have been a few examples of small marine fishes with welldeveloped hypoxia tolerance under temperate conditions. The toadfish Ž Opsanus tau. is able to tolerate 20 h of anoxia at 22 ⬚C ŽUltsch et al., 1981.. The bottom dwelling torpedo ray ŽTorpedo marmorata. can tolerate relatively severe hypoxia ŽHughes, 1978., but this is a temperate rather than a tropical species. A major reason for the rarity of hypoxia-tolerant marine fishes is probably that the marine environments, in contrast to freshwater habitats, seldom expose its inhabitants to more than moderately hypoxic conditions. Moreover, marine fishes can usually choose to escape hypoxic zones. Until recently, no sharks were known to withstand hypoxia, furthermore, no tropical hypoxiatolerant species of elasmobranchs had been reported. It was recently demonstrated that the epaulette shark Ž Hemiscyllium ocellatum, Bonnaterre . is able to endure 3.5 h of severe hypoxia Ž5% air saturation s 0.36 mg O 2 ly1 . at 25 ⬚C. without any impairment to neurological functions such as the righting reflex, ventilatory movements

and rhythmic swimming ŽWise et al., 1998.. Furthermore, in contrast with hypoxia-intolerant animals, the brain of the epaulette shark does not undergo delayed neuronal apoptosis in response to prolonged hypoxia ŽRenshaw and Dyson, 1999.. The apparent hypoxia-tolerance of the epaulette shark is likely to be an adaptation to the repeated occurrence of hypoxia in its habitat. It lives on shallow reef platforms that surrounds islands of the Great Barrier Reef and Torres Strait ŽLast and Stevens, 1994.. During low tide, the water on the reef platform becomes cut off from the surrounding ocean, and at night the shallow water on the reef platform becomes hypoxic due to the respiration of coral reef organisms. During nocturnal low tides, Kinsey and Kinsey Ž1966. measured a fall in water wO 2 x from 6.8 to 2.1 mg ly1 on the Heron Island reef platform, and our own measurements show that during calm conditions, the wO 2 x minima can be as low as 1.2 mg O 2 ly1 . Many fishes appear to leave the reef platform under such conditions, but the epaulette shark remains. In contrast to several other species of sharks, the epaulette relies entirely on buccal pumping for gill ventilation. It has a small downward directed mouth and during the daylight hours it rests on the bottom under coral heads. However, this shark is active at night, even when the water wO 2 x on the reef platform is low. The apparent hypoxia tolerance ŽWise et al., 1998. of the epaulette shark needs to be carefully examined to establish whether it really is hypoxia tolerant because all reports to date have indicated that sharks are intolerant to hypoxia. While hypoxia-tolerant teleosts typically display a low critical O 2 concentration ŽwO 2 x crit ., viz. they are able to maintain their normoxic rate of O 2 consumption Ž V O 2 . down to environmental O 2 levels as low as 10᎐20% of air saturation, the wO 2 x crit of most elasmobranchs appears to be well above 50% of air saturation ŽButler and Taylor, 1975; Chan and Wong, 1977; Piiper et al., 1970.. Moreover, hypoxia-tolerant turtles and teleosts of the genus Carassius elevate their blood glucose levels during hypoxia, apparently to support glycolytic ATP production in order to compensate for the reduction or cessation of oxidative phosphorylation Žreviewed by Lutz and Nilsson, 1997.. Indeed, most teleosts appear to show increased blood glucose levels in response to hypoxia ŽMcDonald and Milligan, 1992.. In contrast, the blood glu-

M.H. Routley et al. r Comparati¨ e Biochemistry and Physiology Part A 131 (2002) 313᎐321

cose level in the dogfish Ž Scyliorhinus canicula. remains low during hypoxia ŽButler et al., 1979., which indicates that elasmobranchs may lack the ability to increase their glucose levels in response to hypoxia. In addition, elasmobranchs appear to have lower haematocrits ŽHct. than do teleosts ŽFange, 1992., and they do not display a rise in ¨ Hct in response to hypoxia ŽButler et al., 1979; Perry and Gilmour, 1996; Short et al., 1979.. This raises the question of whether the respiratory and metabolic responses of the epaulette shark to hypoxia follow the typical elasmobranch pattern, or whether they follow that of other hypoxiatolerant vertebrates viz. by having a low wO 2 x crit , an elevated blood glucose level and an increased Hct. Consequently, we used closed respirometry to examine how progressive hypoxia affected: V O 2 ; ventilation rate; Hct; blood wglucosex; and blood wlactate x in the hypoxia-tolerant epaulette shark, since hypoxic pre-conditioning results in neuronal hypometabolism of cardiorespiratory centres in the brain of this shark ŽMulvey and Renshaw, 2000 .. The onset of such neuronal hypometabolism in response to repeated hypoxia raises the question of whether or not the respiratory and metabolic responses of the epaulette shark to hypoxia could be altered by hypoxic pre-conditioning. In goldfish, hypoxic pre-conditioning has been found to reduce both V O 2 and wO 2 x crit ŽProsser et al., 1957.. More recently, reports that hypoxic pre-conditioning improves the survival of the mammalian brain and heart during subsequent periods of ischemia Že.g. Heurteaux et al., 1995; Murry et al., 1986. have attracted considerable interest.

2. Materials and methods

2.1. Animal collection The epaulette sharks, weighing 636 " 22 g Ž n s 17., were caught by hand from the reef platform surrounding Heron Island on the Great Barrier Reef Ž23⬚ 26⬘S, 151⬚ 55⬘E. during low tide. All animals were kept at Heron Island Research Station in a 10 000-l holding tank continuously sup-

315

plied with fresh seawater Ž25 " 1 ⬚C.. The wO 2 x in the holding tank was close to air saturation Ž6.0᎐7.0 mg O 2 ly1 .. The sharks were fed chopped pilchards and crustaceans but fasted for the 24 h prior to respirometry and for the duration of the experimental period.

2.2. Experimental protocol

Two experimental groups of epaulette sharks were studied: controls Ž n s 10.; and sharks preconditioned to hypoxia Ž n s 7.. Hypoxic pre-conditioning was conducted by placing the epaulette sharks in 200-l tanks fitted with perspex lids. The wO 2 x was maintained at 0.36 mg O 2 ly1 Ž5% of air saturation . by bubbling nitrogen through the water for 120 min, twice daily for the 4 sequential days immediately before the respirometry experiment. The controls were given the same treatment except that the water in the tanks was kept between 80 and 100% of air saturation. Prior to cannulation, the sharks were anaesthetised by adding 5% benzocaine Žin ethanol. to seawater Ž1.5 ml, 5% benzocaine per litre of fresh seawater.. When anaesthesia had commenced, the sharks’ gills were ventilated with seawater via a tube inserted into the mouth. Each epaulette shark was cannulated using PE50 tubing. The cannula was inserted into the dorsal aorta 50 mm caudally from the cloaca using a syringe needle as a guide, as described by Axelsson and Fritsche Ž1994.. The cannula was then flushed with elasmobranch saline ŽSimpson and Sargent, 1985. containing 100 IU heparin mly1 to prevent coagulation. A single animal was placed in a closed respirometer consisting of a perspex tube 998 mm long and 237 mm in diameter with a volume of 44.4 l. The O 2 probe and cannula were inserted through rubber stoppers. The respirometer was covered with towels to mimic their preference for sheltered habitats and to avoid any visual disturbance of the fish. The respirometer contained an immersion pump to maintain water circulation within the chamber. All animals were acclimatised in the respirometer for 3᎐5 h after surgery before commencing the experiment, until their ventilation rate fell to resting level Ži.e. the level seen in undisturbed sharks in the holding tank..

316

M.H. Routley et al. r Comparati¨ e Biochemistry and Physiology Part A 131 (2002) 313᎐321

Longer acclimatisation periods combined with the length of the experiment were found to compromise the patency of the cannula. During the acclimatisation period, seawater continually flowed through the respirometer. At the beginning of the experiment, the seawater inflow and outflow to the respirometer were sealed and the decline in wO 2 x was measured continuously using a WTW323 oxygen meter ŽWTW Wissenschaftlische Technische Werkstatten, Heidelberg, Germany. attached to a chart recorder. The tip of the O 2 probe was fitted with a magnetically driven propeller Žfrom WTW.. The water temperature in the respirometer throughout the experiment was 25 " 1 ⬚C. The epaulette sharks were allowed to consume the O 2 in the respirometer until the wO 2 x fell to 0.3 mg O 2 ly1 , which took an average of 7.7 h. The epaulette sharks showed very little swimming activity after 30 min in the respirometer. The fall in wO 2 x was used to calculate the V O 2 in response to progressive hypoxia. The wO 2 x crit for each epaulette shark was calculated as described by Nilsson Ž1996.. Thus, wO 2 x crit was considered to be the point on the O 2 concentration chart where all subsequent points reflected lower V O 2 ’s. At the conclusion of every respirometry session, ammonia, nitrate and nitrite levels in the respirometer were measured, using test kits supplied by Aquarium Systems, ŽMentor, OH, USA. and were found to be too low to be detected. The ventilation rate of the sharks was measured by counting the number of buccal movements during 1 min every 30 min. Blood samples Ž200 ␮l. were collected at hourly intervals, placed in heparinised eppendorf tubes, and used to measure changes in blood lactate ŽSigma lactate kit, 725-10. and blood glucose ŽSigma glucose kit, 16-10.. Hct was determined by drawing some of the blood sample into a capillary tube, centrifuging at 5000 = g for 10 min, and measuring the proportion of packed red cells to the total sample volume in the capillary tube. After sampling, the cannula was flushed with heparinised elasmobranch saline solution. All data are presented as means " S.E.M. and compared statistically using ANOVA followed by Students t-test, or Mann᎐Whitney U-test if the variances in the data to be compared were significantly different Žas indicated by an F-test.. P-values less than 0.05 were regarded as statistically significant.

3. Results 3.1. Respiratory and metabolic responses of the epaulette shark In non-pre-conditioned epaulette sharks, two phases of V O 2 were observed in response to falling ambient wO 2 x ŽFig. 1a.. Initially, the V O 2 remained constant, with the mean V O 2 during this phase being 83.4" 5.5 mg kgy1 hy1 ŽFig. 1b.. An initial linear phase, in which this routine metabolic rate was maintained, was followed by a non-linear phase characterised by a progressive decline in V O 2 . The transition point between these two phases, the wO 2 x crit , occurred at 2.16" 0.10 mg O 2 ly1 . During the initial linear phase of V O 2 ŽFig. 1c., the ventilation rate of the epaulette sharks increased significantly from 36.4" 4.7 ventilations miny1 at the beginning of the experiment, to 74.0" 2.5 ventilations miny1 at the wO 2 x crit ŽStudents t-test, P- 0.0001.. However, the maximum ventilation rate Ž79.5" 1.2 miny1 . was observed at the beginning of the non-linear phase of V O 2 when the ambient wO 2 x was 1.25 mg O 2 ly1 . As the ambient oxygen level fell further, the rate of ventilation decreased to a final value of 53.3" 6.3 ventilations miny1 at the end of the experiment, when the ambient wO 2 x had fallen to 0.3 mg ly1 ŽStudents t-test, Ps 0.0008, compared to the maximum value.. Blood glucose levels in the epaulette sharks did not change significantly during the experiment ŽFig. 2a.. The blood lactate level ŽFig. 2b. began rising towards the end of the non-linear phase of the V O 2 curve, reaching 6.64" 2.27 mM at the end of the experiment ŽMann᎐Whitney U-test, Ps 0.009, compared to the initial value of 0.99" 0.32 mM.. No significant changes occurred in the Hct during the experiment ŽFig. 2c.. 3.2. Comparison of the respiratory and metabolic responses of pre-conditioned and control epaulette sharks Epaulette sharks which were pre-conditioned to hypoxia prior to respirometry had a significantly lower V O 2 ŽMann᎐Whitney U-test, Ps 0.002. during the initial linear phase when the V O 2 remained stable Ž59.5" 2.2 mg kgy1 hy1 ; Fig. 1d,e., compared to the control sharks in the same phase Ž83.4" 5.5 mg kgy1 hy1 ; Fig. 1a,b..

M.H. Routley et al. r Comparati¨ e Biochemistry and Physiology Part A 131 (2002) 313᎐321

317

Fig. 1. The effect of falling ambient oxygen levels on the rate of O 2 consumption Ž V O 2 . for individual control and pre-conditioned sharks Ža and d, respectively.. The different symbols in Ža. and Žg. represent individual sharks while all other values are means " S.E.M. from 10 Žcontrol. or seven Žpre-conditioned. animals. The mean V O 2 for control Žb. and pre-conditioned Že. sharks. The ventilation rate in response to falling ambient oxygen levels in control Žc., and pre-conditioned sharks Žf..

Also, the variance in the V O 2 during this phase was significantly lower in the pre-conditioned epaulette sharks compared to the controls Ž F-test, Ps 0.008.. This indicates that hypoxic pre-conditioning results in a depression of the routine metabolic rate down towards the basal metabolic rate The wO 2 x crit for pre-conditioned sharks was 1.70 " 0.24 mg O 2 ly1 , and was significantly lower ŽMann᎐Whitney U-test, Ps 0.04. than the wO 2 x crit for controls Ž2.16" 0.10 mg O 2 ly1 .. This could be entirely attributed to the lower V O 2 during displayed by the pre-conditioned sharks during the linear phase. At 1.70" 0.24 mg O 2 ly1 , the wO 2 x crit for pre-conditioned sharks, and the V O 2 of control and pre-conditioned sharks, was not

significantly different, being 63.7" 5.2 mg kgy1 hy1 and 56.2" 1.6 mg kgy1 hy1 , respectively. With regard to the other parameters measured Žventilation, glucose, lactate and Hct; Fig. 2d᎐f., the pre-conditioned epaulette sharks did not differ significantly from the controls.

4. Discussion The results of the present study show that the epaulette shark Žat 25 ⬚C. has a wO 2 x crit between 2.2 mg ly1 Žnon-pre-conditioned animals. and 1.7 mg ly1 Žpre-conditioned animals., which are the lowest wO 2 x crit values determined for any elasmobranch, and establish that the epaulette shark

318

M.H. Routley et al. r Comparati¨ e Biochemistry and Physiology Part A 131 (2002) 313᎐321

Fig. 2. The blood glucose Ža, d., lactate Žb, e. and haematocrit Žc, f. in response to decreasing ambient oxygen levels for control and pre-conditioned sharks, respectively.

is a hypoxia-tolerant vertebrate. Like the epaulette shark, hypoxia-tolerant teleosts have a low wO 2 x crit . In the crucian carp, the wO 2 x crit is 1.4 mg O 2 ly1 at 18 ⬚C ŽNilsson, 1992.. This is similar to that of the congeneric goldfish Ž1.5 mg O 2 ly1 at 22 ⬚C, Prosser et al., 1957. and the European eel Anguilla anguilla L. Ž1.4 mg O 2 ly1 at 25 ⬚C, Cruz-Neto and Steffensen, 1997.. With regard to tropical hypoxia-tolerant teleosts, the African elephant-nose fish Ž Gnathonemus petersii . appears to have a record low wO 2 x crit Ž0.8 mg O 2 ly1 at 26 ⬚C; Nilsson, 1996., while the wO 2 x crit of the Oscar cichlid Astronotus ocellatus, Ž1.6 mg ly1 at 28 ⬚C, Muuse et al., 1998. and the tilapia Oreochromis niloticus Ž1.2᎐1.4 mg O 2 ly1 at 20᎐35 ⬚C; Fernandes and Rantin, 1989. are very similar to that of the epaulette shark. In general, elasmobranchs appear to have a relatively high wO 2 x crit . In the bamboo shark,

Hemiscyllium plagiosum, a close relative of the epaulette shark, the wO 2 x crit is substantially higher at 3.5 mg O 2 ly1 at 23 ⬚C ŽChan and Wong, 1977.. In the dogfish Scyliorhinus canicula, wO 2 xcrit is 4.5 mg ly1 at 17 ⬚C ŽButler and Taylor, 1975. and in S. stellaris the wO 2 x crit is so high Žor absent. that V O 2 shows an immediate fall in response to a fall in ambient wO 2 x ŽPiiper et al., 1970.. This is also the case in the pelagic blacktip reef shark Ž Carcharhinus melanopterus. ŽJ.M. Mulvey, G.E. Nilsson, G.M.C. Renshaw, personal communication.. Unlike the epaulette shark, the wO 2 xcrit of these elasmobranchs is well outside the range of hypoxia-tolerant teleosts since it exceeds 50% of air saturation. This general elasmobranch characteristic is conserved in the shovelnose ray Ž Rhinobatus typus Bennett ., which visits the reef platform at Heron island. This ray is unable to regulate its V O 2 in response to falling wO 2 x or

M.H. Routley et al. r Comparati¨ e Biochemistry and Physiology Part A 131 (2002) 313᎐321

increase its glycolytic lactate production in response to hypoxia ŽM.H. Routley, G.E. Nilsson, G.M.C. Renshaw, personal communication.. In all likelihood, the low wO 2 x crit of the epaulette shark is a set of physiological adaptations to the hypoxic environment on the shallow reef platform during nocturnal low tides which allows it to exploit that niche by maintaining its routine metabolic rate once oxygen levels fall below saturation. The blood glucose level of the epaulette shark remained stable during exposure to severe hypoxia in the respirometer. This is in contrast with other hypoxia-tolerant vertebrates, such as turtles and teleosts Ž Carassius., which elevate their blood glucose levels during hypoxia ŽLutz and Nilsson, 1997, for review.. In fact, it appears that most teleosts increase their blood glucose levels in response to hypoxia ŽMcDonald and Milligan, 1992.. Elevated glucose levels probably facilitate an increased glycolytic ATP production in order to compensate for the slowing down of aerobic ATP production. However, since the epaulette shark showed increased lactate levels during the anaerobic phase of the experiments, glycolytic ATP production appears to be stimulated during hypoxia. One may therefore argue that because the epaulette shark maintained a stable blood glucose level under hypoxic conditions, it may be releasing additional glucose into the blood Žpresumably from liver glycogen stores., in order to balance an increased glucose utilisation by the tissues without actually increasing blood glucose levels. Nevertheless, in the dogfish Ž Scyliorhinus canicula. the blood glucose level also remains low during hypoxia ŽButler et al., 1979., so it is possible that elasmobranchs have a rather limited ability to elevate the release of glucose to the blood during hypoxia. In addition, elasmobranchs appear to have a lower Hct than teleosts, and they do not display a rise in Hct in response to hypoxia ŽButler et al., 1979; Perry and Gilmour, 1996; Short et al., 1979.. The epaulette shark did not show a hypoxia-induced elevation of Hct, and it also seems to be at the lower end of the range of Hct levels for elasmobranchs ŽBaldwin and Wells, 1990.. While a low Hct could have the benefit of reducing blood viscosity ŽBaldwin and Wells, 1990., it would not contribute to the epaulette’s hypoxia-tolerance. In most vertebrates, the spleen is responsible

319

for erythropoiesis and erythrocyte storage. It has been found to act to increase erythrocyte number both in response to chronic hypoxia, by elevating erythropoiesis, and in response to acute hypoxia, by contracting and releasing stored erythrocytes into the circulation ŽFange and Nilsson, 1985.. ¨ However, we could not find any signs of these processes in the epaulette shark; there was no difference in Hct between controls and hypoxia pre-conditioned sharks, or between initial and final Hct values during the respirometry experiments. The epaulette shark may be limited by phylogenetic constraints because adrenaline, which contracts the spleen in many vertebrates, has only little ŽNilsson et al., 1975. or no effects on the dogfish spleen ŽOpdyke and Opdyke, 1971.. Thus, elasmobranchs in general may not possess the ability to change Hct in response to hypoxia. The epaulette shark appears to rely on anaerobic Žglycolytic. ATP production after the wO 2 x crit has been reached; this effect was revealed by a rise in blood lactate levels after prolonged exposure to ambient wO 2 x below the wO 2 x crit . This strategy is likely to be a critical factor in hypoxic survival, but since anaerobic glycolysis yields only approximately 1r10 of the ATP produced through aerobic respiration ŽHochachka and Somero, 1984., a considerable increase in glycolytic flux is probably needed to avoid a fall in cellular ATP levels. A fall in ATP has rapid and catastrophic consequences for cellular survival, especially in organs that have a high level of ATP consumption such as the brain Žsee Lutz and Nilsson, 1997, for a review.. In the present experiments, it took 4᎐5 h for the shark to reduce the ambient wO 2 x from wO 2 x crit down to 0.3 mg O 2 ly1 . Our observations indicate that during this long phase of apparent metabolic depression, the epaulette shark was able to sustain a sufficient level of ATP production, since it retained its righting reflex, indicating that it did not shut down brain function. The epaulette shark displayed a biphasic ventilatory response to hypoxia as has been observed in teleost fish and the dogfish shark ŽHughes, 1973; Ultsch et al., 1981.. In the epaulette shark, there was an initial increase in the ventilatory rate in response to decreasing ambient wO 2 x. This elevated level of gill ventilation is likely to be one factor that underlies the ability to maintain O 2 uptake even though the ambient wO 2 x is falling. The initial increase in ventilation was followed by a significant ventilatory depression, coinciding

320

M.H. Routley et al. r Comparati¨ e Biochemistry and Physiology Part A 131 (2002) 313᎐321

with a decreased V O 2 and an increase in the blood lactate level. Epaulette sharks that were preconditioned following the same protocol had significantly reduced oxidative activity in cardiorespiratory centres ŽMulvey and Renshaw, 2000.. This is not due to a reduction in cerebral perfusion because while epaulette sharks respond to hypoxia with bradycardia, brain blood flow is maintained at normal levels ŽSoderstrom et al., 1999.. Therefore, it seems likely that ventilatory depression observed in the present study is closely linked with the onset of metabolic depression. Metabolic depression is a common response to hypoxia in other hypoxia-tolerant species aimed at reducing ATP use in response to the slowing down of aerobic ATP production Žsee Lutz and Nilsson, 1997 for a review.. Especially after pre-conditioning, the wO 2 x crit of the epaulette shark Ž1.7 mg O 2 ly1 at 25 ⬚C. approached that of hypoxia-tolerant teleosts and reveals that the hypoxia tolerance of the epaulette shark, like that of the goldfish, can be significantly improved by hypoxic pre-conditioning. The V O 2 of pre-conditioned sharks was significantly less than the controls during the initial linear phase, revealing that pre-conditioning had lowered the initial metabolic rate. This may explain the significantly lower wO 2 x crit in the pre-conditioned group of sharks Ž1.7 mg ly1 .. Interestingly, the variance in the V O 2 was lower in the pre-conditioned animals, suggesting that their initial metabolic activity had been suppressed to a level closer to their basal metabolic rate. These data suggest that the epaulette shark use metabolic depression rather than enhanced O 2 uptake as an acclimatisation response to hypoxic survival. Possible mediators of this effect remain to be studied. However, it should be mentioned that adenosine, which appears to be involved in hypoxic pre-conditioning in the mammalian brain ŽHeurteaux et al., 1995., has been found to increase in concentration in the brain of epaulette sharks exposed to anoxia ŽG.M.C. Rehshaw, C.B. Kerrisk and G.E. Nilsson, personal communication.. Hypoxic pre-conditioning of the epaulette shark significantly depressed its V O 2 and lowered its wO 2 x crit . Taken together, our data indicate that the epaulette shark has evolved respiratory and metabolic strategies aimed at maintaining cellular energy homeostasis during hypoxia, which may make it unique among elasmobranchs. Interest-

ingly, hypoxic pre-conditioning can significantly enhance the physiological responses of the epaulette shark to hypoxia by lowering their metabolic rate and depressing V O 2 . Investigation of the underlying regulatory mechanisms may contribute to an understanding of hypoxia tolerance at tropical temperatures.

Acknowledgements We would like to thank James Ulyate for his excellent technical assistance and the staff at the Heron Island Research Station for facilitating the fieldwork. The project was funded by grants from the Sea World Research and Rescue Foundation and the Research Council of Norway. G.E. Nilsson was the recipient of a University of Queensland Travel Grant for International Research. References Axelsson, M., Fritsche, R., 1994. Cannulation techniques. In: Hochachka, P.W., Mommsen, T.P. ŽEds.., Biochemistry and Molecular Biology of Fishes, Analytical Techniques, 3. Elsevier, Amsterdam, pp. 17᎐36. Baldwin, J., Wells, R.M.G., 1990. Oxygen transport potential in tropical elasmobranchs from the Great Barrier Reef: relationship between haematology and blood viscosity. J. Exp. Mar. Biol. Ecol. 144, 145᎐155. Butler, P.J., Taylor, E.W., 1975. The effect of progressive hypoxia on respiration in the dogfish Ž Scyliorhinus canicula. at different seasonal temperatures. J. Exp. Biol. 63, 117᎐130. Butler, P.J., Taylor, E.W., Davison, W., 1979. The effect of long term, moderate hypoxia on acid base balance, plasma catecholamines and possible anaerobic end products in the unrestrained dogfish Scyliorhinus canicula. J. Comp. Physiol. B. 132, 297᎐303. Chan, D.K.O., Wong, T.M., 1977. Physiological adjustments to dilution of the external medium in the lip shark, Hemiscyllium plagiosum ŽBennet.. III Oxygen consumption and metabolic rates. J. Exp. Zool. 200, 97᎐102. Cruz-Neto, A.P., Steffensen, J.F., 1997. The effects of acute hypoxia and hypercapnia on oxygen consumption of the freshwater European eel. J. Fish Biol. 50, 759᎐769. Fernandes, M.N., Rantin, F.T., 1989. Respiratory responses of Oreochromis niloticus ŽPisces, Cichlidae. to environmental hypoxia under different thermal conditions. J. Fish Biol. 35, 509᎐519.

M.H. Routley et al. r Comparati¨ e Biochemistry and Physiology Part A 131 (2002) 313᎐321

Fange, R., 1992. Blood cells, haemopoiesis and lym¨ phoid tissue in fish. Fish Shellfish Immunol. 4, 405᎐411. Fange, R., Nilsson, S., 1985. The fish spleen: structure ¨ and function. Experientia 41, 152᎐158. Heurteaux, C., Lauritzen, I., Widmann, C., Lazdunski, M., 1995. Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive Kq channels in cerebral ischaemic pre-conditioning. Proc. Natl. Acad. Sci. USA 92, 4666᎐4670. Hochachka, P.W., Somero, G.N., 1984. Biochemical Adaptations. Princeton University Press, Princeton, NJ. Hughes, G.M., 1978. On the respiration of Torpedo marmorata. J. Exp. Biol. 73, 85᎐105. Hughes, G.M., 1973. Respiratory responses to hypoxia in fish. Am. Zool. 13, 475᎐489. Kinsey, D.W., Kinsey, B.E., 1966. Diurnal changes in oxygen content of the water over the coral reef platform at Heron I. Aust. J. Mar. Freshwater Res. 18, 23᎐24. Last, P.R., Stevens, J.D., 1994. Sharks and Rays of Australia. CSIRO, Hobart, Australia. Lutz, P.L., Nilsson, G.E., 1997. The Brain Without Oxygen, 2nd ed. R.G. Landes Co.rChapman and HallrSpringer. 207 pp. McDonald, D.G., Milligan, C.L., 1992. Chemical properties of the blood. In: Hoar, W.S., Randall, D.J., Farrell, A.P. ŽEds.., Fish Physiology, The Cardiovascular System, XIIB. Academic Press, San Diego, pp. 55᎐133. Mulvey, J.M., Renshaw, G.M.C., 2000. Neuronal oxidative hypometabolism in the brainstem of the epaulette shark Ž Hemiscyllium ocellatum. in response to hypoxic pre-conditioning. Neurosci. Lett. 290, 1᎐4. Murry, C.E., Jennings, R.B., Reimer, K.A., 1986. Preconditioning with ischemia: a delay of lethal cell injury in ischaemic myocardium. Circulation 74, 1124᎐1136. Muuse, B., Marcon, J., van den Thillart, G., AlmeidaVal, V., 1998. Hypoxia tolerance of Amazon fish respirometry and energy metabolism of the cichlid Astronotus ocellatus. Comp. Biochem. Physiol. 120, 151᎐156. Nilsson, G.E., 1992. Evidence for a role of GABA in metabolic depression during anoxia in crucian carp Ž Carassius carassius L... J. Exp. Biol. 164, 243᎐259. Nilsson, G.E., 1996. Brain and body oxygen requirements of Gnathonemus petersii, a fish with an exceptionally large brain. J. Exp. Biol. 199, 603᎐607.

321

Nilsson, S., Holmgren, S., Grove, D.J., 1975. Effects of drugs and nerve stimulation on the spleen and arteries of two species of dogfish, Scyliorhinus canicula and Squalus acanthias. Acta Physiol. Scand. 95, 219᎐230. Opdyke, D.F., Opdyke, N.E., 1971. Splenic responses to stimulation in Squalus acanthias. Am. J. Physiol. 221, 623᎐625. Perry, S.F., Gilmour, K.M., 1996. Consequences of catecholamine release on ventilation and blood oxygen transport during hypoxia and hypercapnia in an elasmobranch Ž Squalus acanthias. and a teleost Ž Oncorhynchus mykiss.. J. Exp. Biol. 199, 2105᎐2118. Piiper, J., Baumgarten, D., Meyer, M., 1970. Effects of hypoxia upon respiration and circulation in the dogfish Scyliorhinus stellaris. Comp. Biochem. Physiol. 36, 513᎐520. Prosser, C.L., Barr, L.M., Pinc, R.D., Lauer, C.Y., 1957. Acclimation of goldfish to low concentrations of oxygen. Physiol. Zool. 30, 137᎐141. Renshaw, G.M.C., Dyson, S.E., 1999. Increased nitric oxide synthase in the vasculature of the epaulette shark brain following hypoxia. NeuroReport 10, 1707᎐1712. Short, S., Taylor, E.W., Butler, P.J., 1979. The effectiveness of oxygen transfer during normoxia and hypoxia in the dogfish Ž Scyliorhinus canicula L.. before and after cardiac vagotomy. J. Comp. Physiol. B 132, 289᎐295. Simpson, C.M.F., Sargent, J.R., 1985. Inositol lipid turnover and adenosine 3-5 monophosphate in the salt secreting rectal gland of the dogfish Ž Scyliorhinus canicula.. Comp. Biochem. Physiol. 82B, 781᎐786. Soderstrom, V., Renshaw, G.M.C., Nilsson, G.E., 1999. Brain blood flow and blood pressure during hypoxia in the epaulette shark Hemiscyllium ocelatum, a hypoxia tolerant elasmobranch. J. Exp. Biol. 202, 829᎐835. Ultsch, G.R., Jackson, D.C., Moalli, R., 1981. Metabolic oxygen conformity among lower vertebrates: the toadfish revisited. J. Comp. Physiol. 142, 439᎐443. Ultsch, G.R., 1989. Ecology and physiology of hibernation and over-wintering among freshwater fishes, turtles and snakes. Biol. Rev. 64, 435᎐516. Val, A.L., de Almeida-Val, V.M.F., 1995. Fishes of the Amazon and their Environment. Springer, Berlin Heidelberg. Wise, G., Mulvey, J.M., Renshaw, G.M.C., 1998. Hypoxia tolerance in the epaulette shark Ž Hemiscyllium ocellatum.. J. Exp. Zool. 281, 1᎐5.