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Changes in blood metabolites following stress from capture and handling of the marine teleost Girella tricuspidata
Camp. Printed
Biochem.
Vol. 82A. No. 3, pp. 609-612, 1985
Physiol.
in Great
Britain
0
0300-9629/85 $3.00 + 0.00 1985 Pergamon Press Ltd
CHANGES IN BLOOD METABOLITES FOLLOWING STRESS FROM CAPTURE AND HANDLING OF THE MARINE TELEOST GZRELLA TRZCUSPZDATA N. LING and R. M. G. Department
of Zoology,
University
of Auckland,
WELLS
Auckland,
New Zealand.
Telephone:
737-999
(Received 26 February 1985)
Whole blood nucleoside triphosphate (NTP) and lactate in the parore (Girella tricuspiduta, Fam: Kyphosidae) were monitored over a period of 12 hr following capture by gill net. 2. An increase in NTP during the post-capture recovery period was mainly attributable to a significant rise (P < 0.05) in the NTP component guanosine triphosphate (GTP). 3. The rise in GTP levels correlated with the decline in blood lactate (r = -0.72) accumulated during the period of capture stress. 4. It is suggested that metabolism of lactate via the Krebs cycle may be responsible for the rise in GTP.
Abstract-l.
INTRODUCTION
Haematological responses to may differ markedly. Responses
hypoxia
and
Harbour (36”18’S; 174”46’E) using multifilament gill nets (110mm mesh size) set in a shallow mixed weed habitat after dusk and left for 45-90 min. The nets were then lifted and the fish transported immediately to the Leigh Marine laboratory, a distance of 11 km. Upon arrival at the laboratory the fish were anaesthetized with buffered MS-222 (1: 10,000) and cannulated via the caudal vein using PE-10 polyethylene tubing (Intramedic, Clay Adams). Fish were placed in 1500 1 tanks with a continuous supply of fresh sea-water (18 f 2°C). Samples of 0.25 ml whole blood were withdrawn at intervals of 1 hr for the following 12 hr using sodium heparin (400 i.u./ml) as an anticoagulant. A second group was captured in the same manner but left to recover from capture for 5-7 days in sea-water tanks. These fish were then cannulated as above and monitored for a 12 hr period to ascertain the haematological effects of cannulation alone. A further group of aquarium acclimated fish was subjected to induced capture stress by restraint in a landing net for a period of 30 min. Blood was then sampled by acute caudal venepuncture to determine haematological responses during the period of capture stress.
exercise
to hypoxia generally include metabolic control of blood oxygen affinity elicited by a decrease in the ratio of erythrocytic organic phosphates to haemoglobin (Weber et al., 1976; Lykkeboe and Weber, 1978; Weber and Lykkeboe, 1978; Soivio et al., 1980; Tetens and Lykkeboe, 1981) implying increased oxygen loading at the respiratory surfaces. Responses to exercise, however, may involve haemo-concentration and increased blood oxygen carrying capacity, without substantial allosteric phosphate control of blood oxygen affinity (Soivio and Oikari, 1976: Casillas and Smith, 1977; Perrier et al., 1978; Turner et al., 1983a, b). The effect is an implied increase in oxygen delivery to the tissues and removal of CO, and hydrogen ions. Previous studies on the blood chemistry of stressed fish have dealt primarily with aspects of hypoxia and severe exercise with relatively few studies addressing the problem of the effects of capture. The physiological effects of capture stress are partly known for comparatively few species of teleosts. These studies were primarily concerned with post-capture responses to hooking and angling (Bouck and Ball, 1966; Beggs et al., 1980; Wells and Davie, 1985) and trawl net capture (Dando, 1969). The present study serves to examine aspects of blood chemistry and haematology in an active coastal marine teleost subjected to gill net capture. The results are compared with those from earlier studies of marine and freshwater teleosts stressed by hypoxia and exercise.
MATERIALS
Haemoglobin The cyanmethaemoglobin method for estimating haemoglobin concentration (see Dacie and Lewis, 1975) is recommended for use with fish blood (Blaxhall, 1972). Aliquots of 5 ~1 whole blood were added to 1 ml Drabkin’s reagent, shaken and left to stand for 20min to ensure complete conversion of haemoglobin to cyanmethaemoglobin. The solution was then centrifuged to remove cell debris prior to reading absorbance at 540 nm in a Pye Unicam 1750 spectrophotometer. Nucleoside triphosphates Samples of 100~1 freshly drawn whole blood were deproteinized in 100 ~1 12% trichloroacetic acid (TCA). NTP levels were determined by the Sigma NADH+nzymatic technique (Sigma Technical Bulletin No. 366-UV) using 10% of the recommended reagent volumes in semi-micro cuvettes. Reduction in absorbance due to NADH depletion was monitored to a stable baseline using a Pye Unicam 1750 recording spectrophotometer. Thin-layer chromatography (TLC) of 30~1 aliquots of the TCA supematants was used to determine whole blood
AND METHODS
Fish capture und blood collection Parore (Girella tricuspidara Quoy and Gaimard) weighing between 800 and 12OOg were caught in the Whangateau 609
N. LING and R. M. G. WELLS
610
concentrations of adenosine triphosphate (ATP) and guanosine triphosphate (GTP) according to the method of Cashel et al. (1969). Samples were spotted onto polyethyleneimine impregnated plates chromatography (MN300, Machery-Nagel) and developed for 34 hr at 20°C in 1.5 M KH,PO, buffer (pH 3.5) in a glass chromatography tank by ascending chromatography. ATP and GTP spots were identified against Sigma standards under short-wave ultraviolet light and the spots scraped into 1.2 ml of 0.5 M Tris-HCI buffer containing 35 mM MgCI, (pH 7.5). Samples were mechanically mixed for 2 min to elute phosphates from the adsorbent and centrifuged at 12,OOOg for 5min. ATP and GTP concentrations were determined spectrophotometrically by peak absorbance at 259nm and 253 nm respectively.
Samples of 50~1 blood were deproteinized in 100~1 80/;, perchloric acid. Total blood lactate was determined by the Sigma NAD enzymatic technique employing 10% of the recommended reagent volumes. Production of NADH was monitored to a stable endpoint using a recording spectrophotometer.
RESULTS
of TLC revealed that the principal components of NTP were ATP and GTP. The total molar concentrations of ATP and GTP determined by TLC were equal to NTP determined by the enzymatic procedure. NTP concentrations are expressed in molar ratios of haemoglobin thus providing information which is independent of other haematological responses such as haemoconcentration or erythrocyte swelling. Following the cessation of stress a rise in the whole blood concentration of nucleoside triphosphates was observed. NTP levels were significantly higher (P < 0.05) in capture-stressed fish than in cannulated aquarium acclimated fish for most of the period following cannulation and persisting for at least 12 hr. The rise in NTP may be attributed to the significant rise in the levels of GTP (P < 0.05). GTP concentration rose steadily following the cessation of stress to reach a peak some 7-8 hr later (Fig. 1). Application
1.7
1
4 _
1.5 1 I
-I
1
11
3 0 0II
1.3
0.7 1 9
6
3
0
RECOVERY
TIME
12
(Hi
Fig. 2. 12 hr post-operative response of the molar ratio of ATP to Hb following capture and cannulation (a). and cannulation alone (m).
ATP decreased immediately following the period of stress, then subsequently increased to exceed initial levels after IC-12 hr (Fig. 2). The whole blood GTP/ATP ratio showed a significant increase (P < 0.05) during the initial period of recovery with a decline to resting values after 12 hr (Fig. 3). A significant inverse correlation, using analysis of variance (P < 0.05) was found between the periods of maximum increase in GTP and maximal decline in whole blood lactate during the recovery period shown in Fig. 4. No changes in the levels of organic phosphate compounds were observed during the period of induced capture stress (Fig. 5) despite a significant (P < 0.001) rise in blood lactate from 0.62 k 0.06 to 4.32 f 0.24 mM. DISCUSSION
Changes phosphate
in erythrocytic concentrations of organic compounds in fish during or following
1.7 -
.
[L
_-.----_--__
,_--
3 go.9.
. '\
0.7+. 0
.--_-_m' . .
.
.'
,'
/'
/'
F--m
.
.
RECOVERY
r”
0.7
_m--*---;-- ------ --. _____--l -________.
.
.
'.""'.
3
I:
6
9 TIME
( 12
(HI
Fig. 1. 12 hr post-operative response of the molar ratio of GTP to Hb following capture and cannulation (e), and cannulation alone (m).
Fig. 3. 12 hr post-operative response of the molar ratio of GTP to ATP following capture and cannulation (a). and cannulation alone (m).
Blood metabolites
‘X..
-L_..__l__..-~-l--I--~
OJ., 0
3
6 RECOVERY
12
9 T:XECHI
response of whole blood lactate following capture and cannulation (a), and cannulation alone (m).
Fig. 4. 12 hr post-operative
concentration
periods of hypoxia have been described as adaptive modifications of haemoglobin oxygen affinity. A decrease in the molar ratio of NTP to haemoglobin is documented as a response to hypoxia in fish, which may protect oxygen loading at the gills (Weber et al., 1976; Weber and Lykkeboe, 1978). The response of increased NTP/Hb following capture presumably improves oxygen delivery to the tissues since oxygen loading at the gills would not be greatly compromised at constant environmental oxygen levels during recovery and assuming that oxygen flux across the gill epithelium remains constant. The results suggest that during recovery there are changes in the relative proportions of ATP and GTP. Increase in NTP/Hb decreases haemoglobin oxygen affinity by the allosteric role of these compounds in addition to their disruptive influence on the Donnan distribution of protons across the erythrocyte membrane (Wood, 1980). The relatively high proportion
in capture
stressed
fish
611
of GTP following stress, even in the absence of a significant change in total phosphates, would be expected to decrease haemoglobin oxygen affinity because GTP is more potent than ATP as an allosteric modifier of fish haemoglobins (Weber et al., 1976; Peterson and Poluhowich, 1976; Weber and Lykkeboe, 1978; Weber and Wood, 1979). A separate study has shown GTP to have a greater effect than ATP on the depression of haemoglobin oxygen affinity in G. tricuspiduta (Watson, 1983). One mechanism to account for an increase in the relative ratio of GTP to ATP is an increase in erythrocytic phosphokinase activity. Parks et al. (1973) found two phosphokinases in fish blood capable of transferring high-energy phosphates between ATP and GTP. Erythrocytic ATP may act as a reservoir of high-energy phosphate for the more effective haemoglobin oxygen affinity modulator, GTP. Another explanation seems to warrant consideration. Since ATP is formed largely by oxidative phosphorylation and GTP formed via the Krebs Cycle (Wood, 1980) changes in the relative ratio of GTP to ATP may be a purely metabolic consequence of a post-capture lactate accumulation. In order to remove accumulated lactate from the blood as quickly as possible, metabolism via the Krebs Cycle is the most likely course of events. However, metabolism of some blood lactate has been shown to occur via gluconeogenesis (Batty and Wardle, 1979). Since the increase in erythrocytic GTP correlates with the period of maximum decline in blood lactate, metabolism of lactate to pyruvate, and subsequent increase in Krebs cycle activity, may be the reason for this increase. Metabolism of some blood lactate in this manner could provide the energy (GTP and NADH) required for gluconeogenic metabolism of the rest of the lactate load. This speculative explanation of blood metabolite changes following stress would be difficult to verify, however. All of the metabolic pathways involved are complex, and verification of such a theory requires a detailed study of the fate of such glycolytic intermediates at a molecular level. A~kno~~led~enzmts-This No. 141 ‘2 168 from Committee.
study was funded the Auckland University
by grant Research
REFERENCES
0.8
1
PRE STRESS
POSTSTRESS
Fig. 5. Responses of the molar ratios of GTP (o), and ATP (0) to Hb, and of GTP to ATP (m) following 30min
induced capture stress.
Batty R. S. and Wardle C. S. (1979)Restoration of glycogen from lactic acid in the anaerobic swimming muscle of plaice, Pleuronectes plaIe.wa L. J. Fish Biol. 15, 5099519. Beggs G. L., Holeton G. F. and Crossman E. J. (1980) Some physiological consequences of angling stress in muskellunge, Esor musquinongy. J. Fish Biol. 17, 649-659. Blaxhall P. C. (1972) The haematological assessment of the health of freshwater fish. A review of selected literature. J. Fish Biol. 4, 593-604. Bouck G. R. and Ball R. C. (1966) Influence of capture methods on blood characteristics and mortality in the rainbow trout (Salmo gairdnerii). Trans. Am. Fish. Sot. 95, 17&176. Cashel M., Lazzarini R. A. and Kalbacher B. (1969) An improved method for thin-layer chromatography of nucleoside mixtures containing iZP-labelled orthophosphates. J. Chrom. 40, 103-109.
612
N. LING and R. M. G. WELLS
Casillas E. and Smith L. S. (1977) Effect of stress on blood coagulation and haematology in rainbow trout (Sulmo gcrirdnrrii). J. Fish Biol. 10, 481-491. Dacie J. C. and Lewis S. M. (1975) Practical Haematology, 5th edn. Churchill Livingstone, Edinburgh. Dando P. R. (I 969) Lactate metabolism in fish. J. mar. biol. Ass. LJ.K. 49, 209-223. Lykkeboe C. and Weber R. E. (1978) Changes in the respiratory properties of the blood of the carp, Cyprinus car@, induced by diurnal variation in ambient oxygen tension. 1. camp. P~.~.~j~~.lzS, 117-125. Parks R. E., Brown P. R., Cheng Y., Agarwal K. C., Kong C. M., Agarwai P. P. and Parks C. C. (1973) Purine metabolism in primitive vertebrates. Comp. Biochem. Ph_wiol. 45B, 355-364. Perrier C., Terrier M. and Perrier H. (1978) A time course study of the effects of angling stress on cyclic AMP, lactate and glucose plasma levels in the rainbow trout (S&IO gairdnerii Richardson) during a 64hr recovery period. Camp. Biochem. Physiol. 6OA, 211-219. Peterson A. J. and Poluhowich J. J. (1976) The effects of organic phosphates on the oxygenation behaviour of eel multiple hemoglobins. Comp. Biochem. Physiol. SA, 351-354. Soivio A., Nikinmaa M. and Westman K. (1980) The blood oxygen binding properties of hypoxic Saimo gairdnerii. .J. romp. Physioi. 136, 83-87. Soivio A. and Oikari A. (1976) Haematological effects of stress on a teleost, Esox lucius. f. Fish Biol. 8, 397-411. Tetens V. and Lykkeboe G. (1981) Blood respiratory properties of rainbow trout, Saimo gairdnerii: responses to hypoxia acclimation and anoxic incubation of blood in vitro. J. romp. Physiol. 145, 117-125.
Turner J. D.. Wood C. M. and Clark D. (1983a) Lactate and proton dynamics in the rainbow trout (Salmo gairdnerii). J. exp. Biol. 104, 247-268. Turner J. D., Wood C. M. and Hobe H. (1983b) Physiological consequences of severe exercise in the inactive benthic flathead sole (Hippoglossoides ellasodon): a comparison with the active pelagic rainbow trout (Sa/mo gairdnerii). J. exp. Biol. 104, 269-288. Watson G. (1983) Respiratory properties of parore blood: a molecular study. Unpublished MSc. Thesis, University of Auckland. Weber R. E. and Lykkeboe G. (1978) Respiratory adaptations in carp blood; influences of hypoxia, red cell organic phosphates, divalent cations and CO, on haemoglobin-oxygen ahinity. J. camp. Physioi. 128, 127-137. Weber R. E., Lykkeboe G. and Johansen K. (1976) Physiological properties of eel haemoglobin: hypoxic acclimation, phosphate effects and multiplicity. J. exp. Biol. 64, 75-88. Weber R. E. and Wood S. C. (1979) Effects of erythrocyte nucleoside triphosphates on oxygen equilibria. of composite and fractionated hemoglobins from the facultative air-breathing Amazonian catfish Hypostomus and Pterygoplichihys. Comp. Biochem. Physioi. 82, 207-233. . Wells R. M. G. and Davie P. S. (1985) Oxygen binding by the blood and haematological effects of capture stress in two big gamefish: mako shark and striped marlin. Comp. Biochem. Physioi. 81 A, 643-646. Wood S. C. (1980) Adaptation of red cell function to hypoxia and temperature in ectothermic vertebrates. Am. 2001. 20, 163-172.
Biochem.
Vol. 82A. No. 3, pp. 609-612, 1985
Physiol.
in Great
Britain
0
0300-9629/85 $3.00 + 0.00 1985 Pergamon Press Ltd
CHANGES IN BLOOD METABOLITES FOLLOWING STRESS FROM CAPTURE AND HANDLING OF THE MARINE TELEOST GZRELLA TRZCUSPZDATA N. LING and R. M. G. Department
of Zoology,
University
of Auckland,
WELLS
Auckland,
New Zealand.
Telephone:
737-999
(Received 26 February 1985)
Whole blood nucleoside triphosphate (NTP) and lactate in the parore (Girella tricuspiduta, Fam: Kyphosidae) were monitored over a period of 12 hr following capture by gill net. 2. An increase in NTP during the post-capture recovery period was mainly attributable to a significant rise (P < 0.05) in the NTP component guanosine triphosphate (GTP). 3. The rise in GTP levels correlated with the decline in blood lactate (r = -0.72) accumulated during the period of capture stress. 4. It is suggested that metabolism of lactate via the Krebs cycle may be responsible for the rise in GTP.
Abstract-l.
INTRODUCTION
Haematological responses to may differ markedly. Responses
hypoxia
and
Harbour (36”18’S; 174”46’E) using multifilament gill nets (110mm mesh size) set in a shallow mixed weed habitat after dusk and left for 45-90 min. The nets were then lifted and the fish transported immediately to the Leigh Marine laboratory, a distance of 11 km. Upon arrival at the laboratory the fish were anaesthetized with buffered MS-222 (1: 10,000) and cannulated via the caudal vein using PE-10 polyethylene tubing (Intramedic, Clay Adams). Fish were placed in 1500 1 tanks with a continuous supply of fresh sea-water (18 f 2°C). Samples of 0.25 ml whole blood were withdrawn at intervals of 1 hr for the following 12 hr using sodium heparin (400 i.u./ml) as an anticoagulant. A second group was captured in the same manner but left to recover from capture for 5-7 days in sea-water tanks. These fish were then cannulated as above and monitored for a 12 hr period to ascertain the haematological effects of cannulation alone. A further group of aquarium acclimated fish was subjected to induced capture stress by restraint in a landing net for a period of 30 min. Blood was then sampled by acute caudal venepuncture to determine haematological responses during the period of capture stress.
exercise
to hypoxia generally include metabolic control of blood oxygen affinity elicited by a decrease in the ratio of erythrocytic organic phosphates to haemoglobin (Weber et al., 1976; Lykkeboe and Weber, 1978; Weber and Lykkeboe, 1978; Soivio et al., 1980; Tetens and Lykkeboe, 1981) implying increased oxygen loading at the respiratory surfaces. Responses to exercise, however, may involve haemo-concentration and increased blood oxygen carrying capacity, without substantial allosteric phosphate control of blood oxygen affinity (Soivio and Oikari, 1976: Casillas and Smith, 1977; Perrier et al., 1978; Turner et al., 1983a, b). The effect is an implied increase in oxygen delivery to the tissues and removal of CO, and hydrogen ions. Previous studies on the blood chemistry of stressed fish have dealt primarily with aspects of hypoxia and severe exercise with relatively few studies addressing the problem of the effects of capture. The physiological effects of capture stress are partly known for comparatively few species of teleosts. These studies were primarily concerned with post-capture responses to hooking and angling (Bouck and Ball, 1966; Beggs et al., 1980; Wells and Davie, 1985) and trawl net capture (Dando, 1969). The present study serves to examine aspects of blood chemistry and haematology in an active coastal marine teleost subjected to gill net capture. The results are compared with those from earlier studies of marine and freshwater teleosts stressed by hypoxia and exercise.
MATERIALS
Haemoglobin The cyanmethaemoglobin method for estimating haemoglobin concentration (see Dacie and Lewis, 1975) is recommended for use with fish blood (Blaxhall, 1972). Aliquots of 5 ~1 whole blood were added to 1 ml Drabkin’s reagent, shaken and left to stand for 20min to ensure complete conversion of haemoglobin to cyanmethaemoglobin. The solution was then centrifuged to remove cell debris prior to reading absorbance at 540 nm in a Pye Unicam 1750 spectrophotometer. Nucleoside triphosphates Samples of 100~1 freshly drawn whole blood were deproteinized in 100 ~1 12% trichloroacetic acid (TCA). NTP levels were determined by the Sigma NADH+nzymatic technique (Sigma Technical Bulletin No. 366-UV) using 10% of the recommended reagent volumes in semi-micro cuvettes. Reduction in absorbance due to NADH depletion was monitored to a stable baseline using a Pye Unicam 1750 recording spectrophotometer. Thin-layer chromatography (TLC) of 30~1 aliquots of the TCA supematants was used to determine whole blood
AND METHODS
Fish capture und blood collection Parore (Girella tricuspidara Quoy and Gaimard) weighing between 800 and 12OOg were caught in the Whangateau 609
N. LING and R. M. G. WELLS
610
concentrations of adenosine triphosphate (ATP) and guanosine triphosphate (GTP) according to the method of Cashel et al. (1969). Samples were spotted onto polyethyleneimine impregnated plates chromatography (MN300, Machery-Nagel) and developed for 34 hr at 20°C in 1.5 M KH,PO, buffer (pH 3.5) in a glass chromatography tank by ascending chromatography. ATP and GTP spots were identified against Sigma standards under short-wave ultraviolet light and the spots scraped into 1.2 ml of 0.5 M Tris-HCI buffer containing 35 mM MgCI, (pH 7.5). Samples were mechanically mixed for 2 min to elute phosphates from the adsorbent and centrifuged at 12,OOOg for 5min. ATP and GTP concentrations were determined spectrophotometrically by peak absorbance at 259nm and 253 nm respectively.
Samples of 50~1 blood were deproteinized in 100~1 80/;, perchloric acid. Total blood lactate was determined by the Sigma NAD enzymatic technique employing 10% of the recommended reagent volumes. Production of NADH was monitored to a stable endpoint using a recording spectrophotometer.
RESULTS
of TLC revealed that the principal components of NTP were ATP and GTP. The total molar concentrations of ATP and GTP determined by TLC were equal to NTP determined by the enzymatic procedure. NTP concentrations are expressed in molar ratios of haemoglobin thus providing information which is independent of other haematological responses such as haemoconcentration or erythrocyte swelling. Following the cessation of stress a rise in the whole blood concentration of nucleoside triphosphates was observed. NTP levels were significantly higher (P < 0.05) in capture-stressed fish than in cannulated aquarium acclimated fish for most of the period following cannulation and persisting for at least 12 hr. The rise in NTP may be attributed to the significant rise in the levels of GTP (P < 0.05). GTP concentration rose steadily following the cessation of stress to reach a peak some 7-8 hr later (Fig. 1). Application
1.7
1
4 _
1.5 1 I
-I
1
11
3 0 0II
1.3
0.7 1 9
6
3
0
RECOVERY
TIME
12
(Hi
Fig. 2. 12 hr post-operative response of the molar ratio of ATP to Hb following capture and cannulation (a). and cannulation alone (m).
ATP decreased immediately following the period of stress, then subsequently increased to exceed initial levels after IC-12 hr (Fig. 2). The whole blood GTP/ATP ratio showed a significant increase (P < 0.05) during the initial period of recovery with a decline to resting values after 12 hr (Fig. 3). A significant inverse correlation, using analysis of variance (P < 0.05) was found between the periods of maximum increase in GTP and maximal decline in whole blood lactate during the recovery period shown in Fig. 4. No changes in the levels of organic phosphate compounds were observed during the period of induced capture stress (Fig. 5) despite a significant (P < 0.001) rise in blood lactate from 0.62 k 0.06 to 4.32 f 0.24 mM. DISCUSSION
Changes phosphate
in erythrocytic concentrations of organic compounds in fish during or following
1.7 -
.
[L
_-.----_--__
,_--
3 go.9.
. '\
0.7+. 0
.--_-_m' . .
.
.'
,'
/'
/'
F--m
.
.
RECOVERY
r”
0.7
_m--*---;-- ------ --. _____--l -________.
.
.
'.""'.
3
I:
6
9 TIME
( 12
(HI
Fig. 1. 12 hr post-operative response of the molar ratio of GTP to Hb following capture and cannulation (e), and cannulation alone (m).
Fig. 3. 12 hr post-operative response of the molar ratio of GTP to ATP following capture and cannulation (a). and cannulation alone (m).
Blood metabolites
‘X..
-L_..__l__..-~-l--I--~
OJ., 0
3
6 RECOVERY
12
9 T:XECHI
response of whole blood lactate following capture and cannulation (a), and cannulation alone (m).
Fig. 4. 12 hr post-operative
concentration
periods of hypoxia have been described as adaptive modifications of haemoglobin oxygen affinity. A decrease in the molar ratio of NTP to haemoglobin is documented as a response to hypoxia in fish, which may protect oxygen loading at the gills (Weber et al., 1976; Weber and Lykkeboe, 1978). The response of increased NTP/Hb following capture presumably improves oxygen delivery to the tissues since oxygen loading at the gills would not be greatly compromised at constant environmental oxygen levels during recovery and assuming that oxygen flux across the gill epithelium remains constant. The results suggest that during recovery there are changes in the relative proportions of ATP and GTP. Increase in NTP/Hb decreases haemoglobin oxygen affinity by the allosteric role of these compounds in addition to their disruptive influence on the Donnan distribution of protons across the erythrocyte membrane (Wood, 1980). The relatively high proportion
in capture
stressed
fish
611
of GTP following stress, even in the absence of a significant change in total phosphates, would be expected to decrease haemoglobin oxygen affinity because GTP is more potent than ATP as an allosteric modifier of fish haemoglobins (Weber et al., 1976; Peterson and Poluhowich, 1976; Weber and Lykkeboe, 1978; Weber and Wood, 1979). A separate study has shown GTP to have a greater effect than ATP on the depression of haemoglobin oxygen affinity in G. tricuspiduta (Watson, 1983). One mechanism to account for an increase in the relative ratio of GTP to ATP is an increase in erythrocytic phosphokinase activity. Parks et al. (1973) found two phosphokinases in fish blood capable of transferring high-energy phosphates between ATP and GTP. Erythrocytic ATP may act as a reservoir of high-energy phosphate for the more effective haemoglobin oxygen affinity modulator, GTP. Another explanation seems to warrant consideration. Since ATP is formed largely by oxidative phosphorylation and GTP formed via the Krebs Cycle (Wood, 1980) changes in the relative ratio of GTP to ATP may be a purely metabolic consequence of a post-capture lactate accumulation. In order to remove accumulated lactate from the blood as quickly as possible, metabolism via the Krebs Cycle is the most likely course of events. However, metabolism of some blood lactate has been shown to occur via gluconeogenesis (Batty and Wardle, 1979). Since the increase in erythrocytic GTP correlates with the period of maximum decline in blood lactate, metabolism of lactate to pyruvate, and subsequent increase in Krebs cycle activity, may be the reason for this increase. Metabolism of some blood lactate in this manner could provide the energy (GTP and NADH) required for gluconeogenic metabolism of the rest of the lactate load. This speculative explanation of blood metabolite changes following stress would be difficult to verify, however. All of the metabolic pathways involved are complex, and verification of such a theory requires a detailed study of the fate of such glycolytic intermediates at a molecular level. A~kno~~led~enzmts-This No. 141 ‘2 168 from Committee.
study was funded the Auckland University
by grant Research
REFERENCES
0.8
1
PRE STRESS
POSTSTRESS
Fig. 5. Responses of the molar ratios of GTP (o), and ATP (0) to Hb, and of GTP to ATP (m) following 30min
induced capture stress.
Batty R. S. and Wardle C. S. (1979)Restoration of glycogen from lactic acid in the anaerobic swimming muscle of plaice, Pleuronectes plaIe.wa L. J. Fish Biol. 15, 5099519. Beggs G. L., Holeton G. F. and Crossman E. J. (1980) Some physiological consequences of angling stress in muskellunge, Esor musquinongy. J. Fish Biol. 17, 649-659. Blaxhall P. C. (1972) The haematological assessment of the health of freshwater fish. A review of selected literature. J. Fish Biol. 4, 593-604. Bouck G. R. and Ball R. C. (1966) Influence of capture methods on blood characteristics and mortality in the rainbow trout (Salmo gairdnerii). Trans. Am. Fish. Sot. 95, 17&176. Cashel M., Lazzarini R. A. and Kalbacher B. (1969) An improved method for thin-layer chromatography of nucleoside mixtures containing iZP-labelled orthophosphates. J. Chrom. 40, 103-109.
612
N. LING and R. M. G. WELLS
Casillas E. and Smith L. S. (1977) Effect of stress on blood coagulation and haematology in rainbow trout (Sulmo gcrirdnrrii). J. Fish Biol. 10, 481-491. Dacie J. C. and Lewis S. M. (1975) Practical Haematology, 5th edn. Churchill Livingstone, Edinburgh. Dando P. R. (I 969) Lactate metabolism in fish. J. mar. biol. Ass. LJ.K. 49, 209-223. Lykkeboe C. and Weber R. E. (1978) Changes in the respiratory properties of the blood of the carp, Cyprinus car@, induced by diurnal variation in ambient oxygen tension. 1. camp. P~.~.~j~~.lzS, 117-125. Parks R. E., Brown P. R., Cheng Y., Agarwal K. C., Kong C. M., Agarwai P. P. and Parks C. C. (1973) Purine metabolism in primitive vertebrates. Comp. Biochem. Ph_wiol. 45B, 355-364. Perrier C., Terrier M. and Perrier H. (1978) A time course study of the effects of angling stress on cyclic AMP, lactate and glucose plasma levels in the rainbow trout (S&IO gairdnerii Richardson) during a 64hr recovery period. Camp. Biochem. Physiol. 6OA, 211-219. Peterson A. J. and Poluhowich J. J. (1976) The effects of organic phosphates on the oxygenation behaviour of eel multiple hemoglobins. Comp. Biochem. Physiol. SA, 351-354. Soivio A., Nikinmaa M. and Westman K. (1980) The blood oxygen binding properties of hypoxic Saimo gairdnerii. .J. romp. Physioi. 136, 83-87. Soivio A. and Oikari A. (1976) Haematological effects of stress on a teleost, Esox lucius. f. Fish Biol. 8, 397-411. Tetens V. and Lykkeboe G. (1981) Blood respiratory properties of rainbow trout, Saimo gairdnerii: responses to hypoxia acclimation and anoxic incubation of blood in vitro. J. romp. Physiol. 145, 117-125.
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